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Inflammation has a role in the pathogenesis of childhood malnutrition. We investigated the effect of malnutrition and inflammatory challenge on bone marrow composition and bone health. We studied an established murine model of moderate acute malnutrition at baseline and after acute inflammatory challenge with bacterial lipopolysaccharide (LPS), a surrogate of Gram-negative bacterial sepsis, or Leishmania donovani, the cause of visceral leishmaniasis. Both of these infections cause significant morbidity and mortality in malnourished children. Of the 2 stimuli, LPS caused more pronounced bone marrow changes that were amplified in malnourished mice. LPS challenge led to increased inflammatory cytokine expression (Il1b, Il6, and Tnf), inflammasome activation, and inflammatory monocyte accumulation in the bone marrow of malnourished mice. Depletion of inflammatory monocytes in Csfr1-LysMcre-DT malnourished mice significantly reduced the inflammasome activation and IL1-ß production after LPS challenge. The inflammatory challenge also led to increased expansion of mesenchymal stem cells (MSCs), bone marrow adiposity, and expression of genes (Pparg, Adipoq, and Srbp1) associated with adipogenesis in malnourished mice. This suggests that inflammatory challenge promotes differentiation of BM MSCs toward the adipocyte lineage rather than toward bone-forming osteoblasts in the malnourished host. Concurrent with this reduced osteoblastic potential there was an increase in bone-resorbing osteoclasts, enhanced osteoclast activity, upregulation of inflammatory genes, and IL-1B involved in osteoclast differentiation and activation. The resulting weakened bone formation and increased bone resorption would contribute to the bone fragility associated with malnutrition. Lastly, we evaluated the effect of replacing lipid rich in omega-6 fatty acids (corn oil) with lipid-rich in omega-3 fatty acids (fish oil) in the nutrient-deficient diet. LPS-challenged malnourished mice that received dietary fish oil showed decreased expression of inflammatory cytokines and Rankl and reduced osteoclast differentiation and activation in the bone marrow. This work demonstrates that the negative effect of inflammatory challenge on bone marrow is amplified in the malnourished host. Increasing dietary intake of omega-3 fatty acids may be a means to reduce inflammation and improve bone health in malnourished children.

Acute malnutrition, or wasting, is implicated in over half of all deaths in children under five and increases risk of infectious disease. Studies in humans and preclinical models have demonstrated that malnutrition is linked to an immature intestinal microbiota characterized by increased prevalence of Enterobacteriaceae. Observational studies in children with moderate acute malnutrition (MAM) have also observed heightened systemic inflammation and increased circulating bacterial lipopolysaccharides (LPS; endotoxin). However, the mechanisms that underpin the systemic inflammatory state and endotoxemia, and their pathophysiological consequences, remain uncertain. Understanding these pathophysiological mechanisms is necessary to design targeted treatments that will improve the unacceptable rate of failure or relapse that plague current approaches. Here we use a mouse model of MAM to investigate the mechanisms that promote inflammation in the malnourished host. We found that mice with MAM exhibited increased systemic inflammation at baseline, increased translocation of bacteria and bacterial LPS, and an exaggerated response to inflammatory stimuli. An exaggerated response to bacterial LPS was associated with increased acute weight loss. Remarkably, intestinal inflammation and barrier dysfunction was found in the cecum and colon. The cecum showed a dysbiotic microbiota with expansion of Gammaproteobacteria and some Firmicutes, and contraction of Bacteroidetes. These changes were paralleled by an increase in fecal LPS bioactivity. The inflammatory phenotype and weight loss was modulated by oral administration of non-absorbable antibiotics that altered the proportion of cecal Gammaproteobacteria. We propose that the heightened inflammation of acute malnutrition is the result of changes in the intestinal microbiota, intestinal barrier dysfunction in the cecum and colon, and increased systemic exposure to LPS.

Acute malnutrition affects more than 50 million children worldwide. These children are at an increased risk of morbidity and mortality from infectious disease. However, the pathogenesis of acute malnutrition and mechanisms underlying the increased risk and poor outcomes from infection are not well understood. Our objective was to identify differences in inflammation and inflammatory responses between children with moderate acute malnutrition (MAM) and healthy controls (HCs), and search for environmental, pathophysiological, and metabolic factors that may influence this response. Sixteen children with MAM and 16 HCs aged 18–36 months were studied in Nairobi, Kenya. None of the children had symptoms of an infectious disease (fever, diarrhea, or cough) in the 2 weeks before enrollment and sample collection. Demographic and health data were provided by their primary caregivers. Blood samples were collected to measure various biomarkers and the response to an inflammatory stimulus. Children with MAM were more frequently from households with contaminated water, crowding, and unstable income sources. They also had increases in basal inflammation, circulating bacterial lipopolysaccharide (LPS), markers of intestinal damage, and an exaggerated whole blood inflammatory response to LPS. Metabolic changes in children with MAM led to increased plasma levels of long-chain fatty acids, which were found to contribute to the pro-inflammatory state. These exploratory findings suggest convergence of multiple factors to promote dysregulated inflammatory responses and prompt several mechanistic hypotheses that can be pursued to better understand the pathogenesis of MAM.

Peromyscus yucatanicus, the main reservoir of Leishmania (Leishmania) mexicana in the Yucatan peninsula of Mexico, reproduces clinical and histological pictures of LCL in human as well as subclinical infection. Thus, we used this rodent as a novel experimental model. In this work, we analyzed cytokine mRNA expression in P. yucatanicus infected with L. (L.) mexicana. Animals were inoculated with either 2.5×10(6) or 1×10(2) promastigotes and cytokine expressions were analyzed by real-time RT-PCR in skin at 4 and 12weeks post-infection (wpi). Independently of the parasite inoculum none of the infected rodents had clinical signs of LCL at 4wpi and all expressed high IFN-γ mRNA. All P. yucatanicus inoculated with 2.5×10(6) promastigotes developed signs of LCL at 12wpi while the mice inoculated with 1×10(2) remained subclinical. At that time, both IFN-γ and IL-10 were expressed in P. yucatanicus with clinical and subclinical infections. Expressions of TNF-α and IL-4 were significantly higher in clinical animals (2.5×10(6)) compared with subclinical ones (1×10(2)). High TGF-β expression was observed in P. yucatanicus with clinical signs when compared with healthy animals. Results suggested that the clinical course of L. (L.) mexicana infection in P. yucatanicus was associated with a specific local pattern of cytokine production at 12wpi.

Current drug therapies for cutaneous leishmaniasis are often difficult to administer and treatment failure is an increasingly common occurrence. The efficacy of anti-leishmanial therapy relies on a combination of anti-parasite activity of drugs and the patient’s immune response. Previous studies have reported in vitro antimicrobial activity of histamine 1-receptor antagonists (H1RAs) against different pathogens. We used an ex vivo explant culture of lymph nodes from mice infected with Leishmania major to screen H1RAs compounds. Azelastine (AZ) and Fexofenadine (FX) showed remarkable ex vivo efficacy (EC50 = 0.05 and 1.50 μM respectively) and low in vitro cytotoxicity yielding a high therapeutic index. AZ significantly decreased the expression of H1R and the proinflammatory cytokine IL-1ẞ in the ex vivo system, which were shown to be augmented by histamine addition. The anti-leishmanial efficacy of AZ was enhanced in the presence of T cells from infected mice suggesting an immune-modulatory mechanism of parasite suppression. L. major infected BALB/c mice treated per os with FX or intralesionally with AZ showed a significant reduction of lesion size (FX = 69%; AZ = 52%). Furthermore, there was significant parasite suppression in the lesion (FX = 82%; AZ = 87%) and lymph nodes (FX = 81%; AZ = 36%) with no observable side effects. AZ and FX and potentially other H1RAs are good candidates for assessing efficacy in larger studies as monotherapies or in combination with current anti-leishmanial drugs to treat cutaneous leishmaniasis., Author summary Cutaneous leishmaniasis (CL) is a parasitic disease present in more than 90 countries. Different species of Leishmania produce skin ulcers upon infection through the bite of infected sand fly vectors. There are several drugs used to treat CL but most of them are toxic or difficult to administer and there is increasing drug resistance leading to treatment failure. Therefore, new drugs are needed for treating CL. The objective of this study was to determine the anti-leishmanial efficacy of antihistamine drugs. Using cell cultures of lymph nodes obtained from Leishmania major infected mice, we evaluated the parasiticidal activity of the antihistamine drugs azelastine and fexofenadine. Both drugs showed high efficacy against L. major and low toxicity for a human cell line. Treatment of mice infected in the skin with L. major indicated that both azelastine and fexofenadine significantly reduced the size of the lesions and suppressed parasite multiplication. Consequently, these two drugs are good candidates to further evaluate their efficacy as monotherapies or in combination with other anti-leishmanial drugs.

