Fig 1.
Molecular structures of mycobacterium phenolic glycolipids.
The structures of the phenolic glycolipids from M. leprae (PGL I), M. tuberculosis (PGL-tb), and M. bovis (PGL-bovis) highlight the differences in the saccharidic moiety. R = common lipid core. In [14–16].
Fig 2.
PGL I production and bacterial viability are determinants for mycobacterial internalization into Schwann cells.
A. The level of bacterial association of the PKH67-(green) labeled BCG recombinant strains was determined by Flow Cytometry (FL1-A green channel). ST8814 SC were either left uninfected (NI) or were treated with BCG WT, BCG PGL TB, or BCG PGL I. A representative histogram plot of the 48 h incubation experiment is shown. The percentage of bacterial association was determined after 4 h, 24 h, and 48 h of incubation with SC at 33°C, MOI 50:1. B. Association and internalization of live and dead bacilli were determined by flow cytometry after 48 h of incubation at 33°C and MOI 50:1. Bacteria were labeled with PKH67 and the degree of internalization was determined after Trypan Blue quenching. Representative histogram plots show fluorescence at the FL1-A channel. Results were represented as a percentage of the cell population with internalized bacteria. C. Fluorescence microscopy showing the degree of bacterial association of live and dead bacilli after 48 h of incubation with SC at 33°C, MOI 50:1. BCG WT, BCG PGL I, BCG PGL TB, and M leprae were labeled with green fluorescent PKH67. Nuclei were stained with DAPI (blue). Scale 10μm. Quantification of the percentage of cells with associated bacteria in 200 fields per condition and per replicate. D. Primary human Schwann (PHSC) cells were treated with BCG WT, BCG PGL TB, BCG PGL I or M. leprae for 48 h at 33°C, MOI 50:1. Bacteria were previously labeled with PKH67; and the degree of internalization was determined after Trypan Blue quenching. Percentage of cells with internalized bacteria was determined using flow cytometry. E. PHSC were treated with green fluorescent latex beads covered or not with PGL I. The percentage of cells with internalized beads was determined after 48 h of incubation at 33°C and a 50:1 proportion via flow cytometry. Each bar represents the mean ± SEM from at least three independent experiments in triplicate. An ANOVA test followed by Bonferroni as a post-test were performed and used for statistical analyses. ** p<0.01; ***p<0.001. In E) Statistical significance was calculated by Mann Whitney Test **p < 0.01.
Fig 3.
Pre-infection with live BCG PGL I or M. leprae allows BCG WT to enter Schwann cells.
A-B. A higher degree of association and internalization of PKH67-labeled BCG WT (live or irradiated) was observed after pre-infection with live BCG PGL I or M. leprae at MOI 10:1. C. A first stimulus with beads, beads covered with PGL I, and dead M. leprae or dead BCG PLG I did not result in a higher degree of BCG WT internalization. D. Flow cytometric results showed no change in the degree of internalization of PKH67-labeled M. smegmatis after pre-stimulus with M. leprae at MOI 10:1. (NI shows background fluorescence). A-D. Results are represented as mean ± SEM of at least three independent biological replicates; and statistical significance was calculated by ANOVA followed by Bonferroni’s multiple comparison test. *** p<0.001.
Fig 4.
Infection with BCG PGL I or M. leprae induces CD206 expression in Schwann cells.
A. Competition assay suggesting the Mannose Receptor (CD206) as a receptor candidate to mediate the internalization of BCG WT in SC. After pre-infection with M. leprae or BCG PGL I, the addition of mannose at 100 or 1000 μg/mL reduced the BCG WT internalization rate 48 h post-infection. In all experiments, the degree of internalization of PKH67-labeled BCG WT was determined after Trypan Blue quenching by flow cytometry. B. Pre-infection with PGLI-expressing bacteria MOI 10:1 favors the internalization of ManLAM-covered latex beads, which were incubated with SC for 48 h at 33°C and a 50:1 proportion. MFI was determined using the flow cytometry FL1-A channel. Green fluorescent latex beads covered with ManLAM showed a higher degree of internalization in comparison to the control green fluorescent latex beads. C. Normalized relative expression of mrc1 (delta delta Ct) in SC infected for 4 h, 24 h or 48 h with either BCG PGL I, M. leprae, or BCG WT at MOI 50:1. Results are presented in terms of fold changes after normalization with rpl13 mRNA. D. CD206 expression in SC after 24 h of infection was measured by flow cytometry. Immunofluorescent labeling of CD206 was carried out using the FITC conjugated anti-CD206 antibody. The conditions were: uninfected (NI), isotype control, and treatment with BCG WT, BCG PGL I, or M. leprae. A representative histogram plot shows fluorescence at the FL1-A channel. E. Representative images of fluorescence microscopy showing CD206 expression in uninfected SC and M. leprae or BCG PGL I-infected SC after 24 h of incubation. Cells on coverslips were fixed with paraformaldehyde and stained with DAPI (blue) for nuclear localization. Cells were immunolabeled with FITC conjugated anti-CD206 antibody or a FITC conjugated isotype and then examined by fluorescence microscopy. The mean CD206 signal intensity per cell was quantified. Scale bar: 10μm (white line) A-E. Results are represented as mean ± SEM of three or more independent biological replicates. Statistical significance was calculated by ANOVA followed by Bonferroni’s multiple comparison test. *p < 0.05; ** p<0.01; *** p<0.001.
