This work was supported

This work was supported Talazoparib manufacturer by National Institutes of Health grants NIH R01 DK 066917 and a Dana-Farber/Harvard Cancer Center Prostate Cancer SPORE P50CA090381 Development Award (M. A. E.). Conflict of interest: The authors declare no financial or commercial conflict of interest. “
“Mitochondrial components, including mitochondrial DNA (mtDNA), when released extracellularly, can act as “damage-associated molecular pattern” (DAMP) agents and cause inflammation. As many elderly people are characterized by a low-grade, chronic inflammatory status defined “inflamm-aging,” we evaluated if circulating mtDNA can contribute to this phenomenon. Eight hundred and thirty-one Caucasian subjects were enrolled

in the study, including 429 siblings aged 90–104 (90+ siblings). mtDNA plasma levels

increased gradually after the fifth decade of life. In 90+ subjects, mtDNA values of two members of the same sibling relationship were directly correlated, suggesting a role for familiar/genetic background in controlling the levels of circulating mtDNA. The subjects with the highest mtDNA plasma levels had the highest amounts of TNF-α, IL-6, RANTES, and IL-1ra; the subjects selleck chemicals with the lowest mtDNA levels had the lowest levels of the same cytokines. In vitro stimulation of monocytes with mtDNA concentrations similar to the highest levels observed in vivo resulted in an increased production of TNF-α, suggesting that mtDNA can modulate the production of proinflammatory cytokines. Our findings therefore show that circulating mtDNA increases with age, and can significantly contribute to the maintenance of the low-grade, chronic inflammation observed in elderly people. “
“Haemonchus contortus is an economically important gastrointestinal parasite that infects primarily sheep and goats. To survive inside the host, the parasite must overcome the host immune response. Axenfeld syndrome In this study, we have identified and characterized a complement-C3-binding protein (H.c-C3BP)

from this parasite employing biochemical and molecular biology tools. Initially, a truncated form of the protein was isolated from the excretory–secretory products of the parasite using C3–Sepharose column that facilitated its identification by mass spectroscopy. Subsequently, the parent molecule was generated in E. coli, and sequence analysis confirmed it as glyceraldehyde-3-phosphate dehydrogenase (GAPDH). GAPDH reacted with the antiserum raised against the truncated protein, and the truncated protein reacted with anti-GAPDH antiserum. The protein inhibited complement function as measured by haemolytic assay and membrane attack complex (MAC) formation. Sera from H. contortus-infected animals reacted with GAPDH as well as the truncated form of the protein, which further lend support to protein secretion. Thus, the C3-binding property of H. contortus GAPDH is a new function, and it represents a new entity of complement-binding protein. Identification and characterization of H.

albicans infection in humans WT C57BL/6 mice or mice lacking TLR

albicans infection in humans. WT C57BL/6 mice or mice lacking TLR7 or TLR9 were infected i.v. with a low dose (1 × 104 CFU) of C. albicans, a challenge that was found to be sublethal for WT mice

in preliminary experiments. Survival and morbidity were monitored daily. As shown in IWR1 Figure 7A, most of the mice lacking either TLR7 or TLR9 succumbed to infection while all WT mice survived. To ascertain whether increased lethality was associated with a decreased ability of these mice to control in vivo infection, we measured fungal burden in the kidney, the main target of hematogenous C. albicans dissemination, at 5 days after infection with the same C. albicans dose (1 × 104 CFU) used in the lethality experiments. In these experiments, we also tested MyD88−/−, IRF1−/−, and 3d mice in addition of TLR7−/− and TLR9−/− animals. While low CFU numbers were found in kidneys of WT mice, fungal burden was significantly increased in mice lacking

