A Metabolism-Based Quorum Sensing Mechanism Contributes to Termination of Inflammatory Responses.

Recruitment of immune cells with antimicrobial activities is essential to fight local infections but has the potential to trigger immunopathology. Whether the immune system has the ability to sense inflammation intensity and self-adjust accordingly to limit tissue damage remains to be fully established. During local infection with an intracellular pathogen, we have shown that nitric oxide (NO) produced by recruited monocyte-derived cells was essential to limit inflammation and cell recruitment. Mechanistically, we have provided evidence that NO dampened monocyte-derived cell cytokine and chemokine production by inhibiting cellular respiration and reducing cellular ATP:ADP ratio. Such metabolic control operated at the tissue level but only when a sufficient number of NO-producing cells reached the site of infection. Thus, NO production and activity act as a quorum sensing mechanism to help terminate the inflammatory response.

NLRP1-dependent activation of Gasdermin D in neutrophils controls cutaneous leishmaniasis.

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.

Localized Leishmania major infection disrupts systemic iron homeostasis that can be controlled by oral iron supplementation.

Leishmania parasites are heavily dependent on efficient iron acquisition from a tightly regulated host iron pool for survival and virulence. Prior studies uncovered multiple strategies adopted by the parasite to hijack the iron-regulatory network of macrophages. Despite these extensive studies with infected macrophages, there is limited knowledge of the effect of Leishmania infection on systemic iron homeostasis. This issue is particularly relevant for Leishmania major, which causes localized skin infection with minimal lymphatic spread. We show for the first time that L. major infection in the mouse footpad induced influx of iron at the site of infection through blood with simultaneous upregulation of transferrin receptor 1 and downregulation of phagolysosomal iron exporter Nramp1 expression in the footpad tissue. Interestingly, localized L. major infection had far-reaching effects beyond the infection site triggering anemia-like symptoms. This was evident from depleted physiological iron stores from the liver and bone marrow as well as reduced hemoglobin levels and deformed erythrocytes. The infected mice also developed splenomegaly with signs of splenic stress erythropoiesis as indicated by upregulation of several erythroid-related genes. These observations prompted us to provide oral iron supplementations to the L. major-infected mice, which resulted in a drastic reduction of the parasite load and restoration of iron homeostasis.

The c-MET receptor tyrosine kinase contributes to neutrophil-driven pathology in cutaneous leishmaniasis.

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.

Interplay of Trypanosome Lytic Factor and innate immune cells in the resolution of cutaneous Leishmania infection.

Trypanosome Lytic Factor (TLF) is a primate-specific high-density lipoprotein (HDL) complex that, through the cation channel-forming protein apolipoprotein L-1 (APOL1), provides innate immunity to select kinetoplastid parasites. The immunoprotective effects of TLF have been extensively investigated in the context of its interaction with the extracellular protozoan Trypanosoma brucei brucei, to which it confers sterile immunity. We previously showed that TLF could act against an intracellular pathogen Leishmania, and here we dissected the role of TLF and its synergy with host-immune cells. Leishmania major is transmitted by Phlebotomine sand flies, which deposit the parasite intradermally into mammalian hosts, where neutrophils are the predominant phagocytes recruited to the site of infection. Once in the host, the parasites are phagocytosed and shed their surface glycoconjugates during differentiation to the mammalian-resident amastigote stage. Our data show that mice producing TLF have reduced parasite burdens when infected intradermally with metacyclic promastigotes of L. major, the infective, fly-transmitted stage. This TLF-mediated reduction in parasite burden was lost in neutrophil-depleted mice, suggesting that early recruitment of neutrophils is required for TLF-mediated killing of L. major. In vitro we find that only metacyclic promastigotes co-incubated with TLF in an acidic milieu were lysed. However, amastigotes were not killed by TLF at any pH. These findings correlated with binding experiments, revealing that labeled TLF binds specifically to the surface of metacyclic promastigotes, but not to amastigotes. Metacyclic promastigotes of L. major deficient in the synthesis of surface glycoconjugates LPG and/or PPG (lpg1- and lpg5A-/lpg5B- respectively) whose absence mimics the amastigote surface, were resistant to TLF-mediated lysis. We propose that TLF binds to the outer surface glycoconjugates of metacyclic promastigotes, whereupon it kills the parasite in the acidic phagosome of phagocytes. We hypothesize that resistance to TLF requires shedding of the surface glycoconjugates, which occurs several hours after phagocytosis by immune cells, creating a relatively short-lived but effective window for TLF to act against Leishmania.

