Interleukin-12 receptor β2 from grass carp: Molecular characterization and its involvement in Aeromonas hydrophila-induced intestinal inflammation

Interleukin-12 receptor beta 2 (IL-12R beta 2) is a signaling subunit of heterodimeric receptors for IL-12 and IL-35. It plays important regulatory functions in the development of Th1 cells and in the expression of inflammatory cytokines in mammals and other higher vertebrates. However, little is known about IL-12R beta 2 in teleost fish. In this work, we have cloned and characterized IL-12R beta 2 from grass carp (Ctenopharyngodon idella). The full-length cDNA of grass carp IL-12R132 is 2875 bp, which encodes a mature protein with 741 amino acids. This mature protein contains three fibronectin type III domains, a transmembrane helix, and CXW and WSXWS-like motifs that are characteristic of the type I cytokine receptor family. Phylogenetic analysis revealed that cyprinid fish IL-12R beta 2 formed a single branch, clearly separated from those of other vertebrates. We expressed and purified a recombinant grass carp IL-12R beta 2 protein containing major antigenic regions, which was used to raise a polyclonal antibody. The specificity of the antibody was assessed by Western blotting analysis of whole cell lysates from Escherichia coil cells expressing the recombinant IL-12R beta 2, grass carp intestinal intraepithelial lymphocytes, and cultured C. idella kidney cells. To explore the potential regulatory role of IL-12R beta 2 in inflammation, we generated an intestinal inflammation model by anal intubation of fish with Aeromonas hydrophila. Immunohistochemical staining of the inflamed intestines revealed that IL-12R beta 2 expression is consistent with inflammatory cell recruitment during intestinal inflammation. Real-time quantitative PCR revealed that IL-12R beta 2 is widely expressed in normal tissues and is up-regulated in most tissues after infecting with A. hydrophila. We found that IL-12R beta 2, IL-12p35, and interferon-gamma were expressed in similar patterns in the intestines during inflammation. Taken together, our results suggest that IL-12R beta 2 is involved in the regulation of intestinal inflammation.