either TLR7 or TLR9 (Fig. 7B). Notably, fungal burden was even higher in 3d Hydroxychloroquine or IRF1−/− mice compared with TLR7−/− or TLR9−/− mice. Mice lacking MyD88 showed the most severe phenotype of all, with colony counts that were approximately 6 orders of magnitude higher than those of WT controls. Collectively, these data indicated that the TLR7/TLR9/MyD88/IRF1 pathway has a nonredundant role in defenses against C. albicans. Moreover, 3d mice (that are unable to mobilize TLR7/9 and other intracellular TLRs to phagosomes) showed a phenotype

that was similar to that of IRF1−/− mice and intermediary between MyD88−/− (highly susceptible) and TLR9−/− or TLR7−/− (moderately susceptible). Our results, showing an increased susceptibility of TLR9−/− mice to C. albicans infection, were apparently in contrast with those of previous studies showing similar [28, 38] or even decreased [14] susceptibility Histamine H2 receptor of TLR9−/− mice in comparison with WT animals. We hypothesized that these discrepancies could be related to the fact that the cited studies used a higher (1–2 log) challenge doses than the one we used. Therefore, to test this hypothesis, we challenged TLR7- and TLR9- defective mice with a 20-fold higher C. albicans dose than that previously used in the experiments summarized in Fig. 7. Under these conditions, no differences were found in susceptibility to infection between TLR7-or TLR9-deficient mice and WT controls, as measured by kidney colony counts (Supporting Information Fig. 5). This data indicate that the effects of TLR7 or TLR9 deficiency on the outcome of the infection are critically dependent on the challenge dose. The identification of receptors and signal transduction pathways involved in immune responses to fungi is essential to understand the mechanisms underlying the development of mycoses and to devise alternative strategies to control these difficult to treat infections.

To address this possibility, we performed a LUC reporter assay A

To address this possibility, we performed a LUC reporter assay. A pGL3-LUC vector subcloned with the promoter region from –1500 bp to the Prdm1 transcription start site [29] was co-transfected with a pMIG-Egr-2 vector to 293T cells. As shown in Figure 3A, Egr-2 significantly enhanced the activity of the Prdm1 promoter. Next, a ChIP assay was performed with antibodies against Egr-2 to investigate whether Egr-2 directly binds to the promoter region of Blimp-1 in CD4+ T cells. Among four Dabrafenib in vitro promoter regions examined (−3000 bp, −2000 bp, −1000 bp, and +1000 bp from its transcription

site) of Blimp-1, only one region (−1000 bp) showed significant enrichment compared with control, indicating that Egr-2 specially binds to the Blimp-1 promoter, but not to Lag3 and Il10 promoters (Fig. 3B and Supporting Information Fig. 2A). Cretney et al. reported that Blimp-1 binds to intron 1 of the Il10 locus and, together selleck screening library with IFN regulatory factor-4, directly regulates IL-10 expression in CD4+CD25+ Treg cells by the remodeling of active chromatin at the Il10 locus [28]. Our observation suggested that IL-10 regulation with Blimp-1 was controlled by Egr-2. STAT1 and STAT3 have been shown to be crucial for IL-10 production from IL-27-stimulated

naïve CD4+ T cells [17]. We investigated the effect of STAT1 and STAT3 deficiencies on IL-27-induced Egr-2 expression. As shown in Figure 4A and B, Egr-2 induction by IL-27 in CD4+ T cells was impaired by a STAT3 deficiency, but not by a STAT1 deficiency. When we analyzed the induction of Il10 transcription and IL-10 protein expression by IL-27 in STAT1- and STAT3-deficient

