Moreover, Dlg1 loss has been linked to increased rates of cell pr

Moreover, Dlg1 loss has been linked to increased rates of cell proliferation [7]. Given the involvement of Dlg1 in signaling molecule assembly in neural synapses [2, 3], we and others proposed it could also play a role in regulating Ag receptor-mediated signaling in T cells [8-12]. Indeed, several published reports implicate cell polarity proteins in regulation of T-cell development and function. For example, Scribble has been shown to be involved in T-cell migration and immunological synapse formation [9] as well as T-cell development [13], while Par6 and aPKC

may contribute to the ability of T cells to efficiently scan dendritic cells [14], and PALS1 has been implicated in regulation of TCR-driven T-cell proliferation [15]. Recently, several reports suggested a function for Dlg1 as an important scaffolding adaptor involved in modulation of signaling

networks at the immunological synapse [8, 11, Bcl-2 inhibitor 16-18]. Dlg1 was shown to be recruited to the immunological synapse and to colocalize with ZAP70, Lck, Vav1, TCR-ξ, and Kv.1.3 potassium channel, which collectively coordinate signaling cascades from TCR receptor to the nucleus [8, 19]. Nonetheless, MK1775 the requirement and function of Dlg1 in T-cell activation and TCR signal transduction remains to be clarified. Because deletion of Dlg1 from the murine germline is lethal [20], we employed a conditional KO mouse in which Dlg1 loss is restricted to the T-cell lineage only, or all hematopoietic cells. Using this system, we showed that Dlg1 is not required for Ag activation of T cells harboring transgenic TCR in vitro and in vivo. Surprisingly, however, we found that Dlg1 is required for normal regulation of memory T-cell generation in response to immunization with conventional Ag. Our previous studies using RAG-deficient complementation approaches indicated that Dlg1 is dispensable for development of all major αβ-lineage thymocyte subsets [17].

To verify this finding we generated Lck-Cre+ Dlg1flox/flox and Vav1-Cre+ Dlg1flox/flox mice, in which Ribose-5-phosphate isomerase transgenic Cre expression is driven by the Lck [21], or the Vav promoter [22], respectively. We observe efficient deletion of Dlg1 in both models, as ascertained by Western blotting with Dlg1-specific antibodies using lysates from either thymocytes or T-cell blasts obtained from purified and activated peripheral T cells, which show a complete deletion of Dlg1 protein (Supporting Information Fig. 1, and top panel in Fig. 2). We analyzed T-cell development in Dlg1-deficient (Lck-Cre+ Dlg1flox/flox or Vav1-Cre+ Dlg1flox/flox, further referred to as KO) and control (Lck-Cre+ Dlg1flox/+ or Vav1-Cre+ Dlg1flox/+, further referred to as WT) mice and find no obvious abnormalities (Supporting Information Fig. 2). We note, however, that the requirement for Dlg1 in T-cell development has not yet been assessed in thymocytes harboring functionally rearranged TCR transgenes.

In this study, we investigated the effect of stimulation of human

In this study, we investigated the effect of stimulation of human primary cells with bacterial ligands during RSV infection. To determine BYL719 whether microbial ligands for specific PRRs modulate the response to RSV infection, we costimulated human PBMCs with RSV and LTA, LPS, flagellin, CpG, or MDP. LTA (Gram-positive), LPS (Gram-negative),

flagellin (Gram-positive and Gram-negative), CpG (all bacteria), and MDP (mostly Gram-positive) are recognized by TLR2, TLR4, TLR5, TLR9, and NOD2, respectively. The amount of cytokine release after these stimulations can be found in Supporting Information Fig. 1. Of all tested combinations, only costimulation with MDP and RSV was found to modulate the production Selleck GW 572016 of the proinflammatory cytokines TNF-α

