TNF Mice Research Articles

Nanocarriers Targeting Intercellular Adhesion Molecule Enhance Delivery to the Inflamed Brain

Over 600,000 Americans suffer from ischemic stroke annually. Inflammatory consequences of ischemic stroke include upregulation of adhesion molecules (e.g. intercellular adhesion molecule -1, ICAM) by activated endothelial cells. However, drug delivery to the central nervous system remains an emerging challenge. Here we developed anti-ICAM monoclonal antibody conjugated nanocarriers (ICAM/NCs) to target activated endothelial cells in the pathologically-altered neurovasculature. In a mouse model similar to post-stroke neuroinflammation (intra-striatal injection of Tumor Necrosis Factor-alpha, TNF), ICAM/NCs injected intravenously 2 hrs-post TNF injury accumulated in lungs within 30 min (146.7±2.5 % injected dose per gram tissue (%ID/g)). Over 24 hrs, ICAM/NCs slowly accumulated in the brain, resulting in a >4-fold increase in brain uptake (0.22±0.02%ID/g) vs. 30 minutes (0.05±0.01%ID/g), and significant clearance from lungs (Panel A). In addition, dexamethasone (Dex)-loaded ICAM/NCs were protective against TNF induced brain edema, as measured by extravasation of radiolabeled albumin, while free Dex had no protective effects at the same dose (1.5mg/kg) (Panel B). Flow cytometry of single cell suspensions from lungs and brain in TNF mice revealed that ~80% of ICAM/NC-positive cells were leukocytes in the lungs 30 min-post injection (while in naïve animals, ~40% NC-positive cells were leukocytes), and that ~98% of ICAM/NC-positive cells were leukocytes in the brain 24 hr-post injection (Panel C). The flow cytometry data for the lungs and brain indicated that ICAM/NCs target not only endothelial cells but also leukocytes. Targeting to pulmonary leukocytes is further enhanced following pathological challenges. Altogether, our results suggest that delayed brain delivery of ICAM/NCs was partially due to pulmonary leukocytes ferrying NCs from lungs to brain over time. Future work will focus on elucidating the mechanism of brain delivery.

A wake-like state in vitro induced by transmembrane TNF/soluble TNF receptor reverse signaling

Tumor necrosis factor alpha (TNF) has sleep regulatory and brain development roles. TNF promotes sleep in vivo and in vitro while TNF inhibition diminishes sleep. Transmembrane (tm) TNF and the tmTNF receptors (Rs), are cleaved by tumor necrosis factor alpha convertase to produce soluble (s) TNF and sTNFRs. Reverse signaling occurs in cells expressing tmTNF upon sTNFR binding. sTNFR administration in vivo inhibits sleep, thus we hypothesized that a wake-like state in vitro would be induced by sTNFR-tmTNF reverse signaling. Somatosensory cortical neuron/glia co-cultures derived from male and female mice lacking both TNFRs (TNFRKO), or lacking TNF (TNFKO) and wildtype (WT) mice were plated onto six-well multi-electrode arrays. Daily one-hour electrophysiological recordings were taken on culture days 4 through 14. sTNFR1 (0.0, 0.3, 3, 30, 60, and 120 ng/µL) was administered on day 14. A final one-hour recording was taken on day 15. Four measures were characterized that are also used to define sleep in vivo: action potentials (APs), burstiness index (BI), synchronization of electrical activity (SYN), and slow wave power (SWP; 0.25–3.75 Hz). Development rates of these emergent electrophysiological properties increased in cells from mice lacking TNF or both TNFRs compared to cells from WT mice. Decreased SWP, after the three lowest doses (0.3, 3 and 30 ng/µL) of the sTNFR1, indicate a wake-like state in cells from TNFRKO mice. A wake-like state was also induced after 3 ng/µl sTNFR1 treatment in cells from TNFKO mice, which express the TNFR1 ligand, lymphotoxin alpha. Cells from WT mice showed no treatment effects. Results are consistent with prior studies demonstrating involvement of TNF in brain development, TNF reverse signaling, and sleep regulation in vivo. Further, the current demonstration of sTNFR1 induction of a wake-like state in vitro is consistent with the idea that small neuronal/glial circuits manifest sleep- and wake-like states analogous to those occurring in vivo. Finally, that sTNF forward signaling enhances sleep while sTNFR1 reverse signaling enhances a wake-like state is consistent with other sTNF/tmTNF/sTNFR1 brain actions having opposing activities.

