Host M-CSF induced gene expression drives changes in susceptible and resistant mice-derived BMdMs upon Leishmania major infection.

Leishmaniases are a group of diseases with different clinical manifestations. Macrophage-Leishmania interactions are central to the course of the infection. The outcome of the disease depends not only on the pathogenicity and virulence of the parasite, but also on the activation state, the genetic background, and the underlying complex interaction networks operative in the host macrophages. Mouse models, with mice strains having contrasting behavior in response to parasite infection, have been very helpful in exploring the mechanisms underlying differences in disease progression. We here analyzed previously generated dynamic transcriptome data obtained from Leishmania major (L. major) infected bone marrow derived macrophages (BMdMs) from resistant and susceptible mouse. We first identified differentially expressed genes (DEGs) between the M-CSF differentiated macrophages derived from the two hosts, and found a differential basal transcriptome profile independent of Leishmania infection. These host signatures, in which 75% of the genes are directly or indirectly related to the immune system, may account for the differences in the immune response to infection between the two strains. To gain further insights into the underlying biological processes induced by L. major infection driven by the M-CSF DEGs, we mapped the time-resolved expression profiles onto a large protein-protein interaction (PPI) network and performed network propagation to identify modules of interacting proteins that agglomerate infection response signals for each strain. This analysis revealed profound differences in the resulting responses networks related to immune signaling and metabolism that were validated by qRT-PCR time series experiments leading to plausible and provable hypotheses for the differences in disease pathophysiology. In summary, we demonstrate that the host's gene expression background determines to a large degree its response to L. major infection, and that the gene expression analysis combined with network propagation is an effective approach to help identifying dynamically altered mouse strain-specific networks that hold mechanistic information about these contrasting responses to infection.

Stress conditions promote Leishmania hybridization in vitro marked by expression of the ancestral gamete fusogen HAP2 as revealed by single-cell RNA-seq.

Leishmania are protozoan parasites transmitted by the bite of sand fly vectors producing a wide spectrum of diseases in their mammalian hosts. These diverse clinical outcomes are directly associated with parasite strain and species diversity. Although Leishmania reproduction is mainly clonal, a cryptic sexual cycle capable of producing hybrid genotypes has been inferred from population genetic studies and directly demonstrated by laboratory crosses. Experimentally, mating competence has been largely confined to promastigotes developing in the sand fly midgut. The ability to hybridize culture promastigotes in vitro has been limited so far to low-efficiency crosses between two Leishmania tropica strains, L747 and MA37, that mate with high efficiency in flies. Here, we show that exposure of promastigote cultures to DNA damage stress produces a remarkably enhanced efficiency of in vitro hybridization of the L. tropica strains and extends to other species, including Leishmania donovani, Leishmania infantum, and Leishmania braziliensis, a capacity to generate intra- and interspecific hybrids. Whole-genome sequencing and total DNA content analyses indicate that the hybrids are in each case full genome, mostly tetraploid hybrids. Single-cell RNA sequencing of the L747 and MA37 parental lines highlights the transcriptome heterogeneity of culture promastigotes and reveals discrete clusters that emerge post-irradiation in which genes potentially involved in genetic exchange are expressed, including the ancestral gamete fusogen HAP2. By generating reporter constructs for HAP2, we could select for promastigotes that could either hybridize or not in vitro. Overall, this work reveals that there are specific populations involved in Leishmania hybridization associated with a discernible transcriptomic signature, and that stress facilitated in vitro hybridization can be a transformative approach to generate large numbers of hybrid genotypes between diverse species and strains.

Nitric oxide controls proliferation of Leishmania major by inhibiting the recruitment of permissive host cells.

