Molecular identification and characterization of chicken nucleotide-binding oligomerization domain like receptor pyrin domain containing 3 (NLRP3) inflammasome
Abstract
The present study was conducted to determine functional genomics of chicken innate immune cytosolic sensor nucleotide-binding oligomerization domain like receptor pyrin domain containing 3 (NLRP3) inflammasome. RNA was extracted from chicken macrophage cell line HD11 cells or bursa of Fabricius, reversely transcribed to cDNA and subjected to PCR amplification of chicken NLRP3. The PCR amplicons were cloned and sequenced. The expression level of NLRP3 in various chicken tissues was determined by SyBr Green-based chicken NLRP3-specific real-time RT-PCR. Functional characterization of NLRP3 was determined by 12-hour lipopolysaccharide (LPS) and additional 15-minute ATP stimulation of HD11 cells or HD11 cells with knockdown of chicken NLRP3 by small interfering RNA (siRNA) targeting chicken NLRP3. Chicken IL-1β levels in the cell culture supernatants were analyzed and determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western Blotting. The chicken NLRP3 has an open reading frame encompassing 2778 base pairs of nucleotides and encoding a protein of 925 amino acids. There is one pyrin domain (PYD) in the N-terminal region and leucine-rich repeat domain (LRR) in C-terminal region. The domain architecture is conserved among NLRP3 paralogues. Chicken NLRP3 does not share high genetic identity to mammalian NLRP3 and is phylogenetically different from mammalian counterpart. The highest mRNA levels of chicken NLRP3 were present in kidney, bursa and spleen at 9.4x1010, 9.33x10 10 and 8.57x1010 (copy number/µl), respectively. Production of mature chicken IL-1β was detected on the Western blots from the supernatants of HD11 cells stimulated with LPS for 12 hours. Increased amounts of chicken IL-1β were detected in the supernatants of HD11 cells, following stimulation with LPS for 12 hours and exposure to ATP for additional 15 minutes. Production of mature chicken IL-1β was partially abrogated or decreased in the supernatants of chicken NLRP3-knockdown HD11 cells stimulated with LPS and ATP. The result indicated that chicken tissues possess NLRP3 encompassing 2778 base pairs of nucleotides and coding for 925 amino acids. Chicken NLRP3 is phylogenetically different from mammalian NLRP3. Chicken NLRP3 is ubiquitously expressed in chicken tissues. In addition, chicken NLRP3 functions as a cytosolic sensor for LPS and ATP and production and activation of mature chicken IL-1β is chicken NLRP3 dependent.
Degree
M.S.
Advisors
Lin, Purdue University.
Subject Area
Veterinary services|Immunology
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