Drebrin 1 in dendritic cells regulates phagocytosis and cell surface receptor expression through recycling for efficient antigen presentation.

Phagocytosis, macropinocytosis and antigen presentation by dendritic cells (DC) requires reorganization of the actin cytoskeleton. Drebrin (Dbn1) is an actin binding and stabilizing protein with roles in endocytosis, formation of dendrite spines in neurons and coordinating cell-cell synapses in immune cells. However, its role in DC phagocytosis and antigen presentation is unknown. These studies now report that silencing of Dbn1 in DC resulted in restrained cell surface display of receptors, most notably MHC class I and II and co-stimulatory molecules. This, as expected, resulted in impaired antigen-specific T-cell activation and proliferation. Studies additionally revealed that knockdown of Dbn1 in DC impaired macropinocytosis and phagocytosis. However, there was a concomitant increase in fluid-phase uptake, suggesting that Dbn1 is responsible for the differential control of macropinocytosis versus micropinocytosis activities. Taken together, these findings now reveal that Dbn1 plays a major role in coordinating the actin cytoskeletal activities responsible for antigen presentation in DC.

CD40-CD40L cross-talk drives fascin expression in dendritic cells for efficient antigen presentation to CD4+ T cells.

Fascin is an actin-bundling protein that, among immune cells, is restricted to expression in dendritic cells (DCs). Previous reports have suggested that fascin plays an important role in governing DC antigen presentation to CD4+ T cells. However, no report has clearly linked the receptor-ligand engagement that can direct downstream regulation of fascin expression. In this study, bone marrow-derived DCs from wild-type versus CD40-knockout C57BL/6 mice were used to elucidate the mechanisms of fascin expression and activity upon CD40-CD40 ligand (CD40L) engagement. These investigations now show that CD40 engagement governs fascin expression in DCs to promote CD4+ T-cell cytokine production. Absence of CD40 signaling resulted in diminished fascin expression in DCs and was associated with impaired CD4+ T-cell responses. Furthermore, the study found that loss of CD40-CD40L engagement resulted in reduced DC-T-cell contacts. Rescue by ectopic fascin expression in CD40-deficient DCs was able to re-establish sustained contacts with T cells and restore cytokine production. Taken together, these results show that cross-talk through CD40-CD40L signaling drives elevated fascin expression in DCs to support acquisition of full T-cell responses.

Increased thiol levels in antimony-resistant Leishmania infantum isolated from treatment-refractory visceral leishmaniasis in Brazil.

BACKGROUND Treatment-refractory visceral leishmaniasis (VL) has become an important problem in many countries. OBJECTIVES We evaluated the antimony-resistance mechanisms of Leishmania infantum isolated from VL patients refractory or responsive to treatment with pentavalent antimony. METHODS Strains isolated from antimony-refractory patients (in vitro antimony-resistant isolates) and antimony-responsive patients (in vitro antimony-sensitive isolates) were examined. Morphological changes were evaluated by transmission electron microscopy after trivalent antimony exposure. P-glycoprotein (P-gp) efflux pump activity was evaluated using the pump-specific inhibitor verapamil hydrochloride, and the role of thiol in trivalent antimony resistance was investigated using the enzymatic inhibitor L-buthionine sulfoximine. FINDINGS Antimony treatment induced fewer alterations in the cellular structure of L. infantum resistant isolates than in that of sensitive isolates. P-gp efflux activity was not involved in antimony resistance in these isolates. Importantly, the resistant isolates contained higher levels of thiol compared to the sensitive isolates, and inhibition of thiol synthesis in the resistant isolates recovered their sensitivity to trivalent antimony treatment, and enhanced the production of reactive oxygen species in promastigotes exposed to the drug. MAIN CONCLUSIONS Our results demonstrate that isolates from patients with antimony-refractory VL exhibited higher thiol levels than antimony-sensitive isolates. This indicates that redox metabolism plays an important role in the antimony-resistance of New World VL isolates.

Dispositional depression and hostility are associated with inflammatory markers of cardiovascular disease in African Americans.

