The NF-B ansc ip ion ac o c-Rel con ols hos de ense agains Ci obac e
oden ium
Maik Luu1, Rossana Rome o1, Jasmin Bazan 1, El adil Abass2, Sab ina Ha mann1, Hanna
Leis e 1, Flo ence Fische 1, Rouzbeh Mahda i1, Ca los Plaza-Si en 3, Ingo Schmi z3,4, Ul ich
S einho 1*, Alexande Visek una1*
1Ins i u e o Medical Mic obiology and Hygiene, Philipps-Uni e si y Ma bu g, Ma bu g,
Ge many.
2Depa men o Clinical Labo a o y Science, College o Applied Medical Sciences, Imam
Abdul ahman Bin Faisal Uni e si y, Dammam, Saudi A abia.
3Ins i u e o Molecula and Clinical Immunology, O o- on-Gue icke Uni e si y Magdebu g,
Magdebu g, Ge many.
4Sys ems-O ien ed Immunology and In lamma ion Resea ch G oup, Dep . o Expe imen al
Immunology, Helmhol z Cen e o In ec ion Resea ch, B aunschweig, Ge many
Co espondence: D . Alexande Visek una
Ins i u e o Medical Mic obiology and Hygiene,
Philipps-Uni e si y, Ma bu g,
Biomedical Resea ch Cen e (BMFZ)
Hans Mee wein S aße 2, 35032 Ma bu g, Ge many
[email p o ec ed]
Tel: +49 (0)6421 / 2864359
Fax: +49 (0)6421 / 5866420
Au ho ship no e: *AV and *US con ibu ed equally o his wo k
Keywo ds: Ci obac e oden ium, c-Rel, CD4+ T cells, B cells, NF-B
The amily o NF-B ansc ip ion ac o s comp ises i e closely ela ed subuni s ha a e
in ol ed in mul iple aspec s o adap i e and inna e immune esponses. Bac e ial in ec ions and
ch onic in lamma ion ac i a e p edominan ly he canonical NF-B pa hway consis ing o
RelA/p50 and c-Rel/p50 dime s [1]. The ac i a ion o NF-B leads o apid p o easomal
deg ada ion o IB, a main inhibi o y p o ein in ol ed in he egula ion o he canonical NF-B
pa hway, esul ing in ansloca ion o RelA- and c-Rel-con aining dime s in o he nucleus and
induc ion o a ge gene exp ession [2]. The po en ial ole o NF-B in he p o ec i e immune
esponses agains C. oden ium has no been in es iga ed in he de ail. The unc ional analysis o
mice de icien o he p50 subuni o NF-B, which can di e en ially egula e immune esponses
by building ei he homodime s o he e odime s wi h a ious NF-B p o eins, e ealed ha his
p o ein is c ucial o he e adica ion o C. oden ium in ec ion [3]. Fu he , mice lacking he
a ypical IB p o ein IBNS showed impai ed Th17 cell esponses upon in ec ion wi h C.
oden ium [4]. The impo an ole o c-Rel in egula ing he unc ion o immune cells p omp ed
us o in es iga e i his ansc ip ion ac o is c ucial o o ches a ing p o ec i e immune de ense
agains C. oden ium.
To examine he consequences o c-Rel de iciency o he cou se o C. oden ium in ec ion, el-/-
and WT mice we e o ally in ec ed wi h his pa hogen and he bac e ial numbe s (colony o ming
uni s, CFU) we e de e mined in he aeces. While WT mice we e capable o elimina ing he
in ec ion wi hin app oximia ely 20 days, el-/- animals we e no able o clea he pa hogen. O
no e, he as majo i y o c-Rel-de icien mice died be ween days 105 and 120 pos in ec ion (Fig.
