transforming-growth-factor-beta and Leishmaniasis--Cutaneous

Immune responses have a crucial role during the wound healing process in cutaneous leishmaniasis (CL). However, there are several paradoxes in immunity against CL. On the one hand, regulatory cytokines interleukin (IL)-10 and transforming growth factor beta (TGF-β) increase susceptibility to CL through suppression of several proinflammatory cytokines that require for defense against CL. On the other hand, these cytokines play a pivotal role in the acceleration of wound healing process. This review discusses about the dual role of IL-10 and TGF-β during the wound healing process and immunity against CL to offer a new insight about wound healing in CL.

Topics: Animals; Humans; Interleukin-10; Leishmaniasis, Cutaneous; Mice; Th17 Cells; Transforming Growth Factor beta; Wound Healing

1. The course of infection with the protozoan parasite Leishmania is determined in part by its early replication in macrophages, the exclusive host cells for these organisms. Resistance to and recovery from leishmanial infection is related to cell-mediated immune responses in all forms of human and murine leishmaniasis. 2. Factors contributing to the early inhibition or proliferation of Leishmania are poorly understood, but cytokines such as IFN gamma, IL-10 or transforming growth factor beta (TGF-beta) are known to influence the replication of Leishmania in macrophages. 3. TGF-beta is a multipotential cytokine with diverse effects on cells of the immune system, including down-regulation of certain macrophage functions. Infection of murine or human macrophages by Leishmania induces the production of active TGF-beta. Recombinant TGF-beta added to murine or human macrophage cultures leads to increased intracellular replication of Leishmania. Exogenous TGF-beta administered in vivo promotes enhancement of infection, whereas its neutralization by monoclonal antibodies decreases the level of in vitro infection, and protects susceptible mice. 4. Susceptible animals treated with anti-TGF-beta monoclonal antibodies change their immune response, not increasing the expression of IL-4 while increasing the expression of IFN gamma mRNA in their draining lymph nodes. Resistant animals treated with TGF-beta also change their pattern of immune response as indicated by an increase of the important Th2 cytokine IL-10 mRNA in the draining lymph node. 5. TGF-beta has profound effects on the host response to Leishmania in both mouse and man, and probably is an important parasite escape mechanism.

Topics: Animals; Antibodies, Monoclonal; Cytokines; Host-Parasite Interactions; Humans; Leishmania; Leishmaniasis, Cutaneous; Macrophages; Mice; Transforming Growth Factor beta

Visceral leishmaniasis (VL) or kala-azar is known to be associated with a mixed Th1-Th2 response, and effective host defense requires the induction of IFN-gamma and IL-12. We address the role of the differential decline of IL-10 and TGF-beta in response to sodium antimony gluconate (SAG) and amphotericin B (AmB), the therapeutic success of SAG and AmB in Indian VL, and the significance of IL-10 and TGF-beta in the development and progression of post-kazla-azar dermal leishmaniasis (PKDL). In the active disease, PBMC from VL patients showed suppressed Ag-specific lymphoproliferation, IFN-gamma and IL-12 production, and elevation of IL-10 and TGF-beta. Cure corresponded with an elevation in IFN-gamma and IL-12 production and down-regulation of IL-10 and TGF-beta. Both CD4(+) and CD8(+) T cells were involved in IFN-gamma and IL-10 production. Interestingly, the retention and maintenance of residual IL-10 and TGF-beta in some SAG-treated individuals and the elevation of IL-10 and TGF-beta in PKDL, a sequel to kala-azar, probably reflects the role of these cytokines in reactivation of the disease in the form of PKDL. Contrastingly, AmB treatment of VL resulted in negligible TGF-beta levels and absolute elimination of IL-10, reflecting the better therapeutic activity of AmB and its probable role in the recent decline in PKDL occurrences in India. Moreover, elucidation of immune responses in Indian PKDL patients revealed a spectral pattern of disease progression where disease severity could be correlated inversely with lymphoproliferation and directly with TGF-beta, IL-10, and Ab production. In addition, the enhancement of CD4(+)CD25(+) T cells in active VL, their decline at cure, and reactivation in PKDL suggest their probable immunosuppressive role in these disease forms.

Topics: Adolescent; Adult; Amphotericin B; Animals; Antimony Sodium Gluconate; Cells, Cultured; Coculture Techniques; Disease Susceptibility; Female; Humans; India; Interleukin-10; Leishmania donovani; Leishmaniasis, Cutaneous; Leishmaniasis, Visceral; Male; Recurrence; Transforming Growth Factor beta

The innate immune mediators are likely to influence the clinical phenotype of leishmaniasis by primary responses which limit or facilitate the spread of the parasite, as well as by modulating adaptive immunity. This study investigated the response of key innate immune cells in a focus which regularly reports localised cutaneous leishmaniasis (LCL) caused by Leishmania donovani, a species which typically causes visceral disease.. Peripheral blood mononuclear cell (PBMC) derived macrophages and dendritic cells from patients with LCL and healthy controls from endemic and non-endemic areas, were stimulated with soluble Leishmania antigen (SLA). Inflammatory mediators produced by macrophages (TNF-α/TGF-β/IL-10, ELISA; NO, Griess method) and dendritic cells (IL-12p70, IL-10, flowcytometry) and macrophage expression of surface markers of polarization, activation and maturation (flowcytometry) were determined at 24h, 48h and 72h and compared. Study was conducted prospectively from 2015-2019.. Patient derived macrophages and dendritic cells produced higher levels of both pro and anti-inflammatory mediators compared to controls (p<0.05) with the best discrimination for active disease observed at 72h. Data demonstrated an early activation of macrophages and a subsequent pro-inflammatory bias, as indicated by temporal profiles of TNF-α/TGF-β and TNF-α/IL-10 ratios and higher proportions of classical (M1) macrophages. Higher TGF-β levels were observed in cells from patients with ulcerated or persistent lesions. Immune responses by cells derived from controls in endemic and non-endemic regions did not differ significantly from each other.. The overall immunophenotypic profile suggests that LCL observed in the country is the result of a balancing immune response between pro-inflammatory and regulatory mediators. The mediators which showed distinct profiles in patients warrant further investigation as potential candidates for immunotherapeutic approaches. A comparison with visceral leishmaniasis caused by the same species, would provide further evidence on the differential role of these mediators in the resulting clinical phenotype.

Topics: Cytokines; Humans; Interleukin-10; Leishmania donovani; Leishmaniasis, Cutaneous; Leishmaniasis, Visceral; Leukocytes, Mononuclear; Phenotype; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

Leishmania parasites are deposited in the host through sand fly bites along with sand fly saliva. Therefore, salivary proteins are promising vaccine candidates for controlling leishmaniasis. Herein, two immunogenic salivary proteins, PpSP15 from Phlebotomus papatasi and PsSP9 from Phlebotomus sergenti, were selected as vaccine candidates to be delivered by live Leishmania tarentolae as vector. The stepwise in silico protocol advantaged in this study for multi-protein design in L. tarentolae is then described in detail.. All possible combinations of two salivary proteins, PpSP15 and PsSP9, with or without T2A peptide were designed at the mRNA and protein levels. Then, the best combination for the vaccine candidate was selected based on mRNA and protein stability along with peptide analysis.. At the mRNA level, the most favored secondary structure was PpSP15-T2A-PsSP9. At the protein level, the refined three-dimensional models of all combinations were structurally valid; however, local quality estimation showed that the PpSp15-T2A-PsSP9 fusion had higher stability for each amino acid position, with low root-mean-square deviation (RMSD), compared with the original proteins. In silico evaluation confirmed the PpSP15-T2A-PsSP9 combination as a good Th1-polarizing candidate in terms of high IFN-γ production and low IL-10/TGF-β ratio in response to three consecutive immunizations. Potential protein expression was then confirmed by Western blotting.. The approach presented herein is among the first studies to have privileged protein homology modeling along with mRNA analysis for logical live vaccine design-coding multi-proteins.

