Leishmania major: effects of proteophosphoglycan on reactive oxygen species, IL-12, IFN-gamma and IL-10 production in healthy individuals

Protozoan parasites of the genus Leishmania secrete a range of proteophosphoglycans (PPG) known to be important for successful colonization of Leishmania in the sandfly and for virulence in the mammalian host. PPGs are a large family of extensively glycosylated proteins with some unusual and unique features. In this study we purified PPG from culture supernatant of Leishmania major metacyclic promastigotes. In discontinuous SDS-PAGE, PPG could not enter the resolving gel but after mild acid hydrolysis several bands resolved. Agarose gel electrophoresis and immunoblot analysis using monoclonal antibody (WIC 79.3) indicated that the PPG preparation consisted of heterogeneous molecules. Compositional analysis showed that the PPG preparation contained 67% glycan, 28% protein and 5% phosphate. Additionally, the effect of PPG on reactive oxygen species (ROS) production and induction of IL-10, IL-12 and IFN-γ secretion by human peripheral blood mononuclear cells (PBMCs) isolated from healthy individuals was investigated. The water-soluble secreted form of PPG at a concentration of 1 μg glycan/ml seems to be a potent inducer of ROS and IL-10 and to a lesser extent of IFN-γ and IL-12. Cytokines and ROS production was decreased in a dose-dependent manner as the concentration of PPG was increased to 100 μg glycan/ml.     

Leishmania strains causing self-healing cutaneous leishmaniasis have greater susceptibility towards oxidative stress

Abstract

The survival of Leishmania parasites within macrophages is influenced by generation of free radicals. To establish whether generation of free radicals influenced chemotherapeutic response, promastigotes from isolates causing self-healing or delayed/non-self-healing cutaneous leishmaniasis (CL) or visceral leishmaniasis (VL) were evaluated for their susceptibility to nitric oxide (NO), antimony and miltefosine. In a self-healing CL strain of Leishmania major (5ASKH), susceptibility to NO and antimony was higher than other species. Likewise, a Leishmania amazonensis strain, M2269, showed greater susceptibility to NO and antimony than other species but no such correlation was observed with miltefosine. Additionally, 5ASKH and M2269 showed poorer free radical scavenging capacity as also their thiol levels were lower than species causing VL. Collectively, our study suggests that self-healing isolates tend to be more susceptible to oxidative stress.     

Tumor necrosis factor alpha neutralization has no direct effect on parasite burden,but causes impaired IFN-γ production by spleen cells from human visceral leishmaniasis patients

The pro-inflammatory cytokine tumor necrosis factor (TNF)-α has an important role in control of experimental Leishmania donovani infection. Less is known about the role of TNF-α in human visceral leishmaniasis (VL). Evidence for a protective role is primarily based on case reports of VL development in individuals treated with TNF-α neutralizing antibody. In this study, we have evaluated how TNF-α neutralization affects parasite replication and cytokine production in ex vivo splenic aspirates (SA) from active VL patients. The effect of TNF-α neutralization on cell mediated antigen specific responses were also evaluated using whole blood cultures. Neutralization of TNF-α did not affect parasite numbers in SA cultures. Interferon (IFN)-γ levels were significantly reduced, but interleukin (IL)-10 levels were unchanged in these cultures. Leishmania antigen stimulated SA produced significant TNF-α which suggests that TNF-α is actively produced in VL spleen. Further it stimulates IFN-γ production, but no direct effect on parasite replication.     

Leishmania donovani: role of CD2 on CD4+ T-cell function in Visceral Leishmaniasis

In this study, we investigated whether alteration in the CD2 mediated coordination of an immune response was associated with down regulation of CD4 associated Th1 cell response during Visceral Leishmaniasis (VL). Leishmania donovani (Ld) infection in VL patients markedly reduced expression of CD2 cell surface antigen on CD4+ cells. T-cells of VL patients were mostly in G0/G1 stage of the cell cycle (98.20%) with little or no activity of protein kinase C-alpha (PKC-alpha) isoform. However, pre-incubation with activating anti-CD2 monoclonal antibody (MAb) resulted in a corresponding increase up to 2.52-fold in T-cells of G2/M population supported by both activity and expression of PKC-alpha isoform. Furthermore, we observed that co-incubation of T-cell with anti-CD2 increased the lymphocyte-blast population in patients in whom the CD4 cells became more antigen responsive (CD4+ CD69+ cells). Consistent with these observations, it was shown that 59.3% of CD4 cells from patients responded to Ld by producing IFN-gamma. Even in the culture condition, when the T-cells from patients were depleted of APC, IFN-gamma production was noticed after CD2 activation. On the other hand, IL-4 production became low in the anti-CD2 antibody supplemented peripheral blood mononuclear cells (PBMNCs) culture. These findings imply that infection with L. donovani induces less CD2 on the surface of CD4+ T-cells, which once activated orchestrate the protective IFN-gamma dominant host defense mechanism via PKC-mediated signal transduction and cell cycle.     