CD4+ T cells, IL-10 protein induction by IL-27 was abolished both in STAT1 KO and in STAT3 CKO CD4+ T cells, although IL-10 mRNA expression levels were slightly up-regulated by IL-27 in STAT1 KO CD4+ T cells (Fig. 4C and D). These results suggest that IL-27-induced Egr-2 expression in CD4+ T cells is mostly dependent on STAT3, although both STAT1 and STAT3 are important for IL-10 production by IL-27. Next, we investigated the effect of other STAT1 or STAT3 activating cytokines for Egr-2 induction. IL-6 and IFN-γ were selected as the representatives of cytokines activating STAT3- and STAT1-mediated pathways, respectively. As shown in Figure 4E, IL-6 induced Egr-2 expression as effectively as IL-27 Tolmetin in CD4+ T cells, but IFN-γ did not. Interestingly, both IL-10 and Blimp-1 mRNA expressions were also elevated by IL-6, but expression levels seemed to be lower than those by IL-27 (Fig. 4F). IL-6 is a type I cytokine that shares structural homology and a receptor subunit, gp130, with IL-27 and has already been shown to induce IL-10 in CD4+ T cells [17]. These results suggest that Egr-2 is important for IL-10 production mediated both by IL-27 and by IL-6 through the STAT3-dependent pathway. To examine the role of Egr-2 in inflammatory cytokine production, we investigated the production of IFN-γ and IL-17 in response to IL-27 stimulation.

Microvascular knee CTA was performed in nine rats across a major

Microvascular knee CTA was performed in nine rats across a major histocompatibility barrier with both pedicle repair and implantation of host-derived arteriovenous (“a/v”) bundles. In the control group (N = 3), the pedicle was ligated. Immunosuppression was given daily. Joint mobility,

weight-bearing, pedicle patency, bone blood flow, and sprouting from a/v bundles were assessed at 3 weeks. All but the nonrevascularized control knees had full passive motion and full weight find more bearing. One nutrient pedicle thrombosed prematurely. Blood flow was measurable in transplants with patent nutrient pedicles. Implanted a/v bundles produced new vascular networks on angiography. This new rat microsurgical model permits further study of joint allotransplantation. Patency of both pedicles and implanted a/v bundles was maintained, laying a foundation for future studies. © 2010 Wiley-Liss, Inc. Microsurgery, 2010. “
“The effect of sialodacryoadenitis virus (SDAV) infection on axonal regeneration PLX4032 ic50 and functional

recovery was investigated in male Lewis rats. Animals underwent unilateral tibial nerve transection, immediate repair, and treatment with either FK506 (treated) or control vehicle (untreated). Serial walking track analyses were performed to assess functional recovery. Nerves were harvested for morphometric analysis on postoperative day 18 after an SDAV outbreak occurred that affected the 12 experimental animals. Histomorphometry and walking track data were compared against 36 historical controls. Rats infected with SDAV demonstrated severely impaired axonal regeneration and diminished functional recovery. Total fiber counts, nerve density, and percent neural tissue were all significantly reduced in infected animals (P < 0.05). Active SDAV infection severely impaired nerve regeneration

and negated the positive effect of FK506 on nerve regeneration in rats. Immunosuppressive risks must be weighed carefully click here against the potential neuroregenerative benefits in the treatment of peripheral nerve injuries. © 2011 Wiley-Liss, Inc. Microsurgery, 2011. “
“Soft-tissue defects of the distal foot that involve an exposed tendon and bone demonstrate a reconstructive challenge for plastic surgeons. This report investigates the feasibility and reliability of metatarsal artery perforator (MAP)-based propeller flap for reconstruction of the distal foot soft-tissue defects. Between July 2011 and June 2012, six patients underwent distal foot reconstruction with seven MAP-based propeller flaps. Five flaps were based on the third metatarsal artery and two flaps were based on the first metatarsal artery. The flap size ranged from 4 × 2 cm to 8 × 4 cm. All flaps completely survived. Two patients developed transient distal venous congestion, which subsided spontaneously without complications. There were no donor site complications. All patients were ambulating without difficulty within the first month of surgery.