Topics: Amino Acids; Animals; Interleukin-10; Leishmania; Leishmaniasis, Cutaneous; Phlebotomus; Psychodidae; RNA, Messenger; Salivary Proteins and Peptides; Transforming Growth Factor beta; Vaccines, Attenuated

Currently, there is no satisfactory treatment modality available for cutaneous leishmaniasis (CL). The major objective of the present study was to explore the effect of immunomodulator-levamisole in combination with Glucantime in end-stage unresponsive patients with anthroponotic CL (ACL). Twenty end-stage unresponsive patients with ACL were identified for participation in this single-group trial study. Simultaneously, each patient was received a combination of levamisole pills along with Glucantime during the remedy course. Several in vitro complementary experiments were performed to evaluate the mode of action of levamisole and Glucantime alone and in combination using a macrophage model, in vitro MTT assay, flow cytometry and quantitative real time PCR (qPCR). Overall, 75% of the patients showed complete clinical cure, 10% partially improved and the remaining (15%) had underlying chronic diseases demonstrated no response to the treatment regimen. In in vitro studies, there was no cytotoxic effect associated with these drugs in the range of our experiments. The findings by the flow cytometric analysis represented that the highest apoptotic values corresponded to the drugs combination (32.23%) at 200 μg/ml concentration. Finally, the gene expression level of IL-12 p40, iNOS and TNF-α promoted while the level of IL-10 and TGF-β genes reduced as anticipated. The findings clearly indicated that the combination of levamisole and Glucantime should be considered in end-stage unresponsive patients with ACL who have not responded to basic treatments. The immunomodulatory role of levamisole in mounting immune system as documented by the in vitro experiments and further substantiated by this single-group trail study was highlighted.

Topics: Adolescent; Adult; Aged; Animals; Antiprotozoal Agents; Cell Line; Cell Survival; Child; Chronic Disease; Drug Combinations; Drug Therapy, Combination; Female; Humans; Interleukin-10; Interleukin-12 Subunit p40; Leishmania tropica; Leishmaniasis, Cutaneous; Levamisole; Macrophages; Male; Meglumine Antimoniate; Mice; Middle Aged; Nitric Oxide Synthase Type II; Transforming Growth Factor beta; Treatment Outcome; Tumor Necrosis Factor-alpha; Young Adult

Immunization with killed Leishmania promastigotes without adjuvant was considered as safe, but gave variable rates of protection. Taking advantage of the immuno-modulatory effect of caffeic acid (CA), a natural polyphenolic antioxidant, we investigated its potentiating effect in autoclaved Leishmania major (ALM)-induced protection of Leishmania major-infected BALB/c.. First, BALB/c mice were sensitized for 6 weeks either with CA, or ALM alone or combined with caffeic acid (ALM-CA) or Freund's adjuvant (ALM-FA), and subsequently infected with L. major promastigotes. Second, to test the curative effect, CA was given daily for 5 weeks to susceptible mice, starting on week 4 post-infection. Sera, footpads and lymph nodes (LNs) were collected at week 9 post-infection and submitted to biochemical or histological analyses.. Compared to the respective controls, our results showed that CA directly healed footpad lesions and reduced the hallmarks of cutaneous leishmaniasis including oxidative inflammation, parasite load, and phagocytes influx and infestation. In sensitized mice, the protection enhanced gradually from ALM-FA, CA, ALM to ALM-CA in parallel to decreased seric IgGt levels. In contrast to ALM-FA, the combined effect of ALM and CA increased specific isotype IgG2, and decreased IL-17 and MCP-1, and phagocyte influx, as attested by the concomitant reduction in myeloperoxidase (MPO) and α-naphthyl acetate esterase (ANAE) activities. ALM-CA shifted IFN-γ/TGF-β and iNO synthase/arginase1 (iNOS/Arg1) balances in a Th1 immune response that control efficiently cutaneous lesions and LNs hypertrophy, and reactivate the death of infected phagocytes.. Therefore, CA combined with ALM synergizes with L. major antigens for priming innate cells, through early polarization to optimal Th1 response that leads to IFN-γ and iNOS-dependent leishmanicidal activity of neutrophils and macrophages.

Topics: Animals; Arginase; Caffeic Acids; Immunoglobulin G; Interferon-gamma; Leishmania major; Leishmaniasis, Cutaneous; Mice; Mice, Inbred BALB C; Nitric Oxide Synthase; Phagocytes; Transforming Growth Factor beta

In Honduras,

Topics: CD4 Antigens; Central America; Forkhead Transcription Factors; Honduras; Humans; Immunohistochemistry; Interleukin-10; Leishmaniasis, Cutaneous; Skin; Skin Diseases; Transforming Growth Factor beta

Besides the Th1×Th2 paradigm, Treg and Th17 cytokines may play a role in the response to American tegumentary leishmaniasis. Considering the sensitivity and accuracy of qPCR and the lack of studies using this approach, we evaluated mRNA expression for IFN-γ, TNF-α, IL-4, IL-10, IL-6, IL-17A, IL-22, TGF-β, Foxp3 and RORC in peripheral blood mononuclear cells (PBMC) from patients with active disease, after stimulation with L. (V.) braziliensis soluble or insoluble fractions. Our results show that the antigens promoted specific mRNA expression related to the immune response in patients with ATL, and the insoluble fraction seems to stimulate the immune response in a higher intensity. The pro-inflammatory response was also fueled by IFN-γ and TNF-α, probably due to the active disease. IL-4, in certain way, seems to regulate this response along with IL-10 that may be produced by Treg cells, which are supposedly present in the patients' samples due the evidenced expression of Foxp3, in the presence of AgIns. In contrast, down-regulated RORC suggests that the significant levels of IL-6 expressed in response to AgSol were not able to induce an expressive Th17 profile along with TGF-β, which might have predominantly contributed to the development of a regulatory profile in the active disease.

Topics: Adolescent; Adult; Aged; Antigens, Protozoan; Case-Control Studies; Complex Mixtures; Female; Forkhead Transcription Factors; Gene Expression Regulation; Humans; Interferon-gamma; Interleukins; Leishmania braziliensis; Leishmaniasis, Cutaneous; Male; Middle Aged; Nuclear Receptor Subfamily 1, Group F, Member 3; Primary Cell Culture; RNA, Messenger; T-Lymphocytes, Regulatory; Th1 Cells; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

Post-kala-azar dermal leishmaniasis (PKDL) is an important factor in kala-azar transmission; hence its early detection and assessment of effective treatment is very important for disease control. In present study on 60 PKDL cases presented with macular, mixed papulonodular, or erythematous lesions, Leishmania parasites were demonstrated microscopically in 91% of papulonodular and 40% of macular lesions. Cellular infiltrates in skin biopsy imprint smears from lesions were mononuclear cells, 25-300/OIF (oil immersion field), predominantly histiocytes with vacuolation, many lymphocytes, some plasma cells, and Leishmania amastigotes 0-20/OIF. Cases with no demonstrable parasites were diagnosed on the basis of past history of VL, lesion's distribution, cytopathological changes, and positive DAT (86.83%). Following antileishmanial treatment with SAG, papulonodular forms of PKDL lesions disappeared clinically but microscopically the mononuclear cells (20-200/OIF) persisted in the dermal lesions. Response observed in macular PKDL lesions was poor which persisted both clinically and cytopathologically. Follow-up of PKDL will assess the effectivity of treatment as either disappearance of lesions or any relapse. Studies on involvement of immunological factors, that is, certain cytokines (IL-10, TGF-β, etc.) and chemokines (macrophage inflammatory protein, MIP 1-α, etc.) in PKDL, may provide insight for any role in the treatment response.

