Sandfly Fever Sicilian Virus-Leishmania major co-infection modulates innate inflammatory response favoring myeloid cell infections and skin hyperinflammation.

BACKGROUND: The leishmaniases are a group of sandfly-transmitted diseases caused by species of the protozoan parasite, Leishmania. With an annual incidence of 1 million cases, 1 billion people living in Leishmania-endemic regions, and nearly 30,000 deaths each year, leishmaniasis is a major global public health concern. While phlebotomine sandflies are well-known as vectors of Leishmania, they are also the vectors of various phleboviruses, including Sandfly Fever Sicilian Virus (SFSV). Cutaneous leishmaniasis (CL), caused by Leishmania major (L. major), among other species, results in development of skin lesions on the infected host. Importantly, there exists much variation in the clinical manifestation between individuals. We propose that phleboviruses, vectored by and found in the same sandfly guts as Leishmania, may be a factor in determining CL severity. It was reported by our group that Leishmania exosomes are released into the gut of the sandfly vector and co-inoculated during blood meals, where they exacerbate CL skin lesions. We hypothesized that, when taking a blood meal, the sandfly vector infects the host with Leishmania parasites and exosomes as well as phleboviruses, and that this viral co-infection results in a modulation of leishmaniasis. METHODOLOGY/PRINCIPAL FINDINGS: In vitro, we observed modulation by SFSV in MAP kinase signaling as well as in the IRF3 pathway that resulted in a pro-inflammatory phenotype. Additionally, we found that SFSV and L. major co-infection resulted in an exacerbation of leishmaniasis in vivo, and by using endosomal (Toll-like receptor) TLR3, and MAVS knock-out mice, deduced that SFSV's hyperinflammatory effect was TLR3- and MAVS-dependent. Critically, we observed that L. major and SFSV co-infected C57BL/6 mice demonstrated significantly higher parasite burden than mice solely infected with L. major. Furthermore, viral presence increased leukocyte influx in vivo. This influx was accompanied by elevated total extracellular vesicle numbers. Interestingly, L. major displayed higher infectiveness with coincident phleboviral infection compared to L. major infection alone. CONCLUSION/SIGNIFICANCE: Overall our work represents novel findings that contribute towards understanding the causal mechanisms governing cutaneous leishmaniasis pathology. Better comprehension of the potential role of viral co-infection could lead to treatment regimens with enhanced effectiveness.

Metabolic reprogramming of the myeloid lineage by Schistosoma mansoni infection persists independently of antigen exposure.

Macrophages have a defined role in the pathogenesis of metabolic disease and cholesterol metabolism where alternative activation of macrophages is thought to be beneficial to both glucose and cholesterol metabolism during high fat diet induced disease. It is well established that helminth infection protects from metabolic disease, but the mechanisms underlying protection are not well understood. Here, we investigated the effects of Schistosoma mansoni infection and cytokine activation in the metabolic signatures of bone marrow derived macrophages using an approach that integrated transcriptomics, metabolomics, and lipidomics in a metabolic disease prone mouse model. We demonstrate that bone marrow derived macrophages (BMDM) from S. mansoni infected male ApoE-/- mice have dramatically increased mitochondrial respiration compared to those from uninfected mice. This change is associated with increased glucose and palmitate shuttling into TCA cycle intermediates, increased accumulation of free fatty acids, and decreased accumulation of cellular cholesterol esters, tri and diglycerides, and is dependent on mgll activity. Systemic injection of IL-4 complexes is unable to recapitulate either reductions in systemic glucose AUC or the re-programing of BMDM mitochondrial respiration seen in infected males. Importantly, the metabolic reprogramming of male myeloid cells is transferrable via bone marrow transplantation to an uninfected host, indicating maintenance of reprogramming in the absence of sustained antigen exposure. Finally, schistosome induced metabolic and bone marrow modulation is sex-dependent, with infection protecting male, but not female mice from glucose intolerance and obesity. Our findings identify a transferable, long-lasting sex-dependent reprograming of the metabolic signature of macrophages by helminth infection, providing key mechanistic insight into the factors regulating the beneficial roles of helminth infection in metabolic disease.

