Plasmodium hepatocyte interactions : implications for protection against malaria
Abstract
A malária é uma das doenças infecciosas mais importantes a nível mundial, sendo anualmente responsável por mais de 1 milhão de mortes. O agente causador da doença é o parasita intracelular, denominado Plasmodium, que possui um ciclo de vida bastante complexo. A infecção tem início com a inoculação de esporozoítos através da picada de um mosquito fêmea Anopheles, o vector de transmissão da doença. Uma vez na corrente sanguínea, o esporozoíto migra até ao fígado onde infecta a célula hospedeira, o hepatócito. No fígado, o parasita replica-se e desenvolve-se até atingir o seu próximo estado de maturação – o merozoíto. Dada esta complexidade, é natural que o Plasmodium provoque no hospedeiro uma variedade de mecanismos distintos, especialmente ao nível imunitário. Por isso, a resposta imune desenvolvida pelo hospedeiro, contra o parasita, é caracterizada como sendo complexa e específica em relação à espécie e ao estádio do mesmo. De forma a adquirir uma resposta imune protectora contra a malária é necessário que o indivíduo seja infectado consecutivamente durante a vida. Mesmo assim o resultado obtido é apenas uma imunidade parcial contra o parasita. Melhorias significativas têm sido registadas, no que diz respeito à compreensão dos mecanismos de protecção envolvidos na doença, assim como na identificação de novas moléculas que possam ser utilizadas no desenvolvimento de novas vacinas. No entanto, ainda não está disponível uma vacina que seja eficaz conferindo uma protecção total. O uso de esporozoítos irradiados em imunizações induz uma protecção total contra a doença, que é mediada pela activação de linfócitos T CD8+ específicos para antigénios do parasita. O início desta resposta é mediado por células dendríticas, embora a origem dos antigénios intervenientes seja ainda desconhecida. Os esporozoítos irradiados conseguem infectar os hepatócitos. Contudo, não são capazes de progredir para a fase sanguínea da doença. Este desenvolvimento incompleto da fase hepática é uma característica fundamental para que ocorra imunidade. Embora alguns dos mecanismos protectores induzidos pela infecção com esporozoítos irradiados já tenham sido identificados é, ainda, necessário proceder a uma caracterização detalhada dos mesmos. Sendo o fígado um local de extrema importância durante o ciclo de vida do parasita da malária, qualquer descoberta ao nível das interacções que se estabelecem entre o Plasmodium e o hepatócito, terá uma repercussão no melhoramento do processo de indução de uma resposta imune contra a doença. Utilizando um modelo murino, demonstrou-se que os hepatócitos infectados com esporozoítos irradiados entram em apoptose logo após o início da infecção. Durante esta fase as célulasdendríticas são recrutadas para o fígado, local onde fagocitam os corpos apoptóticos provenientes dos hepatóctios que entraram em morte celular. Uma vez que estas células são capazes de apresentar antigénios exógenos e ainda induzir o priming e activação das células T, os resultados por nós obtidos sugerem que os hepatócitos infectados apoptóticos são a fonte de antigénios do parasita utilizada pelas células dendríticas durante a iniciação de uma resposta imune contra a malária. Durante o curso de uma infecção, a morte celular possui um papel fundamental no estabelecimento de uma resposta imune contra um agente patogénico. Os parasitas possuem a capacidade de modular esta resposta através da indução ou inibição da morte da célula hospedeira, de forma a possibilitar o seu desenvolvimento e sobrevivência. Previamente, foi demonstrado que durante a migração dos esporozoítos através dos hepatócitos, as células atravessadas secretam um factor de crescimento específico, o HGF -“hepatocyte growth factor”, que aumenta a susceptibilidade celular à infecção. Esta via de sinalização iniciada pelo HGF através do seu receptor, o MET, provoca uma série de efeitos em diversos tipos de células. Entre eles destaca-se a protecção contra a morte celular programada. Considerando tal facto, estudou-se qual o efeito desta protecção durante a infecção por Plasmodium, tendo como hipótese que a activação da via de sinalização do HGF/MET induziria uma protecção da apoptose nas células infectadas. Os resultados por nós obtidos confirmaram esta teoria. A inibição desta via de sinalização induziu um aumento na quantidade de morte celular observada. Tendo em conta que, usualmente, a activação da sinalização do HGF ocorre segundo o sinal de transdução do PI3K/Akt, testou-se se o bloqueio desta via produzia algum efeito na infecção. De facto, os resultados observados indicam que esta via de sinalização é utilizada durante a infecção quando o HGF/MET é activado. Estas observações demonstram que a inibição da apoptose da célula hospedeira durante a infecção por Plasmodium é necessária para ocorrer doença. Na parte final deste trabalho, procurou-se ainda identificar um gene do parasita responsável pela inibição da morte do hepatócito. Algumas observações preliminares levaram-nos a sugerir que a proteína HSP70 do parasita possa exercer uma função neste processo, daí que sugerimos que no futuro este envolvimento seja mais aprofundado. Assim, os resultados apresentados nesta tese contribuem para um maior esclarecimento e compreensão das interacções que se estabelecem no fígado aquando da infecção por Plasmodium, e para um conhecimento mais alargado da relação entre o parasita e o hepatócito.
Full text
Plasmodium
-Hepa ocy e in e ac ions:
implica ions o p o ec ion agains Mala ia
Pa ícia Rod igues Saa ed a Lei ião
2006
UNIVERSIDADE NOVA DE LISBOA
Ins i u o de Higiene e Medicina T opical
Plasmodium
-Hepa ocy e in e ac ions:
implica ions o p o ec ion agains Mala ia
Pa ícia Rod igues Saa ed a Lei ião
A disse a ion submi ed o ob ain a Doc o o
Philosophy deg ee in Biomedical Sciences, Pa asi ology
speciali y, by he Uni e sidade No a de Lisboa,
Ins i u o de Higiene e Medicina T opical.
Supe ise : Dou o a Ma ia Manuel Mo a
Ins i u o de Medicina Molecula - FML
Ins i u o Gulbenkian de Ciência
Co-Supe ise : Dou o a Ana Rod iguez
Medical and Molecula Pa asi ology Depa amen - NYU
2006
P e ace
This hesis assembles da a ob ained du ing my PhD esea ch p ojec de eloped a he New Yo k
Uni e si y School o Medicine, Depa men o Medical and Molecula Pa asi ology; and a he
Ins i u o Gulbenkian de Ciência, om Augus 2001 o July 2005. The wo k was supe ised by
Doc o Ma ia Manuel Mo a and co-supe ised by Doc o Ana Rod iguez. The inancial suppo was
p o ided by Fundação pa a a Ciência e Tecnologia wi h a PhD ellowship g an
(SFRH/BD/3230/2000).
This hesis is s uc u ed in 7 chap e s, which a e p eceded by a summa y, bo h in Po uguese and
English, ou lining he aims, esul s and ou comes, o his Mala ia esea ch p ojec . The i s chap e
places ou wo k wi hin he Mala ia scien i ic ield gi ing a backg ound and signi icance o i s inpu ,
and also speci ies he objec i es ha we p oposed o accomplish. A gene al in oduc ion o Mala ia
an i s cu en wo ld si ua ion is p esen ed in chap e wo, oge he wi h a li e a u e e iew o he
la e insigh s o li e s age biology and immuni y, in addi ion o he p esen knowledge conce ning
apop osis a hos -pa hogen in e ace. A desc ip ion o he me hods and ma e ials employed o ca y
ou he p esen wo k is done in chap e h ee. The esul s ob ained h oughou his esea ch p ojec
a e p esen ed in he nex h ee chap e s om ou o six. Each one is o ganized as ollow: a sho
speci ic in oduc ion o he pa icula subjec , da a obse ed and analyzed, and a discussion o hose.
Finally, chap e se en encloses an o e all discussion and conclusion o he s udies pe o med,
oge he wi h an addi ional pe spec i e o he ecen highligh s o wha lies ahead wi hin his
scien i ic a ea o Mala ia esea ch. In Appendix a e included he publica ions ha de i ed om his
p ojec .
The da a p esen ed in his disse a ion is he esul o my own wo k and i is s a ed in he ex
whene e da a o eagen s p oduced by o he s as pa o collabo a ions we e used. This wo k has
no been p e iously submi ed o any deg ee a his o any uni e si y.
P e ace|iii
Co e Legend| A cell unde going apop osis wi h gene a ion o apop o ic bodies ha a e engul ed by a phagocy e.
(adap ed om Ke ,1995)
Acknowledgemen s
Once I hea d someone s a e ha “Be e han doing Science i sel , is who you do i wi h!” This complies wha I
belie e has been one o he g ea es accomplishes o hese las yea s, all o you ha I had he p i ilege o li e, wo k
and lea n om. Looking back I now ealize ha i eally made all he di e ence and he e o e I ake his oppo uni y
o acknowledge e e y one.
Fi s I would like o hank Ma ia Manuel Mo a o accep ing me as s uden and gi ing me he oppo uni y o pe o m
his esea ch p ojec . You supe ision, ideas, discussions, pa ience, iendship and you special endless op imism and
en husiasm owa ds Science (e e y hing is always an as ic!), has con ibu ed o he e y ewa ding expe ience ha
u n ou o be hese las yea s. I also would like o hank you o you e o s in p o iding all o us a op scien i ic
en i onmen o wo k. I lea n a lo and I will be always g a e ul o you.
Se e al hanks o Ana Rod iguez o ecei ing me in you lab and accep ing o co-supe ise his p ojec . Thank you
o in oducing me o he ascina ing dend i ic cell wo ld and o showing me he beau y o cell biology. You
guidance, opinions, and discussions, con ibu ed o make his li e ime expe ience.
To P o esso Vi gílio E. do Rosá io and o he Cen o de Malá ia e ou as Doenças T opicais, o all you suppo ,
pa ience wi h he adminis a i e dealings, and o o e ing and pe suading me in o s a ing his PhD. Thank you also
o accep ing o be my uni e si y ad iso and being pa o my scien i ic commi ee. I was om you ha I i s hea
abou he impo ance o wo king wi hin he mala ia esea ch ield.
To P o esso D Ma ia Amélia G ácio, o accep ing me as a PhD s uden a he Ins i u o de Higiene e Medicina
T opical, gi ing me he oppo uni y o p esen and submi his esea ch wo k o a doc o al deg ee. I also would like
o acknowledge D Ru h Nussenzweig o welcoming me a he NYU Medical and Molecula Pa asi ology
Depa men . He sympa hy and example o pe se e ance we e ex ao dina y. To D Michael Pa khouse and D
Sukalyan Cha e jee, o accep ing o be pa o my IGC commi ee and o he supe b discussion o my wo k and an
o e all look a Science as a big pic u e.
To Sónia, my colleague and iend, you in e es , wo k and dedica ion, con ibu ed di ec ly o he ou come o he
HGF wo k p esen ed in his hesis. Thank you so much o you p ecious help and endless hou s a he
mic oscope…wi hou you I would ne e had inished his. Lo e you unique and exquisi e sense o humou and you
a i ude (please do no e e change gi l! Big kiss!).
To he es o my pas and p esen colleagues o he Mala ia Cell Biology g oup, Dani, Lau i a, C is, Ma , Sa, Ana,
B uno, Susana, Ma a, Miguel, Nuno, Sil ia, Iana, Rica do, C is ina, Ca ina and Ana Robe o; I am in deb o each
one o you o all you help, suppo , en husiasm, incen i e, ideas, sugges ions, discussions, pa ience and iendship. I
since ely hink ha we ha e one o kind g oup o people ha is a e o ind. I do no ake you o g an ed, so he e o e I
will always che ish hese yea s and hope o con inue o do so wi h you nea by. Mil ob igadinhos ppl!!!
Fo a li e s age mala ia lab, he mos p ecious and indispensable gi is ou in e eb a e iends. Thus, eno mous big
hanks is en i le o all o he an as ic people ha p oduce, main ain and in ec ou supe b mosqui oes. Wi hou hem
his en i e p ojec would ha e no been possible. So he e is my acknowledge o I e e Ca o-Aguila , Jean Noonan
(amazing Jamaica smile!), Claudia Canas o, Ca a ina Al es, Ca a ina Casimi o, Gee -Jan an Geme and Robe W.
Saue wein. Many hanks also o Simona Co so and Sil ia Gio dano o es ablishing a g ea collabo a ion wi h ou
g oup, and especially o p o iding he HGF/MET cell lines and use ul ad ices. To Susana Cons an ino o a p ecious
echnical help.
To Ca los (La in music ules o cou se!), Jullius, U ashi, Pu nima, Ka hy, Alexand e, Gen, Pho ini, Ge a d, Jessica,
Ka ine, and K is ine; a big hanks o e e y one o you help, echnical assis ance, iendship and o making he
depa men a un se o wo k. To my big apple Po uguese acquain ances: Ma ga ida Ca olo ( he unbea able
mo ning ene gy), i was g ea ha ing you a ound, hanks o e e y hing we sha ed; and Ca a ina Figuei edo, huge
hanks o all you help, ca ing and o jus being like you a e!
To he Ins i u o Gulbenkian de Ciência and o i s c owd, especially o he membe s o he Mala ia g oups and ou
neighbou g oups in ala Gil Eanes, hank you o p o iding an excep ional en i onmen o wo k and ha ing un doing
i , and also o you pe manen a ailabili y o help. As hey say he pen house is always he bes (jus easing, lols)!
Bu , o cou se in any ule he e a e excep ions, so a big hanks o he S ess and T ansc ip ion g oup, Célia, Filipa,
Susaninha, Nuno, Ma isa, and e e yone else o cou se.
I would like o hank Ca ina, Miguel and Tiago o ha ing he pa ience o e iewing his hesis and some o you ha
also ga e you opinion a ce ain poin o his wo k. To Fundação pa a a Ciência e Tecnologia, and o he PRAXIS
XXI p og am o unding me wi h a PhD ellowship.
To C is y, Michelle and Ka hy, he c azy La in gi ls ha ga e a whole new meaning o he Big Apple my li e he e
wi hou you…many hanks o you iendship. To Heloísa, o sha ing he s eng h, since i y and inc edible al o
Aknowledgemen s|
Table o Con en s
P e ace iii
Aknowledgemen s
Sumá io ii
Abs ac ix
Abb e ia ions xi
Table o Con en s xiii
Index o Figu es x
Index o Tables x i
Chap e one
Backg ound, Signi icance & Objec i es 1
1.1 Backg ound and Signi icance 3
1.2 Objec i es 6
Chap e Two
Gene al In oduc ion 7
2.1 Mala ia: his o ical aspec s and cu en global pic u e 9
2.2 Plasmodium li e cycle 11
2.3 Li e s age biology 12
2.3.1 F om skin o li e 13
2.3.2 Reaching he li e 14
2.3.3 Hepa ocy e in asion 16
2.3.4 In ahepa ic de elopmen 17
2.4 Immuni y o mala ia li e s age 17
2.4.1 Na u al in ec ion e sus immuniza ion 18
2.4.2 Exo-e y h ocy ic an igens 19
2.4.3 Ini ia ion o hos immune esponse 20
2.4.4 Hos immune esponses a e immuniza ion 20
2.5 Apop osis a he hos -pa hogen in e ace 23
2.5.1 Cell dea h 23
2.5.2
Pa hways o apop osis 25
2.5.3
In acellula li ing 27
2.5.4
Modula ion o apop osis and pa asi e su i al/dea h
27
2.5.5
Modula ion o apop osis and immuni y 30
Table o Con en s|xiii
Chap e Th ee
Ma e ials and Me hods 31
3.1 Cells 33
3.2 Pa asi es, mosqui oes and mice 33
3.3 Spo ozoi e isola ion and pu i ica ion 33
3.4 In i o in ec ions 33
3.5 Immuno luo escence assays 34
3.6 Cell ea men du ing spo ozoi e in ec ion 35
3.7 Apop osis induc ion assays 35
3.8 De ec ion o apop osis 35
3.9 Dend i ic cell ec ui men o he li e 36
3.10 In i o in ec ion, isola ion and s aining o li e mononuclea cells 36
3.11 De ec ion and quan i ica ion o in i o Plasmodium spo ozoi e in ec ion using Re e se
T ansc ip ion and Real-Time PCR 37
3.12 Inhibi ion o PI3-kinase pa hway du ing Plasmodium in ec ion 37
3.13 Analysis o AKT exp ession du ing Plasmodium in ec ion 37
3.14 Immunop ecip i a ion and Wes e n blo analysis 38
3.15 MET down-modula ion by siRNA in hepa oma cell line 38
3.16 Assembly o DN MET-GFP hepa oma cell line 39
3.17 Isola ion o plasmid DNA 39
3.18 DNA es ic ion enzyme diges ion 39
3.19 DNA Elec opho esis 40
3.20 DNA Ex ac ion om aga ose gels 40
3.21 Cloning PCR p oduc s 40
3.22 HSP70 cons uc 40
3.23 In i o ansien ans ec ion sys em 40
Chap e Fou
Resul s I - Role o dend i ic cells du ing li e s age mala ia and he ini ia ion o
an immune esponse 41
4.1 In oduc ion 43
4.2 Resul s 45
4.2.1 I adia ed Plasmodium spo ozoi es induce in ec ed hepa ocy es apop osis in i o 45
4.2.2 The ole o dend i ic cells du ing P. yoelii in ec ion 48
4.2.2.1 Rec ui men o dend i ic cells o he li e a e mala ia in ec ion 49
4.2.2.2 Plasmodium an igens a e phagocy osed by dend i ic cells and mac ophages 50
4.2.2.3 Es ablishmen o a c oss-p esen a ion assay 53
4.3 Discussion 54
Chap e Fi e
Resul s II - An i-Apop o ic HGF/MET signalling in mala ia in ec ion 59
5.1 In oduc ion 61
5.2 Resul s 63
5.2.1 Spo ozoi e in ec ion p o ec s cells om apop osis 63
5.2.2 HGF/MET signalling enhances in ec ion and con e s p o ec ion 64
5.2.3 MET inac i a ion leads o apop osis du ing in ec ion 66
xi |Table o Con en s
5.2.4 MET down- egula ion leads o apop osis du ing in ec ion 68
5.2.5 An i-apop o ic e ec o HGF/MET signalling is media ed ia
ac i a ion o PI3K pa hway 69
5.2.6 Inhibi ion o PI3K pa hway dec eases Plasmodium in ec ion in i o 70
5.3 Discussion 70
Chap e Six
Resul s III - Does PyHSP70 inhibi in ec ed hepa ocy e apop osis? 73
6.1 In oduc ion 75
6.2 Resul s 78
6.2.1 PyHSP70 exp ession p o ec s hepa ocy es om dea h 78
6.2.2 PyHSP70 knock ou pa asi e 79
6.3 Discussion 79
Chap e Se en
Final Discussion & Conclusion 81
7.1 Final Discussion and Conclusion 83
7.2 Pe spec i es 91
Bibliog aphy 93
Appendix 127
Index o Figu es
Fig. 2.1|Global dis ibu ion o mala ia. 11
Fig. 2.2|Plasmodium li e cycle. 12
Fig. 2.3|Pa hways o apop osis. 26
Fig. 3.1|Schema ic ep esen a ion o he in i o spo ozoi e in ec ion sys em. 34
Fig. 4.1|Time-cou se o an in i o in ec ion wi h non-i adia ed o
i adia ed P. yoelii spo ozoi es. 46
Fig. 4.2|I adia ed P. yoelii spo ozoi es lead o he apop osis o he in ec ed hepa ocy e. 47
Fig. 4.3|Quan i ica ion o apop osis a ea ly ime poin s a e in ec ion. 47
Fig. 4.4|Apop o ic cell in ec ed wi h i adia ed P. yoelii spo ozoi es. 48
Fig. 4.5|Dend i ic cells a e p esen in he li e a e in ec ion wi h Plasmodium spo ozoi es. 49
Table o Con en s|x
Fig. 4.6|Dend i ic cells a e ec ui ed o he li e a e in ec ion wi h P. yoelii spo ozoi es. 50
Fig. 4.7|Phagocy osis o apop o ic hepa ocy es con aining Plasmodium an igens by
dend i ic cells and mac ophages. 51
Fig. 4.8|Hepa ocy e p o eins a e ound in P. yoelii phagocy osed esicles. 52
Fig. 4.9|Caspase-3 is ac i e in P. yoelii phagocy osed apop o ic bodies. 52
Fig. 5.1|Schema ic ep esen a ion o he di e en pa hways ha a e ac i a ed
by HGF/MET signalling. 62
Fig. 5.2|Plasmodium in ec ion p o ec s om dea h. 63
Fig. 5.3|In ec ed cells a e mo e esis an o apop osis. 64
Fig. 5.4|HGF-induced MET ac i a ion enhances in ec ion and p o ec s
in ec ed cells om apop osis. 65
Fig. 5.5|HGF agonis s induce in ec ion and hos cell p o ec ion. 66
Fig. 5.6|Inhibi ion o caspase ac i i y inc eases in ec ion. 66
Fig. 5.7|MET inac i a ion leads o ea ly apop osis o in ec ed cells. 67
Fig. 5.8|Dominan nega i e MET-GFP in ec ed cell unde going apop osis. 68
Fig. 5.9|MET exp ession on sho in e e ing RNA cell line. 68
Fig. 5.10|Down-modula ion o HGF/MET signaling leads o ea ly apop osis o
in ec ed cells. 68
Fig. 5.11|Inhibi ion o PI3K/AKT pa hway in i o leads o a dec ease in in ec ion. 69
Fig. 5.12|In i o inhibi ion o PI3K/AKT pa hway du ing in ec ion. 70
Fig. 6.1|E en s egula ed by HSPs in he mi ochond ial and dea h
ecep o -media ed apop o ic pa hways. 76
Fig. 6.2|P. yoelii HSP70 p o ec s hepa ocy es om apop osis. 79
Index o Tables
Table 3.1|An ibodies used in immuno luo escence s ainings. 35
x i|Table o Con en s
Chap e One
Backg ound,
Signi icance & Objec i es
1.1 Backg ound and Signi icance
Mala ia is one o he mos p e alen and se e e human in ec ious diseases in he wo ld. Recen ly i s
incidence has inc eased and i is es ima ed mo e han 300 million episodes o acu e illness occu in
endemic coun ies and a leas 1 million people die pe yea om his disease (WHO, 2003). Wi h
41% o he wo ld’s popula ion exposed o his h ea , mala ia also imposes an ex eme bu den on o
a ec ed popula ions as economy and de elopmen a e deeply impai ed esul ing om he
symp oms expe ienced by in ec ed indi iduals. Applicable measu es o con ol he disease a y
acco ding o each coun y’s endemici y bu se e al ac o s con ibu e o mala ia esu gence such as
d ug- esis an pa asi e s ains and insec icide- esis an mosqui oes (G eenwood e al., 2005; WHO,
2003).
A e decades o a ela i e lack o a en ion, new e o s a e p esen ly being made o add ess hese
challenges, such as new s a egies ha a e being applied o he de elopmen o an e ec i e accine
(Moo hy e al., 2004; Tong en e al., 2004). Al hough in e ms o public heal h accina ion has
always been a p io i y, i s ill emains an elusi e and complex esea ch ield. A comp ehensi e
knowledge o he hos -pa asi e in e ace would cons i u e a einsu ed gua an ee o a aining his
goal success ully.
Mala ia is caused by he in acellula Apicomplexan pa asi e Plasmodium spp., which holds a complex
li e cycle in ol ing di e en hos s and s ages o in ec ion. The i s s ep in mala ia in ec ion is he
in asion o he li e by Plasmodium spo ozoi es. These a e he in ec i e o m o he pa asi e,
ansmi ed by emale anopheline mosqui oes du ing a blood meal. Inside hepa ocy es, spo ozoi es
eplica e and de elop in o a me ozoi e s a e, a p ocess ha cons i u es he hepa ic s age o he
pa asi e li e cycle. Al hough, being an obliga o y s ep owa ds he es ablishmen o a success ul
mala ia in ec ion, his s age o Plasmodium li e cycle is poo ly unde s ood. This pa asi e speci ici y
owa ds he li e cells indica es ha pa asi e-encoded su ace p o eins and hos su ace ecep o s
a e implica ed in he p ocess o in asion and de elopmen , playing a key ole in he es ablishmen
o in ec ion. Howe e , he mechanisms, as well as he hos and pa asi e molecules, a play du ing
he cou se o in ec ion in he mammalian hos by mala ia spo ozoi es a e no en i ely known
(G une e al., 2003; Mille e al., 2002; Plebanski and Hill, 2000).
