Mononuclear Phagocyte Subsets Research Articles

OMIP-103: A 35-marker imaging mass cytometry panel for the co-detection of HIV and immune cell populations in human formalin fixed paraffin embedded intestinal tissue.

We introduce a 35-marker imaging mass cytometry (IMC) panel for a detailed examination of immune cell populations and HIV RNA in formalin fixed paraffin embedded (FFPE) human intestinal tissue. The panel has broad cell type coverage and particularly excels in delineating subsets of mononuclear phagocytes and T cells. Markers for key tissue structures are included, enabling identification of epithelium, blood vessels, lymphatics, and musculature. The described method for HIV RNA detection can be generalized to other low abundance RNA targets, whether endogenous or pathogen derived. As such, the panel presented here is useful for high parameter spatial mapping of intestinal immune cells and their interactions with pathogens such as HIV.

Characterization of intestinal mononuclear phagocyte subsets in young ruminants at homeostasis and during Cryptosporidium parvum infection.

Cryptosporidiosis is a poorly controlled zoonosis caused by an intestinal parasite, Cryptosporidium parvum, with a high prevalence in livestock (cattle, sheep, and goats). Young animals are particularly susceptible to this infection due to the immaturity of their intestinal immune system. In a neonatal mouse model, we previously demonstrated the importance of the innate immunity and particularly of type 1 conventional dendritic cells (cDC1) among mononuclear phagocytes (MPs) in controlling the acute phase of C. parvum infection. These immune populations are well described in mice and humans, but their fine characterization in the intestine of young ruminants remained to be further explored. Immune cells of the small intestinal Peyer's patches and of the distal jejunum were isolated from naive lambs and calves at different ages. This was followed by their fine characterization by flow cytometry and transcriptomic analyses (q-RT-PCR and single cell RNAseq (lamb cells)). Newborn animals were infected with C. parvum, clinical signs and parasite burden were quantified, and isolated MP cells were characterized by flow cytometry in comparison with age matched control animals. Here, we identified one population of macrophages and three subsets of cDC (cDC1, cDC2, and a minor cDC subset with migratory properties) in the intestine of lamb and calf by phenotypic and targeted gene expression analyses. Unsupervised single-cell transcriptomic analysis confirmed the identification of these four intestinal MP subpopulations in lamb, while highlighting a deeper diversity of cell subsets among monocytic and dendritic cells. We demonstrated a weak proportion of cDC1 in the intestine of highly susceptible newborn lambs together with an increase of these cells within the first days of life and in response to the infection. Considering cDC1 importance for efficient parasite control in the mouse model, one may speculate that the cDC1/cDC2 ratio plays also a key role for the efficient control of C. parvum in young ruminants. In this study, we established the first fine characterization of intestinal MP subsets in young lambs and calves providing new insights for comparative immunology of the intestinal MP system across species and for future investigations on host-Cryptosporidium interactions in target species.

Single-cell and spatial multi-omics highlight effects of anti-integrin therapy across cellular compartments in ulcerative colitis

Ulcerative colitis (UC) is driven by immune and stromal subsets, culminating in epithelial injury. Vedolizumab (VDZ) is an anti-integrin antibody that is effective for treating UC. VDZ is known to inhibit lymphocyte trafficking to the intestine, but its broader effects on other cell subsets are less defined. To identify the inflammatory cells that contribute to colitis and are affected by VDZ, we perform single-cell transcriptomic and proteomic analyses of peripheral blood and colonic biopsies in healthy controls and patients with UC on VDZ or other therapies. Here we show that VDZ treatment is associated with alterations in circulating and tissue mononuclear phagocyte (MNP) subsets, along with modest shifts in lymphocytes. Spatial multi-omics of formalin-fixed biopsies demonstrates trends towards increased abundance and proximity of MNP and fibroblast subsets in active colitis. Spatial transcriptomics of archived specimens pre-treatment identifies epithelial-, MNP-, and fibroblast-enriched genes related to VDZ responsiveness, highlighting important roles for these subsets in UC.

