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Background: In Ethiopia, cutaneous leishmaniasis is mainly caused by Leishmania (L.) aethiopica parasites and presents in three main clinical forms. It is still not clear if the host immune response plays a role in the development of these different presentations. Since neutrophils are likely to be one of the first immune cells present at the site of the sand fly bite, we set up an in vitro model of infection of neutrophils with L. aethiopica and assessed some of the main neutrophil effector functions: association with and internalisation of parasites, apoptosis and ROS production. We used three freshly isolated clinical isolates and one isolate that has been kept in culture for decades., Results: Our results showed by flow cytometry that all four L. aethiopica isolates had the ability to associate with neutrophils. The three clinical isolates of L. aethiopica associated more efficiently with neutrophils than the long-term cultured L. aethiopica. At 18 h, two distinct populations of neutrophils were identified that associated with L. aethiopica, CD15 high and CD15 low neutrophils. Confocal microscopy demonstrated that all isolates can be internalised. Our results also showed that all parasites induced apoptosis in L. aethiopica-associated neutrophils. Moreover, our results showed that after 2 h, L. aethiopica-associated neutrophils upregulated their production of ROS, but to a greater extent with the long-term cultured L. aethiopica. After 18 h of incubation, CD15 low parasite + showed an impaired ability to produce ROS compared to CD15 high parasite + ., Conclusions: Using this in vitro model, our results show that different L. aethiopica parasite isolates, most notably long-term cultured parasites, had differential effects on neutrophil effector functions., Competing Interests: Declarations Ethics approval and consent to participate Ethical approval was obtained from the Faculty Ethic Committee University of Greenwich (FES-FREC-20-01.04.08.CA). Informed written consent was obtained from each participant. Consent for publication Not applicable. Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

The leishmaniases are globally important parasitic diseases for which no human vaccines are currently available. To facilitate vaccine development, we conducted an open-label observational study to establish a controlled human infection model (CHIM) of sand fly-transmitted cutaneous leishmaniasis (CL) caused by Leishmania major. Between 24 January and 12 August 2022, we exposed 14 participants to L. major-infected Phlebotomus duboscqi. The primary objective was to demonstrate effectiveness of lesion development (take rate) and safety (absence of CL lesion at 12 months). Secondary and exploratory objectives included rate of lesion development, parasite load and analysis of local immune responses by immunohistology and spatial transcriptomics. Lesion development was terminated by therapeutic biopsy (between days 14 and 42 after bite) in ten participants with clinically compatible lesions, one of which was not confirmed by parasite detection. We estimated an overall take rate for CL development of 64% (9/14). Two of ten participants had one and one of ten participants had two lesion recurrences 4-8 months after biopsy that were treated successfully with cryotherapy. No severe or serious adverse events were recorded, but as expected, scarring due to a combination of CL and the biopsy procedure was evident. All participants were lesion free at >12-month follow-up. We provide the first comprehensive map of immune cell distribution and cytokine/chemokine expression in human CL lesions, revealing discrete immune niches. This CHIM offers opportunities for vaccine candidate selection based on human efficacy data and for a greater understanding of immune-mediated pathology. ClinicalTrials.gov identifier: NCT04512742 ., Competing Interests: Competing interests P.M.K. and C.J.N.L. are co-authors of a patent protecting the gene insert used in Leishmania candidate vaccine ChAd63-KH (Europe 10719953.1; India 315101). The other authors declare no competing interests., (© 2024. The Author(s).)

Phlebotomus argentipes is the established vector of leishmaniasis in the Indian sub-continent. Antibodies to sand fly salivary antigens are biomarkers for vector-host exposure in leishmaniasis-endemic regions. Ph. argentipes transmits Leishmania donovani in Sri Lanka, primarily causing cutaneous leishmaniasis (CL). Our study compared the performance of salivary gland homogenate (SGH) from a lab-reared local strain of Ph. argentipes females to a composite recombinant salivary biomarker (rPagSP02 + rPagSP06) in a CL-endemic population. Sera from 546 healthy individuals, 30 CL patients, and 15 non-endemic individuals were collected. Western blot analysis of Ph. argentipes SGH identified immunogenic bands between 15 kDa and 67 kDa, with bands of predicted molecular weight ∼of 15 kDa (SP02) and ∼28-30 kDa (SP06) as the major antibody targets. Indirect ELISAs using SGH or rPagSP02 + rPagSP06 antigens showed high sensitivity (96.7%) and specificity (100%), detecting comparable seropositivity in endemic populations. rPagSP02 + rPagSP06 exhibited enhanced discriminatory ability, supported by a strong positive correlation (r = 0.869) with SGH. Our findings indicate that the composite rPagSP02 + rPagSP06 salivary biomarker effectively identifies Ph. argentipes exposure in individuals living in Sri Lanka, showing promising potential for use in surveillance. These findings should be further validated to confirm the epidemiological applications in leishmaniasis-endemic regions., (© 2024. The Author(s).)

Background: Host-related factors highly regulate the increased circulation of neutrophils during Leishmania infection. Platelet-derived Dickkopf-1 (DKK1) is established as a high-affinity ligand to LRP6. Recently, we demonstrated that DKK1 upregulates leukocyte-platelet aggregation, infiltration of neutrophils to the draining lymph node and Th2 differentiation during Leishmania infection, suggesting the potential involvement of the DKK1-LRP6 signalling pathway in neutrophil migration in infectious diseases., Results: In this study, we further explored the potential role of DKK1-LRP6 signalling in the migration and longevity of activated neutrophils in the infection site using BALB/c mice with PMNs deficient in LRP6 (LRP6 NKO ) or BALB/c mice deficient in both PMN LRP6 and platelet DKK1 (LRP6 NKO DKK1 PKO ). Relative to the infected wild-type BALB/c mice, reduced neutrophil activation at the infection site of LRP6 NKO or LRP6 NKO DKK1 PKO mice was noted. The neutrophils obtained from either infected LRP6 NKO or LRP6 NKO DKK1 PKO mice additionally showed a high level of apoptosis. Notably, the level of LRP6 expressing neutrophils was elevated in infected BALB/c mice. Relative to infected BALB/c mice, a significant reduction in parasite load was observed in both LRP6 NKO and LRP6 NKO DKK1 PKO infected mice. Notably, DKK1 levels were comparable in the LRP6 NKO and BALB/c mice in response to infection, indicating that PMN activation is the major pathway for DKK1 in promoting parasitemia. Parasite-specific components also play a crucial role in modulating neutrophil circulation in Leishmania disease. Thus, we further determine the contribution of Leishmania membrane components in the migration of neutrophils to the infection site using null mutants deficient in LPG synthesis ( Δlpg1 - ) or lacking all ether phospholipids (plasmalogens, LPG, and GIPLs) synthesis ( Δads1 - ). Relative to the WT controls, Δads1 - parasite-infected mice showed a sustained decrease in neutrophils and neutrophil-platelet aggregates (for at least 14 days PI), while neutrophils returned to normal in Δlpg1 - parasite-infected mice after day 3 PI., Conclusion: Our results suggest that DKK1 signalling and Leishmania pathogen-associated molecular patterns appear to regulate the migration and sustenance of viable activated neutrophils in the infection site resulting in chronic type 2 cell-mediated inflammation., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Ihedioha, Marcarian, Sivakoses, Beverley, McMahon-Pratt and Bothwell.)

