Title: MicroRNA‐146a‐mediated downregulation of IRAK1 protects mouse and human small intestine against ischemia/reperfusion injury
Abstract: Research Article9 November 2012Open Access MicroRNA-146a-mediated downregulation of IRAK1 protects mouse and human small intestine against ischemia/reperfusion injury Cécilia Chassin Corresponding Author Cécilia Chassin [email protected] Institute of Medical Microbiology and Hospital Epidemiology, Hannover Medical School, Hannover, Germany Present address: ATIP-Avenir Group, INSERM U699, Faculté de Médecine Xavier Bichat, Université Paris 7, Paris, France Search for more papers by this author Cordelia Hempel Cordelia Hempel Institute of Medical Microbiology and Hospital Epidemiology, Hannover Medical School, Hannover, Germany Search for more papers by this author Silvia Stockinger Silvia Stockinger Institute of Medical Microbiology and Hospital Epidemiology, Hannover Medical School, Hannover, Germany Present address: University of Veterinary Medicine Vienna, Institute of Animal Breeding and Genetics, Vienna, Austria Search for more papers by this author Aline Dupont Aline Dupont Institute of Medical Microbiology and Hospital Epidemiology, Hannover Medical School, Hannover, Germany Search for more papers by this author Joachim F. Kübler Joachim F. Kübler Department of Pediatric Surgery, Hannover Medical School, Hannover, Germany Search for more papers by this author Jochen Wedemeyer Jochen Wedemeyer Department of Gastroenterology and Hepatology, Hannover Medical School, Hannover, Germany Present address: Klinikum Region Hannover Robert Koch, Gehrden, Germany Search for more papers by this author Alain Vandewalle Alain Vandewalle INSERM U773, Centre de Recherche Biomédicale Bichat-Beaujon (CRB3), Université Paris 7-Denis Diderot, Paris, France Search for more papers by this author Mathias W. Hornef Corresponding Author Mathias W. Hornef [email protected] Institute of Medical Microbiology and Hospital Epidemiology, Hannover Medical School, Hannover, Germany Search for more papers by this author Cécilia Chassin Corresponding Author Cécilia Chassin [email protected] Institute of Medical Microbiology and Hospital Epidemiology, Hannover Medical School, Hannover, Germany Present address: ATIP-Avenir Group, INSERM U699, Faculté de Médecine Xavier Bichat, Université Paris 7, Paris, France Search for more papers by this author Cordelia Hempel Cordelia Hempel Institute of Medical Microbiology and Hospital Epidemiology, Hannover Medical School, Hannover, Germany Search for more papers by this author Silvia Stockinger Silvia Stockinger Institute of Medical Microbiology and Hospital Epidemiology, Hannover Medical School, Hannover, Germany Present address: University of Veterinary Medicine Vienna, Institute of Animal Breeding and Genetics, Vienna, Austria Search for more papers by this author Aline Dupont Aline Dupont Institute of Medical Microbiology and Hospital Epidemiology, Hannover Medical School, Hannover, Germany Search for more papers by this author Joachim F. Kübler Joachim F. Kübler Department of Pediatric Surgery, Hannover Medical School, Hannover, Germany Search for more papers by this author Jochen Wedemeyer Jochen Wedemeyer Department of Gastroenterology and Hepatology, Hannover Medical School, Hannover, Germany Present address: Klinikum Region Hannover Robert Koch, Gehrden, Germany Search for more papers by this author Alain Vandewalle Alain Vandewalle INSERM U773, Centre de Recherche Biomédicale Bichat-Beaujon (CRB3), Université Paris 7-Denis Diderot, Paris, France Search for more papers by this author Mathias W. Hornef Corresponding Author Mathias W. Hornef [email protected] Institute of Medical Microbiology