Intestinal Stem Cell Niche Defects Result in Impaired 3D Organoid Formation in Mouse Models of Crohn's Disease-like Ileitis.

SummaryIntestinal epithelial barrier dysfunction is a risk factor in the pathogenesis of Crohn’s disease (CD); however, no corrective FDA-approved therapies exist. We used an enteroid (EnO)-based system in two murine models of experimental CD, SAMP1/YitFc (SAMP) and TNFΔARE/+ (TNF). While severely inflamed SAMP mice do not generate EnOs, “inflammation-free” SAMP mice form EnO structures with impaired morphology and reduced intestinal stem cell (ISC) and Paneth cell viability. We validated these findings in TNF mice concluding that inflammation in intestinal tissues impedes EnO generation and suppressing inflammation by steroid administration partially rescues impaired formation in SAMP mice. We generated the first high-resolution transcriptional profile of the SAMP ISC niche demonstrating that alterations in multiple key pathways contribute to niche defect and targeting them may partially rescue the phenotype. Furthermore, we correlated the defects in formation and the rescue of EnO formation to reduced viability of ISCs and Paneth cells.

Anti-TNF effect of combined pravastatin and cilostazol treatment in an in vivo mouse model

Objectives: Pravastatin and cilostazol are used as lipid-lowering and antiplatelet agents, respectively. Regarding their well-known anti-inflammatory effects, the additive effect of the two drugs on anti-TNF functions has not yet been investigated. In the present investigation, the beneficial effect of combined pravastatin and cilostazol on their anti-TNF activities was assessed using an in vivo mouse model.Methods: Mice were pretreated with pravastatin and/or cilostazol (40 mg/kg of each), orally once two hour prior to an LPS (5 mg/kg, i.p.) challenge. One hour post challenge, blood and descending aorta were collected for serum TNF levels and immune cell infiltration analyses. For survival analysis, pravastatin and/or cilostazol (40 mg/kg of each) were administered 30 minutes prior to d-galactosamine administration (700 mg/kg, i.p.) and TNF (10 µg/kg, i.p.) challenge and mice survival was monitored. We also examined the effect of either drug or the combination of drugs on TNF-mediated MAPK and NF-κB signaling, using Western blot analysis.Results: Combined treatment of pravastatin and cilostazol significantly decreased serum TNF release and immune cell infiltration in the descending aorta following LPS administration, compared to each single treatment. Additionally, the combined drugs significantly decreased TNF-mediated mouse mortality and downregulated TNF-induced MAPK and NF-κB activation.Conclusions: These findings suggest that combined pravastatin and cilostazol is more effective for reducing TNF-driven inflammation through their anti-TNF activity than monotherapy.

Immunomodulatory effects of herbal formula of astragalus polysaccharide (APS) and polysaccharopeptide (PSP) in mice with lung cancer

ObjectiveThis study is to investigate the immunomodulatory effects of the herbal formula of astragalus polysaccharide (APS) and polysaccharopeptide (PSP) in mouse models of immunosuppression and lung cancer. MethodsImmune parameters were recorded for these model mice. Peripheral white blood cells (WBC) were detected with the automatic blood cell analyzer. Spleen and thymus indices, and tumor inhibition ratio were obtained. Percentage of peripheral blood CD4+ and CD8+ T lymphocytes were detected by flow cytometry. Serum levels of Th1 (IL-2, TNF, and IFN-γ), Th2 (IL-4, IL-6, and IL-10), and Th17 (IL-17A) were detected with the BD cytometric bead array (CBA) mouseTh1/Th2/Th17 cytokine kit. ResultsCompared with the NS group, the PSP and APS herbal formula significantly improved the WBC, thymus index, spleen index, CD4+/CD8+ ratio, TNF, IFN-γ, IL-2, andIL-17Ainimmunosuppressivemice and lung cancer mice (P<0. 05). On the contrary, IL-10 was relatively low in the PSP+APS herbal formula group (P<0. 05). Besides, the PSP+APS herbal formula group induced comparable tumor inhibiting effect with the AMD group (23.3% and 24.1%, respectively). ConclusionThe PSP+APS herbal formula have immunomodulatory effects and anti-tumor activity in mice with of lung cancer.

