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Multiple sclerosis (MS) is an autoimmune demyelinating disease of the CNS that is linked with both genetic and environmental factors. A Western-style diet rich in fat and simple sugars is hypothesized as a potential factor contributing to the increased incidence of inflammatory autoimmune diseases, such as MS, in developed countries. Although the adverse effects of a high-fat diet in MS have been studied extensively, the effect of a fructose-rich diet (FRD) on MS etiology is unknown. We hypothesized that an FRD will alter the gut microbiome, influence immune populations, and negatively impact disease in experimental autoimmune encephalomyelitis (EAE), an animal model of MS. To test this, we fed C57BL/6 mice either an FRD or normal feed for 4 or 12 wk and analyzed the effect of an FRD on gut microbiota, immune populations, and EAE. An FRD significantly influenced the gut microbiota, with reduced abundance of beneficial bacteria and enrichment of potentially proinflammatory bacteria. We also observed immune modulation in the gut and periphery. Of particular interest was a population of Helios−RORγt+Foxp3+CD4+ T cells that was enriched in the small intestine lamina propria of FRD-fed mice. However, despite gut microbiota and immune modulations, we observed only a subtle effect of an FRD on EAE severity. Overall, our data suggest that in C57Bl6/J mice, an FRD modulates the gut microbiota and immune system without significantly impacting myelin oligodendrocyte glycoprotein 35–55/CFA-induced EAE.

Multiple sclerosis (MS) is a chronic inflammatory disease of the CNS that affects around one million people in the United States. Predisposition or protection from this disease is linked with both genetic and environmental factors. In recent years, gut microbiome has emerged as an important environmental factor in the pathobiology of MS. The gut microbiome supports various physiologic functions, including the development and maintenance of the host immune system, the perturbation of which is known as dysbiosis and has been linked with multiple diseases including MS. We and others have shown that people with MS (PwMS) have gut dysbiosis that is characterized by specific gut bacteria being enriched or depleted. Consequently, there is an emphasis on determining the mechanism(s) through which gut bacteria and/or their metabolites alter the course of MS through their ability to provide protection, predispose individuals, or promote disease progression. Improving our understanding of these mechanisms will allow us to harness the enormous potential of the gut microbiome as a diagnostic and/or therapeutic agent. In this chapter, we will discuss current advances in microbiome research in the context of MS, including a review of specific bacteria that are currently linked with this disease, potential mechanisms of disease pathogenesis, and the utility of microbiome-based therapy for PwMS.

Multiple sclerosis (MS) is an autoimmune disease of the CNS in which the interaction between genetic and environmental factors plays an important role in disease pathogenesis. Although environmental factors account for 70% of disease risk, the exact environmental factors associated with MS are unknown. Recently, gut microbiota has emerged as a potential missing environmental factor linked with the pathobiology of MS. Yet, how genetic factors, such as HLA class II gene(s), interact with gut microbiota and influence MS is unclear. In the current study, we investigated whether HLA class II genes that regulate experimental autoimmune encephalomyelitis (EAE) and MS susceptibility also influence gut microbiota. Previously, we have shown that HLA-DR3 transgenic mice lacking endogenous mouse class II genes (AE-KO) were susceptible to myelin proteolipid protein (91–110)–induced EAE, an animal model of MS, whereas AE-KO.HLA-DQ8 transgenic mice were resistant. Surprisingly, HLA-DR3.DQ8 double transgenic mice showed higher disease prevalence and severity compared with HLA-DR3 mice. Gut microbiota analysis showed that HLA-DR3, HLA-DQ8, and HLA-DR3.DQ8 double transgenic mice microbiota are compositionally different from AE-KO mice. Within HLA class II transgenic mice, the microbiota of HLA-DQ8 mice were more similar to HLA-DR3.DQ8 than HLA-DR3. As the presence of DQ8 on an HLA-DR3 background increases disease severity, our data suggests that HLA-DQ8–specific microbiota may contribute to disease severity in HLA-DR3.DQ8 mice. Altogether, our study provides evidence that the HLA-DR and -DQ genes linked to specific gut microbiota contribute to EAE susceptibility or resistance in a transgenic animal model of MS.

IntroductionPhytoestrogen found in soy, fruits, peanuts, and other legumes, has been identified as a metabolite capable of providing beneficial effect in multiple pathological conditions due to its ability to mimic endogenous estrogen. Interestingly, the health promoting effect of phytoestrogens such as isoflavone is dependent on the presence of gut bacteria with ability to metabolize dietary phytoestrogen into metabolite such as equol which have higher affinity for endogenous estrogen receptors. We have previously shown that patients with multiple sclerosis (MS), a neuroinflammatory disease lack gut bacteria with the ability to metabolize phytoestrogen. We have validated the importance of phytoestrogen and gut bacteria in disease protection utilizing an animal model of MS. Specifically, we have shown that an isoflavone rich diet can protect from neuroinflammatory diseases and that protection was dependent on the ability of gut bacteria to metabolize isoflavone into equol. Additionally, mice on a diet with isoflavone showed an anti-inflammatory response compared to the mice on a phyto-free diet. However, it is unknown how isoflavone and/or equol mediates its protective effect especially the effect of isoflavone on host metabolomics. ObjectivesIn this study we utilized untargeted metabolomics to identify the metabolites modulated by the presence of dietary isoflavone. ResultsWe found that the consumption of isoflavone increases anti-inflammatory monounsaturated fatty acids and beneficial polyunsaturated fatty acids while reducing pro-inflammatory glycerophospholipids, sphingolipids, phenylalanine metabolism, and arachidonic acid derivatives. ConclusionIsoflavone consumption alters the systemic metabolic landscape through resultant increase in monounsaturated fatty acids and beneficial polyunsaturated fatty acids while reducing proinflammatory metabolites and pathways. This highlights a potential mechanism by which an isoflavone diet modulate immune mediated disease.

