Steatotic liver responds with increased hepatocellular injury when exposed to an ischemic-reperfusion insult. Increasing evidence supports the role of immune cells as key mediators of this injury in a normal (lean) state, but data about their role in a steatotic liver are practically nonexistent. The objective of the current study was to delineate the contribution of specific phenotypes of T cells and adhesion molecules in exacerbated cell death in steatotic liver injury. RNA sequencing was performed on isolated steatotic primary hepatocytes, and T-cell markers were assessed in hepatic lymphocytes after ischemia reperfusion injury (IRI) in high-fat diet (HFD)–fed mice. Cluster of differentiation 8 knockout (CD8−/−) and CD4−/−mice along with CD8 and L-selectin antibody–treated mice were fed an HFD, and hepatocellular injury was assessed by histology, propidium iodide injection, and alanine aminotransferase after IRI. RNA sequencing demonstrated a strikingly differential gene profile in steatotic hepatocytes versus lean hepatocytes. After injury, the HFD liver showed increased necrosis, infiltrating CD8+cells, alanine aminotransferase, and proinflammatory cytokines. Hepatic lymphocytes demonstrated increased CD8+/CD62L+(L-selectin) cells in HFD-fed mice after IRI. CD8−/−mice and CD8-depleted C57BL/6 mice demonstrated significant protection from injury, which was not seen in CD4−/−mice. L-selectin blockade also demonstrated significant hepatoprotection from IRI. L-selectin ligand MECA-79 was increased in HFD-fed mice undergoing IRI. Conclusion: Blockade of CD8 and L-selectin, but not CD4, ameliorated hepatocellular injury, confirming that CD8+cells are critical drivers of injury in a steatotic liver; this represents a therapeutic target in steatotic liver injury, underlining the importance of development of therapies specific to a steatotic liver. (Hepatology 2017;66:1258-1274).

Background: Adenosine, an endogenous purine nucleoside, is involved in several physiological functions. We have previously shown that A 2BAR plays a pro-inflammatory role during colitis. Aims: Our goals were to determine if A 2BAR expression was necessary on immune cells/non-immune cells during colitis and if A 2BAR was a suitable target for treating intestinal inflammation. Methods: Wild-type and A 2BAR knockout mice were utilized in bone marrow transplants to explore the importance of immune/non-immune A 2BAR expression during the development of colitis. Additionally, a T-cell transfer model of colitis was used in Rag1 knockout or A 2BAR/RAG1 double knockout recipients. Finally, A 2BAR small interfering RNA nanoparticles were administered to dextran sodium sulphate-treated mice. Results: Wild-type mice receiving wild-type or knockout bone marrow developed severe colitis after dextran sodium sulphate treatment, whereas colitis was significantly attenuated in knockout mice receiving wild-type or knockout bone marrow. Colitis induced in Rag1 knockout animals was attenuated in A 2BAR/RAG1 double knockout recipients. Animals receiving nanoparticles exhibited attenuated parameters of colitis severity compared to mice receiving control nanoparticles. Conclusions: Our results suggest that A 2BAR on non-immune cells plays an important role for the induction of colitis and targeting A 2BAR expression during colitis may be useful for alleviating symptoms of intestinal inflammation.

Objectives To characterize fundamental late tissue effects in the human vocal fold following radiation therapy. To develop a murine model of radiation fibrosis to ultimately develop both treatment and prevention paradigms. Design Translational study using archived human and fresh murine irradiated vocal fold tissue. Methods 1) Irradiated vocal fold tissue from patients undergoing laryngectomy for loss of function from radiation fibrosis were identified from pathology archives. Histomorphometry, immunohistochemistry, and whole-genome microarray as well as real-time transcriptional analyses was performed. 2) Focused radiation to the head and neck was delivered to mice in a survival fashion. One month following radiation, vocal fold tissue was analyzed with histomorphometry, immunohistochemistry, and real-time PCR transcriptional analysis for selected markers of fibrosis. Results Human irradiated vocal folds demonstrated increased collagen transcription with increased deposition and disorganization of collagen in both the thyroarytenoid muscle and the superficial lamina propria. Fibronectin were increased in the superficial lamina propria. Laminin decreased in the thyroarytenoid muscle. Whole genome microarray analysis demonstrated increased transcription of markers for fibrosis, oxidative stress, inflammation, glycosaminoglycan production and apoptosis. Irradiated murine vocal folds demonstrated increases in collagen and fibronectin transcription and deposition in the lamina propria. Transforming growth factor (TGF)-β increased in the lamina propria. Conclusion Human irradiated vocal folds demonstrate molecular changes leading to fibrosis that underlie loss of vocal fold pliability that occurs in patients following laryngeal irradiation. Irradiated murine tissue demonstrates similar findings, and this mouse model may have utility in creating prevention and treatment strategies for vocal fold radiation fibrosis.