Prior research has demonstrated that state depressive symptoms and hostility can modulate inflammatory immune responses and directly contribute to cardiovascular disease (CVD) onset and development. Previous studies have not considered the contribution of dispositional depressive symptoms to the inflammatory process. They have also largely excluded African Americans, despite their disproportionate risk for CVD. The first aim of the study was to examine the impact of state and dispositional depression and hostility on CVD-associated inflammatory biomarkers interleukin-6 (IL-6) and C-reactive protein (CRP) in an African American sample. The second aim was to examine synergistic influences of hostility and state and dispositional depression on IL-6 and CRP. The final aim was to examine whether the relations between state and dispositional depression, hostility, IL-6, and CRP varied as a function of gender and education. Anthropometric measures, blood serum samples, and psychosocial data were collected from 198 African Americans from the Washington, DC metropolitan area. Hierarchical and stepwise regression analyses indicated that (1) increased levels of hostility were associated with increased levels of CRP; (2) hostility and IL-6 were more strongly associated among participants with lower educational attainment; and (3) dispositional depression and CRP were more strongly associated among participants with greater hostility and lower educational attainment. Findings suggest that enduring personality dispositions, such as dispositional depression and hostility, are critical to a thorough assessment of cardiovascular profiles in African Americans. Future studies should investigate causal pathways that link depressive and hostile personality styles to inflammatory activity for African American men and women.

Fascin1 promotes cell migration of mature dendritic cells.

Dendritic cells (DCs) play central roles in innate and adaptive immunity. Upon maturation, DCs assemble numerous veil-like membrane protrusions, disassemble podosomes, and travel from the peripheral tissues to lymph nodes to present Ags to T cells. These alterations in morphology and motility are closely linked to the primary function of DCs, Ag presentation. However, it is unclear how and what cytoskeletal proteins control maturation-associated alterations, in particular, the change in cell migration. Fascin1, an actin-bundling protein, is specifically and greatly induced upon maturation, suggesting a unique role for fascin1 in mature DCs. To determine the physiological roles of fascin1, we characterized bone marrow-derived, mature DCs from fascin1 knockout mice. We found that fascin1 is critical for cell migration: fascin1-null DCs exhibit severely decreased membrane protrusive activity. Importantly, fascin1-null DCs have lower chemotactic activity toward CCL19 (a chemokine for mature DCs) in vitro, and in vivo, Langerhans cells show reduced emigration into draining lymph nodes. Morphologically, fascin1-null mature DCs are flatter and fail to disassemble podosomes, a specialized structure for cell-matrix adhesion. Expression of exogenous fascin1 in fascin1-null DCs rescues the defects in membrane protrusive activity, as well as in podosome disassembly. These results indicate that fascin1 positively regulates migration of mature DCs into lymph nodes, most likely by increasing dynamics of membrane protrusions, as well as by disassembling podosomes.

Influence of Testosterone in Neglected Tropical Diseases: Clinical Aspects in Leprosy and In Vitro Experiments in Leishmaniasis.

Neglected tropical diseases encompass a group of chronic and debilitating infectious diseases that primarily affect marginalized populations. Among these diseases, leprosy and leishmaniasis are endemic in numerous countries and can result in severe and disfiguring manifestations. Although there have been reports indicating a higher incidence of leprosy and leishmaniasis in males, the underlying factors contributing to this observation remain unclear. Therefore, the objective of this study was to examine both clinical and experimental evidence regarding the role of testosterone in leprosy and leishmaniasis. A prospective clinical study was conducted to compare the clinical forms of leprosy and assess circulating testosterone levels. Additionally, the impact of testosterone on Leishmania amazonensis-infected macrophages was evaluated in vitro. The findings demonstrated that serum testosterone levels were higher in women with leprosy than in the control group, irrespective of the multi- or pauci-bacillary form of the disease. However, no differences in testosterone levels were observed in men when comparing leprosy patients and controls. Interestingly, increasing doses of testosterone in macrophages infected with L. amazonensis resulted in a higher proportion of infected cells, decreased CD40 expression on the cell surface, elevated expression of SOCS1, and decreased expression of IRF5. These findings provide biological evidence to support the influence of testosterone on intracellular infections, though the interpretation of clinical evidence remains limited.

