Down-regulation of miR-155 Inhibits Inflammatory Response in Human Pulmonary Microvascular Endothelial Cells Infected with Influenza A Virus by Targeting Sphingosine-1-Phosphate Receptor 1
Influenza A virus is a highly contagious respiratory pathogen that poses a significant threat to human health. While mild infections typically result in self-limited upper respiratory tract disease, severe cases can progress to acute respiratory distress syndrome (ARDS), which is often fatal. The molecular mechanisms underlying the severe complications of influenza A infection are not fully understood, but accumulating evidence suggests that aberrant proinflammatory cytokine production, known as a cytokine storm, plays a key role. Modulation of the influenza A virus-mediated inflammatory response may represent a valid therapeutic strategy to mitigate severe disease outcomes.
Endothelial cells are central to the cytokine storm caused by influenza A virus. These cells, which line the blood vessels, become activated during severe infections, leading to the secretion of cytokines and chemokines that recruit leukocytes and disrupt the alveolar epithelial-endothelial barrier. This cascade of events ultimately results in ARDS. MicroRNA-155 (miR-155) is an important regulator of inflammation, and its role in the inflammatory response to influenza A infection has been the subject of recent investigation. This study explores the role and underlying mechanism of miR-155 in cytokine production in influenza A-infected endothelial cells, with a focus on its interaction with sphingosine-1-phosphate receptor 1 (S1PR1).
The study utilized human pulmonary microvascular endothelial cells (HPMECs) infected with the influenza A virus strain H1N1. The efficiency of H1N1 infection was confirmed using immunofluorescence, which detected the intracellular expression of the influenza virus nucleoprotein (NP) at 8 hours post-infection (p.i.). The expression levels of proinflammatory cytokines and miR-155 were determined using real-time polymerase chain reaction (PCR). A dual-luciferase reporter assay was employed to characterize the interaction between miR-155 and S1PR1, and changes in target protein levels were assessed using Western blot analysis.
The results revealed that miR-155 expression was significantly elevated in HPMECs in response to H1N1 infection. At 24 hours p.i., the fold change of miR-155 expression was 3.875 ± 0.062 compared to 1.043 ± 0.013 at 0 hours p.i. (P = 0.001). Overexpression of miR-155 enhanced the production of multiple proinflammatory cytokines, including interleukin-1β (IL-1β), IL-6, IL-8, CC motif chemokine ligand 2 (CCL2), CCL5, tumor necrosis factor-α (TNF-α), and interferon-β (IFN-β). Conversely, inhibition of miR-155 decreased the levels of these cytokines. For example, the mRNA expression of IL-1β was significantly increased in miR-155 mimic-treated cells compared to negative control (NC) cells (P = 0.001), while inhibition of miR-155 led to a decrease in IL-1β expression (P = 0.005).
The study further identified S1PR1 as a direct target of miR-155 in HPMECs. S1PR1 is a G protein-coupled receptor that plays a role in regulating immune responses and endothelial cell activation. The interaction between miR-155 and S1PR1 was confirmed using a luciferase reporter assay, which showed that miR-155 significantly decreased the luciferase activity of the wild-type S1PR1 3′-untranslated region (UTR) but did not affect the activity of a mutant 3′-UTR. Overexpression of miR-155 reduced S1PR1 protein levels, while inhibition of miR-155 enhanced S1PR1 expression. In H1N1-infected HPMECs, the downregulation of S1PR1 decreased the inhibitory effect of miR-155 blockade on cytokine production and nuclear factor kappa B (NF-κB) activation.
NF-κB is a transcription factor that plays a critical role in the proinflammatory response to influenza. The study found that overexpression of miR-155 increased the phosphorylation of the p65 subunit of NF-κB (phospho-p65) in H1N1-infected HPMECs, while inhibition of miR-155 reduced phospho-p65 levels. The reduction of S1PR1 expression via small interfering RNA (siRNA) attenuated the inhibitory effect of miR-155 blockade on NF-κB activation, suggesting that the function of miR-155 in H1N1-infected HPMECs is mediated by S1PR1.
The findings of this study highlight the role of miR-155 as a positive regulator of the inflammatory response in influenza A-infected endothelial cells. The upregulation of miR-155 in response to H1N1 infection leads to the suppression of S1PR1 and the subsequent activation of NF-κB signaling, resulting in increased cytokine production. Inhibition of miR-155 attenuates this inflammatory response by promoting S1PR1 expression, suggesting that targeting the miR-155/S1PR1 axis may represent a potential therapeutic strategy for mitigating the severe complications of influenza A infection, such as ARDS.
The study also underscores the importance of endothelial cell activation in the pathogenesis of influenza-induced ARDS. Activated endothelial cells secrete cytokines and chemokines that recruit leukocytes and disrupt the alveolar epithelial-endothelial barrier, leading to ARDS. By targeting miR-155 and its downstream effects on S1PR1 and NF-κB signaling, it may be possible to modulate the inflammatory response and prevent the progression to severe disease.
In conclusion, this study demonstrates that miR-155 plays a proinflammatory role in influenza A-infected endothelial cells by targeting S1PR1 and activating NF-κB signaling. Inhibition of miR-155 attenuates cytokine production and NF-κB activation, suggesting that the miR-155/S1PR1 axis may be a promising target for therapeutic intervention in severe influenza A infections. Further research is needed to explore the potential of miR-155 inhibition in vivo and to develop targeted therapies for the prevention and treatment of influenza-induced ARDS.
doi.org/10.1097/CM9.0000000000001036
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