Coxsackie-adenovirus receptor expression is enhanced in pancreas from patients with type 1 diabetes

Coxsackie–adeno i us ecep o exp ession is enhanced in panc eas om pa ien s wi h ype 1 diabe es M Hodik, 1 M Anagandula, 1 J Fuxe, 2 L K og old, 3 K Dahl-Jø gensen, 3 H Hyö y, 4,5 L Sa mien o, 6 G F isk, 1 POD-V Conso ium To ci e: Hodik M, Anagandula M, Fuxe J, e al. Coxsackie–adeno i us ecep o exp ession is enhanced in panc eas om pa ien s wi h ype 1 diabe es. BMJ Open Diabe es Resea ch and Ca e 2016;4:e000219. doi:10.1136/bmjd c-2016- 000219 ▸Addi ional ma e ial is a ailable. To iew please isi he jou nal (h p://dx.doi.o g/ 10.1136/bmjd c-2016- 000219). Recei ed 29 Feb ua y 2016 Re ised 8 June 2016 Accep ed 9 July 2016 Fo numbe ed a ilia ions see end o a icle. Co espondence o D Gun F isk; gun.e gida. [email p o ec ed] ABSTRACT Objec i es: One o he heo ies connec ing en e o i us (EV) in ec ion o human isle s wi h ype 1 diabe es (T1D) is he de elopmen o a e ile ield in he isle s. This implies induc ion o app op ia e p o eins o he i al eplica ion such as he coxsackie– adeno i us ecep o (CAR). The aim o his s udy was o in es iga e o wha ex en CAR is exp essed in human isle s o Lange hans, and wha condi ions ha would change he exp ession. Design: Immunohis ochemis y o CAR was pe o med on pa a in-embedded panc ea ic issue om pa ien s wi h T1D (n=9 ecen onse T1D, n=4 long-s anding T1D), isle au oan ibody-posi i e indi iduals (n=14) and non-diabe ic con ols (n=24) indi iduals. The exp ession o CAR was also examined by e e se ansc ip ion PCR on mic odissec ed isle s (n=5), exoc ine issue (n=5) and on explan ed isle s in ec ed wi h EV o exposed o chemokines p oduced by EV-in ec ed isle cells. Resul s: An inc eased equency o pa ien s wi h T1D and au oan ibody-posi i e indi iduals exp essed CAR in he panc eas (p<0.039). CAR s aining was de ec ed mo e equen ly in panc ea ic isle s om pa ien s wi h T1D and au oan ibody-posi i e subjec s (15/27) compa ed wi h (6/24) non-diabe ic con ols (p<0.033). Also in explan ed isle s cul u ed in UV- ea ed cul u e medium om coxsackie i us B (CBV)-1-in ec ed isle s, he exp ession o he CAR gene was inc eased compa ed wi h con ols. Lase mic odissec ion o panc ea ic issue e ealed ha CAR exp ession was 10- old highe in endoc ine compa ed wi h exoc ine cells o he panc eas. CAR was also exp essed in explan ed isle s and he exp ession le el dec eased wi h ime in cul u e. CBV-1 in ec ion o explan ed isle s clea ly dec eased he exp ession o CAR (p<0.05). In con as , in ec ion wi h echo i us 6 did no a ec he exp ession o CAR. Conclusions: CAR is exp essed in panc ea ic isle s o pa ien s wi h T1D and he exp ession le el o CAR is inc eased in explan ed isle s exposed o p oin lamma o y cy okines/chemokines p oduced by in ec ed isle s. T1D is associa ed wi h inc eased le els o ce ain chemokines/cy okines in he isle s and his migh be he mechanism behind he inc eased exp ession o CAR in TID isle s. INTRODUCTION Type 1 diabe es (T1D) is a li elong disease cha ac e ized by he loss o o se e ely educed numbe o insulin-p oducing βcells in he isle o Lange hans, p esence o isle au oan ibodies and, especially in younge indi iduals, insuli is consis ing o infil a ion o he isle s p edominan ly by CD8+ T cells and mac ophages. The e iology o he disease is unclea bu i has been shown ha gene ic ac o s, especially genes in he human leuco- cy e an igen (HLA) complex, a e o majo impo ance o he pa hogenesis. In addi ion, se e al s udies ha e shown ha en i onmen- al ac o s likely con ibu e o disease de el- opmen . 12 En e o i uses (EVs), pa icula ly he coxsackie i us B (CBV), a e among he main en i onmen al candida es and nume - ous s udies ha e shown associa ion be ween hese gu i uses and T1D by di e en ech- niques. 