Successful Treatment of Human Visceral Leishmaniasis Restores Antigen-Specific IFN-γ, but not IL-10 Production

One of the key immunological characteristics of active visceral leishmaniasis (VL) is a profound immunosuppression and impaired production of Interferon-γ (IFN-γ). However, recent studies from Bihar in India showed using a whole blood assay, that whole blood cells have maintained the capacity to produce IFN-γ. Here we tested the hypothesis that a population of low-density granulocytes (LDG) might contribute to T cell responses hyporesponsiveness via the release of arginase. Our results show that this population is affected by the anticoagulant used to collect blood: the frequency of LDGs is significantly lower when the blood is collected with heparin as compared to EDTA; however, the anticoagulant does not impact on the levels of arginase released. Next, we assessed the capacity of whole blood cells from patients with active VL to produce IFN-γ and IL-10 in response to antigen-specific and polyclonal activation. Our results show that whole blood cells produce low or levels below detection limit of IFN-γ and IL-10, however, after successful treatment of VL patients, these cells gradually regain their capacity to produce IFN-γ, but not IL-10, in response to activation. These results suggest that in contrast to VL patients from Bihar, India, whole blood cells from VL patients from Gondar, Ethiopia, have lost their ability to produce IFN-γ during active VL and that active disease is not associated with sustained levels of IL-10 production following stimulation.     

Increased levels of cortisol are associated with the severity of experimental visceral leishmaniasis in a Leishmania (L.) infantum-hamster model

BackgroundSeveral infectious diseases are associated with hypothalamic-pituitary-adrenal (HPA) axis disorders by elevating circulating glucocorticoids (GCs), which are known to have an immunosuppressive potential. We conducted this study in golden hamsters, a suitable model for human visceral leishmaniasis (VL), to investigate the relationship of Leishmania (L.) infantum infection on cortisol production and VL severity.MethodsL. infantum-infected (n = 42) and uninfected hamsters (n = 30) were followed-up at 30, 120, and 180 days post-infection (dpi). Plasma cortisol was analyzed by radioimmunoassay and cytokines, inducible nitric oxide synthase (iNOS), and arginase by RT-qPCR.ResultsAll hamsters showed splenomegaly at 180 dpi. Increased parasite burden was associated with higher arginase expression and lower iNOS induction. Cortisol levels were elevated in infected animals in all-time points evaluated. Except for monocytes, all other leucocytes showed a strong negative correlation with cortisol, while transaminases were positively correlated. Immunological markers as interleukin (IL)-6, IL-1β, IL-10, and transforming growth-factor-β (TGF-β) were positively correlated to cortisol production, while interferon-γ (IFN-γ) presented a negative correlation. A network analysis showed cortisol as an important knot linking clinical status and immunological parameters.ConclusionsThese results suggest that L. infantum increases the systemic levels of cortisol, which showed to be associated with hematological, biochemical, and immunological parameters associated to VL severity.     

Vaccination with Leishmania infantum Acidic Ribosomal P0 but Not with Nucleosomal Histones Proteins Controls Leishmania infantum Infection in Hamsters

Background

Several intracellular Leishmania antigens have been identified in order to find a potential vaccine capable of conferring long lasting protection against Leishmania infection. Histones and Acid Ribosomal proteins are already known to induce an effective immune response and have successfully been tested in the cutaneous leishmaniasis mouse model. Here, we investigate the protective ability of L. infantum nucleosomal histones (HIS) and ribosomal acidic protein P0 (LiP0) against L. infantum infection in the hamster model of visceral leishmaniasis using two different strategies: homologous (plasmid DNA only) or heterologous immunization (plasmid DNA plus recombinant protein and adjuvant).

Methodology/Principal Findings

Immunization with both antigens using the heterologous strategy presented a high antibody production level while the homologous strategy immunized group showed predominantly a cellular immune response with parasite load reduction. The pcDNA-LiP0 immunized group showed increased expression ratio of IFN-γ/IL-10 and IFN-γ/TGF-β in the lymph nodes before challenge. Two months after infection hamsters immunized with the empty plasmid presented a pro-inflammatory immune response in the early stages of infection with increased expression ratio of IFN-γ/IL-10 and IFN-γ/TGF-β, whereas hamsters immunized with pcDNA-HIS presented an increase only in the ratio IFN-γ/ TGF-β. On the other hand, hamsters immunized with LiP0 did not present any increase in the IFN-γ/TGF-β and IFN-γ/IL-10 ratio independently of the immunization strategy used. Conversely, five months after infection, hamsters immunized with HIS maintained a pro-inflammatory immune response (ratio IFN-γ/ IL-10) while pcDNA-LiP0 immunized hamsters continued showing a balanced cytokine profile of pro and anti-inflammatory cytokines. Moreover we observed a significant reduction in parasite load in the spleen, liver and lymph node in this group compared with controls.

