Differential expression of HLA-DR was used to distinguish macrophages (CD16+DR+) and neutrophils (CD16+DR–) and the expression of galectins Selleck ATM/ATR inhibitor was studied in both subpopulations. A low level of eosinophil counts (< 3%) was observed in samples from both asmathic patients and healthy donors (see Table 2). As shown in Fig. 2a, gal-1 and gal-9 were expressed only on macrophages, while gal-3 expression was detected on both

macrophages and neutrophils. Differential gal expression by macrophages and neutrophils was also confirmed by immunofluorescence staining of sputum cell samples (Fig. 2b). Next, we compared galectin expression between asthma patients and healthy controls. Surface expression of gal-1 and gal-9 was clearly diminished in asthma patients compared with the control group (P < 0·05) (Fig. 3a,b), which is consistent with the Aloxistatin reported action of these proteins as negative regulators of the immune responses [22, 23]. Surface expression of gal-3 was highly variable, and although it tended to be lower in asthmatic patients, this difference did not reach statistical significance (Fig. 3b). Gal-1, gal-9 and especially gal-3 have been linked to allergic conditions. However, we did not find any difference in gal expression between atopic and non-atopic asthma patients, indicating that the lower expression of gal-1 and

gal-9 is independent of atopic status (Fig. 3c). In addition, no significant differences in galectin expression were observed when patients were classified according to the dose of inhaled corticosteroids (Supplementary Table S2). Next, we explored the role of gal-1, gal-3 and gal-9 in the cytokine production induced by LPS. PBMC were stimulated with LPS in the absence or presence of gal-1, gal-3 and gal-9 during 24 h. RT–PCR assays showed that gal-3 reduced the expression of IL-12A induced by LPS (Fig. 4a). When samples were matched it was observed that the reduction of IL-12A

levels occurred in four of five samples tested; however, statistical analysis did Astemizole not show any significant differences (Supplementary Fig. S2a). Gal-9 also caused a mild inhibition of IL-12B in four of five samples included (Fig. 4a and Supplementary Fig. S2b). In addition, we observed a slight increment of TNF-α expression in PBMC stimulated with LPS in the presence of gal-9. However, analysis of matched samples showed that this effect occurs in only three of five samples (Fig. 4a and Supplementary Fig. S2c). Regarding IL-1β, we did not detect any significant difference among treatments (Fig. 4a). Conversely, both gal-1 and gal-9 were able to increase the expression of LPS-induced IL-10 mRNA; in both cases the induction of IL-10 expression was observed in all samples tested (P = 0·01 and P = 0·03, respectively; Fig. 4b and Supplementary Fig. S2d).

Caspase-1

is present in the cytosol of phagocytic cells as an inactive zymogen 4, 5. Upon stimulation of phagocytic cells by pro-inflammatory signals, the procaspase-1 zymogen is activated by self-cleavage at aspartic residues to generate the enzymatically active homodimer of catalytic domains, consisting of a p20 and a p10 subunit 6, 7. Although it has long been recognized that microbial stimuli elicit the secretion of mature IL-1β, the cellular machinery mediating the activation of caspase-1 was only identified in 2002 when Tschopp and colleagues described the inflammasome, a multi-protein complex that induces robust processing c-Met inhibitor of proIL-1β 8. Here we discuss recent findings about caspase-1 activation with an emphasis on the regulation of the NLRC4 and NLRP3 inflammasomes by microbial stimuli. The NLR family is composed of more than 20 family members in mammals which share a tripartite structure consisting of a variable N-terminal domain, a centrally located nucleotide-binding oligomerization domain (NOD) and a C-terminal leucine-rich repeat for upstream sensing. While NOD1 and NOD2 activate NF-κB and MAPK in response to peptidoglycan fragments, Selleck HDAC inhibitor a class of NLR including NLRC4, NLRP1

and NLRP3 function as caspase-1 activators 9. These NLR contain N-terminal CARDs or PYRIN domains that mediate the assembly of the inflammasome through NOD-mediated oligomerization and interaction with caspase-1 via the adaptor ASC 6. Human NLRP1 senses bacterial muramyl dipeptide whereas mouse Nlrp1b recognizes lethal toxin, which is secreted by Bacillus anthracis6. Recently, the HIN-200 family member AIM2 has been shown to be a crucial molecule linking cytosolic double strand DNA to caspase-1 activation 10. AIM2 regulates the host response to vaccinia during viruses, but further work is needed to understand the role of AIM2 in microbial recognition 10. We discuss in more detail in the following two sections the NLRC4 and NLRP3 inflammasomes. Several Gram-negative bacteria, including Salmonella

