Genetic polymorphisms of the <i>IL6</i> and <i>NOD2</i> genes are risk factors for inflammatory reactions in leprosy

<div><p>The pathways that trigger exacerbated immune reactions in leprosy could be determined by genetic variations. Here, in a prospective approach, both genetic and non-genetic variables influencing the amount of time before the development of reactional episodes were studied using Kaplan–Meier survival curves, and the genetic effect was estimated by the Cox proportional-hazards regression model. In a sample including 447 leprosy patients, we confirmed that gender (male), and high bacillary clinical forms are risk factors for leprosy reactions. From the 15 single nucleotide polymorphisms (SNPs) at the 8 candidate genes genotyped (<i>TNF</i>/<i>LTA</i>, <i>IFNG</i>, <i>IL10</i>, <i>TLR1</i>, <i>NOD2</i>, <i>SOD2</i>, and <i>IL6)</i> we observed statistically different survival curves for rs751271 at the <i>NOD2</i> and rs2069845 at the <i>IL6</i> genes (log-rank p-values = 0.002 and 0.023, respectively), suggesting an influence on the amount of time before developing leprosy reactions. Cox models showed associations between the SNPs rs751271 at <i>NOD2</i> and rs2069845 at <i>IL6</i> with leprosy reactions (HR_GT = 0.45, p = 0.002; HR_AG = 1.88, p = 0.0008, respectively). Finally, IL-6 and IFN-γ levels were confirmed as high, while IL-10 titers were low in the sera of reactional patients. Rs751271-GT genotype-bearing individuals correlated (p = 0.05) with lower levels of IL-6 in sera samples, corroborating the genetic results. Although the experimental size may be considered a limitation of the study, the findings confirm the association of classical variables such as sex and clinical forms with leprosy, demonstrating the consistency of the results. From the results, we conclude that SNPs at the <i>NOD2</i> and <i>IL6</i> genes are associated with leprosy reactions as an outcome. <i>NOD2</i> also has a clear functional pro-inflammatory link that is coherent with the exacerbated responses observed in these patients.</p></div

Differential regulation of BECN1 and BCL2 proteins in skin lesion cells of T-lep and L-lep patients.

<p>(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. *<i>P</i> < 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. **<i>P</i> < 0.01, Mann-Whitney test. Scale bar: 10 μm.</p

Baseline characteristics of the patients with leprosy included in the study.

<p>Baseline characteristics of the patients with leprosy included in the study.</p

T1R episodes enhances the levels of autophagy in L-lep patients.

<p>(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). *<i>P</i> < 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. *<i>P</i> < 0.05, **<i>P</i> < 0.01, **<i>P</i> < 0.001, Mann-Whitney test. Scale bar: 50 μm.</p

Autophagy gene-expression profiling of leprosy lesions reveals a differential expression of <i>BECN1</i> in T-lep patients.

<p>(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 <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006103#ppat.1006103.s006" target="_blank">S1 Table</a>). Threshold for statistical significance was established at <i>P</i> < 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.</p

Increase of the autophagy levels in skin lesions of T-lep patients.

<p>(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, <i>M</i>. <i>leprae</i>; 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. **<i>P</i> < 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. *<i>P</i> < 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. *<i>P</i> < 0.05, Mann-Whitney test. Scale bars: 20 and 10 μm.</p

IFN-γ rescues <i>M</i>. <i>leprae</i>-mediated inhibition of autophagosome formation in skin lesion MΦs of L-lep patients.

<p>(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). *<i>P</i> < 0.05, **<i>P</i> < 0.01, Kruskal-Wallis test; ##<i>P</i> < 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 <i>M</i>. <i>leprae</i> (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 <i>M</i>. <i>leprae</i> 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 <i>M</i>. <i>leprae</i> and LC3 were quantified and expressed as percentage of cell area. Results represent the mean ± SEM of four independent experiments. *<i>P</i> < 0.05, **<i>P</i> < 0.01, ***<i>P</i> < 0.001, Kruskal-Wallis test; ##<i>P</i> < 0.01, ###<i>P</i> < 0.001, Mann-Whitney test. Scale bar: 10 μm.</p

<i>M</i>. <i>leprae</i>-mediated inhibition of the autophagic flux in skin-derived L-lep MΦs was restored by IFN-γ.

<p>(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. *<i>P</i> < 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), <i>M</i>. <i>leprae</i> 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 <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006103#ppat.1006103.s002" target="_blank">S2 Fig</a>). 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 <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006103#ppat.1006103.s002" target="_blank">S2 Fig</a>). 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 <i>M</i>. <i>leprae</i>, 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 <i>M</i>. <i>leprae</i>. 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, <i>M</i>. <i>leprae</i> and LC3, and <i>M</i>. <i>leprae</i> and LysoTracker, respectively. *<i>P</i> < 0.05, **<i>P</i> < 0.01; #<i>P</i> < 0.05, ##<i>P</i> < 0.01; °<i>P</i> < 0.05, Mann-Whitney test. Scale bar: 20 μm.</p

Gene-expression profiles of leprosy lesions showed a balanced modulation of autophagy-associated genes between L-lep patients with and without T1R episodes.

<p>(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 <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006103#ppat.1006103.s007" target="_blank">S2 Table</a>). The threshold for statistical significance was <i>P</i> < 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.</p

table_3_Autophagy Impairment Is Associated With Increased Inflammasome Activation and Reversal Reaction Development in Multibacillary Leprosy.XLSX

<p>Leprosy reactions are responsible for incapacities in leprosy and represent the major cause of permanent neuropathy. The identification of biomarkers able to identify patients more prone to develop reaction could contribute to adequate clinical management and the prevention of disability. Reversal reaction may occur in unstable borderline patients and also in lepromatous patients. To identify biomarker signature profiles related with the reversal reaction onset, multibacillary patients were recruited and classified accordingly the occurrence or not of reversal reaction during or after multidrugtherapy. Analysis of skin lesion cells at diagnosis of multibacillary leprosy demonstrated that in the group that developed reaction (T1R) in the future there was a downregulation of autophagy associated with the overexpression of TLR2 and MLST8. The autophagy impairment in T1R group was associated with increased expression of NLRP3, caspase-1 (p10) and IL-1β production. In addition, analysis of IL-1β production in serum from multibacillary patients demonstrated that patients who developed reversal reaction have significantly increased concentrations of IL-1β at diagnosis, suggesting that the pattern of innate immune responses could predict the reactional episode outcome. In vitro analysis demonstrated that the blockade of autophagy with 3-methyladenine (3-MA) in Mycobacterium leprae-stimulated human primary monocytes increased the assembly of NLRP3 specks assembly, and it was associated with an increase of IL-1β and IL-6 production. Together, our data suggest an important role for autophagy in multibacillary leprosy patients to avoid exacerbated inflammasome activation and the onset of reversal reaction.</p