The IL-33-ILC2 pathway protects from amebic colitis.

Entamoeba histolytica is a pathogenic protozoan parasite that causes intestinal colitis, diarrhea, and in some cases, liver abscess. Through transcriptomics analysis, we observed that E. histolytica infection was associated with increased expression of IL-33 mRNA in both the human and murine colon. IL-33, the IL-1 family cytokine, is released after cell injury to alert the immune system of tissue damage. Treatment with recombinant IL-33 protected mice from amebic infection and intestinal tissue damage; moreover, blocking IL-33 signaling made mice more susceptible to amebiasis. IL-33 limited the recruitment of inflammatory immune cells and decreased the pro-inflammatory cytokine IL-6 in the cecum. Type 2 immune responses were upregulated by IL-33 treatment during amebic infection. Interestingly, administration of IL-33 protected RAG2-/- mice but not RAG2-/-γc-/- mice, demonstrating that IL-33-mediated protection required the presence of innate lymphoid cells (ILCs). IL-33 induced recruitment of ILC2 but not ILC1 and ILC3 in RAG2-/- mice. At baseline and after amebic infection, there was a significantly higher IL13+ILC2s in C57BL/J mice, which are naturally resistant to amebiasis, than CBA/J mice. Adoptive transfer of ILC2s to RAG2-/-γc-/- mice restored IL-33-mediated protection. These data reveal that the IL-33-ILC2 pathway is an important host defense mechanism against amebic colitis.

Role of inflammasomes in innate host defense against Entamoeba histolytica.

Intestinal amebiasis is the disease caused by the extracellular protozoan parasite Entamoeba histolytica (Eh) that induces a dynamic and heterogeneous interaction profile with the host immune system during disease pathogenesis. In 90% of asymptomatic infection, Eh resides with indigenous microbiota in the outer mucus layer of the colon without prompting an immune response. However, for reasons that remain unclear, in a minority of the Eh-infected individuals, this fine tolerated relationship is switched to a pathogenic phenotype and advanced to an increasingly complex host-parasite interaction. Eh disease susceptibility depends on parasite virulence factors and their interactions with indigenous bacteria, disruption of the mucus bilayers, and adherence to the epithelium provoking host immune cells to evoke a robust pro-inflammatory response mediated by inflammatory caspases and inflammasome activation. To understand Eh pathogenicity and innate host immune responses, this review highlights recent advances in our understanding of how Eh induces outside-in signaling via Mϕs to activate inflammatory caspases and inflammasome to regulate pro-inflammatory responses.

Gut microbiome communication with bone marrow regulates susceptibility to amebiasis.

The microbiome provides resistance to infection. However, the underlying mechanisms are poorly understood. We demonstrate that colonization with the intestinal bacterium Clostridium scindens protects from Entamoeba histolytica colitis via innate immunity. Introduction of C. scindens into the gut microbiota epigenetically altered and expanded bone marrow granulocyte-monocyte progenitors (GMPs) and resulted in increased intestinal neutrophils with subsequent challenge with E. histolytica. Introduction of C. scindens alone was sufficient to expand GMPs in gnotobiotic mice. Adoptive transfer of bone marrow from C. scindens-colonized mice into naive mice protected against amebic colitis and increased intestinal neutrophils. Children without E. histolytica diarrhea also had a higher abundance of Lachnoclostridia. Lachnoclostridia C. scindens can metabolize the bile salt cholate, so we measured deoxycholate and discovered that it was increased in the sera of C. scindens-colonized specific pathogen-free and gnotobiotic mice, as well as in children protected from amebiasis. Administration of deoxycholate alone increased GMPs and provided protection from amebiasis. We elucidated a mechanism by which C. scindens and the microbially metabolized bile salt deoxycholic acid alter hematopoietic precursors and provide innate protection from later infection with E. histolytica.

Host-antibody inductivity of virulent Entamoeba histolytica and non-virulent Entamoeba moshkovskii in a mouse model.