Decreased memory T-cell response and function in human immunodeficiency virus-infected patients with tegumentary leishmaniasis

The effects of human immunodeficiency virus (HIV) on the immune response in patients with cutaneous leishmaniasis have not yet been fully delineated. This study quantified and evaluated the function of memory T-cell subsets in response to soluble Leishmania antigens (SLA) from patients coinfected with HIV and Leishmania with tegumentary leishmaniasis (TL). Eight TL/HIV coinfected subjects and 10 HIV seronegative subjects with TL were evaluated. The proliferative response of CD4+and CD8+T-cells and naïve, central memory (CM) and effector memory (EM) CD4+T-cells in response to SLA were quantified using flow cytometry. The median cell division indices for CD4+and CD8+T-cells of coinfected patients in response to SLA were significantly lower than those in patients with Leishmania monoinfection (p < 0.05). The proportions of CM and EM CD4+T-cells in response to SLA were similar between the coinfected patients and patients with Leishmania monoinfection. However, the median CM and EM CD4+T-cell counts from coinfected patients were significantly lower (p < 0.05). The reduction in the lymphoproliferative response to Leishmania antigens coincides with the decrease in the absolute numbers of both EM and CM CD4+T-cells in response to Leishmania antigens in patients coinfected with HIV/Leishmania.     

The role of inflammatory and anti-inflammatory cytokines in the pathogenesis of human tegumentary leishmaniasis

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.     

Sub-optimal dose of Sodium Antimony Gluconate (SAG)-diperoxovanadate combination clears organ parasites from BALB/c mice infected with antimony resistant Leishmania donovani by expanding antileishmanial T-cell repertoire and increasing IFN-γ to IL-10 ratio

We demonstrate that the combination of sub-optimal doses of Sodium Antimony Gluconate (SAG) and the diperoxovanadate compound K[VO(O₂)₂(H₂O)], also designated as PV6, is highly effective in combating experimental infection of BALB/c mice with antimony resistant (Sb^R) Leishmania donovani (LD) as evident from the significant reduction in organ parasite burden where SAG is essentially ineffective. Interestingly, such treatment also allowed clonal expansion of antileishmanial T-cells coupled with robust surge of IFN-γ and concomitant decrease in IL-10 production. The splenocytes from the treated animals generated significantly higher amounts of IFN-γ inducible parasiticidal effector molecules like superoxide and nitric oxide as compared to the infected group. Our study indicates that the combination of sub-optimal doses of SAG and PV6 may be beneficial for the treatment of SAG resistant visceral leishmaniasis patients.     

Leishmania (Viannia) braziliensis amastigotes induces the expression of TNFα and IL-10 by human peripheral blood mononuclear cells in vitro in a TLR4-dependent manner

While the role of Toll-like receptors (TLRs) has been investigated in murine models of tegumentary leishmaniasis caused by Leishmania (Viannia) braziliensis, the interaction between TLRs and Leishmania sp. has not been investigated in human cells. The aim of this study was to evaluate the involvement of TLR4 in cytokine production of human peripheral blood mononuclear cells (PBMCs) induced by L. braziliensis, and whether the parasite alters the expression of TLR4 on monocytes/macrophages. Amastigote forms were obtained from mice lesions and PBMCs were isolated from healthy donors. PBMCs were cultured in absence or presence of IFNγ, TLR4 neutralizing antibodies, natural antagonist of TLR4 (Bartonella LPS), TLR4 agonist (E. coli LPS), and amastigote forms. The concentrations of tumor necrosis factor (TNFα) and interleukin 10 (IL-10) were assayed by ELISA and TLR4 expression by flow cytometry. Amastigotes forms of L. braziliensis induced TNFα and IL-10 production only in IFNγ-primed PBMCs. The TNFα and IL-10 production was inhibited by TLR4 neutralization, both with anti-TLR4 antibodies and Bartonella LPS. Interestingly, addition of E. coli LPS further increased TNFα but not IL-10 production induced by L. braziliensis amastigotes. Amastigotes of L. braziliensis strongly reduced membrane TLR4 expression on monocytes/macrophages, apparently by internalization after the infection. The present study reveals that TLR4 drives the production of TNFα and IL-10 induced by L. braziliensis amastigotes and that the parasites decrease TLR4 expression on monocyte surface.     

