HIF-1α modulates sex-specific Th17/Treg responses during hepatic amoebiasis.

BACKGROUND & AIMS: An invasive form of intestinal Entamoeba (E.) histolytica infection, which causes amoebic liver abscess, is more common in men than in women. Immunopathological mechanisms are responsible for the more severe outcome in males. Here, we used a mouse model of hepatic amoebiasis to investigate the contribution of hepatic hypoxia-inducible factor (HIF)-1α to T helper 17 (Th17)/regulatory T cell (Treg) responses in the context of the sex-specific outcome of liver damage. METHODS: C57BL/6J mice were infected intrahepatically with E. histolytica trophozoites. HIF-1α expression was determined by qPCR, flow cytometry and immunohistochemistry. Tregs and Th17 cells were analysed by immunohistochemistry and flow cytometry. Finally, male and female hepatocyte-specific Hif1α knockout mice were generated, and the effect of HIF-1α on abscess development, the cytokine milieu, and Th17/Treg differentiation was examined. RESULTS: E. histolytica infection increased hepatic HIF-1α levels, along with the elevated frequencies of hepatic Th17 and Treg cells. While the Th17 cell population was larger in male mice, Tregs characterised by increased expression of Foxp3 in female mice. Male mice displayed increased IL-6 expression, contributing to immunopathology; this increase in IL-6 expression declined upon deletion of hepatic HIF-1α. In both sexes, hepatic deletion of HIF-1α reduced the Th17 cell frequency; however, the percentage of Tregs was reduced in female mice only. CONCLUSIONS: Hepatic HIF-1α modulates the sex-specific outcome of murine E. histolytica infection. Our results suggest that in male mice, Th17 cells can be modulated by hepatic HIF-1α via IL-6, indicating marked involvement in the immunopathology underlying abscess development. Strong expression of Foxp3 by hepatic Tregs from female mice suggests a potent immunosuppressive function, leading to initiation of liver regeneration. LAY SUMMARY: Infection with the parasite Entamoeba histolytica activates immunopathological mechanisms in male mice, which lead to liver abscesses that are larger than those in female mice. In the absence of the protein HIF-1α in hepatocytes, abscess formation is reduced; moreover, the sex difference in abscess size is abolished. These results suggest that HIF-1α modulates the immune response involved in the induction of immunopathology, resulting in differential disease susceptibility in males and females.

Antileishmanial Effects of Synthetic EhPIb Analogs Derived from the Entamoeba histolytica Lipopeptidephosphoglycan.

With an estimated number of new cases annually of approximately 1.4 million, leishmaniasis belongs to the most important parasitic diseases in the world. Nevertheless, existing drugs against leishmaniasis in general have several drawbacks that urgently necessitate new drug development. A glycolipid molecule of the intestinal protozoan parasite Entamoeba histolytica and its synthetic analogs previously showed considerable immunotherapeutic effects against Leishmania major infection. Here, we designed and synthesized a series of new immunostimulatory compounds derived from the phosphatidylinositol b anchor of Entamoeba histolytica (EhPIb) subunit of the native compound and investigated their antileishmanial activity in vitro and in vivo in a murine model of cutaneous leishmaniasis. The new synthetic EhPIb analogs showed almost no toxicity in vitro Treatment with the analogs significantly decreased the parasite load in murine and human macrophages in vitro In addition, topical application of the EhPIb analog Eh-1 significantly reduced cutaneous lesions in the murine model, correlating with an increase in the production of selected Th1 cytokines. In addition, we could show in in vitro experiments that treatment with Eh-1 led to a decrease in mRNA expression of arginase-1 (Arg1) and interleukin 4 (IL-4), which are required by the parasites to circumvent their elimination by the immune response. The use of the host-targeting synthetic EhPIb compounds, either alone or in combination therapy with antiparasitic drugs, shows promise for treating cutaneous leishmaniasis and therefore might improve the current unsatisfactory status of chemotherapy against this infectious disease.

Trigger-induced RNAi gene silencing to identify pathogenicity factors of Entamoeba histolytica.

