Transcriptional Profiling in Experimental Visceral Leishmaniasis Reveals a Broad Splenic Inflammatory Environment that Conditions Macrophages toward a Disease-Promoting Phenotype.

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.

Impact of Childhood Malnutrition on Host Defense and Infection.

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.

Growth factor and Th2 cytokine signaling pathways converge at STAT6 to promote arginase expression in progressive experimental visceral leishmaniasis.

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.

Cytokine mRNA expression in Peromyscus yucatanicus (Rodentia: Cricetidae) infected by Leishmania (Leishmania) mexicana.

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-ß 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.

Antileishmanial Activity of Disulfiram and Thiuram Disulfide Analogs in an Ex Vivo Model System Is Selectively Enhanced by the Addition of Divalent Metal Ions.

Current treatments for cutaneous and visceral leishmaniasis are toxic, expensive, difficult to administer, and limited in efficacy and availability. Disulfiram has primarily been used to treat alcoholism. More recently, it has shown some efficacy as therapy against protozoan pathogens and certain cancers, suggesting a wide range of biological activities. We used an ex vivo system to screen several thiuram disulfide compounds for antileishmanial activity. We found five compounds (compound identifier [CID] 7188, 5455, 95876, 12892, and 3117 [disulfiram]) with anti-Leishmania activity at nanomolar concentrations. We further evaluated these compounds with the addition of divalent metal salts based on studies that indicated these salts could potentiate the action of disulfiram. In addition, clinical studies suggested that zinc has some efficacy in treating cutaneous leishmaniasis. Several divalent metal salts were evaluated at 1 µM, which is lower than the normal levels of copper and zinc in plasma of healthy individuals. The leishmanicidal activity of disulfiram and CID 7188 were enhanced by several divalent metal salts at 1 µM. The in vitro therapeutic index (IVTI) of disulfiram and CID 7188 increased 12- and 2.3-fold, respectively, against L. major when combined with ZnCl2. The combination of disulfiram with ZnSO4 resulted in a 1.8-fold increase in IVTI against L. donovani. This novel combination of thiuram disulfides and divalent metal ions salts could have application as topical and/or oral therapies for treatment of cutaneous and visceral leishmaniasis.

Deficiency of lymph node-resident dendritic cells (DCs) and dysregulation of DC chemoattractants in a malnourished mouse model of Leishmania donovani infection.

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.

Transcriptional profiling of the spleen in progressive visceral leishmaniasis reveals mixed expression of type 1 and type 2 cytokine-responsive genes.

BACKGROUND: The Syrian golden hamster (Mesocricetus aureus) has been used as a model to study infections caused by a number of human pathogens. Studies of immunopathogenesis in hamster infection models are challenging because of the limited availability of reagents needed to define cellular and molecular determinants. RESULTS: We sequenced a hamster cDNA library and developed a first-generation custom cDNA microarray that included 5131 unique cDNAs enriched for immune response genes. We used this microarray to interrogate the hamster spleen response to Leishmania donovani, an intracellular protozoan that causes visceral leishmaniasis. The hamster model of visceral leishmaniasis is of particular interest because it recapitulates clinical and immunopathological features of human disease, including cachexia, massive splenomegaly, pancytopenia, immunosuppression, and ultimately death. In the microarray a differentially expressed transcript was identified as having at least a 2-fold change in expression between uninfected and infected groups and a False Discovery Rate of <5%. Following a relatively silent early phase of infection (at 7 and 14 days post-infection only 8 and 24 genes, respectively, were differentially expressed), there was dramatic upregulation of inflammatory and immune-related genes in the spleen (708 differentially expressed genes were evident at 28 days post-infection). The differentially expressed transcripts included genes involved in inflammation, immunity, and immune cell trafficking. Of particular interest there was concomitant upregulation of the IFN-γ and interleukin (IL)-4 signaling pathways, with increased expression of a battery of IFN-γ- and IL-4-responsive genes. The latter included genes characteristic of alternatively activated macrophages. CONCLUSIONS: Transcriptional profiling was accomplished in the Syrian golden hamster, for which a fully annotated genome is not available. In the hamster model of visceral leishmaniasis, a robust and functional IFN-γ response did not restrain parasite load and progression of disease. This supports the accumulating evidence that macrophages are ineffectively activated to kill the parasite. The concomitant expression of IL-4/IL-13 and their downstream target genes, some of which were characteristic of alternative macrophage activation, are likely to contribute to this. Further dissection of mechanisms that lead to polarization of macrophages toward a permissive state is needed to fully understand the pathogenesis of visceral leishmaniasis.

