Fig 1: S100A7, S100A8, CCL17, and CCL19 show antimicrobial activity against M. leprae in infected human macrophages.(A–D) MDMs from healthy donors were infected overnight with M. leprae at a MOI of 5:1, followed by addition of 0.1 μM recombinant human S100A7, S100A8, CCL17, and CCL19 for 4 days. M. leprae viability was assessed by qPCR, and the percentage of antimicrobial activity was calculated by assigning 100% bacteria viability to the media control. Rifampin (10 μg/mL) (RIF) was added as a positive control. (E) Lysosome acidification was assessed by LysoTracker staining (green) in MDMs previously stimulated with 0.1 µM recombinant human S100A7, S100A8, CCL17, and CCL19 for 1 hour and then infected with M. leprae labeled with PKH26 (red) at a MOI of 5:1 over night. Leptin (0.1 μM) was used as a negative control. Images were captured using a Leica TCS SP8 Digital Light Sheet Microscope. DAPI (blue) was used to stain the nuclei. Scale bars: 10 μm; original magnification, ×630 with ×4 zoom. Statistical analyses were performed in GraphPad Prism 9.12 using the Friedman test followed by Dunn’s multiple-comparison test (A–D). Data represent the mean ± SEM (n = 6 for A and C) and (n =7, B and D). *P < 0.05 and **P < 0.01.
Fig 2: Protein expression of S100A7, S100A8, CCL17, and CCL19 in RR and pre-RR skin lesions.(A) S100A7 and S100A8 protein expression in a representative pre-RR and RR skin lesion pair (LL1/RR.LL1) evaluated by IHC. (B) CCL17 and CCL19 protein expression in a representative pre-RR and RR skin lesion pair (BL4/RR.BL4) evaluated by IHC. CD68, a macrophage marker, was used as a positive control. Graphs show quantification of S100A7 (n = 6 pairs), S100A8 (n = 5 pairs), CCL17 (n = 4 pairs), and CCL19 (n = 5 pairs) staining 3-amino-9-ethylcarbazole (AEC)/nuclear area. Image J plugin ImmunoRatio was used for quantification, and staining was visualized and images were acquired using a Leica microscope (Leica 250). Scale bars: 25 μm; original magnification, ×200. Statistical analyses were performed in GraphPad Prism 9.12 using the paired t test (S100A7 and CCL19) or ratio paired, 2-tailed t test (S100A8 and CCL17). *P < 0.05.
Fig 3: RNA-FISH shows antimicrobial gene expression in RR and pre-RR skin lesions by different cell populations.(A) RNA-FISH of S100A7 (green) and staining for keratin 14 (KRT14) protein (red) in 1 representative pair of RR and pre-RR skin lesions (BL4/RR.BL4). S100A7 RNA dot quantification (number of dots) was performed on 4 pairs of RR and pre-RR skin lesions. (B) RNA-FISH of S100A8 (green) and protein staining of KRT14 (red) in 1 representative pair of RR and pre-RR skin lesions (BL5/RR.BL5). S100A8 RNA dot quantification (number of dots) was performed on 4 pairs of RR and pre-RR skin lesions. (C) RNA-FISH of CCL17 (red) and LYZ (green), a macrophage marker, in 1 representative pair of RR and pre-RR skin lesions (BL3/RR.BL3). CCL17 RNA dot quantification (number of dots) was performed on 4 pairs of RR and pre-RR skin lesions. (D) RNA-FISH of CCL19 (red) and COL1A1 (green), a fibroblast marker, in 1 representative pair of RR and pre-RR skin lesions (BL4/RR.BL4). CCL19 RNA dot quantification (number of dots) was performed on 4 pairs of RR and pre-RR skin lesions. Cell nuclei were stained with DAPI (blue). Images were acquired with a Leica TCS SP8 Digital Light Sheet microscope, and RNA dot quantification was performed using ImageJ. Scale bars: 10 μm; original magnification, ×630 (A–C) and ×630 with ×3 zoom (D). Statistical analyses were performed in GraphPad Prism 9.12 using the ratio paired t test (A and B) or paired 2-tailed t test (C and D). *P < 0.05 and **P < 0.01.
Fig 4: S100A7, S100A8, CCL17, and CCL19 show direct antimicrobial activity against M. leprae.(A–D) Different concentrations of recombinant human S100A7, S100A8, CCL17, and CCL19 were added to M. leprae (2 × 106 bacilli) in 7H9 broth with 10 mM sodium phosphate, pH 7.2, for 72 hours. Bacteria viability was assessed by qPCR, and rifampin (10 μg/mL) (RIF) was used as a positive control. (E) S100A7 (4.5 μM), S100A8 (9 μM), CCL17 (4.5 μM), and CCL19 (4.5 μM) were added to M. leprae (15 × 106 bacilli) in 7H9 broth with 10 mM sodium phosphate, pH 7.2, for 6, 24, 48, and 96 hours, and bacteria morphology was evaluated by scanning electron microscopy. IL-26 (10 μM) was used as a positive control. Scale bar: 500 nm; original magnification, ×100,000. Statistical analyses were performed in GraphPad Prism 9.12 using repeated-measures of 1-way ANOVA with the Geisser-Greenhouse correction and Dunnett’s multiple-comparison test (A–D). Data represent the mean ± SEM (n = 4). **P < 0.01, ***P < 0.001, and ****P < 0.0001.
Fig 5: Protein expression of S100A7, S100A8, CCL17, and CCL19 in RR and pre-RR skin lesions.(A) S100A7 and S100A8 protein expression in a representative pre-RR and RR skin lesion pair (LL1/RR.LL1) evaluated by IHC. (B) CCL17 and CCL19 protein expression in a representative pre-RR and RR skin lesion pair (BL4/RR.BL4) evaluated by IHC. CD68, a macrophage marker, was used as a positive control. Graphs show quantification of S100A7 (n = 6 pairs), S100A8 (n = 5 pairs), CCL17 (n = 4 pairs), and CCL19 (n = 5 pairs) staining 3-amino-9-ethylcarbazole (AEC)/nuclear area. Image J plugin ImmunoRatio was used for quantification, and staining was visualized and images were acquired using a Leica microscope (Leica 250). Scale bars: 25 μm; original magnification, ×200. Statistical analyses were performed in GraphPad Prism 9.12 using the paired t test (S100A7 and CCL19) or ratio paired, 2-tailed t test (S100A8 and CCL17). *P < 0.05.
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