Transcriptional profiling of human Leishmania braziliensis skin lesions

<p>Normalized, batch corrected Log2 gene expression data and average Log2 FC for 20,942 genes across all 35 skin samples (10 normal, 8 early lesions, 17 late lesions) represented by one or more probesets from the Illumina HT-12v4 beadarray.</p> <p>Abbreviations: FC, fold-change.</p

Comparison of the L. braziliensis transcriptiome with published psoriasis datasets

<p>Comparison of transcriptional responses in L. braziliensis lesions with published psoriasis meta-analysis. Log2 FC in gene expression between skin lesion and normal control skin for 17,070 genes in common between the L. braziliensis data and published psoriasis ‘MAD3’ data. Rank order is shown for both Leishmania and psoriasis data in which genes were ranked from most highly upregulated in lesion (rank = 1) to most downregulated in lesion (rank = 17,070). Abbreviations: FC, fold change</p

Pathway analysis of differentially expressed genes in L. braziliensis lesions

<p>Pathway enrichment analysis. Excel spreadsheet showing pathways and their enrichment score from Fig. 2b found by GSEA to be enriched 16 fold (FDR ≤ 1%) in L. braziliensis lesions, relative to normal skin. Functionally related pathways manually grouped together for simplified representation in Fig. 2b are shown as a separate tab in the spreadsheet. Abbreviations: GSEA, gene set enrichment analysis; FDR, false discovery rate.</p

CD8⁺ T cell cytotoxicity mediates pathology in the skin by inflammasome activation and IL-1β production

<div><p>Deregulated CD8+ T cell cytotoxicity plays a central role in enhancing disease severity in several conditions. However, we have little understanding of the mechanisms by which immunopathology develops as a consequence of cytotoxicity. Using murine models of inflammation induced by the protozoan parasite leishmania, and data obtained from patients with cutaneous leishmaniasis, we uncovered a previously unrecognized role for NLRP3 inflammasome activation and IL-1β release as a detrimental consequence of CD8+ T cell-mediated cytotoxicity, ultimately resulting in chronic inflammation. Critically, pharmacological blockade of NLRP3 or IL-1β significantly ameliorated the CD8+ T cell-driven immunopathology in leishmania-infected mice. Confirming the relevance of these findings to human leishmaniasis, blockade of the NLRP3 inflammasome in skin biopsies from leishmania-infected patients prevented IL-1β release. Thus, these studies link CD8+ T cell cytotoxicity with inflammasome activation and reveal novel avenues of treatment for cutaneous leishmaniasis, as well as other of diseases where CD8+ T cell-mediated cytotoxicity induces pathology.</p></div

Increased IL-1β in <i>L</i>. <i>braziliensis</i> lesions is dependent on CD8 T cell cytotoxicity.

<p>RAG-/- mice were infected with <i>L</i>. <i>braziliensis</i> in the ear, and reconstituted with CD8 T cells or did not receive cells and (a) the course of infection was monitored and representative images of lesions are shown. At 7 weeks post infection mice were euthanized and (b) mRNA levels for <i>IL1a</i> and <i>IL1b</i> were assessed. mRNA data is represented as a fold change (FC) over expression in naïve mice. At 7 weeks post infection, lesions were also digested and used for flow cytometric analysis. Depicted are (c) representative histogram and (d) bar graph of intracellular staining for IL-1β. (e) Ears were cultured for 48 hours and IL-1β release was measured in the supernatants by ELISA. RAG-/- mice were infected with <i>L</i>. <i>braziliensis</i> in the ear, and reconstituted with either WT or perforin-/- CD8 T cells or did not receive cells and (f) course of infection was monitored. At 7 weeks post infection mice were euthanized, lesions were digested and used for flow cytometric analysis. Depicted are (g) representative histogram and (h) bar graph of intracellular staining for IL-1β. Representative data from one of three or more independent experiments (n = 3 to 5 mice per group) with similar results are presented. <i>*p ≤ 0</i>.<i>05</i> or <i>***p ≤ 0</i>.<i>001;</i> ns, non-significant</p

Treatment of mice with NLRP3 inhibitors dampens the immunopathology caused by CD8 T cells.

