TIRAP, TRAM, and Toll-Like Receptors: The Untold Story.

Toll-like receptors (TLRs) are the most studied receptors among the pattern recognition receptors (PRRs). They act as microbial sensors, playing major roles in the regulation of the innate immune system. TLRs mediate their cellular functions through the activation of MyD88-dependent or MyD88-independent signaling pathways. Myd88, or myeloid differentiation primary response 88, is a cytosolic adaptor protein essential for the induction of proinflammatory cytokines by all TLRs except TLR3. While the crucial role of Myd88 is well described, the contribution of other adaptors in mediating TLR signaling and function has been underestimated. In this review, we highlight important results demonstrating that TIRAP and TRAM adaptors are also required for full signaling activity and responses induced by most TLRs.

Interleukin 15 in murine models of colitis.

Inflammatory bowel diseases (IBDs) are characterized by abnormal, non-antigen specific chronic inflammation of unknown etiology. Genome-wide association studies show that many IBD genetic susceptibility loci map to immune function genes and compelling evidence indicate that environmental factors play a critical role in IBD pathogenesis. Clinical and experimental evidence implicate the pro-inflammatory cytokine IL-15 in the pathogenesis of IBD. IL-15 and IL-15α expression is increased in the inflamed mucosa of IBD patients. IL-15 contributes to the maintenance of different cell subsets in the intestinal mucosa. However, very few studies have addressed the role of IL-15 in pre-clinical models of colitis. In this study, we use three well-characterized models of experimental colitis to determine the contribution of IL-15 to pathological intestinal inflammation.

Workflow for Quantitative Proteomic Analysis of Intestinal Organoids Using SILAC.

Stable isotope labeling by amino acids in cell culture (SILAC) is a strategic quantitative mass spectrometry method to analyze multiple protein samples in different conditions simultaneously. In recent years, 3D cell growth culture conditions have been developed to establish intestinal organoids from isolated crypts, which mimic the intestine's cell composition and organization. Organoids, isolated from normal or diseased tissues, can be used to compare cell distribution and differentiation, signaling pathways, and cell responses to pharmacological agents, therapeutic drugs, endogenous or exogenous metabolites, and environmental stresses, among others. Here, we describe the process of generating SILAC organoids from the mouse small intestine.

JAK-STAT Pathway Inhibition Partially Restores Intestinal Homeostasis in Hdac1- and Hdac2-Intestinal Epithelial Cell-Deficient Mice.

We have previously reported that histone deacetylase epigenetic regulator Hdac1 and Hdac2 deletion in intestinal epithelial cells (IEC) disrupts mucosal tissue architecture and barrier, causing chronic inflammation. In this study, proteome and transcriptome analysis revealed the importance of signaling pathways induced upon genetic IEC-Hdac1 and Hdac2 deletion. Indeed, Gene Ontology biological process analysis of enriched deficient IEC RNA and proteins identified common pathways, including lipid metabolic and oxidation-reduction process, cell adhesion, and antigen processing and presentation, related to immune responses, correlating with dysregulation of major histocompatibility complex (MHC) class II genes. Top upstream regulators included regulators associated with environmental sensing pathways to xenobiotics, microbial and diet-derived ligands, and endogenous metabolites. Proteome analysis revealed mTOR signaling IEC-specific defects. In addition to mTOR, the STAT and Notch pathways were dysregulated specifically in jejunal IEC. To determine the impact of pathway dysregulation on mutant jejunum alterations, we treated mutant mice with Tofacitinib, a JAK inhibitor. Treatment with the inhibitor partially corrected proliferation and tight junction defects, as well as niche stabilization by increasing Paneth cell numbers. Thus, IEC-specific histone deacetylases 1 (HDAC1) and 2 (HDAC2) support intestinal homeostasis by regulating survival and translation processes, as well as differentiation and metabolic pathways. HDAC1 and HDAC2 may play an important role in the regulation of IEC-specific inflammatory responses by controlling, directly or indirectly, the JAK/STAT pathway. IEC-specific JAK/STAT pathway deregulation may be, at least in part, responsible for intestinal homeostasis disruption in mutant mice.

Phosphatases in toll-like receptors signaling: the unfairly-forgotten.