Recently, Entamoeba histolytica clones derived from isolate HM-1:IMSS that differ in their pathogenicity were identified. Whereas some clones induce amoebic liver abscesses (ALAs) in animal models of amoebiasis, others provoke only minimal liver lesions. Based on transcriptome studies of pathogenic and nonpathogenic clones, differentially expressed genes associated with reduced or increased liver pathology can be identified. Here, to analyze the influence of these genes on ALA formation in more detail, an RNA interference-trigger mediated silencing approach was used. Using newly identified trigger sequences, the expression of 15 genes was silenced. The respective transfectants were analyzed for their ability to induce liver destruction in the murine model for the disease. Silencing of EHI_180390 (encoding an AIG1 protein) increased liver pathology induced by a nonpathogenic parent clone, whereas silencing of EHI_127670 (encoding a hypothetical protein) decreased the pathogenicity of an initially pathogenic parent clone. Additional phenotypical in vitro analyses of EHI_127670 silencing as well as overexpression transfectants indicated that this molecule has an influence on size, growth, and cysteine peptidase activity of E. histolytica. This work describes an example of how the sole operational method for effective gene silencing in E. histolytica can be used for comprehensive analyses of putative pathogenicity factors.-Matthiesen, J., Lender, C., Haferkorn, A., Fehling, H., Meyer, M., Matthies, T., Tannich, E., Roeder, T., Lotter, H., Bruchhaus, I. Trigger-induced RNAi gene silencing to identify pathogenicity factors of Entamoeba histolytica.

Overexpression of Differentially Expressed Genes Identified in Non-pathogenic and Pathogenic Entamoeba histolytica Clones Allow Identification of New Pathogenicity Factors Involved in Amoebic Liver Abscess Formation.

We here compared pathogenic (p) and non-pathogenic (np) isolates of Entamoeba histolytica to identify molecules involved in the ability of this parasite to induce amoebic liver abscess (ALA)-like lesions in two rodent models for the disease. We performed a comprehensive analysis of 12 clones (A1-A12) derived from a non-pathogenic isolate HM-1:IMSS-A and 12 clones (B1-B12) derived from a pathogenic isolate HM-1:IMSS-B. "Non-pathogenicity" included the induction of small and quickly resolved lesions while "pathogenicity" comprised larger abscess development that overstayed day 7 post infection. All A-clones were designated as non-pathogenic, whereas 4 out of 12 B-clones lost their ability to induce ALAs in gerbils. No correlation between ALA formation and cysteine peptidase (CP) activity, haemolytic activity, erythrophagocytosis, motility or cytopathic activity was found. To identify the molecular framework underlying different pathogenic phenotypes, three clones were selected for in-depth transcriptome analyses. Comparison of a non-pathogenic clone A1np with pathogenic clone B2p revealed 76 differentially expressed genes, whereas comparison of a non-pathogenic clone B8np with B2p revealed only 19 differentially expressed genes. Only six genes were found to be similarly regulated in the two non-pathogenic clones A1np and B8np in comparison with the pathogenic clone B2p. Based on these analyses, we chose 20 candidate genes and evaluated their roles in ALA formation using the respective gene-overexpressing transfectants. We conclude that different mechanisms lead to loss of pathogenicity. In total, we identified eight proteins, comprising a metallopeptidase, C2 domain proteins, alcohol dehydrogenases and hypothetical proteins, that affect the pathogenicity of E. histolytica.

IL-23 prevents IL-13-dependent tissue repair associated with Ly6C(lo) monocytes in Entamoeba histolytica-induced liver damage.

BACKGROUND & AIMS: The IL-23/IL-17 axis plays an important role in the pathogenesis of autoimmune diseases and the pathological consequences of infection. We previously showed that immunopathologic mechanisms mediated by inflammatory monocytes underlie the severe focal liver damage induced by the protozoan parasite, Entamoeba histolytica. Here, we analyze the contribution of the IL-23/IL-17 axis to the induction and subsequent recovery from parasite-induced liver damage. METHODS: IL-23p19(-/-), IL-17A/F(-/-), CCR2(-/-), and wild-type (WT) mice were intra-hepatically infected with E. histolytica trophozoites and disease onset and recovery were analyzed by magnetic resonance imaging. Liver-specific gene and protein expression during infection was examined by qPCR, microarray, FACS analysis and immunohistochemistry. Immuno-depletion and substitution experiments were performed in IL-23p19(-/-) and WT mice to investigate the role of IL-13 in disease outcome. RESULTS: Liver damage in infected IL-23p19(-/-), IL-17A/F(-/-), and CCR2(-/-) mice was strongly attenuated compared with that in WT mice. IL-23p19(-/-) mice showed reduced accumulation of IL-17 and CCL2 mRNA and proteins. Increased numbers of IL-13-producing CD11b(+)Ly6C(lo) monocytes were associated with disease attenuation in IL-23p19(-/-) mice. Immuno-depletion of IL-13 in IL-23p19(-/-) mice reversed this attenuation and treatment of infected WT mice with an IL-13/anti-IL-13-mAb complex supported liver recovery. CONCLUSIONS: The IL-23/IL-17 axis plays a critical role in the immunopathology of hepatic amebiasis. IL-13 secreted by CD11b(+)Ly6C(lo) monocytes may be associated with recovery from liver damage. An IL-13/anti-IL13-mAb complex mimics this function, suggesting a novel therapeutic option to support tissue healing after liver damage.

