TLR4 abrogates the Th1 immune response through IRF1 and IFN-ß to prevent immunopathology during L. infantum infection.

A striking feature of human visceral leishmaniasis (VL) is chronic inflammation in the spleen and liver, and VL patients present increased production levels of multiple inflammatory mediators, which contribute to tissue damage and disease severity. Here, we combined an experimental model with the transcriptional profile of human VL to demonstrate that the TLR4-IFN-ß pathway regulates the chronic inflammatory process and is associated with the asymptomatic form of the disease. Tlr4-deficient mice harbored fewer parasites in their spleen and liver than wild-type mice. TLR4 deficiency enhanced the Th1 immune response against the parasite, which was correlated with an increased activation of dendritic cells (DCs). Gene expression analyses demonstrated that IRF1 and IFN-ß were expressed downstream of TLR4 after infection. Accordingly, IRF1- and IFNAR-deficient mice harbored fewer parasites in the target organs than wild-type mice due to having an increased Th1 immune response. However, the absence of TLR4 or IFNAR increased the serum transaminase levels in infected mice, indicating the presence of liver damage in these animals. In addition, IFN-ß limits IFN-γ production by acting directly on Th1 cells. Using RNA sequencing analysis of human samples, we demonstrated that the transcriptional signature for the TLR4 and type I IFN (IFN-I) pathways was positively modulated in asymptomatic subjects compared with VL patients and thus provide direct evidence demonstrating that the TLR4-IFN-I pathway is related to the nondevelopment of the disease. In conclusion, our results demonstrate that the TLR4-IRF1 pathway culminates in IFN-ß production as a mechanism for dampening the chronic inflammatory process and preventing immunopathology development.

Systems analysis of subjects acutely infected with the Chikungunya virus.

The largest ever recorded epidemic of the Chikungunya virus (CHIKV) broke out in 2004 and affected four continents. Acute symptomatic infections are typically associated with the onset of fever and often debilitating polyarthralgia/polyarthritis. In this study, a systems biology approach was adopted to analyze the blood transcriptomes of adults acutely infected with the CHIKV. Gene signatures that were associated with viral RNA levels and the onset of symptoms were identified. Among these genes, the putative role of the Eukaryotic Initiation Factor (eIF) family genes and apolipoprotein B mRNA editing catalytic polypeptide-like (APOBEC3A) in the CHIKV replication process were displayed. We further compared these signatures with signatures induced by the Dengue virus infection and rheumatoid arthritis. Finally, we demonstrated that the CHIKV in vitro infection of murine bone marrow-derived macrophages induced IL-1 beta production in a mechanism that is significantly dependent on the inflammasome NLRP3 activation. The observations provided valuable insights into virus-host interactions during the acute phase and can be instrumental in the investigation of new and effective therapeutic interventions.

Antimony resistance associated with persistence of Leishmania (Leishmania) infantum infection in macrophages.

Visceral leishmaniasis is a severe disease caused by protozoan parasites that include Leishmania (L.) infantum. The disease is established when parasites subvert the immune response of the host. Notably, chemotherapy-based use of antimonial compounds can partially alleviate disease burden. Unfortunately, the resistance to drug treatments is increasing in areas endemic to the disease. In this report, we investigated immune responses within macrophages infected with antimony-resistant L. infantum isolates from patients with a relapse in the disease. Results revealed that antimony-resistant parasites persist in the first 24 h of infection. Activation of macrophage or blocking of thiol production during infection shows enhanced clearance of parasites, which is coordinately associated with increased production of pro-inflammatory cytokines. Taken together, these results suggest that the mechanism of antimony resistance in L. infantum isolates may be related to a decrease in macrophage microbicidal functions.

Leishmania infantum (syn. L. chagasi) parasites affect the release of soluble CD14 by infected macrophages.