ratti infection at days 10 or 31 post-L  major infection (Figure 

ratti infection at days 10 or 31 post-L. major infection (Figure 2d, e). The comparison of the L. major-specific humoral response revealed also no difference in single and co-infected mice (Figure 3a–e). Especially L. major-specific IgG2b that is associated with a Th1 response was not suppressed but even slightly increased by S. ratti co-infection (Figure 3d). Taken together, these results suggest that a pre-existing nematode infection

did not interfere with the generation of a protective cellular and humoral type-1 response to L. major but increased pro-inflammatory responses in general. Subsequent L. major infection, in contrast, partially suppressed the Th2 polarization induced by pre-existing S. ratti infection. Therefore, we asked whether this changed nematode-induced production of Th2 cytokines would affect clearance of S. ratti infection in these co-infected mice. First, we compared the larval output in the faeces of S. ratti singly and S. ratti/L. major Target Selective Inhibitor Library supplier co-infected mice by

quantitative PCR (Figure 4a). Despite the changed cytokine response, S. ratti/L. major co-infected mice displayed the same larval output with comparable kinetics until the faeces was negative for S. ratti DNA indicating complete clearance of nematode infection (Figure 4b). Re-infection of S. ratti single and S. ratti/L. major co-infected mice with S. ratti again led to similar PLX4032 order larval output that was reduced in comparison with the first infection indicating efficient memory generation (data not shown). Nevertheless, the suppression of nematode-induced Th2 responses by the pro-inflammatory responses elicited by L. major co-infection (Figure 2b, c) strongly suggests that worm expulsion could be affected if L. major infection preceded nematode infection. To prove this hypothesis, we performed co-infection experiments in reversed order. Mice were infected with a high Carnitine palmitoyltransferase II dose of L. major and 14 days later, when L. major-specific Th1 response was established, mice were co-infected with S. ratti iL3 (Figure 5a). Comparison of the larval output in the faeces (Figure 5b) as well as numbers of parasitic adults in the gut (Figure 5c) did not reveal impaired

or delayed clearance of S. ratti infection in co-infected mice. We did observe an increased output of L1 in co-infected mice at the maximum of infection that was not significant (Figure 5b, day 8 p.i.). As neither the kinetics of worm clearance nor the worm burden in the intestine showed significant differences, we chose to analyse the underlying immune responses (Figure 6a). Strikingly, no suppression of CD3-induced or S. ratti antigen-specific proliferation, IL-10 and IL-13 response were observed in this experimental set-up in co-infected mice (Figure 6b–d). Also, the absent IFN-γ response in S. ratti-infected mice was not restored by pre-existing L. major infection (Figure 6e). Finally, no change in the S. ratti-specific humoral response was observed upon co-infection (Figure 6f, g).

Neurons in CA2-4 fields and DG, generally spared from classic NFT

Neurons in CA2-4 fields and DG, generally spared from classic NFT pathology development in AD, exhibited markedly increased UBL immunoreactivity in the nucleoplasm in Braak stages III-IV and V-VI AD cases compared to the Braak 0-I-II group. The reason for this change is unknown, but it may be influenced by age differences

between Braak groups, since the Braak stage 0-I-II (non-AD) group trended toward being younger than both the Braak stage III-IV and Braak stage V-VI AD groups. Other factors, including nucleotide polymorphisms in the ubiquilin gene, may contribute to the observed differences and warrant future clinical-genetic-pathological studies. Genetic abnormalities in Fer-1 price UBL-1 were reported to associate with increased risk[20] and age of onset and duration[21] of AD, although this association was not replicated in all studies.[22] Because Braak staged groups represent a continuum, rather than a stepwise progression, of NFT pathology, the large variability in UBL intensity ratios in the Braak stage III-IV group, particularly in the CA1 region, is likely due to variability in the extent of pathologic changes, and UBL expression, in individual

pyramidal neurons. The functional relevance of the changes in the subcellular localization of UBL, and their association with different types of NFT, is Meloxicam unknown but it may reflect a response, compensatory or dysregulatory, of the ubiquitin-proteosome system to increased cellular stress this website due to accumulation of aggregated and heavily phosphorylated proteins, especially

