The Phenotypic Characterization of the Human Renal Mononuclear Phagocytes Reveal a Co-Ordinated Response to Injury.
Ontology highlight
ABSTRACT: Mammalian tissues contain networks of mononuclear phagocytes (MPh) that sense injury and orchestrate the response to it. In mice, this is affected by distinct populations of dendritic cells (DC), monocytes and macrophages and recent studies suggest the same is true for human skin and intestine but little is known about the kidney. Here we describe the analysis of MPh populations in five human kidneys and show they are highly heterogeneous and contain discrete populations of DC, monocytes and macrophages. These include: plasmacytoid DC (CD303+) and both types of conventional DC-cDC1 (CD141+ cells) and CD2 (CD1c+ cells); classical, non-classical and intermediate monocytes; and macrophages including a novel population of CD141+ macrophages clearly distinguishable from cDC1 cells. The relative size of the MPh populations differed between kidneys: the pDC population was bi-modally distributed being less than 2% of DC in two kidneys without severe injury and over 35% in the remaining three with low grade injury in the absence of morphological evidence of inflammation. There were profound differences in the other MPh populations in kidneys with high and low numbers of pDC. Thus, cDC1 cells were abundant (55 and 52.3%) when pDC were sparse and sparse (12.8-12.5%) when pDC were abundant, whereas the proportions of cDC2 cells and classical monocytes increased slightly in pDC high kidneys. We conclude that MPh are highly heterogeneous in human kidneys and that pDC infiltration indicative of low-grade injury does not occur in isolation but is part of a co-ordinated response affecting all renal DC, monocyte and macrophage populations.
Project description:Conventional markers of macrophages (Mфs) and dendritic cells (DCs) lack specificity and often overlap, leading to confusion and controversy regarding the precise function of these cells in kidney and other diseases. This study aimed to identify the phenotype and function of renal mononuclear phagocytes (rMPs) expressing key markers of both Mфs and DCs. F4/80(+)CD11c(+) cells accounted for 45% of total rMPs in normal kidneys and in those from mice with Adriamycin nephropathy (AN). Despite expression of the DC marker CD11c, these double-positive rMPs displayed the features of Mфs, including Mф-like morphology, high expression of CD68, CD204, and CD206, and high phagocytic ability but low antigen-presenting ability. F4/80(+)CD11c(+) cells were found in the cortex but not in the medulla of the kidney. In AN, F4/80(+)CD11c(+) cells displayed an M1 Mф phenotype with high expression of inflammatory mediators and costimulatory factors. Adoptive transfer of F4/80(+)CD11c(+) cells separated from diseased kidney aggravated renal injury in AN mice. Furthermore, adoptive transfer of common progenitors revealed that kidney F4/80(+)CD11c(+) cells were derived predominantly from monocytes, but not from pre-DCs. In conclusion, renal F4/80(+)CD11c(+) cells are a major subset of rMPs and display Mф-like phenotypic and functional characteristics in health and in AN.
Project description:Background and aimsCrohn's disease [CD] and ulcerative colitis [UC] are distinct forms of inflammatory bowel disease. Heterogeneity of HLA-DR+SIRPα + mononuclear phagocytes [MNPs], including macrophages [MΦ], monocyte-derived [Mono] cells, and dendritic cells [DCs], was reported in gut tissue but not yet investigated in mesenteric lymph nodes [MLNs] of IBD patients. We here compared the phenotype, function, and molecular profile of HLA-DR+SIRPα + MNPs in CD and UC MLNs.MethodsCell distribution, morphology, immune function, and transcriptomic [bulk RNAseq] and high-dimensional protein expression profiles [CyTOF] of HLA-DR+SIRPα + MNPs were examined in MLNs of UC [n = 14], CD [n = 35], and non-IBD [n = 12] patients.ResultsElevated frequencies of CD14+CD64+CD163+ [Mono/MΦ-like] MNPs displaying monocyte/MΦ morphology and phagocytic function were a distinct feature of UC MLNs. In CD, the proportion of CD14-CD64-CD163- [DC-like] cells was augmented relative to Mono/MΦ-like cells; DC-like cells drove naïve T cell proliferation, Th1 polarisation, and Th17 TCM plasticity. Gene expression profile corroborated the nature of DC-like cells, best represented by BTLA, SERPINF, IGJ and, of Mono/MΦ-like cells, defined by CD163, MARCO, MAFB, CD300E, S100A9 expression. CyTOF analysis showed that CD123+ plasmacytoid cells predominated over conventional DCs in DC-like cells. Four CD163+ clusters were revealed in Mono/MΦ-like cells, two of which were enriched in MARCO-CD68dimHLA-DRdim monocyte-like cells and MARCOhiCD68hiHLA-DRhi Mɸ, whose proportion increased in UC relative to CD.ConclusionsDefining the landscape of MNPs in MLNs provided evidence for expansion of CD163+ Mono/MΦ-like cells in UC only, highlighting a distinction between UC and CD, and thus the potential contribution of monocyte-like cells in driving colitis.