and Hospital Epidemiology, Hannover Medical School, Hannover, Germany Search for more papers by this author Author Information Cécilia Chassin *,1,5, Cordelia Hempel1, Silvia Stockinger1,6, Aline Dupont1, Joachim F. Kübler2, Jochen Wedemeyer3,7, Alain Vandewalle4 and Mathias W. Hornef *,1 1Institute of Medical Microbiology and Hospital Epidemiology, Hannover Medical School, Hannover, Germany 2Department of Pediatric Surgery, Hannover Medical School, Hannover, Germany 3Department of Gastroenterology and Hepatology, Hannover Medical School, Hannover, Germany 4INSERM U773, Centre de Recherche Biomédicale Bichat-Beaujon (CRB3), Université Paris 7-Denis Diderot, Paris, France 5Present address: ATIP-Avenir Group, INSERM U699, Faculté de Médecine Xavier Bichat, Université Paris 7, Paris, France 6Present address: University of Veterinary Medicine Vienna, Institute of Animal Breeding and Genetics, Vienna, Austria 7Present address: Klinikum Region Hannover Robert Koch, Gehrden, Germany *Cécilia Chassin, Tel: +33 1 57 27 75 48; Fax: +33 1 57 27 76 61Mathias W. Hornef, Tel: +49 511 532 4540; Fax: +49 511 532 4366 EMBO Mol Med (2012)4:1308-1319https://doi.org/10.1002/emmm.201201298 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions Figures & Info Abstract Intestinal ischemia/reperfusion (I/R) injury causes inflammation and tissue damage and is associated with high morbidity and mortality. Uncontrolled activation of the innate immune system through toll-like receptors (Tlr) plays a key role in I/R-mediated tissue damage but the underlying mechanisms have not been fully resolved. Here, we identify post-transcriptional upregulation of the essential Tlr signalling molecule interleukin 1 receptor-associated kinase (Irak) 1 as the causative mechanism for post-ischemic immune hyper-responsiveness of intestinal epithelial cells. Increased Irak1 protein levels enhanced epithelial ligand responsiveness, chemokine secretion, apoptosis and mucosal barrier disruption in an experimental intestinal I/R model using wild-type, Irak1−/− and Tlr4−/− mice and ischemic human intestinal tissue. Irak1 accumulation under hypoxic conditions was associated with reduced K48 ubiquitination and enhanced Senp1-mediated deSUMOylation of Irak1. Importantly, administration of microRNA (miR)-146a or induction of miR-146a by the phytochemical diindolylmethane controlled Irak1 upregulation and prevented immune hyper-responsiveness in mouse and human tissue. These findings indicate that Irak1 accumulation triggers I/R-induced epithelial immune hyper-responsiveness and suggest that the induction of miR-146a offers a promising strategy to prevent I/R tissue injury. The paper explained PROBLEM: Ischemia/reperfusion (I/R) injury is observed in a variety of clinical conditions such as vascular occlusion, haemorrhagic shock, trauma or following solid organ transplantation and associated with high morbidity and mortality. I/R in the intestine has additionally been implicated in the pathogenesis of necrotizing enterocolitis in preterm delivered neonates. Innate immune hyper-responsiveness mediated by enhanced Tlr signalling has been identified in the pathogenesis of I/R injury but the underlying molecular mechanisms have remained ill-defined and novel prophylactic and therapeutic strategies are needed. RESULTS: Using a mouse model of intestinal I/R injury and human intestinal mucosal biopsies, we observed enhanced protein expression of the essential Tlr signalling molecule Irak1 in ischemic epithelial cells associated with a striking increase in the responsiveness to innate immune stimulation. Enhanced Irak1 expression was associated with increased ligand responsiveness, chemokine secretion, epithelial