Vitamin D regulates lipid metabolism in Mycobacterium tuberculosis infection

Introduction

Effective treatment of tuberculosis, a global infectious disease that kills almost two million people a year, can only be achieved with a protracted, multi-antibiotic regimen; the rapid emergence of antibiotic resistance has been eroding even these less-than-optimal treatment options (1). A hallmark of infection with Mycobacterium tuberculosis, the causative agent of tuberculosis, is the differentiation of infected macrophages into lipid-rich foam cells (2). These cells accumulate lipid droplets, lipid storage organelles (3) that are required for intracellular bacillary growth (4), presumably because they provide nutrients (2). Foam cells are located in the tuberculous granuloma at the interface between the caseous center and the lymphocytic cuff; there they contribute to an environment permissive for mycobacterial dormancy (5). Thus, the remodeling of macrophage lipid metabolism that occurs during M. tuberculosis infection is likely associated with infection outcome. Finding ways to prevent infection-induced dysregulation of macrophage lipid metabolism might help control infection. In the present report, we asked whether the protective role of vitamin D during infection with Mycobacterium tuberculosis (6-8) involves an effect on macrophage lipid metabolism in addition to its well known induction of macrophage antimicrobial functions (9), since vitamin D can have lipid-altering effects (for example, (10)), and the Vitamin D Receptor (VDR) interacts with lipid-sensing regulatory molecules affecting macrophage functions (11).

Materials and Methods

Results and Discussion

We used transcriptomics to explore associations among VDR signaling, tuberculosis, and lipid metabolism. When we analyzed the transcriptome of M. tuberculosis-infected THP-1 cells (15), we found that VDR was highly represented among DNA-binding proteins associated with upregulated genes (Fig. 1A). Re-analysis of human whole-blood transcriptome data from Berry et al (14) identified altered expression of VDR-bound genes associated with latent M. tuberculosis infection and BCG vaccination compared to controls, and with pulmonary tuberculosis compared to latent infection or treated disease (Fig. 1B). Thus, mycobacterial infection leaves a significant fingerprint of VDR-regulated gene expression in leukocytes, and this fingerprint is recapitulated in THP-1 cells. Next, we investigated the link between the VDR signal and the cellular functions associated with infection. Upregulated gene pathways in THP-1 cell infection included Metabolism of lipids and lipoproteins (and its subsets Cholesterol biosynthesis and Sphingolipid metabolism), in addition to well-studied immune responses such as interferon alpha/beta signaling and Toll-like receptor cascades (Supplemental Fig. 1). Further analysis revealed that VDR was the DNA binding protein most significantly associated with upregulation of Metabolism of lipids and lipoproteins (Fig. 1C and Supplemental Fig. 1). VDR was also significant within upregulated lipid metabolism pathways in some of the clinical comparisons in Fig. 1B, including pulmonary TB vs. controls (FDR = 5e-06) and BCG-vaccination vs. controls (FDR = 2.3e-03). Collectively, the transcriptomics data strongly implicate VDR in the regulation of lipid metabolism in M. tuberculosis-infected THP-1 cells and in human tuberculosis.

To assess the mechanistic implications of the transcriptomic results, we experimentally investigated the connection between macrophage lipid metabolism and VDR signaling in the context of M. tuberculosis infection. Since lipid droplets form in M. tuberculosis-infected macrophages (2), we measured lipid droplet content as a readout for altered lipid metabolism in infected THP-1 cells. We found that addition of vitamin D (1,25α-dihydroxyvitamin D3) to the infected cells prevented induction of lipid droplets (Fig. 2A) but had no effect on lipid droplets in uninfected cells (Supplemental Fig. 2A, left panel). Moreover, vitamin D did not concurrently reduce bacterial CFU (Fig. 2B), but did limit bacillary growth at later times post-infection (Supplemental Fig. 2B). Together, the data show that inhibition of lipid droplet induction by vitamin D was dependent on infection and was not merely secondary to reduced bacterial burden.

