For example, phosphorylation

of JIP-1 enhances its ability to bind JNK indicating a feedback mechanism that may be responsible for the changes [46, 47]. Moreover, modifications by AKT1/2 [48] and Siah1 [49] can regulate POSH function and change the composition of the complex. Our data suggests that the POSH SH3.3 domain is dispensable for TCR-mediated NF-κB Dabrafenib supplier activation in CD8+ T cells [26]. Curiously though, POSH binds TAK1, a MAP3K responsible for IKKα/β phosphorylation and NF-κB (and JNK) activation [50]. When this is considered with the role of the Carma1/Bcl10 complex in the regulation of JNK2 (and NF-κB) [28], these data suggest the intriguing possibility that POSH could have a role in regulating JNK2 and NF-κB activity through the sequestration of TAK1. Considered together, these findings provide insight into the complex mechanisms that regulate the JNK pathway. The defect in POSH/JIP-1/JNK1-dependent Eomes expression may be indicative of impaired T-cell memory PARP inhibitor [45]. The loss of Tat-POSH-treated cells between days

9 and 20 supports this idea. Eomes−/− CD8+ effector T cells are both impaired in survival and the ability to re-expand upon rechallenge [41]. Interestingly, CD8+ T cells lacking both Eomes/T-bet acquire effector functions but are unable to mount an effective antitumor response [40]. In apparent contradiction, memory numbers and function were normal in both JNK1−/− and JNK2−/− mice after infection with LCMV [16]. However, the presence of high levels of proinflammatory cytokines in the LCMV-infected mice may have compensated for the lack of JNK activation. Therefore, whether the difference in these outcomes (LCMV versus tumor) is due to the nature of the “pathogen,” the inflammatory milieu, or POSH (and or TCR) independent signals in vivo remains to be determined. Regardless, the differential expression of T-bet and Eomes strongly suggests a mechanism

to explain how the POSH/JIP-1/JNK1 complex contributes to the T-cell effector differentiation program. In summary, our data indicate that the POSH/JIP-1 scaffold complex regulates JNK1 signaling and the development of T-cell effector function. This study highlights a mechanism by which unique scaffold complexes specifically regulate different isoforms of the same protein; JNK1 uses POSH/JIP-1 while JNK2 uses the Carma1/Bcl10 scaffold complex [28]. This provides the cell Smoothened with multiple points of control over the JNK signal pathways. How the POSH/JIP-1 scaffold complex regulates the unique role of JNK during thymic selection, CD4+ T-cell differentiation and the role of POSH in TCR-independent activation of JNK remains an open question. Together, given the various roles of JNK in T cells, inflammatory cells, neurons, and numerous cancers, these data identify POSH as a promising therapeutic target for manipulating cell fates and function. C57BL/6, OT-I, and OT-I Rag−/− mice were maintained in our animal facilities at the University of Missouri.

The finding that VCAM-1+ stroma express 4–1BBL, CCL19, CXCL12, and IL-7 and that adoptively transferred CD8+ memory T cells are often found in

proximity to VCAM-1+CD45− cells in the BM demonstrates the plausibility of the VCAM-1+ stromal cell as Protein Tyrosine Kinase inhibitor the radioresistant cell that provides 4–1BBL to memory CD8+ T cells in the BM. These data support a model in which a radioresistant VCAM-1+ stromal cell attracts the VLA-4+ CD8+ memory T cells via CCL19, where they can receive 4–1BB-4–1BBL induced survival signals. As the VCAM-1-positive stromal population is very abundant in the BM, there may be heterogeneity in the VCAM-1+ stroma with respect to 4–1BBL, cytokines, and chemokines that contribute to CD8+ T-cell memory maintenance. Further analysis will be required to definitively identify the 4–1BBL-expressing radioresistant cell that contributes to CD8+ T-cell memory. C57BL/6 WT mice were obtained from Charles River Laboratories (St. Constant, QC, Canada).

