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The coagulation protein tissue factor (TF) regulates inflammation and angiogenesis via its cytoplasmic domain in infection, cancer and diabetes. While TF is highly abundant in the heart and is implicated in cardiac pathology, the contribution of its cytoplasmic domain to post-infarct myocardial injury and adverse left ventricular (LV) remodeling remains unknown.Methods: Myocardial infarction was induced in wild-type mice or mice lacking the TF cytoplasmic domain (TF increment CT) by occlusion of the left anterior descending coronary artery. Heart function was monitored with echocardiography. Heart tissue was collected at different time-points for histological, molecular and flow cytometry analysis.Results: Compared with wild-type mice, TF increment CT had a higher survival rate during a 28-day follow-up after myocardial infarction. Among surviving mice, TF increment CT mice had better cardiac function and less LV remodeling than wild-type mice. The overall improvement of post-infarct cardiac performance in TF increment CT mice, as revealed by speckle-tracking strain analysis, was attributed to reduced myocardial deformation in the peri-infarct region. Histological analysis demonstrated that TF increment CT hearts had in the infarct area greater proliferation of myofibroblasts and better scar formation. Compared with wild-type hearts, infarcted TF increment CT hearts showed less infiltration of proinflammatory cells with concomitant lower expression of protease-activated receptor-1 (PAR1) -Rac1 axis. In particular, infarcted TF increment CT hearts displayed markedly lower ratios of inflammatory M1 macrophages and reparative M2 macrophages (M1/M2). In vitro experiment with primary macrophages demonstrated that deletion of the TF cytoplasmic domain inhibited macrophage polarization toward the M1 phenotype. Furthermore, infarcted TF increment CT hearts presented markedly higher peri-infarct vessel density associated with enhanced endothelial cell proliferation and higher expression of PAR2 and PAR2-associated pro-angiogenic pathway factors. Finally, the overall cardioprotective effects observed in TF increment CT mice could be abolished by subcutaneously infusing a cocktail of PAR1-activating peptide and PAR2-inhibiting peptide via osmotic minipumps.Conclusions: Our findings demonstrate that the TF cytoplasmic domain exacerbates post-infarct cardiac injury and adverse LV remodeling via differential regulation of inflammation and angiogenesis. Targeted inhibition of the TF cytoplasmic domain-mediated intracellular signaling may ameliorate post-infarct LV remodeling without perturbing coagulation.

Vascular permeability and plasma leakage are immune-pathologies of severe dengue virus (DENV) infection, but the mechanisms underlying the exacerbated inflammation during DENV pathogenesis are unclear. Here, we demonstrate that TLR2, together with its co-receptors CD14 and TLR6, is an innate sensor of DENV particles inducing inflammatory cytokine expression and impairing vascular integrity in vitro. Blocking TLR2 prior to DENV infection in vitro abrogates NF-κB activation while CD14 and TLR6 block has a moderate effect. Moreover, TLR2 block prior to DENV infection of peripheral blood mononuclear cells prevents activation of human vascular endothelium, suggesting a potential role of the TLR2-responses in vascular integrity. TLR2 expression on CD14 + + classical monocytes isolated in an acute phase from DENV-infected pediatric patients correlates with severe disease development. Altogether, these data identify a role for TLR2 in DENV infection and provide insights into the complex interaction between the virus and innate receptors that may underlie disease pathogenesis., The mechanisms underlying immunpathologies in dengue virus (DENV) infection are incompletely understood. Here, authors show that TLR2 recognizes DENV particles inducing cytokine expression and activating vascular endothelium cells in vitro, and that TLR2 expression on monocytes correlates with disease severity in patients.

