All groups experienced increased vitamin D levels at both week 4 and week 8. The 10,000 IU dose group saw the highest increase in vitamin D during 8 weeks of supplementation, from 27.91 ± 8.68 ng/ml to 66.40 ± 4.89 ng/mL. Supplementation of vitamin D 10,000 IU/day for 8 weeks can increase vitamin D levels > 50 ng/dl so that it functions optimally as an immunomodulator. Meanwhile, at other doses it did not reach > 50 ng/ml.

A study conducted by Nurpudji et al. [17] showed an increase in vitamin D levels after the administration of a dose of 10,000 IU for 2 weeks. In the study carried out by Salma et al. [18], increased vitamin D levels were found after vitamin D supplementation. This study showed significant increases in vitamin D levels at weeks 4 and 8 in the group given vitamin D supplementation compared to the placebo group. Vitamin D can decrease due to several factors, such as dark skin color, the use of sunscreen, and UV exposure. In addition, decreased production of vitamin D can also be caused by geographical factors such as: season, latitude, air pollution, fog/clouds, and humidity. Absorption disorders can also affect the decrease in vitamin D such as celiac disease, pancreatic disorders, and obstruction of the biliary tract [6, 19].

Vitamin D deficiency has always been associated with an increased risk of infection with Mycobacterium tuberculosis [20] In a study, Tang et al. [4] found that vitamin D deficiencies correlated with spondylitis tuberculosis type necrosis.

A study by Gibney et al. [21] showed lower levels of vitamin D in patients with latent TB infection than in healthy people. Increased vitamin D can affect the low probability of latent tuberculosis infection. In addition to vitamin D, another study conducted by Orafli et al. [22] found that vitamin A deficiency also plays a role in tuberculosis infection in the Moroccan population. Previous research also found no effect of high doses of vitamin A on the outcome of children with tuberculosis infection. Vitamin A has also been shown not to affect sputum conversion in patients with pulmonary tuberculosis infection. Patients with pulmonary tuberculosis infection who were given OAT treatment found that their vitamin A levels would return to normal even though they were not given supplementation [23]. However, in a study conducted by Visser et al. [24], it was found that supplementation with vitamin A combined with zinc for 8 weeks was considered to have no effect on the outcome of patients with pulmonary tuberculosis infection.

Vitamin D supplementation at doses of 400, 5000, and 10,000 mg/dl for 8 weeks may also affect TLR 2 and TLR 4 levels. There was a difference between TLR 2 and TLR-4 levels of 7 mg/dl to 15 mg/dL at week 8 compared to week 0. The study also found a significant correlation between vitamin D levels for TLR-2 and TLR-4. After supplementation, TLR-2 and TLR-4 levels increased. This increase in TLR indicates an increased immune response that plays a role in the process of fighting M. pneumoniae and tuberculosis. TLR activation by the Mycobacterium antigen will give the cell a response to produce more pro-inflammatory cytokines in macrophages and dendritic cells [13] (Fig. 3, Table 1).

The concentration of vitamin D

Simultaneous activation of the TLR2 and TLR4 signaling pathways induces macrophage apoptosis and induction of apoptosis and necrosis in response to Mycobacterium tuberculosis [25]. Mycobacterial components are recognized by TLR-2 and TLR-4. Several mycobacterial antigens including lipoarabinomannan (LAM), 38-kDa antigen, LpqH, lipomannan (LM), LprG, PhoS1, LprA, phosphatidylinositol mannoside (PIM), and trehalose dimycolate (TDM) activate TLR2, TLR1, or TLR6, whereas TLR4 recognizes 38-kDa and 60/65 heat shock protein (HSP) antigens. TLR 2 and TLR-4 receptors are overexpressed during infection [13, 26]. Activation of TLRs by antigens belonging to mycobacteria leads to intracellular signaling pathways that culminate in pro-inflammatory production in dendritic cells and macrophages through the MAPK and NF-κB pathways [10]. In a study by Wani et.al, TLR-4 (Thr/Ile) and TLR-2 (Del/Del) acted as significant risk factors for extrapulmonary tuberculosis in the ethnic Kashmiri population [27] Salih et al. [28] revealed that the upregulation of TLR- 2 and TLR-4 due to single nucleotide polymorphisms (SNPs) may be involved in the tuberculosis infection process in Sudanese individuals.

In a study conducted by Ali et al. on TLR-2 levels in cases of tuberculosis infection, significant differences in serum TLR2 levels were found in each variation of TB infection cases. In cases of recurrence of tuberculosis infection, TLR-2 serum levels were found to decrease significantly compared to new cases of TB infection [29]. In another study conducted by Hendri et al., it was found that vitamin D levels correlated positively with TLR2 serum expression in systematic lupus erythematosus patients. The higher the levels of vitamin D, the higher the TLR-2 expression in the saliva sample examined [30]. In a study conducted by Eve et al. [20], vitamin D supplementation was found to increase TLR2 levels in pregnant women. Hendri et al. [31] found that vitamin D levels correlated with serum TLR-4 expression in systematic lupus erythematosus patients.

Other studies showed similar results, as Rahmini Shabariah et al. showed a significant increase in serum levels of TLR-2 and TLR-4 in patients with extrapulmonary TB who had post-vaccination scars of Mycobacterium bovis Bacillus Calmette–Guerin (BCG) compared to patients without BCG scars. The formation of scars after BCG vaccination affects the formation of immunity against M infection. TLR-2 and TLR-4 serum scores were higher in patients with extrapulmonary tuberculosis, in addition to serum TLR-2, TLR-4 was higher in those with BCG scars than in those without BCG, and statistical analysis results showed a significant difference. Additionally, evidence suggests that toll-like receptor 2,4 (TLR-2 and TLR-4) is associated with susceptibility to TB by interacting with toll-like interactive protein (TOLLIP) to activate macrophages [32].

Vitamin D has functions in the immune system, cell proliferation, and differentiation. Supplementation of vitamin D to > 100 nmol/L shows an increase in TLR-2 expression and a decrease in the TLR2-profile of cytokine stimulation for TNF, IL-6, and IFN, and this condition will be reversed when vitamin D doses are reduced to < 100 nmol/L (p = 0.002). Optimal vitamin D supplementation can improve TLR-2 expression and increase the body’s ability to fight infections [27].

The side effect of supplementation with vitamin D is intoxication. It is characterized by markedly elevated vitamin D concentrations (usually > 150 ng/ml), hypercalcemia, hypercalciuria and PTH suppression. Clinical manifestations are associated with hypercalcemia and include: weakness, fatigue, confusion, drowsiness, apathy, vomiting, constipation, polyuria, polydipsia, and abnormalities on the electrocardiogram (reduced Q-T interval) [33]. However, in this study, no participant had an intoxication of vitamin D. TLR-4 has other important roles that can be activated by mycobacterial lipopolysaccharides and lipotechoid acids, one of the two immune pathways that the inhibitor can activate through TLR-4. TLR-2 and TLR4 are risk factors for extrapulmonary TB [27].