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1.
Osteoarthritis Cartilage ; 26(8): 1098-1109, 2018 08.
Article in English | MEDLINE | ID: mdl-29857156

ABSTRACT

OBJECTIVE: To determine the contribution of the gut microbiota to the development of injury-induced osteoarthritis (OA). DESIGN: OA was induced using the destabilized medial meniscus (DMM) model in 20 germ-free (GF) C57BL/6J male mice housed in a gnotobiotic facility and 23 strain-matched specific pathogen free (SPF) mice in 2 age groups -13.5 weeks avg age at DMM (17 SPF and 15 GF) and 43 weeks avg age at DMM (6 SPF and 5 GF). OA severity was measured using scores for articular cartilage structure (ACS), loss of safranin O (SafO) staining, osteophyte size, and synovial hyperplasia. Microbiome analysis by 16S rRNA amplicon sequencing was performed on stool samples and LPS and LPS binding protein (LBP) were measured in plasma. RESULTS: Compared to the SPF DMM mice, the maximum (MAX) ACS score per joint was 28% lower (p = 0.036) in GF DMM mice while the SafO sum score of all sections evaluated per joint was decreased by 31% (p = 0.009). The differences between SPF and GF mice in these scores were greater when only the younger mice were included in the analysis. The younger GF DMM mice also had significant reductions in osteophyte size (36%, P = 0.0119) and LBP (27%, P = 0.007) but not synovial scores or LPS. Differences in relative abundance of a number of Operational Taxonomic Units (OTUs) were noted between SPF mice with high vs low maximum ACS scores. CONCLUSIONS: These results suggest factors related to the gut microbiota promote the development of OA after joint injury.


Subject(s)
Gastrointestinal Microbiome , Osteoarthritis/etiology , Tibial Meniscus Injuries/complications , Acute-Phase Proteins , Animals , Carrier Proteins/blood , Cartilage, Articular/pathology , Disease Models, Animal , Gastrointestinal Microbiome/genetics , Germ-Free Life , Interleukin-6/blood , Lipopolysaccharides/blood , Male , Membrane Glycoproteins/blood , Menisci, Tibial/pathology , Mice , Mice, Inbred C57BL , Osteoarthritis/microbiology , RNA, Ribosomal, 16S/genetics
2.
Mucosal Immunol ; 11(1): 120-130, 2018 01.
Article in English | MEDLINE | ID: mdl-28401939

ABSTRACT

The multidrug resistance-1 (MDR1) gene encodes an ATP-dependent efflux transporter that is highly expressed in the colon. In mice, loss of MDR1 function results in colitis with similarities to human inflammatory bowel diseases (IBD). Here, we show that MDR1 has an unexpected protective role for the mitochondria where MDR1 deficiency results in mitochondrial dysfunction with increased mitochondrial reactive oxygen species (mROS) driving the development of colitis. Exogenous induction of mROS accelerates, while inhibition attenuates colitis in vivo; these effects are amplified in MDR1 deficiency. In human IBD, MDR1 is negatively correlated to SOD2 gene expression required for mROS detoxification. To provide direct evidential support, we deleted intestinal SOD2 gene in mice and showed an increased susceptibility to colitis. We exploited the genome-wide association data sets and found many (∼5%) of IBD susceptibility genes with direct roles in regulating mitochondria homeostasis. As MDR1 primarily protects against xenotoxins via its efflux function, our findings implicate a distinct mitochondrial toxin+genetic susceptibility interaction leading to mitochondrial dysfunction, a novel pathogenic mechanism that could offer many new therapeutic opportunities for IBD.


Subject(s)
ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism , Colitis/genetics , Inflammation/genetics , Inflammatory Bowel Diseases/genetics , Intestines/immunology , Mitochondria/physiology , Superoxide Dismutase/genetics , Animals , Disease Models, Animal , Genetic Predisposition to Disease , Homeostasis , Humans , Metabolic Detoxication, Phase I/genetics , Mice , Mice, Inbred C57BL , Mice, Knockout , Reactive Oxygen Species/metabolism
3.
Clin Transl Gastroenterol ; 7(8): e187, 2016 Aug 25.
Article in English | MEDLINE | ID: mdl-27560928

ABSTRACT

OBJECTIVES: Rifaximin has clinical benefits in minimal hepatic encephalopathy (MHE) but the mechanism of action is unclear. The antibiotic-dependent and -independent effects of rifaximin need to be elucidated in the setting of MHE-associated microbiota. To assess the action of rifaximin on intestinal barrier, inflammatory milieu and ammonia generation independent of microbiota using rifaximin. METHODS: Four germ-free (GF) mice groups were used (1) GF, (2) GF+rifaximin, (3) Humanized with stools from an MHE patient, and (4) Humanized+rifaximin. Mice were followed for 30 days while rifaximin was administered in chow at 100 mg/kg from days 16-30. We tested for ammonia generation (small-intestinal glutaminase, serum ammonia, and cecal glutamine/amino-acid moieties), systemic inflammation (serum IL-1ß, IL-6), intestinal barrier (FITC-dextran, large-/small-intestinal expression of IL-1ß, IL-6, MCP-1, e-cadherin and zonulin) along with microbiota composition (colonic and fecal multi-tagged sequencing) and function (endotoxemia, fecal bile acid deconjugation and de-hydroxylation). RESULTS: All mice survived until day 30. In the GF setting, rifaximin decreased intestinal ammonia generation (lower serum ammonia, increased small-intestinal glutaminase, and cecal glutamine content) without changing inflammation or intestinal barrier function. Humanized microbiota increased systemic/intestinal inflammation and endotoxemia without hyperammonemia. Rifaximin therapy significantly ameliorated these inflammatory cytokines. Rifaximin also favorably impacted microbiota function (reduced endotoxin and decreased deconjugation and formation of potentially toxic secondary bile acids), but not microbial composition in humanized mice. CONCLUSIONS: Rifaximin beneficially alters intestinal ammonia generation by regulating intestinal glutaminase expression independent of gut microbiota. MHE-associated fecal colonization results in intestinal and systemic inflammation in GF mice, which is also ameliorated with rifaximin.

