Cholesterol can exist in the form of free cholesterol, but it is more commonly transported to tissues throughout the body, including macrophages, in the form of low-density lipoprotein (LDL). Macrophages uptake cholesterol and its lipoproteins through a family of low-density lipoprotein receptor (LDLR) proteins expressed on cell membranes. Low-density lipoprotein receptor-related protein 2 (Lrp2), a 600 kDa giant membrane glycoprotein, belongs to the LDLR protein family and is primarily responsible for mediating the uptake of intracellular cholesterol. Lrp2 expression was found to be remarkably increased in macrophages infected with H37Rv, and it is positively correlated with the accumulation of intracellular cholesterol (7). Cluster of differentiation 36 (CD36), a transmembrane glycoprotein belonging to the LDLR protein family, forms a channel through the cell membrane and is responsible for mediating the absorption of lipid molecules. Tuberculous pleural effusion has been shown to induce massive accumulation of cholesterol in macrophages and markedly enriches the fluorescence intensity of CD36 on the cell membrane (25). Further evidence from a guinea pig model of Mtb infection confirms the accumulation of oxidized low-density lipoprotein (OxLDL) in extrapulmonary granulomas and pulmonary macrophages of TB guinea pigs. Moreover, macrophages increase the surface expression of scavenger receptor CD36 and lectin-like oxidized low-density lipoprotein receptor-1 (LOX1), promoting the uptake of cholesterol and LDL (26). LOX1 is a membrane receptor protein belonging to the C-type lectin family involved in the absorption and transport of LDL. A study was conducted to analyze granuloma samples from human TB. Transcriptomic analysis revealed a significant upregulation of genes involved in cholesterol and other lipid synthesis, and immunohistochemistry confirmed the high expression of these lipid synthesis proteins in human TB granulomas. Thin-layer chromatography and mass spectrometry analyses revealed that granulomas and foam cells were rich in cholesterol, cholesterol esters, and triglycerides, with LDL being a probable major source of these lipids (9).

In summary, cholesterol is primarily taken up by macrophages in the form of LDL in the body. Macrophages uptake cholesterol and LDL through the expression of LDLR family proteins, such as Lrp2, LOX1, and CD36. Although animal experiments and human samples provide some evidence, further clinical research is needed to validate these findings, especially in observing the accumulation of cholesterol and LDL and the expression changes of receptors in the human body during Mtb infection. Besides, other potential receptors involved in the uptake of cholesterol and LDL, as well as their roles in the development of the disease, can be explored to provide new therapeutic strategies for the prevention and treatment of TB ( Figure 2 ).

Multiple transcriptomic studies have revealed that Mtb infection leads to upregulation of cholesterol synthesis-related enzyme gene expression in macrophages, including acetoacetyl-coenzyme A synthetase (AACS), 3-hydroxy-3-methylglutaryl- coenzyme A synthase 1 (HMGCS1), 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR), mevalonate diphosphate decarboxylase (MVD), and 3β-hydroxysterol Δ24-reductase (DHCR24) (7, 27). AACS is a cytoplasmic enzyme involved in fatty acid biosynthesis that catalyzes the coupling of acetyl-coenzyme A (CoA) to produce acetoacetyl-CoA, which serves as the starting substrate for cholesterol synthesis (28). Therefore, the promotion of AACS gene expression by Mtb may contribute to de novo cholesterol synthesis. HMGCS1 is a key enzyme in the mevalonate pathway of cholesterol biosynthesis, catalyzing the conversion of acetoacetyl-CoA to 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA), which is further reduced to mevalonic acid by HMGCR, a rate-limiting enzyme in cholesterol synthesis that regulates cholesterol production (29). HMGCR and HMGCS1 play pivotal roles in macrophage function. According to previous research, HMGCR knockout was found to significantly inhibit the migration, proliferation, and survival of macrophages. However, another study found that increased expression of HMGCR induces excessive cholesterol synthesis, resulting in decreased membrane fluidity and suppressed phagocytic activity of macrophages (30, 31). When HMGCS1 activity is excessively elevated, it can cause the accumulation of hydroxymethylglutaryl-CoA in macrophages, which result in cell necrosis. Necrosis is a unique form of cell death characterized by cell membrane rupture, cytoplasmic leakage, and organelle damage. In macrophages, cell death induced by HMGCS1 via necrosis pathway exerts irreversible effects on the physiological function of macrophages, thereby impacting the normal immune system function (32). Furthermore, HMGCR and HMGCS1 are key genes in promoting macrophage foam cell formation. Increased activity of HMGCR and HMGCS1 contributes to excessive activation of the cholesterol synthesis pathway, which in turn leads to macrophage lipid droplet formation (33). Therefore, Mtb-induced overexpression of HMGCR and HMGCS1 may inhibit phagocytic activity of macrophages, promote cholesterol accumulation and foam cell formation, and even cause macrophage death.