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.

DC expressing transgene Foxp3 are regulatory APC.

Tolerogenic DC and suppressive Foxp3(+) Treg play important roles in preventing autoimmunity and allograft rejection. We report that (adenovirus mediated) ectopic expression of Foxp3 in human DC (i.e. DC.Foxp3) yields an APC that severely limits T-cell proliferation and type-1 immune responses from the naïve, but not memory, pool of responder T cells in vitro. In marked contrast, the frequencies of type-2 and Treg responses were dramatically increased after stimulation of naïve T cells with DC.Foxp3 versus control DC. DC.Foxp3-induced CD4(+)CD25(+) Treg cells potently suppressed the proliferation of, and IFN-gamma production from, CD4(+) and CD8(+) responder T cells. Notably, the immunosuppressive biology of DC.Foxp3 was effectively normalized by addition of 1-methyl-tryptophan or neutralizing anti-TGF-beta1 Ab during the period of T-cell priming. These data suggest the potential utility of regulatory DC.Foxp3 and/or DC.Foxp3-induced CD4(+)CD25(+) Treg as translational agents for the amelioration or prevention of pathology in the setting of allograft transplantation and/or autoimmunity.

Ectopic T-bet expression licenses dendritic cells for IL-12-independent priming of type 1 T cells in vitro.

T-bet (TBX21) is a transcription factor required for the optimal development of type 1 immune responses. Although initially characterized for its intrinsic role in T cell functional polarization, endogenous T-bet may also be critical to the licensing of type 1-biasing APCs. Here, we investigated whether human dendritic cells (DC) genetically engineered to express high levels of T-bet (i.e., DC.Tbet) promote superior type 1 T cell responses in vitro. We observed that DC.Tbet were selective activators of type 1 effector T cells developed from the naive pool of responder cells, whereas DC.Tbet and control DC promoted type 1 responses equitably from the memory pool of responder cells. Naive T cells primed by (staphylococcal enterotoxin B or tumor-associated protein-loaded) DC.Tbet exhibited an enhancement in type 1- and a concomitant reduction in Th2- and regulatory T cell-associated phenotype/function. Surprisingly, DC.Tbets were impaired in their production of IL-12 family member cytokines (IL-12p70, IL-23, and IL-27) when compared with control DC, and the capacity of DC.Tbet to preferentially prime type 1 T cell responses was only minimally inhibited by cytokine (IL-12p70, IL-23, IFN-gamma) neutralization or receptor (IL-12Rbeta2, IL-27R) blockade during T cell priming. The results of transwell assays suggested the DC.Tbet-mediated effects are predominantly the result of direct DC-T cell contact or their close proximity, thereby implicating a novel, IL-12-independent mechanism by which DC.Tbets promote improved type 1 functional polarization from naive T cell responders. Given their superior type 1 polarizing capacity, DC.Tbet may be suitable for use in vaccines designed to prevent/treat cancer or infectious disease.

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.

Space-time risk cluster of visceral leishmaniasis in Brazilian endemic region with high social vulnerability: An ecological time series study.

BACKGROUND: Despite visceral leishmaniasis (VL) being epidemic in most Brazilian regions, the Northeast region is responsible for the highest morbidity and mortality outcomes within the country. OBJECTIVE: To analyse the spatiotemporal dynamics of VL cases to identify the temporal trends and high-risk areas for VL transmission, as well as the association of the disease with social vulnerability in Brazilian Northeast. METHODS: We carried out an ecological time series study employing spatial analysis techniques using all VL confirmed cases of 1,794 municipalities of Brazilian Northeast between the years 2000 to 2017. The Social Vulnerability Index (SVI) was used to represent the social vulnerability. Incidence rates were standardized and smoothed by the Local Empirical Bayesian Method. Time trends were examined through segmented linear regression. Spatiotemporal analysis consisted of uni- and bivariate Global and Local Moran indexes and space-time scan statistics. RESULTS: Incidence rate remained stable and ranged from 4.84 to 3.52 cases/100,000 inhabitants. There was higher case prevalence between males (62.71%), children and adolescents (63.27%), non-white (69.75%) and urban residents (62.58%). Increasing trends of new cases were observed among adult male subjects (≥ 40 years old) and urban residents. Importantly, VL incidence showed a direct spatial dependence. Spatial and space-time clusters were identified in sertão and meio-norte sub-regions, overlapping with high social vulnerability areas. CONCLUSIONS: VL is a persistent health issue in Brazilian Northeast and associated with social vulnerability. Space-time clustering of VL cases in socially vulnerable municipalities demands intersectoral public policies of surveillance and control, with focus on reducing inequalities and improving living conditions for regional inhabitants.

