TLR4, MyD88, and TNF-α Expression in the Lungs of Akkaraman and Romanov Lambs in Response to LPS and LTA.

Toll-like receptors are involved in the recognition of bacterial toxins, which cause infection in the respiratory system. This study aimed to evaluate microanatomical and histological alterations in the lungs of 24 healthy Akkaraman and Romanov lambs after the administration of lipoteichoic acid (LTA), lipopolysaccharide (LPS), and LTA + LPS and investigate the gene, protein, and immune expression levels of TLR4, MyD88, and TNF-α molecules, known to have immune functions. Microanatomical examinations showed thickened peribronchial and alveolar walls in the lungs of groups LTA, LPS, and LTA + LPS of both breeds due to immune cell infiltration. TLR4, MyD88, and TNF-α immunoexpressions were positive to varying degrees in the cytoplasm and nucleus of the bronchial and bronchiolar luminal epithelial cells, alveolar epithelial cells, and alveolar macrophages. TLR4 and TNF-α protein expressions were statistically different in the LPS-treated Romanov lambs, compared to the other groups. Among the Akkaraman lambs, TLR4 gene expression was significantly higher in group LPS, and among the Romanov lambs, TLR4, MyD88, and TNF-α gene expressions were significantly higher in group LTA + LPS. Therefore, TLR4, MyD88, and TNF-α molecules, involved in the immune response, were found to be expressed at different levels against LTA and LPS in the lungs of two different sheep breeds.

LPS- and LTA-Induced Expression of TLR4, MyD88, and TNF-α in Lymph Nodes of the Akkaraman and Romanov Lambs.

Toll-like receptor (TLR)-mediated inflammatory processes play a critical role in the innate immune response during the initial interaction between the infecting microorganism and immune cells. This study aimed to investigate the possible microanatomical and histological differences in mandibular and bronchial lymph nodes in Akkaraman and Romanov lambs induced by lipopolysaccharide (LPS) and lipoteichoic acid (LTA) and study the gene, protein, and immunoexpression levels of TLR4, myeloid differentiation factor 88 (MyD88), and tumor necrosis factor-α (TNF-α) that are involved in the immune system. Microanatomical examinations demonstrated more intense lymphocyte infiltration in the bronchial lymph nodes of Akkaraman lambs in the LPS and LTA groups compared to Romanov lambs. TLR4, MyD88, and TNF-α immunoreactivities were more intense in the experimental groups of both breeds. Expression levels of MyD88 and TNF-α genes in the bronchial lymph node of Akkaraman lambs were found to increase statistically significantly in the LTA group. TLR4 gene expression level in the mandibular lymph node was found to be statistically significantly higher in the LTA + LPS group. In conclusion, dynamic changes in the immune cell populations involved in response to antigens such as LTA and LPS in the lymph nodes of both breeds can be associated with the difference in the expression level of the TLR4/MyD88/TNF-α genes.

Effect of LPS and LTA stimulation on the expression of TLR-pathway genes in PBMCs of Akkaraman lambs in vivo.

This is the first study investigating the changes in some gene expressions related to the TLR pathway in vivo in sheep. Lipopolysaccharide (LPS) and lipoteichoic acid (LTA) molecules were administrated separately and in combination to the Akkaraman lambs via intranasal route. For this purpose, 28 lambs were distributed into four groups (LPS, LTA, LPS + LTA, and control, n = 7). Blood samples were collected to isolate the peripheral blood mononuclear cells (PBMCs) at 24 h and on day 7. Expression levels of TLR2, TLR4, MyD88, TRAF6, TNF-α, IL-1ß, IL-6, IL-10, NF-κß, and IFN-γ genes were determined by qRT-PCR. Increases were determined in the expression data of TLR2 [LPS (P < 0.05) and LTA + LPS (P < 0.01)], TLR4 [LTA + LPS (P < 0.05)], TNF-α, IL-10 [LTA + LPS (P < 0.05)], and IFN-γ genes in all groups in the mRNA expression analysis of PBMCs isolated at 24 h whereas decreases were determined in the expression levels of these genes on day 7. The combination of LPS + LTA stimulated lamb PBMCs more effectively than separate administration of LPS and LTA at 24 h. Therefore, this article may contribute to the understanding the host-pathogen interaction of respiratory-transmitted bacterial diseases concerning PBMCs at 24 h and on day 7. Also this study may contribute to the dose adjustment for bacterial vaccine studies in sheep. Experimental application doses will be helpful for in vivo and in vitro drug and vaccine development studies in the fields of pharmacology and microbiology.