Development of an ex vivo lymph node explant model for identification of novel molecules active against Leishmania major.

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 ex vivo lymph node explant culture (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 (EC50). We found 24 compounds (7%) that had an in vitro therapeutic index (IVTI; 50% cytotoxic/effective concentration [CC50]/EC50) > 100; 19 of the compounds had an EC50 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.

Progressive visceral leishmaniasis is driven by dominant parasite-induced STAT6 activation and STAT6-dependent host arginase 1 expression.

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<0.001) and increased polyamine synthesis (p<0.05). Increased arginase activity was also evident in macrophages isolated from the spleens of infected hamsters (p<0.05), and arg1 expression was induced by L. donovani in primary hamster peritoneal macrophages (p<0.001) and fibroblasts (p<0.01), and in a hamster fibroblast cell line (p<0.05), without synthesis of endogenous IL-4 or IL-13 or exposure to exogenous cytokines. miRNAi-mediated selective knockdown of hamster arginase 1 (arg1) in BHK cells led to increased generation of nitric oxide and reduced parasite burden (p<0.005). Since many of the genes involved in alternative macrophage activation are regulated by Signal Transducer and Activator of Transcription-6 (STAT6), and because the parasite-induced expression of arg1 occurred in the absence of exogenous IL-4, we considered the possibility that L. donovani was directly activating STAT6. Indeed, exposure of hamster fibroblasts or macrophages to L. donovani resulted in dose-dependent STAT6 activation, even without the addition of exogenous cytokines. Knockdown of hamster STAT6 in BHK cells with miRNAi resulted in reduced arg1 mRNA expression and enhanced control of parasite replication (p<0.0001). Collectively these data indicate that L. donovani infection induces macrophage STAT6 activation and STAT6-dependent arg1 expression, which do not require but are amplified by type 2 cytokines, and which contribute to impaired control of infection.

In situ cytokines (IL-4, IL-10, IL-12, IFN-γ) and chemokines (MCP-1, MIP-1α) gene expression in human Leishmania (Leishmania) Mexicana infection.

Crucial to the defense against Leishmania is the ability of the host to mount a cell-mediated immune response capable of controlling and/or eliminating the parasite. The composition of the cell populations recruited in the early phase of the infection seems to be essential for defining the infection outcomes. The signals that initiate and regulate the early immune response and local accumulation of cell subsets in the skin are poorly understood. We previously studied the in situ expression of cytokine genes in patients with localized cutaneous leishmaniasis (LCL) caused by Leishmania (Leishmania) mexicana. In the present study we examined in situ cytokine (IL-4, IL-10, IL-12, IFN-γ) and chemokine (MCP-1, MIP-1α) gene expression in L. (L.) mexicana active LCL lesions, and in the delayed type hypersensitivity (DTH) skin response to Leishmania antigen in subjects with healed lesion and subclinical infection. Data regarding cytokines were similar to previous studies in patients with active LCL. There were no significant differences in the profile of cytokine and chemokine gene expression in DTH from subjects with healed or subclinical infection. IL-12 gene expression detected in both groups was similar. High expression of MCP-1 was detected in all patients with active LCL. There was no difference in the level of MCP-1 expression between the healed lesion and the subclinical infection groups (p = 0.876). IL-12 and MCP-1 in the absence of IFN-γ might be playing a crucial role in infection outcomes at skin level.

