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Intestinal intraepithelial lymphocytes (IELs) are located at the critical interface between the intestinal lumen, which is chronically exposed to food and microbes, and the core of the body. Using high-resolution microscopy techniques and intersectional genetic tools, we investigated the nature of IEL responses to luminal microbes. We observed that TCRγδ IELs exhibit unique microbiota-dependent location and movement patterns in the epithelial compartment. This behavioral pattern quickly changes upon exposure to different enteric pathogens, resulting in increased interepithelial cell (EC) scanning, expression of antimicrobial genes, and glycolysis. Both dynamic and metabolic changes to γδ IEL depend on pathogen sensing by ECs. Direct modulation of glycolysis is sufficient to change γδ IEL behavior and susceptibility to early pathogen invasion. Our results uncover a coordinated EC-IEL response to enteric infections that modulates lymphocyte energy utilization and dynamics and supports maintenance of the intestinal epithelial barrier. VIDEO ABSTRACT.
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Intestinos/citologia , Intestinos/imunologia , Infecções por Salmonella/imunologia , Linfócitos T/imunologia , Animais , Células Epiteliais/metabolismo , Vigilância Imunológica , Mucosa Intestinal/imunologia , Camundongos , Infecções por Salmonella/microbiologia , Salmonella typhimurium/fisiologiaRESUMO
Gut bacteria are known to affect immune cell development, but most intestinal lymphocytes have no direct contact with luminal bacteria. Two studies by Atarashi et al. and Sano et al. shed light on how bacterial adhesion can cue intestinal epithelial cells to direct differentiation of gut T cells.
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Aderência Bacteriana , Citrobacter rodentium/fisiologia , Infecções por Enterobacteriaceae/imunologia , Infecções por Escherichia coli/imunologia , Escherichia coli O157/fisiologia , Microbioma Gastrointestinal , Interleucinas/metabolismo , Mucosa Intestinal/imunologia , Intestinos/imunologia , Receptores de Interleucina/metabolismo , Proteína Amiloide A Sérica/metabolismo , Células Th17/imunologia , Animais , HumanosRESUMO
Microbial metabolites can be viewed as the cytokines of the microbiome, transmitting information about the microbial and metabolic environment of the gut to orchestrate and modulate local and systemic immune responses. Still, many immunology studies focus solely on the taxonomy and community structure of the gut microbiota rather than its functions. Early sequencing-based microbiota profiling approaches relied on PCR amplification of small regions of bacterial and fungal genomes to facilitate identification of the microbes present. However, recent microbiome analysis methods, particularly shotgun metagenomic sequencing, now enable culture-independent profiling of microbiome functions and metabolites in addition to taxonomic characterization. In this review, we showcase recent advances in functional metagenomics methods and applications and discuss the current limitations and potential avenues for future development. Importantly, we highlight a few examples of key areas of opportunity in immunology research where integrating functional metagenomic analyses of the microbiome can substantially enhance a mechanistic understanding of microbiome-immune interactions and their contributions to health and disease states.
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Microbiota generates millimolar concentrations of short-chain fatty acids (SCFAs) that can modulate host metabolism, immunity and susceptibility to infection. Butyrate in particular can function as a carbon source and anti-inflammatory metabolite, but the mechanism by which it inhibits pathogen virulence has been elusive. Using chemical proteomics, we found that several virulence factors encoded by Salmonella pathogenicity island-1 (SPI-1) are acylated by SCFAs. Notably, a transcriptional regulator of SPI-1, HilA, was acylated on several key lysine residues. Subsequent incorporation of stable butyryl-lysine analogs using CRISPR-Cas9 gene editing and unnatural amino acid mutagenesis revealed that site-specific modification of HilA impacts its genomic occupancy, expression of SPI-1 genes and attenuates Salmonella enterica serovar Typhimurium invasion of epithelial cells, as well as dissemination in vivo. Moreover, a multiple-site HilA lysine acylation mutant strain of S. Typhimurium was resistant to butyrate inhibition ex vivo and microbiota attenuation in vivo. Our results suggest that prominent microbiota-derived metabolites may directly acylate virulence factors to inhibit microbial pathogenesis in vivo.
