An incomplete TCA cycle increases survival of Salmonella Typhimurium during infection of resting and activated murine macrophages

Background

In comparison to the comprehensive analyses performed on virulence gene expression, regulation and action, the intracellular metabolism of Salmonella during infection is a relatively under-studied area. We investigated the role of the tricarboxylic acid (TCA) cycle in the intracellular replication of Salmonella Typhimurium in resting and activated macrophages, epithelial cells, and during infection of mice.

Methodology/Principal Findings

We constructed deletion mutations of 5 TCA cycle genes in S. Typhimurium including gltA, mdh, sdhCDAB, sucAB, and sucCD. We found that the mutants exhibited increased net intracellular replication in resting and activated murine macrophages compared to the wild-type. In contrast, an epithelial cell infection model showed that the S. Typhimurium ΔsucCD and ΔgltA strains had reduced net intracellular replication compared to the wild-type. The glyoxylate shunt was not responsible for the net increased replication of the TCA cycle mutants within resting macrophages. We also confirmed that, in a murine infection model, the S. Typhimurium ΔsucAB and ΔsucCD strains are attenuated for virulence.

Conclusions/Significance

Our results suggest that disruption of the TCA cycle increases the ability of S. Typhimurium to survive within resting and activated murine macrophages. In contrast, epithelial cells are non-phagocytic cells and unlike macrophages cannot mount an oxidative and nitrosative defence response against pathogens; our results show that in HeLa cells the S. Typhimurium TCA cycle mutant strains show reduced or no change in intracellular levels compared to the wild-type [1]. The attenuation of the S. Typhimurium ΔsucAB and ΔsucCD mutants in mice, compared to their increased net intracellular replication in resting and activated macrophages suggest that Salmonella may encounter environments within the host where a complete TCA cycle is advantageous.     

IL-17RA signaling amplifies antibody-induced arthritis

Objective

To investigate the role of IL-17RA signaling in the effector phase of inflammatory arthritis using the K/BxN serum-transfer model.

Methods

Wild-type and Il17ra^−/− mice were injected with serum isolated from arthritic K/BxN mice and their clinical score was recorded daily. Mice were also harvested on days 12 and 21 and ankles were analyzed for cytokine and chemokine mRNA expression by qPCR on day 12 and for bone and cartilage erosions by histology on day 21, respectively. The induction of cytokine and chemokine expression levels by IL-17A in synovial-like fibroblasts was also analyzed using qPCR.

Results

Il17ra^−/− mice were partially protected from clinical signs of arthritis and had markedly fewer cartilage and bone erosions. The expression of several pro-inflammatory mediators, including the chemokines KC/CXCL1, MIP-2/CXCL2, LIX/CXCL5 MIP-1γ/CCL9, MCP-3/CCL7, MIP-3α/CCL20, the cytokines IL-1β, IL-6, RANKL and the matrix metalloproteinases MMP2, MMP3, and MMP13 were decreased in the ankles of Il17ra^−/− mice compared to wild-type mice. Many of these proinflammatory genes attenuated in the ankles of Il17ra^−/− mice were shown to be directly induced by IL-17A in synovial fibroblasts in vitro.

Conclusions

IL-17RA signaling plays a role as an amplifier of the effector phase of inflammatory arthritis. This effect is likely mediated by direct activation of synovial fibroblasts by IL-17RA to produce multiple inflammatory mediators, including chemokines active on neutrophils. Therefore, interrupting IL-17RA signaling maybe a promising pharmacological target for the treatment of inflammatory arthritis.     

MIF participates in Toxoplasma gondii-induced pathology following oral infection

Background

Macrophage migration inhibitory factor (MIF) is essential for controlling parasite burden and survival in a model of systemic Toxoplasma gondii infection. Peroral T. gondii infection induces small intestine necrosis and death in susceptible hosts, and in many aspects resembles inflammatory bowel disease (IBD). Considering the critical role of MIF in the pathogenesis of IBD, we hypothesized that MIF participates in the inflammatory response induced by oral infection with T. gondii.

