OMIP-048 MC: Quantification of calcium sensors and channels expression in lymphocyte subsets by mass cytometry.

Calcium (Ca2+ ) signaling controls T-cell activation and functions. Ca2+ concentrations are locally detected and controlled by Ca2+ -sensors (STIM1 and 2 detecting the depletion from ER stores channels) and Ca2+ -channels (ORAI1-3 in the cell membrane and VDAC1 in the outer mitochondrial membrane). We first validated and titrated antibodies to assess the expression of these Ca2+ -sensors and -channels in human and murine cells, and further devised a 18-antibodies mass cytometry panel to characterize their expression in primary murine lymphocyte subsets.

Decreased expression of the glucocorticoid receptor-GILZ pathway in Kupffer cells promotes liver inflammation in obese mice.

BACKGROUND & AIMS: Kupffer cells (KC) play a key role in the onset of inflammation in non-alcoholic steatohepatitis (NASH). The glucocorticoid receptor (GR) induces glucocorticoid-induced leucine zipper (GILZ) expression in monocytes/macrophages and is involved in several inflammatory processes. We hypothesized that the GR-GILZ axis in KC may contribute to the pathophysiology of obesity-induced liver inflammation. METHODS: By using a combination of primary cell culture, pharmacological experiments, mice deficient for the Gr specifically in macrophages and transgenic mice overexpressing Gilz in macrophages, we explored the involvement of the Gr-Gilz axis in KC in the pathophysiology of obesity-induced liver inflammation. RESULTS: Obesity was associated with a downregulation of the Gr and Gilz, and an impairment of Gilz induction by lipopolysaccharide (LPS) and dexamethasone (DEX) in KC. Inhibition of Gilz expression in isolated KC transfected with Gilz siRNA demonstrated that Gilz downregulation was sufficient to sensitize KC to LPS. Conversely, liver inflammation was decreased in obese transgenic mice specifically overexpressing Gilz in macrophages. Pharmacological inhibition of the Gr showed that impairment of Gilz induction in KC by LPS and DEX in obesity was driven by a downregulation of the Gr. In mice specifically deficient for Gr in macrophages, Gilz expression was low, leading to an exacerbation of obesity-induced liver inflammation. CONCLUSIONS: Obesity is associated with a downregulation of the Gr-Gilz axis in KC, which promotes liver inflammation. The Gr-Gilz axis in KC is an important target for the regulation of liver inflammation in obesity.

CXCR4 dysfunction in non-alcoholic steatohepatitis in mice and patients.

Homing of inflammatory cells to the liver is key in the progression of non-alcoholic steatohepatitis (NASH). An abnormal response of CD4+ T-cells from obese mice to the chemotactic effect of CXCL12 has been reported but the mechanism involved in this process and relevance in patients are unknown. We aimed to explore the mechanism involved in the abnormal chemotaxis of CXC chemokine ligand 12 (CXCL12) in several mouse models of NASH and the relevance in the context of human non-alcoholic fatty liver disease (NAFLD). We assessed chemotactic responsiveness of CD4+ T-cells to CXCL12, the effect of AMD3100, a CXC chemokine receptor 4 (CXCR4) antagonist, in mice and lymphocytes from patients with NAFLD, and the affinity of CXCL12 for CXCR4. CXCL12-promoted migration of CD4+ T-cells from three different mouse models of NASH was increased and dependent of CXCR4. CD4+ T-cells from patients with NASH, but not from patients with pure steatosis, responded more strongly to the chemotactic effect of CXCL12, and this response was inhibited by AMD3100. Treatment with AMD3100 decreased the number of CD4+ T-cells to the liver in ob/ob mice. CXCL12 expression in the liver, CXCR4 and CXCR7 expression in CD4+ T-cells were not increased in three different mouse models of NASH. However, the affinity of CXCL12 for CXCR4 was increased in CD4+ T-cells of ob/ob mice. In conclusion, the CXCL12/CXCR4 pathway contributes in both mice and patients to the enhanced recruitment of CD4+ T-cells in NASH. An increased affinity of CXCL12 to CXCR4 rather than a higher expression of the chemokine or its receptors is involved in this process.

Interleukin-24 inhibits the plasma cell differentiation program in human germinal center B cells.

