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N-Formyl Methionine Peptide-Mediated Neutrophil Activation in Systemic Sclerosis.

ABSTRACT: Exaggerated neutrophil activation and formation of neutrophil extracellular traps (NETs) are reported in systemic sclerosis (SSc) but its involvement in SSc pathogenesis is not clear. In the present study we assessed markers of neutrophil activation and NET formation in SSc patients in relation to markers of inflammation and disease phenotype. Factors promoting neutrophil activation in SSc remain largely unknown. Among the neutrophil activating factors, mitochondrial-derived N-formyl methionine (fMet) has been reported in several autoinflammatory conditions. The aim of the current study is to assess whether SSc patients have elevated levels of fMet and the role of fMet in neutrophil-mediated inflammation on SSc pathogenesis. Markers of neutrophil activation (calprotectin, NETs) and levels of fMet were analyzed in plasma from two SSc cohorts (n=80 and n=20, respectively) using ELISA. Neutrophil activation assays were performed in presence or absence of formyl peptide receptor 1 (FPR1) inhibitor cyclosporin H. Elevated levels of calprotectin and NETs were observed in SSc patients as compared to healthy controls (p<0.0001) associating with SSc clinical disease characteristics. Further, SSc patients had elevated levels of circulating fMet as compared to healthy controls (p<0.0001). Consistent with a role for fMet-mediated neutrophil activation, fMet levels correlated with levels of calprotectin and NETs (r=0.34, p=0.002; r=0.29, p<0.01 respectively). Additionally, plasma samples from SSc patients with high levels of fMet induced de novo neutrophil activation through FPR1-dependent mechanisms. Our data for the first time implicates an important role for the mitochondrial component fMet in promoting neutrophil-mediated inflammation in SSc.

SUBMITTER: Kuley R

PROVIDER: S-EPMC8766990 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Essentials The role of formyl peptide receptor 1 (FPR1) and its ligand, fMLF, in the regulation of platelet function, hemostasis, and thrombosis is largely unknown. Fpr1-deficient mice and selective inhibitors for FPR1 were used to investigate the function of fMLF and FPR1 in platelets. N-formyl-methionyl-leucyl-phenylalanine primes platelet activation and augments thrombus formation, mainly through FPR1 in platelets. Formyl peptide receptor 1 plays a pivotal role in the regulation of platelet function.BackgroundFormyl peptide receptors (FPRs) play pivotal roles in the regulation of innate immunity and host defense. The FPRs include three family members: FPR1, FPR2/ALX, and FPR3. The activation of FPR1 by its high-affinity ligand, N-formyl-methionyl-leucyl-phenylalanine (fMLF) (a bacterial chemoattractant peptide), triggers intracellular signaling in immune cells such as neutrophils and exacerbates inflammatory responses to accelerate the clearance of microbial infection. Notably, fMLF has been demonstrated to induce intracellular calcium mobilization and chemotaxis in platelets that are known to play significant roles in the regulation of innate immunity and inflammatory responses. Despite a plethora of research focused on the roles of FPR1 and its ligands such as fMLF on the modulation of immune responses, their impact on the regulation of hemostasis and thrombosis remains unexplored.ObjectiveTo determine the effects of fMLF on the modulation of platelet reactivity, hemostasis, and thrombus formation.MethodsSelective inhibitors for FPR1 and Fpr1-deficient mice were used to determine the effects of fMLF and FPR1 on platelets using various platelet functional assays.ResultsN-formyl-methionyl-leucyl-phenylalanine primes platelet activation through inducing distinctive functions and enhances thrombus formation under arterial flow conditions. Moreover, FPR1 regulates normal platelet function as its deficiency in mouse or blockade in human platelets using a pharmacological inhibitor resulted in diminished agonist-induced platelet activation.ConclusionSince FPR1 plays critical roles in numerous disease conditions, its influence on the modulation of platelet activation and thrombus formation may provide insights into the mechanisms that control platelet-mediated complications under diverse pathological settings.