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Ácidos Graxos/metabolismo , Regulação Bacteriana da Expressão Gênica , Ilhas Genômicas , Salmonella typhimurium/metabolismo , Virulência , Animais , Arginina/química , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Butiratos/química , Sistemas CRISPR-Cas , Genoma Bacteriano , Genômica , Lisina/química , Camundongos , Camundongos Endogâmicos C57BL , Mutagênese , Mutação , Proteômica/métodos , Salmonella typhimurium/patogenicidade , Transativadores/metabolismo , Fatores de Virulência/metabolismoRESUMO
The intestinal epithelium forms a barrier between the microbiota and the rest of the body. In addition, beyond acting as a physical barrier, the function of intestinal epithelial cells (IECs) in sensing and responding to microbial signals is increasingly appreciated and likely has numerous implications for the vast network of immune cells within and below the intestinal epithelium. IECs also respond to factors produced by immune cells, and these can regulate IEC barrier function, proliferation and differentiation, as well as influence the composition of the microbiota. The mechanisms involved in IEC-microbe-immune interactions, however, are not fully characterized. In this review, we explore the ability of IECs to direct intestinal homeostasis by orchestrating communication between intestinal microbes and mucosal innate and adaptive immune cells during physiological and inflammatory conditions. We focus primarily on the most recent findings and call attention to the numerous remaining unknowns regarding the complex crosstalk between IECs, the microbiota and intestinal immune cells.
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Imunidade nas Mucosas , Inflamação/imunologia , Mucosa Intestinal/imunologia , Microbiota/imunologia , Junções Íntimas/patologia , Imunidade Adaptativa , Animais , Comunicação Celular , Homeostase , Interações Hospedeiro-Patógeno , Humanos , Mucosa Intestinal/microbiologia , Receptores de Reconhecimento de Padrão/metabolismoRESUMO
During primary Ag encounter, T cells receive numerous positive and negative signals that control their proliferation, function, and differentiation, but how these signals are integrated to modulate T cell memory has not been fully characterized. In these studies, we demonstrate that combining seemingly opposite signals, CTLA-4 blockade and rapamycin-mediated mammalian target of rapamycin inhibition, during in vivo T cell priming leads to both an increase in the frequency of memory CD8(+) T cells and improved memory responses to tumors and bacterial challenges. This enhanced efficacy corresponds to increased early expansion and memory precursor differentiation of CD8(+) T cells and increased mitochondrial biogenesis and spare respiratory capacity in memory CD8(+) T cells in mice treated with anti-CTLA-4 and rapamycin during immunization. Collectively, these results reveal that mammalian target of rapamycin inhibition cooperates with rather than antagonizes blockade of CTLA-4, promoting unrestrained effector function and proliferation, and an optimal metabolic program for CD8(+) T cell memory.
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Linfócitos T CD8-Positivos/imunologia , Antígeno CTLA-4/imunologia , Memória Imunológica , Listeriose/imunologia , Linfoma/imunologia , Serina-Treonina Quinases TOR/imunologia , Animais , Anticorpos Monoclonais/farmacologia , Linfócitos T CD8-Positivos/microbiologia , Linfócitos T CD8-Positivos/patologia , Antígeno CTLA-4/antagonistas & inibidores , Antígeno CTLA-4/genética , Diferenciação Celular , Proliferação de Células , Regulação da Expressão Gênica , Listeria monocytogenes/imunologia , Listeriose/genética , Listeriose/microbiologia , Listeriose/patologia , Ativação Linfocitária , Linfoma/genética , Linfoma/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Ovalbumina/genética , Ovalbumina/imunologia , Transdução de Sinais , Sirolimo/farmacologia , Serina-Treonina Quinases TOR/antagonistas & inibidores , Serina-Treonina Quinases TOR/genéticaRESUMO
We report a technique to selectively and continuously label the proteomes of individual cell types in coculture, named cell type-specific labeling using amino acid precursors (CTAP). Through transgenic expression of exogenous amino acid biosynthesis enzymes, vertebrate cells overcome their dependence on supplemented essential amino acids and can be selectively labeled through metabolic incorporation of amino acids produced from heavy isotope-labeled precursors. When testing CTAP in several human and mouse cell lines, we could differentially label the proteomes of distinct cell populations in coculture and determine the relative expression of proteins by quantitative mass spectrometry. In addition, using CTAP we identified the cell of origin of extracellular proteins secreted from cells in coculture. We believe that this method, which allows linking of proteins to their cell source, will be useful in studies of cell-cell communication and potentially for discovery of biomarkers.
