Sara B. Cohen

Insights into inflammatory bowel disease using Toxoplasma gondii as an infectious trigger

Oral infection of certain inbred mouse strains with the protozoan Toxoplasma gondii triggers inflammatory pathology resembling lesions seen during human inflammatory bowel disease, in particular Crohns disease (CD). Damage triggered by the parasite is largely localized to the distal portion of the small intestine, and as such is one of only a few models for ileal inflammation. This is important because ileal involvement is a characteristic of CD in over two‐thirds of patients. The disease induced by Toxoplasma is mediated by Th1 cells and the cytokines tumor necrosis factor‐α and interferon‐γ. Inflammation is dependent upon IL‐23, also identified by genome‐wide association studies as a risk factor in CD. Development of lesions is concomitant with emergence of E. coli that display enhanced adhesion to the intestinal epithelium and subepithelial translocation. Furthermore, depletion of gut flora renders mice resistant to Toxoplasma‐triggered ileitis. Recent findings suggest complex CCR2‐dependent interactions between lamina propria T cells and intraepithelial lymphocytes in fueling proinflammatory pathology in the intestine. The advantage of the Toxoplasma model is that disease develops rapidly (within 7–10 days of infection) and can be induced in immunodeficient mice by adoptive transfer of mucosal T cells from infected donors. We propose that Toxoplasma acts as a trigger setting into motion a series of events culminating in loss of tolerance in the intestine and emergence of pathogenic T cell effectors. The Toxoplasma trigger model is providing new leaps in our understanding of immunity in the intestine.

CXCR3-Dependent CD4+ T Cells Are Required to Activate Inflammatory Monocytes for Defense against Intestinal Infection

Chemokines and their receptors play a critical role in orchestrating immunity to microbial pathogens, including the orally acquired Th1-inducing protozoan parasite Toxoplasma gondii. Chemokine receptor CXCR3 is associated with Th1 responses, and here we use bicistronic CXCR3-eGFP knock-in reporter mice to demonstrate upregulation of this chemokine receptor on CD4+ and CD8+ T lymphocytes during Toxoplasma infection. We show a critical role for CXCR3 in resistance to the parasite in the intestinal mucosa. Absence of the receptor in Cxcr3−/− mice resulted in selective loss of ability to control T. gondii specifically in the lamina propria compartment. CD4+ T cells were impaired both in their recruitment to the intestinal lamina propria and in their ability to secrete IFN-γ upon stimulation. Local recruitment of CD11b+Ly6C/G+ inflammatory monocytes, recently reported to be major anti-Toxoplasma effectors in the intestine, was not impacted by loss of CXCR3. However, inflammatory monocyte activation status, as measured by dual production of TNF-α and IL-12, was severely impaired in Cxcr3−/− mice. Strikingly, adoptive transfer of wild-type but not Ifnγ−/− CD4+ T lymphocytes into Cxcr3−/− animals prior to infection corrected the defect in inflammatory macrophage activation, simultaneously reversing the susceptibility phenotype of the knockout animals. Our results establish a central role for CXCR3 in coordinating innate and adaptive immunity, ensuring generation of Th1 effectors and their trafficking to the frontline of infection to program microbial killing by inflammatory monocytes.

Synergy Between Intraepithelial Lymphocytes and Lamina Propria T Cells Drives Intestinal Inflammation During Infection

Oral infection of C57BL/6 mice with Toxoplasma gondii triggers severe necrosis in the ileum within 7–10 days of infection. Lesion development is mediated by Th-1 cytokines, CD4+ T cells, and subepithelial bacterial translocation. As such, these features share similarity to Crohns disease. Recently, we uncovered a role for intraepithelial lymphocytes (IELs) in mediating pathology after Toxoplasma infection. We show here that αβ and not γδ T-cell IELs mediate intestinal damage. By adoptive transfer of mucosal T cells into naive Rag1−/− mice, we demonstrate that IELs do not function alone to cause inflammatory lesions, but act with CD4+ T lymphocytes from the lamina propria (LP). Furthermore, recipient mice pretreated with broad-spectrum antibiotics to eliminate intestinal flora resisted intestinal disease after transfer of IELs and LP lymphocytes. Our data provide valuable new insights into the mechanisms of intestinal inflammation, findings that have important implications for understanding human inflammatory bowel disease.