Spatial Point Pattern Analysis Identifies Mechanisms Shaping the Skin Parasite Landscape in Leishmania donovani Infection.

Increasing evidence suggests that in hosts infected with parasites of the Leishmania donovani complex, transmission of infection to the sand fly vector is linked to parasite repositories in the host skin. However, a detailed understanding of the dispersal (the mechanism of spread) and dispersion (the observed state of spread) of these obligatory-intracellular parasites and their host phagocytes in the skin is lacking. Using endogenously fluorescent parasites as a proxy, we apply image analysis combined with spatial point pattern models borrowed from ecology to characterize dispersion of parasitized myeloid cells (including ManR+ and CD11c+ cells) and predict dispersal mechanisms in a previously described immunodeficient model of L. donovani infection. Our results suggest that after initial seeding of infection in the skin, heavily parasite-infected myeloid cells are found in patches that resemble innate granulomas. Spread of parasites from these initial patches subsequently occurs through infection of recruited myeloid cells, ultimately leading to self-propagating networks of patch clusters. This combination of imaging and ecological pattern analysis to identify mechanisms driving the skin parasite landscape offers new perspectives on myeloid cell behavior following parasitism by L. donovani and may also be applicable to elucidating the behavior of other intracellular tissue-resident pathogens and their host cells.

Circulating and bone marrow myeloid cells containing Leishmania amastigotes in a case of advanced canine leishmaniosis.

A 5-y-old male Poodle mix was presented with intermittent vomiting, anorexia, and weight loss. Physical examination revealed emaciation, lethargy, dehydration, hypothermia, respiratory distress, and splenomegaly. Based on clinicopathologic, serologic, and parasitologic findings, diagnoses of severe leishmaniosis and dirofilariasis were made. Extracellular, intraneutrophilic, and intramonocytic Leishmania amastigotes were observed on blood smear and buffy coat smear examination. In blood smears, 0.2% of neutrophils were observed to be infected; in buffy coat smears, 0.5% of neutrophils and 0.1% of monocytes were found to be infected. Leishmania amastigotes were also found engulfed by eosinophils and neutrophil precursors in bone marrow aspiration cytology. The detection of Leishmania amastigotes in blood smears is rare, and the clinical significance is uncertain. In circulating blood, Leishmania amastigotes are primarily found phagocytized by neutrophils. Although debatable, there is growing evidence that neutrophils are used as carriers enabling the "silent entry" of the protozoa into macrophages ("Trojan horse" theory). To date, cytologic screening of blood smears for the diagnosis of canine leishmaniosis is not a routine practice. Clinical pathologists and practitioners should be aware that Leishmania amastigotes may be present in neutrophils and less frequently monocytes during blood smear evaluation; neutrophil precursors and eosinophils may also be parasitized in bone marrow specimens.

Gamma Interferon Mediates Experimental Cerebral Malaria by Signaling within Both the Hematopoietic and Nonhematopoietic Compartments.

Experimental cerebral malaria (ECM) is a gamma interferon (IFN-γ)-dependent syndrome. However, whether IFN-γ promotes ECM through direct and synergistic targeting of multiple cell populations or by acting primarily on a specific responsive cell type is currently unknown. Here, using a panel of cell- and compartment-specific IFN-γ receptor 2 (IFN-γR2)-deficient mice, we show that IFN-γ causes ECM by signaling within both the hematopoietic and nonhematopoietic compartments. Mechanistically, hematopoietic and nonhematopoietic compartment-specific IFN-γR signaling exerts additive effects in orchestrating intracerebral inflammation, leading to the development of ECM. Surprisingly, mice with specific deletion of IFN-γR2 expression on myeloid cells, T cells, or neurons were completely susceptible to terminal ECM. Utilizing a reductionist in vitro system, we show that synergistic IFN-γ and tumor necrosis factor (TNF) stimulation promotes strong activation of brain blood vessel endothelial cells. Combined, our data show that within the hematopoietic compartment, IFN-γ causes ECM by acting redundantly or by targeting non-T cell or non-myeloid cell populations. Within the nonhematopoietic compartment, brain endothelial cells, but not neurons, may be the major target of IFN-γ leading to ECM development. Collectively, our data provide information on how IFN-γ mediates the development of cerebral pathology during malaria infection.

