Toll like receptor induces Ig synthesis in Catla catla by activating MAPK and NF-κB signalling.

The molecular crosstalk of proximal innate immune receptor signaling mediated by Toll-like receptors (TLRs) is crucial in generating an adaptive immune response. The extracellular-signal regulated kinases (ERK) participate in propagating intracellular signals initiated by stimulated TLRs to transcription factors eliciting cytokine release. Although ERK signaling has been extensively studied in mammalian counterparts, very little is known about its existence in carps and its role in augmentation of immunoglobulin (Ig) synthesis. Therefore, to gain insights into the efficacy of MAP kinase cascade in orchestrating fish antigen receptor generation, Catla catla fingerlings were induced with various TLR agonists or pathogen associated molecular patterns (PAMPs). Analysis of upstream signaling events revealed that PAMPs stimulated the tissues leading to a significant upregulation (P < 0.001, One-way ANOVA) of different TLRs (TLR2, TLR3, TLR4 and TLR5) followed by activation of MyD88 dependent and independent pathway. Activation of ERK and NF-κB mediated cytokine production consequently triggered the enhanced expression of IgZ and IgM as was evident by qRT-PCR analysis, flow cytometry, immunoblotting and ELISA. Pretreatment with ERK inhibitor (UO126) antagonized PAMPs mediated TLR stimulation, leading to sequential downregulation of MyD88/NF-κB/cytokines via interrupting ERK/NF-κB signaling axis. Together these results demonstrate that TLR stimulation triggers IgZ and IgM production via activation of ERK and NF-κB in C. catla indicating that NF-κB mediated cytokine production and ERK1/2 signaling is not only functional in fish, but may be crucial for generation of Ig repertoire in lower vertebrates.

LrHMGB1 Shares Structural Similarities with Human HMGB1, and Its Expression Is Induced in Bacterial Infection, Antiviral Vaccination, and Pathogen-Associated Molecular Patterns Stimulation.

The high-mobility group box 1 (HMGB1) protein is a highly conserved nonhistone chromosomal protein ubiquitously present in almost all cell types. In the nucleus, it facilitates DNA repair and replication, V(D)J recombination, stabilization of nucleosome, and in the cytoplasm, it regulates autophagy and apoptosis. In addition to these intracellular functions, HMGB1 also facilitates activation of innate immune responses and plays key roles in host defense. To investigate its role in fish, we cloned and characterized HMGB1 in Labeo rohita (LrHMGB1), the most important freshwater fish species in the Indian subcontinent. The full-length cDNA sequence of LrHMGB1 consisted of 787 bp having an open reading frame of 618 bp encoding 205 amino acids (aa) polypeptide, with an estimated molecular mass of 23.61 kDa and isoelectric point (pI) of 5.96. Motif search of LrHMGB1 revealed two homologous DNA-binding domains, the A-box and B-box comprising 8-78 aa and 94-162 aa, respectively, and a negatively charged acidic C-terminal tail (179-204) that consisted of 26 consecutive aspartic and glutamic acid residues. The amino acids sequence of LrHMGB1 protein and the secondary structure having helix (H) and coils (C) in tandem in the A- and B-box region and only coils in the acidic tail region shared significant similarity with mouse and human HMGB1. In addition to the three prominent motifs (A-box, B-box, and the acidic tail), the site of acetylation (lys27-29), phosphorylation (serine38,41,45,52), methylation (lys43), and oxidation (cysteine44,105) in LrHMGB1 was also conserved across the fish species, mouse, and human. LrHMGB1 was expressed during embryogenesis and was widely expressed in various organs/tissues having highest expression in blood. In response to bacterial infection, antiviral vaccination, and pathogen-associated molecular patterns stimulations, LrHMGB1 gene expression was significantly (p < 0.05) induced in various organs and tissues. These results together suggest an evolutionary conserved structure and function of HMGB1 from fish to human.

Molecular cloning, characterization and expression of immunoglobulin D on pathogen challenge and pathogen associated molecular patterns stimulation in freshwater carp, Catla catla.