BACKGROUND: Despite similarities in morphology, gene and protein profiles, Entamoeba histolytica and E. moshkovskii show profound differences in pathogenicity. Entamoeba histolytica infection might result in amoebic dysentery and liver abscess, while E. moshkovskii causes only mild diarrhea. Extensive studies focus on roles of host immune responses to the pathogenic E. histolytica; however, evidence for E. moshkovskii remains scarce. METHODS: To study differences in host-antibody response profiles between E. histolytica and E. moshkovskii, mice were immunized intraperitoneally with different sets of Entamoeba trophozoites as single species, mixed species and combinations. RESULTS: Mice prime-immunized with E. histolytica and E. moshkovskii combination, followed by individual species, exhibited higher IgG level than the single species immunization. Mice immunized with E. moshkovskii induced significantly higher levels and long-lasting antibody responses than those challenged with E. histolytica alone. Interestingly, E. histolytica-specific anti-sera promoted the cytopathic ability of E. histolytica toward Chinese hamster ovarian (CHO) cells, but showed no effect on cell adhesion. There was no significant effect of immunized sera on cytopathic activity and adhesion of E. moshkovskii toward both CHO and human epithelial human colonic (Caco-2) cell lines. Monoclonal-antibody (mAb) characterization demonstrated that 89% of E. histolytica-specific mAbs produced from mice targeted cytoplasmic and cytoskeletal proteins, whereas 73% of E. moshkovskii-specific mAbs targeted plasma membrane proteins. CONCLUSIONS: The present findings suggest that infection with mixed Entamoeba species or E. moshkovskii effectively induces an antibody response in mice. It also sheds light on roles of host antibody response in the pathogenic difference of E. histolytica and E. moshkovskii trophozoites, and cell surface protein modifications of the amoebic parasites to escape from host immune system.

Learning from the research on amebiasis and gut microbiome: Is stimulation by gut flora essential for effective neutrophil mediated protection from external pathogens?

Amebiasis, caused by intestinal infection with Entamoeba histolytica, is one of the leading causes of parasite infection-related mortality and morbidity globally. Although its pathogenesis, including determinant factors of infection outcome, remains unclear, recent clinical data indicate that the gut microbiome plays a role in determining the severity of amebiasis. Recently, we investigated the effects of the gut microbiome on neutrophil mediated protection from E. histolytica infection using a mouse model. We identified that surface expression of CXCR2 on neutrophils was diminished in mice with dysbiosis, which resulted in decreased neutrophil recruitment to the infection site, allowing more aggressive intestinal tissue damage by E. histolytica. Our results indicated that oxidase activity during E. histolytica infection was also diminished after dysbiosis, consistent with the results from prior research. Thus, the gut microbiome plays an important role in regulating neutrophil phenotype when fighting against external pathogens.

IL-17A contributes to reducing IFN-γ/IL-4 ratio and persistence of Entamoeba histolytica during intestinal amebiasis.

Amebiasis is an infectious disease caused by Entamoeba histolytica, an anaerobic protozoan parasite, and is a major public health problem worldwide, particularly in areas with inadequate sanitation and poor hygiene. Th1 responses, represented by interferon gamma (IFN-γ), play a protective role by clearing the amebae from the gut, whereas Th2 responses are responsible for chronic infection. Th17 responses preconditioned by vaccination or by modulating the intestinal microbiome protect mice from the settlement of E. histolytica. However, the role of interleukin-17A (IL-17A), which is upregulated during the natural course of intestinal amebiasis, has not been clarified. The aim of this study was to investigate the role of IL-17A during intestinal amebiasis in a mouse model. IL-17A knockout and wild-type CBA/J mice were challenged intracecally with 2×106E. histolytica trophozoites, and their infection, pathology, and immune responses were monitored. Neither the initial settlement of E. histolytica nor the inflammation of the cecum was affected by the absence of IL-17A for week 1, but the infection rate and parasite burden declined in a late stage of infection, accompanied by an increased IFN-γ/IL-4 ratio. Therefore, IL-17A contributes to the persistence of E. histolytica and modulates the immune response, including the IFN-γ/IL-4 ratio, which may be responsible for the reduction of the parasite burden in the IL-17A knockout mice during the chronic phase of intestinal amebiasis.

Microbiome-mediated neutrophil recruitment via CXCR2 and protection from amebic colitis.