apoptosis, mucosal barrier disruption and tissue destruction in an I/R model whereas Irak1-deficient mice were protected from ischemia-mediated tissue damage. Irak1 protein accumulation under hypoxic conditions was caused by changes in the ubiquitination pattern and Ubc9-mediated transient SUMOylation of Irak1. Importantly, administration of miR-146a or the miR-146a-inducing agent DIM controlled epithelial Irak1 protein levels in mouse and human mucosal tissue by translational repression and protected from I/R injury. IMPACT: We identify Irak1 protein as a major regulator of Tlr-mediated innate immune responsiveness in IECs and show that administration or pharmacological induction of miR-146a represents a new strategy to control innate immune hyper-responsiveness and reduce tissue damage after transient hypoxia or I/R. INTRODUCTION Ischemia/reperfusion (I/R) injury is observed in a variety of diseases, such as vascular occlusion, haemorrhagic shock or trauma. It is also an unavoidable event during organ transplantation. Reperfusion of post-ischemic tissue induces activation of the innate immune system, leading to an inflammatory response that significantly contributes to the hypoxic cell damage (Chen et al, 2003; Mkaddem et al, 2010; Watson et al, 2008). An important role of Toll-like receptor (Tlr) activation in the pathogenesis of I/R injury has been established in post-ischemic hepatic, cardiac, renal tissue damage and haemorrhagic shock (Bamboat et al, 2010; Ellett et al, 2009; Moses et al, 2009; Suzuki et al, 2008; Zanotti et al, 2009). Particularly, a critical role of the Tlr4 signalling pathway has been identified (Moses et al, 2009; Pope et al, 2010; Watson et al, 2008). However, the underlying molecular mechanism of the exaggerated innate immune response following transient ischemia has remained poorly understood. Innate immune signalling at the intestinal epithelium actively contributes to antimicrobial host defence and the maintenance of mucosal homeostasis (Cario et al, 2007; Nenci et al, 2007; Rakoff-Nahoum et al, 2004; Voss et al, 2006; Weiss et al, 2004; Zaph et al, 2007). Given the permanent exposure to the enteric microbiota, efficient control of epithelial innate immune activation is, however, required to prevent inappropriate cell stimulation, tissue inflammation and organ dysfunction (Chassin et al, 2010; Turer et al, 2008; Vereecke et al, 2010; Xiao et al, 2007). We recently demonstrated that downregulation of the essential Tlr signalling molecule interleukin 1 receptor-associated kinase 1 (Irak1) in intestinal epithelial cells (IEC) contributes significantly to protect the immature intestinal epithelium from bacteria-induced tissue damage during the neonatal period (Lotz et al, 2006). Signalling-induced proteasomal degradation and translational repression as a result of enhanced microRNA (miR)-146a expression act in concert to reduce Irak1 and protect the intestinal mucosa against inappropriate stimulation during postnatal colonization (Chassin et al, 2010). The observed tight regulation of Irak1 protein levels prompted us to investigate whether enhanced epithelial Irak1 protein might also occur and contribute to innate immune-mediated tissue damage in relevant clinical conditions. Given that innate immune hyper-responsiveness is found under clinical conditions of I/R, we hypothesized that the accumulation of Irak1 under ischemic conditions might contribute to the enhanced innate immune response after intestinal I/R. Our results identify a direct functional link between oxygen restriction, Irak1 protein accumulation and innate immune-mediated cell damage in mouse and human tissue in vitro and in vivo and characterize the