Over the past 2 decades, pattern recognition receptors (PRRs) have been shown to be on the front line of many illnesses such as autoimmune, inflammatory, and neurodegenerative diseases as well as allergies and cancer. Among PRRs, toll-like receptors (TLRs) are the most studied family. Dissecting TLRs signaling turned out to be advantageous to elaborate efficient treatments to cure autoimmune and chronic inflammatory disorders. However, a broad understanding of TLR effectors is required to propose a better range of cures. In addition to kinases and E3 ubiquitin ligases, phosphatases emerge as important regulators of TLRs signaling mediated by NF-κB, type I interferons (IFN I) and Mitogen-Activated Protein Kinases signaling pathways. Here, we review recent knowledge on TLRs signaling modulation by different classes and subclasses of phosphatases. Thus, it becomes more and more evident that phosphatases could represent novel therapeutic targets to control pathogenic TLRs signaling. Video Abstract.

Distinct Effects of Milk-Derived and Fermented Dairy Protein on Gut Microbiota and Cardiometabolic Markers in Diet-Induced Obese Mice.

BACKGROUND: Recent meta-analyses suggest that the consumption of fermented dairy products reduces type 2 diabetes and cardiovascular disease (CVD) risk, although the underlying mechanisms remain unclear. OBJECTIVE: We evaluated whether dairy protein products modulated gut microbiota and cardiometabolic features in mouse models of diet-induced obesity and CVD. METHODS: Eight-week-old C57BL/6J wild-type (WT) and LDLr-/-ApoB100/100 (LRKO) male mice were fed for 12 and 24 wk, respectively, with a high-fat/high-sucrose diet [66% kcal lipids, 22% kcal carbohydrates (100% sucrose), 12% kcal proteins]. The protein sources of the 4 diets were 100% nondairy protein (NDP), or 50% of the NDP energy replaced by milk (MP), milk fermented by Lactobacillus helveticus (FMP), or Greek-style yogurt (YP) protein. Fecal 16S rRNA gene-based amplicon sequencing, intestinal gene expression, and glucose tolerance test were conducted. Hepatic inflammation and circulating adhesion molecules were measured by multiplex assays. RESULTS: Feeding WT mice for 12 wk led to a 74% increase in body weight, whereas after 24 wk the LRKO mice had a 101.5% increase compared with initial body weight. Compared with NDP and MP, the consumption of FMP and YP modulated the gut microbiota composition in a similar clustering pattern, upregulating the Streptococcus genus in both genotypes. In WT mice, feeding YP compared with NDP increased the expression of genes involved in jejunal (Reg3b, 7.3-fold, P = 0.049) and ileal (Ocln, 1.7-fold, P = 0.047; Il1-ß,1.7-fold, P = 0.038; Nos2, 3.8-fold, P = 0.018) immunity and integrity. In LRKO mice, feeding YP compared with MP improved insulin sensitivity by 65% (P = 0.039). In LRKO mice, feeding with FMP versus NDP attenuated hepatic inflammation (monocyte chemoattractant protein 1, 2.1-fold, P ˂ 0.0001; IL1-ß, 5.7-fold, P = 0.0003; INF-γ, 1.7-fold, P = 0.002) whereas both FMP [vascular adhesion molecule 1 (VCAM1), 1.3-fold, P = 0.0003] and YP (VCAM1, 1.04-fold, P = 0.013; intracellular adhesion molecule 1, 1.4-fold, P = 0.028) decreased circulating adhesion molecules. CONCLUSION: Both fermented dairy protein products reduce cardiometabolic risk factors in diet-induced obese mice, possibly by modulating the gut microbiota.

Potential interaction between timing of infant complementary feeding and breastfeeding duration in determination of early childhood gut microbiota composition and BMI.