Skin under Tnf influence: how regulatory T cells work against macrophages in psoriasis

AbstractTumour necrosis factor (TNF)‐α and interleukin (IL)‐17 are key cytokines driving psoriasis and other inflammatory autoimmune diseases, and thus represent effective targets for anti‐psoriatic therapy. In a recent issue of The Journal of Pathology, Leite Dantas et al explore a mouse model of TNF‐mediated psoriasiform dermatitis and arthritis with doxycyclin‐inducible general overexpression of human TNF (ihTNFtg) mice for the contributions of macrophages and T cells in skin inflammation – with some unexpected and interesting findings. Although T cells are commonly known as major proinflammatory players in psoriasis, in the ihTNFtg mouse model macrophages were the predominant cells causing inflammation, and T cells, represented by Foxp3+ regulatory T cells, mainly formed the opposition to keep inflammation in check. In addition to offering a new perspective on potential alternative initiation mechanisms in psoriatic skin inflammation, this constellation illustrates how cellular networks in inflammatory conditions evolve according to the prevailing cytokine, and may help to explain individual responses to either anti‐TNF‐α or anti‐IL‐17 therapy regimens in psoriasis. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley &amp; Sons, Ltd.

Targeted overexpression of tumor necrosis factor-α increases cyclin-dependent kinase 5 activity and TRPV1-dependent Ca2+ influx in trigeminal neurons.

We reported earlier that TNF-α, a proinflammatory cytokine implicated in many inflammatory disorders causing orofacial pain, increases the activity of Cdk5, a key kinase involved in brain development and function and recently found to be involved in pain signaling. To investigate a potential mechanism underlying inflammatory pain in trigeminal ganglia (TGs), we engineered a transgenic mouse model (TNF) that can conditionally overexpresses TNF-α upon genomic recombination by Cre recombinase. TNF mice were bred with Nav1.8-Cre mouse line that expresses the Cre recombinase in sensory neurons to obtain TNF-α:Nav1.8-Cre (TNF-α cTg) mice. Although TNF-α cTg mice appeared normal without any gross phenotype, they displayed a significant increase in TNF-α levels after activation of NFκB signaling in the TG. IL-6 and MCP-1 levels were also increased along with intense immunostaining for Iba1 and GFAP in TG, indicating the presence of infiltrating macrophages and the activation of satellite glial cells. TNF-α cTg mice displayed increased trigeminal Cdk5 activity, and this increase was associated with elevated levels of phospho-T407-TRPV1 and capsaicin-evocated Ca influx in cultured trigeminal neurons. Remarkably, this effect was prevented by roscovitine, an inhibitor of Cdk5, which suggests that TNF-α overexpression induced sensitization of the TRPV1 channel. Furthermore, TNF-α cTg mice displayed more aversive behavior to noxious thermal stimulation (45°C) of the face in an operant pain assessment device as compared with control mice. In summary, TNF-α overexpression in the sensory neurons of TNF-α cTg mice results in inflammatory sensitization and increased Cdk5 activity; therefore, this mouse model would be valuable for investigating the mechanism of TNF-α involved in orofacial pain.

Mechanisms of Microbe-Host Interaction in Crohn's Disease: Dysbiosis vs. Pathobiont Selection.

Crohn’s disease (CD) is a systemic chronic inflammatory condition mainly characterized by discontinuous transmural pathology of the gastrointestinal tract and frequent extraintestinal manifestations with intermittent episodes of remission and relapse. Genome-wide association studies identified a number of risk loci that, catalyzed by environmental triggers, result in the loss of tolerance toward commensal bacteria based on dysregulated innate effector functions and antimicrobial defense, leading to exacerbated adaptive immune responses responsible for chronic immune-mediated tissue damage. In this review, we discuss the inter-related role of changes in the intestinal microbiota, epithelial barrier integrity, and immune cell functions on the pathogenesis of CD, describing the current approaches available to investigate the molecular mechanisms underlying the disease. Substantial effort has been dedicated to define disease-associated changes in the intestinal microbiota (dysbiosis) and to link pathobionts to the etiology of inflammatory bowel diseases. A cogent definition of dysbiosis is lacking, as well as an agreement of whether pathobionts or complex shifts in the microbiota trigger inflammation in the host. Among the rarely available animal models, SAMP/Yit and TNFdeltaARE mice are the best known displaying a transmural CD-like phenotype. New hypothesis-driven mouse models, e.g., epithelial-specific Caspase8−/−, ATG16L1−/−, and XBP1−/− mice, validate pathway-focused function of specific CD-associated risk genes highlighting the role of Paneth cells in antimicrobial defense. To study the causal role of bacteria in initiating inflammation in the host, the use of germ-free mouse models is indispensable. Unraveling the interactions of genes, immune cells and microbes constitute a criterion for the development of safe, reliable, and effective treatment options for CD.