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Lisina/metabolismo , Proteoma/biossíntese , Proteômica/métodos , Animais , Sequência de Bases , Linhagem Celular , Técnicas de Cocultura/métodos , Humanos , Marcação por Isótopo/métodos , Camundongos , Dados de Sequência Molecular , Análise de Sequência com Séries de Oligonucleotídeos , Organismos Geneticamente Modificados , Proteoma/genética , RNA Mensageiro/química , RNA Mensageiro/genética , Análise de Sequência de DNA , Espectrometria de Massas em TandemRESUMO
CTLA-4, an Ig superfamily molecule with homology to CD28, is one of the most potent negative regulators of T-cell responses. In vivo blockade of CTLA-4 exacerbates autoimmunity, enhances tumor-specific T-cell responses, and may inhibit the induction of T-cell anergy. Clinical trials of CTLA-4-blocking antibodies to augment T-cell responses to malignant melanoma are at an advanced stage; however, little is known about the effects of CTLA-4 blockade on memory CD8(+) T-cell responses and the formation and maintenance of long-term CD8(+) T-cell memory. In our studies, we show that during in vivo memory CD8(+) T-cell responses to Listeria monocytogenes infection, CTLA-4 blockade enhances bacterial clearance and increases memory CD8(+) T-cell expansion. This is followed by an accumulation of memory cells that are capable of producing the effector cytokines IFN-γ and TNF-α. We also demonstrate that in a vaccination setting, blocking CTLA-4 during CD8(+) T-cell priming leads to increased expansion and maintenance of antigen-specific memory CD8(+) T cells without adversely affecting the overall T-cell repertoire. This leads to an increase in memory cell effector function and improved protective immunity against further bacterial challenges. These results indicate that transient blockade of CTLA-4 enhances memory CD8(+) T-cell responses and support the possible use of CTLA-4-blocking antibodies during vaccination to augment memory formation and maintenance.
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Anticorpos Monoclonais/farmacologia , Antígenos CD/imunologia , Linfócitos T CD8-Positivos/imunologia , Memória Imunológica/imunologia , Listeria monocytogenes/imunologia , Listeriose/imunologia , Transferência Adotiva , Animais , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/microbiologia , Antígeno CTLA-4 , Citocinas/biossíntese , Proteínas de Ligação a DNA/genética , Citometria de Fluxo , Memória Imunológica/efeitos dos fármacos , Camundongos , Camundongos KnockoutRESUMO
BACKGROUND: Pancreatic cancer, specifically pancreatic ductal adenocarcinoma (PDAC), continues to pose a significant clinical and scientific challenge. The most significant finding of recent years is that PDAC tumours harbour their specific microbiome, which differs amongst tumour entities and is distinct from healthy tissue. This review aims to evaluate and summarise all PDAC studies that have used the next-generation technique, 16S rRNA gene amplicon sequencing within each bodily compartment. As well as establishing a causal relationship between PDAC and the microbiome. MATERIALS AND METHODS: This systematic review was carried out according to the Preferred Reporting Items for Systematic Reviews and Meta-analysis (PRISMA) guidelines. A comprehensive search strategy was designed, and 1727 studies were analysed. RESULTS: In total, 38 studies were selected for qualitative analysis and summarised significant PDAC bacterial signatures. Despite the growing amount of data provided, we are not able to state a universal 16S rRNA gene microbial signature that can be used for PDAC screening. This is most certainly due to the heterogeneity of the presentation of results, lack of available datasets and the intrinsic selection bias between studies. CONCLUSION: Several key studies have begun to shed light on causality and the influence the microbiome constituents and their produced metabolites could play in tumorigenesis and influencing outcomes. The challenge in this field is to shape the available microbial data into targetable signatures. Making sequenced data readily available is critical, coupled with the coordinated standardisation of data and the need for consensus guidelines in studies investigating the microbiome in PDAC.