Topics: Adolescent; Adult; Child; Female; Humans; Interleukin-10; Leishmania donovani; Leishmaniasis, Cutaneous; Leishmaniasis, Visceral; Male; Middle Aged; Transforming Growth Factor beta; Treatment Outcome

Regulatory T cells (Tregs) are a unique population of CD25+CD4+ T cells that regulate innate and adaptive immune responses and have the ability to control the excessive or misdirected effects of the immune system. This modulation involves different mechanisms, such as the suppression of T cell proliferation and cytokine production, the secretion of suppressive cytokines (IL-10 and TGF-β) and the induction of effector T cell apoptosis in humans with infectious diseases such as Leishmania infections. The aim of this study was to evaluate the expression of Foxp3, IL-10 and TGF-β through immunohistochemistry in 22 skin biopsies of patients with localized cutaneous leishmaniasis (LCL) caused by Leishmania (Viannia) spp. from an endemic area in pre-Amazonian area of Maranhão State, Brazil. The density of these markers was also analyzed according to the species of parasite and the progression of the disease. The cellular density was 234 cells/mm(2) for Foxp3+ cells, 357 cells/mm(2) for TGF-β+ cells and 648 cells/mm(2) for IL-10+ cells in the studied skin lesions. The analysis of the cellular density of these immunological markers in relation to the species of Leishmania demonstrated that lesions caused by L. (V.) braziliensis had a lower density of Foxp3+ cells than lesions caused by L. (Viannia) spp. The expression of IL-10 was also lower in lesions caused by L. (V.) braziliensis. There were no significant differences in TGF-β expression between the two groups. The evaluation of these markers according to the progression of the disease did not reveal any significant differences. These findings suggest that Treg Foxp3+ cells, IL-10, and TGF-β play important roles in the immunopathogenesis of LCL and that these roles differ depending on the causal Leishmania species.

Topics: Adolescent; Adult; Biomarkers; Brazil; Disease Progression; Female; Forkhead Transcription Factors; Humans; Interleukin-10; Leishmania braziliensis; Leishmaniasis, Cutaneous; Male; Middle Aged; Skin; Species Specificity; T-Lymphocytes, Regulatory; Transforming Growth Factor beta; Young Adult

The Yucatan deer mouse, Peromyscus yucatanicus (order Rodentia), is the principal reservoir of Leishmania (Leishmania) mexicana in the Yucatan peninsula of Mexico. Experimental infection results in clinical and histopathological features similar to those observed in humans with cutaneous leishmaniasis (CL) as well as peritoneal macrophage production of nitric oxide. These results support the possible use of P. yucatanicus as a novel experimental model to study CL caused by L. (L.) mexicana. However, immunological studies in these rodents have been limited by the lack of specific reagents. To address this issue, we cloned and analyzed cytokine sequences of P. yucatanicus as part of an effort to develop this species as a CL model. We cloned P. yucatanicus interleukin 4 (IL-4), IL-10, IL-12p35, gamma interferon, transforming growth factor beta and tumor necrosis factor partial cDNAs. Most of the P. yucatanicus sequences were highly conserved with orthologs of other mammalian species and the identity of all sequences were confirmed by the presence of conserved amino acids with possible biological functions in each putative polypeptide. The availability of these sequences is a first step which will allow us to carry out studies characterizing the immune response during pathogenic and nonpathogenic L. (L.) mexicana infections in P. yucatanicus.

Topics: Amino Acid Sequence; Animals; Base Sequence; Cloning, Molecular; Female; Interferon-gamma; Interleukin-10; Interleukin-12 Subunit p35; Interleukin-4; Leishmania mexicana; Leishmaniasis, Cutaneous; Male; Molecular Sequence Data; Peromyscus; Sequence Alignment; Sequence Analysis, DNA; Th1 Cells; Th2 Cells; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

In tegumentary leishmaniasis caused by Leishmania braziliensis, there is evidence that increased production of IFN-γ, TNF-α and absence of IL-10 is associated with strong inflammatory reaction and with tissue destruction and development of the lesions observed in cutaneous leishmaniasis (CL) and mucosal leishmaniasis (ML). We evaluate the role of regulatory cytokines and cytokine antagonists in the downregulation of immune response in L. braziliensis infection. Peripheral blood mononuclear cells from CL and ML were stimulated with soluble Leishmania antigen in the presence or absence of regulatory cytokines (IL-10, IL-27 and TGF-β) or antagonists of cytokines (α-TNF-α and α-IFN-γ). Cytokines production (IL-10, IL-17, TNF-α and IFN-γ) was measured by ELISA. IL-10 and TGF-β downmodulate TNF-α and IL-17 production, whereas IL-27 had no effect in the production of TNF-α, IFN-γ and IL-17 in these patients. Neutralization of TNF-α decreased IFN-γ level and the neutralization of IFN-γ decreased TNF-α level and increased IL-10 production. This study demonstrate that IL-10 and TGF-β are cytokines that appear to be more involved in modulation of immune response in CL and ML patients. IL-10 might have a protective role, since the neutralization of IFN-γ decreases the production of TNF-α in an IL-10-dependent manner.

Topics: Adult; Cells, Cultured; Female; Humans; Inflammation; Interferon-gamma; Interleukin-10; Interleukin-17; Interleukins; Leishmania braziliensis; Leishmaniasis, Cutaneous; Leukocytes, Mononuclear; Male; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

Two different Toll-like receptors (TLRs) have been shown to play a role in host responses to Leishmania infection. TLR-2 is involved in parasite survival in macrophages upon activation by lipophosphoglycan (LPG), a virulence factor expressed by Leishmania. In contrast, activation of TLR-9 has been shown to promote a host-protective response. However, whether there is a relationship between the interaction of LPG and TLR-2, on one hand, with the effect of TLR-9, on the other hand, remains unknown. In this study, we report that in-vitro infection of macrophages with a L. major parasite with high expression levels of LPG results in decreased TLR-9 expression compared to infection with a L. major parasite with lower expression levels of LPG. Addition of anti-LPG as well as anti-TLR-2 antibodies prevents this reduction of TLR-9 expression. Also, the addition of purified LPG to macrophages results in a decrease of TLR-9 expression, which is shown to be mediated by transforming growth factor (TGF)-β and interleukin (IL)-10. Finally, in-vitro treatment of macrophages with anti-LPG and/or anti-TLR-2 antibodies before infection reduces the number of amastigotes in macrophages and co-treatment of mice with anti-TLR-2 antibodies and cytosine-phosphate-guanosine (CpG) reduces footpad swelling and parasite load in the draining lymph nodes, accompanied by an interferon (IFN)-γ-predominant T cell response. Thus, for the first time, we show how interactions between LPG and TLR-2 reduce anti-leishmanial responses via cytokine-mediated decrease of TLR-9 expression.

Topics: Animals; CpG Islands; Gene Expression; Glycosphingolipids; Host-Parasite Interactions; Immune Tolerance; Interleukin-10; Leishmania major; Leishmaniasis, Cutaneous; Macrophage Activation; Macrophages; Mice; Mice, Inbred BALB C; Nitric Oxide Synthase Type II; Toll-Like Receptor 2; Toll-Like Receptor 9; Transforming Growth Factor beta; Virulence

Leishmania braziliensis causes cutaneous (CL) and mucosal (ML) leishmaniasis. In the mouse, Fli1 was identified as a gene influencing enhanced wound healing and resistance to CL caused by Leishmania major. Polymorphism at FLI1 is associated with CL caused by L. braziliensis in humans, with an inverse association observed for ML disease. Here we extend the analysis to look at other wound healing genes, including CTGF, TGFB1, TGFBR1/2, SMADS 2/3/4/7 and FLII, all functionally linked along with FLI1 in the TGF beta pathway. Haplotype tagging single nucleotide polymorphisms (tag-SNPs) were genotyped using Taqman technology in 325 nuclear families (652 CL cases; 126 ML cases) from Brazil. Robust case-pseudocontrol (CPC) conditional logistic regression analysis showed associations between CL and SNPs at CTGF (SNP rs6918698; CC genotype; OR 1.67; 95%CI 1.10-2.54; P=0.016), TGFBR2 (rs1962859; OR 1.50; 95%CI 1.12-1.99; P=0.005), SMAD2 (rs1792658; OR 1.57; 95%CI 1.04-2.38; P=0.03), SMAD7 (rs4464148; AA genotype; OR 2.80; 95%CI 1.00-7.87; P=0.05) and FLII (rs2071242; OR 1.60; 95%CI 1.14-2.24; P=0.005), and between ML and SNPs at SMAD3 (rs1465841; OR 2.15; 95%CI 1.13-4.07; P=0.018) and SMAD7 (rs2337107; TT genotype; OR 3.70; 95%CI 1.27-10.7; P=0.016). Stepwise logistic regression analysis showed that all SNPs associated with CL at FLI1, CTGF, TGFBR2, and FLII showed independent effects from each other, but SNPs at SMAD2 and SMAD7 did not add independent effects to SNPs from other genes. These results suggest that TGFβ signalling via SMAD2 is important in directing events that contribute to CL, whereas signalling via SMAD3 is important in ML. Both are modulated by the inhibitory SMAD7 that acts upstream of SMAD2 and SMAD3 in this signalling pathway. Along with the published FLI1 association, these data further contribute to the hypothesis that wound healing processes are important determinants of pathology associated with cutaneous forms of leishmaniasis.