Blocking Tumor Necrosis Factor α Enhances CD8 T-cell-Dependent Immunity in Experimental Melanoma.

TNF plays a dual, still enigmatic role in melanoma, either acting as a cytotoxic cytokine or favoring a tumorigenic inflammatory microenvironment. Herein, the tumor growth of melanoma cell lines expressing major histocompatibility complex class I molecules at high levels (MHC-I(high)) was dramatically impaired in TNF-deficient mice, and this was associated with enhanced tumor-infiltrating CD8(+) T lymphocytes. Immunodepletion of CD8 T cells fully restored melanoma growth in TNF(-/-) mice. Systemic administration of Etanercept inhibited MHC-I(high) melanoma growth in immunocompetent but not in immunodeficient (IFNγ(-/-), nude, or CD8(-/-)) mice. MHC-I(high) melanoma growth was also reduced in mice lacking TNF-R1, but not TNF-R2. TNF(-/-) and TNF-R1(-/-) mice as well as Etanercept-treated WT mice displayed enhanced intratumor content of high endothelial venules surrounded by high CD8(+) T-cell density. Adoptive transfer of activated TNF-R1-deficient or -proficient CD8(+) T cells in CD8-deficient mice bearing B16K1 tumors demonstrated that TNF-R1 deficiency facilitates the accumulation of live CD8(+) T cells into the tumors. Moreover, in vitro experiments indicated that TNF triggered activated CD8(+) T cell death in a TNF-R1-dependent manner, likely limiting the accumulation of tumor-infiltrating CD8(+) T cells in TNF/TNF-R1-proficient animals. Collectively, our observations indicate that TNF-R1-dependent TNF signaling impairs tumor-infiltrating CD8(+) T-cell accumulation and may serve as a putative target to favor CD8(+) T-cell-dependent immune response in melanoma.

Interferon-gamma–mediated tissue factor expression contributes to T-cell-mediated hepatitis through induction of hypercoagulation in mice

Concanavalin A (Con A) treatment induces severe hepatitis in mice in a manner dependent on T cells, interferon (IFN)-gamma, and tumor necrosis factor (TNF). Treatment with the anticoagulant heparin protects against hepatitis, despite healthy production of IFN-γ and TNF. Here, we investigated molecular and cellular mechanisms for hypercoagulation-mediated hepatitis. After Con A challenge, liver of wild-type (WT) mice showed prompt induction of Ifnγ and Tnf, followed by messenger RNA expression of tissue factor (TF) and plasminogen activator inhibitor-1 (PAI-1), which initiate blood coagulation and inhibit clot lysis, respectively. Mice developed dense intrahepatic fibrin deposition and massive liver necrosis. In contrast, Ifnγ(-/-) mice and Ifnγ(-/-) Tnf(-/-) mice neither induced Pai1 or Tf nor developed hepatitis. In WT mice TF blockade with an anti-TF monoclonal antibody protected against Con A-induced hepatitis, whereas Pai1(-/-) mice were not protected. Both hepatic macrophages and sinusoidal endothelial cells (ECs) expressed Tf after Con A challenge. Macrophage-depleted WT mice reconstituted with hematopoietic cells, including macrophages deficient in signal transducer and activator of transcription-1 (STAT1) essential for IFN-γ signaling, exhibited substantial reduction of hepatic Tf and of liver injuries. This was also true for macrophage-depleted Stat1(-/-) mice reconstituted with WT macrophages. Exogenous IFN-γ and TNF rendered T-cell-null, Con A-resistant mice deficient in recombination-activating gene 2, highly susceptible to Con A-induced liver injury involving TF. Collectively, these results strongly suggest that proinflammatory signals elicited by IFN-γ, TNF, and Con A in both hepatic macrophages and sinusoidal ECs are necessary and sufficient for the development of hypercoagulation-mediated hepatitis.