4.
Aliment Pharmacol Ther ; 43 Suppl 1: 27-36, 2016 Jan.
Article in English | MEDLINE | ID: mdl-26618923

ABSTRACT

BACKGROUND: Gut microbiota dysbiosis contributes to the pathogenesis of inflammatory bowel diseases (IBD). Although the microbiota's role in IBD pathogenesis, specifically Crohn's disease (CD), provides a rationale for antibiotic treatment, antibiotic use in CD remains controversial. Rifaximin, traditionally identified as a nonsystemic bactericidal antibiotic, may be therapeutically beneficial for inducing CD remission. AIM: To examine the role of rifaximin in the management of IBD and its potential mechanisms of action. METHODS: A literature search using the following strategy: ('inflammatory bowel disease' OR 'Crohn's' OR 'ulcerative'), 'rifaximin' AND ('barrier' OR 'translocation' OR 'adhesion' OR 'internalization' OR 'pregnane X'), AND 'pregnane X' AND ('Crohn's' OR 'ulcerative colitis' OR 'inflammatory bowel disease'). RESULTS: In vitro data suggest rifaximin mediates changes in epithelial cell physiology and reduces bacterial attachment and internalisation. In experimental colitis models, rifaximin antagonised the effects of tumour necrosis factor-α on intestinal epithelial cells by activating pregnane X receptor, which inhibits nuclear factor-κB-mediated proinflammatory mediators and induces detoxification genes (e.g. multidrug resistance 1 and cytochrome P450 3A4). Rifaximin also inhibits bacterial translocation into the mesenteric lymph nodes. CONCLUSION: Accumulating evidence suggests that mechanisms of action of rifaximin in IBD may not be limited to direct bactericidal activity; therefore, rifaximin could potentially be redefined as a gut environment modulator.


Subject(s)
Anti-Bacterial Agents/therapeutic use , Inflammatory Bowel Diseases/drug therapy , Rifamycins/pharmacology , Rifamycins/therapeutic use , Colitis, Ulcerative/drug therapy , Crohn Disease/drug therapy , Gastrointestinal Microbiome/drug effects , Humans , Intestinal Mucosa/pathology , NF-kappa B/drug effects , Pregnane X Receptor , Receptors, Steroid/drug effects , Rifaximin , Tumor Necrosis Factor-alpha/drug effects
5.
Mucosal Immunol ; 7(6): 1452-66, 2014 Nov.
Article in English | MEDLINE | ID: mdl-24850428

ABSTRACT

Loss of intestinal epithelial cell (IEC) homeostasis and apoptosis negatively affect intestinal barrier function. Uncontrolled activation of the unfolded protein response (UPR) in IEC contributes to an impaired barrier and is implicated in the pathogenesis of inflammatory bowel diseases. However, the contribution of the UPR target gene C/EBP homologous protein (CHOP), an apoptosis-associated transcription factor, to inflammation-related disease susceptibility remains unclear. Consistent with observations in patients with ulcerative colitis, we show that despite UPR activation in the epithelium, CHOP expression was reduced in mouse models of T-cell-mediated and bacteria-driven colitis. To elucidate the molecular mechanisms of IEC-specific CHOP expression, we generated a conditional transgenic mouse model (Chop(IEC Tg/Tg)). Chop overexpression increased the susceptibility toward dextran sodium sulfate (DSS)-induced intestinal inflammation and mucosal tissue injury. Furthermore, a delayed recovery from DSS-induced colitis and impaired closure of mechanically induced mucosal wounds was observed. Interestingly, these findings seemed to be independent of CHOP-mediated apoptosis. In vitro and in vivo cell cycle analyses rather indicated a role for CHOP in epithelial cell proliferation. In conclusion, these data show that IEC-specific overexpression impairs epithelial cell proliferation and mucosal tissue regeneration, suggesting an important role for CHOP beyond mediating apoptosis.


Subject(s)
Apoptosis/immunology , Cell Cycle/immunology , Colitis, Ulcerative/immunology , Intestinal Mucosa/physiology , Regeneration/immunology , Transcription Factor CHOP/immunology , Animals , Apoptosis/genetics , Cell Cycle/genetics , Colitis, Ulcerative/genetics , Colitis, Ulcerative/pathology , Disease Models, Animal , Mice , Mice, Transgenic , Regeneration/genetics , Transcription Factor CHOP/genetics , Unfolded Protein Response/genetics , Unfolded Protein Response/immunology
6.
Mucosal Immunol ; 4(2): 127-32, 2011 Mar.
Article in English | MEDLINE | ID: mdl-21248723

ABSTRACT

Co-evolution with an extremely complex commensal enteric microbiota has helped shape mammalian mucosal immune responses. A yet incompletely defined subset of intestinal bacteria is required to stimulate chronic, immune-mediated intestinal inflammation, including human Crohn's disease, and intestinal microbiota composition is altered in a characteristic manner by the inflammatory response to create a dysbiotic relationship of protective vs. aggressive bacteria. We pose a number of questions regarding host interactions with the enteric microbiota, including influences of inflammation, host genetics, early environmental exposure, and diet on microbial composition and function, and conversely, the effect of bacterial metabolism, enteric fungi and viruses, and endogenous protective bacterial species on host immune and inflammatory responses. These questions are designed to stimulate research that will promote a better understanding of host-microbial interactions in the intestine and promote targeted novel therapeutic interventions.


