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Principal Investigator: BRIAN KELSALL
Organization: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
Fiscal Year: 2023
Award: $1,757,182
Funding agency: National Institute of Allergy and Infectious Diseases
This project focuses on the roles of different populations of dendritic cells (DC) and macrophages (MP) in immune responses in mucosal tissues. While it is clear that the normal outcome of mucosal antigen exposure can be positive, i.e., the development of intestinal IgA and effector T cell responses, and in some cases the induction of systemic immunity; and/or largely regulatory, i.e., the induction of mucosal tolerance, the details of why one or the other outcome occurs is complex and still poorly understood. Furthermore, the normal mucosal immune response to symbiotic/commensal bacteria, which allows for one to tolerate these organisms without the onset of inflammation, is essential for immune homeostasis, as a defect in this homeostasis results in inflammatory bowel disease (IBD), such as Crohn's disease and ulcerative colitis. Therefore, this project focuses on how immune responses are regulated in mucosal tissues with a focus on the roles of DCs and MPs in this regulation, and on factors that control inflammatory functions of these cells.
In prior studies, we defined antigen-presenting cell populations in the Peyer's patches (PP), and detailed their surface phenotype, function, and migration using in situ immunofluorescence microscopy and mRNA hybridization, flow cytometry, and in vitro assays of cytokine production and T cell differentiation. Furthermore, we delineated for the first-time precise definitions of MPs and DCs in the colon lamina propria (LP) and isolated lymphoid follicles based on the use of a comprehensive array of surface markers, gene expression analysis, and development from defined circulating precursors.
Furthermore, we evaluated gene regulation in resident and inflammatory colon MPs and determined that a major, previously unappreciated level of control of inflammatory cytokine production by intestinal MPs is via post-transcriptional mechanisms, which differentially affect the production of inflammatory cytokines, such as TNFalpha, IL1beta, and IL6, as well as the expression of the inflammasome NLRP3, and regulatory cytokines, such as IL-10. This distinct post-transcriptional regulation of IL-10 and pro-inflammatory cytokines was present in resting and activated cMPs in the steady-state, but lost during experimental colitis, indicating that environmental conditions present in the intestinal LP influence cMPs directly or their differentiation from blood monocytes to influence post-transcriptional gene regulation. Given that the production these pro inflammatory cytokines is essential for tissue inflammation in patients with IBD, these results suggested that the control of cytokines by post-transcriptional mechanisms is essential for controlling susceptibility to IBD.
In addition, we completed studies of single cell mRNA analysis of intestinal myeloid cells in mice and determined a new level of heterogeneity amongst DC and monocyte/MP populations. Thus, we defined 6 populations of monocyte/macrophages and 5 populations of DCs in normal mouse colon. Unique gene expression by these populations allowed for developmental trajectory analysis resulting in the identification of two unique developmental pathways for the development of macrophages from monocyte precursors. These two unique populations were further characterized for unique surface markers, and localized in tissues by immunofluorescence. We found that these two discrete developmental pathways resulted in cells that are either near the lumen of the intestine and thus more exposed to epithelial cell and commensal microbial products, or near the base of the lamina propria near blood vessels and lymphatic drainage. We further identified the ability of these two macrophage populations to differentially sample antigens from the blood, indicating a novel function of macrophages in the intestine, to sample blood derived products. We have further generated novel hypotheses for the functions of these discrete macrophage populations based on their expressions of specific proteins that have unique functions. Finally, we showed that the intestinal microbiota are essential for the differentiation of several but not all macrophage populations, and for not for DC differentiation. We have now made progress in identifying unique markers for both macrophage and DC populations that will allow us to further localize these cells and allow for their isolation to help understand thrive functions.
During the current FY 2023, using cell-specific gene knockout mice and experimental models of inflammatory bowel disease we have continued to explore the role of specific receptors that 1) have known regulatory roles on mononuclear phagocytes, 2) have been identified by GWAS to be susceptibility genes for Crohn's disease or ulcerative colitis, 3) have been identified on intestinal phagocytes in other studies, and 4) that regulate intestinal mononuclear phagocyte function and susceptibility to experimental colitis. We evaluated cell-specific deficiency in EP4, a receptor for prostaglandin E2, in two models of inflammatory bowel disease, the DSS colitis model and the T cell transfer colitis model. We found that in both models EP4 deficiency in CX3CR1+ cells results in more severe disease, and in the former has major early effects on the intestinal barrier. We are currently exploring the mechanisms of this enhanced disease.
