Leanne Mortimer

The immunopathogenesis of Entamoeba histolytica

Amebiasis is the disease caused by the enteric dwelling protozoan parasite Entamoeba histolytica. The WHO considers amebiasis as one of the major health problems in developing countries; it is surpassed by only malaria and schistosomiasis for death caused by parasitic infection. E. histolytica primarily lives in the colon as a harmless commensal, but is capable of causing devastating dysentery, colitis and liver abscess. What triggers the switch to a pathogenic phenotype and the onset of disease is unknown. We are becoming increasingly aware of the complexity of the host-parasite interaction. During chronic stages of amebiasis, the host develops an immune response that is incapable of eliminating tissue resident parasites, while the parasite actively immunosuppresses the host. However, most individuals with symptomatic infections succumb only to an episode of dysentery. Why most halt invasion and a minority progress to chronic disease remains poorly understood. This review presents a current understanding of the immune processes that shape the outcome of E. histolytica infections during its different stages.

Entamoeba histolytica cysteine proteinase 5 binds integrin on colonic cells and stimulates NFκB-mediated pro-inflammatory responses.

Integrins are important mammalian receptors involved in normal cellular functions and the pathogenesis of inflammation and disease. Entamoeba histolytica is a protozoan parasite that colonizes the gut, and in 10% of infected individuals, causes amebic colitis and liver abscess resulting in 105 deaths/year. E. histolytica-induced host inflammatory responses are critical in the pathogenesis of the disease, yet the host and parasite factors involved in disease are poorly defined. Here we show that pro-mature cysteine proteinase 5 (PCP5), a major virulent factor that is abundantly secreted and/or present on the surface of ameba, binds via its RGD motif to αVβ3 integrin on Caco-2 colonic cells and stimulates NFκB-mediated pro-inflammatory responses. PCP5 RGD binding to αVβ3 integrin triggered integrin-linked kinase(ILK)-mediated phosphorylation of Akt-473 that bound and induced the ubiquitination of NF-κB essential modulator (NEMO). As NEMO is required for activation of the IKKα-IKKβ complex and NFκB signaling, these events markedly up-regulated pro-inflammatory mediator expressions in vitro in Caco-2 cells and in vivo in colonic loop studies in wild-type and Muc2−/− mice lacking an intact protective mucus barrier. These results have revealed that EhPCP5 RGD motif is a ligand for αVβ3 integrin-mediated adhesion on colonic cells and represents a novel mechanism that E. histolytica trophozoites use to trigger an inflammatory response in the pathogenesis of intestinal amebiasis.

NLRP3 inflammasome inhibition is disrupted in a group of auto-inflammatory disease CAPS mutations

Inflammasomes are positioned to rapidly escalate the intensity of inflammation by activating interleukin (IL)-1β, IL-18 and cell death by pyroptosis. However, negative regulation of inflammasomes remains poorly understood, as is the signaling cascade that dampens inflammasome activity. We found that rapid NLRP3 inflammasome activation was directly inhibited by protein kinase A (PKA), which was induced by prostaglandin E2 (PGE2) signaling via the PGE2 receptor E-prostanoid 4 (EP4). PKA directly phosphorylated the cytoplasmic receptor NLRP3 and attenuated its ATPase function. We found that Ser295 in human NLRP3 was critical for rapid inhibition and PKA phosphorylation. Mutations in NLRP3-encoding residues adjacent to Ser295 have been linked to the inflammatory disease CAPS (cryopyrin-associated periodic syndromes). NLRP3-S295A phenocopied the human CAPS mutants. These data suggest that negative regulation at Ser295 is critical for restraining the NLRP3 inflammasome and identify a molecular basis for CAPS-associated NLRP3 mutations.

The NLRP3 Inflammasome Is a Pathogen Sensor for Invasive Entamoeba histolytica via Activation of α5β1 Integrin at the Macrophage-Amebae Intercellular Junction

Entamoeba histolytica (Eh) is an extracellular protozoan parasite of humans that invades the colon to cause life-threatening intestinal and extra-intestinal amebiasis. Colonized Eh is asymptomatic, however, when trophozoites adhere to host cells there is a considerable inflammatory response that is critical in the pathogenesis of amebiasis. The host and/or parasite factors that trigger the inflammatory response to invading Eh are not well understood. We recently identified that Eh adherence to macrophages induces inflammasome activation and in the present study we sought to determine the molecular events upon contact that coordinates this response. Here we report that Eh contact-dependent activation of α5β1 integrin is critical for activation of the NLRP3 inflammasome. Eh-macrophage contact triggered recruitment of α5β1 integrin and NLRP3 into the intercellular junction, where α5β1 integrin underwent activation by an integrin-binding cysteine protease on the parasite surface, termed EhCP5. As a result of its activation, α5β1 integrin induced ATP release into the extracellular space through opening of pannexin-1 channels that signalled through P2X7 receptors to deliver a critical co-stimulatory signal that activated the NLRP3 inflammasome. Both the cysteine protease activity and integrin-binding domain of EhCP5 were required to trigger α5β1 integrin that led to ATP release and NLRP3 inflammasome activation. These findings reveal engagement of α5β1 integrin across the parasite-host junction is a key regulatory step that initiates robust inflammatory responses to Eh. We propose that α5β1 integrin distinguishes Eh direct contact and functions with NLRP3 as pathogenicity sensor for invasive Eh infection.