Cyclone: an accessible pipeline to analyze, evaluate, and optimize multiparametric cytometry data.

In the past decade, high-dimensional single-cell technologies have revolutionized basic and translational immunology research and are now a key element of the toolbox used by scientists to study the immune system. However, analysis of the data generated by these approaches often requires clustering algorithms and dimensionality reduction representation, which are computationally intense and difficult to evaluate and optimize. Here, we present Cytometry Clustering Optimization and Evaluation (Cyclone), an analysis pipeline integrating dimensionality reduction, clustering, evaluation, and optimization of clustering resolution, and downstream visualization tools facilitating the analysis of a wide range of cytometry data. We benchmarked and validated Cyclone on mass cytometry (CyTOF), full-spectrum fluorescence-based cytometry, and multiplexed immunofluorescence (IF) in a variety of biological contexts, including infectious diseases and cancer. In each instance, Cyclone not only recapitulates gold standard immune cell identification but also enables the unsupervised identification of lymphocytes and mononuclear phagocyte subsets that are associated with distinct biological features. Altogether, the Cyclone pipeline is a versatile and accessible pipeline for performing, optimizing, and evaluating clustering on a variety of cytometry datasets, which will further power immunology research and provide a scaffold for biological discovery.

Immunoregulatory mechanisms between epithelial clear cells and mononuclear phagocytes in the epididymis.

One of the most intriguing aspects of male reproductive physiology is the ability of the epididymis to prevent the mounting of immune responses against the onslaught of foreign antigens carried by spermatozoa while initiating very efficient immune responses versus stressors. Epithelial clear cells are strategically positioned to work in a concerted manner with region-specific heterogeneous subsets of mononuclear phagocytes to survey the epididymal barrier and regulate the balance between inflammation and immune tolerance in the post-testicular environment. This review aims to describe how clear cells communicate with mononuclear phagocytes to contribute to the unique immune environment in which sperm mature and are stored in the epididymis. A comprehensive systematic review was performed. PubMed was searched for articles specific to clear cells, mononuclear phagocytes, and epididymis. Articles that did not specifically address the target material were excluded. In this review, we discuss the unexpected roles of clear cells, including the transfer of new proteins to spermatozoa via extracellular vesicles and nanotubes as they transit along the epididymal tubule; and we summarize the immune phenotype, morphology, and antigen capturing, processing, and presenting abilities of mononuclear phagocytes. Moreover, we present the current knowledge of immunoregulatory mechanisms by which clear cells and mononuclear phagocytes may contribute to the immune-privileged environment optimal for sperm maturation and storage. Notably, we provide an in-depth characterization of clear cell-mononuclear phagocyte communication networks in the steady-state epididymis and in the presence of injury. This review highlights crucial concepts of mucosal immunology and cellcell interactions, all of which are critical but understudied facets of human male reproductive health.

A novel 3D cell culture model to study the human small intestinal immune landscape.

Several subsets of mononuclear phagocytes and DCs (MDC) populate the small intestine (SI), and these cells reportedly exert specialized functions in anti-microbial immunity and tolerance. Given the specialized phenotype of these cells, differing from other MDC family members, including their putative circulating blood precursors, local intestinal factors play key instructive roles in their differentiation. We designed an SI cell culture model composed of three intestinal epithelial cell (IEC) types, including absorptive enterocytes (E cells), antigen delivering microfold (M) cells, and mucus-producing goblet (G) cells plus T lymphocytes and soluble B cell-derived factors. This model was used to study the differentiation fate of CD34+ hematopoietic progenitor cell-derived monocyte/DC precursors. Progeny cells can be analyzed after a 3-week co-culture period, mimicking the physiologic turn-over time of intestinal MDC. A dominant monocyte differentiation pathway was suppressed, in favor of partial differentiation along DC and macrophage pathways, with low percentages of cells acquired DC or macrophage markers. Moreover, E and G cells play opposing roles in CX3CR1+ vs CD103dim cell differentiation, indicating that both together might counter-balance M/DC differentiation. Thus, SI epithelial cells suppress M/DC differentiation, supporting a key role for exogenous factors in M/DC differentiation.

