Mast cell chymase protects against acute ischemic kidney injury by limiting neutrophil hyperactivation and recruitment.

Here we investigated the role of murine mast cell protease 4 (MCPT4), the functional counterpart of human mast cell chymase, in an experimental model of renal ischemia reperfusion injury, a major cause of acute kidney injury. MCPT4-deficient mice had worsened kidney function compared to wildtype mice. MCPT4 absence exacerbated pathologic neutrophil infiltration in the kidney and increased kidney myeloperoxidase expression, cell death and necrosis. In kidneys with ischemia reperfusion injury, when compared to wildtype mice, MCPT4-deficient mice showed increased surface expression of adhesion molecules necessary for leukocyte extravasation including neutrophil CD162 and endothelial cell CD54. In vitro, human chymase mediated the cleavage of neutrophil expressed CD162 and also CD54, P- and E-Selectin expressed on human glomerular endothelial cells. MCPT4 also dampened systemic neutrophil activation after renal ischemia reperfusion injury as neutrophils expressed more CD11b integrin and produced more reactive oxygen species in MCPT4-deficient mice. Accordingly, after renal injury, neutrophil migration to an inflammatory site distal from the kidney was increased in MCPT4-deficient versus wildtype mice. Thus, contrary to the described overall aggravating role of mast cells, one granule-released mediator, the MCPT4 chymase, exhibits a potent anti-inflammatory function in renal ischemia reperfusion injury by controlling neutrophil extravasation and activation thereby limiting associated damage.

Mast Cells and MCPT4 Chymase Promote Renal Impairment after Partial Ureteral Obstruction.

Obstructive nephropathy constitutes a major cause of pediatric renal progressive disease. The mechanisms leading to disease progression are still poorly understood. Kidney fibrotic lesions are reproduced using a model of partial unilateral ureteral obstruction (pUUO) in newborn mice. Based on data showing significant mast cell (MC) infiltration in patients, we investigated the role of MC and murine MCPT4, a MC-released chymase, in pUUO using MC- (Wsh/sh), MCPT4-deficient (Mcpt4-/-), and wild-type (WT) mice. Measurement of kidney length and volume by magnetic resonance imaging (MRI) as well as postmortem kidney weight revealed hypotrophy of operated right kidneys (RKs) and compensatory hypertrophy of left kidneys. Differences between kidneys were major for WT, minimal for Wsh/sh, and intermediate for Mcpt4-/- mice. Fibrosis development was focal and increased only in WT-obstructed kidneys. No differences were noticed for local inflammatory responses, but serum CCL2 was significantly higher in WT versus Mcpt4-/- and Wsh/sh mice. Alpha-smooth muscle actin (αSMA) expression, a marker of epithelial-mesenchymal transition (EMT), was high in WT, minimal for Wsh/sh, and intermediate for Mcpt4-/- RK. Supernatants of activated MC induced αSMA in co-culture experiments with proximal tubular epithelial cells. Our results support a role of MC in EMT and parenchyma lesions after pUUO involving, at least partly, MCPT4 chymase. They confirm the importance of morphologic impairment evaluation by MRI in pUUO.

Kinesin-1 controls mast cell degranulation and anaphylaxis through PI3K-dependent recruitment to the granular Slp3/Rab27b complex.

Cross-linking of mast cell (MC) IgE receptors (FcεRI) triggers degranulation of secretory granules (SGs) and the release of many allergic and inflammatory mediators. Although degranulation depends crucially on microtubule dynamics, the molecular machinery that couples SGs to microtubule-dependent transport is poorly understood. In this study, we demonstrate that mice lacking Kif5b (the heavy chain of kinesin-1) in hematopoietic cells are less sensitive to IgE-mediated, passive, systemic anaphylaxis. After IgE-induced stimulation, bone marrow-derived MCs from Kif5b knockout mice exhibited a marked reduction in SG translocation toward the secretion site. In contrast, a lack of Kif5b did not affect cytokine secretion, early FcεRI-initiated signaling pathways, or microtubule reorganization upon FcεRI stimulation. We identified Slp3 as the critical effector linking kinesin-1 to Rab27b-associated SGs. Kinesin-1 recruitment to the Slp3/Rab27b effector complex was independent of microtubule reorganization but occurred only upon stimulation requiring phosphatidylinositol 3-kinase (PI3K) activity. Our findings demonstrate that PI3K-dependent formation of a kinesin-1/Slp3/Rab27b complex is critical for the microtubule-dependent movement of SGs required for MC degranulation.

Superparamagnetic nanoparticles as vectors for inner ear treatments: driving and toxicity evaluation.