Gal-lectin-dependent contact activates the inflammasome by invasive Entamoeba histolytica

Entamoeba histolytica (Eh) is an extracellular protozoan parasite of the human colon, which occasionally breaches the intestinal barrier. Eradicating ameba that invades is essential for host survival. A defining but uncharacterized feature of amebic invasion is direct contact between ameba and host cells. This event corresponds with a massive pro-inflammatory response. To date, pathogen recognition receptors (PRRs) that are activated by contact with viable Eh are unknown. Here we show that the innate immune system responds in a qualitatively different way to contact with viable Eh vs. soluble ligands produced by viable or dead ameba. This unique Eh Gal-lectin contact-dependent response in macrophages was mediated by activation of the inflammasome. Soluble native Gal-lectin did not induce inflammasome activation, but was sufficient for transcriptional priming of the inflammasome and non-inflammasome-dependent pro-inflammatory cytokine release. We conclude the inflammasome is a pathogenicity sensor for invasive Eh and identify for the first time a PRR that specifically responds to contact with intact parasites in a manner that accords with scale immune response to parasite invasion.

Entamoeba histolytica Cathepsin-Like Enzymes

Cysteine proteases of the protozoan parasite Entamoeba histolytica are key virulence factors involved in overcoming host defences. These proteases are cathepsin-like enzymes with a cathepsin-L like structure, but cathepsin-B substrate specificity. In the host intestine, amoeba cysteine proteases cleave colonic mucins and degrade secretory immunoglobulin (Ig) A and IgG rendering them ineffective. They also act on epithelial tight junctions and degrade the extracellular matrix to promote Cell death. They are involved in the destruction of red blood cells and the evasion of neutrophils and macrophages and they activate pro-inflammatory cytokines IL- 1β and IL-18. In short, amoeba cysteine proteases manipulate and destroy host defences to facilitate nutrient acquisition, parasite colonization and/or invasion. Strategies to inhibit the activity of amoeba cysteine proteases could contribute significantly to host protection against E. histolytica.

Innate Host Defenses in the Gut

In the human intestine, evolutionary pressures have selected host and parasite mechanisms that maintain spatial separation of Entamoeba histolytica on the luminal side of the mucus–epithelial barrier. The function of the barrier is conferred by many systems acting on multiple levels. Mechanisms that strengthen and maintain stability of the epithelial barrier are critical for preventing disease and keeping E. histolytica infections asymptomatic. It is unknown why invasion happens. Intestinal epithelial cells are in close and continuous proximity to the parasite, and abnormal responses by epithelial cells are suspected to instigate disease. This interaction, however, is poorly understood. When invasion occurs the gut has a second line of innate defenses that rapidly eliminate the parasite: sensing of invasion by resident cells, innate humoral immunity, and recruitment of competent immune cells to sites of invasion. The pathology that arises during invasion, which culminates as amebic dysentery or colitis, is a combined effect of direct damage by trophozoites and collateral damage from host defenses.

The Roles of Interleukin-17 and T Helper 17 Cells in Intestinal Barrier Function

Inflammatory bowel diseases (IBD) are caused by chronic inflammation of the gastrointestinal tract, affecting as many as 1.4 million persons in the United States, and 2.2 million persons in Europe (Loftus, 2004). Crohn’s disease (CD) and ulcerative colitis (UC), the two major forms of IBD, affect different regions of the intestinal tract and have distinct cytokine profiles. In CD, transmural inflammation can occur over the entire length of the gastrointestinal tract, whereas UC inflammation is restricted to the mucosa of the colon. The T helper (Th) paradigm was established by Mosmann et al (1986) who observed distinct cytokine patterns were produced by two types of fully differentiated effector T cells which they termed termed Th1 and Th2 cells. The initial cytokine profiles observed in IBD helped to classify CD as a Th1 disease, due to the increased production of the main Th1 effector cytokine, interferon-gamma (IFN-┛). UC was slightly more difficult to classify because levels of a central Th2 effector cytokine, IL-4, are not increased; however, other Th2 effector cytokines, such as IL-5 and IL-13 are produced at higher levels (Fuss et al, 1996). Therefore, UC is not considered fully Th2, but rather a Th2-like disease (Fuss et al, 2004). Conventional IBD therapies, including corticosteroids and anti-tumor necrosis factor-alpha (TNF-α) therapy, are aimed at reducing nonspecific inflammation. TNF-α is a central proinflammatory cytokine that contributes to the pathology of many autoimmune disorders. Anti-TNF-α was the first biological therapy introduced for patients with IBD in the late 1990s, and corticosteroid-refractory or fistulizing CD and refractory UC generally respond very well to anti-TNF-α treatment (Hoentjen & van Bodegraven, 2009; Rutgeerts et al., 2006). The initial identification of disease-specific inflammatory mediators in CD and UC, Th1 and Th2-associated cytokines respectively, lead to the development of more specific antiinflammatory treatment options, and the efficacies of these new biological agents have in turn helped evolve our understanding of IBD pathogenesis. Using mouse models of intestinal inflammation that resemble CD, and targeting the main cytokine that drives Th1 cellular development, IL-12, with an antibody to the IL-12p40 subunit either prevented the development of colitis, or completely cured established colitis (Liu et al., 2001; Neurath et al, 1995). These observations further supported the link between CD and Th1 responses, in addition to warranting the development of an anti-IL-12p40 antibody for human patients with CD. In clinical trials, anti-IL-12p40 therapy induced clinical responses and remissions in patients with active CD (Mannon et al., 2005; Sandborn et al., 2008), which lead to its acceptance as a new therapy for CD.