Genomic deletion of Bcl6 differentially affects conventional dendritic cell subsets and compromises Tfh/Tfr/Th17 cell responses.

Conventional dendritic cells (cDC) play key roles in immune induction, but what drives their heterogeneity and functional specialization is still ill-defined. Here we show that cDC-specific deletion of the transcriptional repressor Bcl6 in mice alters the phenotype and transcriptome of cDC1 and cDC2, while their lineage identity is preserved. Bcl6-deficient cDC1 are diminished in the periphery but maintain their ability to cross-present antigen to CD8+ T cells, confirming general maintenance of this subset. Surprisingly, the absence of Bcl6 in cDC causes a complete loss of Notch2-dependent cDC2 in the spleen and intestinal lamina propria. DC-targeted Bcl6-deficient mice induced fewer T follicular helper cells despite a profound impact on T follicular regulatory cells in response to immunization and mounted diminished Th17 immunity to Citrobacter rodentium in the colon. Our findings establish Bcl6 as an essential transcription factor for subsets of cDC and add to our understanding of the transcriptional landscape underlying cDC heterogeneity.

Interleukin-10 enhances recruitment of immune cells in the neonatal mouse model of obstructive nephropathy.

Urinary tract obstruction during renal development leads to inflammation, leukocyte infiltration, tubular cell death, and interstitial fibrosis. Interleukin-10 (IL-10) is an anti-inflammatory cytokine, produced mainly by monocytes/macrophages and regulatory T-cells. IL-10 inhibits innate and adaptive immune responses. IL-10 has a protective role in the adult model of obstructive uropathy. However, its role in neonatal obstructive uropathy is still unclear which led us to study the role of IL-10 in neonatal mice with unilateral ureteral obstruction (UUO). UUO serves as a model for congenital obstructive nephropathies, a leading cause of kidney failure in children. Newborn Il-10-/- and C57BL/6 wildtype-mice (WT) were subjected to complete UUO or sham-operation on the 2nd day of life. Neonatal kidneys were harvested at day 3, 7, and 14 of life and analyzed for different leukocyte subpopulations by FACS, for cytokines and chemokines by Luminex assay and ELISA, and for inflammation, programmed cell death, and fibrosis by immunohistochemistry and western blot. Compared to WT mice, Il-10-/- mice showed reduced infiltration of neutrophils, CD11bhi cells, conventional type 1 dendritic cells, and T-cells following UUO. Il-10-/- mice with UUO also showed a reduction in pro-inflammatory cytokine and chemokine release compared to WT with UUO, mainly of IP-10, IL-1α, MIP-2α and IL-17A. In addition, Il-10-/- mice showed less necroptosis after UUO while the rate of apoptosis was not different. Finally, α-SMA and collagen abundance as readout for fibrosis were similar in Il-10-/- and WT with UUO. Surprisingly and in contrast to adult Il-10-/- mice undergoing UUO, neonatal Il-10-/- mice with UUO showed a reduced inflammatory response compared to respective WT control mice with UUO. Notably, long term changes such as renal fibrosis were not different between neonatal Il-10-/- and neonatal WT mice with UUO suggesting that IL-10 signaling is different in neonates and adults with UUO.

Macrophages and fibrosis: how resident and infiltrating mononuclear phagocytes account for organ injury, regeneration or atrophy.

Mononuclear phagocytes (MP), i.e., monocytes, macrophages, and dendritic cells (DCs), are essential for immune homeostasis via their capacities to clear pathogens, pathogen components, and non-infectious particles. However, tissue injury-related changes in local microenvironments activate resident and infiltrating MP towards pro-inflammatory phenotypes that contribute to inflammation by secreting additional inflammatory mediators. Efficient control of injurious factors leads to a switch of MP phenotype, which changes the microenvironment towards the resolution of inflammation. In the same way, MP endorses adaptive structural responses leading to either compensatory hypertrophy of surviving cells, tissue regeneration from local tissue progenitor cells, or tissue fibrosis and atrophy. Under certain circumstances, MP contribute to the reversal of tissue fibrosis by clearance of the extracellular matrix. Here we give an update on the tissue microenvironment-related factors that, upon tissue injury, instruct resident and infiltrating MP how to support host defense and recover tissue function and integrity. We propose that MP are not intrinsically active drivers of organ injury and dysfunction but dynamic amplifiers (and biomarkers) of specific tissue microenvironments that vary across spatial and temporal contexts. Therefore, MP receptors are frequently redundant and suboptimal targets for specific therapeutic interventions compared to molecular targets upstream in adaptive humoral or cellular stress response pathways that influence tissue milieus at a contextual level.

