The Impact of Vitamin A Deficiency on Tuberculosis Progression

BACKGROUND: Although previous studies have shown that vitamin A deficiency is associated with incident tuberculosis (TB) disease, the direction of the association has not been established. We investigated the impact of vitamin A deficiency on TB disease progression. METHODS: We conducted a longitudinal cohort study nested within a randomized clinical trial among HIV-infected patients in Haiti. We compared serial vitamin A levels in individuals who developed TB disease to controls matched on age, gender, follow-up time, and time to antiretroviral therapy initiation. We also evaluated histopathology, bacterial load, and immune outcomes in TB infection in a guinea pig model of dietary vitamin A deficiency. RESULTS: Among 773 participants, 96 developed incident TB during follow-up, 62.5% (60) of whom had stored serum samples obtained 90-365 days before TB diagnosis. In age- and sex- adjusted and multivariate analyses, respectively, incident TB cases were 3.99 times (95% confidence interval [CI], 2.41 to 6.60) and 3.59 times (95% CI, 2.05 to 6.29) more likely to have been vitamin A deficient than matched controls. Vitamin A-deficient guinea pigs manifested more extensive pulmonary pathology, atypical granuloma morphology, and increased bacterial growth after experimental TB infection. Reintroduction of dietary vitamin A to deficient guinea pigs after established TB disease successfully abrogated severe disease manifestations and altered cellular immune profiles. CONCLUSIONS: Human and animal studies support the role of baseline vitamin A deficiency as a determinant of future TB disease progression

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

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

A model of type 2 diabetes in the guinea pig using sequential diet-induced glucose intolerance and streptozotocin treatment

Type 2 diabetes is a leading cause of morbidity and mortality among noncommunicable diseases, and additional animal models that more closely replicate the pathogenesis of human type 2 diabetes are needed. The goal of this study was to develop a model of type 2 diabetes in guinea pigs, in which diet-induced glucose intolerance precedes β-cell cytotoxicity, two processes that are crucial to the development of human type 2 diabetes. Guinea pigs developed impaired glucose tolerance after 8 weeks of feeding on a high-fat, high-carbohydrate diet, as determined by oral glucose challenge. Diet-induced glucose intolerance was accompanied by β-cell hyperplasia, compensatory hyperinsulinemia, and dyslipidemia with hepatocellular steatosis. Streptozotocin (STZ) treatment alone was ineffective at inducing diabetic hyperglycemia in guinea pigs, which failed to develop sustained glucose intolerance or fasting hyperglycemia and returned to euglycemia within 21 days after treatment. However, when high-fat, high-carbohydrate diet-fed guinea pigs were treated with STZ, glucose intolerance and fasting hyperglycemia persisted beyond 21 days post-STZ treatment. Guinea pigs with diet-induced glucose intolerance subsequently treated with STZ demonstrated an insulin-secretory capacity consistent with insulin-independent diabetes. This insulin-independent state was confirmed by response to oral antihyperglycemic drugs, metformin and glipizide, which resolved glucose intolerance and extended survival compared with guinea pigs with uncontrolled diabetes. In this study, we have developed a model of sequential glucose intolerance and β-cell loss, through high-fat, high-carbohydrate diet and extensive optimization of STZ treatment in the guinea pig, which closely resembles human type 2 diabetes. This model will prove useful in the study of insulin-independent diabetes pathogenesis with or without comorbidities, where the guinea pig serves as a relevant model species

Sucrose fed guinea pigs had significantly higher lung and extrapulmonary Mtb lesion burden.

<p>The lung and lesion area was determined from hematoxylin and eosin stained tissue sections for each animal and the data expressed as mean percent involvement for each treatment group. The sucrose-fed guinea pigs had a significantly higher lesion burden compared to the water-fed control group in the lung (A) on day 60 of infection, spleen (B) on days 30 and 60 of infection, and mediastinal lymph node (C) on day 30 of infection. n = 10, *p≤0.05.</p

Sucrose feeding of <i>Mtb</i> infected guinea pigs increased lesion associated AGEs on day 30 and 60 of infection.