Subject(s)
Inflammation/immunology , Inflammation/microbiology , Intestines/immunology , Intestines/microbiology , Metagenome/immunology , Animals , Bacteria/immunology , Fungi/immunology , Humans , Immunity, Mucosal/genetics , Immunity, Mucosal/immunology , Inflammation/genetics , Prebiotics , Probiotics/metabolism , Viruses/immunology
7.
J Infect Dis ; 202(11): 1708-12, 2010 Dec 01.
Article in English | MEDLINE | ID: mdl-20977342

ABSTRACT

The increased incidence and severity of Clostridium difficile infection (CDI) in older adults (age, ≥65 years) corresponds with the emergence of the BI/NAP1 strain, making elucidation of the host immune response extremely important. We therefore infected germ-free C57BL/6 mice aged 7-14 months with a BI/NAP1 strain and monitored the mice for response. Infected mice were moribund 48-72 h after infection and developed gross and histological cecitis and colitis and elevated concentrations of keratinocyte chemoattractant, interleukin 1ß, monocyte chemotactic protein 1, and granulocyte colony-stimulating factor and decreased levels of interferon γ, interleukin 12 p40, interleukin 12 p70, and interleukin 10 compared with controls. We conclude that aged, germ-free C57BL/6 mice are susceptible to fulminant CDI from a BI/NAP1 strain and represent a novel model to further elucidate the host immune response to acute CDI.


Subject(s)
Clostridioides difficile/immunology , Clostridioides difficile/pathogenicity , Enterocolitis, Pseudomembranous/microbiology , Animals , Clostridioides difficile/classification , Colon/microbiology , Colon/pathology , Disease Models, Animal , Enterocolitis, Pseudomembranous/immunology , Enterocolitis, Pseudomembranous/pathology , Germ-Free Life , Granulocyte Colony-Stimulating Factor/analysis , Interferon-gamma/analysis , Interleukin-10/analysis , Interleukin-12/analysis , Interleukin-12 Receptor beta 1 Subunit/analysis , Interleukin-1beta/analysis , Mice , Mice, Inbred C57BL
8.
Clin Exp Rheumatol ; 27(4): 580-6, 2009.
Article in English | MEDLINE | ID: mdl-19772788

ABSTRACT

OBJECTIVES: This report documents a multimodal nanoparticle (MNP) contrast agent, containing embedded luminophores and surface-immobilized gadolinium chelates, as a contrast agent of inflamed synovium in a collagen induced arthritis (CIA) model. METHODS: DBA-1J mice were immunized for CIA and imaged after disease onset by two independent modalities. After intravenous administration of MNP contrast, optical and magnetic resonance images were obtained and clinical disease was scored, which was followed by processing of hindlimbs for immunofluorescence and confocal microscopy. RESULTS: We show a correlation between disease severity and MNP optical luminescence that is dose dependent. Immunofluorescence of hindlimb sections reveal that MNP-labeled cells are monocytes/macrophages within the inflamed synovium. Magnetic resonance (MR) relaxation time maps, which determine the quantitative measure of T1 and T2 values at each imaging voxel, demonstrated a decreasing T2 signal in actively inflamed joints that was more pronounced earlier rather than later during disease. CONCLUSIONS: MNPs containing surface-immobilized gadolinium chelates and embedded luminophores are potential dual-modality contrast agents in inflammatory arthritis and localize to monocytes/macrophages within inflamed synovium.


Subject(s)
Arthritis, Experimental/diagnosis , Contrast Media , Magnetic Resonance Imaging/methods , Metal Nanoparticles , Animals , Arthritis, Experimental/pathology , Contrast Media/chemistry , Edema/pathology , Fluorescent Antibody Technique , Gadolinium/chemistry , Hindlimb/drug effects , Hindlimb/pathology , Injections, Intravenous , Macrophages/drug effects , Macrophages/pathology , Metal Nanoparticles/chemistry , Mice , Mice, Inbred DBA , Microscopy, Confocal , Monocytes/drug effects , Monocytes/pathology , Synovitis/chemically induced , Synovitis/pathology
9.
Am J Physiol Gastrointest Liver Physiol ; 295(5): G1079-91, 2008 Nov.
Article in English | MEDLINE | ID: mdl-18818316

