Clinical significance of blood cell ratios in healthy and sick Leishmania infantum-seropositive dogs.

BACKGROUND: The accuracy of blood cell ratios (BCRs) as cost-effective and easily accessible diagnostic and prognostic markers of inflammatory conditions has been investigated in veterinary medicine in recent years. METHODS: Neutrophil-to-lymphocyte (NLR), monocyte-to-lymphocyte (MLR), and platelet-to-lymphocyte (PLR) ratios were studied in 195 dogs clinically evaluated and tested for anti-Leishmania infantum (Li) antibodies (Li-seronegative (Li-), n = 10; Li-seropositive clinically healthy (Li+healthy), n = 100; Li-seropositive with clinical and/or clinicopathological abnormalities (Li+sick), n = 85). The Li+sick dogs were classified in LeishVet stages IIa/IIb (Li+IIa/IIb) (n = 66) and III/IV (Li+III/IV) (n = 19). BCR relationships with LeishVet clinical stage, antibody levels, and serum protein electrophoretic fraction concentrations were investigated. RESULTS: Higher NLR values were found in Li+, Li+healthy, and Li+IIa/IIb sick dogs compared to Li- dogs (P < 0.001). Higher NLR and MLR were found in Li+sick (NLR, P < 0.001; MLR, P = 0.034) and Li+III/IV dogs (NLR, P < 0.001; MLR, P = 0.005) compared to Li- dogs, and in Li+III/IV dogs (NLR, P = 0.002; MLR, P < 0.001) compared to Li+healthy. All three BCRs were higher in Li+sick (NLR, MLR, P < 0.001; PLR, P = 0.023) and Li+IIa/IIb dogs (NLR P < 0.001; MLR P = 0.001; PLR, P = 0.012) compared to Li+healthy dogs. The BCRs failed to distinguish dogs with moderate (Li+IIa/IIb) and severe or very severe disease (Li+III/IV). BCRs demonstrated weak positive correlations with serum globulin fractions and antibody levels, and weak negative correlations with serum albumin level were found. Li+sick dogs presenting hypoalbuminemia showed higher MLR ratios (P = 0.001) than those with normal albumin values. CONCLUSIONS: This study shows that BCR measures provide useful information for differentiating antibody-positive healthy and sick dogs at diagnosis. Dogs with hypoalbuminemia showed higher MLR values despite monocytosis being very rare.

Interleukin-27 Regulates Adaptative Immune Responses Associated With Control of Parasite Replication in Canine Leishmaniasis.

Interleukin 27 (IL-27) is a cytokine that regulates susceptibility to Leishmania infantum infection in humans and experimental models. This cytokine has not yet been described in canine leishmaniasis (CanL). Therefore, we investigated whether IL-27 has a regulatory role in CanL. The EBI3 and p28 subunits of IL-27 were measured in splenic leukocytes culture supernatant from dogs with CanL and compared to control dogs. We also correlated EBI3 and p28 levels with IL-21, anti-L. infantum antibodies and parasite loads. We performed functional assays followed by IL-27 blockade and measured parasite loads, production of cytokines in splenic leukocytes culture supernatant, and the expression of PD-1, CTLA-4, phospho-Stat-1/3, T-bet, GATA3 and nitric oxide production (NO). Both IL-27 subunits increased in the supernatant of dogs with CanL compared to control dogs. EBI3 and p28 levels showed a moderate positive correlation with IL-21 (r = 0.67, p < 0.0001 and r = 0.45, p < 0.012, respectively), and the EBI3 subunit was positively associated with anti-L. infantum IgG antibodies (r = 0.38, p < 0.040) and parasite load (r = 0.47, p < 0.009). IL-27 and IL-21 participate of immune responses in CanL. IL-27 may be associated with the failure of immunity to control parasite replication via upregulation of the expression of PD-1, CTLA-4, T-bet and NO in splenic leukocytes from dogs with CanL. These findings suggest that the pathways regulated by IL-27 are involved in CanL pathogenesis in the host, and may be targets for new therapies.