Fig 5.
Mannose receptor mrc1 knockdown diminishes BCG WT and M. leprae entry into Schwann cells.
A. Verification of mrc1 gene knockdown. SC were transfected with control siRNA or two siRNA targeting mrc1 for 24 h before infection. Flow cytometry result showing that knockdown with mrc1siRNA reduces CD206 expression in M. leprae-infected SC. Representative histogram plots show fluorescence at the FL1-A channel. SCs were submitted to immunofluorescent labeling of CD206 with the FITC conjugated anti-CD206 antibody (clone15-2, Mouse IgG1, κ1, Biolegend). SC expression of CD206 after 24 h of infection at MOI 50:1, 33°C was measured using the flow cytometry FL1-A (green) channel to determine MFI. B. Flow cytometry result showing the effect of mrc1 knockdown on the degree of association of PKH26-(red) labeled M. leprae or BCG WT. For BCG WT, the previously described co-infection assay was applied. MFI was determined at the FL2-A channel. C. SC on coverslips were stimulated with M. leprae or BCG WT for 48 h. The cells were fixed with paraformaldehyde, stained with DAPI (blue) for nuclear localization, and examined by fluorescence microscopy. The upper panel shows (a-b) BCG WT association with and without control siRNA, (c) co-infected SC with M. leprae and PKH26-labeled BCG WT. The middle panel shows (d) co-infection in the presence of control siRNA, (e-f) co-infection in the presence of two types of mrc1 siRNA. The lower panel shows PKH26-labeled M. leprae associated to SC in the presence of (g) control siRNA, (h- i) tow types of mrc1 siRNA. Scale (white line) represents 10 μm. Results are represented as mean ± SEM of three independent biological replicates. Statistical significance was calculated by ANOVA followed by Bonferroni’s multiple comparison test. *p < 0.05; **p<0.01; ***p<0.001.
Fig 6.
PGL I-producing mycobacterium induces CD206 expression in Schwann cells via PPARγ.
A. Total lysates (20μg per well) from SC cultures were subjected to Western Blotting using specific antibodies against PPARγ and GAPDH. A representative Western blot from three independent experiments is shown. The content of the bands was estimated by densitometric analysis; and relative expression was plotted in arbitrary units (A.U. = arbitrary units). B and C. SC were treated with the PPARγ antagonist GW9662 (5μM) for 30 minutes previous to a 24 h infection with either BCG WT, BCG PGL I, or M. leprae at MOI 50:1. CD206 expression was determined using flow cytometry and fluorescence microscopy by immunolabeling of CD206 with FITC conjugated anti-CD206 antibody (clone15-2, Mouse IgG1, κ1, Biolegend). Treatment with GW9662 decreased CD206 expression in SC. For microscopy, cells on coverslips were fixed with paraformaldehyde and stained with DAPI (blue) for nuclear localization. Images are representative of 3 independent experiments. Scale (white line) represents 10 μm. CD206 mean signal intensity per cell was quantified. D. SC were treated with GW9662 5μM for 30 minutes previous to 48 h stimulation with either uncovered beads or ManLAM-covered beads at a proportion of (50:1). Pre-stimulus with unlabeled M. leprae or BCG PGL I was carried out after treatment with the antagonist and one hour before the second stimulus. Internalization of the green fluorescent beads was determined by flow cytometry (FL1-A channel). Results are represented as mean ± SEM of three independent biological replicates. Statistical significance was calculated by ANOVA followed by Bonferroni’s multiple comparison test. *p < 0.05; **p<0.01; ***p<0.001.
Fig 7.
Crosstalk between PPARγ and CD206 mediates lipid droplet and PGE2 production in Schwann cells infected with PGL I-producing mycobacteria.