Topics: Adult; Brazil; Cohort Studies; Family; Female; Genetic Predisposition to Disease; Haplotypes; Humans; Leishmaniasis, Cutaneous; Linkage Disequilibrium; Logistic Models; Male; Microfilament Proteins; Polymorphism, Single Nucleotide; Receptors, Cytoplasmic and Nuclear; Signal Transduction; Trans-Activators; Transforming Growth Factor beta; Wound Healing

Cutaneous lesions caused by Leishmania braziliensis infection occasionally heal spontaneously, but with antimonials therapy heal rapidly in approximately 3 weeks. However, about 15% of the cases require several courses of therapy. Matrix metalloproteinase-2 (MMP-2) and MMP-9 are gelatinases that have been implicated in other chronic cutaneous diseases and skin re-epithelialization. These enzymes are controlled by their natural inhibitors [tissue inhibitors of metalloproteinase (TIMPs)] and by some cytokines. Uncontrolled gelatinase activity may result in intense tissue degradation and, consequently, poorly healing wounds. The present study correlates gelatinase activity to therapeutic failure of cutaneous leishmaniasis (CL) lesions. Our results demonstrate an association between gelatinase activity and increased numbers of cells making interferon (IFN)-γ, interleukin (IL)-10 and transforming growth factor (TGF)-β in lesions from poor responders. Conversely, high levels of MMP-2 mRNA and enhanced MMP-2 : TIMP-2 ratios were associated with a satisfactory response to antimonials treatment. Additionally, high gelatinolytic activity was found in the wound beds, necrotic areas in the dermis and within some granulomatous infiltrates. These results indicate the importance of gelatinase activity in the skin lesions caused by CL. Thus, we hypothesize that the immune response profile may be responsible for the gelatinase activity pattern and may ultimately influence the persistence or cure of CL lesions.

Topics: Adult; Antimony; Antiprotozoal Agents; Cytokines; Female; Humans; Interferon-gamma; Interleukin-10; Leishmaniasis, Cutaneous; Male; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Meglumine; Meglumine Antimoniate; Organometallic Compounds; Regeneration; Skin; Transforming Growth Factor beta; Treatment Failure

Mast cells (MCs) play an important role in the regulation of protective adaptive immune responses against pathogens. However, it is still unclear whether MCs promote such host defense responses via direct effects on T cells or rather by modifying the functions of antigen-presenting cells. To identify the underlying mechanisms of the immunoregulatory capacity of MCs, we investigated the impact of MCs on dendritic cell (DC) maturation and function. We found that murine peritoneal MCs underwent direct crosstalk with immature DCs that induced DC maturation as evidenced by enhanced expression of costimulatory molecules. Furthermore, the MC/DC interaction resulted in the release of the T-cell modulating cytokines IFN-γ, IL-2, IL-6 and TGF-β into coculture supernatants and increased the IL-12p70, IFN-γ, IL-6 and TGF-β secretion of LPS-matured DCs. Such MC-"primed" DCs subsequently induced efficient CD4+ T-cell proliferation. Surprisingly, we observed that MC-primed DCs stimulated CD4+ T cells to release high levels of IFN-γ and IL-17, demonstrating that MCs promote Th1 and Th17 responses. Confirming our in vitro findings, we found that the enhanced disease progression of MC-deficient mice in Leishmania major infection is correlated with impaired induction of both Th1 and Th17 cells.

Topics: Animals; Antibodies, Monoclonal; B7-1 Antigen; B7-2 Antigen; CD4-Positive T-Lymphocytes; CD40 Antigens; Cell Communication; Cell Proliferation; Dendritic Cells; Disease Progression; Interferon-gamma; Interleukin-1; Interleukin-12; Interleukin-17; Interleukin-2; Leishmania major; Leishmaniasis, Cutaneous; Lymphocyte Activation; Mast Cells; Mice; Mice, Inbred C57BL; Mice, Knockout; Th1 Cells; Th17 Cells; Transforming Growth Factor beta

Stimulation of neutrophils may potentiate immunity to Leishmania major. CpG-containing oligodeoxynucleotide (ODN) has immune stimulatory effects and has been suggested as adjuvants and therapeutics to potentiate efficacy of vaccines and treatments against leishmaniasis. Here, we examined the stimulatory effect of synthetic ODN containing CpG motifs class A and B on cytokine production by neutrophils. Neutrophils from healthy donors responded to CpG-ODN type A, but not to class B, with secretion of IL-8 and following GM-CSF pretreatment with TNF-α production. To test whether neutrophil responses were altered in cutaneous leishmaniasis (CL) and to better understand the role of neutrophils in susceptibility and resistance to disease, we evaluated cytokine responses in GM-CSF preconditioned neutrophils from asymptomatic (Leishmanin skin test positive, LST+) and nonhealing CL individuals to CpG-ODN class A and assessed the expression levels of toll-like receptors (TLR2), 4 and 9. LST+ and healthy donor, but not nonhealing CL neutrophils, responded with TNF-α secretion. Neutrophils from nonhealing CL displayed increased mRNA expression levels of TLR2, 4 and 9 compared to neutrophils from LST+ or healthy donors. Therefore, failure to cure CL is associated with reduced ability of neutrophils to secrete TNF-α and correlates with high TLR 2, 4 and 9 expressions.

Topics: Adolescent; Adult; Animals; Female; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Interleukin-8; Leishmania major; Leishmaniasis, Cutaneous; Male; Mice; Mice, Inbred BALB C; Middle Aged; Neutrophil Activation; Neutrophils; Oligodeoxyribonucleotides; RNA, Messenger; Toll-Like Receptor 2; Toll-Like Receptor 4; Toll-Like Receptor 9; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

Leishmania tropica is the causative agent of Old World anthroponotic cutaneous leishmaniasis, which is characterized by lesions that take an extended period of time to heal, often resulting in disfiguring scars, and are more refractory to treatment than leishmaniasis caused by Leishmania major. Immunologic studies involving experimental animal models of L. tropica infection are virtually nonexistent. In the current study, infectious-stage L. tropica were used to establish dermal infections in C57BL/6 and BALB/c mice. In both strains, the lesions were slow to develop and showed minimal pathology. They nonetheless contained a stable number of between 10(4) and 10(5) parasites for over 1 year, which were efficiently picked up by a natural sand fly vector, Phlebotomus sergenti. Control of parasite growth depended on the development of a Th1 response, as C57BL/6 mice genetically deficient in Th1 cytokines and BALB/c mice treated with Abs to IFN-gamma harbored significantly more parasites. By contrast, IL-10-deficient mice harbored significantly fewer parasites throughout the infection. To further study the immunologic mechanisms that may prevent efficient clearance of the parasites, IL-10 and TGF-beta signaling were abrogated during the chronic phase of infection in wild-type C57BL/6 mice. Distinct from chronic L. major infection, IL-10 blockade alone had no effect on L. tropica, but required simultaneous treatment with anti-TGF-beta Abs to promote efficient parasite clearance from the infection site. Thus, chronic infection with L. tropica appears to be established via multiple suppressive factors, which together maintain the host as a long-term reservoir of infection for vector sand flies.