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Human health and disease have increasingly been shown to be impacted by the gut microbiota, and mouse models are essential for investigating these effects. However, the compositions of human and mouse gut microbiotas are distinct, limiting translation of microbiota research between these hosts. To address this, we constructed the Mouse Gastrointestinal Bacteria Catalogue (MGBC), a repository of 26,640 high-quality mouse microbiota-derived bacterial genomes. This catalog enables species-level analyses for mapping functions of interest and identifying functionally equivalent taxa between the microbiotas of humans and mice. We have complemented this with a publicly deposited collection of 223 bacterial isolates, including 62 previously uncultured species, to facilitate experimental investigation of individual commensal bacteria functions in vitro and in vivo. Together, these resources provide the ability to identify and test functionally equivalent members of the host-specific gut microbiotas of humans and mice and support the informed use of mouse models in human microbiota research.
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Bactérias/classificação , Bactérias/isolamento & purificação , Microbioma Gastrointestinal/fisiologia , Animais , Bactérias/genética , Bactérias/metabolismo , Butiratos/metabolismo , Genoma Bacteriano , Humanos , Metagenoma/genética , Camundongos , Modelos AnimaisRESUMO
Experimental mouse models are central to basic biomedical research; however, variability exists across genetically identical mice and mouse facilities making comparisons difficult. Whether specific indigenous gut bacteria drive immunophenotypic variability in mouse models of human disease remains poorly understood. We performed a large-scale experiment using 579 genetically identical laboratory mice from a single animal facility, designed to identify the causes of disease variability in the widely used dextran sulphate sodium mouse model of inflammatory bowel disease. Commonly used treatment endpoint measures-weight loss and intestinal pathology-showed limited correlation and varied across mouse lineages. Analysis of the gut microbiome, coupled with machine learning and targeted anaerobic culturing, identified and isolated two previously undescribed species, Duncaniella muricolitica and Alistipes okayasuensis, and demonstrated that they exert dominant effects in the dextran sulphate sodium model leading to variable treatment endpoint measures. We show that the identified gut microbial species are common, but not ubiquitous, in mouse facilities around the world, and suggest that researchers monitor for these species to provide experimental design opportunities for improved mouse models of human intestinal diseases.
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Colite , Microbioma Gastrointestinal , Doenças Inflamatórias Intestinais , Animais , Bacteroidetes , Colite/induzido quimicamente , Colite/microbiologia , Modelos Animais de Doenças , Doenças Inflamatórias Intestinais/microbiologia , CamundongosRESUMO
We discovered that Enterococcus faecium (E. faecium), a ubiquitous commensal bacterium, and its secreted peptidoglycan hydrolase (SagA) were sufficient to enhance intestinal barrier function and pathogen tolerance, but the precise biochemical mechanism was unknown. Here we show E. faecium has unique peptidoglycan composition and remodeling activity through SagA, which generates smaller muropeptides that more effectively activates nucleotide-binding oligomerization domain-containing protein 2 (NOD2) in mammalian cells. Our structural and biochemical studies show that SagA is a NlpC/p60-endopeptidase that preferentially hydrolyzes crosslinked Lys-type peptidoglycan fragments. SagA secretion and NlpC/p60-endopeptidase activity was required for enhancing probiotic bacteria activity against Clostridium difficile pathogenesis in vivo. Our results demonstrate that the peptidoglycan composition and hydrolase activity of specific microbiota species can activate host immune pathways and enhance tolerance to pathogens.
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Antígenos de Bactérias/imunologia , Antígenos de Bactérias/metabolismo , Enterococcus faecium/enzimologia , Enterococcus faecium/imunologia , N-Acetil-Muramil-L-Alanina Amidase/química , N-Acetil-Muramil-L-Alanina Amidase/metabolismo , Cristalografia por Raios X , Células HEK293 , Humanos , Proteína Adaptadora de Sinalização NOD2/metabolismo , Peptidoglicano/metabolismo , Conformação ProteicaRESUMO
Commensal intestinal bacteria can prevent pathogenic infection; however, limited knowledge of the mechanisms by which individual bacterial species contribute to pathogen resistance has restricted their potential for therapeutic application. Here, we examined how colonization of mice with a human commensal Enterococcus faecium protects against enteric infections. We show that E. faecium improves host intestinal epithelial defense programs to limit Salmonella enterica serotype Typhimurium pathogenesis in vivo in multiple models of susceptibility. E. faecium protection is mediated by a unique peptidoglycan hydrolase, SagA, and requires epithelial expression of pattern recognition receptor components and antimicrobial peptides. Ectopic expression of SagA in non-protective and probiotic bacteria is sufficient to enhance intestinal barrier function and confer resistance against S. Typhimurium and Clostridium difficile pathogenesis. These studies demonstrate that specific factors from commensal bacteria can be used to improve host barrier function and limit the pathogenesis of distinct enteric infections.
