IRE1α-XBP1 Activation Elicited by Viral Singled Stranded RNA via TLR8 May Modulate Lung Cytokine Induction in SARS-CoV-2 Pneumonia

Initial symptoms of COVID-19 infection depend on viral replication, while hyperinflammation is a hallmark of critical illness and may drive severe pneumonia and death. Among the mechanisms potentially involved in the hyperinflammatory state, we focused on the unfolded protein response, because the IRE1α-XBP1 branch can be activated as result of the endoplasmic reticulum stress produced by the overwhelming synthesis of viral components and synergizes with Toll-like receptor signaling to induce cytokine expression. Viral RNA may trigger the IRE1α-XBP1 branch via TLR7/8 activation and like TLR2 and TLR4 may underpin cytokine expression trough XBP1 splicing ( sXBP1 ). The expression of IL1B , IL6 , and TNF mRNA in bronchoalveolar aspirates (BAAs) were higher in COVID-19 patients under mechanical ventilation and intubation who showed sXBP1 . The scrutiny of monocytic/macrophagic markers during active infection showed a reduction of those involved in antigen presentation and survival, as well as the IFN stimulated gene MX1 . These changes reverted after infection tests turned negative. In contrast, the expression of the mRNA of the serine protease TMPRSS2 involved in S protein priming showed a high expression during active infection. TLR8 mRNA showed an overwhelming expression as compared to TLR7 mRNA, which suggests the presence of monocyte-derived dendritic cells (MDDCs). In vitro experiments in MDDCs activated with ssRNA40, a positive-sense, single-stranded RNA (+ssRNA) like SARS-CoV-2 RNA, induced sXBP1 and the expression of IL-1β, IL-6, and TNF α at mRNA and protein levels. These responses were blunted by the IRE1α ribonuclease inhibitor MKC8866. Given the analogies between the results observed in BAAs and the effects induced by +ssRNA in MDDCs, IRE1α ribonuclease inhibition might be a druggable target in severe COVID-19 disease. ![Figure][1] Author summary COVID-19 pandemics put an unprecedented pressure on health systems. The need of new therapies urged research on the mechanisms triggered by the interaction of SARS-CoV-2 virus with host cells and the ensuing pathophysiology driving pneumonia and multiorgan failure. Hyperinflammation soon appeared as a mechanism involved in mortality that could even proceed after viral infection comes to an end. Hyperinflammation is supported by an inappropriate production of cytokines, and this explains the use of the term cytokine storm to refer to this phase of the disease. Given that insight into the molecular mechanisms driving cytokine storm should focus on the interaction of viral components with immune cells, experiments addressing the effect of viral components on its cognate receptors were carried out. It was observed that viral RNA induces a cytokine pattern like the one observed in bronchoalveolar aspirates of COVID-19 patients with critical disease. Overall, the study revealed that both cell organelle overload and receptors involved in the recognition of viral RNA may team up to induce proinflammatory cytokines. This mechanism can be exploited to develop new treatments for COVID-19 disease. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement This study was funded by Fondo COVID-19 del Instituto de Salud Carlos III/Junta de Castilla y Leon (N.F.). European Commission-NextGenerationEU, through CSIC's Global Health Platform (PTI Salud Global) (project SGL2103016) (M.S.C.). Plan Nacional de Salud y Farmacia Grant SAF2017-83079-R and Grant PID2020-113751RB-I00 funded by MCIN/AEI/ 10.13039/501100011033 (M.S.C.). Junta de Castilla y Leon/Fondo Social Europeo Grants CSI035P17 (M.S.C.) and VA175P20 (N.F.). Proyecto SEAHORSE INFRARED: IR2020-1-UVA05 (JCyL). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: Ethics Committee of Area de Salud Valladolid Este (ref. PI-GR-20-2011 COVID). Bioethical Committee of the Spanish Council of Research I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines and uploaded the relevant EQUATOR Network research reporting checklist(s) and other pertinent material as supplementary files, if applicable. Yes All data produced in the present study are available upon reasonable request to the authors [1]: pending:yes