CX3CR1 deficiency leads to impairment of immune surveillance in the epididymis.

Mononuclear phagocytes (MPs) play an active role in the immunological homeostasis of the urogenital tract. In the epididymis, a finely tuned balance between tolerance to antigenic sperm and immune activation is required to maintain epididymal function while protecting sperm against pathogens and stressors. We previously characterized a subset of resident MPs that express the CX3CR1 receptor, emphasizing their role in antigen sampling and processing during sperm maturation and storage in the murine epididymis. Bacteria-associated epididymitis is the most common cause of intrascrotal inflammation and frequently leads to reproductive complications. Here, we examined whether the lack of functional CX3CR1 in homozygous mice (CX3CR1EGFP/EGFP, KO) alters the ability of MPs to initiate immune responses during epididymitis induced by LPS intravasal-epididymal injection. Confocal microscopy revealed that CX3CR1-deficient MPs located in the initial segments of the epididymis displayed fewer luminal-reaching membrane projections and impaired antigen capture activity. Moreover, flow cytometry showed a reduction of epididymal KO MPs with a monocytic phenotype under physiological conditions. In contrast, flow cytometry revealed an increase in the abundance of MPs with a monocytic signature in the distal epididymal segments after an LPS challenge. This was accompanied by the accumulation of CD103+ cells in the interstitium, and the prevention or attenuation of epithelial damage in the KO epididymis during epididymitis. Additionally, CX3CR1 deletion induced downregulation of Gja1 (connexin 43) expression in KO MPs. Together, our study provides evidence that MPs are gatekeepers of the immunological blood-epididymis barrier and reveal the role of the CX3CR1 receptor in epididymal mucosal homeostasis by inducing MP luminal protrusions and by regulating the monocyte population in the epididymis at steady state as well as upon infection. We also uncover the interaction between MPs and CD103+ dendritic cells, presumably through connexin 43, that enhance immune responses during epididymitis. Our study may lead to new diagnostics and therapies for male infertility and epididymitis by identifying immune mechanisms in the epididymis.

Single-cell transcriptome analyses reveal microglia types associated with proliferative retinopathy.

Pathological angiogenesis is a major cause of irreversible blindness in individuals of all age groups with proliferative retinopathy (PR). Mononuclear phagocytes (MPs) within neovascular areas contribute to aberrant retinal angiogenesis. Due to their cellular heterogeneity, defining the roles of MP subsets in PR onset and progression has been challenging. Here, we aimed to investigate the heterogeneity of microglia associated with neovascularization and to characterize the transcriptional profiles and metabolic pathways of proangiogenic microglia in a mouse model of oxygen-induced PR (OIR). Using transcriptional single-cell sorting, we comprehensively mapped all microglia populations in retinas of room air (RA) and OIR mice. We have unveiled several unique types of PR-associated microglia (PRAM) and identified markers, signaling pathways, and regulons associated with these cells. Among these microglia subpopulations, we found a highly proliferative microglia subset with high self-renewal capacity and a hypermetabolic microglia subset that expresses high levels of activating microglia markers, glycolytic enzymes, and proangiogenic Igf1. IHC staining shows that these PRAM were spatially located within or around neovascular tufts. These unique types of microglia have the potential to promote retinal angiogenesis, which may have important implications for future treatment of PR and other pathological ocular angiogenesis-related diseases.

