RESUMO
Very low-carbohydrate, high-fat ketogenic diets (KDs) induce a pronounced shift in metabolic fuel utilization that elevates circulating ketone bodies; however, the consequences of these compounds for host-microbiome interactions remain unknown. Here, we show that KDs alter the human and mouse gut microbiota in a manner distinct from high-fat diets (HFDs). Metagenomic and metabolomic analyses of stool samples from an 8-week inpatient study revealed marked shifts in gut microbial community structure and function during the KD. Gradient diet experiments in mice confirmed the unique impact of KDs relative to HFDs with a reproducible depletion of bifidobacteria. In vitro and in vivo experiments showed that ketone bodies selectively inhibited bifidobacterial growth. Finally, mono-colonizations and human microbiome transplantations into germ-free mice revealed that the KD-associated gut microbiota reduces the levels of intestinal pro-inflammatory Th17 cells. Together, these results highlight the importance of trans-kingdom chemical dialogs for mediating the host response to dietary interventions.
Assuntos
Microbioma Gastrointestinal/imunologia , Microbioma Gastrointestinal/fisiologia , Intestinos/imunologia , Intestinos/microbiologia , Células Th17/imunologia , Células Th17/fisiologia , Adolescente , Adulto , Animais , Dieta Hiperlipídica/métodos , Dieta Cetogênica/métodos , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microbiota/imunologia , Microbiota/fisiologia , Pessoa de Meia-Idade , Células Th17/microbiologia , Adulto JovemRESUMO
Emerging evidence suggests that the effect of dietary intake on human health and disease is linked to both the immune system and the microbiota. Yet, we lack an integrated mechanistic model for how these three complex systems relate, limiting our ability to understand and treat chronic and infectious disease. Here, we review recent findings at the interface of microbiology, immunology, and nutrition, with an emphasis on experimentally tractable models and hypothesis-driven mechanistic work. We outline emerging mechanistic concepts and generalizable approaches to bridge the gap between microbial ecology and molecular mechanism. These set the stage for a new era of precision human nutrition informed by a deep and comprehensive knowledge of the diverse cell types in and on the human body.
Assuntos
Dieta , Microbioma Gastrointestinal/fisiologia , Imunomodulação/fisiologia , Humanos , Imunidade/fisiologia , Inflamação/patologiaRESUMO
Human gut bacteria perform diverse metabolic functions with consequences for host health. The prevalent and disease-linked Actinobacterium Eggerthella lenta performs several unusual chemical transformations, but it does not metabolize sugars and its core growth strategy remains unclear. To obtain a comprehensive view of the metabolic network of E. lenta, we generated several complementary resources: defined culture media, metabolomics profiles of strain isolates, and a curated genome-scale metabolic reconstruction. Stable isotope-resolved metabolomics revealed that E. lenta uses acetate as a key carbon source while catabolizing arginine to generate ATP, traits which could be recapitulated in silico by our updated metabolic model. We compared these in vitro findings with metabolite shifts observed in E. lenta-colonized gnotobiotic mice, identifying shared signatures across environments and highlighting catabolism of the host signaling metabolite agmatine as an alternative energy pathway. Together, our results elucidate a distinctive metabolic niche filled by E. lenta in the gut ecosystem. Our culture media formulations, atlas of metabolomics data, and genome-scale metabolic reconstructions form a freely available collection of resources to support further study of the biology of this prevalent gut bacterium.
Assuntos
Actinobacteria , Microbioma Gastrointestinal , Humanos , Camundongos , Animais , Biologia de Sistemas , Ecossistema , Actinobacteria/metabolismoRESUMO
Chronic inflammation is a contributing factor to most life-shortening human diseases. However, the molecular and cellular mechanisms that sustain chronic inflammatory responses remain poorly understood, making it difficult to treat this deleterious condition. Using a mouse model of age-dependent inflammation that results from a deficiency in miR-146a, we demonstrate that miR-155 contributed to the progressive inflammatory disease that emerged as Mir146a(-/-) mice grew older. Upon analyzing lymphocytes from inflamed versus healthy middle-aged mice, we found elevated numbers of T follicular helper (Tfh) cells, germinal center (GC) B cells, and autoantibodies, all occurring in a miR-155-dependent manner. Further, Cd4-cre Mir155(fl/fl) mice were generated and demonstrated that miR-155 functions in T cells, in addition to its established role in B cells, to promote humoral immunity in a variety of contexts. Taken together, our study discovers that miR-146a and miR-155 counterregulate Tfh cell development that drives aberrant GC reactions during chronic inflammation.