The ull equi emen s o Plasmodium de elopmen inside hepa ocy es a e s ill unknown. The lack o
an adequa e in i o sys em capable o deli e ing su icien ma e ial has deeply impeded esea ch
wi hin he s udy o hos /pa asi e in e ac ions. Cu en ly, in i o cul i a ion me hods ha e been
de eloped, and hepa oma cell lines a e being used as in i o models o Plasmodium in ec ion o
s udy he molecula and cellula basis o in asion mechanisms and in acellula de elopmen o he
pa asi e. Ne e heless, in ec ion yields emain e y low and he limi ed numbe o in ec i e
Backg ound, Signi icance & Objec i es|3
mosqui oes a ailable impai s a swi p og ess in he unde s anding o mala ia li e s age biology
(Mo a and Rod iguez, 2000; Cal o-Calle e al., 1994; Hollingdale e al., 1983b).
The a ailabili y o he genome sequence o bo h se e al Plasmodium species and hei hos p o ides
a gene ic ool ha g ea ly inc eases he possibili ies o esea ch in his a ea (Cooke and Coppel,
2004).
The li e , besides being he place whe e ampli ica ion and molecula changes o Plasmodium
pa asi es ake place, is also a unique o gan in wha ega ds he hos ’s immune esponses (Knolle and
Ge ken, 2000). The an igenic pool gene a ed wi hin his o gan is equi ed o he induc ion and
main enance o a p o ec i e an i-mala ia immune esponse. Thus, he hepa ic s age may well hold
he sec e o unde s anding he pa asi e’s p e e ence o his o gan wi hin he mammalian hos and,
simul aneously, p o iding e ec i e immune a ge s agains he disease (Baldacci and Ména d, 2004;
F e e , 2004; K zych e al., 2000).
Unlike many o he diseases in which a li elong esis ance o e-in ec ion is induced, mala ia causes
only pa ial immuni y a e se e al yea s o ecu ing in ec ions and illness. None heless, a comple e
esis ance o mala ia can be achie ed by accina ion wi h adia ion-a enua ed spo ozoi es in bo h
mice and humans (Doolan and Ho man, 2000; Weiss, 1990; Clyde e al., 1975; Nussenzweig e al.,
1972; Nussenzweig e al., 1967).
I adia ed spo ozoi es in ec hepa ocy es as no mal spo ozoi es, bu hey do no each a me ozoi e
s age (Schelle e al., 1995; Sigle e al., 1984). Addi ionally, i is known ha i is essen ial ha a
hepa ic s age occu s du ing in ec ion o he e ec i eness o i adia ed-spo ozoi e p o ec ion
(Schelle and Azad, 1995). Al hough, un il now his is he only accine ha con e s comple e
p o ec ion, he mechanism behind he p ocess o he es ablishmen o he immune esponse
associa ed is s ill unclea (Doolan and Ho man, 2000).
In acellula pa hogens ha e he capaci y o modula e he hos cell esponse and exploi i s esou ces
in o de o de elop and eplica e. Pa asi es can manipula e hos cell beha io , including immune
modula ion and egula ion o apop osis (James and G een, 2004). While in ec ed cells a e capable o
ini ia ing hei own dea h, a p ocess called apop osis, which can be used by he o ganism as a
de ense mechanism agains pa hogens, inhibi ion o hos cell apop osis is equen ly used by
pa asi es as a s a egy o su i al (Heussle e al., 2001; Lude e al., 2001).
Du ing he cou se o in ec ion, apop osis o in ec ed hos cells may ei he be induced by he hos
cell esponse o be a di ec esul o pa hogen in asion. In bo h cases, apop o ic dea h esul s in he
o ma ion o apop o ic bodies. These apop o ic bodies a e aken up by phagocy es ha apidly
ecognize and phagocy ose hem, elimina ing he pa asi e oge he wi h he emains o he in ec ed
cell. The pa hogen-de i ed an igens included in apop o ic bodies can be p esen ed by dend i ic cells
in he con ex o bo h class I and class II molecules, which a e ecognized by CD8+ and CD4+ T
4| Chap e One
cells espec i ely, and ac i a e naï e lymphocy es o he ini ia ion o an immune esponse (Schaible
e al., 2003; Sa ill e al., 2002; Rod iguez e al., 1999; Albe e al., 1998a).
Dend i ic cells a e key playe s in ini ia ing immune esponses because hey a e he only cell ype ha
a e able o p ime naï e T cells e icien ly as well as c oss-p esen exogenous an igens (Banche eau e
al., 2000; Mellman e al., 1998).
I has been shown ha dend i ic cells a e able o induce P. yoelii-speci ic CD8+ and CD4+ T cells
(B una-Rome o and Rod iguez, 2001). Addi ionally, dend i ic cells-deple ed mice ailed o induce
cy o oxic T cell esponses wi h a esul in he loss o p iming po en ial o dend i ic cells du ing P.
yoelii in ec ion (Jung e al., 2002).
Al oge he , hese obse a ions sugges an impo an ole o dend i ic cells du ing he immune
esponse ini ia ed agains li e s ages. An ex ended comp ehensi e knowledge o hese molecula
and cellula e en s will p o ide p omising immedia e applica ions in he ield o accine
de elopmen .
Backg ound, Signi icance & Objec i es|5
and de elops in o an oocys o m whe e spo ozoi es a e de eloped (Mille e al., 2002; Gysin, 1998;
Landau and Gau e , 1998). When ully ma u ed, oocys s bu s and elease spo ozoi es which
mig a e in o he mosqui o’s sali a y glands, comple ing Plasmodium’s li e cycle (Fig. 2.2).
Fig. 2.2|Plasmodium li e cycle.
When an in ec ed emale anopheline
mosqui o akes a blood meal,
spo ozoi es en e he hos
bloods eam and a el h ough he
ci cula ion o he li e (A).
Spo ozoi es in ade hepa ocy es and
en e a phase o asexual
ep oduc ion in which hey ampli y
hei numbe housands o imes by
he p oduc ion o me ozoi es (B).
The li e cell up u es and he
me ozoi es a e eleased in o he
blood, a aching o and in ading
e y h ocy es, beginning he
e y h ocy ic cycle. Each me ozoi e in ades an e y h ocy e and eplica es ini ia ing a cycle ha ends wi h he bu s o he ma u e
e y h ocy ic schizon and he elease o new me ozoi es, which will in ec new e y h ocy es. Illness s a s when he ma u e
asexual e y h ocy ic schizon , up u es (C). O he blood s ages di e en ia e in o male and emale sexual s ages pa asi es called
game ocy es. These game ocy es en e a mosqui o as i akes a blood meal. Sexual ep oduc ion occu s in he mosqui o midgu ,
and game es use o ming a mo ile zygo e, he ookine , ha ma u e and mig a es h ough he mosqui o midgu de eloping in o
an oocys , wi hin which spo ozoi es de elop. A e being eleased, no el spo ozoi es a el o he sali a y glands, making he
mosqui o in ec i e (D).
A
B
D
C
A
B
D
C
2.3 Li e s age biology
The disco e y o a mammalian mala ia exo-e y h ocy ic s age was he las missing piece o be illed
in he pa asi e’s li e cycle, when li e schizogony was desc ibed in A ican monkeys (Sho and
Ga nham, 1948). Since no symp oms a e associa ed wi h his pa asi e s age, li le a en ion was gi en
o he e en s ha occu du ing spo ozoi e in asion o hepa ocy e and he in ahepa ic de elopmen .
Howe e , his scena io changed when in 1967, Nussenzweig epo ed ha immunizing mice wi h
adia ion-a enua ed Plasmodium be ghei spo ozoi es p o ec ed hem agains challenge wi h ully
in ec ious spo ozoi es (Nussenzweig e al, 1967). These oden s udies p o ided he impe us o
human s udies, and, du ing he 1970s, Clyde, Rieckmann and colleagues es ablished ha immunizing
human olun ee s wi h he bi es o i adia ed mosqui oes ca ying P. alcipa um spo ozoi es in hei
sali a y glands could p o ec olun ee s agains challenge wi h ully in ec ious P. alcipa um
12| Chap e Two
spo ozoi es (Edelman e al., 1993; He ing on e al., 1991; Rieckmann, 1990; Rieckman e al., 1979;
Clyde e al., 1975; Rieckman e al., 1974; Clyde e al., 1973 a, b)
S ill un il he mid-1980s, knowledge o he biology o mala ia li e s age was es ic ed o he ac
ha hese o ms o he pa asi e could a es in he li e and la e be esponsible o elapses du ing
in ec ions wi h ce ain Plasmodium species, he so-called uninuclea ed “hypnozoi e” o m (Cogswell e
al., 1991; K o oski e al., 1980).
Since hen, expe imen al da a om se e al labo a o ies began o elucida e he nume ous s eps aken
by spo ozoi es om he s a ing poin o in asion o hei de elopmen inside he li e as well as he
immune esponse ha occu s du ing di e en immuniza ion s a egies wi h special emphasis o he
immuniza ion wi h a enua ed pa asi es (Engwe da and Good, 2005; Baldacci and Mena d, 2004).
The s udy o he cell biology o he Plasmodium li e s age is pa icula ly di icul due o lack o a high
in ec ious in i o cul u e sys em, isola ion and numbe o spo ozoi es needed. S ill, he ecen
ad ance in di ec li e obse a ion o pa asi es wi hin he li e ep esen s a use ul esou ce,
p omising an ou b eak o insigh s in o his ield (F e e e al., 2005; Hollingdale e al., 1998; Cal o-
Calle e al., 1994; Hollingdale e al., 1983b).
The easons why Plasmodium has elec ed he li e and he hepa ocy e as a i s cellula home inside
mammalian hos s a e no ully elucida ed. Howe e , i is possible ha he eason is ela ed o he
hepa ocy e’s highly complex me abolism, which is capable o ul illing all pa asi e eplica ion needs
(F e e e al., 2004; Saliba and Ki k, 2001). O he s suppo he idea ha he immunologic
cha ac e is ics o his o gan allow pa asi es o su i e and pu sue in ec ion, ei he by minimal
immune esponses ha a e hough o occu o by he induc ion o immune ole ance. In addi ion,
hepa ocy es a e capable o exp essing majo his ocompa ibili y complex (MHC) class I and class II,
while e y h ocy es a e no (C ispe, 2003; K zych e al., 2000; Rajan, 1997). Ano he aspec o ake
in o accoun is he mo phology o he li e i sel and he ac ha hepa ocy es a e he e ogeneous
and allow easy access o enules and a e ies sepa a ed by he space o Disse (Enomo o e al., 2004;
Wisse e al., 1985).
2.3.1 F om skin o li e
To each he i s and essen ial s op o i s jou ney, he hepa ocy e, Plasmodium spo ozoi es ha e o
a el om he skin inocula ion si e o he li e . Being a highly ascula ized o gan, he skin is he
pe ec place o a mosqui o blood meal, which usually las o 30s in Anopheles and occu s a e a
single p obe o blood aken in small pools o igina ed by capilla y damage. Mosqui o injec ions a e
accompanied by sali a, which has an an i-coagulan ac i i y, acili a ing blood diges ion. In spi e o
he hund eds o spo ozoi es p esen in he mosqui o’s sali a y glands only a small numbe a e
Gene al In oduc ion|13
ansmi ed du ing he bi e o an in ec ed mosqui o (Ma suoka e al., 2002; Ponnudu ai e al., 1991;
Rosenbe gh e al., 1990; Vande be g, 1977; G i i hs and Go don, 1952; Boyd and Ki chen, 1939).
The mig a ion o he spo ozoi e om he si e o bi e o he li e has been an issue unde discussion.
P e ious s udies wi h P. yoelii-in ec ed mosqui oes allowed o eed in mice, p o ided e idence ha
mosqui oes deposi spo ozoi es in an a ascula skin issue a ea and, wi hin 10 minu es pos -
inocula ion, spo ozoi es could be ound in he hos ’s bloods eam, long enough o ind a blood
essel (Sidjanski and Vande be g, 1997). A ecen s udy applying in a i al obse a ions o P. be ghei-
in ec ed mice a he si e o mosqui o bi e e ealed ha pa asi es mig a e widely ac oss he skin
co e ing dis ances o many mic ome e s o se e al minu es be o e eaching ci cula ion (Vande be g
and F e e , 2004). An i-spo ozoi e acqui ed immuni y educes mo ili y speed and could in e e e
wi h skin c ossing (F e e e al., 2005; Vande be g and F e e , 2004). Ei he because hey ind
hemsel es in a new ough en i onmen when hey change om sali a y glands o connec i e issue
o o p e en hos ile encoun e s wi h he hos immune sys em de enses, spo ozoi es ha e a sho li e
span o app oxima ely o 20 minu es o lea e ci cula ion in li e sinusoids and en e he pa enchyma
o in ec hepa ocy es.
The gliding mo ili y p esen ed by spo ozoi es is used du ing in ec ions in i o o hei mig a ion
h ough he de mis, as was p e iously demons a ed in i o (Vande be g and F e e , 2004;
Vande be g, 1974). Plasmodium gliding mo emen in hos cells is cha ac e ized by ails o
ci cumspo ozoi e (CS) p o ein ha a e le behind, simila o wha occu s when hey a e placed in
con ac wi h a i icial su aces (F e e e al., 1998; S ewa and Vande be g, 1991). The eleased CS
p o ein is dis ibu ed h oughou he cy oplasm o he cell and i has been p oposed ha is capable
o inhibi ing ansla ion a he ini ia ion s ep o p o ein syn hesis, as i binds o RNA-associa ed
binding si es on ibosomes (F e e e al., 1998; Hugel e al., 1996). Ne e heless, mosqui o-
ansmi ed spo ozoi es a e mo e in ec i e han spo ozoi es injec ed in a enously, and a e e en
capable o a oiding he an ibody esponse moun ed agains hem (K e li and Dan as, 1999;
Vaughan e al., 1999; Beie e al., 1991; Ponnudu ai e al., 1991).
2.3.2 Reaching he li e
The li e is o ganized in lobules o med by connec i e issue wi h b anches o he po al enule
and he hepa ic a e iole. I possesses a di e se popula ion o cells such as specialized endo helial
cells, Kup e cells (li e esiden mac ophages) and s ella e cells ( a -s o ing cells) (Enomo o e al.,
2004; Sinnis, 1996).
The e a e se e al in e ac ions es ablished be ween spo ozoi e p o eins and he li e cells
componen s. Mala ia spo ozoi es possess an apical complex cons i u ed by sec e o y o ganelles,
unique o Apicomplexa pa asi es and essen ial o he in asion p ocess. The majo componen o
14| Chap e Two
he su ace coa o Plasmodium spo ozoi es is he CS p o ein, which is esponsible o he oocys
ma u a ion and spo ozoi e mo phogenesis (Kappe e al., 2004; Nussenzweig and Nussenzweig,
1989). I is mainly s o ed in he mic onemes (spo ozoi es sec e o y o ganelles) and is con inuously
expo ed o he cell su ace and disca ded a he pa asi e pos e io pole (Tha hy e al., 2002; S ewa
and Vande be g, 1991; Pos huma e al., 1989). I also possesses a glycosyl-phospha idylinosi ol (GPI)
sequence as ancho a he C- e minus o he CS p o ein, which also con ains a species-speci ic
cen al epea egion and wo conse ed mo i s. Speci ic egions wi hin hese mo i s a e esponsible
o he linkage o he glycosaminoglycan o li e sul a ed p o eoglycans and spo ozoi e mo ili y and
in asion (Tewa i e al., 2002; Ying e al., 1997).
Ano he p o ein p esen in he mic onemes ha is also exp essed a he spo ozoi e memb ane
su ace upon sec e ion induced by cell-con ac is he h ombospodin- ela ed adhesi e p o ein
(TRAP) (Bhano e al., 2003; Gan e al., 2000; Temple on and Kaslow, 1997). Besides being
in ol ed in spo ozoi e in asion o sali a y glands and he hepa ocy e, i s i al ole was assessed
when P. be ghei mu an TRAP pa asi es we e shown o lose hei gliding mo ili y (Ma uschewski e al.,
2002; Wengelnik e al., 1999; Sul an e al., 1997).
On he hos side, he molecules ha seem o play an impo an ole du ing in ec ion by allowing
spo ozoi es o ecognize and each he li e a e he p o eoglycans. These molecules, which a e
highly sul a ed and abundan , a e in ol ed in he molecula mechanism o spo ozoi e adhesion,
whe e CS p o ein binds o hepa ocy e memb anes (F e e e al., 1993; Ce ami e al., 1992). Bo h CS
and TRAP a e capable o ecognizing in pa icula he glycosaminoglycan chains (GAG) in he
sul a ed p o eoglycans exp essed by li e cells (Ying e al., 1997; Robson e al., 1995).
When en e ing he li e sinusoids ei he ia he hepa ic a e iole o he po al enule, spo ozoi es
glide along he endo helial cell laye h ough he endo helia, in e ac ing wi h ex acellula ma ix
p o eoglycans ha p o ude om he space o Disse un il hey encoun e a Kup e cell and
ecognize he p o eoglycans exp essed on i s su ace (P adel e al., 2002; P adel and F e e , 2001).
Al hough p e ious wo k wi h P. be ghei in a s sugges ed ha hese cells we e esponsible o pa asi e
phagocy osis emo ing hem om ci cula ion, ecen ly Kup e cells we e shown o be he li e
po al en y o spo ozoi es om a se o expe imen al da a based on in i o models wi h P. be ghei
and P. yoelii spo ozoi e in ec ions and Kup e cells isola ed om a li e s (P adel and F e e , 2001;
Meis e al., 1983). Mo eo e , he de elopmen o mos exo-e y h ocy ic o ms close o he li e
po al enules, whe e he Kup e cells a e loca ed, also sugges s ha he hese cells could well
unc ion as access ga es o he li e (Ve ha e e al., 1985; Sleys e and Knook, 1982).
Thus, he cu en model p oposes ha spo ozoi es ecognize and bind o p o eoglycans exp essed
on he su ace o Kup e cells using hei majo su ace p o eins, CS p o ein and TRAP (Pinzon-
O iz e al., 2001; Ce ami e al., 1992). They ac i ely in ade, sa ely a e se and success ully exi
Gene al In oduc ion|15
Kup e cells p o ec ed by a non- usogenic acuole h ough he space o Disse (F e e e al., 2005;
Meis e al., 1983c).
2.3.3 Hepa ocy e in asion
Hepa ocy e in ec ion occu s a e spo ozoi es exi he space o Disse and gain access o he li e
pa enchyma. Spo ozoi es i s mig a e h ough se e al hepa ocy es be o e in ading a inal one
wi hin a pa asi opho ous acuole. The a e sed cells ha e hei plasma memb ane wounded by he
pa asi e and can ei he su i e, unde going a esealing p ocess, o die (Mo a e al., 2001).
Expe imen al e idence demons a es ha when spo ozoi es in ade hos cells wi hou o ma ion o a
acuole he pa asi es do no de elop, meaning ha mig a ion is a ea u e ha p ecedes in ec ion
(Mo a e al., 2001). This mig a ion o pa asi es lea es behind wounded cells ha could be needed o
po en ia e in ec ion, which would be bene icial o he pa asi e’s de elopmen wi hin in ec ed
hepa ocy es. On he o he hand, one could also suppose ha wounding would inc ease he
gene alized in lamma ion de ec able sho ly a e inocula ion (Khan and Vande be g, 1992). This
pa icula spo ozoi e ea u e can be obse ed ei he in i o o in i o in mice li e s, and i is
unspeci ic as is shown by he ac ha a ange o cell ypes a e sui able o hos ing Plasmodium
mig a ion (Mo a e al., 2001). Mo eo e , du ing pa asi es in i o jou ney om he skin o he li e
hey encoun e di e en issues and p obably mig a e h ough hem (Mo a and Rod iguez, 2004).
The molecula mechanism is s ill no ully elucida ed, bu ecen epo s e ealed a newly disco e ed
spo ozoi e p o ein ha plays a ole in mig a ion. The SPECT p o ein (spo ozoi e mic oneme
p o ein essen ial o cell a e sal), which is localized in spo ozoi es mic onemes, is equi ed o he
pa asi e’s mig a ion in i o. Ye , exo-e y h ocy ic de elopmen was main ained, and a lowe
in ec i i y was shown in i o, sugges ing ha hos cell mig a ion is manda o y o spo ozoi e access
in o he li e pa enchyma (Ishino e al., 2004). Recen ly, ano he SPECT p o ein was desc ibed and a
simila unc ion was shown o a p o ein p esen in mosqui o ookine e in asion in o he midgu
epi helium (Ishino e al., 2005a; Kado a e al., 2004).
The inal in asion is accompanied by he sec e ion o TRAP and he pa asi e inds i sel su ounded
by a pa asi opho ous acuole, inside which i eplica es and de elops (Sil ie e al., 2004; Mo a e al.,
2001; Meis e al., 1983b). De ailed in o ma ion abou how his e en akes place is sca ce, in pa
because o he low in ec i i y o spo ozoi es in i o. By analogy wi h ela ed pa asi es such as
Toxoplasma gondii, i is belie ed ha spo ozoi e in e naliza ion wi h o ma ion o a pa asi opho ous
acuole occu s wi hin a ew seconds and is dependen on he plasma memb ane-associa ed mo o
ha also d i es pa asi e gliding mo ili y (Solda i and Meissne , 2004). In addi ion, he e is an
associa ed in ense sec e o y ac i i y in hei apical end, which is ei he a cons i u i e exocy osis o
16| Chap e Two
molecules om wi hin he apical o ganelles, o a egula ed exocy osis es ic ed o he o ma ion o a
cap (Mo a and Rod iguez, 2004; Mo a e al., 2002).
Mo eo e , mig a ion induces sec e ion o “hepa ocy e g ow h ac o ” (HGF) ha ende he
su oundings mo e adequa e o pa asi e g ow h and a e impe a i e in o de o ob ain a success ul
in ec ion (Ca olo e al., 2003). Expe imen al e idence has sugges ed ha o he hos cell ac o s a e
in ol ed in spo ozoi e in ec ion in he li e , o example, he in e ac ion o he CS p o ein wi h he
low-densi y lipop o ein- ela ed p o ein ecep o o he equi emen o CD81 e aspanin o P.
alcipa um and P. yoelii spo ozoi es o in ade hepa ocy es (Sil ie e al., 2003; Shabkibaei and F e e ,
1996).
2.3.4 In ahepa ic de elopmen
Knowledge conce ning pa asi e de elopmen inside hepa ocy es is s ill e y limi ed conside ing he
la ge ange o mo phological cha ac e is ics ha occu s du ing his in ahepa ic de elopmen .
Hepa ic schizogony, whe e pa asi es g ow, ma u a e and eplica e, las s 2 days o oden species and
5 o 7 days o p ima es, esul ing in 10.000 o 30.000 o me ozoi es om each in ading spo ozoi e
(Meis e al., 1985 a, b; Meis e al., 1983 a, b). F om seize a single gene a ion, me ozoi es indi idualize
in he cy oplasm, and hen sepa a e as isle s wi hin which memb ane o ma ion occu s be o e
up u e in o he bloods eam. An in ec i e mosqui o bi e leads o he o ma ion o a ound 12 exo-
e y h ocy ic schizon s in a ew days, eaching 100 µm in size, con as ing wi h blood s ages ha only
enla ge up o 12 µm, eplica ing in o 8 o 24 e y h ocy ic schizon s. Thus, he pa asi e’s needs o
memb ane and nucleic acid syn hesis du ing he li e s age a e eno mous, almos h ee imes mo e
han o blood s ages schizogony (Hollingdale, 1985). Nu ien s a e ob ained om he hepa ocy e
which as i ha bo s glycogen and se um p o ein ac o ies, allowing he ma u a ion p ocess o
p og ess ending in he elease o me ozoi es ha will in ade e y h ocy es (F e e , 2004; Meis e al.,
1985 a, b).
Howe e , spo ozoi es a e able o unde go pa ial de elopmen o ea ly exo-e y h ocy ic o ms in he
absence o hos cells o o he cell ypes, and p ese e some mo phological and molecula ea u es
p esen ed by egula ones (Wang e al., 2004; Kaise e al., 2003).
