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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.

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.

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.

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.

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.

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.

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.

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.

Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system. Despite IL-17A being linked to the immunopathogenesis of MS, the mechanism of IL-17A in disease development and maintenance is unclear. In the present study, we investigated the association of IL-17A and gut microbiota in disease development and severity, utilizing HLA-DR3 transgenic mice lacking IL-17A (IL-17A−/−). We found that HLA-DR3.IL-17A−/− mice had a higher frequency of CD4+CD25+FoxP3+ Treg cells and develop milder EAE compared to IL-17A suffcient HLA-DR3 mice. Utilizing 16S metagenocmic sequencing and analysis of gut microbiota we observed that HLA-DR3.IL-17A−/− mice had an increased abundance of regulatory T cells promoting bacteria in the gut such as Lactobacillus and Clostridia. Fecal transplantation followed by co-housing of HLA.DR3 mice with HLA-DR3.IL17A−/− resulted in development of milder EAE and an increase in CD4+CD25+FoxP3+ Treg cells in HLA-DR3 mice. Further whole-genome shotgun metagenomic analysis showed modulation of pathogen-associated molecular patterns (PAMPs) and O-antigen pathways linked with the host innate immune responses in HLA-DR3.IL-17A−/− mice. Thus our study suggests that gut microbiota changes due to the absence of IL17A induce regulatory T cells and suppresses disease in an animal model of MS by modulating the host-bacterial dynamics via PRR-PAMPS interactions.

Multiple sclerosis (MS), an autoimmune disease of the central nervous system, has been shown to be influenced by gut bacteria. We and others have shown that MS patients have depletion of isoflavone-metabolizing bacteria compared to healthy individuals. We hypothesize that gut bacteria-mediated isoflavone metabolism induces an anti-inflammatory state and reduction/depletion of these gut bacteria might predispose to MS development and/or severity. To test this, we placed mice on a diet with or without isoflavones and induced EAE, an animal model of MS. Mice on an isoflavone diet developed milder disease compared to those on an isoflavone-free diet. This phenomenon was dependent on the presence of isoflavone-metabolizing gut bacteria as confirmed by antibiotic depletion and bacterial reconstitution. Mice on an isoflavone diet exhibited altered peripheral immune responses, CNS cellular infiltration, and CNS pathology post EAE induction compared to mice on an isoflavone-free diet. Additionally, the gut microbiome of mice on an isoflavone diet and an isoflavone-free diet closely resembles the gut microbiota of healthy individuals and MS patients, respectively. Thus our results suggest that dietary isoflavone metabolites produced with the help of gut bacteria can influence the disease through modulation of the immune system.

Background: In recent years, attention has shifted to modification of tumor response to immunotherapy via the host microbiome. The mechanisms of these associations, causative or consequential, remain incompletely understood. We seek to explore this further with a longitudinal study of lung cancer patient microbiomes and immunotherapy-related adverse effects (AEs) preceding and during immunotherapy. Methods: Patients with lung cancer (LC) treated with immunotherapy (anti-PD-1/L1 agents including pembrolizumab, nivolumab, atezolizumab, and durvalumab) with or without chemotherapy at the University of Iowa from November 2018–April 2019 were consented for this ongoing study. Fecal samples and nasal and buccal swabs were obtained prior to therapy. Patients were treated and monitored per respective disease protocol; responses were recorded per RECIST 1.1 and AEs per CTCAE 5.0. Samples underwent DNA extraction followed by 16S rRNA metagenomic analysis and taxonomic profiling using Divisive Amplicon Denoising Algorithm (DADA)-2 pipeline. Microbiomes from all body sites were compared and correlated to treatment response and AEs. In addition, baseline LC gut microbiomes were compared to fecal samples provided by healthy controls (HC) in the same geographic location. This project is registered on clinicaltrials.gov (NCT03688347). Results: Gut microbiota significantly differed compared to oral and buccal microbiota in all patients. Gut microbiota from LC patients was compared to HC samples from the same geographic area. LC patients exhibited drastically different baseline composition, including dramatic increases in Firmicutes, Actinobacteria, and Verrucomicrobia, and significant decreases in Bacteroidetes, Proteobacteria, and Cyanobacteria. We noticed a clear inversion of Firmicutes/Bacteroidetes ratio between HC and LC patients, differences also reflected at the genus level. NSCLC patients experiencing immunotherapy-related adverse events were found to have at baseline markedly fewer Bifidobacterium and Desulfovibrio (p < 0.05 and FDR = PR) exhibited enriched baseline Clostridiales (phylum Firmicutes, p=0.018) but reduced Rikenellaceae (phylum Bacteroidetes, p=0.016). Conclusion: Our study found promising trends in microbiome constitution. Compared to HC, we found significant differences in baseline LC gut microbiome at phylum and genus levels. More importantly, there are notable differences comparing LC patients who suffered AEs to those with none and between immunotherapy responders vs. nonresponders. Our project marks an important first step in a long-term study that could shed new light on the microbiome’s influence on immunotherapy treatment outcomes. Citation Format: Justin J. Chau, Meeta Yadav, Muhammad Furqan, Keri Mercer, Evan Eastman, Shailesh Shahi, Qun Dai, Carlos Chan, George Weiner, Taher Abu Hejleh, Ashutosh Mangalam, Jun Zhang. The microbiome in lung cancer under immunotherapy: Significant compositional differences associated with treatment response and AEs [abstract]. In: Proceedings of the AACR Special Conference on the Microbiome, Viruses, and Cancer; 2020 Feb 21-24; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2020;80(8 Suppl):Abstract nr B24.

