Corrigendum: miR-548d-3p Is Up-regulated in Human Visceral Leishmaniasis and Suppresses Parasite Growth in Macrophages.

[This corrects the article DOI: 10.3389/fcimb.2022.826039.].

miR-548d-3p Is Up-Regulated in Human Visceral Leishmaniasis and Suppresses Parasite Growth in Macrophages.

Visceral leishmaniasis caused by Leishmania (Leishmania) infantum in Latin America progress with hepatosplenomegaly, pancytopenia, hypergammaglobulinemia, and weight loss and maybe lethal mainly in untreated cases. miRNAs are important regulators of immune and inflammatory gene expression, but their mechanisms of action and their relationship to pathogenesis in leishmaniasis are not well understood. In the present study, we sought to quantify changes in miRNAs associated with immune and inflammatory pathways using the L. (L.) infantum promastigote infected- human monocytic THP-1 cell model and plasma from patients with visceral leishmaniasis. We identified differentially expressed miRNAs in infected THP-1 cells compared with non-infected cells using qPCR arrays. These miRNAs were submitted to in silico analysis, revealing targets within functional pathways associated with TGF-ß, chemokines, glucose metabolism, inflammation, apoptosis, and cell signaling. In parallel, we identified differentially expressed miRNAs in active visceral leishmaniasis patient plasma compared with endemic healthy controls. In silico analysis of these data indicated different predicted targets within the TGF-ß, TLR4, IGF-I, chemokine, and HIF1α pathways. Only a small number of miRNAs were commonly identified in these two datasets, notably with miR-548d-3p being up-regulated in both conditions. To evaluate the potential biological role of miR-548d-3p, we transiently transfected a miR-548d-3p inhibitor into L. (L.) infantum infected-THP-1 cells, finding that inhibition of miR-548d-3p enhanced parasite growth, likely mediated through reduced levels of MCP-1/CCL2 and nitric oxide production. Further work will be required to determine how miR-548d-3p plays a role in vivo and whether it serves as a potential biomarker of progressive leishmaniasis.

miR-548d-3p Alters Parasite Growth and Inflammation in Leishmania (Viannia) braziliensis Infection.

American Tegumentary Leishmaniasis (ATL) is an endemic disease in Latin America, mainly caused in Brazil by Leishmania (Viannia) braziliensis. Clinical manifestations vary from mild, localized cutaneous leishmaniasis (CL) to aggressive mucosal disease. The host immune response strongly determines the outcome of infection and pattern of disease. However, the pathogenesis of ATL is not well understood, and host microRNAs (miRNAs) may have a role in this context. In the present study, miRNAs were quantified using qPCR arrays in human monocytic THP-1 cells infected in vitro with L. (V.) braziliensis promastigotes and in plasma from patients with ATL, focusing on inflammatory response-specific miRNAs. Patients with active or self-healed cutaneous leishmaniasis patients, with confirmed parasitological or immunological diagnosis, were compared with healthy controls. Computational target prediction of significantly-altered miRNAs from in vitro L. (V.) braziliensis-infected THP-1 cells revealed predicted targets involved in diverse pathways, including chemokine signaling, inflammatory, cellular proliferation, and tissue repair processes. In plasma, we observed distinct miRNA expression in patients with self-healed and active lesions compared with healthy controls. Some miRNAs dysregulated during THP-1 in vitro infection were also found in plasma from self-healed patients, including miR-548d-3p, which was upregulated in infected THP-1 cells and in plasma from self-healed patients. As miR-548d-3p was predicted to target the chemokine pathway and inflammation is a central to the pathogenesis of ATL, we evaluated the effect of transient transfection of a miR-548d-3p inhibitor on L. (V.) braziliensis infected-THP-1 cells. Inhibition of miR-548d-3p reduced parasite growth early after infection and increased production of MCP1/CCL2, RANTES/CCL5, and IP10/CXCL10. In plasma of self-healed patients, MCP1/CCL2, RANTES/CCL5, and IL-8/CXCL8 concentrations were significantly decreased and MIG/CXCL9 and IP-10/CXCL10 increased compared to patients with active disease. These data suggest that by modulating miRNAs, L. (V.) braziliensis may interfere with chemokine production and hence the inflammatory processes underpinning lesion resolution. Our data suggest miR-548d-3p could be further evaluated as a prognostic marker for ATL and/or as a host-directed therapeutic target.