2.4 Immuni y o mala ia li e s age
When na u ally exposed o mala ia, humans g adually acqui e immuni y o he pa asi e, al hough
epea ed in ec ions a e equi ed in o de o main ain i . Na u ally acqui ed immuni y is cha ac e ized
by being sho -li ed and s ain-speci ic (H iid, 2005; Day and Ma sh, 1991). The easons o such
Gene al In oduc ion|17
sca ce p o ec ion a e no known. I is media ed by blood-s age speci ic an ibodies and is pa ially T
cell-based (S e enson and Riley, 2004; Good and Doolan, 1999). Bu he con ibu ion o each o he
di e en s ages o he gene al immuni y- ela ed p o ec ion emains poo ly unde s ood. Du ing he
blood s ages i is clea ha he hos de elops mechanisms ei he o neu alize he e ec s o pa asi e-
eleased oxins o o kill he pa asi e o e en o inhibi i s eplica ion. The educed acquisi ion o
mala ia immuni y in na u ally exposed popula ions has also been explained by he ac ha he
pa asi e ac i ely modula es he immune sys em o he hos du ing blood s ages, p e en ing he
de elopmen o speci ic immune esponses (Ocana-Mo gne e al., 2003; U ban e al., 1999;
Plebanski e al., 1997). Conce ning he li e , he ac ha he gu is loca ed close o he li e is one o
he easons why his o gan has such a peculia immunologic p o ile, p edominan ly ole ogenic
when esponding o o eign an igens. Li e dend i ic cells, Kup e cells and sinusoidal endo helia
a e ma u e an igen-p esen ing cells ha unde ake he ask o main aining his immune en i onmen
(Dohe y and O’Fa elly, 2001; Knolle and Ge ken, 2000; Lohse e al., 1996). Toge he wi h an i-
in lamma o y cy okine sec e ion, he li e con ols he in lamma ion induced by in lux o bac e ia
and endo oxins om in es ines, and eaches a s a e o po al ein ole ance. An igen-speci ic
ac i a ed CD8+ T cells a e elimina ed by Fas-induced apop osis conside ing he model o ac i a ion-
induced cell dea h, hus accoun ing o li e ole ance (C ispe, 2003).
Ne e heless, na u ally acqui ed p o ec ion obse ed in in ec ed indi iduals and se e al expe imen al
da a eco ds, aised hopes o he de elopmen and easibili y o an e ec i e accine agains his
disease (Ca alho e al., 2002).
In wha ega ds he li e s age o in ec ion, majo hopes we e aised based on he ac ha s e ile
p o ec i e immuni y was ob ained bo h in mice, monkeys and humans; agains P. be ghei, P. yoelli, P.
knowlesi, P. alcipa um o P. i ax spo ozoi e challenge induced by immuniza ion wi h adia ion-
a enua ed spo ozoi es (Ho man e al., 2002; Gwadz e al., 1979; Clyde, 1975; Clyde e al., 1973 a, b;
Nussenzweig e al., 1969; 1967).
P o ec i e mechanisms agains he li e s age o mala ia in ec ion ha e been he aim o many s udies,
since knowledge conce ning he ype o immune esponses induced owa ds he in ec ed hepa ocy e
a e s ill e y limi ed. The ad an ages o inducing exo-e y h ocy ic s age immuni y would no only be
a dec ease in mo ali y and disease ansmission, bu also a p e en ion o symp oms, since he
pa asi e would be a es ed be o e eaching he e y h ocy ic s age (Td yk and Wal he , 2005; Tsuji
and Za ala, 2003).
2.4.1 Na u al in ec ion e sus immuniza ion
Mimicking ea u es occu ing in na u ally in ec ed indi iduals is he gene al aim o accines which,
in u n, means ha hey mus be capable o inducing an ibodies and T cell esponses, i possible o
18| Chap e Two
mo e han one an igen. In pa icula , a g ea e magni ude in esponses han he one achie ed in
indi iduals ha ha e been in ec ed, oge he wi h anscendence bo h in ime and s ains, would be
desi able o he de elopmen o a accine agains mala ia. Adminis a ion o a cock ail o se e al
an igens has been he p e ailing hypo hesis o many yea s now, bu an expensi e and complex
p oduc would be he likely esul . Ideally, a accine agains he exo-e y h ocy ic s age o he pa asi e
should ul ill wo oles: he induc ion o high i e s o unc ional an ibodies agains spo ozoi es
inocula ed by he in ec ious mosqui o, in o de o s op hem om en e ing he li e ; he
es ablishmen o po en cy o oxic T lymphocy e immunogenici y agains he li e s age o kill all
in ec ed hepa ocy es while no ha ming he hos (Hill, 2006; Doolan and Ho man, 2000; Doolan
and Ho man, 1997).
In ha espec , he disco e y ha immuniza ion wi h adia ion-a enua ed spo ozoi es could lead o
ull p o ec ion pa ed he way o de e mine he ull ange o ac o s in ol ed in his p o ec ion,
namely Plasmodium an igens, ini ia ion o hos immune esponse and immune esponse ypes (Na din
e al., 1999). Gamma-i adia ed spo ozoi es in ec he li e bu a e a es ed he e wi hou pu sing o
blood s ages. La e , i was also ound ha he a ge s o his p o ec ion we e no only he spo ozoi e
bu mainly he in ec ed hepa ocy e (Schelle and Azad, 1995; Suh bie e al., 1990; Weiss, 1990;
Ho man e al., 1989). In ac , spo ozoi es i adia ed wi h a dose ha did no allow hem o each he
li e we e no p o ec i e any mo e (Sil ie e al., 2002; Nussle e al., 1989). S ill, is no clea i his
elies on he p esen a ion o di e en and speci ic an igens o on he way ha an igens a e p esen ed
o he hos immune sys em (Langho ne e al., 2004; K zych e al., 2000).
2.4.2 Exo-e y h ocy ic an igens
Some spo ozoi e an igens a e also exp essed wi hin he newly o med li e s age o ms. In ac , a
limi ed numbe ha e been al eady iden i ied and cha ac e ized as a ge s o CD8+ T cell esponses.
Among hese a e he CS p o ein and he TRAP p o ein (Khusmi h e al., 1994; Roge s e al., 1992;
Weiss e al., 1992). Ano he p o ein ha has also been shown o be a a ge o T cells is he P. yoelii
hepa ocy e expo ed p o ein 17 (PyHEP17), an homolog o he P. alcipa um expo ed p o ein 1
(P EXP) (Doolan e al., 1996).
The i s s ic ly speci ic mala ia li e s age an igen disco e ed was he P. alcipa um li e s age
an igen 1 (P LSA-1), which is exp essed in he acuole lumen and is solely exp essed du ing he
hepa ic s age (Gue in-Ma chand e al., 1987). The LSA-3 an igen was cha ac e ized h ough an ibody
ecogni ion in immunized and non-immunized indi iduals (Daube sies e al., 2000; Connelly e al.,
1997). A ew o he pa asi e p o eins appea a his s age o he pa asi e’s li e cycle, such as
glu amine- ich p o ein and pa asi e hea shock p o ein 70 (Kuma e al., 1993). Mo e pa asi e
Gene al In oduc ion|19
p o eins a e being iden i ied h ough ecen new genomic app oaches, leading o an inc ease in he
numbe o he an igenic epe oi e known in mala ia (G une e al., 2003).
Recen ly, se e al Plasmodium genes, which a e no exp essed in he blood s ages and some a e also
no exp essed in he spo ozoi es, we e shown o possess a i al ole o pa asi e li e de elopmen
(Ishino e al., 2005b; Muelle e al., 2005; an Dijk e al., 2005; K aise e al., 2003).
2.4.3 Ini ia ion o hos immune esponse
Naï e T cell p iming by spo ozoi es can occu by specialized p esen ing cells, mac ophages,
dend i ic cells, and B cells (Riley, 1999; D uilhe e al., 1998; Pape e al., 1997). The e is e idence ha
spo ozoi es a e able o a oid des uc ion by mac ophages, om which hey can exi and induce
dea h (Vande be g e al., 1990; Seguin e al., 1989; Dan o h e al., 1980). In ac , spo ozoi es a e oo
la ge o be in e nalized by B cells, bu eleased ma e ial migh be aken up by speci ic B cells, he eby
ini ia ing ac i a ion o a T cell esponse (Link e al., 1993; S ewa and Vande be g, 1992). Al hough
i has been shown ha he in ec ed li e cell can p esen pa asi e-de i ed pep ides on i s su ace,
exp essed on MHC class I o class II molecules and ecognized by CD8+ and CD4+ T cells,
espec i ely, he hepa ocy e does no no mally exp ess co-s imula o y molecules necessa y o naï e
T cell ac i a ion, which he high le els o In e leukin-10 (IL-10) p esen in he li e , may u he
delay (Renia e al., 1993; Weiss e al., 1993; Weiss, 1990; Ho man e al., 1989). Thus, he possibili y
ha in ec ed hepa ocy es can ac i a e naï e T cells is unlikely (K zych e al., 2000; Vande be g e al.,
1993). Fu he mo e, he exis ence o a pa asi opho ous acuole be ween he pa asi e and he hos
cell poses some ques ions. T a icking o Plasmodium an igens inside he hepa ocy e be ween he
pa asi opho ous acuole and he hos cell memb ane is s ill an unclea issue. This double biological
memb ane cons i u es a ba ie o he anspo o mala ial pep ide epi opes by MHC molecules
(F e e , 2004; G une e al., 2003; Mellman e al., 1998).
2.4.4 Hos immune esponses a e immuniza ion
The p o ec i e mechanisms induced by immuniza ion wi h i adia ed spo ozoi es we e ex ensi ely
s udied. Since he li e is an immune p i ileged si e, he in ec ed hepa ocy e was o decades
conside ed o be p o ec ed om he immune sys em and, he e o e, immuni y was hough o be
an i-spo ozoi e only. Howe e , la e was shown ha his immune esponse is moun ed agains he
in ec ed li e cell (Lau e al., 2001; Ho man and Doolan, 2000; Ho man e al., 1998).
The i s line o e ec o mechanisms igge ed by mala ia in ec ion is an ibody esponse. CS p o ein
is a a ge o he p oduc ion o neu alizing an ibodies ha induce p o ec ion as conse a ion wi hin
each plasmodial species is e y high (Hollingdale e al., 1998; McCu chan e al., 1996; Hollingdale e
al., 1984; Lockye e al., 1989; San o o e al., 1983). An ibodies agains he CS p o ein p o ec ed mice
20| Chap e Two
by ei he blocking spo ozoi e in asion o by killing spo ozoi es hemsel es, ul ima ely leading o
immuni y. This immune esponse mechanism agains spo ozoi es is desc ibed in he li e a u e o
di e en pa asi e species: P. be ghei, P. yoelli and P. i ax (Cha oen i e al., 1991; Wi z e al., 1991;
Webe e al., 1987; Po ocnjak e al., 1980; Yoshida e al., 1980). In oden models he e a e examples
o an ibody p oduc ion agains single epi opes o di e en p o eins, like he P. yoelii HEP17 ha is
capable o emo ing in ec ed hepa ocy es in cul u e (Cha oen i e al., 1995). Hepa ocy e in asion
and de elopmen o P. alcipa um was p e en ed when an an i-P -CS p o ein epea s monoclonal
an ibody was used in i o (Mazie e al., 1986; Hollingdale e al., 1984). Howe e , p o ec ion does no
consis en ly co ela e wi h an i-CS an ibody le els in mala ia-exposed indi iduals o immunized
olun ee s. Besides, no accine has been shown o induce a s ong s e ile immuni y based on he
induc ion o an i-CS an ibodies alone (He ing on e al., 1987; Ho man e al., 1987).
The ole o T cells was i s demons a ed when i adia ed spo ozoi es con e ed p o ec ion in mice,
which we e no capable o making an ibodies (Chen e al., 1977). Addi ionally, spleen cells o
immune T cells om spo ozoi e-immunized mice could con e p o ec ion agains mala ia, when
adop i e ans e was pe o med in i o in o naï e mice (Egan e al., 1987; Ve ha e e al., 1978).
P o ec i e immuni y induced by i adia ed spo ozoi es is comple ely dependen on he p esence o
CD8+ T cells while a equi emen o CD4+ T cells has only been demons a ed in a ew s ains
(Tsuji and Za ala, 2003; Doolan and Ho man, 2000; Weiss e al., 1993; Tsujii e al., 1990).
CD8+ T cells ha e been implica ed as c i ical e ec o cells in his p o ec ion, hus being he co e o
se e al immunologic s udies, which p o ided he ollowing obse a ions. Induc ion o IFN-γ is a
di ec consequence o he CD8+ T cell ac i a ion, IFN-γ p oduc ion p ecedes and ini ia es
p oduc ion o IL-12, and hen IL-12 in u n induces IFN-γ p oduc ion by APCs and/o na u al
kille cells (NK) in a posi i e eedback loop ha ep esen s an impo an ampli ying mechanism.
The IFN-γ hen ac i a es inducible ni ic oxide (NO) syn hase and induces he L-a ginine-dependen
NO pa hway, subsequen ly elimina ing he in ec ed hepa ocy e o he in ahepa ic schizon (Mo o
and Za ala, 2004b; K zych e al., 2000; Doolan and Ho man, 1999; Sedegah e al., 1994; Nussle e
al., 1993; Nussle e al., 1991; Scho ield e al., 1987a). Ne e heless, i adia ed spo ozoi e
immuniza ion induces dis inc mechanisms o p o ec ion in di e en hos s (Doolan and Ho man,
2000).
In i o, CD8+ T cells agains a single epi ope in he ca boxyl e minus o he P. yoelii CS p o ein
elimina ed in ec ed hepa ocy es om cul u e in an an igen-speci ic, MHC- es ic ed manne (Weiss,
1990). P o ec ion agains in ec ion in mice was also achie ed by using CD8+ T cells ha a e speci ic
o an epi ope o he CS p o ein ei he o P. be ghei CS o P. yoelii CS, which could, in some cases, be
abolished by in i o ea men wi h an i-IFN-γ (Weiss e al., 1992; Rod igues e al., 1991; Rome o e al.,
1989). Besides CS p o ein, o he clones agains di e en p o eins could p o ec mice such as TRAP,
Gene al In oduc ion|21
cy okines, sec e ion o oxins, p oduc ion o eac i e oxygen species, a e common ou comes o his
ype o ela ionship. Many examples o s a egies used by i uses and bac e ia p e en ing he dea h
o he hos cell h ough ei he o he apop o ic pa hways ha e been epo ed (Gao and Kwaik, 2000;
G an ille e al., 1998; Liles, 1997).
Analogous mechanisms ha e been p oposed o p o ozoa including ecep o -media ed apop o ic
igge s and s ess-media ed apop osis s imuli, sugges ing ha a close in e ac ion a di e en check
poin s is es ablished wi h he apop o ic machine y o he hos cell. The e o e, he ab oga ion o hos
cell apop osis o en u ns ou o be bene icial o he pa hogen and o esul in a success ul in asion
(Heussle e al., 2001).
While in many cases he mechanisms o esis ance o apop osis a e s ill unknown, in o he s he e
appea o be a a ie y o ways in which he pa hogen ensu es cellula su i al. These include
p oduc ion o molecules ha esemble and mimic Bcl-2 p o ein, he exp ession o inhibi o s o
dea h ecep o signaling pa hway, he gene a ion o caspase inhibi o s and in e e ence wi h nuclea
ac o kB (NFkB) pa hway. So, se e al s udies poin ou o pa asi e-p oduced molecules ha a e
capable o di ec ly ac i a ing apop o ic pa hways in he a ge cells (Ga ilescu and Denke s, 2003;
Hasnain e al., 2003).
Among hese a e examples o pa asi ic in ec ions con e ing p o ec ion o induced-apop osis in
in ec ed-hos cells. Dec eased caspase ac i a ion and educed le els o poly (ADP- ibose)
polyme ase exp ession we e desc ibed in Toxoplasma-in ec ed cells (Goebel e al., 2001; Nash e al.,
1998). On he o he hand, cell iabili y in he absence o g ow h ac o s was shown o be induced
when Leishmania-in ec ed mac ophages soluble media o s we e eleased (Moo e e al., 1994).
Bo h induc ion and inhibi ion mechanisms we e desc ibed in T ypanosoma c uzi-in ec ed cells,
depending on he s imulus elici ed. Hence, an apop o ic pa hway can be induced du ing in ec ion by
adia ion and chemicals and when a di e en pa hway, like Fas o TNF-α, was igge ed, hos cells
became p o ec ed and esis an o dea h (Nakajima-Shimada e al., 2000; Nunes e al., 1998; Lopes e
al., 1995). In addi ion, T ypanosoma is able o p oduce a ans-sialidase ha can in e ac wi h he hos
cell PI3K-Ak pa hway o p omo e su i al in he pe iphe al ne ous sys em and also seems o be
esponsible o cell deple ion in he hymus du ing in ec ion (Mucci e al., 2002; Chen e al., 2001;
Chuenko a e al., 2001).
Theile ia is ano he pa asi ic pa hogen ha is able o sub e he hos o i s own bene i , making use
o ema kable app oaches, esembling a cance cell. This pa asi e places a huge p essu e on he hos
cell by aking con ol o bo h he cell cycle and he apop osis p ocess h ough se e al mechanisms
ha in ol e he ac i ac ion o ansc ip ion ac o s like AP-1, ATF-2, which a e implica ed in JNK
and MAPK pa hways (Heussle e al., 1999; Romashko a and Maka o , 1999; Palme e al., 1997).
NFkB signaling pa hway is no mally associa ed wi h ansc ip ion o p o-in lamma o y media o s
28| Chap e Two
and esis ance o in ec ion. Howe e , i can also induce exp ession o se e al an i-apop o ic p o eins,
including IAPs and c-FLIP, which blocks caspase-8 ac i i y leading o su i al (Ba ke and Gilmo e,
1999). Theile ia-in ec ed cells show an ac i a ed pheno ype as he inhibi o molecule (IkB) is
deg aded by phospho yla ion, main aining he nuclea ansloca ion leading o immo alized-in ec ed
cells. Consequen ly, hese modula ions o he biology o Theile ia-in ec ed cells lead o dea h-
esis ance o bo h ex insic and in insic apop o ic igge s (Kuenzi e al., 2003; Heussle e al., 2002;
Heussle e al., 1999).
O he examples o mi ochond ial pa hway induced-dea h a e also ound in he li e a u e, al hough
no di ec ly in ol ing he in ec ed cell. Du ing acu e T. gondii in ec ion, CD4+ and CD8+ T cells
accumula e in he b ain and exp ess high mRNA le els o Bax and Bad, while in ch onic in ec ion T
lymphocy e exp ession o p o-apop o ic molecules is down egula ed and an i-apop o ic le els o
Bcl-2 and Bcl-xl a e inc eased (Schlu e e al., 2001; Khan e al., 1996).
Simila p ocesses occu wi h P. be ghei le hal in ec ion (a model o oden ce eb al mala ia), whe e
inc eased le els o Bax, Bcl-2, p53, and cy oplasmic cy och ome c in he b ain we e measu ed.
Fu he mo e, al e a ions on mi ochond ia mo phology we e de ec ed, sugges ing ha he
dys unc ion o his o ganelle may play a ole in ce eb al mala ia pheno ypes, al hough i is no clea
which apop o ic pa hway was ollowed (Kuma and Babu, 2002).
Fo ex acellula pa asi es he induc ion o apop osis occu s in cells ha cons i u e hei die o
appea along hei pa hway o in asion. En amoeba his oly ica-in ec ed cells unde go apop osis h ough
a cell lec in-binding su ace con ac mechanism while e idence shows helmin hs pa asi es a ge ing
o hos cells mobilized o a ack hem using also Fas-FasL in e ac ion (Lopez-B iones e al., 2003;
Chen e al., 2002; Jenson e al., 2002; Ku oda e al., 2002; Rumbley e al., 2001; Hus on e al., 2000).
Simila e en s occu in a i us-hos ela ionship. Upon i al in ec ion many cells unde go apop osis,
he eby educing he i al load. The e o e, in e e ing wi h hos cell apop osis would lead o
eplica ion and sp eading o p ogeny. Fo his pu pose, s a egic poin s in he apop o ic pa hways
a e a ge s o i al p o eins ha e ol ed o be able o inhibi o delay he hos ’s p o ec i e ac ions
by a ge ing cells o he immune sys em o he hos cell i sel (G an ille e al., 1998; Ploegh, 1998).
The an i-apop o ic membe s o he Bcl-2 amily, he inac i a ion o umo supp esso p53 and
caspases inhibi ion a e examples o a ge s o his modula ion (Tabakin-Fix e al ., 2005; Li e al.,
2002; Yang e al., 2002; Be in e al., 1996; Shen and Shenk, 1995). In la en i al in ec ions, he hos
p o ein ansla ion machine y is con olled by he i us. Ini ia ion o a i al in ec ion leads o he
shu ing down o he hos ’s p o ein syn hesis, which in u n leads o a decline o me abolic p ocesses
and apop osis induc ion (Clemens, 2005).
Gene al In oduc ion|29
2.5.5 Modula ion o apop osis and immuni y
Cell-speci ic apop osis con ibu es o he egula ion o pa hogen-induced immune esponses (Lude
e al., 2001). As he Fas-FasL signaling pa hway is speci ically in ol ed in apop osis, dis up ion o he
Fas-FasL pa hways can be di ec ly ela ed o an al e a ion in apop osis s eady-s a e (Dock ell, 2003).
S udies wi h knockou mice in ec ed wi h di e en p o ozoa showed ha FasL- igge ed apop osis
plays a ole in esis ance o in ec ion as he lack o unc ion o his molecules induced highe le els
o pa asi emia and lesions ha he mice ailed o esol e (Lopes e al., 1999; Conceicao-Sil a e al.,
1998). Howe e , i is no known i his ou come esul s om in ec ed cell lysis o p e en ion o
uncon olled in lamma o y eac ions a he lesion si e.
P oduc ion o FasL, which, h ough in e ac ion wi h i s ecep o Fas will ini ia e he apop osis
cascade, o ins ance in T- and B-cells, leading o lymphocy openia, occu s in Plasmodium, T. gondii
and Leishmania (Eidsmo e al., 2002; Nishikawa e al., 2002; Wipasa e al., 2001; Ma sumo o e al.,
2000).
When o albumin- o pa asi e-speci ic T cells we e adop i ely ans e ed in o P. yoelii-in ec ed T cell-
de icien nude mice, only he P. yoelii-speci ic T cells unde wen apop osis and demons a ed signs o
inc eased Fas exp ession. These da a sugges a mechanism o elimina e an i-Plasmodium T-cell
e ec o s. Mo eo e , in an acu e blood s age in ec ion wi h P. chabaudi, 60% o he spleen cells
exp ess he Fas molecule (Wipasa e al., 2001; Helmby e al., 2000). In human mala ia, in ec ed
indi iduals exp ess high le els o apop o ic ma ke s in pe iphe al blood mononuclea cells, which in
i o a e sensi i e o apop osis induc ion and ha e ele a ed soluble Fas le els in se um. Thus, his
apop o ic pa hway may also play a ole in in ec ion. In addi ion, FasL le els ha e been shown o
dec ease wi h mala ia chemo he apy (Kemp e al., 2002).
CTLs employ pe o in and g anzyme molecules o di ec ly igge apop osis in in ec ed a ge cells.
Se e al in acellula p o ozoans elici MHC class I- es ic ed CD8+ CTL ac i i y; hese pa asi es
include Toxoplasma gondii, Plasmodium spp., T ypanosoma c uzi and Leishmania majo (Bonelo e al., 2000;
da Conceicao-Sil a e al., 1994; G azzinelli e al., 1993; Nickell e al., 1993; Rod igues e al., 1991).
Howe e , i seems ha pe o in-media ed CTL ac i i y does no p o ec he hos , as pe o in-
knockou mice we e esis an o in ec ion wi h T. gondii and T. c uzi (Nickell and Sha ma, 2000;
Denke s e al., 1997).
Repo s conce ning immuni y- ela ed apop osis desc ibe addi ional immunologic ac o s ha
possess a signi ican ole in he ou come pheno ype. P o-in lamma o y cy okines a e able o media e
apop osis p ocess du ing pa hogenic in ec ions. Howe e , i is di icul o de e mine he biological
signi icance o apop osis d i en by media o s such as IFN-γ, IL-12, NO and TNF-α; because hese
molecules a e, in addi ion, in ol ed in esis ance o pa hogens and a e needed o hos su i al
(Denke s, 2003; Ma ins e al., 1999; G azzinelli e al., 1998; Sil a e al., 1998).
