Sialic acid-modified antigens impose tolerance via inhibition of T-cell proliferation and de novo induction of regulatory T cells.

Sialic acids are negatively charged nine-carbon carboxylated monosaccharides that often cap glycans on glycosylated proteins and lipids. Because of their strategic location at the cell surface, sialic acids contribute to interactions that are critical for immune homeostasis via interactions with sialic acid-binding Ig-type lectins (siglecs). In particular, these interactions may be of importance in cases where sialic acids may be overexpressed, such as on certain pathogens and tumors. We now demonstrate that modification of antigens with sialic acids (Sia-antigens) regulates the generation of antigen-specific regulatory T (Treg) cells via dendritic cells (DCs). Additionally, DCs that take up Sia-antigen prevent formation of effector CD4(+) and CD8(+)T cells. Importantly, the regulatory properties endowed on DCs upon Sia-antigen uptake are antigen-specific: only T cells responsive to the sialylated antigen become tolerized. In vivo, injection of Sia-antigen-loaded DCs increased de novo Treg-cell numbers and dampened effector T-cell expansion and IFN-γ production. The dual tolerogenic features that Sia-antigen imposed on DCs are Siglec-E-mediated and maintained under inflammatory conditions. Moreover, loading DCs with Sia-antigens not only inhibited the function of in vitro-established Th1 and Th17 effector T cells but also significantly dampened ex vivo myelin-reactive T cells, present in the circulation of mice with experimental autoimmune encephalomyelitis. These data indicate that sialic acid-modified antigens instruct DCs in an antigen-specific tolerogenic programming, enhancing Treg cells and reducing the generation and propagation of inflammatory T cells. Our data suggest that sialylation of antigens provides an attractive way to induce antigen-specific immune tolerance.

Notch controls generation and function of human effector CD8+ T cells.

The generation of effector CD8(+) T cells with lytic capacity is crucial for tumor control. Dendritic cells (DCs) provide important signals to promote naive CD8(+) T cell priming and activation of effector T cells. Here, we report that the Notch pathway has an important role in both these processes in human CD8(+) T cells. Activated monocyte-derived DCs express Notch ligands Jagged1 and Delta-like4, whereas naive CD8(+) T cells express Notch2. The role for Notch signaling in CD8(+) T cell priming was determined using an ex-vivo model system in which tumor antigen-specific primary CD8(+) T cell responses were measured. Inhibition of Notch using γ-secretase inhibitors or soluble Delta-like4-Fc during activation reduced expansion of antigen-specific CD8(+) T cells, which was mirrored by decreased frequencies of interferon (IFN)γ-, tumor necrosis factor-α-, and granzymeB-producing CD8(+) T cells. Moreover, T cells primed when Notch signaling was prevented are functionally low-avidity T cells. In addition, Notch partially regulates established effector T cell function. Activation-induced Notch signaling is needed for IFNγ release but not for cytolytic activity. These data indicate that Notch signaling controls human CD8(+) T cell priming and also influences effector T cell functions. This may provide important information for designing new immunotherapies for treatment of cancer.

Transition metal sensing by Toll-like receptor-4: next to nickel, cobalt and palladium are potent human dendritic cell stimulators.

BACKGROUND: Nickel was recently identified as a potent activator of dendritic cells through ligating with human Toll-like receptor (TLR)-4. OBJECTIVES: Here, we studied an extended panel of transition metals neighbouring nickel in the periodic table of elements, for their capacity to activate human monocyte-derived dendritic cells (MoDCs). METHODS: The panel included chromium, cobalt, and palladium, all of which are known to be frequent clinical sensitizers. MoDC activation was monitored by assessment of release of the pro-inflammatory mediator interleukin (IL)-8, a major downstream result of TLR ligation. Results The data obtained in the present study show that cobalt and palladium also have potent MoDC-activating capacities, whereas copper and zinc, but not iron and chromium, have low but distinct MoDC-activating potential. Involvement of endotoxin contamination in MoDC activation was excluded by Limulus assays and consistent stimulation in the presence of polymyxin B. The critical role of TLR4 in nickel-induced, cobalt-induced and palladium-induced activation was confirmed by essentially similar stimulatory patterns obtained in an HEK293 TLR4/MD2 transfectant cell line. CONCLUSIONS: Given the adjuvant role of costimulatory danger signals, the development of contact allergies to the stimulatory metals may be facilitated by signals from direct TLR4 ligation, whereas other metal sensitizers, such as chromium, may rather depend on microbial or tissue-derived cofactors to induce clinical sensitization.