Methodology/Principal Findings

Mif deficient (Mif⁻/⁻) and wild-type mice in the C57Bl/6 background were orally infected with T. gondii strain ME49. Mif⁻/⁻ mice had reduced lethality, ileal inflammation and tissue damage despite of an increased intestinal parasite load compared to wt mice. Lack of MIF caused a reduction of TNF-α, IL-12, IFN-γ and IL-23 and an increased expression of IL-22 in ileal mucosa. Moreover, suppressed pro-inflammatory responses at the ileal mucosa observed in Mif⁻/⁻ mice was not due to upregulation of IL-4, IL-10 or TGF-β. MIF also affected the expression of matrix metalloproteinase-9 (MMP-9) but not MMP-2 in the intestine of infected mice. Signs of systemic inflammation including the increased concentrations of inflammatory cytokines in the plasma and liver damage were less pronounced in Mif⁻/⁻ mice compared to wild-type mice.

Conclusion/Significance

In conclusion, our data suggested that in susceptible hosts MIF controls T. gondii infection with the cost of increasing local and systemic inflammation, tissue damage and death.     

Early interferon-γ production in human lymphocyte subsets in response to nontyphoidal Salmonella demonstrates inherent capacity in innate cells

Background

Nontyphoidal Salmonellae frequently cause life-threatening bacteremia in sub-Saharan Africa. Young children and HIV-infected adults are particularly susceptible. High case-fatality rates and increasing antibiotic resistance require new approaches to the management of this disease. Impaired cellular immunity caused by defects in the T helper 1 pathway lead to intracellular disease with Salmonella that can be countered by IFNγ administration. This report identifies the lymphocyte subsets that produce IFNγ early in Salmonella infection.

Methodology

Intracellular cytokine staining was used to identify IFNγ production in blood lymphocyte subsets of ten healthy adults with antibodies to Salmonella (as evidence of immunity to Salmonella), in response to stimulation with live and heat-killed preparations of the {"type":"entrez-nucleotide","attrs":{"text":"D23580","term_id":"427513","term_text":"D23580"}}D23580 invasive African isolate of Salmonella Typhimurium. The absolute number of IFNγ-producing cells in innate, innate-like and adaptive lymphocyte subpopulations was determined.

Principal Findings

Early IFNγ production was found in the innate/innate-like lymphocyte subsets: γδ-T cells, NK cells and NK-like T cells. Significantly higher percentages of such cells produced IFNγ compared to adaptive αβ-T cells (Student''s t test, P<0.001 and ≤0.02 for each innate subset compared, respectively, with CD4⁺- and CD8⁺-T cells). The absolute numbers of IFNγ-producing cells showed similar differences. The proportion of IFNγ-producing γδ-T cells, but not other lymphocytes, was significantly higher when stimulated with live compared with heat-killed bacteria (P<0.0001).

Conclusion/Significance

Our findings indicate an inherent capacity of innate/innate-like lymphocyte subsets to produce IFNγ early in the response to Salmonella infection. This may serve to control intracellular infection and reduce the threat of extracellular spread of disease with bacteremia which becomes life-threatening in the absence of protective antibody. These innate cells may also help mitigate against the effect on IFNγ production of depletion of Salmonella-specific CD4⁺-T lymphocytes in HIV infection.     

Identification by PCR of Non-typhoidal Salmonella enterica Serovars Associated with Invasive Infections among Febrile Patients in Mali

Background

In sub-Saharan Africa, non-typhoidal Salmonella (NTS) are emerging as a prominent cause of invasive disease (bacteremia and focal infections such as meningitis) in infants and young children. Importantly, including data from Mali, three serovars, Salmonella enterica serovar Typhimurium, Salmonella Enteritidis and Salmonella Dublin, account for the majority of non-typhoidal Salmonella isolated from these patients.

Methods

We have extended a previously developed series of polymerase chain reactions (PCRs) based on O serogrouping and H typing to identify Salmonella Typhimurium and variants (mostly I 4,[5],12:i:-), Salmonella Enteritidis and Salmonella Dublin. We also designed primers to detect Salmonella Stanleyville, a serovar found in West Africa. Another PCR was used to differentiate diphasic Salmonella Typhimurium and monophasic Salmonella Typhimurium from other O serogroup B, H:i serovars. We used these PCRs to blind-test 327 Salmonella serogroup B and D isolates that were obtained from the blood cultures of febrile patients in Bamako, Mali.

