Host Protective Mechanisms to Intestinal Amebiasis

Vesicle-mediated exocytosis of MUC2 is critical in providing protection against tissue damage, and MUC2 regulates antimicrobial peptide production by Paneth cells during Entamoeba histolytica infection.While certain pathobionts can defend the ameba from oxidative stress in the colon, other commensal microbiota can protect the host by controlling the recruitment of neutrophils.Amebic infection downregulates interleukin (IL)-25 production in mice and humans. Exogenous treatment with IL-25 induces eosinophilia to confer protection.Children with a polymorphism in transcription factor cAMP response element modulator (CREM), and CREM knockout mice, are more susceptible to amebiasis.Vaccination with amebic surface protein LecA combined with Toll-like receptor (TLR)4 and TLR7/8 agonists induces a robust mucosal IgA, interferon (IFN)-γ, and IL-17a response to provide protection from amebiasis. The protozoan parasite Entamoeba histolytica is the causative agent of amebiasis, an infection that manifests as colitis and, in some cases, liver abscess. A better understanding of host protective factors is key to developing an effective remedy. Recently, significant advances have been made in understanding the mechanisms of MUC2 production by goblet cells upon amebic infection, regulation of antimicrobial peptide production by Paneth cells, the interaction of commensal microbiota with immune stimulation, and host genetics in conferring protection from amebiasis. In addition to host pathways that may serve as potential therapeutic targets, significant progress has also been made with respect to development of a vaccine against amebiasis. Here, we aim to highlight the current understanding and knowledge gaps critically. The protozoan parasite Entamoeba histolytica is the causative agent of amebiasis, an infection that manifests as colitis and, in some cases, liver abscess. A better understanding of host protective factors is key to developing an effective remedy. Recently, significant advances have been made in understanding the mechanisms of MUC2 production by goblet cells upon amebic infection, regulation of antimicrobial peptide production by Paneth cells, the interaction of commensal microbiota with immune stimulation, and host genetics in conferring protection from amebiasis. In addition to host pathways that may serve as potential therapeutic targets, significant progress has also been made with respect to development of a vaccine against amebiasis. Here, we aim to highlight the current understanding and knowledge gaps critically. Entamoeba histolytica infection accounts for millions of symptomatic infections and about 55 000 deaths worldwide annually [1.Lozano R. et al.Global and regional mortality from 235 causes of death for 20 age groups in 1990 and 2010: A systematic analysis for the Global Burden of Disease Study 2010.Lancet. 2012; 380: 2095-2128Abstract Full Text Full Text PDF PubMed Scopus (8784) Google Scholar]. Infection occurs when amebic cysts (see Glossary) enter the host intestinal lumen via contaminated food and water. In the lumen, the cyst produces trophozoites which can invade intestinal epithelial cells. Trophozoites adhere to intestinal epithelial cells by the parasite's surface Gal/GalNAc lectin which binds to host cell membrane carbohydrates galactose (Gal) and/or N-acetyl-d-galactosamine (GalNAc) [2.Petri W.A. et al.Isolation of the galactose-binding lectin that mediates the in vitro adherence of Entamoeba histolytica.J. Clin. Invest. 1987; 80: 1238-1244Crossref PubMed Scopus (201) Google Scholar]. After adhering to host cells, an ameba uses several cytotoxic mechanisms to induce cell killing and tissue invasion, including apoptosis, phagocytosis, and trogocytosis [3.Begum S. et al.Immune evasion mechanisms of Entamoeba histolytica: Progression to disease.Front. Microbiol. 2015; 6: 1394Crossref PubMed Scopus (38) Google Scholar,4.Ralston K.S. et al.Trogocytosis by Entamoeba histolytica contributes to cell killing and tissue invasion.Nature. 