Tissue factor-dependent chemokine production aggravates experimental colitis.

Tissue factor (TF) is traditionally known as the initiator of blood coagulation, but TF also plays an important role in inflammatory processes. Considering the pivotal role of coagulation in inflammatory bowel disease, we assessed whether genetic ablation of TF limits experimental colitis. To this end, wild-type and TF-deficient (TFlow) mice were treated with 1.5% dextran sulfate sodium (DSS) for 7 d, and effects on disease severity, cytokine production and leukocyte recruitment were examined. Clinical and histological parameters showed that the severity of colitis was reduced in both heterozygous and homozygous TFlow mice compared with controls. Most notably, edema, granulocyte numbers at the site of inflammation and cytokine levels were reduced in TFlow mice. Although anticoagulant treatment with dalteparin of wild-type mice reduced local fibrin production and cytokine levels to a similar extent as in TFlow mice, it did not affect clinical and histological parameters of experimental colitis. Mechanistic studies revealed that TF expression did not influence the intrinsic capacity of granulocytes to migrate. Instead, TF enhanced granulocyte migration into the colon by inducing high levels of the granulocyte chemoattractant keratinocyte-derived chemokine (KC). Taken together, our data indicate that TF plays a detrimental role in experimental colitis by signal transduction-dependent KC production in colon epithelial cells, thereby provoking granulocyte influx with subsequent inflammation and organ damage.

Urokinase plasminogen activator receptor-deficient mice demonstrate reduced hyperoxia-induced lung injury.

Patients with respiratory failure often require supplemental oxygen therapy and mechanical ventilation. Although both supportive measures are necessary to guarantee adequate oxygen uptake, they can also cause or worsen lung inflammation and injury. Hyperoxia-induced lung injury is characterized by neutrophil infiltration into the lungs. The urokinase plasminogen activator receptor (uPAR) has been deemed important for leukocyte trafficking. To determine the expression and function of neutrophil uPAR during hyperoxia-induced lung injury, uPAR expression was determined on pulmonary neutrophils of mice exposed to hyperoxia. Hyperoxia exposure (O2>80%) for 4 days elicited a pulmonary inflammatory response as reflected by a profound rise in the number of neutrophils that were recovered from bronchoalveolar lavage fluid and lung cell suspensions, as well as increased bronchoalveolar keratinocyte-derived chemokine, interleukin-6, total protein, and alkaline phosphatase levels. In addition, hyperoxia induced the migration of uPAR-positive granulocytes into lungs from wild-type mice compared with healthy control mice (exposed to room air). uPAR deficiency was associated with diminished neutrophil influx into both lung tissues and bronchoalveolar spaces, which was accompanied by a strong reduction in lung injury. Furthermore, in uPAR(-/-) mice, activation of coagulation was diminished. These data suggest that uPAR plays a detrimental role in hyperoxia-induced lung injury and that uPAR deficiency is associated with diminished neutrophil influx into both lung tissues and bronchoalveolar spaces, accompanied by decreased pulmonary injury.

Dendritic cell populations in colon and mesenteric lymph nodes of patients with Crohn's disease.