Assuntos
Centro Germinativo/imunologia , Inflamação/imunologia , MicroRNAs/imunologia , Linfócitos T Auxiliares-Indutores/imunologia , Transferência Adotiva , Animais , Autoanticorpos/biossíntese , Autoanticorpos/imunologia , Linfócitos B/imunologia , Antígenos CD4/biossíntese , Diferenciação Celular/imunologia , Modelos Animais de Doenças , Antígeno 2 Relacionado a Fos/genética , Centro Germinativo/citologia , Imunidade Humoral , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , MicroRNAs/genética , Interferência de RNA , RNA Interferente PequenoRESUMO
Identifying regulatory mechanisms that influence inflammation in metabolic tissues is critical for developing novel metabolic disease treatments. Here, we investigated the role of microRNA-146a (miR-146a) during diet-induced obesity in mice. miR-146a is reduced in obese and type 2 diabetic patients and our results reveal that miR-146a-/- mice fed a high-fat diet (HFD) have exaggerated weight gain, increased adiposity, hepatosteatosis, and dysregulated blood glucose levels compared to wild-type controls. Pro-inflammatory genes and NF-κB activation increase in miR-146a-/- mice, indicating a role for this miRNA in regulating inflammatory pathways. RNA-sequencing of adipose tissue macrophages demonstrated a role for miR-146a in regulating both inflammation and cellular metabolism, including the mTOR pathway, during obesity. Further, we demonstrate that miR-146a regulates inflammation, cellular respiration and glycolysis in macrophages through a mechanism involving its direct target Traf6. Finally, we found that administration of rapamycin, an inhibitor of mTOR, was able to rescue the obesity phenotype in miR-146a-/- mice. Altogether, our study provides evidence that miR-146a represses inflammation and diet-induced obesity and regulates metabolic processes at the cellular and organismal levels, demonstrating how the combination of diet and miRNA genetics influences obesity and diabetic phenotypes.
Assuntos
Inflamação/prevenção & controle , Doenças Metabólicas/prevenção & controle , MicroRNAs/genética , MicroRNAs/metabolismo , Animais , Glicemia/metabolismo , Dieta Hiperlipídica/efeitos adversos , Modelos Animais de Doenças , Feminino , Expressão Gênica , Humanos , Hiperglicemia/genética , Hiperglicemia/metabolismo , Hiperglicemia/prevenção & controle , Inflamação/genética , Inflamação/metabolismo , Insulina/sangue , Gordura Intra-Abdominal/metabolismo , Gordura Intra-Abdominal/patologia , Macrófagos/metabolismo , Masculino , Doenças Metabólicas/genética , Doenças Metabólicas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , MicroRNAs/antagonistas & inibidores , NF-kappa B/metabolismo , Obesidade/genética , Obesidade/metabolismo , Obesidade/prevenção & controle , Proteínas Proto-Oncogênicas c-akt/genética , Sirolimo/farmacologia , Serina-Treonina Quinases TOR/antagonistas & inibidores , Serina-Treonina Quinases TOR/genética , Aumento de Peso/efeitos dos fármacos , Aumento de Peso/genéticaRESUMO
FLT3-ITD+ acute myeloid leukemia (AML) accounts for â¼25% of all AML cases and is a subtype that carries a poor prognosis. microRNA-155 (miR-155) is specifically overexpressed in FLT3-ITD+ AML compared with FLT3 wild-type (FLT3-WT) AML and is critical for the growth of FLT3-ITD+ AML cells in vitro. However, miR-155's role in regulating FLT3-ITD-mediated disease in vivo remains unclear. In this study, we used a genetic mouse model to determine whether miR-155 influences the development of FLT3-ITD-induced myeloproliferative disease. Results indicate that miR-155 promotes FLT3-ITD-induced myeloid expansion in the bone marrow, spleen, and peripheral blood. Mechanistically, miR-155 increases proliferation of the hematopoietic stem and progenitor cell compartments by reducing the growth-inhibitory effects of the interferon (IFN) response, and this involves targeting of Cebpb. Consistent with our observations in mice, primary FLT3-ITD+ AML clinical samples have significantly higher miR-155 levels and a lower IFN response compared with FLT3-WT AML samples. Further, inhibition of miR-155 in FLT3-ITD+ AML cell lines using CRISPR/Cas9, or primary FLT3-ITD+ AML samples using locked nucleic acid antisense inhibitors, results in an elevated IFN response and reduces colony formation. Altogether, our data reveal that miR-155 collaborates with FLT3-ITD to promote myeloid cell expansion in vivo and that this involves a multitarget mechanism that includes repression of IFN signaling.
