ABSTRACT
The intestine contains some of the most diverse and complex immune compartments in the body. Here we describe a method for isolating human gut-associated lymphoid tissues (GALTs) that allows unprecedented profiling of the adaptive immune system in submucosal and mucosal isolated lymphoid follicles (SM-ILFs and M-ILFs, respectively) as well as in GALT-free intestinal lamina propria (LP). SM-ILF and M-ILF showed distinct patterns of distribution along the length of the intestine, were linked to the systemic circulation through MAdCAM-1+ high endothelial venules and efferent lymphatics, and had immune profiles consistent with immune-inductive sites. IgA sequencing analysis indicated that human ILFs are sites where intestinal adaptive immune responses are initiated in an anatomically restricted manner. Our findings position ILFs as key inductive hubs for regional immunity in the human intestine, and the methods presented will allow future assessment of these compartments in health and disease.
Subject(s)
Adaptive Immunity/immunology , Immunity, Mucosal/immunology , Intestinal Mucosa/immunology , Intestines/immunology , Lymphoid Tissue/immunology , Adaptive Immunity/genetics , Animals , Flow Cytometry , Gastric Mucosa/immunology , Gastric Mucosa/metabolism , Gastric Mucosa/ultrastructure , Humans , Immunity, Mucosal/genetics , Immunoglobulin A/genetics , Immunoglobulin A/immunology , Immunoglobulin M/genetics , Immunoglobulin M/immunology , Intestinal Mucosa/metabolism , Intestinal Mucosa/ultrastructure , Intestines/ultrastructure , Lymphocytes/immunology , Lymphocytes/metabolism , Lymphoid Tissue/metabolism , Lymphoid Tissue/ultrastructure , Microscopy, Confocal , Microscopy, Electron, Scanning , Peyer's Patches/immunology , Peyer's Patches/metabolism , Peyer's Patches/ultrastructure , Sequence Analysis, DNAABSTRACT
Cross-talk between different components of the intestinal barrier and the immune system may be important in maintaining gut homeostasis. A crucial part of the gut barrier is the mucus layer, a cross-linked gel on top of the intestinal epithelium that consists predominantly of the mucin glycoprotein MUC2. However, whether the mucin layer actively regulates intestinal immune cell responses is not clear. Because recent evidence suggests that intestinal dendritic cells (DCs) may be regulated by the mucus layer, we purified intestinal mucin, incubated it with human DCs, and determined the functional effects. Here we show that expression of the chemokine IL-8 and co-stimulatory DC markers CD86 and CD83 are significantly up-regulated on human DCs in the presence of intestinal mucins. Additionally, mucin-exposed DCs promoted neutrophil migration in an IL-8-dependent manner. The stimulatory effects of mucins on DCs were not due to mucin sample contaminants such as lipopolysaccharide, DNA, or contaminant proteins. Instead, mucin glycans are important for the pro-inflammatory effects on DCs. Thus, intestinal mucins are capable of inducing important pro-inflammatory functions in DCs, which could be important in driving inflammatory responses upon intestinal barrier damage.
Subject(s)
Dendritic Cells/metabolism , Interleukin-8/metabolism , Intestinal Mucosa/metabolism , Mucins/pharmacology , Polysaccharides/chemistry , Animals , Cells, Cultured , Dendritic Cells/cytology , Dendritic Cells/drug effects , Dendritic Cells/immunology , Glycosylation , Humans , Intestinal Mucosa/cytology , Intestinal Mucosa/drug effects , Intestinal Mucosa/immunology , Mice , Mice, Inbred C57BL , Polysaccharides/metabolismABSTRACT
Gut-associated lymphoid tissues (GALT) serve as key priming sites for intestinal adaptive immune responses. Most of our understanding of GALT function and development arises from studies in mice. However, the diversity, structure and cellular composition of GALT differs markedly between mammalian species and the developmental window in which distinct GALT structures develop in large mammals remains poorly understood. Given the importance of pigs as models of human disease, as well as their role in livestock production, we adapted a recently developed protocol for the isolation of human GALT to assess the diversity, development and immune composition of large intestinal GALT in neonatal and adult pigs. We demonstrate that the large intestine of adult pigs contains two major GALT types; multifollicular submucosal GALT that we term submucosal lymphoid clusters (SLC) which develop prenatally, and as yet undescribed mucosal isolated lymphoid follicles (M-ILF), which arise after birth. Using confocal laser microscopy and flow cytometry, we additionally assess the microanatomy and lymphocyte composition of SLC and M-ILF, compare them to jejunal Peyer's patches (PP), and describe the maturation of these structures. Collectively, our results provide a deeper understanding of the diversity and development of GALT within the porcine large intestine.