Conclusions/Significance

Our results suggest that vaccination with L. infantum LiP0 antigen administered in a DNA formulation could be considered a potential component in a vaccine formulation against visceral leishmaniasis.     

IL-17 Mediates Immunopathology in the Absence of IL-10 Following Leishmania major Infection

Leishmaniasis, resulting from infection with the protozoan parasite Leishmania, consists of a wide spectrum of clinical manifestations, from healing cutaneous lesions to fatal visceral infections. A particularly severe form of cutaneous leishmaniasis, termed mucosal leishmaniasis, exhibits decreased IL-10 levels and an exaggerated inflammatory response that perpetuates the disease. Using a mouse model of leishmaniasis, we investigated what cytokines contribute to increased pathology when IL-10-mediated regulation is absent. Leishmania major infected C57BL/6 mice lacking IL-10 regulation developed larger lesions than controls, but fewer parasites. Both IFN-γ and IL-17 levels were substantially elevated in mice lacking the capacity to respond to IL-10. IFN-γ promoted an increased infiltration of monocytes, while IL-17 contributed to an increase in neutrophils. Surprisingly, however, we found that IFN-γ did not contribute to increased pathology, but instead regulated the IL-17 response. Thus, blocking IFN-γ led to a significant increase in IL-17, neutrophils and disease. Similarly, the production of IL-17 by cells from leishmaniasis patients was also regulated by IL-10 and IFN-γ. Additional studies found that the IL-1 receptor was required for both the IL-17 response and increased pathology. Therefore, we propose that regulating IL-17, possibly by downregulating IL-1β, may be a useful approach for controlling immunopathology in leishmaniasis.     

Clinical and laboratory characteristics of hemophagocytic lymphohistiocytosis induced by Leishmania infantum infection

BackgroundVisceral leishmaniasis (VL) could progress to secondary hemophagocytic lymphohistiocytosis (HLH), which is a rare but life-threatening condition with poor prognosis. So far, the clinical and laboratory characteristics of VL associated HLH have not been well elucidated.Method and findingsIn this study, we retrospectively analyzed the clinical and laboratory profiles between 17 patients with VL associated HLH and 27 patients with VL alone admitted at the Beijing Friendship Hospital, Capital Medical University from May 2016 to March 2021. In addition to the identification of Leishmania infection, hemophagocytosis was identified in bone marrow in the most cases of VL associated HLH (15/17). The patients with VL associated HLH had higher chances of bleeding, hepatomegaly, thrombocytopenia, hypertriglyceridemia, hyperferritinemia, hypofibrinogenemia, elevated secretion of soluble IL-2 receptor or lower NK cell activity compared to patients with VL only. Furthermore, patients with VL associated HLH had higher inflammation status associated with higher levels of Th1 (TNF-α, IFN-γ, IL-1beta, IL-6, IL-8, IL-12p70), Th2 (IL-4) and Th17 cytokines (IL-17, IL-23) in the peripheral blood, and higher parasite load (qPCR and parasite culture). All 27 VL cases were totally recovered after being treated with Sodium Stibogluconate, five of the 17 patients with VL associated HLH died even after timely treatment with anti-parasite and immunosuppressive chemotherapy.ConclusionWithout appropriate treatment, visceral leishmaniosis could develop to secondary HLH. The parasite culturing and qPCR detection of bone marrow samples facilitates the diagnosis of VL associated HLH in addition to other findings of HLH. Prompt treatment with anti-Leishmania and immunosuppressive chemotherapy is critical to reduce the mortality of VL associated HLH.     