BACKGROUND: Introducing complementary foods other than breastmilk or formula acutely changes the infant gut microbiota composition. However, it is unknown whether the timing of introduction to complementary foods (early vs. late) in infancy is associated with early childhood gut microbiota and BMI, and if these associations depend on breastfeeding duration. OBJECTIVE: Our primary objective was to investigate whether timing of infant complentary feeding with solid foods is associated with early childhood gut microbiota composition and BMI-z, and whether these associations differ by duration of breastfeeding. METHODS: We used data from a Canadian pre-birth cohort followed till age 5 years. We examined timing of introduction to solid foods with the gut microbiota, determined by 16S rRNA gene sequencing of stool collected at 5 years of age, and age-and-sex specific BMI-z. We conducted analyses before and after stratifying by breastfeeding duration, and adjusted for delivery mode, gestational age and birth weight. RESULTS: Of the 392 children in the analysis, 109 (27.8%) had early (≤4 months) solids. The association between early (vs later) solids and BMI-z at 5 years was modified by breastfeeding status at 4 months (P = .06). Among children breastfed >4 months, early (vs later) solids were associated with differential relative abundance of 6 bacterial taxa, including lower Roseburia, and 0.30 higher BMI-z (95% CI: 0.05, 0.55) at 5 years. In children breastfed <4 months, early solids were associated with differential relative abundance of 9 taxa, but not with child BMI-z. CONCLUSIONS: Early (vs. later) introduction to solid foods in infancy is associated with altered gut microbiota composition and BMI in early childhood, however these associations differ by duration of breastfeeding.

HDAC1 and HDAC2 independently regulate common and specific intrinsic responses in murine enteroids.

Both HDAC1 and HDAC2 are class I deacetylases acting as erasers of lysine-acetyl marks on histones and non-histone proteins. Several histone deacetylase inhibitors, either endogenous to the cell, such as the ketogenic ß-hydroxybutyrate metabolite, or exogenous, such as butyrate, a microbial-derived metabolite, regulate HDAC activity. Different combinations of intestinal epithelial cell (IEC)-specific Hdac1 and/or Hdac2 deletion differentially alter mucosal homeostasis in mice. Thus, HDAC1 and HDAC2 could act as sensors and transmitters of environmental signals to the mucosa. In this study, enteroid culture models deleted for Hdac1 or Hdac2 were established to determine IEC-specific function as assessed by global transcriptomic and proteomic approaches. Results show that Hdac1 or Hdac2 deficiency altered differentiation of Paneth and goblet secretory cells, which sustain physical and chemical protection barriers, and increased intermediate secretory cell precursor numbers. Furthermore, IEC Hdac1- and Hdac2-dependent common and specific biological processes were identified, including oxidation-reduction, inflammatory responses, and lipid-related metabolic processes, as well as canonical pathways and upstream regulators related to environment-dependent signaling through steroid receptor pathways, among others. These findings uncover unrecognized regulatory similarities and differences between Hdac1 and Hdac2 in IEC, and demonstrate how HDAC1 and HDAC2 may complement each other to regulate the intrinsic IEC phenotype.

Role of the p38 MAPK/C/EBPß Pathway in the Regulation of Phenotype and IL-10 and IL-12 Production by Tolerogenic Bone Marrow-Derived Dendritic Cells.

Dendritic cells (DCs) play a major role in innate and adaptive immunity and self-immune tolerance. Immunogenic versus tolerogenic DC functions are dictated by their levels of costimulatory molecules and their cytokine expression profile. The transcription factor C/EBPß regulates the expression of several inflammatory genes in many cell types including macrophages. However, little is known regarding the role of C/EBPß in tolerogenic versus immunogenic DCs functions. We have previously reported that bone marrow-derived DCs generated with GM-CSF (GM/DCs) acquire the signature of semi-mature tolerogenic IL-10-producing DCs as opposed to immunogenic DCs generated with GM-CSF and IL-4 (IL-4/DCs). Here, we show that tolerogenic GM/DCs exhibit higher levels of phosphorylation and enhanced DNA binding activity of C/EBPß and CREB than immunogenic IL-4/DCs. We also show that the p38 MAPK/CREB axis and GSK3 play an important role in regulating C/EBPß phosphorylation and DNA binding activity. Inhibition of p38 MAPK in GM/DCs resulted in a drastic decrease of C/EBPß and CREB DNA binding activities, a reduction of their IL-10 production and an increase of their IL-12p70 production, a characteristic of immunogenic IL-4/DCs. We also present evidence that GSK3 inhibition in GM/DCs reduced C/EBPß DNA binding activity and increased expression of costimulatory molecules in GM/DCs and their production of IL-10. Analysis of GM/DCs of C/EBPß-/- mice showed that C/EBPß was essential to maintain the semimature phenotype and the production of IL-10 as well as low CD4⁺ T cell proliferation. Our results highlight the importance of the p38MAPK-C/EBPß pathway in regulating phenotype and function of tolerogenic GM/DCs.

