Role of protein kinase R in the killing of Leishmania major by macrophages in response to neutrophil elastase and TLR4 via TNF and IFN

In cutaneous leishmaniasis, Leishmania amazonensis activates macrophage double-stranded, RNA-activated protein kinase R (PKR) to promote parasite growth. In our study, Leishmania major grew normally in RAW cells, RAW-expressing dominant-negative PKR (PKR-DN) cells, and macrophages of PKR-knockout mice, revealing that PKR is dispensable for L. major growth in macrophages. PKR activation in infected macrophages with poly I:C resulted in parasite death. Fifty percent of L. major-knockout lines for the ecotin-like serine peptidase inhibitor (ISP2; Δisp2/isp3), an inhibitor of neutrophil elastase (NE), died in RAW cells or macrophages from 129Sv mice, as a result of PKR activation. Inhibition of PKR or NE or neutralization of Toll-like receptor 4 or 2(TLR4 or TLR2) prevented the death of Δisp2/isp3. Δisp2/isp3 grew normally in RAW-PKR-DN cells or macrophages from 129Sv pkr−/−, tlr2−/−, trif−/−, and myd88−/− mice, associating NE activity, PKR, and TLR responses with parasite death. Δisp2/isp3 increased the expression of mRNA for TNF-α by 2-fold and of interferon β (IFNβ) in a PKR-dependent manner. Antibodies to TNF-α reversed the 95% killing by Δisp2/isp3, whereas they grew normally in macrophages from IFN receptor–knockout mice. We propose that ISP2 prevents the activation of PKR via an NE-TLR4-TLR2 axis to control innate responses that contribute to the killing of L. major.—Faria, M. S., Calegari-Silva, T. C., de Carvalho Vivarini, A., Mottram, J. C., Lopes, U. G., Lima, A. P. C. A. Role of protein kinase R in the killing of Leishmania major by macrophages in response to neutrophil elastase and TLR4 via TNFα and IFNβ

Lutzomyia longipalpis Saliva Induces Heme Oxygenase-1 Expression at Bite Sites

Sand flies bite mammalian hosts to obtain a blood meal, driving changes in the host inflammatory response that support the establishment of Leishmania infection. This effect is partially attributed to components of sand fly saliva, which are able to recruit and activate leukocytes. Our group has shown that heme oxygenase-1 (HO-1) favors Leishmania survival in infected cells by reducing inflammatory responses. Here, we show that exposure to sand fly bites is associated with induction of HO-1 in vivo. Histopathological analyses of skin specimens from human volunteers experimentally exposed to sand fly bites revealed that HO-1 and Nrf2 are produced at bite sites in the skin. These results were recapitulated in mice ears injected with a salivary gland sonicate (SGS) or exposed to sand fly bites, indicating that vector saliva may be a key factor in triggering HO-1 expression. Resident skin macrophages were the main source HO-1 at 24–48 h after bites. Additionally, assays in vivo after bites and in vitro after stimulation with saliva both demonstrated that HO-1 production by macrophages was Nrf2-dependent. Collectively, our data demonstrates that vector saliva induces early HO-1 production at the bite sites, representing a major event associated with establishment of naturally-transmitted Leishmania infections

HIV-1 Tat protein enhances the intracellular growth of Leishmania amazonensis via the ds-RNA induced protein PKR

