triggers proinflammatory lesions in the intestine of C57BL/6 mice. This destructive pathology has many features of Crohn’s disease, insofar as both encompass ileal involvement, CD4+
T lymphocyte pathogenic activity, inflammatory cytokine overproduction, and dysbiosis of gut microflora 12
. Moreover, both Crohn’s inflammation and Toxoplasma
-triggered ileitis can be ameliorated by blocking TNF-α 7, 20
. Recently, we uncovered a role for CCR2- and IFN-γ-dependent CD8α+
IEL in gut lesion development during oral T. gondii
. Here, we establish that αβ and not γδ TCR+
IEL mediate intestinal damage. We also show that transfer of IEL together with LP cells from infected mice into naïve Rag1−/−
mice triggers proinflammatory intestinal pathology in the absence of parasites. We show that IEL recruit LP-derived αβTCR+
T cells into intraepithelial regions in partial dependence on CCR2, and that these cells are mediators of intestinal damage. Transfer of IEL alone in this model induced mild inflammation, suggesting that in addition to recruitment of LP cells, they may also directly contribute to damage in the intestine. We also found that endogenous intestinal flora in recipient mice was required for the pathogenic effects of cell transfer. These results, which are summarized in Fig. S6
, are the first to demonstrate that microbial induced interactions between IEL and LP lymphocytes promote proinflammatory cytokine responses and inflammatory lesions in the small intestine.
Important issues that await resolution are the MHC requirements and antigen specificity of the IEL and LPL subsets. The requirement for gut flora in recipient Rag1−/−
mice suggests that one or both subsets could be reactive to intestinal bacteria. With regard to the IEL compartment, some CD8+
IEL subsets display unusual MHC requirements 16
, and it will therefore be important in the future to examine responses of MHC class I and β2-microglobulin knockout mice.
Our results show pathological activity of IEL induced by Toxoplasma
. Nevertheless, under homeostatic conditions the IEL compartment is generally associated with immunoregulation and immune quiescence in the intestinal mucosa 16, 21
. For example, transfer of CD8ααTCRαβ IEL protects immunodeficient mice from subsequent transfer of pathogenic CD4+
. In parallel, it has been shown that γδTCR+
IEL have a protective function in the dextran sodium sulfate mouse colitis model23
. Yet, IEL isolated from Crohn’s disease patients have been shown to display abnormally enhanced cytotoxic activity and overproduction of IFN-γ compared to IEL from normal donors 24, 25
. The latter observations, together with our data, suggest that dysfunctional IEL form part of the constellation that constitutes IBD.
Related to our studies is elegant work by others who examined early mucosal responses during low dose Toxoplasma
infection, a situation that avoids immunopathology, enabling host survival, protective immunity, and persistent infection. Here, IEL protect against ileitis and subsequent parasite challenge after adoptive transfer 26
. In the LP compartment, CD4+
T cells synergize with intestinal epithelial cells resulting in protective proinflammatory responses that control infection 27
. At least part of the beneficial activity of IEL in this model stems from TGF-β production that down-regulates LP CD4+
T lymphocyte responses and controls ileitis 28, 29
. We hypothesize that high dose T. gondii
infection triggers a switch in IEL function from mediators of protection and immunoregulation to effectors of pathology.
In our model, transfer of IEL and LP cells from Toxoplasma
-infected mice triggered fulminant pathology in noninfected Rag1−/−
recipients. It seems most unlikely that parasites were the direct cause of disease in these mice as we could not detect parasites in either the cells injected into the naïve Rag1−/−
or in intestinal tissue isolated 5 days after transfer. Furthermore, Rag1−/−
mice are resistant to lesion development when directly infected with T. gondii
and ref. 30
). In wild-type mice, development of intestinal lesions during Toxoplasma
infection is associated with a shift from gram-positive to predominantly gram-negative bacteria in the small intestine, and fulminant pathology is accompanied by increased adherence and bacterial translocation into the sub-epithelium. Host responses to gut bacteria are implicated in intestinal inflammation because both Tlr4−/−
mice are resistant to lesion development 10, 11, 31
We show here that Ccr2−/− LP cells do not re-locate to the IEL compartment after transfer into Rag1−/− mice. Yet, previously we found that IEL transfer into T. gondii infected Ccr2−/− recipients (where the LP compartment would be CCR2 negative) were able to induce intestinal damage. We speculate that in the latter case other chemokines and cytokines induced as a result of Toxoplasma infection in the recipients compensate for loss of CCR2 responsiveness.
What is the triggering event that drives Toxoplasma
-mediated intestinal lesion development? Possibly, the parasite induces early barrier damage as a result of invasion and egress from epithelial cells, enabling translocation of bacteria and in turn triggering pathogenic responses. Arguing against this scenario are data suggesting that Toxoplasma
crosses the intestinal barrier using a paracellular pathway that involves transmigration without membrane disruption 32
. It is also possible that the parasite directly activates mucosal lymphocytes for pathogenic activity, although this does not fully account for the requirement for bacterial flora in lesion development.
Another possibility is that disease development is a consequence of immunosuppression by Toxoplasma
. The parasite is known to downregulate cytokine and chemokine responses during intracellular macrophage and dendritic cell infection 33–36
. This may have relevance to induction of IBD, because recent data reveal impaired cytokine secretion by macrophages from Crohn’s patients 37
. Older studies have shown impaired neutrophil recruitment in individuals with Crohn’s disease 38, 39
. Thus, a model for Crohn’s pathogenesis is that defective sentinel responses to occasional bacterial ingress in the gut results in failure to recruit neutrophils, leading to loss of control of infection and chronic secondary inflammation 37
. We are currently examining the functional status of intestinal dendritic cells and macrophages at early time points of T. gondii
The results of this study highlight the value of Toxoplasma infection in C57BL/6 mice as a model for induction of Crohn’s like pathology in the ileum. Our results reveal for the first time that LP T cells and IEL synergize to cause inflammatory ileitis. By exploiting Toxoplasma as a trigger, we can distinguish cell and cytokine requirements required for inflammation, and in so doing we can expect to gain further mechanistic insight into immunological factors that are important in human IBD.