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1.
Brain Behav Immun ; 93: 226-237, 2021 03.
Article in English | MEDLINE | ID: mdl-33516921

ABSTRACT

While toll-like receptors (TLRs), which mediate innate immunity, are known to play an important role in host defense, recent work suggest their involvement in some integrated behaviors, including anxiety, depressive and cognitive functions. Here, we investigated the potential involvement of the flagellin receptor, TLR5, in anxiety, depression and cognitive behaviors using male TLR5 knock-out (KO) mice. We aobserved a specific low level of basal anxiety in TLR5 KO mice with an alteration of the hypothalamo-pituitary axis (HPA) response to acute restraint stress, illustrated by a decrease of both plasma corticosterone level and c-fos expression in the hypothalamic paraventricular nucleus where TLR5 was expressed, compared to WT littermates. However, depression and cognitive-related behaviors were not different between TLR5 KO and WT mice. Nor there were significant changes in the expression of some cytokines (IL-6, IL-10 and TNF-α) and other TLRs (TLR2, TLR3 and TLR4) in the prefrontal cortex, amygdala and hippocampus of TLR5 KO mice compared to WT mice. Moreover, mRNA expression of BDNF and glucocorticoid receptors in the hippocampus and amygdala, respectively, was not different. Finally, acute intracerebroventricular administration of flagellin, a specific TLR5 agonist, or chronic neomycin treatment did not exhibit a significant main effect, only a significant main effect of genotype was observed between TLR5 KO and WT mice. Together, those findings suggest a previously undescribed and specific role of TLR5 in anxiety and open original prospects in our understanding of the brain-gut axis function.


Subject(s)
Anxiety , Toll-Like Receptor 5 , Animals , Anxiety/genetics , Anxiety Disorders , Corticosterone , Male , Mice , Mice, Knockout , Toll-Like Receptor 5/genetics
2.
Mucosal Immunol ; 10(6): 1455-1467, 2017 11.
Article in English | MEDLINE | ID: mdl-28327619

ABSTRACT

Regulatory and effector T helper (Th) cells are abundant at mucosal surfaces, especially in the intestine, where they control the critical balance between tolerance and inflammation. However, the key factors that reciprocally dictate differentiation along these specific lineages remain incompletely understood. Here we report that the interleukin-1 (IL-1) family member IL-36γ signals through IL-36 receptor, myeloid differentiation primary response gene 88, and nuclear factor-κBp50 in CD4+ T cells to potently inhibit Foxp3-expressing induced regulatory T cell (Treg) development, while concomitantly promoting the differentiation of Th9 cells via a IL-2-STAT5- (signal transducer and activator of transcription factor 5) and IL-4-STAT6-dependent pathway. Consistent with these findings, mice deficient in IL-36γ were protected from Th cell-driven intestinal inflammation and exhibited increased colonic Treg cells and diminished Th9 cells. Our findings thus reveal a fundamental contribution for the IL-36/IL-36R axis in regulating the Treg-Th9 cell balance with broad implications for Th cell-mediated disorders, such as inflammatory bowel diseases and particularly ulcerative colitis.


Subject(s)
Colitis, Ulcerative/immunology , Colon/immunology , Receptors, Interleukin-1/metabolism , T-Lymphocytes, Helper-Inducer/immunology , T-Lymphocytes, Regulatory/immunology , Animals , Benzofurans , Cell Differentiation , Disease Models, Animal , Forkhead Transcription Factors/metabolism , Humans , Interleukin-2/metabolism , Interleukin-9/metabolism , Mice , Mice, Knockout , Myeloid Differentiation Factor 88/genetics , Myeloid Differentiation Factor 88/metabolism , Quinolines , Receptors, Interleukin-1/genetics , STAT Transcription Factors/metabolism , STAT5 Transcription Factor/metabolism , Signal Transduction , Transcription Factor RelA/genetics , Transcription Factor RelA/metabolism
3.
Mucosal Immunol ; 10(3): 673-684, 2017 05.
Article in English | MEDLINE | ID: mdl-27624780

ABSTRACT

Specific components of the intestinal microbiota are capable of influencing immune responses such that a mutualistic relationship is established. In mice, colonization with segmented filamentous bacteria (SFB) induces T-helper-17 (Th17) cell differentiation in the intestine, yet the effector functions of interleukin (IL)-17A in response to SFB remain incompletely understood. Here we report that colonization of mice with SFB-containing microbiota induced IL-17A- and CXCR2-dependent recruitment of neutrophils to the ileum. This response required adaptive immunity, as Rag-deficient mice colonized with SFB-containing microbiota failed to induce IL-17A, CXCL1 and CXCL2, and displayed defective neutrophil recruitment to the ileum. Interestingly, neutrophil depletion in wild-type mice resulted in significantly augmented Th17 responses and SFB expansion, which correlated with impaired expression of IL-22 and antimicrobial peptides. These data provide novel insight into a dynamic IL-17A-CXCR2-neutrophil axis during acute SFB colonization and demonstrate a central role for neutrophils in limiting SFB expansion.


