Pathogenesis Of Visceral Leishmaniasis Research Articles

Leishmania antigens activated CD4+ T cells expressing CD200R receptors are the prime IL-10 producing phenotype and an important determinant of visceral leishmaniasis pathogenesis

The excessive production of IL-10, an anti-inflammatory cytokine, by Leishmania antigen-activated T cells is supposed to be a key player in the onset and progression of visceral leishmaniasis (VL). The IL-10-producing sources in VL remain unidentified and uncharacterized. In this study, we reveal that antigen-activated CD4+ T cells, i.e., CD44+CD4+ T cells expressing CD200R receptors, are the prime IL-10-producing phenotypes in Leishmania donovani infection-induced pathogenesis. These phenotypes are separate from CD25+Foxp3+CD4+ T regulatory cells, which are classical IL-10-producing phenotypes. In order to ascertain the role of CD200R and CD25 receptors in IL-10 overexpression-associated VL pathogenesis, we abrogated CD200R and CD25 receptor-mediated signaling in the infected mice. The splenic load of parasites and the size of the liver and spleen were significantly reduced in CD200-blocked mice as compared to CD25-blocked mice. Further, the CD200 blocking polarized CD4+ T cells to pro-inflammatory cytokines-producing phenotypes, as we observed a higher frequency of IFN-γ, TNF-α, and IL-12 positive cells as compared to controls including the CD25 blocking. Our findings suggest that in L. donovani infection-induced pathogenesis the expression of CD200R on antigen-activated T cells helps them to acquire IL-10-producing abilities as part of its one of the survival strategies. However, more studies would be warranted to better understand CD200R receptors role in VL pathogenesis and to develop the next generation of therapeutic and prophylactic control measures.

Total serum N-glycans mark visceral leishmaniasis in human infections with Leishmania infantum

Visceral leishmaniasis (VL) is a clinical form of leishmaniasis with high mortality rates when not treated. Diagnosis suffers from invasive techniques and sub-optimal sensitivities. The current (affordable) treatment with pentavalent antimony as advised by the WHO is possibly harmful to the patient. There is need for an improved diagnosis to prevent possibly unnecessary treatment. N-glycan analysis may aid in diagnosis. We evaluated the N-glycan profiles from active VL, asymptomatic infections (ASYMP) and controls from non-endemic (NC) and endemic (EC) areas. Active VL has a distinct N-glycome profile that associates with disease severity. Our study suggests that the observed glycan signatures could be a valuable additive to diagnosis and assist in identifying possible markers of disease and understanding the pathogenesis of VL. Further studies are warranted to assess a possible future role of blood glycome analysis in active VL diagnosis and should aim at disease specificity.

Role of Chemokines in the Pathogenesis of Visceral Leishmaniasis.

Visceral leishmaniasis (VL; also known as kala-azar), caused by the protozoan parasite Leishmania donovani, is characterized by the inability of the host to generate an effective immune response. The manifestations of the disease depend on the involvement of various immune components such as activation of macrophages, cell mediated immunity, secretion of cytokines and chemokines, etc. Macrophages are the final host cells for Leishmania parasites to multiply, and they are the key to a controlled or aggravated response that leads to clinical symptoms. The two most common macrophage phenotypes are M1 and M2. The pro-inflammatory microenvironment (mainly by IL-1β, IL-6, IL-12, IL-23, and TNF-α cytokines) and tissue injury driven by classically activated macrophages (M1-like) and wound healing driven by alternatively activated macrophages (M2-like) in an anti-inflammatory environment (mainly by IL-10, TGF-β, chemokine ligand (CCL)1, CCL2, CCL17, CCL18, and CCL22). Moreover, on polarized Th cells, chemokine receptors are expressed differently. Typically, CXCR3 and CCR5 are preferentially expressed on polarized Th1 cells, whereas CCR3, CCR4, and CCR8 have been associated with the Th2 phenotype. Further, the ability of the host to produce a cell-mediated immune response capable of regulating and/or eliminating the parasite is critical in the fight against the disease. Here, we review the interactions between parasites and chemokines and chemokine receptors in the pathogenesis of VL.

