The intestinal immune system balances immune responses to commensal and harmful antigens in the intestinal lumen to maintain homeostasis. Dysfunction of this system results in intestinal inflammation. Several experimental models of IBD facilitate evaluation of the role of the intestinal immune system, and have identified key regulators and pathways of IBD pathogenesis.

Some mouse strains with inactivation or disruption of TGF-β signaling are susceptible to intestinal inflammation and are used to study the pathogenesis of IBD. In 1993, TGF-β1 germline-null mice were reported to exhibit massive inflammatory lesions in multiple organs, including the colon, at a few weeks of age [33, 34]. Thereafter, several studies have focused on the specific cellular functions mediated by TGF-β signaling. Mice with inactivated TGF-β signaling due to the presence of a dominant-negative mutant under the control of a cell-specific promoter, or with cell-specific disruption of TGF-β signaling by a Cre-lox recombination, have been used in these studies (Table 1).

Table 1 Experimental mouse model targeting TGF-β signaling Full size table

In this section, we review the role of TGF-β in immune cells and the intestinal epithelium in the pathogenesis of IBD, focusing on experimental mouse models. In addition, we discuss the roles of TGF-β and the intestinal microbiota in intestinal immunity.

Immune cells

T-cells

TGF-β regulates multiple immune processes of T-cells. A major function of TGF-β signaling in T-cells is to suppress T-cell proliferation and activation through Treg differentiation. Mice with T-cell-targeted deletion of TGF-β signaling (CD4-Cre Tgfbr2 fl/fl) showed early onset of fatal systemic autoimmunity at 3–5 weeks of age [35, 36]. Furthermore, mice with T-cell–targeted inactivation of TGF-β signaling (CD4-dnTGFβRII) slowly developed systemic autoimmunity with spontaneous severe colitis at 3–4 months of age [37]. Autoimmunity in both mouse strains was characterized by massive infiltration of lymphocytes and the presence of activated T-cells in multiple organs. CD4-Cre Tgfbr2 fl/fl mice lack TGFβRII expression on immature CD4+ thymocytes and mature peripheral CD4+ T-cells, including CD4+ Tregs [35]. As a result, CD4-Cre Tgfbr2 fl/fl mice showed a marked reduction in peripheral CD4+Foxp3+ Tregs. These results suggest that TGF-β signaling in T-cells contributes to intestinal immune tolerance, in part by maintenance of the peripheral Treg cell population. Regarding the effect of TGF-β production by CD4+ T-cells on Treg differentiation, mice with CD4+ T-cell-targeted deletion of TGF-β1 production (CD4-CreTgfb1 fl/−) did not show a reduction in the numbers of peripheral CD4+Foxp3+ Tregs, although TGF-β1-null mice showed lower numbers of Tregs, indicating that TGF-β1 produced by cell types other than T-cells contributes to peripheral Treg differentiation [38].

Another function of TGF-β in T-cells is Th17 differentiation. TGF-β together with IL-6 was reported to induce differentiation of Th17 cells from naïve CD4+ T-cells. Th17 cells produce IL-17 and IFN-γ, which are necessary for mucosal defense against bacteria, but tend to promote intestinal inflammation [39, 40]. Previous studies showed that Th17 development in the intestine was impaired in TGF-β1-null mice and CD4-Cre Tgfb1 fl/− mice [38, 41]. In contrast, Th17 development was not impaired in CD4-Cre Tgfbr1 fl/fl mice or CD4-dnTGFβRII mice [42]. Moreover, the role of Th17 cells in intestinal inflammation in TGF-β-mutant mice is unclear.

A novel function for TGF-β in memory CD8+ T-cells was reported recently. cLck-Cre Tgfbr2 fl/fl mice showed decreased retention of antigen-specific memory CD8+ T-cells in the intestine, partly due to the defective expression of integrins [43].

B-cells

TGF-β in B-cells mediates IgA class-switch and promotes IgA production [44, 45]. Although mice with deletion of TGF-β signaling in B-cells (CD19-Cre Tgfbr2 fl/fl) did not show signs of autoimmunity or colitis, CD19-Cre Tgfbr2 fl/fl mice showed B-cell hyperplasia in Peyer’s patches and decreased B-cell responsiveness with complete serum IgA deficiency [45, 46]. IgA protects against luminal bacteria by neutralization, enhancing phagocytosis and antigen presentation by DCs. IgA also inhibits bacterial adhesion to the epithelium by blocking surface epitopes of bacteria [47]. IgA production was augmented by the interaction between B-cells and DCs in Peyer’s patches through integrin αvβ8-activated TGF-β [48]. This was confirmed by the finding that IgA class-switch by B-cells in Peyer’s patches was impaired in CD11c-Cre Itgb8 fl/fl mice and by treatment with an αvβ8-blocking antibody [48].