The cytokine profile of human NKT cells and PBMCs is dependent on donor sex and stimulus.

Sex-related variations in natural killer T (NKT) cells may influence immunoregulation and outcome of infectious and autoimmune diseases. We analyzed sex-specific differences in peripheral blood NKTs and peripheral blood mononuclear cells (PBMCs) from men and women and determined the frequencies of NKT cells and their subpopulations [CD4(+); CD8(+); double negative (DN)] and the levels of cytokine production following stimulation with the NKT cell ligands α-Galactosylceramide (αGalCer) and Entamoeba histolytica lipopeptidephosphoglycan (Lotter et al. in PLoS Pathog 5(5):e1000434, 2009). Total and DN NKT cells were more abundant in women than in men. In women, αGalCer induced higher production of intracellular IFNγ, IL-4, IL-17 and TNF by CD4(+) and DN(+)NKT cells. Both ligands induced expression of multiple cytokines in PBMCs and influenced the ratio of NKT cell subpopulations during long-term culture. Although the sex-specific differences in frequencies of NKT cells and their subpopulations were marginal, the significant sex-specific differences in cytokine production might influence disease outcomes.

Antimicrobial activity of NK cells to Trypanosoma cruzi infected human primary Keratinocytes.

Infection with the protozoan parasite Trypanosoma cruzi is causative for Chagas disease, which is a highly neglected tropical disease prevalent in Latin America. Humans are primary infected through vectorial transmission by blood-sucking triatomine bugs. The parasite enters the human host through mucous membranes or small skin lesions. Since keratinocytes are the predominant cell type in the epidermis, they play a critical role in detecting disruptions in homeostasis and aiding in pathogen elimination by the immune system in the human skin as alternative antigen-presenting cells. Interestingly, keratinocytes also act as a reservoir for T. cruzi, as the skin has been identified as a major site of persistent infection in mice with chronic Chagas disease. Moreover, there are reports of the emergence of T. cruzi amastigote nests in the skin of immunocompromised individuals who are experiencing reactivation of Chagas disease. This observation implies that the skin may serve as a site for persistent parasite presence during chronic human infection too and underscores the significance of investigating the interactions between T. cruzi and skin cells. Consequently, the primary objective of this study was to establish and characterize the infection kinetics in human primary epidermal keratinocytes (hPEK). Our investigation focused on surface molecules that either facilitated or hindered the activation of natural killer (NK) cells, which play a crucial role in controlling the infection. To simulate the in vivo situation in humans, an autologous co-culture model was developed to examine the interactions between T. cruzi infected keratinocytes and NK cells. We evaluated the degranulation, cytokine production, and cytotoxicity of NK cells in response to the infected keratinocytes. We observed a strong activation of NK cells by infected keratinocytes, despite minimal alterations in the expression of activating or inhibitory ligands on NK cell receptors. However, stimulation with recombinant interferon-gamma (IFN-γ), a cytokine known to be present in significant quantities during chronic T. cruzi infections in the host, resulted in a substantial upregulation of these ligands on primary keratinocytes. Overall, our findings suggest the crucial role of NK cells in controlling acute T. cruzi infection in the upper layer of the skin and shed light on keratinocytes as potential initial targets of infection.

Testosterone affects type I/type II interferon response of neutrophils during hepatic amebiasis.

