Genes differentially expressed between pathogenic and non-pathogenic Entamoeba histolytica clones influence pathogenicity-associated phenotypes by multiple mechanisms.

Recently, two genes involved in amoebic liver abscess formation in a mouse model were identified by their differential expression of non-pathogenic (A1np) and pathogenic (B2p) clones of the Entamoeba histolytica isolate HM:1-IMSS. While overexpression of a gene encoding the metallopeptidase EhMP8-2 reduces the virulence of the pathogenic clone B2p, overexpression of the gene ehi_127670 (ehhp127), encoding a hypothetical protein, increases the virulence of the non-pathogenic clone A1np, while silencing this gene in the pathogenic B2p reduces virulence. To understand the role of both molecules in determining the pathogenicity of E. histolytica, silencing, and overexpression transfectants were characterized in detail. Silencing of ehmp8-2, of the homologous gene ehmp8-1, or both in non-pathogenic A1np trophozoites significantly altered the transcript levels of 347, 216, and 58 genes, respectively. This strong change in the expression profiles caused by the silencing of ehmp8-1 and ehmp8-2 implies that these peptidases regulate the expression of numerous genes. Consequently, numerous phenotypic characteristics, including cytopathic, hemolytic, and cysteine peptidase activity, were altered in response to their silencing. Silencing of ehhp127 in pathogenic B2p trophozoites did not affect the expression of other genes, whereas its overexpression in non-pathogenic A1np trophozoites results in an altered expression of approximately 140 genes. EhHP127 is important for trophozoite motility, as its silencing reduces, while its overexpression enhances movement activity. Interestingly, the specific silencing of ehhp127 also significantly affects cytopathic, cysteine peptidase, and hemolytic activities. All three molecules characterized in this study, namely EhMP8-1, EhMP8-2, and EhHP127, are present in amoeba vesicles. The results show that ehmp8-2 and ehhp127 are not only differentially expressed between pathogenic and non-pathogenic amoebae, but that they also significantly affect amoeba pathogenicity-associated phenotypes by completely different mechanisms. This observation suggests that the regulation of amoeba pathogenicity is achieved by a complex network of molecular mechanisms rather than by single factors.

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.

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.

CCR2 Deficiency Impairs Ly6Clo and Ly6Chi Monocyte Responses in Orientia tsutsugamushi Infection.

Orientia (O.) tsutsugamushi, the causative agent of scrub typhus, is a neglected, obligate intracellular bacterium that has a prominent tropism for monocytes and macrophages. Complications often involve the lung, where interstitial pneumonia is a typical finding. The severity of scrub typhus in humans has been linked to altered plasma concentrations of chemokines which are known to act as chemoattractants for myeloid cells. The trafficking and function of monocyte responses is critically regulated by interaction of the CC chemokine ligand 2 (CCL2) and its CC chemokine receptor CCR2. In a self-healing mouse model of intradermal infection with the human-pathogenic Karp strain of O. tsutsugamushi, we investigated the role of CCR2 on bacterial dissemination, development of symptoms, lung histology and monocyte subsets in blood and lungs. CCR2-deficient mice showed a delayed onset of disease and resolution of symptoms, higher concentrations and impaired clearance of bacteria in the lung and the liver, accompanied by a slow infiltration of interstitial macrophages into the lungs. In the blood, we found an induction of circulating monocytes that depended on CCR2, while only a small increase in Ly6Chi monocytes was observed in CCR2-/- mice. In the lung, significantly higher numbers of Ly6Chi and Ly6Clo monocytes were found in the C57BL/6 mice compared to CCR2-/- mice. Both wildtype and CCR2-deficient mice developed an inflammatory milieu as shown by cytokine and inos/arg1 mRNA induction in the lung, but with delayed kinetics in CCR2-deficient mice. Histopathology revealed that infiltration of macrophages to the parenchyma, but not into the peribronchial tissue, depended on CCR2. In sum, our data suggest that in Orientia infection, CCR2 drives blood monocytosis and the influx and activation of Ly6Chi and Ly6Clo monocytes into the lung, thereby accelerating bacterial replication and development of interstitial pulmonary inflammation.