As geographical location can impact the gut microbiome, it is important to study region-specific microbiome signatures of various diseases. Therefore, we profiled the gut microbiome of breast cancer (BC) patients of the Midwestern region of the United States. The bacterial component of the gut microbiome was profiled utilizing 16S ribosomal RNA sequencing. Additionally, a gene pathway analysis was performed to assess the functional capabilities of the bacterial microbiome. Alpha diversity was not significantly different between BC and healthy controls (HC), however beta diversity revealed distinct clustering between the two groups at the species and genera level. Wilcoxon Rank Sum test revealed modulation of several gut bacteria in BC specifically reduced abundance of those linked with beneficial effect such as Faecalibacterium prausnitzii. Machine learning analysis confirmed the significance of several of the modulated bacteria found by the univariate analysis. The functional analysis showed a decreased abundance of SCFA (propionate) production in BC compared to HC. In conclusion, we observed gut dysbiosis in BC with the depletion of SCFA-producing gut bacteria suggesting their role in the pathobiology of breast cancer. Mechanistic understanding of gut bacterial dysbiosis in breast cancer could lead to refined prevention and treatment.

Background Introduction of gut flora into the biliary system is common owing to biliary stenting in patients with obstructing pancreatic head cancer. We hypothesize that alteration of biliary microbiome modifies bile content that modulates pancreatic cancer cell survival. Methods Human bile samples were collected during pancreaticoduodenectomy. Bacterial strains were isolated from contaminated (stented) bile and identified using 16S ribosomal RNA sequencing. Human pancreatic cancer cells (AsPC1, CFPAC, Panc1) were treated for 24 hours with sterile (nonstented) bile, contaminated (stented) bile, and sterile bile preincubated with 106 colony forming unit of live bacteria isolated from contaminated bile or a panel of bile acids for 24 hours at 37°C, and evaluated using CellTiter-Blue Cell Viability Assay (Promega Corp. Madison, WI). Human bile (30–50 μl/mouse) was coinjected intraperitoneally with 105 Panc02 mouse pancreatic cancer cells in C57BL6/N mice to evaluate the impact of bile on peritoneal metastasis 3 to 4 weeks after tumor challenge. Results While all bile samples significantly reduced peritoneal metastasis of Panc02 cells in mice, some contaminated bile samples had diminished antitumor effect. All sterile bile (n = 4) reduced pancreatic cancer cell survival in vitro. Only 40% (2/5) of contaminated bile samples had significant effect. Preincubation of sterile bile with live Enterococcus faecalis or Streptococcus oralis modified the antitumor effect of sterile bile. These changes were not observed with culture media preincubated with live bacteria, suggesting live gut bacteria can modify the antitumor components present in bile. Conjugated bile acids were more potent than unconjugated cholic acid in reducing pancreatic cancer cell survival. Conclusion Alteration of bile microbiome from biliary stenting has a direct impact on pancreatic cancer cell survival. Further study is warranted to determine if this microbiome shift alters tumor microenvironment.

Metabolic aberrations impact the pathogenesis of multiple sclerosis (MS) and possibly can provide clues for new treatment strategies. Using untargeted metabolomics, we measured serum metabolites from 35 patients with relapsing-remitting multiple sclerosis (RRMS) and 14 healthy age-matched controls. Of 632 known metabolites detected, 60 were significantly altered in RRMS. Bioinformatics analysis identified an altered metabotype in patients with RRMS, represented by four changed metabolic pathways of glycerophospholipid, citrate cycle, sphingolipid, and pyruvate metabolism. Interestingly, the common upstream metabolic pathway feeding these four pathways is the glycolysis pathway. Real-time bioenergetic analysis of the patient-derived peripheral blood mononuclear cells showed enhanced glycolysis, supporting the altered metabolic state of immune cells. Experimental autoimmune encephalomyelitis mice treated with the glycolytic inhibitor 2-deoxy-D-glucose ameliorated the disease progression and inhibited the disease pathology significantly by promoting the antiinflammatory phenotype of monocytes/macrophage in the central nervous system. Our study provided a proof of principle for how a blood-based metabolomic approach using patient samples could lead to the identification of a therapeutic target for developing potential therapy.

