Multiple pathways of programmed cell death are important in liver homeostasis. Hepatocyte death is associated with progression of nonalcoholic fatty liver disease, and inhibition of apoptosis ...partially protects against liver injury in response to a high‐fat diet (HFD). However, the contribution of necroptosis, a caspase‐independent pathway of cell death, to HFD‐induced liver injury is not known. Wild‐type C57BL/6 and receptor interacting protein (RIP) 3−/− mice were randomized to chow or HFD. HFD‐fed C57BL/6 mice increased expression of RIP3, the master regulator of necroptosis, as well as phosphorylated mixed lineage kinase domain‐like, an effector of necroptotic cell death, in liver. HFD did not increase phosphorylated mixed lineage kinase domain‐like in RIP3−/− mice. HFD increased fasting insulin and glucose, as well as glucose intolerance, in C57BL/6 mice. RIP3−/− mice were glucose‐intolerant even on the chow diet; HFD further increased fasting glucose and insulin but not glucose intolerance. HFD also increased hepatic steatosis, plasma alanine aminotransferase activity, inflammation, oxidative stress, and hepatocellular apoptosis in wild‐type mice; these responses were exacerbated in RIP3−/− mice. Importantly, increased inflammation and injury were associated with early indicators of fibrosis in RIP3−/− compared to C57BL/6 mice. Culture of AML12 hepatocytes with palmitic acid increased cytotoxicity through apoptosis and necrosis. Inhibition of RIP1 with necrostatin‐1 or small interfering RNA knockdown of RIP3 reduced palmitic acid‐induced cytotoxicity. Conclusion: Absence of RIP3, a key mediator of necroptosis, exacerbated HFD‐induced liver injury, associated with increased inflammation and hepatocyte apoptosis, as well as early fibrotic responses; these findings indicate that shifts in the mode of hepatocellular death can influence disease progression and have therapeutic implications because manipulation of hepatocyte cell death pathways is being considered as a target for treatment of nonalcoholic fatty liver disease. (Hepatology 2016;64:1518‐1533)
The innate immune system, including monocytes/macrophages, is critical to the progression of alcoholic liver disease (ALD). In response to chronic ethanol, Kupffer cells, the resident macrophage of ...livers, and peripheral monocytes become sensitized to bacterial lipopolysaccharides (LPS), express more pro-inflammatory cytokines and exhibit macrophage M1/M2 hyperpolarization. Since miRNAs play an important role in the regulation of M1/M2 polarization, we hypothesized that miRNAs regulating macrophage polarization would be dysregulated after chronic ethanol consumption. miRNA sequencing data from Kupffer cells isolated from rats fed an ethanol diet vs. control diet and qPCR data from PBMCs isolated from alcoholic hepatitis (AH) patients and healthy controls were used to assess the role of miRNAs in macrophage hyperpolarization in ALD. Differential expression analyses revealed 40 misregulated miRNAs in Kupffer cells from the chronic ethanol-fed rats compared to pair-fed controls. Nine of these miRNAs are known to be associated with macrophage polarization and consist of a mixture of M1- and M2-associated miRNAs, indicative of hyperpolarization. Twenty-three of the 40 differentially expressed miRNAs were localized to miRNA clusters throughout the genome. Correlation analyses revealed that miRNAs in three of these clusters were co-regulated and located within antisense non-coding RNAs. Similar to Kupffer cells from ethanol-fed rats, M1 and M2 polarization markers, as well as sensitivity to LPS, were elevated in PBMCs from AH patients compared to healthy controls. These increases were associated with an up-regulation of polarization-associated miRNAs, including miR-125a-5p, a miRNA associated with hyperpolarization. miR-125a-5p is clustered in the genome with other miRNAs inside a host gene, Spaca6, which was also upregulated in PBMCs, as well as isolated monocytes, from AH patients. Finally, correlation analyses revealed co-regulation of human polarization-associated miRNA clusters. While expression of polarization-associated miRNAs in clusters was upregulated in AH compared to healthy controls, co-regulation of the miRNAs within a cluster was independent of disease state. Together, these results reveal that global changes in miRNA regulation are associated with polarization phenotypes in Kupffer cells from rat after chronic ethanol as well as in PBMCs from patients with AH. Importantly, polarization-associated miRNAs were localized to coordinately regulated clusters.