and IL-1β (21.0- and 9.7-fold increase, respectively) (Fig. 1). In contrast, MDP was not found to have an effect on the IL-10 response to RSV infection, suggesting the effect is limited to pro-inflammatory cytokines. MDP was the only bacterial ligand tested that was able to affect the innate cytokine response to RSV infection, we therefore investigated the underlying mechanism. As NOD2 has been implicated in the recognition of MDP, we made use of the fact that Crohn’s patients homozygous for the 3020insC mutation produce a truncated NOD2 receptor and consequently cannot recognize MDP [[19]]. PBMCs from healthy volunteers and NOD2-deficient patients were stimulated with RSV and MDP. Stimulation with RSV or MDP alone induced low TNF-α and IL-1β responses in both healthy and NOD2-deficient PBMCs (Fig. 2A and B). Following stimulation with RSV and MDP together, only PBMCs from healthy volunteers showed a strong synergistic increase in these cytokines (Fig. 2C). In contrast, no synergistic upregulation in the production of these cytokines was seen in PBMCs from NOD2-deficient volunteers, suggesting that the observed synergy

in cytokine production is dependent on the recognition of Buspirone HCl MDP by NOD2. Our data demonstrated that MDP recognition by NOD2 is essential for the synergy observed. We next aimed at identifying the viral components and receptors involved in this phenotype. Human PBMCs were stimulated with MDP in combination with specific ligands for all receptors currently associated with RSV recognition. The amount of cytokine release after these stimulations can be found in Supporting Information Fig. 2. We found that ssRNA40-LyoVec (NOD2) and R848 (TLR7) did not show a synergistic inflammatory response (Fig. 3). LPS (TLR4) and Poly(I:C)-LyoVec HMW (MDA-5) induced a small increase in the production of TNF-α and IL-1β. The ligands that induced the strongest synergy were Poly(I:C) HMW (TLR3) and Poly(I:C)-LyoVec LMW (RIG-I). These data suggest that the synergistic effects observed with live RSV are likely due to engagement of either RIG-I, TLR3, or a combination of these receptors.

Correspondingly, the Register has very few reports of adverse rea

Correspondingly, the Register has very few reports of adverse reactions caused by green pea or soy, a substantial number of reports regarding lupin Trichostatin A and fenugreek, and many regarding peanut. These data show that there is a need to further investigate cross-allergy in legumes. Most of the work performed on legume allergy has focused on peanut as the major allergenic legume, and information on other

types of legume allergy is limited [4]. As we previously have established mouse models of lupin and fenugreek allergy [25, 26], we used these models to address the clinical cross-allergy between the four most common allergenic legumes: lupin, fenugreek, peanut and soy. We also assessed different serological and cellular responses to explore possible mechanisms related to the cross-allergic reactions. Animals.  Female inbred C3H/HeJ mice (Jackson Laboratories, Bar Harbor, ME, USA), 5 weeks old at the start of the experiments, were used. Several experiments have been combined in this study and an account of the animals with immunizations and challenges is therefore given in Table 1. Female Sprague-Dawley rats, 150–200 g (Taconic M&B A/S, Ry, Denmark) were used to perform the passive cutaneous anaphylaxis (PCA) tests. The animals were housed, 3–4

mice or two rats per cage, on NESTPAK bedding (Datesand Ltd, Manchester, UK) in type III macrolon cages in filter cabinets (Scantainers), exposed to a 12-hr/12-hr light/dark cycle Sitaxentan (30–60 lux in cages), room temperature of 21 ± 2 °C and 35–75% humidity. Pelleted food (RM1; Trametinib concentration SDS, Essex, UK) and tap water ad libitum were given. Before entering the experiments, the animals were allowed to rest for 1 week. The experiments were performed in conformity with the laws and regulations for experiments with live animals in Norway and were approved by the Norwegian Animal Research Authority under the Ministry of Agriculture. Legume extracts.  The National Veterinary Institute of Norway provided all protein extracts. In short, extracts of peanut, lupin and soy were made by extracting

homogenized peanuts, soybeans or lupin (Lupinus angustifolius) in Tris/glycine buffer, pH 8.7, overnight followed by centrifugation. The fenugreek extract was made using an extended protocol utilizing precipitation with (NH4)2SO4, dialysis and freeze-drying [26]. The total protein concentration of the extracts was measured by Lowry’s method. The endotoxin level of the extract was determined with the Limulus Amebocyte Lysate (LAL) Kinetic-QCL Kit (BioWhittaker, Walkersville, MD, USA) and found to be below 0.1 ng/ml for all extracts. Immunizations and challenges.  Immunizations were performed perorally (p.o.) according to the experimental protocols previously established [25, 26]. Briefly, immunizations were performed on days 0, 1, 2, 7, 21 and 28 and challenges on day 35. Lupin immunized mice received 5.

