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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
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
Introduction
Q. Gut microbiota와 immune system의 연관성을 근거로 autoimmune disease를 연구하
고자 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,
Fcg receptors, TNFa, IL-1
Q1. Impact of commensal microbes on the development of autoimmune arthritis
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
7 weeks mice
GF to SPF : 21-day old GF mice
 Transfer into SPF
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
Figure 2. Impact of Commensal Flora on the B and T Cell
compartments of K/BxN Mice
ELISPOT assay : ASC 비교
SPF에서
Fas+PNA-R+(germinal
center B cells)
population의 증가
 activation and
expansion of the anti-GPI
specificities
GF mice에서는 적은 수의
CXCR5+PD-1+ T follicular
helper (Tfh) cells이 보임
B cell
Enzyme conjugated
anti-mouse IgG
substrate
detection
rGPI
Figure 2. Impact of Commensal Flora on the B and T Cell
compartments of K/BxN Mice
CD4+ T cells (isolated from spleen)의
activation state에 차이 없음
그러나 GPI peptide에 대한 splenocytes의 response는
GF 상태의 경우 SPF 보다 낮음
A2. GF에서 자란 K/BxN mice는 B / T cell의
defect 보임
Figure 3. A Defective Th17 Cell Signature in GF K/BxN Mice
Q3.
두 condition의 Th cells의 비교
 CD4+ T cell isolation from spleen
 Microarray-based gene-expression profiling
Volcano plot
SPF에 비해 GF에서
발현 감소
GF에 비해 SPF에서
발현 감소
GF에서 발현이
증가된 gene의
수
FC SPF K/BxN VS. BxN
P value
FC GF K/BxN VS. BxN
FC/FC plot
FC GF VS. SPF
Figure 3. A Defective Th17 Cell Signature in GF K/BxN Mice
각 condition별 spleen에서
B220-CD3+CD4+ cell gating
CCR6+IL-17+ cells
CCR6 :
CCL20의 receptor
Th17 cell의 migration과 effector
function을 조절함
 SPF보다 GF에서 감소
Spleen  CD4+T isolation
 RT-PCR로 IL-4, IFN-g, IL-17의 발현양 비교
 GF condition에서 IL-17이 상대적으로 적게 발현
 SPF에서는 IL17a gene의 induction이 강하게 일어나지만
GF에서는 그렇지 않음
A3. GF K/BxN mice의 Th1과 Th17 cell에서 defect가 보임
Figure 4. Reduction of Arthritis by Neutralization of IL-17
Q4. Th17 cell의 defect가 arthritis와 실제 관련이 있는가
 IL-17 monoclonal Ab를 이용한 neutralization experiment
Anti-IL-17 Ab treatment로 arthritis의 severity, anti-GPI Ab
titer 감소됨을 확인
 이것은 GC의 formation이 잘 되지 않기 때문
Figure 4. Reduction of Arthritis by Neutralization of IL-17
IL-17과 B cell의 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
Gated on B220+
A4.
IL-17은 GC formation을 촉진시켜
GPI autoAb production을 증가시킴
Gated on FAS+PD-1+B220+
Figure 5. Linking Gut and Spleen IL-17 Cells
Q5. Impact of commensal microbes on the production of IL-17 by splenic T cells
Gut에서의 microbial colonization은 소장의 laminal propria(SI-LP) Th17 cell
differentiation을 촉진한다 (Ivanov et al., 2008)
 GF- SI-LP Th17 cell 없음
Nature Reviews Immunology 3, 331-341 (April 2003)
SPF 상태에서 Th17 cell의 발생 시기는?
Splenic Th17의 gut homming Rc의 발현은?
Figure 5. Linking Gut and Spleen IL-17 Cells
SPF mice Th17 cell 발생
SI-LP : 2-3주
Spleen : 3-4 주
4주 : arthritis onset
SPF mice
Splenic Th17 cell의 gut homming Rc(a4b7)
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
SI-LP
Met : metronidazole
Neo: neomycin
Van : vancomycin
Amp : ampicillin
arthritis
neo
none
met
amp
van
GF에서 자란 K/BxN mice에서는
- SPF mice보다 arthritis가 심하지 않음
- Th17 cell의 defect보임
Neomycin이나 vancomycin에 의해서 죽는 microbes
 SI-LP에서 Th17 cell의 differentiation을 유발한다.
 Th17 cell : spleen으로 이동
 GC formation을 촉진
 B cell이 anti-GPI Ab를 secreation
 arthritis 발생
Single bacterial species-SFB(Segmented Filamentous Bacteria)가 Jax SPF
mice의 SI-LP Th17의 development에 충분하다는 보고(Ivanov et al., 2009)
Q6. SFB를 GF mice에 넣어주면 arthritis가 일어날 것인가
 SFB-mono-colonized mice의 fecal을 GF K/BxN mice에 먹임
Figure 7. Triggering of Arthritis in SFB-Colonized GF K/BxN Mice
16s rRNA
A6. Single bacterial species ,
SFB는 Th17 cell의 promotion을
통해 arthritis를 일으킨다.
Total bacteria
Conclsion
•
A single commensal microbe, via its ability to promote a specific Th cell
subset, can drive an auto-immune disease