Topics: Animals; Chronic Disease; Disease Models, Animal; Ear, External; Host-Parasite Interactions; Immunity, Innate; Insect Vectors; Interleukin-10; Leishmania tropica; Leishmaniasis, Cutaneous; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Knockout; Phlebotomus; Transforming Growth Factor beta

Classical activation of macrophages infected with Leishmania species results in expression and activation of inducible NO synthase (iNOS) leading to intracellular parasite killing. Macrophages can contrastingly undergo alternative activation with increased arginase activity, metabolism of arginine along the polyamine pathway, and consequent parasite survival. An active role for parasite-encoded arginase in host microbicidal responses has not previously been documented. To test the hypothesis that parasite-encoded arginase can influence macrophage responses to intracellular Leishmania, a comparative genetic approach featuring arginase-deficient mutants of L. mexicana lacking both alleles of the gene encoding arginase (Deltaarg), as well as wild-type and complemented Deltaarg controls (Deltaarg[pArg]), was implemented. The studies showed: 1) the absence of parasite arginase resulted in a significantly attenuated infection of mice (p<0.05); 2) poorer survival of Deltaarg in mouse macrophages than controls correlated with greater NO generation; 3) the difference between Deltaarg or control intracellular survival was abrogated in iNOS-deficient macrophages, suggesting iNOS activity was responsible for increased Deltaarg killing; 4) consistently, immunohistochemistry showed enhanced nitrotyrosine modifications in tissues of mice infected with Deltaarg compared with control parasites. Furthermore, 5) in the face of decreased parasite survival, lymph node cells draining cutaneous lesions of Deltaarg parasites produced more IFN-gamma and less IL-4 and IL-10 than controls. These data intimate that parasite-encoded arginase of Leishmania mexicana subverts macrophage microbicidal activity by diverting arginine away from iNOS.

Topics: Animals; Arginase; Female; Gene Deletion; Leishmania mexicana; Leishmaniasis, Cutaneous; Macrophages; Mice; Mice, Inbred BALB C; Nitric Oxide; Protozoan Proteins; Transforming Growth Factor beta

Peripheral blood CD16 (Fc receptor for immunoglobulin G III)-positive monocytes have been shown to expand in different pathological conditions, such as cancer, asthma, sepsis, human immunodeficiency virus infection, and AIDS progression, but data in leishmaniasis are lacking. We found that cutaneous leishmaniasis patients (n = 15) displayed a significant increase in the percentage (3.5 vs. 10.1) as well as mean fluorescent intensity (13.5 vs. 29.2) of ex vivo CD16 expression in monocytes as compared with healthy controls. We observed a significant positive correlation between the percentage of ex vivo CD16+ monocytes and lesion size (P = 0.0052, r = 0.75) or active transforming growth factor-beta plasma levels (P = 0.0017, r = 0.78). In addition, two patients with nonhealing lesions during a 3-year follow-up had high (9.1-19.4%) CD16 levels at diagnosis. Our data suggest a deleterious role for CD16 in human leishmaniasis, as well as its possible use as a marker for disease severity and/or adverse disease outcome.

Topics: Acquired Immunodeficiency Syndrome; Adult; Antigens, CD; Biomarkers; Follow-Up Studies; Gene Expression Regulation; GPI-Linked Proteins; HIV; Humans; Leishmaniasis, Cutaneous; Male; Monocytes; Neoplasms; Receptors, IgG; Sepsis; Transforming Growth Factor beta

American tegumentary leishmaniasis has three forms: localized (LCL), found in resistant individuals; diffuse (DCL), found in susceptible individuals; and intermediate cutaneous leishmaniasis (ICL), found in individuals with exacerbated immunity. We evaluated cytokines and inducible nitric oxide synthase (iNOS) in lesions of LCL, ICL and DCL using immunohistochemistry. LCL granulomas showed a preponderance of interferon (IFN)-gamma and interleukin (IL)-12 expression, whereas ICL granulomas had more IL-4-, IL-10- and mainly transforming growth factor (TGF)-beta1-expressing cells. Higher densities of iNOS+ cells were observed in ICL and LCL than in DCL. iNOS was also expressed in keratinocytes of LCL and ICL lesions, and in epidermal dendritic cells of ICL lesions. In LCL and ICL, most keratinocytes expressed IL-12 and a portion expressed IFN-gamma. IL-12+ and IFN-gamma+ dendritic cells were absent or sparse in LCL and ICL epidermis. Our results show the importance of iNOS, IL-12 and INF-gamma in LCL and ICL lesions, emphasizing the existence of a mixed cytokine pattern in ICL different from the Th1 and Th2 responses established in LCL and DCL lesions.

Topics: Cytokines; Dendritic Cells; Humans; Immunohistochemistry; Interferon-gamma; Interleukin-10; Interleukin-12; Interleukin-4; Keratinocytes; Leishmaniasis, Cutaneous; Nitric Oxide Synthase Type II; Th1 Cells; Th2 Cells; Transforming Growth Factor beta; Transforming Growth Factor beta1

Endogenous regulatory T (Treg) cells are involved in the control of infections, including Leishmania infection in mice. Leishmania viannia braziliensis is the main etiologic agent of cutaneous leishmaniasis (CL) in Brazil, and it is also responsible for the more severe mucocutaneous form. Here, we investigated the possible involvement of Treg cells in the control of the immune response in human skin lesions caused by L. viannia braziliensis infection. We show that functional Treg cells can be found in skin lesions of patients with CL. These cells express phenotypic markers of Treg cells--such as CD25, cytotoxic T lymphocyte-associated antigen 4, Foxp3, and glucocorticoid-induced tumor necrosis factor receptor--and are able to produce large amounts of interleukin-10 and transforming growth factor- beta . Furthermore, CD4+CD25+ T cells derived from the skin lesions of 4 of 6 patients with CL significantly suppressed in vitro the phytohemagglutinin-induced proliferative T cell responses of allogeneic peripheral-blood mononuclear cells (PBMCs) from healthy control subjects at a ratio of 1 Treg cell to 10 allogeneic PBMCs. These findings suggest that functional Treg cells accumulate at sites of Leishmania infection in humans and possibly contribute to the local control of effector T cell functions.

Topics: Adolescent; Adult; Aged; Animals; CD4 Antigens; Chemokines, CC; Child; Female; Forkhead Transcription Factors; Humans; Immunosuppression Therapy; Interleukin-10; Leishmania braziliensis; Leishmaniasis, Cutaneous; Lymphocyte Activation; Male; Middle Aged; Phenotype; Receptors, CCR4; Receptors, Chemokine; Receptors, Interleukin-2; Skin; T-Lymphocytes, Regulatory; Transforming Growth Factor beta

Suppressor of cytokine signaling (SOCS)3 is a major negative feedback regulator of signal transducer and activator of transcription (STAT)3-activating cytokines. Transgenic mouse studies indicate that high levels of SOCS3 in T cells result in type 2 T helper cell (Th2) skewing and lead to hypersensitivity to allergic diseases. To define the physiological roles of SOCS3 in T cells, we generated T cell-specific SOCS3 conditional knockout mice. We found that the mice lacking SOCS3 in T cells showed reduced immune responses not only to ovalbumin-induced airway hyperresponsiveness but also to Leishmania major infection. In vitro, SOCS3-deficient CD4+ T cells produced more transforming growth factor (TGF)-beta1 and interleukin (IL)-10, but less IL-4 than control T cells, suggesting preferential Th3-like differentiation. We found that STAT3 positively regulates TGF-beta1 promoter activity depending on the potential STAT3 binding sites. Furthermore, chromatin immunoprecipitation assay revealed that more STAT3 was recruited to the TGF-beta1 promoter in SOCS3-deficient T cells than in control T cells. The activated STAT3 enhanced TGF-beta1 and IL-10 expression in T cells, whereas the dominant-negative form of STAT3 suppressed these. From these findings, we propose that SOCS3 regulates the production of the immunoregulatory cytokines TGF-beta1 and IL-10 through modulating STAT3 activation.

Topics: Animals; Cell Differentiation; Down-Regulation; Interleukin-10; Leishmania major; Leishmaniasis, Cutaneous; Mice; Mice, Knockout; Promoter Regions, Genetic; STAT3 Transcription Factor; Suppressor of Cytokine Signaling 3 Protein; Suppressor of Cytokine Signaling Proteins; T-Lymphocytes, Helper-Inducer; Th2 Cells; Transforming Growth Factor beta; Transforming Growth Factor beta1; Up-Regulation

Previous studies have shown that the in vitro ligation of FcgammaRs with IgG-opsonized Leishmania amastigotes promotes IL-10 production by macrophages. In addition, infection of either BALB/c mice lacking the common gamma-chain of Fc receptors (FcgammaR(-/-)) or mice genetically altered to lack circulating Ab (J(H)D) with Leishmania pifanoi results in reduced and delayed lesion development and a deficit in the recruitment of inflammatory cells into infected lesions. We show in this study that FcgammaR(-/-) mice can control infection with Leishmania major and totally resolve cutaneous lesions. The ability to eventually control infection is not associated with a reduction in lesion inflammation or a reduction in the ability of Leishmania to parasitize cells through week 6 of infection. The immune response in healing FcgammaR(-/-) mice is associated with a reduction in numbers of cells producing Th2-type cytokines, including IL-4 and IL-10, but not an increase in numbers of IFN-gamma-producing cells characteristic of a dominant Th1-type response. Instead, we observe a reduction in levels of IL-10 and TGF-beta within infected lesions, including reduced levels of these cytokines within parasitized macrophages. Together, these results suggest that uptake of opsonized parasites via FcgammaRs may be a strong in vivo stimulus for the production of anti-inflammatory cytokines that play a role in susceptibility to infection.