Identification of CX3CR1+ mononuclear phagocyte subsets involved in HIV-1 and SIV colorectal transmission

SummaryThe difficulty to unambiguously identify the various subsets of mononuclear phagocytes (MNPs) of the intestinal lamina propria has hindered our understanding of the initial events occurring after mucosal exposure to HIV-1.Here, we compared the composition and function of MNP subsets at steady-state and following ex vivo and in vivo viral exposure in human and macaque colorectal tissues.Combined evaluation of CD11c, CD64, CD103, and CX3CR1 expression allowed to differentiate lamina propria MNPs subsets common to both species. Among them, CD11c+ CX3CR1+ cells expressing CCR5 migrated inside the epithelium following ex vivo and in vivo exposure of colonic tissue to HIV-1 or SIV. In addition, the predominant population of CX3CR1high macrophages present at steady-state partially shifted to CX3CR1low macrophages as early as three days following in vivo SIV rectal challenge of macaques.Our analysis identifies CX3CR1+ MNPs as novel players in the early events of HIV-1 and SIV colorectal transmission.

CFTR-mediated monocyte/macrophage dysfunction revealed by cystic fibrosis proband-parent comparisons.

Cystic fibrosis (CF) is an inherited disorder caused by biallelic mutations of the CF transmembrane conductance regulator (CFTR) gene. Converging evidence suggests that CF carriers with only 1 defective CFTR copy are at increased risk for CF-related conditions and pulmonary infections, but the molecular mechanisms underpinning this effect remain unknown. We performed transcriptomic profiling of peripheral blood mononuclear cells (PBMCs) of CF child-parent trios (proband, father, and mother) and healthy control (HC) PBMCs or THP-1 cells incubated with the plasma of these participants. Transcriptomic analyses revealed suppression of cytokine-enriched immune-related genes (IL-1β, CXCL8, CREM), implicating lipopolysaccharide tolerance in innate immune cells (monocytes) of CF probands and their parents. These data suggest that a homozygous as well as a heterozygous CFTR mutation can modulate the immune/inflammatory system. This conclusion is further supported by the finding of lower numbers of circulating monocytes in CF probands and their parents, compared with HCs, and the abundance of mononuclear phagocyte subsets, which correlated with Pseudomonas aeruginosa infection, lung disease severity, and CF progression in the probands. This study provides insight into demonstrated CFTR-related innate immune dysfunction in individuals with CF and carriers of a CFTR mutation that may serve as a target for personalized therapy.

Clinical significance of detection of mononuclear phagocyte subsets in blood and bronchoalveolar lavage fluid (BALF) in pulmonary sarcoidosis.

This study aimed to investigate the clinical significance of the detection of mononuclear phagocytes subsets in pulmonary sarcoidosis blood and bronchoalveolar lavage fluid (BALF). For this purpose, a total of 52 patients with pulmonary sarcoidosis were selected as the study group, 52 healthy people served as the "NC Group a" (peripheral blood mononuclear cell control group), 47 patients with chronic cough and no pulmonary sarcoidosis who underwent bronchoscopy were used as "control group b" (alveolar lavage fluid macrophage control group). Fasting peripheral blood and BALF were collected, and flow cytometry was used to detect monocytes and macrophage subpopulations. The monocytes and macrophage subpopulations of the study group were compared before and after treatment. The results showed that the proportion of CD14++CD16- subgroup of patients with pulmonary sarcoidosis was lower than that of healthy people (74.21±4.10% vs 84.32±4.76%); The proportion of CD14++CD16+ subgroups of patients with pulmonary sarcoidosis was higher than that of healthy people (7.42±4.08% vs 3.95±1.94%); The proportion of CD14+CD16++ subgroups of patients with pulmonary sarcoidosis was higher than that of healthy people in the control group, but the difference was not significant. After 2 months of treatment, the proportion of CD14++CD16- subgroups in peripheral blood mononuclear cells increased, and the proportion of CD14++CD16+ subgroups decreased. The proportion of M1 in patients with pulmonary sarcoidosis was lower than that in patients with non-pulmonary nodules (24.32±11.36% vs 47.03±13.86%); the proportion of M2 in patients with pulmonary sarcoidosis was higher than the proportion of M2 in patients with non-pulmonary nodules (75.40±10.23% vs 52.53±12.01%). After treatment, the proportion of M1 of BALF macrophages in patients with pulmonary sarcoidosis was increased (P<0.05), and the proportion of M2 was reduced (P<0.05). In general, detection of changes in peripheral blood mononuclear cell subpopulations and BALF macrophage subpopulations in patients with pulmonary sarcoidosis has certain clinical significance for the treatment.

