Mucosal immunity and epithelial permeability are two interconnected etiological factors in the pathogenesis of IBD. Increased intestinal permeability could allow luminal antigens to penetrate the intestinal tissue that activates and/or perpetuates the immune response. Abnormal immune reaction could overproduce inflammatory cytokines that regulate TJs and increase the epithelial permeability. In this study, we have identified a novel function of FoxO4 in regulation of mucosal immunity and epithelial permeability. Our results show that the mucosal immune reaction and intestinal epithelial permeability are both upregulated in Foxo4-null mice under physiological conditions, which could provide a mechanism for the exacerbated inflammatory response to TNBS-treatment. How does FoxO4 regulate mucosal immunity and epithelial permeability?
FoxO4 may regulate intestinal mucosal immunity by repressing inflammatory cytokine expression. Cytokines CCL5, CXCL9, TNFα, and IFNγ are upregulated in colons of Foxo4
-null mice under physiological conditions. More importantly, part of the upregulated CCL5 is attributed to epithelial cells. CCL5 is an important chemoattractant cytokine for regulating movement of T cells in the intestine (18
). Upregulation of epithelial derived CCL5 could result in an increased recruitment of T cells in the intestine and play an early role in the onset of IBD. Consistent with this hypothesis, we observed increased number of CD4+ IELs in the Foxo4
-null mice. We have also observed that Foxo4
-deficient macrophages produce higher level of IL-6 in response to LPS than WT controls, suggesting that there might be an additional mechanism for the elevated colitis susceptibility in Foxo4
-null animals that Foxo4
-deficient colonic lymphocytes are more activated.
FoxO4 may regulate intestinal permeability through TJ proteins including occludins, claudins, and ZO proteins. Expression levels and distribution of these TJ proteins influence the epithelial permeability (5
). We have observed downregulation of ZO-1 and claudin-1 in both Foxo4
-deficient epithelial cells in vivo
-knocked down epithelial cells in vitro
, which could provide a structural basis for the increased intestinal epithelial permeability in Foxo4
The effects of FoxO4 on intestinal mucosal immunity and epithelial permeability are likely through NF-κB (16
). Both loss and gain-of function of NF-κB have been shown to increase epithelial permeability through different mechanisms (15
). Nenci et al. showed that NF-κB-inactivated epithelial cells have increased permeability due to an increase in TNFα -mediated epithelial cell death (22
). Upregulation of epithelial permeability in Foxo4
-null mice is likely due to the downregulation of TJ protein levels as a result of increased NF-κB activity in the epithelial cells.
There are several mechanisms by which FoxO4 may inhibit NF-κB. FoxO4 may inhibit NF-κB by upregulating IκB expression indirectly through Foxj1 or compete with IκB for IκB kinase binding (9
). Alternatively, FoxO4 could inactivate NF-κB through direct physical interaction, which could prevent NF-κB from either entering the nucleus or binding to the DNA. No down-regulation of IκB protein level was observed in Foxo4
-deficient epithelial cells compared to WT controls (data not shown), suggesting that inhibition of NF-κB by FoxO4 is likely IκB-independent. We found that FoxO4 indeed interacts with NF-κB both in vivo
and in vitro
. Binding of NF-κB to FoxO4 interferes with its nuclear translocation and its DNA binding activity. Together, our data favor the possibility that FoxO4 inhibits NF-κB through direct physical interaction.
In our TNBS-treated animals, we observed a transient down regulation of mucosal FoxO4 mRNA. Similar decreases of epithelial FoxO4 expression were also observed in UC patients. Although our mouse model of chemically induced colitis may not accurately reflect the immunological and histopathological aspects of IBD in humans, it is tempting to speculate nonetheless that FoxO4 may play an important role in the pathogenesis of human IBD.
In addition, FoxO4 may regulate the colonic mucosal immunity in a NF-κB-independent manner. FoxO4 is known to induce expression of antioxidant enzymes such as superoxide dismutase and catalase in response to oxidative stress signals to scavenge the reactive oxygen species (ROS) in vitro
). It will be worth testing whether such an anti-oxidative stress function of FoxO4 plays a role in the elevated susceptibility of Foxo4-KO
mice to colitis in vivo
FoxO4 activity may be regulated at transcriptional level by inflammatory signals. We have identified several conserved cis
- elements for transcription factors including interferon regulatory factor (IRF) and NF-κB in the promoter region of Foxo4
gene using ECR browser (www.dcode.org
). We have found that NF-κB can activate Foxo4 transcription in a luciferase reporter assay whereas IRF1 inhibits it (WZ and ZPL, unpublished results). Although it remains to be determined, NF-κB could activate transcription of Foxo4 in vivo, providing a mechanism of negative feed-back loop for regulating NF-κB activity. In support of this hypothesis, we found that transcription of Foxo4 was returned to normal after transient downregulation in response to TNBS- treatment. It is noted that many of the proteins that inhibit NF-κB activity including IκB can also be transcriptionally activated by NF-κB (24
In summary, our data support the following mechanistic model. Under physiological conditions, the transcriptional activity of NF-κB in the gut is tightly regulated by FoxO4 so that NF-κB is minimally activated to keep a low-grade chronic inflammation. Inflammatory signals activate NF-κB and in the mean time inactivate FoxO4 possibly through IRFs, releasing its inhibition of NF-κB to allow maximum activation to combat inflammation. Once the inflammation is resolved, FoxO4 expression returns to its normal level to keep NF-κB minimally activated. Although we favor the hypothesis that FoxO4 targets both intestinal epithelial permeability and mucosal cytokine expression in intestinal epithelial cells and lymphocytes, given the complexity of the cell types involved in mucosal immunity, studies with tissue-targeted deletion of Foxo4 will be needed to delineate the mechanisms of Foxo4 function in various cells and tissues.