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The intestinal microbiome modulates host susceptibility to enteric pathogens, but the specific protective factors and mechanisms of individual bacterial species are not fully characterized. We show that secreted antigen A (SagA) from Enterococcus faecium is sufficient to protect Caenorhabditis elegans against Salmonella pathogenesis by promoting pathogen tolerance. The NlpC/p60 peptidoglycan hydrolase activity of SagA is required and generates muramyl-peptide fragments that are sufficient to protect C. elegans against Salmonella pathogenesis in a tol-1-dependent manner. SagA can also be heterologously expressed and secreted to improve the protective activity of probiotics against Salmonella pathogenesis in C. elegans and mice. Our study highlights how protective intestinal bacteria can modify microbial-associated molecular patterns to enhance pathogen tolerance.
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Antígenos de Bactérias/imunologia , Proteínas de Bactérias/imunologia , Enterococcus faecium/imunologia , Microbioma Gastrointestinal/imunologia , Interações Hospedeiro-Patógeno/imunologia , N-Acetil-Muramil-L-Alanina Amidase/imunologia , Infecções por Salmonella/prevenção & controle , Salmonella typhimurium/imunologia , Animais , Caenorhabditis elegans/imunologia , Caenorhabditis elegans/microbiologia , Proteínas de Caenorhabditis elegans , Enterococcus faecium/enzimologia , Camundongos , Camundongos Endogâmicos C57BL , Modelos Biológicos , Proteínas do Tecido Nervoso , ProbióticosRESUMO
Conventional dendritic cells (cDCs) play an essential role in host immunity by initiating adaptive T cell responses and by serving as innate immune sensors. Although both innate and adaptive functions of cDCs are well documented, their relative importance in maintaining immune homeostasis is poorly understood. To examine the significance of cDC-initiated adaptive immunity in maintaining homeostasis, independent of their innate activities, we generated a cDC-specific Cre mouse and crossed it to a floxed MHC class II (MHCII) mouse. Absence of MHCII on cDCs resulted in chronic intestinal inflammation that was alleviated by antibiotic treatment and entirely averted under germ-free conditions. Uncoupling innate and adaptive functions of cDCs revealed that innate immune functions of cDCs are insufficient to maintain homeostasis and antigen presentation by cDCs is essential for a mutualistic relationship between the host and intestinal bacteria.
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Apresentação de Antígeno , Colite/imunologia , Células Dendríticas/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Imunidade Inata , Animais , Doença Crônica , Colite/genética , Colite/microbiologia , Células Dendríticas/patologia , Antígenos de Histocompatibilidade Classe II/genética , Inflamação/genética , Inflamação/imunologia , Inflamação/microbiologia , Camundongos , Camundongos TransgênicosRESUMO
Regulation of CD8 T cell expansion and contraction is essential for successful immune defense against intracellular pathogens. IL-10 is a regulatory cytokine that can restrict T cell responses by inhibiting APC functions. IL-10, however, can also have direct effects on T cells. Although blockade or genetic deletion of IL-10 enhances T cell-mediated resistance to infections, the extent to which IL-10 limits in vivo APC function or T cell activation/proliferation remains unknown. Herein, we demonstrate that primary and memory CD8 T cell responses following Listeria monocytogenes infection are enhanced by the absence of IL-10. Surface expression of the IL-10R is transiently up-regulated on CD8 T cells following activation, suggesting that activated T cells can respond to IL-10 directly. Consistent with this notion, CD8 T cells lacking IL-10R2 underwent greater expansion than wild-type T cells upon L. monocytogenes infection. The absence of IL-10R2 on APCs, in contrast, did not enhance T cell responses following infection. Our studies demonstrate that IL-10 produced during bacterial infection directly limits expansion of pathogen-specific CD8 T cells and reveal an extrinsic regulatory mechanism that modulates the magnitude of memory T cell responses.