C-reactive protein for the diagnosis and prognosis of visceral leishmaniasis caused by Leishmania infantum

Acute-phase reaction (APR) and its marker C-reactive protein (CRP) are heightened in some infectious diseases. APR may contribute to clinical manifestations of systemic inflammation such as hemorrhages, anemia and edema. These symptoms are shared by visceral leishmaniasis (VL), a disease caused by the protozoa Leishmania infantum and L. donovani. The former is zoonotic, hitting mostly children and the immunosuppressed, with higher mortality. As APR and CRP have not been studied in VL caused by L. infantum, we decided to investigate their role as diagnostic and prognostic tools in Brazil. We measured CRP in 136 patients before the treatment of VL and 128 who survived and returned 30 days later and compared it to the clinical presentation, HIV status, and disease severity. Sensitivity for the disease was 97.8% (95%CI: 93.7 - 99.5) and specificity was 85.9% (95%CI: 78.7 - 91.0) with the cut-off of 10mg/L. There was no association of CRP concentration with demographic, clinical and laboratory data. The correlation between pre-and post-treatment levels existed but was poor. Marginal association with the presence of parasites in the bone marrow and death was noticed. The role of APR in the pathogenesis of VL and disease severity remains to be explored. However, the study reveals the significant role of CRP for VL caused by L. infantum and should be routinely required for the diagnosis and follow-up. KEY WORDS: visceral leishmaniasis, kala-azar, acute phase reaction, C-reactive protein, Leishmania infantum, diagnosis, prognosis.  

MicroRNA-21 Deficiency Promotes the Early Th1 Immune Response and Resistance toward Visceral Leishmaniasis.

MicroRNA-21 (miR-21) inhibits IL-12 expression and impairs the Th1 response necessary for control of Leishmania infection. Recent studies have shown that Leishmania infection induces miR-21 expression in dendritic cells and macrophages, and inhibition of miR-21 restores IL-12 expression. Because miR-21 is known to be expressed due to inflammatory stimuli in a wide range of hematopoietic cells, we investigated the role of miR-21 in regulating immune responses during visceral leishmaniasis (VL) caused by Leishmania donovani infection. We found that miR-21 expression was significantly elevated in dendritic cells, macrophages, inflammatory monocytes, polymorphonuclear neutrophils, and in the spleen and liver tissues after L. donovani infection, concomitant with an increased expression of disease exacerbating IL-6 and STAT3. Bone marrow dendritic cells from miR-21 knockout (miR-21KO) mice showed increased IL-12 production and decreased production of IL-10. On L. donovani infection, miR-21KO mice exhibited significantly greater numbers of IFN-γ- and TNF-α-producing CD4+ and CD8+ T cells in their organs that was associated with increased production of Th1-associated IFN-γ, TNF-α, and NO from the splenocytes. Finally, miR-21KO mice displayed significantly more developing and mature hepatic granulomas leading to reduction in organ parasitic loads compared with wild type counterparts. Similar results were noted in L. donovani-infected wild type mice after transient miR-21 depletion. These observations indicate that miR-21 plays a critical role in pathogenesis of VL by suppressing IL-12- and Th1-associated IFN-γ and also inducing disease-promoting induction of the IL-6 and STAT-3 signaling pathway. miR-21 could therefore be used as a potential target for developing host-directed treatment for VL.

Seropositivity of Visceral leishmaniasis on people of VL endemic three districts of Nepal.