Complex molecular mechanisms control B-cell fate to become a memory or a plasma cell. Interleukin-24 (IL-24) is a class II family cytokine of poorly understood immune function that regulates the cell cycle. We previously observed that IL-24 is strongly expressed in leukemic memory-type B cells. Here we show that IL-24 is also expressed in human follicular B cells; it is more abundant in CD27(+) memory B cells and CD5-expressing B cells, whereas it is low to undetectable in centroblasts and plasma cells. Addition of IL-24 to B cells, cultured in conditions shown to promote plasma cell differentiation, strongly inhibited plasma cell generation and immunoglobulin G (IgG) production. By contrast, IL-24 siRNA increased terminal differentiation of B cells into plasma cells. IL-24 is optimally induced by BCR triggering and CD40 engagement; IL-24 increased CD40-induced B-cell proliferation and modulated the transcription of key factors involved in plasma cell differentiation. It also inhibited activation-induced tyrosine phosphorylation of signal transducer and activator of transcription-3 (STAT-3), and inhibited the transcription of IL-10. Taken together, our results indicate that IL-24 is a novel cytokine involved in T-dependent antigen (Ag)-driven B-cell differentiation and suggest its physiologic role in favoring germinal center B-cell maturation in memory B cells at the expense of plasma cells.

IL-24 induces apoptosis of chronic lymphocytic leukemia B cells engaged into the cell cycle through dephosphorylation of STAT3 and stabilization of p53 expression.

Chronic lymphocytic leukemia (CLL) is characterized by the accumulation of long-lived monoclonal B cells mostly arrested at the G(0)/G(1) phase of the cell cycle. CLL cells strongly express intracellular melanoma differentiation-associated gene-7 (MDA7)/IL-24. However, adenovirus-delivered MDA7 was reported to be cytotoxic in several tumor cell lines. We report herein that rIL-24 alone had no effect; however, sequential incubation with rIL-2 and rIL-24 reduced thymidine incorporation by 50% and induced apoptosis of CLL cells in S and G(2)/M phases of the cell cycle, but not of normal adult blood or tonsil B cells. IL-24 stimulated STAT3 phosphorylation in IL-24R1-transfected cells but not in normal or CLL B cells. In contrast, IL-24 reversed the IL-2-induced phosphorylation of STAT3 in CLL, and this effect was neutralized by anti-IL-24 Ab. Phospho- (P)STAT3 inhibition induced by IL-24 was reversed by pervanadate, an inhibitor of tyrosine phosphatases. The addition of rIL-24 to IL-2-activated CLL B cells resulted in increases of transcription, protein synthesis. and phosphorylation of p53. The biological effects of IL-24 were reversed by the p53 inhibitor pifithrin-alpha and partly by the caspase inhibitor zvad. Troglitazone (a protein tyrosine phosphatase, PTP1B activator) phosphatase inhibited PSTAT3 and augmented p53 expression. PSTAT3 is a transcriptional repressor of p53, and therefore IL-24 induction of p53 secondary to PSTAT3 dephosphorylation may be sensed as a stress signal and promote apoptosis in cycling cells. This model explains why IL-24 can protect some resting/differentiated cells and be deleterious to proliferating cells.

Plasmacytoid dendritic cell reconstitution following bone marrow transplantation: subnormal recovery and functional deficit of IFN-alpha/beta production in response to herpes simplex virus.

Infections with herpesviruses were frequent after bone marrow transplantation (BMT) before the preventive use of antiviral drugs, suggesting a deficit of innate immunity. A retrospective phenotypical and functional study was carried out on 25 patients 1-36 months after allogeneic BMT. Leukocyte counts followed a normal reconstitution, including natural killer (NK) cells and monocytes. Plasmacytoid dendritic cell (PDC) counts increased steadily, although they remained below normal values after 2 years. Most patients produced less interferon- alpha/beta (IFN-alphabeta) in vitro than healthy controls after infection with herpes simplex virus type 1 (HSV-1), whereas they responded normally to Sendai virus (SV). In addition, 6 patients had biologic signs of infection with herpesviruses, confirming a specific immunologic deficit against these viruses. IFN production was not correlated to PDC counts or to the occurrence of graft-versus-host disease (GVHD). Because all patients were under immunosuppressive treatment, we investigated the effect of drugs on IFN production by mononuclear cells. Glucocorticoids and cyclosporine A inhibited IFN production by infected leukocytes, with a predominant action on HSV-1-infected PDC. The inability of transplanted patients to mount an efficient immune response to herpesviruses may be partly related to drug toxicity toward cells of the innate immune system.