The disease severity of Entamoeba histolytica infection ranges from asymptomatic to life-threatening. Recent human and animal data implicate the gut microbiome as a modifier of E. histolytica virulence. Here we have explored the association of the microbiome with susceptibility to amebiasis in infants and in the mouse model of amebic colitis. Dysbiosis occurred symptomatic E. histolytica infection in children, as evidenced by a lower Shannon diversity index of the gut microbiota. To test if dysbiosis was a cause of susceptibility, wild type C57BL/6 mice (which are innately resistant to E. histiolytica infection) were treated with antibiotics prior to cecal challenge with E. histolytica. Compared with untreated mice, antibiotic pre-treated mice had more severe colitis and delayed clearance of E. histolytica. Gut IL-25 and mucus protein Muc2, both shown to provide innate immunity in the mouse model of amebic colitis, were lower in antibiotic pre-treated mice. Moreover, dysbiotic mice had fewer cecal neutrophils and myeloperoxidase activity. Paradoxically, the neutrophil chemoattractant chemokines CXCL1 and CXCL2, as well as IL-1ß, were higher in the colon of mice with antibiotic-induced dysbiosis. Neutrophils from antibiotic pre-treated mice had diminished surface expression of the chemokine receptor CXCR2, potentially explaining their inability to migrate to the site of infection. Blockade of CXCR2 increased susceptibility of control non-antibiotic treated mice to amebiasis. In conclusion, dysbiosis increased the severity of amebic colitis due to decreased neutrophil recruitment to the gut, which was due in part to decreased surface expression on neutrophils of CXCR2.

Role of Eosinophils and Tumor Necrosis Factor Alpha in Interleukin-25-Mediated Protection from Amebic Colitis.

The parasite Entamoeba histolytica is a cause of diarrhea in infants in low-income countries. Previously, it was shown that tumor necrosis factor alpha (TNF-α) production was associated with increased risk of E. histolytica diarrhea in children. Interleukin-25 (IL-25) is a cytokine that is produced by intestinal epithelial cells that has a role in maintenance of gut barrier function and inhibition of TNF-α production. IL-25 expression was decreased in humans and in the mouse model of amebic colitis. Repletion of IL-25 blocked E. histolytica infection and barrier disruption in mice, increased gut eosinophils, and suppressed colonic TNF-α. Depletion of eosinophils with anti-Siglec-F antibody prevented IL-25-mediated protection. In contrast, depletion of TNF-α resulted in resistance to amebic infection. We concluded that IL-25 provides protection from amebiasis, which is dependent upon intestinal eosinophils and suppression of TNF-α.IMPORTANCE The intestinal epithelial barrier is important for protection from intestinal amebiasis. We discovered that the intestinal epithelial cytokine IL-25 was suppressed during amebic colitis in humans and that protection could be restored in the mouse model by IL-25 administration. IL-25 acted via eosinophils and suppressed TNF-α. This work illustrates a previously unrecognized pathway of innate mucosal immune response.

Noninvasive intestinal amebiasis: Entamoeba histolytica colonization without invasion.

PURPOSE OF REVIEW: Entamoeba histolytica infection remains a major cause of morbidity and mortality worldwide. Although research with the organism began in the late nineteenth century, our understanding of the natural history of the infection remains incomplete and is heavily based on expert opinion. Most persons infected with E. histolytica are carriers with the organism colonizing the large intestine. Host defense mechanisms that prevent invasive diseases are poorly understood. A timely review could lead to renewed interest. RECENT FINDINGS: We herein review 2012 and 2013 publications related to the epidemiology, diagnosis, management and potential mechanisms that enable noninvasive E. histolytica colonization without invasion. SUMMARY: There are several publications that advance our knowledge in the first three categories listed above, but studies of mechanisms for noninvasive E. histolytica colonization are glaringly few.

Differentiation of Entamoeba histolytica/Entamoeba dispar by the polymerase chain reaction in stool samples of patients with gastrointestinal symptoms in the Sanliurfa Province.