Nitric oxide (NO) is an important antimicrobial effector but also prevents unnecessary tissue damage by shutting down the recruitment of monocyte-derived phagocytes. Intracellular pathogens such as Leishmania major can hijack these cells as a niche for replication. Thus, NO might exert containment by restricting the availability of the cellular niche required for efficient pathogen proliferation. However, such indirect modes of action remain to be established. By combining mathematical modeling with intravital 2-photon biosensors of pathogen viability and proliferation, we show that low L. major proliferation results not from direct NO impact on the pathogen but from reduced availability of proliferation-permissive host cells. Although inhibiting NO production increases recruitment of these cells, and thus pathogen proliferation, blocking cell recruitment uncouples the NO effect from pathogen proliferation. Therefore, NO fulfills two distinct functions for L. major containment: permitting direct killing and restricting the supply of proliferation-permissive host cells.

In vitro and in vivo therapeutic potentials of 6-gingerol in combination with amphotericin B for treatment of Leishmania major infection: Powerful synergistic and multifunctional effects.

The ongoing conventional drugs for leishmaniasis treatment are insufficient. The present study aimed to assess 6-gingerol alone and in combination with amphotericin B on Leishmania major stages using experimental and in vivo murine models. Here, arrays of experimental approaches were designed to monitor and evaluate the 6-gingerol potential therapeutic outcomes. The binding affinity of 6-gingerol and IFN-γ was the basis for docking conformations. 6-Gingerol combined with amphotericin B represented a safe mixture, extremely leishmanicidal, a potent antioxidant, induced a remarkable apoptotic index, significantly increased the expression of the Th1-related cytokines (IL-12p40, IFN-γ, and TNF- α), iNOS, and transcription factors (STAT1, c-Fos, and Elk-1). In contrast, the expression of the Th2-related cytokines was significantly downregulated (p < 0.001). This combination was also potent when the lesion appearance was evaluated following three weeks of treatment. The histopathological and immunohistochemical patterns of the murine model represented clusters of CD4+ and CD8+ T lymphocytes which compressed and deteriorated the macrophages harboring Leishman bodies. The primary mode of action of 6-gingerol and amphotericin B involved broad mechanistic insights providing a coherent basis for further clinical study as a potential drug candidate for CL. In conclusion, 6-gingerol with amphotericin B synergistically exerted anti-leishmanial activity in vitro and in vivo and potentiated macrophages' leishmanicidal activity, modulated Th1- and Th2-related phenotypes improved the histopathological changes in the BALB/c mice infected with L. major. They elevated the leukocyte infiltration into the lesions. Therefore, this combination should be considered for treating volunteer patients with CL in clinical studies.

Leishmanicidal potentials of Gossypium hirsutum extract and its fractions on Leishmania major in a murine model: parasite burden, gene expression, and histopathological profile.

Introduction. Leishmaniasis is a neglected tropical and subtropical disease caused by over 20 protozoan species.Hypothesis. Treatment of this complex disease with traditional synthetic drugs is a major challenge worldwide. Natural constituents are unique candidates for future therapeutic development.Aim. This study aimed to assess the in vivo anti-leishmanial effect of the Gossypium hirsutum extract, and its fractions compared to the standard drug (Glucantime, MA) in a murine model and explore the mechanism of action.Methodology. Footpads of BALB/c mice were infected with stationary phase promastigotes and treated topically and intraperitoneally with G. hirsutum extract, its fractions, or Glucantime, 4 weeks post-infection. The extract and fractions were prepared using the Soxhlet apparatus with chloroform followed by the column procedure.Results. The crude extract significantly decreased the footpad parasite load and lesion size compared to the untreated control group (P<0.05), as revealed by dilution assay, quantitative real-time PCR, and histopathological analyses. The primary mode of action involved an immunomodulatory role towards the Th1 response in the up-regulation of IFN-γ and IL-12 and the suppression of IL-10 gene expression profiling against cutaneous leishmaniasis caused by Leishmania major.Conclusion. This finding suggests that the extract possesses multiple combinatory effects of diverse bioactive phytochemical compositions that exert its mechanisms of action through agonistic-synergistic interactions. The topical extract formulation could be a suitable and unique candidate for future investigation and pharmacological development. Further studies are crucial to evaluate the therapeutic potentials of the extract alone and in combination with conventional drugs using clinical settings.

