Activation of formyl peptide receptor 1 elicits therapeutic effects against collagen-induced arthritis.

Rheumatoid arthritis (RA) is an autoimmune disorder which shows production of autoantibodies, inflammation, bone erosion, swelling and pain in joints. In this study, we examined the effects of an immune-modulating peptide, WKYMVm, that is an agonist for formyl peptide receptors (FPRs). Administration of WKYMVm into collagen-induced arthritis (CIA) mice, an animal model for RA, attenuated paw thickness, clinical scores, production of type II collagen-specific antibodies and inflammatory cytokines. WKYMVm treatment also decreased the numbers of TH 1 and TH 17 cells in the spleens of CIA mice. WKYMVm attenuated TH 1 and TH 17 differentiation in a dendritic cell (DC)-dependent manner. WKYMVm-induced beneficial effects against CIA and WKYMVm-attenuated TH 1 and TH 17 differentiation were reversed by cyclosporin H but not by WRW4, indicating a crucial role of FPR1. We also found that WKYMVm augmented IL-10 production from lipopolysaccharide-stimulated DCs and WKYMVm failed to suppress TH 1 and TH 17 differentiation in the presence of anti-IL-10 antibody. The therapeutic administration of WKYMVm also elicited beneficial outcome against CIA. Collectively, we demonstrate that WKYMVm stimulation of FPR1 in DCs suppresses the generation of TH 1 and TH 17 cells via IL-10 production, providing novel insight into the function of FPR1 in regulating CIA pathogenesis.

Promotion of formyl peptide receptor 1-mediated neutrophil chemotactic migration by antimicrobial peptides isolated from the centipede Scolopendra subspinipes mutilans.

We investigated the effects of two antimicrobial peptides (AMPs) isolated from Scolopendra subspinipes mutilans on neutrophil activity. Stimulation of mouse neutrophils with the two AMPs elicited chemotactic migration of the cells in a pertussis toxin-sensitive manner. The two AMPs also stimulated activation of ERK and Akt, which contribute to chemotactic migration of neutrophils. We found that AMP-stimulated neutrophil chemotaxis was blocked by a formyl peptide receptor (FPR) 1 antagonist (cyclosporin H); moreover the two AMPs stimulated the chemotactic migration of FPR1-expressing RBL-2H3 cells but not of vector-expressing RBL-2H3 cells. We also found that the two AMPs stimulate neutrophil migration in vivo, and that this effect is blocked in FPR1-deficient mice. Taken together, our results suggest that the two AMPs stimulate neutrophils, leading to chemotactic migration through FPR1, and the two AMPs will be useful for the study of FPR1 signaling and neutrophil activation. [BMB Reports 2016; 49(9): 520-525].

Distribution and immunotoxicity by intravenous injection of iron nanoparticles in a murine model.

With the increased application of iron oxide nanoparticles (FeNPs) for biomedical imaging purposes, concerns regarding the onset of the unexpected adverse health effects following exposure have been rapidly raised. In this study, we investigated the tissue distribution and immunotoxicity of FeNPs (2 and 4 mg kg(-1)) over time (2, 4 and 13 weeks) after single intravenous injection. At 13 weeks after a single injection, the iron levels increased in all measured tissues compared to the control, and iron accumulation was notable in the liver, spleen and thymus. These changes were accompanied by changes in levels of redox reaction-related elements, including copper, manganese, zinc and cobalt. In addition, as compared to the control, the number of white blood cells and percentage of neutrophils significantly increased in the treated groups, and the interleukin-8 secretion and lactate dehydrogenase release were clearly elevated in the treated groups along with enhanced expressions of chemotaxis-related proteins. However, expression of antigen presenting related proteins attenuated following accumulation of FeNPs. Taken together, we suggest that FeNPs may primarily induce toxicity in the liver and immune system, and immunotoxicological evaluation should be considered to predict adverse health effects following exposure to NPs.