Double Staining with Fluorescent Tracers to Determine Myeloid Cell Migration of Leishmania-infected Cells from Mouse Skin to Lymphatic Tissues by Flow Cytometry.

Immune cell trafficking in steady-state conditions and inflammatory cell recruitment into injured tissues is crucial for the surveillance of the immune system and the maintenance of body homeostasis. Tracking the cell journey from the infection site in the skin to lymphoid tissues has been challenging, and is typically determined using fluorescent cell tracers, antibodies, or photoconvertible models. Here, we describe the detailed method to track Leishmania-infected myeloid cells migrating from the skin to lymphatic tissues by multiparametric flow cytometry. These methods involve labeling of infective Leishmania donovani parasites with fluorescent cell tracers and phenotyping of myeloid cells with fluorescent antibodies, to determine the infection status of migratory myeloid cells. We also describe the detailed protocol to trace donor monocytes transferred intradermally into recipient mice in Leishmania donovani infection. These protocols can be adapted to study skin-lymphoid tissue migration of dendritic cells, inflammatory monocytes, neutrophils, and other phagocytic myeloid cells in response to vaccine antigens and infection. Key features • Cell-tracking of cell-trace-labeled parasites and monocytes from the skin to lymphatic tissues after transference into donor mice. • Identification of migratory cells labeled with fluorescent cell tracers and antibodies by flow cytometry. • Isolation, labeling, and transference of bone marrow monocytes from donor mice into the skin of recipient mice. • Description of a double-staining technique with fluorescent cell tracers to determine cell and parasite dissemination from the skin to lymphoid tissues.

Malnutrition-related parasite dissemination from the skin in visceral leishmaniasis is driven by PGE2-mediated amplification of CCR7-related trafficking of infected inflammatory monocytes.

People are infected with Leishmania donovani when the parasite is deposited in the dermis during the blood meal of the sand fly vector. Most infected people develop a subclinical latent infection, but some develop progressive visceral leishmaniasis. Malnutrition is a risk factor for the development of active VL. We previously demonstrated increased parasite dissemination from the skin to visceral organs in a murine model of malnutrition. Here we investigated the mechanism of early parasite dissemination. After delivery of L. donovani to the skin, we found enhanced capture of parasites by inflammatory monocytes and neutrophils in the skin of malnourished mice. However, parasite dissemination in malnourished mice was driven primarily by infected inflammatory monocytes, which showed increased CCR7 expression, greater intrinsic migratory capacity, and increased trafficking from skin to spleen. PGE2 production, which was increased at the site of skin infection, increased monocyte CCR7 expression and promoted CCR7-related monocyte-mediated early parasite dissemination in malnourished mice. Parasite dissemination in monocytes was reduced by inhibition of PGE2, knockdown or silencing of CCR7 in monocytes, and depletion of inflammatory monocytes through administration of diphtheria toxin to CSFR1-DTR transgenic mice that have monocyte-specific DT receptor expression. CCR7-driven trafficking of infected inflammatory monocytes through the lymph node was accompanied by increased expression of its ligands CCL19 and CCL21. These results show that the CCR7/PGE2 axis is responsible for the increased trafficking of L. donovani-infected inflammatory monocytes from the skin to the spleen in the malnourished host. Undernutrition and production of PGE2 are potential targets to reduce the risk of people developing VL. Nutritional interventions that target improved immune function and reduced PGE2 synthesis should be studied in people at risk of developing VL.

Hematological and Clinical Features Associated with Initial Poor Treatment Outcomes in Visceral Leishmaniasis Patients with and without HIV Coinfection in Gondar, Northwest Ethiopia.