4–1BB−/− mice [47] extensively backcrossed to the C57BL/6 (n = 10) background were bred in our facility. These mice were previously provided to us by Dr. Byoung S. Kwon (National Cancer Center, Ilsan, Korea). 4–1BBL-deficient (4–1BBL−/−) mice were originally obtained under a materials transfer agreement from Immunex (Amgen, Thousand Oaks, CA, USA) and further backcrossed to the C57BL/6 background in our facility (total n = 9). OT-I

and CD45.1 congenic mice were obtained from Jackson Laboratories (Bar Harbor, ME, USA) and crossed to buy ZD1839 generate CD45.1+/+ or CD45.1+/− OT-I mice. TCRα−/– mice were kindly provided by Dr. Cynthia Guidos (Hospital for Sick Children, Toronto). FoxP3gfp knock-in mice on the C57BL/6 background were kindly provided by Dr. Mohamed Oukka (Harvard Medical School) [48]. ACTB-DsRed transgenic mice expressing DsRed protein under control of the β-actin promoter and backcrossed to B6 mice for five generations (B6.Cg-Tg (ACTB-DsRed*MST) 1Nagy/J) were obtained from the Jackson laboratories and crossed with OT-I mice to obtain OT-I ACTB-DsRed mice (OT-I-DsRed). Mice were maintained under specific pathogen-free conditions in sterile microisolators at the University of Toronto. All mouse experiments were approved from by the University of Toronto animal care committee in accordance with the regulations of the Canadian Council on animal care (University of Toronto approved protocol #20007828). CD8+ T cells with a central memory phenotype were generated by culture with Ag followed by IL-15 using a variation of a previous protocol [7, 29]. In brief, OT-I splenocytes were stimulated with 0.1 μg/mL SIINFEKL peptide and 1 μg/mL of LPS for 1 day, and then the nonadherent cells were rested for 2 days in fresh media (RPMI-1640 with 10% heat-inactivated FCS, 0.03% L-glutamine, antibiotics, and 2-mercaptoethanol).

2). These data confirm that Egr2 is not able to force development of SP cells in the absence of a selection stimulus or alter the process of lineage commitment. To study the initiation of positive selection in more Talazoparib solubility dmso detail, Egr2f/fCD4Cre mice were bred with MHC° mice to provide a source of unsignaled Egr2f/fCD4Cre DP thymocytes. These naïve cells cannot undergo positive selection in situ as peptide antigen cannot be

presented due to the lack of MHC, but they respond in vitro to stimuli, such as TCR crosslinking with anti-CD3, which mimic antigen engagement. To test whether positive selection could be impaired in Egr2f/fCD4Cre mice as a result of defective TCR-proximal signaling, cells were crosslinked with anti-CD3 for 2 min, and levels of phospho-Erk, a sensitive indicator of activation of the MAPK pathway following TCR ligation, were measured by flow cytometry. Figure 4A shows that both WT and Egr2f/fCD4Cre MHC° thymocytes were able to respond to anti-CD3 crosslinking by phosphorylating

Erk to the same extent, Venetoclax order with around 20% of thymocytes staining positive for phospho-Erk. Stimulation of normal and Egr2f/fCD4Cre thymocytes with plate-bound anti-CD3 over 24 h also showed that upregulation of the positive selection markers CD69 (Fig. 4B) and CD5 (Fig. 4C) was unchanged. Therefore, the defect in selection of Egr2f/fCD4Cre thymocytes is unlikely to be due to a failure Histidine ammonia-lyase to initiate selection. To determine at what point following TCR-proximal signaling Egr2 might be acting, we profiled Egr2 mutant thymocytes by staining for TCR-β and CD69. These markers can be used to fine-map the stages of positive selection, which is initiated in CD69− TCR-βlo DP thymocytes, and completed