Phospholipase D2 drives mortality in sepsis by inhibiting neutrophil extracellular trap formation and down-regulating CXCR2.

We determined the function of phospholipase D2 (PLD2) in host defense in highly lethal mouse models of sepsis using PLD2(-/-) mice and a PLD2-specific inhibitor. PLD2 deficiency not only increases survival but also decreases vital organ damage during experimental sepsis. Production of several inflammatory cytokines (TNF, IL-1ß, IL-17, and IL-23) and the chemokine CXCL1, as well as cellular apoptosis in immune tissues, kidney, and liver, are markedly decreased in PLD2(-/-) mice. Bactericidal activity is significantly increased in PLD2(-/-) mice, which is mediated by increased neutrophil extracellular trap formation and citrullination of histone 3 through peptidylarginine deiminase activation. Recruitment of neutrophils to the lung is markedly increased in PLD2(-/-) mice. Furthermore, LPS-induced induction of G protein-coupled receptor kinase 2 (GRK2) and down-regulation of CXCR2 are markedly attenuated in PLD2(-/-) mice. A CXCR2-selective antagonist abolishes the protection conferred by PLD2 deficiency during experimental sepsis, suggesting that enhanced CXCR2 expression, likely driven by GRK2 down-regulation in neutrophils, promotes survival in PLD2(-/-) mice. Furthermore, adoptively transferred PLD2(-/-) neutrophils significantly protect WT recipients against sepsis-induced death compared with transferred WT neutrophils. We suggest that PLD2 in neutrophils is essential for the pathogenesis of experimental sepsis and that pharmaceutical agents that target PLD2 may prove beneficial for septic patients.

Inhibition of lethal inflammatory responses through the targeting of membrane-associated Toll-like receptor 4 signaling complexes with a Smad6-derived peptide.

We have previously reported that Smad6, one of the inhibitory Smads of transforming growth factor-ß (TGF-ß)/bone morphogenetic protein (BMP) signaling, inhibits Toll-like receptor (TLR) 4 signaling by disrupting the Pellino-1-mediated TLR4 signaling complex. Here, we developed Smaducin-6, a novel membrane-tethered palmitic acid-conjugated Smad6-derived peptide composed of amino acids 422-441 of Smad6. Smaducin-6 interacted with Pellino-1, located in the inner membrane, thereby disrupting the formation of IRAK1-, RIP1-, IKKε-mediated TLR4 signaling complexes. Systemic administration of Smaducin-6 showed a significant therapeutic effect on mouse TLR4-mediated inflammatory disease models, cecal-ligation-puncture (CLP)-induced sepsis, and lipopolysaccharide-induced endotoxemia, by inhibiting pro-inflammatory cytokine production and apoptosis while enhancing neutrophil migration and bacterial clearance. Our findings provide clues to develop new peptide-based drugs to target Pellino-1 protein in TLR4 signaling pathway for the treatment of sepsis.

Anti-septic activity of α-cubebenoate isolated from Schisandra chinensis.

Sepsis is a life-threatening, infectious, systemic inflammatory disease. In this study, we investigated the therapeutic effect of α-cubebenoate, a novel compound isolated from Schisandra chinensis against polymicrobial sepsis in a cecal ligation and puncture (CLP) experimental model. Administration of α-cubebenoate strongly enhanced survival in the CLP model. α-cubebenoate administration also markedly blocked CLP-induced lung inflammation and increased bactericidal activity by enhancing phagocytic activity and hydrogen peroxide generation in mouse bone marrow-derived macrophages and neutrophils. Expression of two important inflammatory cytokines, IL-1ß and IL-6, was strongly increased in the CLP model, and this was dramatically blocked by α-cubebenoate. Lymphocyte apoptosis and caspase-3 activation, which are associated with immune paralysis during sepsis, were markedly attenuated by α-cubebenoate. Taken together, our findings indicate that α-cubebenoate, a natural compound isolated from Schisandra chinensis, is a powerful potential anti-septic agent.