Type I interferon (IFN) inhibits, by an unknown mechanism, the replication of human papillomaviruses (HPV), which are major human pathogens, Here, we present evidence that P56 (a protein), the ...expression of which is strongly induced by IFN, double‐stranded RNA and viruses, mediates the anti‐HPV effect of IFN. Ectopic expression of P56 inhibited HPV DNA replication and its ablation in IFN‐treated cells alleviated the inhibitory effect of IFN on HPV DNA replication. Protein–protein interaction and mutational analyses established that the antiviral effect of P56 was mediated by its direct interaction with the DNA replication origin‐binding protein E1 of several strains of HPV, through the tetratricopeptide repeat 2 in the N‐terminal region of P56 and the C‐terminal region of E1. In vivo, the interaction with P56, a cytoplasmic protein, caused translocation of E1 from the nucleus to the cytoplasm. In vitro, recombinant P56, or a small fragment derived from it, inhibited the DNA helicase activity of E1 and E1‐mediated HPV DNA replication. These observations delineate the molecular mechanism of IFN's antiviral action against HPV.
Toll-like receptors (TLRs) recognize specific microbial products and elicit innate immune signals to activate specific transcription factors that induce protective proteins, such as interferon. TLR3 ...is localized to endosomes and recognizes double-stranded RNA (dsRNA), which is generated by virally infected or apoptotic cells. TLR3 has been genetically linked to several human diseases, including some without viral etiology. Unlike other TLRs, TLR3 requires phosphorylation of two specific tyrosine residues in its cytoplasmic domain to recruit the adaptor protein TRIF (Toll-interleukin-1 receptor domain-containing adaptor protein inducing interferon-β) and initiate the antiviral response. We showed that two protein tyrosine kinases, the epidermal growth factor receptor (EGFR) ErbB1 and Src, bound sequentially to dsRNA-activated TLR3 and phosphorylated the two tyrosine residues. In cells lacking EGFR or treated with an inhibitor of EGFR, viral replication was enhanced and induction of antiviral genes was impaired. Thus, these results reveal a connection between antiviral innate immunity and cell growth regulators.
Immunoglobulin A nephropathy (IgAN) is the most common form of glomerulonephritis throughout the world. A majority (approx. 60%) of patients with IgAN experience disease exacerbations associated with ...an acute respiratory or gastrointestinal illness that appears to represent a viral infection. However, the exact mechanism of the disease exacerbation by viral infection is not understood, especially at the cellular and molecular levels. Here we report that glomerular podocytes express the major sensors for double-stranded RNA (dsRNA), a common byproduct of viral replication. In addition to these receptors, Toll-like receptor 3 (TLR3) and retinoic acid-inducible gene 1 (RIG-I)-like helicases (RLHs), podocytes express the collateral proteins required to support intracellular signaling. The pathways that mediate responses to dsRNA are fully functional in podocytes. The transcription factor interferon regulatory factor 3 (IRF3) and nuclear factor kappa B (NF-ĸB) are phosphorylated and translocate to the nucleus, and dsRNA increases synthesis of proteins driven by IRF3 (P54, P56 and P60) or NF-ĸB (interleukin 8 and A20). Furthermore, dsRNA suppresses podocyte cell migration, alters the expression of a panel of podocyte essential proteins (nephrin, podocin and CD2-associated protein or CD2AP) and changes transepithelial albumin flux. These effects are dsRNA sensor-specific: TLR3-/- podocytes do not respond to extracellular dsRNA, while intracellular dsRNA has no effect on podocytes bearing a dominant negative form of the major active RLH. These results demonstrate that innate responses to viruses can disturb podocyte cell function in vitro.