Hypoxia-Inducible Factor Signaling in Macrophages Promotes Lymphangiogenesis in Leishmania major Infection.

Vascular remodeling is a phenomenon seen in the cutaneous lesions formed during infection with Leishmania parasites. Within the lesion, Leishmania major infection leads to the infiltration of inflammatory cells, including macrophages, and is associated with hypoxic conditions and lymphangiogenesis in the local site. This low-oxygen environment is concomitant with the expression of hypoxic inducible factors (HIFs), which initiate the expression of vascular endothelial growth factor-A (VEGF-A) in macrophages during the infection. Here, we found that macrophage hypoxia is elevated in the skin, and the HIF target Vegfa is preferentially expressed at the site of infection. Further, transcripts indicative of both HIF-1α and HIF-2α activation were increased at the site of infection. Given that HIF mediates VEGF-A and that VEGF-A/VEGFR-2 signaling induces lymphangiogenesis, we wanted to investigate the link between myeloid HIF activation and lymphangiogenesis during L. major infection. We show that myeloid aryl hydrocarbon receptor nuclear translocator (ARNT)/HIF/VEGF-A signaling promotes lymphangiogenesis (the generation of newly formed vessels within the local lymphatic network), which helps resolve the lesion by draining away inflammatory cells and fluid. Concomitant with impaired lymphangiogenesis, we find the deletion of myeloid ARNT/HIF signaling leads to an exacerbated inflammatory response associated with a heightened CD4+ Th1 immune response following L. major infection. Altogether, our data suggest that VEGF-A-mediated lymphangiogenesis occurs through myeloid ARNT/HIF activation following Leishmania major infection and this process is critical in limiting immunopathology.

Interdependencies between Toll-like receptors in Leishmania infection.

Multiple pathogen-associated molecular patterns (PAMPs) on a pathogen's surface imply their simultaneous recognition by the host cell membrane-located multiple PAMP-specific Toll-like receptors (TLRs). The TLRs on endosomes recognize internalized pathogen-derived nucleic acids and trigger anti-pathogen immune responses aimed at eliminating the intracellular pathogen. Whether the TLRs influence each other's expression and effector responses-termed TLR interdependency-remains unknown. Herein, we first probed the existence of TLR interdependencies and next determined how targeting TLR interdependencies might determine the outcome of Leishmania infection. We observed that TLRs selectively altered expression of their own and of other TLRs revealing novel TLR interdependencies. Leishmania major-an intra-macrophage parasite inflicting the disease cutaneous leishmaniasis in 88 countries-altered this TLR interdependency unfolding a unique immune evasion mechanism. We targeted this TLR interdependency by selective silencing of rationally chosen TLRs and by stimulation with selective TLR ligands working out a novel phase-specific treatment regimen. Targeting the TLR interdependency elicited a host-protective anti-leishmanial immune response and reduced parasite burden. To test whether this observation could be used as a scientific rationale for treating a potentially fatal L. donovani infection, which causes visceral leishmaniasis, we targeted the inter-TLR dependency adopting the same treatment regimen. We observed reduced splenic Leishman-Donovan units accompanied by host-protective immune response in susceptible BALB/c mice. The TLR interdependency optimizes TLR-induced immune response by a novel immunoregulatory framework and scientifically rationalizes targeting TLRs in tandem and in sequence for redirecting immune responses against an intracellular pathogen.

LmjF.36.3850, a novel hypothetical Leishmania major protein, contributes to the infection.