Principal Findings

We have shown that when used in conjunction with our previously described O-serogrouping PCR, our PCRs are 100% sensitive and specific in identifying Salmonella Typhimurium and variants, Salmonella Enteritidis, Salmonella Dublin and Salmonella Stanleyville. When we attempted to differentiate 171 Salmonella Typhimurium (I 4,[ 5],12:i:1,2) strains from 52 monophasic Salmonella Typhimurium (I 4,[5],12:i:-) strains, we were able to correctly identify 170 of the Salmonella Typhimurium and 51 of the Salmonella I 4,[5],12:i:- strains.

Conclusion

We have described a simple yet effective PCR method to support surveillance of the incidence of invasive disease caused by NTS in developing countries.     

IL-1 and IL-23 mediate early IL-17A production in pulmonary inflammation leading to late fibrosis

Background

Idiopathic pulmonary fibrosis is a devastating as yet untreatable disease. We demonstrated recently the predominant role of the NLRP3 inflammasome activation and IL-1β expression in the establishment of pulmonary inflammation and fibrosis in mice.

Methods

The contribution of IL-23 or IL-17 in pulmonary inflammation and fibrosis was assessed using the bleomycin model in deficient mice.

Results

We show that bleomycin or IL-1β-induced lung injury leads to increased expression of early IL-23p19, and IL-17A or IL-17F expression. Early IL-23p19 and IL-17A, but not IL-17F, and IL-17RA signaling are required for inflammatory response to BLM as shown with gene deficient mice or mice treated with neutralizing antibodies. Using FACS analysis, we show a very early IL-17A and IL-17F expression by RORγt⁺ γδ T cells and to a lesser extent by CD4αβ⁺ T cells, but not by iNKT cells, 24 hrs after BLM administration. Moreover, IL-23p19 and IL-17A expressions or IL-17RA signaling are necessary to pulmonary TGF-β1 production, collagen deposition and evolution to fibrosis.

Conclusions

Our findings demonstrate the existence of an early IL-1β-IL-23-IL-17A axis leading to pulmonary inflammation and fibrosis and identify innate IL-23 and IL-17A as interesting drug targets for IL-1β driven lung pathology.     

Lysate of probiotic Lactobacillus casei DN-114 001 ameliorates colitis by strengthening the gut barrier function and changing the gut microenvironment

Background

Probiotic bacteria can be used for the prevention and treatment of human inflammatory diseases including inflammatory bowel diseases (IBD). However, the nature of active components and exact mechanisms of this beneficial effects have not been fully elucidated. Our aim was to investigate if lysate of probiotic bacterium L. casei DN-114 001 (Lc) could decrease the severity of intestinal inflammation in a murine model of IBD.

Methodology/Principal Findings

The preventive effect of oral administration of Lc significantly reduces the severity of acute dextran sulfate sodium (DSS) colitis in BALB/c but not in SCID mice. In order to analyze how this beneficial effect interferes with well-known phases of intestinal inflammation pathogenesis in vivo and in vitro, we evaluated intestinal permeability using the FITC-labeled dextran method and analysed tight junction proteins expression by immunofluorescence and PCR. We also measured CD4⁺FoxP3⁺ regulatory T cells proportion by FACS analysis, microbiota composition by pyrosequencing, and local cytokine production by ELISA. Lc leads to a significant protection against increased intestinal permeability and barrier dysfunction shown by preserved ZO-1 expression. We found that the Lc treatment increases the numbers of CD4⁺FoxP3⁺ regulatory T cells in mesenteric lymph nodes (MLN), decreases production of pro-inflammatory cytokines TNF-α and IFN-γ, and anti-inflammatory IL-10 in Peyer''s patches and large intestine, and changes the gut microbiota composition. Moreover, Lc treatment prevents lipopolysaccharide-induced TNF-α expression in RAW 264.7 cell line by down-regulating the NF-κB signaling pathway.

Conclusion/Significance

Our study provided evidence that even non-living probiotic bacteria can prevent the development of severe forms of intestinal inflammation by strengthening the integrity of intestinal barrier and modulation of gut microenvironment.     

Antigen-specific B cells reactivate an effective cytotoxic T cell response against phagocytosed Salmonella through cross-presentation

Background

The eradication of facultative intracellular bacterial pathogens, like Salmonella typhi, requires the concerted action of both the humoral immune response and the cytotoxic CD8⁺ T cell response. Dendritic cells (DCs) are considered to orchestrate the cytotoxic CD8⁺ T cell response via cross-presentation of bacterial antigens onto MHC class I molecules. Cross-presentation of Salmonella by DCs however, is accompanied by the induction of apoptosis in the DCs. Besides antibody production, B cells are required to clear Salmonella infection for other unknown reasons.

