The methyltransferase Ezh2 controls cell adhesion and migration through direct methylation of the extranuclear regulatory protein talin.

A cytosolic role for the histone methyltransferase Ezh2 in regulating lymphocyte activation has been suggested, but the molecular mechanisms underpinning this extranuclear function have remained unclear. Here we found that Ezh2 regulated the integrin signaling and adhesion dynamics of neutrophils and dendritic cells (DCs). Ezh2 deficiency impaired the integrin-dependent transendothelial migration of innate leukocytes and restricted disease progression in an animal model of multiple sclerosis. Direct methylation of talin, a key regulatory molecule in cell migration, by Ezh2 disrupted the binding of talin to F-actin and thereby promoted the turnover of adhesion structures. This regulatory effect was abolished by targeted disruption of the interactions of Ezh2 with the cytoskeletal-reorganization effector Vav1. Our studies reveal an unforeseen extranuclear function for Ezh2 in regulating adhesion dynamics, with implications for leukocyte migration, immune responses and potentially pathogenic processes.

Human dermal CD14⁺ cells are a transient population of monocyte-derived macrophages.

Dendritic cells (DCs), monocytes, and macrophages are leukocytes with critical roles in immunity and tolerance. The DC network is evolutionarily conserved; the homologs of human tissue CD141(hi)XCR1⁺ CLEC9A⁺ DCs and CD1c⁺ DCs are murine CD103⁺ DCs and CD64⁻ CD11b⁺ DCs. In addition, human tissues also contain CD14⁺ cells, currently designated as DCs, with an as-yet unknown murine counterpart. Here we have demonstrated that human dermal CD14⁺ cells are a tissue-resident population of monocyte-derived macrophages with a short half-life of <6 days. The decline and reconstitution kinetics of human blood CD14⁺ monocytes and dermal CD14⁺ cells in vivo supported their precursor-progeny relationship. The murine homologs of human dermal CD14⁺ cells are CD11b⁺ CD64⁺ monocyte-derived macrophages. Human and mouse monocytes and macrophages were defined by highly conserved gene transcripts, which were distinct from DCs. The demonstration of monocyte-derived macrophages in the steady state in human tissue supports a conserved organization of human and mouse mononuclear phagocyte system.

The Role of Autophagy in Chemical Proteasome Inhibition Model of Retinal Degeneration.

We recently demonstrated that chemical proteasome inhibition induced inner retinal degeneration, supporting the pivotal roles of the ubiquitin-proteasome system in retinal structural integrity maintenance. In this study, using beclin1-heterozygous (Becn1-Het) mice with autophagic dysfunction, we tested our hypothesis that autophagy could be a compensatory retinal protective mechanism for proteasomal impairment. Despite the reduced number of autophagosome, the ocular tissue morphology and intraocular pressure were normal. Surprisingly, Becn1-Het mice experienced the same extent of retinal degeneration as was observed in wild-type mice, following an intravitreal injection of a chemical proteasome inhibitor. Similarly, these mice equally responded to other chemical insults, including endoplasmic reticulum stress inducer, N-methyl-D-aspartate, and lipopolysaccharide. Interestingly, in cultured neuroblastoma cells, we found that the mammalian target of rapamycin-independent autophagy activators, lithium chloride and rilmenidine, rescued these cells against proteasome inhibition-induced death. These results suggest that Becn1-mediated autophagy is not an effective intrinsic protective mechanism for retinal damage induced by insults, including impaired proteasomal activity; furthermore, autophagic activation beyond normal levels is required to alleviate the cytotoxic effect of proteasomal inhibition. Further studies are underway to delineate the precise roles of different forms of autophagy, and investigate the effects of their activation in rescuing retinal neurons under various pathological conditions.

Biallelic interferon regulatory factor 8 mutation: A complex immunodeficiency syndrome with dendritic cell deficiency, monocytopenia, and immune dysregulation.

BACKGROUND: The homozygous K108E mutation of interferon regulatory factor 8 (IRF8) is reported to cause dendritic cell (DC) and monocyte deficiency. However, more widespread immune dysfunction is predicted from the multiple roles ascribed to IRF8 in immune cell development and function. OBJECTIVE: We sought to describe the effect on hematopoiesis and immunity of the compound heterozygous R83C/R291Q mutation of IRF8, which is present in a patient with recurrent viral infection, granuloproliferation, and intracerebral calcification. METHODS: Variant IRF8 alleles were identified by means of exome sequencing, and their function was tested by using reporter assays. The cellular phenotype was studied in detail by using flow cytometry, functional immunologic assay transcriptional profiling, and antigen receptor profiling. RESULTS: Both mutations affected conserved residues, and R291Q is orthologous to R294, which is mutated in the BXH2 IRF8-deficient mouse. R83C showed reduced nuclear translocation, and neither mutant was able to regulate the Ets/IRF composite element or interferon-stimulated response element, whereas R291Q retained BATF/JUN interactions. DC deficiency and monocytopenia were observed in blood, dermis, and lung lavage fluid. Granulocytes were consistently increased, dysplastic, and hypofunctional. Natural killer cell development and maturation were arrested. TH1, TH17, and CD8+ memory T-cell differentiation was significantly reduced, and T cells did not express CXCR3. B-cell development was impaired, with fewer memory cells, reduced class-switching, and lower frequency and complexity of somatic hypermutation. Cell-specific gene expression was widely disturbed in interferon- and IRF8-regulated transcripts. CONCLUSIONS: This analysis defines the clinical features of human biallelic IRF8 deficiency, revealing a complex immunodeficiency syndrome caused by DC and monocyte deficiency combined with widespread immune dysregulation.

