The aim of this study was to determine the changes in the expression of avian β-defensins (AvBDs) and proinflammatory cytokines and localization of AvBD2 in the intestine of broiler embryos and chicks during growth. The ileum and cecum of embryonic day 19 (ED19) and of day-old (D0) and 7-day-old (D7) chicks were collected. Gene expression levels of 10 AvBDs (AvBD1-8, 10, and 12) and proinflammatory cytokines were analyzed using real-time PCR, and the localization of AvBD2 was examined by immunohistochemistry. Gene expression levels of AvBD1, 2, 6, and 7 in the ileum and of AvBD1 and 4 in the cecum were higher on ED19 than on D7. The expression of AvBD10 in the ileum was higher on D0 than on ED19, whereas the expression levels of AvBD8 and 10 in the cecum were higher on D0 than on ED19, and that of AvBD10 decreased on D7. The expression levels of IL-1β, -6, and -8 in the ileum were higher on D7 than on ED19. The expression levels of IL-1β, -6, and -8 in the cecum were higher on D0 than on ED19, and that of IL-1β and -6 declined on D7. AvBD2-positive cells were localized in the lamina propria beneath epithelial cells of villi and crypts. The number of positive cells in the cecum mucosa was greater on D0 than on ED19 and D7. In conclusion, we suggest that AvBDs are expressed in the ileum and cecum of embryos and chicks at high levels before or just after hatching and decrease by D7. The expression of proinflammatory cytokines in the ileum increases with growth until D7, but is the highest in the cecum around hatching. These AvBDs and proinflammatory cytokines may play roles in host defense in the intestinal mucosa of embryos and neonatal chicks.Keywords: avian β-defensin, broiler embryo and chick, innate immunity, intestine, proinflammatory cytokines IntroductionThe intestine is one of the organs that have a high risk of infection by pathogenic agents. Numerous microbes colonize the cecum soon after hatching (Crhanova et al., 2011). The mucosal innate immune system plays an essential role in defense against microbial infections (Smith and Beal, 2008). The gut-associated lymphoid tissues contain functionally immature T and B cells at the time of hatching, and their functionality is attained during the first 2 weeks post hatching (Bar-Shira et al., 2003).Thus, the adaptive immune system of the neonate is relatively immature, and maternal antibodies in the yolk transported to the chick are believed to play a role in protection against infection (Kovacs-Nolam and Mine, 2012). As it is generally accepted that the innate immune system develops at an early phase, it may play a primary role in the defense against infection in the embryo and chick.Defensins are factors involved in innate immunity that has antimicrobial activity against a variety of microbes, including Gram-negative and -positive bacteria, enveloped viruses, and fungi (Ganz, 2003). In the chicken, fourteen avian β-defensins (AvBD1 to AvBD14) have been identified (Lynn et al., 2007). Lee et al. (2016) reported the antimicrobial activity of AvBD2-4, 6, 7, 11...

The aim of this study was to determine the effects of live probiotics Lactobacillus reuteri (LR) and Clostridium butyricum (CB) on the expression of genes of innate immune system, in broiler chick ileum and cecum. Chicks were administered 500 μl water with or without LR or CB, daily from day 1 to 6 after hatching. The ileum and cecum were collected on day 7 for analysis of gene expression of Toll-like receptors (TLRs), pro-and anti-inflammatory cytokines, and antimicrobial peptides (AMPs) using real-time PCR. The expression of TLR2-1 was upregulated by CB in the ileum and that of TLR5 was upregulated by both LR and CB. Expression of IL-1β and TGFβ2 in the ileum and of TGFβ3 and TGFβ4 in the cecum was upregulated by both LR and CB. The gene expressions of avian β-defensin (AvBD) 1 and cathelicidin (CATH) 3 were upregulated by CB and that of AvBD4 was upregulated by LR in the cecum. However, the expression of CATH2 in the ileum was downregulated by LR. These results suggest that probiotic LR and CB treatments affect a part of the innate defense system in the ileum and cecum by modulating the expression of innate immune molecules including TLRs, pro-and anti-inflammatory cytokines, and AMPs.

The aim of this study was to confirm whether the expression of innate immune molecules in the chick cecum is altered in association with changes in the composition of the intestinal microbiome that are regulated by treatment with antibiotics. Broiler chicks were administered with antibiotics (penicillin and streptomycin) daily, and the composition of the microbiota, expression of innate immune molecules, and localization of antimicrobial peptides in the chick cecum were examined at day 7 and day 14 using real-time PCR and immunohistochemistry. The oral administration of antibiotics caused an increase in the relative frequency of the Enterobacteriaceae family and a decrease in some gram-negative (Barnesiellaceae) and gram-positive bacterial (Clostridiaceae and Erysipelotrichaceae) families. The gene expression levels of immune molecules, including 4 Toll-like receptors ( TLR ) (TLR 2, 4, 5, and 21), inflammation-related cytokines (IL-1β, TGFβ3, TGFβ4, and IL-8), and antimicrobial peptides (avian β-defensins and cathelicidins) showed a tendency to decrease with antibiotic treatment at day 7. However, expression levels of TLR21 and some cytokines (IL-1β, TGFβ3, and IL-8) were higher in the cecum or cecal tonsils of the antibiotic-treated group than in those of the control at day 14. The immunoreactive avian β-defensin 2 and cathelicidin 1 proteins were localized in the leukocyte-like cells in the lamina propria, and they were aggregated in the form of small islands. We conclude that the expression of innate immune molecules, including TLR, inflammation-related cytokines, and antimicrobial peptides, in the cecum are altered in association with changes in the density or composition of the luminal microbiota during the early phase of life in chicks.

Few studies have focused on the regulation of cytokine and avian β-defensin (AvBDs) expression for promoting immune defense in the avian intestine. The aim of this study was to investigate the effects of different Toll-like receptor (TLR) ligands (bacterial patterns) on the expression of proinflammatory cytokines (IL-1β and IL-6) and AvBDs (AvBD1, AvBD4, and AvBD7) in the chick intestine. The ileum and cecum of 3-day-old chicks were collected and examined histologically to identity the cells present in the intestinal mucosa. Other tissues were cultured with or without the TLR2, TLR4, and TLR21 ligands-Pam3CSK4, LPS, and CpG-ODN-for 1 or 3 h. The gene expression profiles of proinflammatory cytokines and AvBDs were determined in these tissues using real-time polymerase chain reaction (PCR). The mucosa of the ileum and cecum contained leukocytes, luminal and crypt epithelial cells, and other enterocytes. Pam3CSK4 tended to downregulate the expression of IL-1β, AvBD1, and AvBD7 in the ileum but upregulated their expression in the cecum. LPS downregulated the expression of IL-1β and IL-6 in both the ileum and the cecum, whereas it upregulated the expression of AvBD1, AvBD4, and AvBD7 in the cecum. CpG-ODN upregulated the expression of IL-6 and AvBD7 in the ileum and IL-1β in the cecum, and downregulated the expression of IL-1β and AvBDs in the ileum. We suggested that the expression levels of proinflammatory cytokines and AvBDs in the chick intestine are affected by TLR2, TLR4, and TLR21 ligands. Thus, these innate immune factors may be modulated by the luminal microbe complex in the intestine.

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