The influence of a copper efflux pump in Histoplasma capsulatum virulence.

During the infectious process, pathogenic microorganisms must obtain nutrients from the host in order to survive and proliferate. These nutritional sources include the metallic nutrient copper. Despite its essentiality, copper in large amounts is toxic. Host defense mechanisms use high copper poisoning as a fungicidal strategy to control infection. Transcriptional analyses showed that yeast cultured in the presence of copper or inside macrophages (24 h) had elevated expression of CRP1, a copper efflux pump, suggesting that Histoplasma capsulatum could be exposed to a high copper environment in macrophages during the innate immune stage of infection. Accordingly, macrophages cultured in high copper are more efficient in controlling H. capsulatum growth. Also, silencing of ATP7a, a copper pump that promotes the copper influx in phagosomes, increases fungal survival in macrophages. The rich copper environment faced by the fungus is not dependent on IFN-γ, since fungal CRP1 expression is induced in untreated macrophages. Appropriately, CRP1 knockdown fungal strains are more susceptible to macrophage control than wild-type yeasts. Additionally, CRP1 silencing decreases fungal burden in mice during the phase of innate immune response (4-day postinfection) and CRP1 is required for full virulence in a macrophage cell lines (J774 A.1 and RAW 264.7), as well as primary cells (BMDM). Thus, induction of fungal copper detoxifying genes during innate immunity and the attenuated virulence of CRP1-knockdown yeasts suggest that H. capsulatum is exposed to a copper-rich environment at early infection, but circumvents this condition to establish infection.

Characterization of T Helper 1 and 2 Cytokine Profiles in Newborns of Mothers with COVID-19.

An infectious disease caused by SARS-CoV-2, COVID-19 greatly affects the pediatric population and is 3 times more prevalent in newborns than in the general population. In newborns, the overexpression of immunological molecules may also induce a so-called cytokine storm. In our study, we evaluated the expression of cytokines in newborns admitted to a neonatal ICU whose mothers had SARS-CoV-2 and symptoms of SARS. The blood of newborns of infected and healthy mothers was collected to identify their Th1 and Th2 cytokine profiles, and via flow cytometry, the cytokines TNF-α, IFN-γ, IL-2, IL-6, and IL-10 were identified. Overexpression was observed in the Th1 and Th2 cytokine profiles of newborns from infected mothers compared with the control group. Statistical analysis also revealed significant differences between the cellular and humoral responses of the infected group versus the control group. The cellular versus humoral responses of the newborns of infected mothers were also compared, which revealed the prevalence of the cellular immune response. These data demonstrate that some cytokines identified relate to more severe symptoms and even some comorbidities. IL-6, TNF-α, and IL-10 may especially be related to cytokine storms in neonates of mothers with COVID-19.

Polymethylmethacrylate Microspheres are Immunologically Inert in Mouse Tissues.

Nowadays, aesthetic concerns have gained attention, especially by patients looking for a less invasive alternative to minor facial corrections. Polymethylmethacrylate (PMMA) is widely used as a soft tissue filler; the demand for this polymer has increased, and along with it, there are some reports of adverse reactions. Such adverse reactions stem from consequences of immune and inflammatory reactions to PMMA. Some animal models have been used to unravel the causes of these reactions, among other factors involving the management of PMMA. The aim of this study was to determine the immunogenic profile of PMMA implantation in different anatomical planes of mice, over up to 360 experimental days. In this study, BALB/c mice were divided into 30 groups for immune evaluation of the interaction between the organism and the polymer; 2% PMMA was implanted subcutaneously, 10% intramuscularly and 30% in periosteal juxtaposition and followed during five experimental days (7, 30, 90, 180 and 360 days after implantation-DAI). Pro- and anti-inflammatory cytokines (IL-2, IL-4, IL-6, IFN-gamma, TNF, IL-17A, IL-10 and TGF-beta) were quantified in all experimental days. There was no statistical difference between the groups analyzed considering the evaluated parameters. Therefore, at all implanted depths, PMMA behaved inertly in a murine model.No Level Assigned This journal requires that authors assign a level of evidence to each submission to which Evidence-Based Medicine rankings are applicable. This excludes Review Articles, Book Reviews, and manuscripts that concern Basic Science, Animal Studies, Cadaver Studies, and Experimental Studies. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

Single nucleotide polymorphisms in genes involved in immune responses and outcome of tegumentary leishmaniasis.