Quantification of Parasite Load in Clinical Samples of Leishmaniasis Patients: IL-10 Level Correlates with Parasite Load in Visceral Leishmaniasis

A rapid and accurate method to detect and quantify Leishmania parasite is urgently needed to facilitate early diagnosis of Leishmaniasis and monitoring of antileishmania therapy. In this study, real-time assay was applied to estimate parasite load in clinical samples of visceral leishmaniasis (VL) and post kala-azar dermal leishmaniasis (PKDL) patients. The mean parasite load in blood of VL patients (n = 31) was 8,372 parasites/ml, while the mean parasite load in bone marrow aspirate (BMA) was 194,962 parasites/million nucleated cells (n = 12). Parasite load was undetectable after treatment with amphotericin B (n = 9) in VL, while a residual parasite burden was detected in 2 of 6 patients following treatment with sodium antimony gluconate. Further, circulating levels of IFN-γ, TNF-α, IL-10, IL-6, IL-4 and IL-2 were analysed in VL patients (n = 29) by Cytometric Bead Array to evaluate correlation with parasitic load. Interestingly, IL-10 levels correlated significantly with parasite load (r = 0.82, P<0.0001). The mean parasite load in dermal lesions of PKDL patients was 9,502 parasites/µg tissue DNA at pre-treatment stage (n = 25), with no detectable parasites after therapy (n = 5). Parasite burden was distinctly higher (P<0.0001) in nodular lesions (n = 12) (19,586 parasites/µg tissue DNA) compared to papular/macular lesions (n = 13, 193 parasites/µg tissue DNA). Further, chronic PKDL lesions showed significantly (P = 0.0166) higher parasite load in comparison with acute lesions. Results indicate that chronic, nodular cases constitute the major parasite reservoir for anthroponotic transmission. Our results establish that the high parasite load in VL is strongly correlated with a high level of IL-10, implicating IL-10 as a marker of disease severity. The assay is applicable for diagnosis as well as prognosis of both VL and PKDL, providing a simple molecular tool to monitor the efficacy of antileishmanial drugs or vaccines.     

Cytokine Responses to Novel Antigens in an Indian Population Living in an Area Endemic for Visceral Leishmaniasis

Background

There are no effective vaccines for visceral leishmaniasis (VL), a neglected parasitic disease second only to malaria in global mortality. We previously identified 14 protective candidates in a screen of 100 Leishmania antigens as DNA vaccines in mice. Here we employ whole blood assays to evaluate human cytokine responses to 11 of these antigens, in comparison to known defined and crude antigen preparations.

Methods

Whole blood assays were employed to measure IFN-γ, TNF-α and IL-10 responses to peptide pools of the novel antigens R71, Q51, L37, N52, L302.06, J89, M18, J41, M22, M63, M57, as well as to recombinant proteins of tryparedoxin peroxidase (TRYP), Leishmania homolog of the receptor for activated C kinase (LACK) and to crude soluble Leishmania antigen (SLA), in Indian patients with active (n = 8) or cured (n = 16) VL, and in modified Quantiferon positive (EHC^+ve, n = 20) or modified Quantiferon negative (EHC^−ve, n = 9) endemic healthy controls (EHC).

Results

Active VL, cured VL and EHC^+ve groups showed elevated SLA-specific IFN-γ, but only active VL patients produced IL-10 and EHC^+ve did not make TNF-α. IFN-γ to IL-10 and TNF-α to IL-10 ratios in response to TRYP and LACK antigens were higher in cured VL and EHC^+ve exposed individuals compared to active VL. Five of the eleven novel candidates (R71, L37, N52, J41, and M22) elicited IFN-γ and TNF-α, but not IL-10, responses in cured VL (55–87.5% responders) and EHC^+ve (40–65% responders) subjects.

Conclusions

Our results are consistent with an important balance between pro-inflammatory IFNγ and TNFγ cytokine responses and anti-inflammatory IL-10 in determining outcome of VL in India, as highlighted by response to both crude and defined protein antigens. Importantly, cured VL patients and endemic Quantiferon positive individuals recognise 5 novel vaccine candidate antigens, confirming our recent data for L. chagasi in Brazil, and their potential as cross-species vaccine candidates.     

