Generation of DC from mouse spleen cell cultures in response to GM-CSF: Immunophenotypic and functional analyses

In all tissues that have been studied to date, dendritic leucocytes constitute only a small proportion of total cells and are difficult both to isolate and purify. This study reports on a method for the propagation of large numbers of dendritic cells (DC) from mouse spleen using granulocyte-macrophage colony-stimulating factor (GM-CSF) and their characteristics. Within a few days of liquid culture in GM-CSF, B10 BR (H-2(k), I-E+) mouse splenocytes formed loosely adherent myeloid cell clusters. Mononuclear progeny released from these clusters at and beyond 4 days exhibited distinct dendritic morphology and strongly expressed leucocyte common antigen (CD45), CD11b, heat-stable antigen, Pgp-1 (CD44) and intercellular adhesion molecule-1 (ICAM-1; CD54). The intensity of expression of the DC-restricted markers NLDC 145 and 33D1, the macrophage marker F4/80, and FcγRII (CDw32) was low to moderate, whereas the cells were negative for CD3, CD45RA and NK1.1. High and moderate levels, respectively, of cell surface staining for major histocompatibility complex (MHC) class II (I-E(k)) and the B7 antigens (counterreceptors of CTLA4, a structural homologue of CD28) were associated with potent stimulation of unprimed, allogeneic T-cells (B10; H-2b, I-E-). DC propagated in a similar fashion from DBA/2 mouse spleen proved to be strong antigen-presenting cells (APC) for MHC-restricted, syngeneic T-helper type 2 (Th2) cell clones specifically responsive to sperm whale myoglobin. Footpad or intravenous injection of GM-CSF-stimulated B10.BR spleen-derived DC into B10 (H-2b, I-E-) recipients resulted in homing of the allogeneic cells to T-cell-dependent areas of lymph nodes and spleen, where they strongly expressed donor MHC class II antigen 1-2 days later. These findings indicate that cells can be propagated from fresh splenocyte suspensions that exhibit distinctive features of DC, namely morphology, motility, cell-surface phenotype, potent allogeneic and syngeneic APC function and in vivo homing ability. Propagation of DC in this manner from progenitors present in lymphoid tissue provides an alternative and relatively convenient source of high numbers of these otherwise difficult to isolate but functionally important APC

Transforming Growth Factor β Blocks Tec Kinase Phosphorylation, Ca2+ Influx, and NFATc Translocation Causing Inhibition of T Cell Differentiation

Transforming growth factor (TGF)-β inhibits T cell proliferation and differentiation. TGF-β has been shown to inhibit the expression of transcription factors such as GATA-3 and T-bet that play important roles in T cell differentiation. Here we show that TGF-β inhibits T cell differentiation at a more proximal step. An early event during T cell activation is increased intracellular calcium levels. Calcium influx in activated T cells and the subsequent activation of transcription factors such as NFATc, events essential for T cell differentiation, are modulated by the Tec kinases that are downstream of the T cell receptor and CD28. We show that in stimulated CD4+ T cells, TGF-β inhibits phosphorylation and activation of the Tec kinase Itk, increase in intracellular Ca2+ levels, NFATc translocation, and activation of the mitogen-activated protein kinase ERK that together regulate T cell differentiation. Our studies suggest that by inhibiting Itk, and consequently Ca2+ influx, TGF-β limits T cell differentiation along both the Th1 and Th2 lineages

Interleukin-17 regulates matrix metalloproteinase activity in human pulmonary tuberculosis.