by the time cells are CD69+TCR-βhi29. Gating thymocytes on the basis of TCR-β and CD69 expression showed that while each of the populations in the maturation sequence – TCR-βloCD69−; TCR-βloCD69+; TCR-βhiCD69+; TCR-βhiCD69− – were present in both Egr2f/f and Egr2f/fCD4Cre mice (Fig. 4D, left and centre panels), there was a statistically significant decrease in the proportion of TCR-βhi Egr2f/fCD4Cre thymocytes in Egr2f/fCD4Cre animals (p=0.023; Fig. 4D, right panel). As the TCR-βlo populations did not differ in terms of CD69 expression (data not shown), this decrease suggests that the defect in Egr2f/fCD4Cre thymocyte development occurs after upregulation of CD69, and hence later on in the process, such that fewer Egr2f/fCD4Cre cells completed positive selection and became TCR-βhi. Comparable staining profiles for Egr2-Tg thymocytes gave the reciprocal phenotype; cells progressed through the first stage of positive selection, upregulating CD69 as normal (Fig. 4E, left and centre panels), but there were significantly more TCR-βhi Egr2 Tg thymocytes than TCR-βhi non-Tg thymocytes (p=0.042; Fig. 4E, right panel).

Domain I, the N-terminal ∼120 residues, is highly basic and is probably involved in the recruitment of the viral RNA during particle formation. Domain II, situated between a.a.∼120 and ∼175, has been predicted to form one or two alpha-helices that are presumed to be involved in the association of Core with membrane proteins and lipids. This domain is not present in the capsid proteins of most of the other members of the Flaviviridae family. It has recently been shown that the cysteine residue at a.a.128 is responsible for the disulfide-bonded dimer of Core and for particle formation (19). Domain III, located at

the C-terminal ∼20 residues, is highly hydrophobic and has been predicted to form an alpha helix. This domain serves as a signal sequence find more Selleckchem Dabrafenib for E1 as described above. The ubiquitin-proteasome pathway, a major route by which selective protein degradation occurs in eukaryotic cells, is involved

in post-translational modification of Core (20–25). Ubiquitin ligase E6AP has been identified as a core-binding protein that enhances its ubiquitylation and degradation. It has been suggested that E6AP-dependent degradation of Core is common to a variety of HCV isolates and plays a critical role in the HCV life cycle (23). Recently, we also demonstrated that proteasomal degradation of Core is mediated by two distinct mechanisms. One leads to polyubiquitylation in which lysine residues in the N-terminal region are preferential ubiquitylation sites. The other is ubiquitin-independent, why but depends on interaction with proteasome activator PA28gamma (24). Although is so far unclear as to whether destabilization of Core via two distinct mechanisms is physiologically significant, it is reasonable to consider that tight control over cellular levels of Core may contribute to restricting its potential for functional activity. E1 and E2 proteins are essential

components of the virion envelope and are necessary for viral entry. These glycosylated proteins extend from a.a. 192–383 (E1) and from a.a. 384–746 (E2) of the polyprotein, and have molecular weights of 33–35 and 70–72 kDa, respectively (26). Intracellular envelope proteins mainly exhibit high-mannose type glycans, consistent with their accumulation in the ER (27), whereas infectious-virion-associated envelope proteins display a mixture of high-mannose and complex types of glycans. It has been shown that E1 and E2 are heavily glycosylated, suggesting that HCV glycoproteins are processed by Golgi-resident glycosidases and glycosyltransferases (28). Complex N-linked glycans have also been detected on the surface of HCV particles isolated from patient sera (29). Based on prediction of membrane topology, it is hairpin structures that pass through the membrane twice, thereby allowing processing by a signal peptide in the ER lumen (30).

These events include phosphorylation of the CD3ζ chain, ZAP70, and LAT 37. Moreover, the Scr-family kinase LCK is inhibited 38, 39 which leads to a modulation of the calcium signaling 39. Therefore, while the inhibition of LFA-1

accumulation by dexamethasone is probably mediated by the inhibition of L-plastin phosphorylation, the additional defective accumulation of the TCR/CD3 complex in dexamethasone-treated T cells might be due to the inhibition of TCR/CD3-induced tyrosine phosphorylation and calcium signaling by dexamethasone. In contrast to other actin-binding proteins, such as cofilin or Arp2/3, the expression of L-plastin is restricted to leukocytes and certain tumors 47, potentially making it a valuable target for immunosuppression. Supporting this assumption, Wang et al. 46 demonstrated that LPL−/− mice showed a less severe experimental autoimmune encephalomyelitis (EAE). Moreover, they found that Alectinib clinical trial L-plastin expression has an important role in delayed, but not immediate