Adipose Tissue Promotes a Serum Cytokine Profile Related to Lower Insulin Sensitivity after Chronic Central Leptin Infusion

Obesity is an inflammatory state characterized by an augment in circulating inflammatory factors. Leptin may modulate the synthesis of these factors by white adipose tissue decreasing insulin sensitivity. We have examined the effect of chronic central administration of leptin on circulating levels of cytokines and the possible relationship with cytokine expression and protein content as well as with leptin and insulin signaling in subcutaneous and visceral adipose tissues. In addition, we analyzed the possible correlation between circulating levels of cytokines and peripheral insulin resistance. We studied 18 male Wistar rats divided into controls (C), those treated icv for 14 days with a daily dose of 12 μg of leptin (L) and a pair-fed group (PF) that received the same food amount consumed by the leptin group. Serum leptin and insulin were measured by ELISA, mRNA levels of interferon-γ (IFN-γ), interleukin-2 (IL-2), IL-4, IL-6, IL-10 and tumor necrosis factor-α (TNF-α) by real time PCR and serum and adipose tissue levels of these cytokines by multiplexed bead immunoassay. Serum leptin, IL-2, IL-4, IFN-γ and HOMA-IR were increased in L and TNF-α was decreased in PF and L. Serum leptin and IL-2 levels correlate positively with HOMA-IR index and negatively with serum glucose levels during an ip insulin tolerance test. In L, an increase in mRNA levels of IL-2 was found in both adipose depots and IFN-γ only in visceral tissue. Activation of leptin signaling was increased and insulin signaling decreased in subcutaneous fat of L. In conclusion, leptin mediates the production of inflammatory cytokines by adipose tissue independent of its effects on food intake, decreasing insulin sensitivity.     

Negative Regulation of Hepatitis C Virus Specific Immunity Is Highly Heterogeneous and Modulated by Pegylated Interferon-Alpha/Ribavirin Therapy

Specific inhibitory mechanisms suppress the T-cell response against the hepatitis C virus (HCV) in chronically infected patients. However, the relative importance of suppression by IL-10, TGF-β and regulatory T-cells and the impact of pegylated interferon-alpha and ribavirin (PegIFN-α/ribavirin) therapy on these inhibitory mechanisms are still unclear. We revealed that coregulation of the HCV-specific T-cell responses in blood of 43 chronic HCV patients showed a highly heterogeneous pattern before, during and after PegIFN-α/ribavirin. Prior to treatment, IL-10 mediated suppression of HCV-specific IFN-γ production in therapy-naive chronic HCV patients was associated with higher HCV-RNA loads, which suggests that protective antiviral immunity is controlled by IL-10. In addition, as a consequence of PegIFN-α/ribavirin therapy, negative regulation of especially HCV-specific IFN-γ production by TGF-β and IL-10 changed dramatically. Our findings emphasize the importance of negative regulation for the dysfunctional HCV-specific immunity, which should be considered in the design of future immunomodulatory therapies.     

Retinoic Acid Correlates with Reduced Serum IL-10 And TGF-β in Allergic Rhinitis

Background:Retinoic acid (RA) plays a key role in naïve T cell differentiation into FOXP3+ Treg cell in the respiratory airways. The present study aims to investigate RA and Treg-related cytokine serum levels, salivary IgA levels, FOXP3 and IL-4 gene expression, and the relationships between RA serum levels and Treg-related cytokines in allergic rhinitis (AR) patients and healthy controls.Methods:Salivary IgA and serum IgE, RA, IL-10, and TGF-β concentrations were measured by ELISA in 37 AR patients and 30 age- and sex-matched healthy controls.Results:IL-10 and TGF-β concentrations were significantly less in AR patients than in healthy controls (p< 0.01 and P< 0.0001, respectively). Salivary IgA was significantly greater in patients than in controls (p< 0.05). RA was not significantly different between patients and controls (p> 0.05); however, a significant positive correlation was found between serum RA and both IL-10 and TGF-β in AR patients.Conclusion:Our data suggest that RA may influence AR risk via affecting the TGF-β and IL-10 production.Key Words: Allergic Rhinitis, Interleukin-10, Obesity, Retinoic Acid, Transforming Growth Factor-β     

The Severity of Visceral Leishmaniasis Correlates with Elevated Levels of Serum IL-6, IL-27 and sCD14

Background

Visceral leishmaniasis (VL) is a severe disease caused by infection with protozoa of the genus Leishmania. Classic VL is characterized by a systemic infection of phagocytic cells and an intense activation of the inflammatory response. It is unclear why 90% of infected individuals do not develop the disease while a minority develop the classical form. Furthermore, among those that develop disease, a small group progresses to more severe form that is unresponsive to treatment. The presence of inflammatory mediators in serum could theoretically help to control the infection. However, there is also a release of anti-inflammatory mediators that could interfere with the control of parasite multiplication. In this study, we took advantage of the spectrum of outcomes to test the hypothesis that the immune profile of individuals infected with Leishmania (L.) infantum is associated with the development and severity of disease.