HIF-1α is a key regulator in potentiating suppressor activity and limiting the microbicidal capacity of MDSC-like cells during visceral leishmaniasis.

Leishmania donovani is known to induce myelopoiesis and to dramatically increase extramedullary myelopoiesis. This results in splenomegaly, which is then accompanied by disruption of the splenic microarchitecture, a chronic inflammatory environment, and immunosuppression. Chronically inflamed tissues are typically hypoxic. The role of hypoxia on myeloid cell functions during visceral leishmaniasis has not yet been studied. Here we show that L. donovani promotes the output from the bone marrow of monocytes with a regulatory phenotype that function as safe targets for the parasite. We also demonstrate that splenic myeloid cells acquire MDSC-like function in a HIF-1α-dependent manner. HIF-1α is also involved in driving the polarization towards M2-like macrophages and rendering intermediate stage monocytes more susceptible to L. donovani infection. Our results suggest that HIF-1α is a major player in the establishment of chronic Leishmania infection and is crucial for enhancing immunosuppressive functions and lowering leishmanicidal capacity of myeloid cells.

Myeloid cells do not contribute to gender-dependent differences in disease outcome in murine cutaneous leishmaniasis.

Gender-associated differences in the outcome of infections are well known. Apart from behavior-released differences in their incidence, immunological factors also contribute to disease outcome. The underlying mechanisms are often unknown. Here, we show that in murine experimental leishmaniasis, female mice develop larger skin lesions that harbor significantly more parasites, exhibit increased parasite dissemination to visceral organs associated with a shift towards T helper (Th) 2 immunity with increased levels of IL-4. Antigen presenting cells (APC) responsible for T cell priming, such as macrophages or dendritic cells, were not involved in the process. Additionally, in adoptive transfer experiments, we show that differences in the lymphoid lineage are also not critical for mediating these gender-dependent effects. In summary, neither myeloid nor lymphoid cells contribute to disease outcome against this important human pathogen, but stromal cells influenced by e.g. hormonal effects in addition to other parts of the immune system might play a role.

GITR Activation Positively Regulates Immune Responses against Toxoplasma gondii.

Toxoplasma gondii is a widespread parasite responsible for causing clinical diseases especially in pregnant and immunosuppressed individuals. Glucocorticoid-induced TNF receptor (GITR), which is also known as TNFRS18 and belongs to the TNF receptor superfamily, is found to be expressed in various cell types of the immune system and provides an important costimulatory signal for T cells and myeloid cells. However, the precise role of this receptor in the context of T. gondii infection remains elusive. Therefore, the current study investigated the role of GITR activation in the immunoregulation mechanisms induced during the experimental infection of mice with T. gondii. Our data show that T. gondii infection slightly upregulates GITR expression in Treg cells and B cells, but the most robust increment in expression was observed in macrophages and dendritic cells. Interestingly, mice infected and treated with an agonistic antibody anti-GITR (DTA-1) presented a robust increase in pro-inflammatory cytokine production at preferential sites of parasite replication, which was associated with the decrease in latent brain parasitism of mice under treatment with DTA-1. Several in vivo and in vitro analysis were performed to identify the cellular mechanisms involved in GITR activation upon infection, however no clear alterations were detected in the phenotype/function of macrophages, Tregs and B cells under treatment with DTA-1. Therefore, GITR appears as a potential target for intervention during infection by the parasite Toxoplasma gondii, even though further studies are still necessary to better characterize the immune response triggered by GITR activation during T. gondii infection.

Contribution of myeloid cell subsets to liver fibrosis in parasite infection.

Accumulation of extracellular matrix components secreted by fibroblasts is a normal feature of wound healing during acute inflammation. However, during most chronic/persistent inflammatory diseases, this tissue repair mechanism is incorrectly regulated and results in irreversible fibrosis in various organs. Fibrosis that severely affects tissue architecture and can cause organ failure is a major cause of death in developed countries. Organ-recruited lymphoid (mainly T cells) and myeloid cells (eosinophils, basophils, macrophages and DCs) have long been recognized in their participation to the development of fibrosis. In particular, a central role for recruited monocyte-derived macrophages in this excessive connective tissue deposit is more and more appreciated. Moreover, the polarization of monocyte-derived macrophages in classically activated (IFNγ-dependent) M1 cells or alternatively activated (IL-4/IL-13) M2 cells, that mirrors the Th1/Th2 polarization of T cells, is also documented to contribute differentially to the fibrotic process. Here, we review the current understanding of how myeloid cell subpopulations affect the development of fibrosis in parasite infections.