Methodology/Principal Findings

Here we show that Salmonella-specific B cells that phagocytose Salmonella upon BCR-ligation reactivate human memory CD8⁺ T cells via cross-presentation yielding a Salmonella-specific cytotoxic T cell response. The reactivation of CD8⁺ T cells is dependent on CD4⁺ T cell help. Unlike the DCs, B cell-mediated cross-presentation of Salmonella does not coincide with apoptosis.

Conclusions/Significance

B cells form a new player in the activation of the cytotoxic effector arm of the immune response and the generation of effective adaptive immunity in Salmonella infection.     

Cell walls of Saccharomyces cerevisiae differentially modulated innate immunity and glucose metabolism during late systemic inflammation

Background

Salmonella causes acute systemic inflammation by using its virulence factors to invade the intestinal epithelium. But, prolonged inflammation may provoke severe body catabolism and immunological diseases. Salmonella has become more life-threatening due to emergence of multiple-antibiotic resistant strains. Mannose-rich oligosaccharides (MOS) from cells walls of Saccharomyces cerevisiae have shown to bind mannose-specific lectin of Gram-negative bacteria including Salmonella, and prevent their adherence to intestinal epithelial cells. However, whether MOS may potentially mitigate systemic inflammation is not investigated yet. Moreover, molecular events underlying innate immune responses and metabolic activities during late inflammation, in presence or absence of MOS, are unknown.

Methods and Principal Findings

Using a Salmonella LPS-induced systemic inflammation chicken model and microarray analysis, we investigated the effects of MOS and virginiamycin (VIRG, a sub-therapeutic antibiotic) on innate immunity and glucose metabolism during late inflammation. Here, we demonstrate that MOS and VIRG modulated innate immunity and metabolic genes differently. Innate immune responses were principally mediated by intestinal IL-3, but not TNF-α, IL-1 or IL-6, whereas glucose mobilization occurred through intestinal gluconeogenesis only. MOS inherently induced IL-3 expression in control hosts. Consequent to LPS challenge, IL-3 induction in VIRG hosts but not differentially expressed in MOS hosts revealed that MOS counteracted LPS''s detrimental inflammatory effects. Metabolic pathways are built to elucidate the mechanisms by which VIRG host''s higher energy requirements were met: including gene up-regulations for intestinal gluconeogenesis (PEPCK) and liver glycolysis (ENO2), and intriguingly liver fatty acid synthesis through ATP citrate synthase (CS) down-regulation and ATP citrate lyase (ACLY) and malic enzyme (ME) up-regulations. However, MOS host''s lower energy demands were sufficiently met through TCA citrate-derived energy, as indicated by CS up-regulation.

Conclusions

MOS terminated inflammation earlier than VIRG and reduced glucose mobilization, thus representing a novel biological strategy to alleviate Salmonella-induced systemic inflammation in human and animal hosts.     

The Q705K polymorphism in NLRP3 is a gain-of-function alteration leading to excessive interleukin-1β and IL-18 production

Background

The Q705K polymorphism in NLRP3 has been implicated in several chronic inflammatory diseases. In this study we determine the functional role of this commonly occurring polymorphism using an in-vitro system.

Principal Findings

NLRP3-WT and NLRP3-Q705K were retrovirally transduced into the human monocytic cell line THP-1, followed by the assessment of IL-1β and IL-18 levels in the cell culture supernatant. THP-1 cells expressing the above NLRP3 variants were sorted based upon Green Fluorescent Protein (GFP) expression. Cytokine response to alum (one of the most widely used adjuvants in vaccines) in the cells stably expressing NLRP3-WT and NLRP3-Q705K were determined. IL-1β and IL-18 levels were found to be elevated in THP-1 cells transduced with NLRP3-Q705K compared to the NLRP3-WT. Upon exposure to alum, THP-1 cells stably expressing NLRP3-Q705K displayed an increased release of IL-1β, IL-18 and TNF-α, in a caspase-1 and IL-1 receptor-dependent manner.

Conclusions

Collectively, these findings show that the Q705K polymorphism in NLRP3 is a gain-of-function alteration leading to an overactive NLRP3 inflammasome. The option of IL-1β blockade may be considered in patients with chronic inflammatory disorders that are unresponsive to conventional treatments.     