Ethiopia is among the countries with a high leishmaniasis burden. In this retrospective review, we aimed to determine hematological and clinical features associated with initial poor treatment outcomes of visceral leishmaniasis (VL) patients. The majority of VL cases in this study had leucopenia (94.3%), thrombocytopenia (87.1%), and anemia (85.9%). HIV coinfection was present in 7.0% (n = 23) of VL cases. At the center, VL patients without HIV coinfection were treated with sodium stibogluconate and paromomycin combination, whereas HIV coinfected cases were treated with AmBisome and miltefosine combination therapy. End-of-treatment cure rates among HIV-positive and HIV-negative visceral leishmaniasis cases, respectively, were 52.2% and 96.9%. Case fatality rates were 34.8% and 2.7% in HIV-positive and HIV-negative cases, respectively. Overall, non-survivors in this study were more likely to have HIV (55.0% vs. 4.1%, p < 0.001), sepsis (15.0% vs. 1.4%, p = 0.019), and dyspnea (40.0% vs. 2.7%, p < 0.001) at admission. In this regard, particular attention to the management of superimposed disease conditions at admission, including sepsis, HIV, and dyspnea, is needed to improve VL patients' treatment outcomes. The inadequacy of the current treatments, i.e., AmBisome and miltefosine combination therapy, for HIV coinfected visceral leishmaniasis patients requires further attention as it calls for new treatment modalities.

A Preliminary Study to Compare Recombinase Polymerase Amplification-Lateral Flow and Quantitative PCR in the Detection of Cutaneous Leishmania in Communities from the Volta Region of Ghana.

Background: Leishmaniasis is a parasitic disease that mostly affects populations in tropical and subtropical countries. In Ghana, cutaneous leishmaniasis (CL) is the most common form of the disease affecting communities of the Volta Region. Conventional parasitological method (microscopy) is the commonly used test for CL diagnosis in many endemic countries, but has low sensitivity in chronic cases. Therefore, there is a clear need for a sensitive and easy-to-use point-of-care diagnostic method like an isothermal recombinase polymerase amplification-lateral flow (RPA-LF) test, suitable for use in austere and low-resource settings for the identification of CL cases. This study compared the efficacy of RPA-LF test with quantitative PCR (qPCR) in detecting Leishmania in suspected CL cases from the Volta Region. Methods: Twenty-five participants between 5 and 14 years were enrolled in the study from whom a total of 26 samples were obtained. Lesion samples were collected using FTA® filter papers applied to ulcerated lesions for molecular diagnosis. DNA isolated from filter papers was used for both the RPA-LF test and qPCR. Results: Twenty-two participants (88%) presented with one or two ulcerated active lesions per individual, while the rest of them had plaques or dried lesions. Among the 26 samples, 19/26 (73%) had concordant results when comparing the two diagnostic methods. Conclusion: Data from this study suggest that the RPA-LF test can be used in addition to a conventional parasitological diagnostic test (microscopy) to detect CL cases in communities of the Volta Region.

Etiological characterization of acute undifferentiated febrile illness in Apartadó and Villeta municipalities, Colombia, during COVID-19 pandemic.

Background: Acute undifferentiated febrile illness (AUFI) is one of the leading causes of illness in tropical regions. Although malaria is the most important cause, other pathogens such as Dengue (DENV), Leptospira and recently, Coronavirus Disease 2019 (COVID-19) have gained importance. In Colombia, few studies aimed to identify the etiology of AUFI. Most of them performed in Apartadó and Villeta municipalities, identifying the active circulation of several pathogens. Thus, we conducted a cross-sectional study in these municipalities to characterize the etiologies of AUFI during COVID-19 pandemic. Methods: An active surveillance was conducted between September and December 2021 in local hospitals of Apartadó and Villeta municipalities. Febrile patients were enrolled after voluntarily agreeing to participate in the study. Ten different etiologies were evaluated through direct, serological, molecular and rapid diagnostic methods. Results: In Apartadó a confirmed etiology was found in 60% of subjects, DENV (25%) being the most frequent, followed by leptospirosis (16.7%), malaria (10%), COVID-19 (8.3%), spotted fever group (SFG) rickettsiosis (6.7%) and Chikungunya (1.7%). In Villeta, a specific etiology was confirmed in 55.4% of patients, of which SFG rickettsiosis (39.3%) was the most frequent, followed by leptospirosis (21.4%), DENV (3.6%) and malaria (1.8%). No cases due to Mayaro, Yellow Fever, Oropouche and Venezuelan Equine Encephalitis viruses were detected. Conclusion: We confirm the relevance of dengue fever, leptospirosis, SFG rickettsiosis, COVID-19 and malaria as causes of AUFI in the municipality of Apartadó, and highlight the great importance of SFG rickettsiosis as the main cause of AUFI in the municipality of Villeta.