2014; 508: 526-530Crossref PubMed Scopus (123) Google Scholar] (Box 1).Box 1Mechanisms of Cell Killing and Tissue InvasionRalston et al. reported a novel method of host cell ingestion and killing by amebae termed 'trogocytosis'. Following adherence of an ameba to a target cell, the parasite starts nibbling a part of the target cell and, over time, ingests multiple bites that eventually lead to cell killing [4.Ralston K.S. et al.Trogocytosis by Entamoeba histolytica contributes to cell killing and tissue invasion.Nature. 2014; 508: 526-530Crossref PubMed Scopus (123) Google Scholar]. The mechanism by which nibbling fragments of the target cell induces killing is not fully understood. Trogocytosis may break the membrane integrity of the target cells. Alternatively, the ameba might also inject acidic lysosomal content during nibbling, which may eventually cause cell death – as inhibiting the lysosomal acidification using weak bases inhibits trogocytosis [82.Gilmartin A.A. et al.Inhibition of amebic lysosomal acidification blocks amebic trogocytosis and cell killing.mBio. 2017; 8e01187-17Crossref PubMed Scopus (13) Google Scholar]. However, this mechanism might not be unique to trogocytosis. Treatment with weak bases also impedes phagocytosis. Interestingly, blocking the amebic cysteine proteases, using the inhibitor E-64d, downregulated trogocytosis but not phagocytosis [83.Gilmartin A.A. et al.Inhibition of amebic cysteine proteases blocks amebic trogocytosis but not phagocytosis.J. Infect. Dis. 2020; 221: 1734-1739Crossref PubMed Scopus (6) Google Scholar]. Although trogocytosis is one of the cell-killing mechanisms in vitro, the contribution of trogocytosis-mediated host cell death in vivo is unknown.Intracellular calcium signaling in the ameba may also be involved in host cell killing. Pretreatment of an ameba with calcium channel blockers, or calcium chelators, significantly reduced the adherence and killing of host cells in vitro [84.Ravdin J.I. et al.Effect of ion channel inhibitors on the cytopathogenicity of Entamoeba histolytica.J. Infect. Dis. 1982; 146: 335-340Crossref PubMed Scopus (41) Google Scholar]. Increased calcium in the host cell induced by the ameba precedes caspase-3-dependent apoptosis. Inhibition of caspase, using pan-caspase inhibitor ZVAD, decreased amebic infection in mice [85.Becker S.M. et al.Epithelial cell apoptosis facilitates Entamoeba histolytica infection in the gut.Am. J. Pathol. 2010; 176: 1316-1322Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar]. At the end of the apoptosis, dead cells are phagocytosed by the ameba. Phagocytosis might help amebae to control excessive inflammation that could be cytotoxic for them. However, the role of phagocytosis in maintaining persistent infection has yet to be definitively determined [86.Vaithilingam A. et al.Entamoeba histolytica cell surface calreticulin binds human C1q and functions in amebic phagocytosis of host cells.Infect. Immun. 2012; 80: 2008-2018Crossref PubMed Scopus (30) Google Scholar]. A significant amount of work has been done to understand amebic phagocytosis. Recent work showed that amebic ESCRT (endosomal sorting complex required for transport) proteins and EhP3 (a homolog of the 14-3-3 family of proteins) are involved in phagocytic cup formation and actin reorganization during phagocytosis [87.Agarwal S. et al.EhP3, a homolog of 14-3-3 family of protein participates in actin reorganization and phagocytosis in Entamoeba histolytica.PLoS Pathog. 2019; 15e1007789Crossref PubMed Scopus (8) Google Scholar,88.Avalos-Padilla Y. et al.The conserved ESCRT-III machinery participates in the phagocytosis of Entamoeba histolytica.Front. Cell. Infect. Microbiol. 2018; 8: 53Crossref PubMed Scopus (20) Google Scholar]. Ralston et al. reported a novel method of host cell ingestion and killing by amebae termed 'trogocytosis'. Following adherence of an ameba to a target cell, the parasite starts nibbling a part of the target cell and, over time, ingests multiple bites that eventually lead to cell killing [4.Ralston K.S. et al.Trogocytosis by Entamoeba histolytica contributes to cell killing and tissue invasion.Nature. 