Immune response to viscerotropic Leishmania: a comprehensive review.

L. donovani and L. infantum infections are associated with a broad clinical spectrum, ranging from asymptomatic cases to visceral leishmaniasis (VL) with high mortality rates. Clinical manifestations such as post-kala-azar dermal leishmaniasis (PKDL) and visceral leishmaniasis-associated hemophagocytic lymphohistiocytosis-mimic (VL-associated HLH-mimic) further contribute to the diversity of clinical manifestations. These clinical variations are intricately influenced by the complex interplay between the host's immune response and the parasite's escape mechanisms. This narrative review aims to elucidate the underlying immunological mechanisms associated with each clinical manifestation, drawing from published literature within the last 5 years. Specific attention is directed toward viscerotropic Leishmania sinfection in patients with inborn errors of immunity and acquired immunodeficiencies. In VL, parasites exploit various immune evasion mechanisms, including immune checkpoints, leading to a predominantly anti-inflammatory environment that favors parasite survival. Conversely, nearly 70% of individuals are capable of mounting an effective pro-inflammatory immune response, forming granulomas that contain the parasites. Despite this, some patients may experience reactivation of the disease upon immunosuppression, challenging current understandings of parasite eradication. Individuals living with HIV and those with inborn errors of immunity present a more severe course of infection, often with higher relapse rates. Therefore, it is crucial to exclude both primary and acquired immune deficiencies in patients presenting disease relapse and VL-associated HLH-mimic. The distinction between VL and HLH can be challenging due to clinical similarities, suggesting that the nosological entity known as VL-associated HLH may represent a severe presentation of symptomatic VL and it should be considered more accurate referring to this condition as VL-associated HLH-mimic. Consequently, excluding VL in patients presenting with HLH is essential, as appropriate antimicrobial therapy can reverse immune dysregulation. A comprehensive understanding of the immune-host interaction underlying Leishmania infection is crucial for formulating effective treatment and preventive strategies to mitigate the disease burden.

Lupeol stimulates iNOS, TNF-α, and IL-10 expression in the U937 cell line infected with old-world Leishmania donovani.

OBJECTIVE: Visceral leishmaniasis is a neglected tropical disease that can be lethal if not treated. The available medicines have severe side effects, such as toxicity and drug resistance. Various investigations are looking into new anti-leishmanial compounds from natural products that have little impact on host cells. Lupeol, a triterpenoid present in the flora of many edible plants, has been shown to have antimicrobial properties. The present study investigated the immunomodulatory effects of lupeol on U937 macrophages infected with Leishmania donovani, focusing on the expression of key cytokines and enzymes involved in the immune response. METHODS: U937 macrophages were infected with Leishmania donovani amastigotes and treated with varying concentrations of lupeol throughout three days. The expression levels of inducible nitric oxide synthase (iNOS), tumor necrosis factor-alpha (TNF-α), and interleukin-10 (IL-10) were measured using real-time polymerase chain reaction (RT-PCR). A positive simulation of gene expression was estimated using ΔΔCT to assess relative expression. RESULTS: The results demonstrated that lupeol significantly upregulated iNOS and TNF-α expression, especially at higher concentrations, indicating enhanced pro-inflammatory and anti-leishmanial activity. Interestingly, IL-10 expression also increased, suggesting a complex immunomodulatory role of lupeol that involves both pro-inflammatory and anti-inflammatory pathways. Pearson correlation analysis revealed a strong association between iNOS and TNF-α (0.97692), as well as a moderate correlation between iNOS and IL-10 (0.51603). CONCLUSION: These findings suggest that lupeol may promote a balanced immune response, enhancing the body's ability to combat L. donovani while potentially mitigating excessive inflammation. Lupeol can potentially serve as a novel therapeutic agent against visceral leishmaniasis.