The blood spots were extracted on ice with 25 mm Tris-HCl, pH 7 4

The blood spots were extracted on ice with 25 mm Tris-HCl, pH 7.4, 15 mm KCl, 1 mm EDTA and 1 mm dithiothreitol, and ADA and purine nucleoside phosphorylase (PNP) activities as well as total protein content were assayed as described previously [12]. An additional aliquot of the extract was treated with perchloric acid, neutralized and analysed for AXP and dAXP content; “percent dAXP” (dAXP/(AXP + dAXP) × 100) was used

to assess dAXP elevation [12]. Cell proliferation assays.  Peripheral blood mononuclear cells (PBMC) from the patient and controls were purified from whole blood using density gradient centrifugation with Ficoll-Hypaque (Sigma Aldrich) and suspended in RPMI 1640 supplemented with 2 mm l-glutamine, 50 U/ml penicillin, 50 μg/ml streptomycin and 10% human serum. PBMC at 2 × 105 from each individual were added in triplicates to 96-well

AZD6738 research buy U-bottom plates (Falcon-Becton Dickinson, San Diego, CA, USA), and cells were stimulated with Phytohaemagglutinin (PHA; Sigma Aldrich) at 5, 10 and 20 μg/ml and cultured in a humidified incubator at 37 °C containing 5% CO2 for 86 h. One μCi of 3H-thymidine (MP Biomedicals, Irving, CA, USA) was added to each well and the cells were cultured for an additional 20 h. Cultures were harvested onto glass fibre filter papers this website (Inotech Biosystems Internacional Inc, Rockville, MD, USA) using an automated multisample Cell Harvester (Inotech Biosystems). Counts per minute (cpm) were measured using a liquid scintillation counter (Plate Chameleon; Multilabel reader, Hidex, Turku, Finland), and the results were expressed as proliferation index (PI), calculated by dividing the mean cpm from the triplicates of stimulated cells by the mean cpm of triplicates Rucaparib manufacturer from unstimulated cells. Complementarity determining region 3 (CDR3) size distribution

analysis of T cells.  Anticoagulated whole blood was collected from the patient and three controls, treated with RNA Stabilization Reagent (Roche Diagnostics GmbH, Mannheim, Germany) and stored at −20 °C until use. Total RNA was isolated using the High Pure RNA Isolation kit (Roche Diagnostics) according to the manufacturer’s instructions, with the exception that stabilized samples were directly added to the filters instead of the initial lysis step. The cDNA was generated from 2 μg of total RNA using the SuperScript II reverse Transcriptase kit (Invitrogen, Carlsbad, CA, USA) and later used as template for PCR using 24 different unlabelled TCR Vβ primers (Gene Probe Technology, Gaithersburg, MD, USA) and a 6-fluorescein phosphoramidite (6-FAM)-labelled Cβ-specific primer (Invitrogen) that recognizes both Cβ1 and Cβ2. PCR conditions included 40 cycles of amplification at 95 °C/2 min, 95 °C for 45 s, 60 °C/45 s and 72 °C/54 s, with a final step at 72 °C/7 min.

It is important to call attention to the fact that CCL25 induced

It is important to call attention to the fact that CCL25 induced the migration of IL-17+ cells without affecting IL-17 production in vitro. Therefore, the modulation of pleural IL-17 levels by CCL25 is actually a result of the in vivo migration of IL-17+ γδ T lymphocytes. IL-17+ γδ T lymphocytes are committed as IL-17 producers within the fetal thymus, via RORγt transcriptional factor- and Notch-Hes1-dependent mechanisms, independently of TCR signaling [[43-46]] and CD27 costimulation [[47]]. In the immune site, their activation is determined by multiple cytokines, among which a chief role is attributed to IL-1β and IL-23 in mice and humans [[48-50]]. It has been shown that TCR γδ+/CCR6+ cells that