2014; 508: 526-530Crossref PubMed Scopus (123) Google Scholar]. The mechanism by which nibbling fragments of the target cell induces killing is not fully understood. Trogocytosis may break the membrane integrity of the target cells. Alternatively, the ameba might also inject acidic lysosomal content during nibbling, which may eventually cause cell death – as inhibiting the lysosomal acidification using weak bases inhibits trogocytosis [82.Gilmartin A.A. et al.Inhibition of amebic lysosomal acidification blocks amebic trogocytosis and cell killing.mBio. 2017; 8e01187-17Crossref PubMed Scopus (13) Google Scholar]. However, this mechanism might not be unique to trogocytosis. Treatment with weak bases also impedes phagocytosis. Interestingly, blocking the amebic cysteine proteases, using the inhibitor E-64d, downregulated trogocytosis but not phagocytosis [83.Gilmartin A.A. et al.Inhibition of amebic cysteine proteases blocks amebic trogocytosis but not phagocytosis.J. Infect. Dis. 2020; 221: 1734-1739Crossref PubMed Scopus (6) Google Scholar]. Although trogocytosis is one of the cell-killing mechanisms in vitro, the contribution of trogocytosis-mediated host cell death in vivo is unknown. Intracellular calcium signaling in the ameba may also be involved in host cell killing. Pretreatment of an ameba with calcium channel blockers, or calcium chelators, significantly reduced the adherence and killing of host cells in vitro [84.Ravdin J.I. et al.Effect of ion channel inhibitors on the cytopathogenicity of Entamoeba histolytica.J. Infect. Dis. 1982; 146: 335-340Crossref PubMed Scopus (41) Google Scholar]. Increased calcium in the host cell induced by the ameba precedes caspase-3-dependent apoptosis. Inhibition of caspase, using pan-caspase inhibitor ZVAD, decreased amebic infection in mice [85.Becker S.M. et al.Epithelial cell apoptosis facilitates Entamoeba histolytica infection in the gut.Am. J. Pathol. 2010; 176: 1316-1322Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar]. At the end of the apoptosis, dead cells are phagocytosed by the ameba. Phagocytosis might help amebae to control excessive inflammation that could be cytotoxic for them. However, the role of phagocytosis in maintaining persistent infection has yet to be definitively determined [86.Vaithilingam A. et al.Entamoeba histolytica cell surface calreticulin binds human C1q and functions in amebic phagocytosis of host cells.Infect. Immun. 2012; 80: 2008-2018Crossref PubMed Scopus (30) Google Scholar]. A significant amount of work has been done to understand amebic phagocytosis. Recent work showed that amebic ESCRT (endosomal sorting complex required for transport) proteins and EhP3 (a homolog of the 14-3-3 family of proteins) are involved in phagocytic cup formation and actin reorganization during phagocytosis [87.Agarwal S. et al.EhP3, a homolog of 14-3-3 family of protein participates in actin reorganization and phagocytosis in Entamoeba histolytica.PLoS Pathog. 2019; 15e1007789Crossref PubMed Scopus (8) Google Scholar,88.Avalos-Padilla Y. et al.The conserved ESCRT-III machinery participates in the phagocytosis of Entamoeba histolytica.Front. Cell. Infect. Microbiol. 2018; 8: 53Crossref PubMed Scopus (20) Google Scholar]. Besides the symptomatic cases, a large number of infections remain asymptomatic [5.Haque R. et al.Entamoeba histolytica infection in children and protection from subsequent amebiasis.Infect. Immun. 2006; 74: 904-909Crossref PubMed Scopus (120) Google Scholar]. Host defense mechanisms presumably contribute to these asymptomatic infections. In recent years, significant advancement has been made in our understanding of the mechanisms through which the MUC2 mucin protects from amebic infection [6.Cornick S. et al.Entamoeba histolytica cysteine proteinase 5 evokes mucin exocytosis from colonic goblet cells via αvβ3 integrin.PLoS Pathog. 2016; 12e1005579Crossref PubMed Scopus (30) Google Scholar, 7.Cobo E.R. et al.MUC2 mucin and butyrate contribute to the synthesis of the antimicrobial peptide cathelicidin in response to Entamoeba histolytica- and dextran sodium sulfate-induced colitis.Infect. Immun. 2017; 85: e00905-e00916Crossref PubMed Scopus (37) Google Scholar, 8.Cornick S. et al.Entamoeba histolytica-induced mucin exocytosis is mediated by VAMP8 and is critical in mucosal innate host defense.mBio. 2017; 8e01323-17Crossref PubMed Scopus (16) Google Scholar]. Epithelial cells, in addition to mucin production, can also promote protection by producing antimicrobial peptides (AMPs) and cytokines [9.Ayala-Sumuano J.T. et al.Toll-like receptor signaling activation by Entamoeba histolytica induces beta defensin 2 in human colonic epithelial cells: its possible role as an element of the innate immune response.PLoS Negl. Trop. Dis. 2013; 7: e2083Crossref PubMed Scopus (13) Google Scholar, 10.Cobo E.R. et al.Entamoeba histolytica induces intestinal cathelicidins but is resistant to cathelicidin-mediated killing.Infect. Immun. 