3–10 In addi ion, expe imen s using isola ed human isle s ha e shown ha CBVs a e able o in ec and eplica e in insulin- p oducing βcells. 411–13 CBV belong o he human en e o i us species B (HEV-B) species. Thei genome is Signi icance o his s udy Wha is al eady known abou his subjec ? ▪I is known ha en e o i us, especially Coxsackie i us B, o en can be de ec ed in human isle s o Lange hans in ype 1 diabe es cases. Wha a e he new indings? ▪I is no known i and o wha ex en isle cells exp ess app op ia e i us ecep o s and i he exp ession di e s be ween ype 1 diabe es cases, p e-diabe ic cases and non-diabe es indi iduals. How migh hese esul s change he ocus o esea ch o clinical p ac ice? ▪Ou inding ha he Coxsackie-Aden i us- Recep o (CAR) is mo e equen ly exp essed in panc eas om p e-diabe ic and in ype 1 dia- be es cases compa ed o con ol indi iduals sugges he p esence o a e ile ield in panc eas in he wo o me g oups. BMJ Open Diabe es Resea ch and Ca e 2016;4:e000219. doi:10.1136/bmjd c-2016-000219 1 Open Access Resea ch g oup.bmj.com on Decembe 1, 2016 - Published by h p://d c.bmj.com/Downloaded om ∼7500 nucleo ides long and p o ec ed by a capsid com- posed o 180 subuni s o he capsid p o eins VP1–VP4. The i ions use a ious ecep o molecules o en e he cell. The main ecep o o CBVs is he igh junc- ion p o ein Coxsackie–adeno i us ecep o (CAR), which belongs o he la ge amily o adhesion molecules; howe e , o he ecep o s ha e also been shown o be impo an o i us in e naliza ion. 14 15 The ull-leng h CAR p o ein comp ises wo ex acellula immunoglobulin-like domains (D1 and D2), one ans- memb ane helix and an in acellula C- e minal domain. 16 17 A leas fi e iso o ms o CAR a e known, wo ansmemb ane iso o ms, which di e only a hei C- e minal, and h ee lacking he ansmemb ane domain ha we e shown o be sec e ed in ans ec ed HeLa cells. 18–20 CBV binds he dis al end o he D1 domain in he canyon o he i us capsid, 21 whe eas he in acellula C domain has been shown o be dispensable o in ec ion. 22 The issue dis ibu ion o CAR in human issues has been mainly s udied a he ansc ip ional le el; CAR is exp essed in a a ie y o human o gans, including he panc eas. 19 23 The p o ein is highly exp essed in he b ain and hea du ing mu ine emb y- onic de elopmen , bu exp ession declines apidly a e bi h. 24 25 Exp ession o he CAR p o ein in human adul panc eas has been demons a ed in duc al epi he- lial cells, 26 isle cells, 27 and exoc ine acina cells (h p:// www.p o eina las.o g) by immunohis ochemis y (IHC). Which o he iso o ms o he CAR p o ein is p esen in panc eas, and he biological unc ions o hese a e no known. A monoclonal an ibody di ec ed agains he ex acellula CAR domain 28 blocked eplica ion o CBV-4 and CBV-5 in explan ed isle s, an indi ec p oo o exp es- sion and unc ion in human isle s. 4 Fu he mo e, he exp ession o he CAR p o ein has been no ed o be inc eased du ing healing and inflamma ion 29 30 and down egula ed in a ious human cance s. 31 The main aim o his s udy was o in es iga e he exp ession o he CAR gene and p o ein in panc ea ic issue, ob ained om di e en g oups o pa ien s wi h T1D, and o compa e i wi h non-diabe ic con ol indi i- duals. We also wan ed o s udy how EV in ec ion o i us-induced inflamma o y media o s egula e he exp ession o CAR in human isle s. MATERIALS AND METHODS Tissue specimen Panc ea ic pa a fin-embedded issue om he panc ea ic head o ail we e ob ained om non-diabe ic dono s wi h isle cell au oan ibodies (n=14), dono s wi h ecen onse T1D (n=9, bo h om o gan dono s and om he DiViD s udy), dono s wi h long-s anding T1D (n=5), and om con ol, au oan ibody-nega i e, non-diabe ic o gan dono s (n=24). Tissues om o gan dono s we e p o- ided by he Ne wo k o Panc ea ic O gan Dono s wi h Diabe es (nPOD) and he Uppsala biobank. Panc eas biopsies