Ectosomes from neutrophil-like cells down-regulate nickel-induced dendritic cell maturation and promote Th2 polarization.

DCs are the first immune cells to be exposed to allergens, including chemical sensitizers, such as nickel, a human TLR4 agonist that induces DC maturation. In ACD, DCs can interact with PMNs that are recruited and activated, leading, in particular, to ectosome release. The objective of this work was to characterize the effects of PMN-Ect on DC functions in an ACD context. We first developed a standardized protocol to produce, characterize, and quantify ectosomes by use of human PLB-985 cells, differentiated into mature PMN (PLB-Ect). We then studied the in vitro effects of these purified ectosomes on human moDC functions in response to NiSO4 and to LPS, another TLR4 agonist. Confocal fluorescence microscopy showed that PLB-Ect was internalized by moDCs and localized in the lysosomal compartment. We then showed that PLB-Ect down-regulated NiSO4-induced moDC maturation, as witnessed by decreased expression of CD40, CD80, CD83, CD86, PDL-1, and HLA-DR and by decreased levels of IL-1ß, IL-6, TNF-α, and IL-12p40 mRNAs. These effects were related to p38MAPK and NF-κB down-regulation. However, no increase in pan-regulatory DC marker genes (GILZ, CATC, C1QA) was observed; rather, levels of effector DC markers (Mx1, NMES1) were increased. Finally, when these PLB-Ect + NiSO4-treated moDCs were cocultured with CD4(+) T cells, a Th2 cytokine profile seemed to be induced, as shown, in particular, by enhanced IL-13 production. Together, these results suggest that the PMN-Ect can modulate DC maturation in response to nickel, a common chemical sensitizer responsible for ADC.

Type I interferon production by plasmacytoid dendritic cells and monocytes is triggered by viruses, but the level of production is controlled by distinct cytokines.

The principal interferon-alpha/beta (IFN-I)-producing cells are plasmacytoid dendritic cell (PDC) precursors belonging to the lymphoid lineage. Monocytes that can differentiate into dendritic cells (DC) also produce IFN-I, although much less than PDC, after interaction with infectious agents. We show that whereas viruses trigger these cells to produce IFN-I, the amount of IFN is tightly controlled by cytokines. Monocytes produced IFN-I in response to Sendai virus (SV) infection, and PDC responded to both SV and herpes simplex virus (HSV). All cytokines tested failed to induce production of IFN-I in the absence of infection. However, among 18 relevant cytokines, incubation of PDC with interleukin-4 (IL-4), IL-15, and IL-7 alone or in combination with IL-3 before infection, enhanced IFN-I secretion. At variance, IL-12 alone or in synergy with granulocyte-macrophage colony-stimulating factor (GM-CSF) was active on SV-infected but not on HSV-infected monocytes. Tumor necrosis factor-alpha (TNF-alpha) and IL-4 inhibited IFN-I production by PDC and monocytes, respectively, and IL-10 strongly inhibited IFN-I production in both cell lineages. The response of PDC to IL-7 and IL-15, which also activate natural killer (NK) cell maturation, further emphasizes the cooperation between these two cell subsets in the control of innate immunity.

Natural phosphorylation of CD5 in chronic lymphocytic leukemia B cells and analysis of CD5-regulated genes in a B cell line suggest a role for CD5 in malignant phenotype.

Chronic lymphocytic leukemia (CLL) results in the accumulation of B cells, presumably reflecting the selection of malignant cell precursors with Ag combined with complex alterations in protein activity. Repeated BCR stimulation of normal B cells leads to anergy and CD5 expression, both of which are features of CLL. Because CD5 is phosphorylated on tyrosine following BCR engagement and negatively regulates BCR signaling in normal B cells, we investigated its phosphorylation status and found it to be naturally phosphorylated on tyrosine but not on serine residues in CLL samples. To analyze the role of CD5, we established a B cell line in which CD5 is phosphorylated. Gene profiling of vector vs CD5-transfected B cells pointed out gene groups whose expression was enhanced: Apoptosis inhibitors (BCL2), NF-kappaB (RELB, BCL3), Wnt, TGFbeta, VEGF, MAPKs, Stats, cytokines, chemokines (IL-10, IL-10R, IL-2R, CCL-3, CCL-4, and CCR7), TLR-9, and the surface Ags CD52, CD54, CD70, and CD72. Most of these gene groups are strongly expressed in CLL B cells as compared with normal B cells. Unexpectedly, metabolic pathways, namely cholesterol synthesis and adipogenesis, are also enhanced by CD5. Conversely, CD5 inhibited genes involved in RNA splicing and processing, ribosome biogenesis, proteasome, and CD80 and CD86 Ags, whose expression is low in CLL. Comparison of CD5- vs tailless CD5-transfected cells further demonstrated the role of CD5 phosphorylation in the regulation of selected genes. These results support a model where CLL cells are chronically stimulated, leading to CD5 activation and cell survival. In addition to CD5 itself, we point to several CD5-induced genes as potential therapeutic targets.

