JNK kinase promotes inflammatory responses by inducing the expression of the inflammatory amplifier TREM1 during influenza a virus infection

Abstract

Since the twentieth century, four influenza pandemics caused by IAV have killed millions of people worldwide. IAV infection could induce acute lung injury mediated by cytokine storms, which is an essential cause of death in critically ill patients. Consequently, it is crucial to explore the regulators and regulatory mechanisms of cytokine storms, which may provide potential drug targets and expand our understanding of acute lung injury. Previous studies have shown that JNK kinase is essential in promoting inflammatory responses during viral infections. In this study, we demonstrated that JNK kinase could regulate the IAV-induced cytokine storms by affecting the expression of pro-inflammatory and anti-inflammatory factors. Further studies revealed that inhibition of JNK kinase activity significantly downregulated the expression of the inflammatory amplifier TREM1. Besides, TREM1 knockdown could significantly inhibit the expression of pro-inflammatory factors. Furthermore, SP600125 is a specific inhibitor of JNK kinase. The results show that TREM1 overexpression reversed the effect of SP600125 treatment on the expression of pro-inflammatory factors. Together, we found that JNK kinase could activate the inflammatory amplifier TREM1 to promote inflammatory responses during influenza A virus infection. These findings may provide some inspiration for subsequent researchers to explore the regulatory mechanisms of cytokine storms induced by emerging viral infections.

Keywords: Inflammatory amplifier; Inflammatory responses; Influenza A virus; JNK kinase; TREM1.

Fig. 1

mRNA expression profiles and biological function of JNK kinase during IAV infection analyzed by functional enrichment. (A) A549 cells were pretreated with 20 μM SP600125 or DMSO as a vehicle control for 2 h and subsequently infected with WSN (MOI = 1). Infected and uninfected cells were collected at 24hpi. Total RNA was extracted for RNA-seq analysis. A bar graph showing the number of differentially expressed mRNAs between two groups. (B) A cluster heat map showing the mRNAs with significantly different expressions between two groups (WSN_SP600125-vs-WSN). (C) A volcano map showing the mRNAs with significantly different expressions between two groups (WSN_SP600125-vs-WSN). (D) A Venn diagram showing the number of differentially expressed mRNAs between two groups and the intersection. (E) A bubble plot showing the top20 enriched KEGG pathways (WSN_SP600125-vs-WSN). (F) KEGG classification diagram (WSN_SP600125-vs-WSN).

Fig. 2

JNK kinase induces the expression of pro-inflammatory factors IL-6, IL-1β, IL-1α, and IL-8 during IAV infection. (A) A549 cells were pretreated with 20 μM SP600125 or DMSO as a vehicle control for 2 h and subsequently infected with WSN. Total RNA was extracted at 24hpi. Real-time polymerase chain reaction (RT-PCR) was performed to detect the expression of IL-6, IL-1β, IL-1α, and IL-8. (B-E) Quantitative real-time polymerase chain reaction (qRT-PCR) was conducted to validate the expression of IL-6 (B), IL-1β (C), IL-1α (D), and IL-8 (E). Data represent means ± standard deviations (n = 3; **, P < 0.01).

Fig. 3

JNK kinase regulates the IAV-induced cytokine storms by affecting the expression of pro-inflammatory and anti-inflammatory factors. (A) Top30 GO enrichment terms (WSN_SP600125-vs-WSN). (B) A heat map showing these pro-inflammatory and anti-inflammatory factors with significantly different expressions between two groups. (C) RT-PCR was performed to validate the expression of CCL3, CCL4, CCL20, CXCL2, CXCL3, IL-23, IL-32, IL-36γ, IL-13, and IL-22. (D-M) qRT-PCR was conducted to validate the expression of CCL3 (D), CCL4 (E), CCL20 (F), CXCL2 (G), CXCL3 (H), IL-23 (I), IL-32 (J), IL-36γ (K), IL-13 (L), and IL-22 (M). Data represent means ± standard deviations (n = 3; **, P < 0.01).

Fig. 4

JNK kinase induces the expression of the inflammatory amplifier TREM1 during IAV infection. (A) A heat map showing the top 30 downregulated differentially expressed genes between two groups (WSN_SP600125-vs-WSN). (B, C) RT-PCR, western blotting, and qRT-PCR were conducted to validate the expression of TREM1, respectively. (D, E) A549 cells were pretreated with SP600125 (10, 20, or 30 μM) or DMSO as a vehicle control for 2 h and subsequently infected with WSN. Western blotting was performed to detect the phosphorylation level of JNK kinase and the expression of TREM1. (F) qRT-PCR was performed to examine the differential expression of TREM1 in A549 cells infected with or without WSN. (G) A549 cells were infected with various multiplicities of infection (MOI) of WSN for a period of 24 h. The expression level of TREM1 was detected by qRT-PCR. Data represent means ± standard deviations (n = 3; **, P < 0.01; ***, P < 0.001).

Fig. 5

TREM1 amplifies the inflammatory responses during IAV infection. (A-N) A549 cells seeded in 24-well plates and 6-well plates were transfected with si-NC/si-TREM1 and then infected with WSN (MOI = 1). Western blotting and qRT-PCR were performed to detect the expression of TREM1 in A549 cells (A, B). qRT-PCR was conducted to detect the expression of IL-6 (C), IL-1β (D), IL-1α (E), IL-8 (F), CCL3 (G), CCL4 (H), CCL20 (I), CXCL2 (J), CXCL3 (K), IL-23 (L), IL-32 (M), and IL-36γ (N). (O) A549 cells underwent transfection with pcDNA3.1/pcDNA3.1-TREM1 for 36 h. The overexpression efficiency of TREM1 in A549 cells was determined by RT-PCR and western blotting. (P, Q) The mRNA expression of pro-inflammatory factors in each group at 24hpi was measured by qRT-PCR. Data represent means ± standard deviations (n = 3; **, P < 0.01).