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NLRC5 knockdown in chicken macrophages alters response to LPS and poly (I:C) stimulation
Ling Lian1, Ceren Ciraci, Guobin Chang
1Department of Animal Science, Iowa State University, Ames, IA 50011, USA.
Background:
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. 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.
Results:
The 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.
Conclusions:
Functional 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.
Insights
Chicken NLRC5 positively regulates IFNA and IFNB gene expression, crucial for innate immunity. This study clarifies NLRC5
Area of Science:
- Immunology
- Molecular Biology
- Avian Biology
Background:
- NLRC5, an NLR family member, recognizes PAMPs and initiates innate immune responses.
- Its role in inflammation is debated, with reported links to type I IFNs, NF-kB, and MHC class I.
- No prior studies investigated NLRC5 function in chickens.
Purpose of the Study:
- To investigate the role of NLRC5 in regulating IFNA, IFNB, IL-6, and MHC class I expression in chicken HD11 macrophages.
- To elucidate NLRC5's function in the chicken innate immune response.
Main Methods:
- Utilized RNAi technology (siRNAs) to knock down NLRC5 expression in chicken HD11 macrophage cell line.
- Stimulated cells with LPS or poly (I:C) and quantified gene expression (NLRC5, IFNA, IFNB, IL-6, MHC class I) via qPCR at various time points post-stimulation.
Main Results:
- NLRC5 knockdown using s3-siRNA significantly down-regulated IFNA and IFNB expression.
- IL-6 and MHC class I expression were not directly affected by NLRC5 knockdown.
- LPS stimulation showed a trend towards restoring NLRC5, IFNA, and IFNB expression, though not statistically significant.
Conclusions:
- Chicken NLRC5 plays a positive regulatory role in IFNA and IFNB expression within the HD11 cell line.
- NLRC5 is important for regulating key inflammatory molecules in chickens.
- Further research on NLRC5 and other NLRs could enhance antiviral strategies and vaccine development in poultry.
