Spatially heterogeneous lipid dysregulation in tuberculous meningitis

Abstract

Tuberculous (TB) meningitis is the deadliest form of extrapulmonary TB which disproportionately affects children and immunocompromised individuals. Studies in pulmonary TB have shown that Mycobacterium tuberculosis can alter host lipid metabolism to evade the immune system. Cholesterol lowering drugs (i.e., statins) reduce the risk of infection, making them a promising host-directed therapy in pulmonary TB. However, the effect of M. tuberculosis infection on the young or adult brain lipidome has not been studied. The brain is the second-most lipid-rich organ, after adipose tissue, with a temporally and spatially heterogeneous lipidome that changes from infancy to adulthood. The young, developing brain in children may be uniquely vulnerable to alterations in lipid composition and homeostasis, as perturbations in cholesterol metabolism can cause developmental disorders leading to intellectual disabilities. To begin to understand the alterations to the brain lipidome in pediatric TB meningitis, we utilized our previously published young rabbit model of TB meningitis and applied mass spectrometry (MS) techniques to elucidate spatial differences. We used matrix assisted laser desorption/ionization-MS imaging (MALDI-MSI) and complemented it with region-specific liquid chromatography (LC)-MS/MS developed to identify and quantify sterols and oxysterols difficult to identify by MALDI-MSI. MALDI-MSI revealed several sphingolipids, glycerolipids and glycerophospholipids that were downregulated in brain lesions. LC-MS/MS revealed the downregulation of cholesterol, several sterol intermediates along the cholesterol biosynthesis pathway and enzymatically produced oxysterols as a direct result of M. tuberculosis infection. However, oxysterols produced by oxidative stress were increased in brain lesions. Together, these results demonstrate significant spatially regulated brain lipidome dysregulation in pediatric TB meningitis.

Keywords: Brain lipidome; Cholesterol; Glycerophospholipid; MALDI-MSI; Oxysterol; Pediatric; Sphingolipid; Tuberculous meningitis.

Fig. 1.

Experimental design. (A) Experimental workflow. After three weeks post-injection, two cohorts of rabbits were utilized for matrix assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI; upper panel) or liquid chromatography (LC)-MS/MS (lower panel). (B) Similar regions of interest (ROI) were used for comparison in MALDI-MSI (left) and LC-MS/MS (right). Other abbreviations: DHB, 2,5-dihydroxybenzoic acid; GM, grey matter; PBS, phosphate buffered saline; WM, white matter.

Fig. 2.

MALDI-MSI of identified lipids. (A) H&E staining of healthy control (top) and M. tuberculosis-infected brain (bottom). (B-G) MALDI images of identified glycerophospholipids, (H-J) identified sphingolipids, and (K-L) identified glycerolipids in healthy control brain (top) and M. tuberculosis-infected brain (bottom). White dotted outline in H&E and red dotted outline in MALDI image correspond to brain lesion. Abbreviations: DG, diradylglycerolipid; HexCer, hexosylceramide; PA, glycerophosphate; PC, glycerophosphocholine; PE, glycerophosphoethanolamine; PG, glycerophosphoglycerol; TG, triadylglycerolipid. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 3.

Sterols are decreased in M. tuberculosis-infected brain. (A) Post-squalene cholesterol biosynthesis pathway demonstrating intermediates measured in LC-MS/MS analysis. (B–C) After three weeks post-injection, the brains of healthy (injected with PBS; grey) and M. tuberculosis-infected (injected with M. tuberculosis H37Rv; blue colors) rabbits were microdissected. Darkest blue (lesion) corresponds to the brain lesion and lighter blue denotes areas farther from brain lesion center (with lightest blue in contralateral, unaffected hemisphere of M. tuberculosis-infected brain). (B) Bloch pathway sterol intermediates. (C) Kandutsch-Russell pathway sterol intermediates. * P < 0.05, ** P < 0.01, *** P < 0.001. Data are represented as median ± interquartile range. Statistical comparisons were made using a Kruskal-Wallis test. Other abbreviations: DHCR7, 7-dehydrocholesterol reductase; DHCR24, 24-dehydrocholesterol reductase or sterol-Δ²⁴-reductase; EBP, emopamil binding protein; LSS, lanosterol synthase; SC5D, lathosterol 5-desaturase. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 4.

Oxysterols are dysregulated in M. tuberculosis-infected brain. (A) Schematic representation of oxysterols produced by enzymatic reactions (B) and reactive oxygen species (ROS, C). (B–C) After three weeks post-injection, the brains of healthy (injected with PBS; grey) and M. tuberculosis-infected (injected with M. tuberculosis H37Rv; blue colors) rabbits were microdissected. Darkest blue (lesion) corresponds to the brain lesion and lighter blue denotes areas farther from brain lesion center (with lightest blue in contralateral, unaffected hemisphere of M. tuberculosis-infected brain). (B) Oxysterols produced by enzymatic reactions are decreased in M. tuberculosis-infected brains. (C) Oxysterols produced by ROS are increased in M. tuberculosis-infected brains. * P < 0.05, ** P < 0.01, *** P < 0.001. Data are represented as median ± interquartile range. Statistical comparisons were made using a Kruskal-Wallis test. Other abbreviations: CH25H, cholesterol-25-hydroxylase; CYP46, cholesterol-24-hydroxylase; CYP27, human sterol-hydroxylase; ROS, reactive oxygen species. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)