Functionally, cluster of differentiation 14 (CD14) is a co-receptor of the complex formed by lipopolysaccharide (LPS) and LPS-binding protein expressed on the membrane of a variety of cells. However, CD14 can be shed from the cell membrane into the circulation as soluble CD14 (sCD14) upon cell activation. Previously, our group reported that elevated sCD14 serum levels were associated with the clinical and laboratory findings in the context of visceral leishmaniasis (VL), but not in the context of LPS stimulation or bacterial infection. In the present study, we investigated the secretion dynamics of sCD14 in the context of Leishmania infantum (syn. L. chagasi) in vitro infection. Macrophages from treated VL patients and delayed-type hypersensitivity positive (DTH+) subjects were infected with L. infantum (syn. L. chagasi) promastigotes, and the infection index was evaluated (number of amastigotes per 100 infected macrophages). Additionally, the levels of sCD14, Inteleukin (IL)10, IL-6 and tumour necrosis factor alpha (TNF-α) were measured in the culture supernatants using the Luminex assay. Interestingly, the release of sCD14 was inversely correlated with the L. infantum (syn. L. chagasi) infection index. Of note, the release of sCD14 was upregulated and downregulated in the context of infected macrophages from DTH+ subjects and treated VL patients, respectively. Additionally, we also observed that the levels of sCD14 in the culture supernatants were positively correlated with the levels of TNF-α, IL-6 and IL-10. Therefore, our data suggest that macrophages from treated VL patients and DTH+ subjects respond differently to L. infantum (syn. L. chagasi) infection in the context of the release of sCD14; therefore, the release of sCD14 may be associated with the outcome of VL.

Interleukin 17A acts synergistically with interferon γ to promote protection against Leishmania infantum infection.

Interleukin 17 (IL-17) is an inflammatory cytokine that plays a protective role against intracellular parasites. The role of IL-17 during Leishmania infection remains controversial and poorly defined. We evaluated whether IL-17 participates in the host immune response to Leishmania infantum. IL-17A is present in sera from patients with visceral leishmaniasis and decreases after successful treatment. In C57BL/6 infected mice, higher production of IL-17A coincided with the peak of parasitism. Il17ra(-/-) mice were more susceptible to infection and also exhibited reduced inflammatory infiltration and interferon γ (IFN-γ)-expressing CD4+ T-cell frequencies than wild-type mice. The frequencies of FoxP3+ CD4+ T cells and interleukin 10 (IL-10)-expressing CD4+ T cells were increased in Il17ra(-/-) mice. We also demonstrated that IL-17A acts synergistically with IFN-γ to potentiate NO production and leishmanicidal activity in infected macrophages. Therefore, our results indicate that L. infantum induces IL-17A production, which promotes the control of parasite replication by strengthening T-helper type 1 responses and NO production and prevents regulatory T-cell and IL-10-expressing T-cell expansion.

Fatty acid profiles in Leishmania spp. isolates with natural resistance to nitric oxide and trivalent antimony.

Fatty acids, especially those from phospholipids (PLFA), are essential membrane components that are present in relatively constant proportions in biological membranes under natural conditions. However, under harmful growth conditions, such as diseases, environmental changes, and chemical exposure, the fatty acid proportions might vary. If such changes could be identified and revealed to be specific for adverse situations, they could be used as biomarkers. Such biomarkers could facilitate the identification of virulence and resistance mechanisms to particular chemotherapeutic agents. Therefore, specific biomarkers could lead to better therapeutic decisions that would, in turn, enhance treatment effectiveness. The objective of this study was to compare the fatty acid profiles of trivalent antimony and nitric oxide (NO)-resistant and -sensitive Leishmania chagasi and Leishmania amazonensis isolates. Fatty acid methyl esters (FAMEs) were obtained from total lipids (MIDI), ester-linked lipids (ELFA), and ester-linked phospholipids (PLFA). FAMEs were analyzed by chromatography and mass spectrometry. Species- or resistance-associated differences in FAME profiles were assessed by nonmetric multidimensional scaling, multiresponse permutation procedures, and indicator species analyses. The isolate groups had different MIDI-FAME profiles. However, neither the ELFA nor PLFA profiles differed between the sensitive and resistant isolates. Levels of the fatty acid 18:1 Δ9c were increased in sensitive isolates (p < 0,001), whereas the fatty acid 20:4 Δ5,8,11,14 showed the opposite trend (p < 0.01). We conclude that these two fatty acids are potential biomarkers for NO and antimony resistance in L. chagasi and L. amazonensis and that they could be helpful in therapeutic diagnoses.

