Abstract
BackgroundNLRC5 is a member of the CARD domain containing, nucleotide-binding oligomerization (NOD)-like receptor (NLR) family, which recognizes pathogen-associated molecular patterns (PAMPs) and initiates an innate immune response leading to inflammation and/or cell death. However, the specific role of NLRC5 as a modulator of the inflammatory immune response remains controversial. It has been reported to be a mediator of type I IFNs, NF-kB, and MHC class I gene. But no study on NLRC5 function has been reported to date in chickens. In the current study, we investigated the role of NLRC5 in the regulation of IFNA, IFNB, IL-6, and MHC class I in the chicken HD11 macrophage cell line, by using RNAi technology. HD11 cells were transfected with one of five siRNAs (s1, s2, s3, negative-siRNA, or a mixture of s1, s2, s3-siRNAs). After 24 hours, cells were exposed to LPS or poly (I:C) or a vehicle control. Gene expression of NLRC5, IFNA, IFNB, IL-6, and MHC class I at 2, 4, 6, and 8 hours post stimulation (hps) was quantified by qPCR.ResultsThe expression of NLRC5, IFNA, IFNB, and IL-6 genes in negative irrelevant transfection controls was up-regulated at 2 hps after LPS treatment compared to the vehicle controls. S3-siRNA effectively knocked down NLRC5 expression at 4 hps, and the expression of IFNA and IFNB (but not IL-6 and MHC class I) was also down-regulated at 4 hps in s3-siRNA transfected cells, compared to negative irrelevant transfection controls. Stimulation by LPS appeared to relatively restore the decrease in NLRC5, IFNA, and IFNB expression, but the difference is not significant.ConclusionsFunctional characterization of chicken NLRC5 in an in vitro system demonstrated its importance in regulating intracellular molecules involved in inflammatory response. The knockdown of NLRC5 expression negatively mediates gene expression of IFNA and IFNB in the chicken HD11 cell line; therefore, NLRC5 likely has a role in positive regulation of IFNA and IFNB expression. No direct relationship was found between NLRC5 knockdown and IL-6 and MHC class I expression. Future studies will further clarify the roles of NLRC5 and other NLRs in infectious diseases of chickens and may increase the efficacy of antiviral vaccine design.
Highlights
NLRC5 is a member of the CARD domain containing, nucleotide-binding oligomerization (NOD)-like receptor (NLR) family, which recognizes pathogen-associated molecular patterns (PAMPs) and initiates an innate immune response leading to inflammation and/or cell death
Cui et al demonstrated that NLRC5 negatively modulated Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) and type I IFN signaling pathways and its absence resulted in elevated expression levels of Tumor necrosis factor (TNF) A, IL-6, and IL-1B [27]
Expression of NLRC5, IFNA, IFNB, and IL-6 in negative irrelevant siRNA transfection controls was up-regulated at 2 hours post LPS stimulation The expression of NLRC5, IFNA, IFNB, and IL-6 was significantly up-regulated, and Major histocompatibility complex (MHC) class I showed an increasing trend, in LPS-treated HD11 cells compared to vehicle controls at 2 hours post stimulation (Figure 1)
Summary
NLRC5 is a member of the CARD domain containing, nucleotide-binding oligomerization (NOD)-like receptor (NLR) family, which recognizes pathogen-associated molecular patterns (PAMPs) and initiates an innate immune response leading to inflammation and/or cell death. There are three classes of PRRs in vertebrates, Toll-like receptors (TLRs), nucleotide-binding oligomerization (NOD)-like receptors (NLRs), and the retinoid acid-inducible gene-I (RIG-I)-like receptors (RLRs) [1,2,3,4,5,6] These molecules showed different subcellular localization, for example, most TLRs identify activating caspase-1 inflammasomes as a response to PAMPs and DAMPs, leading to maturation and secretion of pro-inflammatory cytokines interleukin (IL)-1B and IL-18 [12]. Overexpression of NLRC5 was shown to repress the activation of NF-kB-, type I IFN-, and AP-1-dependent signaling pathways in human embryonic kidney cell line HEK293T, and the knockdown of NLRC5 increased secretion of proinflammatory cytokines in mouse leukaemic monocyte macrophage cell line, RAW264.7 [9]. The effects of NLRC5 in regulating immune-related components and inflammatory responsive pathways are very complex, and likely either cell type- or species-specific [24]
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