Cellular homeostais, that is normally maintained through autophagy, is disrupted in alcoholic liver disease (ALD). Because autophagy and exosome biogenesis share common elements, we hypothesized that increased exosome production in ALD may be linked to disruption of autophagic function. We found impaired autophagy both in ALD and alcoholic hepatitis (AH) mouse models and human livers with ALD as indicated by increased hepatic p62 and LC3‐II levels. Alcohol reduced autophagy flux in vivo in chloroquine‐treated mice as well as in vitro in hepatocytes and macrophages treated with bafilomycin A. Our results revealed that alcohol targets multiple steps in the autophagy pathway. Alcohol‐related decrease in mechanistic target of rapamycin (mTOR) and Ras homolog enriched in brain (Rheb), that initiate autophagy, correlated with increased Beclin1 and autophagy‐related protein 7 (Atg7), proteins involved in phagophore‐autophagosome formation, in ALD. We found that alcohol disrupted autophagy function at the lysosomal level through decreased lysosomal‐associated membrane protein 1 (LAMP1) and lysosomal‐associated membrane protein 2 (LAMP2) in livers with ALD. We identified that micro‐RNA 155 (miR‐155), that is increased by alcohol, targets mTOR, Rheb, LAMP1, and LAMP2 in the authophagy pathway. Consistent with this, miR‐155‐deficient mice were protected from alcohol‐induced disruption of autophagy and showed attenuated exosome production. Mechanistically, down‐regulation of LAMP1 or LAMP2 increased exosome release in hepatocytes and macrophages in the presence and absence of alcohol. These results suggested that the alcohol‐induced increase in exosome production was linked to disruption of autophagy and impaired autophagosome and lysosome function. Conclusion: Alcohol affects multiple genes in the autophagy pathway and impairs autophagic flux at the lysosome level in ALD. Inhibition of LAMP1 and LAMP2 promotes exosome release in ALD. We identified miR‐155 as a mediator of alcohol‐related regulation of autophagy and exosome production in hepatocytes and macrophages.

Disease severity in alcoholic liver disease (ALD) is associated with a significant presence of neutrophils (a type of immune cell) in the liver. It remains unknown how alcohol affects the capacity of neutrophils to control infection, a major hallmark of ALD. We found that binge alcohol drinking impaired important strategies used by neutrophils to contain and resolve infection, resulting in increased liver injury during ALD.

Our study indicates a specific protein signature of ALD EVs and demonstrates a functional role of circulating EVs containing heat shock protein 90 in mediating KC/MØ activation in the liver. (Hepatology 2018;67:1986-2000).

Inflammation promotes the progression of alcoholic liver disease. Alcohol sensitizes KCs to gut-derived endotoxin (LPS); however, signaling pathways that perpetuate inflammation in alcoholic liver disease are only partially understood. We found that chronic alcohol feeding in mice induced miR-155, an inflammatory miRNA in isolated KCs. We hypothesized that miR-155 might increase the responsiveness of KCs to LPS via targeting the negative regulators of LPS signaling. Our results revealed that KCs that were isolated from alcohol-fed mice showed a decrease in IRAK-M, SHIP1, and PU.1, and an increase in TNF-α levels. This was specific to KCs, as no significant differences were observed in these genes in hepatocytes. We found a causal effect of miR-155 deficiency on LPS responsiveness, as KCs that were isolated from miR-155 KO mice showed a greater induction of IRAK-M, SHIP1, and suppressor of cytokine signaling 1 after LPS treatment. C/EBPβ, a validated miR-155 target, stimulates IL-10 transcription. We found a higher induction of C/EBPβ and IL-10 in KCs that were isolated from miR-155 KO mice after LPS treatment. Gain- and loss-of-function studies affirmed that alcohol-induced miR-155 directly regulates IRAK-M, SHIP1, suppressor of cytokine signaling 1, and C/EBPβ, as miR-155 inhibition increased and miR-155 overexpression decreased these genes in LPS or alcohol-pretreated wild-type KCs. HDAC11, a regulator of IL-10, was significantly increased and IL-10 was decreased in KCs that were isolated from alcohol-fed mice. Functionally, knockdown of HDAC11 with small interfering RNA resulted in an IL-10 increase in LPS or alcohol-pretreated Mϕ. We found that acetaldehyde and NF-κB pathways regulate HDAC11 levels. Collectively, our results indicate that the alcohol-induced responsiveness of KCs to LPS, in part, is governed by miR-155 and HDAC11.