tau. Our observation of increased UBL immunoreactivity in X-34-positive eNFT is particularly intriguing considering that ubiquitin, a major component of NFT paired helical filaments in AD,[23, 24] is largely absent from eNFT.[23, 25, 26] These changes may occur in relation to ubiquitin-proteosome dysfunction or, alternatively, they may reflect altered antigenic profiles of these proteins in eNFT.[27] The observation of UBL immunoreactivity in X-34-positive neuritic plaques in advanced Braak stages further suggests a relationship between UBL and tau changes, and warrants further exploration. Furthermore, the source of the fibers that comprise UBL immunoreactive dystrophic neurites, and the significance of these changes in the pathogenesis of neuritic plaques, is unknown. Further investigation is also warranted regarding the observation of UBL immunoreactive cells with the morphological appearance of microglia and oligodendrocytes in the hippocampus of two AD cases, especially when considering that one case had a family history of AD.

The patient did well until 18 months later, when she presented to

The patient did well until 18 months later, when she presented to the Emergency Department with erythema and drainage from a medial malleolar wound. She was again treated with oral cephalexin, and on follow-up, an aspirate was taken from the ankle joint with only bloody return and negative culture results (no growth). Radiographs showed only a possible subtle loosening RG7204 molecular weight of the tibial component of the prosthesis. Nonetheless, based on clinical suspicion, the patient was admitted for intravenous antibiotics and taken to surgery for explantation of the TAR components with the placement of a vancomycin/gentamicin spacer. Intraoperative

irrigation with methylene blue demonstrated a sinus track from the medial malleolar wound to the joint space. Intraoperative cultures were positive only for methicillin-resistant Staphylococcus SCH772984 nmr aureus (MRSA). Explanted specimens are the subject of this report. Tibial and talar components recovered during the implant removal surgery were placed aseptically in sterile specimen bags and placed directly on ice. Additionally, associated reactive

tissue was collected in sterile specimen containers and placed on ice. Two pieces of tissue for RT-PCR were deposited directly into RNase-free tubes containing RNALater® (Ambion) and stored at −20 °C. Postoperatively, the patient was maintained on intravenous vancomycin for 3 weeks, but was changed to daptomycin for a possible antibiotic-induced leucopenia. She subsequently

required re-exploration for persistent wound failure, with replacement of her Progesterone antibiotic-impregnated cement spacer and treatment with tigecycline. Thereafter, her wound ultimately healed and she is now ambulating as tolerated with the cement spacer in place. We used the Ibis T5000 Universal Biosensor System, which is a multiprimer PCR technique used to rapidly identify bacteria associated with clinical specimens (Ecker et al., 2008). The Ibis T5000 is for research use only (RUO) and is not yet approved for use in diagnostic procedures. First, we extracted DNA from the tissue: approximately 1 mm3 of tissue was transferred to a microcentrifuge tube containing lysis buffer (Qiagen) and 20 μg mL−1 proteinase K (Qiagen). The sample was incubated at 55 °C until visual inspection indicated that lysis was achieved. Zirconia/Silica Beads (0.45 g of 0.1 mm diameter, Biospec, PN: 11079101z) were added to the microcentrifuge tube and the sample was homogenized for 10 min at 25 Hz using a Qiagen Tissuelyser (Model MM300, cat# 85210). Nucleic acid from the lysed sample was extracted using the Qiagen DNeasy Tissue kit. Supernatants (200 μL) containing the extracted nucleic acid were removed and aliquoted into the wells of an Ibis Bacterial Surveillance microtiter plate (Abbott, cat# 03N33-01), which is used for broad identification of bacterial species.


