NF-κB signaling induces inductive expression of the downstream molecules and IgD gene in the freshwater carp, Catla catla

Abstract

Toll-like receptors (TLRs) in innate immune system act as primary sensors in detecting the microbial components and activate their signaling cascades to induce NF-κB (nuclear factor NF-κB) towards the augmentation of immunoglobulin (Ig) synthesis. To gain insights into the efficacy of NF-κB pathway in immunoglobulin D (IgD) synthesis in the Indian Major Carp Catla catla, cloning and sequencing of TLR-signaling downstream molecules [TRAF3 (TNF receptor-associated factor 3), NEMO (nuclear factor-kappa B essential modulator), NF-κB and BAFF (B cell activating factor)] were performed by infecting the fish with pathogens. mRNA expression analysis of the downstream molecules and IgD showed significant up-regulation of these genes in kidney (P ≤ 0.001) as compared to spleen (P ≤ 0.05). To ascertain the role of NF-κB pathway in IgD synthesis, the primary cell culture of kidney and spleen in monolayer cell suspension was treated with NF-κB inhibitor (BAY 11-7082) and down-regulation of BAFF, NEMO, NF-κB, and IgD gene was observed. These results highlight the importance of NF-κB signaling pathway in augmenting the IgD gene expression in the freshwater carp, Catla catla.

Keywords: BAFF; Catla catla; IgD; Immunoglobulin; NEMO; NF-κb; TRAF3.

Fig. 1

Basal level gene expression profile of (a) TRAF3 (b) NEMO (c) NF-κB and (d) IgD in kidney and spleen. Expression of gene transcripts in both the tissues was represented as a ratio relative to β-actin (internal control). Liver was chosen as calibrator and the relative expression was expressed as fold changes from the calibrator. All the data are represented as mean ± S.E. (n = 3). Significant difference with respect to the calibrator was determined by one-way ANOVA with ***P < 0.001, **P < 0.01 and *P < 0.05 as significance levels

Fig. 2

Modulation of gene expression of (a) TRAF3 (b) NEMO (c) NF-κB and (d) IgD in the Aeromonas hydrophila challenged fish post 24 h, 48 h and 72 h of infection. β-Actin was used as the internal control for normalizing the values of the target genes. All the data are represented as mean ± S.E. (n = 3). Significant difference with respect to the control was determined by one-way ANOVA with ***P < 0.001, **P < 0.01 and *P < 0.05 as significance levels

Fig. 3

Modulation of gene expression of (a) TRAF3 (b) NEMO (c) NF-κB and (d) IgD in the Streptococcus uberis challenged fish post 24 h, 48 h and 72 h of infection. β-Actin was used as the internal control for normalizing the values of the target genes. All the data are represented as mean ± S.E. (n = 3). Significant difference with respect to the control was determined by One-way ANOVA with ***P < 0.001, **P < 0.01 and *P < 0.05 as significance levels

Fig. 4

Proliferation of primary-cultured kidney and spleen cells of healthy C. catla. Red arrow indicates the initiation of monolayer formation

Fig. 5

Differential gene expression of (BAFF, NEMO, NF-κB and IgD) in primary cell culture of kidney (a, c, e and g) and spleen (b, d, f and h). Primary cell culture was stimulated with PGN and LPS for 4 h and 8 h in presence (treated) or absence (control) of BAY 11-7082. β-actin was used as the internal control for normalizing the values of the target genes. All the data are represented as mean ± S.E. (n = 3). Significant difference with respect to the control was determined by one-way ANOVA with ***P < 0.001, **P < 0.01 and *P < 0.05 as significance levels

Fig. 6

Protein–protein interaction network through STRING software. (a) Protein–protein interaction network of signal transduction immune-related protein. In this network, nodes are the proteins; line and number of lines represent predicted interactions and strength of predicted functional interaction between proteins respectively, (b) screenshot of the STRING shows the supplied set of immune-related proteins involved in signalling pathway. It shows details of protein abbreviation, node colour and interaction of edges