Methodology/Principal Findings

Sera from patients with confirmed diagnosis of VL were evaluated for the presence of numerous molecules, and levels compared with healthy control and asymptomatic infected individuals.

Conclusions/Principal Findings

Although differences were not observed in LPS levels, higher levels of sCD14 were detected in VL patients. Our data suggest that L. infantum may activate the inflammatory response via CD14, stimulating a generalized inflammatory response with production of several cytokines and soluble molecules, including IFN-γ, IL-27, IL-10, IL-6 and sCD14. These molecules were strongly associated with hepatosplenomegaly, neutropenia and thrombocytopenia. We also observed that IL-6 levels greater than 200 pg/ml were strongly associated with death. Together our data reinforce the close relationship of IFN-γ, IL-10, IL-6, TNF-α and IL-27 in the immune dynamics of VL and suggest the direct participation of sCD14 in the activation of the immune response against L. infantum.     

Immune Parameters That Distinguish Multiple Sclerosis Patients from Patients with Other Neurological Disorders at Presentation

Background/AimMultiple sclerosis (MS) is an inflammatory, demyelinating disease of the central nervous system. Effector T helper cells, mainly Th1 and Th17, cytotoxic T-cells, B-cells, macrophages, microglia, and the cytokines they secrete, are implicated in the initiation and maintenance of a deregulated immune response to myelin antigens and the ensuing immune-mediated demyelination. In this study, we investigated whether signature cytokines exist in MS patients at presentation to gain an insight into the underlying immunopathogenic processes at the early stage of the disease.MethodsWe collected serum and cerebrospinal fluid (CSF) samples from 123 patients at presentation, eventually diagnosed with MS or non-inflammatory (NIND) or inflammatory neurological diseases (IND) or symptomatic controls (SC). The levels of cytokines IFN-γ, TNF-α, TGF-β1, IL-2, IL-4, IL-6, IL-10 and IL-17 were measured, and cytokine ratios, such as Th1/Th2, Th1/Th17, and Type-1/Type-2, were calculated. All parameters were tested for their correlations with the intrathecal IgG synthesis.ResultsCytokine levels in CSF were lower than in serum in all the patients, with the exception of IL-6. Serum or CSF cytokine levels of MS patients did not differ significantly from NIND or SC, with the exception of serum IFN-γ and TNF-α that were significantly higher in NIND. IND patients presented with the highest levels of all cytokines in serum and CSF, with the exception of serum IL-10 and CSF IL-17. MS patients had a significantly lower serum Th1/Th2 ratio compared to the NIND and IND groups, and significantly lower serum Type-1/Type-2, IFN-γ/IL-10 and CSF Th1/Th17 ratios compared to IND patients. MS patients had a significantly higher CSF IL-17/IL-10 ratio compared to IND patients. The IgG index was higher in MS patients compared to the control groups; the differences reached statistical significance between the MS and the NIND and SC groups. Reiber-Felgenhauer analysis of the QIgG and QAlb indices revealed higher intrathecal IgG synthesis in MS patients, and higher blood-CSF barrier dysfunction in IND patients. The IgG index correlated with CSF IL-4 in MS patients only.ConclusionsWe found no signature cytokines or profiles thereof in MS patients at presentation. Only IND patients presented with a clear Th1 cytokine polarization in serum and CSF. The parameters that distinguished MS patients from patients with other neurological disorders were IgG intrathecal synthesis, the IgG index and its correlation with CSF IL-4 levels.     

Circulating immune complexes (IC) and IC-induced levels of GM-CSF are increased in sudanese patients with acute visceral Leishmania donovani infection undergoing sodium stibogluconate treatment: implications for disease pathogenesis

