In vitro characterization of N1- and N2-like neutrophil polarization and the putative regulatory role of Pparγ in common carp (Cyprinus carpio L.).

Neutrophils are key regulators of innate immunity and display substantial phenotypic plasticity shaped by the local cytokine and hormonal environment. In mammals, neutrophils can polarize into pro-inflammatory (N1) or anti-inflammatory (N2) phenotypes, but this process has not been comprehensively described in fish. This study investigated whether neutrophils of carp can acquire N1- and N2-like phenotypes and examined the role of peroxisome proliferator-activated receptor gamma (Pparγ) in their polarization. A neutrophil-enriched cell population was isolated from the head kidney of common carp (Cyprinus carpio L.) and stimulated in vitro with recombinant interferon gamma 2 combined with lipopolysaccharide (rcIfnγ2+LPS), recombinant interleukin 4/13b (rcIl-4/13b), or prostaglandin E2 (PGE2). Pro- and anti-inflammatory activities were assessed by analyzing expression of inflammatory markers, production of nitric oxide and reactive oxygen species, and arginase activity. To evaluate the role of Pparγ, cells were additionally treated with its agonist - rosiglitazone or antagonist - GW9662. Stimulation with rcIfnγ2+LPS induced polarization toward a pro-inflammatory N1-like phenotype, characterized by increased expression of inos, il12p35, il1b and cxcr1. In contrast, rcIl-4/13b and PGE2 promoted an anti-inflammatory N2-like phenotype associated with elevated cxcr2, pparg, tgfb and reduced il1b expression. Inhibition of Pparγ modulated gene expression of both pro- and anti-inflammatory markers in a stimulus-dependent manner. These findings provide the first comprehensive characterization of the transcriptional polarization state of neutrophils in common carp and confirm that in fish Pparγ is an important regulator of neutrophil inflammatory plasticity.