The Expression Characteristics, Regulation And The Immunomodulatory Mechanism Of Porcine Antimicrobial Peptide PR-39

PR-39is a gene-encoded, proline-arginine-rich cathelicidin antimicrobial peptide, which was isolated from small intestinal tissues and neutrophil granules in pigs. Several studies revealed that PR-39had exhibited broad-spectrum microbicidal and extensive immunoregulatory activities, including chemotactic activity, anti-inflammation and wound healing. But how PR-39involves in the intestinal mucosal immunity in animals, especially in pigs, is not well understood. Thus, the primary focus of this research was to determine role of PR-39in porcine immunity, by investigating the tissue and developmental expression pattern of PR-39in different pig breeds, comparing the production of PR-39and gut physiology in response to Escherichia coli K88challenge in two different pig breeds, and defining the contribution of PR-39in polarization and immune functions of macrophages in vitro. The major results are as following:1. The breed difference, tissue-specific and developmental expression patterns of antimicrobial peptide PR-39in pigsBreed difference expression of PR-39and several innate immune molecules was compared between Chinese local breed Laiwu, Rongchang, Tibetan pigs, and Danish Landrace pigs. The results showed that Rongchang pigs had significantly higher PR-39mRNA levels in the bone marrow, thymus and duodenum compared with Landrace (p<0.05). Tibetan pigs had significantly higher PR-39mRNA levels in the thymus and liver compared with Landrace pigs (p<0.05). Laiwu pigs had significantly higher PR-39mRNA level in the spleen (p<0.05) compared with Landrace pigs. The breed difference expression of cytokine genes showed that Laiwu and Rongchang pigs had higher IL-la and IL-1(3mRNA levels compared with Landrace pigs (p<0.05), whereas Laiwu and Tibetan pigs had higher IFN-y level compared with Landrace pigs (p<0.05). On the aspect of chemokine genes expression, Rongchang pigs showed higher levels of MCP-1and IL-8compared with Landrace pigs (p<0.05), whereas Tibetan pigs only had the higher MCP-1level compared with Landrace (p<0.05). Lastly, the expression levels of TLR-2,4,5and NOD-1,2in Rongchang pigs were significantly higher than Landrace pigs (p<0.05), whereas the expression levels of those genes in Laiwu and Tibetan pigs were tended to be higher than Landrace pigs.Furthermore, the Tissue-specific and developmental expression pattern of PR-39was determined by real-time PCR in Chinese Jinhua and European Landrace pigs. The PR-39mRNA was predominantly expressed in the bone marrow compared with the spleen, mesenteric lymph node (MLN), liver, kidney and duodeum (p<0.05). Developmental expression pattern of PR-39revealed that the PR-39mRNA level tended to increase from the neonatal to90-day and to decrease between90-and120-day in Jinhua pigs, whereas the PR-39mRNA increased from neonatal to60-day and to decrease between60-and120-day in Landrace pigs. Those results revealed that Chinese local pig breeds had higher expression levels of PR-39antimicrobial peptide and several innate immune molecules, indicating that there could be a relation between PR-39expression and the porcine innate immunity.2. Comparison of the gut function and PR-39gene expression in Jinhua and Landrace pigs challenged with Escherichia coli K88This experiment was conducted to compare the gut function, including the selected gut microbial populations, intestinal morphology, and immunity, and the PR-39gene expression in Jinhua and Landrace pigs, after orally challenge with enterotoxigenic Escherichia coli (ETEC) K88. The results showed that all of Jinhua pigs were the resistant genotype and could prevent the adhesion of ETEC K88to the intestinal epithelia, whereas91.7%(11/12) of the Landrace pigs were the susceptible genotype and resulted in the intestinal colonization of ETEC K88.In the aspect of growth performance, the average daily gain (ADG) of Jinhua and Landrace pigs were decreased by32.1and28.6%, respectively. The ADG of the challenged Jinhua pigs was significantly higher (p<0.05) compared with their Landrace counterparts. In the aspect of incidence of diarrhea, the challenged Jinhua pigs exhibited a significantly (p<0.05) lower incidence of diarrhea compared with their Landrace counterparts. Compared with Jinhua pigs, the effect of the ETEC K88challenge on the incidence of diarrhea was markedly greater in the Landrace pigs (the interaction between breed and ETEC K88challenge; p-0.006).In the aspect of gut microbial population, the Escherichia coli (E.coli) population and the percentage of E. coli in the total bacteria population were increased in response to ETEC K88challenge in both Jinhua and Landrace pigs. The challenged Landrace pigs shed more E. coli (p<0.05) and had higher percentage of E. coli in the total bacteria population in the colon (p<0.05) compared with their Jinhua counterparts. The population of colonic Lactobacillus (p<0.05) was significantly