Leishmania donovani Modulates Macrophage Lipidome During Infection.

Obligate intracellular protozoan parasite, Leishmania donovani, causative agent of visceral leishmaniasis, led to impaired macrophage functions. It is well documented that many of these changes were induced by parasite-mediated reduction in macrophage cholesterol content. Leishmania-mediated alteration in the other lipids has not been explored in detail yet. Here, we found that the expression of key cholesterol biosynthetic genes and total cellular cholesterol were reduced during L. donovani infection. Further, we have also identified that this reduction in the cholesterol led to increased membrane fluidity and inhibition of antigen-presenting potential of macrophages. In addition to this, we studied the relative changes in different lipids in THP-1-derived macrophages during L. donovani infection through liquid chromatography-mass spectrometry. We found that Sphingomyelin (16:0) and ceramide (20:1, 26:0 and 26:1) were significantly reduced in infected macrophages. We further observed that the majority of different sub-classes of phospholipids were downregulated significantly. Overall ratio of phosphatidylcholine versus phosphotidylethanolamine was decreased which indicated the compensatory mechanism of cell in response to cholesterol reduction. The observed Leishmania-mediated alteration in macrophage-lipidome provided the novel insights into mechanism of host-pathogen interactions.

Adaptive and innate immune response of Leishmania infantum infection in Cirneco dell'Etna dog breed.

Leishmania spp. are an intracellular protozoa present in many countries around the world. In Europe, both the parasite and the disease it causes, leishmaniasis, are endemic in the Mediterranean basin. Clinical signs and severity of disease are highly variable depending on the host in both humans and dogs, traditionally considered the main reservoir of the parasite. The reason for these differences is not known, but it has been speculated that some hosts present immune response, related to activation of Th1 and Th17, capable of controlling the spread of the parasite, and that these immune responses are related to the genetic background of the host. The Ibizan hound, an autochthonous canine breed of the Mediterranean basin, has been postulated as a breed resistant to infection, but other canine breeds evolutionarily close to it and native to this region have not been studied. One of them is the Cirneco dell'Etna, native to the island of Sicily in southern Italy. In this study, the immune response against L. infantum infection in this canine breed was analysed. The results showed that infected dogs of this breed present high levels of several cytokines related to Th1 and Th17 immune response, and significant correlation between serum levels of cytokines related to disease resistance. Further studies are necessary in this canine breed to determine the mechanisms of immune response and genetic background related to L. infantum infection control.

Cytokine profiles, blood parasite load and clinical features of visceral leishmaniasis in West Pokot County, Kenya.

Visceral leishmaniasis (VL) is a severe infectious disease caused by protozoan parasites of the Leishmania donovani complex. Blood cytokine concentrations in VL patients can inform us about underlying immunopathogenesis and may serve as a biomarker for treatment effectiveness. However, cytokine levels have not yet been studied in VL patients from Kenya, where case load is high. This study measured the serum cytokine profile, blood parasite load and clinical and haematological features of VL patients from West Pokot County, Kenya, over the course of treatment with sodium stibogluconate and paromomycin (SSG-PM). VL patients recruited at the hospital presented with splenomegaly and weight loss, and frequently had pancytopenia and anaemia. Median Leishmania parasite load in blood, determined with real-time polymerase chain reaction, was 2.6 × 104 parasite equivalents mL−1. Compared to endemic healthy controls, serum interferon gamma (IFN-γ), interleukin 5 (IL-5), IL-6, IL-10, IL-12p70, IL-17A and IL-27 were significantly elevated in untreated VL patients. Severe VL was associated with higher IL-10 and lower IFN-γ levels. After 17 daily injections with SSG-PM, disease symptoms disappeared, leukocyte and thrombocyte counts significantly increased, and blood parasite load decreased to undetectable levels in all VL patients. There was a significant decrease in IL-10 and IL-6, whereas IL-17A levels increased; the remaining cytokines showed no significant concentration change during treatment. In conclusion, the results suggest that SSG-PM treatment of VL patients from West Pokot was effective. Moreover, both inflammatory and regulatory immune responses appeared to decrease during treatment, although the increase in IL-17A could reflect a partial continuation of immune activation.