Leishmaniases are neglected tropical diseases with a broad clinical spectrum. Tegumentary leishmaniasis (TL) is a disease caused by different Leishmania species, transmitted by phlebotomine sand flies and distributed worldwide. TL can present a cutaneous (CL) or mucocutaneous (MCL) clinical form depending on factors inherent to the parasite, the host and the vector. Polymorphisms in the immune response genes are host genetic factors that influence the pathogenesis or control of leishmaniasis. Single nucleotide polymorphisms (SNPs) in immune genes have been evaluated in several countries where leishmaniasis is endemic. In this review, we report studies on SNPs in several immune genes that might be associated with susceptibility or resistance to TL. We summarize studies from around the world and in Brazil, highlight the difficulties of these studies and future analyses needed to enhance our knowledge regarding host genetic factors in TL. Understanding the genetic characteristics of the host that facilitate resistance or susceptibility to leishmaniasis can contribute to the development of immunotherapy schedules for this disease. The current treatment methods are toxic, and no human vaccine is available.

Lipophosphoglycan From Dermotropic New World Leishmania Upregulates Interleukin-32 and Proinflammatory Cytokines Through TLR4 and NOD2 Receptors.

Interleukin-32 (IL-32) is produced during Leishmania infection, but the components of the parasite that induce its production are unknown. An important multivirulence factor of Leishmania spp. protozoa is the lipophosphoglycan (LPG), which plays a crucial role in the host-parasite interaction. Here, the ability of LPGs from two dermotropic Leishmania species to induce IL-32 production was evaluated in human peripheral blood mononuclear cells (PBMCs). Additionally, the potential receptors involved in this activation were assessed. PBMCs from healthy individuals were stimulated with LPGs from L. amazonensis (La) or L. braziliensis (Lb), live promastigotes of La or Lb and E. coli lipopolysaccharide (LPS, TLR4 agonist) as control. Blockers of TLR4 (Bartonella quintana LPS or monoclonal antibody) and Ponatinib (RIPK2 inhibitor, NOD2 pathway) were used to evaluate the receptors. ELISA was performed for IL-32 expression and cytokine (IL-1ß and IL-6) production in cell lysates and in supernatants, respectively. Expression of TLR4 (2 h, 24 h) was assessed by flow cytometry. IL-32γ mRNA transcript was analyzed by qPCR. It was observed that LPG from Leishmania, like whole parasites, induced the production of IL-32, IL-1ß and IL-6. Both LPGs induced the expression of IL32γ mRNA. The production of IL-32 was earlier detected (6 h) and positively associated with the production of IL-1ß and IL-6. The induction of cytokines (IL-32, IL-1ß and IL-6) was dependent on TLR4 and NOD2. The TLR4 was internalized after interaction with LPG. Therefore, our data suggest that LPGs from La and Lb are components of Leishmania able to upregulate IL-32 and other pro-inflammatory cytokines in a TLR4- and NOD2-dependent manner. In addition, LPG-induced IL-32 seems to be necessary for IL-1ß and IL-6 production. To identify the parasite factors and host receptors involved in IL-32 induction is crucial to reveal potential targets for novel strategies to control leishmaniasis.

Protective immune response mediated by neutrophils in experimental visceral leishmaniasis is enhanced by IL-32γ.

Neutrophils are important cells in protection against microbial infections including visceral leishmaniasis (VL). It is well known that IL-32γ increases the protective T helper 17 cell mediated immune response against Leishmania infantum. Thus, in this study we evaluated whether IL-32 γ can increase the protective role of neutrophils against VL. In comparison with wild type (WT) mice, transgenic mice for human IL-32 γ (IL-32 γ Tg) presented a higher frequency and absolute number of neutrophils in both spleen and liver after the establishment of L. infantum infection. The IL-32 concentrations correlated with neutrophil numbers in the infected tissues. The IL-32 γ -induced recruitment of neutrophils was dependent on IL-17, since inhibition of Th17 T cells generation and IL-17 production with digoxin treatment reversed the effects of IL-32 γ. In murine neutrophils, the presence of IL-32 γ enhanced the phagocytosis of L. infantum via CR3. In addition, murine IL-32 γ Tg neutrophils were able to kill L. infantum due to the increased production of ROS when compared with WT neutrophils. In fact, IL-32 γ Tg mice lost their ability to control infection by L. infantum when neutrophils were depleted. In parallel, treatment of human neutrophils with recombinant IL-32 γ increased phagocytosis and ROS-dependent killing of L. infantum, similarly to murine IL-32 γ Tg neutrophils. The data show that IL-32 γ induces neutrophil recruitment to organs affected by VL and increases phagocytosis and killing of L. infantum by neutrophils. Together, data indicate the pivotal axis IL-32 γ -Th17-neutrophils to control VL.