SUMMARY The exact mechanisms underlying the metabolic effects of bariatric surgery remain unclear. Here, we demonstrate, using a combination of direct and indirect calorimetry, an increase in total resting metabolic rate (RMR) and specifically anaerobic RMR after Roux-en-Y gastric bypass (RYGB), but not sleeve gastrectomy (SG). We also show an RYGB-specific increase in splanchnic sympathetic nerve activity and “browning” of visceral mesenteric fat. Consequently, selective splanchnic denervation abolishes all beneficial metabolic outcomes of gastric bypass that involve changes in the endocannabinoid signaling within the small intestine. Furthermore, we demonstrate that administration of rimonabant, an endocannabinoid receptor-1 (CB1) inverse agonist, to obese mice mimics RYGB-specific effects on energy balance and splanchnic nerve activity. On the other hand, arachidonoylethanolamide (AEA), a CB1 agonist, attenuates the weight loss and metabolic signature of this procedure. These findings identify CB1 as a key player in energy regulation post-RYGB via a pathway involving the sympathetic nervous system., In Brief Ye et al. show that energy regulation differs between Roux-en-Y gastric bypass (RYGB) and sleeve gastrectomy (SG) where only RYGB enhances splanchnic nerve activity, to induce visceral fat thermogenesis, and increases resting metabolic rate (RMR). This sympathetic-mediated “browning” of visceral fat seems to be dependent on endocannabinoid receptor-1 (CB1) signaling., Graphical Abstract

Multiple sclerosis (MS) is a chronic neuroinflammatory disease of the central nervous system with unknown etiology. Recently, the gut microbiota has emerged as a potential factor in the development of MS, with a number of studies having shown that patients with MS exhibit gut dysbiosis. The gut microbiota helps the host remain healthy by regulating various functions, including food metabolism, energy homeostasis, maintenance of the intestinal barrier, inhibition of colonization by pathogenic organisms, and shaping of both mucosal and systemic immune responses. Alteration of the gut microbiota, and subsequent changes in its metabolic network that perturb this homeostasis, may lead to intestinal and systemic disorders such as MS. Here we discuss the findings of recent MS microbiome studies and potential mechanisms through which gut microbiota can predispose to, or protect against, MS. These findings highlight the need of an improved understanding of the interactions between the microbiota and host for developing therapies based on gut commensals with which to treat MS.