Microorganisms have long been suspected to influence the outcome of immune-related syndromes, particularly autoimmune diseases. Type 1 diabetes (T1D) results from the autoimmune destruction of the insulin-producing beta cells of pancreatic islets, causing high glycemia levels. Genetics is part of its aetiology, but environmental factors, particularly infectious microorganisms, also play a role. Bacteria, viruses, and parasites influence the outcome of T1D in mice and humans. We used nonobese diabetic (NOD) mice, which spontaneously develop T1D, to investigate the influence of a parasitic infection, leishmaniasis. Leishmania amazonensis is an intracellular eukaryotic parasite that replicates predominantly in macrophages and is responsible for cutaneous leishmaniasis. The implication of Th1 immune responses in T1D and leishmaniasis led us to study this parasite in the NOD mouse model. We previously constructed osteopontin knockout mice with a NOD genetic background and demonstrated that this protein plays a role in the T1D phenotype. In addition, osteopontin (OPN) has been found to play a role in the immune response to various infectious microorganisms and to be implicated in other autoimmune conditions, such as multiple sclerosis in humans and experimental autoimmune encephalomyelitis (EAE) in mice. We present herein data demonstrating the role of OPN in the response to Leishmania in NOD mice and the influence of this parasitic infection on T1D. This exploratory study aimed to investigate the environmental infectious component of the autoimmune response, including Th1 immunity, which is common to both T1D and leishmaniasis., Competing Interests: The authors declare that they have no competing interests., (Copyright: © 2024 Giraud et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Cytotoxic activity is a hallmark of the immunopathogenesis in human cutaneous leishmaniasis (CL). In this study, we identified accumulation of CD4 + granzyme B producing T cells with increased cytotoxic capacity in CL lesions. These cells showed enhanced expression of activating NK receptors (NKG2D and NKG2C), diminished expression of inhibitory NKG2A, along with the upregulation of the senescence marker CD57. Notably, CD4 + T cells freshly isolated from CL lesions demonstrated remarkable capacity to mediate NL-like bystander cytolysis. Phenotypic analyses revealed that lesional CD4 + T cells are mainly composed of late-differentiated effector (CD27-CD45RA-) and terminally differentiated (senescent) TEMRA (CD27-CD45RA+) subsets. Interestingly, the TEMRA CD4 + T cells exhibited higher expression of granzyme B and CD107a. Collectively, our results provide the first evidence that senescent cytotoxic CD4 + T cells may support the skin pathology of human cutaneous leishmaniasis and, together with our previous findings, support the notion that multiple subsets of cytotoxic senescent cells may be involved in inducing the skin lesions in these patients., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Covre, Fantecelle, Garcia de Moura, Oliveira Lopes, Sarmento, Freire-de-Lima, Decote-Ricardo, de Matos Guedes, da Fonsceca-Martins, de Carvalho, de Carvalho, Mosser, Falqueto, Akbar and Gomes.)

Background: Cutaneous leishmaniasis (CL) is a neglected tropical skin disease, caused by the protozoan parasite Leishmania. In Ethiopia, CL is mainly caused by Leishmania aethiopica and can present in different clinical forms. The aim of this study was to assess whether these different forms are associated with differences in parasite genetic and host systemic immune signatures., Methods: Here we analysed the whole genome sequence data for 48 clinical parasite isolates and the systemic immune signature from a cohort of CL patients, who were recruited in Nefas Mewcha, Northern Ethiopia, from January 2019 to January 2022., Results: Our results show that parasites from CL cases with different presentations in a single Ethiopian setting are from the same genetic population based on a permutation test of genome-wide similarity. Furthermore, a logistic regression test for genome wide association did not identify any individual genetic variants significantly associated with disease presentation. We also measured plasma chemokine and cytokine levels of 129 CL patients presenting with different forms of CL. None of the chemokine [eotaxin, eotaxin-3, interleukin (IL)-8, interferon (IFN)-γ-induced protein-10 (IP-10), monocyte chemoattractant protein (MCP)-1, MCP-4, macrophage-derived chemokines (MDC), macrophage inflammatory protein (MIP)-1α, MIP-1β and thymus- and activation-regulated chemokine (TARC)] or cytokine (IFN-γ, IL-1β, interleukin-2, IL-4, IL-6, IL-10, IL-12p70, IL-13, tumor necrosis factor-α) levels measured were significantly different between the different clinical presentations of CL, as measured by Kruskal-Wallis test. We also compared those with healthy nonendemic controls: our results show a chemokine (IP-10, MCP-1, MCP-4, MDC, MIP-1α, MIP-1β and TARC) but not a cytokine immune signature in patients with CL as compared to healthy nonendemic controls, as measured by Mann-Whitney test., Conclusions: The results of our study did not identify a systemic immune signature or parasite genetic factors associated with different clinical presentation of CL., (© 2024. The Author(s).)

Neutrophils rapidly infiltrate sites of infection and possess several microbicidal strategies, such as neutrophil extracellular traps release and phagocytosis. Enhanced neutrophil infiltration is associated with higher susceptibility to Leishmania infection, but neutrophil effector response contribution to this phenotype is uncertain. Here, we show that neutrophils from susceptible BALB/c mice (B/c) produce more NETs in response to Leishmania major than those from resistant C57BL/6 mice (B6), which are more phagocytic. The absence of neutrophil elastase contributes to phagocytosis regulation. Microarray analysis shows enrichment of genes involved in NET formation (mpo, pi3kcg, il1b) in B/c, while B6 shows upregulation of genes involved in phagocytosis and cell death (Arhgap12, casp9, mlkl, FasL). scRNA-seq in L. major-infected B6 showed heterogeneity in the pool of intralesional neutrophils, and we identified the N1 subset as the putative subpopulation involved with phagocytosis. In vivo, imaging validates NET formation in infected B/c ears where NETing neutrophils were mainly uninfected cells. NET digestion in vivo augmented parasite lymphatic drainage. Hence, a balance between NET formation and phagocytosis in neutrophils may contribute to the divergent phenotype observed in these mice., Competing Interests: The authors have declared that no competing interests exist., (Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.)

Background: Laboratory diagnosis of American cutaneous leishmaniasis (ACL) requires a tool amenable to the epidemiological status of ACL in Brazil. Montenegro skin test (MST), an efficient immunological tool used for laboratory diagnosis of ACL, induces delayed-type hypersensitivity (DTH) response to the promastigote antigens of Leishmania; however, human immune responses against infection are modulated by the amastigote of the parasite. Leishmania (V.) lainsoni induces strong cellular immunity in humans; therefore, the antigenic reactivity of its axenic amastigote (AMA antigen) to MST was evaluated for the laboratory diagnosis of ACL., Methods: Among 70 individuals examined, 60 had a laboratory-confirmed diagnosis of ACL; 53 had localized cutaneous leishmaniasis (LCL), and 7 had mucosal leishmaniasis (ML). Patients were treated at the Evandro Chagas Institute's leishmaniasis clinic, Pará State, Brazil. Ten healthy individuals with no history of ACL (control group) were also examined. Leishmania (V.) braziliensis promastigote antigen (PRO) was used to compare the reactivity with that of AMA antigen. Paired Student's t-test, kappa agreement, and Spearman test were used to evaluate the reactivity of AMA and PRO., Results: The mean reactivity of AMA in ACL patients was 19.4 mm ± 13.3, which was higher (P < 0.001) than that of PRO: 12.1 mm ± 8.1. MST reactivity according to the clinical forms revealed that AMA reactivity in LCL and ML, 18.8 mm ± 13.3 and 24.3 mm ± 13.7, was higher (P < 0.001) than that of PRO, 11.8 mm ± 8.2 and 14.6 mm ± 8.4, respectively., Conclusion: AMA reactivity was higher than that of PRO, indicating that AMA is a promising alternative for optimizing MST in the laboratory diagnosis of ACL., (© 2024. The Author(s).)

The clinical manifestations of cutaneous leishmaniasis (CL) depend not only on the infecting species involved, but also on the immune response of the individual. Although not yet well understood in humans, parasite survival and persistence are related to the cytokine profile and T cell proliferation, with the Th1 profile being related to cure, and the Th2 profile to disease progression. Considering the need for studies focused on the species with the highest circulation in the state of Amazonas, this study aimed to analyze the immunoregulation stimulated by soluble antigens (SLAs) of Leishmania (L.) amazonensis and Leishmania (V.) guyanensis in human lymphocytes in vitro, in order to understand the immune response of patients with CL. Lymphoproliferation was evaluated against stimuli of SLAs from L. amazonensis (100 µg/mL), SLAs from L. guyanensis (100 µg/mL) and phytohemagglutinin (10 µg/mL) using a BrdU Cell Proliferation ELISA kit after 72 h of incubation. Quantification of the cytokines IL-1b, IL-6, IL-8, IL-10, IL-12 and TNF was performed using the BD™ cytometric bead array human Th1/Th2/Th17 cytokine kit. Our results demonstrated that soluble antigens from L. amazonensis and L. guyanensis stimulated the lymphoproliferation of PBMCs from patients primo-infected with CL. Among the cytokines dosed, the highest concentrations were of IL-6 and IL-8, thus demonstrating that the soluble antigens evaluated are capable of inducing regulatory mechanisms.

