Gene Expression Analysis of Toll-Like Receptor Pathways in Heterophils from Genetic Chicken Lines that Differ in Their Susceptibility to Salmonella enteritidis.

Michael H Kogut,Igal Y Pevzner,Hsin-I Chiang,Christina L Swaggerty,Huaijun Zhou

doi:10.3389/fgene.2012.00121

Abstract

Previously conducted studies using two chicken lines (A and B) show that line A birds have increased resistance to a number of bacterial and protozoan challenges and that heterophils isolated from line A birds are functionally more responsive. Furthermore, when stimulated with Toll-like receptor (TLR) agonists, heterophils from line A expressed a totally different cytokine and chemokine mRNA expression pattern than heterophils from line B. A large-scale gene expression profile using an Agilent 44K microarray on heterophils isolated from line A and line B also revealed significantly differential expression in many immune-related genes following Salmonella enteritidis (SE) stimulation, which included genes involved in the TLR pathway. Therefore, we hypothesize the differences between the lines result from distinctive TLR pathway signaling cascades that mediate heterophil function and, thus, innate immune responsiveness to SE. Using quantitative RT-PCR on mRNA from heterophils isolated from control and SE-stimulated heterophils of each line, we profiled the expression of all chicken homologous genes identified in a reference TLR pathway. Several differentially expressed genes found were involved in the TLR-induced My88-dependent pathway, showing higher gene expression in line A than line B heterophils following SE stimulation. These genes included the TLR genes TLR4, TLR15, TLR21, MD-2, the adaptor proteins Toll-interleukin 1 receptor domain-containing adaptor protein (TIRAP), Tumor necrosis factor-receptor associated factor 3 (TRAF3), the IκB kinases transforming growth factor-β-activating kinase 1 (TAK1), IKKε and IKKα, the transcription factors NFkB2 and interferon regulatory factor 7, phosphatidylinositol-3 kinase (PI-3K), and the mitogen-activated protein kinase p38. These results indicate that higher expression of TLR signaling activation of both MyD88-dependent and TRIF-dependent pathways are more beneficial to avian heterophil-mediated innate immunity and a complicated regulation of downstream adaptors is involved in stronger induction of a TLR-mediated innate response in the resistant line A. These findings identify new targets for genetic selection of chickens to increase resistance to bacterial infections.

Highlights

Host genetics plays an indispensable role in response to Salmonella colonization of chickens
Upon infection with Salmonella enteritidis (SE), there were no significant differences in Toll-like receptors (TLRs) expression in heterophils between lines A and B with one exception, TLR15 (Table 2) where TLR 15 was differentially expressed in the heterophils from line A following infection with SE when compared to heterophils from line B chickens
After analyzing 16 conserved TLR pathway genes identified from the chicken genome, we found only one gene in each line that was differentially expressed in the non-infected heterophils

Summary

Introduction

Host genetics plays an indispensable role in response to Salmonella colonization of chickens. Line A chickens are more resistant to the pathogen challenges than line B chickens. This resistance was mediated by the predominant avian granulocyte, the heterophil, with heterophils from Line A functionally more responsive and capable of producing a differential cytokine/chemokine profile compared with line B (Ferro et al., 2004; Swaggerty et al, 2004, 2005a). The TLR superfamily represents an evolutionarily conserved signaling system that is a decisive determinant of the innate immune and inflammatory responses. The innate system uses these germ-line encoded receptors to detect evolutionarily conserved microbial proteins, lipids, and nucleic www.frontiersin.org

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