In the present work, we describe a simple, cheap, and unexplored method for "green" synthesis of silver nanoparticles using cell extracts of the cyanobacterium Anabaena doliolum. An attempt was also made to test the antimicrobial and antitumor activities of the synthesized nanoparticles. Analytical techniques, namely UV-vis spectroscopy, X-ray diffraction, Fourier transform infrared (FTIR) spectroscopy, transmission electron microscopy (TEM), and TEMselected area electron diffraction, were used to elucidate the formation and characterization of silver-cyanobacterial nanoparticles (Ag-CNPs). Results showed that the original color of the cell extract changed from reddish blue to dark brown after addition of silver nitrate solution (1 mM) within 1 h, suggesting the synthesis of Ag-CNPs. That the formation Ag-CNPs indeed occurred was also evident from the spectroscopic analysis of the reaction mixture, wherein a prominent peak at 420 nm was noted. TEM images revealed well-dispersed, spherical Ag- CNPs with a particle size in the range of 10-50 nm. The X-ray diffraction spectrum suggested a crystalline nature of the Ag-CNPs. FTIR analysis indicated the utilization of a hydroxyl (-OH) group in the formation of Ag-CNPs. Ag-CNPs exhibited strong antibacterial activity against three multidrug-resistant bacteria. Additionally, Ag-CNPs strongly affected the survival of Dalton's lymphoma and human carcinoma colo205 cells at a very low concentration. The Ag-CNPs-induced loss of survival of both cell types may be due to the induction of reactive oxygen species generation and DNA fragmentation, resulting in apoptosis. Properties exhibited by the Ag-CNP suggest that it may be used as a potential antibacterial and antitumor agent.

Resistance profile to antibiotics and the occurrence of class-1 integron in 23 bacteria isolated from diabetic foot ulcers (DFUs) were analyzed. Among 23 isolates, 16 elicited resistance to at least two antibiotics and seven were sensitive. Majority of the isolates were resistant to spectinomycin and streptomycin (68.75%) followed by trimethoprim (62.50%) and ampicillin (50%). Among the antibiotics resistant isolates, six exhibited resistance to four different groups of antibiotics and one showed resistance to 11 antibiotics (six antibiotic groups). 17 bacterial strains possessed class-1 integrons with an amplicon size of 0.30 to 2.40 kb which suggested their role in conferring resistance to antibiotics. 2-D gel electrophoresis of proteins extracted from Escherichia coli DF39TA grown with antibiotics revealed significant alteration in total proteome as compared to control culture. Five spots showing four fold up regulation were identified by MALDI TOF MS as OmpX, OmpA, OmpA-OmpF, Omp-tolC and chaperone protein DnaK. Changes in abundance of above proteins following growth with antibiotics may be beneficial for antibiotic resistant bacteria to adapt under adverse environmental conditions. Findings of this study suggest that infection with multiple antibiotic-resistant bacteria is common to DFUs and resistance is mediated by class-1 integrons. Key words: Diabetic foot ulcer, antibiotic resistance, polymerase chain reaction (PCR), class-I integron, 2-D gel electrophoresis, Escherichia coli.

Summary The human gut is colonized by a large number of microorganisms (∼10 13 bacteria) that support various physiologic functions. A perturbation in the healthy gut microbiome might lead to the development of inflammatory diseases, such as multiple sclerosis (MS). Therefore, gut commensals might provide promising therapeutic options for treating MS and other diseases. We report the identification of human gut-derived commensal bacteria, Prevotella histicola , which can suppress experimental autoimmune encephalomyelitis (EAE) in a human leukocyte antigen (HLA) class II transgenic mouse model. P. histicola suppresses disease through the modulation of systemic immune responses. P. histicola challenge led to a decrease in pro-inflammatory Th1 and Th17 cells and an increase in the frequencies of CD4 + FoxP3 + regulatory T cells, tolerogenic dendritic cells, and suppressive macrophages. Our study provides evidence that the administration of gut commensals may regulate a systemic immune response and may, therefore, have a possible role in treatment strategies for MS.