Background: The JAK-STAT signaling pathway is a central cascade of signal transduction for the myriad of cytokines in which dysregulation has been implicated in progression of inflammatory and infectious diseases. However, the involvement of this pathway in human cutaneous leishmaniasis (CL) due to Leishmania ( L. ) tropica warrants further investigation., Methods: This study sought to investigate differential gene expression of several cytokines and their associated jak-stat genes in the lesions of L. tropica -infected patients byquantitative Real-Time PCR. Further, the expression of five inhibitory immune checkpoint genes was evaluated., Results: Results showed that the gene expression levelsof both Th1 ( ifng , il12 , il23 ) and Th2 ( il4 , il10 ) types cytokines were increased in the lesion of studied patients. Further, elevated expression levels of il35 , il21 , il27 and il24 genes were detected in the lesions of CL patients. Notably, the expression of the majority of genes involved in JAK/STAT signaling pathway as well as checkpoint genes including pdl1 , ctla4 and their corresponding receptors was increased., Conclusion: Our finding revealed dysregulation of cytokines and related jak-stat genes in the lesion of CL patients. These results highlight the need for further exploration of the functional importance of these genes in the pathogenesis of, and immunity to, CL., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Hadifar, Masoudzadeh, Heydari, Mashayekhi Goyonlo, Kerachian, Daneshpazhooh, Sadeghnia, Tootoonchi, Erfanian Salim, Rafati and Harandi.)

Leishmaniasis, caused by Leishmania (L.) species, remains a neglected infection. Therapeutic vaccination presents a promising strategy for its treatment. In this study, we aimed to develop a therapeutic vaccine candidate using Leishmaniaantigens (SLA) and Toll-like receptor (TLR) 7/8 agonist (R848) encapsulated into the poly (lactic-co-glycolic acid) (PLGA) nanoparticles (NPs). Moreover, TLR1/2 agonist (Pam3CSK4) was loaded onto the NPs. The therapeutic effects of these NPs were evaluated in L. major-infected BALB/c mice. Footpad swelling, parasite load, cellular and humoral immune responses, and nitric oxide (NO) production were analyzed. The results demonstrated that the PLGA NPs (SLA-R848-Pam3CSK4) therapeutic vaccine effectively stimulated Th1 cell responses, induced humoral responses, promoted NO production, and restricted parasite burden and lesion size.Our findings suggest that vaccination with SLA combined with TLR1/2 and TLR7/8 agonists in PLGA NPs as a therapeutic vaccine confers strong protection againstL. majorinfection. These results represent a novel particulate therapeutic vaccine against Old World cutaneous leishmaniasis., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Intracellular pathogens that replicate in host myeloid cells have devised ways to inhibit the cell's killing machinery. Pyroptosis is one of the host strategies used to reduce the pathogen replicating niche and thereby control its expansion. The intracellular Leishmania parasites can survive and use neutrophils as a silent entry niche, favoring subsequent parasite dissemination into the host. Here, we show that Leishmania mexicana induces NLRP1- and caspase-1-dependent Gasdermin D (GSDMD)-mediated pyroptosis in neutrophils, a process critical to control the parasite-induced pathology. In the absence of GSDMD, we observe an increased number of infected dermal neutrophils two days post-infection. Using adoptive neutrophil transfer in neutropenic mice, we show that pyroptosis contributes to the regulation of the neutrophil niche early after infection. The critical role of neutrophil pyroptosis and its positive influence on the regulation of the disease outcome was further demonstrated following infection of mice with neutrophil-specific deletion of GSDMD. Thus, our study establishes neutrophil pyroptosis as a critical regulator of leishmaniasis pathology., Competing Interests: The authors have declared that no competing interests exist., (Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.)

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., Competing Interests: Declaration of competing interest The authors declare that the systematic review was conducted without any conflict of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

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-1β 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-1β 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., (© 2024. The Author(s).)

Currently, no safe vaccine against leishmaniasis is available. So far, different control strategies against numerous reservoir hosts and biological vectors have not been environment-friendly and feasible. Hence, employing medicinal components and conventional drugs could be a promising approach to developing novel therapeutic alternatives. This study aimed to explore diallyl sulfide (DAS), a dynamic constituent of garlic, alone and in a mixture with meglumine antimoniate (MAT as standard drug) using in vitro and animal model experiments against Leishmania major stages. The binding affinity of DAS and four major defense elements of the immune system (iNOS, IFN-ɣ, IL-12, and TNF-α) was used to predict the predominant binding mode for molecular docking configurations. Herein, we conducted a broad range of experiments to monitor and assess DAS and MAT potential treatment outcomes. DAS, combined with MAT, displayed no cytotoxicity and employed a powerful anti-leishmanial activity, notably against the clinical stage. The function mechanism involved immunomodulation through the induction of Th1 cytokine phenotypes, triggering a high apoptotic profile, reactive oxygen species (ROS) production, and antioxidant enzymes. This combination significantly decreased cutaneous lesion diameter and parasite load in BALB/c mice. The histopathological findings performed the infiltration of inflammatory cells associated with T-lymphocytes, particularly CD4+ phenotypes, as determined by biochemical markers in alleviating the amastigote stage and improving the pathological changes in L. major infected BALB/c mice. Therefore, DAS and MAT deserve further advanced therapeutic development and should be considered as possible candidates for treating volunteer cases with cutaneous leishmaniasis in designing an upcoming clinical trial., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Zarrinkar et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

American Tegumentary Leishmaniasis (ATL) is a disease of high severity and incidence in Brazil, in addition to being a worldwide concern in public health. Leishmania amazonensis is one of the etiological agents of ATL, and the inefficiency of control measures, associated with the high toxicity of the treatment and the lack of effective immunoprophylactic strategies, makes the development of vaccines indispensable and imminent. In this light, the present study proposes to elaborate a chimeric protein (rChiP), based on the fusion of multiple epitopes of CD4 + /CD8 + T cells, identified in the immunoproteome of the parasites L. amazonensis and L. braziliensis. The designed chimeric protein was tested in the L. amazonensis murine model of infection using the following formulations: 25 μg of the rChiP in saline (rChiP group) and 25 μg of the rChiP plus 25 μg of MPLA-PHAD® (rChiP+MPLA group). After completing immunization, CD4 + and CD8 + T cells, stimulated with SLa-Antigen or rChiP, showed an increased production of nitric oxide and intracytoplasmic pro-inflammatory cytokines, in addition to the generation of central and effector memory T cells. rChiP and rChiP+MPLA formulations were able to promote an effective protection against L. amazonensis infection determined by a reduction in the development of skin lesions and lower parasitic burden. Reduction in the development of skin lesions and lower parasitic burden in the vaccinated groups were associated with an increase of nitrite, CD4 + /CD8 + IFN-γ + TNF-α + and CD4 + /CD8 + CD44 high CD62L high/low T cells, IgG Total , IgG 2a , and lower rates of IgG 1 and CD4 + /CD8 + IL-10 + . This data suggests that proposed formulations could be considered potential tools to prevent ATL., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Th1 and Th2 cytokines determine the outcome of Leishmania major infection and immune protection depends mainly on memory T cells induced during vaccination. This largely hinges on the nature and type of memory T cells produced. In this study, transgenic Leishmania major strains expressing membrane-associated ovalbumin (mOVA) and soluble ovalbumin (sOVA) were used as a model to study whether fully differentiated Th1/Th2 and Th17 cells can recall immune memory and tolerate pathogen manipulation. Naïve OT-II T cells were polarised in vitro into Th1/Th2 cells, and these cells were transferred adoptively into recipient mice. Following the transferral of the memory cells, the recipient mice were challenged with OVA transgenic Leishmania major and a wild-type parasite was used a control. The in vitro-polarised T helper cells continued to produce the same cytokine signatures after being challenged by both forms of OVA-expressing Leishmania major parasites in vivo. This suggests that antigen-experienced cells remain the same or unaltered in the face of OVA-transgenic Leishmania major. Such ability of these antigen-experienced cells to remain resilient to manipulation by the parasite signifies that vaccines might be able to produce immune memory responses and defend against parasitic immune manipulation in order to protect the host from infection.