<p>AGEs are evaluated by immunohistochemistry on lung tissue sections of <i>Mtb</i> infected guinea pigs. The majority of strong immunoreactivity was associated with TB lesions. Immunoreactivity was evident within the cytoplasm of macrophages forming granulomatous lesions and was also strongly present within central necrosis (N) of primary TB pulmonary lesions. (A) Minimal reactivity was confined to the serum, rare macrophages in the lesions (arrow) and minimally within areas of necrosis (N) of the water-fed control group at 30 days of infection. (B) In contrast, at 30 days of infection, the sucrose-fed animals had strong immunoreactivity in the majority of lesion macrophages (arrow) and in areas of necrosis (N). (C) By day 60 of infection, AGEs began to accumulate within macrophages (arrow) in the water-fed controls but minimal reactivity was present in areas of necrosis (N). (D) The sucrose-fed animals had strong immunoreactivity within the majority of macrophages (arrow) and in areas of necrosis (N) at day 60 of infection. All tissue sections depicted are representative of the mean AGE IHC score for each treatment group at each time point (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046824#pone-0046824-g008" target="_blank">Figure 8</a>). Bar = 100 µm.</p

Sucrose feeding resulted in a more severe pulmonary lesion burden in <i>Mtb</i> infected guinea pigs.

<p>Images represent the animals closest to the mean values for severity of lesion burden as determined by measuring lesion and normal lung area using morphometric analysis (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046824#pone-0046824-g002" target="_blank">Figure 2</a>). Pulmonary lesion severity was similar between water- (A) and sucrose-fed (B) guinea pigs at day 30 of infection but was more severe in the sucrose-fed animals by 60 days of infection (D) compared to the water-fed controls (C). Bar = 1000 µm. Hematoxylin and eosin stain. <i>Insets</i>: High magnification views of the TB lesions delineated on the subgross views of A–D; Bar = 100 µm.</p

Non-Diabetic Hyperglycemia Exacerbates Disease Severity in <em>Mycobacterium tuberculosis</em> Infected Guinea Pigs

<div><p>Hyperglycemia, the diagnostic feature of diabetes also occurs in non-diabetics associated with chronic inflammation and systemic insulin resistance. Since the increased risk of active TB in diabetics has been linked to the severity and duration of hyperglycemia, we investigated what effect diet-induced hyperglycemia had on the severity of <em>Mycobacterium tuberculosis</em> (Mtb) infection in non-diabetic guinea pigs. Post-prandial hyperglycemia was induced in guinea pigs on normal chow by feeding a 40% sucrose solution daily or water as a carrier control. Sucrose feeding was initiated on the day of aerosol exposure to the H37Rv strain of Mtb and continued for 30 or 60 days of infection. Despite more severe hyperglycemia in sucrose-fed animals on day 30, there was no significant difference in lung bacterial or lesion burden until day 60. However the higher spleen and lymph node bacterial and lesion burden at day 30 indicated earlier and more severe extrapulmonary TB in sucrose-fed animals. In both sucrose- and water-fed animals, serum free fatty acids, important mediators of insulin resistance, were increased by day 30 and remained elevated until day 60 of infection. Hyperglycemia mediated by Mtb infection resulted in accumulation of advanced glycation end products (AGEs) in lung granulomas, which was exacerbated by sucrose feeding. However, tissue and serum AGEs were elevated in both sucrose and water-fed guinea pigs by day 60. These data indicate that Mtb infection alone induces insulin resistance and chronic hyperglycemia, which is exacerbated by sucrose feeding. Moreover, Mtb infection alone resulted in the accumulation tissue and serum AGEs, which are also central to the pathogenesis of diabetes and diabetic complications. The exacerbation of insulin resistance and hyperglycemia by Mtb infection alone may explain why TB is more severe in diabetics with poorly controlled hyperglycemia compared to non-diabetics and patients with properly controlled blood glucose levels.</p> </div

Hyperglycemia resulting from Mtb infection was exacerbated early by sucrose treatment.

<p>Random sampling of serum glucose values were compared to the mean glucose level of guinea pigs prior to sucrose feeding or infection (Pre-infection, n = 20) and served as the normal reference value. Serum glucose values of uninfected guinea pigs are similar to normal pre-infection values. Despite a mild increase in serum glucose associated with Mtb infection in the water-fed controls, significant exacerbation of hyperglycemia was only induced in sucrose-fed guinea pigs on day 30 of infection (n = 10). However,this difference was independent of sucrose feeding by day 60 (n = 9) of infection since persistent hyperglycemia was present in both sucrose- and water-fed groups. **p≤0.01.</p