Selective depletion of a CD64-expressing phagocyte subset mediates protection against toxic kidney injury and failure

Dendritic cells (DC), macrophages, and monocytes, collectively known as mononuclear phagocytes (MPs), critically control tissue homeostasis and immune defense. However, there is a paucity of models allowing to selectively manipulate subsets of these cells in specific tissues. The steady-state adult kidney contains four MP subsets with Clec9a-expression history that include the main conventional DC1 (cDC1) and cDC2 subtypes as well as two subsets marked by CD64 but varying levels of F4/80. How each of these MP subsets contributes to the different phases of acute kidney injury and repair is unknown. We created a mouse model with a Cre-inducible lox-STOP-lox-diphtheria toxin receptor cassette under control of the endogenous CD64 locus that allows for diphtheria toxin-mediated depletion of CD64-expressing MPs without affecting cDC1, cDC2, or other leukocytes in the kidney. Combined with specific depletion of cDC1 and cDC2, we revisited the role of MPs in cisplatin-induced kidney injury. We found that the intrinsic potency reported for CD11c+ cells to limit cisplatin toxicity is specifically attributed to CD64+ MPs, while cDC1 and cDC2 were dispensable. Thus, we report a mouse model allowing for selective depletion of a specific subset of renal MPs. Our findings in cisplatin-induced injury underscore the value of dissecting the functions of individual MP subsets in kidney disease, which may enable therapeutic targeting of specific immune components in the absence of general immunosuppression.

Optimal Isolation Protocols for Examining and Interrogating Mononuclear Phagocytes From Human Intestinal Tissue.

The human intestine contains numerous mononuclear phagocytes (MNP), including subsets of conventional dendritic cells (cDC), macrophages (Mf) and monocytes, each playing their own unique role within the intestinal immune system and homeostasis. The ability to isolate and interrogate MNPs from fresh human tissue is crucial if we are to understand the role of these cells in homeostasis, disease settings and immunotherapies. However, liberating these cells from tissue is problematic as many of the key surface identification markers they express are susceptible to enzymatic cleavage and they are highly susceptible to cell death. In addition, the extraction process triggers immunological activation/maturation which alters their functional phenotype. Identifying the evolving, complex and highly heterogenous repertoire of MNPs by flow cytometry therefore requires careful selection of digestive enzyme blends that liberate viable cells and preserve recognition epitopes involving careful selection of antibody clones to enable analysis and sorting for functional assays. Here we describe a method for the anatomical separation of mucosa and submucosa as well as isolating lymphoid follicles from human jejunum, ileum and colon. We also describe in detail the optimised enzyme digestion methods needed to acquire functionally immature and biologically functional intestinal MNPs. A comprehensive list of screened antibody clones is also presented which allows for the development of high parameter flow cytometry panels to discriminate all currently identified human tissue MNP subsets including pDCs, cDC1, cDC2 (langerin+ and langerin-), newly described DC3, monocytes, Mf1, Mf2, Mf3 and Mf4. We also present a novel method to account for autofluorescent signal from tissue macrophages. Finally, we demonstrate that these methods can successfully be used to sort functional, immature intestinal DCs that can be used for functional assays such as cytokine production assays.

A flow cytometry approach reveals heterogeneity in conventional subsets of murine renal mononuclear phagocytes