we e ob ained om he DiViD biobank, Oslo, No way (see online supplemen a y able S1). Human isle and cell cul u es Panc ea ic isle s we e isola ed om he panc eas o o gan dono s acco ding o he p o ocol by Go o e al. 32 Isle s we e hen handpicked unde a ligh mic oscope o u he inc ease pu i y o >90%. Isle s we e cul u ed ee floa ing in six-well pla es o suspension cul u es (Sa s ed , Nümb ech , Ge many) in 3 mL RPMI-1640 5.5 mM glucose (SVA, Uppsala Sweden) supplemen ed wi h 10% e al bo ine se um (FBS) (Gibco, In i ogen, S ockholm, Sweden). G een Monkey Kidney (GMK) cells we e cul u ed in 96-well pla es (Co ning, New Yo k, USA) in Eagle’s minimum essen ial medium (EMEM) (SVA, Uppsala Sweden) supplemen ed wi h 10% FBS (Gibco, In i ogen, S ockholm, Sweden). All cells we e cul u ed a 37°C in 5% CO 2 . P olonged cul u e o isle s Human isle s, isola ed and u he pu ified by handpick- ing, we e cul u ed as desc ibed abo e wi h change o cul u e medium e e y hi d day. To al RNA was ex ac ed on days 1, 4, 8 and 13 (numbe o dono s=2) pos isola- ion o analyze he genes coding o CAR and insulin. E hics The wo k pe o med on human issue was in acco dance wi h he p inciples exp essed in he Decla a ion o Helsinki and he Eu opean Council’s Con en ion on Human Righ s and Biomedicine. Isola ion o human isle s o Lange hans and he esea ch pe o med on hese was app o ed by he Regional E hics Commi ee in Uppsala, Sweden. Collec ion o panc ea ic issue in he DiViD s udy was app o ed by he No wegian Go e nmen Regional E hics Commi ee in Oslo, No way. Vi us Th ee s ains o CBV-1 we e used in he gene exp ession s udy: CBV-1-7-10796 and CBV-1-11-10802 we e isola ed in A gen ina du ing 1983 and 1998, espec i ely, whe eas CBV-1-3-10790 was isola ed in he USA in 1973. All s ains we e ob ained om he Cen e o Disease Con ol and P e en ion (CDC), A lan a, Geo gia, USA. In addi ion, a s ain o echo i us 6 (Echo-6-2C) was included in his s udy. This s ain was isola ed in Cuba 2011 om a pa ien wi h meningi is as a pa o he Public Heal h Su eillance s a egies in Cuba on ci cula - ing EV in meningi is cases. The CBV-1 s ains we e chosen since CBV-1 has been shown o use CAR as i s main ecep o . The Echo-6-2C s ain was chosen since his se o ype does no use CAR as a ecep o and has been shown o bind o CD55/decay accele a ing ac o . 33 Immunohis ochemis y Pa a fin-embedded o malin-fixed panc ea ic issue as well as pa a fin-embedded o malin-fixed explan ed isle s o Lange hans we e sec ioned (5 µm) and d ied on Supe os glass slides (Menzel-Gläze , Fische scien ific, B aunschweig, Ge many), ollowed by de-pa a finiza ion and ehyd a ion in 99.70% e hanol. An igen e ie al 2BMJ Open Diabe es Resea ch and Ca e 2016;4:e000219. doi:10.1136/bmjd c-2016-000219 Isle s udies g oup.bmj.com on Decembe 1, 2016 - Published by h p://d c.bmj.com/Downloaded om was pe o med in TE-bu e , pH 9 (DAKO, Glos up, Denma k) in a s eam boile and pe meabilized in TBS con aining 0.05% TWEEN 20. Endogenous pe oxidase was blocked by he use o a eady- o use pe oxidase blocke (DAKO, Glos up, Denma k). A e insing, he sec ions we e incuba ed wi h p ima y an ibodies, mouse an i-CAR (RmcB clone, Millipo e, Bille ica, Massachuse s, USA) dilu ed 1:1000. Incuba ion wi h hese an ibodies was pe o med a oom empe a u e o 1 hou and he isualiza ion was achie ed wi h he an i- mouse En ision-ki (Dako, Glos up, Denma k) using 3,3’-diaminobenzidine (DAB) as subs a e ch omogen. Fo double s aining wi h CAR and ch omog anin A (mouse, eady o use, Dako, Glos up), he sec ions we e isualized using a Polink DS MM D ki polyme -HRP&AP double s aining ki (Golden B idge, Mukil eo, Washing on, USA). Vi us in ec ion Handpicked human isle s o Lange hans we e inocula- ed wi h any o he i us s ains o ob ain a final concen- a ion o 10 3 issue cul