Comparison of flow cytometry and indirect immunofluorescence assay in the diagnosis and cure criterion after therapy of American tegumentary leishmaniasis by anti-live Leishmania (Viannia) braziliensis immunoglobulin G.

The aim of this study was to compare the techniques of indirect immunofluorescence assay (IFA) and flow cytometry to clinical and laboratorial evaluation of patients before and after clinical cure and to evaluate the applicability of flow cytometry in post-therapeutic monitoring of patients with American tegumentary leishmaniasis (ATL). Sera from 14 patients before treatment (BT), 13 patients 1 year after treatment (AT), 10 patients 2 and 5 years AT were evaluated. The results from flow cytometry were expressed as levels of IgG reactivity, based on the percentage of positive fluorescent parasites (PPFP). The 1:256 sample dilution allowed us to differentiate individuals BT and AT. Comparative analysis of IFA and flow cytometry by ROC (receiver operating characteristic curve) showed, respectively, AUC (area under curve)=0.8 (95% CI=0.64-0.89) and AUC=0.90 (95% CI=0.75-0.95), demonstrating that the flow cytometry had equivalent accuracy. Our data demonstrated that 20% was the best cut-off point identified by the ROC curve for the flow cytometry assay. This test showed a sensitivity of 86% and specificity of 77% while the IFA had a sensitivity of 78% and specificity of 85%. The after-treatment screening, through comparative analysis of the technique performance indexes, 1, 2 and 5 years AT, showed an equal performance of the flow cytometry compared with the IFA. However, flow cytometry shows to be a better diagnostic alternative when applied to the study of ATL in the cure criterion. The information obtained in this work opens perspectives to monitor cure after treatment of ATL.

Evaluation of anti-lived and anti-fixed Leishmania (Viannia) braziliensis promastigote IgG antibodies detected by flow cytometry for diagnosis and post-therapeutic cure assessment in localized cutaneous leishmaniasis.

This study aims to investigate a flow cytometry performance-based methodology to detect anti-live (FC-ALPA-IgG) and anti-fixed (FC-AFPA-IgG) Leishmania (Viannia) braziliensis promastigote IgG as a means to monitor post-therapeutic cure of patients with localized cutaneous leishmaniasis (LCL). Serum samples from 30 LCL patients infected with L. (V.) braziliensis were assayed, comparing the IgG reactivity before and after specific treatment with pentavalent antimonial. Reactivities were reported as the percentage of positive fluorescent parasites (PPFP), using a PPFP of 60% as a cut-off value. In the serum dilution of 1:1024, the positive percentage of LCL serum sample for FC-ALPA-IgG and FC-AFPA-IgG was 86% and 90%, respectively, before treatment. Analysis of ∆PPFP that represents the difference between PPFP after and before treatment appeared as a new approach to monitor post-therapeutic IgG reactivity in LCL. Our data support the perspective of using FC-ALPA and FC-AFPA as a useful serologic tool for diagnosis and for post-therapeutic follow-up of LCL patients.

Mecanismos imunológicos na resposta celular e humoral na Leishmaniose tegumentar americana / Immunological mechanisms in the cellular and humoral response in the american tegumentary leishmaniasis

A leishmaniose tegumentar americana é uma doença antropozonótica, causada por diferentes espécies de Leishmania, cujas manifestações clínicas variam de lesões cutâneas, que tendem para a cura espontânea, até graves lesões mucocutâneas mutilantes. As infecções por Leishmania podem levar a uma resposta imunológica específica por parte do hospedeiro, caracterizada pelo aumento de células T CD4+ e um perfil de citocinas Th1 ou Th2. Infecções por L. major, que é o modelo mais bem estudado, quando ocorrem em camundongos resistentes (como C57BL/6, CBA, C3H) estão associadas a uma resposta Th1, enquanto em camundongos susceptíveis (BALB/c) vinculam-se à resposta do tipo Th2. A resposta imune em humanos não é bem caracterizada como o é em camundongos e envolve o papel das citocinas, das moléculas co-estimulatórias e da saliva do flebótomo. Paralelamente, à existência de uma resposta imunológica por parte do hospedeiro, a sobrevivência e a persistência parasitária dependem de estratégias de escape da resposta imune adaptativa.