CD4+ Th1 and Th17 responses and multifunctional CD8 T lymphocytes associated with cure or disease worsening in human visceral leishmaniasis.

Introduction: In VL, a proinflammatory phenotype is typically associated with enhanced phagocytosis and a Th1 mediated immune response resulting in infection control. In contrast, an anti-inflammatory phenotype, associated with a predominant regulatory response, typically enables intracellular multiplication of Leishmania parasites and disease progression. Methods: To investigate the impact of chemotherapy on Th2 and Th17 immune responses in patients with visceral leishmaniasis (VL), we assessed all combinations of intracellular expression of IFN-γ, IL-10, IL-4 and IL-17 in the CD4+ and CD8+ T cell populations of peripheral blood mononuclear cell (PBMC) samples from patients, after antigenic stimulation with Leishmania lysate, throughout treatment and follow-up. As increases in spleen and liver sizes and decreases in hematocrit, hemogloblin, erythrocytes, monocytes, leukocytes and platelets levels are strongly related to the disease, we studied the correlations between the frequencies of T cells producing the afore mentioned cytokines, individually and in combination, and these variables, as markers of disease or cure. Results: We found that the frequency of IFN-γ-producingCD4+ T cells increased until the end of chemotherapy with Glucantime® or AmBisome ®, while IL-10, IL-4 and IL-17-producing CD4+ T cells peaked on day 7 following the start of treatment. Although the frequency of CD4+IL-17+ cells decreased during treatment an increase was observed after clinical cure. The frequency of CD4+ T cells producing only IFN-γ or IL-17 correlated with blood monocytes levels. Frequencies of double-producers of IFN-γ and IL-10 or IL-4 correlated positively with eosinophils and platelets levels. Together, this suggest that IFN-γ drives the immune response towards Th1 at cure. In contrast, and associated with disease or Th2 response, the frequency of CD4+ IL-10+ cells correlated positively with spleen sizes and negatively with circulating monocyte levels, while the frequency of CD4+ producing both IL-4 and IL-10 correlated negatively with platelets levels. The frequency of CD8+ single-producers of IFN-γ increased from day 21 to 90 while that of single-producers of IL-10 peaked on day 7, of IL-4 on day 30 and of IL-17, on day 180. IFN-γ expression in CD8+ single- and double-producers of cytokines was indicative of an immune response associated with cure. In contrast, frequencies of CD8+ double-producers of IL-4 and IL-10, IL-4 and IL-17 and IL-10 and IL-17 and producers of three and four cytokines, were associated with disease and were low after the cure. Frequencies of CD8+ T cells producing IFN-γ alone or with IL-17 were positively correlated with platelets levels. In contrast, as markers of disease: 1) frequencies of single producers of IL-10 correlated negatively with leukocytes levels, 2) frequencies of double producers of IL-4 and IL-10 correlated negatively with platelet, leukocyte, lymphocyte and circulating monocyte levels, 3) frequencies of triple-producers of IFN-γ, IL-4 and IL-10 correlated negatively with platelet, leukocyte and neutrophil levels and 4) frequencies of producers of IFN-γ, IL-4, IL-10 and IL-17 simultaneously correlated positively with spleen size, and negatively with leukocyte and neutrophil levels. Discussion: Our results confirmed that the clinical improvement of VL patients correlates with the decrease of an IL-4 and IL-10 CD4+Th2 response, the recovery of CD4+ Th1 and Th17 responses and the frequency of CD8+ single-producers of IFN-γ and double producers of IFN-γ and IL-17.

High levels of soluble CD40 ligand and matrix metalloproteinase-9 in serum are associated with favorable clinical evolution in human visceral leishmaniasis.