CD1c+ blood dendritic cells have Langerhans cell potential.

Langerhans cells (LCs) are self-renewing in the steady state but repopulated by myeloid precursors after injury. Human monocytes give rise to langerin-positive cells in vitro, suggesting a potential precursor role. However, differentiation experiments with human lineage-negative cells and CD34(+) progenitors suggest that there is an alternative monocyte-independent pathway of LC differentiation. Recent data in mice also show long-term repopulation of the LC compartment with alternative myeloid precursors. Here we show that, although monocytes are able to express langerin, when cultured with soluble ligands granulocyte macrophage colony-stimulating factor (GM-CSF), transforming growth factor ß (TGFß), and bone morphogenetic protein 7 (BMP7), CD1c(+) dendritic cells (DCs) become much more LC-like with high langerin, Birbeck granules, EpCAM, and E-cadherin expression under the same conditions. These data highlight a new potential precursor function of CD1c(+) DCs and demonstrate an alternative pathway of LC differentiation that may have relevance in vivo.

Donor monocyte-derived macrophages promote human acute graft-versus-host disease.

Myelopoiesis is invariably present and contributes to pathology in animal models of graft-versus-host disease (GVHD). In humans, a rich inflammatory infiltrate bearing macrophage markers has also been described in histological studies. In order to determine the origin, functional properties, and role in pathogenesis of these cells, we isolated single-cell suspensions from acute cutaneous GVHD and subjected them to genotype, transcriptome, and in vitro functional analysis. A donor-derived population of CD11c+CD14+ cells was the dominant population of all leukocytes in GVHD. Surface phenotype and NanoString gene expression profiling indicated the closest steady-state counterpart of these cells to be monocyte-derived macrophages. In GVHD, however, there was upregulation of monocyte antigens SIRPα and S100A8/9 transcripts associated with leukocyte trafficking, pattern recognition, antigen presentation, and costimulation. Isolated GVHD macrophages stimulated greater proliferation and activation of allogeneic T cells and secreted higher levels of inflammatory cytokines than their steady-state counterparts. In HLA-matched mixed leukocyte reactions, we also observed differentiation of activated macrophages with a similar phenotype. These exhibited cytopathicity to a keratinocyte cell line and mediated pathological damage to skin explants independently of T cells. Together, these results define the origin, functional properties, and potential pathogenic roles of human GVHD macrophages.

A humanized mouse model to study mast cells mediated cutaneous adverse drug reactions.

Recently a G-protein-coupled receptor, MAS Related GPR Family Member X2 (MRGPRX2), was identified as a specific receptor on human mast cells responsible for IgE independent adverse drug reactions (ADR). Although a murine homologue, Mrgprb2, has been identified for this receptor, its affinity for many ADR-causing drugs is poor making it difficult to undertake in vivo studies to examine mechanisms of ADR and to develop therapeutic strategies. Here, we have created humanized mice capable of generating MRGPRX2-expressing human MCs allowing for the study of MRGPRX2 MCs-mediated ADR in vitro as well as in vivo. Humanized mice were generated by hydrodynamic-injection of plasmids expressing human GM-CSF and IL-3 into NOD-scid IL2R-γ-/- strain of mice that had been transplanted with human hematopoietic stem cells. These GM/IL-3 humice expressed high numbers of tissue human MCs but the MRGPRX2 receptor expressed in MCs were limited to few body sites including the skin. Importantly, large numbers of MRGPRX2-expressing human MCs could be cultured from the bone marrow of GM/IL-3 humice revealing these mice to be an important source of human MCs for in vitro studies of MRGPRX2-related MCs activities. When GM/IL-3 humice were exposed to known ADR causing contrast agents (meglumine and gadobutrol), the humice were found to experience anaphylaxis analogous to the clinical situation. Thus, GM/IL-3 humice represent a valuable model for investigating in vivo interactions of ADR-causing drugs and human MCs and their sequelae, and these mice are also a source of human MRGPRX2-expressing MCs for in vitro studies.