Phenotypic Analysis of Organoids by Proteomics.

The development of 3D cell cultures into self-organizing organ-like structures named organoids provides a model that better reflects in vivo organ physiology and their functional properties. Organoids have been established from several organs, such as the intestine, prostate, brain, liver, kidney and pancreas. With recent advances in high-throughput and -omics profiling technologies, it is now possible to study the mechanisms of cellular organisation at the systems level. It is therefore not surprising that these methods are now used to characterize organoids at the transcriptomic, proteomic, chromatin state and transcription factor DNA-binding levels. These approaches can therefore provide a wealth of information regarding both the mechanisms involved in different diseases, and those involved in cell responses to different conditions, in a more in vivo setting. The authors provide an overview of the potential applications of quantitative mass spectrometry with organoid culture, and how the use of large-scale proteome measurements is emerging in different organoid systems.

A SILAC-Based Method for Quantitative Proteomic Analysis of Intestinal Organoids.

Organoids have the potential to bridge 3D cell culture to tissue physiology by providing a model resembling in vivo organs. Long-term growing organoids were first isolated from intestinal crypt cells and recreated the renewing intestinal epithelial niche. Since then, this technical breakthrough was applied to many other organs, including prostate, liver, kidney and pancreas. We describe here how to apply a SILAC-based quantitative proteomic approach to measure protein expression changes in intestinal organoids under different experimental conditions. We generated SILAC organoid media that allow organoids to grow and differentiate normally, and confirmed the incorporation of isotopically labelled amino acids. Furthermore, we used a treatment reported to affect organoid differentiation to demonstrate the reproducibility of the quantification using this approach and to validate the identification of proteins that correlate with the inhibition of cellular growth and development. With the combined use of quantitative mass spectrometry, SILAC and organoid culture, we validated this approach and showed that large-scale proteome variations can be measured in an "organ-like" system.

Gata4 is critical to maintain gut barrier function and mucosal integrity following epithelial injury.

The intestinal epithelial barrier is critical to limit potential harmful consequences from exposure to deleterious luminal contents on the organism. Although this barrier is functionally important along the entire gut, specific regional regulatory mechanisms involved in the maintenance of this barrier are poorly defined. Herein, we identified Gata4 as a crucial regulator of barrier integrity in the mouse proximal intestinal epithelium. Conditional deletion of Gata4 in the intestine led to a drastic increase in claudin-2 expression that was associated with an important increase of gut barrier permeability without causing overt spontaneous inflammation. Administration of indomethacin, a non-steroidal anti-inflammatory drug (NSAID) that causes enteritis, led to rapid and restricted proximal small intestinal injuries in Gata4 mutant mice as opposed to control mice. Comparative analysis of gene transcript profiles from indomethacin-challenged control and Gata4 mutant mice identified defects in epithelial cell survival, inflammatory cell recruitment and tissue repair mechanisms. Altogether, these observations identify Gata4 as a novel crucial regulator of the intestinal epithelial barrier and as a critical epithelial transcription factor implicated in the maintenance of proximal intestinal mucosal integrity after injury.

Distinct Roles for Intestinal Epithelial Cell-Specific Hdac1 and Hdac2 in the Regulation of Murine Intestinal Homeostasis.