Vitamin D status and its modulatory effect on interferon gamma and interleukin-10 production by peripheral blood mononuclear cells in culture

ObjectivesWe aimed to investigate the influence of vitamin D on the production of the pro-inflammatory cytokine, interferon gamma (IFN-γ), and the anti-inflammatory cytokine, interleukin-10 (IL-10), in peripheral blood mononuclear cell (PBMC) cultures.MethodsThirty healthy subjects were investigated. Serum levels of calcium, 25(OH) D, and parathyroid hormone (PTH) were assessed. PBMCs were activated in-vitro by phytohemagglutinin (PHA) in the presence and absence of vitamin D3 and then levels of IFN-γ and IL-10 were determined in culture supernatant using enzyme immunoassay.ResultsSerum calcium levels were significantly lower in the vitamin D deficient group while serum PTH levels were significantly higher in the vitamin D deficient group. PTH levels were inversely correlated to both calcium and 25(OH) D levels. In culture, vitamin D inhibited IFN-γ production and increased IL-10 production by PBMCs. Serum vitamin D status had no influence on the amount of cytokine produced in culture.ConclusionsThis study demonstrates that vitamin D modulates IFN-γ and IL-10 production and provides a rationale for evaluating vitamin D as an immunomodulatory agent.     

Leishmania mexicana: participation of NF-kappaB in the differential production of IL-12 in dendritic cells and monocytes induced by lipophosphoglycan (LPG)

Dendritic cells (DC) and macrophages (Mφ) are well known as important effectors of the innate immune system and their ability to produce IL-12 indicates that they possess the potential of directing acquired immunity toward a Th1-biased response. Interestingly, the intracellular parasite Leishmania has been shown to selectively suppress Mφ IL-12 production and are DC the principal source of this cytokine. The molecular details of this phenomenon remain enigmatic. In the present study we examined the effect of Leishmania mexicana lipophosphoglycan (LPG) on the production of IL-12, TNF-α, and IL-10 and nuclear translocation of NF-κB. The results show that LPG induced more IL-12 in human DC than in monocytes. This difference was due in part to nuclear translocation of NF-κB, since LPG induced more translocation in DC than in monocytes. These results suggest that Leishmania LPG impairs nuclear translocation of NF-κB in monocytes with the subsequent decrease in IL-12 production.     

Vaccination with the Leishmania infantum ribosomal proteins induces protection in BALB/c mice against Leishmania chagasi and Leishmania amazonensis challenge

Leishmania chagasi and Leishmania amazonensis are the etiologic agents of different clinical forms of human leishmaniasis in South America. In an attempt to select candidate antigens for a vaccine protecting against different Leishmania species, the efficacy of vaccination using Leishmania ribosomal proteins and saponin as adjuvant was examined in BALB/c mice against challenge infection with both parasite species. Mice vaccinated with parasite ribosomal proteins purified from Leishmania infantum plus saponin showed a specific production of IFN-γ, IL-12 and GM-CSF after in vitro stimulation with L. infantum ribosomal proteins. Vaccinated mice showed a reduction in the liver and spleen parasite burdens after L. chagasi infection. After L. amazonensis challenge, vaccinated mice showed a decrease of the dermal pathology and a reduction in the parasite loads in the footpad and spleen. In both models, protection was correlated to an IL-12-dependent production of IFN-γ by CD4⁺ and CD8⁺ T cells that activate macrophages for the synthesis of NO. In the protected mice a decrease in the parasite-mediated IL-4 and IL-10 responses was also observed. In mice challenged with L. amazonensis, lower levels of anti-parasite-specific antibodies were detected. Thus, Leishmania ribosomal proteins plus saponin fits the requirements to compose a pan-Leishmania vaccine.     

