Understanding the Role of Cellular Proteases, Intrinsic Protease Inhibitors and Reactive Oxygen Species in Murine β-Coronavirus-Induced Neuroinflammation

Sengupta, Sourodip (2022) Understanding the Role of Cellular Proteases, Intrinsic Protease Inhibitors and Reactive Oxygen Species in Murine β-Coronavirus-Induced Neuroinflammation. PhD thesis, Indian Institute of Science Education and Research Kolkata.

Text (PhD thesis of Sourodip Sengupta (14IP009)) 14IP009.pdf - Submitted Version Restricted to Repository staff only Download (7MB)

Abstract

Coronaviruses (CoVs) are a highly diverse group of enveloped viruses having one of the largest positive-sense single-stranded RNA genomes. The first step in successful infection of a host by CoVs involves binding the viral surface spike glycoprotein (S) with the host-cellular entry receptor. The intracellular life cycle begins with releasing the viral genomic RNA in the cytoplasm, followed by immediate translations that give rise to several non-structural proteins that form the viral transcription and replication complex. Profuse replication, transcription, and translation of the CoV particles and their maturation occur mainly in the host cellular membrane organelle, the endoplasmic reticulum (ER). Evidences from past studies demonstrate that infection by CoV disrupts ER homeostasis, leading to ER stress. Cellular ER stress generated during CoV infection induces critical signalling pathways, involving the unfolded protein response (UPR) and intrinsic mitochondrial ROS (reactive oxygen species) production. ER stress and mitochondrial oxidative imbalance are associated with cellular apoptosis and elevated inflammation, which have complex virus replication and pathogenesis effects. Simultaneously, the successful clearance of CoV infection requires the influx of effector cells, destroying the infectious particle, resolving inflammation, and remodelling the extracellular matrix (ECM). Intrinsic cellular proteases such as matrix metalloproteinases or MMPs are needed to complete these events successfully. However, infection-induced tissue damage and immunopathology could be due to elevated level of MMPs and its associated heightened immune response. Tissue damage may further help the dissemination and persistence of pathogen by disrupting tissue barrier to spread and creating an immune privileged infection site not accessible by the immune cells. To prevent excessive tissue damage, tissue inhibitors of metalloproteinases or TIMPs regulate MMP activities and reduce matrix degradation. My research was focused on the regulatory role of MMPs in m--CoV infection and neuroinflammation for the past four years. But at the end of 2019, when SARS-CoV-2 came and became a threat to the human race for the prolonged and enhanced zoonotic potential and its severe lethality, the entire globe is still reeling around designing vaccines, and looking for small molecule therapeutics repurposing drugs, I have redirected my research focus to understand how to protect the human race from the recurring lethal CoV infection by regulating host factors that may provide immediate fortification. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) induced COVID-19 (coronavirus disease 2019) is the third case of zoonotic transmission of CoVs in the human race after SARS-CoV (in 2003) and Middle East respiratory syndrome coronavirus (MERS-CoV; in 2012). So far, seven human CoVs (HCoVs) have been identified. Among the HCoVs, the α-CoVs (NL63 and 229E) and the β-CoVs (OC43 and HKU1) infect the upper respiratory tract and only cause seasonal and mild common cold symptoms. The β-CoVs SARS-CoV, MERS-CoV, and SARS-CoV-2 can infect the lower respiratory tract and cause a severe acute respiratory syndrome with high zoonotic potentials making their study difficult. The periodic surfacing of new HCoVs can be ascribed to their immense genetic diversity, recurrent genetic recombination, and cross-species transmission due to increased human-animal interaction. COVID-19 is considered a respiratory disease as SARS-CoV-2 primarily targets the respiratory system. Common symptoms of COVID-19 such as cough, fever, headache, fatigue, nausea, shortness of breath, and chest pain bears similarities with other viral respiratory diseases. These make the detection of COVID-19 at early stage challenging, allowing the progression of the disease and resulting in severe infection associated with pneumonia. Multidimensional factors such as population density, age, obesity, comorbidity, seasonality, temperature, humidity, social distancing, and critical care capacity have played a significant role in deciding COVID-19 disease incidence and mortality. In addition, the emergence of SARS-CoV-2 mutant strains makes it challenging for available therapeutic drugs and newly developed vaccines to manage COVID-19 effectively. In last year, our studies compelled us to understand how diet can help prevent the SARS-CoV-2 spread and infection and protect from its sustained lethality. Mounting evidence from previous CoV studies and other viral diseases was pertinent in designing antioxidants as antivirals to manage the COVID-19 pandemic effectively. A large body of studies is available in developing antivirals by targeting the fusion, host entry, replication, and assembly of the virus in designing antivirals by targeting the virus fusion, host entry, replication, and assembly. Still, very few studies are available to design antivirals that can target the host factors involved in any process associated with CoV infection. Based on the above observations, we have hypothesized that the cellular proteases and their intrinsic protease inhibitors in combination with ROS may play a role in immune modulation in CoV infection, and dietary factors may enhance immunity by restricting viral infectivity that may help design antivirals against pan β-CoVs. The current study also explored the outcome of ROS inhibition on m-β-CoV infection. In in vitro, ROS inhibition reduced expression of pro-inflammatory genes, essential viral structural genes, and decreased virus replication, suggesting detrimental effect of ROS in virus infection. Furthermore, in vivo studies indicate the need for minimum cellular ROS to maintain immune homeostasis between pro-and anti-inflammation by regulating Treg populations. Potent anti-inflammatory effects of Treg cells have been demonstrated previously in mice infected with neurotropic MHV strain, JHMV. Adoptive transfer of CD4+Foxp3+ Treg cells in immunodeficient mice decreased weight loss, clinical scores, and demyelination upon JHMV infection. In addition to the immunomodulatory role of Treg cells, a regenerative function of Treg cells in promoting oligodendrocyte differentiation and remyelination has also been demonstrated recently in the mice model. Therefore, the extensive pleiotropic nature of ROS and its role in physiological signalling make it difficult to explicitly define its function in terms of simple cause-effect relationships. Findings from the present study indicate that ROS inhibition experiments need to be interpreted with caution. In particular, studies comparing how ROS inhibition affects the immune system in pathological conditions can provide insight into mechanisms of action of ROS.

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