Visceral leishmaniasis (VL) is a life-threatening vector borne disease caused by the Leishmania donovani species complex. In Nepal, it is transmitted to humans by L donovani infected Phlebotomus argentipes sand flies [12]. The pathogenesis of VL is complex, and the clinical presentation ranges from asymptomatic infection to severe and fatal disease. Asymptomatic infection may act as potential reservoirs for sustained transmission of VL in endemic areas. We investigated the sero-prevalence of symptomatic and asymptomatic infection of VL in people of three endemic districts of Nepal by serology targeting family members and neighbors of VL patients. Sero-survey was conducted among 189 people of villages endemic to VL from Palpa, Sarlahi and Saptari districts during 2016 to 2018 using the rK39 rapid diagnostic test (InBios International, Seattle, WA) to detect anti-Leishmania antibodies. Sero-positivity was 35.7% (10/28) in people tested from Sarlahi districts, 6% (3/50) in Saptari district and 1.7% (1/59) from the Palpa district. In Sarlahi, sero-positivity was found to be highest among the age group below 15years (44.5%). All family members of diagnosed VL cases in Saptari and Palpa districts were found to be rK39 test negative. In Sarlahi district, among the ten sero-positive cases, nine were febrile and became symptomatic VL cases after few days and one case remained asymptomatic during the six month follow up. Asymptomatic cases in VL endemic districts of Nepal were found to be sero-positive, screening of people in VL endemic districts would be important for prevention of VL transmission.

An Overview of Immunotherapeutic Approaches Against Canine Visceral Leishmaniasis: What Has Been Tested on Dogs and a New Perspective on Improving Treatment Efficacy

Visceral leishmaniasis (VL), caused by digenetic protozoa of the genus Leishmania, is the most severe form of leishmaniasis. Leishmania infantum is one of the species responsible for VL and the disease caused is considered a zoonosis whose main reservoir is the dog. Canine visceral leishmaniasis (CVL) can lead to the death of the animal if left untreated. Furthermore, the available pharmocologial treatment for CVL presents numerous disadvantages, such as relapses, toxicity, drug resistance, and the fact treated animals continue to be reservoirs when treatment fails to achieve parasitological cure. Moreover, the available VL control methods have not been adequate when it comes to controlling parasite transmission. Advances in immune response knowledge in recent years have led to a better understanding of VL pathogenesis, allowing new treatments to be developed based on immune system activation, often referred to as immunotherapy. In fact, well-defined protocols have been described, ranging from the use of immunomodulators to the use of vaccines. This treatment, which can also be associated with chemotherapy, has been shown to be effective in restoring or inducing an adequate immune response to reduce parasitic burden, leading to clinical improvement. This review focuses on immunotherapy directed at dogs infected by L. infantum, including a literature review of what has already been done in dogs. We also introduce a promising strategy to improve the efficacy of immunotherapy.

Abnormal B-Cell Subset and Blimp-1-Mediated Humoral Responses Associated With Visceral Leishmaniasis Pathogenesis.

B-cells have a spectrum of functions ranging from antibody production to antigen presentation and have additional vital roles in immune mechanisms. There is rudimentary knowledge about the role of B-cells in intracellular infections with contradictory findings. We explored the role of B-cell dysfunctions in visceral leishmaniasis (VL) pathogenesis in terms of the phenotypic and functional properties of B-cells during the course of disease. This study was performed on blood and splenic aspirates (SA) of VL cases pre- and post-treatment. Whole blood was used for flow cytometric studies for determining the profiles of B-cells at different time-points of treatment. Peripheral blood mononuclear cells were used for magnetic purification of B-cells, for transcriptional studies by real-time polymerase chain reaction (RT-PCR). Serum/plasma was used for direct agglutination test for determining parasite-specific antibodies and SA were used for scoring the presence of parasite by microscopic examination. Flow cytometric studies depicted decreased B-cell percentages during the entire course of disease and attainment of exhaustive phenotype with tissue-like memory cell markers, indicative of B-cell dysfunctions in VL. In addition, B-cells had compromised abilities of antigen processing and presentation and altered levels of B-lymphocyte-induced maturation protein-1 (Blimp-1). Blimp-1 expression goes hand in hand with B-cell maturation antigen and transmembrane activator and calcium modulator (TACI) and cyclophilin ligand interactor, suggestive of its role in promoting plasma cell survival and antibody production. Elevated level of VL-specific antibody titre was directly correlated with exhausted phenotype and also with disease severity during VL. This study indicated for impaired B-cell functions during chronic infection which may lead to pathological consequences in human VL.