MVD catalyzes the conversion of mevalonate diphosphate to isopentenyl diphosphate, which is an early step in cholesterol biosynthesis (34). MVD and the mevalonate pathway are crucial for macrophage survival and play an important role in the synthesis and release of inflammatory mediators, contributing to pro-inflammatory responses of macrophages (35). DHCR24, also known as 3β-hydroxysterol Δ24-reductase, is a human flavin adenine dinucleotide (FAD)-dependent oxidoreductase that catalyzes the final step of cholesterol synthesis, converting desmosterol to cholesterol (36). In addition to HMGCR and HMGCS1, DHCR24 is also a key gene in promoting macrophage foam cell formation and possesses antioxidant functions that effectively inhibit oxidative stress (33, 37). Consequently, Mtb infection of macrophages may elevate immune inflammation levels, inhibit oxidative stress, and induce cholesterol accumulation as well as foam cell formation by upregulating MVD and DHCR24 expression. Despite some progress has been made in current studies, most of them have been conducted using in vitro cell culture models, and these findings need to be further validated in vivo. Animal models or clinical samples can provide a better understanding of the impact of Mtb infection on cholesterol anabolism in macrophages ( Figure 2 ).

Accumulation of cholesterol esters (CE) stored in cytoplasmic lipid droplets is a key feature of foam cells, which are central to the development of atherosclerosis and tuberculosis (38). When macrophages are infected with Mtb, not only does it promote cholesterol accumulation, but it also promotes the conversion of cholesterol to cholesterol esters within macrophages, thereby facilitating the formation of lipid droplets and foam cells. Animal pathology studies have shown that macrophages isolated from granulomatous lungs of tuberculosis mice contain large amounts of cholesterol esters, whereas the ester content in resident peritoneal macrophages of normal mice is very low (39). Metabolomics studies of human lung tissue have revealed that large amounts of cholesterol, cholesteryl esters, and triglycerides accumulate in the granulomas of patients with TB. All of these studies provide ample evidence that cholesterol in Mtb-infected macrophages is converted to cholesterol esters for storage. Further mechanistic exploration has revealed that Mtb, especially its cell wall surface lipid trehalose dimycolate, upregulates the expression of adipose differentiation-related protein (ADRP), thereby promoting cholesterol esterification and lipid droplet formation (9). Acyl-CoA: cholesterol acyltransferase (ACAT) is a key enzyme catalyzing cholesterol esterification in macrophages, and it has been found that tuberculosis pleural effusion (TB-PE) induces the expression of ACAT, promoting cholesterol esterification in macrophages. When ACAT is inhibited, the levels of cholesterol esters and triglycerides in TB-PE-treated macrophages are significantly reduced (25). Accordingly, it can be concluded from the existing studies that Mtb and its cell wall surface lipid virulence factors enhance the conversion of cholesterol to cholesterol esters in macrophages by upregulating the expression of ACAT and ADFP, thereby facilitating the formation of lipid droplets and foam cells. Currently, there are still some research gaps and directions that need further exploration. Firstly, the detailed mechanisms underlying the accumulation of cholesterol esters in Mtb-infected macrophages have not been fully understood, including how Mtb regulates the expression and activity of ACAT. In the future, more animal models and patient samples should be utilized to further explore the mechanisms of the accumulation of cholesterol esters in Mtb-infected macrophages ( Figure 2 ).

