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. 2015 Sep;12(5):633-44.
doi: 10.1038/cmi.2014.80. Epub 2014 Sep 1.

Soluble interleukin-6 receptor is elevated during influenza A virus infection and mediates the IL-6 and IL-32 inflammatory cytokine burst

Jun Wang, Qing Wang, Tao Han, Yong-Kui Li, Sheng-Li Zhu, Fang Ao, Jian Feng, Ming-Zhen Jing, Li Wang, Lin-Bai Ye, Ying Zhu

Soluble interleukin-6 receptor is elevated during influenza A virus infection and mediates the IL-6 and IL-32 inflammatory cytokine burst

Jun Wang et al. Cell Mol Immunol. 2015 Sep.

Abstract

Influenza A virus (IAV) infection is a major worldwide public health problem. However, the factors involved in mediating the inflammatory response to this infection and their relationships remain poorly understood. Here, we show that IAV infection stimulates the expression of the soluble IL-6 receptor (sIL-6R), a multifunctional protein involved in IL-6 signaling. Interestingly, sIL-6R expression upregulated the levels of its own ligand, IL-6 and those of the pro-inflammatory cytokine IL-32. shRNA-mediated knockdown of sIL-6R suppressed IL-6 and IL-32, indicating that this regulation is dependent on sIL-6R during IAV infection. Furthermore, our results demonstrate that IL-32 participates in a negative feedback loop that inhibits sIL-6R while upregulating IL-6 expression during IAV infection. Therefore, we show that sIL-6R is a critical cellular factor involved in the acute inflammatory response to viral infection.

