Javier Ochoa-Repáraz

Role of Gut Commensal Microflora in the Development of Experimental Autoimmune Encephalomyelitis

Mucosal tolerance has been considered a potentially important pathway for the treatment of autoimmune disease, including human multiple sclerosis and experimental conditions such as experimental autoimmune encephalomyelitis (EAE). There is limited information on the capacity of commensal gut bacteria to induce and maintain peripheral immune tolerance. Inbred SJL and C57BL/6 mice were treated orally with a broad spectrum of antibiotics to reduce gut microflora. Reduction of gut commensal bacteria impaired the development of EAE. Intraperitoneal antibiotic-treated mice showed no significant decline in the gut microflora and developed EAE similar to untreated mice, suggesting that reduction in disease activity was related to alterations in the gut bacterial population. Protection was associated with a reduction of proinflammatory cytokines and increases in IL-10 and IL-13. Adoptive transfer of low numbers of IL-10-producing CD25+CD4+ T cells (>75% FoxP3+) purified from cervical lymph nodes of commensal bacteria reduced mice and in vivo neutralization of CD25+ cells suggested the role of regulatory T cells maintaining peripheral immune homeostasis. Our data demonstrate that antibiotic modification of gut commensal bacteria can modulate peripheral immune tolerance that can protect against EAE. This approach may offer a new therapeutic paradigm in the treatment of multiple sclerosis and perhaps other autoimmune conditions.

A polysaccharide from the human commensal Bacteroides fragilis protects against CNS demyelinating disease

The intestinal microbiome may have a critical roll in susceptibility or resistance to immune-mediated diseases. Alterations of the gut microflora after oral antibiotic treatment can regulate encephalomyelitis (EAE), an animal model for human multiple sclerosis (MS). We now show that a zwitterionic capsular polysaccharide A (PSA) of Bacteroides fragilis can protect against central nervous system demyelinating disease. Oral administration with purified PSA protected mice against EAE prophylactic and therapeutically. PSA treatment enhanced CD103 expressing dendritic cells (DCs) that accumulated in the cervical lymph nodes. Exposure of naïve DCs to PSA induced the conversion of naïve CD4+ T cells into interleukin (IL)-10-producing FoxP3+Treg cells. Protection against EAE was completely abrogated in IL-10-deficient mice. Our results show an important role for a molecule from human commensal bacteria in protecting against EAE and suggest the possibility for protection in MS.

Central Nervous System Demyelinating Disease Protection by the Human Commensal Bacteroides fragilis Depends on Polysaccharide A Expression

The importance of gut commensal bacteria in maintaining immune homeostasis is increasingly understood. We recently described that alteration of the gut microflora can affect a population of Foxp3+Treg cells that regulate demyelination in experimental autoimmune encephalomyelitis (EAE), the experimental model of human multiple sclerosis. We now extend our previous observations on the role of commensal bacteria in CNS demyelination, and we demonstrate that Bacteroides fragilis producing a bacterial capsular polysaccharide Ag can protect against EAE. Recolonization with wild type B. fragilis maintained resistance to EAE, whereas reconstitution with polysaccharide A-deficient B. fragilis restored EAE susceptibility. Enhanced numbers of Foxp3+Treg cells in the cervical lymph nodes were observed after intestinal recolonization with either strain of B. fragilis. Ex vivo, CD4+T cells obtained from mice reconstituted with wild type B. fragilis had significantly enhanced rates of conversion into IL-10–producing Foxp3+Treg cells and offered greater protection against disease. Our results suggest an important role for commensal bacterial Ags, in particular B. fragilis expressing polysaccharide A, in protecting against CNS demyelination in EAE and perhaps human multiple sclerosis.

Gut, bugs, and brain: Role of commensal bacteria in the control of central nervous system disease

The mammalian gastrointestinal track harbors a highly heterogeneous population of microbial organisms that are essential for the complete development of the immune system. The gut microbes or “microbiota,” coupled with host genetics, determine the development of both local microbial populations and the immune system to create a complex balance recently termed the “microbiome.” Alterations of the gut microbiome may lead to dysregulation of immune responses both in the gut and in distal effector immune sites such as the central nervous system (CNS). Recent findings in experimental autoimmune encephalomyelitis, an animal model of human multiple sclerosis, suggest that altering certain bacterial populations present in the gut can lead to a proinflammatory condition that may result in the development of autoimmune diseases, in particular human multiple sclerosis. In contrast, other commensal bacteria and their antigenic products, when presented in the correct context, can protect against inflammation within the CNS. Ann Neurol 2011

Induction of a regulatory B cell population in experimental allergic encephalomyelitis by alteration of the gut commensal microflora

We have recently shown that alteration of the gut commensal microbiota with antibiotics can modify the susceptibility to autoimmune demyelinating processes of the central nervous system. Treatment of mice with a broad spectrum of antibiotics not only induced significant changes in the regulatory T cell populations of the gut associated lymphoid tissues (GALT) and peripheral lymphoid organs but reduced the susceptibility to EAE, the most widely used animal model for human multiple sclerosis. Here, we show further that oral antibiotic treatment of EAE mice induced a CD5+B cell subpopulation that conferred protection against the disease. Protection was associated with an enhanced frequency of CD5+B cells in distal lymphoid sites such as cervical LN. In vitro stimulation with LPS increased the production of IL-10 by splenic CD5+B cells. Adoptive transfer of CD5+B cells from antibiotic treated mice reduced significantly the severity of EAE by shifting the immune responses from Th1/Th17 towards anti-inflammatory Th2-type responses. Our results demonstrate that this specific B cell population appears to be involved in the immune regulation of autoimmunity, in particular this experimental demyelinating disease of the central nervous system by gut commensal microflora.