Application of allergens onto the sublingual epithelium is used to desensitize allergic individuals, a treatment known as sublingual immunotherapy. However, the response of sublingual epithelial cells to house dust mite allergen and potential tolerance-promoting adjuvants such as Toll-like receptor (TLR) ligands and calcitriol has not been investigated. In order to study this, primary sublingual epithelial cells were isolated from dogs and cultured in vitro. After 24-h incubation with a Dermatophagoides farinae extract, a Dermatophagoides pteronyssinus extract, TLR2 ligands (FSL-1, heat-killed Listeria monocytogenes, Pam3CSK4), a TLR3 ligand (poly I:C), a TLR4 ligand [lipopolysaccharide (LPS)], and calcitriol (1,25-dihydroxyvitamin D3), viability of the cells was analyzed using an MTT test, and their secretion of interleukin 6 (IL-6), IL-10, CXCL8, and transforming growth factor β1 (TGF-β1) was measured by enzyme-linked immunosorbent assay. Additionally, to evaluate its potential effect as an adjuvant, sublingual epithelial cells were incubated with calcitriol in combination with a D. farinae extract followed by measurement of CXCL8 secretion. Furthermore, the effect of D. farinae and calcitriol on the transcriptome was assessed by RNA sequencing. The viability of the sublingual epithelial cells was significantly decreased by poly I:C, but not by the other stimuli. CXCL8 secretion was significantly increased by D. farinae extract and all TLR ligands apart from LPS. Calcitriol significantly decreased CXCL8 secretion, and coadministration with D. farinae extract reduced CXCL8 concentrations to levels seen in unstimulated sublingual epithelial cells. Although detectable, TGF-β1 secretion could not be modulated by any of the stimuli. Interleukin 6 and IL-10 could not be detected at the protein or at the mRNA level. It can be concluded that a D. farinae extract and TLR ligands augment the secretion of the proinflammatory chemokine CXCL8, which might interfere with sublingual desensitization. On the other hand, CXCL8 secretion was reduced by coapplication of calcitriol and a D. farinae extract. Calcitriol therefore seems to be a suitable candidate to be used as adjuvant during sublingual immunotherapy.

The expression of short chain fatty acid receptors FFA2 and FFA3 in pancreatic islets raised interest in using them as drug targets for treating hyperglycemia in humans. This study aims to examine the efficacy of synthetic FFA2- and FFA3-ligands to modulate glucose-stimulated insulin secretion (GSIS) in human pseudoislets which display intact glucose responsiveness. The FFA2-agonists 4-CMTB and TUG-1375 inhibited GSIS, an effect reversed by the FFA2-antagonist CATPB. GSIS itself was not augmented by CATPB. The FFA3-agonists FHQC and 1-MCPC did not affect GSIS in human pseudoislets. For further drug evaluation we used mouse islets. The CATPB-sensitive inhibitory effect of 100 µM 4-CMTB on GSIS was recapitulated. The inhibition was partially sensitive to the Gi/o-protein inhibitor pertussis toxin. A previously described FFA2-dependent increase of GSIS was observed with lower concentrations of 4-CMTB (10 and 30 µM). The stimulatory effect of 4-CMTB on secretion was prevented by the Gq-protein inhibitor FR900359. As in human pseudoislets, in mouse islets relative mRNA levels were FFAR2 > FFAR3 and FFA3-agonists did not affect GSIS. The FFA3-agonists, however, inhibited GSIS in a pertussis toxin-sensitive manner in INS-1E cells and this correlated with relative mRNA levels of Ffar3 > > Ffar2. Thus, in humans, when FFA2-activation impedes GSIS, FFA2-antagonism may reduce glycemia.

