We investigated the type I interferon (IFN‐1)/PKR axis in the outcome of the Leishmania (Leishmania) amazonensis infection, along with the underlying mechanisms that trigger and sustain this signaling pathway. Reporter assays of cell extracts from RAW‐264.7 macrophages infected with L. (L.) amazonensis or HEK‐293T cells cotransfected with TLR2 and PKR promoter constructions were employed. Primary macrophages of TLR2‐knockout (KO) or IFNR‐KO mice were infected, and the levels of PKR, IFN‐1, and superoxide dismutase 1 (SOD1) transcript levels were investigated and compared. Immunohistochemical analysis of human biopsy lesions was evaluated for IFN‐1 and PKR‐positive cells. Leishmania infection increased the expression of PKR and IFN‐β on induction of PKR‐promoter activity. The observed effects required the engagement of TLR2. TLR2‐KO macrophages expressed low IFN‐β and PKR levels postinfection with a reduced parasite load. We also revealed the requirement of PKR signaling for Leishmania‐ induced IFN‐1 expression, responsible for sustaining PKR expression and enhancing infection. Moreover, during infection, SOD1 transcripts increased and were also enhanced when IFN‐1 was added to the cultures. Remarkably, SOD1 expression was abrogated in infected, dominant‐negative PKR‐expressing cells. Finally, lesions of patients with anergic diffuse cutaneous leishmaniasis exhibited higher levels of PKR/IFN‐1‐expressing cells compared to those with single cutaneous leishmaniasis. In summary, we demonstrated the mechanisms and relevance of the IFN‐1/PKR axis in the Leishmania infection.—De Carvalho Vivarini, A., Pereira, R. M. S., Dias Teixeira, K. L., Calegari‐Silva, T. C., Bellio, M., Laurenti, M. D., Corbett, C. E. P., de Castro Gomes, C. M., Soares, R. P., Mendes Silva, A., Silveira, F. T., Lopes, U. G. Human cutaneous leishmaniasis: interferon‐dependent expression of double‐stranded RNA‐kinase (PKR) via TLR2. FASEB J. 25, 4162–4173 (2011). www.fasebj.org

The evolution of Leishmania infection depends on the balance between microbicidal and suppressor macrophage functions. Double‐stranded RNA (dsRNA)‐activated protein kinase R (PKR), a classic antiviral protein, is able to regulate a number of signaling pathways and macrophage functions. We investigated the possible role of PKR in the modulation of Leishmania infection. Our data demonstrated that Leishmania amazonensis infection led to PKR activation and increased PKR levels. Consistently, in macrophages from PKR knockout 129Sv/Ev mice and RAW‐264.7 cells stably expressing a dominant‐negative (DN) construct of PKR (DN‐PKR), L. amazonensis infection was strongly reduced. The treatment of infected macrophages with the synthetic double‐stranded RNA poly(I:C), a potent PKR inductor, increased L. amazonensis intracellular proliferation. This effect was reversed by 2‐aminopurine (2‐AP), a pharmacological inhibitor of PKR, as well as by the expression of DN‐PKR. NO release induced by dsRNA treatment was inhibited by L. amazonensis through NF‐κB modulation. PKR activation induced by dsRNA also resulted in IL‐10 production, whose neutralization with specific antibody completely abrogated L. amazonensis proliferation. Our data demonstrated a new role of PKR in protozoan parasitic infection through IL‐10 modulation.—Pereira, R. M. S., Teixeira, K. L. D., Barreto‐de‐Souza, V. Calegari‐Silva, T. C., De‐Melo, D. B., Soares, D. C., Bou‐Habib, D. C., Silva, A. M., Saraiva, E. M., Lopes, U. G. Novel role for the double‐stranded RNA‐activated protein kinase PKR: modulation of macrophage infection by the protozoan parasite Leishmania. FASEB J . 24, 617–626 (2010). www.fasebj.org