produce IL-17 coexpress CCR9 (which was not observed for γδ+/NK1.1+ cells, which produce IFN-γ) [[6, find protocol www.selleckchem.com/products/voxtalisib-xl765-sar245409.html 34]]. Accordingly, CCL25 induces the specific chemotaxis of Th17 CD4+ T cells polarized by retinoic acid (which is an important regulatory signal in the intestine), but not of Th0 lymphocytes [[51]]. These T cells were shown to express CCR9/α4β7 integrin and preferentially migrate to the intestine and regulate inflammation. Moreover, it has been demonstrated that, throughout Th17 differentiation, CD4+ T lymphocytes are shown to increase the expression of chemokine receptors, including CCR9 [[52]]. Interestingly, IL-17-producing γδ T cells

have been shown to be CD25+ and CD122− [[43, 44]], a phenotype observed by us on γδ T cells recovered from CCL25-stimulated mouse pleura. It is noteworthy that, since CCL25 i.pl. injection failed to trigger CD122+ T-cell migration, the percentage of CD122+ γδ T lymphocyte population in the pleura of CCL25-stimulated mice slightly decreased (SAL 11.8% versus CCL25 5.0% among γδ T lymphocytes). Similar to our data, it has been demonstrated that IL-17-producing γδ T cells did not produce IFN-γ or IL-4

and specifically expressed CD25 but not CD122 (whereas CD122+ γδ T lymphocytes produced IFN-γ). Moreover, IL-17-producing γδ T lymphocyte maintenance was shown to depend on CD25 and IL-2 [[44]]. It has been also shown that CD122lo γδ T cells recovered from mouse spleen, lymph nodes, and thymus produced high levels of IL-17 but small amounts or no IFN-γ upon Quinapyramine TCR in vitro stimulation [[43]]. An inverse correlation between CD122 and CCR9 has also been demonstrated on γδ lymphocytes from mouse thymus [[53]]. This work demonstrates that γδ thymocytes that express high levels of CCR9 are CD122lo, whereas CCR9lo express high levels of CD122. It is important to note that the CCL25 neutralization and α4β7 integrin blockade during allergic pleurisy did not inhibit αβ T lymphocyte recruitment, whereas the i.pl. stimulus with CCL25 selectively triggered γδ T-cell migration. These data corroborate and reinforce the hypothesis that CCL25 is important for the migration of a specific γδ T-cell subset that produces IL-17 during an allergic reaction, via α4β7 integrin.

This demonstrates a STAT4-dependent, IL-12/IL-23-independent path

This demonstrates a STAT4-dependent, IL-12/IL-23-independent pathway of parasite control. Type I IFNs

are important in viral and other infections and can activate STAT4. We found that IFN-α/βR-deficient mice have a nonpersistent, early IFN-γ defect, and a persistent, early IL-10 defect, without changes in serum IL-12 or LN-derived nitric oxide. We found less IL-10 per cell in CD25+CD4+ T cells and possibly fewer IL-10-producing cells in the draining LN of IFN-α/βR-deficient vs. wild-type mice. IFN-α/βR-deficient mice have chronic, nonprogressive disease, like wild-type mice, suggesting that IL-10 and IFN-γ defects may balance each other. Our data indicate that although type I IFNs help promote early Th1 responses, they are not the missing activators of STAT4 responsible for partial control Ceritinib of L. mexicana. Also, the lack of lesion resolution in IFN-α/βR-deficient mice despite lower IL-10 levels indicates that other pathways independent of T cell IL-10 help prevent an IL-12-driven clearance of parasites. Leishmania (L.) mexicana, a New World intracellular protozoan parasite, causes chronic cutaneous infection in mice and humans. The immunology and outcome of infection

of L. mexicana are quite different from those of the better-studied Old World relative, L. major. In C57BL/6 (B6) Selleck BMS-777607 mice, L. major induces a strong Th1 response with interleukin (IL)-12-driven interferon (IFN)-γ from CD4+ T cells. This IFN-γ leads to an upregulation of inducible nitric oxide synthase (iNOS) in infected macrophages, which in turn generates nitric oxide, leading to killing of the intracellular L. major. The outcome is that rapidly growing lesions develop but then heal,