Aims Pulmonary arterial hypertension (PAH) is a devastating disease and treatment options are limited. Urocortin-2 (Ucn-2) has shown promising therapeutic effects in experimental and clinical left ventricular heart failure (HF). Our aim was to analyse the expression of Ucn-2 in human and experimental PAH, and to investigate the effects of human Ucn-2 (hUcn-2) administration in rats with monocrotaline (MCT)-induced pulmonary hypertension (PH). Methods and results Tissue samples were collected from patients with and without PAH and from rats with MCT-induced PH. hUcn-2 (5 μg/kg, bi-daily, i.p., for 10 days) or vehicle was administered to male wistar rats subjected to MCT injection or to pulmonary artery banding (PAB) to induce right ventricular (RV) overload without PAH. Expression of Ucn-2 and its receptor was increased in the RV of patients and rats with PAH. hUcn-2 treatment reduced PAH in MCT rats, resulting in decreased morbidity, improved exercise capacity and attenuated pulmonary arterial and RV remodelling and dysfunction. Additionally, RV gene expression of hypertrophy and failure signalling pathways were attenuated. hUcn-2 treatment also attenuated PAB-induced RV hypertrophy. Conclusions Ucn-2 levels are altered in human and experimental PAH. hUcn-2 treatment attenuates PAH and RV dysfunction in MCT-induced PH, has direct anti-remodelling effects on the pressure-overloaded RV, and improves pulmonary vascular function.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder and the most common cause of dementia. Accumulating experimental evidence shows the important linkage between tumor necrosis factor-α (TNF) and AD, but the exact role of TNF in AD is still not completely understood. Although TNF-inhibitors are successfully used for treating several diseases, total inhibition of TNF can cause side effects, particularly in neurological diseases. This is attributed to the opposing roles of the two TNF receptors. TNF receptor 1 (TNFR1) predominantly mediates inflammatory and pro-apoptotic signaling pathways, whereas TNF receptor 2 (TNFR2) is neuroprotective and promotes tissue regeneration. Therefore, the specific activation of TNFR2 signaling, either by directly targeting TNFR2 via TNFR2 agonists or by blocking TNFR1 signaling with TNFR1-selective antagonists, seems a promising strategy for AD therapy. This mini-review discusses the involvement of TNFR2 and its signaling pathway in AD and outlines its potential application as therapeutic target. A better understanding of the function of TNFR2 may lead to the development of a treatment for AD.

Autoimmunity develops when self-tolerance mechanisms are failing to protect healthy tissue. A sustained reaction to self is generated, which includes the generation of effector cells and molecules that destroy tissues. A way to restore this intrinsic tolerance is through immune modulation that aims at refurbishing this immunologically naive or unresponsive state, thereby decreasing the aberrant immune reaction taking place. One major cytokine has been shown to play a pivotal role in several autoimmune diseases such as rheumatoid arthritis (RA) and multiple sclerosis (MS): tumor necrosis factor alpha (TNF alpha) modulates the induction and maintenance of an inflammatory process and it comes in two variants, soluble TNF (solTNF) and transmembrane bound TNF (tmTNF). tmTNF signals via TNFR1 and TNFR2, whereas solTNF signals mainly via TNFR1. TNFR1 is widely expressed and promotes mainly inflammation and apoptosis. Conversely, TNFR2 is restricted mainly to immune and endothelial cells and it is known to activate the pro-survival PI3K-Akt/PKB signaling pathway and to sustain regulatory T cells function. Anti-TNF alpha therapies are successfully used to treat diseases such as RA, colitis, and psoriasis. However, clinical studies with a non-selective inhibitor of TNFa in MS patients had to be halted due to exacerbation of clinical symptoms. One possible explanation for this failure is the non-selectivity of the treatment, which avoids TNFR2 stimulation and its immune and tissue protective properties. Thus, a receptor-selective modulation of TNFa signal pathways provides a novel therapeutic concept that might lead to new insights in MS pathology with major implications for its effective treatment.

Staphylococcus aureus is a frequent commensal but also a dangerous pathogen, causing many forms of infection ranging from mild to life-threatening conditions. Among its virulence factors are lipoproteins, which are anchored in the bacterial cell membrane. Lipoproteins perform various functions in colonization, immune evasion, and immunomodulation. These proteins are potent activators of innate immune receptors termed Toll-like receptors 2 and 6. This study addressed the specific B-cell and T-cell responses directed to lipoproteins in human S. aureus carriers and non-carriers. 2D immune proteomics and ELISA approaches revealed that titers of antibodies (IgG) binding to S. aureus lipoproteins were very low. Proliferation assays and cytokine profiling data showed only subtle responses of T cells; some lipoproteins did not elicit proliferation. Hence, the robust activation of the innate immune system by S. aureus lipoproteins does not translate into a strong adaptive immune response. Reasons for this may include inaccessibility of lipoproteins for B cells as well as ineffective processing and presentation of the antigens to T cells.

