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1.
J Immunol ; 186(3): 1618-26, 2011 Feb 01.
Article in English | MEDLINE | ID: mdl-21187438

ABSTRACT

Rotavirus is a dsRNA virus that infects epithelial cells that line the surface of the small intestine. It causes severe diarrheal illness in children and ∼500,000 deaths per year worldwide. We studied the mechanisms by which intestinal epithelial cells (IECs) sense rotavirus infection and signal IFN-ß production, and investigated the importance of IFN-ß production by IECs for controlling rotavirus production by intestinal epithelium and virus excretion in the feces. In contrast with most RNA viruses, which interact with either retinoic acid-inducible gene I (RIG-I) or melanoma differentiation-associated gene 5 (MDA5) inside cells, rotavirus was sensed by both RIG-I and MDA5, alone and in combination. Rotavirus did not signal IFN-ß through either of the dsRNA sensors TLR3 or dsRNA-activated protein kinase (PKR). Silencing RIG-I or MDA5, or their common adaptor protein mitochondrial antiviral signaling protein (MAVS), significantly decreased IFN-ß production and increased rotavirus titers in infected IECs. Overexpression of laboratory of genetics and physiology 2, a RIG-I-like receptor that interacts with viral RNA but lacks the caspase activation and recruitment domains required for signaling through MAVS, significantly decreased IFN-ß production and increased rotavirus titers in infected IECs. Rotavirus-infected mice lacking MAVS, but not those lacking TLR3, TRIF, or PKR, produced significantly less IFN-ß and increased amounts of virus in the intestinal epithelium, and shed increased quantities of virus in the feces. We conclude that RIG-I or MDA5 signaling through MAVS is required for the activation of IFN-ß production by rotavirus-infected IECs and has a functionally important role in determining the magnitude of rotavirus replication in the intestinal epithelium.


Subject(s)
Adaptor Proteins, Signal Transducing/physiology , DEAD-box RNA Helicases/physiology , Interferon-beta/biosynthesis , Intestinal Mucosa/immunology , Membrane Proteins/physiology , Nerve Tissue Proteins/physiology , Rotavirus/immunology , Signal Transduction/immunology , Adaptor Proteins, Signal Transducing/deficiency , Animals , Cell Line , Chlorocebus aethiops , DEAD Box Protein 58 , DEAD-box RNA Helicases/deficiency , HT29 Cells , Humans , Interferon-Induced Helicase, IFIH1 , Interferon-beta/physiology , Intestinal Mucosa/enzymology , Intestinal Mucosa/virology , Membrane Proteins/deficiency , Mice , Mice, 129 Strain , Mice, Inbred C57BL , Mice, Knockout , Nerve Tissue Proteins/deficiency , RNA Helicases/genetics , RNA Helicases/physiology , RNA, Viral/biosynthesis , RNA, Viral/genetics , Receptors, Cell Surface , Receptors, Immunologic , Response Elements/immunology , Rotavirus/genetics , Signal Transduction/genetics , Virus Replication/genetics , Virus Replication/immunology
2.
Life Sci ; 82(9-10): 519-28, 2008 Feb 27.
Article in English | MEDLINE | ID: mdl-18215718

ABSTRACT

Intestinal epithelial cells (IEC) have adapted to the presence of commensal bacteria through a state of tolerance that involves a limited response to lipopolysaccharide (LPS). Low or absent expression of two LPS receptor molecules, the myeloid differentiation (MD)-2 receptor, and toll-like receptor (TLR)4 was suggested to underlie LPS tolerance in IEC. In the present study we performed transfections of TLR4 and MD-2 alone or combined in different IEC lines derived from intestinal cancer (Caco-2, HT-29, and SW837). We found that LPS responsiveness increased more than 100-fold when IEC were transfected with MD-2 alone, but not TLR4. The release of interleukin (IL)-8, but also the expression of cyclooxygenase (Cox-)2 and the related secretion of prostaglandin (PG)E2 were coordinately stimulated by LPS in IEC transfected with MD-2 alone. Supernatants collected from MD-2-transfected IEC supported LPS activation of naïve HT-29, providing additional support to the concept that MD-2 alone endows IEC with LPS responsiveness. LPS responsiveness detected at concentrations as low as 110 pg/ml, and maximal values obtained by 10 ng/ml were clearly beyond those evoked by classical stimuli as IL-1beta. In polarized cells, apical LPS stimulation was markedly more efficient than basolateral. Our data contradict previous opinion that both TLR4 and MD-2 limit IEC response to LPS, and emphasize the prominent role of MD-2 in intestinal immune responses to Gram-negative bacteria.


Subject(s)
Epithelial Cells/drug effects , Lipopolysaccharides/pharmacology , Lymphocyte Antigen 96/physiology , Anthracenes/pharmacology , Caco-2 Cells , Cell Line, Tumor , Cells, Cultured , Cyclooxygenase 2/biosynthesis , Dinoprostone/metabolism , Dose-Response Relationship, Drug , Enzyme Inhibitors/pharmacology , Epithelial Cells/metabolism , Epithelial Cells/pathology , Flow Cytometry , Gene Expression , HT29 Cells , Humans , Imidazoles/pharmacology , Interleukin-8/metabolism , Intestines/pathology , Lymphocyte Antigen 96/genetics , Mitogen-Activated Protein Kinases/antagonists & inhibitors , Mitogen-Activated Protein Kinases/metabolism , Monocytes/cytology , Monocytes/drug effects , Monocytes/metabolism , NF-kappa B/metabolism , Pyridines/pharmacology , RNA, Messenger/genetics , RNA, Messenger/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Toll-Like Receptor 4/genetics , Toll-Like Receptor 4/physiology , Transfection
3.
J Immunol ; 179(8): 5425-32, 2007 Oct 15.
Article in English | MEDLINE | ID: mdl-17911629

