Autophagy Is an Innate Mechanism Associated with Leprosy Polarization - PubMed (original) (raw)
Autophagy Is an Innate Mechanism Associated with Leprosy Polarization
Bruno Jorge de Andrade Silva et al. PLoS Pathog. 2017.
Abstract
Leprosy is a chronic infectious disease that may present different clinical forms according to the immune response of the host. Levels of IFN-γ are significantly raised in paucibacillary tuberculoid (T-lep) when compared with multibacillary lepromatous (L-lep) patients. IFN-γ primes macrophages for inflammatory activation and induces the autophagy antimicrobial mechanism. The involvement of autophagy in the immune response against Mycobacterium leprae remains unexplored. Here, we demonstrated by different autophagic assays that LC3-positive autophagosomes were predominantly observed in T-lep when compared with L-lep lesions and skin-derived macrophages. Accumulation of the autophagic receptors SQSTM1/p62 and NBR1, expression of lysosomal antimicrobial peptides and colocalization analysis of autolysosomes revealed an impairment of the autophagic flux in L-lep cells, which was restored by IFN-γ or rapamycin treatment. Autophagy PCR array gene-expression analysis revealed a significantly upregulation of autophagy genes (BECN1, GPSM3, ATG14, APOL1, and TPR) in T-lep cells. Furthermore, an upregulation of autophagy genes (TPR, GFI1B and GNAI3) as well as LC3 levels was observed in cells of L-lep patients that developed type 1 reaction (T1R) episodes, an acute inflammatory condition associated with increased IFN-γ levels. Finally, we observed increased BCL2 expression in L-lep cells that could be responsible for the blockage of BECN1-mediated autophagy. In addition, in vitro studies demonstrated that dead, but not live M. leprae can induce autophagy in primary and lineage human monocytes, and that live mycobacteria can reduce the autophagy activation triggered by dead mycobacteria, suggesting that M. leprae may hamper the autophagic machinery as an immune escape mechanism. Together, these results indicate that autophagy is an important innate mechanism associated with the M. leprae control in skin macrophages.
Conflict of interest statement
The authors have declared that no competing interests exist.
Figures
Fig 1. Increase of the autophagy levels in skin lesions of T-lep patients.
(A to D) Skin lesion biopsies were obtained from the tuberculoid (T-lep) and lepromatous (L-lep) leprosy clinical forms and analyzed as indicated. (A) Presence of autophagosome-like vacuoles in skin lesion cells of leprosy patients. Representative TEM micrographs from T-lep (n = 3) and L-lep (n = 3) patients are shown. N, nucleus; M, mitochondria; Asterisks, M. leprae; arrowheads, double-membrane autophagosomes. Scale bars: 0.2 to 1 μm. (B to D) Increased LC3 expression in skin lesion cells of T-lep patients. (B) Immunohistochemical (IHC) analysis of endogenous LC3. Representative micrographs from T-lep (n = 3) and L-lep (n = 4) patients are shown. IHC images were quantified and data are expressed as arbitrary units (AU). Bars represent the mean values ± SEM. **P < 0.01, Mann-Whitney test. Scale bars: 50 and 25 μm. (C) Protein contents from leprosy lesion cells were analyzed by immunoblotting with anti-LC3. GAPDH antibody was used to verify protein amount loading. Representative blots are shown (T-lep, n = 6; L-lep, n = 3). Densitometric analysis of the blots was performed and the LC3-II/GAPDH ratio expressed as AU. Data are presented as mean ± SEM. *P < 0.05, Mann-Whitney test. (D) Redistribution of LC3 by immunofluorescence microscopy. Leprosy tissue sections were immunolabeled with the anti-LC3 (green) and stained with DAPI to visualize the nuclei (blue). Representative micrographs from T-lep (n = 3) and L-lep (n = 3) patients are shown. The number of fluorescent LC3 puncta was quantified and expressed as percentage of tissue area. Data are presented as mean ± SEM. *P < 0.05, Mann-Whitney test. Scale bars: 20 and 10 μm.
