Novel approach to treat viral pneumonia
Creators: name: Pandey, P
Publisher: name: University Of Tasmania
Year: 2023

description: Pneumonia, an important cause of morbidity and mortality worldwide, is caused by a variety of infectious agents, including viruses such as influenza virus, variola virus (agent of smallpox) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2; agent of COVID-19). No specific treatment is currently available for pneumonia caused by viruses. Treatment of this syndrome with antiviral drugs is ineffective, particularly when initiated after the onset of disease symptoms. In general, patients present to a hospital or seek medical advice only after the onset of symptoms, when antivirals alone are ineffective in improving disease outcomes, mainly because of dysregulated inflammatory response contributing to lung injury, in addition to necrosis and tissue damage caused by virus. Therefore, there is an urgent need to develop novel strategies for the treatment and management of viral pneumonia.<br>In this thesis, I first evaluated the effect of combined treatment in modulating the influenza A virus (IAV)-induced inflammatory response in the human alveolar epithelial A549 cells in vitro to obtain mechanistic insights. IAV infection induced the expression of mRNA transcripts for cytokines and chemokines, namely tumour necrosis factor (TNF), interleukin (IL)-6, IL-8, C-C motif chemokine ligand (CCL)2, CCL5, and C-X-C motif chemokine ligand 10 (CXCL10) at both early and late stages of infection, i.e., 3, 6, 24 and 48 h post-infection. Compared to mock treated cells, both combined treatments, i.e. etanercept (TNF-blockade agent) plus the neuraminidase inhibitor oseltamivir or signal transducer and activator of transcription 3 (STAT3) inhibitor plus oseltamivir, significantly downregulated the expression of IL-8 and CCL5 mRNA levels at 3 h and 48 h posttreatment, respectively. STAT3 inhibitor plus oseltamivir combined treatment was found to be highly effective in suppressing the expression of pro-inflammatory cytokines and chemokines, such as TNF, IL-6, IL-8, CCL2, CCL5 and CXCL10 at 48 h post-treatment. Both etanercept and STAT3 inhibitor modestly reduced the IAV infection-induced protein levels of phosphorylated nuclear factor kappa B p65 (pNF-κB p65) and pSTAT3 in A549 cells, however, their inhibitory effects on the NF-κB and STAT3 signalling pathways were found to be highly pronounced in LPS-stimulated murine macrophage cell line. These results therefore provided the preliminary data needed for subsequent preclinical studies in mice where we tested the potency of different treatment regimens, including combined antiviral and anti-inflammatory therapies in amelioration of viral infection-induced pulmonary pneumonia.<br>Next, I have used two animal models of viral pneumonia, namely mouse models of orthopoxvirus (OPXV) and IAV infection-induced pneumonia to establish proof-of concept that targeting both virus and inflammation after the onset of disease signs might be more effective in reducing morbidity and mortality. I initially focused on anti-TNF therapy for dampening inflammation for two reasons. First, TNF is a key inflammatory cytokine produced very early during the course of viral infections. Second, excessive and prolonged production of TNF is associated with immunopathology and mortality caused by OPXV and IAV pneumonia.<br>Respiratory infection with ectromelia virus (ECTV), an OPXV and the agent of mousepox, causes severe pneumonia in mice, associated with excessive inflammatory cytokine production and lung immunopathology. My study has demonstrated that combined treatment with etanercept and cidofovir (antiviral drug against DNA viruses) is effective in limiting lung pathology and protecting mice from an otherwise lethal infection even when the treatment is initiated after onset of disease signs. Mechanistically, I showed that the combined treatment reduced the lung viral load and significantly downregulated the expression of pro-inflammatory cytokines and chemokines: TNF, IL-6, IL-β, IL-12p40, transforming growth factor beta (TGF-β) and CCL5, which are induced at high levels during OPXV infections. I have established that the soluble form of TNF (sTNF) but not the membrane bound form (mTNF) correlated with disease severity, the levels of which were significantly reduced by the combined treatment. I further demonstrated that the combined treatment mediated its anti-inflammatory effects through inhibition of two important cytokine signalling pathways, namely the NF-κB, and STAT3. Consequently, combined treatment targeting the STAT3 pathway and virus was also found to be effective in reducing disease signs and lung pathology in ECTV-infected mice, and the effects correlated with suppression of TNF, IL-1β, IL-12p40, TGF-β and CXCL10 mRNA transcripts. Interestingly, mRNA levels of TNF, IL-1β, IL-12p40, TGF-β and CCL5 were also down-regulated by the etanercept-cidofovir combination treatment, indicating cross-talk between TNF/NF-κB and STAT3 signalling pathways.<br>Finally, I established that the therapeutic efficacy of combined treatment is not just unique to OPXV pneumonia but rather it has a broader application. Treatment of IAVinfected mice with the combined treatment using etanercept and oseltamivir initiated after the onset of disease signs improved clinical signs, reduced loss in body weight and ameliorated pneumonia through significant reduction in lung inflammation. This anti-inflammatory effect correlated with reductions in levels of expression for TNF, IL-6, IL-1β, IL-12p40, CCL2, CCL5, and CXCL10 mRNA transcripts in the lung. The suppression of multiple cytokines/chemokines by the combined treatment was mediated through inhibition of NF-κB and STAT3 signalling pathways, demonstrating cross-talk between these pathways in the pathogenesis of IAV pneumonia, as observed in ECTV-induced pneumonia. Combined treatment with a STAT3 inhibitor and oseltamivir, administered after the onset of disease signs, also improved clinical signs of disease and ameliorated lung pathology and down-regulated levels of TNF, IL-1β, IL-12p40, TGF-β,CCL2, CCL5, and CXCL10 mRNA transcripts in lungs of IAV-infected mice.<br>This study highlights the NF-κB and STAT3 signalling pathways as the molecular mechanisms underlying IAV- and ECTV-associated hyperinflammatory response and lung immunopathology. In addition, the data indicate that combined treatment targeting both the host inflammatory response and virus will be effective in the treatment of OPXV and influenza pneumonia, particularly when the treatment commencement is delayed after the onset of disease signs. Furthermore, this combined treatment approach has the potential to be used to treat pneumonia caused by other respiratory viruses, such as SARS-CoV, SARS-CoV-2 and Middle East respiratory syndrome coronavirus, where the host’s inflammatory response and virus are major contributors of disease pathogenesis.