Epithelial Sel1L is required for the maintenance of intestinal homeostasis

Endoplasmic reticulum (ER)–associated degradation (ERAD) clears misfolded proteins in the ER. Epithelial ERAD plays an indispensable role in Paneth cell biology and the maintenance of small intestine homeostasis. The findings implicate Sel1L-Hrd1 ERAD as a novel therapeutic target for Crohn’s disease.

β-Catenin Signaling Drives Differentiation and Proinflammatory Function of IRF8-Dependent Dendritic Cells

β-Catenin signaling has recently been tied to the emergence of tolerogenic dendritic cells (DCs). In this article, we demonstrate a novel role for β-catenin in directing DC subset development through IFN regulatory factor 8 (IRF8) activation. We found that splenic DC precursors express β-catenin, and DCs from mice with CD11c-specific constitutive β-catenin activation upregulated IRF8 through targeting of the Irf8 promoter, leading to in vivo expansion of IRF8-dependent CD8α+, plasmacytoid, and CD103+CD11b− DCs. β-Catenin–stabilized CD8α+ DCs secreted elevated IL-12 upon in vitro microbial stimulation, and pharmacological β-catenin inhibition blocked this response in wild-type cells. Upon infections with Toxoplasma gondii and vaccinia virus, mice with stabilized DC β-catenin displayed abnormally high Th1 and CD8+ T lymphocyte responses, respectively. Collectively, these results reveal a novel and unexpected function for β-catenin in programming DC differentiation toward subsets that orchestrate proinflammatory immunity to infection.

Phagocyte Responses to Protozoan Infection and How Toxoplasma gondii Meets the Challenge

