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AP-1 signaling pathway promotes pro-IL-1β transcription to facilitate NLRP3 inflammasome activation upon influenza A virus infection

ABSTRACT NLRP3 inflammasome mainly controls interleukin-1β (IL-1β) secretion, leading to cell death called pyroptosis constituting a major antiviral host defense and inflammatory diseases upon viral infection. The RAF-MEK1/2-ERK1/2 cascade and downstream c-Jun/Fos and Activator protein-1 (AP1) signaling pathway control the degree of inflammatory response. Influenza A virus (IAV) infection is known to stimulate NLRP3 inflammasome activation and inflammatory responses. Nevertheless, the detailed mechanism by which IAV induces NLRP3 inflammasome activation involved in transcription of pro-IL-1β mRNA remains elusive. In our study, we found that IAV infection promotes pro-IL-1β mRNA transcription and activates NLRP3 inflammasome. Detailed studies reveal that type I interferon (IFN-α/IFN-β) as well as U0126 (a selective inhibitor of MEK-1 and MEK-2) typically inhibit IAV-mediated NLRP3 inflammasome activation via downregulating pro-IL-1β mRNA. Moreover, knock-down of c-Jun decreases pro-IL-1β mRNA and inhibits NLRP3 inflammasome activation upon IAV infection. Overall, the findings uncover that AP-1 signaling pathway promotes NLRP3 inflammasome activation upon IAV infection, which provides a new idea for the therapy of NLRP3 inflammasome-associated inflammatory diseases.

IAV infection may cause a highly pathogenic and infectious disease of human and many other animals (such as pigs and chickens). The highly pathogenic IAV caused pandemics, leading to high mortality, such as Spain flu in 1918 and Hong Kong flu in 1997 [16][17][18]. During this process, the tissue damages caused by NLRP3-mediated excessive pro-inflammatory responses in host immune cells was considered as the main factor contributing to high mortality upon IAV infection [19,20]. It is now known that NLRP3 inflammasome is closely involved in IAV-mediated immune responses [21][22][23][24][25][26].
MEK1/2-ERK1/2 signaling pathway is widely known for its essential role in regulating gene expression [27,28]. c-Fos and c-Jun are principal downstream transcriptional factors in the ERK pathway [29]. Activator protein-1 (AP-1), mainly composed of c-Fos and c-Jun, regulates expression levels of pro-inflammatory cytokines [30,31]. However, whether AP-1 is involved in control of NLRP3 inflammasome upon IAV infection requires to be further explored.
Overall, we demonstrate that IAV infection upregulates transcription and maturation of IL-1β. Furthermore, IFN-α/IFN-β suppress NLRP3 inflammasome activation by downregulation of pro-IL-1β mRNA upon IAV infection. Notably, treatment with U0126 and knock-down of c-Jun result in inhibiting IAV infection-mediated NLRP3 inflammasome through downregulating transcription of pro-IL-1β. Thus, we discovered that the AP-1 signaling pathway accelerates activation of NLRP3 inflammasome via upregulating pro-IL-1β mRNA upon IAV infection.

Quantitative real-time PCR
Total RNA extraction was extracted using TRIzol reagent and transcribed into complementary DNA (cDNA). We prepared reaction premix by SYBR Quantitative Real-time PCR (qPCR) kits. The mixture contained 10 μl 2 × SYBR Green mix, 1 μl cDNA template, 1 μl specific primers [0.5 μl forward (F) and reverse (R) primer each], and 8 μl ddH 2 O. All qPCR primers were designed with the available tools online (https://www.ncbi.nlm.nih.gov/). The following primers were used in this study.

ASC oligomerization assay
THP-1 macrophages were lysed in buffer (50 mM Tris, pH7.5, 150 mM NaCl, 1% NP-40, 5 mM EDTA, and 10% glycerol) at 4°C for 30 min. Lysates were centrifugated at 12,000 rpm for 15 min. The supernatants of the lysates were mixed with 5 × SDS loading buffer for the input sample. The pellets of the lysates were washed with PBS and then cross-linked using DSS (at the final concentration of 2 mM) at 37°C for 30 min. The crosslinked pellets were mixed with 2 × SDS loading buffer for ASC oligomerization analysis.