The intestinal epithelium responds to and transmits signals from the microbiota and the mucosal immune system to insure intestinal homeostasis. These interactions are in part conveyed by epigenetic modifications, which respond to environmental changes. Protein acetylation is an epigenetic signal regulated by histone deacetylases, including Hdac1 and Hdac2. We have previously shown that villin-Cre-inducible intestinal epithelial cell (IEC)-specific Hdac1 and Hdac2 deletions disturb intestinal homeostasis. To determine the role of Hdac1 and Hdac2 in the regulation of IEC function and the establishment of the dual knockout phenotype, we have generated villin-Cre murine models expressing one Hdac1 allele without Hdac2, or one Hdac2 allele without Hdac1. We have also investigated the effect of short-term deletion of both genes in naphtoflavone-inducible Ah-Cre and tamoxifen-inducible villin-Cre(ER) mice. Mice with one Hdac1 allele displayed normal tissue architecture, but increased sensitivity to DSS-induced colitis. In contrast, mice with one Hdac2 allele displayed intestinal architecture defects, increased proliferation, decreased goblet cell numbers as opposed to Paneth cells, increased immune cell infiltration associated with fibrosis, and increased sensitivity to DSS-induced colitis. In comparison to dual knockout mice, intermediary activation of Notch, mTOR, and Stat3 signaling pathways was observed. While villin-Cre(ER) Hdac1 and Hdac2 deletions led to an impaired epithelium and differentiation defects, Ah-Cre-mediated deletion resulted in blunted proliferation associated with the induction of a DNA damage response. Our results suggest that IEC determination and intestinal homeostasis are highly dependent on Hdac1 and Hdac2 activity levels, and that changes in the IEC acetylome may alter the mucosal environment.

Identification of GATA-4 as a novel transcriptional regulatory component of regenerating islet-derived family members.

Intestinal epithelial cells are exposed to luminal bacterial threat and require adequate defense mechanisms to ensure host protection and epithelium regeneration against possible deleterious damage. Differentiated intestinal epithelial cells produce antimicrobial and regenerative components that protect against such challenges. Few intestinal specific transcription factors have been identified to control the switching from repression to activation of this class of gene. Herein, we show that gene transcription of some regenerating islet-derived (REG) family members is dependent on the transcription factor GATA-4. Silencing of GATA-4 expression in cultured intestinal epithelial cells identified Reg3ß as a target gene using an unbiased approach of gene expression profiling. Co-transfection and RNA interference assays identified complex GATA-4-interactive transcriptional components required for the activation or repression of Reg3ß gene activity. Conditional deletion of Gata4 in the mouse intestinal epithelium supported its regulatory role for Reg1, Reg3α, Reg3ß and Reg3γ genes. Reg1 dramatic down-modulation of expression in Gata4 conditional null mice was associated with a significant decrease in intestinal epithelial cell migration. Altogether, these results identify a novel and complex role for GATA-4 in the regulation of REG family members gene expression.

Loss of histone deacetylase Hdac1 disrupts metabolic processes in intestinal epithelial cells.

By using acetyl-CoA as a substrate, acetyltransferases and histone deacetylases regulate protein acetylation by adding or removing an acetyl group on lysines. Nuclear-located Hdac1 is a regulator of intestinal homeostasis. We have previously shown that Hdac1 define specific intestinal epithelial cell basal and inflammatory-dependent gene expression patterns and control cell proliferation. We show here that Hdac1 depletion in cellulo leads to increased histone acetylation after metabolic stresses, and to metabolic disturbances resulting in impaired responses to oxidative stresses, AMPK kinase activation and mitochondrial biogenesis. Thus, nuclear Hdac1 may control intestinal epithelial cell metabolism by regulating the supply of acetyl groups.

Shuttling of information between the mucosal and luminal environment drives intestinal homeostasis.

The gastrointestinal tract is a passageway for dietary nutrients, microorganisms and xenobiotics. The gut is home to diverse bacterial communities forming the microbiota. While bacteria and their metabolites maintain gut homeostasis, the host uses innate and adaptive immune mechanisms to cope with the microbiota and luminal environment. In recent years, multiple bi-directional instructive mechanisms between microbiota, luminal content and mucosal immune systems have been uncovered. Indeed, epithelial and immune cell-derived mucosal signals shape microbiota composition, while microbiota and their by-products shape the mucosal immune system. Genetic and environmental perturbations alter gut mucosal responses which impact on microbial ecology structures. On the other hand, changes in microbiota alter intestinal mucosal responses. In this review, we discuss how intestinal epithelial Paneth and goblet cells interact with the microbiota, how environmental and genetic disorders are sensed by endoplasmic reticulum stress and autophagy responses, how specific bacteria, bacterial- and diet-derived products determine the function and activation of the mucosal immune system. We will also discuss the critical role of HDAC activity as a regulator of immune and epithelial cell homeostatic responses.

The acetylome regulators Hdac1 and Hdac2 differently modulate intestinal epithelial cell dependent homeostatic responses in experimental colitis.