Leishmania is a protozoan parasite that resides in mammalian macrophages and inflicts the disease known as leishmaniasis. Although prevalent in 88 countries, an anti-leishmanial vaccine remains elusive. While comparing the virulent and avirulent L. major transcriptomes by microarray, PCR and functional analyses for identifying a novel virulence-associated gene, we identified LmjF.36.3850, a hypothetical protein significantly less expressed in the avirulent parasite and without any known function. Motif search revealed that LmjF.36.3850 protein shared phosphorylation sites and other structural features with sucrose non-fermenting protein (Snf7) that shuttles virulence factors. LmjF.36.3850 was predicted to bind diacylglycerol (DAG) with energy value similar to PKCα and PKCß, to which DAG is a cofactor. Indeed, 1-oleoyl-2-acetyl-sn-glycerol (OAG), a DAG analogue, enhanced the phosphorylation of PKCα and PKCßI. We cloned LmjF.36.3850 gene in a mammalian expression vector and primed susceptible BALB/c mice followed by challenge infection. We observed a higher parasite load, comparable antibody response and higher anti-inflammatory cytokines such as IL-4 and IL-10, while expression of major anti-leishmanial cytokine, IFN-γ, remained unchanged in LmjF.36.3850-vaccinated mice. CSA restimulated LN cells from vaccinated mice after challenge infection secreted comparable IL-4 and IL-10 but reduced IFN-γ, as compared to controls. These observations suggest a skewed Th2 response, diminished IFN-γ secreting Th1-TEM cells and increased central and effector memory subtype of Th2, Th17 and Treg cells in the vaccinated mice. These data indicate that LmjF.36.3850 is a plausible virulence factor that enhances disease-promoting response, possibly by interfering with PKC activation and by eliciting disease-promoting T cells.

Functional partnership between carbonic anhydrase and malic enzyme in promoting gluconeogenesis in Leishmania major.

Leishmania has a remarkable ability to proliferate under widely fluctuating levels of essential nutrients, such as glucose. For this, the parasite is heavily dependent on its gluconeogenic machinery. One perplexing aspect of gluconeogenesis in Leishmania is the lack of the crucial gene for pyruvate carboxylase (PC). PC-catalyzed conversion of pyruvate to oxaloacetate is a key entry point through which gluconeogenic amino acids are funneled into this pathway. The absence of PC in Leishmania thus raises question about the mechanism of pyruvate entry into the gluconeogenic route. In the present study, we report that this task is accomplished in Leishmania major through a novel functional partnership between its mitochondrial malic enzyme (LmME) and carbonic anhydrase 1 (LmCA1). Using a combination of pharmacological inhibition studies with genetic manipulation, we show that both of these enzymes are necessary for promoting gluconeogenesis and supporting parasite growth under glucose-limiting conditions. Functional cross-talk between LmME and LmCA1 was evident when it was observed that the growth retardation caused by inhibition of any one of these enzymes could be protected to a significant extent by overexpressing the other enzyme. We also found that, although LmCA1 exhibited constitutive expression, the LmME protein level was strongly upregulated under low glucose conditions. Notably, both LmME and LmCA1 were found to be important for survival of Leishmania amastigotes within host macrophages. Taken together, our results indicate that LmCA1 by virtue of its CO2 concentrating ability stimulates LmME-catalyzed pyruvate carboxylation, thereby driving gluconeogenesis through the pyruvate-malate-oxaloacetate bypass pathway. Additionally, our study establishes LmCA1 and LmME as promising therapeutic targets.

Characterization of a new Leishmania major strain for use in a controlled human infection model.

Leishmaniasis is widely regarded as a vaccine-preventable disease, but the costs required to reach pivotal Phase 3 studies and uncertainty about which candidate vaccines should be progressed into human studies significantly limits progress in vaccine development for this neglected tropical disease. Controlled human infection models (CHIMs) provide a pathway for accelerating vaccine development and to more fully understand disease pathogenesis and correlates of protection. Here, we describe the isolation, characterization and GMP manufacture of a new clinical strain of Leishmania major. Two fresh strains of L. major from Israel were initially compared by genome sequencing, in vivo infectivity and drug sensitivity in mice, and development and transmission competence in sand flies, allowing one to be selected for GMP production. This study addresses a major roadblock in the development of vaccines for leishmaniasis, providing a key resource for CHIM studies of sand fly transmitted cutaneous leishmaniasis.