Summary Leishmania parasites are the causative agents of leishmaniasis, a neglected tropical disease that causes substantial morbidity and considerable mortality in many developing areas of the world. Recent estimates suggest that roughly 10 million people suffer from cutaneous leishmaniasis (CL), and approximately 76 000 are afflicted with visceral leishmaniasis (VL), which is universally fatal without treatment. Efforts to develop therapeutics and vaccines have been greatly hampered by an incomplete understanding of the parasite's biology and a lack of clear protective correlates that must be met in order to achieve immunity. Although parasites grow and divide preferentially in macrophages, a number of other cell types interact with and internalize Leishmania parasites, including monocytes, dendritic cells and neutrophils. Neutrophils appear to be especially important shortly after parasites are introduced into the skin, and may serve a dual protective and permissive role during the establishment of infection. Curiously, neutrophil recruitment to the site of infection appears to continue into the chronic phase of disease, which may persist for many years. The immunological impact of these cells during chronic leishmaniasis is unclear at this time. In this review we discuss the ways in which neutrophils have been observed to prevent and promote the establishment of infection, examine the role of anti-neutrophil antibodies in mouse models of leishmaniasis and consider recent findings that neutrophils may play a previously unrecognized role in influencing chronic parasite persistence.

SUMMARY The global impact of childhood malnutrition is staggering. The synergism between malnutrition and infection contributes substantially to childhood morbidity and mortality. Anthropometric indicators of malnutrition are associated with the increased risk and severity of infections caused by many pathogens, including viruses, bacteria, protozoa, and helminths. Since childhood malnutrition commonly involves the inadequate intake of protein and calories, with superimposed micronutrient deficiencies, the causal factors involved in impaired host defense are usually not defined. This review focuses on literature related to impaired host defense and the risk of infection in primary childhood malnutrition. Particular attention is given to longitudinal and prospective cohort human studies and studies of experimental animal models that address causal, mechanistic relationships between malnutrition and host defense. Protein and micronutrient deficiencies impact the hematopoietic and lymphoid organs and compromise both innate and adaptive immune functions. Malnutrition-related changes in intestinal microbiota contribute to growth faltering and dysregulated inflammation and immune function. Although substantial progress has been made in understanding the malnutrition-infection synergism, critical gaps in our understanding remain. We highlight the need for mechanistic studies that can lead to targeted interventions to improve host defense and reduce the morbidity and mortality of infectious diseases in this vulnerable population.

Leishmaniasis is a vector-borne zoonotic infection affecting people in tropical and subtropical regions of the world. Current treatments for cutaneous leishmaniasis are difficult to administer, toxic, expensive, and limited in effectiveness and availability. Here we describe the development and application of a medium-throughput screening approach to identify new drug candidates for cutaneous leishmaniasis using an e x vivo l ymph node e xplant c ulture (ELEC) derived from the draining lymph nodes of Leishmania major -infected mice. The ELEC supported intracellular amastigote proliferation and contained lymph node cell populations (and their secreted products) that enabled the testing of compounds within a system that mimicked the immunopathological environment of the infected host, which is known to profoundly influence parasite replication, killing, and drug efficacy. The activity of known antileishmanial drugs in the ELEC system was similar to the activity measured in peritoneal macrophages infected in vitro with L. major . Using the ELEC system, we screened a collection of 334 compounds, some of which we had demonstrated previously to be active against L. donovani , and identified 119 hits, 85% of which were confirmed to be active by determination of the 50% effective concentration (EC 50 ). We found 24 compounds (7%) that had an i n v itro t herapeutic i ndex (IVTI; 50% cytotoxic/effective concentration [CC 50 ]/EC 50 ) > 100; 19 of the compounds had an EC 50 below 1 μM. According to PubChem searchs, 17 of those compounds had not previously been reported to be active against Leishmania . We expect that this novel method will help to accelerate discovery of new drug candidates for treatment of cutaneous leishmaniasis.

We evaluated the importance of neutrophils in the development of chronic lesions caused by L. Viannia spp. using the hamster as experimental model of American Cutaneous Leishmaniasis (ACL). Neutrophils infiltrated the lesion within the first six hours post-infection. Inhibition of this early infiltration using a polyclonal antibody or cyclophosphamide was associated with transient parasite control but the protective effect vanished when lesions became clinically apparent. At lesion onset (approximately 10 days p.i.), there was an increased proportion of both uninfected and infected macrophages, and subsequently a second wave of neutrophils infiltrated the lesion (after 19 days p.i.) This second neutrophil infiltration was associated with lesion necrosis and ulceration (R2 = 0.75) and maximum parasite burden. Intradermal delivery of N-formylmethionyl-leucyl-phenylalanine (fMLP), aimed to increase neutrophil infiltration, resulted in larger lesions with marked necrosis and higher parasite burden than in mock treated groups (p

The low expression of iNOS in hamsters is related to a region of the proximal promoter that lacks an NF-IL6 binding element. IFN-γ/LPS-activated hamster (Mesocricetus auratus) macrophages express significantly less iNOS (NOS2) than activated mouse macrophages, which contributes to the hamsterˈs susceptibility to intracellular pathogens. We determined a mechanism responsible for differences in iNOS promoter activity in hamsters and mice. The HtPP (1.2 kb) showed low basal and inducible promoter activity when compared with the mouse, and sequences within a 100-bp region (−233 to −133) of the mouse and hamster promoters influenced this activity. Moreover, within this 100 bp, we identified a smaller region (44 bp) in the mouse promoter, which recovered basal promoter activity when swapped into the hamster promoter. The mouse homolog (100-bp region) contained a cis-element for NF-IL-6 (−153/−142), which was absent in the hamster counterpart. EMSA and supershift assays revealed that the hamster sequence did not support the binding of NF-IL-6. Introduction of a functional NF-IL-6 binding sequence into the hamster promoter or its alteration in the mouse promoter revealed the critical importance of this transcription factor for full iNOS promoter activity. Furthermore, the binding of NF-IL-6 to the iNOS promoter (−153/−142) in vivo was increased in mouse cells but was reduced in hamster cells after IFN-γ/LPS stimulation. Differences in the activity of the iNOS promoters were evident in mouse and hamster cells, so they were not merely a result of species-specific differences in transcription factors. Thus, we have identified unique DNA sequences and a critical transcription factor, NF-IL-6, which contribute to the overall basal and inducible expression of hamster iNOS.

We postulated that CCR2-driven activation of the transcription factor NF-κB plays a critical role in dendritic cell (DC) maturation (e.g., migration, costimulation, and IL-12p70 production), necessary for the generation of protective immune responses against the intracellular pathogen Leishmania major. Supporting this notion, we found that CCR2, its ligand CCL2, and NF-κB were required for CCL19 production and adequate Langerhans cell (LC) migration both ex vivo and in vivo. Furthermore, a role for CCR2 in upregulating costimulatory molecules was indicated by the reduced expression of CD80, CD86, and CD40 in Ccr2−/− bone marrow-derived dendritic cells (BMDCs) compared with wild-type (WT) BMDCs. Four lines of evidence suggested that CCR2 plays a critical role in the induction of protective immunity against L. major by regulating IL-12p70 production and migration of DC populations such as LCs. First, compared with WT, Ccr2−/− lymph node cells, splenocytes, BMDCs, and LCs produced lower levels of IL-12p70 following stimulation with LPS/IFN-γ or L. major. Second, a reduced number of LCs carried L. major from the skin to the draining lymph nodes in Ccr2−/− mice compared with WT mice. Third, early treatment with exogenous IL-12 reversed the susceptibility to L. major infection in Ccr2−/− mice. Finally, disruption of IL-12p70 in radioresistant cells, such as LCs, but not in BMDCs resulted in the inability to mount a fully protective immune response in bone marrow chimeric mice. Collectively, our data point to an important role for CCR2-driven activation of NF-κB in the regulation of DC/LC maturation processes that regulate protective immunity against intracellular pathogens.

The maintenance of host defense dur- ing pregnancy may depend on heightened innate immunity. We evaluated the immune response of pregnant hamsters during early infection with Leishmania (Viannia) panamensis, a cause of American cutaneous leishmaniasis. At 7 days post-infection, pregnant animals showed a lower parasite burden compared with nonpregnant controls at the cutaneous infection site (P 0.0098) and draining lymph node (P0.02). Resident peritoneal macrophages and neutro- phils from pregnant animals had enhanced Leish- mania killing capacity compared with nonpreg- nant controls (P0.018 each). This enhanced resistance during pregnancy was associated with increased expression of inducible NO synthase (iNOS) mRNA in lymph node cells (P0.02) and higher NO production by neutrophils (P 0.0001). Macrophages from nonpregnant ham- sters infected with L. panamensis released high amounts of NO upon estrogen exposure (P0.05), and addition of the iNOS inhibitor L-N6-(1-iminoethyl) lysine blocked the induction of NO production (P0.02). Infected, nonpreg- nant females treated with estrogen showed a higher percentage of cells producing NO at the infection site than controls (P0.001), which correlated with lower parasite burdens (P 0.036). Cultured macrophages or neutrophils from estrogen-treated hamsters showed signifi- cantly increased NO production and Leishmania killing compared with untreated controls. iNOS was identified as the likely source of estrogen- induced NO in primed and naive macrophages, as increased transcription was evident by real- time PCR. Thus, the innate defense against Leishmania infection is heightened during preg- nancy, at least in part as a result of estrogen- mediated up-regulation of iNOS expression and NO production. J. Leukoc. Biol. 83: 1413-1422; 2008.

