Since 1989, it has been well documented that TFFs are expressed along the length of the normal gastrointestinal tract, with TFF2 expression being primarily localized to the stomach (27
). In previously published experiments using either rats or mice, our group has shown that TFF2 is constitutively expressed in the GI mucosa within specialized epithelial cells and is markedly upregulated in response to inflammation, injury, and repair, as demonstrated using immunohistochemisty of tissue sections (18
). Here we demonstrate that immune cells are an important additional source of TFF2, a novel and potentially important finding, and that immune cell TFF2 is an important regulator of the innate immune response in these cells.
In the gut, TFFs are typically expressed in mucus-secreting cells, and consequently, most of the focus has been on their role in cytoprotection and reparation of the mucosal lining of the gut (29
). However, given that administration of exogenous TFFs reduces inflammation in the gastrointestinal tract (40
), Cook et al. (2
) first explored the possibility of expression of TFFs in nongastric cells. They reported the expression of TFF2 in rat spleen and lymphoid tissues (2
). Thus, the current study extends these findings but also provides the first demonstration that TFF2 functions as an anti-inflammatory peptide with specificity for IL-1β signaling and immune cell cytokine secretion.
In this study, we demonstrate a novel role for TFF2 in the regulation of IL-1 signaling. We confirmed that TFF2 is expressed not only in gastric epithelial cells (18
) but also in spleen cells and that TFF2 deficiency is associated with increased cytokine secretion by macrophages and T-cell proliferation in response to IL-1β. These in vitro responses correlated with increased inflammatory responses to LPS injection, H. felis
infection, and DSS models of inflammatory bowel disease. The effect of TFF2 deficiency on macrophages was highly specific for the IL-1 pathway, since no alteration was seen in the responses of PECs to the TLR4 ligand LPS, which shares many/most of the same signaling pathways.
Several previous observations suggested that TFF2 might play a role in controlling or dampening inflammation. Several groups have reported that application of recombinant trefoils can reduce inflammatory indices in animal models of colitis (16
). For example, luminal application of recombinant TFF2/spasmocytic peptide in a rat model of colitis revealed markedly accelerated colonic mucosal rebuilding and reduced inflammatory indices (40
) through mechanisms that could possibly include inhibition of inducible nitric oxide synthase and NO in monocytes (17
). In addition, TFF2 is clearly upregulated in epithelial cells in chronic inflammatory conditions of the gastrointestinal tract, such as peptic ulcer disease and Crohn's disease, and in other organ systems, such as the lung; in fact, TFF2 may be regulated directly by a variety of cytokines (26
). TFF2 may also function in promoting restitution of the epithelium, and it has always been assumed that such increased epithelial expression functioned mainly in the context of epithelial regeneration and repair. Recently, TFF2 has been shown to affect the development of asthma in mice (26
). However, given our results, it would seem reasonable to suggest that TFF2 may also represent a signal by the epithelium to the immune system to down-regulate or otherwise dampen an acute inflammatory response. Thus, TFF2 would serve as a key player in the epithelial-immune signaling system.
IgG subclass responses have been used as estimates of Th cell type, with IgG1 being produced in Th2 processes and IgG2c (the C57BL/6 allelic equivalent of IgG2a) in Th1 responses of C57BL/6 mice (25
). Thus, the IgG1/IgG2c ratio represents a quantifiable measure of balance between mucosal Th2/Th1 activity, and in previous studies by our group, a low ratio (indicating a predominant Th1 response) has correlated well with the tendency toward neoplastic progression (12
mice, like wild type mice, had a Th1-dominated response to H. felis
and in fact had significantly higher levels of IgG2c than wild-type mice following H. felis
infection. Thus, the Ab response to infection was consistent with an enhanced proinflammatory response in the TFF2−/−
The expression of TFF2 in spleen cells raised the possibility that TFF2 might affect lymphocyte responses. We examined the effect of TFF2 deficiency on spleen cell responses to B- and T-cell mitogens. Splenic B cells proliferate in response to Pokeweed mitogen, while splenic T cells proliferate and secrete cytokines in response to ConA. IL-1β (an inflammatory cytokine which has been linked to human gastrointestinal disease and gastric cancer progression) synergizes with ConA to stimulate T-cell proliferation. T cells are also activated by cross-linking T-cell receptors with plate-bound anti-CD3 Ab.
