Over the years, various anatomical and cellular environments conducive to Mtb infection have been identified, particularly concerning its latency (Cosma et al., 2003; Bussi and Gutierrez, 2019). Pulmonary TB typically presents with classical symptoms such as persistent coughing and mucus production due to lung involvement (Luies and du Preez, 2020). While systemic manifestations like weight loss, fatigue, and loss of appetite are commonly linked to TB progression, the underlying factors driving these metabolic derangements remain unexplored. Our study sheds light on hepatocytes as a previously unrecognized site for Mtb survival and replication, thereby extrapolating the current understanding of TB beyond its pulmonary focus. By combining in vitro, ex vivo, in vivo, and clinical data, we demonstrate that Mtb not only persists within hepatocytes but also induces important metabolic reprogramming, with significant implications for disease progression, symptomatic manifestations, and drug tolerance.
A central finding of our work is the Mtb-mediated activation of peroxisome proliferator-activated receptor gamma (PPARγ) in hepatocytes. PPARγ activation leads to the accumulation of cholesterol esters (CE 16:0, 18:0, 18:1), diacylglycerols (DAGs 36:1, 36:2, 34:1), and triacylglycerols (TAGs 18:1/36:2, 18:0/36:2, 18:0/36:1). These lipid pools contribute to the formation of lipid droplets, which colocalize with intracellular Mtb, serving as a nutrient reservoir that facilitates bacterial persistence and proliferation. The upregulation of key TAG biosynthesis genes such as Tgs1, Tgs4, and Rv1760 in Mtb residing within hepatocytes underscores a transcriptional rewiring within the pathogen to assimilate lipids as a source of their nutrients. Our pharmacological studies using PPARγ agonist and antagonist demonstrated that modulation of PPARγ directly impacts Mtb burden in hepatocytes, confirming the pivotal role of PPARγ in Mtb survival in hepatocytes. The lipid remodeling induced by Mtb infection in hepatocytes is recapitulated in the murine aerosol model, where an increased number of lipid droplets was observed at week 8, accompanied by localized accumulation of immune cells and granuloma-like structures. These findings are consistent with previous reports in macrophages where PPARγ activation enhances lipid biosynthesis, regulates immune responses, and impedes host cell apoptosis (Arnett et al., 2018; Rajaram et al., 2010). Recent findings have further underscored heightened levels of PPARγ in peripheral blood mononuclear cells (PBMCs) from TB patients with elevated cortisol levels and increased disease severity (Díaz et al., 2023). However, hepatocytes are uniquely positioned in systemic lipid regulation, engaging in de novo fatty acid synthesis, TAG synthesis, β-oxidation, and lipoprotein metabolism (Zhang et al., 2022; Bechmann et al., 2012). Mtb-mediated perturbation of these vital processes can lead to a sequela of metabolic disorders such as non-alcoholic fatty liver disease (NAFLD), dyslipidemia, and insulin resistance. In macrophages, heightened intracellular lipid levels have been observed to impede autophagy and the acidification of phagolysosomes, both crucial for bacterial eradication (Lovewell et al., 2016). Despite these parallels, comparative transcriptomic analyses of infected HepG2 cells with THP1 cells highlight differences in pathways such as vacuolar and vesicular transport, xenobiotic metabolism, macroautophagy, and cellular respiration. Furthermore, hepatocytes secrete hepatokines, a class of proteins serving as signaling molecules, with diverse roles in metabolism, inflammation, and energy homeostasis, thereby influencing the host-pathogen interplay (Jensen-Cody and Potthoff, 2021). Intriguingly, a recent investigation demonstrated that hepatic PPARγ activation induces the expression of growth differentiation factor 15 (GDF-15), a crucial regulator of weight loss observed in ketogenic diets (Lu et al., 2024).
Additionally, we suggest that Mtb infection of hepatocytes creates a drug-tolerant environment in the liver due to activation of DMEs, many of which are earlier shown to metabolize the two frontline drugs, isoniazid and rifampicin. Moreover, lipid accumulation in the liver might also indirectly alter the levels of drug metabolizing enzymes as reported in previous studies (Fisher et al., 2009; Rey-Bedon et al., 2022). Upregulation of transcripts is not only observed in infected cells in vitro, but also in the livers of mice eight weeks after Mtb infection. Particularly noteworthy is the upregulation of NAT-2, which controls the rate-limiting step of acetylating isoniazid into acetylisoniazid. This metabolite is further processed into acetylhydrazine and isonicotinic acid, ultimately reducing the drug’s effectiveness against Mtb. Similarly, key rifampicin-metabolizing esterase genes such as Ces1, Ces2, Aadac, and transporter Slco1b2 exhibit upregulation, potentially influencing the drug’s distribution and metabolism in the body. The activation of DMEs can significantly modify the pharmacokinetics and pharmacodynamics of anti-TB medications, resulting in suboptimal drug concentrations and the emergence of drug-resistant strains (Dookie et al., 2018).
Pulmonary TB can lead to miliary through hematogenous dissemination, where Mtb spreads from the infected lungs into blood vessels either from a primary lung focus, reactivated TB, or caseous necrosis. Once in blood vessels, the bacteria seed multiple organs, forming tiny granulomas, characteristic of miliary TB. The liver becomes involved either through direct hematogenous spread or extension from nearby infected lymph nodes, leading to hepatic TB, which presents with granulomas and liver dysfunction. This systemic spread underscore the severity of untreated pulmonary TB and the need for early intervention (Shiloh, 2016; Coleman et al., 2022; Wei et al., 2024; McMullan and Lewis, 2017). Our murine and guinea pig aerosol infection models demonstrated progressive liver involvement starting at week 4, with the presence of lipid-laden hepatocytes, localized immune infiltrates, and granuloma-like structures. Importantly, liver biopsy samples from TB patients revealed ectopic granulomas and Mtb antigen (Ag85B) localization within hepatocytes, highlighting the clinical relevance of our findings. While previous research has explored liver infection by Bacillus Calmette-Guérin (BCG) using an intravenous model in mice, the bacilli were predominantly found within tissue-resident macrophages or Kupffer cells at 15 min and 2 days post-infection (Seiler et al., 2001). However, our study consistently identified the presence of Mtb within hepatocytes in a murine aerosol infection model after the fourth week. Similarly, in a guinea pig aerosol infection model, liver infection was evident, marked by distinct granulomas by week 4. Furthermore, analysis of human biopsy liver samples from pulmonary TB patients revealed ectopic granuloma-like structures within the liver hepatocytes, with the presence of Mtb-specific Ag85B signals within hepatocytes. Together, these findings underscore hepatocytes as a novel niche for Mtb persistence, shedding new light on the pathogenesis of TB. Interestingly, in infected armadillos, M. leprae is shown to infect hepatocytes and hijack liver homeostatic, regeneration pathways to promote de novo organogenesis (Hess et al., 2022).
Although animal models offer valuable insights into Mtb infection and TB pathology, they fall short of fully replicating the complexities of human TB. Several recent studies in COVID-19 patients have shown that hepatocytes get infected with SARs-CoV-2, thereby increasing gluconeogenesis and hyperglycemia (Barreto et al., 2023; Mercado-Gómez et al., 2022). With compelling reports associating hepatic steatosis with the onset of type 2 diabetes mellitus (T2DM), we speculate that TB-induced perturbations in lipid metabolism might predispose chronic TB patients to T2DM and vice versa (Hazlehurst et al., 2016; Dharmalingam and Yamasandhi, 2018). Through our investigation, we propose that future studies in human TB patients might scrutinize this metabolically rich hepatocyte niche to understand multiple organ-wide derangements in TB pathogenesis.