New world Leishmania spp. infection in people living with HIV: Concerns about relapses and secondary prophylaxis.

Coinfection with the human immunodeficiency virus (HIV) and Leishmania impairs immune responses, increases treatment failure and relapse rates in patients with American tegumentary leishmaniasis (ATL), as well as visceral leishmaniasis (VL). There is insufficient data on the treatment, relapse, and secondary prophylaxis in patients coinfected with HIV/Leishmania in Brazil. This study investigated patients with HIV/ATL and HIV/VL to describe the outcome of leishmaniasis in patients assisted at a referral hospital of Brazilian midwestern region. Patients with HIV/ATL (n = 21) mainly presented cutaneous diseases (76.2%) with an overall relapse rate of 28.57% after treatment, whereas HIV/VL (n = 28) patients accounted for 17.5% of the cases. The counts of CD4+ T cells and CD8+ T cells and the CD4+/CD8+ cell ratios at diagnosis or relapses were not significantly different between relapsing and non-relapsing patients. Patients with HIV/ATL or HIV/VL showed high levels of activation markers in CD4+ and CD8+ T cells. The regular use of highly active antiretroviral therapy (HAART) and viral load at the time of diagnosis did not influence the relapse rates. Relapses occurred in 36.4% (4/11) of the patients with HIV/VL receiving secondary prophylaxis and in 5.9% (1/17) of the patients who did not receive secondary prophylaxis (p = 0.06). These data are relevant for the therapeutic management of the patients coinfected with HIV/Leishmania.

The role of IL-32 in Bacillus Calmette-Guérin (BCG)-induced trained immunity in infections caused by different Leishmania spp.

BACKGROUND: Cells of the innate immune system undergo long-term functional reprogramming in response to Bacillus Calmette-Guérin (BCG) exposure via a process called trained immunity, conferring nonspecific protection to unrelated infections. Here, we investigate whether BCG-induced trained immunity is able to protect against infections caused by different Leishmania spp., protozoa that cause cutaneous and mucosal or visceral lesions. METHODS: We used training models of human monocytes with BCG and subsequent infection by L. braziliensis, L. amazonensis and L. infantum, and the vaccination of wild-type and transgenic mice for IL-32γ before in vivo challenge with parasites. RESULTS: We demonstrated that monocytes trained with BCG presented enhanced ability to kill L. braziliensis, L. amazonensis and L. infantum through increased production of reactive oxygen species. Interleukin (IL)-32 appears to play an essential role in the development of trained immunity. Indeed, BCG exposure induced IL-32 production in human primary monocytes, both mRNA and protein. We have used a human IL-32γ transgenic mouse model (IL-32γTg) to study the effect of BCG vaccination in different Leishmania infection models. BCG vaccination decreased lesion size and parasite load in infections caused by L. braziliensis and reduced the spread of L. amazonensis to other organs in both infected wild-type (WT) and IL-32γTg mice. In addition, BCG reduced the parasite load in the spleen, liver and bone marrow of both WT and IL-32γTg mice infected with L. infantum. BCG vaccination increased inflammatory infiltrate in infected tissues caused by different Leishmania spp. In all infections, the presence of IL-32γ was not mandatory, but it increased the protective and inflammatory effects of BCG-induced training. CONCLUSIONS: BCG's ability to train innate immune cells, providing protection against leishmaniasis, as well as the participation of IL-32γ in this process, pave the way for new treatment strategies for this neglected infectious disease.

Comparative EPR spectroscopy analysis of amphotericin B and miltefosine interactions with Leishmania, erythrocyte and macrophage membranes.