Assuntos
Interferons/biossíntese , MicroRNAs/genética , Transtornos Mieloproliferativos/etiologia , Tirosina Quinase 3 Semelhante a fms/genética , Animais , Sistemas CRISPR-Cas , Linhagem Celular Tumoral , Modelos Animais de Doenças , Feminino , Humanos , Leucemia Mieloide Aguda/etiologia , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Mutantes , MicroRNAs/antagonistas & inibidores , Mutação , Células Progenitoras Mieloides/imunologia , Células Progenitoras Mieloides/patologia , Mielopoese/genética , Transtornos Mieloproliferativos/genética , Transtornos Mieloproliferativos/imunologia , Ensaio Tumoral de Célula-TroncoRESUMO
Extracellular vesicles, including exosomes, have recently been implicated as novel mediators of immune cell communication in mammals. However, roles for endogenously produced exosomes in regulating immune cell functions in vivo are just beginning to be identified. In this article, we demonstrate that Rab27a and Rab27b double-knockout (Rab27DKO) mice that are deficient in exosome secretion have a chronic, low-grade inflammatory phenotype characterized by elevated inflammatory cytokines and myeloproliferation. Upon further investigation, we found that some of these phenotypes could be complemented by wild-type (WT) hematopoietic cells or administration of exosomes produced by GM-CSF-expanded bone marrow cells. In addition, chronically inflamed Rab27DKO mice had a blunted response to bacterial LPS, resembling endotoxin tolerance. This defect was rescued by bone marrow exosomes from WT, but not miR-155-/-, cells, suggesting that uptake of miR-155-containing exosomes is important for a proper LPS response. Further, we found that SHIP1 and IRAK-M, direct targets of miR-155 that are known negative regulators of the LPS response, were elevated in Rab27DKO mice and decreased after treatment with WT, but not miR-155-/-, exosomes. Together, our study finds that Rab27-dependent exosome production contributes to homeostasis within the hematopoietic system and appropriate responsiveness to inflammatory stimuli.
Assuntos
Exossomos/metabolismo , Inflamação/imunologia , MicroRNAs/genética , Proteínas rab de Ligação ao GTP/metabolismo , Proteínas rab27 de Ligação ao GTP/metabolismo , Doença Aguda , Animais , Proliferação de Células , Células Cultivadas , Doença Crônica , Citocinas/metabolismo , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Tolerância Imunológica , Mediadores da Inflamação/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células Mieloides/patologia , Proteínas rab de Ligação ao GTP/genética , Proteínas rab27 de Ligação ao GTP/genéticaRESUMO
MicroRNA-155 (miR-155) regulates antitumor immune responses. However, its specific functions within distinct immune cell types have not been delineated in conditional KO mouse models. In this study, we investigated the role of miR-155 specifically within T cells during the immune response to syngeneic tumors. We found that miR-155 expression within T cells is required to limit syngeneic tumor growth and promote IFNγ production by T cells within the tumor microenvironment. Consequently, we found that miR-155 expression by T cells is necessary for proper tumor-associated macrophage expression of IFNγ-inducible genes. We also found that immune checkpoint-blocking (ICB) antibodies against programmed cell death protein 1/programmed death ligand 1 (PD-1/PD-L1) and cytotoxic T lymphocyte-associated protein 4 (CTLA-4) restored antitumor immunity in miR-155 T cell-conditional KO mice. We noted that these ICB antibodies rescued the levels of IFNγ-expressing T cells, expression of multiple activation and effector genes expressed by tumor-infiltrating CD8+ and CD4+ T cells, and tumor-associated macrophage activation. Moreover, the ICB approach partially restored expression of several derepressed miR-155 targets in tumor-infiltrating, miR-155-deficient CD8+ T cells, suggesting that miR-155 and ICB regulate overlapping pathways to promote antitumor immunity. Taken together, our findings highlight the multifaceted role of miR-155 in T cells, in which it promotes antitumor immunity. These results suggest that the augmentation of miR-155 expression could be used to improve anticancer immunotherapies.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Antígeno B7-H1/antagonistas & inibidores , Antígeno CTLA-4/antagonistas & inibidores , Linfócitos do Interstício Tumoral/efeitos dos fármacos , Melanoma/tratamento farmacológico , MicroRNAs/metabolismo , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Animais , Anticorpos Bloqueadores/uso terapêutico , Antineoplásicos Imunológicos/uso terapêutico , Antígeno B7-H1/metabolismo , Antígeno CTLA-4/metabolismo , Linhagem Celular Tumoral , Cruzamentos Genéticos , Vigilância Imunológica/efeitos dos fármacos , Interferon gama/antagonistas & inibidores , Interferon gama/metabolismo , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/metabolismo , Linfócitos do Interstício Tumoral/patologia , Melanoma/imunologia , Melanoma/metabolismo , Melanoma/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , MicroRNAs/genética , Transplante de Neoplasias , Receptor de Morte Celular Programada 1/metabolismo , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Linfócitos T/metabolismo , Linfócitos T/patologia , Carga Tumoral/efeitos dos fármacos , Microambiente Tumoral/efeitos dos fármacosRESUMO