IL-17A expression is localised to both mononuclear and polymorphonuclear synovial cell infiltrates

Introduction

This study examines the expression of IL-17A-secreting cells within the inflamed synovium and the relationship to in vivo joint hypoxia measurements.

Methods

IL-17A expression was quantified in synovial tissue (ST), serum and synovial fluid (SF) by immunohistochemistry and MSD-plex assays. IL-6 SF and serum levels were measured by MSD-plex assays. Dual immunofluorescence for IL-17A was quantified in ST CD15+ cells (neutrophils), Tryptase+ (mast cells) and CD4+ (T cells). Synovial tissue oxygen (tpO₂) levels were measured under direct visualisation at arthroscopy. Synovial infiltration was assessed using immunohistochemistry for cell specific markers. Peripheral blood mononuclear and polymorphonuclear cells were isolated and exposed to normoxic or 3% hypoxic conditions. IL-17A and IL-6 were quantified as above in culture supernatants.

Results

IL-17A expression was localised to mononuclear and polymorphonuclear (PMN) cells in inflamed ST. Dual immunoflourescent staining co-localised IL-17A expression with CD15+ neutrophils Tryptase+ mast cells and CD4+T cells. % IL-17A positivity was highest on CD15+ neutrophils, followed by mast cells and then CD4+T-cells. The number of IL-17A-secreting PMN cells significantly correlated with sublining CD68 expression (r = 0.618, p<0.01). IL-17A SF levels correlated with IL-6 SF levels (r = 0.675, p<0.01). Patients categorized according to tp0₂< or >20mmHg, showed those with low tp0₂<20mmHg had significantly higher IL-17A+ mononuclear cells with no difference observed for PMNs. Exposure of mononuclear and polymorphonuclear cells to 3% hypoxia, significantly induced IL-6 in mononuclear cells, but had no effect on IL-17A expression in mononuclear and polymorphonuclear cells.

Conclusion

This study demonstrates IL-17A expression is localised to several immune cell subtypes within the inflamed synovial tissue, further supporting the concept that IL-17A is a key mediator in inflammatory arthritis. The association of hypoxia with Il-17A expression appears to be indirect, probably through hypoxia-induced pro-inflammatory pathways and leukocyte influx within the joint microenvironment.     

CCL25/CCR9 interactions regulate large intestinal inflammation in a murine model of acute colitis

Background & Aims

CCL25/CCR9 is a non-promiscuous chemokine/receptor pair and a key regulator of leukocyte migration to the small intestine. We investigated here whether CCL25/CCR9 interactions also play a role in the regulation of inflammatory responses in the large intestine.

Methods

Acute inflammation and recovery in wild-type (WT) and CCR9^−/− mice was studied in a model of dextran sulfate sodium (DSS)-induced colitis. Distribution studies and phenotypic characterization of dendritic cell subsets and macrophage were performed by flow cytometry. Inflammatory bowel disease (IBD) scores were assessed and expression of inflammatory cytokines was studied at the mRNA and the protein level.

Results

CCL25 and CCR9 are both expressed in the large intestine and are upregulated during DSS colitis. CCR9^−/− mice are more susceptible to DSS colitis than WT littermate controls as shown by higher mortality, increased IBD score and delayed recovery. During recovery, the CCR9^−/− colonic mucosa is characterized by the accumulation of activated macrophages and elevated levels of Th1/Th17 inflammatory cytokines. Activated plasmacytoid dendritic cells (DCs) accumulate in mesenteric lymph nodes (MLNs) of CCR9^−/− animals, altering the local ratio of DC subsets. Upon re-stimulation, T cells isolated from these MLNs secrete significantly higher levels of TNFα, IFNγ, IL2, IL-6 and IL-17A while down modulating IL-10 production.

Conclusions

Our results demonstrate that CCL25/CCR9 interactions regulate inflammatory immune responses in the large intestinal mucosa by balancing different subsets of dendritic cells. These findings have important implications for the use of CCR9-inhibitors in therapy of human IBD as they indicate a potential risk for patients with large intestinal inflammation.     

Vulnerability of polarised intestinal porcine epithelial cells to mycotoxin deoxynivalenol depends on the route of application

Background and Aims

Deoxynivalenol (DON) is a Fusarium derived mycotoxin, often occurring on cereals used for human and animal nutrition. The intestine, as prominent barrier for nutritional toxins, has to handle the mycotoxin from the mucosa protected luminal side (apical exposure), as well as already absorbed toxin, reaching the cells from basolateral side via the blood stream. In the present study, the impact of the direction of DON exposure on epithelial cell behaviour and intestinal barrier integrity was elucidated.