Lower levels of leptin are associated with severity parameters in visceral leishmaniasis patients.

Visceral leishmaniasis (VL) is the most severe clinical form of leishmaniasis, and if untreated may be fatal. It affects important organs of the immune system and is characterized by a specific immunosuppression, along with intense cellular activation and cytokine storm. Moreover, VL is now recognized as a systemic inflammatory response syndrome (SIRS), in which multiple cytokines and other pro-inflammatory molecules are released. The action of these inflammatory mediators may be considered risk factors for poor prognosis and death. Leptin, a hormone derived from adipose tissue, has been described with several immunoregulatory functions in vitro and in vivo Leishmania infection models, particularly for enhancing the macrophage microbicidal mechanisms. Considering that evaluation of immunologic parameters that may be associated with this clinical scenario may help to decrease VL lethality, we evaluated whether leptin is associated with VL pathogenesis. Thirty-one patients were recruited in the active phase of VL, of which 22 were followed up until one month after therapy (1mpt). Except for creatinine levels, all clinical parameters were altered in active VL patients, especially leucocyte counts and albumin and hemoglobin levels. Also, elevated levels of lipopolysaccharide (LPS), immunoglobulins (Ig)G1 and G3 anti-Leishmania and interleukins (IL)-6 and -10 were higher than in healthy individuals. In contrast, active VL patients presented diminished serum leptin levels and positive correlation with leukocytes counts and hemoglobin and albumin levels. After 1mpt, VL patients showed a significant increase in leptin levels, reaching values similar to healthy volunteers. As expected, only LPS levels remained elevated after 1mpt. These findings suggest that leptin levels are affected in Leishmania infection and the correlation with important parameters associated with the prognosis of VL points to the involvement of this molecule in VL immunopathogenesis. Additional studies are needed to evaluate the possibility of leptin as a prognostic marker of VL.

PDL-1 Blockade Prevents T Cell Exhaustion, Inhibits Autophagy, and Promotes Clearance of Leishmania donovani.

ABSTRACTLeishmania donovani is a causative pathogen of potentially fatal visceral leishmaniasis (VL). Therapeutic agents are available; however, their use is limited because of high cost, serious side effects, and development of antimicrobial resistance. Protective immunity against VL depends on CD4+ Th1 cell-mediated immunity. Studies have shown that progression of VL is due to exhaustion of T cells; however, the mechanism involved is not clearly understood. Here, we examined the role of PD1/PDL-1 in the pathogenesis of VL by using a murine model of VL. Our data indicate that L. donovani is able to elicit initial expansion of gamma interferon-producing CD4+ Th1 and CD8+ T cells at day 7 postinfection (p.i.); however, the frequency of those cells and inflammatory response decreased at day 21 p.i., despite persistence of parasites. Persistent infection-induced expansion of interleukin-10+ FOXP3+ Treg and CD4+ and CD8+ T cells expressing PD1. Blocking of PDL-1 signaling in vivo resulted in restoration of protective type 1 responses by both CD4+ and CD8+ T cells, which resulted in a significant decrease in the parasite burden. Mechanistically, PDL-1 blocking inhibited autophagy, a cellular degradation process hijacked by Leishmania to acquire host cell nutrients for their survival. Inhibition of autophagy was marked by decreased lipidation of microtubule-associated protein 1 light chain 3, a marker of autophagosome formation, and P62 accumulation. Together, our findings show for the first time that anti-PDL-1 antibody is an effective therapeutic approach for restoration of effector arms of protective immunity against VL and subsequent parasite clearance.