enterica serovar Typhimurium, Legionella pneumophila, Pseudomonas aeruginosa and Shigella flexneri induce caspase-1 activation via the NLRC4 inflammasome 11–18. Although NLRC4 contains a CARD that presumably associates directly with that present in pro-caspase-1 19, the adaptor ASC is still required for caspase-1 activation and IL-1β secretion in response to bacterial infection 12, 20. The role of ASC in the NLRC4 inflammasome is still unclear, but it may promote the recruitment and/or dimerization of caspase-1 directly or through unknown factors. Several Gram-negative bacteria that activate the NLRC4 inflammasome require a functional type III secretion system or type IV secretion system to induce caspase-1 activation 6. These bacterial secretion systems form pores in host membranes to inject virulence factors into the host cell cytosol 6.

Higher dialysate calcium may alleviate potential haemodynamic instability yet also risks the development of positive calcium balance, hypercalcaemia and exacerbation of vascular calcification.14 Higher dialysate calcium may be warranted in patients

on long daily haemodialysis. As this form of dialysis is effective in removing more phosphate, the need for calcium-based phosphate binders is reduced, which may result in hypocalcaemia if the dialysate calcium concentration is not appropriately increased. Known pathophysiological effects of magnesium predict the importance of its concentration in dialysate. Magnesium plays a role in myocardial electrical www.selleckchem.com/products/Adrucil(Fluorouracil).html stability and vascular smooth muscle contraction and relaxation.19 Chronic hypermagnesaemia can lead to hypoparathyroidism,20 while the effect of hypomagnesaemia on PTH is controversial. Low

serum magnesium has been implicated in haemodialysis-associated headache.21 The use of magnesium as an inexpensive phosphate binder has necessitated lowering the dialysate magnesium concentration to avoid hypermagnesaemia. Kelber et al.22 showed that a magnesium-free dialysate introduced to maximize use of oral magnesium binders was associated with severe muscle cramps. In the same study, a low magnesium bath in combination with oral magnesium Venetoclax research buy carbonate alleviated these symptoms. Elsharkawy et al.23 found a significant correlation between intradialytic hypotension and a decrease in serum magnesium when using an acetate-based dialysate. Kyriazis et al.24 compared four Leukotriene-A4 hydrolase dialysates with different concentrations of calcium and magnesium and found that increasing

dialysate magnesium concentration could prevent or ameliorate the intradialytic hypotension associated with low calcium dialysate. Thus, low dialysate magnesium may allow the use of magnesium-based phosphate binders, but at the expense of greater intradialytic hypotension, and intolerance of dialysis (See Table 2). Bicarbonate is the principal buffer used in dialysate, with a standard concentration usually within the range of 33–38 mmol/L. Ideally, the dialysate bicarbonate concentration should be low enough to avoid significant post-dialytic alkalosis, yet high enough to prevent predialysis acidosis.25 Daily acid production varies greatly among patients. Inad equate control of acidosis results in protein degradation, insulin resistance, decreased sensitivity of parathyroid glands to calcium and osteomalacia. Conversely, metabolic alkalosis has been shown to decrease cerebral blood flow, impair dialytic phosphate removal and increase neuromuscular excitability leading to paraesthesias and cramps, and has been implicated in post-dialysis fatigue syndrome. Extreme values of plasma bicarbonate (<18 mmol/L or >24 mmol/L) are associated with increased mortality.

