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

National Institute Of Allergy And Infectious Diseases

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Abstract

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. These efforts were initially hampered by the fact that the most useful experimental model in these efforts, the mouse, is not susceptible to infection due to an incompatible sequence of the cellular receptor for virus entry, ACE2. However, several mouse models have now 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 made considerable progress in 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. In addition to standard analyses of these infection models of SARS-Cov2, we also developed a protocol for safe fixation and subsequent multiplex imaging of the lungs of these animals. Remarkably, in the C57Bl/6 mouse model of SARS-CoV-2 lethality, on day two after infection showed almost no inflammatory infiltrate and no evidence of type 1 interferon signaling using anti-pSTAT-1 staining, 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 dynamics of inflammatory processes. We are pursuing these observations to understand how these changes in innate immunity affect later adaptive responses and also if the discoordination of viral spread and innate immunity plays a special role in pathogenesis. The second major initiative is the employment of a lethal influenza infection as a model for severe viral pneumonia for use as a test bed for better understanding other viral pulmonary infections such COVID-19 caused by SARS-Cov2. 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 of the infected animals. No other drug or anti-inflammatory treatments tested alone altered the course of disease appreciably, arguing that either multiple damaging activities are involved and blunting only one is insufficient for a clinical effect, or 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. As with the SARS-Cov2 infection work noted above, current efforts utilize the highly multiplex imaging methods developed in the Lymphocyte Biology Section, LISB, NIAID, NIH to quantitatively probe the state of key cells and structures in the lung during the critical window in which intervention affects death rates to identify possible sites of damage, while treatment strategies involving pairing of anti-virial and anti-immune drugs are being tested for synergy. During the past year, more than a dozen putative treatments aimed at reducing innate or adaptive inflammatory damage during lethal influenza infections were studied in the influenza model we established. These included 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 of these treatments reduced weight loss or led to survival of any of the infected animals, and several worsened disease. Imaging of whole lung lobes using our IBEX method for multiplex staining showed that in all cases, 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 are insufficient to rescue the animals from eventual death. This has led to a change in strategy based on combining arrest of further damage and promoting recovery of functional lung structures. Combination treatments that are designed to achieve these two aims are now under study.