Leptin regulates Granzyme-A, PD-1 and CTLA-4 expression in T cell to control visceral leishmaniasis in BALB/c Mice

Visceral leishmaniasis (VL) is responsible for several deaths in malnourished children accompanied by diminished circulating leptin and impaired cell-mediated immunity. Typically, leptin deficiency is associated with the Th2 polarization that markedly coincides with the pathogenesis of VL. The aim of the present study was to unravel the prophylactic role of leptin in malnutrition-coupled VL mice. Interestingly, we observed that L. donovani infection itself reduces the serum leptin levels in malnutrition. Exogenous leptin restored severe body weight loss and parasite load in the spleen and liver of malnourished infected mice compared to controls. Leptin increases functional CD8+ T-cell population, Granzyme-A expression down-regulates anergic T-cell markers such as PD-1 and CTLA-4. It was also noticed that, leptin suppresses GM-CSF mRNA expression in parasite favored monocytes and reduced arginase activity in bone marrow derived macrophage indicate macrophages dependent T-cell activation and proliferation. Leptin-induced IFN-γ, IL-2, and TNF-α cytokines in the culture supernatant of splenocytes upon soluble leishmanial antigen (SLA) stimulation and significantly up-regulates serum IgG2a titers, which help to generate Th1 immune response in VL. Furthermore, leptin induced a granulomatous response and restored L. donovani induced tissue degeneration in the liver. Altogether, our findings suggest the exogenous leptin can restore T cell mediated immunity in malnourished VL mice.

Some diagnostic aspects, using CD markers, in young Iraqi children with visceral leishmaniasis

     The lymphocytic phenotypes involved in the pathogenesis of visceral leishmaniasis (VL) in Iraqi children have recently been investigated, in a study based on cluster-differentiation (CD) markers . Each case of VL investigated was confirmed serologically by the immunochromatography with rk – 39 antigen strip test (dipstick assay) . A significant decrease was found in number of CD3 and CD4 cells in VL patients compared to the healthy control group, while the results showed that there was a significant increase in CD8 lymphocytes with VL patients in comparison to the control group . The CD4/CD8 ratio was lower in VL patients than that of the control group . There was a relative increase in CD22 of VL patients compared to the control group, but was not significant . There was no significant differnce in all types of CD cells percentage of VL in relation to some clinical parameters such as gender and age . Follow up of the patients showed there was a significant increase in the percentage of CD3, CD4, and CD4/CD8 ratio at the end of therapy . A relative decrease in CD22 but still more than control group .

Anti-parasite therapy drives changes in human visceral leishmaniasis-associated inflammatory balance

Visceral leishmaniasis (VL) remains a major public health problem worldwide. Cytokine balance is thought to play a critical role in the development of this disease. Here, we perform a prospective exploratory study addressing whether simultaneous assessment of circulating levels of different lipid mediators and cytokines could highlight specific pathways involved with VL pathogenesis. VL patients displayed substantial increases in serum levels of Prostaglandin F2α (PGF2α), Leukotriene B4 (LTB4), Resolvin D1 (RvD1), IL-1β, IL-6, IL-8, IL-10, IL-12p70 and TNF-α compared with uninfected endemic control group, while exhibiting decreased levels of TGF-β1. Hierarchical cluster analysis of the prospective changes in the expression level of theses parameters upon anti-Leishmania treatment initiation revealed that the inflammatory profile observed in active disease gradually changed over time and was generally reversed at day 30 of therapy. Furthermore, not only the individual concentrations of most of the inflammatory biomarkers changed upon treatment, but the correlations between those and several biochemical parameters used to characterize VL disease activity were also modified over time. These results demonstrate that an inflammatory imbalance hallmarks active VL disease and open perspective for manipulation of these pathways in future studies examining a potential host-directed therapy against VL.

Leishmania donovani Nucleoside Hydrolase (NH36) Domains Induce T-Cell Cytokine Responses in Human Visceral Leishmaniasis.