underlying molecular mechanisms. Additionally, we provide in vivo evidence that the administration of miR-146a or the pharmacological induction of miR-146a prevent post-ischemic Irak1 upregulation and reduce innate immune hyper-responsiveness and I/R injury. RESULTS Hypoxia increases epithelial IRAK1 protein and innate immune responsiveness The response of intestinal epithelial m-ICcl2 cells to lipopolysaccharide (LPS) critically depends on the innate immune receptor Tlr4 and the signal molecule Irak1 (Chassin et al, 2010; Hornef et al, 2002). Consequently, siRNA-mediated downregulation of Irak1 or Tlr4 almost completely abolished LPS-induced NF-κB reporter activity and the secretion of the proinflammatory chemokine Cxcl2 (Mip-2) (Supporting Information Fig S1A). Conversely, increased expression of Irak1 by transient overexpression significantly enhanced the cellular response (Supporting Information Fig S1B). Enhanced Irak1 expression under certain clinical conditions such as oxygen deprivation might therefore result in innate immune hyper-responsiveness and contribute to immune-mediated tissue damage. A significant, time-dependent increase in Irak1 protein levels was observed during the course of oxygen deprivation (Fig 1A and Supporting Information Fig S1D). Induction of hypoxia was confirmed by enhanced expression of the hypoxia-inducible factor (Hif)-1α analysed by immunoblotting and immunostaining (Fig 1A and Supporting Information Fig S1C). Importantly, hypoxia induced a time-dependent increase in chemokine secretion in the presence of 1 ng/ml LPS, but not in the presence of the Tlr-independent stimulus phorbol myristate acetate (PMA, Fig 1B). Epithelial cells kept for 2 h under hypoxic conditions and subsequently stimulated under normoxic conditions exhibited a 10- to 50-fold increase in ligand sensitivity (Fig 1C and Supporting Information Fig S1E). Of note, oxygen deprivation did not lead to detectable cellular apoptosis during this time (Supporting Information Fig S1F) and the level of the Irak1 protein and innate immune hyper-responsiveness were fully reversible when oxygen deprivation ceased (Fig 1D). Figure 1. Hypoxia increases epithelial Irak1 protein and causes innate immune hyper-responsiveness. *Student's t-test p < 0.05, **p < 0.01, ***p < 0.001 compared to controls (B,C) or hypoxia LPS-treated (D). Values are means ± SEM from 3 to 5 independent experiments, n = 4/group. A.. Time kinetic of Irak1 and Hif-1α protein expression in mICcl2 cells after incubation in hypoxic chambers. B.. mICcl2 cells were incubated in hypoxic chambers for indicated time, and subsequently stimulated under normoxic conditions with 1 ng/ml LPS or 1 µM PMA for 6 h, and the secretion of Cxcl2 was determined. For each data point, n = 4. normoxia: LPS 0.536 ± 0.048 and PMA 0.239 ± 0.091 versus control 0.063 ± 0.008, p = 2 × 10−6 and p = 0.09, respectively; 0.5 h: LPS 0.728 ± 0.117 versus control 0.073 ± 0.033, p = 3 × 10−3; 1 h: LPS 1.156 ± 0.325 versus control 0.094 ± 0.011, p = 6 × 10−4; 1.5 h: LPS 1.336 ± 0.144 versus control 0.065 ± 0.015, p = 2 × 10−6; 2 h: LPS 1.580 ± 0.291 versus control 0.037 ± 0.034, p = 4 × 10−5; 4 h: LPS 1.821 ± 0.425 versus control 0.117 ± 0.082, p = 2 × 10−4). C.. mICcl2 cells were incubated in hypoxic chambers for 2 h and subsequently stimulated with various concentrations of LPS under normoxic conditions for 6 h, and the secretion of Cxcl2 was determined. For each data point, n = 4.0.05: hypoxia 0.188 ± 0.040 versus control 0.032 ± 0.020, p = 4 × 10−4; 0.1: hypoxia 0.316 ± 0.098 versus control 0.059 ± 0.025, p = 0.002; 0.5: hypoxia 0.931 ± 0.080 versus control 0.215 ± 0.034, p = 3 × 10−6; 1: hypoxia 1.455 ± 0.201 versus control 