Altered co-stimulatory and inhibitory receptors on monocyte subsets in patients with visceral leishmaniasis.

Visceral leishmaniasis (VL) is a neglected tropical disease caused by parasites from the Leishmania (L.) donovani complex. VL is characterised by uncontrolled parasite replication in spleen, liver and bone marrow, and by an impaired immune response and high systemic levels of inflammation. Monocytes have been poorly characterised in VL patients. The aim of this study was to evaluate the expression levels of markers involved in the regulation of T cell responses on different subsets of monocytes from the blood of VL patients and healthy non-endemic controls (HNEC). Monocytes can broadly be divided into three subsets: classical, intermediate and non-classical monocytes. Our results show that the percentages of all three subsets stayed similar at the time of VL diagnosis (ToD) and at the end of anti-leishmanial treatment (EoT). We first looked at co-stimulatory receptors: the expression levels of CD40 were significantly increased on classical and intermediate, but not non-classical monocytes, at ToD as compared to EoT and HNEC. CD80 expression levels were also increased on intermediate monocytes at ToD as compared to EoT and HNEC, and on classical monocytes only as compared to HNEC. The levels of CD86 were similar at EoT and ToD and in HNEC on classical and intermediate monocytes, but significantly higher at EoT on non-classical monocytes. We also looked at an inhibitory molecule, PD-L1. Our results show that the expression levels of PD-L1 were significantly higher on all three monocyte subsets at ToD as compared to HNEC, and to EoT on classical and intermediate monocytes. These results show that monocytes from the blood of VL patients upregulate both co-stimulatory and inhibitory receptors and that their expression levels are restored at EoT.

The nexus between Leishmania & HIV: Debilitating host immunity and Hastening Comorbid disease burden.

The scintillating association between Leishmania and HIV has contributed exceptionally towards expansion of Visceral Leishmaniasis (VL) with Acquired Immunodeficiency Syndrome (AIDS). The co-infection poses a grievous threat to elimination of VL and containment of Human Immunodeficiency Virus (HIV). When coinfected, Leishmania and HIV complement each other's proliferation and survival by inducing immunesenescence, T cell fatigue and exhaustion. Antigen presentation is lost, co-stimulatory molecules are diminished whereas co-inhibitory molecules such as CTLA-4, TIGIT, LAG-3 etc. are upregulated to ensure a Th2-baised immune environment. As a consequence, Leishmania-HIV coinfection causes poor outcomes, inflates the spread of Leishmania parasites, enhances the severity of side-effects to drugs, as well as escalate the probability of treatment failure and mortality. What makes control extremely strenuous is that there are frequent episodes of VL relapse with no prognostic markers, no standard immunophenotype(s) and appearance of atypical clinical symptoms. Thus, a standard therapeutic regimen has been difficult to develop and treatment is majorly dependent upon a combination of liposomal Amphotericin B and Miltefosine, a therapy that is expensive and capable of causing drastic side-effects in recipients. As World Health Organization is committed to eliminate both VL and HIV in due course of future, the existing therapeutic interventions require advancements to grapple and overcome this hazardous co-infection. In this context, an overview of HIV-VL co-infection, immunopathology of HIV and Leishmania co-inhabitance, available therapeutic options and their limitations in the treatment of co-infection are discussed in-depth.

Effect of Leishmania infantum infection on B cell lymphopoiesis and memory in the bone marrow and spleen.

