Calcitriol Enhances Pyrazinamide Treatment of Murine Tuberculosis
Tuberculosis (TB) remains a significant global health challenge, particularly in developing countries, where high mortality rates and the emergence of multidrug-resistant Mycobacterium tuberculosis (M.tb) strains underscore the urgent need for improved treatment strategies. The current standard TB therapy is lengthy and can be associated with significant side effects, necessitating the exploration of adjunctive therapies that can enhance the efficacy of existing drugs and shorten treatment duration. Vitamin D, particularly its active metabolite calcitriol (1,25-dihydroxy vitamin D3), has garnered attention for its potential immunomodulatory and antimicrobial properties. This study investigates the synergistic effects of calcitriol and pyrazinamide (PZA), a first-line anti-TB drug, in a murine model of TB, aiming to elucidate the mechanisms by which calcitriol enhances PZA activity and improves treatment outcomes.
The study employed a well-established murine model of TB, using C57BL/6 female mice infected with the M.tb H37Rv strain via aerosol exposure. The mice were divided into four treatment groups: a saline control group, a calcitriol-only group, a PZA-only group, and a combination group receiving both calcitriol and PZA. Treatments were initiated four weeks post-infection and continued for six weeks. The primary outcomes measured included bacterial burden in the lungs and spleen, histopathological assessment of lung lesions, and the expression of inflammatory cytokines and antimicrobial peptides (AMPs).
Calcitriol alone had minimal impact on the bacterial burden in M.tb-infected mice. However, PZA treatment significantly reduced the bacterial load in both the spleen and lungs compared to the saline control group. The combination of calcitriol and PZA further enhanced this effect, resulting in a significantly lower bacterial burden than PZA treatment alone. Specifically, the bacterial load in the spleen was reduced from 4.82 ± 0.22 Log10 CFU/gram in the PZA group to 4.37 ± 0.13 Log10 CFU/gram in the combination group. Similarly, in the lungs, the bacterial load decreased from 5.55 ± 0.15 Log10 CFU/gram in the PZA group to 5.03 ± 0.32 Log10 CFU/gram in the combination group. These findings suggest that calcitriol enhances the bactericidal activity of PZA in vivo.
Histopathological analysis revealed that PZA treatment alone significantly reduced lung lesions compared to the saline control. The combination of calcitriol and PZA further attenuated these lesions, with a gross pathological score of 3.25 ± 0.50 in the combination group compared to 2.50 ± 0.58 in the PZA group. The affected area of the lung was also significantly smaller in the combination group (30.75% ± 6.50%) compared to the PZA group (21.55% ± 2.99%). These results indicate that calcitriol not only enhances the bactericidal effects of PZA but also mitigates the pathological damage caused by M.tb infection.
To understand the immunomodulatory effects of calcitriol, the study examined the expression of key cytokines and AMPs. Calcitriol treatment significantly increased the expression of the anti-inflammatory cytokine interleukin-4 (IL-4) while suppressing the pro-inflammatory cytokine interferon-gamma (IFN-g). Specifically, IL-4 expression increased from 2.80 ± 0.56 fold of control in the saline group to 5.69 ± 0.50 fold of control in the calcitriol group. In contrast, IFN-g expression decreased from 4.13 ± 0.83 fold of control in the saline group to 1.36 ± 0.11 fold of control in the calcitriol group. The combination of calcitriol and PZA further enhanced these effects, with IL-4 expression reaching 6.06 ± 0.51 fold of control and IFN-g expression dropping to 1.59 ± 0.12 fold of control. These findings suggest that calcitriol modulates the immune response to M.tb infection by promoting an anti-inflammatory cytokine profile, which may contribute to the enhanced therapeutic efficacy observed.
In addition to its effects on cytokine expression, calcitriol significantly upregulated the expression of AMPs, including cathelicidin LL-37 and mouse β-defensin-2 (mBD2). Calcitriol treatment increased LL-37 expression from 2.80 ± 0.90 fold of control in the saline group to 10.59 ± 1.03 fold of control, and mBD2 expression from 1.79 ± 0.45 fold of control to 7.92 ± 0.62 fold of control. The combination of calcitriol and PZA further enhanced these effects, with LL-37 expression reaching 11.92 ± 1.13 fold of control and mBD2 expression reaching 9.59 ± 1.09 fold of control. These AMPs are known to have direct antimicrobial activity against M.tb, and their upregulation by calcitriol likely contributes to the observed reduction in bacterial burden.
The study also explored the potential mechanisms underlying the synergistic effects of calcitriol and PZA. Calcitriol is known to induce autophagy, a cellular process that can eliminate intracellular pathogens, including M.tb. Additionally, calcitriol has been shown to enhance the expression of vitamin D receptor (VDR) target genes, which are involved in antimicrobial defense. The combination of these effects with the bactericidal activity of PZA likely results in the enhanced clearance of M.tb observed in the study.
The findings of this study have important implications for the treatment of TB. The addition of calcitriol to standard TB therapy could potentially enhance treatment efficacy, reduce the duration of therapy, and mitigate the immunopathological damage associated with M.tb infection. However, the study also highlights the need for further research to fully elucidate the mechanisms by which calcitriol enhances PZA activity and to determine the optimal dosing and duration of calcitriol supplementation in TB patients.
In conclusion, this study provides compelling evidence that calcitriol enhances the therapeutic efficacy of PZA in a murine model of TB. The combination of calcitriol and PZA results in a significant reduction in bacterial burden and attenuation of lung lesions, likely through the modulation of the immune response and upregulation of AMPs. These findings support the potential use of calcitriol as an adjunctive therapy in the treatment of TB, offering a promising avenue for improving treatment outcomes in patients with this devastating disease.
doi.org/10.1097/CM9.0000000000000394
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