Cutaneous and mucosal leishmaniasis is widely distributed in Central and South America. Leishmania of the Viannia subgenus are the most frequent species infecting humans. L. (V.) braziliensis, L. (V.) panamensis are also responsible for metastatic mucosal leishmaniasis. Conventional or real time PCR is a more sensitive diagnostic test than microscopy, but the cost and requirement for infrastructure and trained personnel makes it impractical in most endemic regions. Primary health systems need a sensitive and specific point of care (POC) diagnostic tool. We developed a novel POC molecular diagnostic test for cutaneous leishmaniasis caused by Leishmania (Viannia) spp. Parasite DNA was amplified using isothermal Recombinase Polymerase Amplification (RPA) with primers and probes that targeted the kinetoplast DNA. The amplification product was detected by naked eye with a lateral flow (LF) immunochromatographic strip. The RPA-LF had an analytical sensitivity equivalent to 0.1 parasites per reaction. The test amplified the principal L. Viannia species from multiple countries: L. (V.) braziliensis (n = 33), L. (V.) guyanensis (n = 17), L. (V.) panamensis (n = 9). The less common L. (V.) lainsoni, L. (V.) shawi, and L. (V.) naiffi were also amplified. No amplification was observed in parasites of the L. (Leishmania) subgenus. In a small number of clinical samples (n = 13) we found 100% agreement between PCR and RPA-LF. The high analytical sensitivity and clinical validation indicate the test could improve the efficiency of diagnosis, especially in chronic lesions with submicroscopic parasite burdens. Field implementation of the RPA-LF test could contribute to management and control of cutaneous and mucosal leishmaniasis., Author Summary Cutaneous leishmaniasis is a parasitic disease transmitted by the bite of sandflies that produces skin ulcers. The severe, disfiguring form of the disease is characterized by parasite dissemination to the mucosa of the nose and palate. Current diagnosis is based on microscopy which has low sensitivity in chronic cases. Molecular methods (PCR) that detect parasite DNA are highly sensitive but costs and personnel training make it impossible to implement it in resource-limited areas. We developed a novel molecular method (RPA-LF) that could be applied in the field because it does not require sophisticated equipment. It is also very sensitive and specific to detect the principal Leishmania species that produce cutaneous leishmaniasis in Latin America. Future field implementation of RPA-LF could have a positive impact on disease management and control.

This unit focuses on the murine model of cutaneous leishmaniasis and models of visceral leishmaniasis in mice and hamsters. Each basic protocol describes the methods used to inoculate parasites and to evaluate infections with regard to lesion progression and visceralization, and quantification of parasite load. Five support protocols are provided; two for the growth and isolation of metacyclic promastigotes from in vitro culture, one for isolation of tissue amastigotes from infected animals, one for cryopreservation of parasites, and one for the preparation of blood agar plates for quantitation of parasite numbers in infected tissue.

The host factors that influence autoimmune arthritides such as rheumatoid arthritis have not been fully elucidated. We previously found that genetic inactivation of CC chemokine receptor 2 (CCR2) in the arthritis-prone DBA/1j mouse strain significantly increases the susceptibility of this strain to autoimmune arthritis induced by immunization with collagen type II (CII) and complete Freund's adjuvant (CFA). Here, we show that following intradermal infection with Mycobacterium avium, a similar arthritis phenotype was detected in Ccr2-null mice in the DBA/1j, but not in the BALB/c background. The failure to develop arthritis in Ccr2-null BALB/c mice occurred in the face of high bacterial burdens and low interferon gamma (IFNgamma) production. By contrast, Ccr2-null DBA/1j mice had low bacterial burdens, produced normal amounts of IFNgamma, and had high titers of autoantibodies against CII. Thus, the Ccr2-null state in an arthritic-prone genetic background leads to increased arthritis susceptibility following infectious (M. avium) and noninfectious (CII/CFA) challenges. Because CCR2 serves as a negative regulator of murine arthritis, caution might need to be exercised while testing CCR2 blockers in human arthritis or other diseases. These findings also indicate that Ccr2-null DBA/1j mice might serve as a valuable model system to uncover the immunological determinants of arthritis and to test novel antiarthritic agents.

Purpose of review The leishmaniases, caused by protozoan parasites of the genus Leishmania, are a significant health problem in many regions of the world. This review highlights the recent advances in the study of leishmaniasis related to parasite biology, disease pathogenesis, clinical evaluation and treatment, and prevention. Recent findings Genetic heterogeneity and clonal diversity is common among Leishmania strains. Gene knockout, overexpression, and re-introduction studies have identified a number of genes that play a role in parasite virulence. Surprisingly, the importance of the surface lipophosphoglycan in parasite virulence appears to differ among Leishmania spp. Studies in experimental animal models have further defined the roles of CD4 and CD8 T cells, IL-4, IL-10, and IL-12 in the control, maintenance, or progression of disease. The effect of Leishmania on dendritic cells and macrophage effector function has also been an important area of investigation. A number of new vaccine candidates have been identified through experimental animal studies. Clinical studies of leishmaniasis have focused on the host determinants of disease (most notably HIV co-infection), serological and DNA-based diagnostic assays, and treatment. Antimony-resistant cases of cutaneous and visceral leishmaniasis have become more common; liposomal amphotericin and oral miltefosine are promising alternative therapies. Summary Significant advances have been made in the areas of pathogenesis, host defence, and treatment of leishmaniasis. A number of new vaccine candidates and potential targets of drug therapy have been identified, but progress from preclinical studies to clinical trials has been slow. Translational research, built upon the solid foundation of existing and ongoing basic investigation, is a high priority.

The different cutaneous leishmaniases are distinct in their etiology, epidemiology, transmission, and geographical distribution. In most instances cutaneous leishmaniasis is limited to one or a few skin ulcers that develop at the site where the parasites were deposited during the bite of the sandfly vector. Lesions typically heal spontaneously after several months but some lesions can be large and follow a chronic, more severe course. Protective immunity is usually acquired following cutaneous infection with Leishmania spp., so prevention of disease through prophylactic immunization appears to be feasible. Since vaccination with live, virulent parasites is associated with an unacceptable rate of adverse events, attention has turned to the use of killed or attenuated parasite vaccines and defined subunit vaccines. Whole parasite vaccines have the advantage of delivering multiple antigenic epitopes that may be necessary for initiation of a broad-based immune response. Persistent or repeated immune-stimulation by parasite antigens and/or sustained expression of interleukin-12 appear to be critical elements in the development of durable immunity. A number of purified or recombinant antigens, when co-administered with a vaccine adjuvant, appear promising as vaccine candidates against cutaneous leishmaniasis. The sustained expression of recombinant Leishmania antigens by vaccination with DNA is an attractive approach because it mimics the persistent antigenic stimulation of subclinical infection. Effective vaccine-induced immunity must generate an antigen-specific memory T cell population that, upon exposure to the infecting parasite, rapidly produces a type 1 effector T cell response that leads to interferon-gamma-mediated activation of infected macrophages to kill the intracellular parasites. This parasite-directed recall response must be prompt and of sufficient magnitude to overcome the subversive effect that the intracellular infection has on macrophage effector function. It is unlikely that vaccination against cutaneous leishmaniasis would induce sterile immunity, but a small number of parasites are likely to persist subclinically.

Malnutrition is thought to contribute to more than one-third of all childhood deaths via increased susceptibility to infection. Malnutrition is a significant risk factor for the development of visceral leishmaniasis, which results from skin inoculation of the intracellular protozoan Leishmania donovani . We previously established a murine model of childhood malnutrition and found that malnutrition decreased the lymph node barrier function and increased the early dissemination of L. donovani . In the present study, we found reduced numbers of resident dendritic cells (conventional and monocyte derived) but not migratory dermal dendritic cells in the skin-draining lymph nodes of L. donovani -infected malnourished mice. Expression of chemokines and their receptors involved in trafficking of dendritic cells and their progenitors to the lymph nodes was dysregulated. C-C chemokine receptor type 2 (CCR2) and its ligands (CCL2 and CCL7) were reduced in the lymph nodes of infected malnourished mice, as were CCR2-bearing monocytes/macrophages and monocyte-derived dendritic cells. However, CCR7 and its ligands (CCL19 and CCL21) were increased in the lymph node and CCR7 was increased in lymph node macrophages and dendritic cells. CCR2-deficient mice recapitulated the profound reduction in the number of resident (but not migratory dermal) dendritic cells in the lymph node but showed no alteration in the expression of CCL19 and CCL21. Collectively, these results suggest that the malnutrition-related reduction in the lymph node barrier to dissemination of L. donovani is related to insufficient numbers of lymph node-resident but not migratory dermal dendritic cells. This is likely driven by the altered activity of the CCR2 and CCR7 chemoattractant pathways.

