1. Gut-Residing Segmented Filamentous Bacteria Drive Autoimmune Arthritis via T Helper 17 Cells Immunity 32, 815–827, June 25, 2010 Hsin-Jung Wu, Ivaylo I. Ivanov, Jaime Darce, Kimie Hattori, Tatsuichiro Shima, Yoshinori Umesaki, Dan R. Littman, Christophe Benoist, and Diane Mathis 2010. 7. 26. Lee, Hye-Yeong

2. Introduction Mammals host trillions of microbes at diverse locations throughout the body, in particular in the gut The total number of genes borne by the gastrointestinal microbiome has been estimated to exceed more than a 100-fold that of the human genome The products of these genes are put to good use by the host, for example in digestion, production of nutrients, detoxification, defense against pathogens, and development of a competent immune system The gastrointestinal microbiome and the immune system are closely tied, each influencing and being influenced by the other GF(Germ Free)-housed individuals and neonates can have –a reduced fraction of peripheral CD4+ T lymphocytes –a systemic tilt toward the T helper 2 (Th2) cell phenotype –defective T and B cell compartments in gut-associated lymphoid tissue –reduced complements of IgG and IgA antibodies

3. Introduction Q. Gut microbiota 와 immune system 의 연관성을 근거로 autoimmune disease 를 연구하고 자 함 Inflammatory arthritis –K/BxN T cell receptor Tg mouse model KRN/B6 x NOD MHC class II restricted TCR Ubiquitously expressed self antigen : Glucose-6-phosphate isomerase(GPI) peptide ; Ag7 Initiation phase –Autoreactive T cells provide help to GPI-specific B cells  autoAb Effector phase –Innate immune system player ; mast cells, neutorphils, complement, Fc  receptors, TNF , IL-1 Q1. Impact of commensal microbes on the development of autoimmune arthritis

4. Figure 1. Attenuation of Arthritis in GF K/xBN Mice SPF(specific pathogen free) or Germ Free 상태의 K/BxN severity  ankle thickness Development of disease  GPI auto Abs GF to SPF : 21-day old GF mice  Transfer into SPF 7 weeks mice

5. A1. Commensal microbes are required for the develpoment of high GPI auto Ab titers and severe arthritis in the K/BxN model Q2. Impact of commensals on the adaptive immune system in K/BxN model  GPI Ab-secreting cells(ASCs) 가 주로 있는 spleen 관찰 http://en.wikipedia.org/wiki/File:Germinal_center.svg

6. Figure 2. Impact of Commensal Flora on the B and T Cell compartments of K/BxN Mice SPF 에서 Fas+PNA-R+(germinal center B cells) population 의 증가  activation and expansion of the anti-GPI specificities GF mice 에서는 적은 수의 CXCR5+PD-1+ T follicular helper (Tfh) cells 이 보임 ELISPOT assay : ASC 비교 rGPI B cell Enzyme conjugated anti-mouse IgG substrate detection

7. Figure 2. Impact of Commensal Flora on the B and T Cell compartments of K/BxN Mice 그러나 GPI peptide 에 대한 splenocytes 의 response 는 GF 상태의 경우 SPF 보다 낮음 CD4+ T cells (isolated from spleen) 의 activation state 에 차이 없음 A2. GF 에서 자란 K/BxN mice 는 B / T cell 의 defect 보임

8. Figure 3. A Defective Th17 Cell Signature in GF K/BxN Mice Q3. 두 condition 의 Th cells 의 비교  CD4+ T cell isolation from spleen  Microarray-based gene-expression profiling FC/FC plot Volcano plot FC SPF K/BxN VS. BxN FC GF K/BxN VS. BxN SPF 에 비해 GF 에 서 발현 감소 GF 에 비해 SPF 에서 발현 감소 P value FC GF VS. SPF GF 에서 발현이 증가된 gene 의 수