with very low persistent parasite burdens (typically <100 per lesion). In L. mexicana infection of B6 mice, a very weak IFN-γ response occurs and parasites induce chronic, but nonprogressive, lesions that plateau in size at around 10–12 weeks post-infection, with higher parasite burdens (generally 107–108 per lesion). Understanding the similarities and differences between these two related parasite infections is instructive in dissecting the immunological mechanisms. We have found that despite these O-methylated flavonoid differing outcomes, many of the pathways involved in control of L. mexicana parallel those seen in L. major infection. B6 mice lacking IFN-γ or iNOS have progressive disease with lesions that do not plateau in size and parasite burdens much higher than in wild-type (WT) mice (1). Interestingly, the Th2 response, as determined by IL-4 production, is not increased in the IFN-γ- and iNOS-deficient mice, showing that susceptibility is because of a lack of an adequate Th1 response rather than an increased Th2 response. We also found that mice lacking STAT4, an important signalling molecule, had progressive disease (1) similar to the increased susceptibility of STAT4 knockout (KO) mice to L. major (2).

IL-1β levels were not affected by corticosteroids As IL-1Ra inhi

IL-1β levels were not affected by corticosteroids. As IL-1Ra inhibits the physiological activities of IL-1β by occupying the IL-1 receptor, we evaluated IL-1Ra in relation to IL-1β through calculation of the IL-1Ra/IL-1β ratio. IPF patients showed a 3·5-fold decrease in the IL-1Ra/IL-1β ratio in BALF (215·7; IQR 58·6–437·9) compared to healthy controls (771·4; IQR 337·4–5210·0), P < 0·0001. A similar decrease

in the IL-1Ra/IL-1β ratio was found in serum from patients (77·9; IQR 51·5–110·9) compared to healthy controls (293·5; IQR 201·1–1054·0), P < 0·0001 (Fig. 1). The IL-1Ra/IL-1β ratio in serum was affected significantly by the use of corticosteroids; the eight patients selleck screening library who were on corticosteroids had a significantly higher IL-1Ra/IL-1β ratio: 101·7 (IQR 77·2–143·4) versus 71·5 (IQR 51·0–102·2), Roxadustat research buy P = 0·01. In BALF there was no significant difference. Table 2 summarizes allelic and genotype frequencies in IPF patients and controls. Both populations were in Hardy–Weinberg equilibrium for all genotypes. One SNP in the IL1RN gene was associated with IPF. The frequency of the rs2637988 allele 2 (G) in the IL1RN gene was increased in the IPF group (47%) compared to the controls (38%), P = 0·04. The best-fitting genetic model was a risk conferred by the carriage of allele 2 compared to non-carriers; odds ratio (OR) 1·95 [95% confidence interval (CI):

1·11–3·42; P = 0·02]. Frequency of the rs408392 allele 2 (T) was increased in IPF patients and showed a trend towards significance; allele 2 occurred in 32% of the IPF patients compared to 26% in controls, P = 0·09. For carriage of allele 2 versus non-carriers, the OR was 1·58 (95% CI: 0·96–2·60, P = 0·07). There was significant linkage disequilibrium between the two SNPs; D′ = 0·94, r2 = 0·46. Additionally, haplotype frequencies were calculated. ZD1839 datasheet Haplotype analysis was of no superior value compared to single SNP analysis. The polymorphisms

in the IL1RN and IL1B genes did not significantly influence BALF or serum IL-1Ra or IL-1β levels in IPF patients and healthy controls. However, differences were seen between genotypes of the rs2637988 polymorphism and the BALF IL-1Ra/IL-1β ratio; AA 1856 (IQR 1421–3730), AG 223·7 (IQR 84·6–384·9), GG 29·3 (IQR 6·95–130), P = 0·005 (Fig. 2). A less significant effect was found when genotypes of the rs408392 polymorphism were compared (P = 0·09). Other SNPs were not associated with the IL-1Ra/IL-1β ratio in serum or BALF. The total cell count and absolute numbers of macrophages, lymphocytes, neutrophils and eosinophils in BALF were increased significantly in IPF patients compared to healthy controls (all P < 0·001; Table 3). The relationship between BALF cellular profiles and IL-1β and IL-1Ra is shown to illustrate the relevance in clinical perspective. In healthy controls, there was no correlation between BALF IL-1β levels or IL-1Ra and absolute neutrophil counts.