A disrupted equilibrium between IL-17A-producing CD4 T-cells (Th17) and CD4+CD25+FoxP3+ regulatory T cells (Tregs) play an important role in the pathobiology of Multiple sclerosis (MS). Gut bacteria help in maintaining immune homeostasis by regulating the balance between anti-inflammatory Tregs and pro-inflammatory Th17 cells. Although, both gut bacteria and Tregs can regulate Th17 cells, the impact of IL-17A on gut microbiota and Tregs is unclear. Utilizing HLA-DR3 transgenic mouse model of MS, we show that IL-17A deficiency (HLA-DR3.IL17A-/- mice) expands Treg-inducing gut bacteria such as Prevotella, Parabacteroides, and Bacteroides and consequently Tregs, resulting in a milder disease in an animal model of MS. Notably, IL-17A sufficient DR3 mice develop milder disease on cohousing with IL-17A-deficient mice, highlighting a dominant role for gut microbiota in inducing Treg and reducing disease severity. Further, we observed an enrichment of bacterial-specific Treg promoting short-chain-fatty-acid metabolic pathways and induction of tolerogenic dendritic cells in HLA-DR3.IL17A-/- mice. Thus, our study shows a novel role of IL-17A in immune homeostasis and inflammation through regulation of the gut microbiota-Treg axis which can be used for the development of gut bacteria as therapeutics for MS.Significance of our workIL-17A a pro-inflammatory cytokine, is linked with pathobiology of multiple inflammatory diseases including multiple sclerosis (MS) and regulated by both gut microbiota and regulatory CD4 T cells (Tregs). However, the importance of IL-17A in the regulation of gut microbiota and Treg is unknown. Here we show that IL-17A can regulate Treg and disease phenotype by modulating gut microbiota and provide a novel mechanism by which immune-mediators such as IL-17A impact the gut microbiota to alter immune cell function and ultimately disease outcomes. Transfer of milder disease phenotype from IL-17A deficient mice to IL-17A sufficient mice on cohousing indicate a dominant role of gut microbiota in disease suppression. Thus, our study lays the foundation for future studies to unravel the interplay between immunological responses and the gut microbiota which will result in the development of microbiota-based therapeutics to treat autoimmune diseases.

The toxicity of many “inert” ingredients of pesticide formulations, such as safeners, is poorly characterized, despite evidence that humans may be exposed to these chemicals. Analysis of ToxCast data for dichloroacetamide safeners with the ToxPi tool identified benoxacor as the safener with the highest potential for toxicity, especially liver toxicity. Benoxacor was subsequently administered to mice via oral gavage for 3 days at concentrations of 0, 0.5, 5, and 50 mg/kg bodyweight (b.w.). Bodyweight-adjusted liver and testes weights were significantly increased in the 50 mg/kg b.w. group. There were no overt pathologies in either the liver or the intestine. 16S rRNA analysis of the cecal microbiome revealed no effects of benoxacor on α- or β-diversity; however, changes were observed in the abundance of certain bacteria. RNAseq analysis identified 163 hepatic genes affected by benoxacor exposure. Benoxacor exposure expressed a gene regulation profile similar to dichloroacetic acid and the fungicide sedaxane. Metabolomic analysis identified 9 serum and 15 liver metabolites that were affected by benoxacor exposure, changes that were not significant after correcting for multiple comparisons. The activity of antioxidant enzymes was not altered by benoxacor exposure. In vitro metabolism studies with liver microsomes and cytosol from male mice demonstrated that benoxacor is enantioselectively metabolized by cytochrome P450 enzymes, carboxylesterases, and glutathione S-transferases. These findings suggest that the minor toxic effects of benoxacor may be due to its rapid metabolism to toxic metabolites, such as dichloroacetic acid. This result challenges the assumption that inert ingredients of pesticide formulations are safe.

Trillions of commensal bacteria colonizing humans (microbiome) have emerged as essential player(s) in human health. The alteration of the same has been linked with diseases including autoimmune disorders such as multiple sclerosis, rheumatoid arthritis, systemic lupus erythematosus, and ankylosing spondylitis. Gut bacteria are separated from the host through a physical barrier such as skin or gut epithelial lining. However, the perturbation in the healthy bacterial community (gut dysbiosis) can compromise gut barrier integrity, resulting in translocation of bacterial contents across the epithelial barrier (leaky gut). Bacterial contents such as lipopolysaccharide and bacterial antigens can induce a systemic inflammatory environment through activation and induction of immune cells. The biggest question in the field is whether inflammation causes gut dysbiosis or dysbiosis leads to disease induction or propagation, i.e., it is inside out or outside in or both. In this review, we first discuss the microbiome profiling studies in various autoimmune disorders, followed by a discussion of potential mechanisms through which microbiome is involved in the pathobiology of diseases. A better understanding of the role of the microbiome in health and disease will help us harness the power of commensal bacteria for the development of novel therapeutic agents to treat autoimmune disorders.

SUMMARYMetabolic aberrations impact the pathogenesis of multiple sclerosis (MS) and possibly can provide clues for new treatment strategies. Using untargeted metabolomics, we measured serum metabolites from 35 relapsing-remitting patients and 14 healthy age-matched controls. Out of 632 known metabolites detected, 60 were significantly altered in relapsing-remitting MS (RRMS). Bioinformatics analysis identified an altered “metabotype” in RRMS patients, represented by 4 changed metabolic pathways of glycerophospholipid, citrate cycle, sphingolipid, and pyruvate metabolism. Interestingly, the common upstream metabolic pathway feeding these 4 pathways is the glycolysis pathway. Real-time bioenergetic analysis of the patient derived peripheral blood mononuclear cells, showed enhanced glycolysis, supporting the altered metabolic state of immune cells. Experimental autoimmune encephalomyelitis mice treated with the glycolytic inhibitor, 2-deoxy-D-glucose ameliorated the disease progression and inhibited the disease pathology significantly by promoting the anti-inflammatory phenotype of monocytes/macrophage in the central nervous system. Our study suggests that targeting glycolysis offers a potential target for MS.