Electron paramagnetic resonance (EPR) spectroscopy of spin labels was used to study the interactions of amphotericin B (AmB) with the plasma membrane of Leishmania (L.) amazonensis promastigotes, human erythrocytes and J774.A1 murine macrophages, in comparison with reported and novel data for miltefosine (MIL). One of the objectives of this work is to look for the relationships between the activities of these two drugs in the Leishmania parasite with their changes in the cell membrane. A spin-labeled stearic acid inserted into the cell membranes showed strong interactions with putative AmB/sterol complexes, characterized by reductions in molecular dynamics. The concentration of the drugs in the plasma membrane that reduced the cell population by 50%, and the membrane-water partition coefficient of the drugs, were assessed. These biophysical parameters enabled estimates of possible therapeutic concentrations of these two drugs in the interstitial fluids of the tissues to be made. AmB displayed higher affinity for the plasma membrane of L. amazonensis than for that of the macrophage and erythrocyte, denoting a preference for a membrane that contains ergosterol. AmB also demonstrated higher hemolytic potential than MIL for measurements on erythrocytes in both PBS and whole blood. For MIL, the EPR technique detected membrane changes induced by the drug in the same concentration range that inhibited the growth of parasites, but in the case of AmB, an 8-fold higher concentration of the IC50 was necessary to observe a reduction in membrane fluidity, suggesting a better localized effect of AmB on the membrane. Taken together, the results demonstrate that the antiproliferative and cytotoxic effects of both drugs are associated with changes in cell membranes.

Antileishmanial activity of the chalcone derivative LQFM064 associated with reduced fluidity in the parasite membrane as assessed by EPR spectroscopy.

A novel chalcone derivative, LQFM064, demonstrated antileishmanial activity against Leishmania (L.) amazonensis, with an IC50 value of ~10 µM for the promastigote form. Electron paramagnetic resonance (EPR) spectroscopy of a spin-labeled stearic acid incorporated in the plasma membrane of L. amazonensis promastigotes revealed that after 2 h of treatment with LQFM064, the parasite showed remarkable reductions in membrane fluidity. The features of the altered EPR spectra were similar to those reported for the erythrocyte membrane, which was suggested to be due to the cross-linking of oxidized hemoglobin with the cytoskeleton spectrin. In comparison to miltefosine (MIL), LQFM064 demonstrated a much lower hemolytic potential against both erythrocytes in PBS and whole blood, less cytotoxicity in J774.A1 macrophages and equivalent ability to kill parasites internalized in J774.A1 macrophages. Measurements of the IC50 values for assays with different cell concentrations enabled the estimation of the membrane-water partition coefficient (KM/W), as well as the concentrations of LQFM064 in membrane (cm50) and aqueous phase (cw50) that reduces the cell population by 50%. From the KM/W and cm50 values it was deduced that LQFM064 has a greater affinity than MIL for the parasite membrane, but the antiproliferative activity of both substances is exerted at a similar concentration in the plasma membrane.

IL-15 enhances the capacity of primary human macrophages to control Leishmania braziliensis infection by IL-32/vitamin D dependent and independent pathways.

How human macrophages can control the intracellular infection with Leishmania is not completely understood. IL-15 and IL-32 are cytokines produced by monocytes/macrophages that can induce antimicrobial mechanisms. Here, we evaluated the effects of recombinant human IL-15 (rhIL-15) on primary human macrophage infection and response to L. braziliensis. Priming with rhIL-15 reduced the phagocytosis of L. braziliensis and increased the killing of the parasites in monocyte-derived macrophages from healthy donors. rhIL-15 induced TNFα and IL-32 in uninfected cells. After infection, the high levels of rhIL-15-induced TNFα and IL-32 were maintained. In addition, there was an increase of NO and an inhibition of the parasite-induced IL-10 production. Inhibition of NO reversed the leishmanicidal effects of rhIL-15. Although rhIL-15 did not increase L. braziliensis-induced reactive oxygen intermediates (ROS) production, inhibition of ROS reversed the control of infection induced by rhIL-15. Treatment of the cells with rhIL-32γ increased microbicidal capacity of macrophages in the presence of high levels of vitamin D (25D3), but not in low concentrations of this vitamin. rhIL-15 together with rhIL-32 lead to the highest control of the L. braziliensis infection in high concentrations of vitamin D. In this condition, NO and ROS mediated rhIL-32γ effects on microbicidal activity. The data showed that priming of human macrophages with rhIL-15 or rhIL-32γ results in the control of L. braziliensis infection through induction of NO and ROS. In addition, rhIL-32γ appears to synergize with rhIL-15 for the control of L. braziliensis infection in a vitamin D-dependent manner.

