Variants of NLRP3 Protein in Haemonchus contortus Infected Sheep: Impact on Immune Cell Responsiveness to LPS In Vitro.

ABSTRACT

Pathogen recognition is an essential component to achieve the desired outcome of host protection. Nod-like receptor pyrin containing domain 3 (NLRP3) is a cytoplasmic pattern recognition receptor (PRR) with a wide array of agonists, such as PAMPs, DAMPs, ATP, bacterial product and viral products. Stimulation of the NLRP3 inflammasome results in proteolytic activation of IL-1ß and IL-18, cell pyroptosis and classically, the induction of proinflammatory responses. St. Croix (STC) sheep have resistance traits exhibiting the appropriate T-helper type 2 immune response ensuing protection during helminth parasitic infection whereas parasite-susceptible Suffolk (SUF) sheep have an impaired response resulting in parasite establishment and adverse symptoms. The objective of these experiments was to determine if NLRP3 protein in H. contortus-infected SUF sheep was defective using the classical activation pathway of NLRP3 inflammasome. Peripheral blood mononuclear cells (PBMCs) derived from H. contortus-infected STC and SUF sheep were isolated from whole blood and treated (MCC950 treatment for 2 h followed by LPS treatment for 3 h, 1400 W treatment for 2 h followed by LPS treatment for 3 h, LPS treatment for 3 h or culture media for 3 h). qPCR analysis of LPS-stimulated PBMC revealed an upregulation in inflammatory associated genes IL-1ß, TLR4, TNFα and NFκB (p < 0.0001) in STC PBMC and downregulation in IFNγ, IL-6 and iNOS for SUF PBMC. Pharmacological inhibition of iNOS in SUF PBMC resulted in an upregulation in the expression of IFNγ. These preliminary data begin to discover a relationship between NLRP3 activation and TLR4 signalling in PBMC of STC and SUF sheep.