allograft rejection in the murine system. Therefore, interference with L-plastin phosphorylation and/or functions may be a sophisticated approach to modulate T-cell immune responses in order to prevent transplant rejection or to treat T-cell-mediated autoimmune diseases in humans. Abs employed were specific for the following markers: CD3 (mouse mAks, clone OKT3 or SK3), CD2 (mouse mAb, clone 3PT2H9, kindly provided by S. F. Schlossman, Dana Farber Cancer Institute, Boston, MA, USA), CXCR4 (R&D Systems, Wiesbaden-Nordenstadt, Germany) CD28 (CD28.2), and CD3-PerCP, LFA-1 (CD18-FITC, CD18-PE or CD11a-FITC), selleck compound CD28-PE (mouse mAb, BD Biosciences, Heidelberg, Germany). The CD3-PeTxR Ab was purchased from Caltag (Buckingham, UK) and the actin antiserum from Sigma-Aldrich (Hamburg, Germany). The GFP Ab was from Clontech. Unconjugated anti-mouse and horseradish peroxidase-conjugated anti-rabbit Abs were purchased from Dianova (Hamburg,

Germany). The L-plastin polyclonal antiserum was produced against recombinant L-plastin protein 8. Phalloidin-AlexaFluor647 and Hoechst33342 was from Invitrogen (Darmstadt, Germany). Dexamethasone was purchased from Calbiochem (Bad Soden, Germany) and Ru486 (mifepristone) was from Sigma-Aldrich. All inhibitors and drugs were reconstituted Montelukast Sodium in DMSO. Thus, the respective controls in the experiments were performed as solvent controls with the relevant concentration of DMSO. In the titration experiment, the highest concentration of DMSO was used as solvent control. Human PBMCs were obtained by Ficoll-Hypaque (Linaris, Wertheim-Bettingen, Germany) density gradient centrifugation of heparinized blood from healthy volunteers upon approval by the local ethics committee. T cells were subsequently isolated with magnetic associated cell sorting using pan T-cell negative isolation kit II (Miltenyi Biotec, Bergisch Gladbach, Germany) 5.

The freed Bcl-2 presumably exerts a prosurvival function, which would enhance the efficacy of the IL-15-induced Bcl-2 increment. Being resident in the intestine epithelium, it may be beneficial for CD8αα+ iIELs to control Bim activity by phosphorylation and dephosphorylation rather than synthesis and degradation, as the former can be achieved in a timely manner in response to the complex environment of the intestinal mucosa. Further studies are needed to test these possibilities. Activation of the Jak3-Jak1-PI3K-Akt-ERK signaling pathway is essential for IL-15-mediated CD8αα+ iIEL survival (Fig. 1). Although ERK activation is downstream of PI3K-Akt,

it was obviously delayed

compared to the activation of PI3K-Akt (Fig. 1C, D and Supporting Information Fig. 6, left panel). Consistently, the reduction of Bcl-2 level by MEK inhibition Ruxolitinib concentration occurred later than that induced by Jak3 or PI3K inhibitor (Fig. 2A). It is possible that ERK1/2 activation was secondary to IL-15 isocitrate dehydrogenase inhibitor stimulation. However, our preliminary experiments using supernatant from 40 h IL-15-treated CD8αα+ iIELs did not support the possibility that IL-15-induced secretory factor(s) activated ERK1/2 in CD8αα+ iIELs (Supporting Information Fig. 6, right panel). Other possible causes for the delayed and sustained ERK1/2 activation includes prolonged activation of upstream kinase and diminished activation of phosphatase. In view of these findings, we propose a stepwise model for IL-15-mediated CD8αα+ iIEL survival (Supporting Information Fig. 7). IL-15 first upregulates prosurvival Bcl-2 and Mcl-1 via activation of the Jak3-Jak1-PI3K-Akt pathway. With elevated Bcl-2, IL-15 induces ERK1/2-mediated phosphorylation of Bim at Ser65 to release Bcl-2 from the Bcl-2-Bim complex and to keep Bim in a phosphorylated Ketotifen state. Activated ERK1/2 also participates in the maintenance of Bcl-2 level. The increase of Bcl-2 abundance and freed Bcl-2 shift the balance of Bcl-2 and Bim function toward promoting CD8αα+ iIEL survival. C57BL/6J (B6) and B6 human (hu) BCL-2