Submitted by sandra infurna ([email protected]) on 2016-04-05T13:40:01Z No. of bitstreams: 1 jairo_temerozo_etal_IOC_2015.pdf: 1066333 bytes, checksum: 1894875d5be6eb5dea98e3f11ac0ce98 (MD5)Approved for entry into archive by sandra infurna ([email protected]) on 2016-04-05T13:55:25Z (GMT) No. of bitstreams: 1 jairo_temerozo_etal_IOC_2015.pdf: 1066333 bytes, checksum: 1894875d5be6eb5dea98e3f11ac0ce98 (MD5)Made available in DSpace on 2016-04-05T13:55:25Z (GMT). No. of bitstreams: 1 jairo_temerozo_etal_IOC_2015.pdf: 1066333 bytes, checksum: 1894875d5be6eb5dea98e3f11ac0ce98 (MD5) Previous issue date: 2015Universidade Federal do Rio Janeiro. Centro de Ciências da Saúde. Instituto de Biofísica Carlos Chagas Filho. Laboratório de Parasitologia Molecular. Rio de Janeiro, RJ, Brasil.Universidade Federal do Rio Janeiro. Centro de Ciências da Saúde. Instituto de Biofísica Carlos Chagas Filho. Laboratório de Parasitologia Molecular. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Pesquisa sobre o Timo. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Pesquisa sobre o Timo. Rio de Janeiro, RJ, Brasil.Universidade Federal do Rio Janeiro. Instituto de Microbiologia Paulo Góes. Laboratório de Imunobiologia de Leishmanioses. Rio de Janeiro, RJ, Brasil.Universidade Federal do Rio Janeiro. Instituto de Microbiologia Paulo Góes. Laboratório de Imunobiologia de Leishmanioses. Rio de Janeiro, RJ, Brasil.Universidade do Estado do Rio de Janeiro. Faculdade de Ciências Médicas. Departamento de Microbiologia. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Pesquisa sobre o Timo. Rio de Janeiro, RJ, Brasil.Universidade Federal do Rio Janeiro. Centro de Ciências da Saúde. Instituto de Biofísica Carlos Chagas Filho. Laboratório de Parasitologia Molecular. Rio de Janeiro, RJ, Brasil.HIV-1 co-infection with human parasitic diseases is a growing public health problem worldwide. Leishmania parasites infect and replicate inside macrophages, thereby subverting host signaling pathways, including the response mediated by PKR. The HIV-1 Tat protein interacts with PKR and plays a pivotal role in HIV-1 replication. This study shows that Tat increases both the expression and activation of PKR in Leishmania-infected macrophages. Importantly, the positive effect of Tat addition on parasite growth was dependent on PKR signaling, as demonstrated in PKR-deficient macrophages or macrophages treated with the PKR inhibitor. The effect of HIV-1 Tat on parasite growth was prevented when the supernatant of HIV-1-infected macrophages was treated with neutralizing anti-HIV-1 Tat prior to Leishmania infection. The addition of HIV-1 Tat to Leishmania-infected macrophages led to inhibition of iNOS expression, modulation of NF-kB activation and enhancement of IL-10 expression. Accordingly, the expression of a Tat construct containing mutations in the basic region (49-57aa), which is responsible for the interaction with PKR, favored neither parasite growth nor IL-10 expression in infected macrophages. In summary, we show that Tat enhances Leishmania growth through PKR signaling

Mycobacterium leprae-induced Insulin-like Growth Factor I attenuates antimicrobial mechanisms, promoting bacterial survival in macrophages