1, A and B). A day 120 o in ec ion, his ological da a demons a ed signi ican c yp hype plasia
and ch onic in il a ion o in lamma o y cells in o he colonic lamina p op ia o mice lacking c-
Rel, while he WT in es ine did no exhibi any immunopa hology due o he clea ance o
in ec ion wi hin i s h ee weeks (Fig. 1C). When we es ed o he localiza ion o C. oden ium
on day 90 pos in ec ion, we ound ha , in con as o he non-de ec able le els o he pa hogen in
WT mice, his bac e ium was de ec ed in all examined ex a-in es inal o gans o el-/- animals
such as panc eas, li e , kidney and spleen, indica ing o ansloca ion and sys emic sp eading o
C. oden ium (Fig. 1D). The high bac e ial load ou side o he in es ine illus a es ha c-Rel is an
essen ial ac o , which es ic s he in ec ion o he in es inal issue. Inna e immune cells such as
dend i ic cells (DCs), mac ophages, neu ophils and inna e lymphoid cells (ILCs) play a cen al
ole in he ea ly phase o in lamma o y esponses du ing in ec ion wi h C. oden ium. No ably,
Rag1-/- el-/- mice in ec ed wi h C. oden ium displayed a p olonged su i al as compa ed o Rag1-
de icien animals (Suppo ing In o ma ion Fig. 1A), sugges ing ha inna e immune esponses
may compensa e o pa ially de ec i e unc ion o in es inal DCs and ILCs in mice lacking c-Rel
ha was p e iously desc ibed [5].
T and B lymphocy es play an essen ial ole du ing he elimina ion o C. oden ium. Rag1-/- mice
lacking ma u e T and B cells a e no able o e adica e his pa hogen [6]. The NF-B signaling
pa hway is known o egula e he ac i i y o lymphocy es, howe e he con ibu ion o indi idual
NF-B subuni s o p o ec i e immune esponses agains pa hogenic bac e ia is only pa ially
unde s ood. To de ine he key e ec o mechanism in ol ed in he c-Rel-media ed de ense agains
C. oden ium in ec ion, we nex examined CD4+ T cells isola ed om he colonic lamina p op ia
a day 12 pos in ec ion. We ound ha he equency o IL-17A+CD4+ and IFN+CD4+ T cells
was signi ican ly inc eased in he colon o WT mice as compa ed o el-/- animals du ing he
cou se o in ec ion. Pa icula ly, he double-posi i e IL-17A+IFN-+CD4+ T cell popula ion,
which is equi ed o he clea ance o C. oden ium, was signi ican ly diminished in mice lacking
he ansc ip ion ac o c-Rel, sugges ing ha a de ec i e T cell esponse con ibu es o he
obse ed pheno ype (Fig. 2, A-D). I is known ha mice lacking c-Rel ha e a subs an ially
impai ed IL-2 p oduc ion and ha low sec e ion o IL-12 and IL-23 may also con ibu e o
de ec i e exp ession o cy okines by T cells du ing in ec ion wi h C. oden ium [7, 8]. When we
supplied IL-2 exogenously in o el-/- T cell cul u es, we obse ed simila p oli e a ion and IFN-
p oduc ion in Th1 cells compa ed o WT con ols (Suppo ing In o ma ion Fig. 1, B and C). O
no e, he nuclea ansloca ion o RelA, a ansc ip ion ac o ela ed o c-Rel, was compa able
be ween WT and el-/- CD4+ T lymphocy es ollowing T cell ac i a ion, sugges ing o an in ac
RelA signaling in he absence o c-Rel (Suppo ing In o ma ion Fig. 1D). Ou p e ious da a
e ealed ha he a ypical IB p o ein IBNS was able o in e ac wi h c-Rel in he nucleus o T
cells [9]. Since IκBNS does no con ain a DNA-binding mo i , his p o ein may equi e in e ac ion
wi h c-Rel o bind o a ge DNA sequences. In e es ingly, bo h p o eins, c-Rel and IκBNS appea
o be equi ed o he clea ance o C. oden ium. I was p e iously shown ha mice lacking c-Rel
we e esis an o collagen-induced a h i is and we e no able o elici a speci ic IgG an ibody
esponse o collagen ype II (CII) [10]. Because c-Rel plays an impo an ole o an ibody
gene a ion, we nex in es iga ed C. oden ium-speci ic an ibody p oduc ion a day 12 pos
in ec ion in se um and colon cul u es. Du ing in ec ion wi h C. oden ium, el-/- mice had only
ma ginal C. oden ium-speci ic IgG an ibody le els in se um and colon as compa ed o high
amoun o bac e ia-speci ic an ibodies in WT animals (Fig 2, E and F). Since clea ance o C.