Allograft inflammatory factor-1 in myeloid cells drives autoimmunity in type 1 diabetes.

Allograft inflammatory factor-1 (AIF1) is a calcium-responsive cytoplasmic scaffold protein that directs hematopoiesis and immune responses within dendritic cells (DC) and macrophages. Although the role of AIF1 in transplant rejection and rheumatoid arthritis has been explored, little is known about its role in type 1 diabetes. Here, we show that in vivo silencing of AIF1 in NOD mice restrained infiltration of immune cells into the pancreas and inhibited diabetes incidence. Analyses of FACS-sorted CD45neg nonleukocyte populations from resected pancreatic islets showed markedly higher expression of insulin in the AIF1-silenced groups. Evaluation of CD45+ leukocytes revealed diminished infiltration of effector T cells and DC in the absence of AIF1. Transcriptional profiling further revealed a marked decrease in cDC1 DC-associated genes CD103, BATF3, and IRF8, which are required for orchestrating polarized type 1 immunity. Reduced T cell numbers within the islets were observed, with concomitant lower levels of IFN-γ and T-bet in AIF1-silenced cohorts. In turn, there was a reciprocal increase in functionally suppressive pancreas-resident CD25+Foxp3+CD4+ Tregs. Taken together, results show that AIF1 expression in myeloid cells plays a pivotal role in promoting type 1 diabetes and that its suppression restrains insulitis by shifting the immune microenvironment toward tolerance.

Pancreatic islets seeded in a novel bioscaffold forms an organoid to rescue insulin production and reverse hyperglycemia in models of type 1 diabetes.

Therapeutic approaches to combat type 1 diabetes (T1D) include donor pancreas transplantation, exogenous insulin administration and immunosuppressive therapies. However, these clinical applications are limited due to insufficient tissue compatible donors, side effects of exogenous insulin administration and/or increased onset of opportunistic infections attributable to induced global immunosuppression. An alternative approach to alleviate disease states is to utilize insulin-producing pancreatic islets seeded in a bioscaffold for implantation into diabetic recipients. The present studies now report that a newly developed cationic polymer biomaterial serves as an efficient bioscaffold for delivery of donor syngeneic pancreatic islet cells to reverse hyperglycemia in murine streptozotocin induced- or non-obese diabetic mouse models of T1D. Intraperitoneal implantation of pancreatic islets seeded within the copolymer bioscaffold supports long-term cell viability, response to extracellular signaling cues and ability to produce soluble factors into the microenvironment. Elevated insulin levels were measured in recipient diabetic mice upon implantation of the islet-seeded biomaterial coupled with reduced blood glucose levels, collectively resulting in increased survival and stabilization of metabolic indices. Importantly, the implanted islet-seeded biomaterial assembled into a solid organoid substructure that reorganized the extracellular matrix compartment and recruited endothelial progenitors for neovascularization. This allowed survival of the graft long-term in vivo and access to the blood for monitoring glucose levels. These results highlight the novelty, simplicity and effectiveness of this biomaterial for tissue regeneration and in vivo restoration of organ functions.

Allograft Inflammatory Factor-1 Governs Hematopoietic Stem Cell Differentiation Into cDC1 and Monocyte-Derived Dendritic Cells Through IRF8 and RelB in vitro.

