Examination of goldfish primary kidney neutrophil extracellular trap (NET) formation and the role of Carassius auratus leukocyte immune-type receptor (CaLITR)3-as an activator of NETosis in fish.

Teleost leukocyte immune-type receptors (LITRs) are a large family of immunoregulatory receptor types that share distant evolutionary relationships with various mammalian immunoregulatory receptor types, including members of the Fc receptor-like family. Carassius auratus (Ca)-LITR3 is a putative stimulatory LITR-type expressed by goldfish immune cells. To investigate the regulatory functions of this putative activating receptor in goldfish, a polyclonal antibody (anti-CaL3.D1 pAb) was generated, which targets an epitope within the D1 domain of CaLITR3. Previous research showed that anti-CaL3.D1 pAb strongly stained the surface of goldfish primary kidney neutrophils (PKNs) and crosslinking of CaLITR3 with anti-CaL3.D1 pAb activated ERK1/2 and p38 in the mitogen-activated protein kinase (MAPK) pathway in PKNs. In the present study, we show that activation of CaLITR3 significantly upregulates transcription of the pro-inflammatory cytokine gene il1β in goldfish PKNs, indicating that beyond ERK1/2 and p38 signaling, CaLITR3 may induce functional effector responses in goldfish PKNs. To further investigate the roles of CaLITR3 as an activator of PKN antimicrobial responses, we examined its ability to trigger NETosis. This process represents a critical antimicrobial mechanism of neutrophils, where activated neutrophils release neutrophil extracellular traps (NETs) composed of decondensed chromatin mixed with granular enzymes to immobilize and kill invading microbes. Since triggering of CaLITR3 with anti-CaL3.D1 pAb activated MAPK pathway proteins in goldfish PKNs, we hypothesized that CaLITR3 is an inducer of NETosis formation in goldfish PKNs. To specifically examine goldfish PKN NETosis, we optimized NETosis assays for the quantitative and visual detection of NET formation in goldfish. Our data showed that goldfish PKNs release NETs in response to both receptor-independent (PMA, calcium ionophore) and receptor-mediated (Zymosan A, heat-killed Aeromonas salmonicida) stimuli. We also showed that CaLITR3 engagement triggers goldfish PKN NET release, implicating this LITR-type as an activator of neutrophil antimicrobial responses in fish.