Abstract
Interleukin-6 (IL-6) is a multipotent cytokine. IL-6 plays a dual role in inflammation through both classical signaling (IL-6 binds membrane IL-6 receptor/IL-6R) and trans-signaling (IL-6 binds soluble IL-6R). However, the regulation of IL-6 activity, especially the regulation of signaling pathways and downstream genes mediated by IL-6 trans-signaling, remains largely unclear in teleost. Grass carp (Ctenopharyngodon idellus) hepatic (L8824) cells, kidney (CIK) cells, and primary hepatocytes were used as test models in this study. First, the biological activity of recombinant blunt snout bream (Megalobrama amblycephala) IL-6 (rmaIL-6) and sIL-6R (rmasIL-6R) was verified by quantitative PCR (qPCR) and western blot. The western blot results showed that rmaIL-6 significantly upregulated signal transducer and activator of transcription 3 (STAT3) phosphorylation in L8824 cells and primary hepatocytes, while rmaIL-6 in combination with rmasIL-6R (rmaIL-6+rmasIL-6R) significantly upregulated STAT3 phosphorylation in all types of cells. Furthermore, maIL-6 and maIL-6+rmasIL-6R could only induce extracellular-signal-regulated kinase 1/2 (ERK1/2) phosphorylation in L8824 cells and CIK cells, respectively. Therefore, IL-6 mainly acts by activating the janus kinase (JAK)/STAT3 pathway rather than the mitogen-activated protein kinase (MEK)/ERK pathway. Finally, the activation of the JAK2/STAT3 pathway was shown to be essential for the generation of socs3a and socs3b induced by IL-6 trans-signaling after treatment by JAK2/STAT3 pathway inhibitors (c188-9 and TG101348). These findings provide functional insights into IL-6 classical signaling and trans-signaling regulatory mechanisms in teleost, enriching our knowledge of fish immunology.
Highlights
Interleukin-6 (IL-6) was first identified in humans as B cell stimulatory factor 2 (BSF-2), which induces B cells to produce immunoglobulin (Ig) [1]
When IL-6 sends signals into cells, it binds to a heterodimeric receptor complex consisting of a high-affinity chain interleukin-6 receptor (IL-6R) and glycoprotein 130, and triggers two major signaling pathways: the extracellular-signal-regulated kinase (ERK)/mitogen-activated protein kinase (MEK) pathway and the Janus kinases (JAK)/signal transducer and activator of transcription 3 (STAT3) pathway [10]
The analysis showed that the similarity in amino acid sequence of IL-6 and soluble IL-6 receptor (sIL-6R) between blunt snout bream and grass carp was as high as 90.91% and 86.89%, respectively (Figure 1A,B)
Summary
Interleukin-6 (IL-6) was first identified in humans as B cell stimulatory factor 2 (BSF-2), which induces B cells to produce immunoglobulin (Ig) [1]. When IL-6 sends signals into cells, it binds to a heterodimeric receptor complex consisting of a high-affinity chain interleukin-6 receptor (IL-6R) and glycoprotein 130 (gp130), and triggers two major signaling pathways: the extracellular-signal-regulated kinase (ERK)/mitogen-activated protein kinase (MEK) pathway and the Janus kinases (JAK)/signal transducer and activator of transcription 3 (STAT3) pathway [10]. The inflammatory cytokine IL-6 upregulates antimicrobial peptides (hamp) expression by activating the IL-6R/JAK2/STAT3 pathway [11]. IL-6 induces the activation of the STAT3/SOCS3 pathway in the liver, but not other downstream pathways such as STAT1, ERK1/2, and PI3K [13]. The JAK2/STAT3 signaling pathway has been shown to promote IL-6 expression [14,15]
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