The multistep differentiation process from hematopoietic stem cells through common myeloid progenitors into committed dendritic cell (DC) subsets remains to be fully addressed. These studies now show that Allograft Inflammatory Factor-1 (AIF1) is required for differentiation of classical DC type 1 (cDC1) subsets and monocyte-derived DC (Mo-DC). Phenotypic studies found that AIF1 expression increased in committed subsets differentiating from common myeloid progenitors (CMP). However, silencing AIF1 expression in hematopoietic stem progenitors restrained the capacity to differentiate into Mo-DC and cDC1 cell subsets under GM-CSF or Flt3-L stimuli conditions, respectively. This was further marked by restrained expression of IRF8, which is critical for development of Mo-DC and cDC1 subsets. As a result, absence of AIF1 restrained the cells at the Lin-CD117+FcγR-CD34+ CMP stage. Further biochemical studies revealed that abrogating AIF1 resulted in inhibition of the NFκB family member RelB expression and p38 MAPK phosphorylation during differentiation of Mo-DC. Lastly, protein binding studies identified that AIF1 interacts with protein kinase C (PKC) to influence downstream signaling pathways. Taken together, this is the first report showing a novel role of AIF1 as a calcium-responsive scaffold protein that supports IRF8 expression and interacts with PKC to drive NFκB-related RelB for successfully differentiating hematopoietic progenitor cells into cDC and Mo-DC subsets under Flt3-L and GM-CSF stimuli, respectively.

IL-10 producing CD8+ CD122+ PD-1+ regulatory T cells are expanded by dendritic cells silenced for Allograft Inflammatory Factor-1.

Allograft Inflammatory Factor-1 (AIF1) is a cytoplasmic scaffold protein that contains Ca2+ binding EF-hand and PDZ interaction domains important for mediating intracellular signaling complexes in immune cells. The protein plays a dominant role in both macrophage- and dendritic cell (DC)-mediated inflammatory responses. This study now reports that AIF1 expression in DC is important in directing CD8+ T cell effector responses. Silencing AIF1 expression in murine CD11c+ DC suppressed antigen-specific CD8+ T cell activation, marked by reduced CXCR3, IFNγ and Granzyme B expression, and restrained proliferation. These primed CD8+ T cells had impaired cytotoxic killing of target cells in vitro. In turn, studies identified that AIF1 silencing in DC robustly expanded IL-10 producing CD8+ CD122+ PD-1+ regulatory T cells that suppressed neighboring immune effector responses through both IL-10 and PD-1-dependent mechanisms. In vivo studies recapitulated bystander suppression of antigen-responsive CD4+ T cells by the CD8+ Tregs expanded from the AIF1 silenced DC. These studies further demonstrate that AIF1 expression in DC serves as a potent governor of cognate T cell responses and present a novel target for engineering tolerogenic DC-based immunotherapies.

Inhibition of Allograft Inflammatory Factor-1 in Dendritic Cells Restrains CD4+ T Cell Effector Responses and Induces CD25+Foxp3+ T Regulatory Subsets.

Allograft inflammatory factor-1 (AIF1) is a cytoplasmic scaffold protein shown to influence immune responses in macrophages and microglial cells. The protein contains Ca2+ binding EF-hand and PDZ interaction domains important for mediating intracellular signaling complexes. This study now reports that AIF1 is expressed in CD11c+ dendritic cells (DC) and silencing of expression restrains induction of antigen-specific CD4+ T cell effector responses. AIF1 knockdown in murine DC resulted in impaired T cell proliferation and skewed polarization away from T helper type 1 and 17 fates. In turn, there was a parallel expansion of IL-10-producing and CD25+Foxp3+ T regulatory subsets. These studies are the first to demonstrate that AIF1 expression in DC serves as a potent governor of cognate T cell responses and presents a novel target for engineering tolerogenic DC-based immunotherapies.

Leishmania infantum Induces the Release of sTREM-1 in Visceral Leishmaniasis.