Toll-like receptor 2 selectively modulates Ras isoforms expression in Leishmania major infection.

Leishmania infection of macrophages results in altered Ras isoforms expression and Toll-like receptor-2 (TLR2) expression and functions. Therefore, we examined whether TLR2 would selectively alter Ras isoforms' expression in macrophages. We observed that TLR2 ligands- Pam3CSK4, peptidoglycan (PGN), and FSL- selectively modulated the expression of Ras isoforms in BALB/c-derived elicited macrophages. Lentivirally-expressed TLR1-shRNA significantly reversed this Ras isoforms expression profile. TLR2-deficient L. major-infected macrophages and the lymph node cells from the L. major-infected mice showed similarly reversed Ras isoforms expression. Transfection of the macrophages with the siRNAs for the adaptors- Myeloid Differentiation factor 88 (MyD88) and Toll-Interleukin-1 Receptor (TIR) domain-containing adaptor protein (TIRAP)- or Interleukin-1 Receptor-Associated Kinases (IRAKs)- IRAK1 and IRAK4- significantly inhibited the L. major-induced down-regulation of K-Ras, and up-regulation of N-Ras and H-Ras, expression. The TLR1/TLR2-ligand Pam3CSK4 increased IL-10 and TGF-ß expression in macrophages. Pam3CSK4 upregulated N-Ras and H-Ras, but down-regulated K-Ras, expression in C57BL/6 wild-type, but not in IL-10-deficient, macrophages. IL-10 or TGF-ß signaling inhibition selectively regulated Ras isoforms expression. These observations indicate the specificity of the TLR2 regulation of Ras isoforms and their selective modulation by MyD88, TIRAP, and IRAKs, but not IL-10 or TGF-ß, signaling.

Semaphorin 3E Promotes Susceptibility to Leishmania major Infection in Mice by Suppressing CD4+ Th1 Cell Response.

Protective immunity to cutaneous leishmaniasis is mediated by IFN-γ-secreting CD4+ Th1 cells. IFN-γ binds to its receptor on Leishmania-infected macrophages, resulting in their activation, production of NO, and subsequent destruction of parasites. This study investigated the role of Semaphorin 3E (Sema3E) in host immunity to Leishmania major infection in mice. We observed a significant increase in Sema3E expression at the infection site at different timepoints following L. major infection. Sema3E-deficient (Sema3E knockout [KO]) mice were highly resistant to L. major infection, as evidenced by significantly (p < 0.05-0.01) reduced lesion sizes and lower parasite burdens at different times postinfection when compared with their infected wild-type counterpart mice. The enhanced resistance of Sema3E KO mice was associated with significantly (p < 0.05) increased IFN-γ production by CD4+ T cells. CD11c+ cells from Sema3E KO mice displayed increased expression of costimulatory molecules and IL-12p40 production following L. major infection and were more efficient at inducing the differentiation of Leishmania-specific CD4+ T cells to Th1 cells than their wild-type counterpart cells. Furthermore, purified CD4+ T cells from Sema3E KO mice showed increased propensity to differentiate into Th1 cells in vitro, and this was significantly inhibited by the addition of recombinant Sema3E in vitro. These findings collectively show that Sema3E is a negative regulator of protective CD4+ Th1 immunity in mice infected with L. major and suggest that its neutralization may be a potential therapeutic option for treating individuals suffering from cutaneous leishmaniasis.

Genetic Iron Overload Hampers Development of Cutaneous Leishmaniasis in Mice.

The survival, growth, and virulence of Leishmania spp., a group of protozoan parasites, depends on the proper access and regulation of iron. Macrophages, Leishmania's host cell, may divert iron traffic by reducing uptake or by increasing the efflux of iron via the exporter ferroportin. This parasite has adapted by inhibiting the synthesis and inducing the degradation of ferroportin. To study the role of iron in leishmaniasis, we employed Hjv-/- mice, a model of hemochromatosis. The disruption of hemojuvelin (Hjv) abrogates the expression of the iron hormone hepcidin. This allows unrestricted iron entry into the plasma from ferroportin-expressing intestinal epithelial cells and tissue macrophages, resulting in systemic iron overload. Mice were injected with Leishmania major in hind footpads or intraperitoneally. Compared with wild-type controls, Hjv-/- mice displayed transient delayed growth of L. major in hind footpads, with a significant difference in parasite burden 4 weeks post-infection. Following acute intraperitoneal exposure to L. major, Hjv-/- peritoneal cells manifested increased expression of inflammatory cytokines and chemokines (Il1b, Tnfa, Cxcl2, and Ccl2). In response to infection with L. infantum, the causative agent of visceral leishmaniasis, Hjv-/- and control mice developed similar liver and splenic parasite burden despite vastly different tissue iron content and ferroportin expression. Thus, genetic iron overload due to hemojuvelin deficiency appears to mitigate the early development of only cutaneous leishmaniasis.