Severe diabetic foot ulcers (DFUs) patients visiting Sir Sunderlal Hospital, Banaras Hindu University, Varanasi, were selected for this study. Bacteria were isolated from swab and deep tissue of 42 patients, for examining their prevalence and antibiotic sensitivity. DFUs of majority of the patients were found infected with Enterococcus spp. (47.61%), Escherichia coli (35.71%), Staphylococcus spp. (33.33%), Alcaligenes spp. (30.95%), Pseudomonas spp. (30.95%), and Stenotrophomonas spp. (30.95%). Antibiotic susceptibility assay of 142 bacteria with 16 antibiotics belonging to eight classes showed the presence of 38 (26.76%) isolates with multidrug resistance (MDR) phenotypes. MDR character appeared to be governed by integrons as class 1 integrons were detected in 26 (68.42%) isolates. Altogether six different arrays of genes (aadA1, aadB, aadAV, dhfrV, dhfrXII, and dhfrXVII) were found within class 1 integron. Gene cassette dhfrAXVII-aadAV (1.6 kb) was present in 12 (3 Gram positive and 9 Gram negative) isolates and was conserved across all the isolates as evident from RFLP analysis. In addition to the presence of class 1 integron, six β-lactamase resistance encoding genes namely bla TEM, bla SHV, bla OXA, bla CTX-M-gp1, bla CTX-M-gp2, and bla CTX-M-gp9 and two methicillin resistance genes namely mecA and femA and vancomycin resistance encoding genes (vanA and vanB) were identified in different isolates. Majority of the MDR isolates were positive for bla TEM (89.47%), bla OXA (52.63%), and bla CTX-M-gp1 (34.21%). To our knowledge, this is the first report of molecular characterization of antibiotic resistance in bacteria isolated from DFUs from North India. In conclusion, findings of this study suggest that class-1 integrons and β-lactamase genes contributed to the MDR in above bacteria.

Multiple sclerosis (MS) is a demyelinating disease of the central nervous system. We and others have shown that gut microbiota play an important role in disease pathogenesis of MS as there is an enrichment or depletion of specific gut bacteria compared to healthy controls (HC). Thus the specific human gut bacteria found depleted or showing lower abundance in MS patients can be used as potential drug to treat MS. Further, we and others have shown that patients with MS have either lower abundance of Prevotella compared to HC or an increased level of Prevotella in patients receiving disease modifying therapies such as Copaxone and/or Interferon beta (IFN-β). We have previously identified a specific strain Prevotella histicola within Prevotella genus which can suppress disease in experimental autoimmune encephalomyelitis (EAE) animal model of MS. In this present study we compared disease suppressing ability of P. histicola to Copaxone or IFN-β in HLA-DR3.DQ8 transgenic mouse model of MS. We observed that P. histicola suppresses EAE as efficiently as Copaxone and IFN-β. P. histicola treated mice showed higher level of CD4+FoxP3+ regulatory T cell frequencies and decreased levels of pro-inflammatory cytokines IFNγ and IL17 suggesting that P. histicola suppresses disease through immunomodulation. Fecal microbiota analysis of different groups showed that group receiving Prevotella histicola had a distinct gut microbiota than the control group suggesting that P. histicola as might suppresses disease through modulation of gut microbiota. In conclusion, our study suggests that human gut commensal Prevotella histicola can provide an alternate treatment option for MS patients.

The polymicrobial nature of diabetic foot infection is a reflection of the immune compromised state of the host.The methods of microbial identification based on colony morphology and biochemical characteristics have limitations as they may not differentiate the diverse microorganisms that infect foot wounds. The aim of the present study was to find out the bacterial diversity in diabetic foot infections at genetic level by finger printing, that is, ERIC-PCR (enterobacterial repetitive intergenic consensus -polymerase chain reaction). Nine patients with infected diabetic foot ulcers were recruited to the study. Pus and tissue samples were collected from the wound site. Aerobic bacteria were isolated employing standard microbiological culture methods and their genetic variability was analyzed using the ERIC-PCR. Sensitivity test for these isolates against commonly used antibiotics were performed using disc diffusion method. The standard microbiological culture technique yielded 38 morphotypes of bacteria and their genetic diversity was confirmed by ERIC-PCR assay. Analysis of the similarity index using NTSYSpc 2.1 software revealed 34 types of banding pattern among these isolates. Based on the similarity index these isolates were divided into 7 groups. As many as 8 types of aerobic bacterial isolates were detected from a single patient using the above technique compared with 2 on routine culture analysis. Genetically diverse isolates showed differential sensitivity pattern against commonly used antibiotics in the assay. The observed diversity at genetic level is attributed to variable sensitivity pattern of these isolates against the class of antibiotics. A molecular technique such as ERIC-PCR is a more sensitive detection method than conventional techniques, the potential of which needs to be fully understood.