Host arginase 1 (arg1) expression is a significant contributor to the pathogenesis of progressive visceral leishmaniasis (VL), a neglected tropical disease caused by the intracellular protozoan Leishmania donovani. Previously we found that parasite-induced arg1 expression in macrophages was dependent on STAT6 activation. Arg1 expression was amplified by, but did not require, IL-4, and required de novo synthesis of unknown protein(s). To further explore the mechanisms involved in arg1 regulation in VL, we screened a panel of kinase inhibitors and found that inhibitors of growth factor signaling reduced arg1 expression in splenic macrophages from hamsters with VL. Analysis of growth factors and their signaling pathways revealed that the Fibroblast Growth Factor Receptor 1 (FGFR-1) and Insulin-like Growth Factor 1 Receptor (IGF-1R) and a number of downstream signaling proteins were activated in splenic macrophages isolated from hamsters infected with L. donovani. Recombinant FGF-2 and IGF-1 increased the expression of arg1 in L. donovani infected hamster macrophages, and this induction was augmented by IL-4. Inhibition of FGFR-1 and IGF-1R decreased arg1 expression and restricted L. donovani replication in both in vitro and ex vivo models of infection. Inhibition of the downstream signaling molecules JAK and AKT also reduced the expression of arg1 in infected macrophages. STAT6 was activated in infected macrophages exposed to either FGF-2 or IGF-1, and STAT6 was critical to the FGFR-1- and IGF-1R-mediated expression of arg1. The converse was also true as inhibition of FGFR-1 and IGF-1R reduced the activation of STAT6 in infected macrophages. Collectively, these data indicate that the FGFR/IGF-1R and IL-4 signaling pathways converge at STAT6 to promote pathologic arg1 expression and intracellular parasite survival in VL. Targeted interruption of these pathological processes offers an approach to restrain this relentlessly progressive disease., Author Summary Visceral leishmaniasis (VL), caused by the intracellular protozoan Leishmania donovani, is a progressive infection that is particularly common in impoverished populations of the world. People die from this disease unless it is treated. We used an experimental infection model that mimics the clinical and pathological features of human VL to study how the parasite causes this severe disease. We found that host macrophages infected with Leishmania donovani are activated in a way that leads to the expression of arginase, an enzyme that counteracts the cell's mechanisms that control the infection. This disease-promoting activation pathway was driven by the convergence of growth factor and cytokine signaling pathways and activation of the transcription factor STAT6. Chemical inhibition of signaling through the fibroblast growth factor receptor-1 (FGFR-1) or insulin-like growth factor-1 receptor (IGF-IR), or genetic knockdown of STAT6 led to reduced expression of arginase and enhanced control of the infection by macrophages. This indicates that the growth factor signaling pathways together with the cytokine pathways promote this disease. Interventions designed to disrupt this signaling could help in the treatment of VL.

The acquisition of immunity following subclinical or resolved infection with the intracellular parasiteLeishmania donovanisuggests that vaccination could prevent visceral leishmaniasis (VL). The LACK (Leishmaniahomolog of receptors for activated C kinase) antigen is of interest as a vaccine candidate for the leishmaniases because of its immunopathogenic role in murineL. majorinfection. Immunization of mice with a truncated (24-kDa) version of the 36-kDa LACK antigen, delivered in either protein or DNA form, was found previously to protect against cutaneousL. majorinfection by redirecting the early T-cell response away from a pathogenic interleukin-4 (IL-4) response and toward a protective Th1 response. The amino acid sequence of theLeishmaniap36(LACK) antigen is highly conserved, but the efficacy of this vaccine antigen in preventing disease caused by strains other thanL. majorhas not been determined. We investigated the efficacy of a p36(LACK) DNA vaccine against VL because of the serious nature of this form of leishmaniasis and because it was unclear whether the LACK vaccine would be effective in a model where there was not a dominant pathogenic IL-4 response. We demonstrate here that although the LACK DNA vaccine induced a robust parasite-specific Th1 immune response (IFN-γ but not IL-4 production) and primed for an in vivo T-cell response to inoculated parasites, it did not induce protection against cutaneous or systemicL. donovanichallenge. Coadministration of IL-12 DNA with the vaccine did not enhance the strong vaccine-induced Th1 response or augment a protective effect.

Active human visceral leishmaniasis (VL) is characterized by a progressive increase in visceral parasite burden, cachexia, massive splenomegaly, and hypergammaglobulinemia. In contrast, mice infected with Leishmania donovani, the most commonly studied model of VL, do not develop overt, progressive disease. Furthermore, mice control Leishmania infection through the generation of NO, an effector mechanism that does not have a clear role in human macrophage antimicrobial function. Remarkably, infection of the Syrian hamster (Mesocricetus auratus) with L. donovani reproduced the clinicopathological features of human VL, and investigation into the mechanisms of disease in the hamster revealed striking differences from the murine model. Uncontrolled parasite replication in the hamster liver, spleen, and bone marrow occurred despite a strong Th1-like cytokine (IL-2, IFN-γ, and TNF/lymphotoxin) response in these organs, suggesting impairment of macrophage effector function. Indeed, throughout the course of infection, inducible NO synthase (iNOS, NOS2) mRNA or enzyme activity in liver or spleen tissue was not detected. In contrast, NOS2 mRNA and enzyme activity was readily detected in the spleens of infected mice. The impaired hamster NOS2 expression could not be explained by an absence of the NOS2 gene, overproduction of IL-4, defective TNF/lymphotoxin production (a potent second signal for NOS2 induction), or early dominant production of the deactivating cytokines IL-10 and TGF-β. Thus, although a Th1-like cytokine response was prominent, the major antileishmanial effector mechanism that is responsible for control of infection in mice was absent throughout the course of progressive VL in the hamster.

The prevailing paradigm is that production of the interleukin (IL)-12 p70 heterodimer, a critical T helper cell type 1 (Th1)–inducing cytokine, depends on the induced transcription of the p40 subunit. Concordant with this paradigm, we found that dendritic cells (DCs) produced IL-12 p70 only after at least 2–4 h of stimulation with lipopolysaccharide plus interferon γ. However, using several complementary experimental approaches, including electron and confocal microscopy, we now show that resting murine and human myeloid cells, including macrophages/DCs and DC-rich tissues, contain a novel source of bioactive IL-12 that is preformed and membrane associated. These preformed, membrane-associated IL-12 p70 stores are released within minutes after in vitro or in vivo contact with Leishmania donovani, an intracellular pathogen. Our findings highlight a novel source of bioactive IL-12 that is readily available for the rapid initiation of Th1 host responses to pathogens such as Leishmania species.

There is growing evidence that chemokines and their receptors regulate the movement and interaction of antigen-presenting cells such as dendritic cells (DCs) and T cells. We tested the hypothesis that the CC chemokine receptor (CCR)2 and CCR5 and the chemokine macrophage inflammatory protein (MIP)-1α, a ligand for CCR5, influence DC migration and localization. We found that deficiency of CCR2 but not CCR5 or MIP-1α led to distinct defects in DC biology. Langerhans cell (skin DC) density in CCR2-null mice was normal, and their ability to migrate into the dermis was intact; however, their migration to the draining lymph nodes was markedly impaired. CCR2-null mice had lower numbers of DCs in the spleen, and this was primarily due to a reduction in the CD8α1 T helper cell type 1 (Th1)-inducing subset of DCs. Additionally, there was a block in the Leishmania major infection–induced relocalization of splenic DCs from the marginal zone to the T cell areas. We propose that these DC defects, in conjunction with increased expression of B lymphocyte chemoattractant, a B cell–specific chemokine, may collectively contribute to the striking B cell outgrowth and Th2 cytokine–biased nonhealing phenotype that we observed in CCR2-deficient mice infected with L. major. This disease phenotype in mice with an L. major–resistant genetic background but lacking CCR2 is strikingly reminiscent of that observed typically in mice with an L. major–susceptible genetic background. Thus, CCR2 is an important determinant of not only DC migration and localization but also the development of protective cell-mediated immune responses to L. major.

SUMMARYWe have previously demonstrated induction and high level expression of IL-1β, IL-6 and tumour necrosis factor-alpha in the myocardium during the acute stage of experimental Trypanosoma cruzi infection (Chagas' disease). The myocardial depressive effects of these cytokines are mediated in part by the induction of nitric oxide synthase (NOS), production of nitric oxide (NO) and formation of peroxynitrite. In this study we investigated the expression, activity and localization of NOS isoforms, and the levels of NO, malondialdehyde (a measure of oxidative stress), and peroxynitrite in rats at 1·5, 5, 10 and 15 days after infection with T. cruzi trypomastigotes. The myocardial inflammatory infiltrate and number of amastigote nests increased over the course of infection. A significant increase in tissue nitrate + nitrite levels, NOS2 mRNA, and NOS2 enzyme activity was observed at all time points in the infected compared with uninfected animals. The enzyme activity of constitutive NOS, tissue malondialdehyde levels, and NOS3 mRNA levels was only transiently increased after infection. The protein levels of the NOS isoforms paralleled their mRNA expression. While no positive nitrotyrosine immunoreactivity was detected in control myocardium, its levels increased in infected animals over time. Thus, by 1·5 days post-infection, when no parasite or immune cell infiltration could be detected, the myocardium expressed high levels of NOS and NO metabolites. Nevertheless, the early production of NO in the myocardium was not sufficient to clear the parasites.