ABSTRACT

Curcumin (diferulolylmethane) demonstrates profound anti-inflammatory effects in intestinal epithelial cells (IEC) and in immune cells in vitro and exhibits a protective role in rodent models of chemically induced colitis, with its presumed primary mechanism of action via inhibition of NF-kappaB. Although it has been demonstrated effective in reducing relapse rate in ulcerative colitis patients, curcumin's effectiveness in Crohn's disease (CD) or in Th-1/Th-17 mediated immune models of CD has not been evaluated. Therefore, we investigated the effects of dietary curcumin (0.1-1%) on the development of colitis, immune activation, and in vivo NF-kappaB activity in germ-free IL-10(-/-) or IL-10(-/-);NF-kappaB(EGFP) mice colonized with specific pathogen-free microflora. Proximal and distal colon morphology showed a mild protective effect of curcumin only at 0.1%. Colonic IFN-gamma and IL-12/23p40 mRNA expression followed similar pattern ( approximately 50% inhibition at 0.1%). Secretion of IL-12/23p40 and IFN-gamma by colonic explants and mesenteric lymph node cells was elevated in IL-10(-/-) mice and was not decreased by dietary curcumin. Surprisingly, activation of NF-kappaB in IL-10(-/-) mice (phospho-NF-kappaBp65) or in IL-10(-/-);NF-kappaB(EGFP) mice (whole organ or confocal imaging) was not noticeably inhibited by curcumin. Furthermore, we demonstrate that IL-10 and curcumin act synergistically to downregulate NF-kappaB activity in IEC and IL-12/23p40 production by splenocytes and dendritic cells. In conclusion, curcumin demonstrates limited effectiveness on Th-1 mediated colitis in IL-10(-/-) mice, with moderately improved colonic morphology, but with no significant effect on pathogenic T cell responses and in situ NF-kappaB activity. In vitro studies suggest that the protective effects of curcumin are IL-10 dependent.


Subject(s)
Colitis/drug therapy , Curcumin/pharmacology , Diet , Interleukin-10/genetics , Th1 Cells/physiology , Animals , Colitis/microbiology , Colitis/pathology , Colon/pathology , Dose-Response Relationship, Drug , Epithelial Cells/drug effects , Epithelial Cells/metabolism , Gene Expression Regulation , Immunohistochemistry , Interleukin-10/metabolism , Intestinal Mucosa/cytology , Mice , Mice, Knockout , NF-kappa B/genetics , NF-kappa B/metabolism , Specific Pathogen-Free Organisms , Spleen/cytology , Th1 Cells/drug effects
10.
J Intern Med ; 263(6): 597-606, 2008 Jun.
Article in English | MEDLINE | ID: mdl-18479259

ABSTRACT

Enteric microbiota can contribute to Crohn's disease and ulcerative colitis in several ways. Pathogenic or functionally altered commensal bacteria with increased mucosal adherence, invasion and intracellular persistence can activate pathogenic T cells and chronic intestinal inflammation. Compositional changes in intestinal microbiota can lead to decreased protective and increased aggressive species. Genetic polymorphisms resulting in increased mucosal permeability, decreased microbial killing, ineffective clearance of bacteria, biased TH1 and TH17 immune responses and loss of immunological tolerance are probably key contributors to IBD. Future therapies for these heterogeneous diseases should be individualized based on the patient-specific subset.


Subject(s)
Antibiosis/genetics , Enterobacteriaceae/pathogenicity , Inflammatory Bowel Diseases/genetics , Inflammatory Bowel Diseases/microbiology , Mutation , Enterobacteriaceae/physiology , Enterobacteriaceae Infections/genetics , Enterobacteriaceae Infections/immunology , Humans , Immunity, Mucosal , Inflammatory Bowel Diseases/immunology
11.
Clin Exp Immunol ; 143(3): 474-83, 2006 Mar.
Article in English | MEDLINE | ID: mdl-16487247

ABSTRACT

HLA-B27 transgenic (TG) rats develop spontaneous colitis when colonized with intestinal bacteria, whereas athymic nude (rnu/rnu) HLA-B27 TG rats remain disease free. The present study was designed to determine whether or not HLA-B27 expression on T cells is required for development of colitis after transfer of mesenteric lymph node (MLN) cells into rnu/rnu HLA-B27 recipients. Athymic nontransgenic (non-TG) and HLA-B27 TG recipients received MLN cells from either TG or non-TG rnu/+ heterozygous donor rats that contain T cells. HLA-B27 TG rnu/rnu recipients receiving either non-TG or TG MLN cells developed severe colitis and had higher caecal MPO and IL-1beta levels, and their MLN cells produced more IFN-gamma and less IL-10 after in vitro stimulation with caecal bacterial lysate compared to rnu/rnu non-TG recipients that remained disease free after receiving either TG or non-TG cells. Interestingly, proliferating donor TG T cells were detectable one week after adoptive transfer into rnu/rnu TG recipients but not after transfer into non-TG recipients. T cells from either non-TG or TG donors induce colitis in rnu/rnu TG but not in non-TG rats, suggesting that activation of effector T cells by other cell types that express HLA-B27 is pivotal for the pathogenesis of colitis in this model.