The role of dermis resident macrophages and their interaction with neutrophils in the early establishment of Leishmania major infection transmitted by sand fly bite.

There is substantial experimental evidence to indicate that Leishmania infections that are transmitted naturally by the bites of infected sand flies differ in fundamental ways from those initiated by needle inocula. We have used flow cytometry and intravital microscopy (IVM) to reveal the heterogeneity of sand fly transmission sites with respect to the subsets of phagocytes in the skin that harbor L. major within the first hours and days after infection. By flow cytometry analysis, dermis resident macrophages (TRMs) were on average the predominant infected cell type at 1 hr and 24 hr. By confocal IVM, the co-localization of L. major and neutrophils varied depending on the proximity of deposited parasites to the presumed site of vascular damage, defined by the highly localized swarming of neutrophils. Some of the dermal TRMs could be visualized acquiring their infections via transfer from or efferocytosis of parasitized neutrophils, providing direct evidence for the "Trojan Horse" model. The role of neutrophil engulfment by dermal TRMs and the involvement of the Tyro3/Axl/Mertk family of receptor tyrosine kinases in these interactions and in sustaining the anti-inflammatory program of dermal TRMs was supported by the effects observed in neutrophil depleted and in Axl-/-Mertk-/- mice. The Axl-/-Mertk-/- mice also displayed reduced parasite burdens but more severe pathology following L. major infection transmitted by sand fly bite.

Application of CRISPR/Cas9-Based Reverse Genetics in Leishmania braziliensis: Conserved Roles for HSP100 and HSP23.

The protozoan parasite Leishmania (Viannia) braziliensis (L. braziliensis) is the main cause of human tegumentary leishmaniasis in the New World, a disease affecting the skin and/or mucosal tissues. Despite its importance, the study of the unique biology of L. braziliensis through reverse genetics analyses has so far lagged behind in comparison with Old World Leishmania spp. In this study, we successfully applied a cloning-free, PCR-based CRISPR-Cas9 technology in L. braziliensis that was previously developed for Old World Leishmania major and New World L. mexicana species. As proof of principle, we demonstrate the targeted replacement of a transgene (eGFP) and two L. braziliensis single-copy genes (HSP23 and HSP100). We obtained homozygous Cas9-free HSP23- and HSP100-null mutants in L. braziliensis that matched the phenotypes reported previously for the respective L. donovani null mutants. The function of HSP23 is indeed conserved throughout the Trypanosomatida as L. majorHSP23 null mutants could be complemented phenotypically with transgenes from a range of trypanosomatids. In summary, the feasibility of genetic manipulation of L. braziliensis by CRISPR-Cas9-mediated gene editing sets the stage for testing the role of specific genes in that parasite's biology, including functional studies of virulence factors in relevant animal models to reveal novel therapeutic targets to combat American tegumentary leishmaniasis.

Assessment of Incubation Period of Cutaneous Leishmaniasis due to Leishmania major in Tunisia.

The period between the infective sandfly bites and appearance of cutaneous leishmaniasis (CL) lesions is still hypothetical and little studied. This work aimed at assessing the incubation time of zoonotic CL (ZCL) due to Leishmania major using a standardized methodology. The retrospective analysis used the epidemiological, clinical, and biological information available in the database recording all the CL cases diagnosed at the Parasitology Department of the Pasteur Institute of Tunis during 2015-2019. It allowed for the selection of 92 privileged observations 1) of confirmed CL cases with presentation suggestive of ZCL form 2) living in northern regions free of ZCL 3) with a single infective trip of less than a week to ZCL foci during transmission season and 4) with accurate dates of travel and onset of lesions. Incubation length computed in this population ranged from 1 to 21 weeks, with a median of 5 weeks (interquartile range: 3-8.5 weeks).

Sterol 14-α-demethylase is vital for mitochondrial functions and stress tolerance in Leishmania major.