To explore the underlying mechanisms, we obtained transcriptome data from infected THP-1 cells with and without vitamin D treatment, and from corresponding controls. Analysis of lipid-annotated gene sets showed that in most cases vitamin D treatment did not alter the direction of the infection-induced changes (Fig. 3A). However, the gene set Lipid binding and its subset Phospholipid binding were significantly upregulated by infection alone but downregulated by vitamin D treatment of infected cells (Fig. 3A). This expression pattern mirrors the effect of infection and vitamin D treatment of infected cells on formation of lipid droplets (Fig. 2A). Further analysis of the downregulated Lipid binding genes for DNA-binding protein annotations identified a strong signal associated with PPARγ in vitamin D-treated, infected cell transcriptomes (Fig. 3B). This result agrees with a role for PPARγ in VDR inhibition of adipocyte differentiation (18).

The downregulation of PPARγ-bound genes in the response of infected cells to vitamin D led us to test relationships between PPARγ- and VDR-signaling. We found that macrophage infection with M. tuberculosis induced PPARγ expression and activity [the latter assessed as expression of the sentinel PPARγ target gene CD36], and that vitamin D abrogated this induction (Fig. 4A). We then tested whether PPARγ agonists reversed the vitamin D inhibition of lipid droplet induction. When M. tuberculosis-infected THP-1 cells were treated with vitamin D and with two PPARγ agonists, Rosiglitazone (a thiazolidinedione) and GW1929 (a nonthiazolidinedione), lipid droplet levels returned to levels comparable to those seen with M. tuberculosis infection alone (Fig. 4B) [lipid droplets were unaffected by agonists alone in uninfected (not shown) or infected cells (Fig. 4B)]. Moreover, the PPARγ agonists also reversed the antimicrobial effect of vitamin D while having very little (if any) effect on bacterial growth when tested alone (Fig. 4C). Thus, PPARγ agonism counteracts both anti-adipogenic and antimicrobial effects of vitamin D. These results agree with pro-adipogenic and bacterial-growth-promoting effects of PPARγ in M. tuberculosis-infected macrophages (Supplemental Fig. 2A,C and (19)). Taken together, our findings show that (i) VDR signaling decreases lipid droplet formation in M. tuberculosis-infected macrophages by limiting the adipogenic function of another nuclear hormone receptor protein, PPARγ, and (ii) the antimicrobial and antiadipogenic effects of VDR both involve reduction in PPARγ signaling.

In conclusion, integrated data from transcriptomic and mechanistic analyses provide evidence for a) a connection between VDR signaling and lipid metabolism in human tuberculosis, BCG vaccination, and macrophage response to M. tuberculosis infection in vitro, b) an antiadipogenic effect of vitamin D in M. tuberculosis-infected macrophages, and c) crosstalk between PPARγ and VDR in both M. tuberculosis-induced dysregulation of macrophage lipid metabolism and macrophage antimicrobial activity. The latter is consistent with the integration of VDR- and PPARγ-signaling pathways seen in other cell types and contexts (for example, (20)). Since VDR does not appear to regulate PPARG directly (21), the crosstalk might involve effects on interactions with the common heterodimerization partner, Retinoid X Receptor, which is required for DNA binding and transcriptional activity of either heterodimer (11), and/or on intracellular levels of the cognate lipid ligands required for transcriptional activity (11). The finding that VDR and PPARγ exert divergent effects on lipid metabolism and bacillary growth in M. tuberculosis-infected human macrophages points to opposing roles of the corresponding signaling pathways on macrophage activation state and permissivity for M. tuberculosis infection. While the data clearly indicate that inhibition of lipid droplet formation by vitamin D occurs in the absence of concurrent antimicrobial effects, mechanistic links might exist between antiadipogenic and antimicrobial functions of vitamin D. These remain to be elucidated.

The present findings have clinical relevance. For example, they point to a need for re-interpretation and de novo investigation of epidemiological links between tuberculosis risk, vitamin D levels, and metabolic abnormalities, including diseases such as diabetes. Moreover, identification of drug-targetable host factors involved in lipid metabolism might lead to treatments that enhance host resistance to M. tuberculosis infection and serve as adjuncts to antituberculosis chemotherapy.

Supplementary Material