<p>WT C57BL/6 mice were infected with <i>L</i>. <i>major</i> in the ear, and 2 weeks later mice were co-infected with 2×10⁵ PFU of LCMV Armstrong strain by i.p. injection. Ten days post LCMV infection mice were treated with MCC950, glyburide or vehicle; (a and b) ear thickness was assessed weekly. Five weeks post infection with <i>L</i>. <i>major</i>, mice were euthanized and the lesions were digested and the (c and d) frequency of neutrophils in the skin was determined directly ex vivo by flow cytometry. (e and f) Number of parasites in the skin was determined at 5 weeks post infection with <i>L</i>. <i>major</i>. Results in mice are data from one experiment with 5 mice per group. <i>*p</i> ≤ <i>0</i>.<i>05</i>, *<i>*p<0</i>.<i>01</i> or ***<i>p</i> ≤ <i>0</i>.<i>001</i></p

Immunopathology caused by CD8 T cells is IL-1β-dependent.

<p>RAG-/- mice were infected with <i>L</i>. <i>braziliensis</i> in the ear, and reconstituted with CD8 T cells or did not receive cells and at 2 weeks post infection mice were treated with (a) anti-IL-1R mAb, (b) anti-IL-1β mAb (c) anti-IL-1α mAb or (f) anakinra; ear thickness was assessed weekly. (d) 4 weeks or (g) 6 weeks post infection mice were euthanized and lesions were digested and used for flow cytometric analysis of intracellular IFN-γ and GzmB on CD8 T cells. (e and h) Parasite burden in the lesions. Graphs are data combined from 2 independent experiments (n = 3 to 5 mice per group in each experiment). C57BL/6 mice were infected with <i>L</i>. <i>major</i> in the ear, and 2 weeks later mice were co-infected with 2×10⁵ PFU of LCMV Armstrong strain by i.p. injection. Ten days post LCMV infection mice were treated with anakinra or were left untreated; (i) ear thickness was assessed weekly. Five weeks post infection with <i>L</i>. <i>major</i>, mice were euthanized and the (j) number of parasites in the skin was determined and lesions were digested and used for flow cytometric analysis of intracellular IFN-γ, and GzmB on (k) CD4 T cells or (l) CD8 T cells. Graphs are data from 2 independent experiments (n = 5 mice per group) with similar results are presented. <i>*p ≤ 0</i>.<i>05</i>, <i>**p ≤ 0</i>.<i>01</i> or <i>***p ≤ 0</i>.<i>001;</i> ns, non-significant</p

TNF enhances MMP-9 production in CL patients.

<p><i>A</i>, PBMC from healthy subjects (HS) <i>(n = 12)</i> and CL patients (<i>n = 19)</i> were cultured in the presence or absence of SLA (5 µg/ml) for 72 hours, and TNF concentrations were determined by ELISA on supernatants. <i>B</i>, PBMC from HS (<i>n = 5</i>) were cultured in the presence or absence of recombinant TNF (5 ng/ml) for 24 hours and the levels of MMP-9 were determined by ELISA. <i>C</i>, PBMC from HS <i>(n = 6)</i> and CL patients <i>(n = 6)</i> was cultured in presence or absence of anti-TNF antibodies (10 µg/ml) for 24 hours. The levels of MMP-9 were determined by ELISA on supernatants. *p<0.05; ***p<0.0005.</p

CL patients have high levels of MMP-9 and low levels of TIMP-1 when compared with healthy subjects.

<p>PBMC were obtained from healthy subjects (HS) (<i>n = 12</i>), early CL (ECL) (<i>n = 19</i>) and CL patients (<i>n = 18</i>), and levels of MMP-9 (<i>A</i>) and TIMP-1 (<i>B</i>), determined by ELISA in culture supernatants of PBMCs upon stimulation with soluble <i>Leishmania</i> antigen (SLA) (5 µg/ml) for 72 hours. <i>C</i>, Ratio between MMP-9 and TIMP-1 in culture supernatants of PBMCs upon stimulation with SLA. The box-and-wickers plot shows the minimum, first quartile, median, third quartile, and maximum values of a set of data. The wickers represent error bars and those represent minimum and maximum values, indicating variability outside the upper and lower quartiles. Data shown from a representative experiment of five performances. *p<0.05; **p<0.005; ***p<0.0005.</p

Monocytes are important source of MMP-9 in CL.

<p>PBMC were obtained from healthy subjects (HS) and CL patients and intracellular labeling to MMP-9 was performed <i>ex-vivo</i>. The frequency of CD4+, CD8+ and CD14+ cells positive to MMP-9 was determined by flow cytometry. The gate strategy was done using all minus one staining. <i>A</i>, representative plots from HS (<i>n</i> = 5). <i>B</i>, representative plots from CL patients (<i>n</i> = 5).</p