0.525 ± 0.075, p = 10−4. D.. mICcl2 cells were left untreated or incubated in hypoxic chambers for 2 h followed for one fraction of cells by overnight incubation in fresh medium in normoxic conditions (recovery). Subsequently, the levels of Irak1 and Cxcl2 secretion were determined after 6 h stimulation with 1 ng/ml LPS. For each data point, n = 4. Hypoxia/recovery + LPS (0.690 ± 0.173) versus Hypoxia + LPS (1.456 ± 0.317), p = 0.005. Download figure Download PowerPoint Hypoxia alters Irak1 ubiquitination and induces Senp1-mediated deSUMOylation Oxygen deprivation did not affect Irak1 mRNA suggesting a solely post-transcriptional mechanism of Irak1 protein upregulation in hypoxic IECs similar to the situation in endotoxin tolerance (Supporting Information Fig S2A; Chassin et al, 2010). Irak1 is ubiquitinated in order to induce both signal transduction and proteasomal degradation. Whereas K48 ubiquitin modification is a marker for non-signalling-prone Irak1 degradation, K63 ubiquitination facilitates signal transduction (Huang et al, 2005; Janssens and Beyaert, 2003; Newton et al, 2008). Interestingly, immunoprecipitation studies revealed that an increased fraction of K63 ubiquitin-modified Irak1 and a decreased fraction of K48 ubiquitin-modified Irak1 was detected in hypoxic m-ICcl2 cells compared to normoxic control cells, whereas both K48- and K63-ubiquitin-conjugated Irak1 was detected after LPS stimulation (Fig 2A and Supporting Information Fig S2B). These findings suggested that the reduction in the amount of K48 ubiquitin-conjugated Irak1 may contribute to the increased level of Irak1 protein in hypoxic cells. The enhanced level of K63-ubiquitin-conjugated Irak1 in turn might reflect a shift from silent Irak1 degradation to signal transduction-prone Irak1, causing a reduction of the signalling threshold upon receptor-ligand engagement. Figure 2. Hypoxia alters the ubiquitination pattern and induces Irak1 SUMOylation. *Student's t-test p < 0.05, ***p < 0.001. Values are means ± SEM, from four independent experiments, n = 4/group. A.. mICcl2 cells were transfected with an ubiquitin-encoding plasmid and incubated in hypoxic chambers or treated with 10 ng/ml LPS for 2 h in the presence of the proteasome inhibitor MG132 (100 nM). Irak1 was immunoprecipitated, and K48 or K63 ubiquitination was individually detected by immunoblot. B,C.. mICcl2 cells were transfected with a plasmid-encoding haemagglutinin (HA)-tagged SUMO, left untreated (co.) or subjected to hypoxia in hypoxic chambers (Hyp.) for 2 h in the presence of 10 mM NEM and 100 nM MG132. Irak1 protein was detected by immunoblot (B), or (C) IRAK1 was immunoprecipitated, and SUMO-HA and Irak1 were detected by immunoblot. D.. mICcl2 cells were left untreated (control), treated with the hypoxia mimetic DMOG (1 mM), or incubated in hypoxic chambers for 2 h and Irak1 levels were determined by immunoblot. E.. mICcl2 cells were left untreated (co.) or incubated in hypoxic chambers (Hyp.) for 2 h, Senp1 was immunoprecipitated, and IRAK1 and SENP1 were detected by immunoblot. F,G.. mICcl2 cells were transfected with siRNA directed against Senp1 or Ubc9, and incubated in hypoxic chambers for 2 h. (F) The level of Irak1 was determined by immunoblot, and (G) the amount of Cxcl2 secretion after stimulation with 1 ng/ml LPS for 6 h was quantified by ELISA. Hypoxia/Senp1 + LPS (0.331 ± 0.092) and Hypoxia/Ubc9 + LPS (2.564 ± 0.751) versus Hypoxia + LPS (1.481 ± 0.122), p = 5 × 10−6 and p = 0.02, respectively. Download figure Download PowerPoint Further analysis of m-ICcl2 cells overexpressing HA-tagged SUMO and incubated in the presence of the deubiquitination inhibitor N-ethylmaleimide (NEM) revealed a form