Subject(s)
Colitis/etiology , HLA-B27 Antigen/metabolism , Adoptive Transfer , Animals , Animals, Genetically Modified , Bacteria/immunology , Cecum/immunology , Cecum/microbiology , Cell Extracts/immunology , Cell Proliferation , Colitis/immunology , Colitis/microbiology , Colitis/pathology , Cytokines/biosynthesis , Disease Models, Animal , HLA-B27 Antigen/genetics , Interleukin-1/immunology , Lymphocyte Activation , Lymphocyte Transfusion , Mesentery , Peroxidase/metabolism , Rats , Rats, Inbred F344 , Rats, Nude
12.
Gut ; 54(2): 207-14, 2005 Feb.
Article in English | MEDLINE | ID: mdl-15647183

ABSTRACT

BACKGROUND AND AIMS: Erroneous thymic selection of developing T lymphocytes may be responsible for the expansion of self reactive T cells or may contribute to the absence of regulatory T cells important in controlling peripheral inflammatory processes. Colitis in bone marrow (BM) transplanted Tgepsilon26 mice is induced by abnormally activated T cells developing in an aberrant thymic microenvironment. We investigated the protective role of regulatory CD4+CD25+ T cells in this model. METHODS: BM from (C57BL/6 x CBA/J) F1 mice was transplanted into specific pathogen free Tgepsilon26 mice (BM-->Tgepsilon26). Transplanted mice received no cells (control), sorted CD4+CD25+, or CD4+CD25- cells from mesenteric lymph nodes (MLN) of normal mice. MLN cell subsets were analysed using membrane markers. Cytokine secretion of MLN cells was measured using intracellular cytokine staining and cytokine secretion in anti-CD3 stimulated cell cultures. Colitis was measured by histological scores. RESULTS: CD4+CD25+ cells were reduced in the MLNs of BM-->Tgepsilon26 mice. Transfer of regulatory CD4CD4+CD25+ but not of CD4+CD25- cells reduced the number of MLN CD4+ T cells in BM-->Tgepsilon26 recipients and increased the number of MLN CD8+ cells, thereby normalising the CD4+/CD8+ ratio. CD4+CD25+ but not CD4+CD25- cell transfer into BM-->Tgepsilon26 mice reduced the number of tumour necrosis factor alpha+ CD4+ cells and increased the secretion of transforming growth factor beta by MLN cells. Transfer of 3 x 10(5) CD4+CD25+ cells after BM transplantation into Tgepsilon26 mice prevented colitis whereas CD4+CD25- cells had no protective effect. CONCLUSIONS: These results suggest that defective selection or induction of regulatory T cells in the abnormal thymus is responsible for the development of colitis in BM-->Tgepsilon26 mice. Transfer of CD4+CD25+ cells can control intestinal inflammation in BM-->Tgepsilon26 mice by normalising the number and function of the MLN T cell pool.


Subject(s)
Bone Marrow Transplantation/immunology , CD4-Positive T-Lymphocytes/immunology , Colitis/prevention & control , Lymphocyte Transfusion , T-Lymphocyte Subsets/immunology , Animals , Bone Marrow Transplantation/adverse effects , CD8-Positive T-Lymphocytes/immunology , Colitis/immunology , Colitis/pathology , Lymph Nodes/immunology , Lymphocyte Activation/immunology , Mice , Mice, Inbred C57BL , Mice, Inbred CBA , Receptors, Interleukin-2/analysis , Tumor Necrosis Factor-alpha/biosynthesis
13.
Immunology ; 112(2): 310-20, 2004 Jun.
Article in English | MEDLINE | ID: mdl-15147574

ABSTRACT

We have previously shown that non-pathogenic Gram negative bacteria induce RelA phosphorylation, nuclear factor (NF)-kappaB transcriptional activity and pro-inflammatory gene expression in intestinal epithelial cells (IEC) in vivo and in vitro. In this study, we investigated the molecular mechanism of immune-epithelial cell cross-talk on Gram-negative enteric bacteria-induced NF-kappaB signalling and pro-inflammatory gene expression in IEC using HT-29/MTX as well as CaCO-2 transwell cultures Interestingly, while differentiated HT-29/MTX cells are unresponsive to non-pathogenic Gram negative bacterial stimulation, interleukin-8 (IL-8) mRNA accumulation is strongly induced in Escherichia coli- but not Bacteroides vulgatus-stimulated IEC cocultured with peripheral blood (PBMC) and lamina propria mononuclear cells (LPMC). The presence of PBMC triggered both E. coli- and B. vulgatus-induced mRNA expression of the Toll-like receptor-4 accessory protein MD-2 as well as endogenous IkappaBalpha phosphorylation, demonstrating similar capabilities of these bacteria to induce proximal NF-kappaB signalling. However, B. vulgatus failed to trigger IkappaBalpha degradation and NF-kappaB transcriptional activity in the presence of PBMC. Interestingly, B. vulgatus- and E. coli-derived lipopolysaccharide-induced similar IL-8 mRNA expression in epithelial cells after basolateral stimulation of HT-29/PBMC cocultures. Although luminal enteric bacteria have adjuvant and antigenic properties in chronic intestinal inflammation, PBMC from patients with active ulcerative colitis and Crohn's disease differentially trigger epithelial cell activation in response to E. coli and E. coli-derived LPS. In conclusion, this study provides evidence for a differential regulation of non-pathogenic Gram-negative bacteria-induced NF-kappaB signalling and IL-8 gene expression in IEC cocultured with immune cells and suggests the presence of mechanisms that assure hyporesponsiveness of the intestinal epithelium to certain commensally enteric bacteria.


