An in vitro Study on Immune Response of Mammary Epithelial Cells (MEC) in Goats against Bacterial Cell Wall Components
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Date
2022-09-12
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Sher-e-Kashmir University of Agricultural Sciences & Technology- Jammu (J&K)
Abstract
The study was aimed to evaluate the immune response of caprine mammary epithelial cells (MECs) as an in vitro model against bacterial PAMPs through NOD mediated pathway. Primary culture of Bakarwali and non-descript goat MECs was established and exposed to bacterial cell wall components(iE-DAP and MDP), and the cell response following exposure was studied through mRNA expression profiling of cellular PRR components. Expression of genes encoding for receptors NOD1 and NOD2; transcription factors RICK and NF-kB; and for pro/ anti-inflammatory cytokines (IL-08, IL-1β, IL-10, GM-CSF) and interferon (IFN) were evaluated by qRT-PCRthrough designed primers at 15 min, 1, 2, 4, 8, 16, 24 and 32 h post exposure. Caprine mammary epithelial cells (MECs) for both Bakarwali and non-descript goat were established andconfirmed through expression of signature β-casein (Csn2), Keratin 18 (Krt18) and Vimentin (Vim) genes. Ligand binding stimulated PRRs in both goat MECs and mediated immune response through the NLR pathway.In general, expression of immune response components was early and intense in MECs derived from Bakarwali goat, and otherwise relatively late, of lower magnitude, but of a more sustained response in non-descript goat MECs. In comparison, stimulation with iE-DAP induces an earlier immune response and reaches maximal at 16-24 h post stimulation, whereas response against MDP reaches peak levels at 24 h post stimulation in both MECs. This implies that there exists breed difference in pattern of innate immune response against bacterial PAMPs mediated by MECs. Further, insights into the genetic variation of caprine NOD1 gene was also evaluated through amplification of different fragments using designed primers. Nucleotide sequence alignment revealed nucleotide changes AG1227 and GA1689 specific to the goat of Jammu and Kashmir and CT1263, CT1286, CT1611 specific to the Caprine species without any amino acid changes. Phylogenetic construct revealed a separate clade for Caprine with closeness to Ovis. Multiple amino acid alignment with CaprineNOD1 showed a single Caprine specific IsoMet11 substitution, with close homology with Ovine. Caprine-Ovine specific amino acid changes included CARD domain (SerPro17), NACHT domain (ValAla347;GlyGlu509) and LRR4 (AgrGly776). The critical binding residues of NACHT domain of NOD1 was analysed further and no caprine specific amino acid changes were observed but shared homology with other ruminants, suggesting that caprine NOD1 are conserved