Subject(s)
Bacteria/immunology , Gastrointestinal Microbiome/immunology , Ileum/immunology , Interleukin-17/metabolism , Neutrophils/immunology , Receptors, Interleukin-8B/metabolism , Th17 Cells/immunology , Adaptive Immunity/genetics , Animals , Antimicrobial Cationic Peptides/metabolism , Bacteria/growth & development , Cell Differentiation , Cell Movement/genetics , Cells, Cultured , Homeodomain Proteins/genetics , Ileum/microbiology , Interleukins/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , Interleukin-22
4.
Mucosal Immunol ; 9(4): 907-16, 2016 07.
Article in English | MEDLINE | ID: mdl-26601902

ABSTRACT

Secretory IgA (SIgA) directed against gut resident bacteria enables the mammalian mucosal immune system to establish homeostasis with the commensal gut microbiota after weaning. Germinal centers (GCs) in Peyer's patches (PPs) are the principal inductive sites where naive B cells specific for bacterial antigens encounter their cognate antigens and receive T-cell help driving their differentiation into IgA-producing plasma cells. We investigated the role of antigen sampling by intestinal M cells in initiating the SIgA response to gut bacteria by developing mice in which receptor activator of nuclear factor-κB ligand (RANKL)-dependent M-cell differentiation was abrogated by conditional deletion of Tnfrsf11a in the intestinal epithelium. Mice without intestinal M cells had profound delays in PP GC maturation and emergence of lamina propria IgA plasma cells, resulting in diminished levels of fecal SIgA that persisted into adulthood. We conclude that M-cell-mediated sampling of commensal bacteria is a required initial step for the efficient induction of intestinal SIgA.


Subject(s)
B-Lymphocytes/immunology , Gastrointestinal Microbiome/immunology , Germinal Center/immunology , Immunoglobulin A, Secretory/metabolism , Intestinal Mucosa/physiology , Peyer's Patches/immunology , T-Lymphocytes, Helper-Inducer/immunology , Animals , Antigen Presentation , Antigens, Bacterial/immunology , Cell Differentiation , Cells, Cultured , Homeostasis , Immunity, Humoral , Immunity, Mucosal , Mice , Mice, Inbred C57BL , Mice, Knockout , Receptor Activator of Nuclear Factor-kappa B/genetics , Symbiosis
5.
Mucosal Immunol ; 5(3): 288-98, 2012 May.
Article in English | MEDLINE | ID: mdl-22318495

ABSTRACT

Bacterial flagellin is a dominant innate immune activator of the intestine. Therefore, we examined the role of the intracellular flagellin receptor, NLRC4, in protecting the gut and/or driving inflammation. In accordance with NLRC4 acting through transcription-independent pathways, loss of NLRC4 did not reduce the rapid robust changes in intestinal gene expression induced by flagellin administration. Loss of NLRC4 did not alter basal intestinal homeostasis nor predispose mice to development of colitis upon administration of an anti-interleukin (IL)-10R monoclonal antibody. However, epithelial injury induced by dextran sulfate sodium in mice lacking NLRC4 resulted in a more severe disease, indicating a role for NLRC4 in protecting the gut. Moreover, loss of NLRC4 resulted in increased mortality in response to flagellate, but not aflagellate Salmonella infection. Thus, despite not being involved in rapid intestinal gene remodeling upon detection of flagellin, NLRC4-mediated inflammasome activation results in production of IL-1ß and IL-18, two cytokines that protect mice from mucosal and systemic challenges.


Subject(s)
Apoptosis Regulatory Proteins/metabolism , Calcium-Binding Proteins/metabolism , Colitis/immunology , Flagellin/metabolism , Intestinal Mucosa/immunology , Receptors, Cytoplasmic and Nuclear/metabolism , Animals , Apoptosis Regulatory Proteins/genetics , Apoptosis Regulatory Proteins/immunology , Calcium-Binding Proteins/genetics , Calcium-Binding Proteins/immunology , Cells, Cultured , Colitis/chemically induced , Cytokines/genetics , Cytokines/metabolism , Dextran Sulfate/administration & dosage , Disease Models, Animal , Flagellin/genetics , Flagellin/immunology , Humans , Immunity, Innate/genetics , Mice , Mice, Knockout , Receptors, Cytoplasmic and Nuclear/genetics , Receptors, Cytoplasmic and Nuclear/immunology
6.
Mucosal Immunol ; 4(1): 102-11, 2011 Jan.
Article in English | MEDLINE | ID: mdl-20844479