30| Chap e Two
Chap e Th ee
MATERIALS and
METHODS
3.1 Cells
Two hepa oma cell lines we e used o in i o cul u e expe imen s: a human hepa oma cell line,
HepG2 (ATCC, HB8065) and a mouse hepa oma cell line Hepa 1-6 (ATCC, CRL-1830) (Da ling on
e al., 1980). Bo h cell lines a e e icien ly in ec ed by oden mala ia pa asi es which a e capable o
unde going comple e de elopmen (Mo a and Rod iguez, 2000; Hollingdale e al., 1983 a,b). This
cons i u es he in i o model sys em used o ep oduce a mala ia li e s age in ec ion du ing he
expe imen al s udies pe o med.
3.2 Pa asi es, mosqui oes and mice
Plasmodium yoelii (17XNL, non-le hal s ain) o Plasmodium be ghei (NK65 o ANKA s ains)
spo ozoi es we e ob ained om in ec ed mosqui oes and we e used ei he o in i o o in i o
expe imen s. Pa asi es we e main ained by al e na e cyclic passages in mosqui oes and mice
(Vande be g and Gwadz, 1980).
Addi ionally, Anopheles s ephensi in ec ed mosqui oes we e also ob ained om he Cen o de Malá ia e
ou as Doenças T opicais (Lisboa, Po ugal) and he Uni e si y Medical Cen e S . Radbound
(Nijmegen, The Ne he lands), and u he main ained in ou depa men s unde adequa e condi ions
(Benedic , 1997). BALB/c o C57/BL6 mouse s ains aged be ween 6-8 weeks we e pu chased om
Taconic Fa ms (Ge man own, New Yo k, US) o Ins i u o Gulbenkian de Ciência (Oei as, Po ugal).
Animals we e b ed and main ained in a pa hogen- ee animal acili y.
3.3 Spo ozoi e isola ion and pu i ica ion
Female Anopheles s ephensi mosqui oes we e ed on in ec ed Plasmodium spp. mice. Spo ozoi es we e
ob ained by dissec ion om hei sali a y glands a days 18-21 pos eeding. Dissec ions o mosqui o
sali a y glands we e pe o med in RPMI 1640 medium (Gibco) con aining 1% mouse se um
(Gibco). The glands we e mechanically dis up ed and homogenized o ee he pa asi es. The deb is
was pelle ed a e spinning a 20g o 5 min a 4ºC. Spo ozoi es we e hen collec ed, coun ed and
main ained on ice un il use (adap ed om Ozaki e al., 1984). Spo ozoi es in ec i i y was dependen
on he pa asi e s ain in use. The numbe o spo ozoi es pe in ec ed mosqui o was de e mined
using a hemacy ome e (Neubaue chambe ). I adia ed spo ozoi es we e submi ed o gamma-
sou ce adia ion equi alen o 20 K ad dose (137Cs sou ce). Hea -killed spo ozoi es we e submi ed
o a hea shock ea men o 56º C o 15-30 min.
3.4 In i o in ec ions
One day p io o in ec ion, 2×105 Hepa 1-6 (o HepG2 cells) we e ha es ed in 24-well pla es o e a
glass co e silp in comple e Dulbecco’s MEM medium (DMEM; Sigma) supplemen ed wi h 10%
Ma e ials and Me hods|33
hea -inac i a ed e al bo ine se um, 100 U/ml penicillin, 0.1 mg/ml s ep omycin, and 2mM
glu amine and g own a 37ºC wi h 5% CO2.
P. yoelii spo ozoi es (105/well) o P. be ghei spo ozoi es (3×104/well) we e added o he monolaye s o
Hepa 1-6 o HepG2 cells espec i ely, cen i uged o 5 min a 1800g and incuba ed a 37ºC wi h 5%
CO2. A di e en ime poin s a e in ec ion, cells we e washed wice wi h Phospha e Bu e ed
Saline (PBS) and ixed in 4% Pa a o maldehyde (PFA) o 20 min a oom empe a u e (RT) (Fig.
3.1).
Plasmodium spo ozoi es
Hepa 1-6 o HepG2Hepa 1-6 o HepG2 In ec ed hepa ocy esIn ec ed hepa ocy es
AB
24h24h
Exo-e y h ocy ic o ms
Fig. 3.1|Schema ic ep esen a ion o he
in i o spo ozoi e in ec ion sys em.
Hepa oma cells we e seeded in 24-well pla es
o e glass co e slips and allowed o g ow
o e nigh a 37ºC in a 5% CO2 a mosphe e. On
he nex day spo ozoi es a e added o he cells
and allowed o de elop in o exo-e y h ocy ic
o ms (EEFs) o 24h (A). The numbe o EEFs
in a co e slip was coun ed unde a luo escence
mic oscope (Plasmodium exo-e y h ocy ic o m,
g een; nucleus, blue) (B).
3.5 Immuno luo escence assays
A e ixa ion wi h PFA, hepa oma cells we e washed wi h PBS and incuba ed o a leas 1h in a
p o ein blocking solu ion (3% Bo ine Se um Albumin, 100mM Glycine, 10% Goa se um; Sigma) o
a oid unspeci ic eac ion, con aining 0.1% Saponin (Sigma) o pe meabiliza ion. Cells we e hen
kep o 45 min a RT wi h he p ima y an ibody agains pa asi e hea shock p o ein 70 (HSP70)
(Tsuji e al., 1994). A e washing wice wi h PBS, cells we e incuba ed o 45 min a RT wi h
seconda y an i-mouse IgG luo escein (FITC)-conjuga ed an ibody (Sigma) dilu ed in blocking
solu ion. Hepa oma cells we e hen washed wice in PBS and incuba ed wi h 4'6-diamidino-2-
phenylindole (DAPI, Sigma) dilu ed in PBS du ing 1 min o s aining he nuclei. A e wa ds, cells
we e washed 3 imes wi h PBS and he co e slip was moun ed on a slide wi h moun ing medium
Mowiol (Calbiochem) and obse ed in a luo escence mic oscope (Leica DM LB2). All he
immuno luo escence assays (IFA) we e pe o med ollowing he p ocedu e desc ibed abo e using
he an ibodies ele an o each assay as lis ed in Table 3.1.
34| Chap e Th ee
Table 3.1|An ibodies used in immuno luo escence s ainings.
Designa ion S aining ea u e O igin
NYSI P. yoelii CS p o ein (Cha oen i e al., 1987)
An i- Mouse IgG FITC o R-PE Mouse IgG Sigma
2E6 P. yoelii HSP70 (Tsuji e al., 1994)
An i-NFkB p65 ( elA) NFkB San a C uz Bio ecnology
An i-albumin Mouse albumin Cappel / ICN I ine
PE-An i-CD11c Dend i ic cell Pha mingen
FITC-An i-Mac3 Mac ophages Pha mingen
An i-Rabbi IgG FITC o Texas Red Rabbi IgG Jackson BioLabs
An i-Ac i e Caspase-3 p18 agmen o caspase-3 P omega
3.6 Cell ea men du ing spo ozoi e in ec ion
The s udy o he e ec o di e en ea men s du ing he cou se o in i o Plasmodium spo ozoi es
in ec ion was ca ied ou acco ding o he e ec unde s udy. The e o e, (i) hepa ocy e g ow h ac o
(HGF) (500 ng/ml, Calbiochem) was added o cells 1h p io in ec ion; (ii) ca bobenzoxy- alyl-
alanyl-aspa yl-[O-me hyl]- luo ome hylke one (Z-VAD-FMK) (20µM, P omega) was also added o
cells 1h p io in ec ion; (iii) he agonis an ibodies DN-30 and DO-24 (1µg/ml) (P a e al.,1998)
we e added o cells 90 min pos in ec ion; (i ) and LY294002 (25µM, Sigma) and PD98059 (30µM,
Sigma) we e added o cells 1h p io in ec ion and ollowed by washes wi h PBS be o e addi ion o
Plasmodium spo ozoi es. Reagen s we e dilu ed in se um- ee DMEM, which was used unde he
same condi ions as he con ol.
3.7 Apop osis induc ion assays
Fo he apop osis induc ion assays, Hepa1-6 o HepG2 cells we e ea ed wi h Tumo Nec osis
Fac o - α (TNF-α, 10ng/ml, R&D Sys ems) and Cycloheximide (CHX, 10µg/ml, Sigma) o
exposed o UV ligh in o de o induce apop osis 18h a e spo ozoi e in ec ion. Hea killed pa asi es
(30min; 56ºC) we e used as con ol. Six hou s la e cells we e ixed and s ained o pa asi e de ec ion
and hos cell apop osis quan i ica ion.
3.8 De ec ion o apop osis
Apop o ic cells we e de ec ed and quan i ied by di e en me hods based on ea u es ha a cell
commonly displays du ing he apop o ic cascade p ocess (Chipuk and G een, 2005).
Ma e ials and Me hods|35
The TUNEL assay using In Si u Cell Dea h De ec ion eagen (Roche) was pe o med acco ding o
he manu ac u e ins uc ions in o de o examine DNA b eaks in cells unde going apop osis.
B ie ly, a e apop osis induc ion cells we e ixed wi h 4% PFA and pe meabilized wi h 0.1% T i on
X-100 in 0.1% sodium ci a e. A e washing wi h PBS, cells we e incuba ed wi h he eac ion
mix u e o 60 min a 37°C. S ained cells we e moun ed wi h Mowiol and analyzed unde a
luo escence mic oscope (Leica DM LB2).
Apop osis was also de ec ed by immuno luo escence assays (see sec ion 3.5). Cells we e s ained wi h
an i-NFkB p65 ( elA) (San a C uz) an ibody o obse e he ansloca ion o he ansc ip ion ac o
NFkB om he cy oplasm o he nucleus o he cell (Ba ke and Gilmo e, 1999), and caspase
ac i i y was measu ed wi h an i-ac i e-caspase-3 an ibody (P omega). Bo h NFkB ansloca ion and
caspase ac i i y a e cha ac e is ic ea u es o apop osis (Tho nbe y and Lazebnik, 1998).
Simul aneously, all cells we e incuba ed wi h DAPI o nuclea mo phology assessmen (see sec ion
3.5).
3.9 Dend i ic cell ec ui men o he li e
G oups o 3 mice we e in ec ed by mosqui o bi e (each anes he ized mouse was in con ac wi h 50 P.
yoelii-in ec ed mosqui oes o 20 min) o by in a enous (i. .) adminis a ion wi h 105 P. yoelii
spo ozoi es in a o al olume o 100 µl (gamma-i adia ed o non-i adia ed) o wi h sali a y glands
o unin ec ed mosqui oes as con ol. A di e en imes a e in ec ion, li e s we e collec ed and
ozen in Tissue-Tek (Saku a), and 15 his ological sec ions (10 µm) om each mouse li e we e
examined wi h PE-an i-CD11c mouse an ibody (Pha migen) o de ec ion and quan i ica ion o
dend i ic cells.
3.10 In i o in ec ion, isola ion and s aining o li e mononuclea cells
BALB/c emale mice o 6-8 weeks o age we e in ec ed wi h P. yoelii gamma-i adia ed (20 K ad),
non-i adia ed, o hea -killed (15 min; 56ºC) spo ozoi es by i. . injec ion (1×106 spo ozoi es/mouse).
Six hou s a e in ec ion he li e o each animal was collec ed, manually homogenized, and passed
h ough a cell s aine in DMEM medium. The cell suspension was washed and esuspended in a
35% Pe coll (Pha macia Bio ech) g adien solu ion and cen i uged (500g; 10 min). The
mononuclea -cell pelle was esuspended in 1ml e y h ocy e lysis bu e (Cappel) o 1 min and
washed 3 imes wi h PBS. Isola ed cells we e coun ed and placed o 1h on poly-L-lysine coa ed
co e slips (~1.5×106 cells/li e we e ob ained). A e ixa ion wi h 2% PFA, mononuclea cells we e
s ained wi h PE-an i-CD11c and FITC-an i-Mac3 an ibodies (Pha migen) o dend i ic cell and
mac ophage labeling, espec i ely. Pa asi es we e s ained wi h an i-P. yoelii HSP70 monoclonal
36| Chap e Th ee
an ibody (2E6). Cells we e also s ained wi h an i-albumin (Cappel/ICN I ine) and an i-ac i e
caspase-3 (P omega). Quan i ica ion was pe o med wi h a luo escence mic oscope.
3.11 De ec ion and quan i ica ion o in i o Plasmodium spo ozoi e in ec ion
using Re e se T ansc ip ion and Real-Time PCR
Fo y hou s pos in ec ion, li e s o mice inocula ed wi h 50.000 Plasmodium spo ozoi es by i. .
injec ion we e mechanically homogenized (Tissue Tea e Blade ) in 4 ml dena u ing solu ion (4M
Guanidium Thiocyana e, 25 mM Sodium Ci a e pH 7, 0.5% N-Lau osyl-sa cosine, 0.1% β-
me cap oe hanol; Sigma) and p ocessed o RNA isola ion using RNeasy Mini ki (Qiagen)
acco ding o he p o ocol supplied by he manu ac u e . To al RNA (1 µg) was e e se- ansc ibed
o cDNA by using a i s s and cDNA syn hesis ki (Roche) in a 20 µl eac ion olume on a PTC
100 p og ammable The mal Con olle (MJ Resea ch, Inc).
A 4 µl sample o he esul ing p oduc was used o eal- ime PCR ampli ica ion o P. be ghei 18S
RNA sequence using SYBRG een I (Ligh cycle -Fas S a DNA mas e SYBRG een I, Roche) as
luo ogenic p obe and speci ic p ime s (5’-AAGCATTAAATAAAGCGAATACATCCTTAC-3’
and 5’-GGAGATTGGTTTTGACGTTTATGTG-3’). Ampli ica ion was pe o med in Roche
Ligh Cycle Quan i a i e Real ime PCR sys em (Roche) wi h he ollowing empe a u e p o ile: 95ºC
o 10 min, and 40 cycles o dena u a ion a 95ºC o 15 sec, annealing a 60ºC o 5 sec and
ex ension a 72ºC o 20 sec. Con ol plasmids wi h s anda d concen a ions o 18S RNA gene
we e used as desc ibed elsewhe e (B uña-Rome o e al., 2001). The amoun o pa asi e 18S RNA
molecules de ec ed in each sample is ep esen ed as he numbe o plasmid-equi alen in
compa ison wi h a s anda d cu e gene a ed wi h plasmid DNA (B uña-Rome o e al., 2001).
3.12 Inhibi ion o PI3-kinase pa hway du ing Plasmodium in ec ion
LY294002 (2.5 mg, PI3-kinase inhibi o , Calbiochem) in DMSO was injec ed in ape i onealy (i.p.).
The con ol g oup was injec ed wi h he same olume o DMSO. Thi y minu es la e , mice we e
in ec ed by i. . injec ion wi h P. be ghei (5x104) spo ozoi es. Real- ime PCR was used o
quan i ica ion o pa asi e load (see sec ion 3.11).
3.13 Analysis o AKT exp ession du ing Plasmodium in ec ion
Mice we e injec ed wi h LY294002 o DMSO (con ol) 30 min p io o in ec ion wi h P. be ghei
spo ozoi es. Th ee hou s a e in ec ion, li e s we e collec ed and cell lysa es we e sepa a ed by
sodium dodecyl sul a e-palyac ylamide gel elec opho esis (SDS-PAGE) and p obed wi h polyclonal
an i-phospho-Ak (phospho-Se -473) and o al Ak an ibodies (Cell Signalling). De ec ion was
Ma e ials and Me hods|37
immuni y es ablishmen and main enance as i ha bou s he sou ce o pa asi e an igens ha ini ia e
an immune esponse in he hos , he in ec ed hepa ocy e (K zych e al., 2000; Schelle and Azad,
1995).
This i adia ed spo ozoi es immune esponse has been cha ac e ized as being dependen o CD8+ T
cells as hey ecognize an epi ope in he CS p o ein and, upon passi e ans e o hese cy o oxic T
cell clones, a high deg ee o p o ec ion was ob ained (Rome o e al., 1989). On he o he hand, in i o
deple ion o hese T cells dec eases d as ically he p o ec i e immuni y agains li e spo ozoi e
challenge (Weiss e al., 1988; Scho ield e al., 1987a).
The a ge o hese mala ia speci ic CD8+ T cells is he in ec ed hepa ocy e (Ho man and Doolan,
2000; Suh bie e al., 1990; Ho man e al., 1989). Since T cells only ecognize an igens a e hey
ha e been p ocessed and p esen ed on cell su aces along wi h majo his ocompa ibili y complex
(MHC) molecules, CD8+ T cells would exclusi ely be ac i a ed by pa asi e-de i ed epi opes in
associa ion wi h MHC class I molecules, gi en ha lymphocy es a e an igen ecogni ion- es ic ed
conce ning MHC molecules (Townsend and Bodme , 1989).
As he hepa ocy e is he only cell ype ha can bea pa asi e de elopmen and is able o exp ess
MHC class I molecules on i s su ace, i is only when he hepa ic s age occu s ha his ype o
immuni y is es ablished (Doolan and Ho man, 2000; Suh bie e al., 1990). Howe e , in o de o
ha e a CD8+ T cell esponse i is equi ed o ha e e icien p iming o naï e T cells ha will be
esponsible o he ini ia ion o he immune esponse i sel . In subsequen in ec ions, a s imula ion
o memo y T cells esiden in he li e also has o occu (Mo o and Za ala, 2004b; Cha e jee e al.,
1999; Riley, 1999).
CD8+ T lymphocy e esponses a e p imed by MHC class I-associa ed pep ides usually gene a ed
om endogenous p o eins in he cy osol o p o essional an igen-p esen ing cells (APC), he
dend i ic cells. They a e known o be highly e icien modula o s o he immune sys em uniquely
specialized in ini ia ing T cell immuni y ei he in i o o in i o (La sson e al., 2001; Banche eau and
S einman, 1998). Du ing mala ia in ec ion, no dend i ic cells a e in ec ed by Plasmodium spo ozoi es
as hey can only eplica e in hepa ocy es. Howe e , speci ic CD8+ T cells eac i e o Plasmodium
an igens we e ound in i adia ed spo ozoi e-immunized hos s, sugges ing ha pa asi e an igens a e
ans e ed o APCs (K zych e al., 2000; Rod igues e al., 1991; Ho man e al., 1989; Rome o e al.,
1989; Weiss e al., 1988). This phenomenon in which dend i ic cells can also p esen exogenous
an igens associa ed wi h MHC class I molecules gene a ing CD8+ T cell epi opes has been
denomina ed as “c oss-p esen a ion” (Hea h and Ca bo ne, 2001). In addi ion, ecen da a
demons a ed ha dend i ic cells a e equi ed o induc ion o his speci ic an i-mala ia CD8+ T cells
esponse a e immuniza ion wi h i adia ed spo ozoi es as in i o deple ion o hese APCs led o he
ab oga ion o Plasmodium speci ic CD8+ T p iming (Jung e al., 2002).
44|Chap e Fou
In ec ion is o en accompanied by he apop o ic dea h o he in ec ed hos cell, which no mally
leads o he o ma ion o apop o ic bodies ha ca y pa hogen an igens wi hin. Se e al s udies ha e
now demons a ed ha c oss-p esen a ion o an igens in apop o ic bodies is an al e na i e way used
by dend i ic cells o c oss-p iming cy o oxic immune esponses agains in acellula pa hogens which
do no in ec di ec ly p o essional APCs (Winau e al., 2005; Schaible e al., 2003; Y lid and Wick,
2000; Albe e al., 1998b). This mechanism o an igen p esen a ion ia up ake o apop o ic in ec ed
cells has u ned ou o be a ool h ough which immuni y is modula ed (Albe , 2004; Fon eneau e
al., 2002). Recen ly, i has been p oposed ha apop osis o hos cells con ibu es o immuni y
induced by i adia ed-pa asi e accines. I adia ion is a po en induce o apop osis and o decades
i adia ed-a enua e pa asi es ha e been used as li e accines which in case o Plasmodium esul ed in
he achie emen o ull p o ec ion agains he disease (James and G een, 2004; Ho man e al., 2002).
The da a p esen ed in his chap e elucida es one mechanism behind he p o ec ion eached wi h
Plasmodium i adia ed-spo ozoi es immuniza ion, highligh ing one sou ce o pa asi e an igen ha
media es he immune esponse. E idence showed ha hepa ocy es in ec ed wi h i adia ed
Plasmodium spo ozoi es unde go apop osis sho ly a e in ec ion. In addi ion, in ec ion wi h
i adia ed spo ozoi es induces he ec ui men o dend i ic cells o he li e o he hos , whe e hey
phagocy ose he apop o ic in ec ed hepa ocy es. Mo eo e , i is p oposed ha hese apop o ic
Plasmodium-in ec ed hepa ocy es cons i u e a sou ce o pa asi e an igens esponsible o he ini ia ion
o a p o ec i e an i-mala ial immune esponse by dend i ic cells.
4.2 Resul s
4.2.1 I adia ed Plasmodium spo ozoi es induce in ec ed hepa ocy es apop osis
in i o
Al hough i is a ac known o some yea s now ha i adia ed spo ozoi es induce p o ec ion, he
mechanism behind his e ec is s ill no unde s ood. Gi en he ou s anding esul s conce ning he
immune esponses ob ained wi h i adia ed spo ozoi es, i s impo ance in he de elopmen o a
hepa ic s age accine is a clea equi emen . The sc u iny o how his immuni y is es ablished is he
ocus o in ense esea ch e o s and an essen ial ask o he con ol o he mala ia pa asi e (K zych
and Schwenk, 2005).
Since he i s desc ip ion o he mala ia exo-e y h ocy ic s age ha a emp s we e made o es ablish
EEF cul u es in i o (Hollingdale e al., 1983 a, b). Se e al s udies we e ca ied ou , using di e en in
i o me hods and pa asi e s ains, pu suing a cul u e sys em whe e he pa asi e ully de elops in o
ma u e in ec ious EEFs (Mo a and Rod iguez, 2000; Cal o-Calle e al., 1994).
Dend i ic cells in li e s age mala ia|45
I is known ha e en high doses o adia ion do no a ec hepa ocy e’s in asion by spo ozoi es in
i o (Nussle e al., 1989). I was also desc ibed p e iously ha a ea ly ime poin s in cul u e he
EEFs om bo h i adia ed and non-i adia ed spo ozoi es a e indis inguishable. Howe e , a ime
poin s 1-2 days pos -in ec ion, a p onounced dec ease in he numbe o i adia ed EEFs was
obse ed (Suh bie e al., 1990; Suh bie e al., 1987; Hollingdale, 1985; Sigle e al., 1984).
In o de o s udy he su i al o hepa ocy es in ec ed wi h i adia ed Plasmodium spo ozoi es a
ea lie imes a e in ec ion, a mu ine hepa oma cell line, Hepa 1-6 (o a human equi alen , HepG2),
was incuba ed wi h ei he i adia ed o non-i adia ed P. yoelii (o P. be ghei) spo ozoi es and in ec ed
cells we e coun ed a di e en ime poin s pos -in ec ion. In ec ed cul u es we e s ained wi h an
an ibody agains HSP70, a p o ein ha is highly exp essed in de eloping pa asi es inside hepa ocy es
bu no in spo ozoi es (Kuma e al., 1993).
The esul e ealed ha he numbe o EEFs ound in cul u e o bo h non-i adia ed and i adia ed
spo ozoi es dec eases wi h he cou se o ime and his educ ion is mo e ma ked in Hepa 1-6
hepa oma cells in ec ed wi h P. yoelii i adia ed spo ozoi es (squa es, Fig. 4.1) a he han when non-
i adia ed spo ozoi es we e used (ci cles; Fig. 4.1). This dec ease s a s occu ing sho ly a e
in ec ion and in he 24h-48h ime ame he numbe o EEFs in hepa ocy es in ec ed wi h non-
i adia ed spo ozoi es s abilizes while i dec eases s eeply in hose in ec ed wi h i adia ed ones.
Fig. 4.1|Time-cou se o an in i o
in ec ion wi h non-i adia ed o
i adia ed P. yoelii spo ozoi es. Hepa 1-6
cells we e in ec ed wi h non-i adia ed
(ci cles) o i adia ed P.yoelii spo ozoi es
(squa es) du ing di e en incuba ion pe iods.
