Functional differences of three CXCL10 homologues in the giant spiny frog Quasipaa spinosa

Chemokines are a superfamily of structurally related chemotactic cytokines exerting significant roles in acting as a bridge between the innate and adaptive immune responses. In this study, we identified three CXC motif chemokine 10 (CXCL10) homologues (QsCXCL10-1, QsCXCL10-2 and QsCXCL10-3) from giant spiny frog Quasipaa spinosa. All three deduced QsCXCL10 proteins contained four conserved cysteine residues as found in other known CXC chemokines. Phylogenetic analysis showed that QsCXCL10-1, 2, 3 and other CXCL10s in amphibian were grouped together to form a separate clade. These three QsCXCL10s were highly expressed in spleen and blood. Upon infection with Staphylococcus aureus or Aeromonas hydrophila, the expressions of QsCXCL10s were markedly increased in spleen and blood during biotic stresses. Meanwhile, the QsCXCL10s transcription in liver could also be up-regulated under abiotic stresses such as cold and heat stresses. The recombinant proteins of frog CXCL10 homologues were produced and purified in E. coli and possessed similar but differential bioactivities. Both rCXCL10-1 and rCXCL10-2 had strong effects on the up-regulation of pro-inflammatory cytokines (TNF-α, IL-1β and IL-8) in vivo, whereas rCXCL10-3 induced a weak expression of these cytokines. Moreover, the rCXCL10-1 and rCXCL10-2 could strongly promote splenocyte proliferation and induce lymphocytes migration, while rCXCL10-3 had limited effects on these biological processes. All three frog chemokines triggered their functional activities by engaging CXC motif chemokine receptor 3 (CXCR3). Taken together, these results revealed that the three QsCXCL10s had similar but differential functional activities in mediating immune responses and host defenses, which might contribute to a better understanding of the functional evolution of CXCL10 in vertebrates.