Submitted by sandra infurna ([email protected]) on 2016-07-02T23:45:33Z No. of bitstreams: 1 katherine_mattos_etal_IOC_2016.pdf: 1295630 bytes, checksum: f98c045639477298b4060493549ed5f8 (MD5)Approved for entry into archive by sandra infurna ([email protected]) on 2016-07-03T00:00:26Z (GMT) No. of bitstreams: 1 katherine_mattos_etal_IOC_2016.pdf: 1295630 bytes, checksum: f98c045639477298b4060493549ed5f8 (MD5)Made available in DSpace on 2016-07-03T00:00:26Z (GMT). No. of bitstreams: 1 katherine_mattos_etal_IOC_2016.pdf: 1295630 bytes, checksum: f98c045639477298b4060493549ed5f8 (MD5) Previous issue date: 2016Submitted by Angelo Silva ([email protected]) on 2016-07-07T11:16:57Z No. of bitstreams: 3 katherine_mattos_etal_IOC_2016.pdf.txt: 65894 bytes, checksum: e5afd9deb877b4e5fae199967e48b1f7 (MD5) katherine_mattos_etal_IOC_2016.pdf: 1295630 bytes, checksum: f98c045639477298b4060493549ed5f8 (MD5) license.txt: 2991 bytes, checksum: 5a560609d32a3863062d77ff32785d58 (MD5)Approved for entry into archive by sandra infurna ([email protected]) on 2016-07-07T12:20:07Z (GMT) No. of bitstreams: 3 license.txt: 2991 bytes, checksum: 5a560609d32a3863062d77ff32785d58 (MD5) katherine_mattos_etal_IOC_2016.pdf: 1295630 bytes, checksum: f98c045639477298b4060493549ed5f8 (MD5) katherine_mattos_etal_IOC_2016.pdf.txt: 65894 bytes, checksum: e5afd9deb877b4e5fae199967e48b1f7 (MD5)Made available in DSpace on 2016-07-07T12:20:07Z (GMT). No. of bitstreams: 3 license.txt: 2991 bytes, checksum: 5a560609d32a3863062d77ff32785d58 (MD5) katherine_mattos_etal_IOC_2016.pdf: 1295630 bytes, checksum: f98c045639477298b4060493549ed5f8 (MD5) katherine_mattos_etal_IOC_2016.pdf.txt: 65894 bytes, checksum: e5afd9deb877b4e5fae199967e48b1f7 (MD5) Previous issue date: 2016Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Microbiologia Celular. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Microbiologia Celular. Rio de Janeiro, RJ, Brasil.Universidade Federal do Rio de Janeiro. Instituto Carlos Chagas Filho. Laboratório de Parasitologia Molecular. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Microbiologia Celular. Rio de Janeiro, RJ, BrasilFundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Microbiologia Celular. Rio de Janeiro, RJ, BrasilInstituto Lauro de Souza Lima. Bauru, SP, Brasil.Instituto Lauro de Souza Lima. Bauru, SP, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Hanseníase. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Microbiologia Celular. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Hanseníase. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Hanseníase. Rio de Janeiro, RJ, Brasil.Universidade Federal do Rio de Janeiro. Instituto Carlos Chagas Filho. Laboratório de Parasitologia Molecular. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Microbiologia Celular. Rio de Janeiro, RJ, Brasil.Mycobacterium leprae (ML), the etiologic agent of leprosy, can subvert macrophage antimicrobial activity by mechanisms that remain only partially understood. In the present study, the participation of hormone insulin-like growth factor I (IGF-I) in this phenomenum was investigated. Macrophages from the dermal lesions of the disseminated multibacillary lepromatous form (LL) of leprosy expressed higher levels of IGF-I than those from the self-limited paucibacillary tuberculoid form (BT). Higher levels of IGF-I secretion by ML-infected macrophages were confirmed in ex vivo and in vitro studies. Of note, the dampening of IGF-I signaling reverted the capacity of ML-infected human and murine macrophages to produce antimicrobial molecules and promoted bacterial killing. Moreover, IGF-I was shown to inhibit the JAK/STAT1-dependent signaling pathways triggered by both mycobacteria and IFN-γ most probably through its capacity to induce the suppressor of cytokine signaling-3 (SOCS3). Finally, these in vitro findings were corroborated by in vivo observations in which higher SOCS3 expression and lower phosphorylation of STAT1 levels were found in LL versus BT dermal lesions. Altogether, our data strongly suggest that IGF-I contributes to the maintenance of a functional program in infected macrophages that suits ML persistence in the host, reinforcing a key role for IGF-I in leprosy pathogenesis