The primordial immunoglobulin class, IgD, was the first non-IgM isotype discovered in teleosts. The crucial roles of IgM and IgZ in imparting systemic and mucosal immunity, respectively, in various fish species have been widely established. However, the putative function of a unique IgD isotype during pathogenic invasions has not been well explored. The present study reports the existence of an IgD ortholog in freshwater carp, Catla catla, and further evaluates its differential expression profile in response to bacterial, parasitic and viral antigenic exposure and pathogen associated molecular patterns (PAMPs) stimulation. The IgD of C. catla (CcIgD) cDNA sequence was found to encode 226 amino acids and confirmed homology with heavy chain delta region of Cyprinidae family members. Phylogenetic analysis of CcIgD exhibited greatest similarity with Ctenopharyngodon idella. qRT-PCR analysis revealed significant upregulation (P < 0.001) of IgD gene expression in kidney with respect to other tissues at 24 hr post-Aeromonas hydrophila challenge. CcIgD gene expression in skin was enhanced following Streptococcus uberis infection and in blood following Argulus infection and inactivated rhabdoviral antigen stimulation. Further, the treatment of bacterial and viral products (PAMPs) also triggered significant (P < 0.05) increases in CcIgD mRNA expression in kidney. These findings indicate the functional importance of teleost IgD in orchestrating tissue specific neutralization of antigens on stimulation with different pathogens and PAMPs.

Molecular cloning and characterization of LrTLR4, analysis of its inductive expression and associated down-stream signaling molecules following lipopolysaccharide stimulation and Gram-negative bacterial infection.

Toll-like receptors (TLRs) play key roles in innate immunity from lower to higher vertebrates. Among various TLR types, TLR4 was reported to recognize LPS in higher vertebrates resulting in the activation of down-stream signaling pathway. Except in some teleosts, function of TLR4 in most fish species including rohu (Labeo rohita) a commercially important fish species in the South-East Asian countries remained unknown. To investigate it, full-length cDNA of Labeo rohita TLR4 (LrTLR4) was cloned, and it consisted of 2729 bp, with a single ORF of 2469 bp encoding a polypeptide of 822 aa with a predicted molecular mass of 94.753 kDa. Structurally, LrTLR4 consisted of 25 LRRs (leucine rich repeat regions), one TM (trans-membrane) domain and one TIR (Toll/interleukin-1 receptor) domain, and was similar to higher vertebrate's TLR4. Phylogenetically, LrTLR4 exhibited highest (85%) identity with the common carp TLR4b amino acids sequence, and formed a separate subgroup in the phylogenetic tree. LrTLR4 was widely expressed in all tested organs/tissues, and amidst the tissues highest expression was detected in blood and the lowest in eye. In response to LPS-stimulation, LrTLR4 was induced with the activation of MyD88-dependent and TRIF-dependent signaling pathway resulting in pro-inflammatory cytokines (interleukin 6 and 8) and type I IFN gene expression. Infection of rohu with a Gram-negative fish pathogen (Aeromonas hydrophila), also activated LrTLR4. Together, these findings suggest the important role of TLR4 in LPS sensing and augmentation of innate immunity against Gram-negative bacterial infection in fish.

Immunoglobulin (Ig) D in Labeo rohita is widely expressed and differentially modulated in viral, bacterial and parasitic antigenic challenges.

Immunoglobulins (Igs) play critical roles in protecting host against diverse pathogenic invasion and diseases. Among all Ig isotypes, IgD is the most recently-evolved and enigmatic molecule detected in all vertebrates species except birds. In South-East Asia, Labeo rohita (rohu) is the leading candidate fish species for freshwater aquaculture, and this article describes about IgD gene expression in rohu following viral, bacterial and parasitic antigenic challenges. The partial cDNA (761bp) of Labeo rohita-IgD (LrIgD) was cloned and submitted in the GenBank with the accession no KT883581. Phylogenetically, LrIgD was closely related to grass carp IgD. Analysis of LrIgD gene expression in juveniles by quantitative real-time PCR (qRT-PCR) assay revealed gradual increase in IgD expression with the advancement of time. In the healthy rohu fingerlings, LrIgD expression occurred predominantly in kidney followed by liver and spleen. In response to rhabdoviral antigenic stimulation, LrIgD expression was significantly enhanced in all tested tissues. In bacterial (Aeromonas hydrophila) infection, transcripts of LrIgD increased more dramatically in liver followed by kidney and gill. In parasitic (Argulus) infection, most significant expression of IgD was noted in the skin, followed by kidney, liver, spleen and gill. These results collectively suggest the key role of IgD in the immune response of rohu during viral, bacterial and parasitic infections.

B cell activating factor is induced by toll-like receptor and NOD-like receptor-ligands and plays critical role in IgM synthesis in Labeo rohita.