Subject(s)
Gram-Negative Bacteria/immunology , Inflammatory Bowel Diseases/immunology , Intestinal Mucosa/immunology , NF-kappa B/metabolism , Animals , Antigens, Bacterial/immunology , Bacteroides/immunology , Caco-2 Cells , Cells, Cultured , Coculture Techniques , Epithelial Cells/immunology , Escherichia coli/immunology , Gene Expression Regulation/immunology , Humans , Immunity, Mucosal , Inflammatory Bowel Diseases/microbiology , Interleukin-8/biosynthesis , Interleukin-8/genetics , Leukocytes, Mononuclear/immunology , Phosphorylation , RNA, Messenger/genetics , Signal Transduction/immunology
14.
Clin Exp Immunol ; 136(1): 30-9, 2004 Apr.
Article in English | MEDLINE | ID: mdl-15030511

ABSTRACT

Germ-free HLA-B27 transgenic (TG) rats do not develop colitis, but colonization with specific pathogen-free (SPF) bacteria induces colitis accompanied by immune activation. To study host-dependent immune responses to commensal caecal bacteria we investigated cytokine profiles in mesenteric lymph node (MLN) cells from HLA-B27 TG versus nontransgenic (non-TG) littermates after in vitro stimulation with caecal bacterial lysates (CBL). Supernatants from CBL-stimulated unseparated T- or B- cell-depleted MLN cells from HLA-B27 TG and non-TG littermates were analysed for IFN-gamma, IL-12, TNF, IL-10 and TGF-beta production. Our results show that unfractionated TG MLN cells stimulated with CBL produced more IFN-gamma, IL-12 and TNF than did non-TG MLN cells. In contrast, CBL-stimulated non-TG MLN cells produced more IL-10 and TGF-beta. T cell depletion abolished IFN-gamma and decreased IL-12 production, but did not affect IL-10 and TGF-beta production. Conversely, neither IL-10 nor TGF-beta was produced in cultures of B cell-depleted MLN. In addition, CD4(+) T cells enriched from MLN of HLA-B27 TG but not from non-TG rats produced IFN-gamma when cocultured with CBL-pulsed antigen presenting cells from non-TG rats. Interestingly, IL-10 and TGF-beta, but not IFN-gamma, IL-12 and TNF were produced by MLN cells from germ-free TG rats. These results indicate that the colitis that develops in SPF HLA-B27 TG rats is accompanied by activation of IFN-gamma-producing CD4(+) T cells that respond to commensal bacteria. However, B cell cytokine production in response to components of commensal intestinal microorganisms occurs in the absence of intestinal inflammation.


Subject(s)
Bacteria/immunology , Colitis/microbiology , Cytokines/biosynthesis , HLA-B27 Antigen/genetics , Animals , Animals, Genetically Modified , Antigen-Presenting Cells/immunology , B-Lymphocytes/immunology , Cecum/microbiology , Cecum/pathology , Cells, Cultured , Colitis/genetics , Colitis/immunology , Cytokines/genetics , Gene Expression , Germ-Free Life , Lymph Nodes/immunology , RNA, Messenger/genetics , Rats , Rats, Inbred F344 , T-Lymphocyte Subsets/immunology
15.
Gut ; 52(12): 1721-7, 2003 Dec.
Article in English | MEDLINE | ID: mdl-14633949

ABSTRACT

BACKGROUND AND AIMS: Multiple rodent models implicate resident intestinal bacteria in the pathogenesis of chronic immune mediated intestinal inflammation. Specific pathogen free (SPF) interleukin 10 gene deficient (IL-10(-/-)) mice develop colitis, which does not occur in the germ free (GF) state. We investigated whether broad or narrow spectrum antibiotics affect onset and progression of disease in various regions of IL-10(-/-) mice. METHODS: Metronidazole, ciprofloxacin, vancomycin-imipenem (50 mg/kg/day), or water (control) was administered orally before (prevention) or two weeks after (treatment) colonisation of GF IL-10(-/-) mice with SPF bacteria. After four weeks, colonic histology scores and cytokine production by colonic explants were determined. Caecal and colonic contents were collected for quantitative bacterial analysis. RESULTS: In the prevention study, all antibiotics decreased inflammation in the caecum and colon. However, in the treatment study, ciprofloxacin and vancomycin-imipenem decreased caecal inflammation, and reduced Escherichia coli and Enterococcus faecalis concentrations, whereas only vancomycin-imipenem lowered direct microscopic bacterial counts. In contrast, metronidazole and vancomycin-imipenem reduced colonic injury and eliminated anaerobic bacteria, including Bacteroides spp. CONCLUSIONS: Both narrow and broad spectrum antibiotics can prevent disease but treatment of established colitis is more selective. Ciprofloxacin is most effective in the treatment of caecal inflammation, metronidazole preferentially treats the colon, whereas vancomycin-imipenem definitively treats both regions. These results suggest that subsets of aerobic or anaerobic bacteria show regional differences in their capacity to mediate experimental colitis in IL-10(-/-) mice.


Subject(s)
Anti-Bacterial Agents/therapeutic use , Bacteria, Aerobic , Bacteria, Anaerobic , Bacterial Infections/prevention & control , Colitis/prevention & control , Animals , Cecum/microbiology , Colitis/microbiology , Colitis/pathology , Colon/microbiology , Gastroenteritis/microbiology , Gastroenteritis/pathology , Gastroenteritis/prevention & control , Interleukin-10/deficiency , Interleukin-10/genetics , Interleukin-10/metabolism , Mice , Mice, Inbred C57BL
16.
Gut ; 52(3): 370-6, 2003 Mar.
Article in English | MEDLINE | ID: mdl-12584218