Topics: Animals; Antibodies, Protozoan; CD4-Positive T-Lymphocytes; Cells, Cultured; Cytokines; Down-Regulation; Female; Genetic Predisposition to Disease; Immunity, Innate; Immunoglobulin G; Interleukin-10; Leishmania major; Leishmaniasis, Cutaneous; Mice; Mice, Inbred BALB C; Mice, Knockout; Protein Subunits; Receptors, IgG; Th1 Cells; Th2 Cells; Transforming Growth Factor beta

While CBA/J mice fail to be permissive to Leishmania amazonensis-driven pathogenic processes, they heal easily following Leishmania major infection. The early-phase events are crucial to the outcome of Leishmania infection and it is known that macrophages (Mphi) are important in infection control. In the present study we investigated the role of Mphi in driving CBA/J susceptibility to L. amazonensis. We performed kinetic studies and compared the capacity of L. amazonensis and L. major to infect Mphi. There was no difference in percentages of infection or parasite burden for 6 h between the two groups. In contrast, after 12 h we observed that infection was about twice as high in L. amazonensis- than in L. major-infected Mphi. In addition, rIFN-gamma added to the cultures induced nitric oxide (NO) production, and did not modify L. amazonensis infection, although the percentage of L. major infection was significantly reduced. This reduction in L. major infection is a TNF-alpha dependent mechanism as L. major-infected Mphi expressed twice as much TNF-alpha mRNA as L. amazonensis-infected cells, and anti-TNF-alpha reversed the IFN-gamma effect. Moreover, rTNF-alpha plus IFN-gamma were able to significantly reduce the percentage of L. amazonensis-infected cells but not to the same extent as in L. major infection. Despite having higher NO production than IFN-gamma-treated cells, AMG addition to IFN-gamma-plus TNF-alpha-treated cells only partially reversed the inhibition in L. major, but not in L. amazonensis infection. Thus, in this study, we demonstrated that L. amazonensis both inactivated and resisted innate and IFN-gamma-induced Mphi killing mechanisms, indicating that the nature of the parasite and its interaction with Mphi could determine immune response polarization.

Topics: Animals; Cells, Cultured; Female; Hydrogen Peroxide; Inflammation; Interferon-gamma; Interleukin-10; Leishmania; Leishmania major; Leishmaniasis; Leishmaniasis, Cutaneous; Macrophages; Male; Mice; Mice, Inbred CBA; Species Specificity; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

The cytokine profile produced by peripheral blood mononuclear cells (PBMC) in response to leishmania antigens and the ability of interleukin-10 (IL-10) and transforming growth factor beta (TGF-beta) to modulate the immune response were evaluated in 21 mucosal leishmaniasis patients. Patients with mucosal disease exhibited increased gamma interferon (IFN-gamma) and tumor necrosis factor alpha (TNF-alpha) secretion and decreased IL-10 secretion compared to patients with classical cutaneous leishmaniasis. CD4(+) Th1 cells were the main source of IFN-gamma and TNF-alpha production in mucosal leishmaniasis patients. Evaluation of cytokine gene expression in PBMC of these patients showed that there was strong up-regulation of IFN-gamma transcripts upon stimulation with leishmania antigen, in contrast to the baseline levels of IL-10 mRNA. IL-10 suppressed IFN-gamma production by 48% in cell cultures from cutaneous leishmaniasis patients and by 86% in cell cultures from healthy subjects stimulated with purified protein derivative, whereas in similar conditions IL-10 suppressed IFN-gamma production by 19% in cell cultures from mucosal leishmaniasis patients stimulated with leishmania antigen. TGF-beta suppressed IFN-gamma levels to a greater extent in healthy subjects than in mucosal leishmaniasis and cutaneous leishmaniasis patients. These data indicate that a poorly modulated T-cell response in mucosal leishmaniasis patients leads to production of high levels of proinflammatory cytokines, such as IFN-gamma and TNF-alpha, as well as a decreased ability of IL-10 and TGF-beta to modulate this response. These abnormalities may be the basis for the pathological findings observed in this disease.

Topics: Animals; Gene Expression Regulation; Humans; Interferon-gamma; Interleukin-10; Leishmania; Leishmaniasis, Cutaneous; Leishmaniasis, Mucocutaneous; Th1 Cells; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha; Up-Regulation

CB6F1 mice display intermediate susceptibility to Leishmania major infection compared with the highly susceptible BALB/c and resistant C57BL/6 parental strains. During early weeks of infection, these mice develop dominant Th2 type responses to L. major, although they eventually exhibit a Th2 to Th1 switch and spontaneously resolve their infections. In this study, we have examined the effects of either IL-12 or anti-TGF-beta therapy on the immune response and course of disease in chronically infected CB6F1 mice. Local treatment with IL-12 inoculated into the parasitized lesion at 4 wk of infection induced a marked increase in IFN-gamma production but did not result in a significant reduction in numbers of parasite or promote more rapid healing. However, local treatment with an Ab to TGF-beta led to both a decrease in parasite numbers and more rapid healing, despite the fact that such treatment did not significantly alter the pattern of IL-4 and IFN-gamma production. Immunohistochemical studies showed that anti-TGF-beta treatment resulted in increased nitric oxide production within parasitized lesions. Our results suggest that TGF-beta may play an important regulatory role during chronic stages of a L. major infection by suppressing macrophage production of nitric oxide and that, in the absence of TGF-beta, even the relatively low levels of IFN-gamma observed in mice with dominant Th2-type responses are sufficient to activate macrophages to destroy amastigotes within parasitized lesions.

Topics: Adjuvants, Immunologic; Animals; Antibodies, Monoclonal; Chronic Disease; Crosses, Genetic; Drug Combinations; Female; Injections, Intralesional; Injections, Subcutaneous; Interferon-gamma; Interleukin-12; Leishmania major; Leishmaniasis, Cutaneous; Lymphocytes; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Nitric Oxide; Transforming Growth Factor beta

Type 2 nitric oxide synthase (NOS2) is required for the Th1-dependent healing of infections with intracellular microbes, including Leishmania major. Here, we demonstrate the expression and define the function of NOS2 during the innate response to L. major. At day 1 of infection, genetic deletion or functional inactivation of NOS2 abolished the IFNgamma and natural killer cell response, increased the expression of TGFbeta, and caused parasite spreading from the skin and lymph node to the spleen, liver, bone marrow, and lung. Induction of NOS2 was dependent on IFNalpha/beta. Neutralization of IFNalpha/beta mimicked the phenotype of NOS2-/- mice. Thus, IFNalpha/beta and NOS2 are critical regulators of the innate response to L. major.

Topics: Animals; Antibodies; Cytokines; Cytotoxicity, Immunologic; Down-Regulation; Female; Humans; Interferon Type I; Interferon-gamma; Isoenzymes; Killer Cells, Natural; Leishmania major; Leishmaniasis, Cutaneous; Macrophages; Male; Mice; Mice, Inbred C57BL; Nitric Oxide Synthase; Rabbits; Th1 Cells; Transforming Growth Factor beta

A vertebrate host becomes infected with Leishmania major when the sand fly vector injects parasites into skin along with saliva. Previous studies showed that salivary gland lysate of the New World sand fly Lutzomyia longipalpis markedly enhanced L. major infection in CBA mice. However, L. major is an Old World parasite transmitted in nature by the Old World sand fly Phlebotomus papatasi. Here we examine the ability of P. papatasi salivary gland lysate to enhance infection (lesion size and parasite burden) by L. major. In addition, we examine the effects of salivary gland lysate on the immune response to L. major by monitoring the levels of cytokine mRNA from the lymph nodes draining cutaneous lesions. We found that P. papatasi salivary gland lysate dramatically exacerbated lesion development in disease-resistant CBA mice. This exacerbation of disease correlated with inhibition of the production of Thl cytokines and associated factors (IFN-gamma, IL-12, and inducible nitric oxide synthase), but with enhancement of the Th2 cytokine IL-4, whereas no changes in the levels of IL-10 and TGF-beta were noted. Importantly, salivary gland lysate directly up-regulated expression of IL-4 mRNA in mice in the absence of infection with L. major.