2012; 80: 143-149Crossref PubMed Scopus (28) Google Scholar, 11.Preet S. et al.Evaluation of amoebicidal potential of paneth cell cryptdin-2 against Entamoeba histolytica.PLoS Negl. Trop. Dis. 2011; 5: e1386Crossref PubMed Scopus (13) Google Scholar]. In addition to the host epithelium, the role of the microbiome has re-emerged as important in determining protection from amebic infection [12.Watanabe K. et al.Microbiome-mediated neutrophil recruitment via CXCR2 and protection from amebic colitis.PLoS Pathog. 2017; 13e1006513Crossref PubMed Scopus (36) Google Scholar, 13.Burgess S.L. et al.Bone marrow dendritic cells from mice with an altered microbiota provide interleukin 17A-dependent protection against Entamoeba histolytica colitis.mBio. 2014; 5e01817-14Crossref PubMed Google Scholar, 14.Ngobeni R. et al.Entamoeba species in South Africa: correlations with the host microbiome, parasite burdens, and first description of Entamoeba bangladeshi outside of Asia.J. Infect. Dis. 2017; 216: 1592-1600Crossref PubMed Scopus (24) Google Scholar, 15.Gilchrist C.A. et al.Role of the gut microbiota of children in diarrhea due to the protozoan parasite Entamoeba histolytica.J. Infect. Dis. 2016; 213: 1579-1585Crossref PubMed Scopus (62) Google Scholar]. Our group's recent work has built a framework for how different members of the bacterial microbiota can communicate with bone marrow progenitor cells, resulting in amebic protection [16.Burgess S.L. et al.Gut microbiome communication with bone marrow regulates susceptibility to amebiasis.J. Clin. Invest. 2020; 130: 4019-4024PubMed Google Scholar]. We also explored the impact of cell-mediated immunity in amebiasis [12.Watanabe K. et al.Microbiome-mediated neutrophil recruitment via CXCR2 and protection from amebic colitis.PLoS Pathog. 2017; 13e1006513Crossref PubMed Scopus (36) Google Scholar,17.Noor Z. et al.Role of eosinophils and tumor necrosis factor alpha in interleukin-25-mediated protection from amebic colitis.mBio. 2017; 8e02329-16Crossref PubMed Scopus (8) Google Scholar]. Building on our early findings – that the amebic surface antigen LecA is critical for colonization – work over the last couple of decades has focused on developing an effective vaccine against amebiasis. By adding liposome-based adjuvants to LecA, the efficacy of the vaccine has been recently improved [18.Abhyankar M.M. et al.Nanoformulation of synergistic TLR ligands to enhance vaccination against Entamoeba histolytica.Vaccine. 2017; 35: 916-922Crossref PubMed Scopus (11) Google Scholar,19.Abhyankar M.M. et al.Adjuvant composition and delivery route shape immune response quality and protective efficacy of a recombinant vaccine for Entamoeba histolytica.npj Vaccines. 2018; 3: 22Crossref PubMed Scopus (18) Google Scholar]. Here, we outline the recent progress on host response to amebiasis. The mucus layer keeps the approximate 100 trillion microorganisms away from the intestinal epithelial layer and confers the first line of defense in the gut against harmful infections. MUC2 mucin is the primary component of the mucus layer and is known to be essential for the protection from detrimental intestinal insults [20.Bergstrom K.S.B. et al.Muc2 protects against lethal infectious colitis by disassociating pathogenic and commensal bacteria from the colonic mucosa.PLoS Pathog. 2010; 6e1000902Crossref PubMed Scopus (348) Google Scholar]. MUC2 mucin was shown to be involved in the maintenance of a healthy microbiota, with transplantation of Muc2–/– microbiota making Muc2+/+ mice more susceptible to dextran sodium sulfate colitis [21.Leon-Coria A. et al.Muc2 mucin and nonmucin microbiota confer distinct innate host defense in disease susceptibility and colonic injury.Cell. Mol. Gastroenterol. Hepatol. 