No abstract

Epigenetic changes of stromal-epithelial interactions are of key importance in the regulation of colorectal carcinoma (CRC) cells and morphologically normal, but genetically and epigenetically altered epithelium in normal adjacent tumor (NAT) areas. Here we demonstrated retained protein expression of well-known Wnt inhibitor, secreted frizzled-related protein 1 (SFRP1) in stromal myofibroblasts and decreasing epithelial expression from NAT tissues towards the tumor. SFRP1 was unmethylated in laser microdissected myofibroblasts and partially hypermethylated in epithelial cells in these areas. In contrast, we found epigenetically silenced myofibroblast-derived SFRP1 in CRC stroma. Our results suggest that the myofibroblast-derived SFRP1 protein might be a paracrine inhibitor of epithelial proliferation in NAT areas and loss of this signal may support tumor proliferation in CRC.

BackgroundEpithelial cells in malignant conditions release DNA into the extracellular compartment. Cell free DNA of tumor origin may act as a ligand of DNA sensing mechanisms and mediate changes in epithelial-stromal interactions.AimsTo evaluate and compare the potential autocrine and paracrine regulatory effect of normal and malignant epithelial cell-related DNA on TLR9 and STING mediated pathways in HT-29 human colorectal adenocarcinoma cells and normal fibroblasts.Materials and MethodsDNA isolated from normal and tumorous colonic epithelia of fresh frozen surgically removed tissue samples was used for 24 and 6 hour treatment of HT-29 colon carcinoma and HDF-α fibroblast cells. Whole genome mRNA expression analysis and qRT-PCR was performed for the elements/members of TLR9 signaling pathway. Immunocytochemistry was performed for epithelial markers (i.e. CK20 and E-cadherin), DNA methyltransferase 3a (DNMT3a) and NFκB (for treated HDFα cells).ResultsAdministration of tumor derived DNA on HT29 cells resulted in significant (p<0.05) mRNA level alteration in 118 genes (logFc≥1, p≤0.05), including overexpression of metallothionein genes (i.e. MT1H, MT1X, MT1P2, MT2A), metastasis-associated genes (i.e. TACSTD2, MACC1, MALAT1), tumor biomarker (CEACAM5), metabolic genes (i.e. INSIG1, LIPG), messenger molecule genes (i.e. DAPP, CREB3L2). Increased protein levels of CK20, E-cadherin, and DNMT3a was observed after tumor DNA treatment in HT-29 cells. Healthy DNA treatment affected mRNA expression of 613 genes (logFc≥1, p≤0.05), including increased expression of key adaptor molecules of TLR9 pathway (e.g. MYD88, IRAK2, NFκB, IL8, IL-1β), STING pathway (ADAR, IRF7, CXCL10, CASP1) and the FGF2 gene.ConclusionsDNA from tumorous colon epithelium, but not from the normal epithelial cells acts as a pro-metastatic factor to HT-29 cells through the overexpression of pro-metastatic genes through TLR9/MYD88 independent pathway. In contrast, DNA derived from healthy colonic epithelium induced TLR9 and STING signaling pathway in normal fibroblasts.

BackgroundSomatostatin (SST) has anti-proliferative and pro-apoptotic effects. Our aims were to analyze and compare the SST expression during normal aging and colorectal carcinogenesis at mRNA and protein levels. Furthermore, we tested the methylation status of SST in biopsy samples, and the cell growth inhibitory effect of the SST analogue octreotide in human colorectal adenocarcinoma cell line.MethodsColonic samples were collected from healthy children (n1 = 6), healthy adults (n2 = 41) and colorectal cancer patients (CRCs) (n3 = 34) for SST mRNA expression analysis, using HGU133 Plus2.0 microarrays. Results were validated both on original (n1 = 6; n2 = 6; n3 = 6) and independent samples ((n1 = 6; n2 = 6; n3 = 6) by real-time PCR. SST expressing cells were detected by immunohistochemistry on colonic biopsy samples (n1 = 14; n2 = 20; n3 = 23). The effect of octreotide on cell growth was tested on Caco-2 cell line. SST methylation percentage in biopsy samples (n1 = 5; n2 = 5; n3 = 9) was defined using methylation-sensitive restriction enzyme digestion.ResultsIn case of normal aging SST mRNA expression did not alter, but decreased in cancer (p<0.05). The ratio of SST immunoreactive cells was significantly higher in children (0.70%±0.79%) compared to CRC (0%±0%) (p<0.05). Octreotide significantly increased the proportion of apoptotic Caco-2 cells. SST showed significantly higher methylation level in tumor samples (30.2%±11.6%) compared to healthy young individuals (3.5%±1.9%) (p<0.05).ConclusionsIn cancerous colonic mucosa the reduced SST production may contribute to the uncontrolled cell proliferation. Our observation that in colon cancer cells octreotide significantly enhanced cell death and attenuated cell proliferation suggests that SST may act as a regulator of epithelial cell kinetics. The inhibition of SST expression in CRC can be epigenetically regulated by promoter hypermethylation.