Methods

A non-transformed intestinal porcine epithelial cell line (IPEC-J2), cultured in membrane inserts, serving as a polarised in vitro model to determine the effects of deoxynivalenol (DON) on cellular viability and tight junction integrity.

Results

Application of DON in concentrations up to 4000 ng/mL for 24, 48 and 72 hours on the basolateral side of membrane cultured polarised IPEC-J2 cells resulted in a breakdown of the integrity of cell connections measured by transepithelial electrical resistance (TEER), as well as a reduced expression of the tight junction proteins ZO-1 and claudin 3. Epithelial cell number decreased and nuclei size was enlarged after 72 h incubation of 4000 ng/mL DON from basolateral. Although necrosis or caspase 3 mediated apoptosis was not detectable after basolateral DON application, cell cycle analysis revealed a significant increase in DNA fragmentation, decrease in G0/G1 phase and slight increase in G2/M phase after 72 hours incubation with DON 2000 ng/mL.

Conclusions

Severity of impact of the mycotoxin deoxynivalenol on the intestinal epithelial barrier is dependent on route of application. The epithelium appears to be rather resistant towards apical (luminal) DON application whereas the same toxin dose from basolateral severely undermines barrier integrity.     

Enhanced susceptibility to lipopolysaccharide-induced arthritis and endotoxin shock in interleukin-32 alpha transgenic mice through induction of tumor necrosis factor alpha

Introduction

The present study assessed the potential functions of interleukin (IL)-32α on inflammatory arthritis and endotoxin shock models using IL-32α transgenic (Tg) mice. The potential signaling pathway for the IL-32-tumor necrosis factor (TNF)α axis was analyzed in vitro.

Methods

IL-32α Tg mice were generated under control of a ubiquitous promoter. Two disease models were used to examine in vivo effects of overexpressed IL-32α: Toll-like receptor (TLR) ligand-induced arthritis developed using a single injection of lipopolysaccharide (LPS) or zymosan into the knee joints; and endotoxin shock induced with intraperitoneal injection of LPS and D-galactosamine. TNFα antagonist etanercept was administered simultaneously with LPS in some mice. Using RAW264.7 cells, in vitro effects of exogenous IL-32α on TNFα, IL-6 or macrophage inflammatory protein 2 (MIP-2) production were assessed with or without inhibitors for nuclear factor kappa B (NFκB) or mitogen-activated protein kinase (MAPK).

Results

Single injection of LPS, but not zymosan, resulted in development of severe synovitis with substantial articular cartilage degradation in knees of the Tg mice. The expression of TNFα mRNA in inflamed synovia was highly upregulated in the LPS-injected Tg mice. Moreover, the Tg mice were more susceptive to endotoxin-induced lethality than the wild-type control mice 48 hours after LPS challenge; but blockade of TNFα by etanercept protected from endotoxin lethality. In cultured bone marrow cells derived from the Tg mice, overexpressed IL-32α accelerated production of TNFα upon stimulation with LPS. Of note, exogenously added IL-32α alone stimulated RAW264.7 cells to express TNFα, IL-6, and MIP-2 mRNAs. Particularly, IL-32α -induced TNFα, but not IL-6 or MIP-2, was inhibited by dehydroxymethylepoxyquinomicin (DHMEQ) and U0126, which are specific inhibitors of nuclear factor kappa B (NFκB) and extracellular signal regulated kinase1/2 (ERK1/2), respectively.

Conclusions

These results show that IL-32α contributed to the development of inflammatory arthritis and endotoxin lethality. Stimulation of TLR signaling with LPS appeared indispensable for activating the IL-32α-TNFα axis in vivo. However, IL-32α alone induced TNFα production in RAW264.7 cells through phosphorylation of inhibitor kappa B (IκB) and ERK1/2 MAPK. Further studies on the potential involvement of IL-32α-TNFα axis will be beneficial in better understanding the pathology of autoimmune-related arthritis and infectious immunity.     

The bile acid receptor GPBAR-1 (TGR5) modulates integrity of intestinal barrier and immune response to experimental colitis

Background

GP-BAR1, a member G protein coupled receptor superfamily, is a cell surface bile acid-activated receptor highly expressed in the ileum and colon. In monocytes, ligation of GP-BAR1 by secondary bile acids results in a cAMP-dependent attenuation of cytokine generation.