Inflammatory responses are essential for the clearance of pathogens and the repair of injured tissues; however, if these responses are not properly controlled chronic inflammation can occur. Chronic inflammation is now recognized as a contributing factor to many age-associated diseases including metabolic disorders, arthritis, neurodegeneration, and cardiovascular disease. Due to the connection between chronic inflammation and these diseases, it is essential to understand underlying mechanisms behind this process. In this review, factors that contribute to chronic inflammation are discussed. Further, we emphasize the emerging roles of microRNAs (miRNAs) and other noncoding RNAs (ncRNA) in regulating chronic inflammatory states, making them important future diagnostic markers and therapeutic targets.
Assuntos
Inflamação/genética , RNA não Traduzido/metabolismo , Animais , Biomarcadores/metabolismo , Doença Crônica , Humanos , Sistema Imunitário/metabolismo , Inflamação/imunologia , Inflamação/patologia , Inflamação/terapia , MicroRNAs/genética , MicroRNAs/metabolismoRESUMO
Neuroblastoma is a common pediatric cancer, where preclinical studies suggest that a mesenchymal-like gene expression program contributes to chemotherapy resistance. However, clinical outcomes remain poor, implying we need a better understanding of the relationship between patient tumor heterogeneity and preclinical models. Here, we generated single-cell RNA-seq maps of neuroblastoma cell lines, patient-derived xenograft models (PDX), and a genetically engineered mouse model (GEMM). We developed an unsupervised machine learning approach ('automatic consensus nonnegative matrix factorization' (acNMF)) to compare the gene expression programs found in preclinical models to a large cohort of patient tumors. We confirmed a weakly expressed, mesenchymal-like program in otherwise adrenergic cancer cells in some pre-treated high-risk patient tumors, but this appears distinct from the presumptive drug-resistance mesenchymal programs evident in cell lines. Surprisingly however, this weak-mesenchymal-like program was maintained in PDX and could be chemotherapy-induced in our GEMM after only 24 hours, suggesting an uncharacterized therapy-escape mechanism. Collectively, our findings improve the understanding of how neuroblastoma patient tumor heterogeneity is reflected in preclinical models, provides a comprehensive integrated resource, and a generalizable set of computational methodologies for the joint analysis of clinical and pre-clinical single-cell RNA-seq datasets.
RESUMO
BACKGROUND: Neuroblastoma is a common pediatric cancer, where preclinical studies suggest that a mesenchymal-like gene expression program contributes to chemotherapy resistance. However, clinical outcomes remain poor, implying we need a better understanding of the relationship between patient tumor heterogeneity and preclinical models. RESULTS: Here, we generate single-cell RNA-seq maps of neuroblastoma cell lines, patient-derived xenograft models (PDX), and a genetically engineered mouse model (GEMM). We develop an unsupervised machine learning approach ("automatic consensus nonnegative matrix factorization" (acNMF)) to compare the gene expression programs found in preclinical models to a large cohort of patient tumors. We confirm a weakly expressed, mesenchymal-like program in otherwise adrenergic cancer cells in some pre-treated high-risk patient tumors, but this appears distinct from the presumptive drug-resistance mesenchymal programs evident in cell lines. Surprisingly, however, this weak-mesenchymal-like program is maintained in PDX and could be chemotherapy-induced in our GEMM after only 24 h, suggesting an uncharacterized therapy-escape mechanism. CONCLUSIONS: Collectively, our findings improve the understanding of how neuroblastoma patient tumor heterogeneity is reflected in preclinical models, provides a comprehensive integrated resource, and a generalizable set of computational methodologies for the joint analysis of clinical and pre-clinical single-cell RNA-seq datasets.
