Molecular characterization and immune response of suppressor of cytokine signaling 5b from redlip mullet (Planiliza haematocheilus): Disclosing its anti-viral potential and effect on cell proliferation.

The suppressor of cytokine signaling (SOCS) proteins family comprising eight proteins (SOCS1-7 and cytokine-inducible SH2-containing (CIS)) are classical feedback inhibitors of cytokine signaling. Although the biological role of CIS and SOCS1-3 have been extensively studied, the biological functions of SOCS4-7 remain unclear. Here, we elucidated the molecular characteristics, expression profile, immune response, anti-viral potential, and effect on cell proliferation of Phsocs5b, a member of the SOCS protein family from redlip mullet (Planiliza haematocheilus); phsocs5b comprised 1695 nucleotides. It was 564 amino acids long with a molecular weight of 62.3 kDa and a theoretical isoelectric point of 8.95. Like SOCS4-7 proteins, Phsocs5b comprised an SH2 domain, SOCS box domain, and a long N-terminal. SH2 domain is highly identical to its orthologs in other vertebrates. Phsocs5b, highly expressed in the brain tissue, was localized in the cytoplasm. Temporal changes in phsocs5b expression were observed following immune stimulation with polyinosinic: polycytidylic acid, lipopolysaccharide, and Lactococcus garvieae. In FHM cells, Phsocs5b overexpression suppressed the viral hemorrhagic septicemia virus (VHSV) infection and epidermal growth factor receptor (egfr) expression but increased the mRNA levels of pi3k, akt, pro-inflammatory cytokines (il1ß and il8), and anti-viral genes (isg15 and ifn). Overall, our findings suggest that Phsocs5b attenuates VHSV infection, either by hindering the cell entry via degradation of Egfr, enhancing pro-inflammatory cytokines and anti-viral factor production, or both. The results also indicated that Phsocs5b could directly activate Pi3k/Akt pathway by itself, thus enhancing the proliferation and migration of cells. Taken together, Phsocs5b may be considered a potential therapeutic target to enhance immune responses while positively regulating the proliferation and migration of cells.

Cytosolic ß-catenin is involved in macrophage M2 activation and antiviral defense in teleosts: Delineation through molecular characterization of ß-catenin homolog from redlip mullet (Planiliza haematocheila).

ß-catenin is a structural protein that makes the cell-cell connection in adherence junctions. Besides the structural functions, it also plays a role as a central transducer of the canonical Wnt signaling cascade, regulating nearly four hundred genes related to various cellular processes. Recently the immune functions of ß-catenin during pathogenic invasion have gained more attention. In the present study, we elucidated the immune function of fish ß-catenin by identifying and characterizing the ß-catenin homolog (PhCatß) from redlip mullet, Planiliza haematocheila. The complete open reading frame of PhCatß consists of 2352 bp, which encodes a putative ß-catenin homolog (molecular weight: 85.7 kDa). Multiple sequence alignment analysis revealed that ß-catenin is highly conserved in vertebrates. Phylogenetic reconstruction demonstrated the close evolutionary relationship between PhCatß and other fish ß-catenin counterparts. The tissue distribution analysis showed the highest mRNA expression of PhCatß in heart tissues of the redlip mullet under normal physiological conditions. While in response to pathogenic stress, the PhCatß transcription level was dramatically increased in the spleen and gill tissues. The overexpression of PhCatß stimulated M2 polarization and cell proliferation of murine RAW 264.7 macrophage. In fish cells, the overexpression of PhCatß resulted in a significant upregulation of antiviral gene transcription and vice versa. Moreover, the overexpression of PhCatß could inhibit the replication of VHSV in FHM cells. Our results strongly suggest that PhCatß plays a role in macrophage activation and antiviral immune response in redlip mullet.

Identification, expression profiling and functional characterization of interleukin 11a ortholog from redlip mullet Liza haematocheila: Insight into its roles in the inflammation and apoptosis regulation.

Interleukin 11 (IL-11) is a secretory cytokine with pleotropic properties, including anti-inflammatory and anti-apoptotic functions. This study aimed to functionally characterize a teleostean IL-11a ortholog from redlip mullet (LhIL-11a) through bioinformatic analysis, transcriptional expression profiling and protein function assays. The deduced LhIL-11a protein sequence is 200 amino acids long, with a predicted molecular weight of 23.168 kDa. Multiple sequence alignment indicates that LhIL-11a has a typical four-bundle architecture of α-helixes as observed in other IL-11s. The identity-similarity matrix show a higher identity between LhIL-11a and other fish IL-11a sequences. Phylogenetic analysis demonstrated that LhIL-11a falls within a clade including other fish counterparts. In the tissue distribution analysis, the highest constitutive expression of LhIL-11a mRNA was observed in the mullet gastrointestinal tract and brain tissues. Following the challenges with LPS, poly I:C and Lactococcus garvie, the transcription levels of LhIL-11a were significantly upregulated in both PBCs and liver. In the biological functional assay, recombinant LhIL-11a protein showed strong activities of suppressing pro-inflammatory cytokines and apoptotic gene expression in mullet kidney cells and reducing LPS stimulated NO production in murine macrophage cells. Overall, the findings in this study provide the experimental clues to understanding the functional roles of fish IL-11a in inflammation and apoptosis regulation during host defense against invading microbial pathogens.