We recently demonstrated how sepsis influences the subsequent development of experimental autoimmune encephalomyelitis (EAE) presented a conceptual advance in understanding the postsepsis chronic immunoparalysis state. However, the reverse scenario (autoimmunity prior to sepsis) defines a high-risk patient population whose susceptibility to sepsis remains poorly defined. In this study, we present a retrospective analysis of University of Iowa Hospital and Clinics patients demonstrating increased sepsis prevalence among multiple sclerosis (MS), relative to non-MS, patients. To interrogate how autoimmune disease influences host susceptibility to sepsis, well-established murine models of MS and sepsis and EAE and cecal ligation and puncture, respectively, were used. EAE, relative to non-EAE, mice were highly susceptible to sepsis-induced mortality with elevated cytokine storms. These results were further recapitulated in LPS and Streptococcus pneumoniae sepsis models. This work highlights both the relevance of identifying highly susceptible patient populations and expands the growing body of literature that host immune status at the time of septic insult is a potent mortality determinant.

Isoflavone-metabolizing gut bacteria ameliorate CNS autoimmunity in mice., The gut microbiota is a potential environmental factor that influences the development of multiple sclerosis (MS). We and others have demonstrated that patients with MS and healthy individuals have distinct gut microbiomes. However, the pathogenic relevance of these differences remains unclear. Previously, we showed that bacteria that metabolize isoflavones are less abundant in patients with MS, suggesting that isoflavone-metabolizing bacteria might provide protection against MS. Here, using a mouse model of MS, we report that an isoflavone diet provides protection against disease, which is dependent on the presence of isoflavone-metabolizing bacteria and their metabolite equol. Notably, the composition of the gut microbiome in mice fed an isoflavone diet exhibited parallels to healthy human donors, whereas the composition in those fed an isoflavone-free diet exhibited parallels to patients with MS. Collectively, our study provides evidence that dietary-induced gut microbial changes alleviate disease severity and may contribute to MS pathogenesis.

Background Toll-like receptor 4 (TLR4) has been suggested as one of the forefront cross-communicators between the intestinal bacteria and the host to regulate inflammatory signals and energy homeostasis. High-fat diet–induced inflammation is mediated by changes in gut microbiota and requires a functional TLR-4, the deficiency of which renders mice resistant to diet-induced obesity and its associated metabolic dysfunction. Furthermore, gut microbiota was suggested to play a key role in the beneficial effects of Roux-en-Y gastric bypass (RYGB), a commonly performed bariatric procedure. Objectives To explore whether TLR4, myeloid differentiation factor 8 (MyD88; 1 of its key downstream signaling regulators) and gut microbiota play an integrative role in RYGB-induced metabolic outcomes. Setting Animal- based study. Method We performed RYGB in TLR4 and MyD88 knock-out (KO) mice and used fecal microbiota transplant (FMT) from RYGB-operated animals to these genetic mouse models to address our questions. Results We demonstrate that RYGB reduces TLR4 expression explicitly in the small and large intestine of C57Blc/6J mice. We also show that TLR4 KO mice have an attenuated glucoregulatory response to RYGB. In addition, we reveal that MyD88 KO mice fail to respond to all RYGB-induced metabolic effects. Finally, fecal microbiota transplant from RYGB-operated mice into TLR4 KO and MyD88 KO naive recipients fails to induce a metabolic phenotype similar to that of the donors, as it does in wild-type recipients. Conclusion TLR4 and MyD88 are required for RYGB-induced metabolic response that is likely mediated by gut microbiome.

SUMMARYAntigenic stimulation promotes T cells metabolic reprogramming to meet increased biosynthetic, bioenergetic, and signaling demands. We show that the one-carbon (1C) metabolism enzyme Methylenetetrahydrofolate Dehydrogenase-2 (MTHFD2) is highly expressed in inflammatory diseases and induced in activated T cells to promote proliferation and produce inflammatory cytokines. In pathogenic Th17 cells, MTHFD2 also prevented aberrant upregulation of FoxP3 and suppressive capacity. Conversely, MTHFD2-deficiency enhanced lineage stability of regulatory T (Treg) cells. Mechanistically, MTHFD2 maintained cellular 10-formyltetrahydrofolate for de novo purine synthesis and MTHFD2 inhibition led to accumulation of the intermediate 5-aminoimidazole carboxamide ribonucleotide that was associated with decreased mTORC1 signaling. MTHFD2 was also required for proper histone de-methylation in Th17 cells. Importantly, inhibiting MTHFD2 in vivo reduced disease severity in Experimental Autoimmune Encephalomyelitis and Delayed-Type Hypersensitivity. MTHFD2 induction is thus a metabolic checkpoint for pathogenic effector cells that suppresses anti-inflammatory Treg cells and is a potential therapeutic target within 1C metabolism.