The cells we e ixed and EEFs we e s ained
wi h an i-HSP70 an ibody o quan i ica ion
unde a luo escence mic oscope.
Time (h)
EEFs
0
50
100
150
200
250
6122448
Time (h)
EEFs
Time (h)
EEFs
0
50
100
150
200
250
6122448
The eason o his dec ease in he numbe o in ec ed cells has been a ibu ed o he degene a ion
o in acellula pa asi es sho ly a e in asion (Suh bie e al., 1990; Sigle e al., 1984). We es ed
whe he his was he case o whe he , al e na i ely, hepa ocy es in ec ed wi h i adia ed spo ozoi es
we e dying by apop osis o he in ec ed cells. To e alua e his hypo hesis, in ec ed cells we e
examined o nuclea mo phology and ac i a ion o NFkB p65 ( elA), a ansc ip ion ac o ha is
ansloca ed o he nucleus a e ini ia ion o ce ain apop o ic pa hways (Ba ke and Gilmo e, 1999).
46|Chap e Fou
Fig. 4.2|I adia ed P. yoelii spo ozoi es lead o he apop osis o he in ec ed hepa ocy e. Hepa 1-6 cells we e
incuba ed wi h P. yoelii i adia ed (a,b) o non-i adia ed (c,d) P. yoelii spo ozoi es. Cells we e ixed a e 6h and s ained wi h
an i-PyHSP70 Ab o de ec EEFs (g een), an i-NFkB o obse e he ansc ip ion ac o ansloca ion om he cy oplasm o he
nucleus o he cell ( ed) and DAPI o e alua e nuclea mo phology (blue).
Bo h he s aining wi h DAPI (blue; Fig. 4.2), showing he i egula nuclea shape, and he
ansloca ion o he ansc ip ion ac o NFkB ( ed; Fig. 4.2) om he cy oplasm o he nucleus,
cons i u e e idence o apop osis in cells in ec ed wi h i adia ed spo ozoi es (Fig. 4.2 a,b), while
in ec ion wi h non-i adia ed spo ozoi es esul s in no signs o apop o ic cells (Fig. 4.2 c,d).
Apop o ic cells we e quan i ied o in ec ion wi h each ype o spo ozoi e and he esul s a e shown
below (Fig. 4.3). Apop osis le el is highe in cells in ec ed wi h i adia ed spo ozoi es in bo h
me hods used o de ec ion.
Con ol Spz I Spz
Apop osis (%)
0
5
10
15
20
25
*
*
Con ol Spz I Spz
Apop osis (%)
Con ol Spz I Spz
Apop osis (%)
0
5
10
15
20
25
*
*
Fig. 4.3|Quan i ica ion o apop osis a ea ly ime
poin s a e in ec ion. Hepa 1-6 cells we e incuba ed wi h
i adia ed o non-i adia ed P. yoelii spo ozoi es. Apop osis
was moni o ed by assessmen o nuclea mo phology (black
ba s) and nuclea ansloca ion o NFkB p65 (whi e ba s).
E o ba s ep esen he s anda d de ia ion o iplica e
samples. As e isks (*) indica e a signi ican di e ence be ween
con ol and i adia ed spo ozoi es (P<0.05).
I could also be obse ed ha EEFs o med by i adia ed spo ozoi es a e much smalle han hose
o med by non-i adia ed ones (Fig. 4.2). I has p e iously been epo ed ha al hough i adia ion
lea es he spo ozoi es capable o in ading and ans o ming in o ophozoi es, de elopmen is
comp omised esul ing in a smalle EEF size. The apid g ow h and eplica ion obse ed in no mal
Dend i ic cells in li e s age mala ia|47
spo ozoi e in ec ions is limi ed when spo ozoi e i adia ion akes place (Schelle e al., 1995; Nussle
e al., 1989; Sigle e al., 1984). Compa ing pa asi e s ains and hos cell ypes, i is e iden ha he
pai o med by hos cell/pa asi e is o ex eme impo ance o achie ing no mal a es o
de elopmen when in i o cul u es o hepa oma cells a e used and could also accoun o di e ences
in EEFs size (Schelle e al., 1994; Hollingdale e al., 1983a).
Apop o ic cell dea h is media ed by molecula pa hways ha culmina e in he ac i a ion o a amily
o cys eine p o eases denomina ed caspases. These p o eases o ches a e he disman ling and
clea ance o he dying cell. Caspase-3 plays a cen al ole in he apop osis cascade and i s ac i a ion
is a conse ed ea u e o he apop o ic p ocess (Chipuk and G een, 2005; Tho nbe y and Lazebnik,
1998). The e o e, apop osis in hepa ocy es in ec ed wi h i adia ed spo ozoi es was also con i med
by de ec ion o caspase-3 ac i a ion (Fig. 4.4). The high deg ee o caspase-3 ac i a ion ( ed; Fig 4.4)
seen in cells in ec ed wi h i adia ed spo o ozoi es (g een; Fig. 4.4) coincides wi h apop o ic cells
ca ying condensed ch oma in obse ed wi h DAPI s aining in blue.
Fig. 4.4|Apop o ic cell in ec ed wi h i adia ed P. yoelii
spo ozoi es. Hepa 1-6 cells we e incuba ed wi h P. yoelii i adia ed
spo ozoi es. 6h pos -in ec ion cells we e ixed and s ained wi h an i-
PyHSP70 Ab o de ec EEFs (g een), an i-ac i e-caspase-3 ( ed) and DAPI o
obse e nuclea mo phology (blue).
Pa asi e i adia ion p obably educes he abili y o de no o syn hesis o pa asi e molecules esponsible
o inhibi ion o apop osis o he in ec ed cell, e en ually leading o he ab oga ion o in ec ion and
cell dea h. Howe e , a lowe bu signi ican amoun o apop osis also occu s du ing a non-i adia ed
spo ozoi e in ec ion, sugges ing ha , in some cases o a ce ain le el, he no mal pa asi e canno
p e en he apop osis o hei hos . This obse a ion aises some ques ions abou whe he his
phenomenon could play a ole du ing he es ablishmen o in ec ion and, speci ically, in gene a ing
immuni y.
4.2.2 The ole o dend i ic cells du ing P. yoelii in ec ion
Dend i ic cells a e an igen-p esen ing cells, capable o ini ia ing and modula ing immune esponses.
In hei imma u e o m, hey a ic om blood in o pe iphe al issues, which hey scan like sen inels
looking o an igens ha hey can cap u e and p ocess. They subsequen ly mig a e o d aining lymph
48|Chap e Fou
nodes, whe e hey a e con e ed in o ma u e and ully ac i a ed cells and p esen an igens o es ing
lymphocy es (Figdo e al., 2004; Banche eau and S einman, 1998).
4.2.2.1 Rec ui men o dend i ic cells o he li e a e mala ia in ec ion
In o de o de e mine i dend i ic cells a e implica ed in he p ocess o es ablishing a li e -s age
immune esponse, in i o assessmen o whe he dend i ic cells we e p esen in he li e du ing
in ec ion was done.
Li e s o mice injec ed wi h i adia ed and non-i adia ed P. yoelii spo ozoi es we e collec ed a
di e en ime poin s pos -injec ion. His ological sec ions s ained wi h an ibody agains mouse
myeloid dend i ic cell ma ke , an i-CD11c, cons i u e e idence o he p esence o dend i ic cells in
he li e sho ly a e in ec ion (Fig. 4.5).
Fig. 4.5|Dend i ic cells a e p esen in he li e a e in ec ion wi h Plasmodium spo ozoi es. His ological li e
sec ions o mice in ec ed wi h mala ia by mosqui o bi e we e s ained wi h an ibodies agains mouse dend i ic cell ma ke an i-
CD11c (g een)(a, ansmi ed ligh o e lay; b). The e is e idence o dend i ic cell p esence nea a blood essel.
The quan i ica ion o hese his ological sec ions showed ha du ing mala ia in ec ion he numbe o
dend i ic cells inc eases in he li e (Fig. 4.6a). Simila esul s we e ob ained when mice we e
subjec ed o bi es om in ec ed mosqui oes (Fig. 4.6b). In bo h cases an inc eased ec ui men o
dend i ic cells ea ly a e in ec ion was obse ed, which was independen o spo ozoi e i adia ion
and in ec ion ou e used. Howe e , when he ec ui men was induced by bi es om in ec ed
mosqui oes, a lowe amoun o dend i ic cells was de ec ed, p obably because o he smalle
numbe s o spo ozoi es injec ed by mosqui o bi e (Medica and Sinnis, 2005; Vaughan e al., 1999;
Vande be g, 1977).
The ec ui men o hese an igen p esen ing cells in he li e indica es ha hey migh play a speci ic
ole du ing Plasmodium in ec ions. The inc ease in he numbe o dend i ic cells in he li e du ing
mala ia in ec ion would allow hese cells o apidly cap u e pa asi e an igens. Howe e , in o de o
be able o p esen hose an igens, hese cells ha e o ecei e a ma u a ion signal and become ully
ac i a ed. The sou ce o hese signals may ha e a ious o igins. Among hem a e he pa hogen i sel
Dend i ic cells in li e s age mala ia|49
o in lamma o y cy okines (Gue monp ez e al., 2002; Medzhi o and Janeway, 2000; Albe e al.,
1998b). The ac ha Plasmodium spo ozoi es mig a e h ough se e al hepa ocy es in he li e be o e
choosing one o in ec , and, in his p ocess, dis up plasma memb anes, causing cell wounding and
nec o ic dea h, could accoun o he o igin o his ma u a ion signal (Mo a e al., 2001; Meis e al.,
1985a). In ac , wounded and dying cells induce local in lamma o y esponses ha can be
esponsible o media ing he dend i ic cell ec ui men o he issues and, consequen ly cause
ma u a ion (Shi e al., 2000; Dieu-Nosjean e al., 1999). I was also desc ibed ha Plasmodium hepa ic
s ages lead o speci ic in lamma o y esponses wi h ec ui men o mononuclea cells (Khan and
Vande be g, 1992). In addi ion, he elease o u ic acid om dying cells would, likewise, p o ide
addi ional ma u a ion signals o dend i ic cells (Shi e al., 2003).
**
** **
Time (h)
DCs/10mm
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0122448
Fig. 4.6|Dend i ic cells a e ec ui ed o he li e a e in ec ion wi h P. yoelii spo ozoi es. Quan i ica ion o
dend i ic cells in his ological sec ions o mice li e s by an i-CD11c s aining. (a) Dend i ic cell numbe in li e s o non-in ec ed
mice (black ba s) and in mice injec ed wi h non-i adia ed (whi e ba s) o i adia ed (s iped ba s) P. yoelii spo ozoi es, a
di e en imes pos -in ec ion. (b) Dend i ic cell numbe in li e s o mice bi en by 50 unin ec ed (black ba s) o in ec ed (whi e
ba s) mosqui oes ob ained a di e en imes pos eeding. E o ba s ep esen he s anda d de ia ion o iplica e his ological
samples, each composed o ≥5 li e sec ions o co e 10 mm2. As e isks (*) indica e a signi ican di e ence be ween he sample
and he con ol (P<0.02).
4.2.2.2 Plasmodium an igens a e phagocy osed by dend i ic cells and mac ophages
As shown abo e in ec ed hepa ocy es unde go apop osis du ing in ec ion. In his p ocess, hey
o igina e apop o ic bodies, which ha e o be clea ed om he li e . Dend i ic cells and mac ophages
a e p o essional an igen-p esen ing phagocy es capable o la ge-scale phagocy osis o apop o ic cells
and clea ance o mic obial pa hogens (Sa ill e al., 2002; Ruba elli e al., 1997). To de e mine
whe he apop o ic hepa ocy es con aining P. yoelii an igens a e phagocy osed by an igen p esen ing
cells in he li e , we isola ed non-pa enchymal mononuclea cells om he li e s o mice immunized
wi h i adia ed spo ozoi es a di e en imes a e injec ion. P. yoelli an igen-ca ying dend i ic cells
and mac ophages we e ound a 6h pos -in ec ion, demons a ing he up ake o Plasmodium an igens
by hese phagocy ic cells (Fig. 4.7).
50|Chap e Fou
Fig. 4.7|Phagocy osis o
apop o ic hepa ocy es
con aining Plasmodium
an igens by dend i ic cells
and mac ophages. Li e non-
pa enchymal mononuclea cells
ob ained 6h a e injec ion o mice
wi h i adia ed P. yoelii spo ozoi es
and s ained wi h di e en an ibodies: dend i ic cell (an i-CD11c, ed; a and b), mac ophage (an i-Mac3, g een; c) and P. yoelii
HSP70 (blue; a, b, c). Plasmodium an igens we e ound inside bo h phagocy ic cells ype.
ac
b
ac
b
An a e age o 3 mac ophages and 2 dend i ic cells in each PyHSP70 acuole-con aining li e we e
ound in 3 di e en expe imen s. The numbe o dend i ic cells con aining hese esicles is p obably
highe in he mouse, bu a e dend i ic cells ha e in e nalized and p ocessed he apop o ic bodies,
hey lea e pe iphe al o gans and mig a e o he lymph nodes, subsequen ly o ha ing ecei ed an
ac i a ion signal. As a esul , he ime ha hey emain in he li e is e y sho . The e o e, he
quan i ica ion ob ained 6h a e immuniza ion migh , in ac , co espond o wha would be expec ed,
because i co esponds o he numbe o phagocy ic cells ha we e s ill in he li e and ha e no ye
mig a ed o he lymph node. Thus, he ime ame o de ec ion o phagocy ic cells ca ying pa asi e
an igens is e y sho and es ic ed as he ime gap be ween hese wo e en s is o app oxima ely 1
h (Sallus o and Lanza ecchia, 2000).
The HSP70-posi i e esicles ound inside mac ophages and dend i ic cells mus be de i ed om
in ec ed hepa ocy es, since his p o ein is no exp essed in spo ozoi es and appea s only in in ec ed
hepa ocy es, app oxima ely 6 h pos -in ec ion (Kuma e al., 1993).
In o de o e alua e he o igin o he phagocy osed Plasmodium an igens, phagocy ic cells we e also
s ained o mouse albumin, a common p o ein known o be ound in he cy osol o hepa ocy es
(Doweiko and Nompleggi, 1991). The esul s demons a ed ha albumin is p esen in he
phagocy osed esicles, con i ming ha in ec ed hepa ocy es, and no dead spo ozoi es, a e he
sou ce o up aken an igens, co esponding o he colocaliza ion obse ed be ween he pa asi e and
albumin (Fig. 4.8). As a con ol, non-pa enchymal li e mononuclea cells we e isola ed om non-
in ec ed con ol mice and p ocessed likewise. No P. yoelii HSP70 o albumin s aining was ound in
hese cells.
Dend i ic cells in li e s age mala ia|51
abcdabcd
Fig. 4.8|Hepa ocy e p o eins a e ound in P. yoelii phagocy osed esicles. Li e non-pa enchymal mononuclea cells
ob ained 6h a e injec ion o mice wi h i adia ed P. yoelii spo ozoi es we e s ained wi h an i-P. yoelii HSP70 (blue; b) and an i-
albumin ( ed; c) an ibodies. Plasmodium esicles colocalized wi h albumin, a p o ein abundan in he li e (b and c o e lay; d).
T ansmi ed ligh iew (a). Cells isola ed om li e s o non-in ec ed con ol mice did no p esen any albumin s aining.
To con i m i hese HSP70-posi i e esicles we e de i ed om apop o ic cells, phagocy ic cells we e
s ained wi h an i-ac i e-capase-3 an ibody. Phagocy es showed e idence ha , indeed, he e is
ac i a ion o caspase-3 in he Plasmodium an igen-con aining esicles implica ing ha dend i ic cells
phagocy ose apop o ic in ec ed hepa ocy es du ing mala ia in ec ions in i o (Fig. 4.9). Non-in ec ed
con ol mice did no exhibi any posi i e s aining o capase-3 ac i a ion.
abcdabcd
Fig. 4.9|Caspase-3 is ac i e in P. yoelii phagocy osed apop o ic bodies. Li e non-pa enchymal mononuclea cells
ob ained 6h a e injec ion o mice wi h i adia ed P. yoelii spo ozoi es we e s ained wi h an i-P. yoelii HSP70 (g een; b) and an i-
ac i e-caspase-3 ( ed; c) an ibodies. Plasmodium esicles colocalized wi h ac i a ed caspase-3 indica ing an apop o ic o igin (b and
c o e lay; d). T ansmi ed ligh iew (a). Cells isola ed om li e s o non-in ec ed con ol mice did no p esen any caspase-3
ac i a ion.
When li e non-pa enchymal mononuclea cells we e analyzed 12 h pos -in ec ion, no cells we e
ound con aining P. yoelii HSP70, sugges ing ha in ec ed hepa ocy e dea h and clea ance occu s
ea ly du ing in i o in ec ions wi h i adia ed spo ozoi es. This is consis en wi h he obse a ion ha
Plasmodium spo ozoi e in ec ion and ini ia ion o he immune esponse occu s apidly, since
ac i a ion o speci ic an i-P. yoelii CD8+ T cells is de ec ed as soon as 8 h a e inocula ion o
spo ozoi es (Ha alla e al., 2003; Ha alla e al., 2002; Sano e al., 2001).
52|Chap e Fou
4.2.2.3 Es ablishmen o a c oss-p esen a ion assay
To ini ia e immune esponses, dend i ic cells need o ac i a e naï e T lymphocy es in o cy o oxic T
lymphocy es by aking up, p ocessing and deg ading an igens in o pep ides which can hen be
p esen ed on MHC class I molecules. Dend i ic cells can p esen an igens acqui ed ei he by
in e naliza ion o syn hesized in hei cy osol. When pa hogens do no in ec hese p o essional
an igen-p esen a ion cells, an igens a e acqui ed ia an exogenous ou e, anspo ed in o he cy osol
and p esen ed by MHC class I molecules h ough a c oss-p esen a ion p ocess (Gil-To eg osa e al.,
2004; Albe e al., 1998 a, b).
Since Plasmodium-in ec ed apop o ic hepa ocy es a e phagocy osed by dend i ic cells and apop o ic
cells cons i u e an e icien exogenous sou ce o an igens, able o induce a mala ia immune
esponse, a c oss-p esen a ion assay capable o p o ing ha dend i ic cells a e he ones esponsible
o his c oss-p esen a ion, a e he an igen up ake p o ided by he apop o ic in ec ed hepa ocy es,
was necessa y.
Se e al a emp s we e made pu sing his aim bu he esul s ob ained we e ei he inconclusi e and
inconsis en o i ep oducible be ween di e en expe imen s o an a ay o di e en easons. Fo
example, he o al cell numbe p esen in a li e (109) and he small p opo ion o hese ha we e
po en ially in ec ed and an igen-p o iding was a huge disad an age on se ing up such an assay.
The e o e, he posi i e esul s ob ained cons i u e p elimina y da a only and a e no shown he e.
Ne e heless, he app oaches ollowed in his p ocess a e desc ibed nex .
Plasmodium ci cumspo ozoi e p o ein cons uc
A gene ic app oach was unde aken o e alua e i dend i ic cells a e capable o c oss-p esen ing
Plasmodium an igens de i ed om apop o ic bodies. A plasmid cons uc was made using he P. yoelii
CS p o ein (kindly supplied by D . Osca B uña-Rome o). Hepa 1-6 cells we e ans ec ed wi h his
cons uc and apop osis was induced by UV ligh . The apop o ic bodies gene a ed in he p ocess
we e incuba ed wi h dend i ic cells o 6h o allow phagocy osis. These cells we e hen injec ed in o
mice and an ELISPOT assay was pe o med nine days pos injec ion o de e mina ion o ac i a ed
CD8+ T cells speci ic o Plasmodium CS p o ein h ough he quan i ica ion o IFN-γ p oduc ion
(Ca alho e al., 2001).
CD8+ T cell clone speci ic o Plasmodium CS
Ano he app oach ha was ca ied ou was based on he use o an immunologic ool o de ec ion
o P. yoelii-speci ic an igen p esen a ion. To obse e i dend i ic cells we e able o ac i a e T cells
a e phagocy osing he pa asi e an igens in he li e , mice we e injec ed wi h i adia ed and hea -
killed spo ozoi es (used as nega i e con ol). Li e cells we e isola ed and placed oge he wi h he
CD8+ T cell clone (Y26) speci ic o an epi ope om P. yoelii CS p o ein (Rod igues e al., 1991).
Dend i ic cells in li e s age mala ia|53
Legend|Model o hepa ocy e in ec ion by Plasmodium spo ozoi es. Spo ozoi es a e se se e al
hepa ocy es, dis up ing hei plasma memb anes. This induces he ac i a ion o spo ozoi e exocy osis and he
sec e ion o HGF om hepa ocy es. Wi hin 2 o 4h, HGF binds o i s ecep o MET, in neighbou ing hepa ocy es,
ansducing signals ha lead o ac in cy oskele on eo ganiza ion and inhibi ion o in ec ed hos cell apop osis. As a
esul , pa asi e de elopmen occu s successu ully.
5.1 In oduc ion
Once hey each he li e , Plasmodium spo ozoi es can in ade hepa ocy es in wo dis inc ways: ei he
by dis up ing hei plasma memb ane o by inducing he o ma ion o a pa asi opho ous acuole
a ound hemsel es. The i s mode o en y allows he pa asi e o mig a e h ough hepa ocy es,
expe iencing a di ec con ac wi h he hos cy oplasm and is ollowed by apid exi o he
spo ozoi es using he same mechanism. Thus, spo ozoi es a e se se e al hepa ocy es be o e
in ading a inal one whe e a acuole is o med by in agina ion o he hos plasma memb ane and
li e in ec ion s a s (Mo a e al., 2001; Mo a and Rod iguez, 2001). This ea u e o Plasmodium’s
in asion p ocess is essen ial since pa asi es become ac i a ed du ing he mig a ion p ocess, inducing
exocy osis o apical o ganelles, a equi emen o acuole o ma ion when he inal hepa ocy e is
in ec ed (Mo a e al., 2002). Mos o he spo ozoi e- a e sed hepa ocy es su i e by esealing hei
plasma memb ane and a oid dea h, bu o as long as up u e pe sis s, g ow h ac o s and o he
p o eins s o ed in he cy osol a e eleased in o he ex acellula en i onmen . Mo eo e , a e
esealing, wounded cells exp ess and sec e e g ow h ac o s, like he hepa ocy e g ow h ac o
(HGF), which ende neighbou ing hepa ocy es mo e suscep ible o in ec ion (Ca olo e al., 2003).
HGF, also known as sca e ac o , was i s desc ibed as a po en mi ogen o hepa ocy es du ing
li e egene a ion as well as mo ili y ac o inducing “sca e ing ac i i y” in pola ized epi helia cells
(Bussolino e al., 1992; Bo a o e al., 1991; S oke e al., 1987; Nakamu a e al., 1986). This
glycop o ein is sec e ed as a biologically ine p ecu so (p o-HGF) ha , unde speci ic condi ions,
such as issue damage, and h ough a p o eoly ic diges ion becomes a bioac i e he e odime ma u e
HGF o m (Za nega and Michalopoulos, 1995).
The ecep o o HGF was iden i ied as a c-me p o o-oncogene p oduc encoding a ecep o
y osine kinase (Bo a o e al., 1991; Naldini e al., 1991; Pa k e al., 1986; Coope e al., 1984).
The MET ecep o has wo main s uc u al cha ac e is ics: a unique mul i unc ional docking si e
h ough which ec ui men o all he downs eam signalling molecules occu s, and one single la ge
adap o p o ein wi h mul iple subs a es, known as Gab1, ha media e mos o he complex cellula
esponses subsequen o ac i a ion (Bi chmeie e al., 2003; Fu ge e al., 2000). Like i s ligand, MET
is also a he e odime wi h an en i ely ex acellula alpha chain and a polypep ide chain wi h an
in acellula y osine kinase domain. The s imula ion o MET esul s in a se ies o biological and
biochemical e ec s wi hin he cell, leading o sca e ing, p oli e a ion and g ow h, enhanced cell
mo ili y, angiogenesis, su i al and he in asion o ex acellula ma ices (T usolino and Comoglio,
2002).