u e in ec ious dose (TCID) 50 . Aliquo s o he cul u e medium we e collec ed on he day o in ec ion and e e y day un il day 3 pos in ec ion (p.i.) o i us eplica ion measu emen by TCID 50 i a- ions, and s o ed a −20°C. On day 3 p.i., all isle s and all cells de ached om he isle s du ing cul u e we e spun down and washed be o e o al RNA was ex ac ed. Isle s inocula ed wi h he CBV-1-11 s ain we e also ha - es ed o wes e n blo (WB) analysis on day 3 p.i. Fo IHC analysis, isle s om he same dono we e inocula ed wi h CBV-1-11 a a final concen a ion o 10 2.5–3 TCID 50 o mock-in ec ed. On day 4 p.i., hey we e fixed and embedded. Analysis o i us eplica ion Vi us eplica ion was de e mined by TCID 50 i a ions o samples o cul u e medium on an in house cell line o i- gina ing om GMK cells, ob ained om Go henbu g Uni e si y, Sweden, as desc ibed p e iously. 34 The samples we e collec ed om he in ec ed isle cul u es on he indica ed days p.i. T ea men wi h i us-induced p oin lamma o y chemokines The e ec o inflamma o y media o s on he exp ession le el o he CAR gene was analyzed by ea ing explan ed isle s wi h UV- ea ed cul u e medium om CBV-1-in ec ed human isle s ( wo dono s) and by adding CXCL10 (Pep o ech, S ockholm, Sweden) a final concen a ions o 0.1, 10 and 50 ng/mL ( ou dono s) o 1 and 4 days. To al RNA was ex ac ed and he exp ession o he gene encoding CAR was analyzed by quan i a i e e e se ansc ip ion PCR. RNA ex ac ion and cDNA syn hesis Th ee days p.i., isola ed human panc ea ic isle s we e washed in phospha e-bu e ed saline (PBS) and lysed by using RLT bu e (Qiagen, Sollen una, Sweden) on QIAsh edde spin columns (Qiagen, Sollen una, Sweden). To al RNA was ex ac ed om he in ec ed and con ol isle s by using he RNAeasy mini ki (Qiagen, Sollen una, Sweden) wi h G DNA elimina o column o emo e he genomic DNA. RNA concen a- ion and quali y we e de e mined wi h Nanod op (The mo Scien ific, B aunschweig, Ge many). Up o 50 ng o o al RNA pe sample was e e se ansc ibed o cDNA in a eac ion mix u e o 0.5 µL andom p ime (300 ng/μL, In i ogen, S ockholm, Sweden), 1 µL RNaseOUT (40 U/µL), 4 µL 5× fi s s and bu e , and 1 µL 0.1 M di hio h ei ol (DTT) acco ding o he manu- ac u e ’s ins uc ions. Reac ion was ca ied ou a 25°C o 10 min, 42°C o 55 min, and 15 min a 75°C. Real- ime PCR The mRNA exp ession le el o genes encoding CAR, insulin, and amylase was analyzed wi h eal- ime PCR. The eac ion mix u e consis ing o 10 µL Sybe G een mas e mix (Applied Biosys ems, S ockholm, Sweden), 2 µL p ime , and 1 µL cDNA was p epa ed acco ding o he manu ac u e ’s ins uc ions. The eac ion was ca ied ou in a 96-well op ical pla e on a S ep one plus Real- ime PCR sys em (Applied Biosys ems, S ockholm, Sweden). The cycling condi ions we e 40 cycles o 15 s a 94°C, 30 s a 55°C and 30 s a 68°C. P edesigned p ime s (Qun i ec , Qiagen, Sollen una Sweden) we e used o de ec ion o CAR and 18s. Also, o amylase and insulin, comme cially a ailable p ime s we e used o analyses o he LMC samples. Real- ime PCR da a we e analyzed by he compa a i e del a c me hod. The exp ession le el o he gene encoding CAR was no malized o he exp ession o he 18s housekeep- ing gene by sub ac ing he 18s c alue om CAR gene c alues. The ela i e gene exp ession le els we e calcula ed by using he 2^- dc o mula and p esen ed as he clus e ed columns in MS Excel. Mel cu e analysis was used o e i y he specifici y o final PCR p oduc s. Wes e n blo Exp ession o he CAR p o ein was analyzed in explan ed human isle s in ec ed wi h CBV-1-11 and mock in ec ed by WB using a polyclonal an ibody (α-CAR20) as p e iously desc ibed, 35 using an an ibody agains calnexin (BioNo dika Sweden AB, S ockholm) as loading con ol. S a is ical analyses Linea eg ession and ORs wi h 95% CIs we e used o es he likelihood o CAR posi