Subject(s)
Immunity, Mucosal , Peyer's Patches , Animals , Intestinal Mucosa , Intestine, Large , Intestines , Lymphoid Tissue , Mammals , Mice , SwineABSTRACT
Gut-associated lymphoid tissues (GALTs) comprise key intestinal immune inductive sites, including the Peyer's patches of the small intestine and different types of isolated lymphoid follicle (ILF) found along the length of the gut. Our understanding of human GALT is limited due to a lack of protocols for their isolation. Here we describe a technique that, uniquely among intestinal cell isolation protocols, allows identification and isolation of all human GALT, as well as GALT-free intestinal lamina propria (LP). The technique involves the mechanical separation of intestinal mucosa from the submucosa, allowing the identification and isolation of submucosal ILF (SM-ILF), LP-embedded mucosal ILF (M-ILF) and LP free of contaminating lymphoid tissue. Individual SM-ILF, M-ILF and Peyer's patch follicles can be subsequently digested for downstream cellular and molecular characterization. The technique, which takes 4-10 h, will be useful for researchers interested in intestinal immune development and function in health and disease.
Subject(s)
Gastrointestinal Tract/physiology , Lymphoid Tissue/physiology , Tissue Culture Techniques/methods , Cell Count , Cell Survival , Colon/physiology , Crohn Disease/pathology , Humans , Immunity, Innate , Intestinal Mucosa/cytology , Leukocyte Common Antigens/metabolismABSTRACT
Gut-associated lymphoid tissues (GALT) are the key antigen sampling and adaptive immune inductive sites within the intestinal wall. Human GALT includes the multi-follicular Peyer's patches of the ileum, the vermiform appendix, and the numerous isolated lymphoid follicles (ILF) which are distributed along the length of the intestine. Our current understanding of GALT diversity and function derives primarily from studies in mice, and the relevance of many of these findings to human GALT remains unclear. Here we review our current understanding of human GALT diversity, structure, and composition as well as their potential for regulating intestinal immune responses during homeostasis and inflammatory bowel disease (IBD). Finally, we outline some key remaining questions regarding human GALT, the answers to which will advance our understanding of intestinal immune responses and provide potential opportunities to improve the treatment of intestinal diseases.
Subject(s)
Intestinal Mucosa/immunology , Intestinal Mucosa/metabolism , Peyer's Patches/physiology , Animals , Biomarkers/metabolism , Disease Susceptibility , Homeostasis , Host-Pathogen Interactions/immunology , Humans , Immunity, Mucosal , Inflammatory Bowel Diseases/diagnosis , Inflammatory Bowel Diseases/etiology , Inflammatory Bowel Diseases/metabolism , Intestinal Mucosa/pathology , Organ Specificity , Peyer's Patches/cytologyABSTRACT
Monocytes are crucial immune cells involved in regulation of inflammation either directly or via differentiation into macrophages in tissues. However, many aspects of how their function is controlled in health and disease are not understood. Here we show that human blood monocytes activate high levels of the cytokine TGFß, a pathway that is not evident in mouse monocytes. Human CD14+, but not CD16+, monocytes activate TGFß via expression of the integrin αvß8 and matrix metalloproteinase 14, which dampens their production of TNFα in response to LPS. Additionally, when monocytes differentiate into macrophages, integrin expression and TGFß-activating ability are maintained in anti-inflammatory macrophages but down-regulated in pro-inflammatory macrophages. In the healthy human intestine, integrin αvß8 is highly expressed on mature tissue macrophages, with these cells and their integrin expression being significantly reduced in active inflammatory bowel disease. Thus, our data suggest that integrin αvß8-mediated TGFß activation plays a key role in regulation of monocyte inflammatory responses and intestinal macrophage homeostasis.