281 ATYPICAL PRESENTATION OF ANTI-GLOMERULAR BASEMENT MEMBRANE DISEASE WITH CO-EXISTING IgA NEPHROPATHY A LECAMWASAM1, A SKENE2, D LEE1, L MCMAHON1 1Department of Renal Medicine, Eastern Health, Melbourne, Victoria; 2Department of Anatomical Pathology, Austin Health,

Melbourne, Victoria, Australia Background: We report a case of atypical presentation of anti-glomerular basement membrane (anti-GBM) find more disease co-existing with IgA nephropathy. Case Report: A 56-year-old Caucasian normotensive man presented with prodromal symptoms for a month. Kidney function deteriorated over 3 weeks with serum creatinine from 134 to 194 μmol/L, while it was normal 14 months prior. Urine microscopy revealed microscopic haematuria but no red cell casts, and spot urine protein-to-creatinine ratio was 0.057 mg/mmol. Anti-GBM antibody titre was 57 units/mL (<20), and anti-neutrophil cytoplasmic antibody was negative. Urgent treatment was commenced consisting of intravenous methylprednisolone, oral cyclophosphamide and plasmapheresis.

Renal biopsy showed 20% crescents. Immunohistochemical studies (IHC) were performed as there was inadequate renal cortex for immunofluorescence (IF) studies. IHC showed mesangial IgA deposits and weak IgG but no observable linear staining, favouring IgA nephropathy

with occasional crescents, and plasmapheresis was ceased. His kidney function worsened, and a second renal biopsy was performed 5 days later showing 41% crescents. Repeat IHC studies identified no IgG deposits and weak mesangial IgA staining. Interestingly, IF studies revealed patchy but linear IgG and mesangial IgA staining consistent with anti-GBM disease with mild IgA nephropathy. Plasmapheresis tuclazepam was reinstituted followed by undetectable circulating anti-GBM antibody, normalisation of kidney function, proteinuria and haematuria at 5 months follow-up. Conclusions: Our case reinforces the importance of strong clinical suspicion for atypical presentation of anti-GBM disease in the context of acute kidney injury and circulating anti-GBM antibody, as early initiation of treatment is paramount for favourable outcomes. Co-existing glomerulonephritis, prodromal symptoms and less rapid deterioration in kidney function are not uncommon. Linear IgG deposits may be more sensitive by IF compared to IHC.

The anti-inflammatory effect of both hBD3 and the mouse orthologu

The anti-inflammatory effect of both hBD3 and the mouse orthologue Defb14 19 was observed in mouse primary BM-derived Mϕ (BMDM), reducing the TNF-α response to LPS (Fig. 1E). hBD2 was not an effective suppressor of the CP-868596 datasheet TNF-α response to LPS in mouse cells (Fig. 1E), whereas hBD3 was more effective than LL37 in all mouse strains tested (Fig. 1F). hBD2 has only approximately 30% amino acid similarity to hBD3, which may explain lack of anti-inflammatory effects. Conversely, Defb14, which is 64% identical to hBD3 20, did demonstrate anti-inflammatory activity. The anti-endotoxic effects of LL37 have been shown to be partly due to direct binding of LL37 to LPS 16, 21. It has previously been shown

that hBD3 does not inhibit endotoxin binding in a Limulus assay 22 and we confirmed this finding (Supporting Information) to demonstrate similar endotoxin this website activity in the presence and absence of hBD3. However, the Limulus assay is not a direct measure of LPS-hBD3 binding; so we also investigated hBD3 effects after LPS stimulation of cells. Figure 2A shows that TNF-α levels were significantly reduced even when hBD3 was added to Mϕ 1 h after LPS. This suggests that even if hBD3 binds LPS to some

extent, most of the hBD3 inhibitory effect is occurring downstream of TLR4 activation by LPS. Further evidence that hBD3 is endowed with general anti-inflammatory properties is shown in Fig. 2B. Stimulation with IFN-γ and CD40L results in Mϕ activation and increased TNF-α, but here we show that hBD3 Cobimetinib mouse inhibited this pro-inflammatory cytokine response in mouse BMDM. This effect was also

