Hyaluronan Ameliorates Viral Pneumonia in Mice and Humans by Inhibiting Transcription Factor E2F1

Abstract

Viral lung infections are a major cause of morbidity and mortality worldwide. Despite significant advances in vaccines and antivirals, there remains a tremendous need for broadly applicable treatments that can be utilized across viral infections. Before infecting epithelial cells, viruses interact with the epithelial glycocalyx, which contains high-molecular weight hyaluronan (HMWHA), a glycosaminoglycan that has beneficial effects in lung injury. In this study, we sought to determine the role of HMWHA in viral pneumonia. We infected mice with influenza or severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and treated them with prophylactic or therapeutic doses of HMWHA or saline control. We performed in vitro experiments of infection with viruses of respiratory and nonrespiratory human and animal cells and evaluated the effect of HMWHA on infection. We analyzed existing databases for expression of hyaluronan and the transcription factor E2F1. Finally, we performed a clinical trial with HMWHA in patients with severe coronavirus disease (COVID-19). Exogenously applied HMWHA improved survival in SARS-CoV-2 and influenza infection in mice by ameliorating inflammation through the inhibition of E2F1. In a clinical study, inhaled HMWHA improved outcomes in patients with severe COVID-19. Furthermore, airway epithelia naturally express HMWHA, which is induced during viral infection and prevents infection through the macromolecular crowding of viruses. Our data provide a mechanistic justification for the use of HMWHA as a broadly effective prophylactic and therapeutic agent in viral airway infection.

Keywords: E2F1; hyaluronan; treatment; viral infection; viral pneumonia.

Figure 1.. Effect of HMWHA in mouse models of SARSCoV2 (panels A-E) and influenza (panels F-J) pneumonia.

A, F. Survival curves. N=7–15 per group. B, G. Lung lavage cell differential. C, H. Lung lavage cytokine expression. D, I. Representative histology and lung injury score quantification by blinded pathologist. E, J. Viral loads. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ANOVA with Tukey post-hoc correction.

Figure 2.. Gene expression analysis in murine and human viral pneumonia.

A. Heatmap of mouse lungs after infection with SARS-CoV2 (top) or Influenza (bottom) and treatment with either HMWHA or saline. B. Six significant enriched gene sets shared between SARS-CoV2 and influenza infection identified by gene set enrichment analysis (top); and top upstream regulators predicted by IPA analysis based on the combined gene list derived from these six gene sets (bottom).. C. E2F target enrichment plot for SARS-CoV2 (top) and influenza (bottom) infection. D. E2F1 gene expression in the lung by treatment after SARS-CoV2. Ctrl=naïve controls. E. Gene expression of E2F1 in circulating blood monocytes in COVID-19 and Influenza pneumonia patients, with associated enrichment plots. *p<0.05, **p<0.01, ****p<0.0001, ANOVA with Tukey post-hoc correction. F. Heatmap with gene expression profile of E2F1 downstream targets by disease severity in human patients. G. E2F1 expression by immunohistochemistry in a human lung from a deceased COVID-19 patient.

Figure 3.. E2F1 promotes inflammation in viral pneumonia.

A. TNFα expression in bone marrow-derived macrophages exposed to the TLR7 agonist imiquimod. Left panel: wildtype macrophages with or without E2F inhibitor; Right panel: wildtype or E2F1-deficient macrophages. **p<0.01, ***p<0.001, Student’s t-test. B. Weight of wildtype or E2F1-deficient mice infected with H1N1-PR8 influenza virus N=18 mice per group. **p<0.01, 2-way ANOVA. C. Cell counts at day 6 post infection. ***p<0.001, Student’s t-test. D. Expression of relevant genes after infection with PR8 influenza virus in lungs of E2F1 wildtype (WT, black circles) and knockout (KO, open circles) mice. Student’s t-test. E. Representative histology of mouse lungs infected with PR8 influenza virus on Day 6 after infection. F. Injury score on Day 6 after infection. *p<0.05, Student’s t-test. G. Expression of E2F1 in mouse monocytes after exposure to short fragment HA (sHA) or HMWHA, or the TLR7 agonist Imiquimod with or without HMWHA. H. CD44 blocking antibodies inhibit E2F1 induction after sHA exposure. I. Exposure to Imiquimod or sHA induces activation of E2F1 in mouse monocytes. J. Gene expression of relevant cytokines in lungs of mice infected with PR8 influenza virus, and treated wither with PBS or HMWHA. Open circles: E2F1 KO mice. Black circles: E2F1 WT mice. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ANOVA with Tukey post-hoc correction.

Figure 4.. Inhibition of viral infection by HMWHA.

A. Inhibition of spike protein attachment to A549 cells. Top left panel: no spike protein. Top right panel: spike protein with vehicle (PBS) pre-incubation. Bottom panel: Spike protein with HMWHA pre-incubation. B. Inhibition of spike-protein-dependent pseudovirion attachment to A549 cells. Top panel: pseudovirions with vehicle (PBS) pre-incubation. Bottom panel: pseudovirions with HMWHA pre-incubation. C. Inhibition of SARS-CoV2 infection of ALI human cells by HMWHA. D. Inhibition of pseudovirion infection, decorated with different SARS-CoV2 spike protein variants, by HMWHA. E. Inhibition of influenza infection of Calu3 cells by HMWHA. F. Quantification of infected cells from ALI cultures infected with RFP-expressing RSV, by HMWHA (HA+INF). G. Inhibition of infection with pseudovirions decorated with SARS-CoV2 spike protein plotted against HA molecular weight. H. Inhibition of infection with pseudovirions decorated with SARS-CoV2 spike protein plotted against HA specific viscosity, using HA solutions of 4 different molecular weights. I. Viral particle movement through mucus with or without HMWHA. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ANOVA with Tukey post-hoc correction.

Figure 5.. Outcomes of clinical trial of inhaled HMWHA in severe COVID-19.

A. Recovery by time after admission in patients with initial P/F ratio<200, treated with saline Placebo (red) or HMWHA (blue). B. Recovery by time after admission in male patients with initial P/F ratio<200, treated with Placebo (red) or HMWHA (blue). C Length of hospital stay in patients with initial P/F ratio<200, treated with Placebo (red) or HMWHA (blue). D. Length of hospital stay in male patients with initial P/F ratio<200, treated with Placebo (red) or HMWHA (blue). E. Duration of O₂ treatment after admission in patients with initial P/F ratio<200, treated with Placebo (red) or HMWHA (blue). F. Duration of O₂ treatment after admission in male patients with initial P/F ratio<200, treated with Placebo (red) or HMWHA (blue). G. Number and percentage of patients with worsening respiratory failure necessitating ICU transfer by treatment group. H. FiO₂ by treatment group on admission and on Day 10, after cessation of treatment period. *p<0.05 Wilcoxon test. I. Hazard ratios for improved outcomes with HMWHA vs. placebo treatment, analyzing patients with admission serum CXCL10 levels above median value (hyperinflammatory, worse outcomes, see Supplemental Figure 7) or below median value (hypoinflammatory, better outcomes).

Figure 6.. Mechanistic summary of HMWHA effects in viral pneumonia prevention and treatment.

HMWHA prevents infection by inhibiting the movement of viruses through the mucus towards their receptors, and ameliorates inflammation by inhibiting the transcription factor E2F1. Bod mechanisms are nonspecific and therefore broadly active against viral infections.