Natural killer (NK) cells include different subsets with diverse effector capacities that are poorly understood in the context of parasitic diseases. Here, we investigated inhibitory and activating receptor expression on NK cells in patients with cutaneous leishmaniasis (CL) and explored their phenotypic and functional heterogeneity based on CD57 and NKG2C expression. The expression of CD57 identified NK cells that accumulated in CL patients and exhibited features of senescence. The CD57+ cells exhibited heightened levels of the activating receptor NKG2C and diminished expression of the inhibitory receptor NKG2A. RNA sequencing analyses based on NKG2C transcriptome have revealed two distinct profiles among CL patients associated with cytotoxic and functional genes. The CD57+NKG2C+ subset accumulated in the blood of patients and presented conspicuous features of senescence, including the expression of markers such as p16, yH2ax, and p38, as well as reduced proliferative capacity. In addition, they positively correlated with the number of days until lesion resolution. This study provides a broad understanding of the NK cell biology during Leishmania infection and reinforces the role of senescent cells in the adverse clinical outcomes of CL., (© The Author(s) 2024. Published by Oxford University Press on behalf of the British Society for Immunology. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

American tegumentary leishmaniasis (ATL) is highly endemic in the Amazon basin and occurs in all South American countries, except Chile and Uruguay. Most Brazilian ATL cases are due to Leishmania (Viannia) braziliensis, however other neglected Amazonian species are being increasingly reported. They belong to the subgenus L. (Viannia) and information on suitable models to understand immunopathology are scarce. Here, we explored the use of the golden hamster Mesocricetus auratus and its macrophages as a model for L. (Viannia) species. We also studied the interaction of parasite glycoconjugates (LPGs and GIPLs) in murine macrophages. The following strains were used: L. (V.) braziliensis (MHOM/BR/2001/BA788), L. (V.) guyanensis (MHOM/BR/85/M9945), L. (V.) shawi (MHOM/BR/96/M15789), L. (V.) lindenbergi (MHOM/BR/98/M15733) and L. (V.) naiffi (MDAS/BR/79/M5533). In vivo infections were initiated by injecting parasites into the footpad and were followed up at 20- and 40-days PI. Parasites were mixed with salivary gland extract (SGE) from wild-captured Nyssomyia neivai prior to in vivo infections. Animals were euthanized for histopathological evaluation of the footpads, spleen, and liver. The parasite burden was evaluated in the skin and draining lymph nodes. In vitro infections used resident peritoneal macrophages and THP-1 monocytes infected with all species using a MOI (1:10). For biochemical studies, glycoconjugates (LPGs and GIPLs) were extracted, purified, and biochemically characterized using fluorophore-assisted carbohydrate electrophoresis (FACE). They were functionally evaluated after incubation with macrophages from C57BL/6 mice and knockouts (TLR2-/- and TLR4-/-) for nitric oxide (NO) and cytokine/chemokine production. All species, except L. (V.) guyanensis, failed to generate evident macroscopic lesions 40 days PI. The L. (V.) guyanensis lesions were swollen but did not ulcerate and microscopically were characterized by an intense inflammatory exudate. Despite the fact the other species did not produce visible skin lesions there was no or mild pro-inflammatory infiltration at the inoculation site and parasites survived in the hamster skin/lymph nodes and even visceralized. Although none of the species caused severe disease in the hamster, they differentially infected peritoneal macrophages in vitro. LPGs and GIPLs were able to differentially trigger NO and cytokine production via TLR2/TLR4 and TLR4, respectively. The presence of a sidechain in L. (V.) lainsoni LPG (type II) may be responsible for its higher proinflammatory activity. After Principal Component analyses using all phenotypic features, the clustering of L. (V.) lainsoni was separated from all the other L. (Viannia) species. We conclude that M. auratus was a suitable in vivo model for at least four dermotropic L. (Viannia) species. However, in vitro studies using peritoneal cells are a suitable alternative for understanding interactions of the six L. (Viannia) species used here. LRV1 presence was found in L. (V.) guyanensis and L. (V.) shawi with no apparent correlation with virulence in vitro and in vivo. Finally, parasite glycoconjugates were able to functionally trigger various innate immune responses in murine macrophages via TLRs consistent with their inflammatory profile in vivo., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Soares et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Type 2 diabetes mellitus is a metabolic disorder that causes chronic high blood sugar levels, and diabetic patients are more susceptible to infections. American cutaneous leishmaniasis is an infectious disease caused by a parasite that affects the skin and mucous membranes, leading to one or multiple ulcerative lesions. Chronic inflammation and functional changes in various organs and systems, including the immune system, are the primary causes of both diseases. Melatonin, an essential immunomodulatory, antioxidant, and neuroprotective agent, can benefit many immunological processes and infectious diseases, including leishmaniasis. Although, limited reports are available on diabetic patients with leishmaniasis. The literature suggests that melatonin may play a promising role in inflammatory disorders. This study was designed to assess melatonin levels and inflammatory mediators in diabetic patients affected by leishmaniasis. Blood samples from 25 individuals were analyzed and divided into four groups: a control group (without any diseases), a Leishmania-positive group, patients with type 2 diabetes mellitus, and patients with a combination of both diseases. This study measured the serum levels of melatonin through ELISA, while IL-4 and TNF-α were measured using flow cytometry, and C-reactive protein was measured through turbidimetry. This study found that patients with leishmaniasis significantly increased TNF-α and decreased melatonin levels. However, the group of diabetic patients with leishmaniasis showed higher melatonin levels than the control group. These observations suggest that TNF-α may influence melatonin production in patients with American cutaneous leishmaniasis, potentially contributing to the inflammatory characteristics of both diseases.

Background: Leishmania braziliensis, a protozoan prevalent in Brazil, is the known causative agent of cutaneous leishmaniasis (CL). The activation of M1 macrophages is a pivotal factor in the host's ability to eliminate the parasite, whereas M2 macrophages may facilitate parasite proliferation. This study analyzed the clinical outcomes of CL and the patients' immunological profiles, focusing on the prevalence of M1 and M2 macrophages, cytokine production, and annexin-A1 (ANXA1) expression in the lesion., Methods: Data were obtained by polymerase chain reaction (PCR) and histopathological, immunofluorescence, and cytokine analyses., Results: Patients with exudative and cellular reaction-type (ECR)-type lesions that healed within 90 days showed a significant increase in M1. Conversely, patients with ECR and exudative and granulomatous reaction (EGR)types, who healed within 180 days, showed an elevated number of M2. Cytokines interferon (IFN)-γ and tumor necrosis factor (TNF)-α were higher in ECR lesions that resolved within 90 days (P<0.05). In contrast, IL-9 and IL-10 levels significantly increased in both ECR and EGR lesions that healed after 180 days (P<0.001). The production of IL-21, IL-23 and TGF-β was increased in patients with ECR or EGR lesions that healed after 180 days (P<0.05). The expression of ANXA1 was higher in M2 within ECR-type lesions in patients who healed after 180 days (P<0.05)., Conclusions: These findings suggest that the infectious microenvironment induced by L. braziliensis affects the differentiation of M1 and M2 macrophages, cytokine release, and ANXA1 expression, thereby influencing the healing capacity of patients. Therefore, histopathological and immunological investigations may improve the selection of CL therapy.