Tumor-derived prostaglandin E2 programs cDC1 dysfunction to impair intratumoral orchestration of anti-cancer T cell responses.

Type 1 conventional dendritic cells (cDC1s) are critical for anti-cancer immunity. Protective anti-cancer immunity is thought to require cDC1s to sustain T cell responses within tumors, but it is poorly understood how this function is regulated and whether its subversion contributes to immune evasion. Here, we show that tumor-derived prostaglandin E2 (PGE2) programmed a dysfunctional state in intratumoral cDC1s, disabling their ability to locally orchestrate anti-cancer CD8+ T cell responses. Mechanistically, cAMP signaling downstream of the PGE2-receptors EP2 and EP4 was responsible for the programming of cDC1 dysfunction, which depended on the loss of the transcription factor IRF8. Blockade of the PGE2-EP2/EP4-cDC1 axis prevented cDC1 dysfunction in tumors, locally reinvigorated anti-cancer CD8+ T cell responses, and achieved cancer immune control. In human cDC1s, PGE2-induced dysfunction is conserved and associated with poor cancer patient prognosis. Our findings reveal a cDC1-dependent intratumoral checkpoint for anti-cancer immunity that is targeted by PGE2 for immune evasion.

Tuning sentinel immune cells.

Neuroimmune interactions in the skin can shape the functions of dendritic cells.

Guidelines for visualization and analysis of DC in tissues using multiparameter fluorescence microscopy imaging methods.

This article is part of the Dendritic Cell Guidelines article series, which provides a collection of state-of-the-art protocols for the preparation, phenotype analysis by flow cytometry, generation, fluorescence microscopy, and functional characterization of mouse and human dendritic cells (DC) from lymphoid organs and various non-lymphoid tissues. Here, we provide detailed procedures for a variety of multiparameter fluorescence microscopy imaging methods to explore the spatial organization of DC in tissues and to dissect how DC migrate, communicate, and mediate their multiple functional roles in immunity in a variety of tissue settings. The protocols presented here entail approaches to study DC dynamics and T cell cross-talk by intravital microscopy, large-scale visualization, identification, and quantitative analysis of DC subsets and their functions by multiparameter fluorescence microscopy of fixed tissue sections, and an approach to study DC interactions with tissue cells in a 3D cell culture model. While all protocols were written by experienced scientists who routinely use them in their work, this article was also peer-reviewed by leading experts and approved by all co-authors, making it an essential resource for basic and clinical DC immunologists.

Guidelines for DC preparation and flow cytometry analysis of mouse nonlymphoid tissues.

This article is part of the Dendritic Cell Guidelines article series, which provides a collection of state-of-the-art protocols for the preparation, phenotype analysis by flow cytometry, generation, fluorescence microscopy and functional characterization of mouse and human dendritic cells (DC) from lymphoid organs and various nonlymphoid tissues. DC are sentinels of the immune system present in almost every mammalian organ. Since they represent a rare cell population, DC need to be extracted from organs with protocols that are specifically developed for each tissue. This article provides detailed protocols for the preparation of single-cell suspensions from various mouse nonlymphoid tissues, including skin, intestine, lung, kidney, mammary glands, oral mucosa and transplantable tumors. Furthermore, our guidelines include comprehensive protocols for multiplex flow cytometry analysis of DC subsets and feature top tricks for their proper discrimination from other myeloid cells. With this collection, we provide guidelines for in-depth analysis of DC subsets that will advance our understanding of their respective roles in healthy and diseased tissues. While all protocols were written by experienced scientists who routinely use them in their work, this article was also peer-reviewed by leading experts and approved by all coauthors, making it an essential resource for basic and clinical DC immunologists.

Ly6D+Siglec-H+ precursors contribute to conventional dendritic cells via a Zbtb46+Ly6D+ intermediary stage.