Dexamethasone Down-Regulates Expression of Triggering Receptor Expressed on Myeloid Cells-1: Evidence for a TNFα-Related Effect

Objectives: To investigate the effect of dexamethasone on triggering receptor expressed on myeloid cells-1 (TREM-1).Methods: Wild-type and tumor necrosis factor (TNF−/−) mice were pre-treated with saline, dexamethasone, or hydrocortisone and exposed to a lethal infection of Pseudomonas aeruginosa. Mortality and TREM-1 on neutrophil membranes was measured after sacrifice. U937 human monocytic cells were stimulated with lipopolysaccharide (LPS) or heat-killed P. aeruginosa without or with dexamethasone or hydrocortisone, and cell-surface TREM-1 and soluble TREM-1 (sTREM-1) were quantified. Expression of TREM-1 and sTREM-1 was also studied in LPS-stimulated U937 cells incubated in the absence or presence of TNFα or anti-TNFα antibody.Results: Pre-treatment with dexamethasone, but not hydrocortisone, prolonged animal survival. Mice pre-treated with dexamethasone showed decreased expression of TREM-1 on neutrophils. In U937 cells, LPS or heat-killed P. aeruginosa induced the expression of TREM-1 and the release of sTREM-1. U937 TREM-1 and sTREM-1 were decreased upon addition of dexamethasone but not hydrocortisone. The suppressive effect of dexamethasone was enhanced in the presence of exogenous TNFα and lost in the presence of anti-TNFα antibody. In TNF−/− mice, dexamethasone suppression of mortality and TREM-1 neutrophil expression was lost. Gene expression of TREM-1 in U937 monocytes was decreased after treatment with dexamethasone.Conclusion: TREM-1/sTREM-1 is a novel site of action of dexamethasone. This action is associated with down-regulation of gene expression and is mediated by TNFα.

P012 Control of mycobacterial infection in “humanized” TNF knock-in mice

Introduction TNF is associated with the development of human immunopathologies and involved in host defense. TNF blockade to treat autoimmune inflammatory diseases revealed a crucial and non-redundant role of TNF for the control to tuberculosis infection. Tuberculosis is a chronic infection and sustained TNF and Th1 immune responses are crucial to control infection. To test and select specific human TNF inhibitors requires a special animal model. Therefore a mouse was developed where murine TNF was replaced by human TNF (human TNF knock-in) and we investigated host resistance to mycobacterial infection. Methods Human TNF KI mice (humanized TNF mice) have been developed (Kruglov et al., in preparation) and studied for their capacity to generate protective cell-mediated immune responses and resistance to Mycobacterium bovis BCG and M. tuberculosis infections. Bone marrow derived macrophages (BMDM) infected with BCG have been analyzed for the ability to respond to in vitro infection. Results BMDM from humanized TNF KI mice were able to produce cytokines and nitrites in response to BCG infection as wild-type macrophages. BCG-induced NF-kB activation was similar in humanized TNF KI and wild-type macrophages whereas TNF KO cells presented impaired NF-kB phosphorylation. Humanized TNF KI mice survived to acute and chronic M. bovis BCG and M. tuberculosis infections as wild-type mice while TNF KO mice rapidly succumbed. Bacterial burden was controlled in humanized TNF KI, while overwhelming infection was evident in TNF KO mice. Bactericidal mechanism such as macrophage iNOS activation was analyzed in the liver of infected mice. iNOS protein levels were decreased in humanized TNF mice compared to wild-type mice at early infection, but these levels became comparable at late infection. The pattern of serum and lung cytokines showed that humanized TNF KI mice were able to activate Th1 type immune responses producing amounts of IFN γ and IL-12 similar to that observed in wild-type mice after an infection. Conclusion This study in humanized TNF mice demonstrates that human TNF can replace the function of mouse TNF during an intracellular bacterial infection providing resistance, NF-kB activation and Th1 immune responses. Therefore, human TNF KI mice may be highly relevant to test novel biologics neutralizing human TNF and their effects on inflammatory and infectious pathologies.

Interplay Between SAFE and RISK Pathways in Sphingosine-1-Phosphate–Induced Cardioprotection