Dendritic cells (DCs) are key cells in innate and adaptive immune responses that determine the pathophysiology of Crohn's disease. Intestinal DCs migrate from the mucosa into mesenteric lymph nodes (MLNs). A number of different markers are described to define the DC populations. In this study we have identified the phenotype and localization of intestinal and MLN DCs in patients with Crohn's disease and non-IBD patients based on these markers. We used immunohistochemistry to demonstrate that all markers (S-100, CD83, DC-SIGN, BDCA1-4, and CD1a) showed a different staining pattern varying from localization in T-cell areas of lymph follicles around blood vessels or single cells in the lamina propria and in the MLN in the medullary cords and in the subcapsular sinuses around blood vessels and in the T-cell areas. In conclusion, all different DC markers give variable staining patterns so there is no marker for the DC.

An arachidonic acid-enriched diet does not result in more colonic inflammation as compared with fish oil- or oleic acid-enriched diets in mice with experimental colitis.

Fish oils (FO) - rich in EPA and DHA - may protect against colitis development. Moreover, inflammatory bowel disease patients have elevated colonic arachidonic acid (AA) proportions. So far, effects of dietary AA v. FO on colitis have never been examined. We therefore designed three isoenergetic diets, which were fed to mice for 6 weeks preceding and during 7 d dextran sodium sulfate colitis induction. The control diet was rich in oleic acid (OA). For the other two diets, 1.0 % (w/w) OA was exchanged for EPA+DHA (FO group) or AA. At 7 d after colitis induction, the AA group had gained weight (0.46 (sem 0.54) g), whereas the FO and OA groups had lost weight (- 0.98 (SEM 0.81) g and - 0.79 (SEM 1.05) g, respectively; P < 0.01 v. AA). The AA group had less diarrhoea than the FO and OA groups (P < 0.05). Weight and length of the colon, histological scores and cytokine concentrations in colon homogenates showed no differences. Myeloperoxidase concentrations in plasma and polymorphonuclear cell infiltration in colon were decreased in the FO group as compared with the OA group. We conclude that in this mice model an AA-enriched diet increased colonic AA content, but did not result in more colonic inflammation as compared with FO- and OA-enriched diets. As we only examined effects after 7 d and because the time point for evaluating effects seems to be important, the present results should be regarded as preliminary. Future studies should further elucidate differential effects of fatty acids on colitis development in time.

Critical appraisal of the current practice in murine TNBS-induced colitis.

There is no standard practice in the induction of colitis by 2,4,6-trinitrobenzene sulfonic acid. In this review the current practice in 2,4,6-trinitrobenzene sulfonic acid colitis is studied using 20 recently published articles. We compare the different protocols, discuss the mechanism of disease and give recommendations for the future use of the model.

Tumor sialylation impedes T cell mediated anti-tumor responses while promoting tumor associated-regulatory T cells.

The increased presence of sialylated glycans on the tumor surface has been linked to poor prognosis, yet the effects on tumor-specific T cell immunity are hardly studied. We here show that hypersialylation of B16 melanoma substantially influences tumor growth by preventing the formation of effector T cells and facilitating the presence of high regulatory T cell (Treg) frequencies. Knock-down of the sialic acid transporter created "sialic acid low" tumors, that grew slower in-vivo than hypersialylated tumors, altered the Treg/Teffector balance, favoring immunological tumor control. The enhanced effector T cell response in developing "sialic acid low" tumors was preceded by and dependent on an increased influx and activity of Natural Killer (NK) cells. Thus, tumor hypersialylation orchestrates immune escape at the level of NK and Teff/Treg balance within the tumor microenvironment, herewith dampening tumor-specific T cell control. Reducing sialylation provides a therapeutic option to render tumors permissive to immune attack.

Innate stimulatory capacity of high molecular weight transition metals Au (gold) and Hg (mercury).