Histone deacetylases (Hdac) remove acetyl groups from proteins, influencing global and specific gene expression. Hdacs control inflammation, as shown by Hdac inhibitor-dependent protection from dextran sulfate sodium (DSS)-induced murine colitis. Although tissue-specific Hdac knockouts show redundant and specific functions, little is known of their intestinal epithelial cell (IEC) role. We have shown previously that dual Hdac1/Hdac2 IEC-specific loss disrupts cell proliferation and determination, with decreased secretory cell numbers and altered barrier function. We thus investigated how compound Hdac1/Hdac2 or Hdac2 IEC-specific deficiency alters the inflammatory response. Floxed Hdac1 and Hdac2 and villin-Cre mice were interbred. Compound Hdac1/Hdac2 IEC-deficient mice showed chronic basal inflammation, with increased basal disease activity index (DAI) and deregulated Reg gene colonic expression. DSS-treated dual Hdac1/Hdac2 IEC-deficient mice displayed increased DAI, histological score, intestinal permeability, and inflammatory gene expression. In contrast to double knockouts, Hdac2 IEC-specific loss did not affect IEC determination and growth, nor result in chronic inflammation. However, Hdac2 disruption protected against DSS colitis, as shown by decreased DAI, intestinal permeability and caspase-3 cleavage. Hdac2 IEC-specific deficient mice displayed increased expression of IEC gene subsets, such as colonic antimicrobial Reg3b and Reg3g mRNAs, and decreased expression of immune cell function-related genes. Our data show that Hdac1 and Hdac2 are essential IEC homeostasis regulators. IEC-specific Hdac1 and Hdac2 may act as epigenetic sensors and transmitters of environmental cues and regulate IEC-mediated mucosal homeostatic and inflammatory responses. Different levels of IEC Hdac activity may lead to positive or negative outcomes on intestinal homeostasis during inflammation.

The histone deacetylase Hdac1 regulates inflammatory signalling in intestinal epithelial cells.

BACKGROUND: It has recently been found that both nuclear epithelial-expressed histone deacetylases Hdac1 and Hdac2 are important to insure intestinal homeostasis and control the mucosal inflammatory response in vivo. In addition, HDAC inhibitors modulate epithelial cell inflammatory responses in cancer cells. However, little is known of the specific role of different HDAC, notably Hdac1, in the regulation of inflammatory gene expression in intestinal epithelial cells (IEC). METHODS: We investigated the role of Hdac1 in non-transformed IEC-6 rat cells infected with lentiviral vectors expressing specific Hdac1 shRNAs, to suppress Hdac1 expression. Proliferation was assessed by cell counting. Deacetylase activity was measured with a colorimetric HDAC assay. Cells were treated with IL-1ß and/or the JQ1 bromodomain acetyl-binding inhibitor. Nuclear protein levels of Hdac1, Hdac2, phosphorylated or unphosphorylated NF-κB p65 or C/EBPß, and NF-κB p50 and actin were determined by Western blot. Chemokine and acute phase protein expression was assessed by semi-quantitative RT-PCR analysis. Secreted cytokine and chemokine levels were assessed with a protein array. Chromatin immunoprecipitation experiments were done to assess RNA polymerase II recruitment. RESULTS: Reduced Hdac1 protein levels led to Hdac2 protein increases and decreased cell proliferation. Hdac1 depletion prolonged nuclear IL-1ß-induced phosphorylation of NF-κB p65 protein on Ser536 as opposed to total p65, and of C/EBPß on Ser105. In addition, semi-quantitative RT-PCR analysis revealed three patterns of expression caused by Hdac1 depletion, namely increased basal and IL-1ß-stimulated levels (Hp, Kng1), increased IL-1ß-stimulated levels (Cxcl2) and decreased basal levels with normal IL-1ß induction levels (Ccl2, Ccl5, Cxcl1, C3). Secreted cytokine and chemokine measurements confirmed that Hdac1 played roles both as an IL-1ß signalling repressor and activator. Hdac1 depletion did not alter the JQ1 dependent inhibition of basal and IL-1ß-induced inflammatory gene expression. Hdac1 depletion led to decreased basal levels of RNA polymerase II enrichment on the Ccl2 promoter, as opposed to the Gapdh promoter, correlating with decreased Ccl2 basal mRNA expression. CONCLUSIONS: Hdac1 is a major nuclear HDAC controlling IL-1ß-dependent inflammatory response in IEC, notably by regulating gene-specific transcriptional responses. Hdac1 may be important in restricting basal and inflammatory-induced gene levels to defined ranges of expression.