Current therapy for leishmaniasis is unsatisfactory. Efficacious and safe oral therapy would be ideal. We examined the efficacy of SCH 56592, an investigational triazole antifungal agent, against cutaneous infection with Leishmania amazonensis and visceral infection with Leishmania donovani in BALB/c mice. Mice were infected in the ear pinna and tail with L. amazonensis promastigotes and were treated with oral SCH 56592 or intraperitoneal amphotericin B for 21 days. At doses of 60 and 30 mg/kg/day, SCH 56592 was highly efficacious in treating cutaneous disease, and at a dose of 60 mg/kg/day, it was superior to amphotericin B at a dose of 1 mg/kg/day. The means of tail lesion sizes were 0.32 ± 0.12, 0.11 ± 0.06, 0.17 ± 0.07, and 0.19 ± 0.08 mm for controls, SCH 56592 at 60 and 30 mg/kg/day, and amphotericin B recipients, respectively ( P = 0.0003, 0.005, and 0.01, respectively). Parasite burden in draining lymph nodes confirmed these efficacy findings. In visceral leishmaniasis due to L. donovani infection, mice treated with SCH 56592 showed a 0.5- to 1-log-unit reduction in parasite burdens in the liver and the spleen compared to untreated mice. Amphotericin B at 1 mg/kg/day was superior to SCH 56592 in the treatment of visceral infection, with a 2-log-unit reduction in parasite burdens in both the liver and spleen. These studies indicate very good activity of SCH 56592 against cutaneous leishmaniasis due to L. amazonensis infection and, to a lesser degree, against visceral leishmaniasis due to L. donovani infection in susceptible BALB/c mice.

We investigated the immune responses in mice lacking CCR2, CCR5, or macrophage inflammatory protein-1α (MIP-1α), a ligand for CCR5, in two situations: following T cell stimulation or after challenge with Leishmania donovani, an intracellular microbe whose control is dependent on a Th1 immune response. Mice deficient in CCR5, MIP-1α, or CCR2 had reduced IFN-γ responses following ligation of the TCR. Reduced IFN-γ responses following PMA and ionomycin were also observed in CD8+ T cells of CCR5−/− and CCR2−/− mice. During the early phases of infection, all three knockout mice had low Ag-specific IFN-γ responses. However, this reduced IFN-γ response was overcome during a state of persistent Ag stimulation (chronic infection), and was not associated with an adverse parasitologic outcome in any of the gene-targeted mouse strains. To the contrary, during the late phase of infection, an exaggerated Ag-specific IFN-γ response was evident in CCR5−/− and MIP-1α−/− mice, and this correlated with an enhanced control of parasite replication. Although granuloma formation was abnormal in each of the knockout mice, there was no correlation between the number or architecture of the granulomas and parasite burden. Collectively, these findings indicate an important role for CCR5, MIP-1α, and CCR2 in granulomatous inflammation, and that CCR5 and MIP-1α, possibly acting through CCR5, might play a deleterious role in the outcome of chronic L. donovani infection. Our data also suggest that there might be cross-talk between TCR and chemokine receptor signaling pathways.

Infections with intracellular pathogens such as Leishmania donovani and Mycobacterium tuberculosis pose serious health problems worldwide. Effective vaccines for these pathogens are not available. Furthermore, despite optimal therapy, disease progression is often seen with several intracellular infections. For these reasons, we initiated studies to develop novel anti-infective vaccine and treatment strategies that couple the potent Ag-presenting capacity of dendritic cells (DC) with paracrine delivery of potent anti-infective cytokines such as IL-12 to local immune response sites. We tested this strategy in a murine model of visceral leishmaniasis. Adoptive transfer of DCs pulsed ex vivo with soluble L. donovani Ags (SLDA) to naive mice induced the Ag-specific production of IFN-γ, and increased the percentage of activation markers on spleen lymphocytes. SLDA-pulsed DCs engineered by retroviral gene transfer techniques to secrete high levels of biologically active murine IL-12 augmented this immune response further. In several different vaccination and immunotherapy protocols, compared with sham-treated mice, animals receiving SLDA-pulsed DCs either before or following infection had 1–3 log lower parasite burdens, and this protection was associated with a pronounced enhancement in the parasite-specific IFN-γ response. The augmentation of this protection by IL-12-engineered DCs was striking. First, live parasites were not detected in the liver of mice vaccinated with IL-12-transduced, SLDA-pulsed DCs. Second, this parasitological response was associated with a nearly normal liver histology. In contrast, parasites and granulomas were found in mice vaccinated with SLDA-pulsed, nontransduced DCs. Collectively, these studies provide the rationale for the development of potent DC-based immunotherapies.

Current therapy for leishmaniasis is unsatisfactory because parenteral antimonial salts and pentamidine are associated with significant toxicity and failure rates. We examined the efficacy of KY62, a new, water-soluble, polyene antifungal, against cutaneous infection with Leishmania amazonensis and against visceral infection with Leishmania donovani in susceptible BALB/c mice. Mice were infected with L. amazonensis promastigotes in the ear pinna and in the tail and were treated with KY62 or amphotericin B. The cutaneous lesions showed a remarkable response to therapy with KY62 at a dose of 30 mg per kg of body weight per day. At this dose, the efficacy of KY62 was equivalent to or better than that of amphotericin B at 1 to 5 mg/kg/day. Mice infected intravenously with 10 7 L. donovani promastigotes and treated with KY62 showed a 4-log reduction in the parasite burden in the liver and spleen compared to untreated mice. These studies indicate potent activity of KY62 against experimental cutaneous leishmaniasis caused by L. amazoniensis and against experimental visceral leishmaniasis caused by L. donovani .

Localized cutaneous leishmaniasis caused by Leishmania major is polymorphic in its clinical presentation and evolution. Clinical and parasitologic features and disease evolution of 112 Tunisian patients was evaluated. The expression of interleukin (IL)-4, IL-6, IL-10, IL-12 (p40), interferon (IFN)-g, and tumor necrosis factor (TNF)-a mRNA was analyzed by reverse transcription ‐ polymerase chain reaction in 73 biopsies. Cytokine mRNA expression varied individually over a wide range; TNF-a, IL-6, and IFN-g were detectable in u90% of lesions, IL-12 and IL-10 in 40% and 70%, respectively, and IL-4 in only 9%. Statistical analysis demonstrated positive association between the level of IL-12 and IFN-g and the presence of parasites in the lesions. Unfavorable evolution of the lesions was positively associated with high IL-10, IL-12, and IFN-g mRNA expression. These results indicate that an unfavorable clinical outcome was not related to an inadequate Th1 cell response and suggest that the macrophage-activating effect of IFN-g may be inhibited by the concomitant expression of IL-10. Human leishmaniasis includes a spectrum of diseases of [3]. In the murine model of leishmaniasis caused by L. major, variable severity, ranging from cutaneous ulcer to a fatal vis- a clear-cut dichotomy between the two functional T helper ceral disease caused by intracellular protozoan parasites of the subsets, Th1 and Th2, has been observed [4]. Th1 cells, which genus Leishmania. Localized cutaneous leishmaniasis (LCL), produce interferon (IFN)-g and interleukin (IL)-2, mediate procaused by Leishmania major, is an endemic zoonosis in North tection in resistant mouse strains (e.g., C57Bl/6), and Th2 cells, Africa and the Middle East [1]. Although often benign and which produce IL-4 and IL-10, promote disease progression in self-healing, LCL is a major public health problem in rural susceptible strains (e.g., BALB/c) [5 ‐ 7]. These cytokines exert areas, where it may cause considerable morbidity. In Tunisia, a potent cross-regulatory activity on the other Th subset. TNFtransmission of L. major by the Phlebotomine sand fly vector a has also been demonstrated to play a protective role in the occurs during the summer months, and active lesions in humans murine model of L. major infection [8, 9], whereas another tend to emerge during the autumn and winter months [2]. The macrophage product, transforming growth factor (TGF)-b ,i s disease is polymorphic in its clinical presentation and evolu- counterprotective [10]. IL-12, produced by B cells, dendritic tion, which may be due to variability in parasite virulence or cells, and macrophages, has been shown to play a major role in the host genetic background and immune response. in the early establishment of Th1-dependent protective immu