i i y in g ouped T1D dono s and non-diabe ic con ols. Owing o low sample sizes, he likelihood o CAR posi i i y in he sepa a e T1D g oups was only measu ed be ween au oan ibody- posi i e dono s and non-diabe ic con ols. Di e ences in gene exp ession le els be ween con ol and i us-in ec ed samples, be ween di e en dono s and be ween he CBV-1 s ains we e analyzed wi h he K uskal-Wallis es (co esponding o he pa ame ic BMJ Open Diabe es Resea ch and Ca e 2016;4:e000219. doi:10.1136/bmjd c-2016-000219 3 Isle s udies g oup.bmj.com on Decembe 1, 2016 - Published by h p://d c.bmj.com/Downloaded om one-way analysis o a iance), when compa ing da a om mo e han wo independen g oups, and he Mann-Whi ney es (co esponding o he pa ame ic independen samples - es ), when compa ing da a om wo independen g oups. Co ela ion analyses be ween he exp ession o amylase and exoc ine issue, insulin and endoc ine issue, and CAR and endoc ine/exoc ine issue we e pe o med wi h Spea man’s . The p<0.05 was conside ed s a is ically significan . All ba s a e p e- sen ed as mean+SEM. RESULTS Immunohis ochemis y CAR posi i i y was significan ly highe in he combined T1D and isle au oan ibody-posi i e g oup (15/27 com- pa ed wi h con ols (6/24) in linea eg ession (p<0.033), wi h an OR o 3.877 and be ween isle au oan ibody-posi i e subjec s and non-diabe ic con ols (p<0.039)). The exac figu e o his highe equency o CAR-posi i e s aining in he endoc ine isle s o ecen onse T1D was 5/9, isle au oan ibody-posi i e 8/14, and long-s anding subjec s wi h T1D 2/4 compa ed wi h con- ols 6/24. To be conside ed posi i e, he sec ion s ained posi i e o CAR in a ew up o se e al isle s. In CAR-nega i e sec ions om ei he g oup o dono s, no s aining o CAR was seen. The posi i e CAR s aining e ealed a memb anous pa e n in he endoc ine issue (figu e 1A), sugges ing he p esence o he ansmem- b aneous pa o CAR. A somewha less in ense cy oplasmic/memb anous CAR s aining was also obse ed in cells in he exoc ine pa o he panc eas in all dono s, wi h CAR-posi i e endoc ine cells. In addi ion, in fi e dono s wi h T1D, posi i e s aining o CAR in duc al epi helial cells was seen. In he majo i y (30/51) o he panc ea ic sec ions (mainly con ols), no s aining o he ex acellula domain o CAR was seen in he endoc ine o exoc ine issue. Vi us eplica ion All EV s ains/se o ypes used o in ec ion eplica ed in he isle s. The mean i us i e inc ease in he cul u e medium du ing 3 days p.i. was 10 1.8+0.28 (CBV-1-11, n=6), 10 2.5+0.4 (CBV-1-3, n=7), 10 2.5+0.40 (CBV-1-7, n=6) and 10 4.5+0.19 (echo i us 6-2C, n=5) TCID 50 /200 µL, espec - i ely (figu e 2). Exp ession o CAR mRNA In unin ec ed con ol isle s (n=3), cul u ed o an ex ended pe iod pos isola ion, he exp ession le el o CAR dec eased linea ly wi h he ime o cul u e (figu e 3). Isle s in ec ed in i o wi h h ee s ains o CBV-1 e ealed a significan (p<0.001) educ ion in CAR coding mRNA compa ed wi h unin ec ed con ol isle s om he same dono s (n=6). The e we e no di e - ences be ween he CBV-1 s ains in ha espec . Howe e , in isle s in ec ed wi h echo i us-6-2C, which does no use CAR as a ecep o , no educ ion o he exp ession o CAR mRNA was seen compa ed wi h con ol (figu e 4), indica ing ha he e ec was no due o i us-induced β-cell dea h. Compa ing he exp ession le el o CAR be ween he di e en CBV-1 s ains and he Echo-6-2C e ealed ha he di e ence was simila o ha o he di - e ence be ween he con ol and he CBV-1-in ec ed isle s. Figu e 2 Mean i us i e inc ease in human isle s, om con ol dono s, in ec ed in i o wi h CBV-1-3 (n=7), CBV-1-7 (n=6), CBV-1-11 (n=6) and Echo-6-2C (n=5). CBV, coxsackie i us B. Figu e 1 (A) Posi i e CAR s aining in one dono wi h GAD65 and IA-2 au oan ibodies. (B) CAR s aining in isola ed human isle s, om con ol o gan dono , in ec ed wi