Monocyte dysregulation: consequences for hepatic infections.

Liver disorders due to infections are a substantial health concern in underdeveloped and industrialized countries. This includes not only hepatotropic viruses (e.g., hepatitis B, hepatitis C) but also bacterial and parasitic infections such as amebiasis, leishmaniasis, schistosomiasis, or echinococcosis. Recent studies of the immune mechanisms underlying liver disease show that monocytes play an essential role in determining patient outcomes. Monocytes are derived from the mononuclear phagocyte lineage in the bone marrow and are present in nearly all tissues of the body; these cells function as part of the early innate immune response that reacts to challenge by external pathogens. Due to their special ability to develop into tissue macrophages and dendritic cells and to change from an inflammatory to an anti-inflammatory phenotype, monocytes play a pivotal role in infectious and non-infectious liver diseases: they can maintain inflammation and support resolution of inflammation. Therefore, tight regulation of monocyte recruitment and termination of monocyte-driven immune responses in the liver is prerequisite to appropriate healing of organ damage. In this review, we discuss monocyte-dependent immune mechanisms underlying hepatic infectious disorders. Better understanding of these immune mechanisms may lead to development of new interventions to treat acute liver disease and prevent progression to organ failure.

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.

Programmed Cell Death Ligand (PD-L)-1 Contributes to the Regulation of CD4+ T Effector and Regulatory T Cells in Cutaneous Leishmaniasis.

Cutaneous Leishmaniasis (CL) affects up to one million people every year and treatments are costly and toxic. The regulation of the host immune response is complex and the knowledge of how CD4+ T cells are activated and maintained during Leishmania infection is still limited. Current therapies aim to target programmed cell death (PD)-1 and programmed cell death ligand (PD-L)-1 in order to boost T cell activity. However, the role of the PD-1/PD-L1 axis during Leishmania infection is still unclear. In this study, we found that patients with active and post-treatment CL displayed different subsets of CD4+PD-1+ T cells. Accordingly, L. major-infected mice upregulated PD-1 on activated CD4+ T effector cells and PD-L1 on resident macrophages and infiltrating monocytes at the site of infection. L. major-infected Pdl1-/- mice expressed lower levels of MHCII and higher levels of CD206 on macrophages and monocytes and, more importantly, the lack of PD-L1 contributed to a reduced frequency of CD4+Ly6Chi T effector cells and an increase of CD4+Foxp3+ regulatory T cells at the site of infection and in draining lymph nodes. Additionally, the lack of PD-L1 was associated with lower production of IL-27 by infiltrating monocytes and lower levels of the Th1 cytokines IFN-γ and TNF-α produced by CD4+ T effector cells. Pdl1-/- mice initially exhibited larger lesions despite having a similar parasite load. Our results describe for the first time how the interruption of the PD-1/PD-L1 axis influences the immune response against CL and suggests that this axis regulates the balance between CD4+Ly6Chi T effector cells and CD4+Foxp3+ regulatory T cells.

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.

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.

Androgen-dependent immune modulation in parasitic infection.

Parasitic infections modulate the immune system of the host, resulting in either immune tolerance or the induction of pro-inflammatory defense mechanisms against the pathogen. In both cases, sex hormones are involved in the regulation of the immune response, as they are present in the systemic circulation and can act on a wide variety of cell types, including immune cells. Men and women have a different milieu of sex hormones, and these hormones play a role in determining immune responses to parasitic infections. Men, who have higher plasma levels of androgens than women, are generally more susceptible to parasitic infections. Many immune cells express the androgen receptor (AR), and the immunologic functions of these cells can be modulated by androgens. In this review, we will highlight the immune cell types that are sensitive to male steroid hormones and describe their roles during three parasitic diseases, amebiasis, leishmaniasis, and helminthiasis.

A Protective Function of IL-22BP in Ischemia Reperfusion and Acetaminophen-Induced Liver Injury.