Lutzomyia longipalpis Saliva Induces Heme Oxygenase-1 Expression at Bite Sites

Submitted by Ana Maria Fiscina Sampaio ([email protected]) on 2018-12-19T16:23:43Z No. of bitstreams: 1 Luz NF. Lutzomyia longipalpis saliva...2018.pdf: 1955626 bytes, checksum: f84660a8c004e77cec0bb334c37bb074 (MD5)Approved for entry into archive by Ana Maria Fiscina Sampaio ([email protected]) on 2018-12-19T16:46:42Z (GMT) No. of bitstreams: 1 Luz NF. Lutzomyia longipalpis saliva...2018.pdf: 1955626 bytes, checksum: f84660a8c004e77cec0bb334c37bb074 (MD5)Made available in DSpace on 2018-12-19T16:46:42Z (GMT). No. of bitstreams: 1 Luz NF. Lutzomyia longipalpis saliva...2018.pdf: 1955626 bytes, checksum: f84660a8c004e77cec0bb334c37bb074 (MD5) Previous issue date: 2018Fundação de Amparo a Pesquisa do Estado da Bahia (FAPESB) e Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), and by the Intramural Research Program of the NIH, National Institute of Allergy and Infectious Diseases. This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) - Finance Code 001. NL is a fellowship recipient from CAPES Brazil; PM is a fellowship recipient from FAPESB; AV is a fellowship recipient from Programa Nacional de Pós-Doutorado/CAPES; CdO, UL, CB and VMB are senior investigators of CNPq.Fundação Oswaldo Cruz. Instituto Gonçalo Moniz. Salvador, BA, Brasil.National Institutes of Health. National Institute of Allergy and Infectious Diseases. Laboratory of Malaria and Vector Research. Vector Molecular Biology Section. Rockville, MD, United States.National Institutes of Health. National Institute of Allergy and Infectious Diseases. Laboratory of Malaria and Vector Research. Vector Molecular Biology Section. Rockville, MD, USA.Federal University of Rio de Janeiro. Carlos Chagas Filho Biophysics Institute. Laboratory of Molecular Parasitology, Center of Health Science. Rio de Janeiro, RJ, Brazil.National Institutes of Health. National Institute of Allergy and Infectious Diseases. Laboratory of Malaria and Vector Research. Vector Molecular Biology Section. Rockville, MD, USA.Fundação Oswaldo Cruz. Instituto Gonçalo Moniz. Salvador, BA, Brasil / Universidade Federal da Bahia. Faculdade de Medicina. Salvador, BA, Brasil.Fundação Oswaldo Cruz. Instituto Gonçalo Moniz. Salvador, BA, Brasil / Universidade Federal da Bahia. Faculdade de Medicina. Salvador, BA, Brasil.National Institutes of Health. National Institute of Allergy and Infectious Diseases. Laboratory of Parasitic Diseases, Immunobiology Section. Bethesda, MD, USA.Fundação Oswaldo Cruz. Teresina, PI, Brasil.National Institutes of Health. National Institute of Allergy and Infectious Diseases. Laboratory of Malaria and Vector Research. Vector Molecular Biology Section. Rockville, MD, USA.Fundação Oswaldo Cruz. Instituto Gonçalo Moniz. Salvador, BA, Brasil / Universidade Federal da Bahia. Faculdade de Medicina. Salvador, BA, Brasil.Fundação Oswaldo Cruz. Instituto Gonçalo Moniz. Salvador, BA, Brasil / Universidade Federal da Bahia. Faculdade de Medicina. Salvador, BA, Brasil.Federal University of Rio de Janeiro. Carlos Chagas Filho Biophysics Institute. Laboratory of Molecular Parasitology, Center of Health Science. Rio de Janeiro, RJ, Brazil.Uniformed Services University of the Health Sciences. Infectious Diseases Division. Bethesda, MD, USA.Fundação Oswaldo Cruz. Instituto Gonçalo Moniz. Salvador, BA, Brasil / Universidade Federal da Bahia. Faculdade de Medicina. Salvador, BA, Brasil / Fundação José Silveira, Bahia. Multinational Organization Network Sponsoring Translational and Epidemiological Research. Salvador, BA, Brasil.Fundação Oswaldo Cruz. Instituto Gonçalo Moniz. Salvador, BA, Brasil / Universidade Federal da Bahia. Faculdade de Medicina. Salvador, BA, Brasil.National Institutes of Health. National Institute of Allergy and Infectious Diseases. Laboratory of Malaria and Vector Research. Vector Molecular Biology Section. Rockville, MD, USA.National Institutes of Health. National Institute of Allergy and Infectious Diseases. Laboratory of Malaria and Vector Research. Vector Molecular Biology Section. Rockville, MD, United States.Fundação Oswaldo Cruz. Instituto Gonçalo Moniz. Salvador, BA, Brasil / Universidade Federal da Bahia. Faculdade de Medicina. Salvador, BA, Brasil.Sand flies bite mammalian hosts to obtain a blood meal, driving changes in the host inflammatory response that support the establishment of Leishmania infection. This effect is partially attributed to components of sand fly saliva, which are able to recruit and activate leukocytes. Our group has shown that heme oxygenase-1 (HO-1) favors Leishmania survival in infected cells by reducing inflammatory responses. Here, we show that exposure to sand fly bites is associated with induction of HO-1 in vivo. Histopathological analyses of skin specimens from human volunteers experimentally exposed to sand fly bites revealed that HO-1 and Nrf2 are produced at bite sites in the skin. These results were recapitulated in mice ears injected with a salivary gland sonicate (SGS) or exposed to sand fly bites, indicating that vector saliva may be a key factor in triggering HO-1 expression. Resident skin macrophages were the main source HO-1 at 24-48 h after bites. Additionally, assays in vivo after bites and in vitro after stimulation with saliva both demonstrated that HO-1 production by macrophages was Nrf2-dependent. Collectively, our data demonstrates that vector saliva induces early HO-1 production at the bite sites, representing a major event associated with establishment of naturally-transmitted Leishmania infections