Infection with Leishmania donovani is associated with IL-10 as well as with GM-CSF. Immune complexes (IC) exert important functions by stimulation of monocytes/macrophage-mediated production of pro- and anti-inflammatory cytokines in rheumatic diseases. In this investigation, we have explored IC-induced cytokine production during Leishmania infection. Sera from 43 patients with visceral leishmaniasis (VL), 17 patients with post-kala-azar dermal leishmaniasis, and 20 healthy Sudanese controls were precipitated with polyethylene glycol (PEG). The PEG precipitates were added to serum-free PBMC for 20 h,whereupon supernatant levels of IL-1beta, IL-6, IL-10, IL-1 receptor antagonist protein, TNF-alpha, TNF receptor p75, and GM-CSF were investigated using ELISA. Circulating levels of C1q-binding IC were also measured in the serum samples. PEG precipitates from Leishmania-infected patients induced significantly higher levels of GM-CSF (p = 0.0037) and IL-10 (p < 0.0001), as well as of IL-6 (p < 0.0001) and IL-1 receptor antagonist (p = 0.0238) as compared with PEG precipitates from controls. Patients with acute VL as well as VL patients receiving sodium stibogluconate treatment displayed significantly increased levels of PEG precipitate-induced GM-CSF. The induction of GM-CSF by circulating IC was especially prominent in acute VL patients receiving sodium stibogluconate treatment; ANOVA revealed significant interaction between disease activity and treatment for PEG precipitate-induced levels of GM-CSF (disease activity, p = 0.0006; treatment, p = 0.0005; interaction, p = 0.0046). Parallel associations were determined for C1q-binding immune complexes, but not for any cytokine other than GM-CSF. The importance of IC-induced GM-CSF in leishmaniasis warrants further study.     

TGF-β and IL-10 Production by HIV-Specific CD8+ T Cells Is Regulated by CTLA-4 Signaling on CD4+ T Cells

Immune dysregulation in HIV-1 infection is associated with increased expression of inhibitory molecules such as CTLA-4, TGF-β, and IL-10. In this study we examined one potential mechanism for regulating TGF-β and IL-10 expression by HIV-specific suppressor CD8+ T cells. No overlap between TGF-β, IL-10, and IFN-γ cytokine production by HIV-specific CD8+ T cells was observed. TGF-β positive and IL-10 positive cells were FOXP3 negative, CD25 negative, and displayed a heterogeneous surface expression of CD127. TGF-β and IL-10 positive CD8+ T cells did not express CTLA-4. Nevertheless, CTLA-4 blockade resulted in a significant decrease in HIV-specific TGF-β positive and IL-10 positive CD8+ T cell responses, and a concomitant increase in HIV-specific IFN-γ positive CD8+ T cell responses. Depletion of CD4+ T cells abrogated the impact of CTLA-4 on HIV-specific TGF-β positive and IL-10 positive CD8+ T cells. Our study suggests that CTLA-4 Signaling on CD4+ T cells regulates the inhibitory functions of the HIV-specific suppressor CD8+ T cells.     

Combination of Liposomal CpG Oligodeoxynucleotide 2006 and Miltefosine Induces Strong Cell-Mediated Immunity during Experimental Visceral Leishmaniasis

Immuno-modulators in combination with antileishmanial drug miltefosine is a better therapeutic approach for treatment of Visceral Leishmaniasis (VL) as it not only reduces the dose of miltefosine but also shortens the treatment regimen. However, immunological mechanisms behind the perceived benefits of this combination therapy have not been investigated in detail. In the present study, we hypothesized that potential use of drugs that target the host in addition to the parasite might represent an alternative strategy for combination therapy. We investigated immune responses generated in Leishmania donovani infected animals (hamsters and mice) treated with combination of CpG-ODN-2006 and miltefosine at short dose regimen. Infected animals were administered CpG-ODN-2006 (0.4 mg/kg, single dose), as free and liposomal form, either alone or in combination with miltefosine for 5 consecutive days and parasite clearance was evaluated at day 4 and 7 post treatment. Animals that received liposomal CpG-ODN-2006 (lipo-CpG-ODN-2006) and sub-curative miltefosine (5 mg/kg) showed the best inhibition of parasite multiplication (∼97%) which was associated with a biased Th1 immune response in. Moreover, compared to all the other treated groups, we observed increased mRNA expression levels of pro-inflammatory cytokines (IFN-γ, TNF-α and IL-12) and significantly suppressed levels of Th2 cytokines (IL-10 and TGF-β) on day 4 post treatment in animals that underwent combination therapy with lipo-CpG-ODN-2006 and sub-curative miltefosine. Additionally, same therapy also induced heightened iNOS mRNA levels and NO generation, increased IgG2 antibody level and strong T-cell response in these hamsters compared with all the other treated groups. Collectively, our results suggest that combination of lipo-CpG-ODN-2006 and sub-curative miltefosine generates protective T-cell response in an animal model of visceral leishmaniasis which is characterized by strong Th1 biased immune response thereby underlining our hypothesis that combination therapy, at short dose regimen can be used as a novel way of treating visceral leishmaniasis.     