The cska-TCRs, in conjunction with surrounding adhesion molecules as LFA1 and CD2 [34, 35], and additional bundling proteins, maintain the specific polar orientation of cytoskeleton structures and a sustained T-cell–APC interaction. These are necessary for optimal cytokine synthesis and polar secretion toward the T-cell–APC interface, events critical for the activation of the T cells and the corresponding BAY 57-1293 APCs, as indicated by expression of CD25 and CD69 on both cell types. The presented model demonstrates the pivotal role of the cska-TCRs in resting T cells and in both early and late processes of T-cell activation. Moreover, our novel results fill the

missing gap that was puzzled by numerous studies, aiming at understanding the mechanism underlying IS formation and maintenance, by showing that the TCR is directly connected to the cytoskeleton and that the cska ζ “guide” the initial activation signal via the TCR toward a subsequent actin-dependent receptor cluster formation. Female BALB/c mice were bred in the Hebrew University SPF facility. ζ KO and transgenic ζ DISTAL and TAIL-LESS mice were kind gift of Dr. see more W.E. Shores from the NIH [13]. Splenocytes were isolated

from 6- to 12-week-old mice. 2B4 T-cell hybridoma and its ζ-deficient variant (MA5.8) expressing full length (FL) and truncated (CT-150 and CT-108) ζ were used. The Abs used are: A2B4 clonotypic Abs, anti-CD3ε, and anti-ζ, as previously described [8], anti-ZAP70 was a gift from L.E. Samelson (NIH), anti-CD3δ, anti-GST-LAT, Non-specific serine/threonine protein kinase and anti-GST were generated in rabbits, anti-Thy1.2 Abs (Serotek), anti-CD3ε, anti-CD28, anti-CD25, anti-CD69, and anti-IL-2 (BD Pharmingen), anti-CD16/32 and H57 (Biolegend),

anti-phosphotyrosine (4G10) (UBI), anti-actin, and anti-pLAT (Abcam), Streptavidin-Cy5 or-allophycocyanin (Jackson Immunoresearch). Polyclonal Abs, “b”, “c”, and “d”, directed against different epitopes within ζ, were generated in rabbits, and H-l46 anti-ζ (Ab “a”) Abs were generously provided by Ralph Kubo, USA. dscf and dicf were separated from tested cells and when indicated, proteins were immunoprecipitated. Samples were separated on 1D or 2D nonreducing/reducing SDS-PAGE and subjected to Western blot analysis. The above-mentioned procedures and those for biotinylation and activation of splenocytes were previously described [10]. Ezrin and IκB were used in all experiments as control proteins to verify efficacy of detergent-insoluble and -soluble fractionation, respectively, and the ratio between dscf and dicf proteins were determined by densitometry analysis. Site-directed mutagenesis of murine ζ was performed using Pfu DNA polymerase (Stratagene) according to the manufacture’s protocol. Double mutated (MUT) cDNA was sequenced and cloned into pcDNA3 (Invitrogen) for transfection or into pGEX6p2 to generate GST recombinant proteins.

2, and Supporting Information Fig. 4A). The majority of TAMs of either population expressed at least one of these two markers that suggest their macrophage commitment. However, we cannot exclude that some of the CD64−MERTK− cells, in particular those belonging to the MHCIIbrightCD11bhiF4/80lo TAM subtype, actually represent tumor-infiltrating DCs. The more mature CD64+MERTK+ cells predominated among CD11bloF4/80hi TAMs but

constituted only a minority of CD11bhiF4/80lo macrophages (Fig. 2) that might reflect a CD11bhiF4/80lo to CD11bloF4/80hi TAM differentiation process. Stat1 deficiency resulted in an expansion of the less differentiated CD64−MERTK− subpopulation on the expense of CD64-positive cells in the CD11bhiF4/80lo TAM subset (Fig. 2), whereas in the case of CD11bhiF4/80lo TAMs there was a shift from CD64-single positive cells to the double-positive subset (Fig. 2). This implies a differential role compound screening assay of STAT1 in macrophage differentiation

depending on the TAM subtype. To examine the contribution of monocytes to the TAM, pool mice were treated with BrdU and appearance of the label was investigated in circulating monocytes and TAMs [7, 11, 12]. In comparison with monocytes, where 75–95% of the population incorporated BrdU+ after 7-day labeling, only 30–40% Deforolimus in vivo of cells in both TAM subsets were BrdU+ (Supporting Information Fig. 5). This alludes to a limited contribution of recruited monocytes to the TAM populations. The frequency of BrdU+ cells was equal in Stat1+/+ and Stat1−/− TAMs. Hence, differences in monocyte recruitment cannot account for the higher abundance of CD11bloF4/80hi