Development of immunoprotection against visceral leishmaniasis (VL) focused on the identification of antigens capable of inducing a Th1 immune response. Alternatively, antigens targeting the CD8 and T-regulatory responses are also relevant in VL pathogenesis and worthy of being included in a preventive human vaccine. We assessed in active and cured patients and VL asymptomatic subjects the clinical signs and cytokine responses to the Leishmania donovani nucleoside hydrolase NH36 antigen and its N-(F1), central (F2) and C-terminal (F3) domains. As markers of VL resistance, the F2 induced the highest levels of IFN-γ, IL-1β, and TNF-α and, together with F1, the strongest secretion of IL-17, IL-6, and IL-10 in DTH+ and cured subjects. F2 also promoted the highest frequencies of CD3+CD4+IL-2+TNF-α−IFN-γ−, CD3+CD4+IL-2+TNF-α+IFN-γ−, CD3+CD4+IL-2+TNF-α−IFN-γ+, and CD3+CD4+IL-2+TNF-α+IFN-γ+ T cells in cured and asymptomatic subjects. Consistent with this, the IFN-γ increase was correlated with decreased spleen (R = −0.428, P = 0.05) and liver sizes (R = −0.428, P = 0.05) and with increased hematocrit counts (R = 0.532, P = 0.015) in response to F1 domain, and with increased hematocrit (R = 0.512, P 0.02) and hemoglobin counts (R = 0.434, P = 0.05) in response to F2. Additionally, IL-17 increases were associated with decreased spleen and liver sizes in response to F1 (R = −0.595, P = 0.005) and F2 (R = −0.462, P = 0.04). Conversely, F1 and F3 increased the CD3+CD8+IL-2+TNF-α−IFN-γ−, CD3+CD8+IL-2+TNF-α+IFN-γ−, and CD3+CD8+IL-2+TNF-α+IFN-γ+ T cell frequencies of VL patients correlated with increased spleen and liver sizes and decreased hemoglobin and hematocrit values. Therefore, cure and acquired resistance to VL correlate with the CD4+-Th1 and Th-17 T-cell responses to F2 and F1 domains. Clinical VL outcomes, by contrast, correlate with CD8+ T-cell responses against F3 and F1, potentially involved in control of the early infection. The in silico-predicted NH36 epitopes are conserved and bind to many HL-DR and HLA and B allotypes. No human vaccine against Leishmania is available thus far. In this investigation, we identified the NH36 domains and epitopes that induce CD4+ and CD8+ T cell responses, which could be used to potentiate a human universal T-epitope vaccine against leishmaniasis.

Transcriptional Profiling in Experimental Visceral Leishmaniasis Reveals a Broad Splenic Inflammatory Environment that Conditions Macrophages toward a Disease-Promoting Phenotype.

Visceral Leishmaniasis (VL), caused by the intracellular protozoan Leishmania donovani, is characterized by relentlessly increasing visceral parasite replication, cachexia, massive splenomegaly, pancytopenia and ultimately death. Progressive disease is considered to be due to impaired effector T cell function and/or failure of macrophages to be activated to kill the intracellular parasite. In previous studies, we used the Syrian hamster (Mesocricetus auratus) as a model because it mimics the progressive nature of active human VL. We demonstrated previously that mixed expression of macrophage-activating (IFN-γ) and regulatory (IL-4, IL-10, IL-21) cytokines, parasite-induced expression of macrophage arginase 1 (Arg1), and decreased production of nitric oxide are key immunopathologic factors. Here we examined global changes in gene expression to define the splenic environment and phenotype of splenic macrophages during progressive VL. We used RNA sequencing coupled with de novo transcriptome assembly, because the Syrian hamster does not have a fully sequenced and annotated reference genome. Differentially expressed transcripts identified a highly inflammatory spleen environment with abundant expression of type I and type II interferon response genes. However, high IFN-γ expression was ineffective in directing exclusive M1 macrophage polarization, suppressing M2-associated gene expression, and restraining parasite replication and disease. While many IFN-inducible transcripts were upregulated in the infected spleen, fewer were induced in splenic macrophages in VL. Paradoxically, IFN-γ enhanced parasite growth and induced the counter-regulatory molecules Arg1, Ido1 and Irg1 in splenic macrophages. This was mediated, at least in part, through IFN-γ-induced activation of STAT3 and expression of IL-10, which suggests that splenic macrophages in VL are conditioned to respond to macrophage activation signals with a counter-regulatory response that is ineffective and even disease-promoting. Accordingly, inhibition of STAT3 activation led to a reduced parasite load in infected macrophages. Thus, the STAT3 pathway offers a rational target for adjunctive host-directed therapy to interrupt the pathogenesis of VL.