Nickel, cobalt and palladium ions can induce an innate immune response by triggering Toll-like receptor (TLR)-4 which is present on dendritic cells (DC). Here we studied mechanisms of action for DC immunotoxicity to gold and mercury. Next to gold (Na3Au (S2O3)2⋅2H2O) and mercury (HgCl2), nickel (NiCl2) was included as a positive control. MoDC activation was assessed by release of the pro-inflammatory mediator IL-8. Also PBMC were studied, and THP-1 cells were used as a substitution for DC for evaluation of cytokines and chemokines, as well as phenotypic, alterations in response to gold and mercury. Our results showed that both Na3Au (S2O3)2⋅2H2O and HgCl2 induce substantial release of IL-8, but not IL-6, CCL2 or IL-10, from MoDc, PBMC, or THP-1 cells. Also gold and, to a lesser extent mercury, caused modest dendritic cell maturation as detected by increased membrane expression of CD40 and CD80. Both metals thus show innate immune response capacities, although to a lower extent than reported earlier for NiCl2, CoCl2 and Na2 [PdCl4]. Importantly, the gold-induced response could be ascribed to TLR3 rather than TLR4 triggering, whereas the nature of the innate mercury response remains to be clarified. In conclusion both gold and mercury can induce innate immune responses, which for gold could be ascribed to TLR3 dependent signalling. These responses are likely to contribute to adaptive immune responses to these metals, as reflected by skin and mucosal allergies.

Single nucleotide polymorphisms in C-type lectin genes, clustered in the IBD2 and IBD6 susceptibility loci, may play a role in the pathogenesis of inflammatory bowel diseases.

BACKGROUND AND AIM: The balance between microbes and host defence mechanisms at the mucosal frontier plays an important, yet unclarified role in the pathogenesis of inflammatory bowel disease (IBD). The importance of microorganisms in IBD is supported by the association of IBD with mutations in pattern recognition receptors (PRRs) such as NOD2 and TLR4. We aimed to examine whether polymorphisms in another type of PRRs, the so-called C-type lectin receptors (CLRs), are associated with IBD. Growing insights into the pathogenetic role of NOD2 mutations in Crohn's disease (CD) and the fact that the majority of CLR-encoding genes are located in IBD susceptibility loci provide strong arguments for further exploration of the role of CLRs in IBD. METHODS: In this study, we selected four single nucleotide polymorphisms (SNPs) in different CLRs to determine whether there could be a role for these CLRs in IBD. Functional SNPs in the genes coding for the candidate CLRs DC-SIGN, LLT1, DCIR and MGL were examined. Genotyping of all SNPs was performed at the Academic Medical Center. In this study, around 1572 samples were included from a maximum of 621 CD patients, 457 ulcerative colitis (UC) patients and 586 healthy controls (HCs). RESULTS AND CONCLUSION: No association was found between our IBD cohort and the candidate SNPs for DC-SIGN (CD/HC: P=0.25 and UC/HC: P=0.36), DCIR (CD/HC: P=0.22 and UC/HC: P=0.41) and MGL (CD/HC: P=0.37 and UC/HC: P=0.25). However, one polymorphism in LLT1 was found to be associated with our CD population (P<0.034). Our UC cohort was not associated with the variation in LLT1 (P=0.33). LLT1 is a ligand for the recently discovered CD161. CD161 is a new surface marker for human interleukin (IL)-17-producing Th17 cells. The Th17 phenotype has been linked to CD by the fact that IL-22, IL-17 and IL-23 receptor levels are increased in CD. The signal transduction pathways involving LLT1 and CD161 are not completely clarified and are currently under investigation in our laboratory.

Glycan-modified liposomes boost CD4+ and CD8+ T-cell responses by targeting DC-SIGN on dendritic cells.