Plasmacytoid and conventional dendritic cells (pDC and cDC) are generated from progenitor cells in the bone marrow and commitment to pDCs or cDC subtypes may occur in earlier and later progenitor stages. Cells within the CD11c+MHCII-/loSiglec-H+CCR9lo DC precursor fraction of the mouse bone marrow generate both pDCs and cDCs. Here we investigate the heterogeneity and commitment of subsets in this compartment by single-cell transcriptomics and high-dimensional flow cytometry combined with cell fate analysis: Within the CD11c+MHCII-/loSiglec-H+CCR9lo DC precursor pool cells expressing high levels of Ly6D and lacking expression of transcription factor Zbtb46 contain CCR9loB220hi immediate pDC precursors and CCR9loB220lo (lo-lo) cells which still generate pDCs and cDCs in vitro and in vivo under steady state conditions. cDC-primed cells within the Ly6DhiZbtb46- lo-lo precursors rapidly upregulate Zbtb46 and pass through a Zbtb46+Ly6D+ intermediate stage before acquiring cDC phenotype after cell division. Type I IFN stimulation limits cDC and promotes pDC output from this precursor fraction by arresting cDC-primed cells in the Zbtb46+Ly6D+ stage preventing their expansion and differentiation into cDCs. Modulation of pDC versus cDC output from precursors by external factors may allow for adaptation of DC subset composition at later differentiation stages.

Comparative Study of the Role of Interepithelial Mucosal Mast Cells in the Context of Intestinal Adenoma-Carcinoma Progression.

Mast cells (MCs) are crucial players in the relationship between the tumor microenvironment (TME) and cancer cells and have been shown to influence angiogenesis and progression of human colorectal cancer (CRC). However, the role of MCs in the TME is controversially discussed as either pro- or anti-tumorigenic. Genetically engineered mouse models (GEMMs) are the most frequently used in vivo models for human CRC research. In the murine intestine there are at least three different MC subtypes: interepithelial mucosal mast cells (ieMMCs), lamina proprial mucosal mast cells (lpMMCs) and connective tissue mast cells (CTMCs). Interepithelial mucosal mast cells (ieMMCs) in (pre-)neoplastic intestinal formalin-fixed paraffin-embedded (FFPE) specimens of mouse models (total lesions n = 274) and human patients (n = 104) were immunohistochemically identified and semiquantitatively scored. Scores were analyzed along the adenoma-carcinoma sequence in humans and 12 GEMMs of small and large intestinal cancer. The presence of ieMMCs was a common finding in intestinal adenomas and carcinomas in mice and humans. The number of ieMMCs decreased in the course of colonic adenoma-carcinoma sequence in both species (p < 0.001). However, this dynamic cellular state was not observed for small intestinal murine tumors. Furthermore, ieMMC scores were higher in GEMMs with altered Wnt signaling (active ß-catenin) than in GEMMs with altered MAPK signaling and wildtypes (WT). In conclusion, we hypothesize that, besides stromal MCs (lpMMCs/CTMCs), particularly the ieMMC subset is important for onset and progression of intestinal neoplasia and may interact with the adjacent neoplastic epithelial cells in dependence on the molecular environment. Moreover, our study indicates the need for adequate GEMMs for the investigation of the intestinal immunologic TME.

Lymph node stromal cells support the maturation of pre-DCs into cDC-like cells via colony-stimulating factor 1.

Conventional dendritic cells (cDCs) arise from committed precursor dendritic cells (pre-DCs) in the bone marrow that continuously seed the periphery. Pre-DCs and other upstream progenitors proliferate and mature in response to Fms-related receptor tyrosine kinase 3 ligand, which is considered the key cytokine for cDC development. However, other cytokines such as stem cell factor and colony-stimulating factor 1 (CSF1) were also shown to induce pre-DC maturation into DC-like cells. Yet, it is still only incompletely understood which cells contribute to cDC development once pre-DCs arrive in peripheral tissues. Here, we analysed the impact of lymph node (LN) fibroblastic stromal cells (FSCs) on the maturation of pre-DCs into cDC-like cells. We could demonstrate that ex vivo isolated LN FSCs co-cultured with pre-DCs induce precursor maturation into DC-like cells, which were capable of efficiently promoting the proliferation of naïve CD4+ T cells. Interestingly, FSCs isolated from distinct LNs induced DC-like cells with highly comparable transcriptomes, characterized by the expression of signature genes of both ex vivo isolated DCs and macrophages. Finally, by performing supplementation and receptor blocking studies, we could demonstrate that CSF1 is a driving factor for LN FSC-mediated pre-DC maturation into DC-like cells. In summary, we could identify CSF1 as a stromal cell-derived factor that has the potential to support the maturation of pre-DCs into cDC-like cells within secondary lymphoid organs.