BACKGROUND: Soluble CD40 ligand (sCD40L) and matrix metalloproteinase 9 (MMP-9) are inflammation markers and have been poorly described in infectious disease. In this prospective study, we describe the sera kinetics of these two molecules in the course of treatment follow up in human visceral leishmaniasis (VL). METHODS: Sera from VL patients were collected before and during follow up of regular Antimony treatment. sCD40L and MMP-9 were measured by Luminex assay. Paired analysis by Wilcoxon signed test was used for comparison of values of the same subjects before and after initiation of treatment. Correlations between clinical data and parasite load with the serum levels of sCD40L and MMP-9 were performed by Spearman test. Tests were considered statistically significant if the probability of a type I error was less than 5% (p-value < 0.05). RESULTS: While sCD40L and MMP-9 were not observed in sera from non endemic controls which are at low risk of Leishmania chagasi infection, elevated levels were observed in sera from VL patients, and an increase in sCD40L and MMP-9 levels were detectable during the follow-up of VL patients undergoing antimony treatment. sCD40L levels were also high in individuals living in endemic settings at high risk of infection (endemic controls). Additionally, negative correlations were found between spleen sizes and MMP-9 before treatment and sCD40L at day 15 of treatment. Negative correlations were also found between parasite load with both sCD40L and MMP-9. CONCLUSION: Serum sCD40L and MMP-9 are identified as new and simple biomarkers in two situations: (i) monitoring the success of therapy and (ii) predicting favorable clinical outcome of human VL.

Dataset of dual RNA-seq mapping in visceral leishmaniasis: Inquiry on parasite transcripts in human blood transcriptome upon Leishmania infantum infection.

This dataset is related to the article "Insight Into the Long Noncoding RNA and mRNA Coexpression Profile in the Human Blood Transcriptome Upon Leishmania infantum Infection" by S.R. Maruyama, C.A. Fuzo, A.E.R. Oliveira, L.A. Rogerio, N.T. Takamiya, G. Pessenda, E.V. de Melo, A.M. da Silva, A.R. Jesus, V. Carregaro, H.I. Nakaya, R.P. Almeida and J.S. da Silva. Frontiers in Immunology, 2022. Through the reuse of raw sequencing data, we generated original dataset by performing a dual RNA-seq mapping procedure to survey the parasite transcripts found in RNA-seq samples from blood of visceral leishmaniasis patients. Diseased patients with active infection displayed the highest number of reads mapped to L. infantum genome. Even after six months later of the treatment, when the patients were considered cured, parasite reads were still detected. Parasite reads were also detected in asymptomatic individuals. The original dual RNA-seq alignment read count data provided here can be further explored to evaluate either host or parasite transcripts.

Co-infection of Leishmania infantum and a Crithidia-related species in a case of refractory relapsed visceral leishmaniasis with non-ulcerated cutaneous manifestation in Brazil.

We report a refractory and relapsed visceral leishmaniasis case in a male child patient followed from 2016 to 2020, whose clinical isolates from multiple relapses were analyzed at the genome level. To the best of our knowledge, it is the first report that both visceral leishmaniasis and non-ulcerated cutaneous leishmaniasis have concomitantly manifested in the same patient. Importantly, sequence analysis revealed that the patient was co-infected with Leishmania infantum and a Crithidia-related parasite, which was previously found in a fatal case of visceral leishmaniasis from the same endemic region.

Association between genetic variants in TREM1, CXCL10, IL4, CXCL8 and TLR7 genes with the occurrence of congenital Zika syndrome and severe microcephaly.

Congenital Zika syndrome (CZS) is a cluster of malformations induced by Zika virus (ZIKV) infection and the underline mechanisms involved in its occurrence are yet not fully understood. Along with epidemiological and environmental factors, the genetic host factors are suggested as important to the CZS occurrence and development, however, few studies have evaluated this. This study enrolled a total of 245 individuals in a case-control association study compound a cohort of high specific interest constituted by 75 mothers who had delivered CZS infants, their 76 infants, and 47 mothers that had delivered healthy infants, and their 47 infants. Sixteen single-nucleotide polymorphisms on TREM1, CXCL10, IL4, CXCL8, TLR3, TLR7, IFNR1, CXCR1, IL10, CCR2 and CCR5 genes were genotyped to investigate their association as risk factors to CZS. The results show an association between C allele at TREM1 rs2234246 and C allele at IL4 rs224325 in mothers infected with ZIKV during pregnancy, with the increased susceptibility to CZS occurrence in their infants and the SNP CXCL8 rs4073 and the G allele at CXCL10 rs4508917 with presence of CZS microcephaly in the infants. Furthermore, the T allele at CXCL8 rs4073 and TRL7 rs179008 SNPs were associated with the severity of microcephaly in children with CZS. These results suggest that these polymorphisms in genes of innate immune responses addressed here are associated to increased risk of occurrence and severity of CZS in pregnant mothers infected with ZIKV and their CZS infants.

