Fig 1.
Mtb infects human and mouse adipocytes in vitro and expresses stress-related genes.
(A-C) Immunofluorescence staining and quantification of extra- and intracellular Mtb-GFP at multiplicity of infection (MOI) of 20 after 24 h in (A) SGBS human cell strain; (B) primary adipocytes from human subcutaneous fat; (C) 3T3-L1 mouse cell line. Blue: DAPI. Original magnification 1,000×. Data are representative of two independent experiments (triplicates). (D-F) log10 Mtb CFUs at indicated time points post-infection with MOI = 5 in the presence or absence of amikacin in (D) SGBS human cell strain, (E) 3T3-L1 mouse cell line, (F) primary adipocytes from human subcutaneous fat; data are representative of two to four independent experiments. (G-H) Expression of Mtb genes dosR, hspX and lat in (G) SGBS human cell strain and (H) 3T3-L1 mouse cell line. Cells were infected with MOI = 5. Steady state transcript levels were normalized to Mtb sigA expression. Means of duplicates from four to six independent experiments are shown. Abbreviations: E, extracellular; I, intracelular; SGBS, Simpson-Golabi-Behmel syndrome.
Fig 2.
Mtb expresses stress-related genes in perigonadal fat at different time points post aerosol-infection of mice.
(A) log10 Mtb CFUs in spleen, lung and perigonadal fat at different time points post aerosol-infection (200 CFUs). Data are representative of four independent experiments (medians). (B) log10 Mtb CFUs in spleen, lung and perigonadal fat at day 28 post aerosol-infection with 50 or 200 CFUs. Data are representative of two independent experiments (median); ** p<0.01 (Mann–Whitney test). (C) log10 Mtb CFUs in spleen, lung and AD and SVF fractions of perigonadal fat at different time points after aerosol infection. Data representative of two independent experiments (medians). (D) Expression of Mtb genes dosR, hspX and lat in the AD and SVF fractions of perigonadal fat at day 28 post aerosol-infection. Results of four independent experiments pooled (medians); *p<0.05 (Mann–Whitney test). (E) PCR specific for Mtb (IS 6110) in perigonadal fat at day 14 post aerosol-infection. Each lane represents a different mouse. Data representative of two independent experiments. (F) Immunofluorescence staining of perigonadal fat of mice infected i.v. with Mtb-GFP at 48 h after infection (arrows, green). Blue: DNA-intercalating dye Draq5. Data representative of two independent experiments. (G) log10 Mtb CFUs in spleen, lung, perigonadal (P) and subcutaneous (SC) fat from uninfected mice that received a subcutaneous injection of pergonadal fat from mice previously infected with 5x106 CFUs of Mtb. Organs were collected 14 days after the transfer of perigonadal fat. Data representative of two independent experiments (medians). Abbreviations: AD, adipose fraction; P, perigonadal; SC, subcutaneous; SVF, stromal vascular fraction.
Fig 3.
Leukocytes infiltrate perigonadal fat post aerosol-infection with Mtb.
(A) Immunohistochemical staining for leukocyte infiltration at day 28 post aerosol infection. Data representative of two independent experiments. (B) Immunofluorescence staining of F4/80+ Arg1+ or iNOS+ cells at day 28 post aerosol- infection. Blue: DAPI. Data representative of two independent experiments. (C) Number of leukocytes per hpf at day 28 post aerosol-infection. Results of two independent experiments pooled (medians); *p<0.05 (Mann–Whitney test). Abbreviations: HE, hematoxilin and eosin staining; hpf, high power field.
Fig 4.
Differential gene expression in perigonadal fat post aerosol-infection with Mtb.
(A-B) Changes in gene expression in perigonadal fat at days 14 and 28 post-infection. Grey dots represent non-significant mean log2 fold increments from uninfected mice (six samples per group). Red dots represent significant mean log2 fold increments from uninfected mice (six samples per group). (A) Cell type-associated changes in gene expression. (B) Pathway-associated changes in gene expression. Data representative of two independent experiments. Abbreviations: d, day; F, female; M, male.
Fig 5.
Genes associated with T cells, cytokines and chemokines are differentially upregulated in perigonadal and subcoutaneous fat post aerosol-infection with Mtb.
(A) Expression of Cd8a, Cd3d, Cd4, Ifng, Il4, Ccl5, Ccr7, Cxcl9, Cxcr3 (left panel) and Tbx21, Rorc and Gata3 (right panel) in perigonadal fat, lung and subcutaneous (SC) fat, as measured with quantitative PCR at day 28 post infection. (B) Expression of genes as in (A) in AD or SVF fractions of perigonadal fat at day 28 post infection. Gene expression is relative to the lower values detected and each group is compared to their own uninfected controls. Data are representative of two to three independent experiments (means); *p<0.05 and **p<0.01 (Student’s t-test). Abbreviations: AD, adipose fraction; SC, subcutaneous; SVF, stromal vascular fraction.
Fig 6.
Mtb-specific CD8+ T cells are present in perigonadal fat post aerosol-infection.
Numbers of CD4+, CD8+ and CD8+ CD44+ TB10.4+ (Mtb-specific) populations in SVF of perigonadal fat (upper panel) and lung (lower panel) at day 28 post infection. Data are representative of two independent experiments (means); *p<0.05, **p<0.01 and ****p<0.0001 (Student´s t-test). Abbreviations: SVF, stromal vascular fraction.
Fig 7.
IFN-γ-producing NK cells are present in perigonadal fat post aerosol- infection.
Numbers of CD4+, CD8+ and NK IFN-γ or IL-4-producing cells at day 28 post infection in SVF of perigonadal fat. Data are representative of two independent experiments (means); *p<0.05 (Student´s t-test). Abbreviations: SVF, stromal vascular fraction.
Fig 8.
Mtb aerosol infection modulates gene expression of Mtb-specific CD8+ T cells and NK cells in perigonadal fat.
Relative gene expression in CD8+ T cells from uninfected mice, Mtb-specific CD8+ T cells (CD8+ CD44+ TB10.4+) and NK cells sorted from (A) perigonadal fat or (B) lung at day 28 post-infection. Results of two independent experiments pooled (means); *p<0.05, **p<0.01 and ***p<0.001 (Student’s t-test). Abbreviations: (-), uninfected.