ABSTRACT

Toll-like receptor-5 (TLR5)-mediated detection of flagellin induces nuclear factor (NF)-κB-mediated transcription of host defense gene expression, whereas recognition of intracellular flagellin by interleukin (IL)-1-converting enzyme protease-activation factor (IPAF) results in maturation/secretion of the inflammasome cytokine IL-1ß. The potent effects of IL-1ß are counter-regulated by secretory IL-1 receptor antagonist (sIL-1Ra). We studied the roles of flagellin receptors in regulating the expression of IL-1ß and sIL-1Ra and their subsequent roles in inflammation. Flagellin induced sIL-1Ra in intestinal epithelia and macrophages in a dose- and time-dependent manner, whereas IL-1ß was only induced in macrophages. In vivo, flagellin-induced sIL-1Ra, but not IL-1ß, was absolutely dependent upon TLR5 expressed on non-hemopioetic cells. Thus, loss of TLR5 increased the IL-1ß/sIL-1Ra ratio on flagellin treatment, which correlated with increased inflammatory pathology in response to this product. Furthermore, the flagellin/TLR5 interaction was important for the induction of sIL-1Ra and limiting inflammatory pathology on Salmonella infection. Finally, reduced sIL-1Ra levels in TLR5KO mice correlated with spontaneous colitis. Taken together, we demonstrate that intestinal epithelia, despite not expressing IL-1ß, secrete sIL-1Ra in a TLR5-dependent manner suggesting that loss of TLR5 may promote inflammation by increasing IL-1ß activity. Thus, optimizing the balance between inflammasome cytokines and their endogenous inhibitors might prove a useful strategy to treat inflammatory disorders.


Subject(s)
Flagellin/immunology , Inflammation/immunology , Interleukin 1 Receptor Antagonist Protein/metabolism , Interleukin-1beta/genetics , Toll-Like Receptor 5/immunology , Animals , Caspase 1/metabolism , Cell Line , Colitis/immunology , Enzyme-Linked Immunosorbent Assay , Gene Expression , Gene Expression Regulation , Inflammasomes , Interleukin 1 Receptor Antagonist Protein/genetics , Interleukin-1beta/metabolism , Intestinal Mucosa/immunology , Intestinal Mucosa/metabolism , Macrophages/immunology , Mice , Mice, Inbred C57BL , Mice, Knockout , NF-kappa B/genetics , NF-kappa B/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Salmonella Infections/immunology , Toll-Like Receptor 5/metabolism
7.
Mucosal Immunol ; 3(6): 622-32, 2010 Nov.
Article in English | MEDLINE | ID: mdl-20664578

ABSTRACT

Rotavirus (RV), a leading cause of severe diarrhea, primarily infects intestinal epithelial cells (IECs) causing self-limiting illness. To better understand innate immunity to RV, we sought to define the extent to which IEC activation of anti-viral responses required viral replication or could be recapitulated by inactivated RV or its components. Using model human intestinal epithelia, we observed that RV-induced activation of signaling events and gene expression typically associated with viral infection was largely mimicked by administration of ultraviolet (UV)-inactivated RV. Use of anti-interferon (IFN) neutralizing antibodies revealed that such replication-independent anti-viral gene expression required type I IFN signaling. In contrast, RV-induction of nuclear factor-κB-mediated interleukin-8 expression was dependent on viral replication. The anti-viral gene expression induced by UV-RV was not significantly recapitulated by RV RNA or RV virus-like particles although the latter could enter IEC. Together, these results suggest that RV proteins mediate viral entry into epithelial cells leading to intracellular detection of RV RNA that generates an anti-viral response.


Subject(s)
Interferon Type I/metabolism , Intestinal Mucosa/metabolism , NF-kappa B/metabolism , Rotavirus Infections/immunology , Rotavirus/physiology , Antibodies, Blocking/pharmacology , Cell Line , Gene Expression Regulation, Viral/immunology , Humans , Immunity, Innate , Interferon Type I/immunology , Interleukin-8/biosynthesis , Interleukin-8/genetics , Interleukin-8/immunology , Intestinal Mucosa/drug effects , Intestinal Mucosa/immunology , Intestinal Mucosa/pathology , Intestinal Mucosa/virology , NF-kappa B/immunology , RNA, Viral/immunology , Rotavirus/pathogenicity , Rotavirus Infections/virology , Signal Transduction/drug effects , Signal Transduction/immunology , Ultraviolet Rays , Virion/immunology , Virus Inactivation , Virus Replication
8.
Mucosal Immunol ; 2(3): 197-205, 2009 May.
Article in English | MEDLINE | ID: mdl-19242410

ABSTRACT

The mucosal immune system is charged with defending the host's vast interfaces with the outside world from the enormous and diverse group of microbes that colonizes these surfaces. A key means by which the mucosal immune system protects the host from such diverse microbes is using germ-line-encoded receptors that target structurally conserved motifs that mediate important bacterial functions. This review focuses on one embodiment of this notion, namely, the mucosal innate immune targeting of flagellin, the primary structural component of flagella, which afford bacteria the ability of directed locomotion. Specifically, we discuss the mechanisms by which flagellin is recognized by the innate immune system, their role in host defense, chronic inflammatory disease, and potential approaches to pharmacologically manipulate these pathways to benefit the host. Discussion will focus on the intestinal tract but will also incorporate key findings in other mucosal surfaces.


