Fig 2: Expression of Ch25h, Cyp7b1, Gpr183, and 25-OHC concentrations in lungs from uninfected (Uninf) and Mycobacterium tuberculosis (Mtb)-infected normoglycemic and dysglycemic mice. (A) The biosynthetic pathway of 25-OHC and 7α,25-OHC. Normoglycemic and dysglycemic mice were infected with ~100 colony-forming units aerosolized Mtb H37Rv, and mRNA expression of (B) Ch25h, Cyp7b1, and Hsd3b7 was measured by quantitative real-time polymerase chain reaction (qRT-PCR) at 3 and 8 weeks postinfection. (C) Concentrations of 25-OHC were measured in the lungs 8 weeks postinfection. Uninfected mice fed a high-fat diet or normal chow diet are represented by the dotted line. Expression of (D) blood and lung Gpr183 mRNA at 3 and 8 weeks postinfection was determined by qRT-PCR. Data are means ± standard error of the mean of n = 9–10 infected mice/group analyzed from 1 experiment. Circles represent normoglycemic mice and squares represent dysglycemic mice. Data analysis was performed by Mann-Whitney U test. *, P < .05; **, P < .01; ***; P < .001; ****, P < .0001. ns, not significant.

Fig 3: Schematic summary of the role of the oxysterols 25-OHC and 7α,25-OHC and GPR183 in Mycobacterium tuberculosis (Mtb)-infected normoglycemic and hyperglycemic mice. The enzymes Cholesterol 25-hydroxylase (CH25H) and Cytochrome P450 Family Subfamily B Member 1 (CYP7B1) are upregulated in the lung upon Mtb infection, resulting in increased production of the oxysterols 25-OHC and 7α,25-OHC, which in turn leads to effective recruitment of GPR183-expressing immune cells to the site of infection. CYP7B1 is predominantly expressed by macrophages in the center of tuberculosis (TB) granuloma surrounded by lymphocytes, and therefore the oxysterol/GPR183 axis may contribute to positioning these immune cells in TB granulomas. In high-fat diet fed hyperglycemic mice, this pathway is altered, with CYP7B1 mRNA and protein expression blunted, which leads to delayed recruitment of macrophages to the lung. (This figure was created with BioRender.).

Fig 4: Mice with dysglycemia have lower CYP7B1 at protein levels compared to normoglycemic mice. (A) Representative images of immunohistochemical labeling of CYP7B1 in lung sections from dysglycemic versus normoglycemic mice either uninfected or 3 weeks and 8 week after Mycobacterium tuberculosis (Mtb) infection. (B) Quantitative analysis of CYP7B1-immunolabeled areas as percentage of total lung section area. Data are means ± standard error of the mean of n = 9–10 infected mice/group analyzed from 1 experiment. Circles represent normoglycemic mice and squares represent dysglycemic mice. Data analysis was performed by Mann-Whitney U test. *, P < .05; **, P < .01; ****, P < .0001. HFD, high-fat diet; NCD, normal chow diet.

Fig 5: Influenza A virus (IAV) infection leads to upregulation of cholesterol 25-hydroxylase (CH25H) and cytochrome P450 family 7 subfamily member B1 (CYP7B1) expression in the lung and production of the oxysterols 25-hydroxycholesterol (25-OHC) and 7α,25-dihydroxycholesterol (7α,25-OHC). a) The biosynthetic pathway of 25-OHC and 7α,25-OHC. b) Experimental design: C57BL/6J mice were infected intranasally (i.n.) with 5500 plaque-forming units (PFU) of IAV. c) mRNA expression of Ch25h, Cyp7b1 and Hsd3b7 was measured by quantitative reverse transcription PCR at 3 days post infection (dpi) (D3) and 7 dpi (D7) normalised to Hprt. d) Quantitative analysis of CH25H, CYP7B1 and HSD3B7 protein labelling by immunohistochemistry (IHC). e) Representative IHC images of CH25H, CYP7B1 and HSD3B7 in lung sections of uninfected or IAV-infected mice. Scale bars: 100 µm (main) and 50 µm (inset). f) Concentrations of 25-OHC and 7α,25-OHC in the lungs (left) and bronchoalveolar lavage fluid (BALF) (right) at 3 dpi and 7 dpi. Data are presented as mean±sd of n=4 uninfected and n=6–10 infected mice per time point. ns: nonsignificant; U/I: mock infected. *: p<0.05; **: p<0.01.