Introduction: Exosomes produced by the protozoan parasite Leishmania (LeishEXO) are well-established drivers of virulence, though mechanisms underlying their exacerbation of experimental leishmaniasis remain elusive. Expression of Annexin A1 (ANXA1), a protein implicated in exosome-mediated pathologies and viral internalization, has been shown to correlate with cutaneous leishmaniasis severity. Given ANXA1's regulation of myeloid cells - the canonical hosts for Leishmania - we studied the potential role of ANXA1 and its receptors FPR1/2 in exerting LeishEXO's effects., Methods: Murine and in vitro ANXA1 -/- models were used to study the generation of protective T H 1 responses during experimental L. major infection with and without LeishEXO. Recruitment of inflammatory cells was assessed using a peritoneal cell recruitment assay and immunophenotyping, and production of inflammatory mediators was measured using a cytokine and chemokine array. Treatment of experimental models with FPR2 antagonist WRW4 and FPR1/2 agonist WKYMVm was used to delineate the role of the FPR/ANXA1 axis in LeishEXO-mediated hyperpathogenesis., Results: We established that ANXA1 deficiency prohibits LeishEXO-mediated pathogenesis and myeloid cell infection, with minimal alterations to adaptive and innate immune phenotypes. FPR2 blockade with WRW4 similarly inhibited leishmanial hyperpathogenesis, while direct activation of FPRs with WKYMVm enhanced infection and recapitulated the LeishEXO-mediated phenotype. This research describes LeishEXO's utilization of the ANXA1/FPR axis to facilitate parasitic internalization and pathogenesis, which may be leveraged in the development of therapeutics for leishmaniasis., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 da Silva Lira Filho, Lafleur, Alvarez, Piccirillo and Olivier.)

The obligate intracellular parasite Leishmania binds several receptors to trigger uptake by phagocytic cells, ultimately resulting in visceral or cutaneous leishmaniasis. A series of signaling pathways in host cells, which are critical for establishment and persistence of infection, are activated during Leishmania internalization. Thus, preventing Leishmania uptake by phagocytes could be a novel therapeutic strategy for leishmaniasis. However, the host cellular machinery mediating promastigote and amastigote uptake is not well understood. Here, using small molecule inhibitors of Mitogen-activated protein/Extracellular signal regulated kinases (MAPK/ERK), we demonstrate that ERK1/2 mediates Leishmania amazonensis uptake and (to a lesser extent) phagocytosis of beads by macrophages. We find that inhibiting host MEK1/2 or ERK1/2 leads to inefficient amastigote uptake. Moreover, using inhibitors and primary macrophages lacking spleen tyrosine kinase (SYK) or Abl family kinases, we show that SYK and Abl family kinases mediate Raf, MEK, and ERK1/2 activity and are necessary for uptake. Finally, we demonstrate that trametinib, a MEK1/2 inhibitor used to treat cancer, reduces disease severity and parasite burden in Leishmania-infected mice, even if it is started after lesions develop. Our results show that maximal Leishmania infection requires MAPK/ERK and highlight potential for MAPK/ERK-mediated signaling pathways to be novel therapeutic targets for leishmaniasis., Competing Interests: Declaration of competing interest The authors have no competing financial interests., (Copyright © 2024 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

Introduction: Pathogenesis of cutaneous leishmaniases involves parasite growth, persistent inflammation, and likely participation of lipoproteins (LP). The cholesteryl ester transfer protein (CETP), involved in LP remodeling, has been shown to participate in the inflammatory response and the evolution of infectious conditions., Methods: We evaluated the impact of the presence of CETP on infection by Leishmania (L.) amazonensis in an experimental model of cutaneous leishmaniasis using C57BL6/J mice transgenic for human CETP (CETP), having as control their littermates that do not express the protein, wild-type (WT) mice. The progression of the lesion after infection in the footpad was monitored for 12 weeks. Two groups of animals were formed to collect the plantar pad in the 4th and 12th week post-infection., Results: The lesion increased from the 3rd week onwards, in both groups, with a gradual decrease from the 10th week onwards in the CETP group compared to the WT group, showing a reduction in parasitism and an improvement in the healing process, a reduction in CD68+ cells, and an increase in CD163+ and CD206, characterizing a population of M2 macrophages. A reduction in ARG1+ cells and an increase in INOS+ cells were observed. During infection, the LP profile showed an increase in triglycerides in the VLDL fraction in the CETP group at 12 weeks. Gene expression revealed a decrease in the CD36 receptor in the CETP group at 12 weeks, correlating with healing and parasite reduction. In vitro , macrophages derived from bone marrow cells from CETP mice showed lower parasite load at 48 h and, a reduction in arginase activity at 4 h accompanied by increased NO production at 4 and 24 h compared to WT macrophages, corroborating the in vivo findings., Discussion: The data indicate that the presence of CETP plays an important role in resolving Leishmania (L.) amazonensis infection, reducing parasitism, and modulating the inflammatory response in controlling infection and tissue repair., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Batista-Dantas, Ozaki, Santana, Nunes, Uscata, Siess-Portugal, Reis, Yamashiro-Kanashiro, Tafuri, Duarte-Neto, Sotto, Goto and Cazita.)

The parasite Leishmania resides as amastigotes within the macrophage parasitophorous vacuoles inflicting the disease Leishmaniasis. Leishmania selectively modulates mitogen-activated protein kinase (MAPK) phosphorylation subverting CD40-triggered anti-leishmanial functions of macrophages. The mechanism of any pathogen-derived molecule induced host MAPK modulation remains poorly understood. Herein, we show that of the fifteen MAPKs, LmjMAPK4 expression is higher in virulent L. major. LmjMAPK4- detected in parasitophorous vacuoles and cytoplasm- binds MEK-1/2, but not MKK-3/6. Lentivirally-overexpressed LmjMAPK4 augments CD40-activated MEK-1/2-ERK-1/2-MKP-1, but inhibits MKK3/6-p38MAPK-MKP-3, phosphorylation. A rationally-identified LmjMAPK4 inhibitor reinstates CD40-activated host-protective anti-leishmanial functions in L. major-infected susceptible BALB/c mice. These results identify LmjMAPK4 as a MAPK modulator at the host-pathogen interface and establish a pathogen-intercepted host receptor signaling as a scientific rationale for identifying drug targets., (Copyright © 2024 Elsevier B.V. All rights reserved.)

