HIF-α signaling regulates the macrophage inflammatory response during Leishmania major infection

Abstract

Cutaneous leishmaniasis (CL) contributes significantly to the global burden of neglected tropical diseases, with 12 million people currently infected with Leishmania parasites. CL encompasses a range of disease manifestations, from self-healing skin lesions to permanent disfigurations. Currently there is no vaccine available, and many patients are refractory to treatment, emphasizing the need for new therapeutic targets. Previous work demonstrated macrophage HIF-α-mediated lymphangiogenesis is necessary to achieve efficient wound resolution during murine L. major infection. Here, we investigate the role of macrophage HIF-α signaling independent of lymphangiogenesis. We sought to determine the relative contributions of the parasite and the host-mediated inflammation in the lesional microenvironment to myeloid HIF-α signaling. Because HIF-α activation can be detected in infected and bystander macrophages in leishmanial lesions, we hypothesize it is the host's inflammatory response and microenvironment, rather than the parasite, that triggers HIF-α activation. To address this, macrophages from mice with intact HIF-α signaling (LysM^CreARNT^f/+) or mice with deleted HIF-α signaling (LysM^CreARNT^f/f) were subjected to RNASequencing after L. major infection and under pro-inflammatory stimulus. We report that L. major infection alone is enough to induce some minor HIF-α-dependent transcriptomic changes, while infection with L. major in combination with pro-inflammatory stimuli induces numerous transcriptomic changes that are both dependent and independent of HIF-α signaling. Additionally, by coupling transcriptomic analysis with several pathway analyses, we found HIF-α suppresses pathways involved in protein translation during L. major infection in a pro-inflammatory environment. Together these findings show L. major induces a HIF-α-dependent transcriptomic program, but HIF-α only suppresses protein translation in a pro-inflammatory environment. Thus, this work indicates the host inflammatory response, rather than the parasite, largely contributes to myeloid HIF-α signaling during Leishmania infection.

Keywords: HIF - 1α; leishmania; leishmaniasis; macrophages; translation.

Figure 1

Single-cell RNASequencing (scRNASeq) shows HIF-α transcriptional targets are elevated in murine lesions during L. major infection. C57BL/6 mice were infected or not with 2×10⁶ L. major parasites intradermally in the ear. At 4 weeks, infected ears and naïve uninfected control ears were digested and subjected to scRNASeq as a part of a previous study (28). (A) Uniform Manifold Approximation and Projection (UMAP) plot revealed 35 distinct cell clusters. Seurat’s FindClusters function identified each cell cluster and cell type designation to the right. To initially define cell clusters both naive and infected groups were combined, but here naive and infected groups are shown to specify transcript expression under each condition. (B) Feature plots of expression distribution for HIF-1α and HIF-2α (gene Epas1). (C) Feature plots of expression distribution for HIF-1α-specific target genes (Nos2, Pgk1, and Ldha). (D) Feature plots of expression distribution for HIF-2α-specific target genes (Arg1 and Oct4 (gene Pou5f1)). Expression levels for each gene are color-coded and overlaid onto UMAP plot. Cells with the highest expression level are colored dark purple.

Figure 2

Infection with L. major induces transcriptional changes that are absent in infected macrophages deficient for HIF-α signaling. Macrophages infected with L. major were lysed and prepped for RNASequencing. (A) A mean-difference plot (MD plot) depicts transcripts upregulated (red) and downregulated (blue) with infection in macrophages with intact HIF-α signaling (ARNT^f/+ P vs. ARNT^f/+) where P indicates parasites. (B) An MD plot shows upregulated and downregulated transcripts in infected macrophages deficient for HIF-α signaling (ARNT^f/f P vs. ARNT^f/f). (C, D) KEGG analysis was performed to identify the top enriched pathways during infection with L. major in macrophages with or without HIF-α signaling. Upregulated pathways are shown in red and downregulated pathways are shown in blue.

Figure 3

Inflammation related transcriptomic changes are both HIF-α dependent and independent. Macrophages were infected or infected and treated with LPS/IFNγ and prepped for subsequent analysis utilizing RNASeq. (A, B) An MD plot illustrates transcriptomic changes during infection and stimulation with LPS/IFNγ in HIF-α competent (ARNT^f/+ PI vs. ARNT^f/+P) and HIF-α deficient macrophages (ARNT^f/f PI vs. ARNT^f/f P). Here the PI indicates parasites and inflammatory stimuli, LPS/IFNγ, and P describes parasite infection alone. Red dots identify upregulated transcripts and blue dots identify downregulated transcripts. (C, D) Enriched pathways were identified using KEGG analysis for both comparisons. Red pathways indicate upregulation while blue pathways are downregulated.

Figure 4

HIF-α mediates DEGs induced by L. major parasites. Macrophages both with and without HIF-α signaling were cultured in media alone or infected with L. major parasites for 8 hours before being prepped for RNASequencing. (A) An MD plot illustrates transcripts inhibited by HIF-α under basal conditions. (B) An MD plot shows transcripts mediated by HIF-α during L. major infection. (C) KEGG pathway analysis identified enriched pathways in macrophages without HIF-α signaling under basal conditions. (D) KEGG pathway analysis identified enriched pathways in macrophages without HIF-α signaling during infection. Red pathways are upregulated and blue pathways are downregulated.

Figure 5

HIF-α signaling suppresses translational pathways under inflammatory conditions. RNASeq and pathway analysis on macrophages with and without intact HIF-α signaling infected with L. major and treated with pro-inflammatory stimuli. (A) DEGs upregulated (red) and downregulated (blue) in infected macrophages treated with LPS/IFNγ without HIF-α signaling compared to macrophages with intact HIF-α signaling under the same conditions. (B) KEGG analysis identified enriched pathways in infected macrophages without HIF-α signaling stimulated with LPS/IFNγ. (C) MSigDB pathway analysis defined upregulated pathways in red and downregulated pathways in blue in the infected macrophages without HIF-α signaling compared to macrophages with intact HIF-α signaling. (D) Ingenuity pathway analysis (IPA) was run to determine upregulated and downregulated pathways (red and blue respectively). (E) Heatmap plots of each upregulated or downregulated pathway defined by the IPA with individual altered DEGs represented in each pathway.

Figure 6

HIF-α stabilization decreases macrophage translation during L. major infection. Macrophages derived from C57BL/6 mice were cultured in media, infected with L. major, treated with DMOG, or infected and treated with DMOG before RNA was isolated and prepped for quantitative PCR. (A) Relative expression of ribosomal protein transcripts is shown for macrophages infected or not and treated or not with DMOG. Data is pooled from two independent experiments. (B) Macrophages were cultured in media, with or without L. major, and with or without DMOG and labeled with puromycin to assess translation activity via flow cytometry. Representative flow plots of Puro⁺ macrophages. Macrophages were gated as CD45⁺CD11b⁺CD64⁺Ly6G^-. (C) Quantification of (B). Data is pooled from two experiments where n=10. Significance was determined using a student’s unpaired t-test *p<0.05, **p<0.01, ***p<0.001 and for (C) significance is relative to the DMOG + L. major group.