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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.
Assuntos
Inflamação/imunologia , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Metilenotetra-Hidrofolato Desidrogenase (NADP)/metabolismo , Purinas/biossíntese , Linfócitos T Reguladores/imunologia , Células Th17/imunologia , Animais , Diferenciação Celular , Citocinas/metabolismo , Metilação de DNA , Modelos Animais de Doenças , Humanos , Mediadores da Inflamação/metabolismo , Ativação Linfocitária , Metilenotetra-Hidrofolato Desidrogenase (NADP)/genética , Camundongos , Camundongos Transgênicos , Mutação/genética , Transdução de SinaisRESUMO
Multiple sclerosis (MS) is a chronic and progressive autoimmune disease of the central nervous system (CNS), with both genetic and environmental factors contributing to the pathobiology of the disease. While human leukocyte antigen (HLA) genes have emerged as the strongest genetic factor, consensus on environmental risk factors are lacking. Recently, trillions of microbes residing in our gut (microbiome) have emerged as a potential environmental factor linked with the pathobiology of MS as PwMS show gut microbial dysbiosis (altered gut microbiome). Thus, there has been a strong emphasis on understanding the factors (host and environmental) regulating the composition of the gut microbiota and the mechanism(s) through which gut microbes contribute to MS disease, especially through immune system modulation. A better understanding of these interactions will help harness the enormous potential of the gut microbiota as a therapeutic approach to treating MS.
Assuntos
Disbiose , Microbioma Gastrointestinal , Esclerose Múltipla , Esclerose Múltipla/microbiologia , Esclerose Múltipla/imunologia , Humanos , Disbiose/microbiologia , Animais , Bactérias/genética , Bactérias/classificação , Bactérias/metabolismoRESUMO
The widespread use of glyphosate, a broad-spectrum herbicide, has resulted in significant human exposure, and recent studies have challenged the notion that glyphosate is safe for humans. Although the link between disease states and glyphosate exposure is increasingly appreciated, the mechanistic links between glyphosate and its toxic effects on human health are poorly understood. Recent studies have suggested that glyphosate may cause toxicity through modulation of the gut microbiome, but evidence for glyphosate-induced gut dysbiosis and its effect on host physiology at doses approximating the U.S. Acceptable Daily Intake (ADI = 1.75 mg/kg body weight) is limited. Here, utilizing shotgun metagenomic sequencing of fecal samples from C57BL/6 J mice, we show that glyphosate exposure at doses approximating the U.S. ADI significantly impacts gut microbiota composition. These gut microbial alterations were associated with effects on gut homeostasis characterized by increased proinflammatory CD4+IL17A+ T cells and Lipocalin-2, a known marker of intestinal inflammation.
Assuntos
Microbioma Gastrointestinal , Herbicidas , Camundongos , Humanos , Animais , Camundongos Endogâmicos C57BL , Herbicidas/toxicidade , Homeostase , GlifosatoRESUMO
UNLABELLED: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: The authors propose that it is not mandatory to supplement Metronidazole in antibiotic regime for treatment of DFU.
RESUMO
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.
Assuntos
Microbioma Gastrointestinal , Esclerose Múltipla , Humanos , Microbioma Gastrointestinal/fisiologia , Disbiose , Esclerose Múltipla/etiologia , Esclerose Múltipla/terapia , Bactérias , Progressão da DoençaRESUMO
Background: Multiple sclerosis (MS) is an inflammatory and demyelinating disease of the CNS. The etiology of MS is complex, and results from the interaction of multiple environmental and genetic factors. Although human leukocyte antigen-HLA alleles such as HLA-DR2 and -DR3 are considered the strongest genetic factors, the environmental factors responsible for disease predisposition are not well understood. Recently, diet and gut microbiota have emerged as an important environmental factors linked to the increased incidence of MS. Especially, western diets rich in protein and fat have been linked to the increased incidence of obesity. Numerous clinical data indicate a role of obesity and gut microbiota in MS; however, the mechanistic link between gut microbiota and obesity in the pathobiology of MS remains unclear. The present study determines the mechanisms driving MS severity in the context of obesity utilizing a high-fat diet (HFD) induced obese HLA-DR3 class-II transgenic mouse model of MS. Methods: HLA-DR3 transgenic mice were kept on a standard HFD diet or Normal Chow (NC) for eight weeks. Gut microbiota composition and functional analysis were performed from the fecal DNA of mice. Experimental autoimmune encephalomyelitis-EAE (an animal model of MS) was induced by immunization with the proteolipid protein-PLP91-110 peptide in complete Freud's Adjuvant (CFA) and pertussis toxin. Results: We observed that HFD-induced obesity caused gut dysbiosis and severe disease compared to mice on NC. Amelioration of disease severity in mice depleted of gut microbiota suggested an important role of gut bacteria in severe EAE in obese mice. Fecal microbiota analysis in HFD mice shows gut microbiota alterations with an increase in the abundance of Proteobacteria and Desulfovibrionaceae bacteria and modulation of various bacterial metabolic pathways including bacterial hydrogen sulfide biosynthetic pathways. Finally, mice on HFD showed increased gut permeability and systemic inflammation suggesting a role gut barrier modulation in obesity induced disease severity. Conclusions: This study provides evidence for the involvement of the gut microbiome and associated metabolic pathways plus gut permeability in obesity-induced modulation of EAE disease severity. A better understanding of the same will be helpful to identify novel therapeutic targets to reduce disease severity in obese MS patients.