Multiple sclerosis (MS) is an inflammatory, demyelinating disease of the central nervous system with unknown etiology. The interaction of both genetic and environmental factors plays an important role in MS pathogenesis. Recently, gut microbiota has emerged as a potential environmental factor in MS pathology due to its ability to modulate host immune response. Yet, it is unknown whether genetic factors, such as HLA class II gene(s), can influence gut microbiota which then can modulate MS through regulation of the host immune response. In the present study, we investigated the involvement of gut microbiota in HLA class II genes mediated susceptibility and resistance to MS/experimental autoimmune encephalomyelitis-EAE (an animal model of MS). Previously, we showed that HLA-DR3 transgenic mice lacking endogenous mouse class II genes (AE-KO) were susceptible to proteolipid protein-PLP91–110 induced EAE, whereas AE-KO.HLA-DQ8 transgenic mice were resistant to PLP91–110. Surprisingly, HLA-DR3.DQ8 double transgenic mice showed increased EAE severity compared with HLA-DR3 mice. Distinct microbiota in HLA-DR3, HLA-DQ8, and HLA-DR3.DQ8 transgenic mice compared to AE-KO mice suggested an important role of HLA class-II gene in shaping the gut microbiota composition. We also observed that gut microbiota of HLA-DQ8 mice was more similar to HLA-DR3.DQ8 than HLA-DR3. As the presence of HLA-DQ8 on an HLA-DR3 background increases disease severity, our data suggest that HLA-DQ8 restricted microbiota may contribute to disease severity in HLA-DR3.DQ8 mice. Altogether, our study provides evidence that the HLA-DR and -DQ genes linked to specific gut microbiota may contribute to EAE susceptibility or resistance in a transgenic animal model of MS. The authors acknowledge funding from the National Multiple Sclerosis Society (RG 5138A1/1T), NIAID/NIH (1R01AI137075-01), a Carver Trust Medical Research Initiative Grant, and the University of Iowa Environmental Health Sciences Research Center, NIEHS/NIH (P30 ES005605). SA was supported by the Emory Warner Fellowship, which provides medical students at the Carver College of Medicine the opportunity to take a full year out of their medical school curriculum to work in a laboratory in the University of Iowa Department of Pathology.

Evaluation of sepsis-induced immunoparalysis has highlighted how decreased lymphocyte number/function contribute to worsened infection/cancer. Yet, an interesting contrast exists with autoimmune disease development, wherein diminishing pathogenic effectors may benefit the post-septic host. Within this framework, the impact of cecal ligation and puncture (CLP)-induced sepsis on the development of experimental autoimmune encephalomyelitis (EAE) was explored. Notably, CLP mice have delayed onset and reduced disease severity, relative to sham mice. Reduction in disease severity was associated with reduced number, but not function, of autoantigen (MOG)-specific pathogenic CD4 T cells in the CNS during disease and draining lymph node during priming. Numerical deficits of CD4 T cell effectors are associated with the loss of MOG-specific naive precursors. Critically, transfer of MOG-TCR transgenic (2D2) CD4 T cells after, but not before, CLP led to EAE disease equivalent to sham mice. Thus, broad impairment of antigenic responses, including autoantigens, is a hallmark of sepsis-induced immunoparalysis., eLife digest Sepsis is a life-threatening condition that can happen when the immune system overreacts to an infection and begins to damage tissues and organs in the body. It causes an extreme immune reaction called a cytokine storm, where the body releases uncontrolled levels of cytokines, proteins that are involved in coordinating the body’s response to infections. This in turn activates more immune cells, resulting in hyperinflammation. People who survive sepsis may have long-lasing impairments in their immune system that may leave them more vulnerable to infections or cancer. But scientists do not know exactly what causes these lasting immune problems or how to treat them. The fact that people are susceptible to cancer and infection after sepsis may offer a clue. It may suggest that the immune system is not able to attack bacteria or cancer cells. One way to explore this clue would be to test the effects of sepsis on autoimmune diseases, which cause the immune system to attack the body’s own cells. For example, in the autoimmune disease multiple sclerosis, the immune system attacks and destroys cells in the nervous system. If autoimmune disease is reduced after sepsis, it would suggest the cell-destroying abilities of the immune system are lessened. Using this approach, Jensen, Jensen et al. show that sepsis reduces the number of certain immune cells, called CD4 T cells, which are are responsible for an autoimmune attack of the central nervous system. In the experiments, mice that survived sepsis were evaluated for their ability to develop a multiple sclerosis-like disease. Mice that survived sepsis developed less severe or no autoimmune disease. After sepsis, these animals also had fewer CD4 T cells. However, when these immune cells were reinstated, the autoimmune disease emerged. The experiments help explain some of the immune system changes that occur after sepsis. Jensen, Jensen et al. suggest that rather than being completely detrimental, these changes may help to block harmful autoimmune responses. The experiments may also hint at new ways to combat autoimmune diseases by trying to replicate some of the immune-suppressing effects of sepsis. Studying the effect of sepsis on other autoimmune diseases in mice might provide more clues.

Trillions of microbes such as bacteria, fungi, and viruses exist in the healthy human gut microbiome. Although gut bacterial dysbiosis has been extensively studied in multiple sclerosis (MS), the significance of the fungal microbiome (mycobiome) is an understudied and neglected part of the intestinal microbiome in MS. The aim of this study was to characterize the gut mycobiome of patients with relapsing-remitting multiple sclerosis (RRMS), compare it to healthy controls, and examine its association with changes in the bacterial microbiome. We characterized and compared the mycobiome of 20 RRMS patients and 33 healthy controls (HC) using Internal Transcribed Spacer 2 (ITS2) and compared mycobiome interactions with the bacterial microbiome using 16S rRNA sequencing. Our results demonstrate an altered mycobiome in RRMS patients compared with HC. RRMS patients showed an increased abundance of Basidiomycota and decreased Ascomycota at the phylum level with an increased abundance of Candida and Epicoccum genera along with a decreased abundance of Saccharomyces compared to HC. We also observed an increased ITS2/16S ratio, altered fungal and bacterial associations, and altered fungal functional profiles in MS patients compared to HC.This study demonstrates that RRMS patients had a distinct mycobiome with associated changes in the bacterial microbiome compared to HC. There is an increased fungal to bacterial ratio as well as more diverse fungal-bacterial interactions in RRMS patients compared to HC. Our study is the first step towards future studies in delineating the mechanisms through which the fungal microbiome can influence MS disease.

