Mycobacterium tuberculosis (M. tb), the causative agent of tuberculosis (TB), is responsible for causing significant morbidity and mortality, especially among individuals with compromised immune systems. We have previously shown that the supplementation of liposomal glutathione (L-GSH) reduces M. tb viability and enhances a Th-1 cytokine response, promoting granuloma formation in human peripheral blood mononuclear cells in vitro. However, the effects of L-GSH supplementation in modulating the immune responses in the lungs during an active M. tb infection have yet to be explored. In this article, we report the effects of L-GSH supplementation during an active M. tb infection in a mouse model of pulmonary infection. We determine the total GSH levels, malondialdehyde (MDA) levels, cytokine profiles, granuloma formation, and M. tb burden in untreated and L-GSH-treated mice over time. In 40 mM L-GSH-supplemented mice, an increase in the total GSH levels was observed in the lungs. When compared to untreated mice, the treatment of M. tb-infected mice with 40 mM and 80 mM L-GSH resulted in a reduction in MDA levels in the lungs. L-GSH treatment also resulted in a significant increase in the levels of IL-12, IFN-γ, IL-2, IL-17, and TNF-α in the lungs, while down-regulating the production of IL-6, IL-10, and TGF-β in the lungs. A reduction in M. tb survival along with a decrease in granuloma size in the lungs of M. tb-infected mice was observed after L-GSH treatment. Our results show that the supplementation of mice with L-GSH led to increased levels of total GSH, which is associated with reduced oxidative stress, increased levels of granuloma-promoting cytokines, and decreased M. tb burden in the lung. These results illustrate how GSH can help mitigate M. tb infection and provide an insight into future therapeutic interventions.

The Coronavirus disease 2019 (COVID-19) pandemic disproportionately affects immunocompetent and immunocompromised individuals, with the latter group being more vulnerable to severe disease and death. However, the differential pathogenesis of SARS-CoV-2 in the context of a specific immunological niche remains unknown. Similarly, systematic analysis of disease pathology in various extrapulmonary organs in immunocompetent and immunocompromised hosts during SARS-CoV-2 infection is not fully understood. We used a hamster model of SARS-CoV-2 infection, which recapitulates the pathophysiology of patients with mild-to-moderate COVID-19, to determine the dynamics of SARS-CoV-2 replication and histopathology at organ-level niches and map how COVID-19 symptoms vary in different immune contexts. Hamsters were intranasally infected with low (LD) or high (HD) inoculums of SARS-CoV-2, and the kinetics of disease pathology and viral load in multiple organs, antibody response, inflammatory cytokine expression, and genome-wide lung transcriptome by RNAseq analysis were determined and compared against corresponding responses from chemically induced immunocompromised hamsters. We observed transient body weight loss proportional to the SARS-CoV-2 infectious dose in immunocompetent hamsters. The kinetics of viral replication and peak viral loads were similar between LD and HD groups, although the latter developed more severe disease pathology in organs. Both groups generated a robust serum antibody response. In contrast, infected immunocompromised animals showed more prolonged body weight loss and mounted an inadequate SARS-CoV-2-neutralizing antibody response. The live virus was detected in the pulmonary and extrapulmonary organs for extended periods. These hamsters also had persistent inflammation with severe bronchiolar-alveolar hyperplasia/metaplasia. Consistent with the differential disease presentation, distinct changes in inflammation and immune cell response pathways and network gene expression were seen in the lungs of SARS-CoV-2-infected immunocompetent and immunocompromised animals.

Mycobacterium tuberculosis (M. tb), the causative agent of tuberculosis (TB), continues to be a global health burden. We have reported that patients with marked deficiency in the production of glutathione (GSH) had impaired granulomatous effector responses against M. tb infection, which were restored when supplementing patients with liposomal GSH (lGSH). However, the effects of GSH deficiency in the lung parenchyma in altering granuloma formation and effector responses against M. tb infection remain unexplored. We aim to elucidate the effects of diethyl maleate (DEM)-induced GSH deficiency during an active M. tb infection in an in vivo mouse model. We assessed for total and reduced GSH levels, malondialdehyde (MDA) levels, cytokine profiles, granuloma formation and M. tb burden. DEM administration significantly diminished total and reduced GSH levels in the lungs and plasma and increased MDA levels in infected mice compared to sham-treated controls. DEM treatment was also associated with an increase in IL-6, TNF-α and ill-formed granulomas in infected mice. Furthermore, M. tb survival was significantly increased along with a higher pulmonary and extrapulmonary bacterial load following DEM treatment. Overall, GSH deficiency led to increased oxidative stress, impaired granuloma response, and increased M. tb survival in infected mice. These findings can provide insight into how GSH deficiency can interfere with the control of M. tb infection and avenues for novel therapeutic approaches.

