Proteomic Analysis Reveals a Predominant NFE2L2 (NRF2) Signature in Canonical Pathway and Upstream Regulator Analysis of Leishmania-Infected Macrophages.

Juliana Perrone Bezerra De Menezes,Pablo Ivan Pereira Ramos,Gregory A Buck,Amanda Do Amor Divino Rebouças,Amanda Lopes Lorentz,Ulisses Gazos Lopes,Áislan De Carvalho Vivarini,Ricardo Khouri,Marco Aurélio Krieger,Jonilson Berlink Lima,Fabrício Klerynton Marchini,Antônio Luis De Oliveira Almeida Petersen ,Taís Fontoura De Almeida ,Jerônimo Nunes Rugani ,Valéria M Borges ,Flávia R L Mendes ,Camila Victória Sousa Oliveira ,Nívea F Luz ,Rodrigo Pedro Soares,Patrícia Sampaio Tavares Veras

doi:10.3389/fimmu.2019.01362

Abstract

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.

Highlights

Experimental murine models have been used to elucidate mechanisms related to host immunity that regulate disease development
Animals were kept and handled in accordance with the norms recommended by the International Guiding Principles for Biomedical Research Involving Animals; all experimental protocols complied with these guidelines, as well as all resolutions established by the Brazilian National Council for the Control of Animal Experimentation (CONCEA)
Kinetic analysis of BMMØ infection involving L. amazonensis or L. major promastigotes revealed that the percentage of infected MØ and the number of parasites per infected cell were similar in both infected cell groups at 1.5 and 3 h after parasites were added to cell cultures (Mann-Whitney, p < 0.05, Figures 1A,B)

Summary

Introduction

Experimental murine models have been used to elucidate mechanisms related to host immunity that regulate disease development. Proteomics is an advanced large-scale technique used to identify and characterize global protein expression, allowing for the targeting of molecules in an infected sample in comparison to an uninfected reference control, both in intracellular microbes and host cells. This advanced technology has proven to be highly efficient in the study of individual molecules, as it permits the identification of hundreds or even thousands of signature proteins that characterize elements of microbial and host response during infection [1, 3,4,5]. Some proteins have demonstrated potential as possible biomarkers for future prophylactic and therapeutic interventions [11, 13]

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Conclusion