It has been shown that the addition of erythrocytes to cultured s

It has been shown that the addition of erythrocytes to cultured slanDC blocks their capacity

to produce IL-12 and TNF-α via the interaction of CD47 on erythrocytes and the corresponding ligand signal regulatory protein α on slanDC.4 After slanDC leave the bloodstream and infiltrate the tissue, as it was shown in inflamed skin of AD lesions, the control by erythrocytes is lost. Our study suggests that histamine might take over this control function in the tissue because we could show that histamine reduces the highly pro-inflammatory capacity of slanDC, particularly via activation of the H4R. To be sure that histamine stimulation does not reduce cytokine production in general, we investigated IL-10 as a Depsipeptide cell line cytokine LEE011 supplier that is associated with anti-inflammatory actions. Interleukin-10 reduces the production of IL-2, TNF-α, IFN-γ and co-stimulatory molecules and was shown to counteract the inflammatory response in allergic contact dermatitis.24 In our study we could not observe a significant effect of histamine receptor activation on the release of IL-10. As a result, it can be assumed that H4R and H2R stimulation on slanDC specifically down-regulate the production of the pro-inflammatory mediators TNF-α and IL-12, whereas the level of the anti-inflammatory mediator IL-10 is not affected. The differential regulation of cytokine release by histamine might

be explained by varying signalling processes involved. For example, it was shown that the activation of mitogen-activated protein kinase signalling mediates histamine-induced down-regulation of IL-12p70 in monocytes,15 but on the other hand induces IL-10 production in monocytes.25 Our observations fit the current understanding

of the role of the H4R on antigen-presenting cells. Several studies have shown that the H4R on DC has an anti-inflammatory role: on MoDC, monocytes and inflammatory dendritic epidermal cells the production of IL-12 and CCL2 was down-regulated after H4R activation.15–17 In response to the reduced presence of these mediators, CHIR-99021 in vivo Th1 polarization is impaired and a lower number of macrophages and T cells is attracted to the site of the immune response, respectively. We can draw the conclusion that the stimulation of the H4R on DC, and as shown here in particular on slanDC, could greatly reduce the inflammatory responses taking place in the course of inflammatory skin diseases like AD and H4R agonists therefore might represent potential therapeutic tools in these kinds of diseases. This study was supported by grants from the Deutsche Forschungsgemeinschaft DFG: Gu434/5-1 and GRK1441/1 and the European Community (COST action BM0806). Maria Gschwandtner was supported by a grant from the Hannover Biomedical Research School. The authors declare no conflict of interest.

Our initial results indicate that administering antigens in the e

Our initial results indicate that administering antigens in the ear is an efficient pathway for inducing the proliferation of specific CD4+ T cells in dCLNs. This could be due to antigen transportation by DCs. However, migrating DCs were not strictly required for the presentation of low antigen doses. Thus, it is possible that in our model, the delivery of free antigens by lymphatic vessels to the LNs occurs in addition to antigen transportation that is mediated by DCs, as previously

reported in other experimental models 25. The increased proliferation of HEL-specific T cells by co-administration of HEL and CT in the ear was also observed with other DC activators, and one possible explanation is the phenotypic changes that occur in DCs (e.g. changes in CD86 www.selleckchem.com/products/AZD0530.html and CD40 expression). The activation of DCs by CT has been reported both in vitro 26 and in vivo 27. Here, we report the activation of skin DCs by CT and also with the CTB. This is in contrast with a previous report where spleen DCs were activated by the CT but not by CTB 21. It has been reported that LCs remain in the epidermis for 48 h, even in the presence of Th1-polarizing adjuvants 7. In our experiments, 24 h after