Leishmania parasites infect macrophages, causing a wide spectrum of clinical manifestations ranging from cutaneous to visceral forms. In search of novel therapeutic targets, we performed comprehensive in vitro and ex vivo mapping of the signaling pathways upstream and downstream of antioxidant transcription factor Nrf2 in cutaneous leishmaniasis (CL), by combining functional assays in human and murine macrophages with a systems biology analysis of in situ (skin biopsies) CL patient samples. First, we show the PKR pathway controls the expression and activation of Nrf2 in Leishmania amazonensis infection in vitro. 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 Sod1 Nrf2-dependent gene and reduced parasite load. L. amazonensis counteracted the Nrf2 inhibitor Keap1 through the up-regulation of p62 via PKR. This Nrf2/Keap1 observation was confirmed in situ in skin biopsies from Leishmania-infected patients. Next, we explored the ex vivo transcriptome in CL patients, as compared to healthy controls. We found the ARE/Nrf2 signaling pathway was significantly upregulated in CL, including downstream target p62. In silico enrichment analysis confirmed upstream signaling by Interferon and PI3K/Akt, and validated our in vitro findings. Our integrated in vitro, ex vivo and in silico approach establish Nrf2 as a central player in human cutaneous leishmaniasis and reveal Nrf2/PKR crosstalk and PI3K/Akt pathways as potential therapeutic targets.

Leishmania amazonensis activates the NF-κB transcriptional repressor homodimer (p50/p50) and promotes nitric oxide synthase (iNOS) downregulation. We investigated the role of PI3K/Akt in p50/p50 NF-κB activation and the effect on iNOS expression in L. amazonensis infection. The increased occupancy of p50/p50 on the iNOS promoter of infected macrophages was observed and we demonstrated that both p50/p50 NF-κB induction and iNOS downregulation in infected macrophages depended on PI3K/Akt activation. Importantly, the intracellular growth of the parasite was also impaired during PI3K/Akt signalling inhibition and in macrophages knocked-down for Akt 1 expression. It was also observed that the increased nuclear levels of p50/p50 in L. amazonensis -infected macrophages were associated with reduced phosphorylation of 907 Ser p105, the precursor of p50. Corroborating these data, we demonstrated the increased levels of phospho- 9 Ser GSK3β in infected macrophages, which is associated with GSK3β inhibition and, consequently, its inability to phosphorylate p105. Remarkably, we found that the levels of pPTEN 370 Ser , a negative regulator of PI3K, increased due to L. amazonensis infection. Our data support the notion that PI3K/Akt activity is sustained during the parasite infection, leading to NF-κB 105 phosphorylation and further processing to originate p50/p50 homodimers and the consequent downregulation of iNOS expression.

Endoplasmic reticulum (ER) stress triggers the integrated ER‐stress response (IERSR) that ensures cellular survival of ER stress and represents a primordial form of innate immunity. We investigated the role of IERSR during Leishmania amazonensis infection. Treatment of RAW 264.7 infected macrophages with the ER stress‐inducing agent thapsigargin (TG; 1 μM) increased L. amazonensis infectivity in an IFN1‐α receptor (IFNAR)‐dependent manner. In Western blot assays, we showed that L. amazonensis activates the inositol‐requiring enzyme (IRE1)/ X‐box binding protein (XBP)‐1‐splicing arms of the IERSR in host cells. In chromatin immunoprecipitation (ChIP) assays, we showed an increased occupancy of enhancer and promoter sequences for the Ifnb gene by XBP1 in infected RAW 264.7 cells. Knocking down XBP1 expression by transducing RAW 264.7 cells with the short hairpin XBP1 lentiviral vector significantly reduced the parasite proliferation associated with impaired translocation of phosphorylated IFN regulatory transcription factor (IRF)‐3 to the nucleus and a decrease in IFN1‐β expression. Knocking down XBP1 expression also increased NO concentration, as determined by Griess reaction and reduced the expression of antioxidant genes, such as heme oxygenase (HO)‐1, that protect parasites from oxidative stress. We conclude that L. amazonensis activation of XBP1 plays a critical role in infection by protecting the parasites from oxidative stress and increasing IFN1‐β expression.—Dias‐Teixeira, K. L., Calegari‐Silva, T. C., Dos Santos, G. R. R. M., Vitorino dos Santos, J., Lima, C., Medina, J. M., Aktas, B. H., Lopes, U. G. The integrated endoplasmic reticulum stress response in Leishmania amazonensis macrophage infection: the role of X‐box binding protein 1 transcription factor. FASEB J. 30, 1557–1565 (2016). www.fasebj.org