OBJECTIVE: We aimed to diagnose amebiasis and also identify Entamoeba histolytica (E. histolytica) and Entamoeba dispar (E. dispar) in patients with gastrointestinal symptoms in an endemic region in Turkey. METHODS: Stool samples obtained from 181 patients with gastrointestinal symptoms from the Harran University Hospital of Sanliurfa were examined for the diagnosis of amebiasis by the three methods which are as follows:- In house polymerase chain reaction (PCR) targeting the 135 base pair region located on the small-subunit ribosomal RNA (SSU rRNA) gene to differentiate E. histolytica from E. dispar; and the commercial kit, RIDASCREEN® stool ELISA, that identifies Entamoeba sensu lato antigen and microscopical examination of Trichrome stained smears of stool samples. RESULTS: Positivity for E. histolytica/E. dispar complex was found to be 79 (43.6%) by microscopy versus 83 (45.9%) by PCR out of 181 stool samples. A total of 45 patients were found to be positive by the antigen detection method. PCR and microscopy were both positive in 59 samples. The number of patients infected with E. dispar (39.8%) was found to be higher than E. histolytica (3.3%) while 5 patients (2.8%) had mixed E. histolytica+E. dispar infections according to PCR results. CONCLUSION: Routine diagnosis of amebiasis by a combination of microscopy and antigen detection technique should be complemented with a PCR assay as a reference test for sensitive differentiation of both species.

Immunogenicity and protective efficacy of heparan sulphate binding proteins of Entamoeba histolytica in a guinea pig model of intestinal amoebiasis.

Entamoeba histolytica infection is associated with considerable morbidity and mortality in the form of intestinal and extraintestinal amoebiasis. No vaccine is yet available for amoebiasis. Heparan Sulphate Binding Proteins (HSBPs) from E. histolytica were evaluated for immunogenicity and protective efficacy in a Guinea pig model. Animals were immunized subcutaneously with 30µg of HSBP by three weekly inoculations. The immunogenicity of HSBP was determined by antibody response (IgG, IgM and IgA), splenocyte proliferation assay and in vitro direct amoebicidal assay with splenic lymphocytes and monocytes from vaccinated and control animals. The efficacy of the vaccine was evaluated by challenge infection to vaccinated and control animals by intra-caecal inoculation of E. histolytica trophozoites and comparing gross and histopathological findings in caeca of these animals. HSBP was found to induce specific anti-amoebic response as seen by specific antibody production and direct amoebicidal activity of splenocytes. The vaccine also showed partial protection against challenge infection in vaccinated animals as shown by mild/absent lesions and histopathological findings.

Amoebic toxic colitis: analysis of factors related to mortality.

BACKGROUND: Toxic or fulminant colitis due to Entamoeba histolytica infrequently presents but is very serious. Unfortunately, there are numerous contradictory factors related to mortality. METHODS: We analyzed several cases of E. histolytica infection to determine the factors related to mortality. We included patients >15 years of age who were histopathologically diagnosed with amoebic toxic colitis and treated from January 2000 through December 2006. We evaluated demographic, clinical, laboratorial, surgical, and histopathological characteristics. RESULTS: We examined 24 patients and recorded 12 deaths (50%). Twenty patients underwent surgery within a mean time of 24 hours (range: 8-120 hours). Tenesmus and intestinal perforation were determined to be statistically significant (P<0·05) by univariate analysis. Three models of logistic regression were able to determine three statistically significant factors that affected mortality: (1) tenesmus and a lymphocyte count <1·5×10(3) cell/µl; 2) depth of invasion beyond the mucosa and a lymphocyte count <1·5×10(3) cell/µl; 3) time spent with symptoms and perforation. CONCLUSIONS: The mortality rate determined in this study is similar to previously reported series. A low lymphocyte count, significant depth of invasion, and intestinal perforation were determined to be the factors related to increased mortality, while tenesmus and limited amount of time spent with symptoms were associated with survival. Quick diagnosis and appropriate treatment are important factors that reduce mortality.

Leptin and mucosal immunity.

Enhanced susceptibility to infection has long been recognized in children with congenital deficiency of leptin or its receptor. Studies in mice have demonstrated that leptin deficiency affects both the innate and acquired immune systems. Here, we review recent studies that demonstrate the impact on immunity of a common non-synonomous polymorphism of the leptin receptor. In a Bangladesh cohort of children, the presence of two copies of the ancestral Q223 allele was significantly associated with resistance to amebiasis. Children and mice with at least one copy of the leptin receptor 223R mutation were more susceptible to amebic colitis. Leptin signaling in the intestinal epithelium and downstream STAT3 (signal transducer and activator of transcription 3) and SHP2 (Src homology phosphatase 2) signaling were required for protection in the murine model of amebic colitis. Murine models have also implicated leptin in protection from other infections, including Mycobacterium tuberculosis, Klebsiella pneumoniae, and Streptococcus pneumoniae. Thus, the role of leptin signaling in infectious disease and specifically leptin-mediated protection of the intestinal epithelium will be the focus of this review.

