Localized skin inflammation during cutaneous leishmaniasis drives a chronic, systemic IFN-γ signature

Abstract

Cutaneous leishmaniasis is a localized infection controlled by CD4+ T cells that produce IFN-γ within lesions. Phagocytic cells recruited to lesions, such as monocytes, are then exposed to IFN-γ which triggers their ability to kill the intracellular parasites. Consistent with this, transcriptional analysis of patient lesions identified an interferon stimulated gene (ISG) signature. To determine whether localized L. braziliensis infection triggers a systemic immune response that may influence the disease, we performed RNA sequencing (RNA-seq) on the blood of L. braziliensis-infected patients and healthy controls. Functional enrichment analysis identified an ISG signature as the dominant transcriptional response in the blood of patients. This ISG signature was associated with an increase in monocyte- and macrophage-specific marker genes in the blood and elevated serum levels IFN-γ. A cytotoxicity signature, which is a dominant feature in the lesions, was also observed in the blood and correlated with an increased abundance of cytolytic cells. Thus, two transcriptional signatures present in lesions were found systemically, although with a substantially reduced number of differentially expressed genes (DEGs). Finally, we found that the number of DEGs and ISGs in leishmaniasis was similar to tuberculosis-another localized infection-but significantly less than observed in malaria. In contrast, the cytolytic signature and increased cytolytic cell abundance was not found in tuberculosis or malaria. Our results indicate that systemic signatures can reflect what is occurring in leishmanial lesions. Furthermore, the presence of an ISG signature in blood monocytes and macrophages suggests a mechanism to limit systemic spread of the parasite, as well as enhance parasite control by pre-activating cells prior to lesion entry.

Fig 1. Transcriptional profiling identifies an Interferon-stimulated gene (ISG) signature in the peripheral blood of patients infected with Leishmania braziliensis.

(A) Principal component analysis showing principal component 1 (PC1) and PC2 for RNA-seq data from the peripheral blood of CL patients (yellow triangles, n = 51) and HS (blue circles, n = 14). A PERMANOVA statistical test was used to calculate distances between the groups, Pr(>F) = .006. (B) Gene set enrichment analysis (GSEA) showing normalized enrichment scores (NES) of the top 10 pathways from the Reactome Pathway Database enriched in the peripheral blood of CL patients relative to HS. (C) Volcano plot highlighting overrepresented genes (n = 51) in the peripheral blood from CL patients relative to healthy subjects, FDR≤0.01 and logFC≥1. The Interferome database was used to identify Interferon-stimulated genes (ISGs), represented in red circles (n = 42, 82%). Other genes are colored in blue (n = 9, 18%).

Fig 2. An Interferon-stimulated type II gene (ISG) systemic signature parallel the local immune response in the CL lesion.

(A) Volcano plots with differentially expressed genes in the peripheral blood (left) and lesion (right) in CL patients relative to HS. Genes were colored according to their ISG classification: gray, not an ISG; purple, an ISG exclusively induced by type I Interferons; red, an ISG exclusively induced by type II Interferon; and yellow, an ISG induced by both type I and II Interferons. Horizontal and vertical lines indicate FDR = 0.01 and logFC = 1, respectively). (B) Histograms show the number of overrepresented type I and type II ISGs from volcano plots above in A. (C) IFN-γ levels in the serum of 17 CL patients infected with L. braziliensis and ten healthy subjects (HS). Wilcoxon rank-sum test was used for statistical analysis. *P<0.05. (D) A Spearman correlation between the 51-gene peripheral blood leishmanial signature enrichment score (ES) and the levels of IFN-γ in the serum of CL patients. ρ, Spearman’s rho correlation coefficient; ****P>0.0001.

Fig 3. A cytotoxicity transcriptional signature is observed in the peripheral blood of patients infected with L. braziliensis.

(A) Expression of genes encoding cytolytic granules (GZMA, GZMB, GZMH, GNLY, and PRF1) in the peripheral blood of HS and CL patients. Wilcoxon rank-sum test was used for statistical analysis, *P<0.05, **P<0.01, ***P<0.001 and ****P < .0001. Gene expression is represented as counts per million (CPM) in log2 scale. (B) GSEA enrichment plots showing three cytotoxicity-related pathways from the Biocarta and KEGG Pathway Databases enriched in the CL peripheral blood relative to HS. Pathway name is indicated in parenthesis, next to the pathway database source. Light blue box indicates genes included in the Leading Edge subgroup from each pathway that were enriched and differentially expressed in CL relative to HS. Genes are shown in ranked order according to their running enrichment score. NES, normalized enrichment score; FDR, false discovery rate.

Fig 4. Peripheral blood cytotoxicity and ISG signatures are associated with CTL/NK cells and monocytes/monocyte-like macrophages, respectively.

(A) Median of MCP-counter abundance scores for cell types that were differentially enriched between CL patients and HS. NK cells, cytotoxic lymphocytes (CTL), monocytes and macrophage/monocytes were differentially enriched by Wilcoxon rank-sum test, P<0.05. (B) A correlation matrix between the MCP-counter abundance scores and the expression of the 51 genes overexpressed in the blood of CL patient samples, in log2 CPM. Spearman’s rho (ρ) correlation coefficient ranges from 1 (red) to -1 (yellow). Correlations where P<0.05 are not represented in the plot (blank spots).

Fig 5. A peripheral blood signature of cytotoxicity distinguishes CL from malaria and TB patients.

(A) Principal component analysis showing principal component 1 (PC1) and PC2 for RNA-seq data from the peripheral blood of patients with active tuberculosis (left, n = 21) and malaria (right, n = 65) and study controls (HS in blue, n = 12 and n = 16, respectively). (B) Top, Volcano plots showing DEGs in TB and malaria, with type I and II ISGs indicated (horizontal and vertical lines denote FDR = 0.01 and logFC = 1, respectively). Bottom, the number of overrepresented genes in the blood of active TB and malaria patients. (C) CL peripheral blood enrichment score per sample in the TB dataset and malaria dataset by ssGSEA. Wilcoxon rank-sum test was used for statistical analysis. ****P<0.0001; ns, non-significant.

Fig 6. The CTL/NK cells signature observed in the peripheral blood of CL is unique when compared to the blood of patients with active TB and malaria.

(A) Expression of genes encoding associated with cytolysis (GZMA, GZMB, GZMH, GNLY, and PRF1) in the peripheral blood of patients with active TB (top) and malaria (bottom) and study controls (healthy subjects, HS). Wilcoxon rank-sum test was used for statistical analysis, *P<0.05, **P<0.01, ****P<0.0001. CPM, counts per million log2 scale. (B) Median of MCP-counter scores between patients with active TB or malaria (yellow) and study controls (blue). Wilcoxon rank-sum test was used for statistical analysis, **P<0.01, ****P<0.0001.