Microbiota-sensitive epigenetic signature predicts inflammation in Crohn's disease.

Altered response to the intestinal microbiota strongly associates with inflammatory bowel disease (IBD); however, how commensal microbial cues are integrated by the host during the pathogenesis of IBD is not understood. Epigenetics represents a potential mechanism that could enable intestinal microbes to modulate transcriptional output during the development of IBD. Here, we reveal a histone methylation signature of intestinal epithelial cells isolated from the terminal ilea of newly diagnosed pediatric IBD patients. Genes characterized by significant alterations in histone H3-lysine 4 trimethylation (H3K4me3) showed differential enrichment in pathways involving immunoregulation, cell survival and signaling, and metabolism. Interestingly, a large subset of these genes was epigenetically regulated by microbiota in mice and several microbiota-sensitive epigenetic targets demonstrated altered expression in IBD patients. Remarkably though, a substantial proportion of these genes exhibited H3K4me3 levels that correlated with the severity of intestinal inflammation in IBD, despite lacking significant differential expression. Collectively, these data uncover a previously unrecognized epigenetic profile of IBD that can be primed by commensal microbes and indicate sensitive targets in the epithelium that may underlie how microbiota predispose to subsequent intestinal inflammation and disease.

Brushed nasal epithelial cells are a surrogate for bronchial epithelial CFTR studies.

Recent advances in the management of cystic fibrosis (CF) target underlying defects in the CF transmembrane conductance regulator (CFTR) protein, but efficacy analyses remain limited to specific genotype-based subgroups. Patient-derived model systems may therefore aid in expanding access to these drugs. Brushed human nasal epithelial cells (HNEs) are an attractive tissue source, but it remains unclear how faithfully they recapitulate human bronchial epithelial cell (HBE) CFTR activity. We examined this gap using paired, brushed HNE/HBE samples from pediatric CF subjects with a wide variety of CFTR mutations cultured at the air-liquid interface. Growth and structural characteristics for the two cell types were similar, including differentiation into mature respiratory epithelia. In electrophysiologic analysis, no correlation was identified between nasal and bronchial cultures in baseline resistance or epithelial sodium channel (ENaC) activity. Conversely, robust correlation was demonstrated between nasal and bronchial cultures in both stimulated and inhibited CFTR activity. There was close correlation in modulator-induced change in CFTR activity, and CFTR activity in both cell types correlated with in vivo sweat chloride measurements. These data confirm that brushed HNE cell cultures recapitulate the functional CFTR characteristics of HBEs with fidelity and are therefore an appropriate noninvasive HBE surrogate for individualized CFTR analysis.

Generation of Human Nasal Epithelial Cell Spheroids for Individualized Cystic Fibrosis Transmembrane Conductance Regulator Study.

While the introduction of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) modulator drugs has revolutionized care in Cystic Fibrosis (CF), the genotype-directed therapy model currently in use has several limitations. First, rare or understudied mutation groups are excluded from definitive clinical trials. Moreover, as additional modulator drugs enter the market, it will become difficult to optimize the modulator choices for an individual subject. Both of these issues are addressed with the use of patient-derived, individualized preclinical model systems of CFTR function and modulation. Human nasal epithelial cells (HNEs) are an easily accessible source of respiratory tissue for such a model. Herein, we describe the generation of a three-dimensional spheroid model of CFTR function and modulation using primary HNEs. HNEs are isolated from subjects in a minimally invasive fashion, expanded in conditional reprogramming conditions, and seeded into the spheroid culture. Within 2 weeks of seeding, spheroid cultures generate HNE spheroids that can be stimulated with 3',5'-cyclic adenosine monophosphate (cAMP)-generating agonists to activate CFTR function. Spheroid swelling is then quantified as a proxy of CFTR activity. HNE spheroids capitalize on the minimally invasive, yet respiratory origin of nasal cells to generate an accessible, personalized model relevant to an epithelium reflecting disease morbidity and mortality. Compared to the air-liquid interface HNE cultures, spheroids are relatively quick to mature, which reduces the overall contamination rate. In its current form, the model is limited by low throughput, though this is offset by the relative ease of tissue acquisition. HNE spheroids can be used to reliably quantify and characterize CFTR activity at the individual level. An ongoing study to tie this quantification to in vivo drug response will determine if HNE spheroids are a true preclinical predictor of patient response to CFTR modulation.

Disruption of Epithelial HDAC3 in Intestine Prevents Diet-Induced Obesity in Mice.