DCs

Mice with deletion of TGF-β signaling in DCs (CD11c-Cre Tgfbr2 fl/fl) developed spontaneous colitis with multiple organ autoimmunity, similar to CD4-Cre Tgfbr2 fl/fl and TGF-β1-null mice [49, 50]. Spontaneous colitis in CD11c-Cre Tgfbr2 fl/fl mice was characterized by loss of goblet cells with lymphocytic infiltration and systemic autoimmunity due to altered Treg differentiation, activated T-cells and B-cells, and increased secretion of inflammatory cytokines such as TNF-α and IFN-γ [49]. We also examined colitis in CD11c-Cre Tgfbr2 fl/fl mice and found enhanced expression of Notch ligands on DCs, goblet cell depletion, a thinner mucus layer, and dysbiosis (Fig. 1b) [50]. These results reveal the critical role played by TGF-β signaling by DCs in colonic homeostasis.

DCs are also important as a source and activator of TGF-β in the intestine. Intestinal DCs produce TGF-β and IL-10, which are major suppressors of intestinal immunity [51]. Previous reports showed that CD103+ tolerogenic DCs produce TGF-β and retinoic acid, which contributes to Treg differentiation [52, 53]. Intestinal DCs also contribute to TGF-β activation. Mice with DC-specific deletion of integrin β8 (CD11c-Cre Itgb8 fl/fl) developed spontaneous colitis due to a lack of TGF-β activation by αvβ8 in DCs, whereas T-cell-specific deletion of integrin β8 (CD4-Cre Itgb8 fl/fl) did not result in the development of colitis [54].

In contrast to CD103+ tolerogenic DCs, E-cadherin+ inflammatory DCs promoted intestinal inflammation through aberrant IL-17 production by CD4+ T-cells [55]. E-cadherin is an adhesion molecule expressed in the intestinal epithelium, and also by subsets of monocytes, DCs, and macrophages [55,56,57]. E-cadherin+ DCs were increased in a T-cell-transfer murine colitis model, especially in the inflamed intestine. Indeed, adoptive transfer of E-cadherin+ BMDCs into T-cell-restored Rag1−/− mice exacerbated colitis, with an increased Th17 response [55]. TGF-β-deficient mice (DO11.10 Tgfb1 −/−) showed an increased frequency of E-cadherin+ DCs, indicating that TGF-β limits the accumulation of E-cadherin on DCs [55]. However, the molecular mechanisms underlying exacerbation of colitis by E-cadherin+ DCs are unclear, and data regarding E-cadherin expression in intestinal DCs from IBD patients are lacking.

Macrophages

Mice with TGF-β signaling inactivation in macrophages (CD68-dnTGFβRII) did not develop spontaneous colitis, but exhibited susceptibility to DSS-induced colitis with reduced IL-10 production [58]. TGF-β signaling in macrophages suppressed IL-33 production and protected against intestinal inflammation [58, 59]. It has also been reported that TGF-β downregulates the expression of innate response receptors, such as that for LPS (CD14), on human intestinal macrophages. This contributed to the development of an “inflammatory anergy” macrophage phenotype, which is characterized by a lack of proinflammatory cytokine production under inflammatory stimuli but retention of phagocytic and bactericidal activity [60].

Intraepithelial lymphocytes (IELs)

TGF-β production and signaling by T-cells are important for IEL development [61]. IELs reside in the intestinal epithelial layer and play a role in mucosal defense. The majority of TCRαβ+ IELs are divided into subsets expressing CD8αα+ or CD8αβ+ [62]. A previous study demonstrated that Tgfb1−/−, Smad3ex8/ex8, and CD4-Cre Tgfbr1 fl/fl mice showed reduced numbers of TCRαβ+CD8αα+ IELs, whereas mice with TGF-β1-overexpressing T-cells (CD4-Cre β1glo) showed increased numbers of TCRαβ+CD8αα+ IELs, suggesting that TGF-β controls the generation and retention of CD8αα+ IELs via CD8α expression [61, 63]. Another study investigated TGF-β production by TCRαβ+CD8αβ+ IELs. Upon infection by Toxoplasma gondii, TGF-β produced by IELs interacted with the lamina propria CD4+ T-cells and reduced intestinal inflammation by downregulating IFN-γ production [62].