Cyclosporin-A inhibits ERK phosphorylation in B cells by modulating the binding of Raf protein to Bcl2.

Extracellular signal-related kinase (ERK) signaling is regulated by sequential phosphorylation of upstream kinases including Raf. We report herein that ERK phosphorylation is inhibited by a short incubation with Cyclosporin-A (CsA) in anti-IgM activated Daudi B cells. As Bcl2, through its BH4 domain, was previously shown to bind both Calcineurin (Can) and Raf proteins, we hypothesized that CsA inhibited Can binding to Bcl2 allowing the latter to bind more Raf at the mitochondria thereby diverting it from activating the ERK cascade. In support of this less Bcl2 coprecipitated with Can heterodimer in total lysates of cells treated with CsA as compared to controls. In parallel, Raf1 was augmented in both the mitochondrial fractions of cells treated with CsA and in Bcl2 immunoprecipitates under CsA. Finally, introduction of a Bcl2 BH4 domain into Daudi cells augmented ERK phosphorylation in unstimulated cells and this augmentation was unsensitive to CsA. We therefore suggest that CsA indirectly inhibited ERK activation through sequestration of Raf1, at the mitochondria.

Human CD5 promotes B-cell survival through stimulation of autocrine IL-10 production.

CD5 is a negative regulator of B-cell receptor (BCR) signaling that is up-regulated after BCR stimulation and likely contributes to B-cell tolerance in vivo. However, CD5 is constitutively expressed on the B-1 subset of B cells. Contrary to CD5(-) B-2 B cells, B-1 B cells are long-lived because of autocrine interleukin-10 (IL-10) production through unknown mechanisms. We demonstrate herein a direct relationship between CD5 expression and IL-10 production. Human peripheral blood CD5(+) B cells produce more IL-10 than CD5(-) B cells after BCR activation. Introducing CD5 into CD5(-) B cells induces the production of IL-10 by activating its promoter and the synthesis of its mRNA. The cytoplasmic domain of CD5 is sufficient for this process. CD5 also protects normal human B cells from apoptosis after BCR stimulation while reducing the BCR-induced Ca(2+) response. We conclude that CD5 supports the survival of B cells by stimulating IL-10 production and by concurrently exerting negative feedback on BCR-induced signaling events that can promote cell death.

CD5-negative regulation of B cell receptor signaling pathways originates from tyrosine residue Y429 outside an immunoreceptor tyrosine-based inhibitory motif.

CD5 is a cell surface receptor that negatively regulates B cell function, but whose relationship to the immunoreceptor tyrosine-based inhibitory motif (ITIM) family of B cell inhibitory receptors is unclear. Using Fcgamma type IIB receptor-CD5 chimeras encompassing the cytoplasmic domain of CD5, we previously showed that a particular region of the molecule containing two tyrosine residues, Y429 and Y441, in an amino acid stretch similar to the Src autophosphorylation motif and a putative ITIM, respectively, antagonized early signaling events triggered through the B cell receptor (BCR). In this study, we provide evidences that only Y429 is mandatory for the inhibition by CD5 of the calcium response activated via the BCR. This residue also efficiently controls inhibition of the Ras/extracellular signal-related kinase-2 pathway. Analyzing the membrane translocation of the AKT protooncogene using its 3'-phosphoinositide-specific pleckstrin homology domain fused to the green fluorescent protein as a probe, we also show that CD5 strongly impairs its cellular redistribution and demonstrate the role played by Y429 in this process. We finally report that Y429 controls almost exclusively CD5 phosphorylation as well as inhibition of BCR-triggered IL-2 production upon coaggregation of the two receptors. Thus, CD5 uses an ITIM-independent strategy, centered on Y429, the major tyrosine-phosphorylated residue in its cytoplasmic domain, to inhibit BCR activation.