B-cell activating factor (BAFF), an important member of the tumor necrosis factor superfamily, plays critical roles in the modulation of B-cell functions and enhancement of immune response in the host. Like higher vertebrates, the important role of BAFF in boosting immune response against diverse pathogens was also envisaged in fishes. We therefore, studied BAFF in rohu (Labeo rohita), a freshwater food fish species of highest economic importance in the Indian subcontinent. Full-length rohu-BAFF- cDNA comprised of 804bp nucleotide long ORF, encoding 267 amino acid residues, and shared high structural similarity with human-BAFF. It was expressed in the embryonic developmental stages suggesting its key role in immune response at the early life of fish. In Aeromonas hydrophila infection and rhabdoviral antigen stimulation, BAFF-gene expression in rohu was induced across the organs/tissues. Stimulation of un-treated healthy rohu fish leukocytes, and viral or bacterial or BSA (bovine serum albumin) antigen stimulated rohu fish leukocytes with recombinant-BAFF (r-BAFF) resulted in enhanced expression of immunoglobulin (Ig)M. Both in-vitro and in-vivo treatment with toll-like receptor (TLR)- ligand (poly I:C) or nod-like receptor (NLR)- ligands (iE-DAP and MDP) resulted in TLR and NLR activation and BAFF-gene expression. This is the first report showing BAFF-expression by innate immune receptor-ligands and its critical role in enhancing adaptive immune response in fish.

Hypoxic stress: impact on the modulation of TLR2, TLR4, NOD1 and NOD2 receptor and their down-stream signalling genes expression in catla (Catla catla).

The damage-associated molecular patterns (DAMPs) released from the damaged tissue/cells are recently reported as endogenous ligands to activate toll-like receptors (TLRs) and nucleotide binding and oligomerization domain (NOD) receptors signaling pathways. In the aquatic environment, reduction in dissolved oxygen (DO) concentration causes hypoxic stress resulting in tissue damage and patho-biological changes in fish. We envisaged the critical role of TLR and NOD receptors in recognizing DAMPs as endogenous ligands during hypoxic stress in fish. Catla (Catla catla) fingerlings (avg. wt ~56 g) was exposed to hypoxic stress (DO: 1-3 mg/L) for 1 and 24 h. After the designated time course, total RNA was extracted from gill, liver, kidney and blood, and modulation of TLRs (TLR2 and TLR4), NOD (NOD1 and NOD2) receptors, MyD88 (myeloid differentiation primary response gene 88), RICK (receptor interacting serine-threonine protein kinase-2), interleukin (IL)-6, IL-8 and IL-10 gene expression were analyzed by quantitative reverse transcriptase PCR assay. Significant (p < 0.05) up-regulation of some DAMPs {high-mobility group box 1 and heat shock protein-70}, TLRs and NOD receptors genes expressions were observed in the hypoxic fish tissues as compared to the control. Further investigation revealed inductive expression of MyD88, RICK, IL-6, IL-8 and IL-10 genes in the TLRs and NODs activated tissues of the hypoxic fish. These data together may suggest the important role of TLRs and NOD receptors signaling pathway in sterile inflammation and pathobiology of fish in hypoxic stress, and warrant further study to investigate the role of TLR and NOD receptors in abiotic stress management in aquaculture.

Characterization and Inductive Expression Analysis of Interferon Gamma-Related Gene in the Indian Major Carp, Rohu (Labeo rohita).

Interferon gamma (IFN-γ) is one of the key cytokines that plays a major role against viral and intracellular bacterial infection. In addition to the IFN-γ gene, teleost fish possess a second copy known as IFN-γ-related (IFN-γrel) gene. This report describes structural and functional properties of IFN-γrel gene in the Indian major carp, rohu (Labeo rohita), a commercially important freshwater fish species in the Indian subcontinent. The rohu IFN-γrel gene consisted of four exons with three intervening introns and phylogenetically closely related to grass carp. The full-length IFN-γrel cDNA comprised 927 bp nucleotides with a single open reading frame of 504 bp, encoding 167 amino acids (aa) polypeptide with a signal peptide of 24 aa. The mature rohu IFN-γrel protein was 143 aa with a predicted molecular weight of 16.85 kDa. Basal expression analysis of IFN-γrel showed its wide range of expression in all examined tissues: The highest was in the skin and the lowest was in the liver. In response to LPS, poly I:C, iE-DAP, muramyl dipeptide stimulations, and bacterial infections, IFN-γrel gene expression was significantly (p<0.05) induced in treated fish tissues as compared with their control. The IFN-γrel was expressed as recombinant protein (rIFN-γrel) and confirmed through western blot. Stimulation of peripheral blood leukocytes with rIFN-γrel protein resulted in the activation of IFN-γ receptor and marked induction of inducible nitric oxide synthase gene expression. These results together may suggest the important role of IFN-γrel as an antimicrobial cytokine in fish.