Mononuclear phagocytes (MNPs) participate in inflammation and repair after kidney injury, reflecting their complex nature. Dissection into refined functional subunits has been challenging and would benefit understanding of renal pathologies. Flow cytometric approaches are limited to classifications of either different MNP subsets or functional state. We sought to combine these two dimensions in one protocol that considers functional heterogeneity in each MNP subset. We identified five distinct renal MNP subsets based on a previously described strategy. In vitro polarization of bone marrow-derived macrophages (BMDM) into M1- and M2-like cells suggested functional distinction of CD86 + MHCII + CD206- and CD206 + cells. Combination of both distinction methods identified CD86 + MHCII + CD206- and CD206 + cells in all five MNP subsets, revealing their heterologous nature. Our approach revealed that MNP composition and their functional segmentation varied between different mouse models of kidney injury and, moreover, was dynamically regulated in a time-dependent manner. CD206 + cells from three analyzed MNP subsets had a higher ex vivo phagocytic capacity than CD86 + MHCII + CD206- counterparts, indicating functional uniqueness of each subset. In conclusion, our novel flow cytometric approach refines insights into renal MNP heterogeneity and therefore could benefit mechanistic understanding of renal pathology.

IRF8-Dependent Type I Conventional Dendritic Cells (cDC1s) Control Post-Ischemic Inflammation and Mildly Protect Against Post-Ischemic Acute Kidney Injury and Disease.

Post-ischemic acute kidney injury and disease (AKI/AKD) involve acute tubular necrosis and irreversible nephron loss. Mononuclear phagocytes including conventional dendritic cells (cDCs) are present during different phases of injury and repair, but the functional contribution of this subset remains controversial. Transcription factor interferon regulatory factor 8 (IRF8) is required for the development of type I conventional dendritic cells (cDC1s) lineage and helps to define distinct cDC1 subsets. We identified one distinct subset among mononuclear phagocyte subsets according to the expression patterns of CD11b and CD11c in healthy kidney and lymphoid organs, of which IRF8 was significantly expressed in the CD11blowCD11chigh subset that mainly comprised cDC1s. Next, we applied a Irf8-deficient mouse line (Irf8 fl/fl Clec9a cre mice) to specifically target Clec9a-expressing cDC1s in vivo. During post-ischemic AKI/AKD, these mice lacked cDC1s in the kidney without affecting cDC2s. The absence of cDC1s mildly aggravated the loss of living primary tubule and decline of kidney function, which was associated with decreased anti-inflammatory Tregs-related immune responses, but increased T helper type 1 (TH1)-related and pro-inflammatory cytokines, infiltrating neutrophils and acute tubular cell death, while we also observed a reduced number of cytotoxic CD8+ T cells in the kidney when cDC1s were absent. Together, our data show that IRF8 is indispensable for kidney cDC1s. Kidney cDC1s mildly protect against post-ischemic AKI/AKD, probably via suppressing tissue inflammation and damage, which implies an immunoregulatory role for cDC1s.

FC 042IRF8 IN CDC1 IS PROTECTIVE IN POST-ISCHEMIC ACUTE KIDNEY INJURY

Abstract Background and Aims Post-ischemic acute tubular necrosis is a common cause of acute kidney disease (AKD) and subsequent chronic kidney disease. In AKD, mononuclear phagocytes (MPCs) including conventional dendritic cells (cDCs) are present during the different phases of kidney injury, repair and regeneration. The contribution of cDCs to AKD is still poorly understood. Hence, we hypothesized that transcription factor interferon regulatory factor 8 (IRF8)-specific cDCs regulate the immune response in AKD. Method AKD was induced by unilateral ischemia reperfusion injury in IRF8-deficient Clec9a-specific DCs (IRF8fl/fl Clec9acre/cre or cre/wt) and wild type C57BL/6 mice. Immune phenotyping of leukocytes in kidney and spleen and mRNA expression profiling were performed, as well as kidney function evaluated. For in vitro studies, IRF8 small interfering RNA transfection technology on bone marrow-derived DCs was used. Results In the healthy kidney and lymphoid organ (e.g., spleen and kidney draining lymph node), we identified four MPC subsets according to the diverse expression patterns of CD11b and CD11c. Of which, IRF8 was specifically expressed in the CD11blow CD11chigh R4 subset (containing mainly cDC1). During AKD, IRF8 deficiency in cDCs reduced the number of MHCII+ DCs accumulating among tubulointerstium space without affecting cDC2 or CD64+ DCs, while completely abolished cDC1 in post-ischemic kidney (See Figure). This was accompanied with a decrease in the surface expression of chemokine receptor CCR7 and CCR9, reduction in the number of kidney CD4- CD8+ T cells and Tregs, but a moderate increase in TH1-related and pro-inflammatory cytokines and infiltrating neutrophils in the kidney of mice with IRF8-deficient cDCs. This was in line with reduced kidney function, marked by aggravated GFR loss, elevated plasma BUN level, kidney atrophy, pathological tubular injury and living proximal tubule loss. In vitro, bone marrow-derived IRF8-deficient DCs showed an impaired ability to repair “artificially injured” tubular epithelial cells (TECs), accompanied with less phagocytosis capacity and maturation capacity under necrotic TECs soup or histone stimulation. Conclusion Our data show that the restricted depletion of IRF8 in cDCs reduces the number of infiltrating kidney cDC1, which drives tissue inflammation and damage, and ultimately aggravates post-ischemic AKD. Thus, cDC1s are having a protective role in AKD.