We studied the role of two powerful molecular signalling mechanisms involved in the cardioprotective effect of sphingosine-1-phosphate (S1P), a major component of high density lipoprotein (HDL) against myocardial ischaemic-reperfusion injury, namely the RISK pathway (Akt/Erk), including its downstream target FOXO-1 and, the SAFE pathway (TNF/STAT-3). Control hearts from wildtype, TNF deficient (TNF(-/-)) or cardiomyocyte STAT-3 deficient (STAT-3(-/-)) male mice were perfused on a Langendorff apparatus (35 min global ischaemia and 45 min reperfusion). S1P (10 nM) was given at the onset of reperfusion for the first 7 min, with/without STAT-3 or Akt inhibitors, AG490 and wortmannin (W), respectively. S1P reduced myocardial infarct size in wildtype hearts (39.3±4.4% in control vs 17.3±3.1% in S1P-treated hearts; n≥6; p<0.05) but not in STAT-3(-/-) or TNF(-/-) mice (34.2±4.3% in STAT-3(-/-) and 34.1±2.0% in TNF(-/-) mice; n≥6; p=ns vs. their respective control). Both STAT-3 and Akt inhibitors abolished the protective effects of S1P (33.7±3.3% in S1P + AG490 and 36.6±4.9% in S1P + W; n=6; p=ns vs. their respective control). Increased nuclear levels of phosphorylated STAT-3 (pSTAT-3), Akt and FOXO-1 were observed at 15 min reperfusion in wildtype mice with Western Blot analysis (53% STAT-3, 47% Akt, 41% FOXO-1; p<0.05 vs control) but not in STAT-3-/- mice or in wiltype hearts treated with the Akt inhibitor. Interestingly, an activation of pSTAT-3 was noticed in the mitochondria at 7 min but not 15 min of reperfusion. In conclusion, S1P activates both the SAFE and RISK pathways, therefore suggesting a dual protective signalling in S1P-induced cardioprotection.

Catechols in caffeic acid phenethyl ester are essential for inhibition of TNF‐mediated IP‐10 expression through NF‐κB‐dependent but HO‐1‐ and p38‐independent mechanisms in mouse intestinal epithelial cells

Caffeic acid phenethyl ester (CAPE) is an active constituent of honeybee propolis inhibiting nuclear factor (NF)-κB. The aims of our study were to provide new data on the functional relevance and mechanisms underlying the role of CAPE in regulating inflammatory processes at the epithelial interface in the gut and to determine the structure/activity relationship of CAPE. CAPE significantly inhibited TNF-induced IP-10 expression in intestinal epithelial cells. Using various analogues, we demonstrated that substitution of catechol hydroxyl groups and addition of one extra hydroxyl group on ring B reversed the functional activity of CAPE to inhibit IP-10 production. The anti-inflammatory potential of CAPE was confirmed in ileal tissue explants and embryonic fibroblasts derived from TNF(ΔARE/+) mice. Interestingly, CAPE inhibited both TNF- and LPS-induced IP-10 production in a dose-dependent manner, independently of p38 MAPK, HO-1 and Nrf2 signaling pathways. We found that CAPE did not inhibit TNF-induced IκB phosphorylation/degradation or nuclear translocation of RelA/p65, but targeted downstream signaling events at the level of transcription factor recruitment to the gene promoter. This study reveals the structure-activity effects and anti-inflammatory potential of CAPE in the intestinal epithelium.

Enterococcus faecalis Metalloprotease Compromises Epithelial Barrier and Contributes to Intestinal Inflammation

Matrix metalloproteases (MMPs) mediate pathogenesis of chronic intestinal inflammation. We characterized the role of the gelatinase (GelE), a metalloprotease from Enterococcus faecalis, in the development of colitis in mice. Germ-free, interleukin-10-deficient (IL-10(-/-)) mice were monoassociated with the colitogenic E faecalis strain OG1RF and isogenic, GelE-mutant strains. Barrier function was determined by measuring E-cadherin expression, transepithelial electrical resistance (TER), and translocation of permeability markers in colonic epithelial cells and colon segments from IL-10(-/-) and TNF(ΔARE/Wt) mice. GelE specificity was shown with the MMP inhibitor marimastat. Histologic analysis (score 0-4) of E faecalis monoassociated IL-10(-/-) mice revealed a significant reduction in colonic tissue inflammation in the absence of bacteria-derived GelE. We identified cleavage sites for GelE in the sequence of recombinant mouse E-cadherin, indicating that it might be degraded by GelE. Experiments with Ussing chambers and purified GelE revealed the loss of barrier function and extracellular E-cadherin in mice susceptible to intestinal inflammation (IL-10(-/-) and TNF(ΔARE/Wt) mice) before inflammation developed. Colonic epithelial cells had reduced TER and increased translocation of permeability markers after stimulation with GelE from OG1RF or strains of E faecalis isolated from patients with Crohn's disease and ulcerative colitis. The metalloprotease GelE, produced by commensal strains of E faecalis, contributes to development of chronic intestinal inflammation in mice that are susceptible to intestinal inflammation (IL-10(-/-) and TNF(ΔARE/Wt) mice) by impairing epithelial barrier integrity.