2020; (Published online July 10, 2020. https://doi.org/10.1016/j.jcmgh.2020.07.003)Abstract Full Text Full Text PDF PubMed Scopus (1) Google Scholar]. The protective role of the host's mucin layer against E. histolytica is well studied. Chadee et al. isolated and purified colonic mucus from rat and human colon and showed that even the crude mucus could inhibit amebic adherence to Chinese hamster ovary (CHO) cells by up to 70% and prevented killing of rat colonic epithelial cells by amebic trophozoites by up to 40% [22.Chadee K. et al.Rat and human colonic mucins bind to and inhibit adherence lectin of Entamoeba histolytica.J. Clin. Invest. 1987; 80: 1245-1254Crossref PubMed Scopus (198) Google Scholar]. In vivo studies using closed colonic loops in Muc2–/– mice revealed severe colonic disease and a substantially higher expression of inflammatory cytokines compared with wild-type (WT) mice [23.Kissoon-Singh V. et al.Entamoeba histolytica exacerbates epithelial tight junction permeability and proinflammatory responses in Muc2–/– mice.Am. J. Pathol. 2013; 182: 852-865Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar]. It will be important to follow up these studies. While the colonic loop model has been useful in unraveling host–parasite interactions, it does not mimic the natural infection model in several ways. In this model, infection is forced to be confined in a certain portion of the colon. Also, mice are harvested after only several hours of amebic challenge, which does not allow long-term colonization. To understand the role of MUC2 in long-term colonization with ameba, a different model with several days of established infection is required (Box 2).Box 2Mouse Model and Current ChallengesFinding an animal model that mimics human-like amebiasis was a limiting factor in the study of host defense mechanisms. Houpt and colleagues challenged C3H, CBA, C57BL/6, and BALB/c mice with log-phase trophozoites through intracecal laparotomy. While CBA and C3H strains remain infected for up to several weeks after the challenge – with colitis evidenced by immune infiltration, epithelial ulceration, and submucosal edema – the C57BL/6 and BALB/c strains were resistant to amebic infection [41.Guo X. et al.Persistence of Entamoeba histolytica infection in CBA mice owes to intestinal IL-4 production and inhibition of protective IFN-γ.Mucosal Immunol. 2008; 1: 139-146Crossref PubMed Scopus (47) Google Scholar,89.Houpt E.R. et al.The mouse model of amebic colitis reveals mouse strain susceptibility to infection and exacerbation of disease by CD4+ T Cells.J. Immunol. 2002; 169: 4496-4503Crossref PubMed Scopus (110) Google Scholar]. Discoveries from the mouse model include the findings that deletion of CREM, the leptin receptor, and CD74, each individually increased susceptibility to infection [36.Guo X. et al.Leptin signaling in intestinal epithelium mediates resistance to enteric infection by Entamoeba histolytica.Mucosal Immunol. 2011; 4: 294-303Crossref PubMed Scopus (81) Google Scholar,39.Wojcik G.L. et al.Genome-wide association study reveals genetic link between diarrhea-associated Entamoeba histolytica infection and inflammatory bowel disease.mBio. 2018; 9e01668-18Crossref PubMed Scopus (9) Google Scholar,90.Farr L. et al.CD74 Signaling links inflammation to intestinal epithelial cell regeneration and promotes mucosal healing.Cell. Mol. Gastroenterol. Hepatol. 2020; 10: 101-112Abstract Full Text Full Text PDF PubMed Scopus (5) Google Scholar]. It is important to mention here that children with polymorphisms in the leptin receptor and the CREM gene were found to have more symptomatic amebic infections [35.Duggal P. et al.A mutation in the leptin receptor is associated with Entamoeba histolytica infection in children.J. Clin. Invest. 2011; 121: 1191-1198Crossref PubMed Scopus (107) Google Scholar,39.Wojcik G.L. et al.Genome-wide association study reveals genetic link between diarrhea-associated Entamoeba histolytica infection and inflammatory bowel disease.mBio. 2018; 9e01668-18Crossref PubMed Scopus (9) Google Scholar].Resistance to amebiasis in WT C57BL/6 mice might be orchestrated by innate signaling in the epithelial layer or due to a difference in the structure or function of the indigenous microbiota. The role of hematopoietic cells was ruled out as adoptive transfer of bone marrow cells from CBA mice did not make C57BL/6 mice susceptible [91.Hamano S. et al.Resistance of C57BL/6 mice to amoebiasis is mediated by nonhemopoietic cells but requires hemopoietic IL-10 production.J. Immunol. 