Modulation of TLR2, TLR4, TLR5, NOD1 and NOD2 receptor gene expressions and their downstream signaling molecules following thermal stress in the Indian major carp catla (Catla catla).

Toll-like receptors (TLRs) and nucleotide binding and oligomerization domain (NOD) receptors are pattern recognition receptors (PRRs) that recognize pathogen-associated molecular patterns (PAMPs) and play crucial role in innate immunity. In addition to PAMPs, PRRs recognize endogenous molecules released from damaged tissue or dead cells [damage-associated molecular patterns (DAMPs)] and activate signaling cascades to induce inflammatory processes. In the aquatic environment, large variation in seasonal and diurnal water temperature causes heat and cold stresses in fish, resulting in tissue injury and mortality of fish. In the Indian subcontinent, catla (Catla catla) is an economically important freshwater fish species and is prone to thermal stresses. To investigate the response of pattern recognition receptors in thermal stress, we analyzed TLRs (TLR2, TLR4 and TLR5) and NOD (NOD1 and NOD2) receptors gene expression in catla following heat and cold stress. Analysis of tissue samples (gill, liver, kidney and blood) of the thermal stressed and control fish by quantitative real-time PCR (qRT-PCR) assay revealed significant (p < 0.05) induction of TLR2, TLR4 and NOD2 gene expression in majority of the tested tissues of the treated fish as compared to the control. The expression of TLR5 and NOD1 gene was also induced in the heat and cold stressed fish, but mostly restricted in the blood. The downstream signaling molecule of TLR and NOD signaling pathway viz., MyD88 (myeloid differentiation primary response gene 88) and RICK (receptor interacting serine-threonine protein kinase-2) was also induced in the thermal stressed fish suggesting the engagement of TLR and NOD signaling pathway during thermal stress.

Toll-like receptor 22 in Labeo rohita: molecular cloning, characterization, 3D modeling, and expression analysis following ligands stimulation and bacterial infection.

Toll-like receptors (TLRs) are a class of innate immune receptors that sense pathogens or their molecular signatures and activate signaling cascades to induce a quick and non-specific immune response in the host. Among various types of TLRs, TLR22 is exclusively present in teleosts and amphibians and is expected to play the distinctive role in innate immunity. This report describes molecular cloning, three-dimensional (3D) modeling, and expression analysis of TLR22 in rohu (Labeo rohita), the most commercially important freshwater fish species in the Indian subcontinent. The open reading frame (ORF) of rohu TLR22 (LrTLR22) comprised of 2,838 nucleotides (nt), encoding 946 amino acid (aa) residues with the molecular mass of ∼ 107.6 kDa. The secondary structure of deduced LrTLR22 exhibited the presence of signal peptide (1-22 aa), 18 leucine-rich repeat (LRR) regions (79-736 aa), and TIR domain (792-935 aa). The 3D model of LrTLR22-LRR regions together elucidated the horse-shoe-shaped structure having parallel ß-strands at the concave surface and few α-helices at the convex surface. The TIR domain structure revealed alternate presence of five α-helices and ß-sheets. Phylogenetically, LrTLR22 was closely related to common carp and exhibited significant similarity (92.2 %) and identity (86.1 %) in their amino acids. In rohu, TLR22 was constitutively expressed in all embryonic developmental stages, and tissue-specific analysis illustrated its expression in all examined tissues, highest was in liver and lowest in brain. In vivo modulation of TLR22 gene expression was analyzed by quantitative real-time PCR (qRT-PCR) assay following stimulation with lipopolysaccharide (LPS), synthetic double stranded RNA (polyinosinic-polycytidylic acid), and bacterial (Aeromonas hydrophila) RNA. Among these ligands, bacterial RNA most significantly (p < 0.05) induced TLR22 gene expression in most of the tested tissues. In A. hydrophila infection, induction of TLR22 gene expression was also observed in majority of the tested tissues. Together, these data suggested that in addition to sensing other microbial signatures, TLR22 can recognize bacterial RNA and may play the important role in augmenting innate immunity in fish.