transgenic (B6-Tg (BCL2) 36Wehi/J) mice were purchased from the Jackson Laboratories. Il15ra−/− mice were generated in our lab [43] and backcrossed to B6 for 24 generations. RNA polymerase II-driven huMCL-1 transgenic mice in the B6 background were generated in Dr. S.-F. Yang-Yen’s lab [44]; Bim−/− mice were kindly provided by Dr. Jeffery C. Y. Yen (Institute of Biomedical Science, Academia Sinica, Taiwan). All mice were raised in a specific pathogen-free facility at the Institute of Molecular Biology, Academia Sinica. The mice were used at 8–22 weeks of age. All mice experiments were approved by the Institutional Animal Care and Use Committee at Academia Sinica and conformed to the relevant regulations.

These cross-reactive T cells were found to be subdominant during the primary response, and the sequence of infection influenced the

selleck chemical hierarchy of these subdominant cross-reactive T cells after secondary heterologous challenge 32, 33. In our model, the immunodominant CD8+ T-cell epitope was found to be cross-reactive, but to differing degrees, following either JEV or WNV infection. Our detailed characterization of these epitope-specific responses did not demonstrate an alteration in epitope hierarchy, but rather differences in cytokine profiles and T-cell phenotype. As previous studies have elucidated a role for subdominant cross-reactive CD4+ and CD8+ T cells in protection as well as immunopathology, future experiments will address Dabrafenib in vitro the role of the two cross-reactive CD4+ T-cell epitopes we identified and subdominant cross-reactive CD8+ T-cell epitopes along with the immunodominant cross-reactive CD8+ T-cell epitope in secondary heterologous JEV and WNV infections 10, 11. Here, we have shown that primary infections with JEV and WNV give rise to functionally and phenotypically distinct CD8+ T-cell responses. These

differences are due to the infecting virus (JEV versus WNV) rather than the stimulating variant (WNV S9 versus JEV S9) or viral pathogenicity. The JEV/WNV cross-reactive CD4+ and CD8+ T-cell epitopes we have identified will be useful tools to study the pathogenesis of sequential heterologous flavivirus infections. Flaviviruses continue to emerge into new geographic regions of the world, giving rise

to the possibility of new patterns of sequential infection with unknown outcomes (e.g. WNV into dengue- and yellow fever virus-endemic regions of South America). Altered CD8+ T-cell effector functions between flaviviruses may to lead to immunopathology or protection upon a secondary flavivirus infection. Additional experiments are needed to determine whether cross-reactivity else occurs between other members of the flavivirus family and its possible impact on disease outcome. JEV strain SA14-14-2 was provided by Dr. Thomas Monath (Acambis, Inc.). JEV strain Beijing was provided by Dr. Alan Barrett (University of Texas Medical Branch, Galveston, TX, USA). WNV strain 3356 was provided by Dr. Kristen Bernard (Wadsworth Center, Albany, NY, USA). Flaviviruses were propagated and titered in Vero cells (ATCC). The EL-4 T-cell lymphoma cell line (H-2b) served as target cells. Peptide (15–19mer) arrays corresponding to the entire proteome of WNV were obtained through the NIH Biodefense and Emerging Infections Research Resources Repository, NIAID, NIH (BEI Resources, Manassas, VA, USA). Peptide truncations (>70 or >90% purity) were obtained from AnaSpec (San Jose, CA, USA) and 21st Century Biochemicals (Marlborough, MA, USA).

