Molecular Evolution Of Avian Host Defense Peptides And Their Expression Induced By Butyrate And 1,25?OH?₂D₃

Host defense peptides?HDPs?constitute a critical component of immunity in virtually all species of life.They are broadly active against a range of pathogens and capable of modulating the host immune by inducing chemotaxis and activation of various cells.BecauseHDPs kills microbes primarily through physical electrostatic interactions and membrane disruption,it is difficult for microbes to gain resistance to HDPs.In modern poultry production,animals are threated by a variety of pathogens.The routine use of antibiotics at subtherapeutic doses for disease prevention in animal production practices is suspected to be a major force that drives the emergence of antibiotic-resistant pathogens.Therefore,endogenous HDPs may have potential to be developed as novel antibiotic alternatives.Here,we aim to explore the gene evolution and induced regulation of avian HDPs.Firstly,we searched Avian ?-defensins?AvBDs?through 17 phylogenetically divergent bird species and conduct cross-species analysis of the AvBD gene family.Secondly,chicken HD11 macrophages,PBMCs and jejunal explants were used as model to examine a possible synergy between 1,25?OH?₂D₃ and butyrate in HDP induction in poultry.Thirdly,in order to explain molecular mechanism of the induction of HDPs by 1,25?OH?₂D₃,butyrate and their combination.We sequenced the transcriptomes of HD 11 cell line under different treatments.Last,multiple inhibitors were added in HD 11 cell line to explore role of related pathways and proteins on the expression of avian HDPs induced by 1,25?OH?₂D₃,butyrate and their combination.The main results are list as follow:1.259 novel AvBD genes were identified from 17 phylogenetically divergent bird species.In each species,the AvBD genes appear to be clustered in a single syntenic chromosomal region.Phylogenetic analysis suggested that all 13 AvBD gene lineages existed before the bird species were seperated from each other.Furthermore,most AvBD lineages formed well-supported clusters with reptilian P-defensins,implying that they were present even before the bird-reptile split.However,species-specific AvBD genes such as the paralogs of AvBD1 and AvBD3 are also prevalent.Additionally,positive selections were detected in multiple AvBD gene lineages.2.We revealed a strong synergy between 1,25?OH?₂D₃ and butyrate.While 10-8 M 1,25?OH?₂D₃ and 2 mM butyrate gave an approximately 2.5-and 600-fold induction of AvBD9,respectively,a combination of both compounds resulted in a 4,900-fold increase in AvBD9 expression in HD 11 cells.Similarly,a strong synergy between 1,25?OH?₂D₃ and butyrate in AvBD9 induction was also observed in chicken PBMCs and jejunal explants.In addition to AvBD9,multiple,but not all,other chicken HDP genes were also synergistically induced.Importantly,1,25?OH?₂D₃ and butyrate suppressed lipopolysaccharide-induced interleukin?IL?-1? expression.3.Compared with controls,10-8 M of 1,25?OH?₂D₃,2 mM butyrate and the combination of both caused 132,4,670 and 4,777 deferentially expressed genes?DEGs?,respectively.These DEGs were involved in a variety of functions and signaling pathways.Of these,two transcriptional factor?FOS and SMAD3?showed strong synergistic effect under the treatments of 1,25?OH?₂D₃ and butyrate.Together with the analysis of KEGG pathways,we speculate that the synergy effects of 1,25?OH?₂D₃ and butyrate on the expression of avian HDPs may got through MAPK and TGF-P signaling pathways in HD11 cells.4.Inhibition of ERK in the MAPK signaling pathway could further significantly increase the expression of AvBD9 under the butyrate and butyrate plus 1,25?OH?₂D₃ treatments.In contrast,the inhibition of TGF-? pathway would significantly decrease the AvBD9 expression levels in butyrate and the combination of 1,25?OH?₂D₃ plus butyrate treatments.At the same time,we also observed that inhibition of the NF-?B,P38,JNK and PKA signaling pathways could also significantly reduce the expression of HDPs in butyrate plus 1,25?OH?₂D₃ treatment.Our results showed that not only ERK and TGF-?signaling pathways but also the NF-?B,P38,JNK and PKA signaling pathways were involved in the synergistic induced HDPs expression by 1,25?OH?₂D₃ and butyrate in birds.Identification and analysis of the complete repertoires of AvBDs in multiple avian species has led to a better understanding of bird innate immune mechanism as well as pave the way for the discovery of novel antimicrobials.Meanwhile,dietary modulation of endogenous HDP synthesis by 1,25?OH?₂D₃ and butyrate has potential to be developed as a promising alternative approach to antibiotics for poultry use.Collectively,the results are expected to provide information for better utilize of avian HDPs as well as development of antibiotic alternatives in poultry production.