Mast Cell Degranulation Exacerbates Skin Rejection by Enhancing Neutrophil Recruitment

Recent evidences indicate an important role of tissue inflammatory responses by innate immune cells in allograft acceptance and survival. Here we investigated the role of mast cells (MC) in an acute male to female skin allograft rejection model using red MC and basophil (RMB) mice enabling conditional MC depletion. Kinetic analysis showed that MCs markedly accelerate skin rejection. They induced an early inflammatory response through degranulation and boosted local synthesis of KC, MIP-2, and TNF. This enhanced early neutrophil infiltration compared to a female-female graft-associated repair response. The uncontrolled neutrophil influx accelerated rejection as antibody-mediated depletion of neutrophils delayed skin rejection. Administration of cromolyn, a MC stabilizer and to a lesser extent ketotifen, a histamine type I receptor antagonist, and absence of MCPT4 chymase also delayed graft rejection. Together our data indicate that mediators contained in secretory granules of MC promote an inflammatory response with enhanced neutrophil infiltration that accelerate graft rejection

Phospholipid scramblase 1 amplifies anaphylactic reactions in vivo.

Mast cells are critical actors of hypersensitivity type I (allergic) reactions by the release of vasoactive and proinflammatory mediators following their activation by aggregation of the high-affinity receptor for immunoglobulin E (FcεRI). We have previously identified Phospholipid Scramblase 1 (PLSCR1) as a new molecular intermediate of FcεRI signaling that amplifies degranulation of the rat mast cell line RBL-2H3. Here we characterized primary mast cells from Plscr1-/- mice. The absence of PLSCR1 expression did not impact mast cell differentiation as evidenced by unaltered FcεRI expression, general morphology, amount of histamine stored and expression of FcεRI signal effector molecules. No detectable mast cell deficiency was observed in Plscr1-/- adult mice. In dose-response and time-course experiments, primary cultures of mast cells (bone marrow-derived mast cells and peritoneal cell-derived mast cells) generated from Plscr1-/- mice exhibited a reduced release of β-hexosaminidase upon FcεRI engagement as compared to their wild-type counterparts. In vivo, Plscr1-/- mice were protected in a model of passive systemic anaphylaxis when compared to wild-type mice, which was consistent with an observed decrease in the amounts of histamine released in the serum of Plscr1-/- mice during the reaction. Therefore, PLSCR1 aggravates anaphylactic reactions by increasing FcεRI-dependent mast cell degranulation. PLSCR1 could be a new therapeutic target in allergy

Basophils contribute to pristane-induced Lupus-like nephritis model

International audienceLupus nephritis (LN), one of the most severe outcomes of systemic lupus erythematosus (SLE), is initiated by glomerular deposition of immune-complexes leading to an inflammatory response and kidney failure. Autoantibodies to nuclear antigens and autoreactive B and T cells are central in SLE pathogenesis. Immune mechanisms amplifying this autoantibody production drive flares of the disease. We previously showed that basophils were contributing to LN development in a spontaneous lupus-like mouse model (constitutive Lyn −/− mice) and in SLE subjects through their activation and migration to secondary lymphoid organs (SLOs) where they amplify autoantibody production. In order to study the basophil-specific mechanisms by which these cells contribute to LN development, we needed to validate their involvement in a genetically independent SLE-like mouse model. Pristane, when injected to non-lupus-prone mouse strains, induces a LN-like disease. In this inducible model, basophils were activated and accumulated in SLOs to promote autoantibody production. Basophil depletion by two distinct approaches dampened LN-like disease, demonstrating their contribution to the pristane-induced LN model. These results enable further studies to decipher molecular mechanisms by which basophils contribute to lupus progression

Transcriptomic Signature and Growth Factor Regulation of Castration-Tolerant Prostate Luminal Progenitor Cells