h a CBV-1 s ain. CAR, coxsackie–adeno i us ecep o ; CBV, coxsackie i us B. 4BMJ Open Diabe es Resea ch and Ca e 2016;4:e000219. doi:10.1136/bmjd c-2016-000219 Isle s udies g oup.bmj.com on Decembe 1, 2016 - Published by h p://d c.bmj.com/Downloaded om CBV-1-11 and CBV-1-3 di e ed somewha mo e compa ed wi h Echo-6-2C (p<0.009) han CBV-1-7 (p<0.004). In explan ed isle s cul u ed in UV- ea ed cul u e medium om in ec ed isle s om o gan dono s, a clea inc ease o CAR mRNA was seen a e 4 days o cul u e compa ed wi h isle s cul u ed in UV- ea ed isle cul u e medium om con ols (figu e 5). Wes e n blo The educ ion o he CAR gene seen in isle s in ec ed wi h s ains o CBV-1 was also confi med on he p o ein le el. The e was a dec eased exp ession o he CAR p o ein ela i e o he housekeeping p o ein in isle s in ec ed wi h CBV-1-11 compa ed wi h ha in mock- in ec ed isle s om he same dono (figu e 6). DISCUSSION In his s udy, we ound ha equency o CAR exp ession was inc eased in he panc ea ic endoc ine issue om au oan ibody-posi i e non-diabe ic dono s and subjec s wi h T1D compa ed wi h con ols. This indica es ha CAR may p omo e i us in ec ion and up ake in endo- c ine cells o panc ea ic isle s, p e iously shown o be pe missi e o i us in ec ion bo h in i o 4 and in i o. 12 The eason o he highe pe cen age CAR posi i i y in pa ien s wi h T1D and p ediabe es is no known, bu s udies ha e shown ha CAR exp ession can be induced by inflamma ion and issue damage. 29 CAR has been shown o be an inducible p o ein and inc eased exp es- sion o CAR has been epo ed in myocy es in dila ed ca diomyopa hy 30 and in hea s om subjec s wi h myo- ca dial in a c ion. 24 Inc eased CAR exp ession has also been associa ed wi h an ongoing inflamma ion 29 and du ing o ma ion o cell- o-cell con ac due o issue des uc ion. 24 25 In ac , we ound ha UV- ea ed cul u e supe na an s o CBV-in ec ed isle s induced simila CAR exp ession on cul u e isle s, sugges ing ha Figu e 4 Mean CAR mRNA exp ession le els in isola ed human isle s om con ol dono s in ec ed in i o wi h di e en EV s ains. CAR exp ession le els we e dec eased in all isle s in ec ed wi h s ains o CBV-1 compa ed wi h he mock-in ec ed con ol (p<0.05). In isle s in ec ed wi h he non-CAR using E6-2C s ain, no a ec on he CAR exp ession le el was seen. Ba s ep esen mean±SEM. CAR, coxsackie– adeno i us ecep o ; CBV, coxsackie i us B; E6-2C, Echo-6-2C; EV, en e o i us. Figu e 3 mRNA exp ession le els o he gene encoding CAR in isola ed human isle s om con ol dono s, cul u ed o p olonged ime (n=2). Isle s we e cul u ed o 1, 4, 8 and 12 days and CAR mRNA exp ession le els we e quan i ied wi h qPCR. CAR, coxsackie–adeno i us ecep o ; qPCR, quan i a i e PCR. Figu e 5 mRNA exp ession le els o he gene encoding CAR in explan ed human isle s om con ol dono s, cul u ed in UV- ea ed cul u e medium de i ed om CBV-in ec ed human isle s and om mock in ec ed isle s. CAR mRNA exp ession le els we e quan i ied wi h qPCR on day 4 o cul u e. CAR, coxsackie–adeno i us ecep o ; CBV, coxsackie i us B; qPCR, quan i a i e PCR; UV, ul a iole . Figu e 6 Wes e n blo analysis o explan ed human isle s om con ol dono s in ec ed in i o wi h CBV-1-11 e ealed dec eased exp ession o he CAR p o ein compa ed wi h mock-in ec ed isle s om he same dono (n=1). CAR, coxsackie–adeno i us ecep o ; CBV, coxsackie i us B. BMJ Open Diabe es Resea ch and Ca e 2016;4:e000219. doi:10.1136/bmjd c-2016-000219 5 Isle s udies g oup.bmj.com on Decembe 1, 2016 - Published by h p://d c.bmj.com/Downloaded om CBV in ec ion in isle s o Lange hans could be one pos- sible cause o CAR exp ession in isle s. This supe na an did no con ain in ec ious i us pa icles, sugges ing ha he e ec was due o some inflamma o y media o s induced by he i us in in ec ed isle s. P e ious s udies ha e shown ha CBV in ec ion o human isle s induces se e al cy okines and chemokines. 