Subject(s)
Flagellin/immunology , Immunity, Innate , Intestinal Mucosa/immunology , Mucous Membrane/immunology , Animals , Epithelial Cells/immunology , Humans , Intestinal Mucosa/microbiology , Macrophages/immunology , Mucous Membrane/microbiology , Toll-Like Receptor 5/immunology
9.
J Immunol ; 167(4): 1882-5, 2001 Aug 15.
Article in English | MEDLINE | ID: mdl-11489966

ABSTRACT

Flagellin, the structural component of bacterial flagella, is secreted by pathogenic and commensal bacteria. Flagellin activates proinflammatory gene expression in intestinal epithelia. However, only flagellin that contacts basolateral epithelial surfaces is proinflammatory; apical flagellin has no effect. Pathogenic Salmonella, but not commensal Escherichia coli, translocate flagellin across epithelia, thus activating epithelial proinflammatory gene expression. Investigating how epithelia detect flagellin revealed that cell surface expression of Toll-like receptor 5 (TLR5) conferred NF-kappaB gene expression in response to flagellin. The response depended on both extracellular leucine-rich repeats and intracellular Toll/IL-1R homology region of TLR5 as well as the adaptor protein MyD88. Furthermore, immunolocalization and cell surface-selective biotinylation revealed that TLR5 is expressed exclusively on the basolateral surface of intestinal epithelia, thus providing a molecular basis for the polarity of this innate immune response. Thus, detection of flagellin by basolateral TLR5 mediates epithelial-driven inflammatory responses to Salmonella.


Subject(s)
Drosophila Proteins , Flagellin/pharmacology , Gene Expression Regulation , Intestinal Mucosa/metabolism , Intestinal Mucosa/pathology , Membrane Glycoproteins/biosynthesis , Membrane Glycoproteins/genetics , Receptors, Cell Surface/biosynthesis , Receptors, Cell Surface/genetics , Animals , COS Cells , Cell Line , Colon , Gene Expression Regulation/immunology , HeLa Cells , Humans , Inflammation/genetics , Inflammation/immunology , Inflammation/microbiology , Intestinal Mucosa/immunology , Intestinal Mucosa/microbiology , Membrane Glycoproteins/physiology , NF-kappa B/metabolism , Receptors, Cell Surface/physiology , Toll-Like Receptor 5 , Toll-Like Receptors , Transfection
10.
J Clin Invest ; 107(7): 861-9, 2001 Apr.
Article in English | MEDLINE | ID: mdl-11285305

ABSTRACT

Adenosine is formed in the intestinal lumen during active inflammation from neutrophil-derived 5' AMP. Using intestinal epithelial cell line T84, we studied the effect of adenosine on the secretion of IL-6, a proinflammatory cytokine involved in neutrophil degranulation and lymphocyte differentiation. Stimulation of T84 monolayers with either apical or basolateral adenosine induces A2b receptor-mediated increase in IL-6 secretion, which is polarized to the apical (luminal) compartment. In addition, Salmonella typhimurium, TNF-alpha, and forskolin, known inducers of IL-6 secretion in intestinal epithelial cells, also stimulate IL-6 secretion into the apical compartment. We show that IL6 promoter induction by adenosine occurs through cAMP-mediated activation of nuclear cAMP-responsive element-binding protein (CREB). We also show that IL-6 released in the luminal (apical) compartment achieves a sufficient concentration to activate neutrophils (from which the adenosine signal originates), since such IL-6 is found to induce an intracellular [Ca(++)] flux in neutrophils. We conclude that adenosine released in the intestinal lumen during active inflammation may induce IL-6 secretion, which is mediated by cAMP/CREB activation and occurs in an apically polarized fashion. This would allow sequential activation of neutrophil degranulation in the lumen -- a flow of events that would, in an epithelium-dependent fashion, enhance microbicidal activity of neutrophils as they arrive in the intestinal lumen.


