Nucleotide-binding oligomerization domain-2 inhibits toll-like receptor-4 signaling in the intestinal epithelium

Abstract

Background & aims: Factors that regulate enterocyte apoptosis in necrotizing enterocolitis (NEC) remain incompletely understood, although Toll-like receptor-4 (TLR4) signaling in enterocytes plays a major role. Nucleotide-binding oligomerization domain-2 (NOD2) is an immune receptor that regulates other branches of the immune system, although its effects on TLR4 in enterocytes and its role in NEC remain unknown. We now hypothesize that activation of NOD2 in the newborn intestine inhibits TLR4, and that failure of NOD2 signaling leads to NEC through increased TLR4-mediated enterocyte apoptosis.

Methods: The effects of NOD2 on enterocyte TLR4 signaling and intestinal injury and repair were assessed in enterocytes lacking TLR4 or NOD2, in mice with intestinal-specific wild-type or dominant-negative TLR4 or NOD2, and in mice with NEC. A protein array was performed on NOD2-activated enterocytes to identify novel effector molecules involved.

Results: TLR4 activation caused apoptosis in newborn but not adult small intestine or colon, and its intestinal expression was influenced by NOD2. NOD2 activation inhibited TLR4 in enterocytes, but not macrophages, and reversed the effects of TLR4 on intestinal mucosal injury and repair. Protection from TLR4-induced enterocyte apoptosis by NOD2 required a novel pathway linking NOD2 with the apoptosis mediator second mitochondria-derived activator of caspase/direct inhibitor of apoptosis-binding protein with low PI (SMAC-DIABLO), both in vitro and in vivo. Strikingly, activation of NOD2 reduced SMAC-DIABLO expression, attenuated the extent of enterocyte apoptosis, and reduced the severity of NEC.

Conclusions: These findings reveal a novel inhibitory interaction between TLR4 and NOD2 signaling in enterocytes leading to the regulation of enterocyte apoptosis and suggest a therapeutic role for NOD2 in the protection of intestinal diseases such as NEC.

Figure 1. TLR4 activation leads to enterocyte apoptosis in the newborn small intestine and is negatively influenced by NOD2 expression

A. Immuno-confocal micrographs showing cleaved caspase 3 (green, arrows indicate apoptotic enterocytes), actin (red) and nuclei (blue) in the small bowel or colon of newborn or adults treated with saline or LPS (1mg/kg, 16hrs) as indicated. B: Quantification of apoptosis under conditions in A. *p<0.05 vs. saline. C. Quantitative RT-PCR showing expression of i: TLR4 (solid bars) and NOD2 (open bars) in wild-type or NOD2-deficient IEC-6 cells *p<0.05 vs. open bars; ii: TLR4 in IEC-6 cells transfected with adenoviruses expressing either GFP (grey) or wild-type-NOD2 (black), *p<0.05vs GFP. D i–iii: Micrographs of the terminal ileum in newborn mice that were either breast fed (control, i) or induced to develop NEC and administered adenoviruses expressing GFP (ii) or dominant-negative NOD2 (iii); iv: RT-PCR showing expression of iNOS pertaining to i–iii. *p<0.05 vs. ctrl, **p<0.05 vs. GFP. E. i: RT-PCR showing expression of TLR4 pertaining to panel D; ii: quantitative RT-PCR for TLR4 and NOD2 in mice with and without NEC,*p<0.05 vs. control. All mice experiments performed over 3 times with> 8 mice/group.

Figure 2. NOD2 activation limits TLR4 signaling in enterocytes both in vivo and in vitro

A. IEC-6 cells or wild-type mice were treated with LPS±MDP as indicated. i: extent of NFkB activation IEC-6 cells as measured by ratio NFkB p65-subunit in nucleus vs. cytoplasm, ii: IL-6 release from IEC-6 cells by ELISA; iii: serum IL-6; iv: RT-PCR showing fold change in IL-6 in small intestinal mucosa. *p<0.05 vs. LPS alone in each case B: Confocal micrographs of IEC-6 cells corresponding to panel Ai immunostained for NFkB (green), actin(red), nucleus(blue), size bar=10μm. C i–iv: Confocal micrographs of IEC-6 cells treated as indicated and immunostained for TLR4 (green) and nuclei(blue); v: quantification of the surface expression of TLR4 using ImageJ software representative of 3 separate experiments. *p<0.05 vs. LPS alone.