Recombinant protein KR95 as an alternative for serological diagnosis of human visceral leishmaniasis in the Americas.

In the Americas, visceral leishmaniasis (VL) is caused by the protozoan Leishmania infantum, leading to death if not promptly diagnosed and treated. In Brazil, the disease reaches all regions, and in 2020, 1,933 VL cases were reported with 9.5% lethality. Thus, an accurate diagnosis is essential to provide the appropriate treatment. Serological VL diagnosis is based mainly on immunochromatographic tests, but their performance may vary by location, and evaluation of diagnostic alternatives is necessary. In this study, we aimed to evaluate the performance of ELISA with the scantily studied recombinant antigens, K18 and KR95, comparing their performance with the already known rK28 and rK39. Sera from parasitologically confirmed symptomatic VL patients (n = 90) and healthy endemic controls (n = 90) were submitted to ELISA with rK18 and rKR95. Sensitivity (95% CI) was, respectively, 83.3% (74.2-89.7) and 95.6% (88.8-98.6), and specificity (95% CI) was 93.3% (85.9-97.2) and 97.8% (91.8-99.9). For validation of ELISA with the recombinant antigens, we included samples from 122 VL patients and 83 healthy controls collected in three regions in Brazil (Northeast, Southeast, and Midwest). When comparing the results obtained with the VL patients' samples, significantly lower sensitivity was obtained by rK18-ELISA (88.5%, 95% CI: 81.5-93.2) compared with rK28-ELISA (95.9%, 95% CI: 90.5-98.5), but the sensitivity was similar comparing rKR95-ELISA (95.1%, 95% CI: 89.5-98.0), rK28-ELISA (95.9%, 95% CI: 90.5-98.5), and rK39-ELISA (94.3%, 95% CI: 88.4-97.4). Analyzing the specificity, it was lowest with rK18-ELISA (62.7%, 95% CI: 51.9-72.3) with 83 healthy control samples. Conversely, higher and similar specificity was obtained by rKR95-ELISA (96.4%, 95% CI: 89.5-99.2), rK28-ELISA (95.2%, 95% CI: 87.9-98.5), and rK39-ELISA (95.2%, 95% CI: 87.9-98.5). There was no difference in sensitivity and specificity across localities. Cross-reactivity assessment, performed with sera of patients diagnosed with inflammatory disorders and other infectious diseases, was 34.2% with rK18-ELISA and 3.1% with rKR95-ELISA. Based on these data, we suggest using recombinant antigen KR95 in serological assays for VL diagnosis.

sTREM-1 and TNF-α levels are associated with the clinical outcome of leprosy patients.

Leprosy reaction (LR) and physical disability (PD) are the most significant clinical complications of leprosy. Herein, we assessed the circulating serum-sTREM-1 and TNF-α levels and their genetic polymorphisms in leprosy. Serum-sTREM-1 and TNF-α levels were measured in leprosy patients (LP) before treatment (n = 51) and from their household contacts (HHCs; n = 25). DNA samples were genotyped using TREM-1 rs2234246 and TNF-α rs1800629-SNP in 210 LPs and 168 endemic controls. The circulating sTREM-1 and TNF-α levels are higher in the multibacillary form. The ROC curve of the serum-sTREM-1 levels was able to differentiate LR from non-LR and PD from non-PD. Similarly, LPs with serum-sTREM-1 levels >210 pg/ml have 3-fold and 6-fold higher chances of presenting with LR and PD, respectively. Genotypes CC+CT of the TREM-1 were associated with leprosy. Taken together, our analyses indicated that sTREM-1 and TNF-α play an important role in the pathogenesis of leprosy and provide promising biomarkers to assist in the diagnosis of leprosy complications.