The Severe Acute Respiratory Syndrome-Coronavirus-2 (SARS-CoV-2) causes Coronavirus disease-2019 (COVID-19), which is an ongoing pandemic that has significantly affected the health, economy, and socio-economic status of individuals worldwide. Laboratory research using in vitro, ex vivo and in vivo models has been accelerated to understand the pathogenesis of SARS-CoV-2 infection. However, such experimental research involving SARS-CoV-2 is restricted to biocontainment/safety level-3 (BSL-3) settings, due to the high pathogenicity of this virus. Since many of the downstream analyses of SARS-CoV-2-infected biological samples need to be conducted in a non-BSL3 setting, it is important to ensure that the samples are fully decontaminated and safe for subsequent analysis. Here, we report the effectiveness of standard procedures used to fix cells and tissues for pathological analysis, including 2% or 4% paraformaldehyde, 50%–70% ethanol, 10% neutral buffered formalin and ultrafiltration using membranes with a molecular weight cut-off (MWCO) ranging from 3 to 30 kDa, for inactivating or eliminating SARS-CoV-2. We validated these methods in experimental laboratory samples, such as viral inoculum in cell culture media, SARS-CoV-2 infected host cells and animal tissue lysates. We found that 15 minutes’ treatment of viral inoculum (10⁵ plaque-forming units; PFU) or SARS-CoV-2 infected cells with paraformaldehyde or 70% ethanol resulted in complete inactivation of the virus. The treatment of infected hamster lung tissues with 10% neutral buffered formalin also fully inactivated the virus. However, only 3 kDa ultracentrifuge filter was effective in eliminating the virus to an undetectable limit in the filtrate. Our validated methods are useful for decontaminating biological samples to reduce infection risk and safe handling in BSL2 facilities.

The human response to Mycobacterium tuberculosis (Mtb) infection is affected by the availability of iron (Fe), which is necessary for proper immune cell function and is essential for the growth and virulence of bacteria. Increase in host Fe levels promotes Mtb growth and tuberculosis (TB) pathogenesis, while Fe-supplementation to latently infected, asymptomatic individuals is a significant risk factor for disease reactivation. However, the effect of Fe-supplementation on the host immunity during latent Mtb infection remains unclear, due partly to the paucity in availability of animal models that recapitulate key pathophysiological features seen in humans. We have demonstrated that rabbits can develop non-progressive latency similar to infected humans. In this study, using this model we have evaluated the effect of Fe-supplementation on the bacterial growth, disease pathology, and immune response. Systemic and lung Fe parameters, gene expression profile, lung bacterial burden, and disease pathology were determined in the Mtb-infected/Fe- or placebo-supplemented rabbits. Results show that Fe-supplementation to Mtb-infected rabbits did not significantly change the hematocrit and Hb levels, although it elevated total Fe in the lungs. Expression of selected host iron- and immune-response genes in the blood and lungs was perturbed in Mtb-infected/Fe-supplemented rabbits. Iron-supplementation during acute or chronic stages of Mtb infection did not significantly affect the bacterial burden or disease pathology in the lungs. Data presented in this study is of significant relevance for current public health policies on Fe-supplementation therapy given to anemic patients with latent Mtb infection.

Despite the availability of antibiotic therapy, tuberculosis (TB) is prevailing as a leading killer among human infectious diseases, which highlights the need for better intervention strategies to control TB. Several animal model systems, including mice, guinea pigs, rabbits, and non-human primates have been developed and explored to understand TB pathogenesis. Although each of these models contributes to our current understanding of host-Mycobacterium tuberculosis (Mtb) interactions, none of these models fully recapitulate the pathological spectrum of clinical TB seen in human patients. Recently, humanized mouse models are being developed to improvise the limitations associated with the standard mouse model of TB, including lack of necrotic caseation of granulomas, a pathological hallmark of TB in humans. However, the spatial immunopathology of pulmonary TB in humanized mice is not fully understood. In this study, using a novel humanized mouse model, we evaluated the spatial immunopathology of pulmonary Mtb infection with a low-dose inoculum. Humanized NOD/LtSscidIL2Rγ null mice containing human fetal liver, thymus, and hematopoietic CD34+ cells and treated with human cytokines were aerosol challenged to implant <50 pathogenic Mtb (low dose) in the lungs. At 2 and 4 weeks post infection, the tissue bacterial load, disease pathology, and spatial immunohistology were determined in the lungs, liver, spleen, and adipose tissue using bacteriological, histopathological, and immunohistochemical techniques. The results indicate that implantation of <50 bacteria can establish a progressive disease in the lungs that transmits to other tissues over time. The disease pathology in organs correspondingly increased with the bacterial load. A distinct spatial distribution of T cells, macrophages, and natural killer cells were noted in the lung granulomas. The kinetics of spatial immune cell distribution were consistent with the disease pathology in the lungs. Thus, the novel humanized model recapitulates several key features of human pulmonary TB granulomatous response and can be a useful preclinical tool to evaluate potential anti-TB drugs and vaccines.