Sterol 14-α-demethylase (C14DM) is a key enzyme in the biosynthesis of sterols and the primary target of azoles. In Leishmania major, genetic or chemical inactivation of C14DM leads to accumulation of 14-methylated sterol intermediates and profound plasma membrane abnormalities including increased fluidity and failure to maintain ordered membrane microdomains. These defects likely contribute to the hypersensitivity to heat and severely reduced virulence displayed by the C14DM-null mutants (c14dm‾). In addition to plasma membrane, sterols are present in intracellular organelles. In this study, we investigated the impact of C14DM ablation on mitochondria. Our results demonstrate that c14dm‾ mutants have significantly higher mitochondrial membrane potential than wild type parasites. Such high potential leads to the buildup of reactive oxygen species in the mitochondria, especially under nutrient-limiting conditions. Consistent with these mitochondrial alterations, c14dm‾ mutants show impairment in respiration and are heavily dependent on glucose uptake and glycolysis to generate energy. Consequently, these mutants are extremely sensitive to glucose deprivation and such vulnerability can be rescued through the supplementation of glucose or glycerol. In addition, the accumulation of oxidants may also contribute to the heat sensitivity exhibited by c14dm‾. Finally, genetic or chemical ablation of C14DM causes increased susceptibility to pentamidine, an antimicrobial agent with activity against trypanosomatids. In summary, our investigation reveals that alteration of sterol synthesis can negatively affect multiple cellular processes in Leishmania parasites and make them vulnerable to clinically relevant stress conditions.

Chemical inhibition of ß-glucocerebrosidase does not affect phagocytosis and early containment of Leishmania by murine macrophages.

Gaucher disease is a lysosomal storage disease in which a genetic deficiency in ß-glucocerebrosidase leads to the accumulation of glycosphingolipids in lysosomes. Macrophages are amongst the cells most severely affected in Gaucher disease patients. One phenotype associated with Gaucher macrophages is the impaired capacity to fight bacterial infections. Here, we investigate whether inhibition of ß-glucocerebrosidase activity affects the capacity of macrophages to phagocytose and act on the early containment of human pathogens of the genus Leishmania. Towards our aim, we performed in vitro infection assays on macrophages derived from the bone marrow of C57BL/6 mice. To mimic Gaucher disease, macrophages were incubated with the ß-glucocerebrosidase inhibitor, conduritol B epoxide (CBE), prior to contact with Leishmania. This treatment guaranteed that ß-glucocerebrosidase was fully inhibited during the contact of macrophages with Leishmania, its enzymatic activity being progressively recovered along the 48 h that followed removal of the inhibitor. Infections were performed with L. amazonensis, L. infantum, or L. major, so as to explore potential species-specific responses in the context of ß-glucocerebrosidase inactivation. Parameters of infection, recorded immediately after phagocytosis, as well as 24 and 48 h later, revealed no noticeable differences in the infection parameters of CBE-treated macrophages relative to non-treated controls. We conclude that blocking ß-glucocerebrosidase activity during contact with Leishmania does not interfere with the phagocytic capacity of macrophages and the early onset of leishmanicidal responses.

Exploring Lutzomyia longipalpis Sand Fly Vector Competence for Leishmania major Parasites.

Lutzomyia longipalpis sand flies are the major natural vector of Leishmania infantum parasites, responsible for transmission of visceral leishmaniasis in the New World. Several experimental studies have demonstrated the ability of Lu. longipalpis to sustain development of different Leishmania species. However, no study had explored in depth the potential vector competence of Lu. longipalpis for Leishmania species other than L. infantum. Here, we show that Lu. longipalpis is a competent vector of L. major parasites, being able to acquire parasites from active cutaneous leishmaniasis lesions, sustain mature infections, and transmit them to naive hosts, causing disease.

Development and Characterization of an Avirulent Leishmania major Strain.