oden ium in ec ion is c ucially dependen on humo al immune esponses o bac e ial p o eins,
we conclude ha c-Rel-media ed gene a ion o C. oden ium-speci ic an ibodies is he key s ep in
p e en ion o sys emic sp eading and elimina ion o his bac e ium. Thus, c-Rel is equi ed o
p o ec i e an ibac e ial esponses and o limi a ion o C. oden ium coloniza ion o in es inal
issues.
Acknowledgmen s: This s udy was suppo ed by he Von Beh ing-Rön gen-S i ung (Ul ich
S einho und Maik Luu), Mancho S i ung (Rossana Rome o), Loewe cen e no el d ug a ge s
agains po e y ela ed neglec ed opical in ec ious disease (DRUID, Ul ich S einho und
Rouzbeh Mahda i), S udiens i ung des deu schen Volkes (Maik Luu) and FAZIT-S i ung
(Hanna Leis e and Alexande Visek una).
Con lic o in e es : The au ho s decla e no comme cial o inancial con lic o in e es .
Re e ences
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5Visek una, A. e al., Mucosal Immunol 2015. 8: 307-315.
6Vallance, B. A. e al., In ec Immun 2002. 70: 2070-2081.
7Liou, H. C. e al., In Immunol 1999. 11: 361-371.
8Reinha d, K. e al., Eu J Immunol 2011. 41: 1388-1398.
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10 Campbell, I. K. e al., J Clin In es 2000. 105: 1799-1806.
Figu e 1: Lack o c-Rel esul s in enhanced suscep ibili y o in ec ion wi h C. oden ium. (A)
WT and el-/- mice we e in ec ed wi h 1x1010 CFU o C. oden ium and bac e ial i e s we e
measu ed a indica ed ime poin s (CFU pe g s ool). Da a a e shown o one ou o wo
indi idual expe imen s, each wi h 6 mice pe g oup. (B) Su i al o WT and el-/- mice was
analyzed o e he indica ed ime a e in ec ion wi h C. oden ium. Da a a e pooled om wo
indi idual expe imen s (n = 12 mice pe g oup). (C) H&E-s ained c yosec ions o colon o WT
and el-/- mice a day 120 pos C. oden ium in ec ion (le panel). Scale ba ep esen s 100 µm.
Righ panel displays he his opa hological sco ing o colonic issue om in ec ed WT and el-/-
mice on day 120. Da a a e one ep esen a i e o wo indi idual expe imen s, each wi h 6 mice pe
g oup. Da a a e means ± SEM, ***P < 0.001 (S uden ´s - es ). (D) Bac e ial i e s (CFU) in
indica ed o gans o WT and el-/- mice we e analysed on day 90 pos C. oden ium in ec ion. n.d.
= no de ec able. Da a a e one ep esen a i e o wo expe imen s, each wi h 6 mice pe g oup.

Figu e 2: Impac o c-Rel on CD4+ T cell ac i i y and an ibody p oduc ion du ing C. oden ium
in ec ion. (A-D) WT and el-/- mice we e in ec ed wi h C. oden ium and colonic lamina p op ia-
de i ed CD4+ T cells we e analyzed by low cy ome y o in acellula cy okine p oduc ion on
day 12 pos in ec ion. The equency o IL-17A and IFN- o ga ed CD4+ T cells is illus a ed in
ep esen a i e do plo s and diag ams. Da a ep esen one ep esen a i e ou o wo indi idual
expe imen s, each wi h 4 mice pe g oup. (E and F) C. oden ium-speci ic IgG an ibody i e s
we e analyzed in se um and colon on day 12 pos in ec ion o WT and el-/- and mice. Da a a e
shown o one ou o wo indi idual expe imen s, each wi h 4-5 mice pe g oup. Resul s a e
ep esen ed as he mean ± SEM, ***P < 0.001 (S uden ´s - es ).