Properly regulated epithelial cell survival has emerged as a critical mechanism for maintaining mucosal barrier function. A role for STAT5b in IEC survival has not previously been proposed.27
Our global gene analysis identified activation of proapoptotic networks in the colon of STAT5b-deficient mice which led to a fivefold increase in the frequency of apoptotic CECs. TNFα is felt to be a primary mediator of CEC death and barrier dysfunction in IBD, as TNFα neutralisation increases CEC survival and reduces permeability. We found that, following STAT5b knockdown, the cellular Bax/Bcl-2 ratio and TNFα-dependent caspase-3 activation were substantially increased in the T84 colon epithelial cell line. It is therefore likely that STAT5b deficiency increases the susceptibility of CECs to TNFα-induced cell death, which in turn directly causes an increase in permeability.
We employed complementary techniques to test for differences in barrier function between WT and STAT5b-deficient mice, and identified a selective increase in colonic transcellular permeability. Our previous publication showed that there are differences in the structure of the TJP complex between the ileum and colon, which include differential distribution and expression of TJP components including ZO-1, 2 and 3 as well as occludin phosphorylation and claudin.15,28
This may contribute to the differential effect of STAT5b deficiency upon permeability among different intestinal segments. In addition, the quantity and composition of the enteric flora vary between the ileum and colon, which may in turn interact with the observed alterations in TLR2/4 abundance to affect barrier function.
While NF-κB exerts primarily a proinflammatory effect in the gut, recent studies utilising targeted deletion of components of the NF-κB activation pathway in IECs have highlighted unexpected roles in epithelial homeostasis and response to injury.29,30
TNBS administration led to a significant increase in CEC NF-κB in STAT5b-deficient mice compared with WT controls. The effects upon IEC survival are dependent upon the precise context in which NF-κB is activated. On the one hand, NF-κB blockade inhibits apoptosis during the resolution of inflammation in vivo.31
On the other hand, NEMO (NF-κB essential modulator) deficiency leading to reduced NF-κB activation sensitises IECs to TNFα-induced apoptosis.29
We found that STAT5b-deficient CECs have higher phosphoserine p65 and lower PPARγ nuclear abundance. As PPARγ has been shown to promote nuclear export of p65 in IECs, this probably contributes to ongoing NF-κB activation. Constitutive activation of NF-κB may then induce local release of proinflammatory factors including IL1α which contribute to ongoing CEC apoptosis, in the context of a primary imbalance in the cellular Bcl-2/Bax ratio. Further studies utilising transgenic mice deficient in both STAT5b and epithelial p65 will be required to determine the net effect of NF-κB activation in this context. However, it is important to note that our data suggest that STAT5b exerts a non-redundant effect upon CEC survival, which cannot be replaced by NF-κB activation.
A prior report has indicated that STAT5b may also directly inhibit NF-κB signalling, by squelching limiting co-activators.8
We found that, following STAT5b knockdown, TNFα activation of NF-κB was enhanced in the T84 colon epithelial cell line. GH treatment prevented TNFα activation of NF-κB; this effect was prevented by STAT5b knockdown. GH has been shown to reduce the severity of colitis due to both TNBS or dextan sodium sulfate (DSS) administration and IL10 deficiency.24,32
To determine whether STAT5b might also inhibit NF-κB activity in vivo, we induced colitis in WT and STAT5b-deficient mice and treated them with GH to activate STAT5b. We found that GH treatment reduced NF-κB activation and histological disease severity in WT mice. The beneficial effect of GH was abrogated in STAT5b-deficient mice. These data suggest that STAT5b is a critical negative regulator of NF-κB activation in CECs, and therefore contributes to maintenance of mucosal tolerance by limiting epithelial inflammatory responses.
Bacterial components including lipopolysaccharide (LPS, via TLR4) and lipoteichoic acids (LTAs, via TLR2) can induce inflammatory or homeostatic responses in the gut.11
Under basal conditions, TLR2 is expressed at a higher level in the proximal colon, while TLR4 is expressed at a higher level in the distal colon.9
Although the expression of TLR2 is low in CECs, it can be upregulated by bacteria during acute infections.9,33
Upregulation of TLR2 in human IBD and in experimental colitis has been linked to increased NF-κB activation leading to chronic mucosal inflammation, while recent animal studies have also suggested a role for basal intestinal homeostasis.34
For example, TLR2-mediated tight junction assembly determines susceptibility to intestinal injury and inflammation.34
More importantly, suppression of TLR2 signalling in mice overexpressing NOD2 led to a protection from TNBS-induced colitis.12
TLR4 also mediates both proinflammatory and homeostatic responses to acute gut injury.10,11
TLR4 is required for the intestinal epithelial response to injury following DSS administration; furthermore, it was shown to limit bacterial translocation during colitis.35,36
However, TLR signalling via MyD88 is also essential for development of colitis in IL10-deficient mice,10
We found that TLR2 abundance was increased in STAT5b-deficient mice, in conjunction with upregulation of the TLR signalling molecule Myd88. Under these conditions, TLR4 expression was reduced. Therefore, the reciprocal increase in TLR2 and reduction in TLR4 mRNA expression with STAT5b deficiency may contribute directly to the constitutive NF-κB activation and susceptibility to TNBS colitis which we have observed. This will need to be formally tested in TLR2-and TLR4-deficient mice.
Prior studies have demonstrated that deletion of STAT5b alone does not significantly affect systemic adaptive immunity. However, combined deletion of STAT5a and STAT5b reduces survival of regulatory T cells (Treg) and leads to autoimmunity including spontaneous colitis.37,38
In agreement with this, we did not detect significant differences in the frequency of splenic or MLN effector (CD44+
) or regulatory (CD4+CD25+
) T cell populations in STAT5b-deficient mice under basal conditions (Supplementary fig 5
). However, Cohen et al
have reported that accumulation and regulatory function of CD4+
T cells are reduced in human STAT5b deficiency,39
and that STAT5b propagates an IL2-mediated signal which is required for the in vivo accumulation of functional Treg.40
Ongoing studies will determine whether alterations in Treg accumulation and/or function contribute to the increase in mucosal inflammation observed in the STAT5b-deficient mice with TNBS colitis. It is reported that natural killer (NK) cell-mediated proliferation was affected by STAT5b deficiency41
; however, we did not test the significant alteration of NK cells in STAT5b-deficient mice.
In summary, we have found that STAT5b is a critical molecular regulator of mucosal barrier function in the gut, via effects upon CEC survival, TLR expression and NF-κB activation. Studies using STAT5b bone marrow chimeras or epithelial-specific STAT5b-deficient mice will be required to test formally the effect of STAT5b deficiency in epithelial versus bone marrow-derived cells. It will be of interest to determine whether genetic polymorphisms affecting STAT5b abundance or activation contribute to susceptibility to IBD. Targeted activation of STAT5b with agents including GH may provide a useful therapeutic approach for restoring mucosal barrier function.