Topics: Animals; Down-Regulation; Female; Interferon-gamma; Interleukin-10; Interleukin-12; Interleukin-4; Leishmania major; Leishmaniasis, Cutaneous; Lymph Nodes; Mice; Mice, Inbred CBA; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Phlebotomus; RNA, Messenger; Salivary Glands; Th1 Cells; Th2 Cells; Transforming Growth Factor beta; Up-Regulation

By using a primary in vitro response specific for Leishmania major, normal T cells from resistant CBA/CaH-T6J and susceptible BALB/c mice commit to a Th1 and a Th2 response, respectively. Since commitment occurred, we measured the production of gamma interferon (IFN-gamma), interleukin-1 (IL-1), IL-2, IL-4, IL-5, IL-10, and IL-12, prostaglandin E2 (PGE2), transforming growth factor beta (TGF-beta), and nitric oxide in the first 7 days of the response to identify factors that are critical for Th1 and Th2 development. While cells from resistant CBA mice produced more IFN-gamma, IL-10, and nitric oxide, cells from susceptible BALB/c mice produced more IL-1alpha, IL-5, PGE2, and TGF-beta. Although substantial amounts of IL-12 were detected, IL-12 did not associate with either Th1 or Th2 development. We did not anticipate that cells from resistant CBA mice would make more IL-10 in vitro. However, this also occurred in vivo since CBA mice produced substantial amounts of IL-10 following infection with L. major. Moreover, adding anti-IL-10 to primary in vitro responses enhanced production of IFN-gamma and nitric oxide by cells from CBA and BALB/c mice. Therefore, IL-10 cannot be regarded as a cytokine that associates with susceptibility to infection with L. major. Finally, the data presented here suggest that a collection of factors that can be produced by accessory cells influence Th commitment (e.g., IL-1, PGE2, and TGF-beta favor Th2 development).

Topics: Animals; Dinoprostone; Female; In Vitro Techniques; Interferon-gamma; Interleukin-1; Interleukin-10; Interleukin-12; Interleukin-2; Interleukin-4; Interleukin-5; Leishmania major; Leishmaniasis, Cutaneous; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Inbred CBA; Nitric Oxide; Species Specificity; Th1 Cells; Th2 Cells; Transforming Growth Factor beta

Treatment of nonhealing forms of human leishmaniasis with antimonial drugs in combination with gamma interferon (IFN-gamma) may promote healing more effectively than conventional drug therapy. Although the natures of immune responses in patients prior to treatment are often unclear, it is generally assumed that such therapy also promotes a switch from a Th2-type response to a dominant Th1-type response. We have examined the efficacy of IFN-gamma therapy, in combination with drug therapy, to promote healing and a Th2-to-Th1 switch in highly susceptible BALB/c mice infected with Leishmania major. Short-term treatment with the antileishmanial drug sodium stibogluconate failed to significantly alter the course of disease or the immune response when it was given during the third and fourth weeks of infection. IFN-gamma therapy, administered over the same time period, also failed to induce cure or a Th1 dominant response. In contrast, mice treated with a combination of drug and IFN-gamma therapy resolved their infections and developed Th1-type responses. However, administration of an antibody to interleukin 12 (IL-12) reversed the therapeutic effects of therapy with drug plus IFN-gamma, suggesting that IFN-gamma promotes cure through an IL-12-dependent mechanism. Analysis of mRNA levels within parasitized lesions suggests that drug treatment plus IFN-gamma treatment, in addition to reducing parasite numbers, results in reduced levels of IL-4, IL-10, and transforming growth factor beta transcripts but increased levels of transcripts of the p40 chain of IL-12 and inducible nitric oxide synthase, which catalyzes the production of nitric oxide. Together, these results suggest that such immunotherapy may promote the development of a protective Th1-type response in susceptible mice by a mechanism which involves both suppression of regulatory cytokines and enhancement of IL-12 and nitric oxide production.

Topics: Animals; Antibodies, Protozoan; Antimony Sodium Gluconate; Antiprotozoal Agents; Chronic Disease; Immunoglobulin G; Interferon-gamma; Interleukin-12; Leishmaniasis, Cutaneous; Mice; Mice, Inbred BALB C; Transforming Growth Factor beta

Mice with a genetically engineered deficiency for either IL-4 or IFN-gamma R1 (single mutants), and IL-4/IFN-gamma R1 (double mutants) on the Balb/c and 129Sv background were used to study the course of infection with Leishmania major. In contrast to genetically resistant 129Sv wildtype mice, IL-4/IFN-gamma R1 double mutant mice developed fetal disease with parasite dissemination to visceral organs similar to mice lacking IFN-gamma R1 only. Balb/c mice, which are exquisitely susceptible to L. major, were rendered resistant to infection by disruption of the IL-4 gene. As compared to homozygous IL-4+/- mice, heterozygous IL-4+/- mice, heterozygous IL-4+/- animals consistently developed smaller lesions with less ulceration and necrosis, indicating the likelihood of gene-dosage effects. This implicates that the magnitude of the IL-4 response determines the severity of disease. CD4+ T cells of IL-4-deficient mice showed impaired Th2 cell development, as assessed by quantitative RT-PCR of characteristic cytokines. Development of resistance is not explained by default Th1 development, because this was observed only at very late stages of infection. Moreover, the induction of inflammatory cytokines (e.g., IL-1 alpha, IL-1 beta, TNF-alpha, IL-12) together with iNOS in the lesion and draining lymph nodes was not altered in the absence of IL-4.

Topics: Animals; Immunity, Innate; Interferon-gamma; Interleukin-4; Leishmania major; Leishmaniasis, Cutaneous; Mice; Mice, Inbred BALB C; Nitric Oxide Synthase; Transcription, Genetic; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

Transforming growth factor (TGF)-beta has several downregulatory functions on the immune system: inhibition of interleukin-2 receptor induction, decrease of interferon-gamma-induced class II antigen expression, inhibition of macrophage activation, as well as cytotoxic and lymphokine-activated killer cell generation. TGF-beta has also been recognized as an important immunoregulator in murine leishmaniasis, for which it increases susceptibility to disease. In the present study we evaluate the involvement of TGF-beta in human leishmaniasis in vitro and in patients with cutaneous leishmaniasis. Human macrophages produce active TGF-beta after infection by Leishmania amazonensis (480 +/- 44.7 pg/ml; mean +/- SEM), L. donovani chagasi (295 +/- 7.6 pg/ml), or L. braziliensis (196 +/- 15.7 pg/ml). When TGF-beta was added to cultures of human macrophages infected with L. braziliensis it led to an increase of approximately 50% in parasite numbers as compared with untreated cultures. Exogenous TGF-beta added to macrophage cultures was able to reverse the effect of interferon-gamma in controlling Leishmania growth. Even at 100 IU/ml interferon-gamma the presence of TGF-beta increases the number of intracellular parasites. On the other hand, TNF-alpha at high concentration (100 IU/ml) totally blunts the suppressive effect of TGF-beta. Immunostaining for TGF-beta was observed in the dermis, produced by fibroblasts and occasionally by inflammatory cells in the biopsies from human leishmaniasis lesions, being present in most of the biopsies taken from patients with early cutaneous leishmaniasis (less than 2 months of ulcer development) and in cases of active mucosal leishmaniasis. Taken together these observations suggest an important role for TGF-beta in human leishmaniasis, with its production by infected macrophages being probably related to parasite establishment in the early stages of the disease.

