Download *Proinflammatory T-cell responses to gut microbiota promote

“Proinflammatory T-cell responses to gut
microbiota promote experimental
autoimmune encephalomyelitis”
Lee YK, Menezes JS, Umesaki Y, & Mazmanian SK
PNAS. 2011 Mar; 108 (1): 4615-4622.
Introduction
Multiple sclerosis (MS)
• Autoimmune disease
• T-cells enter CNS,
attack myelin sheath
• Genetic precursors
• High rates of
discordance in MZ
twins (≥ 70%)
http://www.ncbi.nlm.nih.gov/pubmedhealth/
PMH0001747/
Gut microbiota
• 100 trillion cells
• Primarily in gastrointestinal (GI) tract
• Confirmed role in GI immune system
development and modulation
Role of gut microbiota in MS
• MS associated with microbial contact
• More interaction with commensals
• Noninfectious symbionts modulate CD4+ T-cell
responses in the intestine
– Reduced intestinal Th17 cells in GF mice
– Microbiota directs Th17 differentiation
– Th1 and Th17 response to infectious agents
Hypothesis
The commensal gut microbiota modulates
extraintestinal immune function
in a model of multiple sclerosis.
Experimental Design
Results
Interpretation(s)
Experimental autoimmune
encephalomyelitis (EAE)
• Mouse model of MS
• Induced by immunization with CNS antigens
– Myelin oligodendrocyte glycoprotein (MOG)
– Adjuvants: Freund’s adjuvant, pertussis toxin
• Immune cells enter CNS and destroy myelin
sheath
• Th1 and Th17 cells highly associated with EAE
development
Is EAE development affected in
germ-free mice?
• Germ-free (GF) mice vs.
specific pathogen-free (SPF) mice
• Induce EAE via inoculation with MOG/CFA
• Score EAE development
Attenuated EAE in GF mice
Attenuated EAE in GF mice
Why do GF mice display reduced EAE
incidence and symptoms?
Reduced myelin sheath erosion
H&E stain
Myelin basic
protein
Interpretations
• Reduced EAE in GF mice is due to lack of
inflammation in the CNS
• Microbiota plays a role in the induction of EAE
Are GF T-cells inherently inactive?
• GF mice have developmental deficits for some
inflammatory T-cell subsets
• Harvested CD4+ cells 8-10 days after
immunization
• Reinoculated with MOG peptide
• Transferred to Rag1-/- SPF mice
T-cells from GF mice can be activated
to induce EAE
Interpretations
• CD4+ T-cells from GF mice are not inherently
unresponsive
• Microbes actively control the inflammatory
response of T-cells in the CNS
Does the gut microbiota affect the
proinflammatory profile of T-cells?
• Drained lymph nodes to harvest Th1 and Th17
cells 8d post-immunization
• Stained cells for IL-17A and IFNγ
– Cell-sorting
• Harvested cytokines
– ELISA
Flow cytometry
ELISA
Interpretations
• Th1 and Th17 proinflammatory responses are
reduced in the absence of the microbiota
Do certain microbes regulate
extraintestinal immune response?
• Segmented filamentous bacteria (SFBs)
– “Uniquely able to induce Th17 cell differentiation
in the small intestine” (p.4618)
• Inoculated GF mice with SFBs
Intermediate EAE development in
GF-SFB mice
Conclusions
• The microbiota influences the extraintestinal
development of EAE, a mouse model of MS,
through regulation of proinflammatory
responses of Th1 and Th17
• SFBs in particular regulate EAE development
Conclusions
• The microbiota influences the extraintestinal
development of EAE, a mouse model of MS,
through regulation of proinflammatory
responses of Th1 and Th17
• SFBs in particular regulate EAE development
• Reasonable, considering other autoimmune
diseases in GF mice
• Relatively novel paradigm
Future directions
• Investigate differences in T-cell activation after
transfer to Rag1-/- mice, inoculation with SFB
– Role of microbiota in early immune system
development
– Effect of additional microbial species
• Reduce EAE development in SPF mice via
regulation of SFBs