Antileishmanial and cytotoxic activities of ionic surfactants compared to those of miltefosine.

Using the electron paramagnetic resonance (EPR) of spin-labeled stearic acid and a spin label chemically attached to the membrane proteins, the interaction of miltefosine (MIL) and the ionic surfactants sodium dodecyl sulfate (SDS, anionic), cetyltrimethylammonium chloride (CTAC, cationic) and N-hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (HPS, zwitterionic) with the plasma membrane of Leishmania (L.) amazonensis promastigotes was studied. The spin-label EPR data indicated that the four compounds studied have the ability to increase the molecular dynamics of membrane proteins to a large extent. Compared to the other compounds, SDS produced the smallest increases in dynamics and demonstrated the lowest antileishmanial activity and cytotoxicity to J774.A1 macrophages. The activities of the other three compounds were not different from each other, but CTAC had a stronger activity against L. amazonensis promastigotes at higher cellular concentrations (> 1 × 109 cells/mL) and was the most effective against L. amazonensis-infected macrophages. However, CTAC was also the most cytotoxic to macrophages. By measuring the IC50/CC50 values for assays of different cell concentrations, we estimated the membrane-water partition coefficient (KM/W) as well as the concentrations in the membrane (cm50) and aqueous phase (cw50) of the compounds at their IC50/CC50. Compared to the other compounds, SDS showed the lowest value of KM/W and the highest value of cm50. In all experiments in this study, the data for the zwitterionic molecules HPS and MIL were not significantly different.

Alterations in monocyte subsets and cytokine production after TLR activation in American Cutaneous Leishmaniasis.

Phenotypic and functional aspects of monocytes from Localized Cutaneous Leishmaniasis (LCL) patients were evaluated. The frequencies of monocyte subsets and TLR2/TLR4 expression were evaluated in fresh peripheral blood whereas cytokine production was evaluated in whole blood cell cultures stimulated with TLR agonists or Leishmania braziliensis antigen (Ag). CD16+ monocytes frequency was increased in patients compared with controls. A TLR4 agonist (LPS) induced expression of TNF and IL-10 in monocyte subsets of patients and controls. The CD14+ CD16+ monocytes expressed higher levels of these cytokines than CD14+ CD16- cells. The levels of secreted TNF were higher in whole blood cell cultures from patients than controls after LPS/TLR4 or Ag stimulation. Whereas in controls there was a positive correlation between TNF and IL-10 levels, this was not observed in stimulated cell cultures from patients. The high levels of LPS-induced TNF were associated with the number of lesions and the percentages of CD14hi CD16+ monocytes. The levels of TLR2-induced TNF were also associated with number of lesions. All monocyte subsets from patients expressed higher levels of TLR2 and TLR4 than controls. Data suggest that systemically activated monocytes contribute for an imbalance in pro- and anti-inflammatory cytokine production during LCL, participating in the immunopathogenesis of the disease.

Interferon-Beta Treatment Differentially Alters TLR2 and TLR4-Dependent Cytokine Production in Multiple Sclerosis Patients.