ABSTRACT

Intestinal epithelial cells (IECs) are a first line of defense against microbial pathogens that enter the host through the intestinal tract. Moreover, viral pathogens that infect the host via the intestinal epithelium are an important cause of morbidity and mortality. However, the mechanisms by which viral pathogens activate antiviral defense mechanisms in IECs are largely unknown. The synthetic dsRNA analog polyinosinic-polycytidylic acid and infection with live virus were used to probe the molecules that are activated and the mechanisms of signaling in virus-infected human IECs. Polyinosinic-polycytidylic acid activated IFN regulatory factor 3 dimerization and phosphorylation, increased activity of the IFN-stimulated response element, induced a significant increase in IFN-beta mRNA transcripts and IFN-beta secretion, and up-regulated the expression of IFN-regulated genes in IECs. Those responses were dependent upon activation of the dsRNA binding protein retinoic acid inducible gene I (RIG-I) and the RIG-I interacting protein IFN promoter stimulator-1, but not on dsRNA-activated protein kinase or TLR3, which also were expressed by IECs. Virus replication and virus-induced cell death increased in IECs in which RIG-I was silenced, consistent with the importance of the RIG-I signaling pathway in IEC antiviral innate immune defense mechanisms.


Subject(s)
Adaptor Proteins, Signal Transducing/physiology , Immunity, Innate , Intestinal Mucosa/immunology , Intestinal Mucosa/metabolism , Receptors, Retinoic Acid/physiology , Signal Transduction/immunology , Cells, Cultured , HT29 Cells , Humans , Interferon Regulatory Factor-3/metabolism , Interferon Type I/biosynthesis , Interferon Type I/metabolism , Interferon-beta/metabolism , Intestinal Mucosa/cytology , Poly I-C/pharmacology , Response Elements , Up-Regulation/immunology , Vesicular stomatitis Indiana virus/immunology
4.
J Virol ; 81(3): 1297-304, 2007 Feb.
Article in English | MEDLINE | ID: mdl-17079279

ABSTRACT

Previous studies demonstrated that the induction of the heat shock protein Hsp70 in response to viral infection is highly specific and differs from one cell to another and for a given virus type. However, no clear consensus exists so far to explain the likely reasons for Hsp70 induction within host cells during viral infection. We show here that upon rotavirus infection of intestinal cells, Hsp70 is indeed rapidly, specifically, and transiently induced. Using small interfering RNA-Hsp70-transfected Caco-2 cells, we observed that Hsp70 silencing was associated with an increased virus protein level and enhanced progeny virus production. Upon Hsp70 silencing, we observed that the ubiquitination of the main rotavirus structural proteins was strongly reduced. In addition, the use of proteasome inhibitors in infected Caco-2 cells was shown to induce an accumulation of structural viral proteins. Together, these results are consistent with a role of Hsp70 in the control of the bioavailability of viral proteins within cells for virus morphogenesis.


Subject(s)
Gene Expression Regulation, Viral/physiology , HSP70 Heat-Shock Proteins/physiology , Rotavirus/physiology , Viral Proteins/metabolism , Caco-2 Cells/metabolism , Caco-2 Cells/virology , HSP70 Heat-Shock Proteins/genetics , Humans , Rotavirus/growth & development , Viral Proteins/biosynthesis
5.
J Biol Chem ; 278(24): 21601-6, 2003 Jun 13.
Article in English | MEDLINE | ID: mdl-12682040

ABSTRACT

Accumulating evidence suggests that some heat shock proteins (Hsps), in particular the 72-kDa inducible Hsp70, associate to the cell membrane and might be secreted through an unknown mechanism to exert important functions in the immune response and signal transduction. We speculated that specialized structures named lipid rafts, known as important platforms for the delivery of proteins to the cell membrane, might be involved in the unknown mechanism ensuring membrane association and secretion of Hsp70. Lipid rafts are sphingolipid-cholesterol-rich structures that have been mainly characterized in polarized epithelial cells and can be isolated as detergent-resistant microdomains (DRMs). Analysis of soluble and DRM fractions prepared from unstressed Caco-2 epithelial cells revealed that Hsp70, and to a lesser extent calnexin, were present in DRM fractions. Increased expression of Hsps, through heat shock or by using drugs acting on protein trafficking or intracellular calcium level, induced an efficient translocation to DRM. We also found that Hsp70 was released by epithelial Caco-2 cells, and this release dramatically increased after heat shock. Drugs known to block the classical secretory pathway were unable to reduce Hsp70 release. By contrast, release of the protein was affected by the raft-disrupting drug methyl-beta-cyclodextrin. Our data suggest that lipid rafts are part of a mechanism ensuring the correct functions of Hsps and provide a rational explanation for the observed membrane association and release of Hsp70.


Subject(s)
Detergents/pharmacology , HSP70 Heat-Shock Proteins/biosynthesis , HSP70 Heat-Shock Proteins/genetics , Membrane Microdomains/metabolism , Alkaline Phosphatase/metabolism , Blotting, Western , Caco-2 Cells , Cell Membrane/metabolism , Cholesterol/metabolism , Dipeptidyl Peptidase 4/chemistry , Electrophoresis, Polyacrylamide Gel , Endoplasmic Reticulum/metabolism , Enzyme-Linked Immunosorbent Assay , Epithelial Cells/metabolism , Flow Cytometry , Golgi Apparatus/metabolism , Hot Temperature , Humans , Protein Structure, Tertiary , Temperature , Time Factors , Tumor Cells, Cultured
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