Figure 3. Activation of NOD2 reverses the inhibitory effects of TLR4 on enterocyte migration in vitro and in vivo

A: Micrographs demonstrating IEC-6 cells migrating into a scraped wound after treatment with media or LPS±MDP, at the beginning (T=0) and the end (T=0) of migration. The position of the scrape is indicated by a dashed line. B. Micrographs of terminal ileum immunostained with BrdU 24h after injection of BrdU into newborn mice that were also injected with saline (i), LPS (ii), LPS+MDP (iii) or MDP (iv). Arrows show leading BrdU-positive enterocytes. C–D: Quantification of migration in vitro (C) and in vivo (D). *p<0.005 vs. LPS alone in each case;bar=100μm.

Figure 4. Activation of NOD2 reverses the effects of TLR4 activation on enterocyte apoptosis in vitro and in vivo and in NEC

A–B: Quantification of apoptosis in either (A) wild-type or (B) TLR4^−/− IEC-6 cells treated with LPS±MDP as indicated. C–D: Apoptosis in the terminal ileum of newborn mice injected with C: saline or LPS±MDP, or D: induced to develop NEC and administered adenoviruses expressing GFP, dominant-negative (dn)-NOD2 or wild-type (wt)-NOD2. *p<0.05 vs. solid bar, 3 separate experiments. E: Confocal micrographs of terminal ileum of newborn mice corresponding to C stained with cleaved-caspase-3 (red), nuclei (blue), actin (green). Arrows=apoptotic cells; bar=100μm. F: Confocal micrographs of terminal ileum corresponding to D; i, tissue stained with actin (green), nuclei (blue); ii, tissue stained with GFP (green), cleaved-caspase-3 (red), nuclei (blue); iii–iv, tissue stained with cleaved-caspase-3(red), actin(green), nuclei(blue), size bar=100μm;arrows=apoptotic cells; representative of 3 separate experiments, over 8 mice/group.

Figure 5. MDP reverses TLR4-induced enterocyte apoptosis through activation of SMAC-diablo

A. i: Expression of SMAC-diablo and beta-actin by SDS-PAGE in IEC-6 cells LPS±MDP after being either untransfected or transfected with siRNA to SMAC-diablo or control-siRNA. ii: Quantification of SMAC-diablo:f-actin in 3 separate blots similar to those in i,*p<0.05 vs control; **p<0.05 vs. LPS. B: Apoptosis in IEC-6 cells transfected with control-siRNA or SMAC-diablo-siRNA and treated with LPS±MDP. *p<0.005 vs. control, both transfection conditions. C: quantification of intestinal mucosal expression of SMAC-diablo in wild-type and NOD2 KO pertaining to D and E,*p<0.05 vs control; **p<0.05 vs. LPS. D–E: Confocal micrographs of terminal ileum of wild-type(D) or NOD2^−/− mice(E) treated with saline (i), LPS (ii), LPS±MDP(iii) or MDP (iv) and stained for SMAC-diablo(green) and nuclei(blue). Size bar=100μm. representative of 3 separate experiments, over 8 mice/group.

Figure 6. NOD2 activation regulates SMAC-diablo expression in enterocytes in NEC

A: SDS-PAGE showing SMAC-diablo expression in terminal ileum from 3 separate mice with and without NEC, blots were re-probed for actin; quantification is shown; *p<0.05, n=3 similar blots. B: i: SMAC-diablo quantitative RT-PCR in terminal ileum mucosa in mice without (control) and with NEC that were injected with saline or MDP. *p<0.05 vs. solid bar; ii–iv: quantification of SMAC-diablo expression in enterocytes corresponding to panels C–E; *p<0.05 **p<0.005 black vs. white bar; *** black vs. grey bar. C–E: Confocal micrographs of terminal ileum of newborn mice either breast fed or induced to develop NEC and stained for SMAC-diablo (red) or nuclei (blue). As indicated, mice with NEC were either treated with MDP or saline (C), administered by gavage adenoviruses expressing either GFP, wild-type or dominant negative NOD2 (D), or administered by gavage adenoviruses expressing either GFP, wild-type or dominant negative TLR4 (E). Size bar=100μm. representative of 3 separate experiments, over 8 mice/group.

Figure 7. NOD2 activation with MDP reduces intestinal injury and severity of NEC

A. H&E (i–iv) and confocal micrographs (v–viii, red=cleaved caspase-3, blue=nuclei, green=actin) from terminal ilea of newborn mice with and without NEC and injected with either saline or MDP; arrows=apoptotic cells; size bar=100μm. B–D: Severity of NEC as determined by B: RT-PCR for expression of IL-6, iNOS and TLR4 within the intestinal mucosa, C: Histological grading and D: apoptosis; *p<0.05 vs. NEC-saline. n=5 experiments, >20 mice/group.