OBJECTIVE: Multiple sclerosis (MS) is a multifactorial chronic disease that affects the central nervous system (CNS). Toll-like receptors (TLRs) play a central role in cytokine production after pathogen- and danger-associated molecular patterns (PAMPs and DAMPs) and contribute to CNS damage in MS patients. Here, we evaluated the effects of interferon (IFN)-ß treatment in TLR2 and TLR4-dependent cytokine production and mRNA expression in whole-blood cell cultures from MS patients. METHODS: We evaluated cytokine production by ELISA from whole-blood cell culture supernatants and mRNA expression by real-time polymerase chain reaction in peripheral blood mononuclear cells (PBMCs). RESULTS: In patients treated with IFN-ß, tumor necrosis factor (TNF)-α production after exposure to TLR2 agonist (Pam3Cys) was lower than in healthy controls and untreated MS patients. However, IFN-ß treatment had no significant effect on TNF-α production after TLR4 agonist (LPS) stimulation. On the other hand, interleukin (IL)-10 production was increased in TLR4- but not in TLR2-stimulated whole-blood cell culture from MS patients under IFN-ß treatment when compared to the controls. No differences in TNF-α or IL-10 mRNA expression in PBMCs from healthy controls and untreated or treated MS patients were detected, although PBMCs from treated patients presented higher levels of IL-32γ mRNA than those from controls. CONCLUSIONS: Our data suggest that IFN-ß treatment alters the TLR-dependent immune response of PBMCs from MS patients. This may contribute to the beneficial effects of IFN-ß treatment.

In vitro antileishmanial and cytotoxic activities of nerolidol are associated with changes in plasma membrane dynamics.

The sesquiterpene nerolidol is a membrane-active compound that has demonstrated antitumor, antibacterial, antifungal and antiparasitic activities. In this study, we used electron paramagnetic resonance (EPR) spectroscopy and biophysical parameters determined via cell culture assays to study the mechanisms underlying the in vitro antileishmanial activity of nerolidol. The EPR spectra of a spin-labeled stearic acid indicated notable interactions of nerolidol with the cell membrane of Leishmania amazonensis amastigotes. The nerolidol IC50 values in L. amazonensis amastigotes and promastigotes were found to depend on the cell concentration used in the assay. This dependence was described by an equation that considers various cell suspension parameters, such as the 50% inhibitory concentrations of nerolidol in the cell membrane (cm50) and the aqueous phase (cw50) and the membrane-water partition coefficient of nerolidol (KM/W). Via cytotoxicity (CC50) and hemolytic potential (HC50) data, these parameters were also determined for nerolidol in macrophages and erythrocytes. With a cw50 of 125 µM, macrophages were less sensitive to nerolidol than amastigotes and promastigotes, which had mean cw50 values of 56 and 74 µM, respectively. The estimated cm50 values of nerolidol for amastigotes and promastigotes and macrophages were between 2.6 and 3.0 M, indicating substantial accumulation of nerolidol in the cell membrane. In addition, the spin-label EPR data indicated that membrane dynamic changes occurred in L. amazonensis amastigotes at concentrations similar to the nerolidol IC50 value.

Promastigote parasites cultured from the lesions of patients with mucosal leishmaniasis are more resistant to oxidative stress than promastigotes from a cutaneous lesion.

Human leishmaniasis caused by Leishmania (Viannia) braziliensis can be presented as localized cutaneous leishmaniasis (LCL) or mucosal leishmaniasis (ML). Macrophages kill parasites using nitric oxide (NO) and reactive oxygen species (ROS). The aim of this study was to evaluate the ability of parasites obtained from patients with LCL or ML to produce and resist NO or ROS. Promastigotes and amastigotes from LCL or ML isolates produced similar amounts of NO in culture. Promastigotes from ML isolates were more resistant to NO and H2O2 than LCL parasites in a stationary phase, whereas amastigotes from LCL isolates were more resistant to NO. In addition, in the stationary phase, promastigote isolates from patients with ML expressed more thiol-specific antioxidant protein (TSA) than LCL isolates. Therefore it is suggested that infective promastigotes from ML isolates are more resistant to microbicidal mechanisms in the initial phase of infection. Subsequently, amastigotes lose this resistance. This behavior of ML parasites can decrease the number of parasites capable of stimulating the host immune response shortly after the infection establishment.

Leishmania (Viannia) guyanensis in tegumentary leishmaniasis.

Leishmania (Viannia) guyanensis is a causal agent of American tegumentary leishmaniasis (ATL). This protozoan has been poorly investigated; however, it can cause different clinical forms of ATL, ranging from a single cutaneous lesion to severe lesions that can lead to destruction of the nasopharyngeal mucosa. L. (V.) guyanensis and the disease caused by this species can present unique aspects revealing the need to better characterize this parasite species to improve our knowledge of the immunopathological mechanisms and treatment options for ATL. The mechanisms by which some patients develop a more severe form of ATL remain unclear. It is known that the host immune profile and parasite factors may influence the clinical manifestations of the disease. Besides intrinsic parasite factors, Leishmaniavirus RNA 1 (LRV1) infecting L. guyanensis can contribute to ATL immunopathogenesis. In this review, general aspects of L. guyanensis infection in humans and mouse models are presented.

