Cecropin anisaxin-2S has in vitro immunomodulatory, but not antiproliferative and antiviral properties

Helminthic host defense peptides (HDP) are pleiotropic, multifunctional effector molecules of helminth immunity, efficient against Gram-negative and Gram-positive bacteria. Among them, anisaxin-2S (A-2S), membranolytic cecropin-like HDPs produced by the zoonotic nematodes of the genus <i>Anisakis</i>, shows remarkable efficacy even against multidrug-resistant Gram-negative bacteria, yet its immunomodulatory, antiproliferative and antiviral properties have not been elucidated. Therefore, we tested A-2S immunomodulation in the common carp (<i>Cyprinus carpio</i>) blood cells exposed to two pathogens, the zoonotic bacterium <i>Aeromonas hydrophila</i> and the fish parasite <i>Sphaerospora molnari</i>, and in carp <i>in vivo</i> challenged with the parasite. Furthermore, the A-2S antiproliferative activity was tested <i>in vitro</i> in human bladder and lung cancer cell line, while the antiviral protection was tested in common carp brain cell culture exposed to carp rhabdovirus, alloherpesvirus and paramyxovirus, and in a human immortalized myelogenous leukemia cell line infected with tick-borne encephalitis virus. A-2S exerts an immunostimulatory effect on fish blood cells through upregulation of cytokine expression, with the proinflammatory or anti-inflammatory repertoire conditioned by the presence or absence of co-stimulatory antigen. Surprisingly, in the majority of assays conducted, red blood cells demonstrate equal or even stronger regulation of innate immunity genes compared to white blood cells, along with a more extensive repertoire of differentially expressed markers. In contrast, A-2S has only a limited anticancer activity in human bladder cancer and lung adenocarcinoma cells and limited antiviral activity against the three fish viruses and a human tick-borne encephalitis virus. This study provides the first evidence of red blood cell and platelet immunomodulation by an antimicrobial peptide and highlights the induction of a cytokine repertoire. However, future research should address the study's limitations, including the need for longer <i>in vitro</i> assays (e.g., 3-4 days), testing different white blood cell lineages, to better understand antigen-processing interactions, and evaluating the anticipated adaptive immune response. Powerful antimicrobial activity of A-2S, coupled with immunostimulatory properties, warrant further pursuing of preclinical trials with this anisaxin.