of Irak1 with increased molecular size under hypoxic but not normoxic conditions (Fig 2B). This high-molecular weight form of Irak1 was also detected using an anti-SUMO antibody (Fig 2C) suggesting that Irak1 SUMOylation occurs under hypoxic conditions. This resembles the situation of the Hif-1α molecule that, under normoxic conditions, is proteasomally degraded after hydroxylation (Shao et al, 2004; Ulrich, 2007). Under hypoxic conditions, Ubc9-mediated SUMOylation and ubiquitination of Hif-1α facilitate proteasomal degradation. DeSUMOylation by the SUMO-specific protease (Senp)1, however, prevents the degradation and leads to enhanced protein levels in hypoxic cells (Cheng et al, 2007). Similarly, inhibition of hydroxylation with the cell permeable prolyl-4-hydroxylase inhibitor DMOG led to Irak1 accumulation under normoxic conditions (Fig 2D; Shao et al, 2004; Ulrich, 2007). Also, a direct molecular interaction was observed between Irak1 and Senp1 in hypoxic but not normoxic m-ICcl2 cells (Fig 2E). siRNA-mediated knockdown of Senp1 mRNA prevented both the hypoxia-induced accumulation of Irak1 protein (Fig 2F) and innate immune hyper-responsiveness (Fig 2G). In contrast, Irak1 accumulation and innate immune hyper-responsiveness were still observed under hypoxic conditions after siRNA-mediated silencing of the SUMO-conjugating enzyme Ubc9. Importantly, accumulation of Irak1 under hypoxic conditions was independent of Hif-1α or Hif-2α expression suggesting the presence of two similar but functionally independent processes (Supporting Information Fig S2C). I/R injury is associated with enhanced epithelial Irak1 and requires Tlr4- and Irak1-dependent signalling We then tested whether enhanced Irak1 expression also occurs in the intestinal epithelium of mice subjected to I/R in vivo. IECs isolated from an intestinal loop after transient interruption of the mesenteric blood flow for 30 min followed by restoration of the vascular flow for 60 min exhibited markedly increased Irak1 protein levels but no significant change in Irak1 mRNA (Fig 3A and Supporting Information Fig S3A). Non-ischemic intestinal segments from the same animal were used as controls. Importantly, the increase in Irak1 was also associated with enhanced innate immune responsiveness. Significantly enhanced Cxcl2 mRNA was found in high Irak1-expressing IECs isolated from post-ischemic wild-type intestinal segments exposed to LPS in vitro (Fig 3B and Supporting Information Fig S3B) or after intraluminal injection of LPS in vivo (Fig 3C). The level of Cxcl2 mRNA was significantly enhanced in IECs isolated from post-ischemic intestinal segments of wild-type but not Tlr4−/− or Irak1−/− mice (Fig 3D). Also, the severity of tissue damage, mucosal translocation of intraluminally administered FITC dextran and the number of TUNEL-positive apoptotic IECs after I/R was markedly reduced in the absence of Tlr4 or Irak1 (Fig 3E–G and Supporting Information Fig S3C). I/R-induced epithelial cell death was associated with phosphorylation of c-jun N-terminal kinase (Jnk) and bcl2-associated x protein (Bax) and nuclear translocation of apoptosis-inducing factor (Aif) in isolated IECs from wild-type but not Irak1-deficient mice (Fig 3H and I). No detectable staining for active caspase 3 was detected (Supporting Information Fig S3D). These results are consistent with the previously established model of mitochondrion-dependent apoptosis through Jnk-mediated phosphorylation of Bax with subsequent release and nuclear translocation of Aif in the presence of enhanced levels of epithelial Irak1 protein (Kim et al, 2006; Takada et al, 2008). Figure 3. Ischemia-induced Irak1 accumulation leads to innate immune