Investigation Of HNP-1-induced Activation Of PDC Cells And Its Implication In Rheumatoid Arthritis

Background and Objectives: Defensins are endogenous, small, cysteine-rich antimicrobial peptides that are produced by leukocytes and epithelial cells. Substantial evidence indicates that defensins participate in both the innate and adaptive antimicrobial immunity of the host. Human neutrophil peptide 1(HNP-1) is a predominant α-defensin in the azurophilic granules of human neutrophils. Although initially identified as an antimicrobial peptide, HNP-1 has subsequently been found to have multiple effects on leukocytes of the immune system. HNP-1 is chemotactic for human T cells in vitro and recruits human T cells into sites of injection in vivo. HNP-1 has the capacity to induce the production of TNF-α in human monocytes in vitro or in the respiratory tract in vivo. HNP-1 is reported to induce phenotypic and functional maturation of human monocyte-derived DCs. Owing to the possession of both DC-chemotactic and-activating effects, HNP-1 is classified as a member of the alarmin family, endogenous mediators capable of recruiting and activating DCs and consequently activating innate and adaptive immune responses. However, it is not determined whether it can activate plasmacytoid dendritic cells(p DCs). In the first part of this study, we studied activation of human p DC induced by HNP-1. Next, we studied the molecular mechanisms of p DC activation by observing IRFs and NF-κB nuclear translocation. Furthermore, we investigated the role of HNP-1 in vivo. In the second part of this study, we detected the expression of HNP-1 in rheumatoid arthritis synovial fluid to explore the clinical significance of HNP-1, providing new ideas and targets for the clinical treatment of rheumatoid arthritis.Methods: 1. HNP-1 induced activation of CAL-1 cells and primary p DCs were analyzed by measuring h IFN-α protein levels using ELISA. 2. HNP-1 induced activation of CAL-1 cells and primary p DCs were collected for m RNA measurements and analyzed by Real Time PCR. 3. Western blot was used to detect the IRF-1 and NF-κB p50, p65 protein expression in CAL-1 cells. 4. si RNA knockdown study was used to induced IRF-1 protein expression downregulation in CAL-1 cells. 5. Flow cytometry was used to detect the concentration of neutrophils and p DCs.Results: 1. HNP-1 induced activation of p DC production of IFN-α and IFNβ1 in dose-dependent manner. HNP-1 was an activator of p DC. 2. HNP-1 promoted ODN-induced p DC production of IFN-α and IFNβ1. 3. HNP-1 stimulated CAL-1 cells production of IFN-α. HNP-1 promoted ODN-induced CAL-1 cells production of IFN-α. 4. HNP-1 triggered NF-κB and IRF-1 signaling in CAL-1 cells. 5. Blockade of NF-κB activation inhibited HNP-1-induced production of IFN-α, IFNβ1 and IL-6 in p DC. 6. IRF1 Knockdown inhibited HNP-1 induced expression of IFN-α, IFNβ1 and IL-6 in CAL-1 cells. 7. Animal experiments confirmed HNP-1 could activate human p DC cells not only in vitro but also in vivo. 8. Neutrophils and p DC cells in synovial fluid of rheumatoid arthritis patients increased significantly, and the synovial fluid also contained a lot of HNP-1 protein.Conclusions: 1. HNP-1 induced p DC activation in vitro. 2. HNP-1 triggered NF-κB and IRF-1 signaling. 3. NF-κB and IRF-1 played important roles in HNP-1-induced p DC activation. 4. HNP-1 stimulated p DC activation in SCID mice. 5. Neutrophils, HNP1, and p DCs were co-presented in synovial fluid of rheumatoid arthritic patients.