The Syrian golden hamster ( Mesocricetus auratus ) is uniquely susceptible to a variety of intracellular pathogens and is an excellent model for a number of human infectious diseases. The molecular basis for this high level of susceptibility is unknown, and immunological studies related to this model have been limited by the lack of available reagents. In this report we describe the cloning and sequence analysis of portions of the Syrian hamster interleukin 2 (IL-2), IL-4, gamma interferon (IFN-γ), tumor necrosis factor alpha, IL-10, IL-12p40, and transforming growth factor β cDNAs. In addition, we examined the cytokine response to infection with the intracellular protozoan Leishmania donovani in this animal model. Sequence analysis of the hamster cytokines revealed 69 to 93% homology with the corresponding mouse, rat, and human nucleotide sequences and 48 to 100% homology with the deduced amino acid sequences. The hamster IFN-γ, compared with the mouse and rat homologs, had an additional 17 amino acids at the C terminus that could decrease the biological activity of this molecule and thus contribute to the extreme susceptibility of this animal to intracellular pathogens. The splenic expression of these genes in response to infection with L. donovani , the cause of visceral leishmaniasis (VL), was determined by Northern blotting. VL in the hamster is a progressive, lethal disease which very closely mimics active human disease. In this model there was pronounced expression of the Th1 cytokine mRNAs, with transcripts being detected as early as 1 week postinfection. Basal expression of IL-4 in uninfected hamsters was prominent but did not increase in response to infection with L. donovani . IL-12 transcript expression was detected at low levels in infected animals and paralleled the expression of IFN-γ. Expression of IL-10, a potent macrophage deactivator, increased throughout the course of infection and could contribute to the progressive nature of this infection. These initial studies are the first to examine the molecular immunopathogenesis of a hamster model of VL infection and indicate that progressive disease in this model of VL is not associated with early polarization of the splenic cellular immune response toward a Th2 phenotype and away from a Th1 phenotype.

Infection with the protozoan Leishmania donovani can cause serious visceral disease or subclinical infection in humans. To better understand the pathogenesis of this dichotomy, we have investigated the host cellular immune response to cutaneous or visceral infection in a murine model. Mice infected in the skin developed no detectable visceral parasitism, whereas intravenous inoculation resulted in hepatosplenomegaly and an increasing visceral parasite burden. Spleen cells from mice with locally controlled cutaneous infection showed strong parasite-specific proliferative and gamma interferon (IFN-γ) responses, but spleen cells from systemically infected mice were unresponsive to parasite antigens. The in situ expression of IFN-γ, interleukin-4 (IL-4), IL-10, IL-12, and inducible nitric oxide synthase (iNOS) mRNAs was determined in the spleen, draining lymph node (LN), and cutaneous site of inoculation. There was considerably greater expression of IFN-γ and IL-12 p40 mRNAs in the LN draining a locally controlled cutaneous infection than in the spleen following systemic infection. Similarly, there was a high level of IFN-γ production by LN cells following subcutaneous infection but no IFN-γ production by spleen cells following systemic infection. Splenic IL-4 expression was transiently increased early after systemic infection, but splenic IL-10 transcripts increased throughout the course of visceral infection. IL-4 and IL-10 mRNAs were also increased in the LN following cutaneous infection. iNOS mRNA was detected earlier in the LN draining a cutaneous site of infection compared to the spleen following systemic challenge. Thus, locally controlled cutaneous infection was associated with antigen-specific spleen cell responsiveness and markedly increased levels of IFN-γ, IL-12, and iNOS mRNA in the draining LN. Progressive splenic parasitism was associated with an early IL-4 response, markedly increased IL-10 but minimal IL-12 expression, and delayed expression of iNOS.

Visceral Leishmaniasis (VL), caused by the intracellular protozoan Leishmania donovani, is characterized by relentlessly increasing visceral parasite replication, cachexia, massive splenomegaly, pancytopenia and ultimately death. Progressive disease is considered to be due to impaired effector T cell function and/or failure of macrophages to be activated to kill the intracellular parasite. In previous studies, we used the Syrian hamster (Mesocricetus auratus) as a model because it mimics the progressive nature of active human VL. We demonstrated previously that mixed expression of macrophage-activating (IFN-γ) and regulatory (IL-4, IL-10, IL-21) cytokines, parasite-induced expression of macrophage arginase 1 (Arg1), and decreased production of nitric oxide are key immunopathologic factors. Here we examined global changes in gene expression to define the splenic environment and phenotype of splenic macrophages during progressive VL. We used RNA sequencing coupled with de novo transcriptome assembly, because the Syrian hamster does not have a fully sequenced and annotated reference genome. Differentially expressed transcripts identified a highly inflammatory spleen environment with abundant expression of type I and type II interferon response genes. However, high IFN-γ expression was ineffective in directing exclusive M1 macrophage polarization, suppressing M2-associated gene expression, and restraining parasite replication and disease. While many IFN-inducible transcripts were upregulated in the infected spleen, fewer were induced in splenic macrophages in VL. Paradoxically, IFN-γ enhanced parasite growth and induced the counter-regulatory molecules Arg1, Ido1 and Irg1 in splenic macrophages. This was mediated, at least in part, through IFN-γ-induced activation of STAT3 and expression of IL-10, which suggests that splenic macrophages in VL are conditioned to respond to macrophage activation signals with a counter-regulatory response that is ineffective and even disease-promoting. Accordingly, inhibition of STAT3 activation led to a reduced parasite load in infected macrophages. Thus, the STAT3 pathway offers a rational target for adjunctive host-directed therapy to interrupt the pathogenesis of VL., Author Summary Visceral leishmaniasis (VL) is a neglected parasitic disease that is caused by the intracellular protozoan Leishmania donovani. Patients with this disease suffer from muscle wasting, enlargement of the spleen, reduced blood counts and ultimately will die without treatment. Progressive disease is considered to be due to impaired cellular immunity, with T cell or macrophage dysfunction, or both. We studied the Syrian hamster as an infection model because it mimics the progressive nature of human disease. We examined global changes in gene expression in the spleen and splenic macrophages during experimental VL and identified a highly inflammatory spleen environment with abundant expression of interferon and interferon-response genes that would be expected to control the infection. However, the high level of IFN-γ expression was ineffective in mediating a protective macrophage response, restraining parasite replication and halting progression of disease. We found that IFN-γ itself stimulated parasite growth in splenic macrophages and induced expression of counter-regulatory molecules, which may paradoxically make the host more susceptible. These data give insights into the nature of the immune response that promotes the infection, and identifies potential targets for therapeutic intervention.

Visceral leishmaniasis (VL), caused by infection with the intracellular protozoan Leishmania donovani, is a chronic progressive disease with a relentlessly increasing parasite burden in the spleen, liver and bone marrow. The disease is characterized by fever, splenomegaly, cachexia, and pancytopenia, and progresses to death if not treated. Control of Leishmania infection is mediated by Th1 (IFNγ-producing) CD4+ T cells, which activate macrophages to produce nitric oxide and kill intracellular parasites. However, despite expansion of CD4+ T cells and increased IFNγ expression in the spleen, humans with active VL do not control the infection. We used an experimental model of chronic progressive VL in hamsters, which mimics clinical and pathological features seen in humans, to better understand the mechanisms that lead to progressive disease. Transcriptional profiling of the spleen during chronic infection revealed expression of markers of both T cell activation and inhibition. CD4+ T cells isolated from the spleen during chronic progressive VL showed mixed expression of Th1 and Th2 cytokines and chemokines, and were marginally effective in controlling infection in an ex vivo T cell-macrophage co-culture system. Splenic CD4+ T cells and macrophages from hamsters with VL showed increased expression of inhibitory receptors and their ligands, respectively. Blockade of the inhibitory receptor PD-L2 led to a significant decrease in parasite burden, revealing a pathogenic role for the PD-1 pathway in chronic VL. PD-L2 blockade was associated with a dramatic reduction in expression of host arginase 1, but no change in IFNγ and inducible nitric oxide synthase. Thus, the expression of counter-regulatory molecules on splenic CD4+ T cells and macrophages promotes a more permissive macrophage phenotype and attenuates intracellular parasite control in chronic progressive VL. Host-directed adjunctive therapy targeting the PD-1 regulatory pathway may be efficacious for VL.

The outcomes of Leishmania infection are determined by host immune and nutrition status, parasite species, and co-infection with other pathogens. While subclinical infection and self-healing cutaneous leishmaniasis (CL) are common, uncontrolled parasite replication can lead to non-healing local lesions or visceral leishmaniasis (VL). It is known that infection control requires Th1-differentiation cytokines (IL-12, IL-18, and IL-27) and Th1 cell and macrophage activation. However, there is no generalized consensus for the mechanisms of host susceptibility. The recent studies on regulatory T cells and IL-17-producing cells help explain the effector T cell responses that occur independently of the known Th1/Th2 cell signaling pathways. This review focuses on the immunopathogenesis of non-healing American CL and progressive VL. We summarize recent evidence from human and animal studies that reveals the mechanisms of dysregulated, hyper-responses to Leishmania braziliensis, as well as the presence of disease-promoting or the absence of protective responses to Leishmania amazonensis and Leishmania donovani. We highlight immune-mediated parasite growth and immunopathogenesis, with an emphasis on the putative roles of IL-17 and its related cytokines as well as arginase. A better understanding of the quality and regulation of innate immunity and T cell responses triggered by Leishmania will aid in the rational control of pathology and the infection.