Project description:Recent reports have highlighted greater complexity, plasticity, and functional diversity of mononuclear phagocytes (MPCs), including monocytes, macrophages, and dendritic cells (DCs), in our organs than previously understood. The functions and origins of MPCs resident within healthy organs, especially in the kidney, are less well understood, whereas studies suggest they play roles in disease states distinct from recruited monocytes. We developed an unbiased approach using flow cytometry to analyze MPCs residing in the normal mouse kidney, and identified five discrete subpopulations according to CD11b/CD11c expression as well as F4/80, CD103, CD14, CD16, and CD64 expression. In addition to distinct marker profiles, these subpopulations have different lineages and expression of genes involved in tissue homeostasis, including angiogenesis. Among them, the CD11b(int)CD11c(int) F4/80(high) subpopulation notably exhibited high capacity to produce a representative anti-inflammatory cytokine, IL-10. Each subpopulation had different degrees of both macrophage (phagocytosis) and DC (Ag presentation) capacities, with a tendency to promote differentiation of regulatory T cells, whereas two of these showed expression of transcription factors reported to be highly expressed by classical DCs, and proclivity to exit the kidney following stimulation with LPS. In summary, resident kidney MPCs comprise discrete subpopulations, which cannot be simply classified into the conventional entities, and they produce anti-inflammatory and tissue-homeostatic factors to differing degrees.
Project description:The phosphatidylserine receptor TIM4, encoded by TIMD4, mediates the phagocytic uptake of apoptotic cells. We applied anti-chicken TIM4 mAbs in combination with CSF1R reporter transgenes to dissect the function of TIM4 in the chick (Gallus gallus). During development in ovo, TIM4 was present on the large majority of macrophages, but expression became more heterogeneous posthatch. Blood monocytes expressed KUL01, class II MHC, and CSF1R-mApple uniformly. Around 50% of monocytes were positive for surface TIM4. They also expressed many other monocyte-specific transcripts at a higher level than TIM4- monocytes. In liver, highly phagocytic TIM4hi cells shared many transcripts with mammalian Kupffer cells and were associated with uptake of apoptotic cells. Although they expressed CSF1R mRNA, Kupffer cells did not express the CSF1R-mApple transgene, suggesting that additional CSF1R transcriptional regulatory elements are required by these cells. By contrast, CSF1R-mApple was detected in liver TIM4lo and TIM4- cells, which were not phagocytic and were more abundant than Kupffer cells. These cells expressed CSF1R alongside high levels of FLT3, MHCII, XCR1, and other markers associated with conventional dendritic cells in mice. In bursa, TIM4 was present on the cell surface of two populations. Like Kupffer cells, bursal TIM4hi phagocytes coexpressed many receptors involved in apoptotic cell recognition. TIM4lo cells appear to be a subpopulation of bursal B cells. In overview, TIM4 is associated with phagocytes that eliminate apoptotic cells in the chick. In the liver, TIM4 and CSF1R reporters distinguished Kupffer cells from an abundant population of dendritic cell-like cells.
Project description: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.
Project description:Mononuclear phagocytes (MPs) play a crucial role in tissue homeostasis; however, MPs also contribute to tumor progression and resistance to immune checkpoint blockade (ICB). Targeting MPs could be an effective strategy to enhance ICB efficacy. We report that protein kinase C delta (PKCδ), a serine/threonine kinase, is abundantly expressed by MPs in human and mouse tumors. PKCδ-/- mice displayed reduced tumor progression compared to wild types, with increased response to anti-PD-1. Tumors from PKCδ-/- mice demonstrated TH1-skewed immune response including increased antigen presentation and T cell activation. Depletion of MPs in vivo altered tumor growth in control but not PKCδ-/- mice. Coinjection of PKCδ-/- M2-like macrophages with cancer cells into wild-type mice markedly delayed tumor growth and significantly increased intratumoral T cell activation compared to PKCδ+/+ controls. PKCδ deficiency reprogrammed MPs by activating type I and type II interferon signaling. Thus, PKCδ might be targeted to reprogram MPs to augment ICB efficacy.