Scope: In the present study, the direct interaction of commonly consumed fibers with epithelial or dendritic cells (DCs) was studied.Methods and results: The fibers were characterized for their sugar composition and chain length profile. When in direct contact, fibers activate DCs only mildly. This was different when DCs and fibers were co-cultured together with supernatants from human epithelial cells (Caco spent medium). Caco spent medium enhanced the production of IL-12, IL-1Ra, IL-6, IL-8, TNF-alpha, MCP-1 (monocyte chemotactic protein), and MIP-1 alpha but this was strongly attenuated by the dietary fibers. This attenuating effect on proinflammatory cytokines was dependent on the interaction of the fibers with Toll-like receptors as it was reduced by Pepinh-myd88. The interaction of galacto-oligosaccharides, chicory inulin, wheat arabinoxylan, barley beta-glucan with epithelial cells and DCs led to changes in the production of the Th1 cytokines in autologous T cells, while chicory inulin, and barley beta-glucan reduced the Th2 cytokine IL-6. The Treg-promoting cytokine IL-10 was induced by galacto-oligosaccharides whereas chicory inulin decreased the IL-10 production.Conclusions: Our results suggest that dietary fibers can modulate the host immune system not only by the recognized mechanism of effects on microbiota but also by direct interaction with the consumer's mucosa. This modulation is dietary fiber type dependent.

Background: Nasal vaccination is considered to be a promising alternative for parenteral vaccination against influenza virus as it is non-invasive and offers the opportunity to elicit strong antigen-specific responses both systemic and locally at the port of entry of the pathogen. Previous studies showed that non-living bacterium-like particles (BLPs) from the food-grade bacterium Lactococcus lact-is are effective stimulators of local and systemic immune responses when administered intranasally. Moreover, in vitro, BLPs specifically interact with human Toll-like receptor 2 (TLR2), suggestive of a role for TLR2 dependent immune activation by BLPs.Methods: In the present study, we examined the role of TLR2 in vivo in immune activation after nasal administration of BLP mixed with split influenza vaccine (BLP-SV) of influenza A virus (IAV) using TLR2 knockout mice.Results: The systemic Th1 cell and subsequent B-cell responses induced after intranasal BLP-SV vaccination depended on the interaction of BLPs with TLR2. Notably, the BLP-SV-induced class switch to IgG2c depended on the interaction of BLP with TLR2. Local induced IAV-specific Th1 cell responses and the mucosal B-cell responses also depended on interaction of BLP with TLR2. Strongly reduced SIgA levels were observed in TLR2 knockout mice both in the nasal and vaginal lavages. In addition, detailed analysis of the T-cell response revealed that nasal BLP-SV vaccination promoted Th1/Th17 immune responses that coincided with increased IAV-specific IgG2c antibody production.Discussion: Altogether these results indicate that nasal BLP-SV vaccination induces IAV-specific T-cell and B-cell responses, both systemically and at the site of virus entry in a TLR2-dependent manner. (C) 2014 The Authors. Published by Elsevier Ltd.

Tumor necrosis factor (TNF) is a cytokine produced by macrophages and T lymphocytes that acts through two distinct receptors, TNFR1 (60 kD, CD120a) and TNFR2 (80 kD, CD120b), to affect cellular proliferation, differentiation, survival, and cell death. In addition to its proinflammatory actions in mucosal tissue, TNF is important for liver regeneration. Keratin 8 (K8) and keratin 18 (K18) form intermediate filaments characteristic of liver and other single cell layered, internal epithelia and their derivative cancers. K8-deficient (K8(-)) mice, which escape embryonic lethality, develop inflammatory colorectal hyperplasia, mild liver abnormalities, and tolerate hepatectomy poorly. We show that normal and malignant epithelial cells deficient in K8 and K18 are approximately 100 times more sensitive to TNF-induced death. K8 and K18 both bind the cytoplasmic domain of TNFR2 and moderate TNF-induced, Jun NH(2)-terminal kinase (JNK) intracellular signaling and NFkappaB activation. Furthermore, K8(-) and K18(-) mice are much more sensitive to TNF dependent, apoptotic liver damage induced by the injection of concanavalin A. This moderation of the effects of TNF may be the fundamental function of K8 and K18 common to liver regeneration, inflammatory bowel disease, hepatotoxin sensitivity, and the diagnostic, persistent expression of these keratins in many carcinomas.