Loss of downregulated in adenoma (DRA) impairs mucosal HCO3− secretion in murine ileocolonic inflammation

Ileocolonic luminal pH has been reported to be abnormally low in inflammatory bowel disease (IBD) patients, and one of the causative factors may be reduced epithelial HCO(3)(-) secretory rate (J(HCO3)(-)). Disturbances in J(HCO3)(-) may occur due to inflammation-induced changes in the crypt and villous architecture, or due to the effect of proinflammatory cytokines on epithelial ion transporters. To discriminate between these possibilities, the tumor necrosis factor alpha (TNF-α) overexpressing (TNF(+/ΔARE)) mouse model was chosen, which displays high proinflammatory cytokine levels in both ileum and colon, but develops only mild colonic histopathology and diarrhea. HCO(3)(-) secretion, mRNA expression, immunohistochemistry, and fluid absorptive capacity were measured in ileal and mid-colonic mucosa of TNF(+/ΔARE) and wildtype (WT) (TNF(+/+)) mice in Ussing chambers, and in anesthetized mice in vivo. The high basal J(HCO3)(-) observed in WT ileal and mid-colonic mucosa were luminal Cl(-) -dependent and strongly decreased in TNF(+/ΔARE) mice. Downregulated in adenoma (DRA) mRNA and protein expression was strongly decreased in TNF(+/ΔARE) ileocolon, whereas cystic fibrosis transmembrane conductance regulator (CFTR), Na(+) /H(+) exchanger 3 (NHE3), Na(+) /HCO(3)(-) cotransporter (NBC), and epithelial sodium channel (ENaC) expression was not significantly altered. This indicates that the severe defect in ileocolonic J(HCO3)(-) was due to DRA downregulation. Fluid absorption was severely depressed in the ileum but only mildly affected in the mid-distal colon, preventing the development of overt diarrhea. Even mild ileocolonic inflammation may result in a decrease of epithelial HCO(3)(-) secretion, which may contribute to alterations in surface pH, intestinal flora, and mucus barrier properties.

Tumor necrosis factor-alpha is an endogenous inhibitor of Na+-K+-2Cl- cotransporter (NKCC2) isoform A in the thick ascending limb.

The effects of TNF gene deletion on renal Na(+)-K(+)-2Cl(-) cotransporter (NKCC2) expression and activity were determined. Outer medulla from TNF(-/-) mice exhibited a twofold increase in total NKCC2 protein expression compared with wild-type (WT) mice. This increase was not observed in TNF(-/-) mice treated with recombinant human TNF (hTNF) for 7 days. Administration of hTNF had no effect on total NKCC2 expression in WT mice. A fourfold increase in NKCC2A mRNA accumulation was observed in outer medulla from TNF(-/-) compared with WT mice; NKCC2F and NKCC2B mRNA accumulation was similar between genotypes. The increase in NKCC2A mRNA accumulation was attenuated when TNF(-/-) mice were treated with hTNF. Bumetanide-sensitive O(2) consumption, an in vitro correlate of NKCC2 activity, was 2.8 ± 0.2 nmol·min(-1)·mg(-1) in medullary thick ascending limb tubules from WT, representing ∼40% of total O(2) consumption, whereas, in medullary thick ascending limb tubules from TNF(-/-) mice, it was 5.6 ± 0.3 nmol·min(-1)·mg(-1), representing ∼60% of total O(2) consumption. Administration of hTNF to TNF(-/-) mice restored the bumetanide-sensitive component to ∼30% of total O(2) consumption. Ambient urine osmolality was higher in TNF(-/-) compared with WT mice (2,072 ± 104 vs. 1,696 ± 153 mosmol/kgH(2)O, P < 0.05). The diluting ability of the kidney, assessed by measuring urine osmolality before and after 1 h of water loading also was greater in TNF(-/-) compared with WT mice (174 ± 38 and 465 ± 81 mosmol/kgH(2)O, respectively, P < 0.01). Collectively, these findings suggest that TNF plays a role as an endogenous inhibitor of NKCC2 expression and function.