Circadian clocks guide dendritic cells into skin lymphatics.

Migration of leukocytes from the skin to lymph nodes (LNs) via afferent lymphatic vessels (LVs) is pivotal for adaptive immune responses1,2. Circadian rhythms have emerged as important regulators of leukocyte trafficking to LNs via the blood3,4. Here, we demonstrate that dendritic cells (DCs) have a circadian migration pattern into LVs, which peaks during the rest phase in mice. This migration pattern is determined by rhythmic gradients in the expression of the chemokine CCL21 and of adhesion molecules in both mice and humans. Chronopharmacological targeting of the involved factors abrogates circadian migration of DCs. We identify cell-intrinsic circadian oscillations in skin lymphatic endothelial cells (LECs) and DCs that cogovern these rhythms, as their genetic disruption in either cell type ablates circadian trafficking. These observations indicate that circadian clocks control the infiltration of DCs into skin lymphatics, a process that is essential for many adaptive immune responses and relevant for vaccination and immunotherapies.

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.

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 (Irf8fl/flClec9acre 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.

Environmental signals rather than layered ontogeny imprint the function of type 2 conventional dendritic cells in young and adult mice.

Conventional dendritic cells (cDC) are key activators of naive T cells, and can be targeted in adults to induce adaptive immunity, but in early life are considered under-developed or functionally immature. Here we show that, in early life, when the immune system develops, cDC2 exhibit a dual hematopoietic origin and, like other myeloid and lymphoid cells, develop in waves. Developmentally distinct cDC2 in early life, despite being distinguishable by fate mapping, are transcriptionally and functionally similar. cDC2 in early and adult life, however, are exposed to distinct cytokine environments that shape their transcriptional profile and alter their ability to sense pathogens, secrete cytokines and polarize T cells. We further show that cDC2 in early life, despite being distinct from cDC2 in adult life, are functionally competent and can induce T cell responses. Our results thus highlight the potential of harnessing cDC2 for boosting immunity in early life.

A Simple and Effective Flow Cytometry-Based Method for Identification and Quantification of Tissue Infiltrated Leukocyte Subpopulations in a Mouse Model of Peripheral Arterial Disease.

Arteriogenesis, the growth of a natural bypass from pre-existing arteriolar collaterals, is an endogenous mechanism to compensate for the loss of an artery. Mechanistically, this process relies on a locally and temporally restricted perivascular infiltration of leukocyte subpopulations, which mediate arteriogenesis by supplying growth factors and cytokines. Currently, the state-of-the-art method to identify and quantify these leukocyte subpopulations in mouse models is immunohistology. However, this is a time consuming procedure. Here, we aimed to develop an optimized protocol to identify and quantify leukocyte subpopulations by means of flow cytometry in adductor muscles containing growing collateral arteries. For that purpose, adductor muscles of murine hindlimbs were isolated at day one and three after induction of arteriogenesis, enzymatically digested, and infiltrated leukocyte subpopulations were identified and quantified by flow cytometry, as exemplary shown for neutrophils and macrophages (defined as CD45+/CD11b+/Ly6G+ and CD45+/CD11b+/F4/80+ cells, respectively). In summary, we show that flow cytometry is a suitable method to identify and quantify leukocyte subpopulations in muscle tissue, and provide a detailed protocol. Flow cytometry constitutes a timesaving tool compared to histology, which might be used in addition for precise localization of leukocytes in tissue samples.

The Kidney Contains Ontogenetically Distinct Dendritic Cell and Macrophage Subtypes throughout Development That Differ in Their Inflammatory Properties.