Visceral leishmaniasis (VL) is a systemic transmissible disease that remains to be a major global health problem. The inflammatory response during VL is characterized by the release of several cytokines and other pro-inflammatory mediators. Triggering Receptor Expressed on Myeloid Cells (TREM) are a group of evolutionarily conserved membrane-bound surface receptors expressed on neutrophils and monocytes. Engagement of TREM-1 directs intracellular signaling events that drive cytokine production, degranulation, and phagocytosis. In certain inflammatory-associated diseases, TREM-1 can also be found as a soluble form (sTREM-1), which can negatively regulate TREM-1 receptor signaling. In these studies, we now find that high levels of circulating sTREM-1 correlate directly with VL disease severity. In particular, high levels of sTREM-1 were observed in non-survivor VL patients. Furthermore, these levels of sTREM-1 positively correlated with liver size and negatively correlated with leukocyte counts and hemoglobin concentration. Moreover, we found that neutrophils exposure in vitro to Leishmania infantum modulates TREM-1, DAP12, and IL-8 gene expression, while also increasing release of sTREM-1. Finally, results revealed that higher sTREM-1 levels are associated with increasing parasite ratio. Taken together, these studies suggest that L. infantum may modulate TREM-1 in neutrophils and high levels of this molecule is associated with severe VL.

sCD163 levels as a biomarker of disease severity in leprosy and visceral leishmaniasis.

BACKGROUND: CD163, receptor for the haptoglobin-hemoglobin complex, is expressed on monocytes/macrophages and neutrophils. A soluble form of CD163 (sCD163) has been associated with the M2 macrophage phenotype, and M2 macrophages have been shown to down-modulate inflammatory responses. In particular, previous studies have shown that M2 is closely associated with the most severe clinical presentation of leprosy (i.e. lepromatous leprosy (LL)), as well as tuberculosis. We hypothesized that sCD163 correlates with severity of diseases caused by intracellular pathogens. METHODOLOGY/PRINCIPAL FINDINGS: To assess this hypothesis, sCD163 levels were measured in the serum of leprosy and visceral leishmaniasis (VL) patients stratified by severity of the clinical presentation. sCD163 levels were significantly higher in patients with these diseases than those observed in healthy control individuals. Further analyses on infection and disease status of leprosy and VL patients revealed a clear association of sCD163 levels with clinical parameters of disease severity. In vitro culture assays revealed that Leishmania infection induced CD163 expression on the surface of both monocyte/macrophages and neutrophils, suggesting these cells as possible sources of sCD163. FACS analyses shows that the cells expressing CD163 produces both TNF-α and IL-4. CONCLUSIONS/SIGNIFICANCE: Taken together, our results reveal sCD163 as a potential biomarker of severity of diseases caused by intracellular pathogens M. leprae and Leishmania spp. and have a modulatory role, with a mix of an inflammatory property induced by TNF-α release, but that potentially induces an anti-inflammatory T cell response, related to IL-4 release.

Increased thiol levels in antimony-resistant Leishmania infantum isolated from treatment-refractory visceral leishmaniasis in Brazil

BACKGROUND Treatment-refractory visceral leishmaniasis (VL) has become an important problem in many countries. OBJECTIVES We evaluated the antimony-resistance mechanisms of Leishmania infantum isolated from VL patients refractory or responsive to treatment with pentavalent antimony. METHODS Strains isolated from antimony-refractory patients (in vitro antimony-resistant isolates) and antimony-responsive patients (in vitro antimony-sensitive isolates) were examined. Morphological changes were evaluated by transmission electron microscopy after trivalent antimony exposure. P-glycoprotein (P-gp) efflux pump activity was evaluated using the pump-specific inhibitor verapamil hydrochloride, and the role of thiol in trivalent antimony resistance was investigated using the enzymatic inhibitor L-buthionine sulfoximine. FINDINGS Antimony treatment induced fewer alterations in the cellular structure of L. infantum resistant isolates than in that of sensitive isolates. P-gp efflux activity was not involved in antimony resistance in these isolates. Importantly, the resistant isolates contained higher levels of thiol compared to the sensitive isolates, and inhibition of thiol synthesis in the resistant isolates recovered their sensitivity to trivalent antimony treatment, and enhanced the production of reactive oxygen species in promastigotes exposed to the drug. MAIN CONCLUSIONS Our results demonstrate that isolates from patients with antimony-refractory VL exhibited higher thiol levels than antimony-sensitive isolates. This indicates that redox metabolism plays an important role in the antimony-resistance of New World VL isolates.