Cancer immunotherapy requires potent tumor-specific CD8(+) and CD4(+) T-cell responses, initiated by dendritic cells (DCs). Tumor antigens can be specifically targeted to DCs in vivo by exploiting their expression of C-type lectin receptors (CLR), which bind carbohydrate structures on antigens, resulting in internalization and antigen presentation to T-cells. We explored the potential of glycan-modified liposomes to target antigens to DCs to boost murine and human T-cell responses. Since DC-SIGN is a CLR expressed on DCs, liposomes were modified with DC-SIGN-binding glycans Lewis (Le)(B) or Le(X). Glycan modification of liposomes resulted in increased binding and internalization by BMDCs expressing human DC-SIGN. In the presence of LPS, this led to 100-fold more efficient presentation of the encapsulated antigens to CD4(+) and CD8(+) T-cells compared to unmodified liposomes or soluble antigen. Similarly, incubation of human moDC with melanoma antigen MART-1-encapsulated liposomes coated with Le(X) in the presence of LPS led to enhanced antigen-presentation to MART-1-specific CD8(+) T-cell clones. Moreover, this formulation drove primary CD8(+) T-cells to differentiate into high numbers of tetramer-specific, IFN-γ-producing effector T-cells. Together, our data demonstrate the potency of a glycoliposome-based vaccine targeting DC-SIGN for CD4(+) and CD8(+) effector T-cell activation. This approach may offer improved options for treatment of cancer patients and opens the way to in situ DC-targeted vaccination.

Endogenous MCP-1 promotes lung inflammation induced by LPS and LTA.

Monocyte chemoattractant protein 1 (MCP-1) plays an important role in leukocyte recruitment to sites of infection and inflammation. In addition, MCP-1 may attenuate inflammation by virtue of its capacity to inhibit the production of proinflammatory cytokines. We here investigated the role of MCP-1 in lung inflammation induced by lipopolysaccharide (LPS) or lipoteichoic acid (LTA), constituents of the gram-negative and gram-positive bacterial cell wall, respectively. Healthy humans demonstrated elevated MCP-1 concentrations in their bronchoalveolar lavage fluid (BALF) 6h after inhalation of LPS. Similarly, intranasal administration of LPS or LTA to mice resulted in a rise in BALF MCP-1 levels. Murine alveolar macrophage-like cells released significant amounts of MCP-1 upon stimulation with LPS or LTA in vitro. Compared to Wt mice, MCP-1(-/-) mice demonstrated lower TNF-α levels and a diminished neutrophil influx into their bronchoalveolar space after either LPS or LTA instillation. After intrapulmonary delivery of LPS MCP-1(-/-) mice had decreased interleukin-6 and KC concentrations and less severe lung inflammation upon histopathological examination. Remarkably, MCP-1 deficiency was associated with an early enhancement of interleukin-10 release in BALF after both LPS and LTA instillation. These data suggest that MCP-1 is a proinflammatory mediator during pulmonary inflammation induced by either LPS or LTA.

The enteric nervous system as a regulator of intestinal epithelial barrier function in health and disease.

The intestinal epithelia proliferate and differentiate along the crypt villus axis to constitute a barrier cell layer separating some 10¹³ potentially harmful bacteria from a sterile mucosal compartment. Strict regulatory mechanisms are required to maintain a balance between the appropriate uptake of luminal food components and proteins, while constraining the exposure of the mucosal compartment to luminal antigens and microbes. The enteric nervous system is increasingly recognized as such a regulatory housekeeper of the epithelial barrier integrity, in addition to its ascribed immunomodulatory potential. Inflammation affects both epithelial integrity and barrier function and, in turn, loss of barrier function perpetuates inflammatory conditions. The observation that inflammatory conditions affect enteric neurons may add to the dysregulated barrier function in chronic disease. Here, we review the current understanding of the regulatory role of the nervous system in the maintenance of barrier function in healthy state, or during pathological conditions of, for instance, stress-induced colitis, surgical trauma or inflammation. We will discuss the clinical potential for advances in understanding the role of the enteric nervous system in this important phenomenon.

Selective alpha7 nicotinic acetylcholine receptor agonists worsen disease in experimental colitis.