BACKGROUND: Mononuclear phagocytes (MPs), including macrophages, monocytes, and dendritic cells (DCs), are phagocytic cells with important roles in immunity. The developmental origin of kidney DCs has been highly debated because of the large phenotypic overlap between macrophages and DCs in this tissue. METHODS: We used fate mapping, RNA sequencing, flow cytometry, confocal microscopy, and histo-cytometry to assess the origin and phenotypic and functional properties of renal DCs in healthy kidney and of DCs after cisplatin and ischemia reperfusion-induced kidney injury. RESULTS: Adult kidney contains at least four subsets of MPs with prominent Clec9a-expression history indicating a DC origin. We demonstrate that these populations are phenotypically, functionally, and transcriptionally distinct from each other. We also show these kidney MPs exhibit unique age-dependent developmental heterogeneity. Kidneys from newborn mice contain a prominent population of embryonic-derived MHCIInegF4/80hiCD11blow macrophages that express T cell Ig and mucin domain containing 4 (TIM-4) and MER receptor tyrosine kinase (MERTK). These macrophages are replaced within a few weeks after birth by phenotypically similar cells that express MHCII but lack TIM-4 and MERTK. MHCII+F4/80hi cells exhibit prominent Clec9a-expression history in adulthood but not early life, indicating additional age-dependent developmental heterogeneity. In AKI, MHCIInegF4/80hi cells reappear in adult kidneys as a result of MHCII downregulation by resident MHCII+F4/80hi cells, possibly in response to prostaglandin E2 (PGE2). RNA sequencing further suggests MHCII+F4/80hi cells help coordinate the recruitment of inflammatory cells during renal injury. CONCLUSIONS: Distinct developmental programs contribute to renal DC and macrophage populations throughout life, which could have important implications for therapies targeting these cells.

Tissue clonality of dendritic cell subsets and emergency DCpoiesis revealed by multicolor fate mapping of DC progenitors.

Conventional dendritic cells (cDCs) are found in all tissues and play a key role in immune surveillance. They comprise two major subsets, cDC1 and cDC2, both derived from circulating precursors of cDCs (pre-cDCs), which exited the bone marrow. We show that, in the steady-state mouse, pre-cDCs entering tissues proliferate to give rise to differentiated cDCs, which themselves have residual proliferative capacity. We use multicolor fate mapping of cDC progenitors to show that this results in clones of sister cDCs, most of which comprise a single cDC1 or cDC2 subtype, suggestive of pre-cDC commitment. Upon infection, a surge in the influx of pre-cDCs into the affected tissue dilutes clones and increases cDC numbers. Our results indicate that tissue cDCs can be organized in a patchwork of closely positioned sister cells of the same subset whose coexistence is perturbed by local infection, when the bone marrow provides additional pre-cDCs to meet increased tissue demand.

Correction: Syk Signaling in Dendritic Cells Orchestrates Innate Resistance to Systemic Fungal Infection.

[This corrects the article DOI: 10.1371/journal.ppat.1004276.].

Clec9a-Mediated Ablation of Conventional Dendritic Cells Suggests a Lymphoid Path to Generating Dendritic Cells In Vivo.

Conventional dendritic cells (cDCs) are versatile activators of immune responses that develop as part of the myeloid lineage downstream of hematopoietic stem cells. We have recently shown that in mice precursors of cDCs, but not of other leukocytes, are marked by expression of DNGR-1/CLEC9A. To genetically deplete DNGR-1-expressing cDC precursors and their progeny, we crossed Clec9a-Cre mice to Rosa-lox-STOP-lox-diphtheria toxin (DTA) mice. These mice develop signs of age-dependent myeloproliferative disease, as has been observed in other DC-deficient mouse models. However, despite efficient depletion of cDC progenitors in these mice, cells with phenotypic characteristics of cDCs populate the spleen. These cells are functionally and transcriptionally similar to cDCs in wild type control mice but show somatic rearrangements of Ig-heavy chain genes, characteristic of lymphoid origin cells. Our studies reveal a previously unappreciated developmental heterogeneity of cDCs and suggest that the lymphoid lineage can generate cells with features of cDCs when myeloid cDC progenitors are impaired.