2006; 177: 1208-1213Crossref PubMed Scopus (46) Google Scholar]. Hematopoietic cells, while not responsible for the difference in resistance between CBA and C57Bl/6 mice, are important in defense against amebiasis. For example, IL-10 knockout C57BL/6 mice are susceptible to amebiasis. Transferring the IL-10-sufficient bone marrow cells to IL-10-deficient mice was adequate to protect them from amebiasis [91.Hamano S. et al.Resistance of C57BL/6 mice to amoebiasis is mediated by nonhemopoietic cells but requires hemopoietic IL-10 production.J. Immunol. 2006; 177: 1208-1213Crossref PubMed Scopus (46) Google Scholar]. These data suggest that hematopoietic IL-10 is critical for the protection of C57BL/6 mice. C57BL/6 resistance to amebic colitis has been a challenge in amebiasis research as most of the genetically engineered mouse models have a C57BL/6 background. However, for specific questions, this limitation may be overcome by using double-knockout mice generated by crossing a mouse deficient in a gene of interest with IL-10 knockout (KO) mice. In addition to IL-10 KO mice, C57BL/6 RAG1 KO mice are also sensitive to amebiasis [16.Burgess S.L. et al.Gut microbiome communication with bone marrow regulates susceptibility to amebiasis.J. Clin. Invest. 2020; 130: 4019-4024PubMed Google Scholar]. This observation is interesting because it suggests that adaptive immunity may play a critical role in early protection from amebiasis indirectly via the altered microbiota of RAG1 mice [92.Kwon O. et al.Altered gut microbiota composition in Rag1-deficient mice contributes to modulating homeostasis of hematopoietic stem and progenitor cells.Immune Netw. 2015; 15: 252-259Crossref PubMed Google Scholar]. Cohousing WT with RAG1 mice might make WT mice susceptible to amebiasis. In addition, antibiotic-induced dysbiosis renders C57Bl/6 mice susceptible to amebiasis [12.Watanabe K. et al.Microbiome-mediated neutrophil recruitment via CXCR2 and protection from amebic colitis.PLoS Pathog. 2017; 13e1006513Crossref PubMed Scopus (36) Google Scholar]. These approaches may help researchers to utilize genetic models without resorting to losing a key immune pathway (as one would using an IL-10 KO mouse) to study host pathways that affect amebic infection. Finding an animal model that mimics human-like amebiasis was a limiting factor in the study of host defense mechanisms. Houpt and colleagues challenged C3H, CBA, C57BL/6, and BALB/c mice with log-phase trophozoites through intracecal laparotomy. While CBA and C3H strains remain infected for up to several weeks after the challenge – with colitis evidenced by immune infiltration, epithelial ulceration, and submucosal edema – the C57BL/6 and BALB/c strains were resistant to amebic infection [41.Guo X. et al.Persistence of Entamoeba histolytica infection in CBA mice owes to intestinal IL-4 production and inhibition of protective IFN-γ.Mucosal Immunol. 2008; 1: 139-146Crossref PubMed Scopus (47) Google Scholar,89.Houpt E.R. et al.The mouse model of amebic colitis reveals mouse strain susceptibility to infection and exacerbation of disease by CD4+ T Cells.J. Immunol. 2002; 169: 4496-4503Crossref PubMed Scopus (110) Google Scholar]. Discoveries from the mouse model include the findings that deletion of CREM, the leptin receptor, and CD74, each individually increased susceptibility to infection [36.Guo X. et al.Leptin signaling in intestinal epithelium mediates resistance to enteric infection by Entamoeba histolytica.Mucosal Immunol. 2011; 4: 294-303Crossref PubMed Scopus (81) Google Scholar,39.Wojcik G.L. et al.Genome-wide association study reveals genetic link between diarrhea-associated Entamoeba histolytica infection and inflammatory bowel disease.mBio. 2018; 9e01668-18Crossref PubMed Scopus (9) Google Scholar,90.Farr L. et al.CD74 Signaling links inflammation to intestinal epithelial cell regeneration and promotes mucosal healing.Cell. Mol. Gastroenterol. Hepatol. 2020; 10: 101-112Abstract Full Text Full Text PDF PubMed Scopus (5) Google Scholar]. It is important to mention here that children with polymorphisms in the leptin receptor and the CREM gene were found to have more symptomatic amebic infections [35.Duggal P. et al.A mutation in the leptin receptor is associated with Entamoeba histolytica infection in children.J. Clin. Invest. 