36 37 We es ed he e ec o one o he mos abundan ly p oduced chemo- kines, CXCL10, on isle s, bu i ailed o induce CAR. Thus, u he s udies a e needed o iden i y i us-induced subs ances ha media e induc ion o CAR on human isle s. In addi ion o i al ac o s, o he ac o s, such as he au oimmune p ocess i sel , may be in ol ed. Rega dless o he cause o he induc ion o he splice a ian o CAR ha is used by CBV o in ec ion, induc- ion o his p o ein may make isle mo e suscep ible o CBV in ec ion and also p omo e he sp ead o ongoing in ec ion o o he isle s. 17 The exp ession o CAR in isle s om dono s wi h isle au oan ibodies o T1D sugges s ha isle s in hese indi i- duals a e po en ially mo e suscep ible o CBV in ec ion. In humans, in con as o in mice, i seems ha isle o Lange hans a e he a ge issue o CBV. In con as o ou findings in humans, in si u hyb idiza ion in panc e- a ic issue om mice has ailed o show any exp ession o CAR mRNA in he isle s 38 ha migh explain why mouse isle s a e no mally spa ed du ing CBV in ec ion. 38 39 Mos s udies ha e ailed o de ec EV in human exo- c ine issue, ha is, in neona es wi h sys emic EV in ec- ions. 4 In i o s udies, in which p ima y human exoc ine cells we e inocula ed wi h wo s ains o CBV-5, ailed o show any eplica ion and no i us pa icles could be de ec ed by elec on mic oscopy in he cells. 12 S aining o he ex acellula domain o CAR on pan- c ea ic issue sec ions e ealed ha mos non-diabe ic dono s did no exp ess he ex acellula domain o CAR. In con as , CAR (p o ein and gene) was exp essed in explan ed isle s om all dono s es ed. P olonged cul u e o isola ed isle s e ealed ha he exp ession o he CAR gene dec eased wi h ime o cul u e, sugges ing ha he diges i e p ocess o he panc eas du ing isle isola ion migh cause an induc ion o CAR. In i o in ec ion o isle s wi h EVs e ealed ha in isle s in ec ed wi h CAR using i uses, he exp ession o CAR was dec eased, in con as in ec ion wi h a non-CAR using EV, he exp ession le el o CAR was no a ec ed. One possible explana ion o he educed exp ession could be a species-specific down egula ion o i us ecep o s, which migh lead o inhibi ion o in ec- ion by a second i us, he so-called supe in ec ion exclusion. 40 This phenomenon has also been desc ibed o se e al o he i uses 41 and migh a o a slow- g owing, pe sis en s ain. Whe he he down egula ion is due o s imula ion o ce ain cy okines, which in i o ha e been shown o dec ease he exp ession le el o he CAR gene 42 and also he CAR p o ein by p omo ion o u no e o CAR p o ein h ough ubiqui- in–p o easome and nuclea ac o kappa-ligh -chain- enhance o ac i a ed B cells (NFkB)-dependen pa h- ways 43 o some o he ac o s, emains o be elucida ed. A mo e likely explana ion migh be ha CAR is down- egula ed by hsa-miR-466d; his has ecen ly been shown in a oden b-cell model challenged wi h CBV-4. 44 In con as o he dec eased exp ession o CAR du ing an acu e CBV in ec ion in isle endoc ine cells, he exp ession o CAR in indi iduals wi h acu e EV in ec ion in myocy es was inc eased. 30 Also in EV-posi i e cases wi h dila ed ca diomyopa hy, a disease wi h a ch onic EV in ec ion, he exp ession o he CAR p o ein was inc eased compa ed wi h con ols. 45 Sys emic EV in ec- ion has du ing some epidemics esul ed in se ocon e - sion o isle - ela ed au oan ibodies, indica ing ha i al eplica ion has aken place in he isle cells, wi h elease o in acellula p o eins as a esul . 