Reduction of host cell mitochondrial activity as Mycobacterium leprae's strategy to evade host innate immunity

Abstract

Leprosy is a much-feared incapacitating infectious disease caused by Mycobacterium leprae or M lepromatosis, annually affecting roughly 200,000 people worldwide. During host-pathogen interaction, M leprae subverts the immune response, leading to development of disease. Throughout the last few decades, the impact of energy metabolism on the control of intracellular pathogens and leukocytic differentiation has become more evident. Mitochondria play a key role in regulating newly-discovered immune signaling pathways by controlling redox metabolism and the flow of energy besides activating inflammasome, xenophagy, and apoptosis. Likewise, this organelle, whose origin is probably an alphaproteobacterium, directly controls the intracellular pathogens attempting to invade its niche, a feature conquered at the expense of billions of years of coevolution. In the present review, we discuss the role of reduced host cell mitochondrial activity during M leprae infection and the consequential fates of M leprae and host innate immunity. Conceivably, inhibition of mitochondrial energy metabolism emerges as an overlooked and novel mechanism developed by M leprae to evade xenophagy and the host immune response.

Keywords: HIF-1α; LACC1; PKRN; cholesterol; free fatty acid and xenophagy; immunometabolism; inflammasome; leprosy; parkin.

Figure 1:

Contribution of cellular organelles to the field of medicine over the years and the involvement of mitochondria in Mycobacteria. (A) Timeline of original publications between January 1970 and October 30, 2020 obtained from the PubMed database by looking up the term “medicine” in combination with either “nucleus”, “peroxisom*”, “lysosom*”, “mitochondri*”, “endoplasmic reticulum”, or “golgi”. (B) Timeline of original publications between January 1960 and October 30, 2020 obtained from the PubMed database by researching the term “mitochondri*” in combination with “mycobacteri*”.

Figure 2.. During infection, M. leprae drastically reduces mitochondrial content and activity in axons, Schwann cells, and primary monocytes.

A) Transmission Electron Microscopy of human nerve biopsies from non-leprosy and leprosy patients showing swollen mitochondria (arrow heads) in the nerve. Leprosy patient nerves were compared to those of patients diagnosed with neuropathies not related to leprosy such as diabetes (non-leprosy). By comparing both tissues, it is possible to observe that axonal mitochondrial swelling is indicative of both a leprosy-specific cytopathologic condition as well as a reduction in axonal mitochondrial content (arrow). This last datum is more clearly demonstrated in (B), in which the number of mitochondria was determined per axonal area among 20 images of 8 patients, confirming that leprosy patients present a remarkable reduction in axonal mitochondrial content. C) Mitochondrial DNA reduction in the nerves of leprosy patients confirms these observations as a conserved cytopathologic feature, also produced in vitro, seen in (D), in which after 72h infection, a human Schwann cell culture also presents a drastic reduction in mitochondrial DNA. E) The monocytic mitochondrial membrane potential was also reduced after 48h infection, as determined by TMRM fluorescence intensity registered via fluorescence microscopy. Membrane potential was calculated by measuring the fluorescence intensity ratio between cells exposed to TMRM and TMRM plus CCCP (uncoupled) while the axonal area was identified by ImageJ. Primary human monocytes were obtained and cultured as previously described. All results are expressed as the mean ± SEM (standard error of the mean). Statistical significance was calculated by the nonparametric, two-tailed Mann-Whitney test in which: *** p<0.0001, **p<0.001 and * p <0.01. All analyses and experiments were performed as previously described. F) An in silico reanalysis of microarray data showing cholesterol synthesis upregulation (log2 fold change) in Schwann cells 48h after infection. Data were extracted from a microarray dataset (GSE35423) published elsewhere. Genes were categorized according to Reactome v. 74, except for LACC1 (FAMIN), which was included irrespectively. Data were analyzed as previously described. The false discovery rate (FDR) was controlled using the Benjamini and Hochberg method for the tested gene subset. Gene symbols are specified according to HGNC.

Figure 3.. The interplay between lipid metabolism and mitochondrial function during M. leprae infection.

After M. leprae binds to the host-cell surface receptors such as CD206 and TLR2/6, the bacilli are internalized, inducing the production and secretion of IGF-1, which, through IGF-1 receptor activation, increases glucose uptake via the GLUT1 receptor, promoting SREBP nuclear translocation. Inside the nucleus, SREBP upregulates the genes expression of the ones involved in lipogenesis, de novo lipid synthesis, cholesterol biosynthesis, and cholesterol uptake, ultimately leading to the intracellular lipid accumulation in the form of lipid droplets and free fatty acids. In an infected host cell, glucose is shunted to the pentoses pathway to generate NADPH for lipid synthesis. The accumulation of free fatty acids in the cytosol opens the mPTP, thereby reducing mitochondrial electron potential (ΔΨ). Citrate deviation to lipids reduces α-ketoglutarate synthesis, which, combined with the inhibition of the mitochondrial OCG transporter by cholesterol, drastically decreases α-ketoglutarate availability to PHDs, leading to impaired HIF-1α hydrolyzation. Once stabilized, HIF-1α accumulates in cytosol and invades the nucleus, initiating the gene transcription involved in burst glycolysis and limiting OXPHOS by, firstly, reducing mitogenesis and then promoting mitophagy. Key xenophagic enzymes like PKRN and LRRK2, and proteins TBK1, Rab, and ubiquitin are deviated to mitophagy, resulting in: i) reduced xenophagy efficiency; ii) reduced mROS formation, impairing inflammasomal activation, and iii) inhibited β-oxidation favoring lipid accumulation. Altogether, this cascade of events directly contributes to bacillus survival. Abbreviations: ACLY, ATP citrate lyase; HMGR, 3-hydroxy-3-methylglutaryl-CoA reductase; CD206, Cluster of Differentiation 206; FAMIN/LACC1 (fatty acid metabolism–immunity nexus); GLUT1, Glucose Transporter 1; HIF-1α, Hypoxia-inducible factor 1-alpha; IGF-1, insulin-like growth factor 1; IGF-1R, insulin-like growth factor 1 Receptor; LDLR, low density lipoprotein receptor; LDL-oxCho; Oxidized LDL-cholesterol; LDL-Cho, LDL-Cholesterol; LRRK2, Leucine-rich repeat kinase 2; PHDs, prolyl hydroxylase domain proteins; PKRN, parkin; PPARγ, peroxisome proliferator-activated receptor gamma; SREBP, sterol regulatory element-binding protein; mPTP, mitochondrial permeability transition pore; OCG, 2-oxoglutarate carrier; OXPHOS, oxidative phosphorylation; ΔΨm, mitochondrial membrane potential; mROS, mitochondrial reactive oxygen species; TLR2/6, Toll-like receptor 2/6.