Aims

To investigate the role GP-BAR1 in regulating intestinal homeostasis and inflammation-driven immune dysfunction in rodent models of colitis.

Methods

Colitis was induced in wild type and GP-BAR1^−/− mice by DSS and TNBS administration. Potential GP-BAR1 agonists were identified by in silico screening and computational docking studies.

Results

GP-BAR1^−/− mice develop an abnormal morphology of colonic mucous cells and an altered molecular architecture of epithelial tight junctions with increased expression and abnormal subcellular distribution of zonulin 1 resulting in increased intestinal permeability and susceptibility to develop severe colitis in response to DSS at early stage of life. By in silico screening and docking studies we identified ciprofloxacin as a GP-BAR1 ligand. In monocytes, ciprofloxacin increases cAMP concentrations and attenuates TNFα release induced by TLR4 ligation in a GP-BAR1 dependent manner. Treating mice rendered colitic by TNBS with ciprofloxacin and oleanolic acid, a well characterized GP-BAR1 ligand, abrogates signs and symptoms of colitis. Colonic expression of GP-BAR1 mRNA increases in rodent models of colitis and tissues from Crohn''s disease patients. Flow cytometry analysis demonstrates that ≈90% of CD14+ cells isolated from the lamina propria of TNBS-treated mice stained positively for GP-BAR1.

Conclusions

GP-BAR1 regulates intestinal barrier structure. Its expression increases in rodent models of colitis and Crohn''s disease. Ciprofloxacin is a GP-BAR1 ligand.     

Iridoid glycosides fraction of Folium syringae leaves modulates NF-κB signal pathway and intestinal epithelial cells apoptosis in experimental colitis

Background and Aims

Iridoid glycosides (IG), the major active fraction of F. syringae leaves has been demonstrated to have strong anti-inflammatory properties to ulcerative colitis (UC) in our previous study. The aim of this study was to investigate whether IG modulates the inflammatory response in experimental colitis at the level of NF-κB signal pathway and epithelial cell apoptosis.

Methods

UC in rats was induced by administration with dextran sulfate sodium (DSS) in drinking water. The inflammatory damage was assessed by disease activity index (DAI), macroscopic findings, histology and myeloperoxidase (MPO) activity. The effect of IG on pro-inflammatory cytokines TNF-α, IL-8, COX-2 and regulatory peptide TGF-β1 was measured. Epithelial cell apoptosis and the protein and mRNA expressions of Fas/FasL, Bcl-2/Bax, caspase-3, NF-κB p65, IκBα, p-IκBα and IKKβ were detected by TUNEL method, immunohistochemistry, Western blotting and real-time quantitative PCR, respectively.

Results

IG significantly ameliorated macroscopic damage and histological changes, reduced the activity of MPO, and strongly inhibited epithelial cell apoptosis. Moreover, IG markedly depressed TNF-α, IL-8, COX-2 and TGF-β1 levels in the colon tissues in a dose-dependent manner. Furthermore, IG significantly blocked of NF-κB signaling by inhibiting IκBα phosphorylation/degradation and IKKβ activity, down-regulated the protein and mRNA expressions of Fas/FasL, Bax and caspase-3, and activated Bcl-2 in intestinal epithelial cells.

Conclusions

These results demonstrated for the first time that IG possessed marked protective effects on experimental colitis through inhibition of epithelial cell apoptosis and blockade of NF-κB signal pathway.     

Synovial DKK1 expression is regulated by local glucocorticoid metabolism in inflammatory arthritis

Introduction

Inflammatory arthritis is associated with increased bone resorption and suppressed bone formation. The Wnt antagonist dickkopf-1 (DKK1) is secreted by synovial fibroblasts in response to inflammation and this protein has been proposed to be a master regulator of bone remodelling in inflammatory arthritis. Local glucocorticoid production is also significantly increased during joint inflammation. Therefore, we investigated how locally derived glucocorticoids and inflammatory cytokines regulate DKK1 synthesis in synovial fibroblasts during inflammatory arthritis.