46 I such an in ec ion, in a subg oup o in ec ed indi iduals, p og essed o a ch onic phase wi h a low-g ade i al eplica ion and ongoing issue damage, CAR migh be induced by EV in ec ion. This has been shown ega ding myoca di is and i s p og ession in some indi iduals o dila ed myopa hy. 47 In summa y, his s udy shows ha he CAR p o ein is exp essed in isle s o Lange hans in a highe pe cen age o indi iduals wi h T1D o a isk o T1D compa ed wi h non-diabe ic indi iduals. Inc eased exp ession o CAR is also seen a e ea men wi h p oinflamma o y cy o- kines/chemokines, and induc ion o his p o ein in pan- c ea ic isle s in i o migh induce a e iled field and his may also p omo e he sp ead o CBV o o he isle and play a ole in i us-induced T1D. Au ho a ilia ions 1 Depa men o Immunology, Gene ics and Pa hology, Uppsala Uni e si y, The Rudbeck Labo a o y, Uppsala, Uppland, Sweden 2 Depa men o Mic obiology, Tumo and Cell biology, Ka olinska Ins i u e , S ockholm, Sweden 3 Di ision o Paedia ic and Adolescen Medicine, Oslo Uni e si y Hospi al, Oslo and Facul y o Medicine, Uni e si y o Oslo, Oslo, No way 4 Depa men o Vi ology, Uni e si y o Tampe e, Tampe e, Finland 5 FimlabLabo a o ies, Pi kanmaa Hospi al Dis ic , Tampe e, Finland 6 Au oimmuni y Uni , Depa men o Clinical Sciences, Skåne Uni e si y Hospi al, Lund Uni e si y, Malmo, Sweden Acknowledgemen s The au ho s hank Inga Hansson o he echnical assis ance wi h he IHC. Collabo a o s POD-V Conso ium, h p://www.jd npod.o g/publica ions/ npod- i al-wo k-g oup/. Funding This wo k was suppo ed by unding om he Eu opean Union’s Se en h F amewo k P og amme PEVNET (FP7/2007–2013) unde g an ag eemen numbe 261441 and om a Diabe es Resea ch Wellness Founda ion Non-clinical Resea ch Fellowship, Ba ndiabe es onden, Swedish diabe es associa ion and Ne wo k o Panc ea ic O gan Dono s wi h Diabe es (nPOD), a collabo a i e ype 1 diabe es esea ch p ojec sponso ed by he Ju enile Diabe es Resea ch Founda ion In e na ional (JDRF). The DiViD s udy was unded by he No weigian Sou h Eas Hel h Region, The No o No disk Founda ion and PEVNET. O gan P ocu emen O ganiza ions (OPO) pa ne ing wi h nPOD o p o ide esea ch esou ces a e lis ed a h p://www.jd npod.o g/ ou -pa ne s.php. 6BMJ Open Diabe es Resea ch and Ca e 2016;4:e000219. doi:10.1136/bmjd c-2016-000219 Isle s udies g oup.bmj.com on Decembe 1, 2016 - Published by h p://d c.bmj.com/Downloaded om Compe ing in e es s None decla ed. 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Vi ology 2008;375:480–91. 8BMJ Open Diabe es Resea ch and Ca e 2016;4:e000219. doi:10.1136/bmjd c-2016-000219 Isle s udies g oup.bmj.com on Decembe 1, 2016 - Published by h p://d c.bmj.com/Downloaded om 1 diabe es ypeenhanced in panc eas om pa ien s wi h adeno i us ecep o exp ession is−Coxsackie L Sa mien o, G F isk and POD-V Conso ium M Hodik, M Anagandula, J Fuxe, L K og old, K Dahl-Jø gensen, H Hyö y, doi: 10.1136/bmjd c-2016-000219 2016 4: BMJ Open Diab Res Ca e h p://d c.bmj.com/con en /4/1/e000219 Upda ed in o ma ion and se ices can be ound a : These include: Re e ences #BIBLh p://d c.bmj.com/con en /4/1/e000219 This a icle ci es 46 a icles, 17 o which you can access o ee a : Open Access h p://c ea i ecommons.o g/licenses/by-nc/4.0/non-comme cial. See: p o ided he o iginal wo k is p ope ly ci ed and he use is non-comme cially, and license hei de i a i e wo ks on di e en e ms, pe mi s o he s o dis ibu e, emix, adap , build upon his wo k Commons A ibu ion Non Comme cial (CC BY-NC 4.0) license, which This is an Open Access a icle dis ibu ed in acco dance wi h he C ea i e se ice Email ale ing box a he op igh co ne o he online a icle. Recei e ee email ale s when new a icles ci e his a icle. Sign up in he Collec ions Topic A icles on simila opics can be ound in he ollowing collec ions (3)Isle s udies No es h p://g oup.bmj.com/g oup/ igh s-licensing/pe missions To eques pe missions go o: h p://jou nals.bmj.com/cgi/ ep in o m To o de ep in s go o: h p://g oup.bmj.com/subsc ibe/ To subsc ibe o BMJ go o: g oup.bmj.com on Decembe 1, 2016 - Published by h p://d c.bmj.com/Downloaded om