hyper-responsiveness and Tlr4- and Irak1-dependent tissue injury. ***Student's t-test p < 0.001, **p < 0.01, compared to LPS-treated (B–C), WT (D), or WT I/R (F). Values are means ± SEM from 4 to 5 independent experiments. Magnification ×100. A.. Wild-type mice (n = 12) were subjected to ischemia for 30 min (I), and IECs were isolated. Irak1 protein was determined by immunoblot. B.. An ischemic segment and an unaffected control segment of the intestine were removed and incubated ex vivo for 2 h in the presence of 100 ng/ml LPS at 37°C. IECs were isolated and Cxcl2 mRNA was quantified by real-time PCR. I/R + LPS (71.66 ± 1.56) versus I/R (44.20 ± 2.12), p = 0.004. n = 4 for each data point. C.. After 30 min ischemia, 200 µl of a solution of 100 ng/ml LPS was injected intraluminally during a 1 h reperfusion period. IECs were then isolated, and Cxcl2 mRNA was quantified by real-time RT-PCR. I/R + LPS (90.87 ± 20.70) versus I/R (15.32 ± 3.67), p = 3 × 10−5. n = 4 for each data point. D–F.. Wild-type (WT), Tlr4−/−, and Irak1−/− mice (n = 10 for each group) were subjected to I/R. (D) IECs were isolated, and Cxcl2 mRNA was quantified by real-time RT-PCR. Tlr4−/− (1.72 ± 1.91) and Irak1−/− (2.67 ± 2.70) versus WT (21.23 ± 5.08), p = 5 × 10−6and p = 10−5, respectively. (E) H&E staining was performed using formalin-fixed tissue sections from I/R-treated or untreated (Co) intestinal segments. (F) Permeability of the intestinal barrier was measured by injecting 200 µl of a 25 mg/ml FITC-dextran solution into the intestinal lumen during ischemia (I/R) or into untreated control sections (Co). Subsequently, blood samples were collected and the fluorescence intensity was measured. Tlr4−/− I/R (44.03 ± 7.06) and Irak1−/− I/R (41.05 ± 13.64) versus WT I/R (119.95 ± 7.00), p = 5 × 10−6. n = 5 for each data point. G.. TUNEL staining was performed using formalin-fixed tissue sections from I/R-treated or untreated (Co) intestinal segments. H.. Expression of phospho-jnk (P-Jnk), Jnk, phosphor-Bax and Bax in IECs were assessed by Western blotting. I.. The translocation of Aif was measured in nuclear extract (NE) and cytosolic extract (CE) of IECs (TATA binding protein Tbp and Gapdh expression were used as nuclear or cytosolic loading control respectively). Download figure Download PowerPoint Induction of microRNA-146a lowers Irak1 and protects mice against intestinal I/R injury We and others have previously demonstrated that Irak1 in vivo is regulated on the post-transcriptional level by miR-146a (Boldin et al, 2011; Carthew and Sontheimer, 2009; Chassin et al, 2010). miR-146a in turn was recently shown to be induced by the naturally occurring substance 3,3′-diinodolylmethane (DIM; Li et al, 2010). In accordance, a significant increase in miR-146a was found in m-ICcl2 cells after the administration of DIM under both normoxic and hypoxic conditions (Fig 4A). DIM didn't have any effect on the expression of additional miRs known to be involved in the regulation of inflammation or I/R injury such as Let-7a, miR-21, miR-155 and miR-29a in our model (Supporting Information Fig S4B–E). Importantly, DIM also reversed the effect of hypoxia on epithelial Irak1 protein expression (Fig 4B). Furthermore, the increased secretion of Cxcl2 by hypoxic m-ICcl2 cells decreased after transfection of miR-146a or incubation with DIM but not in the presence of control microRNA (miRco) (Fig 4C). Also in vivo, the enhancement of Irak1 protein levels after I/R was abolished following the intraluminal injection of DIM but not of the solvent control (mock) (Fig 4D). Intramural injection of DIM or miR-146a also significantly reduced the Cxcl2 mRNA levels after I/R (Fig 4E) and diminished the I/R-mediated