ABSTRACT

BACKGROUND AND AIMS: Bacteroides vulgatus induces colitis in gnotobiotic HLA-B27 transgenic (TG) rats while broad spectrum antibiotics prevent and treat colitis in specific pathogen free (SPF) TG rats although disease recurs after treatment ends. Lactobacilli treat human pouchitis and experimental colitis. We investigated if Lactobacillus rhamnosus GG (L GG) can prevent colitis in TG rats monoassociated with B vulgatus and if L GG or Lactobacillus plantarum 299v (LP 299v) can treat established colitis in SPF TG rats and prevent recurrent disease after antibiotics were stopped. METHODS: Germfree B27 TG rats were monoassociated with B vulgatus for four weeks following two weeks of colonisation with L GG or no bacteria. SPF B27 TG rats received oral vancomycin and imipenem for two weeks, or water alone, followed by four weeks of treatment with oral L GG, LP 299v, or water only. Disease activity was quantified by blinded gross and histological scores, caecal myeloperoxidase (MPO) activity, and levels of interleukin (IL)-1 beta, tumour necrosis factor (TNF), transforming growth factor beta, and IL-10. RESULTS: L GG did not prevent colitis in B vulgatus co-associated TG rats or treat established disease in SPF rats. However, L GG but not LP 299v prevented colitis relapse in antibiotic treated rats with reduced gross and histological scores, caecal MPO, IL-1 beta, and TNF whereas caecal IL-10 was increased. CONCLUSIONS: L GG does not prevent colitis in gnotobiotic TG rats or treat established disease in SPF rats, but is superior to LP 299v in the prevention of recurrent colitis. These studies suggest that antibiotics and probiotic agents provide synergistic therapeutic effects, perhaps mediated by altered immunomodulation with selective activity of different lactobacillus species.


Subject(s)
Anti-Bacterial Agents , Bacteroides Infections/therapy , Colitis/therapy , Drug Therapy, Combination/therapeutic use , Lactobacillus , Probiotics/therapeutic use , Animals , Animals, Genetically Modified , Bacteroides Infections/pathology , Cecum/immunology , Cecum/microbiology , Cecum/pathology , Colitis/immunology , Colitis/microbiology , Cytokines/metabolism , HLA-B27 Antigen , Intestinal Mucosa/immunology , Rats , Rats, Inbred F344 , Recurrence , Specific Pathogen-Free Organisms
17.
Immunology ; 104(2): 127-34, 2001 Oct.
Article in English | MEDLINE | ID: mdl-11683951

ABSTRACT

Interleukin-2-deficient (IL-2-/-) mice develop a spontaneous, progressive, CD4+ T-cell-mediated colitis with an age-related decrease in the number of B lymphocytes. The aim of this study was to determine the mechanisms of B-cell loss in IL-2-/- mice. Serum immunoglobulin G1 (IgG1) levels in 8-week-old IL-2-/- mice were above normal but then decreased dramatically with advancing age. Between 8 and 11 weeks of age, the number of B-cell progenitors (B220+ IgM-) in the bone marrow of IL-2-/- mice was less than half of those in IL-2+/+ littermates. By 22 weeks of age, very few progenitor cells remained in the bone marrow of most mice, and spleens were almost devoid of B cells. Likewise, B1 cells were not present in the peritoneal cavity of aged IL-2-/- mice. Flow cytometry analysis of B-cell differentiation in the bone marrow suggested a progressive loss of B cells from the most mature to the least mature stages, which was not dependent on IL-2 receptor-alpha (IL-2Ralpha) expression. B cells transferred from normal animals had similar survival rates in IL-2-/- and wild-type mice. We conclude that conventional B cells in older IL-2-/- mice are lost by attrition owing to a derangement in B-cell development. Because B1 cells are less dependent on the bone marrow, a separate mechanism for their loss is suggested.


Subject(s)
B-Lymphocytes/immunology , Interleukin-2/immunology , Animals , Ascitic Fluid/immunology , B-Lymphocytes/pathology , Bone Marrow Cells/immunology , Cell Differentiation/immunology , Cell Survival/immunology , Flow Cytometry , Hematopoietic Stem Cells/immunology , Immunoglobulin G/biosynthesis , Immunoglobulin G/blood , Interleukin-2/deficiency , Mice , Mice, Inbred C57BL , Spleen/immunology , T-Lymphocyte Subsets/immunology
18.
Immunol Cell Biol ; 79(5): 444-53, 2001 Oct.
Article in English | MEDLINE | ID: mdl-11564152

ABSTRACT

Differences in IL-1beta mRNA expression, stability and translation between non-adherent monocytes and those stimulated by adherence suggest that cytokine regulation is coupled to the function and assembly of cytoskeletal structures. In situ hybridization studies were performed to visualize expression and positioning of IL-1beta mRNA in adherently cultivated monocytes. IL-1beta mRNA expression was heterogeneous with high transcript levels found in spread or polarized cells. Transcripts were compartmentalized to the perinuclear region in spread cells, and partially redistributed with polarization. In contrast to mRNA distribution in other motile cell populations, IL-1beta mRNA did not localize to the distal or proximal actin cytoskeleton. Perinuclear confinement of transcripts required intact actin microfilaments. Treatment with cytoskeleton disruption and detergent extraction suggested that most non-translated IL-1beta mRNA was associated with intermediate filaments. In monocytes stimulated by LPS, IL-1beta, but not IL-1Ra transcripts were redistributed and partially associated, yet not bound to actin microfilaments. The present study demonstrates that IL-1beta mRNA expression and localization in adherent monocytes is interrelated with the cytoskeletal rearrangement upon adherence, spreading and polarization.


