Evidence for Oxidative Stress and Defective Antioxidant Response in Guinea Pigs with Tuberculosis

The development of granulomatous inflammation with caseous necrosis is an important but poorly understood manifestation of tuberculosis in humans and some animal models. In this study we measured the byproducts of oxidative stress in granulomatous lesions as well as the systemic antioxidant capacity of BCG vaccinated and non-vaccinated guinea pigs experimentally infected with Mycobacterium tuberculosis. In non-vaccinated guinea pigs, oxidative stress was evident within 2 weeks of infection as measured by a decrease in the serum total antioxidant capacity and blood glutathione levels accompanied by an increase in malondialdehyde, a byproduct of lipid peroxidation, within lesions. Despite a decrease in total and reduced blood glutathione concentrations, there was an increase in lesion glutathione by immunohistochemistry in response to localized oxidative stress. In addition there was an increase in the expression of the host transcription factor nuclear erythroid 2 p45-related factor 2 (Nrf2), which regulates several protein and non-proteins antioxidants, including glutathione. Despite the increase in cytoplasmic expression of Nrf2, immunohistochemical staining revealed a defect in Nrf2 nuclear translocation within granulomatous lesions as well as a decrease in the expression of the Nrf2-regulated antioxidant protein NQO1. Treating M. tuberculosis–infected guinea pigs with the antioxidant drug N-acetyl cysteine (NAC) partially restored blood glutathione concentrations and the serum total antioxidant capacity. Treatment with NAC also decreased spleen bacterial counts, as well as decreased the lung and spleen lesion burden and the severity of lesion necrosis. These data suggest that the progressive oxidative stress during experimental tuberculosis in guinea pigs is due in part to a defect in host antioxidant defenses, which, we show here, can be partially restored with antioxidant treatment. These data suggest that the therapeutic strategies that reduce oxidant-mediated tissue damage may be beneficial as an adjunct therapy in the treatment and prevention of tuberculosis in humans

Increased Virulence of an Epidemic Strain of Mycobacterium massiliense in Mice

Chronic pulmonary disease and skin/soft tissue infections due to non-tuberculous mycobacteria (NTM) of the Mycobacterium chelonae-abscessus-massiliense group is an emerging health problem worldwide. Moreover, the cure rate for the infections this group causes is low despite aggressive treatment. Post-surgical outbreaks that reached epidemic proportions in Brazil recently were caused by M. massiliense isolates resistant to high-level disinfection with glutaraldehyde (GTA). Understanding the differences in the virulence and host immune responses induced by NTM differing in their sensitivity to disinfectants, and therefore their relative threat of causing outbreaks in hospitals, is an important issue.We compared the replication and survival inside macrophages of a GTA-susceptible reference Mycobacterium massiliense clinical isolate CIP 108297 and an epidemic strain from Brazil, CRM-0019, and characterized the immune responses of IFNγ knockout mice exposed to a high dose aerosol with these two isolates. CRM-0019 replicated more efficiently than CIP 108297 inside mouse bone marrow macrophages. Moreover, the animals infected with CRM-0019 showed a progressive lung infection characterized by a delayed influx of CD4+ and CD8+ T cells, culminating in extensive lung consolidation and demonstrated increased numbers of pulmonary CD4+ Foxp3+ regulatory T cells compared to those infected with the reference strain. Immunosuppressive activity of regulatory T cells may contribute to the progression and worsening of NTM disease by preventing the induction of specific protective immune responses.These results provide the first direct evidence of the increased virulence in macrophages and mice and pathogenicity in vivo of the Brazilian epidemic isolate and the first observation that NTM infections can be associated with variable levels of regulatory T cells which may impact on their virulence and ability to persist in the host

Uptake and Accumulation of Oxidized Low-Density Lipoprotein during Mycobacterium tuberculosis Infection in Guinea Pigs

The typical host response to infection of humans and some animals by M. tuberculosis is the accumulation of reactive oxygen species generating inflammatory cells into discrete granulomas, which frequently develop central caseous necrosis. In previous studies we showed that infection of immunologically naïve guinea pigs with M. tuberculosis leads to localized and systemic oxidative stress that results in a significant depletion of serum total antioxidant capacity and the accumulation of malondialdehyde, a bi-product of lipid peroxidation. Here we show that in addition, the generation of excessive reactive oxygen species in vivo resulted in the accumulation of oxidized low density lipoproteins (OxLDL) in pulmonary and extrapulmonary granulomas, serum and lung macrophages collected by bronchoalveolar lavage. Macrophages from immunologically naïve guinea pigs infected with M. tuberculosis also had increased surface expression of the type 1 scavenger receptors CD36 and LOX1, which facilitate the uptake of oxidized host macromolecules including OxLDL. Vaccination of guinea pigs with Bacillus Calmette Guerin (BCG) prior to aerosol challenge reduced the bacterial burden as well as the intracellular accumulation of OxLDL and the expression of macrophage CD36 and LOX1. In vitro loading of guinea pig lung macrophages with OxLDL resulted in enhanced replication of bacilli compared to macrophages loaded with non-oxidized LDL. Overall, this study provides additional evidence of oxidative stress in M. tuberculosis infected guinea pigs and the potential role OxLDL laden macrophages have in supporting intracellular bacilli survival and persistence

The Efficacy of the BCG Vaccine against Newly Emerging Clinical Strains of Mycobacterium tuberculosis.

