Vascular remodeling is a phenomenon seen in the cutaneous lesions formed during infection with Leishmania parasites. Within the lesion, Leishmania major infection leads to the infiltration of inflammatory cells, including macrophages, and is associated with hypoxic conditions and lymphangiogenesis in the local site. This low-oxygen environment is concomitant with the expression of hypoxic inducible factors (HIFs), which initiate the expression of vascular endothelial growth factor-A (VEGF-A) in macrophages during the infection. Here, we found that macrophage hypoxia is elevated in the skin, and the HIF target Vegfa is preferentially expressed at the site of infection. Furthermore, transcripts indicative of both HIF-1α and HIF-2α activation were increased at the site of infection. Given that HIF mediates VEGF-A and that VEGF-A/VEGFR-2 signaling induces lymphangiogenesis, we wanted to investigate the link between myeloid HIF activation and lymphangiogenesis during L. major infection. We show that myeloid aryl hydrocarbon receptor nuclear translocator (ARNT)/HIF/VEGF-A signaling promotes lymphangiogenesis (the generation of newly formed vessels within the local lymphatic network), which helps resolve the lesion by draining away inflammatory cells and fluid. Concomitant with impaired lymphangiogenesis, we find the deletion of myeloid ARNT/HIF signaling leads to an exacerbated inflammatory response associated with a heightened CD4+ Th1 immune response following L. major infection. Altogether, our data suggest that VEGF-A-mediated lymphangiogenesis occurs through myeloid ARNT/HIF activation following Leishmania major infection and this process is critical in limiting immunopathology.

Leishmanial skin lesions are characterized by inflammatory hypoxia alongside the activation of hypoxia-inducible factors, HIF-1α and HIF-2α, and subsequent expression of the HIF-α target VEGF-A during Leishmania major infection. However, the factors responsible for HIF-α activation are not known. We hypothesize that hypoxia and proinflammatory stimuli contribute to HIF-α activation during infection. RNA-Seq of leishmanial lesions revealed that transcripts associated with HIF-1α signaling were induced. To determine whether hypoxia contributes to HIF-α activation, we followed the fate of myeloid cells infiltrating from the blood and into hypoxic lesions. Recruited myeloid cells experienced hypoxia when they entered inflamed lesions, and the length of time in lesions increased their hypoxic signature. To determine whether proinflammatory stimuli in the inflamed tissue can also influence HIF-α activation, we subjected macrophages to various proinflammatory stimuli and measured VEGF-A. While parasites alone did not induce VEGF-A, and proinflammatory stimuli only modestly induced VEGF-A, HIF-α stabilization increased VEGF-A during infection. HIF-α stabilization did not impact parasite entry, growth, or killing. Conversely, the absence of ARNT/HIF-α signaling enhanced parasite internalization. Altogether, these findings suggest that HIF-α is active during infection, and while macrophage HIF-α activation promotes lymphatic remodeling through VEGF-A production, HIF-α activation does not impact parasite internalization or control.

Leishmanial skin lesions are characterized by inflammatory hypoxia alongside the activation of hypoxia inducible factors, HIF-1a and HIF-2a, and subsequent expression of the HIF-a target VEGF-A during Leishmania major infection. However, the factors responsible for HIF-a activation are not known. We hypothesize hypoxia and pro-inflammatory stimuli contribute to HIF-a activation during infection. RNASeq on leishmanial lesions found transcripts associated with HIF-1a signaling are induced. To determine whether hypoxia contributes to HIF-a activation, we followed the fate of myeloid cells infiltrating from the blood and into hypoxic lesions. Recruited myeloid cells experience hypoxia when they enter inflamed lesions, and the length of time in lesions increases their hypoxic signature. To determine whether pro-inflammatory stimuli in the inflamed tissue can also influence HIF-a activation, we subjected macrophages to various pro-inflammatory stimuli and measured VEGF-A. While parasites alone did not induce VEGF-A, and pro-inflammatory stimuli only modestly induce VEGF-A, HIF- stabilization increases VEGF-A during infection. HIF-a stabilization does not impact parasite entry, growth or killing. Alternatively, the absence of ARNT/HIF- signaling enhances parasite internalization. Altogether, these findings suggest HIF-a is active during infection, and while macrophage HIF-a activation promotes lymphatic remodeling through VEGF-A production, HIF-a activation does not impact parasite internalization or control.