The clinicopathological features of the hamster model of visceral leishmaniasis (VL) closely mimic active human disease. Studies in humans and hamsters indicate that the inability to control parasite replication in VL could be related to ineffective classical macrophage activation. Therefore, we hypothesized that the pathogenesis of VL might be driven by a program of alternative macrophage activation. Indeed, the infected hamster spleen showed low NOS2 but high arg1 enzyme activity and protein and mRNA expression (p, Author Summary Visceral leishmaniasis (VL), caused by the intracellular protozoan Leishmania donovani, is a progressive, potentially fatal infection found in many resource-poor regions of the world. We initiated these studies of an experimental model of VL to better understand the molecular and cellular determinants underlying this disease. We found that host macrophages or fibroblasts, when infected with Leishmania donovani or exposed to products secreted by the parasite, are permissive to infection because they fail to metabolize arginine to generate nitric oxide, the effector molecule needed to kill the intracellular parasites. Instead, the infected host cells are activated in a way that leads to the expression of arginase, an enzyme that metabolizes arginine to produce polyamines, which support parasite growth. This detrimental activation pathway was dependent on the parasite-induced activation of the transcription factor STAT6, but contrary to the previously accepted paradigm, did not require (but was amplified by) the presence of polarized Th2 cells or type 2 cytokines. Knockdown of host arginase or STAT6 enhanced control of the infection, indicating that this activation pathway has a critical role in the pathogenesis of the disease. Interventions designed to inhibit the STAT6-arginase-polyamine pathway could help in the treatment or prevention of VL.

The nature of the host cellular immune response largely determines the expression of disease following infection with the intracellular protozoans Leishmania spp. In experimental animals control and resolution of infection are mediated by gamma interferon and tumor necrosis factor alpha (TNF-alpha), whereas disease progression is associated with the production of interleukin 4 (IL-4), IL-5, IL-10, and transforming growth factor beta (TGF-beta). We have analyzed the profile of cytokine gene expression directly in the lesions of 13 patients with localized cutaneous leishmaniasis due to Leishmania mexicana. All but one patient had a single lesion, and the time of evolution ranged from 8 days to 18 months. Cytokine gene expression was quantitated by reverse transcriptase PCR and interpolation from a standard curve. Gamma interferon, TNF-alpha, IL-1 alpha, IL-6, IL-10, and TGF-beta gene expression was present in all samples. IL-3 and IL-4 gene expression was barely detectable in 1 and 3 of 13 samples, respectively. IL-2 and IL-5 mRNAs were not found. A significant increase in the expression of IL-1 alpha, TNF-alpha, IL-10, and TGF-beta was observed in late lesions (> or = 4 months) compared with that in early lesions (< or = 2 months). Because of their inhibitory effects on macrophage function, the expression of IL-10 and TGF-beta may play a role in the immunopathogenesis of chronic cutaneous leishmaniasis.

We previously described a murine model of malnutrition that mimicked features of moderate human malnutrition, and led to increased dissemination of Leishmania donovani. In this study, we investigated the effect of malnutrition on macrophage production of cytokines, prostaglandins (PGs), and leukotrienes (LTs). Using either LPS or calcium ionophore A23187 as a stimulus, macrophages from the malnourished mice produced a 3-fold higher PG/LT ((PGE(2)+6-keto-PGF(1alpha))/(LTB(4)+cysteinyl leukotrienes)) ratio than macrophages from well-nourished mice. LPS-stimulated macrophages from the malnourished mice produced decreased levels of TNF-alpha, GM-CSF, and IL-10, but similar levels of IL-6 and NO compared to well-nourished mice. A complex crosstalk between the eicosanoids and cytokines in the LPS-stimulated macrophages from the malnourished mice was evident by the following: (1) high levels of PG secretion despite low levels of TNF-alpha; (2) supplemental IL-10 modulated the excessive PG production; (3) GM-CSF rectified the PG/LT ratio, but did not correct the abnormal cytokine profile; and (4) inhibitors of cyclooxygenase decreased the PG/LT ratio, but did not affect TNF-alpha. Thus, in this model of malnutrition, there is a relative increase in anti-inflammatory PGs compared to pro-inflammatory LTs, which may contribute to immunodeficiency.

Recombinant Eimeria antigen (rEA) has been shown to have potent anticancer and antiviral activity in respective mouse disease models, presumably through robust immune stimulation that occurs via TLR11, a pattern recognition receptor that recognizes profilin-like proteins expressed on apicomplexan protozoans. Comparable immunostimulatory activity in other species has yet to be demonstrated. Since rEA is known to be highly effective in treating Punta Toro virus (PTV) infection in mice, its ability to elicit protective immunity in the hamster PTV infection model was investigated. rEA was given alone, or in combination with IL-18 or IL-2, and virally challenged hamsters were observed for mortality. Cytokine transcript profiles for IL-12p40, IL-21, IFN-gamma and TNF-alpha were assessed to evaluate the induction of these inflammatory mediators known to be induced in mice following exposure to rEA. A dose of 100 microg of rEA, given once 4 h prior to viral challenge, and a second time on day 3 of the infection, was found to be the most effective prophylactic therapy protecting 60% of treated hamsters from mortality, compared to only 5-10% observed in animals receiving placebo. Increased expression of IFN-gamma and IL-12p40 was evident following treatment with rEA. The data suggest that rEA does induce host antiviral responses in hamsters that result in significant protection from death, although determining the most appropriate dose for intervention in other species, including humans, will likely be challenging.

Progressive disease in the hamster model of visceral leishmaniasis, caused by Leishmania donovani, in contrast to infection in mice, mimics the progressive disease observed in untreated humans. During progressive infection in hamsters, there was a vigorous type 1 cellular immune response, which is typically associated with control of infection, suggesting that there was ineffective IFN-γ-mediated macrophage activation. Indeed, at the site of infection, hamsters did not express NO synthase 2 (NOS2), which is the primary mechanism for control of infection in mice. Furthermore, in striking contrast to mouse macrophages, IFN-γ-activated hamster macrophages did not did not express NOS2 nor generate NO, and were unable to restrict the replication of intracellular L. donovani. The absent hamster NOS2 expression was not the result of NOS2 gene deletion and the NOS2 cDNA had an intact open reading frame. Furthermore, the impaired transcription of NOS2 mRNA was selective and not due to global impairment of IFN-γ signaling (members of the IFN-γ-signaling pathway were expressed and functional and IFN-γ up-regulated several primary and secondary response genes). Strikingly, the proximal hamster NOS2 promoter, like the human ortholog, had >20-fold less basal and IFN-γ/LPS-inducible activity than the corresponding mouse promoter. Thus, reduced basal and IFN-γ-induced activity of the hamster NOS2 transcriptional unit, which is unique to this small animal and similar to the human counterpart, accompanies the inability of the animal to control an intracellular pathogen.

Interleukin-12 (IL-12) is a heterodimeric cytokine that is a principal mediator of the innate immune response and modulator of acquired cell-mediated immunity. Administration of exogenous IL-12 can direct the host adaptive T cell response toward a type 1 phenotype. The co-administration of IL-12 with vaccine antigens has been shown to augment the vaccine-induced T(H)1 response and protection against intracellular pathogens. We show here that a canine IL-12 DNA, constructed by fusing the p35 and p40 subunit cDNAs with an interspacing linker, generated stable IL-12 transcripts when placed under control of a strong constitutive promoter. The protein expressed from this fused cDNA was fully functional in promoting a type 1 (IFN-gamma) and suppressing a type 2 (IL-4) cytokine response following both in vitro transfection of a canine cell line and in vivo delivery to dogs. This DNA construct may be useful as an adjuvant for vaccines that target tumors or intracellular pathogens of the dog.

The Syrian golden hamster (Mesocricetus auratus) is highly susceptible to a number of intracellular pathogens. Interferon-gamma (IFN-gamma), the primary macrophage-activating cytokine, plays a key role in the host defense against intracellular pathogens. The hamster IFN-gamma cDNA encodes a 174 amino acid protein that has an additional 17 amino acids at the carboxyl-terminus compared to IFN-gamma of mice and rats. A homologous C-terminal tail is also found in other non-murine rodents. The biological activity of hamster IFN-gamma had not been investigated previously so we first demonstrated the activity of native IFN-gamma in assays of IFN-gamma-induced receptor signaling and antiviral activity against vesicular stomatitis virus. We then tested the hypothesis that the C-terminal tail of hamster IFN-gamma could influence its biological activity. A truncated hamster IFN-gamma, in which the C-terminal 17 aa were removed by insertion of a stop codon at the position corresponding to the stop codon in the mouse sequence, had approximately 10-fold greater activity than the full length protein when measured in the two bioassays. Polyclonal and monoclonal anti-hamster IFN-gamma antibodies specifically inhibited this biological activity. Collectively, these data indicate that this unique structural feature influences the biological activity of hamster IFN-gamma.