The receptor Slamf1 on the surface of myeloid lineage cells controls susceptibility to infection by Trypanosoma cruzi.

Trypanosoma cruzi, the protozoan parasite responsible for Chagas' disease, causes severe myocarditis often resulting in death. Here, we report that Slamf1-/- mice, which lack the hematopoietic cell surface receptor Slamf1, are completely protected from an acute lethal parasite challenge. Cardiac damage was reduced in Slamf1-/- mice compared to wild type mice, infected with the same doses of parasites, as a result of a decrease of the number of parasites in the heart even the parasitemia was only marginally less. Both in vivo and in vitro experiments reveal that Slamf1-defIcient myeloid cells are impaired in their ability to replicate the parasite and show altered production of cytokines. Importantly, IFN-γ production in the heart of Slamf1 deficient mice was much lower than in the heart of wt mice even though the number of infiltrating dendritic cells, macrophages, CD4 and CD8 T lymphocytes were comparable. Administration of an anti-Slamf1 monoclonal antibody also reduced the number of parasites and IFN-γ in the heart. These observations not only explain the reduced susceptibility to in vivo infection by the parasite, but they also suggest human Slamf1 as a potential target for therapeutic target against T. cruzi infection.

Cutting edge: mast cells critically augment myeloid-derived suppressor cell activity.

Myeloid-derived suppressor cells (MDSCs) are primarily recognized for their immunosuppressive properties in malignant disease. However, their interaction with other innate immune cells and their regulation of immune responses, such as in parasitic infection, necessitate further characterization. We used our previously published mouse model of MDSC accumulation to examine the immunoregulatory role of MDSCs in B16 melanoma metastasis and Nippostrongylus brasiliensis infection. In this study, we demonstrate that the activity of MDSCs is dependent on the immune stimuli and subset induced. Monocytic MDSCs predictably suppressed antitumor immune responses but granulocytic MDSCs surprisingly enhanced the clearance of N. brasiliensis infection. Intriguingly, both results were dependent on MDSC interaction with mast cells (MCs), as demonstrated by adoptive-transfer studies in MC-deficient (Kit(Wsh)(/)(Wsh)) mice. These findings were further supported by ex vivo cocultures of MCs and MDSCs, indicating a synergistic increase in cytokine production. Thus, MCs can enhance both immunosuppressive and immunosupportive functions of MDSCs.

Myeloid-derived suppressor cells help protective immunity to Leishmania major infection despite suppressed T cell responses.

Th1/Th2 cytokines play a key role in immune responses to Leishmania major by controlling macrophage activation for NO production and parasite killing. MDSCs, including myeloid precursors and immature monocytes, produce NO and suppress T cell responses in tumor immunity. We hypothesized that NO-producing MDSCs could help immunity to L. major infection. Gr1(hi)(Ly6C(hi)) CD11b(hi) MDSCs elicited by L. major infection suppressed polyclonal and antigen-specific T cell proliferation. Moreover, L. major-induced MDSCs killed intracellular parasites in a NO-dependent manner and reduced parasite burden in vivo. By contrast, treatment with ATRA, which induces MDSCs to differentiate into macrophages, increased development of lesions, parasite load, and T cell proliferation in draining LNs. Altogether, these results indicate that NO-producing MDSCs help protective immunity to L. major infection, despite suppressed T cell proliferation.

Myeloid-derived suppressor cells in parasitic infections.

Myeloid-derived suppressor cells (MDSC) are a heterogeneous population of immature myeloid cells that share a common property of suppressing immune responses. Several helminth and protozoan parasite species have developed efficient strategies to increase the rate of medullary or extramedullary myelopoiesis and to induce the expansion and accumulation of immature myeloid cells such as MDSC. In this review, we examine current knowledge on the factors mediating enhanced myelopoiesis and MDSC induction and recruitment during parasitic infections and how the MDSC phenotype and mechanism of immune modulation and suppression depends on the factors they encounter within the host. Finally, we place MDSC expansion in the context of the critical balance between parasite elimination and pathogenicity to the host and suggest attractive avenues for future research.