cells in Stat1+/+ tumors (Supporting Information Fig. 5C). As another approach to investigate the impact of monocyte Methisazone influx on TAM accumulation, circulating monocytes were removed with liposomal clodronate given i.v. [16, 26] (Supporting Information Fig. 6). Interestingly, the percentages of the two TAM populations were not affected by monocyte depletion at both studied time points (7 and 11 days; Fig. 3A). We tested whether marrow-derived precursors contribute to TAMs in a longer time frame. For this purpose, we transferred whole CD45.2+ BM into unconditioned MMTVneu CD45.2− recipients bearing newly detected tumor lesions and assessed the presence of CD45.2+ cells among monocytes and tissue-resident macrophages 2 and 5 weeks thereafter [13]. The donor-origin cells were persistently present in blood and BM over a period of at least 5 weeks, constituting 4–8% of leukocytes (Supporting Information Fig. 7A and 8). For both TAM populations at the longest time point, chimerism was clearly detectable (Fig. 3B), signifying that TAMs relied on marrow hematopoiesis other than lung alveolar macrophages (CD11blo/−F4/80+) [11-13] that exhibited no contribution of donor-origin precursors (Supporting Information Fig. 7C and 9).

Experimental infection. Mycobacterium avium strain 2447 (smooth transparent variant kindly provided by Dr F. Portaels from the Institute of Tropical Medicine, Antwerp, Belgium) was grown in Middlebrook 7H9 medium containing 0.05% Tween 80 at 37 °C until mid-log phase of growth. Bacteria were harvested by centrifugation and resuspended in saline containing 0.05% Tween 80. The bacterial suspensions were briefly sonicated with a Branson

sonifier to disrupt bacterial clumps, diluted, and stored in aliquots at −70 °C until use. Intravenous infection with M. avium was performed through the tail lateral vein with 106 CFU per animal. At specific time-points (4, 8 and 20 weeks post infection), the organ bacterial load was determined as previously described [21]. Briefly, mice were anaesthetized and killed with isoflurane (Abbott, IL, USA). The organs were removed in aseptic conditions, homogenized, and serial dilutions were Neratinib price prepared in distilled sterile water with 0.05% Tween 80 and plated onto Middlebrook 7H10 agar medium. The numbers of CFU were

counted after 1 week of incubation at 37 °C. Flow cytometry.  Single cell suspensions were prepared from the spleen and the thymus of each mouse. Spleen erythrocytes were lysed with a haemolytic solution (155 mm NH4Cl, 10 mm KHCO3, Selleckchem Gefitinib pH 7.2). For each staining, 5 × 105 cells from each organ were incubated with a specific set of antibodies for 20 min at 4 °C. Cell surface markers were analysed using anti-CD25 APC or Pe (clone PC61), anti-CD11b PE (clone M1/70), anti-CD3 FITC, PE or APC (clone 145-2C11), anti-CD4 FITC or PECy5 (clone RM4-5), anti-CD62L FITC (clone MEL-14), anti-CD44 PE (clone IM7), anti-CD19 FITC (clone 6D5), anti-NK-1.1 FITC (clone PK136), anti-CD8 FITC, APC or APCCy7 (clone 53-6.7) and anti-Ly-6G/Ly-6C PECy5 (Gr-1;

clone RB6-8C5) (all from Biolegend, San Diego, CA, USA). Cells were RANTES fixed with 2% formaldehyde after staining. The analysis of the cell populations was based on the acquisition of 30,000 events using CellQuest software on a FACscalibur flow cytometer or a FACSAria cell sorter (Becton Dickinson, NJ, USA). Data analysis was performed using FlowJo software (Tree Star, Inc, Ashland, OR, USA). Detection of IFN-γ in serum samples.  Mice were anaesthetized with isoflurane (Abbott, IL, USA), and retro-orbital bleeding was performed before killing. Blood was allowed to clot and serum was collected after centrifugation and frozen at −80 °C until use. Quantification of IFN-γ was done by a two-side sandwich ELISA using anti-IFN-γ-specific affinity-purified mAbs (R4-6A2 as capture and biotinylated AN-18 as detecting mAbs), and the standard curves were generated with known amounts of IFN-γ (Peprotech, Rocky Hill, NJ, USA). The sensitivity of the assay was 20 pg/ml. Statistical analysis.  All data are presented as means + SD.