evident in C3H/HeJ Mϕ, which lack functional TLR4, demonstrating that hBD3 is not simply inhibiting stimulation by endotoxin contamination. The anti-inflammatory effect was not evident when cells were exposed to PAM3CSK4 a TLR1/2 agonist (Fig. 2C). This suggests that hBD3 has an effect on signalling molecules that are used by TLR4 and CD40 but not TLR1/2. This differs from LL-37, which has been shown to inhibit pro-inflammatory responses via both TLR4 and TLR1/2. 16. As TLR4 and TLR1/2 signalling both involve MyD88 it is possible that hBD3 is affecting components of the non-MyD88 pathway (such as TRAM and TRIF) downstream of TLR4. Next, we wished to see whether hBD3 could reduce the accumulation of TNF-α in mice following exposure to LPS. We injected 16 mg/kg LPS into male Balb/c mice with and without 10 μg of hBD3 and measured serum TNF-α levels 1 h later. We found that the group injected with hBD3 and LPS had significantly reduced levels of TNF-α compared with mice receiving LPS alone (Fig. 2D). This result demonstrates that hBD3 inhibits LPS-stimulated TNF-α production in vivo as well as in vitro. The extent of inhibition afforded by hBD3 was comparable to that conferred by 1 μg IL-10, which protects mice from endotoxic shock 23, so hBD3 may provide similar protection. hBD3 is a promiscuous ligand which interacts with CCR6 and another unknown Mϕ receptor 14, 24.

Rather, the combined effects of PGE2 and other MSC-associated med

Rather, the combined effects of PGE2 and other MSC-associated mediators may be necessary to additionally regulate

the production of Th17-promoting factors by ancillary cell populations such as dendritic cells and monocyte/macrophages 7, 12. In conclusion, this study provides novel evidence that MSC-derived PGE2 is highly induced in Th17-MSC co-cultures and mediates a potent suppressive effect on primary and secondary Th17 induction via the EP4 receptor. We propose that further characterisation of the interactions between Th17 Fulvestrant cells and MSCs, including the nature of the contact-dependent signal responsible for COX-2 up-regulation, will identify Compound Library additional opportunities for manipulation of the Th17 differentiation program. Furthermore, suppression of IL-17A production by effector-memory Th17 cells derived from a site of “sterile inflammation” indicates the potential for MSCs to ameliorate tissue damage associated with maladaptive acute or chronic Th17 activation if delivered in the correct context. Eight- to 12-wk-old female C57BL/6 (B6) and BALB/c mice were purchased from Harlan Laboratories UK (Bicester, UK) and housed in a specific pathogen-free facility. All animal procedures were carried out under licence

from the Irish Department of Health and Children and approved by the NUI Galway Animal Care Research Ethics Committee. Mouse MSC cultures were carried out in supplemented Iscove’s modified Dulbecco’s medium (see Supplemental Methods for details of media and buffer compositions) (Sigma-Aldrich, St. Louis, USA). Th17 cell culture was carried out in supplemented Dulbecco’s modified Eagle medium. Reagents used included

a range of antibody preparations (see through Supplemental Methods), recombinant mouse TGF-β1 and IL-6 (Peprotech, Rocky Hill, NJ, USA), mouse CD3/CD28 T-cell expander beads (Dynabeads®, Invitrogen), Indomethacin and PGE2 (Sigma-Aldrich), and COX-2-selective inhibitor (NS-398), selective EP1 antagonist (SC-51322), selective EP2 antagonist (AH 6809), selective EP4 antagonist (L-161,982) and selective EP4 agonist (L-902,688) (all from Cayman Chemicals, Ann Arbor, MI, USA). Mouse MSCs were isolated from bone marrow according to the method described by Peister et al. 41. Tri-lineage differentiation capacity was determined using standard chondrogenic, adipogenic and osteogenic differentiation assays (Supplemental Fig. S1) 18. All experiments were carried out with passage 5–MSCs grown to 80% confluence in T175 tissue culture flasks (Nunc-Fisher Scientific) and detached with trypsin solution (Sigma-Aldrich). Renal cortical fibroblasts were prepared according Alvarez et al. 42.