Alcoholic liver disease (ALD) develops in approximately 20% of alcoholic patients, with a higher prevalence in females. ALD progression is marked by fatty liver and hepatocyte necrosis, as well as ...apoptosis, inflammation, regenerating nodules, fibrosis, and cirrhosis. ALD develops via a complex process involving parenchymal and nonparenchymal cells, as well as recruitment of other cell types to the liver in response to damage and inflammation. Hepatocytes are damaged by ethanol, via generation of reactive oxygen species and induction of endoplasmic reticulum stress and mitochondrial dysfunction. Hepatocyte cell death via apoptosis and necrosis are markers of ethanol-induced liver injury. We review the mechanisms by which alcohol injures hepatocytes and the response of hepatic sinusoidal cells to alcohol-induced injury. We also discuss how recent insights into the pathogenesis of ALD will affect the treatment and management of patients.
The phosphoprotein P of paramyxoviruses is known to play more than one role in genome transcription and replication. Phosphorylation of P at the NH
2
terminus by cellular casein kinase II has been ...shown to be necessary for transcription of the genome in some of the viruses, while it is dispensable for replication. The phosphorylation null mutant of rinderpest virus P protein, in which three serine residues have been mutated, has been shown earlier to be non-functional in an in vivo minigenome replication/transcription system. In this work, we have shown that the phosphorylation of P protein is essential for transcription, whereas the null mutant is active in replication of the genome in vivo. The null mutant P acts as a transdominant repressor of transcriptional activity of wild-type P and as an activator of replication carried out by wild-type P protein. These results suggest the phosphorylation status of P may act as a replication switch during virus replication. We also show that the phosphorylation null mutant P is capable of interacting with L and N proteins and is able to form a tripartite complex of L-(N-P) when expressed in insect cells, similar to wild-type P protein.
Increased inflammatory signaling by Kupffer cells contributes to alcoholic liver disease (ALD). Here we investigated the impact of small, specific‐sized hyaluronic acid of 35 kD (HA35) on ...ethanol‐induced sensitization of Kupffer cells, as well as ethanol‐induced liver injury in mice. Unbiased analysis of microRNA (miRNA) expression in Kupffer cells identified miRNAs regulated by both ethanol and HA35. Toll‐like receptor 4 (TLR4)‐mediated signaling was assessed in primary cultures of Kupffer cells from ethanol‐ and pair‐fed rats after treatment with HA35. Female C57BL6/J mice were fed ethanol or pair‐fed control diets and treated or not with HA35. TLR4 signaling was increased in Kupffer cells by ethanol; this sensitization was normalized by ex vivo treatment with HA35. Next generation sequencing of Kupffer cell miRNA identified miRNA 181b‐3p (miR181b‐3p) as sensitive to both ethanol and HA35. Importin α5, a protein involved in p65 translocation to the nucleus, was identified as a target of miR181b‐3p; importin α5 protein was increased in Kupffer cells from ethanol‐fed rats, but decreased by HA35 treatment. Overexpression of miR181b‐3p decreased importin α5 expression and normalized lipopolysaccharide‐stimulated tumor necrosis factor α expression in Kupffer cells from ethanol‐fed rats. In a mouse model of ALD, ethanol feeding decreased miR181b‐3p in liver and increased expression of importin α5 in nonparenchymal cells. Treatment with HA35 normalized these changes and also protected mice from ethanol‐induced liver and intestinal injury. Conclusion: miR181b‐3p is dynamically regulated in Kupffer cells and mouse liver in response to ethanol and treatment with HA35. miR181b‐3p modulates expression of importin α5 and sensitivity of TLR4‐mediated signaling. This study identifies a miR181b‐3p–importin α5 axis in regulating inflammatory signaling pathways in hepatic macrophages. (Hepatology 2017;66:602–615).
Basement membranes are thin connective tissue structures composed of organ-specific assemblages of collagens, laminins, proteoglycan-like perlecan, nidogens, and other components. Traditionally, ...basement membranes are thought of as structures which primarily function to anchor epithelial, endothelial, or parenchymal cells to underlying connective tissues. While this role is important, other functions such as the modulation of growth factors and cytokines that regulate cell proliferation, migration, differentiation, and fibrosis are equally important. An example of this is the critical role of both the epithelial basement membrane and Descemet’s basement membrane in the cornea in modulating myofibroblast development and fibrosis, as well as myofibroblast apoptosis and the resolution of fibrosis. This article compares the ultrastructure and functions of key basement membranes in several organs to illustrate the variability and importance of these structures in organs that commonly develop fibrosis.
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