Identification, tissue characterization, and innate immune role of Angiogenin-4 expression in young broiler chickens

Intestinal epithelial cells are major producers of antimicrobial proteins, which play an important role in innate immunity. In addition to defensins, the Ribonuclease A superfamily includes important antimicrobial proteins involved in host-defense mechanisms in vertebrates. Angiogenin-4 (Ang4), a member of this RNase superfamily, has been demonstrated to be secreted by Paneth cells in mice. We have successfully cloned and characterized a new chicken gene (chAng4), found for the first time in a nonmammalian species, from intestinal epithelial and lymphoid cells. Characterization of chAng4 revealed 99% nucleotide and 97% amino acid sequence homology to mouse Ang4. Similar functional regions were identified, suggesting a role in innate immunity and regulation of gut microbiota. Furthermore, the mRNA expression pattern of chAng4 was studied in broilers in the presence or absence of beneficial bacteria (probiotics) and organic acids. The results showed that one-day-old chickens expressed low levels of Ang4 in almost all the evaluated tissues (crop, proventriculus, duodenum, jejunum, ileum, and cecal tonsils), except in the bursa of Fabricius that presented the highest expression level. The addition of probiotics and organic acids for either 7 or 14 consecutive days demonstrated a direct effect of probiotics and organic acids on chAng4 expression; moreover, broilers receiving probiotics and organic acids for only 7 D showed higher levels of chAng4 expression compared with those treated for 14 D. Broilers without treatment had a constant high level of expression in cecal tonsils and bursa. In conclusion, we were able to identify and characterize a new antimicrobial gene in chickens (chAng4) throughout the gastrointestinal tract. chAng4 mRNA gene expression was associated with the presence of naturally occurring and supplemented (probiotic) bacteria. The encoded protein might have a potential bactericidal effect against intestinal nonpathogenic and pathogenic microbes, modulating the intestinal microbiota and the innate immunity, and thereby may help minimize the use of antibiotics in poultry feed.