Differences in immune response between men and women may influence the outcome of infectious diseases. Intestinal infection with Entamoeba histolytica leads to hepatic amebiasis, which is more common in males. Previously, we reported that innate immune cells contribute to liver damage in males in the murine model for hepatic amebiasis. Here, we focused on the influences of sex and androgens on neutrophils in particular. Infection associated with neutrophil accumulation in the liver was higher in male than in female mice and further increased after testosterone treatment in both sexes. Compared with female neutrophils, male neutrophils exhibit a more immature and less activated status, as evidenced by a lower proinflammatory N1-like phenotype and deconvolution, decreased gene expression of type I and type II interferon stimulated genes (ISGs) as well as downregulation of signaling pathways related to neutrophil activation. Neutrophils from females showed higher protein expression of the type I ISG viperin/RSAD2 during infection, which decreased by testosterone substitution. Moreover, ex vivo stimulation of human neutrophils revealed lower production of RSAD2 in neutrophils from men compared with women. These findings indicate that sex-specific effects on neutrophil physiology associated with maturation and type I IFN responsiveness might be important in the outcome of hepatic amebiasis.

The Properties of Proinflammatory Ly6Chi Monocytes Are Differentially Shaped by Parasitic and Bacterial Liver Infections.

In the past, proinflammatory CD11b+Ly6Chi monocytes were predominantly considered as a uniform population. However, recent investigations suggests that this population is far more diverse than previously thought. For example, in mouse models of Entamoeba (E.) histolytica and Listeria (L.) monocytogenes liver infections, it was shown that their absence had opposite effects. In the former model, it ameliorated parasite-dependent liver injury, whereas in the listeria model it exacerbated liver pathology. Here, we analyzed Ly6Chi monocytes from the liver of both infection models at transcriptome, protein, and functional levels. Paralleled by E. histolytica- and L. monocytogenes-specific differences in recruitment-relevant chemokines, both infections induced accumulation of Ly6C+ monocytes at infection sites. Transcriptomic analysis revealed a high similarity between monocytes from naïve and parasite-infected mice and a clear proinflammatory phenotype of listeria-induced monocytes. This was further reflected by the upregulation of M2-related transcription factors (e.g., Mafb, Nr4a1, Fos) and higher CD14 expression by Ly6Chi monocytes in the E. histolytica infection model. In contrast, monocytes from the listeria infection model expressed M1-related transcription factors (e.g., Irf2, Mndal, Ifi204) and showed higher expression of CD38, CD74, and CD86, as well as higher ROS production. Taken together, proinflammatory Ly6Chi monocytes vary considerably depending on the causative pathogen. By using markers identified in the study, Ly6Chi monocytes can be further subdivided into different populations.

High Content Analysis of Macrophage-Targeting EhPIb-Compounds against Cutaneous and Visceral Leishmania Species.

An immunostimulatory glycolipid molecule from the intestinal protozoan parasite Entamoeba histolytica (Eh) and its synthetic analogs derived from its phosphatidylinositol-b-anchor (EhPIb) previously showed considerable immunotherapeutic effects against Leishmania major infection in vitro and in vivo. Here, we describe a high content screening assay, based on primary murine macrophages. Parasites detection is based on a 90 kDA heat shock protein-specific staining, enabling the detection of several Leishmania species. We validated the assay using L. major, L. braziliensis, L. donovani, and L. infantum as well as investigated the anti-leishmanial activity of six immunostimulatory EhPIb-compounds (Eh-1 to Eh-6). Macrophages infected with dermotropic species were more sensitive towards treatment with the compounds as their viability showed a stronger reduction compared to macrophages infected with viscerotropic species. Most compounds caused a significant reduction of the infection rates and the parasite burdens depending on the infecting species. Only compound Eh-6 was found to have activity against all Leishmania species. Considering the challenges in anti-leishmanial drug discovery, we developed a multi-species screening assay capable of utilizing non-recombinant parasite strains, and demonstrated its usefulness by screening macrophage-targeting EhPIb-compounds showing their potential for the treatment of cutaneous and visceral leishmaniasis.

An Alcohol Dehydrogenase 3 (ADH3) from Entamoeba histolytica Is Involved in the Detoxification of Toxic Aldehydes.