CT or CTB inoculation in the ear, the number of LCs in the epidermis was reduced, suggesting that LCs could be mobilized from the dermis at this time point in the presence of strong adjuvants such as CT. Our results show robust expression of IFN-γ, IL-2 and TNF-α in CD4+ T cells after immunization with HEL and CT, whereas IL-4 and IL-5 were not detectable, which is AZD2281 cell line in contrast with previous reports that indicated a Th2 cytokine response after ear immunization 10, 11; this also argues against the occurrence of dominant Th2 responses toward

antigens that are co-administered with CT in mucosae 16, 17. In the skin, both Th1 and Th2 cytokines have been reported following immunization with OVA and CT 24. Our experiments are in agreement with a Th1 cytokine response following skin immunization 12, 13IL-17 expression by CD4+ T cells was also observed following skin immunization with CT as has been reported using other strategies of immunization in the skin 13, 14. Recently, a dominant Th17 response was reported following intranasal immunization with Clomifene OVA together with either CT or CTB 19. In our study, the IL-17 production by CD4+ T cells can be explained by the high expression levels of TGF-β that was observed in the Langerin+ DCs that are present in the dermis of mice that were inoculated with CT in the ear in addition to the presence of IL-6 expressed by dermal cells (Supporting Information Fig. 5) since the combination of TGF-β and IL-6 has been reported as crucial in the Th17 differentiation 28. Interestingly, we also observed IFN-γ and IL-17 CD4+ T-cell differentiation after immunization with the CTB subunit, which argues against previous data that indicated that the adjuvant role of CT is mediated by CT α subunit activity 21.

[118, 119] Similar to

[118, 119] Similar to buy BMS-354825 some of the EAE models, stimulation of type I NKT cells with αGalCer results in disease exacerbation associated with a Th1 cytokine release profile.[118-121] In the latter cases, type I NKT cell activation by αGalCer or its analogues may lead to the tolerization of APC populations. In turn, this outcome may inhibit the activity of most Th1/Th17/Th2 secreting effector cells and thereby lead to protection from autoimmune disease. Generally, activation of type II NKT cells with self-glycolipid

sulphatide may control both antigen-induced and spontaneously arising autoimmune disease. During EAE, sulphatide-reactive type II NKT cells, but not type I NKT cells, are increased several fold in the CNS. This greater abundance of type II NKT cells in the CNS inverts the usual ratio of type II : type I NKT cells (type II NKT cells, 3–4%; and type I NKT cells, 0·6–0·9%) and affords selleck protection from EAE.[27, 61] Furthermore, administration of sulphatide to activate type II NKT cells decreases the number of IFN-γ- and IL-17-secreting myelin basic protein and proteolipid protein-reactive encephalitogenic CD4+

T cells. The net outcome is protection from EAE via a CD1d-dependent regulatory pathway (Maricic et al., submitted). This type II NKT-mediated immunoregulatory pathway results in (i) inactivation of type I NKT cells that now function as regulatory T cells, (ii) tolerization

of conventional DCs, (iii) tolerization of microglia in the CNS and (iv) inhibition of the effector Rebamipide functions of pathogenic MHC-restricted CD4+ T cells. As APCs that activate pathogenic Th1 and Th17 cells in lymphoid organs and the CNS are tolerized following sulphatide administration, activation of type II NKT cells induced by sulphatide is much more potent in the regulation of autoimmune demyelination than only the inactivation of type I NKT cells by αGalCer (Maricic et al., submitted). Activation of type II NKT cells by sulphatide was recently reported to protect NOD mice from type 1 diabetes.[28, 89] Pre-treatment of NOD mice with the C24:0 but not C18:0 sulphatide analogue was found to protect against the transfer of type 1 diabetes.[89] These data suggest that the longer C24:0 sulphatide analogue should be examined for its therapeutic value in clinical trials in human subjects at risk for or newly diagnosed with type 1 diabetes. Our preliminary studies suggest that activation of type II NKT cells following administration of sulphatide significantly prevents lupus nephritis in (NZB × NZW) F1 mice, indicating that the protective capacity of sulphatide activated type II NKT cells can counteract potentially pathogenic type I NKT cells.