hyper-responsiveness of the intestinal epithelium to LPS (Fig 4F). Epithelial uptake of microRNA after luminal exposure was confirmed using fluorescently conjugated miR control (Mimic co; Supporting Information Fig S4A). Finally, lipid oxidation as a consequence of oxidative injury, enhanced translocation of FITC dextran through the intestinal mucosa, intestinal tissue damage, Jnk and Bax phosphorylation, Aif nuclear expression and epithelial apoptosis were all significantly lower in post-ischemic intestinal segments after the intraluminal administration of miR-146a or DIM than in mock-treated, post-ischemic tissue segments (Fig 4G–K). Figure 4. DIM-mediated induction of miR-146a diminishes the hypoxia-induced hyper-sensitivity towards LPS and protects against I/R injury in mice. ***Student's t-test p < 0.001, **p < 0.01 compared to control (A), LPS-treated (C), I/R-treated (E, G, H), or I/R and LPS-treated (F). Values are means ± SEM from 4 to 5 separated experiments. Magnification ×100. A,B.. mICcl2 cells were incubated in hypoxic chambers for 2 h in the absence or presence of 25 µM diindolylmethane (DIM), and the levels of miR-146a (for each point, n = 4; Control: DIM 3.65 ± 0.58 versus Co 1.03 ± 0.64, p = 9 × 10−4; Hyp: DIM 3.99 ± 0.34 versus Co 1.21 ± 0.37, p = 3 × 10−5) (A) and Irak1 (B) were determined by real-time PCR and immunoblot, respectively. C.. mICcl2 cells were transfected with microRNA (miR)-146a mimic, an anti-miR-146a (anti-miR) or miR control (miRco), and/or treated with DIM and subjected to hypoxia for 2h. Cxcl2 mRNA was quantified by real-time RT-PCR after stimulating the cells with 1 ng/ml LPS for 6h (for each point, n = 4; miR-146a/LPS 0.440 ± 0.184 and DIM/LPS 0.475 ± 0.194 versus LPS 1.1516 ± 0.363, p = 10−3 and p = 0.002, respectively). D.. 200 µl of 25 µM DIM or the solvent control DMSO (Mock) was intraluminally injected into the small intestine of WT mice (n = 10 for each group). Subsequently, the intestinal tissue was subjected to ischemia for 30 min followed by reperfusion for 1 h. IECs were isolated and Irak1 protein was determined by immunoblotting. E.. 100 nM miR-146a mimic, 100 nM miR control (miRco), 25 µM DIM or the solvent control (Mock) was intraluminally injected into the small intestine of WT mice (n = 10 for each group). Subsequently, the intestinal tissue was subjected to ischemia for 30 min, followed by reperfusion for 1 h. IECs were isolated and Cxcl2 mRNA was quantified by real-time PCR. miR-146a/IR 3.17 ± 1.17 and DIM/IR 3.59 ± 0.85 versus IR 21.24 ± 2.20, p = 6 × 10−6 and p = 5 × 10−6, respectively. F.. Epithelial Cxcl2 mRNA in response to intraluminal injection of 100 ng/ml LPS during the reperfusion period after pretreatment with miRco, miR-146a, DIM or Mock was determined by quantitative PCR. miR-146a/IR/LPS 34.90 ± 5.98 and DIM/IR/LPS 42.19 ± 8.88 versus IR/LPS 80.51 ± 6.09, p = 4 × 10−5 and p = 4 × 10−4, respectively. For each data point, n = 5. G.. Lipid peroxidation, used as an indirect index of the oxidative injury induced by the reactive oxygen species, was determined by measuring the malonedialdehyde (MDA) concentration with the thiobarbiturate reaction. miR-146a/IR 0.2633 ± 0.0198 and DIM/IR 0.2467 ± 0.715 versus IR 0.5304 ± 0.0446, p = 3 × 10−5 and p = 5 × 10−4, respectively. For each data point, n = 5. H.. The permeability of the intestinal barrier was measured by intraluminal injection of 200 µl 25 mg/ml FITC-dextran during the ischemia period after pretreatment with miRco, miR-146a, DIM or Mock. Blood samples were collected after reperfusion, and the fluorescence intensity was determined. miR-146a/IR 35.66 ± 12.99 and DIM/IR 45.16 ± 7.99 versus IR 122.61 ± 3.51, p = 9 × 10−4 and p = 2 × 10