Epithelium and extracellular matrix

Previous studies have unraveled the roles of TGF-β in intestinal epithelial homeostasis associated with mucosal integrity, wound healing, and consequent fibrosis. Mouse models with disruption of TGF-β signaling in the intestinal epithelium (such as Villin-CreER Tgfbr2 fl/fl mice) showed increased susceptibility to DSS-induced colitis [64], although spontaneous colitis did not occur in these strains, in contrast to TGF-β1-null mice (Table 1). A recent study revealed that TGF-β-containing extracellular vesicles released by epithelial cells induced Treg differentiation and inhibited colitis by binding to EpCAM+ epithelial cells [21]. Therefore, epithelial cells also play an important role in immune homeostasis in a TGF-β-dependent manner.

TGF-β modulates the barrier function of the epithelium by regulating the expression levels of tight-junction proteins and adhesion molecules [65]. A previous in vitro study using an intestinal monolayer cell line reported that TGF-β enhanced intestinal epithelial barrier function by inducing production of the tight junction protein Claudin-1, and by preventing the pathogenic bacteria-induced reduction of levels of the tight-junction proteins Claudin-2, Occludin, and ZO-1 [66].

As wound healing progresses in injured tissues, the provisional extracellular matrix is replaced by a newly formed matrix, which is rich in collagen synthesized by fibroblasts migrating into the wound [67]. The extracellular matrix is composed of collagens, non-collagenous glycoproteins (including fibronectin) and proteoglycans. In CD patients, chronic transmural intestinal inflammation can result in intestinal fibrosis and fistula, which require surgical resection [68]. Intestinal strictures in CD patients, which are usually caused by chronic inflammation and healing, were associated with an increased TGF-β transcript level and excessive accumulation of extracellular matrix proteins, such as collagens and fibronectin [22, 69]. Myofibroblasts isolated from intestinal strictures of CD patients overexpressed collagen III, and TGF-β1 promoted collagen III production by myofibroblasts [69]. Moreover, pirfenidone, an anti-fibrogenic drug used for the treatment of fibrotic diseases, suppressed intestinal fibrosis in a DSS-induced colitis model by inhibiting TGF-β signaling [70, 71].

Microbiota

The intestinal lumen harbors trillions of microbes of diverse taxa, including both commensal and harmful bacteria; this microbial ecosystem is termed the microbiota. The microbiota plays a mutualistic role in intestinal homeostasis by modulating the host immune systems through their own physiological processes and metabolites [72]. Some commensal bacterial strains exert an immunomodulatory effect in a manner involving TGF-β.

Clostridium

Clostridium is a major genus in the intestinal microbiota and includes several commensal taxa. Microbial transplantation experiments using germ-free mice showed that some strains of Clostridium cluster IV and XIVa induced TGF-β release from the intestinal epithelium and Treg differentiation [73,74,75]. Clostridium cluster IV and XIVa were reported to be less abundant in IBD patients than in healthy controls [76]. Another study showed that administration of Clostridium butyricum (cluster I) as a probiotic [77] promoted Treg differentiation through TGF-β1 produced by lamina propria DCs in a TGF-β-Smad and TLR-ERK-AP1 pathway-dependent manner [15].

Bacteroides

Bacteroides is a Gram-negative, obligate anaerobic bacterial genus that comprises a considerable proportion of the normal intestinal flora. The abundance of the genus Bacteroides is decreased in IBD patients compared to healthy controls [78]. Bacteroides fragilis was reported to induce Treg differentiation [79]. B. fragilis monocolonization of germ-free mice restored TGF-β2 and IL-10 production by Tregs and elicited mucosal tolerance in the intestine. This TGF-β2 production was dependent on polysaccharide A of B. fragilis [79]. In contrast, another study reported that luminal commensal bacteria, such as Bacteroides vulgatus and Bacteroides thetaiotaomicron, are responsible for the development of colitis in mice with T-cell-specific inactivation of TGF-β and IL-10 signaling [80]. Importantly, these two Bacteroides species did not induce colitis in hosts with intact TGF-β signaling, suggesting that TGF-β signaling suppresses the proinflammatory effects of commensal bacteria.

Enterobacteriaceae

The commensal Gram-negative Enterobacteriaceae comprise a minor proportion of the intestinal microbiota; however, overgrowth of Enterobacteriaceae occurs in most colitis models and IBD patients, and may be associated with the promotion of intestinal inflammation [8, 72, 81]. The mechanisms underlying the induction and promotion of colitis by Enterobacteriaceae are unclear; however, increased oxidative stress due to, for example, increased ROS and NOS generation caused by bacterial stimulation of TLRs, contributes to dysbiosis [82, 83]. We previously reported overgrowth of Enterobacteriaceae in CD11c-cre Tgfbr2 fl/fl mice. TGF-β signaling of DCs was essential for control of the luminal Enterobacteriaceae through the interactions with epithelial cells [50].