Assuntos
Encefalomielite Autoimune Experimental , Sulfeto de Hidrogênio , Esclerose Múltipla , Animais , Dieta Hiperlipídica/efeitos adversos , Modelos Animais de Doenças , Disbiose/microbiologia , Encefalomielite Autoimune Experimental/genética , Antígeno HLA-DR2 , Antígeno HLA-DR3/genética , Humanos , Camundongos , Camundongos Obesos , Camundongos Transgênicos , Obesidade/microbiologia , Toxina Pertussis , Proteolipídeos , Índice de Gravidade de DoençaRESUMO
The etiopathogenesis of multiple sclerosis (MS) is strongly affected by environmental factors such as diet and the gut microbiota. An isoflavone-rich (ISO) diet was previously shown to reduce the severity of MS in the animal model experimental autoimmune encephalomyelitis (EAE). Translation of this concept to clinical trial where dietary isoflavones may be recommended for MS patients will require preliminary evidence that providing the isoflavone-rich diet to people with MS (PwMS) who lack phytoestrogen-metabolizing bacteria has beneficial effects. We have previously shown that the gut microbiota of PwMS resembles the gut microbiota of mice raised under a phytoestrogen-free (phyto-free) diet in that it lacks phytoestrogen-metabolizing bacteria. To investigate the effects of phytoestrogens on the microbiota inflammatory response and EAE disease severity we switched the diet of mice raised under a phyto-free (PF) diet to an isoflavone-rich diet. Microbiota analysis showed that the change in diet from one that is ISO to one that is PF reduces beneficial bacteria such as Bifidobacterium species. In addition we observed functional differences in lipopolysaccharide (LPS) biosynthesis pathways. Moreover LPS extracted from feces of mice fed an ISO diet induced increased production of anti-inflammatory cytokines from bone marrow-derived macrophages relative to fecal-LPS isolated from mice fed a PF diet. Eventually mice whose diet was switched from a PF diet to an ISO diet trended toward reduced EAE severity and mortality. Overall we show that an isoflavone-rich diet specifically modulates LPS biosynthesis of the gut microbiota imparts an anti-inflammatory response and decreases disease severity.
Assuntos
Encefalomielite Autoimune Experimental , Microbioma Gastrointestinal , Isoflavonas , Animais , Citocinas/metabolismo , Dieta , Encefalomielite Autoimune Experimental/microbiologia , Inflamação , Isoflavonas/farmacologia , Lipopolissacarídeos/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Fitoestrógenos/farmacologiaRESUMO
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.
Assuntos
Ascomicetos , Esclerose Múltipla , Micobioma , Ascomicetos/genética , Bactérias/genética , Disbiose/microbiologia , Fungos/genética , Humanos , Micobioma/genética , RNA Ribossômico 16S/genéticaRESUMO
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.
Assuntos
Encefalomielite Autoimune Experimental/genética , Microbioma Gastrointestinal/genética , Antígenos HLA-D/genética , Antígenos de Histocompatibilidade Classe II/genética , Esclerose Múltipla/genética , Polimorfismo Genético , Animais , Bactérias/classificação , Bactérias/genética , Modelos Animais de Doenças , Encefalomielite Autoimune Experimental/metabolismo , Predisposição Genética para Doença/genética , Antígenos HLA-D/metabolismo , Antígenos HLA-DQ/genética , Antígenos HLA-DQ/metabolismo , Antígeno HLA-DR3/genética , Antígeno HLA-DR3/metabolismo , Antígenos de Histocompatibilidade Classe II/metabolismo , Humanos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Esclerose Múltipla/metabolismo , FenótipoRESUMO
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.