Metronidazole is the drug of choice for anaerobic infection in diabetic foot ulcers (DFU) for a majority of clinicians. The present study was conducted to determine if Metronidazole is really making a difference in the healing of DFU.Deep tissue samples from the wound area of 61 diabetic foot patients were tested for anaerobic bacterial infection (Peptostreptococcus productus, Bacteroides, and Clostridium) by polymerase chain reaction (PCR). PCR-positive patients were randomized into 2 groups: Metronidazole and non-Metronidazole. Antibiotics for the control of infection were given in both groups as per clinical condition of patients. Treatment outcome was assessed by complete healing of the wound.Out of 61 patients, PCR detected evidence of anaerobic infection in 32 (52%), while culture methods detected only 5 (8%) (Clostridium spp.), hence emphasizing the significance of the PCR technique over culture methods in detection of microbes. In this study, Clostridium was found with maximum prevalence of n (75%), followed by Bacteroides with n (53.1%), and Peptostreptococcus productus with n (40.6 %). Across all Wagner Ulcer Classification grades, Clostridium was the most prevalent anaerobe, and significantly associated with wound age and total leukocyte count. No difference was noted in wound healing in both groups at the end of 16 weeks.The authors propose that it is not mandatory to supplement Metronidazole in antibiotic regime for treatment of DFU.

Our recent multiple sclerosis (MS) microbiome study indicates an important role of gut microbiota in pathogenesis of MS, an autoimmune disease of central nervous system. Gut microbiota help in maintaining immune-homeostasis by regulating balance between CD4+CD25+FoxP3+ regulatory T cells (Tregs) and pro-inflammatory Th1/Th17 cells. A shift of balance towards Th1/Th17 cells leading to inflammation and demyelination in central nervous system (CNS), is suggested to be responsible of disease initiation and/or relapses in MS. Therefore, gut commensals capable of restoring the microbiome to a healthy state could provide novel therapeutic options for treating autoimmune diseases including MS. Here, we report identification of human gut-derived commensal bacteria, Prevotella histicola (P. histicola), which can suppress an autoimmune disease in HLA class-II transgenic model of experimental autoimmune encephalomyelitis (EAE), an experimental model of MS. P. histicola suppresses disease through modulation of systemic immune response. P. histicola challenge led to a decrease in pro-inflammatory Th1 (IFNγ) and Th17 (IL17) cytokines, and increase in the frequencies of CD4+FoxP3+ Tregs, tolerogenic dendritic cells and suppressive macrophages. We also observed that mice with EAE had a altered gut microbiota compared to naïve mice and treatment with P. histicola restored gut flora to a normal state which further support an important role of gut microbiota in EAE/MS. Our study provides compelling evidence that administration of gut commensals may regulate a systemic immune response and have a possible role in the treatment of autoimmune diseases.

Klebsiella pneumoniae strain DF12SA (HQ114261) was isolated from diabetic foot wounds. The strain showed resistance against ampicillin, kanamycin, gentamicin, streptomycin, spectinomycin, trimethoprim, tetracycline, meropenem, amikacin, piperacillin/tazobactam, augmentin, co-trimoxazole, carbapenems, penicillins and cefoperazone, and was sensitive to clindamycin. Molecular characterization of the multidrug-resistance phenotype revealed the presence of a class 1 integron containing two genes, a dihydrofolate reductase (DHFR) (PF00186), which confers resistance to trimethoprim; and aminoglycoside adenyltransferase (AadA) (PF01909), which confers resistance to streptomycin and spectinomycin. A class 1 integron in K. pneumoniae containing these two genes was present in eight (18.18%) out of 44 different diabetic foot ulcer (DFU) patients. Hence, there is a need to develop therapeutics that inhibit growth of multidrug resistant K. pneumoniae in DFU patients and still achieve amputation control. Am attempt was made to create a 3D model and find a suitable inhibitor using an in silico study. Rational drug design/testing requires crystal structures for DHFR and AadA. However, the structures of DHFR and AadA from K. pneumoniae are not available. Modelling was performed using Swiss Model Server and Discovery Studio 3.1. The PDBSum server was used to check stereo chemical properties using Ramachandran plot analysis of modeled structures. Clindamycin was found to be suitable inhibitor of DHFR and AadA. A DockingServer based on Autodock & Mopac was used for docking calculations. The amino acid residues Ser(32), Ile(46), Glu(53), Gln(54), Phe(57), Thr(72), Met(76), Val(78), Leu(79), Ser(122), Tyr(128), Ile(151) in case of DHFR and Phe(34), Asp(60), Arg(63), Gln(64), Leu(68), Glu(87), Thr(89), Val(90) for AadA were found to be responsible for positioning clindamycin into the active site. The study identifies amino acid residues crucial to 'DHFR and AadA -drug' and 'DHFR and AadA -inhibitor' interactions that might be useful in the ongoing search for a versatile DHFR and AadA -inhibitor.