Th17 immunity in the colon is controlled by two novel subsets of colon-specific mononuclear phagocytes

Abstract Intestinal immunity is coordinated by specialized populations of mononuclear phagocytes. Although the cell subsets that constitute these populations are thought uniform throughout the intestine, small and large intestine have distinct anatomy, microbial composition, and immunological demands. Whether these distinctions demand organ-specific mononuclear phagocyte populations with dedicated organ-specific roles in immunity is unknown. Here we implement a new strategy to subset murine intestinal mononuclear phagocytes and identify two novel subsets which are colon-specific: a macrophage subset and a Th17-inducing dendritic cell (DC) subset. Colon-specific DCs and macrophages co-expressed CD24 and CD14, and surprisingly, both were dependent on the transcription factor IRF4. Novel IRF4-dependent CD14+CD24+ macrophages were markedly distinct from conventional macrophages and failed to express classical markers including CX3CR1, CD64 and CD88, and surprisingly expressed little IL-10, which was otherwise robustly expressed by all other intestinal macrophages. We further found that colon-specific CD14+CD24+ mononuclear phagocytes were essential for Th17 immunity in the colon, and provide definitive evidence that colon and small intestine have distinct antigen presenting cell requirements for Th17 immunity. Our findings reveal unappreciated organ-specific diversity of intestine-resident mononuclear phagocytes and organ-specific requirements for Th17 immunity.

Th17 Immunity in the Colon Is Controlled by Two Novel Subsets of Colon-Specific Mononuclear Phagocytes.

Intestinal immunity is coordinated by specialized mononuclear phagocyte populations, constituted by a diversity of cell subsets. Although the cell subsets constituting the mononuclear phagocyte network are thought to be similar in both small and large intestine, these organs have distinct anatomy, microbial composition, and immunological demands. Whether these distinctions demand organ-specific mononuclear phagocyte populations with dedicated organ-specific roles in immunity are unknown. Here we implement a new strategy to subset murine intestinal mononuclear phagocytes and identify two novel subsets which are colon-specific: a macrophage subset and a Th17-inducing dendritic cell (DC) subset. Colon-specific DCs and macrophages co-expressed CD24 and CD14, and surprisingly, both were dependent on the transcription factor IRF4. Novel IRF4-dependent CD14+CD24+ macrophages were markedly distinct from conventional macrophages and failed to express classical markers including CX3CR1, CD64 and CD88, and surprisingly expressed little IL-10, which was otherwise robustly expressed by all other intestinal macrophages. We further found that colon-specific CD14+CD24+ mononuclear phagocytes were essential for Th17 immunity in the colon, and provide definitive evidence that colon and small intestine have distinct antigen presenting cell requirements for Th17 immunity. Our findings reveal unappreciated organ-specific diversity of intestine-resident mononuclear phagocytes and organ-specific requirements for Th17 immunity.