Project description:CD4+ T cells are at the nexus of the innate and adaptive arms of the immune system. However, little is known about the evolutionary history of CD4+ T cells, and it is unclear whether their differentiation into specialized subsets is conserved in early vertebrates. In this study, we have created transgenic zebrafish with vibrantly labeled CD4+ cells allowing us to scrutinize the development and specialization of teleost CD4+ leukocytes in vivo. We provide further evidence that CD4+ macrophages have an ancient origin and had already emerged in bony fish. We demonstrate the utility of this zebrafish resource for interrogating the complex behavior of immune cells at cellular resolution by the imaging of intimate contacts between teleost CD4+ T cells and mononuclear phagocytes. Most importantly, we reveal the conserved subspecialization of teleost CD4+ T cells in vivo. We demonstrate that the ancient and specialized tissues of the gills contain a resident population of il-4/13b-expressing Th2-like cells, which do not coexpress il-4/13a Additionally, we identify a contrasting population of regulatory T cell-like cells resident in the zebrafish gut mucosa, in marked similarity to that found in the intestine of mammals. Finally, we show that, as in mammals, zebrafish CD4+ T cells will infiltrate melanoma tumors and obtain a phenotype consistent with a type 2 immune microenvironment. We anticipate that this unique resource will prove invaluable for future investigation of T cell function in biomedical research, the development of vaccination and health management in aquaculture, and for further research into the evolution of adaptive immunity.
Project description:The human mononuclear phagocyte (MP) system, which includes dendritic cells, monocytes, and macrophages, is a critical regulator of innate and adaptive immune responses. During embryonic development, MPs derive sequentially in yolk sac progenitors, fetal liver, and bone marrow haematopoietic stem cells. MPs maintain tissue homeostasis and confer protective immunity in post-natal life. Recent evidence - primarily in animal models - highlight their critical role in coordinating the remodeling, maturation, and repair of target organs during embryonic and fetal development. However, the molecular regulation governing chemotaxis, homeostasis, and functional diversification of resident MP cells in their respective organ systems during development remains elusive. In this review, we summarize the current understanding of the development and functional contribution of tissue MPs during human organ development and morphogenesis and its relevance to regenerative medicine. We outline how single-cell multi-omic approaches and next-generation ex-vivo organ-on-chip models provide new experimental platforms to study the role of human MPs during development and disease.
Project description:BackgroundDietary high salt (HS) is a leading risk factor for mortality and morbidity. Serum sodium transiently increases postprandially but can also accumulate at sites of inflammation affecting differentiation and function of innate and adaptive immune cells. Here, we focus on how changes in extracellular sodium, mimicking alterations in the circulation and tissues, affect the early metabolic, transcriptional, and functional adaption of human and murine mononuclear phagocytes.MethodsUsing Seahorse technology, pulsed stable isotope-resolved metabolomics, and enzyme activity assays, we characterize the central carbon metabolism and mitochondrial function of human and murine mononuclear phagocytes under HS in vitro. HS as well as pharmacological uncoupling of the electron transport chain under normal salt is used to analyze mitochondrial function on immune cell activation and function (as determined by Escherichia coli killing and CD4+ T cell migration capacity). In 2 independent clinical studies, we analyze the effect of a HS diet during 2 weeks (URL: http://www.clinicaltrials.gov. Unique identifier: NCT02509962) and short-term salt challenge by a single meal (URL: http://www.clinicaltrials.gov. Unique identifier: NCT04175249) on mitochondrial function of human monocytes in vivo.ResultsExtracellular sodium was taken up into the intracellular compartment, followed by the inhibition of mitochondrial respiration in murine and human macrophages. Mechanistically, HS reduces mitochondrial membrane potential, electron transport chain complex II activity, oxygen consumption, and ATP production independently of the polarization status of macrophages. Subsequently, cell activation is altered with improved bactericidal function in HS-treated M1-like macrophages and diminished CD4+ T cell migration in HS-treated M2-like macrophages. Pharmacological uncoupling of the electron transport chain under normal salt phenocopies HS-induced transcriptional changes and bactericidal function of human and murine mononuclear phagocytes. Clinically, also in vivo, rise in plasma sodium concentration within the physiological range reversibly reduces mitochondrial function in human monocytes. In both a 14-day and single meal HS challenge, healthy volunteers displayed a plasma sodium increase of [Formula: see text] and [Formula: see text] respectively, that correlated with decreased monocytic mitochondrial oxygen consumption.ConclusionsOur data identify the disturbance of mitochondrial respiration as the initial step by which HS mechanistically influences immune cell function. Although these functional changes might help to resolve bacterial infections, a shift toward proinflammation could accelerate inflammatory cardiovascular disease.