Tuberculosis (TB) is characterized by extensive pulmonary matrix breakdown. Interleukin-17 (IL-17) is key in host defence in TB but its role in TB-driven tissue damage is unknown. We investigated the hypothesis that respiratory stromal cell matrix metalloproteinase (MMP) production in TB is regulated by T-helper 17 (TH -17) cytokines. Biopsies of patients with pulmonary TB were analysed by immunohistochemistry (IHC), and patient bronchoalveolar lavage fluid (BALF) MMP and cytokine concentrations were measured by Luminex assays. Primary human airway epithelial cells were stimulated with conditioned medium from human monocytes infected with Mycobacterium tuberculosis (Mtb) and TH -17 cytokines. MMP secretion, activity, and gene expression were determined by ELISA, Luminex assay, zymography, RT-qPCR, and dual luciferase reporter assays. Signalling pathways were examined using phospho-western analysis and siRNA. IL-17 is expressed in TB patient granulomas and MMP-3 is expressed in adjacent pulmonary epithelial cells. IL-17 had a divergent, concentration-dependent effect on MMP secretion, increasing epithelial secretion of MMP-3 (p < 0.001) over 72 h, whilst decreasing that of MMP-9 (p < 0.0001); mRNA levels were similarly affected. Both IL-17 and IL-22 increased fibroblast Mtb-dependent MMP-3 secretion but IL-22 did not modulate epithelial MMP-3 expression. Both IL-17 and IL-22, but not IL-23, were significantly up-regulated in BALF from TB patients. IL-17-driven MMP-3 was dependent on p38 MAP kinase and the PI3K p110α subunit. In summary, IL-17 drives airway stromal cell-derived MMP-3, a mediator of tissue destruction in TB, alone and with monocyte-dependent networks in TB. This is regulated by p38 MAP kinase and PI3K pathways. © 2017 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland

GM-CSF Inhibits c-Kit and SCF Expression by Bone Marrow-Derived Dendritic Cells.

Stem cell factor (SCF), the ligand of c-kit, is a key cytokine for hematopoiesis. Hematopoietic precursors express c-kit, whereas differentiated cells of hematopoietic lineage are negative for this receptor, with the exception of NK cells, mast cells, and a few others. While it has long been recognized that dendritic cells (DCs) can express c-kit, several questions remain concerning the SCF/c-kit axis in DCs. This is particularly relevant for DCs found in those organs wherein SCF is highly expressed, including the bone marrow (BM). We characterized c-kit expression by conventional DCs (cDCs) from BM and demonstrated a higher proportion of c-kit+ cells among type 1 cDC subsets (cDC1s) than type 2 cDC subsets (cDC2s) in both humans and mice, whereas similar levels of c-kit expression were observed in cDC1s and cDC2s from mouse spleen. To further study c-kit regulation, DCs were generated with granulocyte-macrophage colony-stimulating factor (GM-CSF) from mouse BM, a widely used protocol. CD11c+ cells were purified from pooled non-adherent and slightly adherent cells collected after 7 days of culture, thus obtaining highly purified BM-derived DCs (BMdDCs). BMdDCs contained a small fraction of c-kit+ cells, and by replating them for 2 days with GM-CSF, we obtained a homogeneous population of c-kit+ CD40hi MHCIIhi cells. Not only did BMdDCs express c-kit but they also produced SCF, and both were striking upregulated if GM-CSF was omitted after replating. Furthermore, a small but significant reduction in BMdDC survival was observed upon SCF silencing. Incubation of BMdDCs with SCF did not modulate antigen presentation ability of these cells, nor it did regulate their membrane expression of the chemokine receptor CXCR4. We conclude that the SCF/c-kit-mediated prosurvival circuit may have been overlooked because of the prominent use of GM-CSF in DC cultures in vitro, including those human DC cultures destined for the clinics. We speculate that DCs more prominently rely on SCF in vivo in some microenvironments, with potential implications for graft-versus-host disease and antitumor immunity

Antigen-driven T cell-macrophage interactions mediate the interface between innate and adaptive immunity in histidyl-tRNA synthetase-induced myositis

IntroductionPrevious work in humans has demonstrated that both innate and adaptive immune signaling pathways contribute to the pathogenesis of idiopathic inflammatory myopathy (IIM), a systemic autoimmune disease targeting muscle as well as extra-muscular organs. To better define interactive signaling networks in IIM, we characterized the cellular phenotype and transcriptomic profiles of muscle-infiltrating cells in our established murine model of histidyl-tRNA synthetase (HRS)-induced myositis.MethodsMyositis was induced in wild type (WT) and various congenic/mutant strains of C57BL/6 mice through intramuscular immunization with recombinant HRS. Histopathological, immunohistochemical, flow cytometric, and transcriptomic assessments were used to characterize the functional relationship between muscle-infiltrating cell populations in these strains lacking different components of innate and/or adaptive immune signaling.ResultsRAG1 KO mice developed markedly reduced muscle inflammation relative to WT mice, demonstrating a key requirement for T cells in driving HRS-induced myositis. While the reduction of mononuclear cell infiltrates in CD4-Cre.MyD88fl/fl conditional knockout mice and OT-II TCR transgenic mice highlighted roles for both innate and TCR-mediated/adaptive immune signaling in T cells, diminished inflammation in Lyz2-Cre.MyD88fl/fl conditional knockout mice underscored the importance of macrophage/myeloid cell populations in supporting T cell infiltration. Single cell RNA sequencing-based clustering of muscle-infiltrating subpopulations and associated pathway analyses showed that perturbations of T cell signaling/function alter the distribution and phenotype of macrophages, fibroblasts, and other non-lymphoid cell populations contributing to HRS-induced myositis.DiscussionOverall, HRS-induced myositis reflects the complex interplay between multiple cell types that collectively drive a TH1-predominant, pro-inflammatory tissue phenotype requiring antigen-mediated activation of both MyD88- and TCR-dependent T cell signaling pathways