Statistical analyses
For data with a normal distribution and homogeneity of variance, the difference between two groups was statistically analyzed by a two-tailed Student t test. Data are expressed as means ± SD. The evaluation of statistical significance was according to the P value. *, P < 0.05; **, P < 0.01; ***, P < 0.001; NS, not-significant, P > 0.05.
To further investigate the relationship between IAVinduced pro-IL-1β mRNA and IAV-mediated NLRP3 inflammasome activation, si-IL-1β#2 or si-NC were delivered into THP-1 macrophages, and then the cells were treated with H3N2 at different time points. The knock-down efficiency of si-IL-1β#2 was identified through detecting mRNA level of pro-IL-1β (Figure 3 (e)). The results indicated that IAV-induced pro-IL-1β mRNA occurred prior to IAV-mediated NLRP3 inflammasome activation (Figure 3(e-g)), and IAV-induced pro-IL-1β mRNA indeed facilitated IAV-mediated NLRP3 inflammasome activation by ASC oligomerization (Figure 3(e)) and resulted in the secretion of IL-1β protein (Figure 3(g)). Thus, IAV-induced pro-IL-1β mRNA facilitates IAV-mediated NLRP3 inflammasome activation. . the precursor and maturation of IL-1β, coupled with inter control GAPDH, were analyzed by using indicated antibodies by Western blotting (c). (d) THP-1 macrophages were treated with live H3N2 (MOI = 2), LPS (1 μg/ml) for 6 h, or LPS (1 μg/ml) for 6 h with ATP (5 mM) for 30 min, respectively. Lysates and ASC oligomerization were detected by indicated antibodies. (e-g) NLRP3 (e) and ASC (f) mRnas were assured by qPCR and NLRP3 (g), while ASC (g) proteins were detected by Western blotting in macrophages infected by lentiviruses expressing the indicated shRNA. (h) H3N2 (MOI = 2) infected macrophages stably expressing shRNA against target genes. IL-1β protein were detected by ELISA. (i-l) H3N2 (MOI = 2) infected macrophages for 24 h, and then the cells were treated with Ac-YVADcmk (1 mm) for 6 h. Secreted IL-1β was detected by ELISA (i). the mRNA levels of the indicated genes (i-k) were analyzed by qPCR.
To further explore function of U0126, macrophages were treated with DMSO or U0126, pro-IL-1β transcription was markedly inhibited by U0126 (Figure 5(i)), while NLRP3 transcription was mildly attenuated by U0126 ( Figure 5(j)). Meanwhile, the protein expression of pro-IL-1β was also markedly inhibited by U0126, and NLRP3 protein was mildly attenuated by U0126 ( Figure 5  (k)). These results suggested that U0126 repressed IAV- Figure 5. U0126 specifically inhibits IAV-induced NLRP3 inflammasome activation by downregulating pro-IL-1β mRNA. (a-h) H3N2 (MOI = 2) infected THP-1 macrophages for 24 h, and then the cells were stimulated with U0126 (10 μM) or Bay11-7082 (20 μM) for 6 h. the mRNA levels of the indicated genes (a-e) were analyzed by qPCR. Secreted IL-1β protein was detected by ELISA assay (f). Pro-IL-1β and GAPDH proteins were detected by Western blotting (g). Pro-Caspase-1, mature Caspase-1, and GAPDH proteins were detected by Western blotting (h). THP-1 macrophages were treated with DMSO or U0126 (10 μM). the mRNA levels of the indicated genes (i-j) were analyzed by qPCR, while the indicated protein levels were analyzed by Western blotting (k). mediated secreted IL-1β protein via mainly downregulating pro-IL-1β mRNA. It's well known that NF-κB is critical for pro-IL-1β transcription in inflammasome activation [2], which is consistent with our results of the inhibition of Bay-11-7082 on production of pro-IL-1β mRNA. Unlike Bay-11-7082, U0126 displayed a comparative superiority on the suppression of transcription and maturation of IL-1β, indicating that AP-1 signaling pathway played a dominant role in transcription and maturation of IL-1β induced by H3N2.
AP-1, the main substrate of the multiple MAPK pathways, is stimulated by a series of stimuli, including stress and cytokines [44]. Interestingly, C3G4 (cyanidin-3-glucoside) activated NLRP3 inflammasome through AP-1 signaling pathway in the epithelial cells [45]. PRRSV enhances secreted IL-1β by AP-1 signaling pathway in microglia [46]. Our findings presented in this study show that AP-1 contributes to H3N2mediated NLRP3 inflammasome activation, which extends functional scope of AP-1 in the regulation of NLRP3 inflammasome.
Previous study suggested that NF-κB is essential to accelerate the transcription of pro-IL-1β and NLRP3 [2]. It was also reported that the MyD88/ERK/AP-1 signaling pathway promotes IL-1β production by upregulating pro-IL-1β mRNA, while an inhibitor of NF-κB, BAY11-7082, had no significant influence on promotion of secreted IL-1β during PRRSV infection [46]. Inversely, NF-κB signaling and MAPK signaling promote the NLRP3 inflammasome activation during ischemic stroke in neurons [47]. Our results uncovered that NF-κB is critical for upregulating NLRP3 and pro-Caspase-1 mRNAs, whereas AP-1 is indeed important for upregulating mRNA levels of pro-IL-1β, NLRP3, and pro-Caspase-1. Notably, compared with NF-κB signaling pathway, AP-1 functions a prominent position in upregulating IL-1β transcription and maturation upon IAV infection. These findings uncovered that the mRNA levels of pro-IL-1β, NLRP3, or pro-Caspase-1 were regulated by different signaling pathways, especially the different types of stimuli.
Type I IFN significantly eliminates NLRP3 inflammasome by especially downregulating pro-IL-1β mRNA, which may control the threshold of NLRP3 inflammasome-modulated IL-1β production upon IAV infection. Our results demonstrate that AP-1 plays a superior position in NLRP3 inflammasomemediated IL-1β secretion upon IAV infection relative to NF-κB. We assumed that AP-1 promotes NLRP3 inflammasome-mediated IL-1β activation by targeting the first signal during RNA virus infection, although the assumption still requires it to be further explored. Figure 7. A proposed mechanism by which AP-1 signaling activates NLRP3 inflammasome upon IAV infection. Two intracellular signaling pathways, AP-1 and NF-κB, are involved in the occurrence of the downstream events through two types of mechanisms upon IAV infection. In in 2 the first signal, activation of NLRP3 inflammasome is primed by transcription of pro-IL-1β through ERK/c-Jun/AP-1 signaling pathway, and IL-1β protein is subsequently accumulated upon IAV infection. In in2 the second signal, IAV infection activates the NLRP3 inflammasome by facilitating the binding of ASC to NLRP3 and providing a platform for pro-Caspase-1 activation, allowing the maturation and secretion of IL-1β to undergo inflammatory responses.
In short, we demonstrate that AP-1 plays an essential role in H3N2-activated NLRP3 inflammasome by upregulating pro-IL-1β mRNA transcription. Furthermore, AP-1 signaling pathway may provide a potential target for therapeutic interventions on IAVcaused inflammatory diseases.

Disclosure statement
No potential conflict of interest was reported by the author(s).