Parasite Detection in Visceral Leishmaniasis Samples by Dye-Based qPCR Using New Gene Targets of Leishmania infantum and Crithidia.

Visceral leishmaniasis (VL) is a neglected disease considered a serious public health problem, especially in endemic countries. Several studies have discovered monoxenous trypanosomatids (Leptomonas and Crithidia) in patients with VL. In different situations of leishmaniasis, investigations have examined cases of co-infection between Leishmania spp. and Crithidia spp. These coinfections have been observed in a wide range of vertebrate hosts, indicating that they are not rare. Diagnostic techniques require improvements and more robust tools to accurately detect the causative agent of VL. This study aimed to develop a real-time quantitative dye-based PCR (qPCR) assay capable of distinguishing Leishmania infantum from Crithidia-related species and to estimate the parasite load in samples of VL from humans and animals. The primer LinJ31_2420 targets an exclusive phosphatase of L. infantum; the primer Catalase_LVH60-12060_1F targets the catalase gene of Crithidia. Therefore, primers were designed to detect L. infantum and Crithidia sp. LVH60A (a novel trypanosomatid isolated from VL patients in Brazil), in samples related to VL. These primers were considered species-specific, based on sequence analysis using genome data retrieved from the TriTryp database and the genome assembling of Crithidia sp. LVH60A strain, in addition to experimental and clinical data presented herein. This novel qPCR assay was highly accurate in identifying and quantifying L. infantum and Crithidia sp. LVH60A in samples obtained experimentally (in vitro and in vivo) or collected from hosts (humans, dogs, cats, and vectors). Importantly, the screening of 62 cultured isolates from VL patients using these primers surprisingly revealed that 51 parasite cultures were PCR+ for Crithidia sp. In addition, qPCR assays identified the co-infection of L. infantum with Crithidia sp. LVH60A in two new VL cases in Brazil, confirming the suspicion of co-infection in a previously reported case of fatal VL. We believe that the species-specific genes targeted in this study can be helpful for the molecular diagnosis of VL, as well as for elucidating suspected co-infections with monoxenous-like trypanosomatids, which is a neglected fact of a neglected disease.

Association between asthma, rhinitis and atopic dermatitis with leprosy: A case-control study.

Background Considering the cross-regulation of Th1 and Th2 responses, we hypothesised that atopic diseases (Th2) inhibit the protective Th1 immune response to Mycobacterium leprae and exacerbates leprosy. Objective In this study, we aimed to evaluate the association between leprosy and atopic diseases. Methods To evaluate the association of atopic diseases with leprosy, we conducted a case-control study that included leprosy patients (n = 333) and their household contacts (n = 93). The questionnaire from the International Study of Asthma and Allergies in Childhood, which is validated in several countries for epidemiological diagnosis of atopic diseases, was applied to determine the occurrence of atopic diseases, allergic rhinitis, asthma, and atopic dermatitis among leprosy patients and the household contacts. Results Considering clinical and epidemiological data, among the leprosy group 51.6% (n = 172) were determined to have at least one atopic disease, while atopy was observed less frequently at 40.86% among household contacts (n = 38). When two or more atopic diseases were assessed, the frequency was significantly higher among the leprosy patients than in the household contacts (21.9% vs. 11.8%; P-value = 0.03). Likewise, the frequency of asthma was significantly higher among leprosy patients (21%) than in the household contacts (10.8%; P-value = 0.02). Thus, our analyses revealed an association of atopic diseases with leprosy, with a significant linear increase in the occurrence of leprosy with an increase in the number of atopic diseases (P-value = 0.01). Limitation Due to the difficulties in recruiting household contacts that have prolonged contact with patients, but are not genetically related to the patient, the household contacts group is smaller than the leprosy patient group. Conclusion The data reveal an association between atopic diseases and leprosy outcomes. This knowledge could improve the treatment of leprosy patients with co-incident atopic diseases.