Subject(s)
Adenosine/metabolism , Interleukin-6/metabolism , Intestinal Mucosa/metabolism , Neutrophils/metabolism , Signal Transduction/physiology , Activating Transcription Factors , Adenosine/pharmacology , Animals , Blood Proteins/metabolism , COS Cells , Cell Line , Chlorocebus aethiops , Colforsin/metabolism , Colforsin/pharmacology , Cyclic AMP Response Element-Binding Protein/metabolism , Dose-Response Relationship, Drug , Epithelial Cells/cytology , Epithelial Cells/metabolism , Intestinal Mucosa/cytology , Purinergic P1 Receptor Antagonists , Receptor, Adenosine A2B , Receptors, Purinergic P1/metabolism , Salmonella typhimurium/metabolism , Time Factors , Transcription Factors/metabolism , Transcription, Genetic , Tumor Necrosis Factor-alpha/metabolism , Tumor Necrosis Factor-alpha/pharmacology
12.
J Clin Invest ; 107(1): 99-109, 2001 Jan.
Article in English | MEDLINE | ID: mdl-11134185

ABSTRACT

This study investigated whether soluble paracrine factors mediated Salmonella-induced IL-8 expression in polarized model intestinal epithelia. We found that the basolateral media of model epithelia that had been apically infected with Salmonella typhimurium for a short period (10 minutes) could activate IL-8 secretion in virgin model epithelia, demonstrating that a proinflammatory factor (PIF) was indeed present. Initial characterization found that PIF was a heat-stable protein with a molecular mass of about 50 kDa that acts on the basolateral, but not apical, surface of model intestinal epithelia to elicit IL-8 secretion. PIF was not present in the media of model epithelia stimulated with other inducers of IL-8 secretion (TNF-alpha or carbachol) but was present in S. typhimurium supernatants, indicating PIF is of bacterial origin. PIF was purified from bacterial culture supernatants by anion/cation exchange chromatography and SDS-PAGE and found by using microsequencing to be the protein flagellin. In support of this finding, flagellin-deficient S. typhimurium mutants did not secrete detectable levels of PIF (i.e., a bioactivity that induced IL-8 secretion when placed basolaterally on model epithelia). Furthermore, viable flagellin-deficient mutant organisms (fliC/fljB and flhD) failed to elicit IL-8 secretion when added apically to model intestinal epithelia. These findings indicate that translocation of flagellin across epithelia, subsequent to apical epithelial-S. typhimurium interaction, is likely a major means of activating a mucosal inflammatory response.


Subject(s)
Flagellin/metabolism , Inflammation/etiology , Intestinal Mucosa/microbiology , Salmonella typhimurium/pathogenicity , Cell Line , Epithelium/immunology , Epithelium/microbiology , Flagellin/genetics , Humans , Inflammation/immunology , Inflammation/microbiology , Inflammation Mediators/metabolism , Interleukin-8/metabolism , Intestinal Mucosa/immunology , Models, Biological , Mutation , Salmonella typhimurium/genetics , Salmonella typhimurium/physiology
13.
Curr Opin Investig Drugs ; 2(10): 1395-400, 2001 Oct.
Article in English | MEDLINE | ID: mdl-11890354

ABSTRACT

Genetics Institute has developed and launched oprelvekin (rhIL-11; Neumega), a recombinant form of human IL-11. In November 1997, the FDA cleared oprelvekin for the prevention of severe thrombocytopenia and the reduction of the need for platelet transfusions following myelosuppressive chemotherapy in susceptible patients with non-myeloid malignancies 12703021. The product was launched at the end of 1997 [312556]. By December 1999, phase III trials for Crohn's disease (CD) were underway [363007]. Genetics Institute had commenced a 150-patient phase II trial for mild-to-moderate CD and mucositis and the company planned to file regulatory procedures for the indication of CD in 1999 [271210]. An oral formulation for this indication has been developed. Oprelvekin is also undergoing phase I clinical trials for colitis [396157], phase II clinical trials for rheumatoid arthritis [413835] and clinical trials for psoriasis [299644]. In March 1997, Wyeth-Ayerst became the licensee for Europe, Africa, Latin America and Asia (with the exception of Japan). Genetics Institute holds marketing rights for North America [239273]. In Japan, oprelvekin is being developed by Genetics Institute and Yamanouchi; phase III trials have commenced [295049] and were ongoing in May 2001 [411763]. In April 1996, analysts at Yamaichi estimated launch in 2001 and maximum annual sales of over yen 10 billion [215896]. In January 1998, Morgan Stanley Dean Witter predicted Yamanouchi's share of sales to be yen 1 billion in 2001, rising to yen 2 billion in 2002 [315458]. Sales of oprelvekin were US $34 million for Genetics institute in fiscal 2000 while, in July 2001, Credit Suisse First Boston estimated that this figure will be US $30 million and US $34 million in 2001 and 2002, respectively [416883].