Introduction: The gut microbiome helps to maintain a person's healthy state while perturbations in its function often leading to the development of inflammatory diseases including multiple sclerosis (MS). Consequently, gut-commensals which restore homeostasis have the potential to become novel therapeutic options for treating MS. MS patients have presented gut dysbiosis with a reduction in bacteria belonging to the Prevotella genus. Notably, increased levels of Prevotella are observed when disease-modifying therapies are used. Additionally, Prevotella histicola, an anaerobic bacterium derived from the human, can suppress disease in mice with experimental autoimmune encephalomyelitis, a preclinical MS model. Areas covered: This review compares MS microbiome studies from different geographical regions to identify common gut bacteria. Literature on the potential use of P. histicola as a therapy for MS and the next steps for developing microbial monotherapies in MS is also discussed. Expert commentary: Recent findings presenting an inverse correlation between Prevotella and MS disease severity and ability of P. histicola to suppress disease in preclinical models suggest that P. histicola might provide an additional treatment option for MS patients. However, rigorous testing in well-designed control trials should be performed to determine the safety and efficacy P. histicola in MS patients.

Bacterial and opportunistic infections are a major cause of morbidity and mortality in patients with systemic lupus erythematosus (SLE) and antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis owing to treatment with immunosuppressants. Commonly used laboratory tests are unreliable in differentiating infection from active disease patients. Fc receptor (FcγR1 or CD64) expression on neutrophils and soluble TREM-1 (triggering receptor expressed on monocytes) are potential biomarkers of bacterial infections. Our aim was to measure the clinical usefulness of quantitative CD64 measurement on neutrophils and soluble TREM-1 measurements in differentiating bacterial infection from active disease in patients with SLE and ANCA vasculitis. Patients with bacterial infection (n = 25), active disease (n = 51), and healthy controls (n = 20) were included. Neutrophil CD64 expression using flow cytometry and sTREM-1 and procalcitonin levels by ELISA were studied. The percentage of neutrophils with CD64 expression and their mean fluorescence intensity in patients with infection (68.8 (56.9-86.5)%, 1037 (229-1828)) were significantly (p 0.05) higher as compared to those without infection (7.7 (2.6-13.1)%, 456 (20-968)) and controls (7.05 (1.4-9.5)%, 99.5 (54.7-140.7)). The sensitivity and specificity of CD64 expression on neutrophils to diagnose bacterial infection (using a cutoff value of 30%) was 85% and 84%, respectively, whereas the sensitivity and specificity of procalcitonin was 75% and 85%, respectively. There was no significant difference in soluble TREM-1 levels between the two groups. Quantitative measurement of CD64 on neutrophils can distinguish between systemic infection and the flare of autoimmune diseases.

Summary Antigenic stimulation promotes T cell metabolic reprogramming to meet increased biosynthetic, bioenergetic, and signaling demands. We show that the one-carbon (1C) metabolism enzyme methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) regulates de novo purine synthesis and signaling in activated T cells to promote proliferation and inflammatory cytokine production. In pathogenic T helper-17 (Th17) cells, MTHFD2 prevented aberrant upregulation of the transcription factor FoxP3 along with inappropriate gain of suppressive capacity. MTHFD2 deficiency also promoted regulatory T (Treg) cell differentiation. Mechanistically, MTHFD2 inhibition led to depletion of purine pools, accumulation of purine biosynthetic intermediates, and decreased nutrient sensor mTORC1 signaling. MTHFD2 was also critical to regulate DNA and histone methylation in Th17 cells. Importantly, MTHFD2 deficiency reduced disease severity in multiple in vivo inflammatory disease models. MTHFD2 is thus a metabolic checkpoint to integrate purine metabolism with pathogenic effector cell signaling and is a potential therapeutic target within 1C metabolism pathways.

SUMMARY Osteopontin (OPN) has been considered a potential biomarker of graft-versus-host disease (GVHD). However, the function of OPN in GVHD is still elusive. Using a mouse model of acute GVHD (aGVHD), we report that OPN generated by CD4+ T cells is sufficient to exert a beneficial effect in controlling aGVHD through limiting gastrointestinal pathology, a major target organ of aGVHD. CD4+ T cell-derived OPN works on CD44 expressed in intestinal epithelial cells (IECs) and abates cell death of IECs. OPN also modulates gut microbiota with enhanced health-associated commensal bacteria Akkermansia. Importantly, we use our in vivo mouse mutant model to specifically express OPN isoforms and demonstrate that secreted OPN (sOPN), not intracellular OPN (iOPN), is solely responsible for the protective role of OPN. This study demonstrates that sOPN generated by CD4+ T cells is potent enough to limit aGVHD., Graphical abstract, In brief The role of osteopontin (OPN) derived from CD4+ T cells in acute graft-versus-host disease (aGVHD) is unknown. Aggarwal et al. show that CD4+ T cell-derived secreted OPN is protective in aGVHD by modulating the gut microbiome and limiting cell death of intestinal epithelial cells by the sOPN-CD44 axis.

The human gut contains trillions of bacteria (microbiome) that play a major role in maintaining a healthy state for the host. Perturbation of this healthy gut microbiome might be an important environmental factor in the pathogenesis of inflammatory autoimmune diseases such as multiple sclerosis (MS). Others and we have recently reported that MS patients have gut microbial dysbiosis (altered microbiota) with the depletion of some and enrichment of other bacteria. However, the significance of gut bacteria that show lower or higher abundance in MS is unclear. The majority of gut bacteria are associated with certain metabolic pathways, which in turn help in the maintenance of immune homeostasis of the host. Here we discuss recent MS microbiome studies and the possible mechanisms through which gut microbiome might contribute to the pathogenesis of MS.