Dual Immune Modulatory Effect of Vitamin A in Human Visceral Leishmaniasis

Vitamin A supplementation has shown to prevent mortality by diarrheal and respiratory diseases in several countries. Nevertheless, there are few studies investigating the effect of vitamin A in visceral leishmaniasis (VL), although there are reports of its deficiency in children with symptomatic VL in Brazil and Bangladesh. This study analyzed the effect of vitamin A on a subset of Treg cells and monocytes isolated from symptomatic VL and from healthy children residing in an endemic area for VL in Northeast Brazil. Serum retinol concentrations correlated inversely with IL-10 and TGF-β productions in CD4⁺CD25^highFoxp3⁺ T cells isolated from children with VL stimulated with leishmanial antigens. All-trans retinoic acid in vitro induced IL-10 in CD4⁺CD25^highFoxp3⁺ T cells; IL-10 and TGF-β production in CD4⁺CD25⁻Foxp3⁻ T cells, and IL-10 in monocytes isolated from healthy children. However, the use of all-trans retinoic acid together with leishmanial antigens in vitro prevented increases in IL-10 production in Treg cells and monocytes isolated from VL children. Strikingly, those results show a potential dual role of vitamin A in the immune system: improvement of a regulatory profile in cells from healthy children after leishmanial stimulation and down modulation of IL-10 in Treg cells and monocytes during symptomatic VL. Therefore, the use of vitamin A concomitant to VL therapy might be useful in improving recovery from disease status caused by Leishmania infantum infection and warrants additional study.     

Arginase Activity - A Marker of Disease Status in Patients with Visceral Leishmaniasis in Ethiopia

The underlying mechanisms resulting in the profound immune suppression characteristic of human visceral leishmaniasis (VL) are not fully understood.Here, we tested the hypothesis that arginase, an enzyme associated with immunosuppression, is higher in patients with VL and contributes to impaired T cell responses. We recruited patients with VL before and after treatment and healthy controls and measured the arginase metabolism in the blood of these individuals. Our results show that arginase activity is significantly higher in the blood of patients with active VL as compared to controls. These high levels of arginase decline considerably once the patients are successfully treated. We identified the phenotype of arginase-expressing cells among PBMCs as neutrophils and show that their frequency was increased in PBMCs of patients before treatment; this coincides with reduced levels of L-arginine in the plasma and decreased expression levels of CD3ζ in T cells.     

Oral Administration of Transforming Growth Factor-β1 (TGF-β1) Protects the Immature Gut from Injury via Smad Protein-dependent Suppression of Epithelial Nuclear Factor κB (NF-κB) Signaling and Proinflammatory Cytokine Production

Inflammatory immune responses play an important role in mucosal homeostasis and gut diseases. Nuclear factor κB (NF-κB), central to the proinflammatory cascade, is activated in necrotizing enterocolitis (NEC), a devastating condition of intestinal injury with extensive inflammation in premature infants. TGF-β is a strong immune suppressor and a factor in breast milk, which has been shown to be protective against NEC. In an NEC animal model, oral administration of the isoform TGF-β1 activated the downstream effector Smad2 in intestine and significantly reduced NEC incidence. In addition, TGF-β1 suppressed NF-κB activation, maintained levels of the NF-κB inhibitor IκBα in the intestinal epithelium, and systemically decreased serum levels of IL-6 and IFN-γ. The immature human fetal intestinal epithelial cell line H4 was used as a reductionistic model of the immature enterocyte to investigate mechanism. TGF-β1 pretreatment inhibited the TNF-α-induced IκBα phosphorylation that targets the IκBα protein for degradation and inhibited NF-κB activation. Chromatin immunoprecipitation (ChIP) assays demonstrated decreased NF-κB binding to the promoters of IL-6, IL-8, and IκBα in response to TNF-α with TGF-β1 pretreatment. These TGF-β1 effects appear to be mediated through the canonical Smad pathway as silencing of the TGF-β central mediator Smad4 resulted in loss of the TGF-β1 effects. Thus, TGF-β1 is capable of eliciting anti-inflammatory effects by inhibiting NF-κB specifically in the intestinal epithelium as well as by decreasing systemic IL-6 and IFN-γ levels. Oral administration of TGF-β1 therefore can potentially be used to protect against gastrointestinal diseases.