BACKGROUND AND PURPOSE: In various models vagus nerve activation has been shown to ameliorate intestinal inflammation, via nicotinic acetylcholine receptors (nAChRs) expressed on immune cells. As the alpha7 nAChR has been put forward to mediate this effect, we studied the effect of nicotine and two selective alpha7 nAChR agonists (AR-R17779, (-)-spiro[1-azabicyclo[2.2.2] octane-3,5'-oxazolidin-2'-one and GSK1345038A) on disease severity in two mouse models of experimental colitis. EXPERIMENTAL APPROACH: Colitis was induced by administration of 1.5% dextran sodium sulphate (DSS) in drinking water or 2 mg 2,4,6-trinitrobenzene sulphonic acid (TNBS) intrarectally. Nicotine (0.25 and 2.50 micromol.kg(-1)), AR-R17779 (0.6-30 micromol.kg(-1)) or GSK1345038A (6-120 micromol.kg(-1)) was administered daily by i.p. injection. After 7 (DSS) or 5 (TNBS) days clinical parameters and colonic inflammation were scored. KEY RESULTS: Nicotine and both alpha7 nAChR agonists reduced the activation of NF-kappaB and pro-inflammatory cytokines in whole blood and macrophage cultures. In DSS colitis, nicotine treatment reduced colonic cytokine production, but failed to reduce disease parameters. Reciprocally, treatment with AR-R17779 or GSK1345038A worsened disease and led to increased colonic pro-inflammatory cytokine levels in DSS colitis. The highest doses of GSK1345038A (120 micromol.kg(-1)) and AR-R17779 (30 micromol.kg(-1)) ameliorated clinical parameters, without affecting colonic inflammation. Neither agonist ameliorated TNBS-induced colitis. CONCLUSIONS AND IMPLICATIONS: Although nicotine reduced cytokine responses in vitro, both selective alpha7 nAChR agonists worsened the effects of DSS-induced colitis or were ineffective in those of TNBS-induced colitis. Our data indicate the need for caution in evaluating alpha7 nAChR as a drug target in colitis.

The PPARgamma agonist rosiglitazone impairs colonic inflammation in mice with experimental colitis.

Various animal models showed that peroxisome proliferator-activated receptor (PPAR)gamma agonists, when given as a gavage shortly preceding colitis induction, protect against inflammatory bowel disease (IBD). We have examined the effects of 16 days rosiglitazone treatment via the diet prior to dextran sodium sulphate (DSS)-induced colitis in mice. After 7 days DSS in the drinking water, rosiglitazone-fed mice had lost significantly more weight than control mice. Rosiglitazone-treated mice had more diarrhea, weight of colon and spleen were increased, and length of colon was decreased. Histology showed that rosiglitazone-treated mice had more severe colitis, mainly caused by more ulceration, crypt loss, and edema. Immunofluorescence showed a loss of tight junction structure Zonula Occludens protein 1 (ZO-1) in colons of rosiglitazone-treated mice as compared to control mice. Also, serum amyloid P component (SAP) concentrations in plasma were increased. However, concentrations of tumor necrosis factor (TNF)-alpha and interferon (IFN)-gamma in colon homogenates, and TNF-alpha in spleen homogenates were significantly decreased, whereas interleukin (IL)-10 in spleen homogenates was increased. Other cytokines (IL-2, IL-4, IL-6, IL-12p70 and monocyte chemotactic protein (MCP)-1) and myeloperoxidase (MPO) concentrations showed no differences. In conclusion, 16 days pretreatment with rosiglitazone impaired DSS-induced colitis in mice.

Apoptosis as a therapeutic paradigm in inflammatory bowel diseases.

Evidence is increasing that a defect in apoptosis is involved in the pathogenesis of inflammatory bowel diseases (IBD), including Crohn's disease (CD) and ulcerative colitis (UC). CD seems to be the cause of an intrinsic defect in the apoptotic pathway of (autoreactive) T cells, resulting in excessive T cell responses. In UC, an increased rate of apoptosis of epithelial cells is observed. In this review we will describe apoptotic mechanisms and their association to IBD. In addition, we will review how specific therapeutic approaches interact at different levels with the apoptotic pathway.