Leishmania major causes cutaneous leishmaniasis. An antileishmanial vaccine for humans is unavailable. In this study, we report development of two attenuated L. major strains-5ASKH-HP and LV39-HP-by continuous culture (high passage) of the corresponding virulent strains (low passage). Both avirulent strains showed similar changes in proteome profiles when analyzed by surface-enhanced laser desorption ionization mass spectrometry. Liquid chromatography-mass spectrometry and microarray characterization of 5ASKH strains revealed substantially altered gene and protein expression profiles, respectively. Both virulent and avirulent L. major strains grew comparably in culture, but the avirulent strain survived significantly less in BALB/c-derived peritoneal macrophages. Both attenuated strains failed to infect BALB/c mice and elicited IFN-γ, but not IL-4 and IL-10, responses. 5ASKH-HP parasites failed to induce significant infection even in severely immunocompromised- SCID or inducible NO synthase-, CD40-, or IL-12-deficient mice, indicating attenuation. The avirulent strain induced less IL-10, but higher IL-12, in macrophages. The avirulent strain failed to reduce CD40 relocation to the detergent-resistant membrane domain and to inhibit CD40-induced phosphorylation of the kinases Lyn and protein kinase C-ß and MAPKs MKK-3/6 and p38MAPK or to upregulate MEK-1/2 and ERK-1/2 in BALB/c-derived peritoneal macrophages. The virulent and the avirulent strains reciprocally modulated CD40-induced Ras-mediated signaling through PI-3K and Raf-1. Avirulent 5ASKH-primed BALB/c mice were protected against virulent L. major challenge infection. The loss of virulence accompanied by substantially altered proteome profiles and the elicitation of host-protective immune responses indicate plausibly irreversible attenuation of the L. major strain and its potential use as a vaccine strain.

Pathological roles of MRP14 in anemia and splenomegaly during experimental visceral leishmaniasis.

Myeloid-related protein 14 (MRP14) belongs to the S100 calcium-binding protein family and is expressed in neutrophils and inflammatory macrophages. Increase in the number of MRP14+ cells or serum level of MRP14 is associated with various diseases such as autoimmune diseases and infectious diseases, suggesting the involvement of the molecule in pathogenesis of those diseases. In this study, to examine the pathological involvement of MRP14 during cutaneous and visceral leishmaniasis, wild-type (WT) and MRP14 knockout (MRP14KO) mice were infected with Leishmania major and L. donovani. Increase in the number of MRP14+ cells at the infection sites in wild-type mice was commonly found in the skin during L. major infection as well as the spleen and liver during L. donovani infection. In contrast, the influence of MRP14 to the pathology seemed different between the two infections. MRP14 depletion exacerbated the lesion development and ulcer formation in L. major infection. On the other hand, the depletion improved anemia and splenomegaly but not hepatomegaly at 24 weeks of L. donovani infection. These results suggest that, distinct from its protective role in CL, MRP14 is involved in exacerbation of some symptoms during VL.

Aryl Hydrocarbon Receptor-Signaling Regulates Early Leishmania major-Induced Cytokine Expression.

The early inflammatory skin micromilieu affects resistance in experimental infection with Leishmania major. We pursue the concept that macrophages, which take up parasites during early infection, exert decisive influence on the inflammatory micromilieu after infection. In order to analyze their distinctive potential, we identified differentially regulated genes of murine granuloma macrophages (GMΦ) from resistant and susceptible mice after their infection with metacyclic Leishmania major. We found induction of several cytokines in GMΦ from both strains and a stronger upregulation of the transcription factor aryl hydrocarbon receptor (AhR) in GMΦ from resistant mice. Using both an AhR agonist and antagonist we demonstrated that AhR is involved in Leishmania-induced production of TNF in macrophages. In vivo, single local injection of an AhR agonist in early lesions of susceptible mice caused an increased induction of Tnf and other cytokines in the skin. Importantly, local agonist treatment led to a reduction of disease severity, reduced parasite loads and a weaker Th2 response. Our results demonstrate that local activation of AhR has a beneficial effect in experimental leishmaniasis.