Data from: Proteomic analysis reveals a predominant NFE2L2 (NRF2) signature in the ​canonical pathway and upstream regulator analysis of Leishmania-infected macrophages

CBA mice macrophages (MØ) control infection by Leishmania major and are susceptive to Leishmania amazonensis, suggesting that both parasite species induce distinct responses that play important roles in infection outcome. To evaluate the MØ responses to infection arising from these two Leishmania species, a proteomic study using a Multidimensional Protein Identification Technology (MudPIT) approach with liquid chromatography tandem mass spectrometry (LC-MS/MS) was carried out on CBA mice bone-marrow MØ (BMMØ). Following SEQUEST analysis, which revealed 2,838 proteins detected in BMMØ, data mining approach found six proteins significantly associated with the tested conditions. To investigate their biological significance, enrichment analysis was performed using Ingenuity Pathway Analysis (IPA). A three steps IPA approach revealed 4 Canonical Pathways (CP) and 7 Upstream Transcriptional Factors (UTFs) strongly associated with the infection process. NRF2 signatures were present in both CPs and UTFs pathways. Proteins involved in iron metabolism, such as heme oxigenase 1 (HO-1) and ferritin besides sequestosome (SQSMT1 or p62) were found in the NRF2 CPs and the NRF2 UTFs. Differences in the involvement of iron metabolism pathway in Leishmania infection was revealed by the presence of HO-1 and ferritin. Noteworty, HO-1 was strongly associated with L. amazonensis infection, while ferritin was regulated by both species. As expected, higher HO-1 and p62 expressions were validated in L. amazonensis-infected BMMØ, in addition to decreased expression of ferritin and nitric oxide production. Moreover, BMMØ incubated with L. amazonensis LPG also expressed higher levels of HO-1 in comparison to those stimulated with L. major LPG. In addition, L. amazonensis-induced uptake of holoTf was higher than that induced by L. major in BMMØ, and holoTf was also detected at higher levels in vacuoles induced by L. amazonensis. Taken together, these findings indicate that NRF2 pathway activation and increased HO-1 production, together with higher levels of holoTf uptake, may promote permissiveness to L. amazonensis infection. In this context, differences in protein signatures triggered in the host by L. amazonensis and L. major infection could drive the outcomes in distinct clinical forms of leishmaniasis