Role of Cytokines in the Pathogenesis of Visceral Leishmaniasis.

Infection in humans with Leishmania manifests into a spectrum of diseases. The manifestations of the disease depend on the resultant evasion of the parasite to immune responses namely macrophages, which is an exclusive host of leishmania. The B cells valiantly mount antibody responses, however to no avail as the Leishmania parasites occupy the intracellular niches of the macrophages. Extensive studies have been documented on the role of cell-mediated immunity (CMI) in protection and counter survival strategies of the parasites leading to down-regulation of CMI. The present review attempts to discuss the cytokines in progression or resolution of visceral form of leishmaniasis or kala-azar, predominantly affecting the Indian subcontinent. The components/cytokine(s) responsible for the regulation of the critical balance of Th1/Th2/Th9/Th17/Treg cells has been discussed in the perspective. Therefore, any strategy involving the treatment of VL needs to consider the balance and regulation of CD4+ T cell function.

Visceral Leishmaniasis Patients Display Altered Composition and Maturity of Neutrophils as well as Impaired Neutrophil Effector Functions.

Immunologically, active visceral leishmaniasis (VL) is characterized by profound immunosuppression, severe systemic inflammatory responses, and an impaired capacity to control parasite replication. Neutrophils are highly versatile cells, which play a crucial role in the induction as well as the resolution of inflammation, the control of pathogen replication, and the regulation of immune responses. Neutrophil functions have been investigated in human cutaneous leishmaniasis; however, their role in human VL is poorly understood. In the present study we evaluated the activation status and effector functions of neutrophils in patients with active VL and after successful anti-leishmanial treatment. Our results show that neutrophils are highly activated and have degranulated; high levels of arginase, myeloperoxidase, and elastase, all contained in neutrophils’ granules, were found in the plasma of VL patients. In addition, we show that a large proportion of these cells are immature. We also analyzed effector functions of neutrophils that are essential for pathogen clearance and show that neutrophils have an impaired capacity to release neutrophil extracellular traps, produce reactive oxygen species, and phagocytose bacterial particles, but not Leishmania parasites. Our results suggest that impaired effector functions, increased activation, and immaturity of neutrophils play a key role in the pathogenesis of VL.

Type I interferon signalling suppresses anti-parasitic CD4+ T cell responses during visceral leishmaniasis

Abstract Many pathogens, including viruses, bacteria, and protozoan parasites, suppress cell mediated immune responses through activation of type I Interferon (IFN-1) signalling. However the role of IFN-1 during Leishmania donovani infection causing visceral leishmaniasis (VL) is not well known. Here we report that IFN-1 play an important role in the pathogenesis of VL by impairing parasite clearance and suppressing pro-inflammatory cytokine production. Mice lacking type-1 IFN signalling (B6.IFNαR1−/− mice) had enhanced pro-inflammatory cytokine production and better control of parasite burden in liver as well as spleen compared to wild type C57BL/6 mice. IFN-1 signalling suppressed the CD4+ derived IFN-γ production and prevented Th1 response from controlling parasite replication. Experiment using bone marrow chimeric mice revealed that IFN-1 did not suppress CD4+ T via directly acting to this cell type. Using conventional dendritic cell (cDC) specific IFN-1 deficient mice (CD11c Cre-IFNARfl/fl), we observed that IFN-1 hampered CD4+ T cells response acting via dendritic cells. Studies in VL patients supported these findings and showed enhanced accumulation of mRNA encoding type I IFN signature genes in peripheral blood mononuclear cells (PBMCs) that were reduced following successful drug therapy. Critically, we also showed, using a whole blood assay, that blockade of type-1 IFN signalling enhanced antigen specific IFN-γ production, and that this response was HLA-II restricted. Together, these results identify the type-1 IFN signalling pathways as a potential therapeutic target to treat VL by stimulating anti-parasitic CD4+ T cell responses.