In addition, in a separate set of experiments we explored the role of specific commensal bacteria in the generation of IgA responses to model intestinal vaccines. We found that the presence of a particular bacteria, segmented filamentous bacteria (SFB) is able to enhance IgA responses to vaccination with cholera toxin or infection with reovirus, a model intestinal infection in mice. We used single cell mRNA analysis combined with analysis of cell populations using flow-cytometry, and both mRNA and protein expression by isolated cells identified in scRNAseq studies. We found an increase in the proportions of certain conventional dendritic cell (cDC) populations that influence early IgA B cell class switching in the subepithelial dome region of the PP, and a separate role for IL-6 in the LP correlated with the enhanced IgA production with SFB colonization. We are currently further exploring the mechanisms by which this enhancement of the IgA response occurs in the presence of this commensal bacteria, and whether it is due to changes in antigen uptake and processing by intestinal myeloid cells.
Furthermore, we identified and sequenced a commensal fungal organism in the stomach of mice that influences protection against systemic Candida albicans infection. We are currently studying the mechanisms of protection.
Finally, we have characterized the cDC populations in the the colon of mice by scRNAseq analysis, and identified unique populations that appear to represent key developmental intermediates for cDC1 and cDC2 populations, as well as potential receptors for environmental factors driving their differentiation. We are testing these hypotheses using mouse models of infections that drive cDC1 or cDC2 differentiation in wild-type mice, and mice deficient in candidate developmental factors.
These studies have implications for understanding the immunopathogenesis of inflammatory bowel disease and immune responses to oral vaccines.
Terms: <(TNF)-α><Affect><Antigen Presentation Pathway><Antigen Processing and Presentation><Antigen-Presenting Cells><Antigens><Automobile Driving><Autoregulation><B blood cells><B cell><B cell differentiation factor><B cell stimulating factor 2><B cells><B-Cell Differentiation Factor><B-Cell Differentiation Factor Gene><B-Cell Differentiation Factor-2><B-Cell Stimulatory Factor 2 Gene><B-Cell Stimulatory Factor-2><B-Cells><B-Lymphocytes><B-cell><BCDF><BSF-2><BSF-2 Gene><BSF2><BSF2 Gene><Bacteria><Beta-2 Gene Interferon><Blood><Blood Reticuloendothelial System><Blood Sample><Blood Vessels><Blood monocyte><Blood specimen><Body Tissues><C albicans><C. albicans><C.albicans><CSIF><CSIF-10><Cachectin><Candida albicans><Cell Body><Cell Differentiation><Cell Differentiation process><Cell Function><Cell Isolation><Cell Process><Cell Segregation><Cell Separation><Cell Separation Technology><Cell physiology><Cells><Cellular Function><Cellular Physiology><Cellular Process><Cholera Enterotoxin CT><Cholera Exotoxin><Cholera Toxin><Choleragen><Class Switching><Class Switchings><Colitis><Colon><Complex><Crohn disease><Crohn's><Crohn's disease><Crohn's disorder><Cytokine Synthesis Inhibitory Factor><DSS colitis><DSS model><DSS mouse model><DSS-induced acute colitis><DSS-induced colitis><Defect><Dendritic Cells><Development><Dinoprostone><Disease><Disorder><Disseminated candidiasis><Disseminated candidosis><EP4><Environmental Factor><Environmental Risk Factor><Epididymal Secretory Protein E4><Epithelial Cells><Experimental Models><Exposure to><Flow Cytofluorometries><Flow Cytofluorometry><Flow Cytometry><Flow Microfluorimetry><Flow Microfluorometry><GI microbiota><GWA study><GWAS><Gastrointestinal microbiota><Gene Action Regulation><Gene Expression><Gene Expression Monitoring><Gene Expression Pattern Analysis><Gene Expression Profiling><Gene Expression Regulation><Gene Regulation><Gene Regulation Process><Generations><Goals><Granulomatous Enteritis><HE4><HPGF><HSF Gene><Hepatocyte Stimulatory Factor Gene><Hepatocyte-Stimulating Factor><Heterogeneity><Homeostasis><Hybridoma Growth Factor><Hybridoma Growth Factor Gene><IFN-beta 2><IFNB2><IFNB2 Gene><IL-10><IL-6><IL-6 Gene><IL10><IL10A><IL6><IL6 Protein><IL6 gene><IgA><Immune><Immune response><Immunes><Immunity><Immunofluorescence><Immunofluorescence Immunologic><Immunofluorescence Microscopy><Immunoglobulin A><Immunoglobulin Class Switching><Immunoglobulin Class Switchings><Immunological response><Immunosuppression><Immunosuppression Effect><Immunosuppressive Effect><In Situ><Infection><Infection with V cholerae><Infection with V. cholerae><Infection with Vibrio cholerae><Inflammasome><Inflammation><Inflammatory><Inflammatory