MET exp ession occu s in he no mal epi helium o almos e e y issue, al hough o he cell ypes
such as endo helial cells, hema opoie ic cells, mic oglial cells, neu ons, and a a ie y o umo cell
An i-Apo o ic HGF/MET signalling|61
lines also exp ess his ecep o (Za nega and Michalopoulos, 1995). Al e a ions in he HGF/MET
signalling ha e eme ged as c ucial ea u es o many human malignancies sugges ing ha in e e ing
wi h ac i a ion o his pa hway migh hold he apeu ic alue. Thus, se e al s a egies ha e been
de eloped in o de o manipula e he ansduc ion signals in ol ed, ei he by cons i u i e ac i a ion
o inhibi ion o di e en deg ees (Co so e al., 2005).
Fig. 5.1|Schema ic ep esen a ion o he
di e en pa hways ha a e ac i a ed by
HGF/MET signalling. Upon HGF s imula ion,
wo ecep o molecules dime ize and c oss-
phospho yla e each o he , he eby c ea ing binding
si es o signalling molecules wi hin he cell. F om
he ac i a ion o each ansduce esul s an a ay o
biological ac i i ies ha a e he e summa ized
(adap ed om Mo a e al., 2004).
HGF/MET signalling enhances Plasmodium li e in ec ion and is equi ed no o in asion bu a he
a ea ly s ages o pa asi e de elopmen wi hin he hos cell. In i o inac i a ion o MET inhibi s P.
be ghei li e in ec ion, con i ming he ele ance o his mechanism o in ec ion. Fu he mo e, MET
signals induce he eo ganiza ion o hos cell’s ac in cy oskele on which is belie ed o ha e a
con ibu ion o he inal ou come o Plasmodium spo ozoi e in ec ion (Mo a and Rod iguez, 2004;
Ca olo e al., 2003).
A hos -pa hogen in e ac ion igge s a a ie y o esponses ha o ce he pa hogen o de elop
s a egies o manipula e he hos . As al eady men ion, di ec , as well as indi ec , e ec s o pa hogens
and hei p oduc s modula e hos cell dea h (Lude e al., 2001). The induc ion o apop osis upon
in ec ion esul s om a complex in e ac ion o pa asi e p o eins wi h cellula hos p o eins. In ec ed
cells cons i u e a h ea o he hos and he e o e a e no mally des oyed. On he o he hand,
ab oga ion o hos cell apop osis is o en bene icial o he pa hogens as hey a e able o exploi hos
esou ces in o de o sa is y hei needs and ex ending hei li e inside he hos , which ou comes in a
success ul in asion (Hasnain e al., 2003).
Inhibi ion o apop osis by bac e ia and o he in acellula pa asi es such as Leishmania and
Toxoplasma is well documen ed (Heussle e al., 2001; Gao and Kwaik, 2000; Nash e al., 1998; Moo e
and Ma lashewski, 1994). Howe e , he genes in ol ed in his p ocess ha e no ye been iden i ied
(Payne e al., 2003).
62|Chap e Fi e
Al hough he mechanism h ough which he HGF/MET axis elici s inhibi ion o apop osis is no
comple ely unde s ood, he e is no doub ha HGF is a s ong p omo e o cell su i al, since his
biological esponse was obse ed in li e de elopmen h ough gene abla ion o o e exp ession
s udies in ansgenic mice (Amicone e al., 1997; Maina e al., 1996; Schmid e al., 1995). In addi ion,
he an i-apop o ic e ec o HGF has also been epo ed on a ious cell ypes, om epi helia o
umo s (Xiau e al., 2001; Ba delli e al., 1999; Fan e al., 1998).
In his chap e , an addi ional ole is p oposed o HGF in he main enance o Plasmodium in ec ion:
p o ec ion om apop osis. The esul s showed ha apop osis inhibi ion on in ec ed cells is
media ed by HGF/MET signalling ia he PI3K pa hway and allows pa asi e pe sis ence wi hin he
li e un il ull de elopmen is eached.
5.2 Resul s
5.2.1 Spo ozoi e in ec ion p o ec s cells om apop osis
To de e mine whe he Plasmodium in ec ed cells we e p o ec ed om apop osis, HepG2 cells we e
incuba ed wi h P. be ghei spo ozoi es (li e o hea -killed, as con ol) and ea ed 18h la e wi h TNF
and Cycloheximide (CHX) o induce apop osis. Six hou s pos -induc ion, apop osis was quan i ied
using di e en me hods o e alua e dis inc apop osis ea u es. In ec ed cells a e mo e p o ec ed
om apop osis han non-in ec ed ones, as shown when ac i a ion o caspase-3 is measu ed ( ed;
Fig. 5.2) and nuclea mo phology analyzed a e s aining wi h DAPI (blue; Fig. 5.2). Apop osis was
also quan i ied by TUNEL assay o assessmen o DNA b eaks, a common apop o ic ma k (s iped
ba s; Fig. 5.3). Simila da a we e ob ained using a di e en cell ype (Hepa 1-6 cells) and a di e en
induc o o apop osis (UV ligh ; Fig. 5.3 b). In his case a highe le el o p o ec ion han he one
obse ed when in ec ed cells we e induced wi h TNF and CHX was obse ed. Taken oge he , hese
da a cons i u e e idence ha P. be ghei spo ozoi e in ec ion p o ec s hos cells om apop osis.
Fig. 5.2|Plasmodium in ec ion p o ec s om dea h. A hos cell in ec ed wi h
P. be ghei spo ozoi es no showing signs o apop osis and su ounded by apop o ic
non-in ec ed cells, as de ec ed by an i-PyHSP70 (g een), DAPI (blue) and an i-
ac i e-caspase-3 an ibody ( ed)
An i-Apo o ic HGF/MET signalling|63
apop o
Fig. 5.3|In ec ed
cells a e mo e
esis an o
sis. HepG2
cells we e
incuba ed wi h P.
be ghei spo ozoi es
(hea -killed
spo ozoi es we e
used as con ol) and ea ed 18h la e wi h TNF and Cicloheximide o induce apop osis. (a) Apop osis was quan i ied 6h pos -
induc ion by nuclea mo phology ollowing DAPI s aining (black ba s), ac i e caspase-3 (whi e ba s) and TUNEL de ec ion (s ip
ba s). (b) Hepa 1-6 cells we e incuba ed wi h P. be ghei spo ozoi es (hea -killed spo ozoi es we e used as con ol) and exposed o
UV ligh 18h pos -in ec ion. A e 6h, apop o ic cells we e coun ed by TUNEL de ec ion. (a, b) In ec ed cells we e s ained wi h
an i-PyHSP70 an ibody o de ec EEFs in cul u e.
Con ol
Apop o ic cells (%)
In ec ed cells
a
0
10
20
30
40
50
60 b
Con ol
Apop o ic cells (%)
In ec ed cells
0
10
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30
40
50
60
70
80
Con ol
Apop o ic cells (%)
In ec ed cells
a
0
10
20
30
40
50
60
Con ol
Apop o ic cells (%)
In ec ed cellsCon ol
Apop o ic cells (%)
In ec ed cells
a
0
10
20
30
40
50
60 b
Con ol
Apop o ic cells (%)
In ec ed cells
0
10
20
30
40
50
60
70
80 b
Con ol
Apop o ic cells (%)
In ec ed cells
0
10
20
30
40
50
60
70
80
5.2.2 HGF/MET signalling enhances in ec ion and con e s p o ec ion om
apop osis
Se e al cy okines and g ow h ac o s a e capable o inducing di e en cell mechanisms and essen ial
cell unc ions such as p oli e a ion, di e en ia ion, chemo axis and su i al. One o hese molecules
is he hepa ocy e g ow h ac o (HGF) (T usolino and Comoglio, 2002). Binding o HGF o i s
ecep o c-Me induces he ac i a ion o a y osine kinase-dependen cascade o e en s ha esul s in
phospho yla ion o a se ies o esidues and leads o he ec ui men o in acellula signaling
molecules which igge a ange o biological esponses wi hin he cell (Mo a e al., 2004). The
p esence o his g ow h ac o du ing a mala ia in ec ion enhances he in ec ion ou come and is
media ed by HGF-induced MET ac i a ion (Ca olo e al., 2003).
Taking his in o accoun , i was hypo hesized ha he inc ease in he in ec ion is caused by HGF-
induced p o ec ion om apop osis. To es his possibili y, i s , he HGF e ec on Plasmodium-
spo ozoi e in ec ed cells was de e mined. In o de o do so, HepG2 cells we e ea ed wi h HGF
(500 ng/ml) 1h p io o in ec ion wi h P. be ghei spo ozoi es. Con ol cells did no unde go HGF
ea men . A e 6h, cells we e s ained o pa asi e de ec ion (an i-HSP70 an ibody) and apop osis
quan i ica ion by nuclea mo phology (DAPI) o in ec ed hepa ocy es. The esul s showed ha he
p esence o HGF leads o an inc ease in in ec ion and o a educ ion in he numbe o apop o ic
in ec ed cells, sugges ing ha HGF-induced MET ac i a ion is p o ec ing he hos cell om dea h
(Fig. 5.4).
64|Chap e Fi e
0
50
100
150
200
250
300
Con ol HGF
EEFs
Con ol HGF
Apop o ic cells (%)
0
5
10
15
20
25
30
35
ab
0
50
100
150
200
250
300
Con ol HGF
EEFs
Con ol HGF
Apop o ic cells (%)
0
5
10
15
20
25
30
35
0
50
100
150
200
250
300
Con ol HGF
EEFs
0
50
100
150
200
250
300
Con ol HGF
EEFs
Con ol HGF
EEFs
Con ol HGF
Apop o ic cells (%)
0
5
10
15
20
25
30
35
Con ol HGF
Apop o ic cells (%)
Con ol HGF
Apop o ic cells (%)
0
5
10
15
20
25
30
35
ab
Fig. 5.4|HGF-induced MET ac i a ion enhances in ec ion and p o ec s in ec ed cells om apop osis. (a, b)
HepG2 cells we e ea ed wi h HGF o 1h, and incuba ed wi h P. be ghei spo ozoi es. Hepa ocy es we e ixed a e 24h
(in ec ion, a) o 6h (apop osis, b), and s ained wi h an i-HSP70 an ibody ( o isualize he pa asi e) and wi h DAPI (nuclea
mo phology) o quan i y apop osis.
In addi ion, wo monoclonal an ibodies, DO-24 and DO-30, ha ac on he ex acellula domain o
he MET ecep o we e used o assess he same ques ion wi h a di e en app oach. These agonis
monoclonal an ibodies ha e been desc ibed o ha e di e se e ec s on cells and o ac i a e a ious
pa hways (P a e al., 1998). While DO-24 is a ull agonis , capable o inducing a s ong esponse
om he ecep o and igge wha is known as he comple e in asi e g ow h p og am, including cell
su i al, he DO-30 an ibody has a pa ial e ec , causing a weak ac i a ion o MET and inducing
mo ili y only (P a e al., 1998).
To e alua e he e ec o hese agonis s on in ec ion, HepG2 cells we e in ec ed wi h P. be ghei
spo ozoi es 2h a e addi ion o he an ibodies. The numbe o EEFs was quan i ied by
immuno luo escence s aining wi h an i-HSP70 an ibody o pa asi e de ec ion. Resul s show ha
bo h an ibodies lead o an inc ease in in ec ion, al hough o di e en ex en s. Apop osis
quan i ica ion ei he by nuclea mo phology and caspase-3 ac i i y e ealed a co ela ion be ween
he inc emen in in ec ion and a educ ion o in ec ed hepa ocy e dea h (Fig. 5.5). The ull agonis
DO-24 had a s onge e ec han he pa ial agonis DN-30. The caspase-3 ac i i y is in ol ed in
la e s age apop osis jus i ying why his pe cen age is lowe al hough showing he same end
(Th ombe y and Lazebnick, 1998). These esul s demons a e ha signaling h ough HGF/MET
pa hway du ing a mala ia in ec ion is di ec ly implica ed in he p o ec ion om apop osis o he
in ec ed hos cell.
An i-Apo o ic HGF/MET signalling|65
Con ol DN-30
EEFs
DO-24
0
50
100
150
200
250
300
350
400
450
ab
Con ol DN-30 DO-24
Apop o ic cells (%)
0
5
10
15
20
25
30
35
40
45
50
Con ol DN-30
EEFs
DO-24
0
50
100
150
200
250
300
350
400
450
Con ol DN-30
EEFs
DO-24Con ol DN-30
EEFs
DO-24
0
50
100
150
200
250
300
350
400
450
ab
Con ol DN-30 DO-24
Apop o ic cells (%)
0
5
10
15
20
25
30
35
40
45
50
Con ol DN-30 DO-24
Apop o ic cells (%)
Con ol DN-30 DO-24
Apop o ic cells (%)
0
5
10
15
20
25
30
35
40
45
50
Fig. 5.5|HGF agonis s induce in ec ion and hos cell p o ec ion. HepG2 cells we e p e- ea ed o 2h wi h he agonis
an i-MET monoclonal an ibodies DN-30 o DO-24 (1µg/ml) and a e wa ds in ec ed wi h P. be ghei spo ozoi es. (a) The numbe
o in ec ed cells (EEFs) was quan i ied 24h pos in ec ion by coun ing an i-Plasmodium-HSP70 posi i e cells. (b) Apop osis was
quan i ied 6h pos in ec ion by DAPI (black ba s) and caspase-3 ac i i y (whi e ba s). Un ea ed-in ec ed cells we e used as
con ol.
In o de o analyze he beha io o in ec ed cells in he p esence o an apop osis inhibi o , we used
Z-VAD-FMK, a cell pe meable b oad-spec um caspase inhibi o ha binds co alen ly o ac i e
caspases in li ing cells, is non-cy o oxic and has been shown o inhibi apop osis andomly (Chipuk
and G een, 2005; Al-Olayan e al., 2002). HepG2 cells we e ea ed wi h Z-VAD-FMK (20µM) o
wi h HGF (500 ng/ml) 1h p io o in ec ion wi h P. be ghei spo ozoi es. The chemically induced
inhibi ion o apop osis led o a highe in ec ion han ha obse ed ei he wi h HGF o wi h
un ea ed cells, lending u he suppo o he p e ious esul s (Fig. 5.6).
0
500
1000
1500
2000
2500
Con ol HGF ZVAD
EEFs
0
500
1000
1500
2000
2500
Con ol HGF ZVAD
EEFs
Fig. 5.6|Inhibi ion o caspase ac i i y
inc eases in ec ion. 1h p io o in ec ion
wi h P. be ghei spo ozoi es, HepG2 cells we e
ea ed ei he wi h Z-VAD-FMK (20µM) o
wi h HGF (500 ng/ml). The numbe o
in ec ed cells (EEFs) was de e mined 24h pos
in ec ion a e s aining wi h an i-HSP70
an ibody. As con ol, un ea ed in ec ed cells
we e quan i ied.
5.2.3 MET inac i a ion leads o apop osis du ing in ec ion
The HGF-media ed signaling cascade occu s h ough a y osine kinase ecep o , c-Me . I s ac i i y is
essen ial o he p og ess o he downs eam ca aly ic p ocesses. In o de o de e mine he e ec o
MET signaling impai men on in ec ed hepa ocy es, a s able ans ec ed HepG2 MET-dominan -
nega i e- o m cell line (DN MET-GFP) was used. Fo his pu pose, a len i i us exp essing a
66|Chap e Fi e
chime ic cons uc con aining he ex acellula and ansmemb ane domains o me used o g p
sequences was ans ec ed in o HepG2 cells. The p oduc o his cons uc is exp essed a he
plasma memb ane and binds o HGF bu i is unable o ansduce signals in o he cell since i lacks
he kinase domain and he y osines ac ing as docking si es o in acellula subs a es. This chime ic
ecep o beha es as a dominan nega i e, because i dime izes wi h endogenous MET and
consequen ly p e en s i s ac i a ion (Ca olo e al., 2003; Gio dano e al., 2002; Vigna and Gio dano,
2000). Because cells ans ec ed wi h his len i i us become luo escen , i is possible o de e mine
he in ec ion le el o single- ansduced cells 24h a e in ec ion.
As p e iously men ioned, he ac i a ion o he MET ecep o is essen ial o he in ec ion o
hepa ocy es by Plasmodium spo ozoi es (Ca olo e al., 2003). The impai men o he signal abolishes
comple ely he in ec ion 24h pos - ansduc ion, as no indi idual cells ansduced wi h MET-GFP
we e ound o be in ec ed wi h Plasmodium (Fig. 5.7a). The numbe o apop o ic cells co ela es wi h
MET inac i a ion as in hese cells apop osis inc eased o 68% while con ol cells (GFP- ansduced
only) show a 21% le el o dea h, as de e mined by obse a ion o nuclea mo phology 6h pos -
in ec ion ollowing DAPI s aining clea ly shows apop osis ea u es in MET-GFP ansduced cells
(Fig. 5.7b; Fig. 5.8). These esul s s ongly sugges ha MET signaling is equi ed o hos cell
apop osis inhibi ion and in ha way o hos cell su i al and in ec ion success h ough an an i-
apop o ic mechanism.
0
100
200
300
400
500
600
700
800
Con ol MET-GFP
EEFs
ab
Con ol MET-GFP
Apop o ic cells (%)
0
10
20
30
40
50
60
70
80
0
100
200
300
400
500
600
700
800
Con ol MET-GFP
EEFs
ab
0
100
200
300
400
500
600
700
800
Con ol MET-GFP
EEFs
0
100
200
300
400
500
600
700
800
Con ol MET-GFP
EEFs
ab
Con ol MET-GFP
Apop o ic cells (%)
0
10
20
30
40
50
60
70
80
Con ol MET-GFP
Apop o ic cells (%)
Con ol MET-GFP
Apop o ic cells (%)
0
10
20
30
40
50
60
70
80
Fig. 5.7|MET inac i a ion leads o ea ly apop osis o in ec ed cells. HepG2 cells we e ansduced wi h a len i i us
exp essing MET-GFP o GPF alone (con ol). The dominan nega i e hepa ocy e MET mu an cell and con ol lines we e
in ec ed wi h P. be ghei spo ozoi es. 24h pos -in ec ion he numbe o EEFs was de e mined (a) and 6h pos -in ec ion apop osis
was quan i ied by nuclea mo phology obse a ion (b). All P ≤ 0,001.
I is likely ha all in ec ed cells unde go apop osis in he absence o a unc ional MET ecep o
wi hin 24h, as no in ec ed cells we e ound a his ime poin . No inc ease in apop osis o non-
in ec ed ansduced cells was obse ed.
An i-Apo o ic HGF/MET signalling|67
abab
Fig. 5.8|Dominan nega i e MET-GFP in ec ed cell unde going
apop osis. (a) HepG2 cell 6h a e in ec ion wi h P. be ghei spo ozoi es ( ed,
an i-HSP70) wi h a no mal nuclea mo phology (blue, DAPI). (b) In ec ed cell
exp essing MET-GFP (g een) 6h pos -in ec ion wi h a pa asi e s a ing o
de elop ( ed, an i-HSP70) and exhibi ing apop osis ea u es de ec ed by nuclea
mo phology wi h DAPI (blue).
5.2.4 MET down- egula ion leads o apop osis du ing in ec ion
As shown abo e he impai men o HGF/MET signaling using a dominan nega i e o m o MET
leads o highe le els o apop osis and p omo es he ea ly dea h o he in ec ed cell. To con i m
hese esul s we also ab oga ed MET exp ession using RNA in e e ence. HepG2 cells we e
ansduced wi h a len i i us ec o exp essing a sho in e e ing RNA (siRNA) oligonucleo ide
a ge ed a MET. A con ol sequence was used ha did no cause any educ ion on MET exp ession
as de ec ed by Wes e n blo analysis (Fig. 5.9).
Con ol siRNA MET siRNACon ol siRNA MET siRNA
Fig. 5.9|MET exp ession on sho in e e ing RNA cell line. Wes e n
blo analysis o MET exp ession in HepG2 cells ansduced wi h len i i uses
exp essing con ol siRNA oligonucleo ide (le lane) o MET siRNA
oligonucleo ide ( igh lane) o knockdown me .
The down-modula ion o MET ac i i y showed simila esul s as he ones ob ained o he dominan
nega i e cell line, ha is, an in e se co ela ion be ween a dec ease in in ec ion (Fig. 5.10a) and an
inc ease in apop osis o in ec ed cells (Fig. 5.10b). This e idence con i ms he ela ion be ween
hepa ocy e su i al and HGF/MET ac i a ion, indica ing ha i s an i-apop o ic ac i i y is essen ial
du ing Plasmodium in ec ion.
Con ol siRNA MET siRNA
EEFs
0
100
200
300
400
500
600
700
800
900
Con ol siRNA
Apop o ic cells (%)
MET siRNA
0
5
10
15
20
25
30
35
ba
Con ol siRNA MET siRNA
EEFs
0
100
200
300
400
500
600
700
800
900
Con ol siRNA
Apop o ic cells (%)
MET siRNA
0
5
10
15
20
25
30
35
Con ol siRNA MET siRNA
EEFs
0
100
200
300
400
500
600
700
800
900
Con ol siRNA MET siRNA
EEFs
Con ol siRNA MET siRNA
EEFs
0
100
200
300
400
500
600
700
800
900
Con ol siRNA
Apop o ic cells (%)
MET siRNA
0
5
10
15
20
25
30
35
Con ol siRNA
Apop o ic cells (%)
MET siRNACon ol siRNA
Apop o ic cells (%)
MET siRNA
0
5
10
15
20
25
30
35
ba
Fig. 5.10|Down-modula ion o HGF/MET signaling leads o ea ly apop osis o in ec ed cells. Len i i us
enginee ed o exp ess MET-speci ic siRNA oligonucleo ides we e used o ansduce HepG2 cells. Hepa ocy es ans ec ed wi h a
siRNA speci ic o he me gene we e in ec ed wi h P. be ghei spo ozoi es. As con ol, hepa ocy es we e ans ec ed wi h a siRNA
o an un ela ed sequence. (a) In ec ion was quan i ied 24h pos -spo ozoi e addi ion by coun ing he numbe o EEFs and (b) 6h
pos -in ec ion apop osis was quan i ied by nuclea mo phology obse a ion wi h DAPI s aining. All P ≤ 0,001.
68|Chap e Fi e
5.2.5 An i-apop o ic e ec o HGF/MET signalling is media ed ia ac i a ion o
PI3K pa hway
In a signalling cascade, a se ies o subsequen ly biological e en s ake pa in he p ocess o eaching
a a ge o accomplishing a ce ain le el o ac i a ion o induc ion. In HGF cascade he su i al
pa hway is media ed by a phosphoinosi ide 3-Kinase (PI3K)/Ak , whose ac i i y di ec s he cell o a
dea h inhibi ion s a e (Webs e and Anwe , 2001; Xiau e al., 2001).
To de e mine i his pa hway is implica ed in mala ia in ec ion, HepG2 cells we e ea ed wi h a
speci ic PI3K inhibi o (LY294002) 1h p io in ec ion wi h P. be ghei spo ozoi es. Addi ionally, a
mi ogen-ac i a ed p o ein kinase (MAPK) inhibi o (PD98059) was used o e alua e i his pa hway
was in ol ed in p o ec ion om apop osis. An an i-apop o ic ole has also been desc ibed o
MAPK pa hway, since once ac i a ed by HGF, i was capable o escuing cells om apop osis,
al hough o much smalle ex en (Xiau e al., 2001).
As shown below in Figu e 5.11 when cells a e ea ed wi h ei he o hese inhibi o s he e is a
signi ican dec ease in in ec ion. This e ec is s onge when he PI3K ac i i y is inhibi ed. When
apop osis was quan i ied a e 6h on he same in ec ion expe imen , a signi ican inc ease was
de ec ed in he apop o ic cells numbe , only when cells we e p e ea ed wi h he PI3K inhibi o .
These esul s show ha , al hough bo h PI3K and MAPK pa hways seem o be in ol ed in
Plasmodium in ec ion, only PI3K/Ak ia appea s o be impo an o hos cell p o ec ion om
apop osis.
