Mechanisms of immunopathology of COVID-19/ARDS, and strategies to mitigate detrimental inflammatory responses

点击翻译按钮获取中文摘要

The SARS-CoV-2 global pandemic has a continuing demand for greater understanding of the mechanisms of disease and the development of therapeutics to complement success in vaccination. Several mouse models have emerged that can be used, albeit with limitations, as models of severe SARS-Cov2 infection. Therefore we have initiated two major efforts to employ mouse models that can be utilized in the efforts to understand dysregulation of innate and adaptive immunity associated with severe viral pneumonia. The ultimate goal is to define these processes as they relate to SARS-CoV-2 to identify points of intervention that can be targeted therapeutically. The first major initiative is to develop mouse models of SARS-CoV-2 infection that model human disease. To achieve this, we have partnered with Jackson Laboratories to genetically engineer mice that express a humanized ACE2 gene to enable virus replication in tissues. We have tested 4 separate strategies to humanize ACE2 at the endogenous locus, or as a transgene. We have also tested mouse backgrounds for susceptibility. The ultimate goal will be to fully characterize the host responses as they relate to pathology in these models, and then utilize the models for testing biologics that block various events in inflammatory cascades. During this year, we finalized establishing 12 models of SARS-CoV-2 infection in 10 strains of mice. In humans, the range of disease phenotypes observed is extreme, from asymptomatic to critical, and is dependent on age, sex, genetics and metabolic status. The 10 strains of mice used included the 8 founder strains of the Collaborative Cross (B6, A/J, 129SJ, NZO, NOD, PWK, CAST and WSB) with additional strains Balb/c and DSB. Together, these strains represent over 90% of the genetic diversity within Mus musculus and have enabled us to model distinct disease phenotypes including a) sensitive mice with high sustained virus replication in lung and CNS (B6,A/J), b) resistant mouse strains associated with lower peak virus titer and earlier control of replication in the lung with no or low dissemination to other organs (PWK, NZO), and c) sex bias where resistance is independent of virus titer in the lung suggesting a sex-differences in host response (CAST, NOD, WSB). Cytokine analysis in the BAL revealed that resistance to disease in males was associated with high IFNb expression at 3dpi. Cytokine profiles generally modeled human responses with lethality associated with sustained high IP-10, as well as increasing MCP3, TNFa, IL-10, RANTES, IFNg and IL1b. Taken together, these mouse models represent a powerful tool to understand mechanisms of immune-mediated control and pathology following SARS-CoV-2 infection. The work was published (PMID: 37491352) and additional follow up data is being analysed including RNAseq data from lungs and brain of all models, as well as additional spatial transcriptomics of lungs. We have developed a protocol for safe fixation and subsequent multiplex imaging of the lungs of SAR-COv2 infected animals. Remarkably, in the K18-hACE2 transgenic mouse model of SARS-CoV-2 lethality, lungs on day two after infection showed almost no inflammatory infiltrate and no evidence of type 1 interferon signaling, whereas the influenza-infected animals showed robust innate immune cell infiltrates and interferon signaling at this time point. This reinforces existing evidence that coronaviruses potently suppress type 1 interferon responses and markedly change the inflammatory process. We are pursuing these observations across a more complete time course, using more markers to identify cell types and cell states, to better understand how these changes in innate immunity affect later adaptive responses and if the discoordination of viral spread and innate immunity plays a special role in pathogenesis. However, because cerebral infection plays a major role in lethality in this model, we are seeking alternatives as a substrate for testing therapeutics, and have recently obtained a mouse adapted version of SARS-CoV-2 that causes pulmonary dysfunction. This strain is presently being titrated to establish the proper model for transferring interventions from the influenza model described below. The second major initiative is the employment of a lethal influenza infection as a model for severe viral pneumonia. Ongoing studies involve (i) tests of interventions in the lethal influenza model that might have clinical utility and (ii) molecular, cell, and tissue level studies aimed at better understanding the underlying mechanism(s) of tissue damage and why interventions that constrain viral replication or innate immunity often fail after an early point in infection but well before death of the host. Using a severe influenza infection model that bypasses early nasopharyngeal replication and leads to rapid deep lung infection, we found that only very early treatment with the anti-viral oseltamivir phosphate (Tamiflu) could prevent death. Among 50 single or combined treatments covering many of the agents tested or used clinically for COVID-19 treatment (anti-IL-6, PANAM-G3, PMX205, inosine Pranobex, anti-PSGL1, ruxolitinib, inbrutinib, acalabrutinib, dypridamole, baricitinib, colchicine, silvelestat, AZD5059, anti-IL-6R, anti-CCL2, and Zileuton among others), none reduced weight loss or led to survival of any of the infected animals, and several worsened disease. These findings argue that either (i) multiple damaging activities are involved and blunting only one is insufficient for a clinical effect, and/or (ii) that irreversible tissue damage occurs early and once this occurs, interfering independently with viral replication or host immunity does not play a major role in preventing eventual death. Imaging of whole lung lobes using our IBEX method for multiplex staining showed that in this influenza infection model, there was early infiltration by neutrophils, extensive spread of the virus, loss of pro-surfactant and associated type 2 pneumocytes, alveolar disruption, and myeloid cell bronchiolar plugging, followed by later arrival of T cells in concert with marked loss of viable lung tissue. While some treatments modified the balance and extent of cell infiltrates, none prevented the damage and parenchymal loss. From these data, we developed the hypothesis that the infected animals rapidly pass a tipping point with respect to residual functional pulmonary capacity and that after this point, interference with inflammatory processes alone is insufficient to rescue the animals. This led to a change in strategy based on combining arrest of further damage and promoting recovery of functional lung structures by enabling more effective repair of damaged pulmonary epithelium. Results from other laboratories indicated that type 1 interferons can inhibit pneumocyte proliferation, while other studies suggest that late arrival of cytotoxic T cells in the lung can cause loss of residual functional epithelial cells. We therefore combined low dose Tamiflu administered late in the course of infection in combination with one of two additional treatments that either promote pneumocyte replication (blocking of interferon signaling) or limit further immune destruction (depletion of CD8+ T cells) can rescue mice from death. Remarkably, these combinations prevented death in more than 50% of the infected animals and we are combining Paxlovid in place of Tamiflu with each of these two immune interventions to examine if this combination is effective in the mouse adapted SARS-CoV-2 model.