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.

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.

Galectin-3 plays a protective role in Leishmania (Leishmania) amazonensis infection.

Leishmania (L.) amazonensis is one of the species responsible for the development of cutaneous leishmaniasis in South America. After entering the vertebrate host, L. (L.) amazonensis invades mainly neutrophils, macrophages and dendritic cells. Studies have shown that gal-3 acts as a pattern recognition receptor. However, the role of this protein in the context of L. (L.) amazonensis infection remains unclear. Here, we investigated the impact of gal-3 expression on experimental infection by L. (L.) amazonensis. Our data showed that gal-3 plays a role in controlling parasite invasion, replication and the formation of endocytic vesicles. Moreover, mice with gal-3 deficiency showed an exacerbated inflammatory response. Taken together, our data shed light to a critical role of gal-3 in the host response to infection by L. (L.) amazonensis.

TLR2 dimer-specific ligands selectively activate protein kinase C isoforms in Leishmania infection.

Of the thirteen Toll-like receptors (TLRs) in mice, TLR2 has a unique ability of forming heterodimers with TLR1 and TLR6. Such associations lead to selective cellular signalling and cellular responses such as cytokine expression. One of the signalling intermediates is protein kinase C (PKC); of which, eight isoforms are expressed in macrophages. Leishmania-a protozoan parasite that resides and replicates in macrophages-selectively modulates PKC-α, PKC-ß, PKC-δ and PKC-ζ isoforms in macrophages. As TLR2 plays significant roles in Leishmania infection, we examined whether these PKC isoforms play selective roles in TLR2 signalling and TLR2-induced anti-leishmanial functions. We observed that the TLR2 ligands-Pam3 CSK4 (TLR1/2), PGN (TLR2/2) and FSL (TLR2/6)-differentially phosphorylated and translocated PKC-α, PKC-ß, PKC-δ and PKC-ζ isoforms to cell membrane in uninfected and L. major-infected macrophages. The PKC isoform-specific inhibitors differentially altered IL-10 and IL-12 expression, Th1 and Th2 responses and anti-leishmanial effects in macrophages and in BALB/c mice. While PKC isoforms' inhibitors had insignificant effects on the Pam3CSK4-induced anti-leishmanial functions, PGN-induced pro-leishmanial effects were enhanced by PKC-(α + ß) inhibitors, whereas PKC-(δ + Î¶) inhibitors enhanced the anti-leishmanial effects of FSL. These results indicated that the ligand-induced TLR2 dimerization triggered differential dose-dependent and kinetic profiles of PKC isoform activation and that selective targeting of PKC isoforms using their respective inhibitors in combination significantly modulated TLR2-induced anti-leishmanial functions. To the best of our knowledge, this is the first demonstration of TLR2 dimer signalling through PKC isoforms and TLR2-induced PKC isoform-targeted anti-leishmanial therapy.

Transcriptomic landscape of skin lesions in cutaneous leishmaniasis reveals a strong CD8+ T cell immunosenescence signature linked to immunopathology.

The severity of lesions that develop in patients infected by Leishmania braziliensis is mainly associated with a highly cytotoxic and inflammatory cutaneous environment. Recently, we demonstrated that senescent T and NK cells play a role in the establishment and maintenance of this tissue inflammation. Here, we extended those findings using transcriptomic analyses that demonstrate a strong co-induction of senescence and pro-inflammatory gene signatures in cutaneous leishmaniasis (CL) lesions. The senescence-associated signature was characterized by marked expression of key genes such as ATM, Sestrin 2, p16, p21 and p38. The cell type identification from deconvolution of bulk sequencing data showed that the senescence signature was linked with CD8+ effector memory and TEMRA subsets and also senescent NK cells. A key observation was that the senescence markers in the skin lesions are age-independent of patients and were correlated with lesion size. Moreover, a striking expression of the senescence-associated secretory phenotype (SASP), pro-inflammatory cytokine and chemokines genes was found within lesions that were most strongly associated with the senescent CD8 TEMRA subset. Collectively, our results confirm that there is a senescence transcriptomic signature in CL lesions and supports the hypothesis that lesional senescent cells have a major role in mediating immunopathology of the disease.

