Identification of Immunoglobulin IgZ and IgM and the Role of Innate Immune Signaling on their Synthesis in Catla Catla

Abstract

This thesis illustrates the existence of newly identified immunoglobulin (Ig) heavy chain isotype, IgZ, universally known isotype, IgM and their functional basis against pathogenic manifestation in the most primitive vertebrate model of fish. The study elucidates the molecular crosstalk between TLR mediated immune signalling in immunoglobulin activation involving crucial role of MAP kinase cascade. Further, it provides insights into the therapeutic activities of an immunomodulator from lactic acid bacterial group in facilitation of immunoprophylaxis for fish health management. Partial IgZ and IgM heavy chain constant region sequences of three different heterologous species-Ctenopharygodon idella, Danio rerio and Cyprinus carpio were obtained from NCBI database to identify Ig isotypes in the Indian major carp, Catla catla. Bioinformatic and phylogenetic analysis of 417bp nucleotide sequence of Catla catla (CcIgZ) and 615bp of Catla catla (CcIgM) with a putative 139 and 205 amino acid sequence respectively showed homology with corresponding constant region domains of other teleosts. 5´ and 3´ RACE cloning was performed using nested primers to extend newly identified partial CcIgZ nucleotide sequence upto 933bp. Transcript expression of IgZ and IgM was evaluated in immunologically relevant tissues in response to Aeromonas hydrophila, Streptococcus uberis, Argulus sp. and rhabdoviral antigen stimulation in a time dependent manner. Basal level of both CcIgZ and CcIgM mRNA expression was found to be relatively higher in kidney. Bacterial induction triggered significant increase in CcIgZ mRNA expression in intestine (P<0.001) followed by spleen (P<0.01), whereas Argulus heavily infected groups showed higher expression in gill (P<0.01) and blood (P<0.01). However, rhabdoviral antigenic stimulation triggered relatively higher IgZ expression in intestine followed by gill. CcIgM was relatively up-regulated in blood (P<0.001) on bacterial and parasitic stimulations. Thus, the thesis provides the first evidence of existence of IgZ ortholog in C. catla and its comparative expression analysis with IgM against pathogenic assault. Further, to delineate the role of TLR dependent pathway in Ig activation, C. catla fingerlings were induced with various pathogen associated molecular patterns (PAMPs). 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 was observed on PAMPs induction. Subsequent stimulation of ERK, NF-κB mediated cytokine production enhanced expression of IgZ and IgM 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 NF-κB/cytokines via interrupting ERK/NF-κB signaling axis. Together these results demonstrated that TLR stimulation triggered 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 against pathogenic infiltration. The findings provide avenues for designing better prophylactic interventions in fish health management. Increased use of chemotherapeutants and antibiotics has led to resurgence of antimicrobial resistance in aquaculture practices. Thus, screening of immunomodulators such as probiotics and their mechanism of attenuation of potent fish pathogen, A. hydrophila-induced mortality in C. catla was investigated. The thesis reports that A. hydrophila markedly induced cell injuries, increased ROS/RNS and cytokine production, DNA damage leading to apoptosis as evidenced from increased apoptotic fractions in flow cytometry using Annexin V/PI assay in Catla Thymus Macrophage (CTM) cells. Probiotic, Lactobacillus acidophilus in CTM cells and in in-vivo treatment attenuated A. hydrophila-induced apoptosis by abrogating ROS/RNS production and regulated TLR-mediated Ig expression.