 cholesterol synthesis in vivo, statins have pleiotropic action [32]. This is the reason how statins prevent the entry of tubercle bacilli in the alveolar macrophages and ultimately, prevent the host from M. TB infection. Apart from this, deficiency of vitamin D3 has been observed to increase the risk of M. TB in various populations [33]. Although statins diminish the levels of cholesterol and are expected to cause deficiency of vitamin D3 which is a downstream product of cholesterol, surprisingly statins either do not alter the levels of vitamin D3 or sometimes cause an increment in its concentration [34][35]. Hence, statins are reported to act as vitamin D3 analogs [36]. This production of vitamin D positively influences pancreatic beta-cell function in a chronic diabetic state, which itself is a known risk factor for M. TB infection due to immunosuppression [37]. It is an established fact that the cholesterol content in the macrophage membrane is relatively higher in diabetics when compared with the normal population [5]. This might be an important factor predisposing diabetics to M. TB infection [9]. Therefore, statins via reducing membrane cholesterol levels can minimize the occurrence of M. TB infection among the diabetic population as well [5]. The anti-tuberculous (anti-TB) effects of statins are summarized in Figure 1.

What do studies suggest regarding anti-TB effects of statin therapy?
In Vitro Studies The first study to highlight the potential effects of statins on infection by M. TB was conducted 20 years ago by Montero et al., where it was observed that fluvastatin modulates the release of type 1 T helper (Th1) and type 2 T helper (Th2) cytokines and consequently activates caspase-1 or leads to the secretion of interleukin (IL)-1β, IL-18 and interferon gamma (IFNγ) [38]. The peripheral blood mononuclear cells (PBMCs) were observed to be stimulated synergistically when infected with M. TB and treated with fluvastatin. The results suggested that statins could potentiate the host response against M. TB [38]. In 2009, Lu et al. reported that lovastatin and fluvastatin interfere with the formation of lipid rafts by inhibiting tyrosine phosphorylation and expression of monosialotetrahexosylganglioside (GM1) and cluster of differentiation 69 (CD69) in gamma delta (γδ) T cells. This leads to inhibition of γδ T cells induced by M. TB antigens [39]. However, both of these studies could not assess the effect of statins on mycobacterial growth and immune response of the host [38][39].
Parihar et al. studied the host protective mechanisms in individuals with hypercholesterolemia [12]. The study concluded that PBMCs and monocyte-derived macrophages (MDMs) from patients with familial hypercholesterolemia receiving statin therapy for at least six months were more resistant to mycobacterial infections than the cells of healthy non-statin users. In the same study, bovine bone marrow-derived macrophages (BMDMs) were treated with 50µM of simvastatin and were infected with M. TB for four hours at 37 °C. The results depicted a significant decrease in bacterial growth without alterations in cellular viability. In addition, they also conducted metabolic rescue experiments through confocal microscopy and western blot which demonstrated that statins decrease membrane cholesterol levels and promote phagosomal maturation and autophagy in macrophages. The effect of statins on inhibition of M. TB growth was reversed by phagosome or autophagosome maturation inhibitors like mevalonate. This study suggested the possibility of studying the supplementary role of statins with first-line anti-TB drugs [12]. The results concluded that bactericidal effects in the cells infected by M. leprae were secondary to phagosomal arrest [40].
To evaluate the tuberculocidal effect of simvastatin alone or in combination with first-line anti-TB drugs, Skerry et al. performed an experiment on J774 macrophages [41]. After 2 days of infection, a significant increase in tuberculocidal activity of isoniazid (INH) was observed with exposure to 5µM simvastatin as compared to INH alone. Similarly, simvastatin (25 mg/kg) enhanced bacterial killing when added with the standard oral regimen of RIF (10 mg/kg), INH (10 mg/kg), and pyrazinamide (PZA) (150 mg/kg). This additive bactericidal effect lost statistical significance by day five. Further, it was noted that simvastatin alone lacks anti-TB activity during the acute stage of infection. The study suggested exploring the optimal dose of statin and the ability of combination treatment to accelerate the time required to achieve a stable cure [41].
