|dc.description.abstract||The host immune system orchestrates a defense mechanism against any invading pathogen with a combination of many mechanisms including antibody production, T-cell response, and memory response. The manner and extent of host immune response play a key role in deciding the fate of any infection, whether it manifests only as a mild disease or a lethal one. While a timely and robust immune response is necessary for a successful defense against a pathogen, in some cases a dysregulated immune response can also cause increased immunopathology. This work aims to explore different aspects of immune response across the disease severity spectrum of tuberculosis and COVID-19. Tuberculosis (TB), caused by Mycobacterium tuberculosis, is among the oldest known infectious disease to mankind. Despite over a century’s research on tuberculosis, it is not been successfully managed on a global scale. In late 2019, a coronavirus, SARS-CoV-2 induced COVID-19 emerged as the newest pandemic claiming over 6 million lives in only 2.5 years. Despite their many differences, host genetics and immune response are often the major deciding factors of the outcome of infection in both tuberculosis and COVID-19. Upon getting exposed to Mycobacterium tuberculosis the host can either completely clear the bacteria, or successfully prevent it from replicating and causing disease but fail to remove it completely (latent tuberculosis), or fail to control the infection leading to symptomatic and contagious disease (active tuberculosis). Stages of the disease have also been documented that fall in between latent and active tuberculosis based on the bacterial burden and state of symptoms, such as incipient tuberculosis and subclinical tuberculosis. In the case of COVID-19, most of the patients successfully mounted an immune response to clear the virus with no to mild symptoms (asymptomatic and mild). On the other hand for several others, especially those with a compromised immune response caused by aging or other comorbidities, the disease caused more serious symptoms often requiring external oxygen support (moderate), ICU and ventilation (severe), and even causing organ failure and septic shock (critical and fatal). Understanding different aspects of the host response are critical to finding strategies for successful management of both of these diseases. With the rapid technological advances in different unbiased data generation such as genomics, transcriptomics, and proteomics and sharing of data among the scientific community, the systems-level analysis provides unique opportunities to study a disease from a global perspective and identify the most important perturbations in a system in an unbiased manner. This thesis describes a systems biology approach to understanding the host and pathogen biology across the spectrum of these two infectious respiratory diseases - tuberculosis and COVID-19.
The first part of the thesis (Chapters 2-4) focuses on tuberculosis from a host and pathogen perspective. The whole blood transcriptome can capture systemic immune perturbations in a disease. Although traditional transcriptome analysis has been successful in elucidating different aspects of immune response in active tuberculosis, no clear pattern of systemic host response has so far been linked with latent tuberculosis, as molecular correlates of latent infection have been hard to identify. This work (Chapter 2) uses individualized response network analysis (an in-house algorithm to mine information on important biological processes for large-scale data) to find global perturbations in the host response to latent tuberculosis as compared to uninfected individuals. We specifically focus on the heterogeneity in such responses observed across latent tuberculosis cohorts. We identified the most frequently perturbed immune pathways despite the heterogeneity that are used by the host to maintain the latency of tuberculosis, such as the interferon-γ/interleukin-12 axis, tumor necrosis factor α (TNFα), epidermal growth factor receptor (EGFR) signaling, transforming growth factor β (TGFβ) signaling, etc. The analysis identified patterns of perturbation of these immune response pathways among latent tuberculosis patients despite the high heterogeneity in the gene expression profiles, grouped them into immune subtypes, and identified the subtype most likely to undergo reactivation into active TB. On the other end of the TB spectrum is the active disease. The immune response undergoes significantly large perturbations at both systemic and localized levels in active tuberculosis. Among the different pathways that are activated only in active tuberculosis patients, but not in latent or uninfected cases, pro-inflammatory leukotriene molecules play a significant role. Next, we studied the Mtb pathogen physiology in latent tuberculosis, where the bacteria stays in a dormant non-replicating yet viable condition. Since most anti-TB drugs target essential processes in actively replicating mycobacteria, their efficacy is highly reduced against dormant Mtb. Thus an ideal drug for latent tuberculosis treatment should target cellular processes that are essential for the survival of the non-replicating pathogen. In Chapter 3 we utilized systems modeling of dormant Mtb using flux balance analysis and network analysis to understand the dormancy mechanisms and identify 6 potential drug targets specific for latent tuberculosis treatment. These targets could be associated to lead molecules from approved drugs from DrugBank, showing the possibility of drug-repurposing for these targets. Following this, the focus was shifted to active TB (Chapter 4) and on the role of leukotriene B4 in this stage of tuberculosis. The important effector molecules of leukotriene B4 signaling in tuberculosis patients, STAT1/2 and NADPH oxidase, are identified with transcriptome integrated response network analysis. Further, the identified downstream mediators of leukotriene B4 signaling are experimentally validated in Mtb infected macrophages. The potential for targeting this pathway as a possible mode of host-directed therapy has also been assessed which showed that inhibition of leukotriene B4 signaling significantly reduces bacterial growth in infected THP-1 cells. The same was also seen in murine infection models.