Acute liver injury can be secondary to a variety of causes, including infections, intoxication, and ischemia. All of these insults induce hepatocyte death and subsequent inflammation, which can make acute liver injury a life-threatening event. IL-22 is a dual natured cytokine which has context-dependent protective and pathogenic properties during tissue damage. Accordingly, IL-22 was shown to promote liver regeneration upon acute liver damage. However, other studies suggest pathogenic properties of IL-22 during chronic liver injury. IL-22 binding protein (IL-22BP, IL-22Ra2) is a soluble inhibitor of IL-22 that regulates IL-22 activity. However, the significance of endogenous IL-22BP in acute liver injury is unknown. We hypothesized that IL-22BP may play a role in acute liver injury. To test this hypothesis, we used Il22bp-deficient mice and murine models of acute liver damage induced by ischemia reperfusion and N-acetyl-p-aminophenol (acetaminophen) administration. We found that Il22bp-deficient mice were more susceptible to acute liver damage in both models. We used Il22 × Il22bp double-deficient mice to show that this effect is indeed due to uncontrolled IL-22 activity. We could demonstrate mechanistically increased expression of Cxcl10 by hepatocytes, and consequently increased infiltration of inflammatory CD11b+Ly6C+ monocytes into the liver in Il22bp-deficient mice upon liver damage. Accordingly, neutralization of CXCL10 reversed the increased disease susceptibility of Il22bp-deficient mice. In conclusion, our data indicate that IL-22BP plays a protective role in acute liver damage, via controlling IL-22-induced Cxcl10 expression.

Synthetic analogs of an Entamoeba histolytica glycolipid designed to combat intracellular Leishmania infection.

Intracellular pathogens belonging to the genus Leishmania have developed effective strategies that enable them to survive within host immune cells. Immunostimulatory compounds that counteract such immunological escape mechanisms represent promising treatment options for diseases. Here, we demonstrate that a lipopeptidephosphoglycan (LPPG) isolated from the membrane of a protozoan parasite, Entamoeba histolytica (Eh), shows considerable immunostimulatory effects targeted against Leishmania (L.) major, a representative species responsible for cutaneous leishmaniasis (CL). Treatment led to a marked reduction in the number of intracellular Leishmania parasites in vitro, and ameliorated CL in a mouse model. We next designed and synthesized analogs of the phosphatidylinositol anchors harbored by EhLPPG; two of these analogs reproduced the anti-leishmanial activity of the native compound by inducing production of pro-inflammatory cytokines. The use of such compounds, either alone or as a supportive option, might improve the currently unsatisfactory treatment of CL and other diseases caused by pathogen-manipulated immune responses.

Employing BINOL-Phosphoroselenoyl Chloride for Selective Inositol Phosphorylation and Synthesis of Glycosyl Inositol Phospholipid from Entamoeba histolytica.

The chemical synthesis of glycosyl inositol phospholipids from Entamoeba histolytica is reported. The key feature of this synthesis is a regioselective phosphorylation reaction that occurs through desymmetrization of a myo-inositol derivative with phosphoroselenoyl chloride. A new protecting-group strategy was developed that utilizes allyl and alloc groups to synthesize complex glycolipids bearing unsaturated lipids. These developments provided an efficient synthetic route for various complex inositol phospholipids and their analogues. Furthermore, the binding affinity of the synthetic inositol phospholipids with mouse CD1d molecules has been evaluated, as well as the immunostimulatory activity.

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.

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.

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.

Sex differences in H7N9 influenza A virus pathogenesis.

Sex, gender and age have an impact on incidence and severity of several infectious diseases. Here, we analyzed reported human cases of avian H7N9 influenza A virus infections for potential sex-dependent incidence and mortality. We report that females in their reproductive years display an increased tendency to die of H7N9 influenza than males (female-to-male ratio=1.2). Next, we challenged this potential sex-dependent difference in influenza disease outcome using a mouse infection model. In general, female mice underwent more severe disease than male mice upon infection with various influenza A virus subtypes, such as H7N9, 2009 pH1N1 and H3N2. However, morbidity and mortality were most significantly affected in H7N9 influenza virus infected female mice associated with an increased inflammatory host response. Thus, our mouse infection model described here might assist future investigations on the underlying mechanisms of sex-dependent disease outcome upon zoonotic H7N9 influenza virus infection. Moreover, our findings might help to guide patient management strategies and current vaccine recommendations.