Visceral leishmaniasis (VL) is characterized by an uncontrolled infection of internal organs such as the spleen, liver and bone marrow (BM) and can be lethal when left untreated. No effective vaccination is currently available for humans. The importance of B cells in infection and VL protective immunity has been controversial, with both detrimental and protective effects described. VL infection was found in this study to increase not only all analyzed B cell subsets in the spleen but also the B cell progenitors in the BM. The enhanced B lymphopoiesis aligns with the clinical manifestation of polyclonal hypergammaglobulinemia and the occurrence of autoantibodies. In line with earlier reports, flow cytometric and microscopic examination identified parasite attachment to B cells of the BM and spleen without internalization, and transformation of promastigotes into amastigote morphotypes. The interaction appears independent of IgM expression and is associated with an increased detection of activated lysosomes. Furthermore, the extracellularly attached amastigotes could be efficiently transferred to infect macrophages. The observed interaction underscores the potentially crucial role of B cells during VL infection. Additionally, using immunization against a fluorescent heterologous antigen, it was shown that the infection does not impair immune memory, which is reassuring for vaccination campaigns in VL endemic areas.

MR1 blockade drives differential impact on integrative signatures based on circuits of circulating immune cells and soluble mediators in visceral leishmaniasis.

Introduction: Visceral leishmaniasis (VL) is an important tropical and neglected disease and represents a serious global health problem. The initial interaction between the phagocytes and the parasite is crucial to determine the pathogen's capacity to initiate infection and it shapes the subsequent immune response that will develop. While type-1 T-cells induce IL-6, IL-1ß, TNF-α, and IL-12 production by monocytes/macrophages to fight the infection, type-2 T-cells are associated with a regulatory phenotype (IL-10 and TGF-ß) and successful infection establishment. Recently, our group demonstrated the role of an important Th1/Th17 T-cell population, the mucosal-associated invariant T (MAIT) cells, in VL. MAIT cells can respond to L. infantum by producing TNF-α and IFN-γ upon MR1-dependent activation. Objective and methods: Here, we describe the impact of the MR1-blockage on L. infantum internalization on the functional profile of circulating neutrophils and monocytes as well as the impact of the MR1-blockage on the soluble mediator signatures of in vitro whole blood cultures. Results: Overall, our data showed that VL patients presents higher percentage of activated neutrophils than asymptomatic and non-infected controls. In addition, MR1 blockade led to lower TNF-α and TGF-ß production by non-activated neutrophils from asymptomatic individuals. Moreover, TNF-α and IL-10 production by monocytes was higher in VL patients. In the analysis of soluble mediators produced in vitro, MR1-blockade induced a decrease of IFN-γ and an increase of IL-10, IL-27 and IL-33 in the cell cultures of AS group, a cytokine pattern associated with type 2 deleterious response. Discussion and conclusion: These data corroborate the hypothesis that MR1-restricted responses are associated to a protective role during Leishmania infection.

Unraveling molecular mechanistic disparities in pathogenic visceral Leishmania resistance between reptiles and mammals through comparative transcriptomic analyses.

Leishmaniasis is one of the most important neglected tropical parasitic diseases, manifesting various clinical forms depending on the parasite species and the genetic background of the host. In order to elucidate the underlying mechanisms of reptilian defense against pathogenic Leishmania species and to delineate the global gene expression profile alterations during host-pathogen interaction, we established experimental animal and cell models using both heterothermic lizards (Phrynocephalus przewalskii) and homothermic mammals (BALB/c mice) infected with pathogenic Leishmania infantum (high virulence HCZ strain) and Leishmania donovani (low virulence 801 strain). Overall, the lizards didn't show any obvious clinical symptoms or immune responses in vivo. Using RNA-seq methodology, differentially expressed genes identified in the HCZ and 801-comparison groups of P. przewalskii were primarily associated with arginine biosynthesis, the MAPK signaling pathway and the PI3K-Akt signaling pathway. In contrast, higher parasite loads, exacerbated hepatic inflammatory lesions and enhanced immune responses were observed in BALB/c mice, with DEGs predominantly associated with immunological diseases, innate and adaptive immune responses. By integrating transcriptional data from reptile and mammalian hosts, we elucidated the pivotal role of amino acid metabolism and lipid metabolism in parasite control. In contrast to findings from animal experiments, Leishmania parasites effectively infected peritoneal macrophages of lizards in vitro, demonstrating a high infection rate. Furthermore, we used RT-qPCR to detect changes in cytokine expression in macrophages and found that Th1-type cytokines were significantly upregulated in lizards, facilitating the clearance of the HCZ strain 24 hours post-infection. Conversely, cytokine expression was generally suppressed in BALB/c mice, allowing immune evasion by the parasites.

