Modulation of fish immune response by interferon regulatory factor 4 in redlip mullet (Liza haematocheilus): Delineation through expression profiling, antiviral assay, and macrophage polarization analysis.

Interferon regulatory factor 4 (IRF4) is a crucial member of IRF family, which acts as an imperative transcription factor in the development and maturation of multiple lineages of blood cells and also plays a pivotal role in host defense against microbial infections. In the present study, we aimed to investigate the detailed structural and functional aspects of a redlip mullet IRF4 homolog (LhIRF4). The LhIRF4 open reading frame consists of 1347 base pairs encoding 449 amino acids, with the DNA-binding domain sharing significant homology with that of other vertebrate IRF4 homologs. The highest transcription levels of LhIRF4 were observed in the mullet intestine and spleen under normal physiological conditions. Furthermore, a time-dependent upregulation of LhIRF4 transcription was observed in the spleen and head kidney tissues upon pathogenic challenges. When overexpressed in mullet cells, LhIRF4 was localized to the nucleus and significantly stimulated the transcription of several host antiviral genes. Moreover, the overexpression of LhIRF4 strongly inhibited the replication of viral hemorrhagic septicemia virus (VHSV) in vitro. The function of LhIRF4 in regulation of macrophage M2 polarization has also been evidently demonstrated in RAW 264.7 cells. Taken together, our findings indicate the profound role of LhIRF4 in modulating immune responses against microbial infections in redlip mullet.

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.

Immune responses, subcellular localization, and antiviral activity of interferon-induced protein 35 (IFP35) in rock bream (Oplegnathus fasciatus).

Interferon-induced protein 35 kDa (IFP35) has been demonstrated to play important roles in antiviral defense, inflammatory response and cancer progression. However, its precise function in teleost fish remains to be elucidated. Herein, we functionally characterized the rock bream (Oplegnathus fasciatus) IFP35 (OfIFP35) to understand its expression pattern, subcellular localization, antiviral activity, and regulation of downstream genes. OfIFP35 consists of an 1107 bp open reading frame encoding 368 amino acids, including two N-myc-interactor (Nmi)/IFP35 domains (NIDs). The predicted molecular weight of OfIFP35 was 42 kDa, with a theoretical isoelectric point (pI) of 5.10. Evolutionary conservation of IFP35 was analyzed using multiple, pairwise alignments and phylogenetic tree analysis. OfIFP35 in rock bream was found to be highest expressed in the gills. Immune challenges with iridovirus, polyinosinic:polycytidylic acid, lipopolysaccharide, and live bacteria (Streptococcus iniae and Edwardsiella tarda) significantly upregulated its mRNA expression in gill and liver tissues of the rock bream. GFP-tagged OfIFP35 was localized in the cytoplasm of FHM cells, and its overexpression significantly suppressed VHSV transcription in vitro. Moreover, the analysis of downstream gene expression revealed that OfIFP35 could activate the type I interferon pathway. Collectively, these findings indicate that OfIFP35 is important for the immune system of rock bream as it promotes defense responses during viral infections.

Molecular and functional characterization of a mitochondrial glutathione reductase homolog from redlip mullet (Liza haematocheila): Disclosing its antioxidant properties in the fish immune response mechanism.

Glutathione reductase (GSHR) is a biologically important enzyme involved in the conversion of oxidized glutathione (GSSG) into its reduced form, reduced glutathione (GSH), with the catalytic activity of NADPH. Most animals and aquatic organisms, including fish, possess high levels of this enzyme system to neutralize oxidative stress in cells. The current study was conducted to broaden our knowledge of GSHR in fish by identifying a mitochondrial isoform of this enzyme (LhGSHRm) in redlip mullet, Liza haematocheila, and clarifying its structure and function. The complete open reading frame of LhGSHRm consists of 1527 base pairs, encoding 508 amino acids, with a predicted molecular weight of 55.43 kDa. Multiple sequence alignment revealed the conservation of important amino acids in this fish. Phylogenetic analysis demonstrated the closest evolutionary relationship between LhGSHRm and other fish GSHRm counterparts. In tissue distribution analysis, the highest mRNA expression of LhGSHRm was observed in the gill tissue under normal physiological conditions. Following pathogenic challenges, the LhGSHRm transcription level was upregulated in a time-dependent manner in the gill and liver tissues, which may modulate the immune reaction against pathogens. rLhGSHRm showed considerable glutathione reductase activity in an enzyme assay. Further, the biological activity of rLhGSHRm in balancing cellular oxidative stress was observed in both disk diffusion and DPPH assays. Collectively, these results support that LhGSHRm has profound effects on modulating the immune reaction in fish to sustain precise redox homeostasis.