Fig 1.
M. leprae infection leads to induction of cell fate pathways in MDMs.
(A). Heatmap of clustered genes induced by M. leprae at different time points. Color green indicates downregulated genes and color red indicates upregulated genes. (B). Top Canonical Pathways significantly enriched in the M. leprae induced-gene signature by IPA core analysis. (C). Most expressed upstream regulators by fold-change (FC) in the M. leprae induced-gene signature. (D). IPA Disease and Functions analysis of the M. leprae induced-gene signature. The p-value is calculated by Fisher’s Exact Test and measures the significant overlap between the dataset genes and the genes that belong to a canonical pathway, upstream regulator or the ‘Disease and Function’ categories in the IPA knowledge database. Adjusted p-values (padj) were calculated using Bonferroni correction. Ratios represent the number of genes in the dataset that appear in an IPA term divided by the total number of genes of the same term. Genes of the canonical pathway, upstream regulator or ‘Disease and Functions’ analyses were selected based on their functional relevance and displayed in boxes. # genes represent the exact number of molecules in our dataset regulated by an upstream regulator or found in the Disease and Functions categories.
Fig 2.
M. leprae infection induces NUPR1 gene expression in MDMs.
(A). NUPR1 DESeq normalized counts fold change at different time points post-M. leprae infection in MDMs. (B). NUPR1 gene expression measured by qPCR in M. leprae infected MDMs at different time points (n = 8). Statistical analyses were performed using repeated measures one way-ANOVA with the Geisser-Greenhouse correction followed by Bonferroni’s multiple comparisons test.
Fig 3.
NUPR1 gene expression is induced by M. leprae and type I interferon.
(A). Overlap of the M. leprae induced gene signature (fold change >2) with IFN-β and IFN-γ specific gene signatures from cytokine-stimulated MDMs. (B). Number of IFN-β and IFN-γ specific genes found in the M. leprae induced gene signature. (C). Fold change enrichment (see Materials and methods) of IFN-β and IFN-γ specific genes found in the M. leprae induced gene signature. (D). -log10 enrichment p-value of IFN-β and IFN-γ specific genes found in the M. leprae induced gene signature calculated by hypergeometric test. (E). NUPR1 fold change of DESeq normalized counts from RNA-seq data of MDMs stimulated with IFN-β and IFN-γ (n = 5) at 24h. (F). NUPR1 gene expression fold change in IFN-β (285 u/ml) and IFN-γ (1.5 ng/ml) stimulated MDMs measured by qPCR at 2, 6 and 24h (n = 5). Statistical analyses were performed using the Two-Sample T test (E) and repeated measures two way-ANOVA with the Geisser-Greenhouse correction followed by Bonferroni’s multiple comparisons test (F).
Fig 4.
M. leprae induction of NUPR1 is dependent on type I IFN signaling.
(A). Effect of different doses of IFN-β on the induction of NUPR1 gene expression in MDMs measured by qPCR (n = 4). (B). Effect of IFNAR blocking with different doses of IFN-β on NUPR1 gene expression in MDMs measured by qPCR (n = 3). (C) and (D). Evaluation of the effect of M. leprae infection on NUPR1 gene expression during blockage of IFN-β signaling at 24 and 48h measured by qPCR (n = 6). Statistical analyses were performed using repeated measures one way-ANOVA (A), repeated measures two way-ANOVA with the Geisser-Greenhouse correction (B) and repeated measures two way-ANOVA (C and D) followed by Bonferroni’s multiple comparisons test (A-D).
Fig 5.
NUPR1 is highly expressed in L-lep skin lesions.
(A). Overlap of M. leprae induced gene signature (Fold change >2) with lepromatous (L-lep) and tuberculoid (T-lep) skin lesion specific gene signatures (FC>2; p<0.05; probe intensity average>100) from leprosy skin lesion microarray data [8]. (B). NUPR1 normalized probe intensity in leprosy lesion microarray data in L-lep and T-lep samples. (C). NUPR1 gene expression in L-lep and T-lep skin lesions measured by qPCR (n = 5). (D). NUPR1 protein expression in L-lep (n = 4) and T-lep (n = 4) skin lesions measured by immunohistochemistry. Scale bars (50μm), original magnification 400x. (E). Quantification of NUPR1 staining in L-lep and T-lep skin lesions by ImmunoRatio. Statistical analyses were performed using the Welch’s T test (B and C) and the Two-sample t-test (E).