A. Competition assay suggesting cross-talk between PPARγ and CD206 in SCs. The addition of mannose at 100 μg/mL reduced BCG PGL I-induced PPARγ expression 48 h post-infection. PPARγ detection was performed using the specific rabbit polyclonal antibody (H-100) SC-7196 (Santa Cruz Inc., USA) followed by incubation with IgG anti-rabbit conjugated to Alexa Fluor 594 (Molecular Probes, USA) for immunofluorescence detection. Cells on coverslips were fixed with paraformaldehyde and stained with DAPI (blue) for nuclear localization. Representative fluorescence microscopy images showing the expression and localization of PPARγ after addition of mannose. Mean signal intensity per cell was quantified. Scale bar: 10μm (white line). B and C. Impact of the transcription factor PPARγ and the CD206 receptor on LD formation induced by M.leprae and BCG PGL I in SCs. Representative fluorescence microscopy images showing the effect of PPARγ antagonist GW9662 (5 μM) (B) and of mannose 100 μg/mL (C) on LD induction. The LD induction was estimated by microscopy after fluorescent Oil Red O staining and quantification of ORO-stained LDs was plotted as measurement of LD area/cell. The pretreatment with GW9662 or mannose respectively, reduced the LD formation induced by M.leprae or BCG PGL I 48 h post-infection. Scale bar: (A) 20μm (B) 10 μm and (C) 20 μm (white line). Data are shown as mean±SEM of three (7B) and five (7C) different experiments performed in triplicate. D and E. EIA was used to analyze the effect of the addition of GW9962 or mannose on the levels of PGE2 in the supernatants from the LD induction experiments. The results are the mean ± SEM from at least three independent experiments performed in triplicate. Statistical significance was calculated by ANOVA followed by Bonferroni’s multiple comparison test. *p < 0.05; ** p<0.01; ***p<0.001. F. M. leprae viability measured by qRT-PCR using the ratio of 16S rRNA/16S DNA 48 h after infection of ST8814 pre-treated or not with 100 μg/mL mannose. Data are shown as mean±SEM of six different experiments performed in triplicate. Statistical significance was calculated by Mann Whitney Test *p < 0.05.
Fig 8.
Crosstalk between PPARγ and CD206 mediates IL-8 production in Schwann cells infected with PGL I-producing mycobacteria.
A. SCs were transfected for 24 h with control siRNA or siRNA targeting mrc1, followed by infection with M. leprae or BCG PGL I for 48 h. Supernatants were analyzed for IL-8 production by ELISA. B and C. Alternatively, cells were pretreated with mannose 100 μg/mL or the PPARγ antagonist GW9662 (5 μM) respectively, 30 minutes before infection. Cells were then infected with M. leprae or BCG PGL I for 48 h. Supernatants were analyzed for IL-8. The results are the mean ± SEM from at least three independent experiments performed in triplicate. Statistical significance was calculated by ANOVA followed by Bonferroni’s multiple comparison test. *p < 0.05; ** p<0.01; ***p<0.001.
Fig 9.
CD206 colocalizes with Schwann cells in nerve lesions of leprosy patients.
Nerve biopsies were labeled with antibodies for the SC-specific marker S100 (red image), and the mannose receptor CD206 (green image) and then visualized by fluorescence microscopy. Nuclei were labeled with DAPI (blue image). A-D. Serial sections of a leprosy patient nerve biopsies were analyzed. A. Wade staining showing M.leprae. Scale bars, 20μm. B-D. Tissue sections were visualized by fluorescence microscopy showing CD206/S100 co-staining SCs. Images are representative of a total of five patients. E-G. Serial sections of a nerve biopsy from a patient with a non-leprosy peripheral neuropathy. The merged image in G. shows no CD206/S100 co-localization.Images are representative of a total of three patients. Scale bar, 20μm. Insets: magnified views of CD206/S100 staining in leprosy (insets 1 and 2) and non-leprosy (insets 3 and 4) nerve biopsies. Scale bar, 10μm.
Fig 10.
M. leprae infected Schwann cells express CD206 in leprosy nerve lesions.
Nerves biopsies were labeled with antibodies for mannose receptor CD206 (green image), for the SC-specific marker MBP (red image) and for M.leprae (anti-LAM; yellow image). Nuclei were labeled with DAPI (blue). The serial section of a leprosy patient nerve biopsy was analyzed by fluorescence microscopy. The images (representative of two patients) show the expression profile of the CD206 mannose receptor, the MBP SC marker and the location of M. leprae. The merged image shows CD206/MBP/LAM co-staining in SCs (white arrows). Scale bar, 20μm.
Fig 11.
A model proposing a key role for the PGL I-induced CD206/PPARγ crosstalk in M. leprae neuropathogenesis.
The first step (signal 1) of the pathway activation initiates through PGL I recognition via laminin-2 [12] allowing bacterial internalization. Bacterial sensing by TLR6 also contributes to bacterial entry [22]. Additionally, the recognition of M. leprae ManLAM by baseline levels of CD206 allows some bacterial entry and weak activation of PPARγ, with subsequent CD206 upregulation. The higher expression of CD206 results in increasing bacterial sensing by this pathway, triggering a stronger second signal (signal 2), where internalized M. leprae promotes the amplification of CD206/ PPARγ crosstalk, inducing the accumulation of LDs and their recruitment to bacterium-containing phagosomes. The higher levels of LDs promote the production of PGE-2 and IL-10 [22], which favors the inhibition of SC´s antimicrobial mechanisms. CD206/ PPARγ crosstalk also induces IL-8 secretion that may be involved in demyelination and neuroinflammation. Narrow arrows indicate the initials steps involved in M. leprae-SC interaction. Wide arrows indicate the major steps involved in the amplification loop of the PPAR/CD206 crosstalk. DG = Dystroglycan, α2 LN = alpha 2 laminin, RXR = Retinoid X Receptor.