Fig 2. IFN-γ rescues M. _leprae_-mediated inhibition of autophagosome formation in skin lesion MΦs of L-lep patients.
(A) Macrophages (MΦs) were isolated from skin lesions of tuberculoid (T-lep) and lepromatous (L-lep) patients and treated with IFN-γ (10 ng/mL) or rapamycin (200 ng/mL) for 18 h. Cells were fixed and stained with the anti-LC3 antibody (green) and DAPI (blue). Non-stimulated (N.S.) MΦs from T-lep lesions showed enhanced LC3 puncta formation as compared to L-lep MΦs. IFN-γ treatment increased the number of LC3 puncta in both T-lep and L-lep skin-derived MΦs. The increase was more prominent in T-lep MΦs. A similar increase of LC3-positive vesicles was observed after addition of rapamycin to the cultures of T-lep and L-lep MΦs. Immunofluorescence images were quantified and bars represent the mean values of the number of LC3 puncta per cell ± SEM (T-lep, n = 3; L-lep, n = 3). *P < 0.05, **P < 0.01, Kruskal-Wallis test; ##P < 0.01, Mann-Whitney test. Scale bar: 25 μm. (B) Monocyte-derived THP-1 MΦs were infected or stimulated with live or dead PKH26-labeled M. leprae (red), respectively, at MOIs of 2, 10 and 50 mycobacteria per cell for 18 h. Cells were fixed and immunofluorescence for LC3 (green) and DAPI (blue) was performed. Stimulation with dead M. leprae directly triggers autophagy. In contrast, infection with live mycobacteria does not induce LC3 puncta accumulation in THP-1 MΦs. Rapamycin (200 ng/mL) treatment was used as a positive control. The number of LC3 puncta/cell was calculated. Colocalization profiles between M. leprae and LC3 were quantified and expressed as percentage of cell area. Results represent the mean ± SEM of four independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, Kruskal-Wallis test; ##P < 0.01, ###P < 0.001, Mann-Whitney test. Scale bar: 10 μm.
Fig 3. Live M. leprae inhibits autophagosome formation in primary human monocytes.
Blood-derived monocytes from healthy donors were cultured in the presence or absence of live (blue), dead (red), or both live and dead PKH-labeled M. leprae, at MOIs of 10 and 50 mycobacteria per cell for 18 h. Cells were fixed and immunofluorescence for LC3 (green) and DAPI (cyan) was performed through optical sectioning using structured illumination. LC3 puncta formation in monocytes is inhibited by live, but not dead M. leprae. Rapamycin (200 ng/mL) treatment was used as a positive control. N.S., Non-stimulated cells. The number of LC3 puncta/cell was calculated. Colocalization profiles between M. leprae and LC3 were quantified and expressed as percentage of cell area. Results represent the mean ± SEM of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, Kruskal-Wallis test; #P < 0.05, ##P < 0.01, ###P < 0.001, Mann-Whitney test. Scale bar: 20 μm.
Fig 4. M. _leprae_-mediated inhibition of the autophagic flux in skin-derived L-lep MΦs was restored by IFN-γ.