Analysis of HLA Alleles in Different Cohorts of Patients Infected by L. infantum from Southern Spain.

Leishmaniasis is an infectious disease caused by protozoa of the genus Leishmania, which is endemic in certain areas of Europe, such as southern Spain. The disease manifests in various clinical phenotypes, including visceral, cutaneous, mucosal, or asymptomatic leishmaniasis. This diversity in clinical outcomes may be influenced by the host immune response, with human leukocyte antigen (HLA) molecules playing a crucial role in determining susceptibility and progression of the infection. This study explores the association between specific HLA variants and Leishmania infantum infection. We recruited four cohorts: a control group, asymptomatic individuals, patients with symptomatic disease, and cohabitants of infected individuals. HLA typing was performed for all participants, followed by an association analysis with infection status and disease progression. Our findings indicate that the HLA-B*38 and HLA-C*03 alleles are associated with protection against L. infantum infection. These results contribute to a better understanding of the disease's progression, offer potential for new therapeutic approaches such as vaccines, and expand the existing knowledge in the literature.

Combining Killed Vaccine Candidate with Different Adjuvants to Determine Prophylactic Potential against Leishmaniasis.

Visceral Leishmaniasis is a serious public health problem caused by Leishmania species parasites. Approximately 500 thousand people get Visceral Leishmaniasis (VL) every year. An effective and reliable vaccine against the disease has still not been formulated. Choosing the right adjuvant is important to increase immunogenicity in vaccines prepared with total antigens. In this study, we investigate the ideal adjuvant for use in vaccine formulations against VL. For this purpose, Leishmania antigens (FTLA) obtained from L. infantum parasites by the freeze-thaw method and three different adjuvants (alum-saponin and calcium phosphate) were used. The effectiveness of the formulations was investigated in vitro by cell viability analysis and determination of nitric oxide and cytokine production abilities in J774 macrophage cells. According to the study results, it was determined that formulations prepared with calcium phosphate produced 72% more NO and approximately 7.2 times more IL-12 cytokine. The results obtained showed that calcium phosphate salts can be used as ideal adjuvants in vaccine research against leishmaniasis.

Differences in the Cellular Immune Response during and after Treatment of Sudanese Patients with Post-kala-azar Dermal Leishmaniasis, and Possible Implications for Outcome.

BACKGROUND: The host cellular immune response associated with two treatments for post-kala-azar dermal leishmaniasis (PKDL) - paromomycin plus miltefosine (Arm 1), and liposomal amphotericin B plus miltefosine (Arm 2) - was examined in Sudanese patients before treatment (D0), at the end of treatment (D42), and during the post-treatment period (D180). METHODS: Whole blood samples were stimulated with soluble Leishmania antigen for 24 h (whole blood assay [WBA]) and the concentrations of Th1/Th2/Th17-associated cytokines, IP-10, PDL-1 and granzyme B were determined. RESULTS: The Arm 1 treatment (98.2% cure rate) induced a Th1/Th2/Th17 response, while the Arm 2 treatment (80% cure rate) induced a Th1/Th2 response. Five Arm 2 patients relapsed and showed lower IFN-γ, TNF and IL-1ß concentrations at D0 than non-relapsers in this Arm. In patients with low-IFN-γ-production at D0, Arm 1 treatment led to a better host immune response and clinical outcome than Arm 2 treatment. CONCLUSIONS: A Th1/Th2/Th17 response was associated with a higher cure rate. Patients with low IFN-γ, TNF and IL-1ß before treatment are more likely to relapse if they undergo Arm 2-type treatment. Determining IFN-γ, TNF and IL-10 levels prior to treatment could help predict patients at higher risk of relapse/recovery from PKDL. TRIAL REGISTRATION: ClinicalTrials.gov NCT03399955, Registered 17 January 2018, https://clinicaltrials.gov/study/ NCT03399955.