Vaccination of dogs, the domestic reservoir of Leishmania chagasi , could not only decrease the burden of canine visceral leishmaniasis (VL), but could also indirectly reduce the incidence of human VL. Intramuscular vaccination of foxhounds with a Leishmania multicomponent (10 antigen) DNA vaccine resulted in antigen-induced lymphoproliferative and IFN-γ (but not IL-4) responses. This response was not augmented by co-administration of canine IL-12 or GM-CSF DNA adjuvants. The multicomponent DNA vaccine also induced a delayed type hypersensitivity (DTH) response to viable L. donovani promastigotes and led to a reduction of parasite burden in an in vitro intracellular infection model, and in the draining lymph node of dogs early after cutaneous challenge. Thus, the multicomponent DNA vaccine was effective in priming dogs for a parasite-specific type 1 cellular immune response, which was able to restrict parasite growth.

We have described previously a murine model of multinutrient undernutrition that reproduced the features of moderate human malnutrition and led to increased early dissemination of Leishmania donovani. Peritoneal cells from these malnourished mice produced decreased NO after stimulation with IFN-γ/LPS. We hypothesized that malnutrition may cause a deficit in NF-κB activation, a principal transcription pathway for inducible NO synthase and proinflammatory cytokines. Macrophages from malnourished mice, stimulated with IFN-γ/LPS, showed increased IL-6 production and decreased IL-10 and TNF-α production. Neutralization of TNF-α in macrophage cultures from the control mice mimicked the effect of malnutrition on NO and IL-10 production, whereas supplemental TNF-α added to cultures of macrophages from malnourished mice increased NO secretion. NF-κB nuclear binding activity in macrophages from the malnourished mice was reduced early after stimulation, but increased to supranormal values by 16- or 24-h poststimulation. Blocking NO production in the macrophages from the control mice reproduced the effect of malnutrition on the late activation of NF-κB, whereas supplemental NO decreased the late NF-κB activation in the malnourished mice. Thus, in macrophages from the malnourished mice, initial deficits in NF-κB activity probably lead to decreased TNF-α, which results in decreased NO; however, IL-6 is regulated independently from NF-κB and TNF-α. The late activation of NF-κB in the macrophages from malnourished mice is due to absence of negative feedback from NO.

SUMMARY We determined that the site of inoculation (foot or snout) influences the clinical evolution and immune responses of hamsters infected with Leishmania (Viannia) panamensis . Hamsters infected in the snout showed (i) a more rapid and severe lesion evolution at multiple time points ( P < 0·05), (ii) a more extensive inflammatory infiltrate and tissue necrosis, (iii) a higher tissue parasite burden, (iv) a higher antibody titre ( P < 0·01), but lower antigen-specific spleen cell proliferative response ( P = 0·02), and (v) a slower response to anti-leishmanial drug treatment ( P < 0·002). In both inoculation groups there was co-expression of type 1 (IFN- γ and IL-12) and some type 2 (IL-10 and TGF- β , but not IL-4) cytokines in the cutaneous lesions and spleen. Early in the course of infection, hamsters infected in the snout showed higher expression of splenic IL-10 ( P = 0·04) and intralesional IFN- γ ( P = 0·02) than foot infections. No expression of IL-12p40 or IL-4 was detected. During the chronic phase, snout lesions expressed more IFN- γ ( P = 0·001), IL-12p40 ( P = 0·01), IL-10 ( P = 0·009) and TGF- β ( P = 0·001), and the level of expression of each of these cytokines correlated with lesion size ( P ≤ 0·01). These results suggest that the site of infection influences the clinical outcome in experimental cutaneous leishmaniasis, and that the expression of macrophagedeactivating type 2 cytokines and/or an exaggerated type 1 proinflammatory cytokine response may contribute to lesion severity.

Acute and chronic inflammation cause many changes in total body iron metabolism including the sequestration of iron in phagocytic cells of the reticuloendothelial system. This change in iron metabolism contributes to the development of the anemia of inflammation. MTP1, the duodenal enterocyte basolateral iron exporter, is also expressed in the cells of the reticuloendothelial system (RES) and is likely to be involved in iron recycling of these cells. In this study, we use a lipopolysaccharide model of the acute inflammation in the mouse and demonstrate that MTP1 expression in RES cells of the spleen, liver, and bone marrow is down-regulated by inflammation. The down-regulation of splenic expression of MTP1 by inflammation was also observed in a Leishmania donovani model of chronic infection. The response of MTP1 to lipopolysaccharide (LPS) requires signaling through the LPS receptor, Toll-like receptor 4 (TLR4). In mice lacking TLR4, MTP1 expression is not altered in response to LPS. In addition, mice lacking tumor necrosis factor-receptor 1a respond appropriately to LPS with down-regulation of MTP1, despite hyporesponsiveness to tumor necrosis factor-alpha signaling, suggesting that this cytokine may not be required for the LPS effect. We hypothesize that the iron sequestration in the RES system that accompanies inflammation is because of down-regulation of MTP1.

In regions where leishmaniasis is endemic, clinical disease is usually reported more frequently among males than females. This difference could be due to disparate risks of exposure of males and females, but gender-related differences in the host response to infection may also play a role. Experimental studies of the influence of gender onLeishmaniainfection have not included parasites of the subgenusViannia, which is the most common cause of cutaneous leishmaniasis in the Americas. Mice are not readily susceptible to infection byLeishmania(Viannia) spp., but cutaneous infection of hamsters withL.(V.)panamensisorL.(V.)guyanensisresulted in chronic lesions typical of the human disease caused by these parasites. Strikingly, infection of male hamsters resulted in significantly greater lesion size and severity, an increased rate of dissemination to distant cutaneous sites, and a greater parasite burden in the draining lymph node than infection in female animals. Two lines of evidence indicated this gender-related difference in disease evolution was determined at least in part by the sex hormone status of the animal. First, prepubertal male animals had smaller and/or less severe cutaneous lesions than adult male animals. Second, infection of testosterone-treated female animals resulted in significantly larger lesions than in untreated female animals. The increased severity of disease in male compared to female animals was associated with significantly greater intralesional expression of interleukin-4 (IL-4) (P= 0.04), IL-10 (P= 0.04), and transforming growth factor β (TGF-β) (P< 0.001), cytokines known to promote disease in experimental leishmaniasis. There was a direct correlation between the expression of TGF-β mRNA and lesion size (Spearman's correlation coefficient = 0.873;P< 0.001). These findings demonstrate an inherent risk of increased disease severity in male animals, which is associated with a more permissive immune response.

Infection with the protozoan Leishmania donovani in humans is usually subclinical. Parasites probably persist for the life of the host and the low-level infection is controlled by the cellular immune response. To better understand the mechanisms related to the control of infection, we studied the evolution and architecture of the splenic cellular immune response in a murine model that is most representative of human subclinical infection. Following systemic inoculation with L. donovani, the parasites were primarily localized to the macrophage-rich splenic red pulp. There was an initial increase in the numbers of T cells and dendritic cells in the periarteriolar lymphoid sheath and marginal zone, but the red pulp (where parasitized macrophages were prominent) remained free of these cells until later in the course of infection. Thus, T cells did not colocalize with parasitized red pulp macrophages until later in the course of infection. Early in the course of infection, IL-10 production within the marginal zone and TGF-beta production by cells in the red pulp were prominent. These macrophage-inhibitory cytokines may contribute to the establishment of the infection and early parasite replication. By day 28 of infection, when the visceral parasite burden began to decline, the number of IL-10-producing spleen cells was back to the baseline level, but IFN-gamma production was higher and the number of IL-12-producing cells was increased dramatically. At this time T cells and dendritic cells had moved out of the lymphoid follicle and marginal zone into the red pulp where the parasites were located. These findings therefore suggest that control of infection is associated with IFN-gamma and IL-12 production and migration of T cells and dendritic cells to the site of chronic parasitism.

In neurocysticercosis (NCC), it is thought that the long-term survival of the parasite within the human brain is due in part to the ability of the cestode to suppress the local immune response. When the parasite dies, the immunosuppression is apparently lost and a strong local inflammatory response then develops. In contrast, little is known about the immunologic response that may occur in the peripheral immune system of these patients. In this study, the status of the peripheral (extracerebral) cellular and humoral response was evaluated in patients with a history of NCC. The in vitro proliferation of peripheral blood mononuclear cells to mitogens and foreign antigens was similar in patients and controls. Importantly, a substantive response was elicited by two Taenia solium metacestode antigens. In addition, 8 of 10 patients had a detectable humoral response to the antigenic glycoproteins of the cestode. Considering both the cellular and humoral response, all of the patients with NCC presented an active peripheral immunity.