Con ol LY294002 PD 98059
EEFs
0
200
400
600
800
1000
1200
1400
1600
1800
2000
0
5
10
15
20
25
30
35
40
45
Con ol LY294002 PD 98059
Apop o ic cells (%)
ab
*
*
*
Con ol LY294002 PD 98059
EEFs
0
200
400
600
800
1000
1200
1400
1600
1800
2000
Con ol LY294002 PD 98059
EEFs
Con ol LY294002 PD 98059
EEFs
0
200
400
600
800
1000
1200
1400
1600
1800
2000
0
5
10
15
20
25
30
35
40
45
Con ol LY294002 PD 98059
Apop o ic cells (%)
0
5
10
15
20
25
30
35
40
45
Con ol LY294002 PD 98059
Apop o ic cells (%)
Con ol LY294002 PD 98059
Apop o ic cells (%)
ab
*
*
*
Fig. 5.11|Inhibi ion o PI3K/AKT pa hway in i o leads o a dec ease in in ec ion. HepG2 cells we e p e ea ed
wi h LY294002 (25µM, PI3K inhibi o ) and wi h PD98059 (30µM, MAPK inhibi o ) o 1h. Inhibi o s we e washed be o e
in ec ion wi h P. be ghei spo ozoi es. Con ol cells we e incuba ed wi h DMSO. (a) 24h pos -in ec ion cells we e ixed, s ained
and EEFs we e coun ed (PLY ≤ 0,001; PPD = 0,003). (b) 6h pos -in ec ion cells we e ixed and apop o ic cells quan i ied by DAPI
s aining (PLY = 0,002; PPD = 0,09).
An i-Apo o ic HGF/MET signalling|69
Vi ulen s ains o some p o ozoa exp ess highe amoun s o HSPs as compa ed o low i ulen
s ains. Du ing Toxoplasma gondii in ec ion, exp ession o HSP70 is induced in i ulen s ains, a ac
ha seems o p o ide p o ec ion o hese s ains since pa asi es a e able o pe sis as achyzoi es
wi hou he equi emen o he encys a ions obse ed in a i ulen s ains (Dobbin e al., 2002;
Lyons and Johnson, 1995).
HSPs a e also esponsible o he pa hogen’s adap a ion o he hos en i onmen . Pa asi es ha
possess a biphasic cycle in ol ing wo di e en hos s ha e o adap o he empe a u e changes ha
occu be ween he ec o and he mammalian en i onmen s. The esponse o his empe a u e shi
has been shown o in ol e he p oduc ion o HSP70, bo h in Leishmania majo and in T ypanosoma
b ucei, which allows he pa asi e o di e en ia e in o p oli e a i e o ms (Polla, 1991; Shapi a e al.,
1988; an de Ploeg e al., 1985).
B A
C
Fig. 6.1|E en s egula ed by HSPs in he mi ochond ial and dea h
ecep o -media ed apop o ic pa hways. Ex acellula signals o
s esses con e ge o egula e he mi ochond ia-media ed pa hway o caspase
ac i a ion and cell dea h. (A) Hea shock p o eins in e ene a mul iple
poin s wi hin his pa hway bo h ups eam and downs eam o he associa ed
mi ochond ial changes o egula e he engagemen and/o p og ession o
apop o ic e en s. (C) Liga ion o cell su ace dea h ecep o s, e.g. Fas and
TNFR1, by he app op ia e ligand engages mul iple in acellula signals
leading o caspase ac i a ion and cell dea h o NFkB-media ed su i al. Many
elemen s o hese pa hways a e egula ed by he ac i i ies o HSPs o help
main ain cellula su i al ollowing dea h ecep o liga ion. HSP-media ed
inhibi ion is indica ed (T-ba s) and HSP-media ed po en ia ion o a signaling
pa hway is depic ed as a di ec in e ac ion be ween he HSP and i s a ge
(+) (adap ed om Bee e, 2004).
76|Chap e Six
HSPs o mala ia pa asi es we e cloned and shown o be ex emely simila o homologous
mammalian ones (Syin and Goldman, 1996; Bonne oy e al., 1994; Su and Wellems, 1994; Kuma e
al., 1991). Wi hin he di e en Plasmodium species se e al homologs o HSP70 we e iden i ied, like
genes encoded by P. chabaudi and P. cynomolgi ha p esen a 97% o sequence homology, indica ing a
close ela ion be ween hese molecules (Kuma and Zheng, 1998; Ecke e al., 1992; Ma ei e al.,
1989; Sheppa d e al., 1989).
P. be ghei (PbHSP70) and P. alcipa um (P HSP70) HSP70s a e exp essed in he blood and li e s ages
o hese pa asi es, bu no in spo ozoi es (Tsuji e al., 1994; Kuma e al., 1993; Bianco e al., 1986;
Renia e al., 1990).
Addi ional s udies ha e shown ha du ing a na u al in ec ion, Plasmodium s ess p o eins a e a ge s
o he hos immune sys em. Immune esponses o mala ial HSPs ha e been demons a ed in
pa ien s wi h mala ia (Beh e al., 1992; Kuma e al., 1993; Bianco e al., 1986). P HSP70 is in ac a
a ge o an ibody-dependen esponse and is ecognized by T cells om in ec ed indi iduals (Beh
e al., 1992; Kuma e al., 1993). Like mammalian HSP70s, he P. alcipa um p o ein has he EEVD
cha ac e is ic mo i a i s C- e minus. The p esence o an addi ional mo i (GGMP epea s) is
hough o be esponsible o inducing his immune esponse in he hos , since his mo i is only
obse ed in p o eins o pa asi ic o igin (Kuma and Zheng, 1998).
Simila o o he pa asi es, P. alcipa um showed a ma ked inc ease in HSP70 exp ession when
submi ed o highe empe a u es. This hea shock esponse was shown o be ansien since once
he s ess was emo ed he induc ion was ep essed. Such beha iou could indica e a p o ec i e
unc ion du ing mala ia e e episodes, esul ing in he in i o su i al o he pa asi e (Biswas and
Sha ma, 1994; Joshi e al., 1992; Kuma e al., 1991).
Howe e , his cy op o ec ion o he mala ia pa asi e h ough i s ac ion as a molecula chape one
equi es u he s udies, since he biochemical and chape one p ope ies o P HSP70 a e no o ally
unde s ood. Due o an ex emely A/T- ich genome and he usage o codons ha a e a ely used in
Esche icia coli, i has been di icul o o e exp ess Plasmodium p o eins (Ma ambo e al., 2004; Baca and
Hol, 2000). Recen ly, u he s udies we e pe o med o add ess he ac i i y o P HSP70 in i o and
i s ole as a chape one. Da a demons a ed he p esence o a basal ATPase ac i i y which was
he mo-inducible and shown o be cy op o ec i e (Shonhai e al., 2005).
O he amilies o hea shock p o eins a e also p esen in mala ia pa asi es. HSP90, he mos
abundan chape one in mammalian cells, plays an essen ial ole in he olding o p o eins ha ac on
cell cycle egula ion and signal ansduc ion. The PyHSP90 is di e en ly exp essed h oughou he
li e cycle, highly on blood asexual s ages and e y sca cely on game ocy es (Zhang e al., 1999).
P HSP90 was shown o be essen ial o pa asi e iabili y in blood s ages, since ea men wi h
PyHSP70 as an apop osis inhibi o |77
geldanamycin, a highly speci ic inhibi o o HSP90, was able o ab oga e pa asi e g ow h
(Banuma hy e al., 2003; Kuma e al., 2003).
O e all, he induc ion o HSPs upon cellula exposu e o s ess is clea ly ela ed o he in ol emen
o hese p o eins in inducing su i al and allowing cells o ole a e and eco e om damage (Bee e,
2005). Such e idence has also been implica ed in umou esis ance o chemo he apeu ic s a egies
and ca cinogenesis (C eagh e al., 2000).
As men ioned in chap e II o his hesis, se e al eco ds show e idence ha cell dea h is a
conse ed mechanism h oughou e olu ion and is also p esen in pa asi ic p o ozoa (Depon e and
Becke , 2004). Mo eo e , Plasmodium in ec ion does lead o hos cell dea h inhibi ion. Howe e , he
plasmodial p o eins esponsible o his inhibi o y unc ion a e s ill unknown. The e o e, like o he
pa hogens, Plasmodium mus ca y an i-apop o ic genes wi h homology o cellula egula o s wi h
simila unc ions o genes no di ec ly coding o an i-apop o ic p o eins, bu able o modula e he
exp ession o cellula apop osis- egula o s genes in hepa ocy es. Consequen ly a sea ch o
homologues o known apop o ic inhibi o s wi hin Plasmodium genome da abases could unco e
possible candida es wi h his abili y in he pa asi e. Fu he mo e, membe s o hese amilies a e
p esen in a wide ange o o ganisms and all con ain de ined and conse ed mo i s (Hu d and
Ca e , 2004).
This chap e ep esen s a pilo a emp o s udy he unc ion o PyHSP70 as a possible candida e
esponsible o he inhibi ion o apop osis in in ec ed hepa ocy es. The p elimina y esul s p esen ed
he e suppo his hypo hesis. Hepa oma cells ans ec ed wi h a Plasmodium HSP70 gene a e
p o ec ed agains apop osis, sugges ing ha Plasmodium HSPs may accoun o pa asi e su i al
du ing li e s age in ec ion.
6.2 Resul s
6.2.1 PyHSP70 exp ession p o ec s hepa ocy es om dea h
As p e iously shown, Plasmodium spo ozoi es a e capable o p o ec ing he hepa ocy e om
unde going apop osis by inhibi ing hos cell dea h (see chap e V).
To e alua e whe he a Plasmodium HSP70 has any ole du ing li e in ec ion, apop osis o
hepa ocy es ans ec ed wi h a cons uc exp essing PyHSP70 was induced by UV ligh exposu e.
Resul s showed a dec ease in apop osis in he p esence o HSP70, while cells ans ec ed wi h a
g een luo escence p o ein (g p) cons uc o ano he i ele an gene (con ol), p esen ed much
highe le els o cell dea h (Fig. 6.2).
78|Chap e Six
The inhibi ion o apop osis induced by PyHSP70 o e exp ession in hepa ocy es sugges s ha his
p o ein could ha e a p o ec i e unc ion du ing li e in ec ion, p e en ing hepa ocy es om
unde going apop osis.
0
10
20
30
40
50
60
70
80
Py Hsp 70 GFP Con ol UnT ans ec ed
Apop osis (%)
0
10
20
30
40
50
60
70
80
Py Hsp 70 GFP Con ol UnT ans ec ed
Apop osis (%)
Fig. 6.2|P. yoelii HSP70 p o ec s
hepa ocy es om apop osis. Hepa 1-6 cells
we e ans ec ed wi h di e en DNA plasmid
cons uc s exp essing P. yoelii HSP70
(PyHSP70), g een luo escence p o ein (g p), an
i ele an gene (con ol). 24h pos - ans ec ion
apop osis was induced by UV ligh . A e 6h,
cells we e s ained wi h DAPI and apop o ic cells
we e quan i ied.
In o de o es ablish a s able cell line exp essing his p o ein ha would allow a b oade applica ion
o his ool o u he s udies, an a emp was made o assemble a cons uc wi h a mammalian
ec o ha would addi ionally p o ide he possibili y o con ol gene exp ession by induc ion
h ough a Zeocin esis ance gene (In i ogen). A e selec ion he esul s ob ained we e no
sa is ac o y since he expec ed app op ia e exp ession was no achie ed. A likely explana ion o
hese obse a ions is he al eady known di icul y inhe en o he cloning o Plasmodium p o eins due
o i s A/T– ich genome ha con e s p oblems o p o ein exp ession expe imen s.
6.2.2 PyHSP70 knock ou pa asi e
To analyze and alida e he ac ual ole o HSP70 in apop osis inhibi ion, i is essen ial ha
unc ional s udies a e made using pa asi es in which exp ession o his p o ein has been knocked
ou . The i s s ep o achie e his goal, is o p oduce a cons uc ha will block p o ein exp ession,
which can be accomplished by cloning only a ac ion o he gene sequence. This expe imen is s ill
unde p og ess and a inal conclusion was no ye possible.
6.3 Discussion
Many pa hogens like schis osomes, leishmania, ypanosomes and mala ia plasmodia, ha e o ace
sudden empe a u e shi s du ing ansmission om ec o o he hos . The inc ease in empe a u e
imposes a hea shock o pa asi es, which leads o he induc ion o HSPs. Mo eo e , he hos -pa asi e
in e ace is apop osis dependen since his p ocess can be used by he hos as a de ence mechanism
PyHSP70 as an apop osis inhibi o |79
o by he pa asi e o pe pe ua e in ec ion (Polla, 1991). The e o e, he exis ence o mechanisms able
o induce o inhibi apop osis is essen ial wi hin his dual ela ionship and he balance be ween hem
dic a es hos cell a e. Fu he mo e, in o de o con ol pa hogenesis and egula e homeos a ic
de elopmen , i seems in ui i e ha all species should ha bou cellula p o ec i e mechanisms and
con e sely membe s o he apop o ic machine y eady o induce cell dea h. Se e al cha ac e is ic
ea u es o apop osis ha e al eady been desc ibed o p o ozoan pa asi es indica ing ha hey
possess cellula mechanism o cell dea h simila o ha o mul icellula euka yo es (A noul e al.,
2002). Fo P. be ghei, caspase-like ac i i y was de ec ed in he cy oplasm o ookine es along wi h
apop o ic mo phological and biochemical ea u es (Al-Olayan e al., 2002). The e o e, oge he wi h
he exis ence o apop o ic induce s wi hin Plasmodium, he e should also be apop osis inhibi o s.
The p esence o HSP70 in Plasmodium species makes i an ob ious candida e o his ask. In
addi ion, one can expec ha mo e cellula p ocesses will be disco e ed ha depend on he
chape one ac i i y o HSP70. I s abundance and an igenici y, which igge s po en immune
esponses, makes i a accine candida e wo h in es iga ing.
80|Chap e Six
Chap e Se en
Final Discussion &
Conclusion
7.1 Final Discussion and Conclusion
When an in acellula pa asi e depends on a hos in o de o comple e i s li e cycle, a close
in e ac ion mus be es ablished be ween he hos and he pa asi e. Con ined inside a hos cell he
pa asi e ensu es a sou ce o nu ien s and, a he same ime, a loca ion whe e i can a e he hos ’s
immune sys em. Th oughou in ec ion, he na u e o his ela ionship is dic a ed by he di e en
pa asi e-speci ic equi emen s along i s li e cycle. Howe e , hos -pa asi e in e ac ions also e lec he
balance be ween he pa hogen i ulence mechanisms and hos de ences, which exe a majo e ec
in he inal ou come o in ec ion as well.
Fo mala ia pa asi es, his ela ionship is highly complex and di e se, in acco d wi h he demands o
he li e cycle o Plasmodium. Since he li e is he i s a ge o Plasmodium pa asi es and his si e o
in ec ion is obliga o y o disease p og ession, i is essen ial o e eal and comp ehend he basic
biological in e ac ions es ablished be ween he pa asi e and i s hos cell, he hepa ocy e (Lau e al.,
2001; Shin e al., 1982).
Fo a long ime his was a ha d ask due o he limi ed expe imen al accessibili y o he models and
molecula ools a ailable. Howe e , hese di icul ies a e being o e come and he possibili ies o
esea ch in his a ea a e inc easing g ea ly, as a consequence o he de elopmen o new echnologies
and gene ic ools (Amino e al., 2005; F e e e al., 2005; Ga dine e al., 2005). Ne e heless, he e
has always been he p oblem o ob aining enough quan i ies o spo ozoi es ha would allow li e
in ec ion o be mimicked in i o (Luke and Ho man, 2003). Toge he wi h he low in ec i i y a es
ha a e ob ained expe imen ally, he e is a a he ema kable poo knowledge o he mechanisms
in ol ed in in asion and in acellula de elopmen used by he pa asi e (Kappe e al., 2004; D uilhe
e al., 1998).
Usually, i is conside ed ha hos manipula ion is a s a egy used by in acellula pa asi es. How
pa asi es manage o e ade hos de ences has always been a hub unde a con inuous su ey.
An igenic a ia ion, esis ance o immunological a acks and escape o sa e compa men s a e
common s a egies used (Zamb ano-Villa e al., 2002). Mo eo e , i is becoming common knowledge
ha di e en pa asi es make use o simila s a egies o sub e hos signalling pa hways in hei own
bene i , such as ac i a ing some in o de o exploi hem o in asion and de elopmen o inhibi ing
o he s in o de o p e en hos ile esponses (Kahn e al., 2002).
Du ing i s pe manence in he li e , spo ozoi es unde go a p ocess o eplica ion and de elopmen
ha exploi s esou ces p o ided by he hepa ocy e. In addi ion, as is he case wi h any in acellula
pa hogen, pa asi es de elop ways o con olling hos cell beha iou owa ds inc easing hei own
su i al and inducing changes in he hos ha can ul ima ely elici e ec s in immune modula ion and
egula ion o apop osis (James and G een, 2004; Hasnain e al., 2003). Fo Plasmodium pa asi es bo h
o hese e ec s happen du ing in ec ion bu none o hem ha e been ye comple ely elucida ed.
Final Discussion & Conclusion|83
Aiming a cla i ying some essen ial ea u es wi hin Plasmodium-hepa ocy e in e ac ions, he main goal
o he p ojec p esen ed he e was o de e mine he ole o hepa ocy e apop osis in he inhibi ion o
de elopmen o exo-e y h ocy ic o ms and in he ini ia ion o p o ec i e immune esponses o
mala ia by dend i ic cells.
Plasmodium li e s ages a e conside ed o be a singula phase o he pa asi e li e cycle in e ms o
immuni y. Besides cons i u ing a c ucial s ep o i s su i al, hey a e also impe a i e o he
induc ion, es ablishmen and main enance o an e ec o an i-pa asi ic immune esponse (Doolan
and Ho man, 2000). Al hough cha ac e ized as mul i- ac o ial, his p o ec ion is essen ially
dependen on Plasmodium an igen-speci ic T cells, in pa icula e ec o and memo y CD8+ T cells,
which a e esponsible o he elimina ion o he in ec ed hepa ocy e and, in addi ion, a e
indispensable o enhancing he e icacy o a long las ing immuni y agains mala ia (Mo o and
Za ala, 2004a).
Na u al ecu en in ec ions cause only pa ial immuni y, which is media ed by an ibodies di ec ed o
he blood-s age o he pa asi e (H iid, 2005; Day and Ma sh, 1991). Only i adia ed spo ozoi e
immuniza ion leads o comple e esis ance o s e ile immuni y in bo h mice and humans a e
epea ed immuniza ions (Ho man e al., 2002). These a enua ed pa asi es in ec hepa ocy es in a
simila way compa ed o no mal ones. Howe e , hey ail o comple e a ull in ec ion, p esumably
because hei DNA was damaged upon adia ion and p o ein syn hesis was a ec ed (Suh bie e al.,
1990). Ne e heless, p o ec ion is ob ained when li e a enua ed spo ozoi es each he li e while
inac i a ed spo ozoi es o hei ex ac s ha e consis en ly ailed o achie e he same goal. Di e en
s udies also suppo he hypo hesis ha he e is a equi emen o in a-hepa ic de elopmen o
p o ec ion o be induced, as emo al o li e pa asi es by p imaquine ea men abolishes p o ec ion
(K zych e al., 2000; Schelle and Azad, 1995; Ba es e al., 1990).
Disco e ed mo e han 30 yea s ago he easibili y o an an i-mala ia accine h ough hese i adia ed
spo ozoi es immuniza ions p o ed ha p o ec ion agains Plasmodia was possible. Why his is so is
s ill unde deba e. Ye , a b oad use o his ype o accine was conside ed an impossible app oach o
unde ake, because o he impossibili y o ob aining high numbe s o spo ozoi es, as hey canno be
cul u ed in i o (Luke and Ho man, 2003). Thus, ep oducing his induced immuni y became an
al e na i e app oach. Consequen ly, o unde s anding he mechanisms occu ing in he li e ei he
du ing a na u al in ec ion o in model sys ems o p o ec i e immuni y such as i adia ed spo ozoi es
immuniza ions will acili a e he exploi a ion o hese esponses o a expedi e p og ess in accine
de elopmen .
Spo ozoi es p e e en ially in ec he li e , whe e immune esponses end o be biased owa ds
immunological ole ance (C ispe, 2003). This unique ea u e may con ibu e o he success o
Plasmodium in ec ion o his o gan, since i allows he pa asi e o de elop and p omo es condi ions
84|Chap e Se en
ha seem o i he pa asi e’s needs. Besides his, se e al hypo heses we e p oposed o explain why
a enua ed spo ozoi es a e so e icien . Op ions a e based on spo ozoi e ac i i y a e i adia ion, i s
in e ac ion wi h he hepa ocy e, he subsequen ini ia ion o he immune esponse and i s pe sis ence
wi hin he hepa ocy e. As a esul he e is a high le el o IFN-γ p oduc ion when challenge is
induced in immunized mice and humans, sugges ing ha an enhanced e iciency in T cell p iming
e en s akes place. Gamma-i adia ion induces a pa ial delay in he ma u a ion o he pa asi e,
leading o i s ex ended pe manence in he li e , and gene a ing a depo o an igen, which seems
necessa y o he es ablishmen o a local an igen-speci ic p o ec i e immuni y (K zych e al., 2000).
Howe e , di e en sys ems such as i al in ec ions p esen an independency be ween an igen
pe sis ence and he main enance o memo y CD8+ T cells esponses (Whe y e al., 2004; Muelle e
al., 2002).
Ne e heless, in endemic a eas, na u ally exposed indi iduals do no acqui e long e m p o ec ion,
indica ing a pe sis en lack o memo y. This issue is s ill no ully unde s ood and emains a a he
con o e sial subjec . Ye a ew causes a e appoin ed by some au ho s ha could explain such
e idence. The ole an en i onmen ha domina es on he li e is a key ea u e, as is he
seques a ion o he li e -s age an igens wi hin hepa ocy es, and he ela i ely sho du a ion o he
li e in ec ion (S uik and Riley, 2004). Polymo phisms a he egions ecognized by he CD8+ T
cells induce an an agonis ic e ec ha in e e es wi h p iming and su i al o memo y T cells, an
e en designa ed by al e ed pep ide ligand, which is appoin ed as ano he explana ion o lack o
memo y (Plebanski e al., 1999; 1997). Fu he mo e, he mix u e o di e en Plasmodium s ains in
endemic a eas can agg a a e he dynamic in e ac ions o a ian -speci ic T cell esponses esul ing in
immune in e e ence (Zimme man e al., 2004; B uce and Day, 2002).
The ini ia ion o a speci ic immune esponse agains a disease equi es he ac i a ion o T cells by
p o essional an igen-p esen ing cells (Mellman e al., 1998). In some biological sys ems he iden i y
o hese cells is s ill o be de e mined. Howe e , in mos cases, dend i ic cells a e he ones ha
e icien ly ac i a e naï e T cells because o hei unique abili y o induce p ima y immune esponses
h ough p iming (She e al., 2003; B uña-Rome o and Rod iguez, 2001).
Du ing pa hogenic in ec ions, apop osis o in ec ed cells could ei he be caused by he hos ’s
immune esponse o be a di ec e ec induced by he pa hogen (Ga ilescu and Denke s, 2003).
Bu , in any case, he esul is he o ma ion o apop o ic bodies ha a e loaded wi h an igens de i ed
om he pa hogen. In o de o educe he isk o an in lamma o y esponse hese apop o ic cells
ha e o be emo ed (Sa ill and Fadok, 2000). Phagocy es, such as mac ophages and imma u e
dend i ic cells, ha e he abili y o cap u e hese esicles and p esen enclosed an igens o ac i a e T
lymphocy es (Albe e al., 1998 a, b). Howe e , only dend i ic cells ha e he capaci y o p esen ing
Final Discussion & Conclusion|85
Plasmodium an igens p esen ed in he con ex o hos cell apop osis a e an al e na i e mechanism
in ol ed in he induc ion o p o ec i e immuni y by i adia ed spo ozoi es. Ou indings a e being
ein o ced by ou coming esul s. Clea ly he consequences o apop osis a e being ede ined specially
a he immunological le el. Ce ainly wi h all he e idence aising om di e en in ec ion models i
would be e y misleading o iew apop o ic dea h as single simplis ic e en . An opposi e ou look is
aking his common cellula p ocess o a le el o impo ance ha o su e will wo h o con inuous
s udying (Res i o, 2000).
In he u u e, and making use o bo h pa asi e and hos ansc ip ome and p o eome, an ex ensi e
s udy will help o e eal mo e pa asi e s a egies used o in luence hos cell apop osis du ing
in ec ion, ei he by ac i a ing an i-apop o ic mechanisms o by inhibi ing p o-apop o ic ones
(Coope and Ca ucci, 2004).