A novel delivery platform for therapeutic peptides.

Although many peptides have therapeutic effects against diverse disease, their short half-lives in vivo hurdle their application as drug candidates. To extend the short elimination half-lives of therapeutic peptides, we developed a novel delivery platform for therapeutic peptides using an anti-hapten antibody and its corresponding hapten. We selected cotinine because it is non-toxic, has a well-studied metabolism, and is physiologically absent. We conjugated WKYMVm-NH2, an anti-sepsis therapeutic peptide, to cotinine and showed that the conjugated peptide in complex with an anti-cotinine antibody has a significantly improved in vivo half-life while retaining its therapeutic efficacy. We suggest that this novel delivery platform for therapeutic peptides will be very useful to develop effective peptide therapeutics.

A membrane-tethering pepducin that inhibits formyl peptide receptor 2-induced signaling.

Since formyl peptide receptor 2 (FPR2) plays a key role in the regulation of innate immune response and inflammation, it has been a hot topic to develop molecules which inhibit FPR2-induced cellular responses. In this study, we investigated the effect of an FPR2-derived pepducin in human neutrophils and human umbilical vein endothelial cells (HUVECs). The pepducin (F2pal-12) selectively inhibited FPR2 agonists (MMK-1 and serum amyloid A)-stimulated neutrophil chemotaxis. MMK-1-stimulated superoxide anion production was also inhibited by F2pal-12. HUVECs also express FPR2; FPR2 agonists-stimulated HUVECs migration and tube formation were also selectively inhibited by F2pal-12 but not by scrambled control pepducin. Since FPR2 mediates inflammatory response by inducing chemotactic migration of inflammatory cells, F2pal-12 can be used as a useful material to modulate FPR2-mediated inflammatory responses.

Oxidized low-density lipoprotein-induced foam cell formation is mediated by formyl peptide receptor 2.

The increased level of LDL and its modification into oxLDL has been regarded as an important risk factor for the development of cardiovascular diseases such as atherosclerosis. Although some scavenger receptors including CD36 and RAGE have been considered as target receptors for oxLDL, involvement of other receptors should be investigated for oxLDL-induced pathological responses. In this study, we found that oxLDL-induced foam cell formation was inhibited by formyl peptide receptor 2 (FPR2) antagonist WRW(4). oxLDL also stimulated calcium signaling and chemotactic migration in FPR2-expressing RBL-2H3 cells but not in vector-expressing RBL-2H3 cells. Moreover, oxLDL stimulated TNF-α production, which was also almost completely inhibited by FPR2 antagonist. Our findings therefore suggest that oxLDL stimulates macrophages, resulting in chemotactic migration, TNF-α production, and foam cell formation via FPR2 signaling, and thus likely contributes to atherogenesis.

The immune-stimulating peptide WKYMVm has therapeutic effects against ulcerative colitis.

In this study, we examined the therapeutic effects of an immune-stimulating peptide, WKYMVm, in ulcerative colitis. The administration of WKYMVm to dextran sodium sulfate (DSS)-treated mice reversed decreases in body weight, bleeding score and stool score in addition to reversing DSS-induced mucosa destruction and shortened colon. The WKYMVm-induced therapeutic effect against ulcerative colitis was strongly inhibited by a formyl peptide receptor (FPR) 2 antagonist, WRWWWW, indicating the crucial role of FPR2 in this effect. Mechanistically, WKYMVm effectively decreases intestinal permeability by stimulating colon epithelial cell proliferation. WKYMVm also strongly decreases interleukin-23 and transforming growth factor-ß production in the colon of DSS-treated mice. We suggest that the potent immune-modulating peptide WKYMVm and its receptor FPR2 may be useful in the development of efficient therapeutic agents against chronic intestinal inflammatory diseases.