Human Interleukin-32γ Plays a Protective Role in an Experimental Model of Visceral Leishmaniasis in Mice.

Visceral leishmaniasis (VL) is a chronic parasitic disease caused by Leishmania infantum in the Americas. During VL, several proinflammatory cytokines are produced in spleen, liver, and bone marrow. However, the role of interleukin-32 (IL-32) has not been explored in this disease. IL-32 can induce production of proinflammatory cytokines in innate immune cells and polarize the adaptive immune response. Herein, we discovered that L. infantum antigens induced expression of mRNA mainly for the IL-32γ isoform but also induced low levels of the IL-32ß transcript in human peripheral blood mononuclear cells. Furthermore, infection of human IL-32γ transgenic mice (IL-32γTg mice) with L. infantum promastigote forms increased IL-32γ expression in the spleen and liver. Interestingly, IL-32γTg mice harbored less parasitism in the spleen and liver than wild-type (WT) mice. In addition, IL-32γTg mice showed increased granuloma formation in the liver compared to WT mice. The protection against VL was associated with increased production of nitric oxide (NO), interferon gamma (IFN-γ), IL-17A, and tumor necrosis factor alpha by splenic cells restimulated ex vivo with L. infantum antigens. In parallel, there was an increase in the number of Th1 and Th17 T cells in the spleens of IL-32γTg mice infected with L. infantum IL-32γ induction of IFN-γ and IL-17A expression was found to be essential for NO production by splenic cells of infected animals. These data indicate that IL-32γ potentiates the Th1/Th17 immune response during experimental VL, thus contributing to the control of L. infantum infection.

The NOD2 receptor is crucial for immune responses towards New World Leishmania species.

American Tegumentary Leishmaniasis is a chronic infection caused by Leishmania protozoan. It is not known whether genetic variances in NOD-like receptor (NLR) family members influence the immune response towards Leishmania parasites and modulate intracellular killing. Using functional genomics, we investigated whether genetic variants in NOD1 or NOD2 influence the production of cytokines by human PBMCs exposed to Leishmania. In addition, we examined whether recognition of Leishmania by NOD2 contributes to intracellular killing. Polymorphisms in the NOD2 gene decreased monocyte- and lymphocyte-derived cytokine production after stimulation with L. amazonensis or L. braziliensis compared to individuals with a functional NOD2 receptor. The phagolysosome formation is important for Leishmania-induced cytokine production and upregulation of NOD2 mRNA expression. NOD2 is crucial to control intracellular infection caused by Leishmania spp. NOD2 receptor is important for Leishmania recognition, the control of intracellular killing, and the induction of innate and adaptive immune responses.

Platelet-activating factor increases reactive oxygen species-mediated microbicidal activity of human macrophages infected with Leishmania (Viannia) braziliensis.

Platelet-activating factor (PAF) is produced by macrophages during inflammation and infections. We evaluated whether PAF is able to modulate the infection of human macrophages by Leishmania braziliensis, the main Leishmania sp. in Brazil. Monocyte-derived macrophages were incubated with promastigote forms in absence or presence of exogenous PAF. We observed that the treatment of macrophages with low concentrations of PAF prior to infection increased the phagocytosis of L. braziliensis. More importantly, exogenous PAF reduced the parasitism when it was added before, during or after infection. In addition, treatment with a PAF antagonist (PCA 4248) resulted in a significant increase of macrophage infection in a concentration-dependent manner, suggesting that endogenous PAF is important to control L. braziliensis infection. Mechanistically, while exogenous PAF increased production of reactive oxygen species (ROS) treatment with PCA 4248 reduced oxidative burst during L. braziliensis infection. The microbicidal effects of exogenous PAF were abolished when macrophages were treated with apocynin, an NADPH oxidase inhibitor. The data show that PAF promotes the production of ROS induced by L. braziliensis, suggesting that this lipid mediator may be relevant to control L. braziliensis infection in human macrophages.