An intestinal commensal symbiosis factor controls neuroinflammation via TLR2-mediated CD39 signalling

The mammalian immune system constitutively senses vast quantities of commensal bacteria and their products through pattern recognition receptors, yet excessive immune reactivity is prevented under homeostasis. Intestinal microbiome can influence host susceptibility to extra-intestine autoimmune disorders. Here we report that polysaccharide A (PSA), a symbiosis factor for human intestinal commensal Bacteroides fragilis, protects against central nervous system demyelination and inflammation during experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis, through toll-like receptor 2 (TLR2). TLR2 mediates tissue-specific expansion of a critical regulatory CD39+ CD4 T cell subset by PSA. Ablation of CD39 signaling abrogates PSA control of EAE manifestations and inflammatory cytokine responses. Further, CD39 confers immune-regulatory phenotypes to total CD4 T cells and Foxp3+ CD4 Tregs. Importantly, CD39-deficient CD4 T cells show an enhanced capability to drive EAE progression. Our results demonstrate the therapeutic potential and underlying mechanism by which an intestinal symbiont product modulates CNS-targeted demyelination.

A commensal symbiotic factor derived from Bacteroides fragilis promotes human CD39+Foxp3+ T cells and Treg function

Polysaccharide A (PSA) derived from the human commensal Bacteroides fragilis is a symbiosis factor that stimulates immunologic development within mammalian hosts. PSA rebalances skewed systemic T helper responses and promotes T regulatory cells (Tregs). However, PSA-mediated induction of Foxp3 in humans has not been reported. In mice, PSA-generated Foxp3+ Tregs dampen Th17 activity thereby facilitating bacterial intestinal colonization while the increased presence and function of these regulatory cells may guard against pathological organ-specific inflammation in hosts. We herein demonstrate that PSA induces expression of Foxp3 along with CD39 among naïve CD4 T cells in vitro while promoting IL-10 secretion. PSA-activated dendritic cells are essential for the mediation of this regulatory response. When cultured with isolated Foxp3+ Tregs, PSA enriched Foxp3 expression, enhanced the frequency of CD39+HLA-DR+ cells, and increased suppressive function as measured by decreased TNFα expression by LPS-stimulated monocytes. Our findings are the first to demonstrate in vitro induction of human CD4+Foxp3+ T cells and enhanced suppressive function of circulating Foxp3+ Tregs by a human commensal bacterial symbiotic factor. Use of PSA for the treatment of human autoimmune diseases, in particular multiple sclerosis and inflammatory bowel disease, may represent a new paradigm in the approach to treating autoimmune disease.

A commensal bacterial product elicits and modulates migratory capacity of CD39+ CD4 T regulatory subsets in the suppression of neuroinflammation

Tolerance established by host-commensal interactions regulates host immunity at both local mucosal and systemic levels. The intestinal commensal strain Bacteroides fragilis elicits immune tolerance, at least in part, via the expression capsular polysaccharide A (PSA). How such niche-specific commensal microbial elements regulate extra-intestinal immune responses, as in the brain, remains largely unknown. We have recently shown that oral treatment with PSA suppresses neuro-inflammation elicited during experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis. This protection is dependent upon the expansion of immune-regulatory CD4 T cells (Treg) expressing CD39, an ectonucleotidase. Here, we further show that CD39 modulation of purinergic signals enhances migratory phenotypes of both total CD4 T cells and Foxp3+ CD4 Tregs at central nervous system (CNS) lymphoid-draining sites in EAE in vivo and promotes their migration in vitro. These changes are noted during PSA treatment, which leads to heightened accumulation of CD39+ CD4 Tregs in the CNS. Deficiency of CD39 abrogates accumulation of Treg during EAE, and is accompanied by elevated Th1/Th17 signals in the CNS and in gut-associated lymphoid tissues. Our results demonstrate that immune-modulatory commensal bacterial products impact the migratory patterns of CD4 Treg during CNS autoimmunity via the regulation of CD39. These observations provide clues as to how intestinal commensal microbiome is able to modulate Treg functions and impact host immunity in the distal site.

Augmentation of regulatory B cell activity in experimental allergic encephalomyelitis by glatiramer acetate

We recently showed that B cells reduce CNS inflammation in mice with experimental allergic encephalomyelitis (EAE). Here, we demonstrate that adoptively transferred CD5/CD19+ B cells protect against EAE severity. Furthermore, we show that glatiramer acetate (GA), a therapeutic for relapsing multiple sclerosis treatment, amplifies this effect. Transfer of GA-conditioned B cells leads to increased production of immunoregulatory cytokines and reduced CNS inflammation, as well as decreased expression of the chemokine receptor, CXCR5, and elevated BDNF expression in the CNS. Thus B cells can protect against EAE, and GA augments this effect in maintaining immune homeostasis and controlling EAE disease progression.

Increased expression of B cell-associated regulatory cytokines by glatiramer acetate in mice with experimental autoimmune encephalomyelitis

B cells are of increasing importance as a target for multiple sclerosis treatment. Here we show that GA treatment of mice with experimental autoimmune encephalomyelitis (EAE) biases cytokine production by B cells towards cytokines associated with regulation in MS including interleukin (IL)-4, -10 and -13 and reduces pro-inflammatory IL-6, IL-12, and TNF alpha levels. GA also down-regulates expression of B cell-activating factor (BAFF) of the TNF family and a proliferation-inducing ligand (APRIL), as well as the BAFF receptor in mice with EAE. Thus, GA impacts both B cell survival and B cell cytokine production during CNS inflammatory disease in an EAE model.