Subject(s)
Arthritis, Rheumatoid/drug therapy , Inflammatory Bowel Diseases/drug therapy , Interleukin-11/therapeutic use , Recombinant Proteins/therapeutic use , Thrombocytopenia/drug therapy , Animals , Clinical Trials, Phase I as Topic , Clinical Trials, Phase II as Topic , Humans , Interleukin-11/adverse effects , Interleukin-11/metabolism , Interleukin-11/pharmacology , Interleukin-11/toxicity , Recombinant Proteins/adverse effects , Recombinant Proteins/metabolism , Recombinant Proteins/pharmacology , Recombinant Proteins/toxicity , Structure-Activity Relationship
14.
Curr Opin Investig Drugs ; 2(10): 1401-6, 2001 Oct.
Article in English | MEDLINE | ID: mdl-11890355

ABSTRACT

Alicaforsen (ISIS-2302) is an RNase H-dependent antisense inhibitor of the intercellular adhesion molecule ICAM-1 under development by Isis Pharmaceuticals, for the potential treatment of a variety of inflammatory disorders [175741]. As of April 1997 it was in phase III trials for Crohn's disease (CD); however, the trial failed and, in December 1999, the company suspended development for this indication [352801]. In October 2000, the company re-initiated development in CD [384820] and new phase III trials had begin by May 2001 [409704]. In August 2000, phase II studies of alicaforsen in an enema formulation for ulcerative colitis and a topical formulation for psoriasis were ongoing [378715]. Development of the compound for the potential treatment of rheumatoid arthritis (RA) was discontinued in 1999 [347579]. By the end of 1998, alicaforsen was in phase II trials for kidney transplant rejection. At this time, these trials were expected to finish in mid-1999 [343460]. However, they were ongoing in September 1999, although no further development has been reported for this indication since that time [338672]. In February 1995, Isis Pharmaceuticals and Boehringer Ingelheim (BI) signed a collaborative agreement on cell adhesion inhibitors, including alicaforsen [174111]. By early 1999, Isis and BI were to decide on the next developmental step for alicaforsen following further analyses of its performance against CD [292915], [315439]. Their joint development agreement was terminated in 1999; Isis regained rights to the product and by September 1999 was in talks to license alicaforsen to another partner for CD [338672]. In June 2000, Cytogenix entered into a sponsored research agreement with Baylor College of Medicine at the Texas Medical Center Houston for the use of its ssDNA expression system for the development of antisense strategies directed against intercellular adhesion molecules for the purpose of reducing lung inflammation and injury in disease states and conditions [369677]. US-05514788, and other patents, cover antisense cell adhesion molecule inhibitors [212289], [234792].


Subject(s)
Arthritis, Rheumatoid/drug therapy , Asthma/drug therapy , Colitis, Ulcerative/drug therapy , Crohn Disease/drug therapy , Gastrointestinal Agents/therapeutic use , Graft Rejection/prevention & control , Immunosuppressive Agents/therapeutic use , Oligodeoxyribonucleotides, Antisense/pharmacology , Oligodeoxyribonucleotides, Antisense/therapeutic use , Psoriasis/drug therapy , Thionucleotides/pharmacology , Thionucleotides/therapeutic use , Animals , Clinical Trials, Phase I as Topic , Clinical Trials, Phase II as Topic , Clinical Trials, Phase III as Topic , Gastrointestinal Agents/adverse effects , Gastrointestinal Agents/metabolism , Gastrointestinal Agents/pharmacology , Gastrointestinal Agents/toxicity , Humans , Immunosuppressive Agents/adverse effects , Immunosuppressive Agents/metabolism , Immunosuppressive Agents/pharmacology , Immunosuppressive Agents/toxicity , Oligodeoxyribonucleotides, Antisense/adverse effects , Oligodeoxyribonucleotides, Antisense/metabolism , Oligodeoxyribonucleotides, Antisense/toxicity , Phosphorothioate Oligonucleotides , Structure-Activity Relationship , Thionucleotides/adverse effects , Thionucleotides/metabolism , Thionucleotides/toxicity
15.
Science ; 289(5484): 1560-3, 2000 Sep 01.
Article in English | MEDLINE | ID: mdl-10968793

ABSTRACT

Epithelia of the vertebrate intestinal tract characteristically maintain an inflammatory hyporesponsiveness toward the lumenal prokaryotic microflora. We report the identification of enteric organisms (nonvirulent Salmonella strains) whose direct interaction with model human epithelia attenuate synthesis of inflammatory effector molecules elicited by diverse proinflammatory stimuli. This immunosuppressive effect involves inhibition of the inhibitor kappaB/nuclear factor kappaB (IkappaB/NF-kappaB) pathway by blockade of IkappaB-alpha degradation, which prevents subsequent nuclear translocation of active NF-kappaB dimer. Although phosphorylation of IkappaB-alpha occurs, subsequent polyubiquitination necessary for regulated IkappaB-alpha degradation is completely abrogated. These data suggest that prokaryotic determinants could be responsible for the unique tolerance of the gastrointestinal mucosa to proinflammatory stimuli.