Subject(s)
Cell Adhesion , Cytoskeleton/metabolism , Interleukin-1/genetics , Monocytes/metabolism , Actins/metabolism , Cell Size , Cells, Cultured , Colchicine/pharmacology , Culture Media, Serum-Free , Cytochalasin D/pharmacology , Cytoskeleton/ultrastructure , Humans , In Situ Hybridization , Interleukin-1/metabolism , Lipopolysaccharides/pharmacology , Microtubules/metabolism , Monocytes/drug effects , Monocytes/ultrastructure , Nucleic Acid Synthesis Inhibitors/pharmacology , Polyribosomes/metabolism , Protein Synthesis Inhibitors/pharmacology , Puromycin/pharmacology , RNA, Messenger/genetics , RNA, Messenger/metabolism , Vimentin/metabolism
19.
Inflamm Res ; 50(3): 149-55, 2001 Mar.
Article in English | MEDLINE | ID: mdl-11339503

ABSTRACT

OBJECTIVE AND DESIGN: We studied the ability of bradykinin (BK) receptor antagonists type 1 and 2 (B1-RA, B2-RA) to prevent acute inflammation. MATERIAL: A peptidoglycan-polysaccharide (PG-APS)-induced model of arthritis in the Lewis rat was analyzed. TREATMENT: Four groups of animals were studied for 5 days. Treatment was administered subcutaneously (s.c.) 1 mg/kg every 12 h. Group I received PG-APS and was treated with the B2-RA, CP-0597 (DArg-Arg-Pro-Hyp-Gly-Thi-Ser-DTic-NChg-Arg). Group II received PG-APS and was treated with a combined B1 and B2-RA, B9430 (DArg-Arg-Pro-Hyp-Gly-Igl-Ser-Dlgl-Oic-Arg). Group III received PG-APS and albumin control. Group IV received albumin control. METHODS: Joint diameter, liver weight, hematocrit, white blood count and plasma concentrations of prekallikrein, high molecular weight kininogen, HK and IL-beta were measured. Groups were compared by ANOVA. RESULTS: Acute arthritis and hepatomegaly were attenuated in the B2-RA-treated animals (p<0.05). Weight loss was more pronounced in the B1/B2-RA-treated animals. Anemia induced by PG-APS was prevented by B2-RA and B1/B2-RA treatment (p<0.001). A marked decrease in plasma HK to 64% of normal was found in the disease-untreated animals, which was completely normalized by B2-RA treatment and partially attenuated by the B1/B2-RA (78%). The decrease in plasma prekallikrein levels was prevented by combined B1/B2-RA treatment (p<0.05). Finally, elevated plasma IL-1beta levels were lowered by B1/B2-RA treatment and were below detection limits with the B2-RA treatment. CONCLUSIONS: These results indicate that the systemic inflammation is due in part to BK generation which can be blocked by B2-RA, while inhibiting the B1 receptor prevents an anti-inflammatory response.


Subject(s)
Arthritis/drug therapy , Bradykinin Receptor Antagonists , Inflammation/prevention & control , Oligopeptides/therapeutic use , Peptidoglycan/toxicity , Acute Disease , Animals , Arthritis/chemically induced , Bradykinin/analogs & derivatives , Bradykinin/physiology , Bradykinin/therapeutic use , Female , Interleukin-1/blood , Kallikreins/blood , Neutrophils/physiology , Rats , Rats, Inbred Lew , Receptor, Bradykinin B2
20.
Infect Immun ; 69(4): 2277-85, 2001 Apr.
Article in English | MEDLINE | ID: mdl-11254584

ABSTRACT

Resident bacteria are incriminated in the pathogenesis of experimental colitis and inflammatory bowel diseases. We investigated the relative roles of various enteric bacteria populations in the induction and perpetuation of experimental colitis. HLA-B27 transgenic rats received antibiotics (ciprofloxacin, metronidazole, or vancomycin-imipenem) in drinking water or water alone in either prevention or treatment protocols. Mice were treated similarly with metronidazole or vancomycin-imipenem before or after receiving 5% dextran sodium sulfate (DSS). Germfree transgenic rats were colonized with specific-pathogen-free enteric bacteria grown overnight either in anaerobic or aerobic atmospheres. Nontransgenic rats colonized with anaerobic bacteria served as negative controls. Although preventive metronidazole significantly attenuated colitis in transgenic rats and DSS-treated mice, it had no therapeutic benefit once colitis was established. Ciprofloxacin also partially prevented but did not treat colitis in B27 transgenic rats. In both animal models vancomycin-imipenem most effectively prevented and treated colitis. Germfree transgenic rats reconstituted with enteric bacteria grown under anaerobic conditions had more aggressive colitis than those associated with aerobic bacteria. These results suggest that a subset of resident luminal bacteria induces colitis, but that a complex interaction of commensal aerobic and anaerobic bacteria provides the constant antigenic drive for chronic immune-mediated colonic inflammation.


Subject(s)
Colitis/microbiology , Enterobacteriaceae/physiology , Animals , Bacteroides/isolation & purification , Ciprofloxacin/pharmacology , Colitis/drug therapy , Enterobacteriaceae/drug effects , Female , HLA-B27 Antigen/physiology , Imipenem/pharmacology , Interleukin-1/analysis , Metronidazole/pharmacology , Mice , Mice, Inbred BALB C , Rats , Rats, Inbred F344 , Vancomycin/pharmacology
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