Conclusion Super paramagnetic nanoparticles (MNP) are a promising vector to achieve controlled drug delivery into the cochlea. Objective The goal of the study was to evaluate the toxicological risk of MNP upon the inner ear. Methods Fe3O4-MNP displacement was studied in various catheter materials, shape, and solvent with a local magnetic field. EC5V cells (derived from the inner ear) were cultured with MNP (100 and 500 nm) at various concentrations or without MNP. Cell survival was assessed with a flow cytometry analysis. Localization of MNP within the cells was studied with confocal microscopy. In vivo, a single intra-cochlear administration of 200 nm MNP (3 × 10(10)MNP/mL, n = 8; 1.5 × 10(12) MNP/mL, n = 6) or saline (n = 14) was performed in guinea pigs. Hearing thresholds were assessed with auditory brainstem responses at Day 7. Results MNP could be concentrated at different locations of the catheter with sequential activation of solenoids. MNP were internalized in the cytoplasm, but not in the nuclei nor in endosomes at 48 h. After 48 h of incubation, no difference for cell survival between the groups was observed, whatever the MNP concentration. A size effect was observed with less survival in the 100 nm group. In guinea pigs at day 7, hearing threshold shift was not different in the three groups.

Mast cells in renal inflammation and fibrosis: lessons learnt from animal studies.

Mast cells are hematopoietic cells involved in inflammation and immunity and have been recognized also as important effector cells in kidney inflammation. In humans, only a few mast cells reside in kidneys constitutively but in progressive renal diseases their numbers increase substantially representing an essential part of the interstitial infiltrate of inflammatory cells. Recent data obtained in experimental animal models have emphasized a complex role of these cells and the mediators they release as they have been shown both to promote, but also to protect from disease and fibrosis development. Sometimes conflicting results have been reported in similar models suggesting a very narrow window between these activities depending on the pathophysiological context. Interestingly in mice, mast cell or mast cell mediator specific actions became also apparent in the absence of significant mast cell kidney infiltration supporting systemic or regional actions via draining lymph nodes or kidney capsules. Many of their activities rely on the capacity of mast cells to release, in a timely controlled manner, a wide range of inflammatory mediators, which can promote anti-inflammatory actions and repair activities that contribute to healing, but in some circumstances or in case of inappropriate regulation may also promote kidney disease.

Vesicular trafficking and signaling for cytokine and chemokine secretion in mast cells.

Upon activation mast cells (MCs) secrete numerous inflammatory compounds stored in their cytoplasmic secretory granules by a process called anaphylactic degranulation, which is responsible for type I hypersensitivity responses. Prestored mediators include histamine and MC proteases but also some cytokines and growth factors making them available within minutes for a maximal biological effect. Degranulation is followed by the de novo synthesis of lipid mediators such as prostaglandins and leukotrienes as well as a vast array of cytokines, chemokines, and growth factors, which are responsible for late phase inflammatory responses. While lipid mediators diffuse freely out of the cell through lipid bilayers, both anaphylactic degranulation and secretion of cytokines, chemokines, and growth factors depends on highly regulated vesicular trafficking steps that occur along the secretory pathway starting with the translocation of proteins to the endoplasmic reticulum. Vesicular trafficking in MCs also intersects with endocytic routes, notably to form specialized cytoplasmic granules called secretory lysosomes. Some of the mediators like histamine reach granules via specific vesicular monoamine transporters directly from the cytoplasm. In this review, we try to summarize the available data on granule biogenesis and signaling events that coordinate the complex steps that lead to the release of the inflammatory mediators from the various vesicular carriers in MCs.

Munc18-2 and syntaxin 3 control distinct essential steps in mast cell degranulation.

Mast cell degranulation requires N-ethylmaleimide-sensitive factor attachment protein receptors (SNARE) and mammalian uncoordinated18 (Munc18) fusion accessory proteins for membrane fusion. However, it is still unknown how their interaction supports fusion. In this study, we found that small interfering RNA-mediated silencing of the isoform Munc18-2 in mast cells inhibits cytoplasmic secretory granule (SG) release but not CCL2 chemokine secretion. Silencing of its SNARE-binding partner syntaxin 3 (STX3) also markedly inhibited degranulation, whereas combined knockdown produced an additive inhibitory effect. Strikingly, while Munc18-2 silencing impaired SG translocation, silencing of STX3 inhibited fusion, demonstrating unique roles of each protein. Immunogold studies showed that both Munc18-2 and STX3 are located on the granule surface, but also within the granule matrix and in small nocodazole-sensitive clusters of the cytoskeletal meshwork surrounding SG. After stimulation, clusters containing both effectors were detected at fusion sites. In resting cells, Munc18-2, but not STX3, interacted with tubulin. This interaction was sensitive to nocodazole treatment and decreased after stimulation. Our results indicate that Munc18-2 dynamically couples the membrane fusion machinery to the microtubule cytoskeleton and demonstrate that Munc18-2 and STX3 perform distinct, but complementary, functions to support, respectively, SG translocation and membrane fusion in mast cells.

Mast cell chymase protects against renal fibrosis in murine unilateral ureteral obstruction.