Multiple sclerosis (MS) is a chronic demyelinating disorder of the central nervous system (CNS). Its corresponding animal model, experimental autoimmune encephalomyelitis (EAE), is widely used to understand disease pathogenesis and test novel therapeutic agents. However, existing methods to score EAE disease severity are subjective and often vary between individual researchers, making it difficult to translate findings across different studies. An enhanced automated method of disease scoring would eliminate subjectivity and reduce operator-dependent errors. Here, we used an Infra-Red Activity Monitoring System (IRAMS) to measure murine locomotor activity as a surrogate measure of disease severity and compared it to standard EAE scoring methods. In mice immunized with CNS-specific myelin antigens, we observed an inverse correlation between disease severity and mouse activity, with the IRAMS showing enhanced disease scoring compared to standard EAE scoring methods. Relative to standard EAE scoring methods, IRAMS showed comparable measurement of disease relapses and remissions in the SJL/J-relapsing-remitting model of EAE, and could comparably assess the therapeutic efficiency of the MS drug, Copaxone (Glatiramer acetate-GA). Thus, the IRAMS is a method to measure disease severity in EAE without subjective bias and is a tool to consistently assess the efficacy of novel therapeutic agents for MS.

The human gut is colonized by trillions of bacteria that support physiologic functions such as food metabolism, energy harvesting, and regulation of the immune system. Perturbation of the healthy gut microbiome has been suggested to play a role in the development of inflammatory diseases, including multiple sclerosis (MS). Environmental and genetic factors can influence the composition of the microbiome; therefore, identification of microbial communities linked with a disease phenotype has become the first step towards defining the microbiome's role in health and disease. Use of 16S rRNA metagenomic sequencing for profiling bacterial community has helped in advancing microbiome research. Despite its wide use, there is no uniform protocol for 16S rRNA-based taxonomic profiling analysis. Another limitation is the low resolution of taxonomic assignment due to technical difficulties such as smaller sequencing reads, as well as use of only forward (R1) reads in the final analysis due to low quality of reverse (R2) reads. There is need for a simplified method with high resolution to characterize bacterial diversity in a given biospecimen. Advancements in sequencing technology with the ability to sequence longer reads at high resolution have helped to overcome some of these challenges. Present sequencing technology combined with a publicly available metagenomic analysis pipeline such as R-based Divisive Amplicon Denoising Algorithm-2 (DADA2) has helped advance microbial profiling at high resolution, as DADA2 can assign sequence at the genus and species levels. Described here is a guide for performing bacterial profiling using two-step amplification of the V3-V4 region of the 16S rRNA gene, followed by analysis using freely available analysis tools (i.e., DADA2, Phyloseq, and METAGENassist). It is believed that this simple and complete workflow will serve as an excellent tool for researchers interested in performing microbiome profiling studies.

Background Though the gut microbiome has been associated with efficacy of immunotherapy (ICI) in certain cancers, similar findings have not been identified for microbiomes from other body sites and their correlation to treatment response and immune related adverse events (irAEs) in lung cancer (LC) patients receiving ICIs. Methods We designed a prospective cohort study conducted from 2018 to 2020 at a single-center academic institution to assess for correlations between the microbiome in various body sites with treatment response and development of irAEs in LC patients treated with ICIs. Patients must have had measurable disease, ECOG 0–2, and good organ function to be included. Data was collected for analysis from January 2019 to October 2020. Patients with histopathologically confirmed, advanced/metastatic LC planned to undergo immunotherapy-based treatment were enrolled between September 2018 and June 2019. Nasal, buccal and gut microbiome samples were obtained prior to initiation of immunotherapy +/− chemotherapy, at development of adverse events (irAEs), and at improvement of irAEs to grade 1 or less. Results Thirty-seven patients were enrolled, and 34 patients were evaluable for this report. 32 healthy controls (HC) from the same geographic region were included to compare baseline gut microbiota. Compared to HC, LC gut microbiota exhibited significantly lower α-diversity. The gut microbiome of patients who did not suffer irAEs were found to have relative enrichment of Bifidobacterium (p = 0.001) and Desulfovibrio (p = 0.0002). Responders to combined chemoimmunotherapy exhibited increased Clostridiales (p = 0.018) but reduced Rikenellaceae (p = 0.016). In responders to chemoimmunotherapy we also observed enrichment of Finegoldia in nasal microbiome, and increased Megasphaera but reduced Actinobacillus in buccal samples. Longitudinal samples exhibited a trend of α-diversity and certain microbial changes during the development and resolution of irAEs. Conclusions This pilot study identifies significant differences in the gut microbiome between HC and LC patients, and their correlation to treatment response and irAEs in LC. In addition, it suggests potential predictive utility in nasal and buccal microbiomes, warranting further validation with a larger cohort and mechanistic dissection using preclinical models. Trial registration ClinicalTrials.gov, NCT03688347. Retrospectively registered 09/28/2018.