reduced in response to ETEC K88challenge in Landrace pigs and was significantly affected by the interaction between breed and ETEC K88challenge (p<0.05).In the aspect of intestinal morphology, both pig breeds tended to exhibit greater villous atrophy and crypt depth reduction in all of the intestinal segments after challenge. ETEC K88caused the most obvious intestinal damage in the ileum. Challenged Jinhua pigs showed significantly reduced villous height and villi/crypt ratio compared with the non-challenged counterpart (p<0.05). In the aspect of the expression of tight junction proteins, the expression of tight junction proteins decreased in response to ETEC K88challenge in both pig breeds. In the jejunum, challenged Jinhua pigs showed significantly decreased expression of occludin, ZO-1and ZO-2(p<0.05) compared with the non-challenged counterpart. Challenged Landrace pigs showed significantly decrased expression of occludin comapred with the non-challenged counterpart. The expression of occludin and ZO-2was significantly affected by the breed (p<0.001),ETEC K88challenge (p<0.05), and their interaction (p<0.05). The expression of ZO-1was only significantly affected by the breed (p<0.05) and ETEC K88challenge (p<0.05). The decreased expression of the tight junction protein mRNAs was more pronounced in the challenged Jinhua pigs compared with their Landrace counterparts. However, the challenged Jinhua pigs still exhibited a significantly higher expression level of occludin compared with the challenged Landrace pigs.In the aspect of the serum immunoglobulin, challenged Jinhua pigs showed significantly higher serum IgA and IgG level (p<0.05) compared with the non-challenged counterpart.The serum IgA and IgG level of challenged Landrace pigs tended to be higher compared with the non-challenged counterpart. Challenged Jinhua pigs had significantly higher IgG level compared with their Landrace counterparts (p <0.05).In the asepct of the levels of intesitnal cytokine production, proinflammatory cytokines, including IFN-y, TNF-a, and IL-6and the secretion of sIgA were positively altered whereas the levels of the anti-inflammatory cytokine IL-4and TGF-β were negatively altered by ETEC K88challenge in both breeds. Jinhua pigs exhibited significantly higher levels of IFN-y and TGF-P (p<0.05) in the challenged group. The ETEC K88chanllenge up-regulated PR-39mRNA levels in both Jinhua and Landrace pigs. In the bone marrow and the spleen, Jinhua pigs responded stronger to ETEC K88challenge than Landrace pigs on the expression of PR-39. Those results showed that Jinhua pigs were the ETEC K88resistant genotype, and appeared to show better growth performance, a lower incidence of diarrhea in response to ETEC K88challenge and a higher Lactobacillus population, a higher percentage of Lactobacillus in the total bacteria population, a higher ratio of Lactobacillus to E. coli, and higher levels of tight junction proteins with and without challenge, which could be related to the activation extent of the mucosal immunity and the higher expression level of PR-39in Jinhua pigs. These findings revealed that the endogenous production of PR-39could be induced by bacterial challenge in pigs, and could involved in the regulation of gut microbiota, gut barrier function and mucosal immunity during bacterial infection.3. In vitro modulatory functions of antimicrobial peptide PR-39on polarization and immune responses in porcine macrophageIn this experiment, the effects of PR-39on the polarization and immune functions of macrophages were tested. Structural disruption of macrophages, and increased LDH release and production of proinflammatory mediators-TNF-a, IL-6, IL-8and COX-2(p<0.05) was observed in porcine macrophages after ETEC infection. Pretreatment with PR-39significantly alleviated proinflammatory responses and cytotoxicity caused by ETEC infection. Co-stimulation of PR-39with IFN-y significantly enhanced the expression of Ml marker gene IL-12p40, TNF-a, IL-6and STAT-1(p<0.05) in M1macrophages, suggesting that PR-39drived macrophage polarization into the M1state. Co-stimulation of PR-39with IL-4and M-CSF significantly reduced the expression of M2marker gene IL-10and TGF-β(p<0.05), but enhanced the expression of IL-12p40and COX-2(p<0.05), suggesting that PR-39to some extent suppressed the M2polarization and directed M2macrophage differentiated to macrophages with proinflammatory markers. The synergistic effects of PR-39with either IFN-y or IL-4and M-SCF on macrophage phagocytosis and bacterial adhesion and invasion were further tested. The results showed that PR-39significantly increased phagocytosis in both M1and M2macrophages, and significantly inhibited the adhesion and invasion of ETEC into the macrophages. Together, those data demonstrated that PR-39could prevent macrophages from the ammation and cell damage caused by ETEC infection, and enhance the phagocytosis and bacteria-killing activities of macrophages through the mechanism of directing macrophage M1polarization.