Systems Approach Reveals Nuclear Factor Erythroid 2-Related Factor 2/Protein Kinase R Crosstalk in Human Cutaneous Leishmaniasis

Abstract

<i>Leishmania</i> parasites infect macrophages, causing a wide spectrum of human diseases, from cutaneous to visceral forms. In search of novel therapeutic targets, we performed comprehensive <i>in vitro</i> and <i>ex vivo</i> mapping of the signaling pathways upstream and downstream of antioxidant transcription factor [nuclear factor erythroid 2-related factor 2 (Nrf2)] in cutaneous leishmaniasis (CL), by combining functional assays in human and murine macrophages with a systems biology analysis of <i>in situ</i> (skin biopsies) CL patient samples. First, we show the PKR pathway controls the expression and activation of Nrf2 in <i>Leishmania amazonensis</i> infection <i>in vitro</i>. Nrf2 activation also required PI3K/Akt signaling and autophagy mechanisms. Nrf2- or PKR/Akt-deficient macrophages exhibited increased levels of ROS/RNS and reduced expression of <i>Sod1</i> Nrf2-dependent gene and reduced parasite load. <i>L. amazonensis</i> counteracted the Nrf2 inhibitor Keap1 through the upregulation of p62 <i>via</i> PKR. This Nrf2/Keap1 observation was confirmed <i>in situ</i> in skin biopsies from <i>Leishmania</i>-infected patients. Next, we explored the <i>ex vivo</i> transcriptome in CL patients, as compared to healthy controls. We found the antioxidant response element/Nrf2 signaling pathway was significantly upregulated in CL, including downstream target p62. <i>In silico</i> enrichment analysis confirmed upstream signaling by interferon and PI3K/Akt, and validated our <i>in vitro</i> findings. Our integrated <i>in vitro, ex vivo</i>, and <i>in silico</i> approach establish Nrf2 as a central player in human cutaneous leishmaniasis and reveal Nrf2/PKR crosstalk and PI3K/Akt pathways as potential therapeutic targets.