Assuntos
Neuroblastoma , RNA-Seq , Análise de Célula Única , Neuroblastoma/genética , Neuroblastoma/patologia , Humanos , Animais , Análise de Célula Única/métodos , Camundongos , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Resistencia a Medicamentos Antineoplásicos/genética , Transcriptoma , Análise da Expressão Gênica de Célula ÚnicaRESUMO
Diet can protect from autoimmune disease; however, whether diet acts via the host and/or microbiome remains unclear. Here, we use a ketogenic diet (KD) as a model to dissect these complex interactions. A KD rescued the experimental autoimmune encephalomyelitis (EAE) mouse model of multiple sclerosis in a microbiota-dependent fashion. Dietary supplementation with a single KD-dependent host metabolite (ß-hydroxybutyrate, ßHB) rescued EAE whereas transgenic mice unable to produce ßHB in the intestine developed more severe disease. Transplantation of the ßHB-shaped gut microbiota was protective. Lactobacillus sequence variants were associated with decreased T helper 17 (Th17) cell activation in vitro . Finally, we isolated a L. murinus strain that protected from EAE, which was phenocopied by the Lactobacillus metabolite indole lactic acid. Thus, diet alters the immunomodulatory potential of the gut microbiota by shifting host metabolism, emphasizing the utility of taking a more integrative approach to study diet-host-microbiome interactions.
RESUMO
Eggerthella lenta is a prevalent human gut Actinobacterium implicated in drug, dietary phytochemical, and bile acid metabolism and associated with multiple human diseases. No genetic tools are currently available for the direct manipulation of E. lenta. Here, we construct shuttle vectors and develop methods to transform E. lenta and other Coriobacteriia. With these tools, we characterize endogenous E. lenta constitutive and inducible promoters using a reporter system and construct inducible expression systems, enabling tunable gene regulation. We also achieve genome editing by harnessing an endogenous type I-C CRISPR-Cas system. Using these tools to perform genetic knockout and complementation, we dissect the functions of regulatory proteins and enzymes involved in catechol metabolism, revealing a previously unappreciated family of membrane-spanning LuxR-type transcriptional regulators. Finally, we employ our genetic toolbox to study the effects of E. lenta genes on mammalian host biology. By greatly expanding our ability to study and engineer gut Coriobacteriia, these tools will reveal mechanistic details of host-microbe interactions and provide a roadmap for genetic manipulation of other understudied human gut bacteria.
Assuntos
Actinobacteria , Animais , Humanos , Actinobacteria/metabolismo , Bactérias/metabolismo , Eubacterium/metabolismo , Fatores de Transcrição/metabolismo , Sistemas CRISPR-Cas/genética , Mamíferos/metabolismoRESUMO
Bacterial activation of T helper 17 (Th17) cells exacerbates mouse models of autoimmunity, but how human-associated bacteria impact Th17-driven disease remains elusive. We show that human gut Actinobacterium Eggerthella lenta induces intestinal Th17 activation by lifting inhibition of the Th17 transcription factor Rorγt through cell- and antigen-independent mechanisms. E. lenta is enriched in inflammatory bowel disease (IBD) patients and worsens colitis in a Rorc-dependent manner in mice. Th17 activation varies across E. lenta strains, which is attributable to the cardiac glycoside reductase 2 (Cgr2) enzyme. Cgr2 is sufficient to induce interleukin (IL)-17a, a major Th17 cytokine. cgr2+ E. lenta deplete putative steroidal glycosides in pure culture; related compounds are negatively associated with human IBD severity. Finally, leveraging the sensitivity of Cgr2 to dietary arginine, we prevented E. lenta-induced intestinal inflammation in mice. Together, these results support a role for human gut bacterial metabolism in driving Th17-dependent autoimmunity.