Association of Phlebotomus guggisbergi with Leishmania major and Leishmania tropica in a complex transmission setting for cutaneous leishmaniasis in Gilgil, Nakuru county, Kenya.

BACKGROUND: Phlebotomus (Larroussius) guggisbergi is among the confirmed vectors for cutaneous leishmaniasis (CL) transmission in Kenya. This scarring and stigmatizing form of leishmaniasis accounts for over one million annual cases worldwide. Most recent CL epidemics in Kenya have been reported in Gilgil, Nakuru County, where the disease has become a public health issue. However, little is known about the factors that drive its transmission. Here, we sought to determine the occurrence, distribution and host blood feeding preference of the vectors, and to identify Leishmania species and infection rates in sandflies using molecular techniques. This information could lead to a better understanding of the disease transmission and improvement of control strategies in the area. METHODOLOGY/ PRINCIPAL FINDINGS: An entomological survey of sandflies using CDC light traps was conducted for one week per month in April 2016, and in June and July 2017 from five villages of Gilgil, Nakuru county; Jaica, Sogonoi, Utut, Gitare and Njeru. Sandflies were identified to species level using morphological keys and further verified by PCR analysis of cytochrome c oxidase subunit I (COI) gene. Midguts of female sandflies found to harbour Leishmania were ruptured and the isolated parasites cultured in Novy-MacNeal-Nicolle (NNN) media overlaid with Schneider's insect media to identify the species. Leishmania parasite screening and identification in 198 randomly selected Phlebotomus females and parasite cultures was done by PCR-RFLP analysis of ITS1 gene, nested kDNA-PCR and real-time PCR-HRM followed by sequencing. Bloodmeal source identification was done by real-time PCR-HRM of the vertebrate cytochrome-b gene. A total of 729 sandflies (males: n = 310; females: n = 419) were collected from Utut (36.6%), Jaica (24.3%), Sogonoi (34.4%), Njeru (4.5%), and Gitare (0.1%). These were found to consist of nine species: three Phlebotomus spp. and six Sergentomyia spp. Ph. guggisbergi was the most abundant species (75.4%, n = 550) followed by Ph. saevus sensu lato (11.3%, n = 82). Sandfly species distribution across the villages was found to be significantly different (p<0.001) with Jaica recording the highest diversity. The overall Leishmania infection rate in sandflies was estimated at 7.07% (14/198). Infection rates in Ph. guggisbergi and Ph. saevus s.l. were 9.09% (12/132) and 3.57% (2/56) respectively. L. tropica was found to be the predominant parasite in Gilgil with an overall infection rate of 6.91% (13/188) in Ph. guggisbergi (n = 11) and Ph. saevus s.l. (n = 2) sandflies. However, PCR analysis also revealed L. major infection in one Ph. guggisbergi specimen. Bloodmeal analysis in the 74 blood-fed sandflies disclosed a diverse range of vertebrate hosts in Ph. guggisbergi bloodmeals, while Ph. saevus s.l. fed mainly on humans. CONCLUSIONS/ SIGNIFICANCE: The high infection rates of L. tropica and abundance of Ph. guggisbergi in this study confirms this sandfly as a vector of L. tropica in Kenya. Furthermore, isolation of live L. tropica parasites from Ph. saevus s.l. suggest that there are at least three potential vectors of this parasite species in Gilgil; Ph. guggisbergi, Ph. aculeatus and Ph. saevus s.l. Molecular identification of L. major infections in Ph. guggisbergi suggested this sandfly species as a potential permissive vector of L. major, which needs to be investigated further. Sandfly host preference analysis revealed the possibility of zoonotic transmissions of L. tropica in Gilgil since the main vector (Ph. guggisbergi) does not feed exclusively on humans but also other vertebrate species. Further investigations are needed to determine the potential role of these vertebrate species in L. tropica and L. major transmission in the area.