br Materials and methods br

Materials and methods
Results
Discussion It is known that MAPKs and Akt signaling can regulate cell migration [[24], [25], [26]]. In this study we found that dTAT-WKYMVm stimulates ERK, p38 and Akt phosphorylation in naïve CD4 T cell (Fig. 3A). However their activation did not affect naïve CD 4 T cell migration induced by dTAT-WKYMVm (Fig. 3B). Previous reports have indicated that regulation of T cell differentiation by annexin A1 is mediated by ERK and Akt phosphorylation of FPR's downstream targets [10,27]. We also examined the role of intracellular FPRs on naïve CD4 T cell differentiation. Isolated naïve CD4 T cells were treated with differentiation cytokines in the absence or presence of dTAT-WKYMVm for 5 days. Differentiated cells were then stained with specific T cell marker Cytochrome Oxidase Activity Colorimetric Assay Kit and subjected to flow cytometry analysis. Our results showed that dTAT-WKYMVm did not induce naïve CD4 T cell differentiation into any type of T cells (data not shown). Annexin A1-induced ERK and Akt phosphorylation was mediated by FPR2 signal. However, naïve CD4 T cells were not present in intracellular FPR2. Therefore, dTAT-WKYMVm-induced MAPK and Akt phosphorylation might be involved in other functions of CD4 T cells. Although some studies have described the expression of FPR in CD4 T cells [[9], [10], [11]], we could not observe the surface expression of FPRs in naïve CD4 T cells. Instead, we found that FPR1 and FPR3 are expressed on the surface of anti-CD3 and anti-CD28 stimulated CD4 T cells. In addition, the expression of surface FPRs was observed in CD4 T cells from sepsis patients. Unlike to anti-CD3 and anti-CD28 stimulated CD4 T cells, CD4 T cells derived from sepsis patients expressed FPR2 (Fig. 4D). Since activated CD4 T cells expressed FPR1 and FPR3 on the cell surface, we examined whether these surface FPR1 and FPR3 could mediate chemotactic migration of CD4 T cells by WKYMVm. Unexpectedly, we could not observe any chemotactic migration of activated CD4 T cells stimulated with anti-CD3 and anti-CD28 after treatment with WKYMVm (data not shown). These results suggest that surface expressed FPR1 and FPR3 might not regulate chemotactic migration of anti-CD3 and anti-CD28-activated CD4 T cells. Immunological function of surface expressed FPR1 and FPR3 in anti-CD3 and anti-CD28 stimulated CD4 T cells should be investigated in the near future.
Acknowledgments This work was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT and Future Planning (NRF-2015R1A2A1A10054567, NRF-2016R1A2B4013128).
Introduction The immune system can recognize invading pathogens and effectively remove them by innate immunity and adaptive immunity. In innate immune response, sentinel cells such as tissue residing macrophages can detect invading pathogen-associated molecular patterns using several pattern recognition receptors [1], [2]. Activated macrophages then produce several inflammatory cytokines and chemokines, leading to recruitment of leukocytes from peripheral blood vessel into infected area [3]. Neutrophils are the first leukocytes recruited into the event area after sensing cytokines and chemokines [4]. Neutrophils can also detect pathogen-derived molecules such as N-formyl peptides to mediate innate immune response [4], [5]. Many extracellular stimuli can regulate neutrophil activity in innate immune response. Identifying molecules that can stimulate neutrophils would be important to control innate immunity. Centipede Scolopendra subspinipes mutilans has been used in oriental medicine to treat several human diseases, including rheumatoid arthritis, lymphadenopathy, and carcinoma [6], [7]. This centipede also possesses antimicrobial ingredients that can protect lung and intestine against infection [8]. However, molecular identities of bioactive components in this centipede that can modulate disease pathogenesis have not been extensively revealed due to limited information about its genome analysis. In this study, we systematically analyzed the genome of Scolopendra subspinipes mutilans to identify novel antimicrobial peptides (AMPs) according to a previous report [9]. We identified scolopendrasin X (sequence: MKKFHCLKKICKGLCAKL-CONH2), a novel AMP from Scolopendra subspinipes mutilans. It strongly stimulated mouse neutrophils, resulting in calcium increase, chemotactic migration, and superoxide anion production. We also found that formyl peptide receptor (FPR)2, an important classical chemoattractant receptor, could mediate scolopendrasin X-induced neutrophil activation.