Species-Specific Antimonial Sensitivity in Leishmania Is Driven by Post-Transcriptional Regulation of AQP1

Leishmania is a digenetic protozoan parasite causing leishmaniasis in humans. The different clinical forms of leishmaniasis are caused by more than twenty species of Leishmania that are transmitted by nearly thirty species of phlebotomine sand flies. Pentavalent antimonials (such as Pentostam or Glucantime) are the first line drugs for treating leishmaniasis. Recent studies suggest that pentavalent antimony (Sb(V)) acts as a pro-drug, which is converted to the more active trivalent form (Sb(III)). However, sensitivity to trivalent antimony varies among different Leishmania species. In general, Leishmania species causing cutaneous leishmaniasis (CL) are more sensitive to Sb(III) than the species responsible for visceral leishmaniasis (VL). Leishmania aquaglyceroporin (AQP1) facilitates the adventitious passage of antimonite down a concentration gradient. In this study, we show that Leishmania species causing CL accumulate more antimonite, and therefore exhibit higher sensitivity to antimonials, than the species responsible for VL. This species-specific differential sensitivity to antimonite is directly proportional to the expression levels of AQP1 mRNA. We show that the stability of AQP1 mRNA in different Leishmania species is regulated by their respective 3’-untranslated regions. The differential regulation of AQP1 mRNA explains the distinct antimonial sensitivity of each species.     

Leishmania major: Interleukin-13 increases the infection-induced hyperalgesia and the levels of interleukin-1β and interleukin-12 in rats

Interleukin-13 (IL-13) is a powerful anti-inflammatory cytokine that was previously shown to be a susceptibility factor for Leishmania major (L. major) infection. In this study we report a different role for IL-13 in rats infected with L. major; rIL-13 stimulates expression of pro-inflammatory cytokines and IL-12 which is a key cytokine in protective immunity against Leishmania. Infected rats received daily injections of rIL-13 for eight consecutive days which resulted in increased pain perception for the first week post-infection assessed by thermal pain tests. This hyperalgesia was accompanied by a sustained early up-regulation of interleukin-1β followed by an up-regulation of IL-12p70. Real-time PCR showed no negative impact for rIL-13 upon the clearance of the parasites from the infection sites and blood.     

Protective immunity against Leishmania major induced by Leishmania tropica infection of BALB/c mice

Leishmania (L.) tropica is a causative agent of human cutaneous and viscerotropic leishmaniasis. Immune response to L. tropica in humans and experimental animals are not well understood. We previously established that L. tropica infection induces partial protective immunity against subsequent challenge infection with Leishmania major in BALB/c mice. Aim of the present study was to study immunologic mechanisms of protective immunity induced by L. tropica infection, as a live parasite vaccine, in BALB/c mouse model. Mice were infected by L. tropica, and after establishment of the infection, they were challenged by L. major. Our findings shows that L. tropica infection resulted in protection against L. major challenge in BALB/c mice and this protective immunity is associated with: (1) a DTH response, (2) higher IFN-γ and lower IL-10 response at one week post-challenge, (3) lower percentage of CD4⁺ lymphocyte at one month post-challenge, and (4) the source of IFN-γ and IL-10 were mainly CD4⁻ lymphocyte up to one month post-challenge suggesting that CD4⁻ lymphocytes may be responsible for protection induced by L. tropica infection in the studied intervals.     

Association of interleukin-18 gene variants with susceptibility to visceral leishmaniasis in Iranian population

Host resistance to Leishmania infection is mediated by cellular immune responses leading to macrophage activation and parasite killing. Interleukin-18 (IL-18) known as interferon-γ (IFN-γ) inducing factor, stimulates IFN-γ production by T cells. Taking into account the important role of IL-18 in the defense against visceral leishmaniasis (VL) and the known effect of IL-18 gene polymorphisms on its production, the aim of this study was to investigate the probable relationship between IL-18 gene polymorphisms and the susceptibility to VL. The study groups included 118 pediatric patients who suffered from VL and 156 non-relative healthy people as the controls from the same endemic area. IL-18 gene polymorphisms at the positions ?656 G/T, ?137 G/C and +105A/C (codon 35/3) were analyzed by polymerase chain reaction-restricted fragment length polymorphism (PCR–RFLP). The results showed that the frequency of T allele at the position -656 was significantly higher in the controls, compared with that in the patients (P = 0.047), but it couldn’t tolerate Bonferroni correction. Regarding the IL-18 genotypes, there was no significant difference between the patients and controls. Although the frequencies of ATG single haplotype and AGG/ATG double haplotype were significantly higher in the controls (P = 0.043) and the patients (P = 0.044), respectively, the two P values couldn’t tolerate Bonferroni correction. Furthermore, a strong linkage disequilibrium was observed among the ?656, ?137 and +105 single nucleotide polymorphisms of IL-18 gene (all Ps < 0.001). In conclusion, this study suggests that the inheritance of T allele at the position ?656 may be considered as a genetic factor for resistance to VL.     