Bowel Diseases><Inflammatory Bowel Disorder><Innate Immunity><Interleukin 10 Precursor><Interleukin 6 (Interferon, Beta 2) Gene><Interleukin-10><Interleukin-6><Interleukin-6 Gene><Intestinal><Intestines><Isotype Switching><Isotype Switchings><KO mice><Knock-out Mice><Knockout Mice><Lamina Propria><Lymphoid Follicle><MGI-2><Macrophage><Macrophage-Derived TNF><Major Epididymis-Specific Protein E4><Marrow monocyte><Messenger RNA><Methods><Mice><Mice Mammals><Modeling><Molecular><Monocyte-Derived TNF><Mononuclear><Mucosa><Mucosal Immune Responses><Mucosal Tissue><Mucous Membrane><Murine><Mus><Myeloid Cells><Myeloid Differentiation-Inducing Protein><Mφ><Native Immunity><Natural Immunity><Non-Specific Immunity><Nonspecific Immunity><Null Mouse><Organism><Outcome><PGE2><PGE2 alpha><PGE2alpha><Pathogenicity><Pathway interactions><Patients><Peyer's Patches><Phagocytes><Phagocytic Cell><Phenotype><Physiologic><Physiological><Physiological Homeostasis><Plasmacytoma Growth Factor><Play><Population><Post-Transcriptional Control><Post-Transcriptional Regulation><Predisposition><Predisposition gene><Production><Prostaglandin E2><Prostaglandin E2 alpha><Prostaglandin E2alpha><Prostaglandin Receptor><Proteins><Putative Protease Inhibitor WAP5><Receptor Protein><Regulation><Regulatory T-Lymphocyte><Reovirus><Respiratory Enteric Orphan Viruses><Rest><Role><Sampling><Stomach><Subcellular Process><Surface><Susceptibility><Susceptibility Gene><Systemic candida><Systemic candida infections><Systemic candidiasis><T cell differentiation><T cell response><T-Cells><T-Lymphocyte><TNF><TNF A><TNF Alpha><TNF gene><TNF-α><TNFA><TNFα><Teff cell><Testing><Time><Tissues><Transcript Expression Analyses><Transcript Expression Analysis><Treg><Tumor Necrosis Factor><Tumor Necrosis Factor-alpha><Ulcerated Colitis><Ulcerative Colitis><V cholerae infection><V. cholerae infection><Vaccination><Vaccines><Veiled Cells><Vibrio cholerae infection><WAP Four-Disulfide Core Domain Protein 2><WAP5><WFDC2><WFDC2 gene><Wild Type Mouse><accessory cell><acquired immunity><amebocyte><analyze gene expression><base><bases><bowel><cell sorting><cholera infection><colitis-induced dysbiosis><commensal bacteria><commensal bacterial species><commensal flora><commensal microbes><commensal microbiota><commensal microflora><cytokine><dJ461P17.6><developmental><dextran sulfate sodium colitis><dextran sulfate sodium induced colitis><dextran sulfate sodium model><dextran sulfate sodium mouse model><driving><effector T cell><eleocolitis><enteral infection><enteric infection><enteric microbial community><enteric microbiota><enteric pathogen infection><enteropathogen infection><enteropathogenic infection><environmental risk><experiment><experimental research><experimental study><experiments><flow cytophotometry><gastric><gastrointestinal microbial flora><gene expression analysis><gene expression assay><genome wide association><genome wide association scan><genome wide association studies><genome wide association study><genomewide association scan><genomewide association studies><genomewide association study><gut commensal><gut community><gut flora><gut microbe community><gut microbial community><gut microbial composition><gut microbial consortia><gut microbiota><gut microbiotic><gut microflora><host response><immune suppression><immune suppressive activity><immune suppressive function><immune system response><immunogen><immunoresponse><immunosuppressive activity><immunosuppressive function><immunosuppressive response><in vitro Assay><infected with cholera><infected with enteropathogen><infection with cholera><inflammatory disease of the intestine><inflammatory disorder of the intestine><interferon beta 2><intestinal autoinflammation><intestinal barrier><intestinal flora><intestinal infection><intestinal microbes><intestinal microbiota><intestinal microflora><intestinal mucosal barrier><intestinal tract microflora><intestine infection><knockout gene><living system><lymphatic drainage><mRNA><mRNA Expression><microbial products><migration><monocyte><mouse model><murine model><novel><oral vaccine><pathogen><pathway><population based><post-transcriptional gene regulation><posttranscriptional><posttranscriptional control><posttranscriptional regulation><predisposing gene><protein expression><receptor><regional enteritis><regulatory T-cells><repair><repaired><response><social role><susceptibility allele><susceptibility locus><susceptibility variant><thymus derived lymphocyte><transcriptional profiling><uptake><vascular><whole genome association analysis><whole genome association studies><whole genome association study><wildtype mouse>