CCR2 plays a critical role in dendritic cell maturation: possible role of CCL2 and NF-kappa B.

We postulated that CCR2-driven activation of the transcription factor NF-kappaB 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-kappaB 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-gamma 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-kappaB in the regulation of DC/LC maturation processes that regulate protective immunity against intracellular pathogens.

Water insecurity, food insecurity and social capital associated with a group-led microfinance programme in semi-rural Kenya.

ABSTRACTSocial capital predicts many positive health outcomes, including food and water access and sufficiency. Hence, increasing social capital has emerged as one potential strategy to improve food and water security. In this study, we investigate whether social capital generated through participation in a community-based microlending programme based in semi-rural Kenya is associated with water and food insecurity, and explore the interconnectedness of water and food insecurity through mediation analysis. Randomly-selected women participants of the community-based programme (n = 400) were interviewed in June 2018 and again in June 2019. Survey measures included water insecurity, food insecurity and an index of social capital constructs, namely group cohesion, trust, expectations of mutual support, sense of belonging and frequency of attendance in the programme. Random effects linear regression showed that an increase the social capital index was associated with lower water and food insecurity. The mediation analysis indicated that the association between social capital and food insecurity was completely mediated by water insecurity. This study demonstrates the need for further investigation into how social capital-generating programmes can contribute to systems approaches for collaborative food and water security programmes, especially among rural communities in low- and middle-income countries.

Inflammatory stimuli alter bone marrow composition and compromise bone health in the malnourished host.

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.

CCR7/dendritic cell axis mediates early bacterial dissemination in Orientia tsutsugamushi-infected mice.

Scrub typhus is a life-threatening zoonosis caused by the obligate intracellular bacterium Orientia tsutsugamushi (Ot) that is transmitted by the infected larvae of trombiculid mites. However, the mechanism by which Ot disseminates from the bite site to visceral organs remains unclear; host innate immunity against bacterial dissemination and replication during early infection is poorly understood. In this study, by using an intradermal infection mouse model and fluorescent probe-labeled Ot, we assessed the dynamic pattern of innate immune cell responses at the inoculation site. We found that neutrophils were the first responders to Ot infection and migrated into the skin for bacterial uptake. Ot infection greatly induced neutrophil activation, and Ot-neutrophil interaction remarkably promoted cell death both in vitro and in vivo. Depletion of neutrophils did not alter bacterial dissemination in mice, as evidenced by similar bacterial burdens in the skin and draining lymph nodes (dLN) at day 3, as well as in the lungs and brains at day 14, as compared to the control mice. Instead, dendritic cells (DCs) and macrophages played a role as a Trojan horse and transmitted Ot from the skin into dLN. Importantly, the absence of homing receptor CCR7 or neutralization of its ligand, CCL21, significantly impaired DC migration, resulting in reduced bacterial burdens in dLN. Taken together, our study sheds light on a CCR7/dendritic cell-mediated mechanism of early Ot dissemination and provides new insights into therapeutic and vaccine development strategies for scrub typhus.

Pathologic Inflammation in Malnutrition Is Driven by Proinflammatory Intestinal Microbiota, Large Intestine Barrier Dysfunction, and Translocation of Bacterial Lipopolysaccharide.

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.