American tegumentary leishmaniasis (ATL) diagnosis is an open question, and the search for a solution is urgent. The available tests that detect the etiological agent of the infection are specific for ATL diagnosis. However, they present disadvantages, such as low sensitivity and the need for invasive procedures to obtain the samples. Immunological methods (leishmanin skin test and search for anti-Leishmania antibodies) are good alternatives to the etiological diagnosis of ATL. Presently, we face problems with disease confirmation due to the discontinuity in the production of leishmanin skin test antigen, particularly in resource-poor settings. Aiming to diagnose ATL, we validated rLb6H-ELISA for IgG antibodies using 1,091 samples from leishmaniasis patients and healthy controls, divided into four panels, living in 19 Brazilian endemic and non-endemic states. The rLb6H-ELISA showed a sensitivity of 98.6% and a specificity of 100.0%, with the reference panel comprising 70 ATL patient samples and 70 healthy controls. The reproducibility evaluation showed a coefficient of variation of positive samples ≤ 8.20% for repeatability, ≤ 17,97% for reproducibility, and ≤ 8.12% for homogeneity. The plates sensitized with rLb6H were stable at 4°C and -20°C for 180 days and 37°C for seven days, indicating 12 months of validity. In samples of ATL patients from five research and healthcare centers in endemic and non-endemic areas, rLb6H-ELISA showed a sensitivity of 84.0%; no significant statistical difference was observed among the five centers (chi-square test, p = 0.13). In samples of healthy controls from four areas with different endemicity, a specificity of 92.4% was obtained; lower specificity was obtained in a visceral leishmaniasis high endemicity locality (chi-square test, p<0.001). Cross-reactivity was assessed in 166 other disease samples with a positivity of 13.9%. Based on the good diagnostic performance and the reproducibility and stability of the antigen, we suggest using ELISA-rLb6H to diagnose ATL., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Valencia-Portillo et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Cutaneous leishmaniasis caused by Leishmania parasites exhibits a wide range of clinical manifestations. Although parasites influence disease severity, cytolytic CD8+ T cell responses mediate disease. Although these responses originate in the lymph node, we found that expression of the cytolytic effector molecule granzyme B was restricted to lesional CD8+ T cells in Leishmania-infected mice, suggesting that local cues within inflamed skin induced cytolytic function. Expression of Blimp-1 (Prdm1), a transcription factor necessary for cytolytic CD8+ T cell differentiation, was driven by hypoxia within the inflamed skin. Hypoxia was further enhanced by the recruitment of neutrophils that consumed oxygen to produce ROS and ultimately increased the hypoxic state and granzyme B expression in CD8+ T cells. Importantly, lesions from patients with cutaneous leishmaniasis exhibited hypoxia transcription signatures that correlated with the presence of neutrophils. Thus, targeting hypoxia-driven signals that support local differentiation of cytolytic CD8+ T cells may improve the prognosis for patients with cutaneous leishmaniasis, as well as for other inflammatory skin diseases in which cytolytic CD8+ T cells contribute to pathogenesis.

The parasite Leishmania (Viannia) braziliensis is widely distributed in Brazil and is one of the main species associated with human cases of different forms of tegumentary leishmaniasis (TL) such as cutaneous leishmaniasis (CL) and mucosal leishmaniasis (ML). The mechanisms underlying the pathogenesis of TL are still not fully understood, but it is known that factors related to the host and the parasite act in a synergistic and relevant way to direct the response to the infection. In the host, macrophages have a central connection with the parasite and play a fundamental role in the defense of the organism due to their ability to destroy intracellular parasites and present antigens. In the parasite, some intrinsic factors related to the species or even the strain analyzed are fundamental for the outcome of the disease. One of them is the presence of Leishmania RNA Virus 1 (LRV1), an endosymbiont virus that parasitizes some species of Leishmania that triggers a cascade of signals leading to a more severe TL phenotype, such as ML. One of the strategies for understanding factors associated with the immune response generated after Leishmania/host interaction is through the analysis of molecular patterns after infection. Thus, the gene expression profile in human monocyte-derived macrophages obtained from healthy donors infected in vitro with L. braziliensis positive (LbLRV1+) and negative (LbLRV1-) for LRV1 was evaluated. For this, the microarray assay was used and 162 differentially expressed genes were identified in the comparison LbLRV1+ vs. LbLRV1-, 126 upregulated genes for the type I and II interferons (IFN) signaling pathway, oligoadenylate synthase OAS/RNAse L, non-genomic actions of vitamin D3 and RIG-I type receptors, and 36 down-regulated. The top 10 downregulated genes along with the top 10 upregulated genes were considered for analysis. Type I interferon (IFNI)- and OAS-related pathways results were validated by RT-qPCR and Th1/Th2/Th17 cytokines were analyzed by Cytometric Bead Array (CBA) and enzyme-linked immunosorbent assay (ELISA). The microarray results validated by RT-qPCR showed differential expression of genes related to IFNI-mediated pathways with overexpression of different genes in cells infected with LbLRV1+ compared to LbLRV1- and to the control. No significant differences were found in cytokine levels between LbLRV1+ vs. LbLRV1- and control. The data suggest the activation of gene signaling pathways associated with the presence of LRV1 has not yet been reported so far. This study demonstrates, for the first time, the activation of the OAS/RNase L signaling pathway and the non-genomic actions of vitamin D3 when comparing infections with LbLRV1+ versus LbLRV1- and the control. This finding emphasizes the role of LRV1 in directing the host's immune response after infection, underlining the importance of identifying LRV1 in patients with TL to assess disease progression., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Felipin et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Cytolytic CD8+ T cells mediate immunopathology in cutaneous leishmaniasis without controlling parasites. Here, we identify factors involved in CD8+ T cell migration to the lesion that could be targeted to ameliorate disease severity. CCR5 was the most highly expressed chemokine receptor in patient lesions, and the high expression of CCL3 and CCL4, CCR5 ligands, was associated with delayed healing of lesions. To test the requirement for CCR5, Leishmania-infected Rag1-/- mice were reconstituted with CCR5-/- CD8+ T cells. We found that these mice developed smaller lesions accompanied by a reduction in CD8+ T cell numbers compared to controls. We confirmed these findings by showing that the inhibition of CCR5 with maraviroc, a selective inhibitor of CCR5, reduced lesion development without affecting the parasite burden. Together, these results reveal that CD8+ T cells migrate to leishmanial lesions in a CCR5-dependent manner and that blocking CCR5 prevents CD8+ T cell-mediated pathology., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Amorim Sacramento et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Improving the diagnostic technology used to detect tegumentary leishmaniasis (TL) is essential in view of it being a widespread, often neglected tropical disease, with cases reported from the Southern United States to Northern Argentina. Recombinant proteins, recombinant multiepitope proteins, and synthetic peptides have been extensively researched and used in disease diagnosis. One of the benefits of applying these antigens is a measurable increase in sensitivity and specificity, which improves test accuracy. The present review aims to describe the use of these antigens and their diagnostic effectiveness. With that in mind, a bibliographic survey was conducted on the PudMed platform using the search terms "tegumentary leishmaniasis" AND "diagno", revealing that recombinant proteins have been described and evaluated for their value in TL diagnosis since the 1990s. However, there was a spike in the number of publications using all of the antigens between 2013 and 2022, confirming an expansion in research efforts to improve diagnosis. Moreover, all of the studies involving different antigens had promising results, including improved sensitivity and specificity. These data recognize the importance of doing research with new technologies focused on developing quick, more effective diagnostic kits as early diagnosis facilitates treatment., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Background: Cutaneous leishmaniasis (CL) is a parasitic disease with a significant burden in the Old World countries., Objective: In the current study, some of the primary biochemical properties and IFN-γ inducing epitopes with specific binding capacity to human and mouse MHC alleles were predicted for Leishmania major gp46 antigenic protein., Methods: Several online servers were used to predict physico-chemical traits, allergenicity, antigenicity, transmembrane domain and signal peptide, subcellular localization, post-translational modifications (PTMs), secondary and tertiary structures, tertiary model refining with validations. Also, IEDB web server was used to predict mouse/human cytotoxic T-lymphocyte (CTL) and helper T-lymphocyte (HTL) epitopes., Results: The 33.25 kDa protein was stable, hydrophilic, antigenic, while non-allergenic, with enhanced thermotolerance and 45 PTM sites. The secondary structure encompassed a random coil, followed by extended strands and helices. Ramachandran-based analysis of the refined model showed 73.1%, 21.6%, 3.4% and 1.9% of residues in the most favored, additional allowed, generously-allowed and disallowed regions, respectively. Epitope screening demonstrated 4 HTL epitopes against seemingly protective HLA alleles, 5 HTL epitopes against the HLA reference set, 3 human CTL epitopes and a number of mouse MHC-restricted epitopes., Conclusion: This paper provides insights into the bioinformatics characteristics of the L. major gp46 protein as a promising vaccine candidate., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Cutaneous leishmaniasis exhibits a spectrum of clinical presentations dependent upon the parasites' persistence and host immunopathologic responses. Although cytolytic CD8 T cells cannot control the parasites, they significantly contribute to pathologic responses. In a murine model of cutaneous leishmaniasis, we previously found that NKG2D plays a role in the ability of cytolytic CD8 T cells to promote disease in leishmanial lesions. Here, we investigated whether NKG2D plays a role in human disease. We found that NKG2D and its ligands were expressed within lesions from L. braziliensis-infected patients and that IL-15 and IL-1β were factors driving NKG2D and NKG2D ligand expression, respectively. Blocking NKG2D reduced degranulation by CD8 T cells in a subset of patients. Additionally, our transcriptional analysis of patients' lesions found that patients who failed the first round of treatment exhibited higher expression of KLRK1, the gene coding for NKG2D, than those who responded to treatment. These findings suggest that NKG2D may be a promising therapeutic target for ameliorating disease severity in cutaneous leishmaniasis caused by L. braziliensis infection., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Sacramento et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The heterogeneity of CD4+ T cells has been investigated since the late 1970s, when their Th1 and Th2 subsets were coined. Later studies on the cutaneous form of the Leishmaniasis were focused on the experimental models of Leishmania major infection using the susceptible BALB/c and the resistant C57BL/6 mice. At the early 21st century, the regulatory T-cells subpopulation was introduced and its role in concomitant immunity, responsible for lifelong resistance of the host to the reinfection was proposed. Subsequent studies, mainly focused on the visceral form of the infection pointed to the role of IL-17, produced by Th17 subset of CD4+ T cells that along the neutrophils were shown to have important yet equivocal functions in protection against or exacerbation of the infection. Altogether, the current knowledge indicates that the above four subsets could orchestrate the immune, the regulatory and the inflammatory responses of the host against different forms of leishmaniases.