Insight Into the Long Noncoding RNA and mRNA Coexpression Profile in the Human Blood Transcriptome Upon Leishmania infantum Infection.

Visceral leishmaniasis (VL) is a vector-borne infectious disease that can be potentially fatal if left untreated. In Brazil, it is caused by Leishmania infantum parasites. Blood transcriptomics allows us to assess the molecular mechanisms involved in the immunopathological processes of several clinical conditions, namely, parasitic diseases. Here, we performed mRNA sequencing of peripheral blood from patients with visceral leishmaniasis during the active phase of the disease and six months after successful treatment, when the patients were considered clinically cured. To strengthen the study, the RNA-seq data analysis included two other non-diseased groups composed of healthy uninfected volunteers and asymptomatic individuals. We identified thousands of differentially expressed genes between VL patients and non-diseased groups. Overall, pathway analysis corroborated the importance of signaling involving interferons, chemokines, Toll-like receptors and the neutrophil response. Cellular deconvolution of gene expression profiles was able to discriminate cellular subtypes, highlighting the contribution of plasma cells and NK cells in the course of the disease. Beyond the biological processes involved in the immunopathology of VL revealed by the expression of protein coding genes (PCGs), we observed a significant participation of long noncoding RNAs (lncRNAs) in our blood transcriptome dataset. Genome-wide analysis of lncRNAs expression in VL has never been performed. lncRNAs have been considered key regulators of disease progression, mainly in cancers; however, their pattern regulation may also help to understand the complexity and heterogeneity of host immune responses elicited by L. infantum infections in humans. Among our findings, we identified lncRNAs such as IL21-AS1, MIR4435-2HG and LINC01501 and coexpressed lncRNA/mRNA pairs such as CA3-AS1/CA1, GASAL1/IFNG and LINC01127/IL1R1-IL1R2. Thus, for the first time, we present an integrated analysis of PCGs and lncRNAs by exploring the lncRNA-mRNA coexpression profile of VL to provide insights into the regulatory gene network involved in the development of this inflammatory and infectious disease.

Use of N-acetylcysteine as treatment adjuvant regulates immune response in visceral leishmaniasis: Pilot clinical trial and in vitro experiments.

This investigation aimed to assess the effect of N-acetylcysteine (NAC) as an adjuvant treatment to alleviate visceral leishmaniasis (VL). The present work includes both blinded randomized clinical intervention and experimental in vitro studies. The clinical trial included 60 patients with VL randomly allocated into two groups: a test group (n = 30) treated with meglumine antimoniate plus NAC (SbV + NAC) and a control group (n = 30) treated with meglumine antimoniate only (SbV). The primary outcome was clinical cure (absence of fever, spleen and liver sizes reduction, and hematological improvement) in 180 days. The cure rate did not differ between the groups; both groups had similar results in all readout indices. The immunological parameters of the patients treated with SbV + NAC showed higher sCD40L in sera during treatment, and the levels of sCD40L were negatively correlated with Interleukin-10 (IL-10) serum levels. In addition, data estimation showed a negative correlation between the sCD40L levels and the spleen size in patients with VL. For the in vitro experiments, peripheral blood mononuclear cells (PBMCs) or PBMC-derived macrophages from healthy donors were exposed to soluble Leishmania antigen (SLA) or infected with stationary promastigotes of Leishmania infantum in the presence or absence of NAC. Results revealed that NAC treatment of SLA-stimulated PBMCs reduces the frequency of monocytes producing IL-10 and lowers the frequency of CD4+ and CD8+ T cells expressing (pro-)inflammatory cytokines. Together, these results suggest that NAC treatment may modulate the immune response in patients with VL, thus warranting additional investigations to support its case use as an adjuvant to antimony therapy for VL.

Case Report: Visceral Leishmaniasis and Hemophagocytic Lymphohistiocytosis: Three Clinical Cases, Three Different Patterns.