Recently, a putative alcohol dehydrogenase 3, termed EhADH3B of the Entamoeba histolytica isolate HM-1:IMSS was identified, which is expressed at higher levels in non-pathogenic than in pathogenic amoebae and whose overexpression reduces the virulence of pathogenic amoebae. In an in silico analysis performed in this study, we assigned EhADH3B to a four-member ADH3 family, with ehadh3b present as a duplicate (ehadh3ba/ehadh3bb). In long-term laboratory cultures a mutation was identified at position 496 of ehadh3ba, which codes for a stop codon, which was not the case for amoebae isolated from human stool samples. When using transfectants that overexpress or silence ehadh3bb, we found no or little effect on growth, size, erythrophagocytosis, motility, hemolytic or cysteine peptidase activity. Biochemical characterization of the recombinant EhADH3Bb revealed that this protein forms a dimer containing Ni2+ or Zn2+ as a co-factor and that the enzyme converts acetaldehyde and formaldehyde in the presence of NADPH. A catalytic activity based on alcohols as substrates was not detected. Based on the results, we postulate that EhADH3Bb can reduce free acetaldehyde released by hydrolysis from bifunctional acetaldehyde/alcohol dehydrogenase-bound thiohemiacetal and that it is involved in detoxification of toxic aldehydes produced by the host or the gut microbiota.

Androgens predispose males to monocyte-mediated immunopathology by inducing the expression of leukocyte recruitment factor CXCL1.

Hepatic amebiasis, predominantly occurring in men, is a focal destruction of the liver due to the invading protozoan Entamoeba histolytica. Classical monocytes as well as testosterone are identified to have important functions for the development of hepatic amebiasis in mice, but a link between testosterone and monocytes has not been identified. Here we show that testosterone treatment induces proinflammatory responses in human and mouse classical monocytes. When treated with 5α-dihydrotestosterone, a strong androgen receptor ligand, human classical monocytes increase CXCL1 production in the presence of Entamoeba histolytica antigens. Moreover, plasma testosterone levels of individuals undergoing transgender procedure correlate positively with the TNF and CXCL1 secretion from their cultured peripheral blood mononuclear cells following lipopolysaccharide stimulation. Finally, testosterone substitution of castrated male mice increases the frequency of TNF/CXCL1-producing classical monocytes during hepatic amebiasis, supporting the hypothesis that the effects of androgens may contribute to an increased risk of developing monocyte-mediated pathologies.

Magnetic resonance imaging of pathogenic protozoan parasite Entamoeba histolytica labeled with superparamagnetic iron oxide nanoparticles.

OBJECTIVES: The aim of this study was to establish a noninvasive tracking of the pathogenic parasite Entamoeba histolytica (Eh) after superparamagnetic iron oxide (SPIO) labeling by magnetic resonance imaging (MRI) on a single-cell level in vitro and in vivo in a mouse model for amebic liver abscess (ALA). MATERIALS AND METHODS: Local institutional review committee on animal care approved all animal experiments. Entamoeba histolytica trophozoites were labeled with SPIO nanoparticles (SPIO-Eh). The uptake of SPIO by Eh was optimized using flow cytometry and visualized by bright field, fluorescence, and transmission electron microscopy. The viability of SPIO-Eh was assessed in vitro by determination of growth and ingestion rate of red blood cells. Migration of SPIO-Eh was proven by in vitro MRI in a preclinical 7 T MRI system using continually repeated MRI scans. In vivo distribution of SPIO-Eh within the mouse liver was assessed qualitatively and quantitatively by serial respiration-triggered T2*-weighted MRI, T2-weighted MRI, and R2* MR relaxometry up to 5 days after injection and correlated with immunohistology of the liver sections after removal. RESULTS: Entamoeba histolytica can be efficiently labeled with SPIO without influence on parasite growth rate or phagocytic capacity. In vitro dynamic MRI allowed real-time migration monitoring and determination of velocity of single SPIO-Eh. In vivo SPIO-Eh showed signal decrease in T2*-weighted and increase of R2* in ALA formations. Motility of SPIO-Eh was necessary to induce ALA formations. CONCLUSIONS: The present study demonstrates the feasibility of an efficient magnetic labeling and a noninvasive in vitro and in vivo MR tracking of the pathogenic protozoan Eh in a mouse model for ALA, thus representing in future a noninvasive imaging tool to study parasite, as well as on host-specific pathomechanisms.