To date, most new vaccines against Mycobacterium tuberculosis, including new recombinant versions of the current BCG vaccine, have usually been screened against the laboratory strains H37Rv or Erdman. In this study we took advantage of our recent work in characterizing an increasingly large panel of newly emerging clinical isolates [from the United States or from the Western Cape region of South Africa], to determine to what extent vaccines would protect against these [mostly high virulence] strains. We show here that both BCG Pasteur and recombinant BCG Aeras-422 [used here as a good example of the new generation BCG vaccines] protected well in both mouse and guinea pig low dose aerosol infection models against the majority of clinical isolates tested. However, Aeras-422 was not effective in a long term survival assay compared to BCG Pasteur. Protection was very strongly expressed against all of the Western Cape strains tested, reinforcing our viewpoint that any attempt at boosting BCG would be very difficult to achieve statistically. This observation is discussed in the context of the growing argument made by others that the failure of a recent vaccine trial disqualifies the further use of animal models to predict vaccine efficacy. This viewpoint is in our opinion completely erroneous, and that it is the fitness of prevalent strains in the trial site area that is the centrally important factor, an issue that is not being addressed by the field

OxLDL accumulates in pulmonary alveolar macrophages of <i>M. tuberculosis</i>-infected guinea pigs.

<p>A and B represent OxLDL immunostaining in BAL cells collected from <i>M. tuberculosis</i> infected guinea pigs at day 30 and day 60 after infection respectively (1000× magnification). Predominantly macrophages (arrows) and occasionally granulocytes (arrowhead) show intracellular staining.</p

<i>M. tuberculosis</i> infection of guinea pigs results in progressive lung lesions that are less severe in BCG vaccinated animals.

<p>The lung lesion burden increases with time in guinea pigs infected by aerosol with the H37Rv strain of <i>M. tuberculosis</i>. BCG vaccination prior to challenge decreases the rate and severity of lung granulomas as determined by lesion scores. The bars represent median values plus range (when present) for each group (n = 5). The asterisks denote statistically significant increase compared to day 5 after infection (* = p<0.05, *** = p<0.001 and **** = p<0.0001).</p

Serum OxLDL levels increase in guinea pigs infected with <i>M. tuberculosis</i> infection.

<p>In guinea pigs sham-vaccinated with saline, serum OxLDL levels increased with the progression of disease as determined by a competitive ELISA. BCG vaccination of guinea pigs prior to virulent challenge abrogated the increase in serum OxLDL levels. Data is expressed as the mean values for each treatment group (n = 5). The asterisks denote statistically significant increase compared to the naive animals (* = p<0.05 and *** = p<0.001).</p

OxLDL loaded guinea pig pulmonary alveolar macrophages support intracellular growth of <i>M. tuberculosis in vitro</i>.

<p>A. Immunofluorescence of normal guinea pig alveolar macrophages show no evidence of OxLDL cytoplasmic staining. B. Uninfected alveolar macrophages loaded with OxLDL show strong cytoplasmic immunofluorescence staining after 3 days of treatment. C. Alveolar macrophages loaded with LDL for 3 days and infected with <i>M. tuberculosis</i> (stained with rhodamine/auramine) show minimal to no positive staining for OxLDL. D. Co-localization of <i>M. tuberculosis</i> stained with rhodamine/auramine within alveolar macrophages loaded with OxLDL for 3 days <i>in vitro</i>.</p

Activities of TMC207, Rifampin, and Pyrazinamide against Mycobacterium tuberculosis Infection in Guinea Pigs▿

The experimental compound TMC207 is showing promise against infections caused by Mycobacterium tuberculosis both in a variety of animal studies and in the field. In this study, we used the guinea pig model, a species that shows several similarities to human tuberculosis, including the hallmark of primary granuloma necrosis, to determine the efficacy of a combination regimen combining TMC207 with rifampin and pyrazinamide. This drug regimen rapidly reduced the bacterial load in the lungs to undetectable levels by 8 weeks of treatment. This reduction was associated with a substantial improvement in lung pathology, but despite this effect areas of residual necrosis still remained. In the draining lymph nodes, however, tissue damage was rapid and not significantly reversed by the drug treatment. Approximately 10 to 11 months after the treatment had ended, the animals began to trigger a Karnovsky scale indicating bacterial regrowth and potential relapse, an event confirmed by the new development of both pulmonary and extrapulmonary granulomatous lesions. Interestingly, a similar rate of relapse was also seen in animals receiving 24 weeks of rifampin, pyrazinamide, and isoniazid standard chemotherapy. These data indicate that TMC207 could be a useful addition to current treatment regimens for tuberculosis