Background: Leishmania (L) parasite, the causative agent of zoonotic cutaneous leishmaniasis (ZCL), effectively stimulates the mammalian cells to mount strong humoral responses by enhancing T-helper-2 (Th2)-associated cytokines for its survival. The best strategy to decrease the intensity of infection in the host is induction of cellular immunity., Methods: We evaluated the effects of the empty bacterial pcDNA3 plasmid on mice infected with L. major and quantified the immune mediators including IFN-γ, IL-4, IL-10, IgG2a, IgG1, arginase activity and nitric oxide (NO) in the mice. Moreover, the footpad lesion size and parasite load were assessed., Results: We observed that pcDNA3 could modulate the immune responses in favor of host cells and decrease the disease severity. Th2- associated mediators, including arginase, IL-4, and IL-10 are downregulated, while cellular responses are upregulated in line with an increase in the levels of nitric oxide (NO) and interfero-gamma (IFN-γ). Interestingly, pcDNA3 induced specific Th1-associated antibodies, IgG2a isotype; however, it suppressed the production of humoral IgG1. The stimulation of the immune response by the empty pcDNA3 is able to shift the immune function to predominant cellular responses caused by Th1, and it had a positive effect on the treatment of zoonotic cutaneous leishmaniasis (ZCL)., Conclusions: Altogether, we introduced the pcDNA3 as a potential interfering factor in the modulation of the immune system against ZCL. Since this vector has been widely used as a control group in different studies, we suggest that the potential function of the empty vector should be deeply assessed, as it exerts anti-parasitic effects on mice infected with L. major., Competing Interests: The authors have declared that no competing interests exist.

IL-4 and IL-13 cytokines have been associated with a non-healing phenotype in murine leishmaniasis in L. mexicana -infected BALB/c mice as demonstrated in IL-4 -/- , IL-13 -/- and IL-4Rα -/- global knockout mouse studies. However, it is unclear from the studies which cell-type-specific IL-4/IL-13 signaling mediates protection to L. mexicana . Previous studies have ruled out a role for IL-4-mediated protection on CD4 + T cells during L. mexicana infections. A candidate for this role may be non-lymphocyte cells, particularly DCs, as was previously shown in L. major infections, where IL-4 production drives dendritic cell-IL-12 production thereby mediating a type 1 immune response. However, it is unclear if this IL-4-instruction of type 1 immunity also occurs in CL caused by L. mexicana , since the outcome of cutaneous leishmaniasis often depends on the infecting Leishmania species. Thus, BALB/c mice with cell-specific deletion of the IL-4Rα on CD11c + DCs (CD11c cre IL-4Rα -/lox ) were infected with L. mexicana promastigotes in the footpad and the clinical phenotype, humoral and cellular immune responses were investigated, compared to the littermate control. Our results show that CL disease progression in BALB/c mice is independent of IL-4Rα signaling on DCs as CD11c cre IL-4Rα -/lox mice had similar footpad lesion progression, parasite loads, humoral responses (IgE, IgG1, IgG 2a/b), and IFN-γ cytokine secretion in comparison to littermate controls. Despite this comparable phenotype, surprisingly, IL-4 production in CD11c cre IL-4Rα -/lox mice was significantly increased with an increasing trend of IL-13 when compared to littermate controls. Moreover, the absence of IL-4Rα signaling did not significantly alter the frequency of CD4 and CD8 lymphocytes nor their activation, or memory phenotype compared to littermate controls. However, these populations were significantly increased in CD11c cre IL-4Rα -/lox mice due to greater total cell infiltration into the lymph node. A similar trend was observed for B cells whereas the recruitment of myeloid populations (macrophages, DCs, neutrophils, and Mo-DCs) into LN was comparable to littermate IL-4Rα -/lox mice. Interestingly, IL-4Rα-deficient bone marrow-derived dendritic cells (BMDCs), stimulated with LPS or L. mexicana promastigotes in presence of IL-4, showed similar levels of IL-12p70 and IL-10 to littermate controls highlighting that IL-4-mediated DC instruction was not impaired in response to L. mexicana . Similarly, IL-4 stimulation did not affect the maturation or activation of IL-4Rα-deficient BMDCs during L. mexicana infection nor their effector functions in production of nitrite and arginine-derived metabolite (urea). Together, this study suggests that IL-4 Rα signaling on DCs is not key in the regulation of immune-mediated protection in mice against L. mexicana infection., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Osero, Cele, Aruleba, Maine, Ozturk, Lutz, Brombacher and Hurdayal.)

Competing Interests: The authors have declared that no competing interests exist.

Neutrophils are the first line of defence against invading pathogens. Although neutrophils are well-known professional killers, some pathogens including Leishmania (L.) parasites survive in neutrophils, using these cells to establish infection. Manipulation of neutrophil recruitment to the infection site is therefore of interest in this cutaneous disease. The c-MET tyrosine kinase receptor was shown to promote neutrophil migration to inflamed sites. Here, we investigated the importance of c-MET expression on neutrophils in their recruitment to the infection site and the role of c-Met expression in the pathology of leishmaniasis. Following infection with L. mexicana, mice with conditional deletion of c-MET in neutrophils controlled significantly better their lesion development and parasite burden compared to similarly infected wild type mice. Our data reveal a specific role for c-MET activation in Leishmania-induced neutrophil infiltration, a process correlating with their negative role in the pathology of the diseases. We further show that c-MET phosphorylation is observed in established cutaneous lesions. Exposure to L. mexicana upregulated c-Met expression predominantly in infected neutrophils and c-Met expression influenced ROS release by neutrophils. In addition, pharmacological inhibition of c-MET, administrated once the lesion is established, induced a significant decrease in lesion size associated with diminished infiltration of neutrophils. Both genetic ablation of c-MET in neutrophils and systemic inhibition of c-MET locally resulted in higher levels of CD4+T cells producing IFNγ, suggesting a crosstalk between neutrophils and these cells. Collectively, our data show that c-MET activation in neutrophils contributes to their recruitment following infection, and that L. mexicana induction of c-MET on neutrophils impacts the local pathology associated with this disease. Our results suggest a potential use for this inhibitor in the control of the cutaneous lesion during this parasitic infection., Competing Interests: The authors have declared that no competing interest exist.