Assuntos
Colite/metabolismo , Microbioma Gastrointestinal/fisiologia , Ativação Linfocitária/fisiologia , Células Th17/metabolismo , Actinobacteria , Animais , Bactérias/metabolismo , Colite/imunologia , Citocinas , Suplementos Nutricionais , Modelos Animais de Doenças , Feminino , Humanos , Doenças Inflamatórias Intestinais/microbiologia , Interleucina-17/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/metabolismoRESUMO
Intercellular communication is critical for homeostasis in mammalian systems, including the gastrointestinal (GI) tract. Exosomes are nanoscale lipid extracellular vesicles that mediate communication between many cell types. Notably, the roles of immune cell exosomes in regulating GI homeostasis and inflammation are largely uncharacterized. By generating mouse strains deficient in cell-specific exosome production, we demonstrate deletion of the small GTPase Rab27A in CD11c+ cells exacerbated murine colitis, which was reversible through administration of DC-derived exosomes. Profiling RNAs within colon exosomes revealed a distinct subset of miRNAs carried by colon- and DC-derived exosomes. Among antiinflammatory exosomal miRNAs, miR-146a was transferred from gut immune cells to myeloid and T cells through a Rab27-dependent mechanism, targeting Traf6, IRAK-1, and NLRP3 in macrophages. Further, we have identified a potentially novel mode of exosome-mediated DC and macrophage crosstalk that is capable of skewing gut macrophages toward an antiinflammatory phenotype. Assessing clinical samples, RAB27A, select miRNAs, and RNA-binding proteins that load exosomal miRNAs were dysregulated in ulcerative colitis patient samples, consistent with our preclinical mouse model findings. Together, our work reveals an exosome-mediated regulatory mechanism underlying gut inflammation and paves the way for potential use of miRNA-containing exosomes as a novel therapeutic for inflammatory bowel disease.
Assuntos
Antígenos CD11 , Colite , Exossomos , Inflamação , Células Mieloides , Animais , Antígenos CD11/genética , Antígenos CD11/imunologia , Colite/genética , Colite/imunologia , Exossomos/genética , Exossomos/imunologia , Inflamação/genética , Inflamação/imunologia , Doenças Inflamatórias Intestinais/imunologia , Intestinos/imunologia , Lipídeos , Mamíferos/genética , Mamíferos/imunologia , Camundongos , MicroRNAs/imunologia , Proteínas Monoméricas de Ligação ao GTP/imunologia , Células Mieloides/imunologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/imunologia , Fator 6 Associado a Receptor de TNF/imunologiaRESUMO
Mechanistic insights into the role of the human microbiome in the predisposition to and treatment of disease are limited by the lack of methods to precisely add or remove microbial strains or genes from complex communities. Here, we demonstrate that engineered bacteriophage M13 can be used to deliver DNA to Escherichia coli within the mouse gastrointestinal (GI) tract. Delivery of a programmable exogenous CRISPR-Cas9 system enables the strain-specific depletion of fluorescently marked isogenic strains during competitive colonization and genomic deletions that encompass the target gene in mice colonized with a single strain. Multiple mechanisms allow E. coli to escape targeting, including loss of the CRISPR array or even the entire CRISPR-Cas9 system. These results provide a robust and experimentally tractable platform for microbiome editing, a foundation for the refinement of this approach to increase targeting efficiency, and a proof of concept for the extension to other phage-bacterial pairs of interest.
Assuntos
Bacteriófago M13/genética , Proteína 9 Associada à CRISPR/genética , Sistemas CRISPR-Cas , Deleção Cromossômica , Cromossomos Bacterianos , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Escherichia coli/genética , Microbioma Gastrointestinal , Edição de Genes , Animais , Proteína 9 Associada à CRISPR/metabolismo , Escherichia coli/crescimento & desenvolvimento , Fezes/microbiologia , Feminino , Regulação Bacteriana da Expressão Gênica , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Estudo de Prova de ConceitoRESUMO
Immunomodulatory drugs can inhibit bacterial growth, yet their mechanism of action, spectrum, and clinical relevance remain unknown. Methotrexate (MTX), a first-line rheumatoid arthritis (RA) treatment, inhibits mammalian dihydrofolate reductase (DHFR), but whether it directly impacts gut bacteria is unclear. We show that MTX broadly alters the human gut microbiota. Drug sensitivity varied across strains, but the mechanism of action against DHFR appears conserved between mammalian and bacterial cells. RA patient microbiotas were sensitive to MTX, and changes in gut bacterial taxa and gene family abundance were distinct between responders and non-responders. Transplantation of post-treatment samples into germ-free mice given an inflammatory trigger led to reduced immune activation relative to pre-treatment controls, enabling identification of MTX-modulated bacterial taxa associated with intestinal and splenic immune cells. Thus, conservation in cellular pathways across domains of life can result in broad off-target drug effects on the human gut microbiota with consequences for immune function.