Methods

We examined expression and regulation of DKK1 in primary cultures of human synovial fibroblasts isolated from patients with inflammatory arthritis. The effect of TNFα, IL-1β and glucocorticoids on DKK1 mRNA and protein expression was examined by real-time PCR and ELISA. The ability of inflammatory cytokine-induced expression of the glucocorticoid-activating enzyme 11beta-hydroxysteroid dehydrogenase type 1 (11β-HSD1) to sensitise fibroblasts to endogenous glucocorticoids was explored. Global expression of Wnt signalling and target genes in response to TNFα and glucocorticoids was assessed using a custom array.

Results

DKK1 expression in human synovial fibroblasts was directly regulated by glucocorticoids but not proinflammatory cytokines. Glucocorticoids, but not TNFα, regulated expression of multiple Wnt agonists and antagonists in favour of inhibition of Wnt signalling. However, TNFα and IL-1β indirectly stimulated DKK1 production through increased expression of 11β-HSD1.

Conclusions

These results demonstrate that in rheumatoid arthritis synovial fibroblasts, DKK1 expression is directly regulated by glucocorticoids rather than TNFα. Consequently, the links between synovial inflammation, altered Wnt signalling and bone remodelling are not direct but are dependent on local activation of endogenous glucocorticoids.     

Expression of human beta-defensins in children with chronic inflammatory bowel disease

Background

Human beta-defensins (hBDs) are antimicrobial peptides known to play a major role in intestinal innate host defence. Altered mucosal expression of hBDs has been suggested to be implicated in chronic inflammatory bowel disease pathogenesis. However, little is known about expression of these peptides in children.

Methods

Intestinal biopsies were obtained from the duodenum (n = 88), terminal ileum (n = 90) and ascending colon (n = 105) of children with Crohn''s disease (n = 26), ulcerative colitis (n = 11) and healthy controls (n = 16). Quantitative real-time (RT) PCR was performed and absolute mRNA copy numbers analyzed for hBD1-3 as well as inflammatory cytokines IL-8 and TNF-alpha.

Results

Significant induction of hBD2 and hBD3 was observed in the inflamed terminal ileum and ascending colon of IBD children. In the ascending colon induction of hBD2 was found to be significantly lower in children with Crohn''s disease compared to ulcerative colitis. A strong correlation was found between inducible defensins hBD2 and 3 and the inflammatory cytokines IL-8 and TNF-alpha, both in the terminal ileum and ascending colon.

Conclusion

Our study demonstrates distinct changes in hBD expression throughout the intestinal tract of children with IBD, lending further support for their potential role in disease pathogenesis.     

Modification of Salmonella Typhimurium motility by the probiotic yeast strain Saccharomyces boulardii

Background

Motility is an important component of Salmonella enterica serovar Typhimurium (ST) pathogenesis allowing the bacteria to move into appropriate niches, across the mucus layer and invade the intestinal epithelium. In vitro, flagellum-associated motility is closely related to the invasive properties of ST. The probiotic yeast Saccharomyces boulardii BIOCODEX (S.b-B) is widely prescribed for the prophylaxis and treatment of diarrheal diseases caused by bacteria or antibiotics. In case of Salmonella infection, S.b-B has been shown to decrease ST invasion of T84 colon cell line. The present study was designed to investigate the impact of S.b-B on ST motility.

Methodology/Principal Findings

Experiments were performed on human colonic T84 cells infected by the Salmonella strain 1344 alone or in the presence of S.b-B. The motility of Salmonella was recorded by time-lapse video microscopy. Next, a manual tracking was performed to analyze bacteria dynamics (MTrackJ plugin, NIH image J software). This revealed that the speed of bacterial movement was modified in the presence of S.b-B. The median curvilinear velocity (CLV) of Salmonella incubated alone with T84 decreased from 43.3 µm/sec to 31.2 µm/sec in the presence of S.b-B. Measurement of track linearity (TL) showed similar trends: S.b-B decreased by 15% the number of bacteria with linear tract (LT) and increased by 22% the number of bacteria with rotator tract (RT). Correlation between ST motility and invasion was further established by studying a non-motile flagella-deficient ST strain. Indeed this strain that moved with a CLV of 0.5 µm/sec, presented a majority of RT and a significant decrease in invasion properties. Importantly, we show that S.b-B modified the motility of the pathogenic strain SL1344 and significantly decreased invasion of T84 cells by this strain.

Conclusions

This study reveals that S.b-B modifies Salmonella''s motility and trajectory which may account for the modification of Salmonella''s invasion.