The Prevalence of Gingival Dendritic Cell Subsets in Periodontal Patients

As the most potent cells activating and polarizing naive T cells, dendritic cells (DCs) are of major importance in the induction of immunity and tolerance. DCs are a heterogeneous population of antigen-presenting cells that are widely distributed in lymphoid and nonlymphoid tissues. Murine studies have highlighted the important role of oral DCs and Langerhans cells (LCs) in orchestrating the physiological homeostasis of the oral mucosa. DCs are also critically involved in pathological conditions such as periodontal diseases, in which gingival DCs appear to have special localization and function. While the characterization of human DCs in health and disease has been extensively investigated in various tissues, this topic was rarely studied in human gingiva. Here, we employed an up-to-date approach to characterize by flow cytometry the gingival DCs of 27 healthy subjects and 21 periodontal patients. Four distinct subsets of mononuclear phagocytes were identified in healthy gingiva: conventional DC type 1 (cDC1), cDC2, plasmacytoid DCs (pDCs), and LCs. In periodontitis patients, the frequencies of gingival LCs and pDCs were dysregulated, as LCs decreased, whereas pDCs increased in the diseased gingiva. This shift in the prevalence of DCs was accompanied by increased expression of the proinflammatory cytokines interleukin (IL)–1β, interferon (IFN)–α, and IFN-γ, while the anti-inflammatory cytokine IL-10 was suppressed. We further found that smoking, a known risk factor of periodontitis, specifically reduces gingival LCs in healthy individuals, indicating a possible role of LCs in the elevated severity of periodontitis in smokers. Collectively, this work reveals the various DC subsets residing in the human gingiva and the impact of periodontitis, as well as smoking, on the prevalence of each subset. Our findings provide a foundation toward understanding the role of human DCs in orchestrating physiological oral immunity and set the stage for the evaluation and modulation of shifts in immunity associated with periodontitis.

A NOVEL LANGERIN EXPRESSING TYPE 2-CONVENTIONAL DENDRITIC CELL IS SIGNIFICANTLY DECREASED IN CROHN’S DISEASE

Abstract Crohn’s disease is a chronic relapsing auto-inflammatory condition of the gastrointestinal tract that primarily affects young individuals with an increasing incidence rate. There is still no cure for Crohn’s disease and current treatment-options are limited to controlling inflammatory molecules such as TNF. Unfortunately, these biologics can produce significant side effects and not all patients respond to anti-TNF treatment. New treatments are therefore urgently needed. Targeting the cells and molecules of the immune system still represents the most promising approach which is why we have conducted an in-depth study of the immune cells and molecules associated with a healthy intestinal immune system and compared that to what is happening in Crohn’s disease. Our group has privileged access to large human ileum explants as well as draining lymph node samples isolated from patients undergoing colorectal surgery to remove Crohn’s affected tissue. We have designed and optimised enzymatic tissue digestion protocols to isolate these cells in an immature state with minimal receptor cleavage. We have also developed high-parameter flow and mass cytometry panels to comprehensively identify and characterise all known subsets of mononuclear phagocytes (MNP), innate lymphoid cells, mucosal-associated invariant T cells, natural killer cells and T and B lymphocytes. We have discovered that the newly described Langerin+ type 2-conventional dendritic cell (cDC2) is significantly decreased in Crohn’s affected ileum compared to healthy ileum. Furthermore, we have shown this decrease corresponds with lower TGF-b levels, a known driver of Langerin expression. After 7 days of co-culture, sorted Langerin+ cDC2 induced significantly higher levels of IL-17 and IL-22 in allogenic naïve CD4+ T cells compared to other MNP subsets, including cDC2 which did not express Langerin. These differentiated T cells expressed high levels of RORt and aryl hydrocarbon receptor (AHR) – transcription factors that are associated with CD4+ T helper 17 cells, implying that they may play a crucial role in intestinal barrier repair and regeneration that is absent in Crohn’s disease. Together these results suggest that Langerin+ cDC2 may have an anti-inflammatory role in human tissue and their reduction in Crohn’s disease may contribute to the pathogenesis of this disease, highlighting a potential therapeutic target for Crohn’s disease.