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Principal Investigator: BRIAN KELSALL
Organization: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
Fiscal Year: 2020
Award: $1,630,774
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; and demonstrated the dual capacity of Ly6Chi blood monocytes to differentiate into either regulatory MP or inflammatory DCs in the colon, and that the balance of these immunologically antagonistic cell types is dictated by micro-environmental conditions.
Furthermore, we evaluated gene regulation in resident and inflammatory colon MPs. We determined that a major, previously unappreciated level of control of inflammatory cytokine production by intestinal MPs is via post-transcriptional mechanisms. From freshly isolated cells levels of mRNA for the pro inflammatory cytokines proIL-1-beta, TNF-alpha, and IL-6, together with the inflammasome NLRP3 were very high, while protein levels were low to non-existent. In contrast, mRNA and protein levels of IL-10, a major suppressive cytokine, were both high. Furthermore, activation of cMPs resulted in low levels of pro inflammatory cytokine production, and poor NLRP3 activation, but high production of 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.
Furthermore, we demonstrated that the polyubiquitin/proteosome pathway is important for the control of both NLRP3 and pro-IL1-beta protein levels in cMPs. This was the first data showing that NLRP3 leaves can be controlled by degradation in a relevant cell type in vivo.
During FY 2020, 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.
We are also currently exploring the role of specific receptors that have known regulatory roles on mononuclear phagocytes, have been identified by GWAS to be susceptibility genes for Crohn's disease or ulcerative colitis, and have been identified on intestinal phagocytes in other studies, on intestinal mononuclear phagocyte function and susceptibility to experimental colitis using cell-specific gene knockout mice and experimental models of inflammatory bowel disease.
Finally, in a separate set of experiments we are exploring 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 are currently exploring the mechanisms by which this enhancement of IgA responses occurs in the presence of this commensal bacteria, and whether it is due to changes in antigen uptake and processing by intestinal myeloid cells.
These studies have implications for understanding the immunopathogenesis of inflammatory bowel disease and immune responses to oral vaccines.
Terms: <(TNF)-α><20S Catalytic Proteasome><20S Core Proteasome><20S Proteasome><20S Proteosome><Antigen Presentation Pathway><Antigen Processing and Presentation><Antigen-Presenting Cells><Antigens><Autoregulation><B cell differentiation factor><B cell stimulating factor 2><B-Cell Differentiation Factor><B-Cell Differentiation Factor-2><B-Cell Stimulatory Factor-2><BCDF><BSF-2><BSF2><Bacteria><Beta Proprotein Interleukin 1><Blood><Blood Reticuloendothelial System><Blood Sample><Blood Vessels><Blood monocyte><Blood specimen><Body Tissues><CSIF><CSIF-10><Cachectin><Cell Body><Cell Differentiation><Cell Differentiation process><Cell Function><Cell Process><Cell physiology><Cells><Cellular Function><Cellular Physiology><Cellular Process><Cholera Enterotoxin CT><Cholera Exotoxin><Cholera Toxin><Choleragen><Colitis><Colon><Complex><Crohn disease><Crohn's><Crohn's disease><Crohn's disorder><Cytokine Synthesis Inhibitory Factor><Data><Defect><Dendritic Cells><Development><Disease><Disorder><Epithelial Cells><Equilibrium><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><Gene Transcription><Generations><Genetic Transcription><Goals><Granulomatous Enteritis><HPGF><Hepatocyte-Stimulating Factor><Heterogeneity><Homeostasis><Hybridoma Growth Factor><IFN-beta 2><IFNB2><IL-1 beta><IL-1 β><IL-1-b><IL-10><IL-1β><IL-6><IL1-Beta><IL1-β><IL10><IL10A><IL1B Protein><IL1F2><IL1β><IL6 Protein><IgA><Immune><Immune response><Immunes><Immunity><Immunochemical Immunologic><Immunofluorescence><Immunofluorescence Immunologic><Immunofluorescence Microscopy><Immunoglobulin A><Immunologic><Immunological><Immunological response><Immunologically><Immunologics><Immunosuppression><Immunosuppression Effect><Immunosuppressive Effect><In Situ><Infection with V cholerae><Infection with V. cholerae><Infection with Vibrio cholerae><Inflammasome><Inflammation><Inflammatory><Inflammatory Bowel Diseases><Inflammatory Bowel Disorder><Inflammatory Intestinal Disease><Inflammatory Intestinal Disorder><Innate Immunity><Interleukin 10 Precursor><Interleukin 1beta><Interleukin-1 beta><Interleukin-10><Interleukin-1β><Interleukin-6><Intestinal><Intestines><Lamina Propria><Lymphoid Follicle><MGI-2><Macropain><Macrophage-Derived TNF><Macroxyproteinase><Marrow monocyte><Messenger RNA><Methods><Mice><Mice Mammals><Modeling><Molecular><Monocyte-Derived TNF><Mononuclear><Mucosa><Mucosal Immune Responses><Mucosal Tissue><Mucous Membrane><Multicatalytic Proteinase><Murine><Mus><Myeloid Cells><Myeloid Differentiation-Inducing Protein><Native Immunity><Natural Immunity><Non-Specific Immunity><Nonspecific Immunity><Organism><Outcome><Pathogenicity><Pathway interactions><Patients><Peyer's Patches><Phagocytes><Phagocytic Cell><Phenotype><Physiologic><Physiological><Physiological Homeostasis><Plasmacytoma Growth Factor><Play><Polyubiquitin><Population><Post-Transcriptional Control><Post-Transcriptional Regulation><Predisposition><Predisposition gene><Preinterleukin 1 Beta><Production><Prosome><Proteasome><Proteasome Endopeptidase Complex><Proteins><Proteosome><RNA Expression><Receptor Protein><Regulation><Regulatory T-Lymphocyte><Reoviridae><Reovirus><Respiratory Enteric Orphan Viruses><Rest><Role><Sampling><Subcellular Process><Surface><Susceptibility><Susceptibility Gene><T cell differentiation><T cell response><TNF><TNF A><TNF Alpha><TNF gene><TNF-α><TNFA><TNFα><Time><Tissues><Transcript Expression Analyses><Transcript Expression Analysis><Transcription><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><accessory cell><acquired immunity><amebocyte><balance><balance function><base><bowel><cell type><cholera infection><commensal bacteria><commensal bacterial species><commensal flora><commensal microbes><commensal microbiota><commensal microflora><cytokine><developmental><effector T cell><eleocolitis><enteral infection><enteric infection><enteric microbial community><enteric microbiota><enteric pathogen infection><enteropathogen infection><enteropathogenic infection><experiment><experimental research><experimental study><flow cytophotometry><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><immunogen><immunoresponse><in vitro Assay><in vivo><infected with cholera><infected with enteropathogen><infection with cholera><interferon beta 2><intestinal flora><intestinal infection><intestinal microbes><intestinal microbiota><intestinal microflora><intestinal tract microflora><intestine infection><knockout gene><living system><lymphatic drainage><mRNA><macrophage><microbial><migration><monocyte><mouse model><multicatalytic endopeptidase complex><murine model><novel><oral vaccine><pathogen><pathway><poly-ubiquitin><population based><post-transcriptional gene regulation><posttranscriptional control><posttranscriptional regulation><predisposing gene><receptor><regional enteritis><regulatory T-cells><repair><repaired><response><social role><susceptibility allele><susceptibility locus><susceptibility variant><transcriptional profiling><uptake><vascular><whole genome association analysis><whole genome association studies><whole genome association study>