Topics: Animals; Cells, Cultured; Humans; Immunohistochemistry; Interferon-gamma; Leishmania braziliensis; Leishmaniasis, Cutaneous; Macrophages; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

CD8+ T cells and lysis of parasitized macrophages seem to be important in the resistance to murine leishmaniasis. In the present study, we evaluated peripheral blood mononuclear cell (PBMC) from patients with either cutaneous (CL) or mucosal (ML) leishmaniasis in cell lysis assays using 51-Cr-labeled Daudi or K562 cells, or autologous antigen-pulsed macrophages as targets. Results are reported as lytic units (number of cells required for 30% lysis) per million PBMC. Exposure of patient PBMC (n = 12) to lysate from Leishmania amazonensis promastigotes led to an increase in cytotoxic activity compared to unstimulated patient cells against Daudi (81.8 +/- 14.9 vs 13.6 +/- 5 lytic units (LU) per million PBMC; mean +/- SEM) and K562 (65.7 +/- 8.4 vs 13.1 +/- 5 LU/10(6) PBMC). ML had higher responses than CL in both targets (80.4 +/- 11.0 vs 46.4 +/- 11.6 LU/10(6) PBMC for K562, and 104.3 +/- 23.8 vs 59.3 +/- 14.3 LU/10(6) PBMC for Daudi). Normal control PBMC, stimulated with L. amazonensis antigen had 6.32 +/- 3.72 LU/10(6) PBMC against Daudi cells and 9.06 +/- 2.78 LU/10(6) PBMC against K562. The cell responsible for lysis of the K562 cells was characterized as NK, by means of cell separation employing magnetic beads coupled to antibodies. Addition of recombinant TGF-beta or recombinant human IL-10 reduced L. amazonensis-induced cytotoxicity by 90% and 70%, respectively. Cytotoxicity of antigen-stimulated PBMC was also demonstrated against autologous L. amazonensis antigen-pulsed macrophages in the range of 6.7 to 41.7 LU/10(6) PBMC.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Antibodies, Protozoan; Antigens, Protozoan; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Cytotoxicity, Immunologic; Dose-Response Relationship, Immunologic; Humans; Interferon-gamma; Interleukin-10; Killer Cells, Natural; Leishmania mexicana; Leishmaniasis, Cutaneous; Leukocytes, Mononuclear; Macrophages; Mice; T-Lymphocytes, Cytotoxic; Transforming Growth Factor beta; Tumor Cells, Cultured

Previous studies with inhibitors of inducible nitric oxide synthase (iNOS) suggested that high-output production of nitric oxide (NO) is an important antimicrobial effector pathway in vitro and in vivo. Here, we investigated the tissue expression of iNOS in mice after infection with Leishmania major. Immunohistochemical staining with an iNOS-specific antiserum revealed that in the cutaneous lesion and draining lymph nodes (LN) of clinically resistant mice (C57BL/6), iNOS protein is found earlier during infection and in significantly higher amounts than in the nonhealing BALB/c strain. Similar differences were seen on the mRNA level as quantitated by competitive polymerase chain reaction. Anti-CD4 treatment of BALB/c mice not only induced resistance to disease, but also restored the expression of iNOS in the tissue. In situ, few or no parasites were found in those regions of the skin lesion and the draining LN which were highly positive for iNOS. By double labeling experiments, macrophages were identified as iNOS expressing cells in vivo. In the lesions of BALB/c mice, cells staining positively for transforming growth factor beta (TGF-beta), a potent inhibitor of iNOS in vitro, were strikingly more prominent than in C57BL/6, whereas no such difference was found for interleukin 4 or interferon gamma (IFN-gamma). In vitro, production of NO was approximately threefold higher in C57BL/6 than in BALB/c macrophages after stimulation with IFN-gamma. We conclude that the pronounced expression of iNOS in resistant mice is an important mechanism for the elimination of Leishmania in vivo. The relative lack of iNOS in susceptible mice might be a consequence of macrophage deactivation by TGF-beta and reduced responsiveness to IFN-gamma.

Topics: Amino Acid Oxidoreductases; Animals; Base Sequence; Cells, Cultured; Female; Interferon-gamma; Leishmania major; Leishmaniasis, Cutaneous; Macrophages; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Molecular Sequence Data; NADPH Dehydrogenase; Nitric Oxide Synthase; RNA, Messenger; Transforming Growth Factor beta

The nature of the host cellular immune response largely determines the expression of disease following infection with the intracellular protozoans Leishmania spp. In experimental animals control and resolution of infection are mediated by gamma interferon and tumor necrosis factor alpha (TNF-alpha), whereas disease progression is associated with the production of interleukin 4 (IL-4), IL-5, IL-10, and transforming growth factor beta (TGF-beta). We have analyzed the profile of cytokine gene expression directly in the lesions of 13 patients with localized cutaneous leishmaniasis due to Leishmania mexicana. All but one patient had a single lesion, and the time of evolution ranged from 8 days to 18 months. Cytokine gene expression was quantitated by reverse transcriptase PCR and interpolation from a standard curve. Gamma interferon, TNF-alpha, IL-1 alpha, IL-6, IL-10, and TGF-beta gene expression was present in all samples. IL-3 and IL-4 gene expression was barely detectable in 1 and 3 of 13 samples, respectively. IL-2 and IL-5 mRNAs were not found. A significant increase in the expression of IL-1 alpha, TNF-alpha, IL-10, and TGF-beta was observed in late lesions (> or = 4 months) compared with that in early lesions (< or = 2 months). Because of their inhibitory effects on macrophage function, the expression of IL-10 and TGF-beta may play a role in the immunopathogenesis of chronic cutaneous leishmaniasis.

Topics: Base Sequence; Chronic Disease; Cytokines; Humans; Interleukin-10; Leishmaniasis, Cutaneous; Molecular Sequence Data; RNA, Messenger; Transforming Growth Factor beta

Transforming growth factor beta (TGF-beta) has potent down-regulating effects on macrophages and is thus capable of influencing the fate of intramacrophage parasites, including leishmanias. We report the development of a mouse model for the study of the human pathogen Leishmania braziliensis and demonstrate, both in vitro and in vivo, a key regulatory role for TGF-beta in the pathogenesis of infection with this parasite. Recombinant TGF-beta added to cultures of murine peritoneal macrophages led to increased intracellular L. braziliensis replication, whereas addition of neutralizing anti-TGF-beta monoclonal antibody decreased levels of infection. Macrophages infected with L. braziliensis produced biologically active TGF-beta, with a direct correlation between amounts of TGF-beta induced by two parasite isolates and their relative virulence. In vivo, treatment with recombinant TGF-beta rendered avirulent parasites virulent and activated latent L. braziliensis infection. Activation of parasite replication was observed in mice which had been infected with L. braziliensis 15 weeks previously but had not developed lesions or had healed lesions, depending on the parasite isolate used to infect the mice. The exacerbation of L. braziliensis infection in vivo was associated with an increase of interleukin 10 mRNA in the draining lymph node. These results demonstrate that TGF-beta is able to alter the course of in vitro and in vivo infections with L. braziliensis, the latter being characterized by an increase in interleukin 10, an important Th2 helper-T-cell cytokine.

Topics: Actins; Animals; Base Sequence; Humans; Interferon-gamma; Interleukin-2; Interleukin-4; Leishmania braziliensis; Leishmaniasis, Cutaneous; Macrophages; Mice; Mice, Inbred BALB C; Molecular Sequence Data; Oligodeoxyribonucleotides; Polymerase Chain Reaction; Recombinant Proteins; RNA; Transforming Growth Factor beta; Virulence

The course of infection with the protozoan parasite Leishmania is determined in part by their early replication in macrophages, the exclusive host cells for these organisms. Although factors contributing to the survival of Leishmania are not well understood, cytokines influence the course of infection. Transforming growth factor-beta (TGF-beta) is a multipotential cytokine with diverse effects on cells of the immune system, including down-regulation of certain macrophage functions. Leishmanial infection induced the production of active TGF-beta, both in vitro and in vivo. TGF-beta was important for determining in vivo susceptibility to experimental leishmanial infection.

Topics: Actins; Animals; Base Sequence; Disease Susceptibility; Interferon-gamma; Interleukin-4; Leishmania; Leishmaniasis, Cutaneous; Macrophages; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Molecular Sequence Data; Oligodeoxyribonucleotides; Polymerase Chain Reaction; Transforming Growth Factor beta