Assuntos
Encefalomielite Autoimune Experimental , Isoflavonas , Esclerose Múltipla , Animais , Bactérias/metabolismo , Dieta , Encefalomielite Autoimune Experimental/metabolismo , Humanos , Isoflavonas/metabolismo , Isoflavonas/farmacologia , Esclerose Múltipla/tratamento farmacológicoRESUMO
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.
Assuntos
Doença Enxerto-Hospedeiro/prevenção & controle , Receptores de Hialuronatos/metabolismo , Osteopontina/fisiologia , Linfócitos T/metabolismo , Animais , Células Epiteliais/metabolismo , Feminino , Doença Enxerto-Hospedeiro/etiologia , Doença Enxerto-Hospedeiro/metabolismo , Doença Enxerto-Hospedeiro/patologia , Intestinos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
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 naïve 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.
Assuntos
Derivação Gástrica , Microbioma Gastrointestinal , Fator 88 de Diferenciação Mieloide/metabolismo , Receptor 4 Toll-Like/metabolismo , Animais , Derivação Gástrica/métodos , Microbioma Gastrointestinal/fisiologia , Camundongos , Fator 88 de Diferenciação Mieloide/genética , Obesidade/cirurgia , Receptor 4 Toll-Like/genéticaRESUMO
Gut microbiota has emerged as an important environmental factor in the pathobiology of multiple sclerosis (MS), an inflammatory demyelinating disease of the central nervous system (CNS). Both genetic and environmental factors have been shown to play an important role in MS. Among genetic factors, the human leukocyte antigen (HLA) class II allele such as HLA-DR2, DR3, DR4, DQ6, and DQ8 show the association with the MS. We have previously used transgenic mice expressing MS susceptible HLA class II allele such as HLA-DR2, DR3, DQ6, and DQ8 to validate significance of HLA alleles in MS. Although environmental factors contribute to 2/3 of MS risk, less is known about them. Gut microbiota is emerging as an imporatnt environmental factor in MS pathogenesis. We and others have shown that MS patients have distinct gut microbiota compared to healthy control (HC) with a lower abundance of Prevotella. Additionally, the abundance of Prevotella increased in patients receiving disease-modifying therapies (DMTs) such as Copaxone and/or Interferon-beta (IFNß). We have previously identified a specific strain of Prevotella (Prevotella histicola), which can suppress experimental autoimmune encephalomyelitis (EAE) disease in HLA-DR3.DQ8 transgenic mice. Since Interferon-ß-1b [IFNß (Betaseron)] is a major DMTs used in MS patients, we hypothesized that treatment with the combination of P. histicola and IFNß would have an additive effect on the disease suppression. We observed that treatment with P. histicola suppressed disease as effectively as IFNß. Surprisingly, the combination of P. histicola and IFNß was not more effective than either treatment alone. P. histicola alone or in combination with IFNß increased the frequency and number of CD4+FoxP3+ regulatory T cells in the gut-associated lymphoid tissue (GALT). Treatment with P. histicola alone, IFNß alone, and in the combination decreased frequency of pro-inflammatory IFN-γ and IL17-producing CD4+ T cells in the CNS. Additionally, P. histicola alone or IFNß alone or the combination treatments decreased CNS pathology, characterized by reduced microglia and astrocytic activation. In conclusion, our study indicates that the human gut commensal P. histicola can suppress disease as effectively as commonly used MS drug IFNß and may provide an alternative treatment option for MS patients.
Assuntos
Anti-Inflamatórios/farmacologia , Encefalomielite Autoimune Experimental/prevenção & controle , Microbioma Gastrointestinal , Interferon beta/farmacologia , Intestinos/microbiologia , Prevotella/fisiologia , Animais , Astrócitos/efeitos dos fármacos , Astrócitos/imunologia , Astrócitos/metabolismo , Astrócitos/microbiologia , Sistema Nervoso Central/efeitos dos fármacos , Sistema Nervoso Central/imunologia , Sistema Nervoso Central/metabolismo , Sistema Nervoso Central/microbiologia , Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/metabolismo , Encefalomielite Autoimune Experimental/microbiologia , Feminino , Fatores de Transcrição Forkhead/metabolismo , Cadeias beta de HLA-DQ/genética , Cadeias HLA-DRB1/genética , Humanos , Interferon gama/metabolismo , Interleucina-17/metabolismo , Tecido Linfoide/efeitos dos fármacos , Tecido Linfoide/imunologia , Tecido Linfoide/metabolismo , Tecido Linfoide/microbiologia , Masculino , Camundongos Transgênicos , Microglia/efeitos dos fármacos , Microglia/imunologia , Microglia/metabolismo , Microglia/microbiologia , Linfócitos T Reguladores/efeitos dos fármacos , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Linfócitos T Reguladores/microbiologiaRESUMO
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.