Subject(s)
DNA-Binding Proteins/metabolism , I-kappa B Proteins , Intestinal Mucosa/metabolism , Intestinal Mucosa/microbiology , NF-kappa B/metabolism , Salmonella/physiology , Trans-Activators , Cell Nucleus/metabolism , Cysteine Proteinase Inhibitors/pharmacology , Cytoskeletal Proteins/metabolism , Dimerization , Humans , Inflammation Mediators/pharmacology , Interleukin-8/genetics , Interleukin-8/metabolism , Leupeptins/pharmacology , Ligases/metabolism , NF-KappaB Inhibitor alpha , NF-kappa B/genetics , Phosphorylation , Salmonella/pathogenicity , Salmonella typhimurium/pathogenicity , Salmonella typhimurium/physiology , Transcription Factor RelA , Tumor Cells, Cultured , Tumor Necrosis Factor-alpha/pharmacology , Ubiquitin-Protein Ligases , Ubiquitins/metabolism , beta Catenin
17.
J Clin Invest ; 105(1): 79-92, 2000 Jan.
Article in English | MEDLINE | ID: mdl-10619864

ABSTRACT

Interactions between the enteric pathogen Salmonella typhimurium and the luminal surface of the intestine provoke an acute inflammatory response, mediated in part by epithelial cell secretion of the chemokine IL-8 and other proinflammatory molecules. This study investigated the mechanism by which this pathogen induces IL-8 secretion in physiologically polarized model intestinal epithelia. IL-8 secretion induced by both the prototypical proinflammatory cytokine TNF-alpha and S. typhimurium was NF-kappaB dependent. However, NF-kappaB activation and IL-8 secretion induced by S. typhimurium, but not by TNF-alpha, was preceded by and required an increase in intracellular [Ca(2+)]. Additionally, agonists that increased intracellular [Ca(2+)] by receptor-dependent (carbachol) or independent (thapsigargin, ionomycin) means also induced IL-8 secretion. Furthermore, the ability of S. typhimurium mutants to induce IkappaB-alpha degradation, NF-kappaB translocation, and IL-8 transcription and secretion correlated precisely with their ability to induce an intracellular [Ca(2+)] increase in model intestinal epithelia, but not with their ability to invade these cells. Finally, S. typhimurium, but not TNF-alpha, induced a Ca(2+)-dependent phosphorylation of IkappaB-alpha. These results indicate that S. typhimurium-induced activation of NF-kappaB-dependent epithelial inflammatory responses proceeds by a Ca(2+)-mediated activation of an IkappaB-alpha kinase. These observations raise the possibility that pharmacologic intervention of the acute inflammatory response can be selectively matched to the specific class of initiating event.


Subject(s)
Calcium/physiology , I-kappa B Proteins , Interleukin-8/biosynthesis , Intestinal Mucosa/metabolism , Intestinal Mucosa/microbiology , NF-kappa B/physiology , Salmonella typhimurium/physiology , DNA-Binding Proteins/physiology , Humans , Interleukin-1/pharmacology , NF-KappaB Inhibitor alpha , Phosphorylation , Tumor Necrosis Factor-alpha/pharmacology
18.
Am J Physiol ; 276(4): C988-94, 1999 04.
Article in English | MEDLINE | ID: mdl-10199831

ABSTRACT

The eicosanoid lipoxin A4 (LXA4) is biosynthesized in vivo by cells present at inflammatory sites and appears to be an endogenous anti-inflammatory mediator. Further, in the presence of aspirin, the 15-epimer of LXA4 (15-epi-LXA4) is biosynthesized and may mediate some of aspirin's desirable bioactions. LXA4, 15-epi-LXA4, and their stable analogs inhibit inflammation in established animal models, indicating that these compounds may be useful for treating inflammatory disease states. To investigate the cellular mechanisms by which these lipid mediators downregulate inflammation, we investigated whether these eicosanoids could influence receptor-mediated degranulation of human neutrophils, an event thought to play a major causative role in several inflammatory disease states. LXA4, 15-epi-LXA4, and their stable analogs potently (IC50 < 1 nM) and selectively downregulated neutrophil release of azurophilic granule contents but did not affect other neutrophil secretory functions. Thus the cellular basis of action of these natural off-switches to inflammation appears to involve downregulation of neutrophil azurophilic granule release.


Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Aspirin/pharmacology , Cell Degranulation/drug effects , Hydroxyeicosatetraenoic Acids/pharmacology , Lipoxins , Neutrophils/physiology , Cell Degranulation/physiology , Dose-Response Relationship, Drug , Humans , In Vitro Techniques , Kinetics , Leukocyte Elastase/metabolism , Neutrophils/drug effects , Receptors, IgG/drug effects , Receptors, IgG/physiology , Superoxides/blood
19.
Blood ; 93(5): 1738-48, 1999 Mar 01.
Article in English | MEDLINE | ID: mdl-10029604

ABSTRACT

Analysis of peptide binding to human neutrophils (PMN) using phage display techniques has revealed cell-specific motifs reactive with the PMN surface. Phage libraries displaying either linear 9-mer or cyclic 10-mer and 6-mer peptides were incubated with normal human neutrophils followed by elution of bound phage with low pH (pH 2.2) and non-ionic detergent. Three rounds of selection generated several related peptide sequences that bound with high avidity to PMN. Using the linear 9-mer library, PMN-binding phage expressed peptides with the motif (G/A)PNLTGRW. The binding of phage bearing this motif was highly specific since no binding was observed on lymphocytes, fibroblasts, epithelial, or endothelial cells. Functional assays revealed that phage bearing the sequence FGPNLTGRW induced a pertussis toxin-sensitive increase in PMN cytosolic calcium analogous to that observed with Galphai coupled receptors. Other prominent motifs identified included phage bearing the consensus DLXTSK(M/L)X(V/I/L), where X represents a non-conserved position. Phage with this motif bound exclusively to a sub population of human PMN that comprised approximately 50% of the total and did not elicit a calcium response. The binding of such phage to PMN was prevented by co-incubation with competing peptides displaying identical or similar sequences (IC50 range from 0.6 micromol/L to 50 micromol/L for DLXTSK and GPNLTG, respectively). We speculate that these techniques will be useful in identifying functional cell-specific binding motifs and contribute to the development of new therapeutic and diagnostic strategies in human disease.


Subject(s)
Neutrophils/metabolism , Peptides/metabolism , Amino Acid Sequence , Bacteriophages , Calcium/metabolism , Humans , Molecular Sequence Data , Peptide Library , Peptides/genetics , Protein Binding , Sequence Analysis
20.
Infect Immun ; 67(2): 608-17, 1999 Feb.
Article in English | MEDLINE | ID: mdl-9916066

ABSTRACT

Intestinal epithelial cells respond to Salmonella typhimurium by internalizing this pathogen and secreting, in a polarized manner, an array of chemokines which direct polymorphonuclear leukocyte (PMN) movement. Notably, interleukin-8 (IL-8) is secreted basolaterally and directs PMN through the lamina propria, whereas pathogen-elicited epithelial chemoattractant (PEEC) is secreted apically and directs PMN migration across the epithelial monolayer to the intestinal lumen. While most studies of S. typhimurium pathogenicity have focused on the mechanism by which this bacterium invades its host, the enteritis characteristically associated with salmonellosis appears to be more directly attributable to the PMN movement that occurs in response to this pathogen. Therefore, we sought to better understand the relationship between S. typhimurium invasion and epithelial promotion of PMN movement. First, we investigated whether S. typhimurium becoming intracellular was necessary or sufficient to induce epithelial promotion of PMN movement. Blocking S. typhimurium invasion by preventing, with cytochalasin D, the epithelial cytoskeletal rearrangements which mediate internalization did not reduce the epithelial promotion of PMN movement. Conversely, bacterial attainment of an intracellular position was not sufficient to induce model epithelia to direct PMN transmigration, since neither basolateral invasion by S. typhimurium nor apical internalization of an invasion-deficient mutant (achieved by inducing membrane ruffling with epidermal growth factor) induced this epithelial cell response. These results indicate that specific interactions between the apical surface of epithelial cells and S. typhimurium, rather than simply bacterial invasion, mediate the epithelial direction of PMN transmigration. To further investigate the means by which S. typhimurium induces epithelia to direct PMN movement, we investigated whether the same signaling pathways regulate secretion of IL-8 and PEEC. IL-8 secretion, but not PEEC secretion, was activated by phorbol myristate acetate and blocked by an inhibitor (mg-132) of the proteosome which mediates NF-kappabeta activation. Further, secretion of IL-8, but not PEEC, was activated by an entry-deficient (HilDelta) S. typhimurium mutant or by basolateral invasion of a wild-type strain. Together, these results indicate that distinct signaling pathways mediate S. typhimurium invasion, induction of IL-8 secretion, and induction of PEEC secretion in model intestinal epithelia.


Subject(s)
Intestinal Mucosa/immunology , Neutrophils/immunology , Salmonella typhimurium/immunology , Animals , Cell Movement , Cells, Cultured , Chemokines/metabolism , Epithelial Cells/immunology , Humans , Interleukin-8/metabolism , Intestinal Mucosa/cytology , Neutrophils/cytology , Rabbits , Signal Transduction
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