Multiple sclerosis (MS) is an inflammatory, demyelinating disease of the central nervous system, and among all the genes associated with MS, HLA class II genes have the highest relative risk. Although IL-17A is thought to be a major cytokine in the pathogenesis of MS, the exact role of IL-17A in the MS is still poorly understood. Specifically, it is unclear whether IL-17A is required for disease initiation or disease severity or in both. Therefore, in the present study, utilizing HLA-DR3 transgenic mice lacking IL-17A (HLA-DR3.IL-17A−/−), we investigated the importance of IL-17A in disease development and progression in experimental autoimmune encephalomnyelitis (EAE) an animal model of MS. We observed that HLA-DR3.IL-17A−/− mice had a higher frequency of CD4+CD25+FoxP3+ regulatory T cells (Treg)s and developed milder EAE compared to IL-17 sufficient HLA-DR3 mice. Further characterization of mechanism identified a novel role of IL-17A in regulating the disease progression through modulation of immunoregulatory responses. We also identified an important role of gut microbiota in IL-17A dependent disease regulation specifically depletion of gut bacteria with ability to promote Treg population in IL-17A sufficient HLA-DR3 mice. Fecal transplantation of HLA.DR3 mice with HLA-DR3. IL17A−/− showed milder EAE and an increase in Treg cells in HLA-DR3 mice confirming a role of gut microbiota in shaping Treg repertoire and function. Additionally, we observed higher frequency of metabolic pathway involved with Treg promoting short chain fatty acid (SCFA) metabolism in HLA-DR3.IL17A−/− mice. Thus our study shows a novel role of IL-17A in immune homeostasis and inflammation by regulating levels of Tregs through modulation of gut microbiota.

Objective The gut microbiome regulates host immune homeostasis. Rheumatoid arthritis (RA) is associated with intestinal dysbiosis. This study was undertaken to test the ability of a human gut-derived commensal to modulate immune response and treat arthritis in a humanized mouse model. Methods We isolated a commensal bacterium, Prevotella histicola, that is native to the human gut and has systemic immune effects when administered enterally. Arthritis-susceptible HLA–DQ8 mice were immunized with type II collagen and treated with P histicola. Disease incidence, onset, and severity were monitored. Changes in gut epithelial proteins and immune response as well as systemic cellular and humoral immune responses were studied in treated mice. Results When treated with P histicola in prophylactic or therapeutic protocols, DQ8 mice exhibited significantly decreased incidence and severity of arthritis compared to controls. The microbial mucosal modulation of arthritis was dependent on regulation by CD103+ dendritic cells and myeloid suppressors (CD11b+Gr-1+ cells) and by generation of Treg cells (CD4+CD25+FoxP3+) in the gut, resulting in suppression of antigen-specific Th17 responses and increased transcription of interleukin-10. Treatment with P histicola led to reduced intestinal permeability by increasing expression of enzymes that produce antimicrobial peptides as well as tight junction proteins (zonula occludens 1 and occludin). However, the innate immune response via Toll-like receptor 4 (TLR-4) and TLR-9 was not affected in treated mice. Conclusion Our results demonstrate that enteral exposure to P histicola suppresses arthritis via mucosal regulation. P histicola is a unique commensal that can be explored as a novel therapy for RA and may have few or no side effects.

The AMP-activated protein kinase, AMPK, is an energy-sensing, metabolic switch implicated in various metabolic disorders; however, its role in inflammation is not well defined. We have previously shown that loss of AMPK exacerbates experimental autoimmune encephalomyelitis (EAE) disease severity. In this study, we investigated the mechanism through which AMPK modulates inflammatory disease like EAE. AMPKα1 knockout (α1KO) mice with EAE showed severe demyelination and inflammation in the brain and spinal cord compared with wild-type due to higher expression of proinflammatory Th17 cytokines, including IL-17, IL-23, and IL-1β, impaired blood–brain barrier integrity, and increased infiltration of inflammatory cells in the CNS. Infiltrated CD4 cells in the brains and spinal cords of α1KO with EAE were significantly higher compared with wild-type EAE and were characterized as IL-17 (IL-17 and GM-CSF double-positive) CD4 cells. Increased inflammatory response in α1KO mice was due to polarization of macrophages (Mϕ) to proinflammatory M1 type phenotype (IL-10lowIL-23/IL-1β/IL-6high), and these M1 Mϕ showed stronger capacity to induce allogenic as well as Ag-specific (myelin oligodendrocyte glycoprotein [MOG]35–55) T cell response. Mϕ from α1KO mice also enhanced the encephalitogenic property of MOG35–55–primed CD4 T cells in B6 mice. The increased encephalitogenic MOG-restricted CD4+ T cells were due to an autocrine effect of IL-1β/IL-23–mediated induction of IL-6 production in α1KO Mϕ, which in turn induce IL-17 and GM-CSF production in CD4 cells. Collectively, our data indicate that AMPK controls the inflammatory disease by regulating the M1 phenotype–Th17 axis in an animal model of multiple sclerosis.

Two zoonotic coronaviruses (CoVs)-SARS-CoV and MERS-CoV-have crossed species to cause severe human respiratory disease. Here, we showed that induction of airway memory CD4(+) T cells specific for a conserved epitope shared by SARS-CoV and MERS-CoV is a potential strategy for developing pan-coronavirus vaccines. Airway memory CD4(+) T cells differed phenotypically and functionally from lung-derived cells and were crucial for protection against both CoVs in mice. Protection was dependent on interferon-γ and required early induction of robust innate and virus-specific CD8(+) T cell responses. The conserved epitope was also recognized in SARS-CoV- and MERS-CoV-infected human leukocyte antigen DR2 and DR3 transgenic mice, indicating potential relevance in human populations. Additionally, this epitope was cross-protective between human and bat CoVs, the progenitors for many human CoVs. Vaccine strategies that induce airway memory CD4(+) T cells targeting conserved epitopes might have broad applicability in the context of new CoVs and other respiratory virus outbreaks.