Ezh2 Controls Skin Tolerance through Distinct Mechanisms in Different Subsets of Skin Dendritic Cells.

Ezh2, a well-established epigenetic repressor, can down-regulate leukocyte inflammatory responses, but its role in cutaneous health remains elusive. Here we demonstrate that Ezh2 controls cutaneous tolerance by regulating Langerhans cell (LC) transmigration across the epidermal basement membrane directly via Talin1 methylation. Ezh2 deficiency impaired disassembly of adhesion structures in LCs, leading to their defective integrin-dependent emigration from the epidermis and failure in tolerance induction. Moreover, mobilization of Ezh2-deficient Langerin- dermal dendritic cells (dDCs) via high-dose treatment with a weak allergen restored tolerance, which is associated with an increased tolerogenic potential of Langerin- dDCs likely due to epigenetic de-repression of Aldh in the absence of Ezh2. Our data reveal novel roles for Ezh2 in governing LC- and dDC-mediated host protection against cutaneous allergen via distinct mechanisms.

Bat-mouse bone marrow chimera: a novel animal model for dissecting the uniqueness of the bat immune system.

Bats are an important animal model with long lifespans, low incidences of tumorigenesis and an ability to asymptomatically harbour pathogens. Currently, in vivo studies of bats are hampered due to their low reproduction rates. To overcome this, we transplanted bat cells from bone marrow (BM) and spleen into an immunodeficient mouse strain NOD-scid IL-2R-/- (NSG), and have successfully established stable, long-term reconstitution of bat immune cells in mice (bat-mice). Immune functionality of our bat-mouse model was demonstrated through generation of antigen-specific antibody response by bat cells following immunization. Post-engraftment of total bat BM cells and splenocytes, bat immune cells survived, expanded and repopulated the mouse without any observable clinical abnormalities. Utilizing bat's remarkable immunological functions, this novel model has a potential to be transformed into a powerful platform for basic and translational research.

A Novel Human Systemic Lupus Erythematosus Model in Humanised Mice.

Mouse models have contributed to the bulk of knowledge on Systemic Lupus Erythematosus (SLE). Nevertheless, substantial differences exist between human and mouse immune system. We aimed to establish and characterise a SLE model mediated by human immune system. Injection of pristane into immunodeficient mice reconstituted with human immune system (humanised mice) recapitulated key SLE features, including: production of human anti-nuclear autoantibodies, lupus nephritis, and pulmonary serositis. There was a reduction in the number of human lymphocytes in peripheral blood, resembling lymphopenia in SLE patients. Concurrently, B cells and T cells were systemically hyperactivated, with a relative expansion of CD27+ and CD27-IgD- memory B cells, increased number of plasmablasts/plasma cells, and accumulation of effector memory T cells. There was also an increased production of human pro-inflammatory cytokines, including: IFN-γ, IL-8, IL-18, MCP-1, and IL-6, suggesting their role in SLE pathogenesis. Increased expression of type I IFN signature genes was also found in human hepatocytes. Altogether, we showed an SLE model that was mediated by human immune system, and which recapitulated key clinical and immunological SLE features. The advancements of humanised mice SLE model would provide an in vivo platform to facilitate translational studies and pre-clinical evaluations of human-specific mechanisms and immunotherapies.

Mapping the human DC lineage through the integration of high-dimensional techniques.

Dendritic cells (DC) are professional antigen-presenting cells that orchestrate immune responses. The human DC population comprises two main functionally specialized lineages, whose origins and differentiation pathways remain incompletely defined. Here, we combine two high-dimensional technologies-single-cell messenger RNA sequencing (scmRNAseq) and cytometry by time-of-flight (CyTOF)-to identify human blood CD123+CD33+CD45RA+ DC precursors (pre-DC). Pre-DC share surface markers with plasmacytoid DC (pDC) but have distinct functional properties that were previously attributed to pDC. Tracing the differentiation of DC from the bone marrow to the peripheral blood revealed that the pre-DC compartment contains distinct lineage-committed subpopulations, including one early uncommitted CD123high pre-DC subset and two CD45RA+CD123low lineage-committed subsets exhibiting functional differences. The discovery of multiple committed pre-DC populations opens promising new avenues for the therapeutic exploitation of DC subset-specific targeting.

Isolation of Human Skin Dendritic Cell Subsets.