Samples were analysed using negative electrospray ionization (ESI). The ion spray voltage was set at −4500 V. The source temperature was Selleck Ibrutinib set at 400°C. Nitrogen was used as the nebulizer and auxiliary gas and was set at 20, 50 and 50 arbitrary units for the curtain gas, the ion source gas 1 and the ion source gas 2, respectively. MS/MS spectra of

15-epi-LXA4 showed the same fragmentation pattern as the published [31] and commercial source (data not shown) spectra. Moreover, LC-MS/MS analysis confirmed 15-epi-LXA4 stability and no changes in height peak and area were observed during the time of the in-vitro assay conditions and using the 15-epi-LXA4 concentration reported to show biological activity (data not shown). The synthetic ROCK inhibitor peptide WKYMVm (Trp-Lys-Tyr-Met-Val-D-Met-NH2) was purchased from Tocris Bioscience (Bristol, UK). IL-8 was purchased from Peprotech (Rocky Hill, NJ, USA). Montelukast, MK-571, compound 43 and SCH527123 were synthesized at the Medicinal Chemistry Department in Almirall R&D Centre (Sant Feliu de Llobregat, Barcelona, Spain). Human Chinese

hamster ovary (CHO)-FPR2/ALX (ES-610-C) and human CHO-CysLT1 (ES-470-C) cell lines were purchased from Perkin Elmer (Waltham, MA, USA). Surface expression of the receptor FPR2/ALX was monitored by flow cytometry using a commercial monoclonal antibody against the receptor. Results clearly show high levels of receptor expression in FPR2/ALX-recombinant CHO cells compared to non-transfected CHO cells (increased 40-fold in mean expression). In addition, information on Bmax of recombinant cell lines by a radioligand saturation binding assay was provided by Perkin Elmer Cyclic nucleotide phosphodiesterase and confirmed activity of both receptors in the recombinant cells. Ham’s F12 culture medium supplemented with 100 IU/ml penicillin and 400 μg/ml G418 was used to grow the cells. FPR2/ALX cell membrane preparation was performed from FPR2/ALX stable transfected CHO cells purchased from Perkin-Elmer. Adherent-growing CHO-h FPR2/ALX cells were washed in cold phosphate-buffered saline (PBS), harvested by scraping

and collected by centrifugation at 1500 g for 5 min. The cell pellet was washed twice with cold PBS and resuspended in homogenization buffer [15 mM Tris-HCl, pH 7·5, 2 mM MgCl2, 0·3 mM ethylenediamine teraacetic acid (EDTA), 1 mM ethylene glycol tetraacetic acid (EGTA)]. The cells were then lysed with an Ultraturrax homogenizer. Intact cells and nuclei were removed by centrifugation at 1000 g for 5 min. The cell membranes in the supernatant were then pelleted by centrifugation at 40 000 g for 25 min and resuspended in storage buffer (50 mM Tris-HCl pH 7·4, 0·5 mM EDTA, 10 mM MgCl2, 10% sucrose), aliquoted, quick-frozen in liquid N2 and stored at −80°C. Protein concentration in membrane preparations was determined using the DC Protein Assay kit (Bio-Rad, Hercules, CA, USA).

Some affected infants, for instance, evolve myocardial disease only later in life [39, 40]. Furthermore, we have shown that the EFE detected echocardiographically often underestimates the degree of EFE based on the examination of corresponding pathological specimens [39]. That Rapamycin the more diffuse myocardial disease represents a separate manifestation of NLE is suggested by our observations of isolated EFE and cardiomyopathy, in the absence of conduction abnormalities [40]. Histologically, we have shown maternal autoantibody-induced EFE and cardiomyopathy to be associated with diffuse disarray of myocardial fibres with IgG deposition in all, IgM deposition and even T cell subset activation,

the latter findings suggestive of a foetal immune response contributing to the disease process [39]. Early in the disease course, there may be evidence of acute inflammation with lymphocytic infiltrates in keeping with an acute myocarditis [42, 43]. Why more diffuse myocardial disease occurs in some but not all foetuses Apoptosis inhibitor and infants with maternal autoimmune-mediated AVB remains unclear, but variability in the foetal immune response may