Leishmaniasis continues to afflict known and newer endemic sites despite global efforts towards its control and elimination. In this regard, the emergence of newer endemic sites with unusual disease formats is recognized wherein Leishmania donovani complex classically known to cause visceral disease is demonstrated to cause cutaneous manifestation. In this context, atypical cutaneous leishmaniasis (CL) cases caused by L. donovani genetic variants from the newer endemic state of Himachal Pradesh (HP) in India are beginning to be understood in terms of parasite determinants. The atypical CL manifestation further needs to be explored to define host immune correlates with a possible role in driving the unusual disease progression. In the given study, we performed comprehensive systemic-immune profiling of the atypical CL patients from the study area in HP, India, in comparison with the classical visceral leishmaniasis (VL) patients from the northeast region of India. The systemic immune response was studied using ELISA-based assessment of Th1, Th2, Th17, Treg, and Th22 specific plasma cytokine expression pattern and parasite-specific total serum IgG/IgG subclasses. The specified immune correlates are known to exhibit heterogeneous association with the different infecting parasite species, infection load, and co-lateral host immunopathology in classical CL and VL. In the atypical CL patient group, altered expression of IL-10 emerged as the key finding that could potentially fine-tune the Th1/Th17/Th22 effector cytokine axis towards a localized cutaneous manifestation. A reduced expression of IL-10 along with a high IFN-γ/IL-10 ratio as a readout of effective parasite killing defined atypical cutaneous outcome. In contrast, high circulatory IL-10 levels and a depressed IFN-γ/IL-10 ratio were seen in classical VL patients in line with an ineffective parasite-killing cytokine response. Overall, the study highlights new knowledge on host immune correlates in terms of cytokine expression pattern and IgG subclasses that underline atypical disease manifestation such that L. donovani , a generally visceralizing parasite species cause skin localized cutaneous lesions., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Thakur, Madaan, Jain, Shankar, Negi, Chauhan, Sundar, Singh and Jain.)

Leishmaniasis is a disease caused by the protozoan parasite Leishmania and is known to affect millions of individuals worldwide. In recent years, we have established the critical role played by Leishmania zinc-metalloprotease GP63 in the modulation of host macrophage signalling and functions, favouring its survival and progression within its host. Leishmania major lacking GP63 was reported to cause limited infection in mice, however, it is still unclear how GP63 may influence the innate inflammatory response and parasite survival in an in vivo context. Therefore, we were interested in analyzing the early innate inflammatory events upon Leishmania inoculation within mice and establish whether Leishmania GP63 influences this initial inflammatory response. Experimentally, L. major WT (L. majorWT), L. major GP63 knockout (L. majorKO), or L. major GP63 rescue (L. majorR) were intraperitoneally inoculated in mice and the inflammatory cells recruited were characterized microscopically and by flow cytometry (number and cell type), and their infection determined. Pro-inflammatory markers such as cytokines, chemokines, and extracellular vesicles (EVs, e.g. exosomes) were monitored and proteomic analysis was performed on exosome contents. Data obtained from this study suggest that Leishmania GP63 does not significantly influence the pathogen-induced inflammatory cell recruitment, but rather their activation status and effector function. Concordantly, internalization of promastigotes during early infection could be influenced by GP63 as fewer L. majorKO amastigotes were found within host cells and appear to maintain in host cells over time. Collectively this study provides a clear analysis of innate inflammatory events occurring during L. major infection and further establish the prominent role of the virulence factor GP63 to provide favourable conditions for host cell infection., Competing Interests: The authors have declared that no competing interests exist.

Cutaneous leishmaniasis (CL) patients present an exacerbated inflammatory response associated with tissue damage and ulcer development. Increasing numbers of patients have exhibited treatment failure, which remains not well understood. We hypothesized that adjuvant anti-inflammatory therapy would benefit CL patients. The aim of the present study was to investigate the contribution of Notch signalling and gamma-secretase activity to the inflammatory response observed in CL patients. Notch signalling is a molecular signalling pathway conserved among animal species. Gamma-secretase forms a complex of proteins that, among other pathways, modulates Notch signalling and immune response. We found that Notch 1 cell receptor signalling protects against the pathologic inflammatory response, and JLK6, a gamma-secretase inhibitor that does not interfere with Notch signalling, was shown to decrease the in-vitro inflammatory response in CL. Our data suggest that JLK6 may serve as an adjuvant treatment for CL patients.

Cutaneous leishmaniasis (CL) is caused by Leishmania donovani in Sri Lanka. Pentavalent antimonials (e.g., sodium stibogluconate [SSG]) remain first-line drugs for CL with no new effective treatments emerging. We studied whole blood and lesion transcriptomes from Sri Lankan patients with CL at presentation and during SSG treatment. From lesions but not whole blood, we identified differential expression of immune-related genes, including immune checkpoint molecules, after onset of treatment. Using spatial profiling and RNA-FISH, we confirmed reduced expression of programmed death-ligand 1 (PD-L1) and indoleamine 2,3-dioxygenase 1 (IDO1) proteins on treatment in lesions of a second validation cohort and further demonstrated significantly higher expression of these checkpoint molecules on parasite-infected compared with noninfected lesional CD68+ monocytes and macrophages. Crucially, early reduction in PD-L1 but not IDO1 expression was predictive of rate of clinical cure (HR = 4.88) and occurred in parallel with reduction in parasite load. Our data support a model whereby the initial anti-leishmanial activity of antimonial drugs alleviates checkpoint inhibition on T cells, facilitating immune-drug synergism and clinical cure. Our findings demonstrate that PD-L1 expression can be used as a predictor of rapidity of clinical response to SSG treatment in Sri Lanka and support further evaluation of PD-L1 as a host-directed therapeutic in leishmaniasis.

Infection with the same species of Leishmania (L)donovani causes different manifestations including visceral leishmaniasis (VL) and post kala-azar dermal leishmaniasis (PKDL), indicating that the host-related immunological parameters perform a decisive role in the pathogenesis of diseases. As PKDL is a reservoir of the parasite, a better understanding of the host immune responses is necessary to restrict the L. donovani transmission. The proper local production of Th1 cell-related cytokines (including IFN-γ, TNF-α and IL-12), Th17 cell-derived cytokines (such as IL-17A, IL-17F and IL-22), and CD8 + cytotoxic T lymphocyte (CTL)-derived IFN-γ are protective against PKDL. However, dominant production of regulatory CD4 + T cell-derived cytokines (such as IL-10 and TGF-β), Th2 cell-derived cytokines (such as IL-4/IL-13), M2 macrophage-derived cytokines (such as IL-4 and IL-10), keratinocyte-derived IL-10, regulatory CD8 + T cell-derived IL-10, and dendritic cell-derived IL-10, IL-27 and IL-21 can contribute to the parasite persistence and PKDL development. Understanding of the T cell-related cytokine network within PKDL lesions gives rise to novel insights concerning the role of each cytokine in the protection or susceptibility to disease. Manipulation of the cytokine network can be considered as an interesting immunotherapeutic strategy for the treatment of L. donovani-mediated PKDL., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Leishmaniasis is an unresolved global health problem with a high socio-economic impact. Data generated in mouse models has revealed that the Th1 response, with IL-12, IFN-γ, TNF-α, and IL-2 as prominent cytokines, predominantly controls the disease progression. Premised on these findings, all examined vaccine formulations have been aimed at generating a long-lived memory Th1 response. However, all vaccine formulations with the exception of live Leishmania inoculation (leishmanization) have failed to sufficiently protect against sand fly delivered infection. It has been recently unraveled that sand fly dependent factors may compromise pre-existing Th1 memory. Further scrutinizing the immune response after leishmanization has uncovered the prominent role of early (within hours) and robust IFN-γ production (Th1 concomitant immunity) in controlling the sand fly delivered secondary infection. The response is dependent upon parasite persistence and subclinical ongoing primary infection. The immune correlates of concomitant immunity (Resident Memory T cells and Effector T subsets) mitigate the early effects of sand fly delivered infection and help to control the disease. In this review, we have described the early events after sand fly challenge and the role of Th1 concomitant immunity in the protective immune response in leishmanized resistant mouse model, although leishmanization is under debate for human use. Undoubtedly, the lessons we learn from leishmanization must be further implemented in alternative vaccine approaches., (Copyright © 2020 Elsevier Ltd. All rights reserved.)