The intracellular protozoan Toxoplasma gondii is arguably the most successful parasite on the planet. It exploits an uncommonly wide host range that encompasses essentially all warm-blooded animals including both mammalian and avian species. Sexual reproduction in the intestine of the definitive host, the cat, results in fecal shedding of up to 108 highly infectious oocysts. The presence of felines from equatorial latitudes to sub-arctic regions of the globe ensures widespread distribution of the parasite. Moreover, unlike closely related apicomplexans such as the Plasmodia, passage through the definitive host is not obligatory to complete the life-cycle, because T. gondii can be transmitted from one intermediate host to the next through predation and carnivorism [1]. While Toxoplasma causes asymptomatic infection in most hosts, the parasite may emerge as an opportunistic infection under immunodeficient conditions such as in AIDS patients and during congenital infection. This danger underscores the importance of the encounter between T. gondii and the host immune system in determining the success of this particular host-parasite interaction. It is well understood that complete evasion of immunity (or for that matter passive failure to trigger immunity) results in rampant infection and host death, an outcome undesirable for both host and parasite. At the same time, we are learning in greater detail the mechanisms employed by the host immune system to destroy Toxoplasma. The parasite must obviously avoid this outcome of immunity to ensure persistence. The global success of T. gondii (over 109 asymptomatic infections in humans alone) suggests that the parasite employs sophisticated molecular strategies to balance evasion versus activation of the host immune response. As summarized in Figure 1, the multiple ways this unifying principle plays out is revealed in studies on infection in phagocytes of innate immunity, namely dendritic cells (DC), macrophages, and neutrophils. These cells are among the first to encounter and be infected by Toxoplasma after the parasite crosses the intestinal epithelium. The studies together form a platform from which we can further understand the complex relationship between microbial pathogens and cells of the innate immune system. Figure 1 Integrating phagocyte function with T. gondii infection. Dendritic Cells Are Sentinels and Trojan Horses Early on it was recognized that DC were an early source of IL-12 driving protective Th1 responses to Toxoplasma. Further studies using an intraperitoneal infection model showed that ablation of CD11c+ DC results in failure to mount protective immunity and death during infection [2]. With the discovery of Toll-like receptors (TLR) and their ligands in the late 1990s, attention turned to the role of this system in sensing protozoan pathogens, in particular T. gondii [3]. Indeed, mice lacking MyD88, a central adaptor of TLR signaling, are extremely susceptible to infection. More specifically, it has been shown using cell-specific gene-deleted mouse strains that MyD88 expression in CD11c+ dendritic cells is required to resist Toxoplasma infection [4]. There is evidence for involvement of mouse TLR2, TLR4, TLR9, and TLR11 in the innate immune response to T. gondii [3]. Of these receptors, deletion of TLR11 has the most dramatic effect on loss of host resistance [5]. However, Tlr11−/− mice fail to recapitulate the extreme susceptibility phenotype of Myd88−/− mice. This has led to the suggestion that multiple TLR function together to provide optimal resistance, or alternatively that other untested TLR serve as the major MyD88-dependent receptor mediating protective immunity. Toxoplasma profilin (TgPRF), an actin polymerizing molecule, is the ligand recognized by TLR11 [5]. In DC, TgPRF stimulates TLR11-dependent IL-12 production. Interestingly, it was recently found that this response occurs through phagocytic uptake of parasite material followed by TLR11 activation from within the endolysosome [6]. Surface-expressed glycosylphosphatidylinositol moieties purified from tachyzoites have also been found to mediate TLR2 and TLR4 activation [3], although the in vivo importance of this phenomenon is not clear. While the TLR11/TgPRF interaction is significant in the rodent response to Toxoplasma, the importance of TgPRF in human infection is uncertain since we do not express functional TLR11. In addition to TLR-dependent recognition of Toxoplasma, there is clear evidence for MyD88-independent resistance. This is because Myd88−/− mice develop strong (albeit delayed) Th1 responses during oral infection, and the same mouse strain develops protective immunity following intraperitoneal infection with attenuated parasites [7]. In this regard, it was recently shown that release of tachyzoite dense granule protein GRA5 into the host cytoplasm by intracellular parasites bypasses MyD88 to activate NFκB and stimulate IL-12 synthesis [8]. The relative roles that profilin and GRA5 assume during normal infection have not yet been determined. However, GRA5 IL-12 inducing properties are parasite strain-specific, in that only one lineage (Type II) of the three predominant strains found in Europe and North America possess this activity [8]. On the other hand, there is no evidence that profilin acts in a parasite-strain-dependent manner. Another function of DC during the response to Toxoplasma is to serve as early reservoirs of infection [9], [10]. It has been suggested that parasites utilize DC in a “Trojan horse” strategy to disseminate throughout the host. Upon in vitro infection, DC acquire a hypermotility phenotype that is dependent upon host cell G-protein signaling triggered by the parasite. Intraperitoneal inoculation of tachyzoite-harboring DC spreads infection more rapidly than injection of extracellular parasites alone, suggesting that DC hypermotility promotes dissemination during in vivo infection, although whether a similar phenomenon occurs during oral infection is not clear [11]. Interestingly, hypermotility and in vivo dissemination of infected DC occur most efficiently with Type II Toxoplasma, the strain most frequently found in human infection [12].

Impact of Toxoplasma gondii on Dendritic Cell Subset Function in the Intestinal Mucosa

The function of mucosal dendritic cell (DC) subsets in immunity and inflammation is not well understood. In this study, we define four DC subsets present within the lamina propria and mesenteric lymph node compartments based on expression of CD103 and CD11b. Using IL-12p40 YFP (Yet40) reporter mice, we show that CD103+CD11b− mucosal DCs are primary in vivo sources of IL-12p40; we also identified CD103−CD11b− mucosal DCs as a novel population producing this cytokine. Infection was preferentially found in CD11b+ DCs that were negative for CD103. Lamina propria DCs containing parasites were negative for IL-12p40. Instead, production of the cytokine was strictly a property of noninfected cells. We also show that vitamin A metabolism, as measured by ALDH activity, was preferentially found in CD103+CD11b+ DC and was strongly downregulated in all mucosal DC subsets during infection. Finally, overall apoptosis of lamina propria DC subsets was increased during infection. Combined, these results highlight the ability of intestinal Toxoplasma infection to alter mucosal DC activity at both the whole population level and at the level of individual subsets.