contribute [39,44]. Finally, in addition to myocardial disease, pericardial effusion without other signs of hydrops has been reported in some affected foetuses and could suggest the presence of pericarditis [45]. The outcome of clinically manifested diffuse myocardial disease associated Decitabine manufacturer with maternal autoantibodies in the absence of intervention is very poor with a greater than 80% rate of demise or need for cardiac transplantation [14, 39–41]. In an effort to improve the outcome of this difficult pathology, we have recently prospectively treated a small cohort of foetuses and infants with EFE, most with complete AVB, with intraumbilical, maternal/transplacental or post-natal intravenous immunoglobulin and corticosteroids and have observed a 78% survival rate at a follow-up of 3 years [46]. Other strategies suggested for

the treatment of these foetuses and infants include intrauterine pacing, maternal and infant plasmapheresis, early dual (AV) chamber pacing and even biventricular pacing have not as yet been evaluated in a series of affected patients. Prospective randomized trial of the use of these strategies may help clarify their role and efficacy in the treatment of EFE; however, the clinical disease is so rare that this makes such an initiative difficult. In addition to AVB, several other electrophysiological abnormalities have been reported in the foetus and infant with maternal autoimmune-mediated cardiac disease. These abnormalities include both transient and persistent sinus node dysfunction, long QT interval, ventricular and atrial ectopy, ventricular and junctional tachycardia, and atrial flutter (Fig. 3).

We further examined IL-23 production by K5-PLCε-TG keratinocytes because it was reported that IL-23 could induce acanthosis in mouse models 26, 30. The ELISA for IL-23 heterodimer demonstrated that cultured K5-PLCε-TG keratinocytes released a small

but substantially increased amount of IL-23 compared Rucaparib in vivo to WT keratinocytes (Fig. 7B). Immunohistological analysis of the skin showed that keratinocytes, as well as epidermal CD205+ DC, were positive for IL-23 in the K5-PLCε-TG mouse skin at P26 (Fig. 7C). In particular, keratinocytes located in the upper epidermal layer rather than those in the basal layer produced a substantial amount of IL-23 (Fig. 7C), which is likely to account for our data that the amount of IL-23 released from the proliferative keratinocytes in Talazoparib culture was rather small (Fig. 7B). At P6, epidermal keratinocytes of K5-PLCε-TG mice expressed a higher level of IL-23 p19 compared to those of WT mice (Fig. 7D). This difference became more pronounced at P9 and P26 even taking account of the difference in their epidermal thickness. In contrast, IL-23 was below the detection limit at 15 wk although PLCε remained overexpressed (Figs. 5 and 7D). The role of IL-23 in the symptom development of K5-PLCε-TG mice was examined by neutralizing antibody-mediated blockade of IL-23 (Fig. 8A). As expected, blocking of

IL-23 suppressed the skin symptoms, especially accumulation of inflammatory cells, around the site of the antibody injection (Fig. 8B and Supporting Information Fig. 7). Further, immunostainig for CD4 and Th cytokines demonstrated that the number of CD4+ T cells, particularly those producing IL-22, was significantly reduced after IL-23 blockade (Fig. 8C and D). These results demonstrated that IL-23 plays a crucial role in the symptom development in K5-PLCε-TG mice. We next studied the effect of FK506 on the symptom development in the K5-PLCε-TG mouse

skin. As above indicated, administration of FK506 resulted in disappearance of adherent silvery scales in K5-PLCε-TG mice whereas it failed to block acanthosis (Fig. 9A and B), which could be accounted for by its growth-promoting activity 22. Examination of the skin sections indicated that the FK506 treatment markedly suppressed the infiltration Etofibrate of CD4+ T cells as well as MPO+ neutrophils (Fig. 9C). Among CD4+ T cells, those producing IL-22 rather than those producing IFN-γ were considerably affected by the FK506 treatment (Fig. 9D), which was compatible with the qRT-PCR data showing the entire abrogation of Th17 cytokines (Fig. 9E). These results suggested an important role of IL-22-producing CD4+ T cells in the development of the skin symptoms in K5-PLCε-TG mice. In this study, we show that K5-PLCε-TG mice spontaneously develop dermatitis over the whole body.