Diabetic foot ulcer (DFU) is a common and devastating complication in diabetes. Antimicrobial resistance mediated by extended-spectrum β-lactamases (ESBLs) production by bacteria is considered to be a major threat for foot amputation. The present study deals with the detection of Escherichia coli and the prevalence of bla(TEM), bla(SHV) and bla(OXA) genes directly from biopsy and swab of foot ulcers of diabetic patients. In total, 116 DFU patients were screened, of which 42 suffering with severe DFUs were selected for this study. Altogether 16 E. coli strains were successfully isolated from biopsy and/or swab samples of 15 (35.71%) patients. ESBL production was noted in 12 (75%) strains. Amplification of β-lactamase genes by multiplex PCR showed the presence of bla(CTX-M) like genes in 10 strains, bla(TEM) and bla(OXA) in 9 strains each, and bla(SHV) in 8 of the total 16 strains of E. coli. Out of the ten antibiotics tested, E. coli strains were found to be resistant to ampicillin (75%), cefoxitin (56.25%), cefazolin (50%), meropenem (37.5%), cefoperazone (25%), cefepime (31.25%), ceftazidime (56.25%), and cefotaxime (68.75%) but all showed sensitivity (100%) to clindamycin and piperacillin-tazobactam. 3D models of the most prevalent variants of β-lactamases namely TEM-1, SHV-1, OXA-1, and ESBL namely CTX-M-15 were predicted and docking was performed with clindamycin and piperacillin-tazobactam to reveal the molecular basis of drug sensitivity. Docking showed the best docking score with significant interactions, forming hydrogen bond, Van der Waals and polar level interaction with active site residues. Findings of the present study may provide useful insights for the development of new antibiotic drugs and may also prevent ESBLs-mediated resistance problem in DFU. The novel multiplex PCR assay designed in this study may be routinely used in clinical diagnostics of E. coli and associated bla(TEM), bla(SHV), and bla(OXA) like genes.

114 diazotrophic bacteria from the rice rhizosphere of five districts of Eastern Uttar Pradesh (India) were isolated and screened for plant growth promoting (PGP) activities employing standard microbiological and biochemical techniques. All these isolates showed nitrogenase activity in the range of 0.23 to 1.72 μmol C 2 H 4 mg -1 protein h -1 . Further analysis showed that 84 (73.68%) isolates were Indole-3-acetic acid (IAA) producer; the value of IAA production ranged from 10.1 to 353.0 μg IAA mg -1 protein. IAA production occurred solely in the medium supplemented with tryptophan. P solubilization activity was observed in 28 (24.56%) isolates, the activity being in the range of 38.50 to 321.0 P released μg mg -1 protein. 45 (39.46%) isolates were capable of producing siderophore, the range of production being 4.50 to 223.26 μg mg -1 protein. Analysis of molecular diversity was made by amplified ribosomal DNA restriction analysis (ARDRA) and denaturating gradient gel electrophoresis (DGGE), which exhibited distinct differences among all the isolates. Of the 114 isolates, twenty one (21) isolates showed multiple plant growth promoting traits and were potent in terms of PGP activities. These isolates were identified on the basis of 16S rDNA sequencing and belonged to the genera Pantoea, Bacillus, Microbacterium, Pseudomonas, Sphingomonas, Ancylobacter, Enterobacter, Advenella, γ-proteobacterium strain VA3S1, Rhizobium and Agrobacterium . Findings of this study suggest that certain isolates may be exploited for developing a potential source of biofertilizer. Key words: Plant growth promoting rhizobacteria, N 2 fixation, amplified ribosomal DNA restriction analysis, indole-3-acetic acid, siderophore, denaturing gradient gel electrophoresis, temperature gradient gel electrophoresis