Background Neurogenic inflammation plays a major role in the pathogenesis of inflammatory bowel disease (IBD). We examined the role of neuropeptide Y (NPY) and neuronal nitric oxide synthase (nNOS) in modulating colitis. Methods Colitis was induced by administration of dextran sodium sulphate (3% DSS) or streptomycin pre-treated Salmonella typhimurium (S.T.) in wild type (WT) and NPY (NPY−/−) knockout mice. Colitis was assessed by clinical score, histological score and myeloperoxidase activity. NPY and nNOS expression was assessed by immunostaining. Oxidative stress was assessed by measuring catalase activity, glutathione and nitrite levels. Colonic motility was assessed by isometric muscle recording in WT and DSS-treated mice. Results DSS/S.T. induced an increase in enteric neuronal NPY and nNOS expression in WT mice. WT mice were more susceptible to inflammation compared to NPY−/− as indicated by higher clinical & histological scores, and myeloperoxidase (MPO) activity (p<0.01). DSS-WT mice had increased nitrite, decreased glutathione (GSH) levels and increased catalase activity indicating more oxidative stress. The lower histological scores, MPO and chemokine KC in S.T.-treated nNOS−/− and NPY−/−/nNOS−/− mice supported the finding that loss of NPY-induced nNOS attenuated inflammation. The inflammation resulted in chronic impairment of colonic motility in DSS-WT mice. NPY –treated rat enteric neurons in vitro exhibited increased nitrite and TNF-α production. Conclusions NPY mediated increase in nNOS is a determinant of oxidative stress and subsequent inflammation. Our study highlights the role of neuronal NPY and nNOS as mediators of inflammatory processes in IBD.

BACKGROUND: Crohn's disease is a lifelong disease characterized by chronic inflammation of the gastrointestinal tract. Defining the cellular and transcriptional composition of the mucosa at different stages of disease progression is needed for personalized therapy in Crohn's. METHODS: Ileal biopsies were obtained from (1) control subjects (n = 6), (2) treatment-naïve patients (n = 7), and (3) established (n = 14) Crohn's patients along with remission (n = 3) and refractory (n = 11) treatment groups. The biopsies processed using 10x Genomics single cell 5' yielded 139 906 cells. Gene expression count matrices of all samples were analyzed by reciprocal principal component integration, followed by clustering analysis. Manual annotations of the clusters were performed using canonical gene markers. Cell type proportions, differential expression analysis, and gene ontology enrichment were carried out for each cell type. RESULTS: We identified 3 cellular compartments with 9 epithelial, 1 stromal, and 5 immune cell subtypes. We observed differences in the cellular composition between control, treatment-naïve, and established groups, with the significant changes in the epithelial subtypes of the treatment-naïve patients, including microfold, tuft, goblet, enterocyte,s and BEST4+ cells. Surprisingly, fewer changes in the composition of the immune compartment were observed; however, gene expression in the epithelial and immune compartment was different between Crohn's phenotypes, indicating changes in cellular activity. CONCLUSIONS: Our study identified cellular and transcriptional signatures associated with treatment-naïve Crohn's disease that collectively point to dysfunction of the intestinal barrier with an increase in inflammatory cellular activity. Our analysis also highlights the heterogeneity among patients within the same disease phenotype, shining a new light on personalized treatment responses and strategies.

We have reported that epithelial adenosine 2B receptor (A2BAR) mRNA and protein are up-regulated in colitis, which we demonstrated to be regulated by tumor necrosis factor α (TNF-α). Here, we examined the mechanism that governs A2BAR expression during colitis. A 1.4-kb sequence of the A2BAR promoter was cloned into the pFRL7 luciferase vector. Anti-microRNA (miRNA) was custom-synthesized based on specific miRNA binding sites. The binding of miRNA to the 3′-untranslated region (UTR) of A2BAR mRNA was examined by cloning this 3′-UTR downstream of the luciferase gene in pMIR-REPORT. In T84 cells, TNF-α induced a 35-fold increase in A2BAR mRNA but did not increase promoter activity in luciferase assays. By nuclear run-on assay, no increase in A2BAR mRNA following TNF-α treatment was observed. Four putative miRNA target sites (miR27a, miR27b, miR128a, miR128b) in the 3′-UTR of the A2BAR mRNA were identified in T84 cells and mouse colon. Pretreatment of cells with TNF-α reduced the levels of miR27b and miR128a by 60%. Over expression of pre-miR27b and pre-miR128a reduced A2BAR levels by >60%. Blockade of miR27b increased A2BAR mRNA levels by 6-fold in vitro. miR27b levels declined significantly in colitis-affected tissue in mice in the presence of increased A2BAR mRNA. Collectively, these data demonstrate that TNF-α-induced A2BAR expression in colonic epithelial cells is post-transcriptionally regulated by miR27b and miR128a and show that miR27b influences A2BAR expression in murine colitis.

