Neutrophil functions can be regulated by IL-35, which is mainly expressed in IL-15Rα+ cells in grass carp (Ctenopharyngodon idella)

IL-35 plays a key role in regulatory T (Treg) and regulatory B (Breg) cell functions in mammals. CD25 has been demonstrated as one of the markers of Treg cells, and CD19(+)CD25(hi)CD71(hi) cells have been verified as a type of Breg cells in humans. These results indicate that there is a close relationship between IL-35 and CD25(+) cells. In mammals, CD25 (alias IL-2R alpha) has been identified as having high affinity and specificity for IL-2 binding, and is closely linked and structurally related to IL-15R alpha, which having high affinity for IL-15 binding. In teleost, IL15R alpha can bind to both IL-2 and IL-15, with higher affinity to IL-15 than IL-2, and has been termed a CD25-like molecule in some research studies. To date, no studies of IL-35 and IL-15R alpha have been documented in fish. In this work, five isoforms of IL-15R alpha were cloned from grass carp, and a monoclonal antibody to the protein was developed. The results of flow cytometry and quantitative real-time PCR analyses demonstrated that grass carp IL-35 subunit genes EBI3a and IL-12p35 were mainly expressed in IL-15R alpha(+) cells, while the expression levels of IL-10 and TGF-beta in IL-15R alpha(+) and IL-15R alpha- cells were insignificant. Recombinant grass carp IL-35 (rgcIL-35) could increase the proportion of IL-15R alpha(+) cells in leukocytes, and a certain proportion of IL-15R alpha(+) cells also appeared in myeloid cell subset II after stimulation with rgcIL-35. Meanwhile, the migration, phagocytic ability, and bactericidal ability of grass carp neutrophils were significantly decreased after stimulation with certain concentrations of rgcIL-35. Moreover, neutrophil apoptosis could be significantly inhibited by rgcIL-35.