Only non-esterified or free cholesterol (FC) can efflux from the cell to the extracellular space, thus the excessively accumulated cholesterol esters need to be effectively degraded by cells to prevent pathological progression. Cholesterol esters are hydrolyzed by cholesterol esterase within the cells, resulting in the formation of free cholesterol and fatty acids (38). Cholesterol ester hydrolysis contributes to the reduction of lipid load in macrophages, diminishing inflammation and related diseases, and is crucial for macrophage function (40). Inhibition of cholesterol ester hydrolysis leads to dysregulation of cholesterol content in microdomains, which prolongs the activation of immune cell signaling pathways and promotes immune cell proliferation, infiltration, and foam cell formation (41). A study using transcriptomic analysis and in vitro validation demonstrated that macrophage receptor with collagenous structure (MARCO) and collectin sub-family member 12 (COLEC12), which are involved in phagocytosis, as well as carboxylesterase 1 (CES1), the cholesterol ester hydrolase, were downregulated in alveolar macrophages (AMs) from TB patients compared to controls, indicating impaired cholesterol ester degradation and reduced free cholesterol, leading to excessive accumulation of cholesterol esters and adverse progression of pulmonary TB (8). Further research can further validate the key role of CES1 in TB progression through protein detection and functional experiments, and explore the specific mechanisms by which Mtb regulates the downregulation of macrophage CES1 using cell and animal models ( Figure 2 ).

In addition to cholesterol esters, cholesterol can also be converted into oxidized cholesterol forms in macrophages, with 25-hydroxycholesterol(25HC)and 1,25 dihydroxyvitamin D (1,25(OH)₂D) being two typical oxidized cholesterol derivatives. Mtb infection induces macrophage activation, and activated macrophages are a rich source of oxidized cholesterol (42–45). In macrophage models of Mtb infection, oxidized cholesterol typically exerts an antimicrobial effect. Studies have shown that the production of 25HC is significantly increased in activated macrophages, which in turn inhibits cholesterol biosynthesis and accumulation, while cholesterol accumulation promotes the growth of Mtb (21, 46). An increase in 25HC concentrations was shown in lungs of mice infected with Mtb (24). Furthermore, 7α,25-dihydroxycholesterol can increase Mtb phagocytosis and reduces intracellular Mtb growth in primary human monocytes (47). Multiple studies have demonstrated the potent anti-Mtb infection activity of 1,25(OH)₂D (48). 1,25(OH)₂D inhibits Mtb growth by suppressing cholesterol and lipid droplet formation (49). Additionally, 1,25(OH)₂D promotes the production of antimicrobial peptides and the maturation of phagolysosomes in macrophages, thereby enhancing an effective cellular immune response against Mtb (13, 50). In conclusion, Mtb infection may promote the conversion of cholesterol into oxidized cholesterol in macrophages, and oxidized cholesterol plays a promoting role in macrophage-mediated anti-Mtb infection. Future research needs to explore the underlying mechanisms and target points by which Mtb infection promotes the production of oxidized cholesterol, as well as develop new therapeutic strategies for anti-Mtb infection based on oxidized cholesterol ( Figure 2 ).

Even though cholesterol esters can be degraded to cholesterol, macrophages themselves are unable to degrade cholesterol. Therefore, when cholesterol accumulates excessively in macrophages, it ultimately needs to be eliminated from the cells through efflux. ATP-binding cassette (ABC) transporters are a class of transmembrane proteins that utilize ATP hydrolysis to mediate cholesterol efflux, including genes such as ABCA1, ABCG1, and ABCA5 (51). In vitro studies have shown that infection of macrophages with the Mtb strain H37Ra inhibits the expression of ABCG1 and promotes the formation of lipid bodies and foam cells. In contrast, overexpression of ABCG1 in macrophages through lentiviral transduction can significantly reverse foam cell formation (52). Researchers have constructed a macrophage model infected with Bacille Calmette-Guérin (BCG) and found that BCG infection inhibit the expression of macrophage ABCG1, ABCA1, and ABCA5, promoting intracellular cholesterol accumulation and further inducing macrophage autophagy to suppress BCG infection (53). Further animal experiments have provided more compelling evidence, as it was found that the expression of ABCA1 and ABCG1, which are involved in cholesterol efflux, was significantly down-regulated in peritoneal macrophages and lung tissue sections of mice infected with H37Ra (7). In conclusion, Mtb infection has a direct regulatory effect on cholesterol efflux in macrophages by downregulating the expression of ABC transporters, inhibiting cholesterol efflux, and promoting cholesterol accumulation and foam cell formation. Current research mainly focuses on in vitro experiments and animal experiments, and further human-related studies, including clinical research and proteomic studies, are needed to assess the impact of Mtb infection on ABC transporters and cholesterol metabolism, as well as to explore the relevant molecular mechanisms. Additionally, while overexpression of ABC transporters can reverse foam cell formation, further studies are needed to investigate their potential therapeutic efficacy in the treatment of TB ( Figure 2 ).