Nicotinamide mitigates visceral leishmaniasis by regulating inflammatory response and enhancing lipid metabolism.

BACKGROUND: Currently, treatment regimens for visceral leishmaniasis (VL) are limited because of the presence of numerous adverse effects. Nicotinamide, a readily available and cost-effective vitamin, has been widely acknowledged for its safety profile. Several studies have demonstrated the anti-leishmanial effects of nicotinamide in vitro. However, the potential role of nicotinamide in Leishmania infection in vivo remains elusive. METHODS: In this study, we assessed the efficacy of nicotinamide as a therapeutic intervention for VL caused by Leishmania infantum in an experimental mouse model and investigated its underlying molecular mechanisms. The potential molecular mechanism was explored through cytokine analysis, examination of spleen lymphocyte subsets, liver RNA-seq analysis, and pathway validation. RESULTS: Compared to the infection group, the group treated with nicotinamide demonstrated significant amelioration of hepatosplenomegaly and recovery from liver pathological damage. The NAM group exhibited parasite reduction rates of 79.7% in the liver and 86.7% in the spleen, respectively. Nicotinamide treatment significantly reduced the activation of excessive immune response in infected mice, thereby mitigating hepatosplenomegaly and injury. Furthermore, nicotinamide treatment enhanced fatty acid ß-oxidation by upregulating key enzymes to maintain lipid homeostasis. CONCLUSIONS: Our findings provide initial evidence supporting the safety and therapeutic efficacy of nicotinamide in the treatment of Leishmania infection in BALB/c mice, suggesting its potential as a viable drug for VL.

Activation of neutrophils excels the therapeutic potential of Mycobacterium indicus pranii and heat-induced promastigotes against antimony-resistant Leishmania donovani infection.

Repurposing drugs and adjuvants is an attractive choice of present therapy that reduces the substantial costs, chances of failure, and systemic toxicity. Mycobacterium indicus pranii was originally developed as a leprosy vaccine but later has been found effective against Leishmania donovani infection. To extend our earlier study, here we reported the immunotherapeutic modulation of the splenic and circulatory neutrophils in favour of hosts as neutrophils actually serve as the pro-parasitic portable shelter to extend the Leishmania infection specifically during the early entry into the hosts' circulation. We targeted to disrupt this early pro-parasitic incidence by the therapeutic combination of M. indicus pranii and heat-induced promastigotes against antimony-resistant L. donovani infection. The combination therapy induced the functional expansion of CD11b+Ly6CintLy6Ghi neutrophils both in the post-infected spleen, and also in the circulation of post-treated animals followed by the immediate Leishmania infection. More importantly, the enhanced expression of MHC-II, phagocytic uptake of the parasites by the circulatory neutrophils as well as the oxidative burst were induced that limited the chances of the very early establishment of the infection. The enhanced expression of pro-inflammatory cytokines, like IL-1α and TNF-α indicated resistance to the parasite-mediated takeover of the neutrophils, as these cytokines are critical for the activation of T cell-mediated immunity and host-protective responses. Additionally, the induction of essential transcription factors and cytokines for early granulocytic lineage commitment suggests that the strategy not only contributed to the peripheral activation of the neutrophils but also promoted granulopoiesis in the bone marrow.