Allergen uptake, activation, and IL-23 production by pulmonary myeloid DCs drives airway hyperresponsiveness in asthma-susceptible mice.

Maladaptive, Th2-polarized inflammatory responses are integral to the pathogenesis of allergic asthma. As regulators of T cell activation, dendritic cells (DCs) are important mediators of allergic asthma, yet the precise signals which render endogenous DCs "pro-asthmatic", and the extent to which these signals are regulated by the pulmonary environment and host genetics, remains unclear. Comparative phenotypic and functional analysis of pulmonary DC populations in mice susceptible (A/J), or resistant (C3H) to experimental asthma, revealed that susceptibility to airway hyperresponsiveness is associated with preferential myeloid DC (mDC) allergen uptake, and production of Th17-skewing cytokines (IL-6, IL-23), whereas resistance is associated with increased allergen uptake by plasmacytoid DCs. Surprisingly, adoptive transfer of syngeneic HDM-pulsed bone marrow derived mDCs (BMDCs) to the lungs of C3H mice markedly enhanced lung IL-17A production, and rendered them susceptible to allergen-driven airway hyperresponsiveness. Characterization of these BMDCs revealed levels of antigen uptake, and Th17 promoting cytokine production similar to that observed in pulmonary mDCs from susceptible A/J mice. Collectively these data demonstrate that the lung environment present in asthma-resistant mice promotes robust pDC allergen uptake, activation, and limits Th17-skewing cytokine production responsible for driving pathologic T cell responses central to the development of allergen-induced airway hyperresponsiveness.

Alternatively activated myeloid cells limit pathogenicity associated with African trypanosomiasis through the IL-10 inducible gene selenoprotein P.

Uncontrolled inflammation is a major cause of tissue injury/pathogenicity often resulting in death of a host infected with African trypanosomes. Thus, comparing the immune response in hosts that develop different degrees of disease severity represents a promising approach to discover processes contributing to trypanosomiasis control. It is known that limitation of pathogenicity requires a transition in the course of infection, from an IFN-gamma-dependent response resulting in the development of classically activated myeloid cells (M1), to a counterbalancing IL-10-dependent response associated with alternatively activated myeloid cells (M2). Herein, mechanisms and downstream effectors by which M2 contribute to lower the pathogenicity and the associated susceptibility to African trypanosomiasis have been explored. Gene expression analysis in IL-10 knockout and wild-type mice, that are susceptible and relatively resistant to Trypanosoma congolense infection, respectively, revealed a number of IL-10-inducible genes expressed by M2, including Sepp1 coding for selenoprotein P. Functional analyses confirm that selenoprotein P contributes to limit disease severity through anti-oxidant activity. Indeed, Sepp1 knockout mice, but not Sepp1(Delta)(240-361) mice retaining the anti-oxidant motif but lacking the selenium transporter domain of selenoprotein P, exhibited increased tissue injury that associated with increased production of reactive oxygen species and increased apoptosis in the liver immune cells, reduced parasite clearance capacity of myeloid cells, and decreased survival. These data validate M2-associated molecules as functioning in reducing the impact of parasite infection on the host.

Leishmania donovani infection of human myeloid dendritic cells leads to a Th1 response in CD4+ T cells from healthy donors and patients with kala-azar.

The role played by dendritic cells (DCs) in Leishmania donovani infection is poorly understood. Here, we report that L. donovani amastigotes efficiently infect human peripheral-blood monocyte-derived DCs. Opsonization with normal human serum enhanced the infectivity of amastigotes and promastigotes only marginally. Surface attachment versus internalization was distinguished by incubation of DCs with live, fluorescein isothiocyanate-labeled parasites, followed by quenching with crystal violet. Infection with amastigotes was accompanied by DC maturation, as was evident from the up-regulation of maturation-associated cell-surface markers, the nuclear translocation of RelB, and the release of cytokines. Amastigote-primed DCs produced inflammatory cytokines in response to subsequent treatment with interferon- gamma or anti-CD40 monoclonal antibody. When cocultured, amastigote-infected DCs induced T helper cell type 1 (Th1) responses both in naive allogeneic CD4(+) T cells and in autologous CD4(+) T cells from patients with kala-azar and up-regulated the expression of T-bet. Our data reveal that infection with L. donovani amastigotes induces a Th1 cytokine milieu in both DCs and T cells.