Cholesterol depletion associated with Leishmania major infection alters macrophage CD40 signalosome composition and effector function.

CD40, a costimulatory molecule expressed on macrophages, induces expression of interleukin 12 (IL-12) in uninfected macrophages and IL-10 in macrophages infected with Leishmania major. IL-12 suppresses, whereas IL-10 enhances, L. major infection. The mechanisms that regulate this difference in CD40-induced cytokine production remain unclear, but it is known that L. major depletes cholesterol. Here we show that cholesterol influenced the assembly of distinct CD40 signalosomes. Depletion of membrane cholesterol inhibited the assembly of an IL-12-inducing CD40 signalosome containing the adaptors TRAF2, TRAF3 and TRAF5 and the kinase Lyn and promoted the assembly of an IL-10-inducing CD40 signalosome containing the adaptor TRAF6 and the kinase Syk. Thus, cholesterol depletion might represent an immune-evasion strategy used by L. major.

The different faces of the NLRP3 inflammasome in cutaneous Leishmaniasis: A review.

Cutaneous leishmaniasis (CL) is a vector-borne parasitic disease caused by Protozoa of the genus Leishmania. Clinical manifestations of this disease are the result of a complex interplay of diverse factors, including the genetic background and the immune status of the host. Understanding the impact of these factors on the CL pathology may provide new targets to manage the infection and improve clinical outcome. The NLRP3 inflammasome, an innate immune complex of several cell types, seems to be involved in the CL physiopathology. Current studies of its role show contradictory effects of this complex on the evolution of Leishmania infection in mice and humans. In this review, we discuss the data regarding different roles of the NLRP3 inflammasome in murine and human CL.

Interleukin-11 receptor expression on monocytes is dispensable for their recruitment and pathogen uptake during Leishmania major infection.

Interleukin-11 (IL-11) is an important member of the IL-6 family of cytokines. IL-11 activates its target cells via binding to a non-signaling α-receptor (IL-11R), which results in recruitment and activation of a gp130 homodimer. The cytokine was initially described as an anti-inflammatory protein, but has recently gained attention as a potent driver in certain types of cancer and different fibrotic conditions. Leishmania spp. are a group of eukaryotic parasites that cause the disease leishmaniasis. They infect phagocytes of their hosts, especially monocytes recruited to the site of infection, and are able to replicate within this rather harsh environment, often resulting in chronic infections of the patient. However, the molecular mechanisms underlying parasite and host cell interactions and factors of the immune cells that are crucial for Leishmania uptake are so far largely unspecified. Recently, increased IL-11 expression in the lesions of patients with cutaneous leishmaniasis has been reported, but the functional relevance is unknown. In this study, we show that monocytes express IL-11R on their cell surface. Furthermore, using an adoptive transfer model of IL-11R-/- monocytes, we analyze the contribution of IL-11 signaling on monocyte recruitment and monocyte infection in a mouse model of cutaneous leishmaniasis and find that IL-11 signaling is dispensable for monocyte recruitment and pathogen uptake during Leishmania major infection.

Resolution of Cutaneous Leishmaniasis and Persistence of Leishmania major in the Absence of Arginase 1.

Arginase (Arg) 1 is expressed by hematopoietic (e.g., macrophages) and nonhematopoietic cells (e.g., endothelial cells) and converts l-arginine into ornithine and urea. The enzyme is implicated in tissue repair but also antagonizes the production of NO by type 2 NO synthase in myeloid cells and thereby impedes the control of intracellular parasites such as Leishmania major In this study, we tested whether Arg1 is required for spontaneous healing of acute cutaneous leishmaniasis in C57BL/6 mice and for lifelong parasite persistence in draining lymph nodes (dLNs) of healed mice. In vitro, bone marrow-derived macrophages and lymphoid endothelial cells readily expressed Arg1 in response to IL-4 and/or IL-13, whereas skin or dLN fibroblasts failed to do so, even during hypoxia. In vivo, Arg1 was found in skin lesions and, to a much lower extent, also in dLNs of acutely infected C57BL/6 mice but became undetectable at both sites after healing. Deletion of Arg1 in hematopoietic and endothelial cells using Tie2Cre+/-Arg1fl/fl C57BL/6 mice abolished the expression of Arg1 in skin lesions and dLNs but did not affect development and resolution of skin lesions, parasite burden, NO production, or host cell tropism of L. major during the acute or persistent phase of infection. Similar to wild-type controls, parasites persisting in Arg1-deficient mice favored NO synthase 2-negative areas and mainly resided in myeloid cells and fibroblasts. We conclude that Arg1 expression by hematopoietic and endothelial cells is completely dispensable for clinical resolution of cutaneous leishmaniasis and for long-term persistence of L. major.

