Genetic control of chicken heterophil function in advanced intercross lines: associations with novel and with known Salmonella resistance loci and a likely mechanism for cell death in extracellular trap production

Heterophils, the avian polymorphonuclear leukocyte and the counterpart of mammalian neutrophils, generate the primary innate response to pathogens in chickens. Heterophil performance against pathogens is associated with host disease resistance, and heterophil gene expression and function are under genetic control. To characterize the genomic basis of heterophil function, heterophils from F13 advanced intercross chicken lines (broiler × Leghorn and broiler × Fayoumi) were assayed for phagocytosis and killing of Salmonella enteritidis, oxidative burst, and extracellular trap production. A whole-genome association analysis of single nucleotide polymorphisms at 57,636 loci identified genomic locations controlling these functional phenotypes. Genomic analysis revealed a significant association of extracellular trap production with the SAL1 locus and the SLC11A1 gene, which have both been previously associated with resistance to S. enteritidis. Fine mapping supports SIVA1 as a candidate gene controlling SAL1-mediated resistance and indicates that the proposed cell-death mechanism associated with extracellular trap production, ETosis, likely functions through the CD27/Siva-1-mediated apoptotic pathway. The SLC11A1 gene was also associated with phagocytosis of S. enteritidis, suggesting that the Slc11a1 protein may play an additional role in immune response beyond depleting metal ions to inhibit intracellular bacterial growth. A region of chromosome 6 with no characterized genes was also associated with extracellular trap production. Further characterization of these novel genes in chickens and other species is needed to understand their role in polymorphonuclear leukocyte function and host resistance to disease

Structure, function and immunolocalization of a proton-coupled amino acid transporter (hPAT1) in the human intestinal cell line Caco-2

The human orthologue of the H+-coupled amino acid transporter (hPAT1) was cloned from the human intestinal cell line Caco-2 and its functional characteristics evaluated in a mammalian cell heterologous expression system. The cloned hPAT1 consists of 476 amino acids and exhibits 85 % identity with rat PAT1. Among the various human tissues examined by Northern blot, PAT1 mRNA was expressed most predominantly in the intestinal tract. When expressed heterologously in mammalian cells, hPAT1 mediated the transport of α-(methylamino)isobutyric acid (MeAIB). The cDNA-induced transport was Na+-independent, but was energized by an inwardly directed H+ gradient. hPAT1 interacted with glycine, l-alanine, l-proline, α-aminoisobutyrate (AIB) and γ-aminobutyrate (GABA), as evidenced from direct transport measurements and from competition experiments with MeAIB as a transport substrate. hPAT1 also recognized the d-isomers of alanine and proline. With serine and cysteine, though the l-isomers did not interact with hPAT1 to any significant extent, the corresponding d-isomers were recognized as substrates. With proline and alanine, the affinity was similar for l- and d-isomers. However, with cysteine and serine, the d-isomers showed 6- to 8-fold higher affinity for hPAT1 than the corresponding l-isomers. These functional characteristics of hPAT1 closely resemble those that have been described previously for the H+-coupled amino acid transport system in Caco-2 cells. Furthermore, there was a high degree of correlation (r2 = 0.93) between the relative potencies of various amino acids to inhibit the H+-coupled MeAIB transport measured with native Caco-2 cells and with hPAT1 in the heterologous expression system. Immunolocalization studies showed that PAT1 was expressed exclusively in the apical membrane of Caco-2 cells. These data suggest that hPAT1 is responsible for the H+-coupled amino acid transport expressed in the apical membrane of Caco-2 cells