A type I IFN-dependent pathway induced by Schistosoma mansoni eggs in mouse myeloid dendritic cells generates an inflammatory signature.

Schistosomes are helminth parasites that display a dual impact on the immune system of their hosts. Although the larval stage, also known as schistosomulum, appears to subvert the host defenses, the egg stage induces strong inflammatory reactions. Given the pivotal role of dendritic cells (DC) in initiating and regulating immune responses, we compared the distinct transcriptional programs induced in immature mouse DC by S. mansoni eggs or schistosomula. Although SLA abrogated the transcription of many genes implicated in DC functions, eggs caused myeloid DC to produce IFN-beta. Autocrine/paracrine signaling through the type I IFN receptor in response to eggs was necessary for the induction of known IFN-responsive genes and enhanced the synthesis of key inflammatory products. Taken as a whole, our data provide molecular insights into the immune evasion mechanism of schistosomula and suggest an unexpected role for type I IFN in the innate response to helminth eggs.

Synthesis and antiprotozoal activity of naphthofuranquinones and naphthothiophenequinones containing a fused thiazole ring.

The synthesis of tetracyclic quinones 10a,b, 14a,b, 19a,b and 20a,b is described. The preparations involve regioselective Diels-Alder reactions via trapping the thiazole o-quinodimethane 9 with several benzofuranquinones and benzothiophenequinones. The structure of the regioisomers was assigned through 2D NMR 1H-13C HMBC experiments performed on 10a and 14a. Compounds 10a,b, 14a as well as phenol 1 and the starting quinones 2, 5, 7 and 15 are evaluated against Leishmania sp., Toxoplasma gondii and THP-1 cells. Almost all the tested compounds exhibit significant antiprotozoal activities with lower cytotoxicities than the reference compounds. Among them, quinones 2 and 14a possess the best activities towards L. donovani and T. gondii with the lowest toxicities.

Theileria annulata: attenuation of a schizont-infected cell line by prolonged in vitro culture is not caused by the preferential growth of particular host cell types.

Flow cytometry and monoclonal antibodies to bovine leucocyte surface antigens were used to identify the types of host cells that the sporozoites of Theileria annulata infect in cattle, to determine whether virulent schizont-infected cell lines (lines) differed phenotypically from avirulent lines, and to establish whether attenuation in vitro was accompanied by the preferential growth of particular host cell types. The surface antigens of four pairs of T. annulata (Ta) (Hisar) lines derived ex vivo and in vitro, including the virulent ex vivo-derived Ta Hisar S45 line, were consistent with a myeloid origin for all lines, irrespective of their derivation. The profiles of lines derived from cattle inoculated with a virulent line showed that the schizonts liberated from inoculated cells had transferred to myeloid cells. A number of other lines infected with different stocks of T. annulata expressed myeloid markers; a single line expressed CD21, a B cell marker. During prolonged in vitro culture, the parasites in the ex vivo (virulent)- and in vitro (avirulent)-derived Ta Hisar S45 myeloid lines became clonal, as defined by glucose phosphate isomerase (GPI) polymorphism, and the virulent line became attenuated. The two lines retained phenotypic profiles indicative of a myeloid origin but coexpressed some lymphoid antigens (CD2, CD4, CD8), although not CD3. Cloned schizont-infected lines, representing the three parasite GPI isotypes which constituted the virulent line, expressed similar patterns of myeloid and lymphoid markers to the virulent parent line. Some schizont-infected clones failed to establish as lines during the early weeks of culture because the cells died as the parasites differentiated into merozoites at 37 degrees C, the temperature at which schizont-infected cells normally grow exponentially. These results provided no evidence that prolonged culture induces preferential growth or loss of particular host cell types. However, a number of the alterations in host cell surface antigens induced by prolonged culture were shown to be linked to permanent changes in the parasite genome.