Abstract
Influenza A virus (IAV), an RNA virus, remains a global public health threat, infecting a diverse range of hosts, such as humans, pigs, and poultry. Due to its constant evolution through a high mutation rate and genetic reassortment, IAV causes seasonal epidemics, pandemics, and zoonotic outbreaks of influenza, commonly known as the flu. The severity of the flu varies among individuals, with some cases progressing to severe pneumonia. Several viral and host factors contribute to the severity of flu in humans. One such factor is the hyperactivation of the proinflammatory immune response to infection. The host possesses complex innate immune response networks that are activated upon viral infection to counteract the pathogen. Immediately after infection, host cells sense IAV infection through different pattern recognition receptors (PRRs) and stimulate various innate immune response cascades. Among these, the NLRP3 inflammasome, which belongs to the NOD-like receptors (NLRs) class of PRRs, plays a vital role in sensing IAV. The goal of NLRP3 inflammasome-mediated signalling is to induce the proinflammatory death of infected cells and promote viral clearance. However, hyperactivation of this pathway can lead to hyperinflammation, which is detrimental to flu patients.
The regulatory mechanisms of NLRP3 inflammasome-mediated signalling in IAVinfected cells have yet to be elucidated. In heterologous conditions, NLRP3 inflammasome components undergo various post-translational modifications that regulate their signalling. One such protein modification is acetylation, which is regulated by histone acetyltransferases (HATs) and histone deacetylases (HDACs). Previously, our group, demonstrated through manipulation of the expression and activity of HDACs and HATs that acetylation negatively regulates the host innate response against IAV. However, acetylation's role in IAV-induced NLRP3 inflammasome signalling has not been previously investigated.
The aim of this study was to decipher the role of acetylation in NLRP3 inflammasomemediated signalling during IAV infection. The NLRP3 inflammasome signalling pathway is initiated by the priming of NLRP3, caspase-1, and proinflammatory cytokines genes. Priming is followed by activation, which involves the oligomerisation of NLRP3, ASC, and pro-caspase-1, to form a complex that leads to the release of cleaved caspase-1, which further cleaves pro-interleukins into their active secreted forms. To investigate this, we assessed IAV-induced priming and activation of the NLRP3 inflammasome in human lung epithelial cells (A549). The NLRP3 inflammasome priming and activation were examined using RT-qPCR, western blotting, and confocal microscopy. Priming and activation of the IAV-induced NLRP3 inflammasome were reduced upon treatment with HDACs’ inhibitors (TSA and TBA). Consistent with this, confocal microscopy images revealed a reduction in NLRP3 puncta formation following inhibitor treatment, suggesting the involvement of acetylation. Furthermore, siRNA-mediated depletion of individual HDACs, HDAC2 and HDAC6 were found to positively regulate the priming and activation of the IAVinduced NLRP3 inflammasome. Moreover, from confocal imaging, HDAC2 and HDAC6 were found to promote IAV-induced NLRP3-GFP puncta formation in A549 cells.
Overall, these findings indicate that acetylation negatively regulates NLRP3 inflammasome signalling during IAV infection. This study provides critical insights into the fine-tuning of NLRP3 inflammasome signalling in response to IAV and design new therapeutic strategies to treat severe influenza cases, especially those caused by highly pathogenic avian influenza (HPAI) viruses.