Project description:Systemic sclerosis (SSc) is a chronic autoimmune disease characterized by fibrosis, alteration in the microvasculature and immunologic abnormalities. It has been hypothesized that an abnormal redox state could regulate the persistent fibrotic phenotype in SSc patients. N-Formyl peptide receptors (FPRs) are chemotactic receptors overexpressed in fibroblasts derived from SSc patients. In this study, we demonstrated that stimulation of FPRs promotes the generation of reactive oxygen species (ROS) in skin fibroblasts. In fibroblast cells, ROS production was due to FPRs interaction with the urokinase receptor (uPAR) and to β1 integrin engagement. FPRs cross-talk with uPAR and integrins led to Rac1 and ERKs activation. FPRs stimulation increased gp91phox and p67phox expression as well as the direct interaction between GTP-Rac1 and p67phox, thus promoting assembly and activation of the NADPH oxidase complex. FPRs functions occur through interaction with a specific domain of uPAR (residues 88SRSRY92) that can be exposed on the cell membrane by protease-mediated receptor cleavage. Immunohistochemistry analysis with a specific anti-SRSRY antibody showed increased expression of uPAR in a cleaved form, which exposes the SRSRY sequence at its N-terminus (DIIDIII-uPAR88-92) in skin biopsies from SSc patients. As expected by the increased expression of both FPRs and DII-DIII-uPAR88-92, fibroblasts derived from SSc patients showed a significantly increase in ROS generation both at a basal level than after FPRs stimulation, as compared to fibroblasts from normal subjects. C37, a small molecule blocking the interaction between FPRs and uPAR, and selumetinib, a clinically approved MAPKK/ERK inhibitor, significantly inhibited FPRs-mediated ROS production in fibroblasts derived from SSc patients. Thus, FPRs, through the interaction with the uPA/uPAR system, can induce ROS generation in fibroblasts by activating the NADPH oxidase, playing a role in the alteration of the redox state observed in SSc.

Project description:IntroductionPreviously, we discovered similar esophageal gene expression patterns in patients with systemic sclerosis (SSc) and eosinophilic esophagitis (EoE) where eosinophil/mast cell-targeted therapies are beneficial. Because SSc and EoE patients experience similar esophageal symptoms, we hypothesized that eosinophil/mast cell-directed therapy may potentially benefit SSc patients. Herein, we determine the association between esophageal mast cell quantities, gene expression and clinical parameters in order to identify SSc patients who may benefit from eosinophil/mast cell-directed therapy.MethodsEsophageal biopsies from SSc patients and healthy participants were stained for tryptase, a mast cell marker, and associations with relevant clinical parameters including 24h esophageal pH testing were assessed. Intra-epithelial mast cell density was quantified by semi-automated microscopy. Microarray data were utilized for functional and gene set enrichment analyses and to identify intrinsic subset (IS) assignment, an SSc molecular classification system that includes inflammatory, proliferative, limited and normal-like subsets.ResultsEsophageal biopsies from 40 SSc patients (39 receiving proton pump inhibition) and eleven healthy participants were studied. Mast cell numbers in both the upper esophagus (rs = 0.638, p = 0.004) and the entire (upper + lower) esophagus (rs = 0.562, p = 0.019) significantly correlated with acid exposure time percentage. The inflammatory, fibroproliferative, and normal-like ISs originally defined in skin biopsies were identified in esophageal biopsies. Although esophageal mast cell numbers in SSc patients and healthy participants were similar, gene expression for mast cell-related pathways showed significant upregulation in the inflammatory IS of SSc patients compared to patients classified as proliferative or normal-like.DiscussionEsophageal mast cell numbers are heterogeneous in SSc patients and may correlate with acid exposure. Patients with inflammatory IS profiles in the esophagus demonstrate more tryptase staining. Mast cell targeted therapy may be a useful therapeutic approach in SSc patients belonging to the inflammatory IS, but additional studies are warranted.

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N-Formyl Methionine Peptide-Mediated Neutrophil Activation in Systemic Sclerosis.

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