2011; 121: 1191-1198Crossref PubMed Scopus (107) Google Scholar,39.Wojcik G.L. et al.Genome-wide association study reveals genetic link between diarrhea-associated Entamoeba histolytica infection and inflammatory bowel disease.mBio. 2018; 9e01668-18Crossref PubMed Scopus (9) Google Scholar]. Resistance to amebiasis in WT C57BL/6 mice might be orchestrated by innate signaling in the epithelial layer or due to a difference in the structure or function of the indigenous microbiota. The role of hematopoietic cells was ruled out as adoptive transfer of bone marrow cells from CBA mice did not make C57BL/6 mice susceptible [91.Hamano S. et al.Resistance of C57BL/6 mice to amoebiasis is mediated by nonhemopoietic cells but requires hemopoietic IL-10 production.J. Immunol. 2006; 177: 1208-1213Crossref PubMed Scopus (46) Google Scholar]. Hematopoietic cells, while not responsible for the difference in resistance between CBA and C57Bl/6 mice, are important in defense against amebiasis. For example, IL-10 knockout C57BL/6 mice are susceptible to amebiasis. Transferring the IL-10-sufficient bone marrow cells to IL-10-deficient mice was adequate to protect them from amebiasis [91.Hamano S. et al.Resistance of C57BL/6 mice to amoebiasis is mediated by nonhemopoietic cells but requires hemopoietic IL-10 production.J. Immunol. 2006; 177: 1208-1213Crossref PubMed Scopus (46) Google Scholar]. These data suggest that hematopoietic IL-10 is critical for the protection of C57BL/6 mice. C57BL/6 resistance to amebic colitis has been a challenge in amebiasis research as most of the genetically engineered mouse models have a C57BL/6 background. However, for specific questions, this limitation may be overcome by using double-knockout mice generated by crossing a mouse deficient in a gene of interest with IL-10 knockout (KO) mice. In addition to IL-10 KO mice, C57BL/6 RAG1 KO mice are also sensitive to amebiasis [16.Burgess S.L. et al.Gut microbiome communication with bone marrow regulates susceptibility to amebiasis.J. Clin. Invest. 2020; 130: 4019-4024PubMed Google Scholar]. This observation is interesting because it suggests that adaptive immunity may play a critical role in early protection from amebiasis indirectly via the altered microbiota of RAG1 mice [92.Kwon O. et al.Altered gut microbiota composition in Rag1-deficient mice contributes to modulating homeostasis of hematopoietic stem and progenitor cells.Immune Netw. 2015; 15: 252-259Crossref PubMed Google Scholar]. Cohousing WT with RAG1 mice might make WT mice susceptible to amebiasis. In addition, antibiotic-induced dysbiosis renders C57Bl/6 mice susceptible to amebiasis [12.Watanabe K. et al.Microbiome-mediated neutrophil recruitment via CXCR2 and protection from amebic colitis.PLoS Pathog. 2017; 13e1006513Crossref PubMed Scopus (36) Google Scholar]. These approaches may help researchers to utilize genetic models without resorting to losing a key immune pathway (as one would using an IL-10 KO mouse) to study host pathways that affect amebic infection. E. histolytica needs to cleave the mucus surface and attach to intestinal epithelial cells to establish an infection. Although several cysteine proteases, including EhCP1, EhCP2, and EhCP5, are expressed by E. histolytica, only the role of EhCP5 in amebiasis is well established. In vitro studies have revealed that the C-terminal cysteine-rich domain of MUC2 is cleaved off by EhCP5 [6.Cornick S. et al.Entamoeba histolytica cysteine proteinase 5 evokes mucin exocytosis from colonic goblet cells via αvβ3 integrin.PLoS Pathog. 2016; 12e1005579Crossref PubMed Scopus (30) Google Scholar,24.Lidell M.E. et al.Entamoeba histolytica cysteine protease cleave the MUC2 mucin in its C-terminal domain and dissolve the protective colonic mucus gel.Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 9298-9303Crossref PubMed Scopus (196) Google Scholar]. Since MUC2 is a major component of the mucin layer, EhCP5 facilitates mucin degradation by cleaving it, which allows E. histolytica to overcome the mucin barrier. EhCP5-deficient E. histolytica trophozoites were unabl