Background: The molecular and cellular mechanisms that drive castration-resistant prostate cancer (CRPC) remain poorly understood. LSCmed cells defines an FACS-enriched population of castration-tolerant luminal progenitor cells that has been proposed to promote tumorigenesis and CRPC in Pten-deficient mice. The goals of this study were to assess the relevance of LSCmed cells through the analysis of their molecular proximity with luminal progenitor-like cell clusters identified by single-cell (sc)RNA-seq analyses of mouse and human prostates, and to investigate their regulation by in silico-predicted growth factors present in the prostatic microenvironment. Methods: Several bioinformatic pipelines were used for pan-transcriptomic analyses. LSCmed cells isolated by cell sorting from healthy and malignant mouse prostates were characterized using RT-qPCR, immunofluorescence and organoid assays. Results: LSCmed cells match (i) mouse luminal progenitor cell clusters identified in scRNA-seq analyses for which we provide a common 15-gene signature including the previously identified LSCmed marker Krt4, and (ii) Club/Hillock cells of the human prostate. This transcriptional overlap was maintained in cancer contexts. EGFR/ERBB4, IGF-1R and MET pathways were identified as autocrine/paracrine regulators of progenitor, proliferation and differentiation properties of LSCmed cells. The functional redundancy of these signaling pathways allows them to bypass the effect of receptor-targeted pharmacological inhibitors. Conclusions: Based on transcriptomic profile and pharmacological resistance to monotherapies that failed in CRPC patients, this study supports LSCmed cells as a relevant model to investigate the role of castration-tolerant progenitor cells in human prostate cancer progression

Small AntiMicrobial Peptide with In Vivo Activity Against Sepsis

Antimicrobial peptides (AMPs) are considered as potential therapeutic sources of future antibiotics because of their broad-spectrum activities and alternative mechanisms of action compared to conventional antibiotics. Although AMPs present considerable advantages over conventional antibiotics, their clinical and commercial development still have some limitations, because of their potential toxicity, susceptibility to proteases, and high cost of production. To overcome these drawbacks, the use of peptides mimics is anticipated to avoid the proteolysis, while the identification of minimalist peptide sequences retaining antimicrobial activities could bring a solution for the cost issue. We describe here new polycationic -amino acids combining these two properties, that we used to design small dipeptides that appeared to be active against Gram-positive and Gram-negative bacteria, selective against prokaryotic versus mammalian cells, and highly stable in human plasma. Moreover, the in vivo data activity obtained in septic mice reveals that the bacterial killing effect allows the control of the infection and increases the survival rate of cecal ligature and puncture (CLP)-treated mice

Mast Cell Degranulation Exacerbates Skin Rejection by Enhancing Neutrophil Recruitment

Recent evidences indicate an important role of tissue inflammatory responses by innate immune cells in allograft acceptance and survival. Here we investigated the role of mast cells (MC) in an acute male to female skin allograft rejection model using red MC and basophil (RMB) mice enabling conditional MC depletion. Kinetic analysis showed that MCs markedly accelerate skin rejection. They induced an early inflammatory response through degranulation and boosted local synthesis of KC, MIP-2, and TNF. This enhanced early neutrophil infiltration compared to a female-female graft-associated repair response. The uncontrolled neutrophil influx accelerated rejection as antibody-mediated depletion of neutrophils delayed skin rejection. Administration of cromolyn, a MC stabilizer and to a lesser extent ketotifen, a histamine type I receptor antagonist, and absence of MCPT4 chymase also delayed graft rejection. Together our data indicate that mediators contained in secretory granules of MC promote an inflammatory response with enhanced neutrophil infiltration that accelerate graft rejection

Basophils and IgE contribute to mixed connective tissue disease development

International audienceBackground: Mixed connective tissue disease (MCTD) is a rare and complex autoimmune disease that presents mixed features with other connective tissue diseases, such as systemic lupus erythematosus, systemic sclerosis, and myositis. It is characterized by high levels of anti-U1 small nuclear ribonucleoprotein 70k autoantibodies and a high incidence of life-threatening pulmonary involvement. The pathophysiology of MCTD is not well understood, and no specific treatment is yet available for the patients. Basophils and IgE play a role in the development of systemic lupus erythematosus and thus represent new therapeutic targets for systemic lupus erythematosus and other diseases involving basophils and IgE in their pathogenesis.Objective: We sought to investigate the role of basophils and IgE in the pathophysiology of MCTD.Methods: Basophil activation status and the presence of autoreactive IgE were assessed in peripheral blood of a cohort of patients with MCTD and in an MCTD-like mouse model. Basophil depletion and IgE-deficient animals were used to investigate the contribution of basophils and IgE in the lung pathology development of this mouse model.Results: Patients with MCTD have a peripheral basopenia and activated blood basophils overexpressing C-C chemokine receptor 3. Autoreactive IgE raised against the main MCTD autoantigen U1 small nuclear ribonucleoprotein 70k were found in nearly 80% of the patients from the cohort. Basophil activation and IgE anti-U1 small nuclear ribonucleoprotein 70k were also observed in the MCTD-like mouse model along with basophil accumulation in lymph nodes and lungs. Basophil depletion dampened lung pathology, and IgE deficiency prevented its development.Conclusions: Basophils and IgE contribute to MCTD pathophysiology and represent new candidate therapeutic targets for patients with MCTD