(A) Protein contents from skin lesion cells of tuberculoid (T-lep) and lepromatous (L-lep) patients were examined for intracellular levels of SQSTM1/p62 and NBR1 by ELISA. SQSTM1/p62 and NBR1 strongly accumulated in L-lep samples while, in T-lep samples, only low levels were detected. Bars represent the mean values ± SEM of the SQSTM1/p62 (T-lep, n = 5; L-lep, n = 4) and NBR1 (T-lep, n = 6; L-lep, n = 4) levels. *P < 0.05, Mann-Whitney test. (B) Macrophages (MΦs) were isolated from skin lesions of L-lep patients and incubated in full medium with 10 ng/mL IFN-γ or 200 ng/mL rapamycin, or in starvation media (PBS). Eighteen hours after incubation, cells were loaded with 500 nM LysoTracker (red) for 30 min and then fixed and labeled for LC3 (green), M. leprae LAM (blue), and DAPI (white). In non-stimulated (N.S.) MΦs from L-lep lesions reduced colocalization profiles were seen among LC3, LysoTracker, and LAM, as compared to T-lep MΦs (data in S2 Fig). Activation of autophagy by IFN-γ treatment induces the colocalization of LC3-positive vesicles with LysoTracker stained lysosomes and LAM in both L-lep and T-lep MΦs (data in S2 Fig). Starvation and rapamycin-induced autophagy were also able to promote the autophagic flux in L-lep MΦs. Arrowheads, indicate three-channel colocalization profiles. Colocalization analysis of immunofluorescence images was performed as indicated and expressed as percentage of cell area. Results represent the mean ± SEM of three independent experiments. Scale bar: 10 μm. (C) Blood-derived monocytes from healthy donors were cultured in the presence or absence of live (blue), dead (red), or both live and dead PKH-labeled M. leprae, at MOI of 50 mycobacteria per cell. Eighteen hours after incubation, cells were loaded with 500 nM LysoTracker (white) for 30 min and then fixed and labeled for LC3 (green) and DAPI (cyan), and imaged through optical sectioning using structured illumination. The autophagic flux in monocytes is inhibited by live, but not dead M. leprae. Arrowheads, indicate three-channel colocalization profiles. Colocalization analysis of immunofluorescence images was performed as indicated and expressed as percentage of cell area. Results represent the mean ± SEM of three independent experiments. *, #, and °, indicate colocalization profiles between LC3 and LysoTracker, M. leprae and LC3, and M. leprae and LysoTracker, respectively. *P < 0.05, **P < 0.01; #P < 0.05, ##P < 0.01; °P < 0.05, Mann-Whitney test. Scale bar: 20 μm.
Fig 5. Autophagy gene-expression profiling of leprosy lesions reveals a differential expression of BECN1 in T-lep patients.
(A and B) Purified mRNAs from skin lesions of tuberculoid (T-lep) and lepromatous (L-lep) patients were analyzed by RT-qPCR autophagy array. (A) Autophagy processes-related genes differentially expressed were sub-categorized. The expression fold values of the significantly upregulated genes in T-lep lesions are tabulated (full data are available in S1 Table). Threshold for statistical significance was established at P < 0.05. (B) Heat map showing analysis of changes in expression of autophagy processes-related genes in leprosy patients. Each row represents one donor. Asterisks indicate genes with differential expression. Heat map data are representative of four T-lep and seven L-lep samples. (C) Autophagy gene interaction network in T-lep and L-lep skin lesions. Genes with a differential expression in leprosy lesions by autophagy PCR array analysis were visualized by STRING. The action network view. In this view, colored lines and arrow styles between genes indicate the various types of interactions. Network nodes represent genes. Edges represent gene-gene associations. Interactions among autophagy-associated genes were more predominant in T-lep than L-lep patients. Interaction maps are representative of four T-lep and seven L-lep samples.
Fig 6. Differential regulation of BECN1 and BCL2 proteins in skin lesion cells of T-lep and L-lep patients.