CCR9+ T cells contribute to the resolution of the inflammatory response in a mouse model of intestinal amoebiasis.

Analysis of the mechanisms underlying the inflammatory response in amoebiasis is important to understand the immunopathology of the disease. Mucosal associated effector and regulatory T cells may play a role in regulating the inflammatory immune response associated to Entamoeba histolytica infection in the colon. A subpopulation of regulatory T cells has recently been identified and is characterized by the expression of the chemokine receptor CCR9. In this report, we used CCR9 deficient (CCR9(-/-)) mice to investigate the role of the CCR9(+) T cells in a murine model of E. histolytica intestinal infection. Intracecal infection of CCR9(+/+), CCR9(+/-) and CCR9(-/-) mice with E. histolytica trophozoites, revealed striking differences in the development and nature of the intestinal inflammatory response observed between these strains. While CCR9(+/+) and CCR9(+/-) mice were resistant to the infection and resolved the pathogen-induced inflammatory response, CCR9(-/-) mice developed a chronic inflammatory response, which was associated with over-expression of the cytokines IFN-γ, TNF-α, IL-4, IL-6 and IL-17, while IL-10 was not present. In addition, increased levels of CCL11, CCL20 and CCL28 chemokines were detected by qRT-PCR in CCR9(-/-) mice. E. histolytica trophozoites were identified in the lumen of the cecum of CCR9(-/-) mice at seven days post infection (pi), whereas in CCR9(+/+) mice trophozoites disappeared by day 1 pi. Interestingly, the inflammation observed in CCR9(-/-) mice, was associated with a delayed recruitment of CD4(+)CD25(+)FoxP3(+) T cells to the cecal epithelium and lamina propria, suggesting that this population may play a role in the early regulation of the inflammatory response against E. histolytica, likely through IL-10 production. In support of these data, CCR9(+) T cells were also identified in colon tissue sections obtained from patients with amoebic colitis. Our data suggest that a population of CCR9(+)CD4(+)CD25(+)FoxP3(+) T cells may participate in the control and resolution of the inflammatory immune response to E. histolytica infection.

Entamoeba histolytica induces intestinal cathelicidins but is resistant to cathelicidin-mediated killing.

The enteric protozoan parasite Entamoeba histolytica is the cause of potentially fatal amebic colitis and liver abscesses. E. histolytica trophozoites colonize the colon, where they induce inflammation, penetrate the mucosa, and disrupt the host immune system. The early establishment of E. histolytica in the colon occurs in the presence of antimicrobial human (LL-37) and murine (CRAMP [cathelin-related antimicrobial peptide]) cathelicidins, essential components of the mammalian innate defense system in the intestine. Studying this early step in the pathogenesis of amebic colitis, we demonstrate that E. histolytica trophozoites or their released proteinases, including cysteine proteinase 1 (EhCP1), induce intestinal cathelicidins in human intestinal epithelial cell lines and in a mouse model of amebic colitis. Despite induction, E. histolytica trophozoites were found to be resistant to killing by these antimicrobial peptides, and LL-37 and CRAMP were rapidly cleaved by released amebic cysteine proteases. The cathelicidin fragments however, did maintain their antimicrobial activity against bacteria. Degradation of intestinal cathelicidins is a novel function of E. histolytica cysteine proteinases in the evasion of the innate immune system in the bowel. Thus, early intestinal epithelial colonization of invasive trophozoites involves a complex interplay in which the ultimate outcome of infection depends in part on the balance between degradation of cathelicidins by amebic released cysteine proteinases and upregulation of proinflammatory mediators which trigger the inflammatory response.

Association of malnutrition with amebiasis.

Observation of a cohort of preschool children in Dhaka, Bangladesh, is beginning to reveal the contributions of environment, host, and parasite to amebiasis. Reviewed here are the associations and interactions of malnutrition, IgA and interferon-gamma, human leukocyte antigen alleles, and parasite genotypes to the outcome of infection. Future efforts aimed at understanding the mechanisms of these effects are described.

Expression of immune modulator cytokines in human fulminant amoebic colitis.