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Figures

Figure 1
Figure 1
IAV activates sIL-6R expression in different cell types. (a) sIL-6R mRNA levels were determined by real-time RT-PCR in A549 cells and human PBMCs infected with IAV (MOI=1) for the indicated length of time. (b) The level of sIL-6R mRNA was determined by real-time RT-PCR in MRC-5 cells infected with IAV (MOI=1) for 4 h. (c) sIL-6R protein levels were determined by western blot analysis of A549 cells and human PBMCs with (20 nM) or without TAPI for 8 h and were then infected with IAV (MOI=1) for 12 h. (d) A549 cells were incubated with (20 nM) or without TAPI for 8 h and were then infected with IAV (MOI=1) for 4 h. The levels of sIL-6R and TNF-α protein in the culture supernatants were measured by ELISA. Data shown are mean±s.e.; n=3. *P<0.05; ns, not significant. IAV, influenza A virus; MOI, multiplicity of infection; PBMC, peripheral blood mononuclear cell; sIL-6R, soluble interleukin-6 receptor; TNF, tumor-necrosis factor.
Figure 2
Figure 2
Screen of IL-6R-stimulated cytokine promoter activity during IAV infection. (a) A549 cells were transfected with pCMV-Tag2B-sIL-6R for 24 h, and sIL-6R in the culture supernatants was quantitated by ELISA. (b) A549 cells were transfected with shRNA-sIL-6R or shRNA-control for 24 hand infected with IAV (MOI=1) for 6 h. The levels of sIL-6R protein in the culture supernatants were quantitated by ELISA. (c) Luciferase reporter plasmids for the indicated cytokines and a Renilla control (pRL–TK) were cotransfected into A549 cells with pCMV-Tag2B-sIL-6R or a control vector (pCMV–Tag2B) for 24 h. The luciferase activity was measured as described in the section on ‘Materials and methods'. (d) Luciferase reporter plasmids and a Renilla control (pRL–TK) were cotransfected with shRNA-sIL-6R or shRNA-control for 24 h into A549 cells that were infected with IAV (MOI=1) for 6 h, and the luciferase activity was measured. The results are expressed as the mean±s.e.m. of three independent experiments performed in triplicate and normalized according to the Renilla control reporter activity. n=3. *P<0.05. IAV, influenza A virus; sIL-6R, soluble interleukin-6 receptor; MOI, multiplicity of infection.
Figure 3
Figure 3
Induction of sIL-6R, IL-32 and IL-6 expression by IAV infection. (a) sIL-6R, IL-32 and IL-6 mRNA levels were quantitated by real-time RT-PCR in A549 cells infected with IAV (MOI=1) or control-treated (heat-inactivated IAV) for 6 h or 24 h. (b) sIL-6R, IL-32 and IL-6 mRNA levels were quantitated by real-time RT-PCR in human PBMCs infected with IAV (MOI=1) or control-treated (heat-inactivated IAV) for 2 h or 6 h. (c) sIL-6R, IL-32, and IL-6 mRNA levels were quantitated by real-time RT-PCR in MRC-5 cells infected with IAV (MOI=1) or control-treated (heat-inactivated IAV) for 2 h or 6 h. Data shown are mean±s.e.; n=3. **P<0.01; *P<0.05. IAV, influenza A virus; MOI, multiplicity of infection; PBMC, peripheral blood mononuclear cell; sIL-6R, soluble interleukin-6 receptor.
Figure 4
Figure 4
sIL-6R upregulates IL-6 expression during IAV infection. (a–c) A549 cells (a) and human PBMCs (b) were incubated with recombinant human IL-6Rα protein (40 ng/ml) for the indicated times before the cell culture supernatants were collected. The levels of secreted IL-6 protein were detected by ELISA. (c) MRC-5 cells were incubated with IL-6Rα at the indicated doses, and the levels of IL-6 mRNA (top graphs) and secreted protein (bottom graphs) were determined by real-time RT-PCR and ELISA, respectively. (d) A549 cells were transfected with pCMV-sIL-6R for the indicated times before the levels of IL-6 mRNA (top graphs) and secreted protein (bottom graphs) were determined by real-time RT-PCR and ELISA, respectively. (e) A549 cells were transfected with shRNA-sIL-6R or shRNA-control for 24 or 48 h and infected with IAV (MOI=1) for 6 h. The levels of IL-6 mRNA (top graphs) and secreted protein (bottom graphs) were determined by real-time RT-PCR and ELISA, respectively. Data shown are mean±s.e.; n=3. **P<0.01; *P<0.05; ns, not significant; N.D., not detected. IAV, influenza A virus; MOI, multiplicity of infection; PBMC, peripheral blood mononuclear cell; sIL-6R, soluble interleukin-6 receptor.
Figure 5
Figure 5
sIL-6R upregulates IL-32 expression during IAV infection. (a) A549 cells were incubated with recombinant human IL-6Rα (40 ng/ml) for the indicated times, and the level of IL-32 mRNA was examined by real-time RT-PCR. (b) A549 cells were incubated with IL-6Rα at the indicated doses, and the level of IL-32 protein was examined by western blotting. (c) MRC-5 cells were incubated with IL-6Rα at the indicated doses, and the level of IL-32 mRNA was examined by real-time RT-PCR. (d and e) A549 cells were transfected with pCMV-sIL-6R for the indicated times; the IL-32 mRNA levels were determined by real-time RT-PCR (d), and the IL-32 protein was assessed by western blotting (e). (f and g) A549 cells were transfected with shRNA-sIL-6R or shRNA-control for 24 h (f) or 48 h (g) and infected with IAV (MOI=1) for 6 h. The levels of IL-32 mRNA (f) were quantitated by real-time RT-PCR, and western blot analysis was performed to assess the IL-32 levels in the cell lysates (g, upper panel). IAV NP mRNA was detected by semiquantitative RT-PCR (g, lower panel). Data shown are mean±s.e.; n=3. *P<0.05; ns, not significant. IAV, influenza A virus; MOI, multiplicity of infection; sIL-6R, soluble interleukin-6 receptor.
Figure 6
Figure 6
Effect of IL-32 on IAV-induced sIL-6R expression. (a) A549 cells and MRC-5 cells were incubated with recombinant human IL-32γ protein at the indicated concentrations for 4 h, and the level of sIL-6R mRNA was quantitated by real-time RT-PCR. (b) A549 cells were incubated with recombinant human IL-32γ protein (40 ng/ml) for the indicated times, and the sIL-6R protein was analyzed by western blotting. (c) A549 cells were transfected with pCMV-IL-32 for 24 h or 48 h and then infected with IAV (MOI=1) for 6 h. At 24 h post-transfection, the level of sIL-6R mRNA was detected by real-time RT-PCR (top graphs). At 48 h post-transfection, the sIL-6R protein level was measured by ELISA (middle graphs), whereas the level of IL-32 protein was determined by western blotting (bottom graphs). (d) A549 cells were transfected with shRNA-IL-32 or shRNA-control and infected with IAV (MOI=1) for 6 h. The level of sIL-6R mRNA was quantitated by real-time RT-PCR (top graphs), and the sIL-6R protein level was measured by ELISA (middle graphs). The knockdown efficiency of IL-32 was determined by western blotting (bottom graphs). (e) A549 cells were transfected with pCMV-IL-32 for 24 h, and the level of IL-6 mRNA was quantitated by real-time RT-PCR. (f) A549 cells were co-transfected with pCMV-sIL-6R and shRNA-IL-32 or shRNA-control for 24 h and infected with IAV (MOI=1) for 6 h; the level of IL-6 mRNA was then quantitated by real-time RT-PCR. Data shown are mean±s.e.; n=3. *P<0.05. IAV, influenza A virus; MOI, multiplicity of infection; sIL-6R, soluble interleukin-6 receptor.
Figure 7
Figure 7
Schematic of the proposed model for the regulation of IL-32 and IL-6 expression by sIL-6R in response to IAV infection. IAV, influenza A virus; sIL-6R, soluble interleukin-6 receptor.
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