T cells from TFF2−/− mice exhibited an enhanced proliferative response to ConA and ConA plus IL-1 and to anti-CD3-coated plates. Thymocytes, on the other hand, were unaffected by TFF2 deficiency. Thus, the hyperproliferative response of the TFF2−/− T cells was limited to mature cells and/or peripheral lymphoid tissues, i.e., mature T lymphocytes in the spleen but not immature T cells in the thymus. The sensitivity to TFF2 follows the pattern of TFF2 gene expression in these lymphoid organs. In addition to excess proliferation, TFF2−/− lymphocytes secreted increased levels of IL-2 and IL-4, suggesting a generalized enhancement of T-lymphocyte activation. Although TFF2 deficiency markedly increased T-cell proliferative responses in the spleen, TFF2 deficiency did not affect B-cell proliferation induced by Pokeweed mitogen (data not shown). Thus, mature T cells may be the major target of TFF2 regulation in the spleen.
Our current study suggests that in addition to excess cytokine production and lymphocyte proliferation, a major phenotype associated with a deficiency of TFF2 is unrestrained IL-1R signaling. In fact, the TFF2−/− mouse resembled in some ways a mouse with constitutive IL-1R activation. IL-1β signals via the type I IL-1R. The cytoplasmic domain of IL-1RI contains a Toll-interleukin 1 resistance (TIR) domain which mediates receptor signaling via MyD88-dependent pathways leading to cytokine secretion. Like IL-1R, TLR4 utilizes TIR domain-mediated and MyD88-dependent signaling cascades. Thus, LPS-induced signaling via TLR4 engages many of the same downstream adapters that are triggered by IL-1β/IL-1R engagement. To determine if TFF2 deficiency globally enhanced TIR signaling, we examined the response of wild-type and TFF2 knockout macrophages to LPS. Interestingly, TFF2−/− mice were hyperresponsive to IL-1 receptor signaling but not to LPS signaling, suggesting a selective enhancement of IL-1R-induced pathways.
Interleukin 1 receptor signaling has been shown to be particularly important in chronic inflammatory diseases of the gastrointestinal tract, including both inflammatory bowel disease and Helicobacter pylori
gastritis. IL-1R signaling induces the expression and secretion of multiple inflammatory cytokines and chemokines, including IL-6, monocyte chemoattractant protein 1, and tumor necrosis factor alpha, as well as autocrine secretion of IL-1β. These inflammatory cytokines were up-regulated in resident peritoneal macrophages from older TFF2 knockout mice, suggesting an ongoing inflammatory response in vivo. Mononuclear cells isolated from the intestinal mucosa of patients with Crohn's disease or ulcerative colitis show enhanced production of IL-1β (24
), and patients with ulcerative colitis show an increased carrier rate for the intron 2 IL-1 receptor antagonist polymorphism (39
). Infection with H. pylori
also results in a strong inflammatory response led by the early release of IL-1β along with numerous other cytokines and chemokines (5
). Further, we have noted that H. felis
infection in C57BL/6 mice induces a significant IL-1β response (12
). However, the progression of gastric inflammation to gastric atrophy and cancer is closely related to the overall balance between pro- and anti-inflammatory cytokines and specifically to the expression of IL-1β. Thus, proinflammatory polymorphisms in the IL-1B (511 T/T) and IL-1RN (*2) genes are associated with an increased risk for gastric carcinoma in H. pylori
-infected individuals (6
). H. pylori
-infected carriers of these polymorphisms have been shown to exhibit higher mucosal levels of IL-1β and more severe inflammation than H. pylori
-infected noncarriers (19
). Recently, mice lacking SIGIRR/Tir8, an important regulator of IL-1 signaling, were shown to exhibit increased susceptibility to inflammatory bowel disease (15
). Given the central role of IL-1 in the inflammatory process and the potential harm associated with excessive IL-1-dependent signaling, it would be reasonable to speculate that vertebrates have evolved diverse mechanisms, including TFF2, to counterregulate the response to IL-1β. The recognition of trefoil peptides as a novel class of cytokines may provide new insight into the modulation of the immune system.