The second part (Chapters 5-6) of the thesis focuses on COVID-19. Despite the large number of systems-level studies being performed on cohorts from China, North America, and European countries, similar studies from India have been very limited, despite the huge number of cases and fatalities observed. Understanding the immune response in different severities in the Indian population would provide important insights into the disease manifestation in this distinctly different genetic and environmental background. In Chapter 5, an Indian cohort has been generated at the early stages of different COVID-19 severities, and whole blood transcriptome has been utilized to study the differences between their systemic immune response. This study identified some of the critical differences among perturbed pathways between mild and severe COVID-19 at the early stage of disease onset. Suppression of the systemic immune system was observed in the early stages of severe COVID-19 patients. Timely mounting of robust type-I interferon response and classical complement pathway was found to be critical for reduced severity whereas repressed class I
MHC mediated antigen presentation and overall activation in translation pathways were key features in more severe patients. During the outbreak of COVID-19 in 2020 and 2021, another major concern faced by the clinicians in the over-burdened healthcare facilities was the presence of bacterial coinfection with COVID-19 which increased morbidity. It was a major challenge to correctly diagnose this condition to decide whether or not to administer antibiotics. The efficacy of the gold standard test for bacterial infection, the culture sensitivity test, was severely affected due to the widespread use of antibiotics in COVID-19 patients, leading to presumptive bacterial coinfection diagnosis and prescription of high doses of antibiotics in suspected coinfection cases. The unreliability of these tests could lead to both overprescriptions of antibiotics in a false-positive diagnosis and lack of necessary treatment in a false-negative diagnosis. In Chapter 6, the differences between the immune responses in a COVID-19 patient and a suspected coinfection case were explored. From a pool of host blood genes (identified in a previous meta-analysis) known to be perturbed only in confirmed bacterial infections but not in viral infections, a 9-gene signature was identified as a diagnostic tool to increase the confidence of bacterial co-infection diagnosis in COVID-19 patients. The gene signature and the score formulated from the same showed high accuracy, sensitivity, and specificity in distinguishing probable bacterial coinfection cases from only COVID-19 patients.
In summary, this work provides systems-level insights into the systemic immune responses in different stages of tuberculosis and COVID-19 diseases. The heterogeneous systemic host response in latent tuberculosis was successfully classified into subtypes. Our analysis also led to the identification of key effector molecules of leukotriene B4 signaling in active tuberculosis and explored its potential as a mode of host-directed therapy. The study of the dormant pathogen allowed us to identify 6 potential drug targets against latent tuberculosis. Together these works open up new possibilities in TB treatment and can be explored further for their translational potential. On the other hand, this thesis explored the key differences between the systemic immune responses in mild and severe COVID-19 at early stages and identified the key responses whose timely activation is necessary for lower disease severity. At the same time, a 9-gene signature and score were also identified from COVID-19 patients’ blood transcriptome that can assist in the diagnosis of bacterial coinfection in culture-negative cases.||en_US