Leptin induces the phagocytosis and protective immune response in Leishmania donovani infected THP-1 cell line and human PBMCs

Visceral leishmaniasis (VL) is an infectious disease responsible for several deaths in malnourished children due to impaired cell-mediated immunity, which is accompanied by low circulating leptin levels. The cytokine function of leptin is implicated for several immune regulation activities such as hematopoiesis, angiogenesis, innate and adaptive immunity. Its deficiency associated with polarization of Th2 response, which coincides with VL pathogenesis. To determine the cytokine role of leptin in case of experimental VL, we tested the leptin associated Th1/Th2 type cytokine profile at mRNA level from Leishmania donovani infected human monocytic leukemia cell line (THP-1) and peripheral blood mononuclear cells (PBMCs). We also tested the effect of leptin on macrophages activation (viz. studying the phosphorylation of signaling moieties), phagocytic activity and intracellular reactive oxygen species (ROS) production during infection. We observed that leptin induced Th1 specific response by upregulation of IL-1α, IL-1β, IL-8 and TNF-α in THP-1 and IFN-γ, IL-12 and IL-2 in PBMCs. We also observed the downregulation of Th2 type cytokine i.e. IL-10 in THP-1 and unaltered expression of cytokines i.e. TGF-β, IL-10 and IL-4 in PBMCs. In addition, leptin stimulates the macrophages by inducing phosphorylation of Erk1/2 and Akt which are usually dephosphorylated in L. donovani infection. In concordance, leptin also induces the macrophage phagocytic activity by enhancing the intracellular ROS generation which helps in phagolysosome formation and oxidative killing of the parasite. In compilation, leptin is able to maintain the defensive environment against L. donovani infection through the classical macrophage activity.

Hepatic stellate cells regulate liver immunity to visceral leishmaniasis through P110δ-dependent induction and expansion of regulatory T cells in mice.

Visceral leishmaniasis (VL) is associated with severe immune dysfunction and if untreated leads to death. Because the liver is one of the primary target organs in VL, unraveling the mechanisms governing the local hepatic immune response is important for understanding the immunopathogenesis of VL. We previously reported that mice with inactivating knockin mutation in the p110δ gene (p110δ(D910A) ) are resistant to VL, due in part to impaired regulatory T-cell (Treg) expansion. In this study, we investigated the mechanism of this resistance by focusing on hepatic stellate cells (HSCs), which are known to regulate Treg induction and expansion. We show that HSCs are infected with Leishmania donovani in vivo and in vitro and that this infection leads to the production of interleukin-2, interleukin-6, and transforming growth factor-β, cytokines known to induce Tregs. We further demonstrate that L. donovani infection leads to expansion of HSCs in a p110δ-dependent manner and that this correlated with proliferation of hepatic Tregs in vivo. In vitro studies clearly show that L. donovani-infected HSCs induce CD4(+) T cells to become Tregs and expand Tregs in a p110δ-dependent manner. Targeted depletion of HSCs during infection caused a dramatic reduction in liver Treg numbers and proliferation, which was associated with a decrease in interleukin-10 production by hepatic T cells and a more efficient parasite control. These results demonstrate the critical role of HSCs in the pathogenesis of VL and suggest that the enhanced resistance of p110δ(D910A) mice to L. donovani infection is due in part to impaired expansion and inability of their HSCs to induce and expand Tregs in the liver.