Dendritic cells (DCs) are specialized leukocytes with antigen-processing and antigen-presenting functions. DCs can be divided into distinct subsets by anatomical location, phenotype and function. In human, the two most accessible tissues to study leukocytes are peripheral blood and skin. DCs are rare in human peripheral blood (<1 % of mononuclear cells) and have a less mature phenotype than their tissue counterparts (MacDonald et al., Blood. 100:4512-4520, 2002; Haniffa et al., Immunity 37:60-73, 2012). In contrast, the skin covering an average total surface area of 1.8 m(2) has approximately tenfold more DCs than the average 5 L of total blood volume (Wang et al., J Invest Dermatol 134:965-974, 2014). DCs migrate spontaneously from skin explants cultured ex vivo, which provide an easy method of cell isolation (Larsen et al., J Exp Med 172:1483-1493, 1990; Lenz et al., J Clin Invest 92:2587-2596, 1993; Nestle et al., J Immunol 151:6535-6545, 1993). These factors led to the extensive use of skin DCs as the "prototype" migratory DCs in human studies. In this chapter, we detail the protocols to isolate DCs and resident macrophages from human skin. We also provide a multiparameter flow cytometry gating strategy to identify human skin DCs and to distinguish them from macrophages.

Human skin dendritic cells in health and disease.

Dendritic cells (DCs) are specialized antigen presenting cells abundant in peripheral tissues such as skin where they function as immune sentinels. Skin DCs migrate to draining lymph node where they interact with naïve T cells to induce immune responses to microorganisms, vaccines, tumours and self-antigens. In this review, we present the key historical developments and recent advances in human skin DC research. We also integrate the current understanding on the origin and functional specializations of DC subsets in healthy skin with findings in inflammatory skin diseases focusing on psoriasis and atopic eczema. A comprehensive understanding of the dynamic changes in DC subsets in health and disease will form a strong foundation to facilitate the clinical translation of DC-based therapeutic and vaccination strategies.

A three-dimensional atlas of human dermal leukocytes, lymphatics, and blood vessels.

Dendritic cells (DCs), macrophages (Mφ), and T cells are major components of the skin immune system, but their interstitial spatial organization is poorly characterized. Using four-channel whole-mount immunofluorescence staining of the human dermis, we demonstrated the three-dimensional distribution of CD31(+) blood capillaries, LYVE-1(+) lymphatics, discrete populations of CD11c(+) myeloid DCs, FXIIIa(+) Mφ, and lymphocytes. We showed phenotypic and morphological differences in situ between DCs and Mφ. DCs formed the first dermal cellular layer (0-20 µm beneath the dermoepidermal junction), Mφ were located deeper (40-60 µm), and CD3(+) lymphocytes were observed throughout (0-60 µm). Below this level, DCs, T cells, and the majority of Mφ formed stable perivascular sheaths. Whole-mount imaging revealed the true extent of dermal leukocytes previously underestimated from cross-section views. The total area of apical dermis (0-30 µm) contained approximately 10-fold more myeloid DCs than the entire blood volume of an average individual. Surprisingly, <1% of dermal DCs occupied lymphatics in freshly isolated skin. Dermal DCs rapidly accumulated within lymphatics, but Mφ remained fixed in skin explants cultured ex vivo. The leukocyte architecture observed in normal skin was distorted in inflammation and disease. These studies illustrate the micro-anatomy of dermal leukocytes and provide further insights into their functional organization.

Activated dectin-1 localizes to lipid raft microdomains for signaling and activation of phagocytosis and cytokine production in dendritic cells.

Lipid rafts are plasma membrane microdomains that are enriched in cholesterol, glycosphingolipids, and glycosylphosphatidylinositol-anchored proteins and play an important role in the signaling of ITAM-bearing lymphocyte antigen receptors. Dectin-1 is a C-type lectin receptor (CLR) that recognizes beta-glucan in the cell walls of fungi and triggers signal transduction via its cytoplasmic hemi-ITAM. However, it is not known if similar to antigen receptors, Dectin-1 would also signal via lipid rafts and if the integrity of lipid raft microdomains is important for the physiological functions mediated by Dectin-1. We demonstrate here using sucrose gradient ultracentrifugation and confocal microscopy that Dectin-1 translocates to lipid rafts upon stimulation of dendritic cells (DCs) with the yeast derivative zymosan or beta-glucan. In addition, two key signaling molecules, Syk and PLCgamma2 are also recruited to lipid rafts upon the activation of Dectin-1, suggesting that lipid raft microdomains facilitate Dectin-1 signaling. Disruption of lipid raft integrity with the synthetic drug, methyl-beta-cyclodextrin (betamD) leads to reduced intracellular Ca2+ flux and defective Syk and ERK phosphorylation in Dectin-1-activated DCs. Furthermore, betamD-treated DCs have significantly attenuated production of IL-2, IL-10, and TNFalpha upon Dectin-1 engagement, and they also exhibit impaired phagocytosis of zymosan particles. Taken together, the data indicate that Dectin-1 and perhaps also other CLRs are recruited to lipid rafts upon activation and that the integrity of lipid rafts is important for the signaling and cellular functions initiated by this class of innate receptors.