Gene expression modulation in chicken macrophages exposed to Mycoplasma synoviae or Escherichia coli.

Mycoplasma synoviae and Escherichia coli are two avian pathogens that exhibit markedly different mechanisms for infection and pathogenicity and may be expected to manipulate the host innate immune response differently. The aim of this study was to determine the extent of modulated genes and make a comparison between the transcriptomes of chicken macrophages exposed to either M. synoviae type strain WVU 1853 (MS) or avian pathogenic E. coli strain V-G (APEC). To analyze temporal gene expression profile of monocyte-derived macrophages (MDM) and HD11 cell line macrophages after each exposure, two avian immunity microarrays were used: the avian macrophage microarray (AMM) and the avian innate immunity microarray (AIIM). The quantity of MS-modulated genes was estimated in three experiments, using both microarrays. A cross-section revealed 14 AMM/AIIM genetic elements that were modulated in both types of macrophages. Additionally, to compare immunomodulatory activity of MS and APEC, MDM were exposed to each pathogen and gene modulation was detected by AIIM microarray. This study revealed 157 elements uniquely modulated by MS and 1603 elements uniquely modulated by APEC. AIIM microarray analysis also revealed a core set of 146 elements modulated by both pathogens, with generally higher induction/repression levels after APEC exposure. Validation of selected gene expression was done by quantitative real time RT-PCR. The study shows higher transcription levels of IL-1beta, IL-6, iNOS, NCF1, peroxiredoxin 1 and cathepsin L genes after MDM exposure to APEC than after exposure to MS. Surprisingly, complement component C3 gene was repressed after MDM exposure to APEC, while being induced after exposure to MS.

Construction and application of an avian intestinal intraepithelial lymphocyte cDNA microarray (AVIELA) for gene expression profiling during Eimeria maxima infection.

Intestinal intraepithelial lymphocytes (IELs) are the primary immune effector cells in the gut and play a critical role in eliciting protective immunity to enteric pathogens such as Eimeria, the etiologic agent of avian coccidiosis. In this study, a microarray of genes expressed by intestinal IELs from Eimeria-infected chickens was constructed using the expressed sequence tag (EST) strategy. The avian intestinal IEL cDNA microarray (AVIELA) contained duplicates of 9,668 individual ESTs (6,654 known genes and 3,014 unique singletons of unknown identity) and was used to analyze gene expression profiles during primary and secondary Eimeria maxima infections. Following primary inoculation with E. maxima, the expression levels of 74 genes were significantly altered more than two-fold over the 3-day infection period (51 up-regulated, 23 down-regulated). Following secondary infection, the expression levels of 308 genes were significantly altered (62 up-regulated, 246 down-regulated). Pathway gene analysis indicated that many of the modulated genes were related to apoptosis, JAK/STAT, MAPK, interleukin, and TLR signaling pathways, and involving innate and adaptive immune responses. This chicken IEL microarray will provide a valuable resource for future transcriptional profiling of the genes involved in protective immunity to chicken enteric pathogens.

Unique responses of the avian macrophage to different species of Eimeria.

Coccidiosis is recognized as the major parasitic disease of poultry and is caused by the apicomplexan protozoa Eimeria. Increasing evidence shows the complexity of the host immune response to Eimeria and microarray technology presents a powerful tool for the study of such an intricate biological process. Using an avian macrophage microarray containing 4906 unique gene elements, we identified important host genes whose expression changed following infection of macrophages with sporozoites of Eimeria tenella (ET), Eimeria acervulina (EA), and Eimeria maxima (EM). This approach enabled us to identify a common core of 25 genetic elements whose transcriptional expression is induced or repressed by exposure to Eimeria sporozoites and to identify additional transcription patterns unique to each individual Eimeria species. Besides inducing the expression of IL-1beta, IL-6, and IL-18 and repressing the expression of IL-16, Eimeria treated macrophages were commonly found to induce the expression of the CCL chemokine family members macrophage inflammatory protein (MIP)-1beta (CCLi1), K203 (CCLi3), and ah221 (CCLi7). However, the CXCL chemokine K60 (CXCLi1) was found to be induced by macrophage exposure to E. tenella but was repressed upon macrophage exposure to E. maxima and E. acervulina. Fundamental analysis of avian chemokine and cytokine expression patterns offers insight into the unique avian immunological responses to these related but biologically unique pathogens.

Gene expression profiling of avian macrophage activation.

Through the process of phagocytosis, the macrophage is responsible for the clearance and destruction of both intracellular and extracellular pathogens. When stimulated, macrophages undergo a process of activation involving an increase in size and motility, enhanced phagocytic, bactericidal, and tumoricidal activity, and up-regulation of several cell-surface markers. One well characterized method of mammalian macrophage activation involves the Toll-like receptor (TLR) pathway. TLRs are surface molecules that function as direct receptors for microbial components. Binding of ligand to TLRs results in activation of transcription factors that regulate genes involved in microbial killing, apoptosis, and antigen recognition, as well as pro- and anti-inflammatory cytokines and chemokines. We have constructed a 4906-element (14,718 spot) avian macrophage-specific cDNA microarray (AMM). The AMM contains 16 of the approximately 44 genes identified within the mammalian TLR pathway. This array was used to examine the transcriptional response of avian macrophages to Gram-negative bacteria and their cell wall components and to evaluate the contribution of the avian TLR pathway to that response. Of the elements on the AMM, 981 (20%) exhibited significant (greater than two-fold, p < 0.01) changes in expression during phagocytosis of Escherichia coli and 243 (5%) exhibited significant expression changes during exposure to lipopolysaccharide (LPS). A unique set of overlapping elements (154), were observed to exhibit significant changes in expression for both phagocytosis and LPS stimulation, representing a set of core response elements. Of these elements, 63% were commonly induced, while 32% were commonly repressed. Both LPS and bacteria were found to induce NFkappabeta and several end products of the TLR pathway.