Role of cytokine gene polymorphisms on prognosis in hepatocellular carcinoma after radical surgery resection

This study aimed to determine whether SNPs of cytokine genes influence survival of hepatocellular carcinoma (HCC) patients after radical surgery resection. We evaluated 14 SNPs of eight cytokine genes in 263 patients treated with radical surgery resection of HCC. Categorical variables were compared by the χ² test and Fisher's exact test. The Kaplan–Meier methods with log-rank test and Cox regression models were used to compare survival of resected HCC patients according to the genotype. Among the 14 studied SNPs of cytokine genes, only the TNF-α-863 (CA + CC) genotypes were revealed to be significant independent predictors of prolonged overall survival (OS) after HCC radical surgery resection (HR: 0.586, 95% CI: 0.355–0.968), considering for other clinical factors in a Cox proportional hazard model. Meanwhile, no significant association was found between the 14 SNPs and relapse-free survival (RFS) of resected HCC patients. In addition, combination analysis with the Th1 cytokine (IFN-γ, IL-2, IL-12B, TGF-β1) or Th2 cytokine (IL-6, IL-10) genetic polymorphisms by the Kaplan–Meier method and Cox multivariate analysis did not reveal any significant association between OS and RFS of resected HCC patients.     

Host peroxisomal properties are not restored to normal after treatment of visceral leishmaniasis with sodium antimony gluconate

Reason for post-kala-azar dermal leishmaniasis (PKDL) is yet to be established. Earlier it was observed that morphology and biochemical properties of host peroxisomes were impaired during Leishmania infection. As peroxisome is known to be involved in various metabolic pathways to monitor normal function of the host cells, it is essential that Leishmania-induced dysfunction of this organelle should totally be repaired during treatment of visceral leishmaniasis (VL). In this paper it has been shown that resumption of normal peroxisomal function could not be attained when one of the existing drugs sodium antimony gluconate (SAG) was used for chemotherapy against VL. Although Leishmania parasite was found to be completely eliminated from host liver and spleen after SAG treatment, normal activities of peroxisomal catalase and superoxide dismutase could not be restored. Also unusual peptides were found to be present due to abnormal proteolytic cleavage of proteins. It is proposed that peroxisomal disorder which exists even after successful chemotherapy of VL may be figured out as one of the possible reasons to develop PKDL. It may also be pointed out that continued effect of peroxisomal disorder even after complete treatment of this parasitic disease may also lead to genetic disorders not yet been explored in post-kala-azar patients.     

Cathepsin B-like and cell death in the unicellular human pathogen Leishmania

In several studies reporting cell death (CD) in lower eukaryotes and in the human protozoan parasite Leishmania, proteolytic activity was revealed using pan-caspase substrates or inhibitors such as carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone (Z-VAD-FMK). However, most of the lower eukaryotes do not encode caspase(s) but MCA, which differs from caspase(s) in its substrate specificity and cannot be accountable for the recognition of Z-VAD-FMK. In the present study, we were interested in identifying which enzyme was capturing the Z-VAD substrate. We show that heat shock (HS) induces Leishmania CD and leads to the intracellular binding of Z-VAD-FMK. We excluded binding and inhibition of Z-VAD-FMK to Leishmania major metacaspase (LmjMCA), and identified cysteine proteinase C (LmjCPC), a cathepsin B-like (CPC) enzyme, as the Z-VAD-FMK binding enzyme. We confirmed the specific interaction of Z-VAD-FMK with CPC by showing that Z-VAD binding is absent in a Leishmania mexicana strain in which the cpc gene was deleted. We also show that parasites exposed to various stress conditions release CPC into a soluble fraction. Finally, we confirmed the role of CPC in Leishmania CD by showing that, when exposed to the oxidizing agent hydrogen peroxide (H₂O₂), cpc knockout parasites survived better than wild-type parasites (WT). In conclusion, this study identified CPC as the substrate of Z-VAD-FMK in Leishmania and as a potential additional executioner protease in the CD cascade of Leishmania and possibly in other lower eukaryotes.