Visceral leishmaniasis (VL) is a neglected tropical disease with more than 30,000 cases annually reported worldwide. In Brazil, about 3,700 cases are annually reported. The VL clinical presentation is variable, from asymptomatic to severe cases with a high risk of death. We reported three cases of VL with clinical sign similarities but distinct development. All cases had bone marrow hemophagocytosis and hemophagocytic lymphohistiocytosis (HLH) criteria. HLH is a rare condition that may have secondary causes, including infectious and parasitic diseases, like VL. The delayed recognition of the secondary HLH (sHLH) association to VL may cause unfavorable outcomes and death.

Multifunctional, TNF-α and IFN-γ-Secreting CD4 and CD8 T Cells and CD8High T Cells Are Associated With the Cure of Human Visceral Leishmaniasis.

Visceral leishmaniasis (VL) is a chronic and often fatal disease caused by protozoans of the genus Leishmania that affects millions of people worldwide. Patients with symptomatic VL have an impaired anti-Leishmania-specific CD4+ T-cell response, which is reversed after clinical cure. In contrast, the quality of the CD4+ and CD8+ T-cell responses involved in resistance and/or cure of VL relies on the capability of these cells to activate polyfunctional and memory responses, which are associated with the simultaneous production of three cytokines: IFN-γ, IL-2, and TNF-α. Models for the development of CD4 and CD8 T-cell quality in memory and protection to leishmaniasis have been described previously. We aimed to assess the functionality of the T cells involved in the recovery of the immune suppression throughout the VL treatment. Therefore, we cultured peripheral blood mononuclear cells (PBMCs) from VL patients and healthy controls in vitro with soluble Leishmania antigen (SLA). Cell surface markers and intracellular cytokine production were determined on days 7, 14, 21, 30, 60, 90, and 180 after the beginning of chemotherapy. We observed that the frequencies of CD4+TNF-α+IFN-γ+ and the multifunctional CD4+IL-2+TNF-α+IFN-γ+, together with CD4+TNF-α+ and CD4+IFN-γ+ T cells, increased throughout and at the end of the treatment, respectively. In addition, enhanced frequencies of CD8+IL-2+TNF-α+IFN-γ+ and CD8+TNF-α+IFN-γ T cells were also relevant in the healing process. Noteworthy, the frequencies of the CD4+ and CD8 central-memory T cells, which produce IL-2, TNF-α, and IFN-γ and ensure the memory response against parasite reinfection, are significantly enhanced in cured patients. In addition, the subset of the non-functional CD8Low population is predominant in VL untreated patients and decreases along the chemotherapy treatment. In contrast, a CD8High subset increased towards the cure. Furthermore, the cure due to treatment with meglumine antimoniate or with liposomal amphotericin B was associated with the recovery of the T-cell immune responses. We described the evolution and participation of functional T cells during the treatment of patients with VL. Our results disclosed that the clinical improvement of patients is significantly associated with the participation of the CD4+ and CD8+ cytokine-secreting T cells.

Leishmania donovani infection suppresses Allograft Inflammatory Factor-1 in monocytes and macrophages to inhibit inflammatory responses.

Macrophages and monocytes are important for clearance of Leishmania infections. However, immune evasion tactics employed by the parasite results in suppressed inflammatory responses, marked by deficient macrophage functions and increased accumulation of monocytes. This results in an ineffective ability to clear parasite loads. Allograft Inflammatory Factor-1 (AIF1) is expressed in myeloid cells and serves to promote immune responses. However, AIF1 involvement in monocyte and macrophage functions during parasitic infections has not been explored. This study now shows that Leishmania donovani inhibits AIF1 expression in macrophages to block pro-inflammatory responses. Mice challenged with the parasite had markedly reduced AIF1 expression in splenic macrophages. Follow-up studies using in vitro approaches confirmed that L. donovani infection in macrophages suppresses AIF1 expression, which correlated with reduction in pro-inflammatory cytokine production and increased parasite load. Ectopic overexpression of AIF1 in macrophages provided protection from infection, marked by robust pro-inflammatory cytokine production and efficient pathogen clearance. Further investigations found that inhibiting AIF1 expression in bone marrow cells or monocytes impaired differentiation into functional macrophages. Collectively, results show that AIF1 is a critical regulatory component governing monocyte and macrophage immune functions and that L. donovani infection can suppress the gene as an immune evasion tactic.