Subject(s)
Immune Tolerance , Inflammatory Bowel Diseases/immunology , Integrins/immunology , Macrophages/immunology , Monocytes/immunology , Transforming Growth Factor beta/immunology , Adolescent , Adult , Aged , Female , Humans , Inflammatory Bowel Diseases/pathology , Intestines/immunology , Intestines/pathology , Macrophages/pathology , Male , Middle Aged , Monocytes/pathology , Tumor Necrosis Factor-alpha/immunologyABSTRACT
Background: Macrophages are pivotal in coordinating a range of important processes in the intestines, including controlling intracellular infections and limiting damaging inflammation against the microbiota. However, it is not clear how gut macrophages, relative to recruited blood monocytes and other myeloid cells, contribute to the intestinal inflammatory milieu, nor how macrophages and their monocyte precursors mediate recruitment of other immune cells to the inflamed intestine. Methods: Myeloid cell populations isolated from colonic inflammatory bowel disease (IBD) or murine dextran sulphate sodium (DSS) induced colitis were assessed using flow cytometry and compared to healthy controls. In addition, mRNA expression profiles in human and murine colon samples, and in macrophages and monocytes from healthy and inflamed murine colons, were analysed by quantitative PCR (qPCR) and mRNA microarray. Results: We show that the monocyte:macrophage balance is disrupted in colon inflammation to favour recruitment of CD14+HLA-DRInt cells in humans, and Ly6CHi monocytes in mice. In addition, we identify that murine blood monocytes receive systemic signals enabling increased release of IL-1ß prior to egress from the blood into the colon. Further, once within the colon and relative to other myeloid cells, monocytes represent the dominant local source of both IL-1ß and TNF. Finally, our data reveal that, independent of inflammation, murine colon macrophages act as a major source of Ccl7 and Ccl8 chemokines that trigger further recruitment of their pro-inflammatory monocyte precursors. Conclusions: Our work suggests that strategies targeting macrophage-mediated monocyte recruitment may represent a promising approach for limiting the chronic inflammation that characterises IBD.
Subject(s)
Colitis/immunology , Colon/immunology , Macrophage Activation/immunology , Macrophages/immunology , Monocytes/immunology , Animals , Chemokine CCL7/immunology , Chemokine CCL8/immunology , Dextran Sulfate/immunology , Female , Humans , Inflammation/immunology , Inflammatory Bowel Diseases/immunology , Interleukin-1beta/immunology , Mice , Mice, Inbred C57BL , Tumor Necrosis Factors/immunologyABSTRACT
Regulation of an immune response requires complex crosstalk between cells of the innate and adaptive immune systems, via both cell-cell contact and secretion of cytokines. An important cytokine with a broad regulatory role in the immune system is transforming growth factor-ß (TGF-ß). TGF-ß is produced by and has effects on many different cells of the immune system, and plays fundamental roles in the regulation of immune responses during homeostasis, infection and disease. Although many cells can produce TGFß, it is always produced as an inactive complex that must be activated to bind to the TGFß receptor complex and promote downstream signalling. Thus, regulation of TGFß activation is a crucial step in controlling TGFß function. This review will discuss how TGFß controls diverse immune responses and how TGFß function is regulated, with a focus on recent work highlighting a critical role for the integrin αvß8 expressed by dendritic cells in activating TGFß.