Investigating host inflammatory and metabolic mechanisms as novel targets against respiratory infectious diseases
Creators: name: Erlich, Jonathan
Publisher: name: RMIT University
Year: 2024
Subjects: classificationCode: 320211
subject: Infectious diseases

description: Lower respiratory tract infections are the fourth highest cause of mortality world-wide, and most common cause of infection related deaths (WHO, 2018). These include influenza and influenza-related co-morbidities, of which around 10% of cases are fatal (WHO, 2018). Understanding the processes that lead to macrophage activation is a necessary stepping-stone in the development of therapeutics against influenza. Upon activation, glycolysis is up-regulated in macrophages as their primary source of ATP production, providing a rapid but ultimately inefficient method of energy production. As established in the literature, manipulation of the metabolic pathways provides several therapeutic avenues for cancer. We are now, however, beginning to unravel the role of metabolism in other disease states such as chronic inflammation, heart disease and autoimmune disorders, and even infectious diseases, which are already being explored in acute inflammation models caused by infectious pathogens. In one study, glycolytic inhibition by 2-deoxy-d-glucose (2DG), a hexokinase inhibitor, significantly improved survival rates in a sepsis mouse model (1). As a viral pathogen, influenza replication is highly dependent on the rapid generation of energy within the cell. Moreover, replication is dependent on availability of nucleotides and riboses for the building of virions, as well as amino acids to generate viable viral proteins through induction of the pentose phosphate pathway (PPP). Another product of the PPP is NADPH, which is an important cofactor for glutathione. NADPH, however, can also act as the electron donor for NADPH oxidase (NOX) 2 to generate reactive oxygen species (ROS) during the oxidative burst. In short, not only does the glycolytic switch provide the ideal conditions for viral replication, but it also fuels the cytokine storm that is commonly associated with the pathology of influenza.
In this thesis, we investigated the role of key modulators of the host response including Toll-like receptor (TLR) signalling, ROS pathways, metabolism and endosomal trafficking in the pathogenesis of IAV infection using pre-clinical mouse model systems. These evolutionary conserved pathways provide novel opportunities for the development of therapeutic strategies and understanding disease pathogenesis. 

In Chapter 2, we treated mice with the TLR7 agonist imiquimod both intranasally, and topically utilising its commercially available form, Aldara, prior to IAV infection to examine if TLR7 activation can prevent IAV associated pathogenesis. Imiquimod attenuated IAV-induced bodyweight loss, airway neutrophilic and eosinophilic inflammation, which were most likely the result of decreased pro-inflammatory cytokines IL-6, CXCL2, CCL3 and IL-1ß. Imiquimod treatment also resulted in a significant decrease in lung viral titres. Histological assessment of the lung showed significant improvements in peribronchiolar inflammation and a reduction in inflammatory cell infiltration and this was associated with reduced lung resistance, tissue damping and tissue hysteresivity in infected mice. We observed a faster, localised antibody response in imiquimod-treated mice than the IAV infected controls, but no differences were observed in the systemic antibody response that most likely was attributed to the route of intranasal delivery enhancing a local mucosal antibody response.
In Chapter 3, we explored metabolic reprogramming as a critical component of LPS-activated macrophages, and whether glycolysis, and the interconnected PPP, mediate the NOX2-dependent oxidative burst response. We observed significant reductions in NOX2-dependent ROS production and inflammation following administration of the hexokinase inhibitor 2DG, as well as the glucose-6-phosphate dehydrogenase inhibitor 6-aminonicotinamide (6AN). This observation was followed by significant reductions in IL-1ß and, surprisingly, IFN-ß. When we stimulated macrophages with recombinant IFN-ß, we showed an increased oxidative stress response which was confirmed by the reduced oxidative burden in LPS-stimulated macrophages following IFN-a/ß receptor (IFNAR) blockade. 

In Chapter 4, we assessed the alterations in inflammation, redox, TLR signalling, metabolic and endosomal pathways between the sexes due to IAV infection. We observed a reduction in lymphocytic and neutrophilic inflammation at day 3 post-IAV infection in male mice over female mice, which was not replicated at the day 7 timepoint. Conversely, there was evidence of increased inflammatory cytokine storm within the lungs of female mice compared to males infected with IAV. Flow cytometric analysis of immune cells isolated from the lung revealed an enhanced T cell-mediated immune response in female mice. We additionally observed a significant increase in oxidative stress in female mice at the day 3 p.i. timepoint, but not at day 7. IAV promoted increased metabolic rewiring in both sexes, with females experiencing higher glycolytic adaptation to infection over their male counterparts. We also show evidence for a greater capacity for endosomal remodelling, measured through key genes in endosomal trafficking representing the early and late endosomes, in male mice over female mice late in IAV infection.

One of the major findings from Chapter 4 was a significant upregulation in Immune-Responsive Gene 1/Cis-aconitate decarboxylase 1 (IRG1/ACOD1) expression, which is responsible for the generation of the anti-inflammatory and antioxidant metabolite itaconate. ACOD1-/- mice had reduced disease severity measured through bodyweight loss as well as neutrophilic airway inflammation compared to the WT. We found that WT mice had an increased NOX2-dependent oxidative burst response compared to the ACOD1-/- strain, which was associated with an increase protein expression of the ß-splice variant of NOX2.

This thesis builds upon a foundation of literature, outlining key alterations in evolutionarily- conserved pathways which influenza utilizes to drive host pathogenesis. What we show is that TLR activation by the virus, and their downstream consequences such as metabolic and redox signalling pathways, provide significant opportunities for development of therapeutics to treat respiratory viral infections. Personalised medicine is quickly gathering traction and is proving to be more suitable in the treatment of chronic diseases such as cancer. These series of experiments provide a foundational framework for adapting the principles of personalised medicine for the treatment of respiratory tract infections through identification of novel targets, as well as predicting the efficacy of theoretical drugs against these targets across both sexes.