Human fulminant amoebic colitis (FAC) is characterized by ulceration and inflammation of the colon. The specific mixture of pro-inflammatory and anti-inflammatory cytokines may participate in either the host defense or in the pathogenesis of amoebic colitis. Therefore, we studied the expression of IL-8, IL-10, IL-4, TGF-beta and IFN-gamma in human FAC patients and controls through immunohistochemistry analysis. The number of cells expressing IL-8, IL-4 and IL-10 was significantly enhanced in all FAC samples compared to the control samples. However, the expression of TGF- beta in patients was low in the colonic mucosa and high in the lamina propria compared with the control. No expression of IFN-gamma was found in the controls or FAC samples. The production of IL-8 by intestinal epithelial cells may play a role in the pathogenesis of amoebic infection, because this cytokine attracts neutrophils, which lead to an inflammatory reaction that results in tissue damage. The predominant expression of the macrophage down-regulating cytokines, IL-4, IL-10 and TGF-beta, or the Th2-type immune response could inhibit a cell-mediated immune response, which in turn would facilitate parasite invasion in these tissues.

Polymorphonuclear neutrophil infiltration intensity as consequence of Entamoeba histolytica density in amebic colitis.

BACKGROUND: It has been suggested that the damage observed in invasive amebiasis is related to interactions between polymorphonuclear leukocytes (PMN) and Entamoeba histolytica trophozoites. We analyzed the relation between infiltrating inflammatory cell populations and E. histolytica density in intestinal amebic lesions. METHODS: Biopsies obtained endoscopically from patients with amebic colitis were analyzed to describe their morphologic abnormalities. Cellular populations and E. histolytica trophozoites were measured quantitatively in order to assess the correlation between infiltrating inflammatory cell populations and parasite density. RESULTS: Amebic lesions were most often located in the colon (55%). The histopathologic diagnoses were colitis in 31%, erosive colitis in 26%, and ulcerated colitis in 24%. The predominant cellular populations found in the lesions were lymphocytes (59.8%) (3,672 +/- 2,413/mm(2)) followed by PMN (17%) (1,038 +/- 1,171 PMN/mm(2)) (p < 0.01). A higher density of PMN infiltration was observed in severe cases. Cellular populations predictive of the presence of E. histolytica trophozoites (p = 0.047) were PMN (p = 0.019) and lymphocytes (p = 0.033). The highest association was found between E. histolytica trophozoites and PMN (p = 0.0221). CONCLUSIONS: Neutrophils and lymphocytes, particularly the former, are associated significantly with the density of parasites. Our findings support the theory that PMN interaction with E. histolytica contributes to the pathogenesis of amebic intestinal lesions.

Broad early immune response of porcine epithelial jejunal IPI-2I cells to Entamoeba histolytica.

Amoebiasis caused by Entamoebahistolytica triggers an acute inflammatory response at early stages of intestinal infection. The patho-physiological study of intestinal amoebiasis requires the development of powerful animal models. Swine provide robust model for human diseases and they could be used to study intestinal amoebiasis. Here, we introduce an in vitro model of swine intestinal epithelial cell (IPI-2I) co-cultured with E. histolytica. Intestinal epithelial cells (IECs) have crucial roles in sensing pathogens and initiating innate immune response, which qualitatively influence adaptive immune response against them. The contact between the two cells induces marked macroscopic lesions of IEC monolayer and striking alteration of the IPI-2I cell phenotype including blebbing, such as loss of attachment before to be phagocyte by the trophozoite. Increase in Lactate Dehydrogenase (LDH) levels in the culture supernatant of IECs was observed when ameba is present and could reflect the cellular cytotoxicity exerted by the parasite. Using quantitative real-time PCR, we identified the up-regulation of cytokines/chemokines implicated in neutrophil chemoattraction and inflammation, such as CCL2, CCL20, CXCL2, CXCL3, GM-CSF, IL1 alpha, IL6 and IL8, in response to the parasite that can further regulate the immunoregulatory functions of the immune cells of the host. The study points a cardinal role of these pro-inflammatory compounds as central mediators in the interaction IECs/ameba and suggests mechanisms by which they coordinate intestinal immune response. This will focus future efforts on delineating the molecular and cellular mechanisms of other cell partners by the way of in vivo infection of swine.