Assuntos
Endocanabinoides/uso terapêutico , Derivação Gástrica/métodos , Sistema Nervoso Simpático/fisiologia , Animais , Endocanabinoides/farmacologia , Feminino , Humanos , Masculino , CamundongosRESUMO
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.
Assuntos
Microbioma Gastrointestinal/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Reação em Cadeia da Polimerase/métodos , RNA Ribossômico 16S/genética , Análise de Sequência de RNA/métodos , Animais , Fezes/química , Fezes/microbiologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microbiota/genética , RNA Ribossômico 16S/análiseRESUMO
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.
Assuntos
Encefalomielite Autoimune Experimental/patologia , Locomoção/fisiologia , Esclerose Múltipla Recidivante-Remitente/patologia , Animais , Sistema Nervoso Central/efeitos dos fármacos , Sistema Nervoso Central/patologia , Modelos Animais de Doenças , Encefalomielite Autoimune Experimental/tratamento farmacológico , Feminino , Acetato de Glatiramer/farmacologia , Humanos , Locomoção/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos , Camundongos Transgênicos , Esclerose Múltipla Recidivante-Remitente/tratamento farmacológico , Bainha de Mielina/efeitos dos fármacos , Bainha de Mielina/patologiaRESUMO
Multiple sclerosis (MS) is a demyelinating disease of the central nervous system. We and others have shown that there is enrichment or depletion of some gut bacteria in MS patients compared to healthy controls (HC), suggesting an important role of the gut bacteria in disease pathogenesis. Thus, specific gut bacteria that are lower in abundance in MS patients could be used as a potential treatment option for this disease. In particular, we and others have shown that MS patients have a lower abundance of Prevotella compared to HC, whereas the abundance of Prevotella is increased in patients that receive disease-modifying therapies such as Copaxone® (Glatiramer acetate-GA). This inverse correlation between the severity of MS disease and the abundance of Prevotella suggests its potential for use as a therapeutic option to treat MS. Notably we have previously identified a specific strain, Prevotella histicola (P. histicola), that suppresses disease in the animal model of MS, experimental autoimmune encephalomyelitis (EAE) compared with sham treatment. In the present study we analyzed whether the disease suppressing effects of P. histicola synergize with those of the disease-modifying drug Copaxone® to more effectively suppress disease compared to either treatment alone. Treatment with P. histicola was as effective in suppressing disease as treatment with Copaxone®, whereas the combination of P. histicola plus Copaxone® was not more effective than either individual treatment. P. histicola-treated mice had an increased frequency and number of CD4+FoxP3+ regulatory T cells in periphery as well as gut and a decreased frequency of pro-inflammatory IFN-γ and IL17-producing CD4 T cells in the CNS, suggesting P. histicola suppresses disease by boosting anti-inflammatory immune responses and inhibiting pro-inflammatory immune responses. In conclusion, our study indicates that the human gut commensal P. histicola can suppress disease as efficiently as Copaxone® and may provide an alternative treatment option for MS patients.
Assuntos
Encefalomielite Autoimune Experimental/terapia , Acetato de Glatiramer/uso terapêutico , Imunossupressores/uso terapêutico , Prevotella , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/patologia , Encefalomielite Autoimune Experimental/microbiologia , Encefalomielite Autoimune Experimental/patologia , Microbioma Gastrointestinal , Antígeno HLA-DR3/genética , Humanos , Camundongos Transgênicos , Esclerose Múltipla/microbiologia , Esclerose Múltipla/patologia , Esclerose Múltipla/terapia , Medula Espinal/efeitos dos fármacos , Medula Espinal/patologiaRESUMO
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.
Assuntos
Microbioma Gastrointestinal , Esclerose Múltipla/metabolismo , Esclerose Múltipla/microbiologia , Animais , Autoimunidade , Ácidos e Sais Biliares/metabolismo , Colina/metabolismo , Disbiose/complicações , Ácidos Graxos Voláteis/metabolismo , Humanos , Mucinas/metabolismo , Esclerose Múltipla/imunologia , Fitoestrógenos/metabolismo , Triptofano/metabolismoRESUMO
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.
Assuntos
Bactérias/imunologia , Disbiose/microbiologia , Microbioma Gastrointestinal/imunologia , Homeostase/imunologia , Esclerose Múltipla/microbiologia , Bactérias/classificação , Disbiose/imunologia , Humanos , Esclerose Múltipla/imunologiaRESUMO
The human gut is colonized by a large number of microorganisms (â¼1013 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.