Leishmania infantum infection modulates messenger RNA, microRNA and long non-coding RNA expression in human neutrophils in vitro.

In the Americas, L. infantum (syn. chagasi) is the main cause of human visceral leishmaniasis. The role of neutrophils as part of the innate response to Leishmania spp. infection is dubious and varies according to the species causing the infection. Global expression of coding RNAs, microRNAs and long non-coding RNAs changes as part of the immune response against pathogens. Changes in mRNA and non-coding RNA expression resulting from infection by Leishmania spp. are widely studied in macrophages, but scarce in neutrophils, the first cell to encounter the trypanosomatid, especially following infection by L. infantum. Herein, we aimed to understand the expression patterns of coding and non-coding transcripts during acute in vitro infection of human neutrophils by L. infantum. We isolated neutrophils from whole blood of healthy male donors (n = 5) and split into groups: 1) infected with L. infantum (MOI = 5:1), and 2) uninfected controls. After 3 hours of exposure of infected group to promastigotes of L. infantum, followed by 17 hours of incubation, total RNA was extracted and total RNA-Seq and miRNA microarray were performed. A total of 212 genes were differentially expressed in neutrophils following RNA-Seq analysis (log2(FC)±0.58, FDR≤0.05). In vitro infection with L. infantum upregulated the expression of 197 and reduced the expression of 92 miRNAs in human neutrophils (FC±2, FDR≤0.01). Lastly, 5 downregulated genes were classified as lncRNA, and of the 10 upregulated genes, there was only 1 lncRNA. Further bioinformatic analysis indicated that changes in the transcriptome and microtranscriptome of neutrophils, following in vitro infection with L. infantum, may impair phagocytosis, apoptosis and decrease nitric oxide production. Our work sheds light on several mechanisms used by L. infantum to control neutrophil-mediated immune response and identifies several targets for future functional studies, aiming at the development of preventive or curative treatments for this prevalent zoonosis.

Deciphering the intricate dynamics of inflammasome regulation in visceral and post-kala-azar dermal leishmaniasis: A meta-analysis of consistencies.

Post Kala-azar dermal leishmaniasis (PKDL) arises as a significant dermal sequel following Visceral leishmaniasis (VL) caused by protozoan parasite Leishmania donovani (LD). PKDL acts as a significant constrain for VL elimination serving as a crucial reservoir for LD. PKDL patients exhibit depigmented macular and papular lesions on their skin, which results in social discrimination due to loss of natural skin color. Inflammatory reactions, prevalent in both VL and PKDL, potentially lead to tissue damage in areas harboring the parasite. Disruption of the immune-inflammasomal network not only facilitates LD persistence but also leads to the skin hypopigmentation seen in PKDL, impacting social well-being. Activation of inflammasomal markers like STAT1, NLRP1, NLRP3, AIM2, CASP11, and NLRP12 have been identified as a common host-defense mechanism across various Leishmania infections. Conversely, Leishmania modulates inflammasome activation to sustain its presence within the host. Nevertheless, in specific instances of Leishmania infection, inflammasome activation can worsen disease pathology by promoting parasite proliferation and persistence. This study encompasses recent transcriptomic analyses conducted between 2016 and 2023 on human and murine subjects afflicted with VL/PKDL, elucidating significant alterations in inflammasomal markers in both conditions. It offers a comprehensive understanding how these markers contribute in disease progression, drawing upon available literature for logical analysis. Furthermore, our analysis identifies validated miRNA network that could potentially disrupt this crucial immune-inflammasomal network, thereby offering a plausible explanation on how secreted LD-factors could enable membrane-bound LD, isolated from the host cytoplasm, to modulate cytoplasmic inflammasomal markers. Insights from this study could guide the development of host-directed therapeutics to impede transmission and address hypopigmentation, thereby mitigating the social stigma associated with PKDL.