As p e iously men ioned, he a ge ing o mala ia pa asi es bo h h ough accine o an i-mala ia
d ugs is mos ly pe o med using componen s o he pa asi e. Those a e in ol ed in he hos -pa asi e
in e ac ions and a e essen ial o su i al and de elopmen o on he o he hand in ol ed in disease
pa hology. Ano he s a egy ha could be unde aken is o ac on and exploi he hos molecules
ha in e ac wi h he pa asi e componen s o molecules p oduced by i . In his con ex , HGF/MET
signalling should be conside a d ug a ge candida e agains mala ia since an in e en ion based on
he ools a ailable should lead o a success ul p e en ion o in ec ion.
P esen ly, in he new pos -genomic e a is essen ial ha a clea iew o he elemen s ha a e in ol ed
ei he in basic unde s anding o Plasmodium species biology o in applied wo k aimed a con ol and
p e en ion o he disease is achie ed. Taking a s ep o wa d wi hin esea ch would leads us o he
applica ion o sys ems-biology o mala ia, since an in eg a ion o se e al da a se s would aised he
chances o being success ul bo h in accine and d ug de elopmen agains he disease (Young and
Winzele , 2005).
92|Chap e Se en
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APPENDIX
1576 •JID 2005:191 (15 May) •Lei iao e al.
MAJOR ARTICLE
Apop o ic Plasmodium-In ec ed Hepa ocy es P o ide
An igens o Li e Dend i ic Cells
Pa icia Lei iao,
1,a
Ma ia M. Mo a,
2
and Ana Rod iguez
1
1
Depa men o Medical and Molecula Pa asi ology, New Yo k Uni e si y School o Medicine, New Yo k;
2
Ins i u o Gulbenkian de Cieˆncia,
Oei as, Po ugal
(See he edi o ial commen a y by James, on pages 1573–5.)
Mala ia s a s wi h in ec ion o he hos li e by Plasmodium spo ozoi es. Inocula ion wi h adia ion-a enua ed
Plasmodium spo ozoi es induces comple e p o ec ion agains mala ia. P o ec ion is media ed by dend i ic cells
(DCs) and CD8
+
T cells, bu he sou ce o pa asi e an igens media ing his esponse emains unclea . He e,
we show ha hepa ocy es in ec ed wi h i adia ed Plasmodium spo ozoi es unde go apop osis sho ly a e
in ec ion. In ec ion wi h i adia ed spo ozoi es induces he ec ui men o DCs o he li e , whe e hey phago-
cy ose apop o ic in ec ed hepa ocy es con aining pa asi e an igens. We p opose ha apop o ic Plasmodium-
in ec ed hepa ocy es p o ide a sou ce o pa asi e an igens o he ini ia ion o he p o ec i e immune esponse.
Comple e p o ec ion agains mala ia in humans and
mice can be ob ained by inocula ion wi h i adia ed
Plasmodium spo ozoi es ha induce speci ic CD8
+
T
cells [1]. I adia ed spo ozoi es in ec hepa ocy es in
i o, as no mal spo ozoi es do; howe e , i adia ed spo-
ozoi es do no p og ess u he o blood-s age in ec-
ions and, he e o e, do no induce mala ia-associa ed
pa hology [2]. Du ing spo ozoi e in ec ion, no “p o-
essional” an igen-p esen ing cells (i.e., cells ha may
s imula e no only memo y T cells bu also nai e T
cells) a e in ec ed by Plasmodium spo ozoi es, since hey
eplica e only in hepa ocy es. Ne e heless, speci ic
CD8
+
T cells eac i e o Plasmodium an igens a e ound
in i adia ed spo ozoi e–immunized hos s [3, 4], sug-
ges ing ha pa asi e an igens a e ans e ed o p o es-
sional an igen-p esen ing cells [5]. Dend i ic cells (DCs)
a e able o p esen exogenous an igens associa ed wi h
Recei ed 27 Sep embe 2004; accep ed 18 No embe 2004; elec onically
published 4 Ap il 2005.
P esen ed in pa : 43 d Mee ing o Ame ican Socie y o Cell Biology, San
F ancisco, CA, 13–17 Decembe 2003.
Financial suppo : Na ional Ins i u es o Heal h (g an R01 AI049432 o A.R.);
Fundac¸aopa aaCieˆncia e Tecnologia, Po ugal (suppo o M.M.M. and Ph.D.
ellowship SFRH/BD/3230/2000 o P.L.).
a
P esen a ilia ion: Ins i u o Gulbenkian de Cieˆncia, Oei as, Po ugal.
Rep in s o co espondence: D . Ana Rod iguez, Dep . o Medical and Molecula
Pa asi ology, New Yo k Uni e si y School o Medicine, 341 E. 25 h S ., New Yo k,
NY 10010 ([email p o ec ed]).
The Jou nal o In ec ious Diseases 2005;191:1576–81
2005 by he In ec ious Diseases Socie y o Ame ica. All igh s ese ed.
0022-1899/2005/19110-0002$15.00
majo his ocompa ibili y complex (MHC) class I mol-
ecules and ac i a e CD8
+
T cells, a p ocess called “c oss-
p esen a ion” [6]. In e es ingly, DCs a e equi ed o
induc ion o speci ic an imala ia CD8
+
T cells a e ac-
cina ion wi h i adia ed spo ozoi es [7].
In ahepa ocy ic de elopmen o i adia ed spo ozo-
i es appea s o be equi ed o induc ion o p o ec ion,
since in ec ed hepa ocy es and hei ex ac s ha e been
shown o induce signi ican p o ec ion [8], whe eas in-
ac i a ed spo ozoi es and hei ex ac s ha e consis en ly
ailed o achie e he same goal [9, 10]. Tha hepa ocy e
in ec ion is equi ed o induc ion o a cy o oxic esponse
in he hos sugges s ha he sou ce o Plasmodium an-
igens o he ini ia ion o an immune esponse is no
indi idual spo ozoi es bu in ec ed cells [11].
Du ing pa hogenic in ec ions, apop osis o in ec ed
cells esul s in he o ma ion o apop o ic bodies loaded
wi h pa hogen an igens. C oss-p esen a ion o an igens
in apop o ic bodies phagocy osed by DCs has been p o-
posed as a mechanism o an igen p esen a ion in cy-
o oxic immune esponses agains in acellula pa ho-
gens [12, 13]. I has also been p oposed ha apop osis
o hos cells con ibu es o immuni y induced by i -
adia ed-pa asi e accines [14]. He e, we show ha ap-
op o ic in ec ed hepa ocy es a e phagocy osed by DCs
in he li e o he hos , p o iding a sou ce o Plas-
modium an igens o he ini ia ion o an imala ia im-
mune esponses.
Apop o ic Plasmodium-In ec ed Cells •JID 2005:191 (15 May) •1577
MATERIALS AND METHODS
Hos cells, pa asi es, and mice. Hepa 1-6 (ATCC CRL-1830),
a hepa oma cell line de i ed om a C57L/J mouse ha is e -
icien ly in ec ed by oden mala ia pa asi es, was used o in
i o hepa ocy e cul u es. Plasmodium yoelii (cell line 17X NL)
and Plasmodium be ghei (pa asi e line NK65) spo ozoi es we e
isola ed om sali a y glands o in ec ed Anopheles s ephensi
mosqui oes. Spo ozoi es we e i adia ed wi h a gsou ce (20
k ad). BALB/c mice we e pu chased om Taconic.
In i o in ec ions. One day be o e in ec ion, Hepa
5
2⫻10
1-6 cells/mL we e pla ed in 24-well pla es (Cos a ) o e glass
co e slips in Dulbecco’s MEM (Sigma) supplemen ed wi h 10%
hea -inac i a ed e al bo ine se um, 100 U/mL penicillin, 0.1
mg/mL s ep omycin, and 2 mmol/L l-glu amine a 37C wi h
5% CO
2
. A o al o spo ozoi es we e added o Hepa 1-
5
1⫻10
6 cell co e slips on pla es, cen i uged o 5 min a 1800 g, and
incuba ed o di e en du a ions. Cells we e washed wice wi h
PBS and ixed in 4% pa a o maldehyde.
In i o in ec ion, isola ion, and s aining o li e mononuclea
cells. Two mice pe g oup we e in ec ed wi h P. yoelii g-i a-
dia ed, noni adia ed, o hea -killed (15 min; 56C) spo ozoi es
by in a enous (i ) injec ion (1⫻10
6
spo ozoi es/mouse). Six
hou s a e injec ion, li e s we e emo ed and mechanically ho-
mogenized. The cell suspension was washed and esuspended in
a 35% Pe coll (Pha macia Bio ech) g adien solu ion and cen-
i uged a 500 g o 10 min. The mononuclea -cell pelle ob-
ained was esuspended in 1 mL o e y h ocy e lysis bu e o
1 min and washed. Isola ed cells we e coun ed and placed o 1
h on poly-l-lysine co e slips (∼cells/li e we e ob-
6
1.5 ⫻10
ained). A e ixa ion, cells we e incuba ed wi h phycoe y h in
(PE)–an i-CD11c and luo escein isio hiocyana e (FITC)–an i-
Mac3 (Pha migen) o s aining o DCs and mac ophages, e-
spec i ely. Pa asi es we e s ained wi h an i-Hsp70 monoclonal
an ibody (2E6). Cells we e also s ained wi h an i-albumin(Cap-
pel/ICN I ine) and an i–ac i a ed caspase-3 (P omega).
De ec ion o apop osis. Apop o ic cells we e de ec ed and
quan i ied by s aining wi h an i–NF-kB p65 ( elA) (San a C uz
Bio echnology), o examina ion o he ansloca ion o NF-kB
om he cy oplasm o he nucleus o he cell, o wi h an i–
ac i a ed caspase-3 (P omega). All cells we e also incuba ed
wi h 4
6
-diamidino-2-phenylindole (DAPI) o nuclea s ain-
ing, o allow mo phological de ec ion o apop osis.
DC ec ui men o he li e . G oups consis ing o 3 mice
we e in ec ed by mosqui o bi e (each anes he ized mouse was
in con ac wi h 50 P. yoelii–in ec ed mosqui oes o 20 min)
o by i injec ion wi h P. yoelii spo ozoi es (g-i adia ed
5
1⫻10
o noni adia ed) o wi h sali a y glands o unin ec ed mos-
qui oes as a con ol. A di e en imes a e in ec ion, li e s
we e emo ed and ozen, and 15 his ological sec ions om
each mouse we e examined wi h PE–an i-CD11c (Pha migen)
o de ec ion and quan i ica ion o DCs.
RESULTS
Du ing in i o in ec ion o hepa ocy es by Plasmodium spo-
ozoi es, a dec ease in he numbe o de eloping pa asi es is
obse ed 2–3 days a e in ec ion. This dec ease is mo e p o-
nounced o i adia ed spo ozoi es han o noni adia ed ones
[2, 15]. To s udy he su i al o hepa ocy es in ec ed wi h i -
adia ed Plasmodium spo ozoi es a ea lie imes a e in ec ion,
we s ained Plasmodium-in ec ed hepa ocy e cul u es wi h an
an i-Hsp70 an ibody, a molecule ha is highly exp essed in
de eloping pa asi es inside hepa ocy es bu no in spo ozoi es
[16]. We ound ha he numbe o Hepa 1-6 hepa oma cells
in ec ed wi h P. yoelii and P. be ghei (da a no shown) i adia ed
spo ozoi es was lowe han he numbe in ec ed wi h noni -
adia ed spo ozoi es and ha he numbe o in ec ed cells had
al eady dec eased sho ly a e in ec ion ( igu e 1A).
The dec ease in he numbe o in ec ed hepa ocy es has been
a ibu ed o degene a ion o in acellula pa asi es [2]. We
wan ed o de e mine whe he he educ ion in he numbe o
in ec ed hepa ocy es is due o hepa ocy e apop o ic dea h ig-
ge ed by degene a ed pa asi es. Apop osis in in ec ed hepa o-
cy e cul u es was i s moni o ed by assessmen o nuclea mo -
phology and ac i a ion o NF-kB p65 ( elA), a ansc ip ion
ac o ha is ansloca ed o he nucleus ea ly a e ini ia ion
o ce ain apop o ic pa hways [18] ( igu e 1B). We obse ed
ha hepa ocy es in ec ed wi h i adia ed spo ozoi es unde go
apop osis 6 h a e in ec ion ( igu e 1C). Apop osis in hepa-
ocy es in ec ed wi h i adia ed spo ozoi es was also con i med
by de ec ion o caspase-3 ac i a ion ( igu e 1D). I adia ion
p obably inhibi s he syn hesis o pa asi e ac o s ha a e nec-
essa y o he p e en ion o apop osis o in ec ed hepa ocy es,
esul ing in an abo i e in ec ion and cell dea h. Howe e , a
lowe bu signi ican amoun o apop osis was also ound in
hepa ocy es in ec ed wi h no mal spo ozoi es, sugges ing ha ,
in some cases, no mal spo ozoi e in ec ion canno p e en he
apop osis o hos cells.
In esponse o in lamma o y signals, imma u e DCs a e e-
c ui ed om he blood in o pe iphe al issues, whe e hey e -
icien ly phagocy ose o eign an igens. To de e mine whe he
li e in ec ion by P. yoelii spo ozoi es induces he ec ui men
o DCs, li e s o mice injec ed wi h i adia ed o noni adia ed
spo ozoi es we e ob ained a di e en imes a e injec ion. His-
ological sec ions showed a ec ui men o DCs ea ly a e in-
ec ion ( igu e 2A). When mice we e subjec ed o bi es om
in ec ed mosqui oes, a simila e ec was obse ed ( igu e 2B).
The ec ui men o DCs induced by bi es om in ec ed mos-
qui oes is o a lowe magni ude, p obably because o he smalle
numbe s o spo ozoi es injec ed by mosqui o bi e [19].
Inc eased numbe s o DCs in he li e du ing mala ia in-
ec ion would allow apid cap u e o pa asi e an igens. How-
e e , o ac i a e DCs o an igen p esen a ion, hese cells need
o ecei e a ma u a ion signal, which can be p o ided by he
1578 •JID 2005:191 (15 May) •Lei iao e al.
Figu e 1. In ec ed hepa ocy e apop osis. A, In ec ion o Hepa 1-6 cells by i adia ed (whi e ci cles) o noni adia ed (black ci cles) Plasmodium
yoelii spo ozoi es, quan i ied a di e en imes a e in ec ion. In ec ion was quan i ied by coun ing he no. o in ec ed cells wi h de eloping pa asi es
in duplica ed samples. Va ia ion be ween duplica es was !8%. Shown a e he esul s o 1 expe imen ha was ep esen a i e o 3. B, Hepa 1-6
cells we e incuba ed wi h P. yoelii i adia ed (uppe panels) o con ol (lowe panels) spo ozoi es o 6 h. In acellula pa asi es we e s ained wi h
an i-Hsp70 an ibody (g een). The size o he pa asi e esicle is smalle in i adia ed pa asi es [17]. Apop osis was moni o ed by assessmen o nuclea
mo phology (4
6
-diamidino-2-phenylindole [DAPI] s aining, blue) and nuclea ansloca ion o NF-kB p65 ( ed) [18]. C, Quan i ica ion o apop o ic in ec ed
cells by nuclea mo phology (black ba s) and NF-kB ansloca ion (whi e ba s). E o ba s ep esen he SD o iplica e samples. As e isks (*) indica e
a signi ican di e ence be ween con ol and i adia ed spo ozoi es ( ). D, Apop osis was also de ec ed by an i–ac i a ed caspase-3 s ainingP!.05
( ed) in in ec ed cells con aining in acellula pa asi es (g een, ma ked by an a owhead).
pa hogen i sel o by in lamma o y lymphokines [20]. Be o e
in ec ion, Plasmodium spo ozoi es mig a e h ough se e al he-
pa ocy es in he li e , dis up ing hei plasma memb anes and
causing cell wounding and nec o ic dea h [21, 22]. Wounded
and dying cells induce local in lamma o y esponses ha me-
dia e he ec ui men o DCs o issues [23]. In ac , Plasmodium
li e in ec ion induces an in lamma o y esponse and he e-
c ui men o mononuclea cells [24]. Release o u ic acid om
dying cells would p o ide an addi ional ma u a ion signal o
DCs [25].
Apop o ic cells a e apidly phagocy osed in he body by mac-
ophages and DCs [26]. To de e mine whe he apop o ic he-
pa ocy es con aining P. yoelii an igens a e phagocy osed by an-
igen-p esen ing cells in he li e , we isola ed nonpa enchymal
mononuclea cells om he li e s o mice immunized wi h
i adia ed spo ozoi es, a di e en imes a e injec ion. We
Apop o ic Plasmodium-In ec ed Cells •JID 2005:191 (15 May) •1579
Figu e 2. Phagocy osis o apop o ic hepa ocy es con aining Plasmodium an igens by dend i ic cells (DCs) and mac ophages in he li e . Aand B,
Quan i ica ion o DCs in his ological sec ions o mouse li e s. A, DCs in unin ec ed mice (black ba s) and in mice a e di e en imes o in ec ion
wi h P. yoelii noni adia ed (whi e ba s) o i adia ed (s iped ba s) spo ozoi es. B, DCs in mice bi en by 50 unin ec ed (black ba s) o in ec ed (whi e
ba s) mosqui oes. E o ba s ep esen he SD o iplica e his ological samples, each composed o ⭓5 li e sec ions o co e 10 mm
2
. As e isks (*)
indica e a signi ican di e ence be ween he sample and he con ol a ime0h( ).C–M, Li e nonpa enchymal mononuclea cells, ob ainedP!.02
6 h a e injec ion o mice wi h P. yoelii i adia ed spo ozoi es and s ained wi h di e en ma ke s: DC ma ke (CD11c, ed), mac ophage ma ke (mac-
3, g een), and Plasmodium Hsp70 (blue). DCs (Cand D) and mac ophages (E) we e ound o con ain esicles posi i e o Hsp70. F–I, Albumin/Hsp70
s aining, in he same mic oscope ield showing a cell in ansmi ed ligh (F), s aining o albumin ( ed, G), Plasmodium Hsp70 (blue, H), and an o e lay
o albumin and Hsp70 s aining (I). J–M, Apop osis/Plasmodium s aining, in he same mic oscope ield showing cells in ansmi ed ligh (J), s aining
o ac i a ed caspase-3 ( ed, K), Plasmodium Hsp70 (g een, L), and an o e lay o caspase-3 and Hsp70 (M).
ound ha , 6 h a e injec ion, mac ophages and DCs in he
li e ca y esicles ha con ain P. yoelii Hsp70 ( igu e 2C–2E).
In 3 sepa a e expe imen s, we ound an a e age o 3 mac o-
phages and 2 DCs in each li e con aining Hsp70-posi i e es-
icles. The numbe o DCs con aining hese esicles is p obably
highe in he mouse, bu DCs apidly lea e pe iphe al o gans
and mig a e o he lymph nodes a e ecei ing ac i a ion sig-
nals. The Hsp70-posi i e esicles ound inside mac ophagesand
DCs mus be de i ed om in ec ed hepa ocy es, since his p o-
ein is no exp essed in spo ozoi es and appea s only in in ec ed
hepa ocy es ∼6 h a e in ec ion [16]. In addi ion, we ound
ha P. yoelii Hsp70-con aining esicles also con ain mouse al-
bumin, a p o ein p esen in he cy osol o hepa ocy es ( igu e
2F–2I), con i ming ha hese esicles a e de i ed om in ec ed
hepa ocy es. No P. yoelii Hsp70 o albumin s aining was p esen
in nonpa enchymal li e mononuclea cells isola ed om non-
in ec ed con ol mice. To con i m ha hese Hsp70-posi i e
esicles we e de i ed om apop o ic cells, we s ained cells wi h
an i–ac i a ed caspase-3. We ound ha esicles posi i e o
Plasmodium Hsp70 we e also posi i e o ac i a ed caspase-3
1580 •JID 2005:191 (15 May) •Lei iao e al.
( igu e 2J–2M), con i ming ha DCs phagocy ose apop o ic
in ec ed hepa ocy es du ing mala ia in ec ions in i o.
When li e nonpa enchymal mononuclea cells we e ana-
lyzed 12 h a e in ec ion, no cells we e ound con aining P.
yoelii Hsp70, which sugges s ha in ec ed hepa ocy e dea h and
clea ance occu s ea ly du ing in i o in ec ions wi h i adia ed
spo ozoi es. This is consis en wi h he obse a ion ha Plas-
modium spo ozoi e in ec ion and ini ia ion o he immune e-
sponse occu s apidly, since ac i a ion o speci ic an i–P. yoelii
CD8
+
T cells is de ec ed as soon as 8 h a e inocula ion o
spo ozoi es [27].
DISCUSSION
P o ec ion agains mala ia can be achie ed by immuniza ionwi h
i adia ed spo ozoi es [28]. This disco e y aised expec a ions o
he de elopmen o a mala ia accine; howe e , because o he
lack o an in i o sys em o he gene a ion o la ge numbe s
o spo ozoi es, b oad use o an i adia ed spo ozoi e accine
agains mala ia is no conside ed easible. I is impo an o un-
de s and he mechanisms unde lying p o ec ion induced by i -
adia ed spo ozoi es, since his e icien p o ec ion has no ye
been eplica ed by o he o ms o human an imala ia accines.
CD8
+
T cells and DCs media e immuni y induced by i adia ed
spo ozoi es [4, 7], bu he mechanisms in ol ed in an igen p e-
sen a ion o T cells emain unclea , since he low numbe o
Plasmodium-in ec ed hepa ocy es in he li e has p e en ed he
de elopmen o di ec an igen-p esen a ion s udies.
We ound ha bo h mac ophages and DCs phagocy ose in-
ec ed hepa ocy e apop o ic bodies bu ha , in Plasmodium li e
in ec ion, only DCs a e equi ed o ac i a ion o CD8
+
T cells
in i o [7]. DC phagocy osis igge s c oss-p esen a ion o an-
igens wi hin he phagosome and esul s in e icien CD8
+
T
cell ac i a ion [29, 30]. Apop o ic hepa ocy es con aining Plas-
modium an igens p o ide an op imal sou ce o an igens o DC
c oss-p esen a ion, since hey a e o he app op ia e size (1–10
mm) o be in e nalized by phagocy osis. This mechanism would
also explain he equi emen o li e in ec ion o achie e p o-
ec ion agains his s age o he pa asi e, as well as he ac i a ion
o CD8
+
T cells by an obliga e in acellula pa asi e ha can
de elop only inside hepa ocy es, which a e nonp o essional an-
igen-p esen ing cells. The in lamma o y en i onmen induced
du ing spo ozoi e in ec ion in he li e [24] will also a o his
p ocess, by inducing he ec ui men o la ge numbe s o DCs
and p o iding ma u a ion signals o hese cells.
DCs can acqui e i us-in ec ed dead o dying cells as exoge-
nous sou ces o an igens o p esen a ion on MHC class I and
II molecules o ini ia e T cell esponses [12]. This pa hway is
hough o be c i ical o he de elopmen o e ec i e an i i al
immuni y in i o [13]. Recen ly, DC p esen a ion o Mycobac-
e ium ube culosis an igens de i ed om apop o ic in ec ed mac-
ophages has been desc ibed as an al e na i e pa hway o T cell
ac i a ion [31]. Like Plasmodium spo ozoi es in hepa ocy es, My-
cobac e ium is secluded in an in acellula compa men du ing
in ec ion, which sugges s ha p esen a ion o an igens om ap-
op o ic in ec ed cells may ep esen a common mechanism o
immune ac i a ion o esicle-con ained pa hogens.
Expe imen al li e accines based on i adia ed pa asi es ha e
been used as a model o p o ec ion in mala ia [1] and schis-
osomiasis [32]. I is possible ha i adia ion o hese pa asi es
blocks hei abili y o neu alize he apop o ic machine y o
hos cells, allowing hem o esume hei na u al endency o
unde go apop osis upon being in ec ed. Fu he wo k is e-
qui ed o demons a e ha phagocy osis o apop o ic in ec ed
hos cells esul s in e icien p esen a ion o pa asi e an igens
o T cells.
Acknowledgmen
We hank V. E. do Rosa´ io (Cen o de Mala ia e ou as Doenc¸as T opicais).
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