Mast cell release of chymase is important in tissue remodeling and may participate in inflammation leading to fibrosis and organ failure. Here we analyzed the function of chymase in unilateral ureteral obstruction, an established accelerated model of renal tubulointerstitial fibrosis. Mice deficient in mouse mast cell protease 4 (mMCP4), the functional counterpart of human chymase, had increased obstruction-induced fibrosis when compared to wild-type mice indicating a protective effect of mMCP4. Engraftment of mast cell-deficient Kit(Wsh/Wsh) mice with wild type, but not mMCP4-deficient mast cells, restored protection confirming the role of mMCP4. Kidneys of mMCP4-deficient mice had higher levels of renal tubular damage, interstitial fibrosis, collagen deposition, increased α-smooth muscle actin, and decreased E-cadherin expression compared to the kidneys of wild-type mice. Further analysis showed an elevated inflammatory response in mMCP4-deficient mice with increased levels of kidney-infiltrating macrophages and T cells and local profibrotic TGF-ß1 and CCL2. Granulated and degranulated mast cells and mMCP4 were mainly found in the kidney capsule, respectively, before and after ureteral obstruction. Analysis of mMCP4 substrates showed that it mediates its anti-fibrotic actions by degrading interstitial deposits of fibronectin, a known promoter of inflammatory cell infiltration and adhesion. Thus, mast cell released mMCP4 has anti-fibrotic potential in acutely induced obstructive nephropathy.

Critical role of the neutrophil-associated high-affinity receptor for IgE in the pathogenesis of experimental cerebral malaria.

The role of the IgE-FcεRI complex in malaria severity in Plasmodium falciparum-hosting patients is unknown. We demonstrate that mice genetically deficient for the high-affinity receptor for IgE (FcεRIα-KO) or for IgE (IgE-KO) are less susceptible to experimental cerebral malaria (ECM) after infection with Plasmodium berghei (PbANKA). Mast cells and basophils, which are the classical IgE-expressing effector cells, are not involved in disease as mast cell-deficient and basophil-depleted mice developed a disease similar to wild-type mice. However, we show the emergence of an FcεRI(+) neutrophil population, which is not observed in mice hosting a non-ECM-inducing PbNK65 parasite strain. Depletion of this FcεRI(+) neutrophil population prevents ECM, whereas transfer of this population into FcεRIα-KO mice restores ECM susceptibility. FcεRI(+) neutrophils preferentially home to the brain and induce elevated levels of proinflammatory cytokines. These data define a new pathogenic mechanism of ECM and implicate an FcεRI-expressing neutrophil subpopulation in malaria disease severity.

Inhibitory ITAM signaling traps activating receptors with the phosphatase SHP-1 to form polarized "inhibisome" clusters.

The ability of immunoreceptor tyrosine-based activation motif (ITAM)-bearing receptors to inhibit, rather than activate, signaling by other receptors is a regulatory mechanism of immune homeostasis. However, it remains unclear how inhibitory ITAM (ITAMi) receptor signaling and Src homology 2 (SH2) domain-containing phosphatase-1 (SHP-1), which is recruited to ITAMs, target multiple heterologous activating responses without coaggregating with the associated activating receptors. We found that ITAMi signaling triggered by the binding of monomeric ligands to the type I immunoglobulin A (IgA) Fc receptor (FcαRI) induced its dynamic cosegregation with heterologous activating receptors, signaling effectors, and the inhibitory phosphatase SHP-1 into polarized intracellular clusters that we call "inhibisomes." Formation of inhibisomes was preceded by the recruitment of FcαRI and SHP-1 into lipid rafts. Cosegregation required the depolymerization of actin, which depended on SHP-1, and inhibisome formation was abolished by knockdown of SHP-1 and by actin-depolymerizing drugs. Thus, SHP-1- and actin depolymerization-dependent spatiotemporal compartmentalization of ITAMi-containing receptors into lipid rafts, regions associated with intracellular signaling, represents a key event in the integration of ITAMi-mediated inhibitory signals.

Mouse mast cell protease-4 deteriorates renal function by contributing to inflammation and fibrosis in immune complex-mediated glomerulonephritis.

Mast cells exert protective effects in experimental antiglomerular basement membrane-induced glomerulonephritis (GN), yet the responsible mediators have not been identified. In this study, we investigated the role of mouse mast cell protease (mMCP)-4, the functional homolog of human chymase, using mMCP-4-deficient mice. Compared with wild type animals, mMCP-4-deficient mice exhibited lower proteinuria, blood creatinine, and blood urea nitrogen levels, indicating an aggravating role of mMCP-4. Kidney histology confirmed less severe renal damage in mMCP-4-deficient mice with reduced deposits, glomerular and interstitial cellularity, and fibrosis scores. High amounts of mMCP-4 were detected in renal capsules, but not in the whole kidney, from wild type mice. Its expression in renal capsules was markedly decreased after GN induction, suggesting that locally released enzyme by degranulated mast cells could contribute to the functional and physiopathological hallmarks of GN. Supporting a proinflammatory role, glomerular and interstitial macrophage and T cell infiltration, levels of proinflammatory TNF and MCP-1 mRNA, and the expression of the profibrotic peptide angiotensin II together with type I collagen were markedly downregulated in kidneys of mMCP-4-deficient mice. We conclude that mMCP-4 chymase, contrary to the global anti-inflammatory action of mast cells, aggravates GN by promoting kidney inflammation. These results highlight the complexity of mast cell-mediated inflammatory actions and suggest that chymase inhibition may represent a novel therapeutic target in GN.