Eicosanoid control over antigen presenting cells in asthma

Asthma is a common lung disease affecting 300 million people worldwide. Allergic asthma is recognized as a prototypical Th2 disorder, orchestrated by an aberrant adaptive CD4+ T helper (Th2/Th17) cell immune response against airborne allergens, that leads to eosinophilic inflammation, reversible bronchoconstriction, and mucus overproduction. Other forms of asthma are controlled by an eosinophil-rich innate ILC2 response driven by epithelial damage, whereas in some patients with more neutrophilia, the disease is driven by Th17 cells. Dendritic cells (DCs) and macrophages are crucial regulators of type 2 immunity in asthma. Numerous lipid mediators including the eicosanoids prostaglandins and leukotrienes influence key functions of these cells, leading to either pro- or anti-inflammatory effects on disease outcome. In this review, we will discuss how eicosanoids affect the functions of DCs and macrophages in the asthmatic lung and how this leads to aberrant T cell differentiation that causes disease

Rapid Host Defense against Aspergillus fumigatus Involves Alveolar Macrophages with a Predominance of Alternatively Activated Phenotype

The ubiquitous fungus Aspergillus fumigatus is associated with chronic diseases such as invasive pulmonary aspergillosis in immunosuppressed patients and allergic bronchopulmonary aspergillosis (ABPA) in patients with cystic fibrosis or severe asthma. Because of constant exposure to this fungus, it is critical for the host to exercise an immediate and decisive immune response to clear fungal spores to ward off disease. In this study, we observed that rapidly after infection by A. fumigatus, alveolar macrophages predominantly express Arginase 1 (Arg1), a key marker of alternatively activated macrophages (AAMs). The macrophages were also found to express Ym1 and CD206 that are also expressed by AAMs but not NOS2, which is expressed by classically activated macrophages. The expression of Arg1 was reduced in the absence of the known signaling axis, IL-4Rα/STAT6, for AAM development. While both Dectin-1 and TLR expressed on the cell surface have been shown to sense A. fumigatus, fungus-induced Arg1 expression in CD11c+ alveolar macrophages was not dependent on either Dectin-1 or the adaptor MyD88 that mediates intracellular signaling by most TLRs. Alveolar macrophages from WT mice efficiently phagocytosed fungal conidia, but those from mice deficient in Dectin-1 showed impaired fungal uptake. Depletion of macrophages with clodronate-filled liposomes increased fungal burden in infected mice. Collectively, our studies suggest that alveolar macrophages, which predominantly acquire an AAM phenotype following A. fumigatus infection, have a protective role in defense against this fungus

ILC2s—Trailblazers in the Host Response Against Intestinal Helminths

Group 2 innate lymphoid cells (ILC2s) were first discovered in experimental studies of intestinal helminth infection—and much of our current knowledge of ILC2 activation and function is based on the use of these models. It is perhaps not surprising therefore that these cells have also been found to play a key role in mediating protection against these large multicellular parasites. ILC2s have been intensively studied over the last decade, and are known to respond quickly and robustly to the presence of helminths—both by increasing in number and producing type 2 cytokines. These mediators function to activate and repair epithelial barriers, to recruit other innate cells such as eosinophils, and to help activate T helper 2 cells. More recent investigations have focused on the mechanisms by which the host senses helminth parasites to activate ILC2s. Such studies have identified novel stromal cell types as being involved in this process—including intestinal tuft cells and enteric neurons, which respond to the presence of helminths and activate ILC2s by producing IL-25 and Neuromedin, respectively. In the current review, we will outline the latest insights into ILC2 activation and discuss the requirement for—or redundancy of—ILC2s in providing protective immunity against intestinal helminth parasites