Novel development in extraintestinal manifestations and spondylarthropathy

The important co-existence of spondylarthritis (SpA) and inflammatory bowel disease (IBD) within the same individual suggests common etiopathogenic mechanisms. This is supported by intriguing similarities between both diseases at the subclinical and molecular level. The recent advances in IBD genetics have led to the identification of common pathways involved in both IBD and SpA, including bacterial recognition and ER stress. This offers the opportunity to develop potential new therapeutic strategies for both diseases. Transgenic animals which develop both joint and gut inflammation (like the TNF(ΔARE) mice and the HLA-B27 transgenic rats) are a very useful tool to test such novel therapeutics and to get further mechanistic insight into the pathogenetic link between SpA and IBD. This review will focus on the recent scientific progress in our understanding of the link between SpA and IBD. Based on this, potential novel therapeutic strategies are discussed.

Limited Role for Lymphotoxin α in the Host Immune Response toMycobacteriumtuberculosis

The contribution of lymphotoxin (LT)α in the host immune response to virulent Mycobacterium tuberculosis and Mycobacterium bovis bacillus Calmette-Guérin infections was investigated. Despite their ability to induce Th1 cytokine, IFN-γ, and IL-12 pulmonary response, "conventional" LTα(-/-) mice succumb rapidly to virulent M. tuberculosis aerosol infection, with uncontrolled bacilli growth, defective granuloma formation, necrosis, and reduced pulmonary inducible NO synthase expression, similar to TNF(-/-) mice. Contributions from developmental lymphoid abnormalities in LTα(-/-) mice were excluded because hematopoietic reconstitution with conventional LTα(-/-) bone marrow conferred enhanced susceptibility to wild-type mice, comparable to conventional LTα(-/-) control mice. However, conventional LTα(-/-) mice produced reduced levels of TNF after M. bovis bacillus Calmette-Guérin infection, and their lack of control of mycobacterial infection could be due to a defective contribution of either LTα or TNF, or both, to the host immune response. To address this point, the response of "neo-free" LTα(-/-) mice with unperturbed intrinsic TNF expression to M. tuberculosis infection was investigated in a direct comparative study with conventional LTα(-/-) mice. Strikingly, although conventional LTα(-/-) mice were highly sensitive, similar to TNF(-/-) mice, neo-free LTα(-/-) mice controlled acute M. tuberculosis infection essentially as wild-type mice. Pulmonary bacterial burden and inflammation was, however, slightly increased in neo-free LTα(-/-) mice 4-5 mo postinfection, but importantly, they did not succumb to infection. Our findings revise the notion that LTα might have a critical role in host defense to acute mycobacterial infection, independent of TNF, but suggest a contribution of LTα in the control of chronic M. tuberculosis infection.

Characterization of CCX282-B, an Orally Bioavailable Antagonist of the CCR9 Chemokine Receptor, for Treatment of Inflammatory Bowel Disease

The chemokine system represents a diverse group of G protein-coupled receptors responsible for orchestrating cell recruitment under both homeostatic and inflammatory conditions. Chemokine receptor 9 (CCR9) is a chemokine receptor known to be central for migration of immune cells into the intestine. Its only ligand, CCL25, is expressed at the mucosal surface of the intestine and is known to be elevated in intestinal inflammation. To date, there are no reports of small-molecule antagonists targeting CCR9. We report, for the first time, the discovery of a small molecule, CCX282-B, which is an orally bioavailable, selective, and potent antagonist of human CCR9. CCX282-B inhibited CCR9-mediated Ca(2+) mobilization and chemotaxis on Molt-4 cells with IC(50) values of 5.4 and 3.4 nM, respectively. In the presence of 100% human serum, CCX282-B inhibited CCR9-mediated chemotaxis with an IC(50) of 33 nM, and the addition of α1-acid glycoprotein did not affect its potency. CCX282-B inhibited chemotaxis of primary CCR9-expressing cells to CCL25 with an IC(50) of 6.8 nM. CCX282-B was an equipotent inhibitor of CCL25-directed chemotaxis of both splice forms of CCR9 (CCR9A and CCR9B) with IC(50) values of 2.8 and 2.6 nM, respectively. CCX282-B also inhibited mouse and rat CCR9-mediated chemotaxis. Inhibition of CCR9 with CCX282-B results in normalization of Crohn's disease such as histopathology associated with the TNF(ΔARE) mice. Analysis of the plasma level of drug associated with this improvement provides an understanding of the pharmacokinetic/pharmacodynamic relationship for CCR9 antagonists in the treatment of intestinal inflammation.