PLSCR1 amplifies degranulation <i>in vitro</i> in primary culture of BMMC and PCMC.

<p>Wild-type (WT) and <i>Plscr1</i>^<i>-/-</i> (KO) BMMC (A, C, D) and PCMC (B) were sensitized for 24 hours with anti-DNP IgE. After two washes, the IgE-sensitized cells were stimulated with different doses of specific antigen (DNP-HSA) for 30 minutes (A, B) or at different times to the optimal antigen dose of 10 ng/ml (C). Statistical analysis was done by a Two-way ANOVA followed by Sidak’s multiple comparisons test. Data of n independent experiments with n = 12 (A), n = 5 (B) and n = 6 (C) are presented as mean ± s.e.m. *: P <0.05; **: P <0.01; ***: P <0.001. (D) WT and KO BMMC generated in the presence of IL3 and SCF (black bars) or of IL3 alone (open bars) were stimulated for 30 min with 10 ng/ml DNP-HSA. Data of n independent experiments with n = 7 (IL3 + SCF) and n = 3 (IL3) are presented as mean ± s.e.m. Statistical analysis was done by an unpaired Student <i>t</i> test. **: p < 0.01.</p

Sensitivity of <i>Plscr1</i>^<i>-/-</i> mice to histamine.

<p>Thermal probes were inserted under the dorsal skin of wild-type (WT) and <i>Plscr1</i>^<i>-/-</i> (KO) mice. The following day mice were injected i.v. with 5 μmol histamine. The drop in their body temperature was monitored using a thermal probe reader. Data are presented as ± s.e.m. with n = 6 mice per group (3 experiments). Statistical analysis was done using a two-way ANOVA. ns: non significant.</p

<i>Ex vivo</i> and <i>in vivo</i> analysis of <i>Plscr1</i>^<i>-/-</i> mast cells.

<p>(A) Histamine content of peritoneal mast cells from WT and <i>Plscr1</i>^<i>-/-</i> (KO) mice was measured by EIA and expressed as pg per mast cell. Values obtained for each individual mouse are shown. Data are presented as mean ± s.e.m.. Statistical analysis was done by an unpaired Student <i>t</i> test and showed no significant difference. (B) Number of peritoneal mast cells (PMC). The peritoneal cavity of WT and <i>Plscr1</i>^<i>-/-</i> (KO) mice was washed and the number of PMC per mouse was counted after staining with toluidine blue. Data are presented as mean ± s.e.m.. Statistical analysis done by an unpaired Student <i>t</i> test showed no significant difference. (C) The surface expression of FcεRIα chain by WT and <i>Plscr1</i>^<i>-/-</i> peritoneal mast cells was analyzed by flow cytometry. Blue line: WT, red line: <i>Plscr1</i>^<i>-/-</i>, gray line: isotype controls. Representative data of three independent experiments. (D) Number and tissue localization of <i>Plscr1</i>^<i>-/-</i> mast cells. Left panels: Representative histological analysis of the ear skin and of the stomach submucosa of wild-type (WT), <i>Plscr1</i>^<i>-/-</i> (KO) and Kit^{<i>W-sh/W-sh</i>} (W^sh) mice with toluidine blue staining. Arrowheads: mast cells, stained in purple. Selected mast cells (empty arrowheads) are shown under higher magnification in the insets. Scale bar: 100μm. Right panels: Quantitative analysis of countable mast cells in these tissues using CaloPix software. Data are presented as mean + s.e.m. of four mice. Statistical analysis done by an unpaired Student <i>t</i> test showed no significant difference.</p