In 2016, Dutta et al. studied the adjuvant effect of simvastatin with INH, RIF, and PZA on the duration of corrective treatment [42]. To determine the effects of statins on the activity of firstline anti-TB drugs and intracellular RIF concentration, M. TB-infected THP-1 macrophages were exposed to simvastatin. It was concluded that simvastatin significantly enhances the bactericidal activity of first-line drugs without altering intracellular RIF concentrations. A reduction in the time required to achieve culture-negative lungs from 4.5 to 3.5 months was observed with adjuvant treatment (60 mg/kg simvastatin plus 10 mg/kg INH plus 10 mg/kg RIF plus 150 mg/kg PZA). However, simvastatin did not alter plasma or lung lesion cholesterol levels [42].
Guerra-De-Blas et al. analyzed the effects of simvastatin on the treatment of M. TB infection [43]. Direct quantification of M. TB growth was determined using PBMCs infected with M. TB H37Rv at multiplicity of infection (MOI) of 0.1. Although no direct antimicrobial activity was observed, simvastatin decreased the growth of M. TB in PBMCs, increased the proportion of natural killer (NK) T cells in culture and expression of co-stimulatory molecules in monocytes, promoted the secretion of cytokines and activated autophagy in monocytes resulting in a significant depletion in bacterial load. The study also suggested that further research should be conducted to define statin-induced anti-inflammatory mechanisms in TB treatment [43].
To determine whether statins can enhance the activity of anti-TB drugs against intracellular bacilli in macrophages, Dutta et al. conducted another study in 2019 [13]. They performed experiments with the M. TB H37Rv and THP-1 macrophages infected at MOI of 20. Pravastatin (7.8 μM), simvastatin (0.2 μM) and fluvastatin (0.032 μM) enhanced the activity of first-line anti-TB drugs (0.006 μM INH plus 0.0055 μM RIF and 81.23 μM PZA). However, atorvastatin and mevastatin showed no effect on mycobacterial growth at non-toxic doses. Among all statins, pravastatin exhibited the most potent adjunctive activity with the least toxicity by modulating phagosomal maturation characteristics in THP-1 macrophages. These observations concluded that pravastatin can be an attractive candidate for host-directed, adjunctive TB therapy [13]. All in vitro studies are summarized in Table 1.    [12][13], [38][39][40][41][42][43] In Vivo Studies

Author
All in vivo studies depicting the effects of statins on infection by M. TB have been performed on mice. The first study to investigate M. TB infection in a statin-treated experimental model was performed on C57BL/6 mice (8-12 weeks of age) [12]. The mice were treated intraperitoneally with 20 mg/kg of simvastatin or rosuvastatin and phosphate-buffered saline control every two days for six weeks and were infected by low-dose aerosol-based M. TB H37Rv. After monitoring the disease progression, it was concluded that both statins showed a protective response in the infected mice. About 10-fold reduction in bacillary burden was recorded in the spleen, liver, and lungs of mice infected and treated with simvastatin as compared to untreated control models [12]. In 2019, Dutta et al. conducted another experiment on female C3HeB/FeJ mice, five to six weeks of age to determine the anti-TB activity of statins in mice [13]. The mice were inoculated by aerosols of M. TB H37Rv calibrated to deliver ~10 2 CFU/mouse lung. Six weeks after infection, mice were treated with INH (10 mg/kg), RIF (10 mg/kg), PZA (150 mg/kg) and ethambutol (EMB) (100 mg/kg) supplemented with simvastatin (90 mg/kg), fluvastatin (15 mg/kg) and pravastatin (50 and 90 mg/kg). The treatment was administered once daily for five days a week for eight weeks. Satisfactory results were observed as the adjunctive therapy with statins significantly reduced bacillary burdens in the lungs of mice. The number of lung CFU decreased to 1.28 log 10, 1.16 log 10, 0.78 log 10, and 0.90 log 10 by adjunctive treatment with simvastatin (90 mg/kg), pravastatin (90 mg/kg), pravastatin (50 mg/kg) and fluvastatin (15 mg/kg), respectively [13]. All in vivo studies are summarized in Table 2.   [12][13], [40], [42] Retrospective Studies Lee et al. [45] conducted a retrospective cohort study in Taiwan which included 13,981 patients with type 2