9. Figure 3. A Defective Th17 Cell Signature in GF K/BxN Mice Spleen  CD4+T isolation  RT-PCR 로 IL-4, IFN-g, IL-17 의 발현양 비교  GF condition 에서 IL-17 이 상대적으로 적게 발현 각 condition 별 spleen 에서 B220-CD3+CD4+ cell gating  CCR6+IL-17+ cells CCR6 : CCL20 의 receptor Th17 cell 의 migration 과 effector function 을 조절함  SPF 보다 GF 에서 감소  SPF 에서는 IL17a gene 의 induction 이 강하게 일어나지만 GF 에서는 그렇지 않음 A3. GF K/BxN mice 의 Th1 과 Th17 cell 에서 defect 가 보임

10. Figure 4. Reduction of Arthritis by Neutralization of IL-17 Q4. Th17 cell 의 defect 가 arthritis 와 실제 관련이 있는가  IL-17 monoclonal Ab 를 이용한 neutralization experiment Anti-IL-17 Ab treatment 로 arthritis 의 severity, anti-GPI Ab titer 감소됨을 확인  이것은 GC 의 formation 이 잘 되지 않기 때문

11. Figure 4. Reduction of Arthritis by Neutralization of IL-17 IL-17 과 B cell 의 관계 ; Transfer experiment 1x10^7 B cells (not expressing or expressing IL-17R) + arthritis K/BxN splenocytes  transfer into irradiated BxN.Rag1-/-  B cell reconstitution 확인 CD45.1+CD45.2+ : from K/BxN CD45.2+ : IL17R-/- or WT A4. IL-17 은 GC formation 을 촉진시켜 GPI autoAb production 을 증가시킴 Gated on B220+ Gated on FAS+PD-1+B220+

12. Figure 5. Linking Gut and Spleen IL-17 Cells Q5. Impact of commensal microbes on the production of IL-17 by splenic T cells Gut 에서의 microbial colonization 은 소장의 laminal propria(SI-LP) Th17 cell differentiation 을 촉진한다 (Ivanov et al., 2008)  GF- SI-LP Th17 cell 없음 SPF 상태에서 Th17 cell 의 발생 시기는 ? Splenic Th17 의 gut homming Rc 의 발현은 ? Nature Reviews Immunology 3, 331-341 (April 2003)

13. Figure 5. Linking Gut and Spleen IL-17 Cells SPF mice Th17 cell 발생 SI-LP : 2-3 주 Spleen : 3-4 주 4 주 : arthritis onset SPF mice Splenic Th17 cell 의 gut homming Rc(  7)

14. Figure 6. Effects of Various Antibiotics Sensitivity of SI-LP or splenic Th17 cells to antibiotics  SPF K/BxN : from birth to 5 weeks of age, treated with antibiotics Met : metronidazole Neo: neomycin Van : vancomycin Amp : ampicillin arthritis neo none met amp van SI-LP

15. GF 에서 자란 K/BxN mice 에서는 - SPF mice 보다 arthritis 가 심하지 않음 - Th17 cell 의 defect 보임 Neomycin 이나 vancomycin 에 의해서 죽는 microbes  SI-LP 에서 Th17 cell 의 differentiation 을 유발한다.  Th17 cell : spleen 으로 이동  GC formation 을 촉진  B cell 이 anti-GPI Ab 를 secreation  arthritis 발생 Single bacterial species-SFB(Segmented Filamentous Bacteria) 가 Jax SPF mice 의 SI-LP Th17 의 development 에 충분하다는 보고 (Ivanov et al., 2009) Q6. SFB 를 GF mice 에 넣어주면 arthritis 가 일어날 것인가  SFB-mono-colonized mice 의 fecal 을 GF K/BxN mice 에 먹임

16. Figure 7. Triggering of Arthritis in SFB-Colonized GF K/BxN Mice A6. Single bacterial species, SFB 는 Th17 cell 의 promotion 을 통해 arthritis 를 일으킨다. Total bacteria 16s rRNA

17. Conclsion A single commensal microbe, via its ability to promote a specific Th cell subset, can drive an auto-immune disease