(A) Protein contents from tuberculoid (T-lep) and lepromatous (L-lep) lesion cells were analyzed by western blot with anti-BECN1 and anti-BCL2 antibodies. GAPDH antibody was used to verify protein amount loading. Densitometric band-intensity analysis of the blots was realized and the BECN1/GAPDH (T-lep, n = 6; L-lep, n = 5) and BCL2/GAPDH (T-lep, n = 4; L-lep, n = 4) ratios were expressed as arbitrary units (AU). Bars represent the mean values ± SEM. *P < 0.05, Mann-Whitney test. (B) Macrophages (MΦs) were isolated from skin lesions of T-lep and L-lep patients and cultured for 7 days in full medium. Cells were fixed and labeled for BECN1 (red), BCL2 (green) and DAPI (blue). Cytoplasmatic BCL2 colocalizes with BECN1 in L-lep MΦs, but not in T-lep MΦs. Colocalization profiles between cytosolic BCL2 dots and BECN1 were quantified and expressed as percentage of cell area. The expression of nuclear compartment-associated BCL2 was excluded from the analysis. Results represent the mean ± SEM from one of three immunofluorescence experiments that yielded similar results. **P < 0.01, Mann-Whitney test. Scale bar: 10 μm.
Fig 7. Gene-expression profiles of leprosy lesions showed a balanced modulation of autophagy-associated genes between L-lep patients with and without T1R episodes.
(A and B) Purified mRNAs from skin lesions of lepromatous (L-lep) patients with or without type 1 reactional (T1R) episodes were analyzed by RT-qPCR autophagy array. (A) Differentially expressed autophagy processes-related genes were sub-categorized. The expression fold values of the significantly upregulated genes in T1R lesions were tabulated (full data are available in S2 Table). The threshold for statistical significance was P < 0.05. (B) Heat map showing analysis of changes in the expression of autophagy processes-related genes in leprosy patients. Each row represents one donor. Asterisks indicate genes with differential expression. Heat map data are representative of seven L-lep and seven T1R samples. (C) Autophagy gene interaction network in L-lep and T1R skin lesions. Genes with a differential expression in leprosy lesions according to autophagy PCR array analysis were visualized by STRING. The action network view. In this view, colored lines and arrow styles between genes indicate the various types of interactions. Network nodes represent genes. Edges represent gene-gene associations. Interactions among autophagy processes-related genes were more evident in L-lep than T1R patients. Interaction maps are representative of seven L-lep and seven T1R samples.
Fig 8. T1R episodes enhances the levels of autophagy in L-lep patients.
(A) Immunohistochemistry (IHC) for endogenous LC3. Increase of the LC3 expression in skin lesion cells of L-lep patients undergoing T1R episodes. IHC images were quantified and data are expressed as arbitrary units (AU). Bars represent the mean values ± SEM (L-lep, n = 4; T1R, n = 3). *P < 0.05, Mann-Whitney test. Scale bars: 50 and 25 μm. (B) Macrophages (MΦs) were isolated from skin lesions of L-lep and T1R patients and treated with IFN-γ (10 ng/mL) for 18 h. Cells were fixed and stained with the anti-LC3 antibody (green) and DAPI (blue). Non-stimulated (N.S.) T1R MΦs showed more enhanced LC3 puncta formation than L-lep MΦs. IFN-γ stimulation led to accumulation of LC3 dots in both L-lep and T1R MΦs, but to a lesser extent in MΦs derived from L-lep patients without T1R lesions. Immunofluorescence images were quantified and bars represent the mean values of the number of LC3 puncta per cell ± SEM of three independent experiments. *P < 0.05, **P < 0.01, **P < 0.001, Mann-Whitney test. Scale bar: 50 μm.
References
- World Health Organization, Global leprosy update, 2015: time for action, accountability and inclusion. Weekly epidemiological record, 2016. 91(35): p. 405–420. - PubMed
- Ridley DS, Jopling WH. Classification of leprosy according to immunity. A five-group system. Int J Lepr Other Mycobact Dis. 1966;34: 255–273. - PubMed
- Yamamura M, Uyemura K, Deans RJ, Weinberg K, Rea TH, Bloom BR, et al. Defining protective responses to pathogens: cytokine profiles in leprosy lesions. Science. 1991;254: 277–279. - PubMed
Publication types
MeSH terms
Substances
LinkOut - more resources
Full Text Sources
Other Literature Sources
Medical
Miscellaneous