Fpr which belongs the family of the formyl peptide

Fpr1, which belongs the family of the formyl peptide receptors with FPRL1 and FPRL2, is the principal receptor for formylated peptides which prototype is the tri-peptide fMLF. Fpr1 is highly expressed on neutrophils and macrophages [6,30], promotes their migration into the mucosa and lumen in response to formyl peptides derived from resident bacteria during colitis. Moreover, mitochondria derived N-formylated peptides are potent agonist for Fpr1 [31] and can control cell migration following host cell damage. Additionally to expression in phagocytes, FPR1 has been also localized in normal human colonic epithelium, in particular along the lateral membranes of crypt epithelial spectinomycin [32]. In this study we compared the progression of DNBS induced colitis in Fpr1 KO mice compared to WT animals. Our data demonstrate that absence of Fpr1 renders mice significantly less susceptible to the development of the DNBS induced colitis compared to controls. Such a strong outcome is reliant to a variety of cellular and tissutal changes that seem under the control of this receptor. One of the initial responses upon DNBS administration induces free radical production and indeed oxidative stress plays a causal role in the pathophysiology of IBD [33]. It is well known that ROS also stimulates cells to elicit formyl peptide receptor, so, in this way they can modulate oxidant/antioxidant balance [9]. Once FRP1 is activated, guanosine diphosphate (GDP) is converted to guanosine triphosphate (GTP) which induces dissociation of the α subunits from the βγ activating phospholipase C β (PLCβ) [16]. PLCβ hydrolysis 4,5-bisphosphate (PIP2) into inositol 1,4,5-trisphosphate (IP3) which releases calcium from endoplasmatic reticulum. This event leads to the activation of the calmodulin (CaM)/calcineurin pathway, and DAG which in turn activates PKC isoforms. PKC is responsible of the NF-κB translocation to the nucleus. NF-κB is usually located in the cytoplasm as an inactive ternary complex with the inhibitor protein IkB-α. After application of external stimuli, IkB-α is degraded relasing NF-κB from the complex and allowing migration into the nucleus, where it activates the transcription of target genes. The absence of Fpr1 both reduced the IkB-α degradation and inhibited the nuclear NF-κB translocation induced by ROS production due to the DNBS colonic administration. We propose that such an effect may impact on several of the observations made with the Fpr1 KO mice and linked to reduced colon inflammation and damage. Studies on this nuclear transcription factor revealed that the activation of its signalling pathway in turn promotes the expression of pro-inflammatory cytokines (such as tumour necrosis factor-α [TNF-α], interleukin-1β [IL-1β] and interleukin-6 [IL-6]) the colonic mucosa [34,35]. Accumulating literature has shown that they are produced by immunocompetent cells, which facilitate inflammation initiation and propagation [36]. TNF-α is produced by macrophages and intestinal epithelial cells, it promotes the activation of other cells and induces secretion of other cytokine and chemokine expression [37]. IL-1β is an indispensable cytokine that manages immune cells and its expression is correlated with the severity of inflammation [37]. IL-6 is a key pro-inflammatory cytokine with pleiotropic effects, it is released by several cell types. The overexpression of IL-6 is critical in intestinal mucosal lesions [37]. Our experiments demonstrated that the induction of colitis by DNBS caused inflammatory infiltration and damage of the colonic mucosa and amplified the mucosal concentration of IL-1β, TNF-α and IL-6. By contrast, the absence of Fpr1 reduced the mucosal infiltration of the pro-inflammatory cytokines in knockout mice. These findings also revealed an up-regulation of TGF-β expression in the bowels of DNBS treated WT animals. This result is well in line with the dysregulated TGF-β signalling detected in IBD patients, in particular TGF-β expression is up-regulated in lamina propria lymphocytes of active IBD patients [38,39]. Its expression is directly regulated by NF-κB translocation [40], but it can be also promoted by STAT3 pathway, which activated by IL-6 overexpression in the smooth muscle cells [41]. TGF-β is a multifunctional cytokine produced by several cell types [42] which binds specific receptors to modulate mucosal immune reactions [43]. The bioactions and overall function of TGF-β may be still unclear. It can help to define this question by the observation that in absence of Fpr1, through the NF-κB pathway, TGF-β signalling was restored. Indeed, TGF-β is also an immune-suppressive cytokine regulating T-cell development and growth [[44], [45], [46]]. Recent evidences suggest that TGF-β induces de novo Foxp3 expression and has an important role in the generation and expansion of FOXP3+ve regulatory T cells (Tregs), contributing to intestinal immune tolerance [47]. T cells are mainly divided into two populations of CD8+ and CD4+ cells, and both populations contain Tregs [[48], [49], [50]]. Since Tregs frequency decreased in the peripheral blood during disease, proposing that a numerical reduction in Tregs should contribute to IBD pathogenesis [[51], [52], [53]], we may unveil a new pathway whereby Fpr1 activation can contribute to this aspect of the pathogenesis. This dysfunctional immune response to DNBS colitis involves also Th1, Th2, and Th17 cells [54]. GATA-3 is known as a Th2 lineage commitment transcription factor [55,56] and many studies have described UC as a Th2 driven disease [57,58]. Therefore, our study revealed positive correlation between the deletion of Fpr1 and reduced FOXP3 and GATA3 levels, CD4 and CD8 expressions implying important modulatory properties on both Tregs and T cells. It has been described that the expression of CD45 is indispensable for the activation of T cells [59]. CD45 is a receptor‐linked protein tyrosine phosphatase, expressed on all leucocytes. Interestingly, in T cells CD45 has been shown to associate with numerous molecules, both membranes associated and intracellular; these include components of the CD4/CD8. Here we showed that, upon DNBS-induced colitis, Fpr1 KO animals exposed reduced CD45 expression, well in line with the reduced staining of CD4 and CD8. Polymorphonuclear leukocytes (PMNs) play an important role in the immune response to inflammation. They are found at the inflammatory sites, which they get after extravasation from the bloodstream. Endothelial cells are the major regulators of the neutrophil traffic, managing rolling and adhesion from the vasculature to the tissue. P-selectin is recruited on the cell surface of endothelial cells and allows the leukocytes to roll along the endothelium [[60], [61], [62]]. ICAM-1 is constitutively expressed on endothelial cell surfaces and is involved neutrophil adhesion [63]. Hypoxic or injured cells upregulate its expression [64,65]. Moreover, neutrophils trans endothelial migration appears to be deeply dependent on leukocyte integrins, even at high flow rates, and requires the expression of CD11 [66]. In accordance with these findings and with the increased neutrophil recruitment, we observed the up-regulation of ICAM-1, CD11 and P-selectin levels on endothelial vascular wall of DNBS injected WT animals. De novo synthesis of these adhesion molecules occurs downstream NF-κB activation [67]. As such it was reassuring that absence of Fpr1 through reduced activation of the NF-κB pathway yielded lower extent of neutrophils and endothelial cell interaction both for rolling – reliant on P-selectin – and adhesion – sustained by ICAM-1 and CD11. Such microscopic observations under flow corroborate with the reduced leukocytes infiltration, as assessed by MPO activity, immunofluorescence for MPO/ICAM-1 and MPO/CD11, in Fpr1 KO mice bearing colon tissue injury upon DNBS treatment. This process is induced and regulated by several chemoattractant that work to recruit the phagocytic cells to the damaged site, such as bacterial-derived formylated peptides and interleukin-8 (IL-8). The chemoattractant are recognised by cell-surface receptors and it is widely accepted that Fpr1 is one of the most extensively studied receptors involved in neutrophil chemotaxis [6]. In in vitro study a significantly reduced chemotactic response to IL-8 was shown in peripheral blood leukocytes from Fpr1 KO animals in comparison to WT polymorphonuclear leukocyte. Altogether, this reduced tissue damage which may also include necrosis, observed in the absence of Fpr1 was matched by our study of Bax and Bcl-2 protein expression. In our hands, we identified pro-apoptotic changes including up-regulation of the pro-apoptotic Bax protein and down-regulation of anti-apoptotic Bcl-2 protein, in DNBS injected animals. Interestingly, Fpr1 plays a central role in the apoptotic damage [68]. In conclusion, our data clearly demonstrate that DNBS-induced colitis influences proinflammatory signal transduction pathways by modulating the intracellular Formyl peptide receptor system. We provided additional understanding of the mechanism for the activation of the Fpr1. Mice with a targeted deletion of Fpr1 are significantly less vulnerable to the pathologic features of the secondary damage associated with colitis compared to WT controls, opening the therapeutic opportunity for receptor selective antagonists as new tools to control inflammation and tissue damage of the colon.