A hallmark of LL is the accumulation of Virchow's foamy macrophages. However, the origin and nature of these lipids, as well as their function and contribution to leprosy disease, remain unclear. We herein show that macrophages present in LL dermal lesions are highly positive for ADRP, suggesting that their foamy aspect is at least in part derived from LD (also known as lipid bodies) accumulation induced during ML infection. Indeed, the capacity of ML to induce LD formation was confirmed in vivo via an experimental model of mouse pleurisy and in in vitro studies with human peripheral monocytes and murine peritoneal macrophages. Furthermore, infected cells were shown to propagate LD induction to uninfected, neighboring cells by generating a paracrine signal, for which TLR2 and TLR6 were demonstrated to be essential. However, TLR2 and TLR6 deletions affected LD formation in bacterium-bearing cells only partially, suggesting the involvement of alternative receptors of the innate immune response besides TLR2/6 for ML recognition by macrophages. Finally, a direct correlation between LD formation and PGE(2) production was observed, indicating that ML-induced LDs constitute intracellular sites for eicosanoid synthesis and that foamy cells may be critical regulators in subverting the immune response in leprosy.

SummaryThe predilection of Mycobacterium leprae (ML) for Schwann cells (SCs) leads to peripheral neuropathy, a major concern in leprosy. Highly infected SCs in lepromatous leprosy nerves show a foamy, lipid-laden appearance; but the origin and nature of these lipids, as well as their role in leprosy, have remained unclear. The data presented show that ML has a pronounced effect on host-cell lipid homeostasis through regulation of lipid droplet (lipid bodies, LD) biogenesis and intracellular distribution. Electron microscopy and immunohistochemical analysis of lepromatous leprosy nerves for adipose differentiation-related protein expression, a classical LD marker, revealed accumulating LDs in close association to ML in infected SCs. The capacity of ML to induce LD formation was confirmed in in vitro studies with human SCs.Moreover, via confocal and live-cell analysis, it was found that LDs are promptly recruited to bacterial phagosomes and that this process depends on cytoskeletal reorganization and PI3K signalling. ML-induced LD biogenesis and recruitment were found to be independent of TLR2 bacterial sensing. Notably, LD recruitment impairment by cytoskeleton drugs decreased intracellular bacterial survival. Altogether, our data revealed SC lipid accumulation in ML-containing phagosomes, which may represent a fundamental aspect of bacterial pathogenesis in the nerve.

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The mechanisms responsible for nerve injury in leprosy need further elucidation. We recently demonstrated that the foamy phenotype of Mycobacterium leprae-infected Schwann cells (SCs) observed in nerves of multibacillary patients results from the capacity of M. leprae to induce and recruit lipid droplets (LDs; also known as lipid bodies) to bacterial-containing phagosomes. In this study, we analyzed the parameters that govern LD biogenesis by M. leprae in SCs and how this contributes to the innate immune response elicited by M. leprae. Our observations indicated that LD formation requires the uptake of live bacteria and depends on host cell cytoskeleton rearrangement and vesicular trafficking. TLR6 deletion, but not TLR2, completely abolished the induction of LDs by M. leprae, as well as inhibited the bacterial uptake in SCs. M. leprae-induced LD biogenesis correlated with increased PGE2 and IL-10 secretion, as well as reduced IL-12 and NO production in M. leprae-infected SCs. Analysis of nerves from lepromatous leprosy patients showed colocalization of M. leprae, LDs, and cyclooxygenase-2 in SCs, indicating that LDs are sites for PGE2 synthesis in vivo. LD biogenesis Inhibition by the fatty acid synthase inhibitor C-75 abolished the effect of M. leprae on SC production of immunoinflammatory mediators and enhanced the mycobacterial-killing ability of SCs. Altogether, our data indicated a critical role for TLR6-dependent signaling in M. leprae–SC interactions, favoring phagocytosis and subsequent signaling for induction of LD biogenesis in infected cells. Moreover, our observations reinforced the role of LDs favoring mycobacterial survival and persistence in the nerve. These findings give further support to a critical role for LDs in M. leprae pathogenesis in the nerve.

We recently showed that Mycobacterium leprae (ML) is able to induce lipid droplet formation in infected macrophages. We herein confirm that cholesterol (Cho) is one of the host lipid molecules that accumulate in ML-infected macrophages and investigate the effects of ML on cellular Cho metabolism responsible for its accumulation. The expression levels of LDL receptors (LDL-R, CD36, SRA-1, SR-B1, and LRP-1) and enzymes involved in Cho biosynthesis were investigated by qRT-PCR and/or Western blot and shown to be higher in lepromatous leprosy (LL) tissues when compared to borderline tuberculoid (BT) lesions. Moreover, higher levels of the active form of the sterol regulatory element-binding protein (SREBP) transcriptional factors, key regulators of the biosynthesis and uptake of cellular Cho, were found in LL skin biopsies. Functional in vitro assays confirmed the higher capacity of ML-infected macrophages to synthesize Cho and sequester exogenous LDL-Cho. Notably, Cho colocalized to ML-containing phagosomes, and Cho metabolism impairment, through either de novo synthesis inhibition by statins or depletion of exogenous Cho, decreased intracellular bacterial survival. These findings highlight the importance of metabolic integration between the host and bacteria to leprosy pathophysiology, opening new avenues for novel therapeutic strategies to leprosy.