Background & Aims: We used patient-derived organoids (PDOs) to study the epithelial-specific transcriptional and secretome signatures of the ileum during Crohn's disease (CD) with varying phenotypes to screen for disease profiles and potential druggable targets. Methods: RNA sequencing was performed on isolated intestinal crypts and 3-week-old PDOs derived from ileal biopsies of CD patients (n = 8 B1, inflammatory; n = 8 B2, stricturing disease) and non-inflammatory bowel disease (IBD) controls (n = 13). Differentially expressed (DE) genes were identified by comparing CD vs control, B1 vs B2, and inflamed vs non-inflamed. DE genes were used for computational screening to find candidate small molecules that could potentially reverse B1and B2 gene signatures. The secretome of a second cohort (n = 6 non-IBD controls, n = 7 CD, 5 non-inflamed, 2 inflamed) was tested by Luminex using cultured organoid conditioned medium. Results: We found 90% similarity in both the identity and abundance of protein coding genes between PDOs and intestinal crypts (15,554 transcripts of 19,900 genes). DE analysis identified 814 genes among disease group (CD vs non-IBD control), 470 genes different between the CD phenotypes, and 5 false discovery rate correction significant genes between inflamed and non-inflamed CD. The PDOs showed both similarity and diversity in the levels and types of soluble cytokines and growth factors they released. Perturbagen analysis revealed potential candidate compounds to reverse B2 disease phenotype to B1 in PDOs. Conclusions: PDOs are similar at the transcriptome level with the in vivo epithelium and retain disease-specific gene expression for which we have identified secretome products, druggable targets, and corresponding pharmacologic agents. Targeting the epithelium could reverse a stricturing phenotype and improve outcomes.

Current understanding is that receptor interacting serine/threonine protein kinase 1 (RIPK1) can lead to two distinct forms of cell death: RIPK3-mediated necroptosis or caspase 8 (Casp8)-mediated apoptosis. Here, we report that RIPK1 signaling is indispensable for protection from hepatocellular injury in a steatotic liver undergoing ischemia reperfusion injury (IRI) but not in the lean liver. In lean liver IRI, RIPK1-mediated cell death is operational, leading to protection in RIP1 kinase-dead knock-in (RIPK1K45A) mice and necrostatin-1s (Nec1s)-treated lean wild-type (WT) mice. However, when fed a high-fat diet (HFD), RIPK1K45A-treated and Nec1s-treated WT mice undergoing IRI demonstrate exacerbated hepatocellular injury along with decreased RIPK1 ubiquitylation. Furthermore, we demonstrate that HFD-fed RIPK3-/-/Casp8-/- mice show protection from IRI, but HFD-fed RIPK3-/-/Casp8-/+ mice do not. We also show that blockade of RIPK1 leads to increased Casp8 activity and decreases mitochondrial viability. Conclusion: Although more studies are required, we provide important proof of concept for RIPK1 inhibition leading to distinctive outcomes in lean and steatotic liver undergoing IRI. Considering the rising incidence of nonalcoholic fatty liver disease (NAFLD) in the general population, it will be imperative to address this critical difference when treating patients with RIPK1 inhibitors. This study also presents a new target for drug therapy to prevent hepatocellular injury in NAFLD.

A fatty liver is known to have impairment of microcirculation, which is worsened after ischemia reperfusion injury (IRI). This makes most fatty grafts unsuitable for transplantation, and in the absence of real time assessment of microcirculation this selection has been at best, random. The goal of this study was to demonstrate the utility of a contrast enhanced ultrasound model in quantitative assessment of the microcirculation of a fatty liver. We subjected fatty mice to IRI and blood flow dynamics were assessed before and after the injury. There was a significant increase in the resistive and pulsatility index of the extra hepatic artery and a significant decrease in velocity of the portal vein. There was also a quantifiable decrease in the intrahepatic blood volume, blood flow, time to peak flow, and perfusion index of mice with fatty liver suggesting that a fatty liver develops hemodynamic abnormalities after IRI, leading to increased hepatocellular injury. Importantly, these abnormalities can be reliably quantified by using a contrast, enhanced Doppler ultrasound, an inexpensive technique with multiple clinical applications. It can be used to assess the quality of the fatty liver donor graft prior to organ retrieval; for determining live donor candidacy, for making post IRI recovery prognosis, and for assessing the effectiveness of therapeutic interventions.