Sexual dimorphism in NLR transcripts and its downstream signaling protein IL-1ꞵ in teleost Channa punctata (Bloch, 1793)

Abstract

Nucleotide-binding oligomerization domain-like receptors (NOD-like receptors or NLRs) are a family of intracellular pattern recognition receptors (PRRs) that initiates as well as regulate inflammatory responses. NLRs are characterized by a centrally located nucleotide binding domain and a leucine rich repeat domain at the C-terminal responsible for the recognition of intracellular microbe-associated molecular patterns (MAMPs) and danger-associated molecular patterns (DAMPs). In the present study in adult spotted snakehead we have investigated the sex-dependent tissue distribution of NLRs known to be associated with inflammation in teleost namely NOD1, NOD2, NLRC3, NLRC5, and NLRX1. Further, the sexual dimorphism in the expression of NLR transcript as well as the pro-inflammatory protein IL-1β was explored in fish under normal conditions, and in fish exposed to bacterial lipopolysaccharide (LPS). The NLRs show ubiquitous and constitutive expression in all the tissues. Moreover, a prominent disparity between males and females was observed in the basal expression of these genes in various tissues. The sexual dimorphism in NLR expression was also prominent when fish were exposed to LPS. Similarly, IL-1β exhibited sexual dimorphism in both normal as well as LPS-exposed fish.

Figure 1

Variation in expression of (a) ssNOD1, (b) ssNOD2 in various tissues of both male and female C. punctata during preparatory phase. Tissues namely, anterior, mid and posterior brain, head kidney, spleen, skin, gills, eye, muscle, heart, trunk kidney, stomach, liver, intestine and gonad from both male and female (N = 8) were excised out. qPCR was carried out and the data was normalized using 18S rRNA and β-actin genes as reference. The relative fold change was calculated following the 2^−∆∆Ct method with female as reference. Student’s unpaired t-test was employed to calculate significant difference (p < 0.05, female vs. male) for each tissue. Data is shown as a fold change in gene expression (Mean ± SEM). Asterisks indicate significant difference (p < 0.05, female vs male).

Figure 2

Tissue-dependent variation in expression of (a) ssNLRC3, (b) ssNLRC5 and (c) ssNLRX1 in both male and female C. punctata. Tissues of interest, namely, brain (anterior, mid and posterior), immune organs (head kidney, spleen, skin, gills and intestine), eye, muscle, heart, trunk kidney, stomach, liver, and gonad from male and female were excised out during preparatory phase. For gene expression analysis, qPCR was performed with two technical replicates. After normalizing the data with 18S rRNA and β-actin reference genes, female was set as a control for calculating fold change data. Student’s unpaired t-test was employed to calculate significant difference for each tissue. Data is shown as fold change in gene expression (Mean ± SEM, N = 8 for each sex). Asterisks indicate significant difference (p < 0.05, female vs. male).

Figure 3

Differential expression of splenic NLRs in male and female C. punctata in response to LPS. Differential expression of NLRs in lymphoid organ, spleen of male and female C. punctata was studied in response to LPS injection or 1X PBS (control) for 6 h. The data was normalized with 18S rRNA and β-actin genes and control was set as a reference for calculating fold change data. Significant difference between the groups was calculated employing two-way analysis of variance followed by Tukey’s range test. White bars represent male whereas black bars represent female. Data is represented as fold change in gene expression (Mean ± SEM, N = 8 for each group). ‘*’ indicate significant difference (p < 0.05, control vs LPS treated) and ‘#’ indicates a significant difference between male and female.

Figure 4

Differential expression of IL-1β in splenic lysate of male and female C. punctata in response to lipopolysaccharide (LPS). Male and female spotted snakeheads received injections of LPS (1 mg/mL) or 1 × PBS (control) for 6 h. (A) Total splenic protein was isolated and analyzed using SDS-PAGE and Western blotting using anti-IL-1β antibody. β-actin-antibody (approximately 42 kDa) was used as a loading control. Quantitative estimation of image using Image J software was done for (B) precursor pro-IL-1β protein expression and (C) mature IL-1β protein expression using Microsoft Excel, where β-actin was used for normalization and statistical significance was analyzed using GraphPad Prism8 software, where two-way ANOVA was applied followed by Tukey’s range test (p < 0.05) for any significant change. These results are representative of three independent experiments. ‘*’ indicate significant difference (p < 0.05, control vs LPS treated) and ‘#’ indicates a significant difference between male and female.