“Proinflammatory T-cell responses to gut microbiota promote experimental autoimmune encephalomyelitis” Lee YK, Menezes JS, Umesaki Y, & Mazmanian SK PNAS Mar; 108 (1):

Introduction

Multiple sclerosis (MS) Autoimmune disease T-cells enter CNS, attack myelin sheath Genetic precursors High rates of discordance in MZ twins (≥ 70%) PMH /

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

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