BACKGROUND & AIMS: Intestinal microbiota modulate metabolism and associate closely with epithelial cells in the intestine. In intestinal epithelial cells (IECs), histone deacetylase 3 (HDAC3) integrates microbiota-derived signals to control intestinal homeostasis. We investigated whether HDAC3 in IECs regulates metabolism and the development of obesity in mice. METHODS: Adult C57BL/6 (control) mice and mice with constitutive or inducible IEC-specific disruption of Hdac3 (HDAC3ΔIEC mice) were placed on a standard chow or high-fat diet (HFD, 60% kcal from fat). We measured body composition, weight, glucose tolerance, and energy expenditure. IECs were isolated from small intestine and gene expression, and lipid levels were analyzed. HDAC3 levels were determined in 43 pediatric patient ileal biopsy samples and compared with body weight. RESULTS: Control mice fed an HFD gained weight, became obese, and had reduced glucose tolerance with increased serum insulin, whereas HFD-fed HDAC3ΔIEC mice did not develop obesity. Serum levels of triglycerides were reduced in HDAC3ΔIEC mice, and these mice had less liver fat and smaller adipocytes, compared with HFD-fed control mice. HDAC3ΔIEC mice had similar food intake and activity as control mice, but higher energy expenditure because of increased catabolism. IECs from HDAC3ΔIEC mice had altered expression levels of genes that regulate metabolism in response to the microbiota (such as Chka, Mttp, Apoa1, and Pck1) and accumulated triglycerides compared with IECs from control mice. The microbiota-derived short-chain fatty acid butyrate was decreased in obese mice. Butyrate significantly reduced the activity of HDAC3 and increased Pck1 expression in only control IECs. Administration of butyrate to control mice with diet-induced obesity, but not HDAC3ΔIEC mice, led to significant weight loss. Disruption of HDAC3 in IECs of mice after they became obese led to weight loss and improved metabolic profile. Levels of HDAC3 in intestinal biopsy samples correlated with patient weight. CONCLUSIONS: We found that epithelial HDAC3 promotes development of diet-induced obesity in studies of mice and that butyrate reduces activity of HDAC3 in IECs to prevent diet-induced obesity. This pathway might be manipulated to prevent or reduce obesity-associated disease.

Chronic ß2AR stimulation limits CFTR activation in human airway epithelia.

Traditional pulmonary therapies for cystic fibrosis (CF) target the downstream effects of CF transmembrane conductance regulator (CFTR) dysfunction (the cause of CF). Use of one such therapy, ß-adrenergic bronchodilators (such as albuterol), is nearly universal for airway clearance. Conversely, novel modulator therapies restore function to select mutant CFTR proteins, offering a disease-modifying treatment. Recent trials of modulators targeting F508del-CFTR, the most common CFTR mutation, suggest that chronic ß-agonist use may undermine clinical modulator benefits. We therefore sought to understand the impact of chronic or excess ß-agonist exposure on CFTR activation in human airway epithelium. The present studies demonstrate a greater than 60% reduction in both wild-type and modulator-corrected F508del-CFTR activation following chronic exposure to short- and long-acting ß-agonists. This reduction was due to reduced cellular generation of cAMP downstream of the ß-2 adrenergic receptor-G protein complex. Our results point towards a posttranscriptional reduction in adenylyl cyclase function as the mechanism of impaired CFTR activation produced by prolonged ß-agonist exposure. ß-Agonist-induced CFTR dysfunction was sufficient to abrogate VX809/VX770 modulation of F508del-CFTR in vitro. Understanding the clinical relevance of our observations is critical for CF patients using these drugs, and for investigators to inform future CFTR modulator drug trials.

Epithelial Histone Deacetylase 3 Instructs Intestinal Immunity by Coordinating Local Lymphocyte Activation.

Mucosal tissues are constantly in direct contact with diverse beneficial and pathogenic microbes, highlighting the need for orchestrating complex microbial signals to sustain effective host defense. Here, we show an essential role for intestinal epithelial cell expression of histone deacetylase 3 (HDAC3) in responding to pathogenic microbes and activating protective innate immunity. Mice lacking HDAC3 in intestinal epithelial cells were more susceptible to Citrobacter rodentium when under tonic stimulation by the commensal microbiota. This impaired host defense reflected significantly decreased IFNγ production by intraepithelial CD8+ T cells early during infection. Further, HDAC3 was necessary for infection-induced epithelial expression of the IFNγ-inducing factor IL-18, and administration of IL-18 restored IFNγ activity to resident CD8+ T cells and reduced infection. Thus, HDAC3 mediates communication between intestinal epithelial cells and resident lymphocytes, revealing that epithelial priming by an epigenetic modifier may direct mucosal regulation of host defense against pathogenic microbes.