Discovery and Validation of Lmj_04_BRCT Domain, a Novel Therapeutic Target: Identification of Candidate Drugs for Leishmaniasis.

Since many of the currently available antileishmanial treatments exhibit toxicity, low effectiveness, and resistance, search and validation of new therapeutic targets allowing the development of innovative drugs have become a worldwide priority. This work presents a structure-based drug discovery strategy to validate the Lmj_04_BRCT domain as a novel therapeutic target in Leishmania spp. The structure of this domain was explored using homology modeling, virtual screening, and molecular dynamics studies. Candidate compounds were validated in vitro using promastigotes of Leishmania major, L. amazonensis, and L. infantum, as well as primary mouse macrophages infected with L. major. The novel inhibitor CPE2 emerged as the most active of a group of compounds against Leishmania, being able to significantly reduce the viability of promastigotes. CPE2 was also active against the intracellular forms of the parasites and significantly reduced parasite burden in murine macrophages without exhibiting toxicity in host cells. Furthermore, L. major promastigotes treated with CPE2 showed significant lower expression levels of several genes (α-tubulin, Cyclin CYCA, and Yip1) related to proliferation and treatment resistance. Our in silico and in vitro studies suggest that the Lmj_04_BRCT domain and its here disclosed inhibitors are new potential therapeutic options against leishmaniasis.

Deletion of transketolase triggers a stringent metabolic response in promastigotes and loss of virulence in amastigotes of Leishmania mexicana.

Transketolase (TKT) is part of the non-oxidative branch of the pentose phosphate pathway (PPP). Here we describe the impact of removing this enzyme from the pathogenic protozoan Leishmania mexicana. Whereas the deletion had no obvious effect on cultured promastigote forms of the parasite, the Δtkt cells were not virulent in mice. Δtkt promastigotes were more susceptible to oxidative stress and various leishmanicidal drugs than wild-type, and metabolomics analysis revealed profound changes to metabolism in these cells. In addition to changes consistent with those directly related to the role of TKT in the PPP, central carbon metabolism was substantially decreased, the cells consumed significantly less glucose, flux through glycolysis diminished, and production of the main end products of metabolism was decreased. Only minor changes in RNA abundance from genes encoding enzymes in central carbon metabolism, however, were detected although fructose-1,6-bisphosphate aldolase activity was decreased two-fold in the knock-out cell line. We also showed that the dual localisation of TKT between cytosol and glycosomes is determined by the C-terminus of the enzyme and by engineering different variants of the enzyme we could alter its sub-cellular localisation. However, no effect on the overall flux of glucose was noted irrespective of whether the enzyme was found uniquely in either compartment, or in both.

TNF contributes to T-cell exhaustion in chronic L. mexicana infections of mice through PD-L1 up-regulation.

Leishmania mexicana can produce chronic infections leading to exhausted T cell phenotypes, mediated by PD-1/PD-L1. Little is known on mechanisms that induce these inhibitory molecules in chronic leishmaniasis. We analyzed factors that contribute to exhausted phenotypes in chronic L. mexicana infections of mice. Our results show that draining lymph node cells express enhanced levels of PD-1/PD-L1. T lymphocytes producing low cytokine levels were also found. L. mexicana infection of dendritic cells (DCs) produced elevated amounts of TNF and showed up-regulation of PD-L1 expression. We provide evidence that T cells of chronic L. mexicana infections in mice are functionally exhausted due to chronic TNF production, which leads to PD-L1 up-regulation in DCs. We conclude that TNF has a fundamental role in promoting T cell exhaustion during chronic L. mexicana infections, which contributes to the inability of T cells to proliferate and produce pro-inflammatory cytokines, thus favoring disease progression.