Assuntos
Bactérias/metabolismo , Microbioma Gastrointestinal/efeitos dos fármacos , Trato Gastrointestinal/imunologia , Metotrexato/metabolismo , Metotrexato/farmacologia , Animais , Artrite Reumatoide/imunologia , Doenças Autoimunes , Bactérias/classificação , Bactérias/genética , Bactérias/isolamento & purificação , Feminino , Microbioma Gastrointestinal/imunologia , Humanos , Metabolômica , Camundongos , Camundongos Endogâmicos C57BL , Filogenia , Purinas/metabolismo , Pirimidinas/metabolismo , RNA Ribossômico 16S/genética , Tetra-Hidrofolato Desidrogenase , TranscriptomaRESUMO
OBJECTIVE: Although oral methotrexate (MTX) remains the anchor drug for rheumatoid arthritis (RA), up to 50% of patients do not achieve a clinically adequate outcome. In addition, there is a lack of prognostic tools for treatment response prior to drug initiation. This study was undertaken to investigate whether interindividual differences in the human gut microbiome can aid in the prediction of MTX efficacy in new-onset RA. METHODS: We performed 16S ribosomal RNA gene and shotgun metagenomic sequencing on the baseline gut microbiomes of drug-naive patients with new-onset RA (n = 26). Results were validated in an additional independent cohort (n = 21). To gain insight into potential microbial mechanisms, we conducted ex vivo experiments coupled with metabolomics analysis to evaluate the association between microbiome-driven MTX depletion and clinical response. RESULTS: Our analysis revealed significant associations of the abundance of gut bacterial taxa and their genes with future clinical response (q < 0.05), including orthologs related to purine and MTX metabolism. Machine learning techniques were applied to the metagenomic data, resulting in a microbiome-based model that predicted lack of response to MTX in an independent group of patients. Finally, MTX levels remaining after ex vivo incubation with distal gut samples from pretreatment RA patients significantly correlated with the magnitude of future clinical response, suggesting a possible direct effect of the gut microbiome on MTX metabolism and treatment outcomes. CONCLUSION: Taken together, these findings are the first step toward predicting lack of response to oral MTX in patients with new-onset RA and support the value of the gut microbiome as a possible prognostic tool and as a potential target in RA therapeutics.
Assuntos
Antirreumáticos/uso terapêutico , Artrite Reumatoide/tratamento farmacológico , Microbioma Gastrointestinal/genética , Metotrexato/uso terapêutico , Administração Oral , Adulto , Antirreumáticos/metabolismo , Artrite Reumatoide/microbiologia , Artrite Reumatoide/fisiopatologia , Bacteroidetes/genética , Bacteroidetes/metabolismo , Clostridiales/genética , Clostridiales/metabolismo , Estudos de Coortes , Escherichia/genética , Escherichia/metabolismo , Euryarchaeota/genética , Euryarchaeota/metabolismo , Feminino , Firmicutes/genética , Firmicutes/metabolismo , Humanos , Aprendizado de Máquina , Masculino , Metabolômica , Metagenômica , Metotrexato/metabolismo , Pessoa de Meia-Idade , Prognóstico , RNA Ribossômico 16S , Shigella/genética , Shigella/metabolismo , Resultado do TratamentoRESUMO
Despite the recognition, nearly a century ago, that the human microbiome plays a clinically relevant role in drug disposition, mechanistic insights, and translational applications are still limited. Here, we highlight the recent re-emergence of "pharmacomicrobiomics," which seeks to understand how inter-individual variations in the microbiome shape drug efficacy and side effect profiles. Multiple bacterial species, genes, and enzymes have already been implicated in the direct biotransformation of drugs, both from targeted case studies and from systematic computational and experimental analyses. Indirect mechanisms are also at play; for example, microbial interactions with the host immune system can have broad effects on immunomodulatory drugs. Finally, we discuss multiple emerging strategies for the precise manipulation of complex microbial communities to improve treatment outcomes. In the coming years, we anticipate a shift toward a more comprehensive view of precision medicine that encompasses our human and microbial genomes and their combined metabolic activities.