Atorvastatin Potentially Reduces Mycobacterial Severity through Its Action on Lipoarabinomannan and Drug Permeability in Granulomas

Abstract

The majority of preclinical research has shown that Mycobacterium tuberculosis can modify host lipids in various ways. To boost its intramacrophage survival, M. tuberculosis causes host lipids to build up, resulting in the development of lipid-laden foam cells. M. tuberculosis binds to and enters the macrophage via the cell membrane cholesterol. Aggregation of cholesterol in the cell wall of M. tuberculosis and an increase in vascularity at the granuloma site reduce the permeability of rifampicin and isoniazid concentrations. However, very few studies have assessed the effect of statins on drug penetration. Here, we used atorvastatin, a 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor, to observe its effect on the bacterial burden by increasing the drug concentration at the infection site. We looked into how atorvastatin could be used in conjunction with first-line drugs to promote drug permeation. In this study, we detected an accumulation of drugs at the peripheral sites of the lungs and impaired drug distribution to the diseased sites. The efficacy of antituberculosis drugs, with atorvastatin as an adjunct, on the viability of M. tuberculosis cells was demonstrated. A nontoxic statin dosage established phenotypic and normal granuloma vasculature and showed an additive effect with rifampicin and isoniazid. Our data show that statins help to reduce the tuberculosis bacterial burden. Our findings reveal that the bacterial load is connected with impaired drug permeability resulting from lipid accumulation in the bacterial cell wall. Statin therapy combined with antituberculosis medications have the potential to improve treatment in tuberculosis patients. IMPORTANCE Mycobacterium tuberculosis binds to and enters the macrophage via the cell membrane cholesterol. M. tuberculosis limits phagosomal maturation and activation without engaging in phagocytosis. Aggregation of cholesterol in the cell wall of M. tuberculosis and an increase in the vascularity at the granuloma site reduce the permeability of rifampicin and isoniazid concentrations. However, very few studies have assessed the effect of statins on drug penetration, which can be increased through a reduction in cholesterol and vascularity. Herein, we used atorvastatin, a 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor, to observe its effect on bacterial burden through increasing the drug concentration at the infection site. Our main research goal is to diminish mycobacterial dissemination and attenuate bacterial growth by increasing drug permeability.

Keywords: Mycobacterium tuberculosis; atorvastatin; chemokines; drug distribution; drug penetration; granuloma; statins; tuberculosis; vasculature.

FIG 1

In guinea pigs, atorvastatin improved the anti-TB activity of the first-line regimen. (A) Timeline showing the treatments given. Compared to the control, atorvastatin 5 mg/kg administered alone after 2 weeks of aerosol infection had no effect on the course of acute infection at lungs. When combined with rifampicin/isoniazid and atorvastatin, the lung bacillary load was dramatically reduced compared to conventional therapy alone. Results are shown as the mean CFU plus the SD of lung (B) and spleen (C) per gram (log₁₀) from five animals per group, at each time point, or as the proportion of lung exhibiting inflammatory involvement. Dissemination of bacilli was reduced in the spleen in atorvastatin treated guinea pigs, when compared with M. tuberculosis infected guinea pigs without treatment, but not significantly. Statistical comparison between the groups was carried out by employing an unpaired t test. INF, infection; ns, not significant. Es, early stage; TS, terminal stage.

FIG 2

Atorvastatin downregulates LAM expression. (A) Graph showing the antigen levels of LAM in different groups. One-way ANOVA was used to compare the treated conditions to the infection control preceding multiple comparisons. At 2 and 4 weeks, the statin-treated groups showed a significant (P ≤ 0.03) reduction in LAM expression. (B) Graph showing the anti-LAM antibody responses in different groups, revealing enhanced colocalization of LAM in M. tuberculosis-infected guinea pigs. M. tuberculosis colocalizes quantitatively with LAM. Following atorvastatin therapy, the markers significantly (P ≤ 0.01) decreased. There was a considerable increase in the LAM profile between 4 and 6 weeks after M. tuberculosis infection (P ≤ 0.01, based on an unpaired t test; n = 5). (C) Quantitative analysis of Western blot bands. According to a qualitative analysis, expression of LAM antigens was considerably reduced in the treated groups (lanes 8 to 12) compared to the M. tuberculosis-infected groups.

FIG 3

Immunofluorescence-stained slides showing the expression of VEGF in different groups. One-way ANOVA was used to compare the treated conditions to the infection control preceding multiple comparisons. (A) Vascularity in the lungs is normal in the healthy guinea pigs. (B) VEGF expression was significantly upregulated in the M. tuberculosis-infected guinea pig lungs. (C) Treatment with atorvastatin reduced the expression of VEGF significantly (P < 0.01). (D, E) Treatment with anti-TB drugs attenuated the level of VEGF antibodies (VEGFA) (D), which was further reduced in guinea pigs treated with anti-TB drugs and atorvastatin combined (E). (F) Similar results were observed for VEGF mRNA expression. VEGF expression was dramatically decreased after treatment with atorvastatin (P ≤ 0.01). VEGF mRNA levels were further reduced in guinea pigs treated with anti-TB drugs and the atorvastatin combination, bringing the angiogenic factor levels back to normal. ANOVA was performed for multiple comparisons, and significant (P ≤ 0.05) changes were found. C_T, threshold cycle.

FIG 4

To reach the bacilli inside the macrophages, antituberculosis drugs must overcome barriers such as lesions in the lungs, with higher cellular composition and vascularization. From the blood, the drug enters the interstitial space of lesions and then penetrates them. (A to E) The distribution of drug concentration decreased in plasma (A, B), lungs (C, D), and granulomas (E), respectively; atorvastatin-treated guinea pigs combined with first-line drugs showed effective penetration of the drugs into the lesions. P ≤ 0.01 was considered statistically significant. In plasma and lung tissue, no significant difference in isoniazid concentration was seen. C_max, maximum concentration of drug in serum.

FIG 5

Drug action is always involved in the biochemical processes taking place in the body, so the biochemical parameters of liver function tests (LFTs) were measured after atorvastatin therapy, and the risk factors were observed. Biochemical testing of serum samples from atorvastatin-treated M. tuberculosis-infected guinea pigs provided critical insights into the drug. Biochemical variables were compared between three groups of guinea pigs. We evaluated plasma for liver enzymes to determine the status of liver functions during drug therapy. Alanine aminotransferase (ALT) levels in atorvastatin-treated guinea pigs were significantly increased compared to M. tuberculosis-infected guinea pigs without treatment. SGOT, serum glutamic-oxaloacetate transaminase; SGPT, serum glutamate pyruvate transaminase; A/G ratio, albumin/globulin ratio.