HDAC1 and HDAC2 restrain the intestinal inflammatory response by regulating intestinal epithelial cell differentiation.

Acetylation and deacetylation of histones and other proteins depends on histone acetyltransferases and histone deacetylases (HDACs) activities, leading to either positive or negative gene expression. HDAC inhibitors have uncovered a role for HDACs in proliferation, apoptosis and inflammation. However, little is known of the roles of specific HDACs in intestinal epithelial cells (IEC). We investigated the consequences of ablating both HDAC1 and HDAC2 in murine IECs. Floxed Hdac1 and Hdac2 homozygous mice were crossed with villin-Cre mice. Mice deficient in both IEC HDAC1 and HDAC2 weighed less and survived more than a year. Colon and small intestinal sections were stained with hematoxylin and eosin, or with Alcian blue and Periodic Acid Schiff for goblet cell identification. Tissue sections from mice injected with BrdU for 2 h, 14 h and 48 h were stained with anti-BrdU. To determine intestinal permeability, 4-kDa FITC-labeled dextran was given by gavage for 3 h. Microarray analysis was performed on total colon RNAs. Inflammatory and IEC-specific gene expression was assessed by Western blot or semi-quantitative RT-PCR and qPCR with respectively total colon protein and total colon RNAs. HDAC1 and HDAC2-deficient mice displayed: 1) increased migration and proliferation, with elevated cyclin D1 expression and phosphorylated S6 ribosomal protein, a downstream mTOR target; 2) tissue architecture defects with cell differentiation alterations, correlating with reduction of secretory Paneth and goblet cells in jejunum and goblet cells in colon, increased expression of enterocytic markers such as sucrase-isomaltase in the colon, increased expression of cleaved Notch1 and augmented intestinal permeability; 3) loss of tissue homeostasis, as evidenced by modifications of claudin 3 expression, caspase-3 cleavage and Stat3 phosphorylation; 4) chronic inflammation, as determined by inflammatory molecular expression signatures and altered inflammatory gene expression. Thus, epithelial HDAC1 and HDAC2 restrain the intestinal inflammatory response, by regulating intestinal epithelial cell proliferation and differentiation.

The histone H3K27 methylation mark regulates intestinal epithelial cell density-dependent proliferation and the inflammatory response.

Polycomb-group proteins form multimeric protein complexes involved in transcriptional silencing. The Polycomb Repressive complex 2 (PRC2) contains the Suppressor of Zeste-12 protein (Suz12) and the histone methyltransferase Enhancer of Zeste protein-2 (Ezh2). This complex, catalyzing the di- and tri-methylation of histone H3 lysine 27, is essential for embryonic development and stem cell renewal. However, the role of Polycomb-group protein complexes in the control of the intestinal epithelial cell (IEC) phenotype is not known. We show that Suz12 and Ezh2 were differentially expressed along the intestinal crypt-villus axis. ShRNA-mediated Suz12 depletion in the IEC-6 rat crypt-derived cell line decreased Ezh2 expression and H3K27 di-trimethylation. Suz12-depleted cells achieved higher cell densities after confluence, with increased cyclin D2 and cyclin D3 protein levels, and increased STAT3 activation in post-confluent cells. Suz12 depletion specifically increased mostly developmental, cell adhesion and immune response gene expression, including neuronal and inflammatory genes. Suz12 depletion directly and indirectly de-regulated the IL-1ß-dependent inflammatory response, as demonstrated by decreased MAPK p38 activation as opposed to JNK activation, and altered basal and stimulated expression of inflammatory genes, including transcription factors such as C/EBPß. Of note, this positive effect on cell proliferation and inflammatory gene expression was revealed in the absence of the cyclin-dependent kinase inhibitor p16, a main target negatively regulated by PRC2. These results demonstrate that the PRC2 complex, in addition to keeping in check non-IEC differentiation pathways, insures the proper IEC response to cell density as well as to external growth and inflammatory signals, by controlling specific signaling pathways.