e. indwelling lines, port-a-cath and sustained/severe thrombopenia). Biofilms on catheters may be a source of persistent candidaemia. Patients needing their line devices therefore should receive agents capable of acting against biofilm-associated cells. Of note, echinocandin antifungals and amphotericin B lipid formulations have demonstrated high https://www.selleckchem.com/screening/mapk-library.html antifungal activity in fungal

biofilms.74,75 In a recent in vitro investigation, the MIC90 of anidulafungin against a series of 30 C. albicans isolates was even lower in biofilms than in planktonic cultures and caspofungin MIC90 increased by only two dilution steps, whereas an azole antifungal was virtually inactive against sessile Candida, as expected.74 In patients with persistently Candida-positive blood culture, several potential causes for failure of pathogen eradication must be considered. This primarily includes inadequate choice or dosage of antifungal therapy (e.g. fluconazole 400 mg day−1 in patients with C. glabrata infection).76 Note that fluconazole has been found to be associated with elevated rates of persistent candidaemia in the comparator arms of several randomised comparative

trials (see below). Echinocandins consistently had persistence rates of 10% or lower. Sources of Selleckchem GS 1101 persistent candidaemia include dissemination from foci of fungal infection (e.g. from endocarditis vegetations, septic thrombosis or intra-abdominal abscess), and inadequate catheter handling. Central venous catheters should be removed or replaced whenever possible. The new catheter must be placed by a new venous puncture site rather than via

a guidewire inserted into the pre-existing one, potentially colonised catheter. Given the high incidence and poor prognosis of invasive Candida infections in severely ill ICU patients, antifungal prophylaxis appears as an attractive option in selected patient sets. In a meta-analysis of published trials, Vardakas et al. [77] Amine dehydrogenase came to the conclusion that prophylactic use of azoles in high-risk surgical ICU patients is associated with a reduction of fungal infections but not in crude mortality. Neither was an overall survival benefit observed in other meta-analyses and the underlying original studies.78,79 The risk groups treated in the analysed trials included patients with bacterial septic shock, abdominal surgery or gastrointestinal tract leakage, fungal colonisation before enrolment, diabetes, solid tumours, presence of central and peripheral venous catheters for more than 3 days, exposure to antibiotics, and intubation or mechanical ventilation. In a well-performed randomised double-blind trial with gastrointestinal perforation or anastomosis leakage as a clearly defined risk factor, Eggimann et al. [9] observed a significant reduction of Candida peritonitis in patients (n = 43) receiving fluconazole (4%) vs. placebo (35%).

“
“Aspergillus is a saprophytic fungus, which mainly becomes pathogenic in immunosuppressed hosts. A failure of click here host defences results in a diverse set of illnesses, ranging from chronic colonisation, aspergilloma, invasive disease and hypersensitivity. A key concept in immune responses to Aspergillus species is that host susceptibility determines the morphological form,

antigenic structure and physical location of the fungus. Traditionally, innate immunity has been considered as a first line of defence and activates adaptive immune mechanisms by the provision of specific signals; innate and adaptive immune responses are intimately linked. The T-helper cell (TH1) response is associated with increased production of inflammatory cytokines IFN-γ, IL-2 and IL-12 and stimulation of antifungal effector cells. Alternatively, TH2-type responses Y-27632 ic50 are associated with suppression of antifungal effector cell activity, decreased production of IFN-γ and increased concentrations of IL-4 and IL-10, which promote humoral responses to Aspergillus. The host’s defensive capacity is defined by the sum of resistance and tolerance. Resistance displays the ability to limit fungal burden and elimination of the pathogen, and tolerance means the ability to limit host damage

caused by immune response. “
“For anthropophilic tinea capitis (TC), household spread and asymptomatic scalp carriage (ASC) is considered an important route of transmission and incomplete clearance. To investigate ASC in household contacts of patients diagnosed with TC in

a tertiary hospital in Athens, Greece, we retrospectively reviewed the medical files of household contacts that were screened for ASC from 1997 to 2011. Only 34 household contacts of 15 index cases agreed to come for screening. Thirty-three (97%) household contacts were asymptomatic scalp carriers. The most commonly isolated species was Trichophyton violaceum (59%). There was a statistically significant association of ASC with the isolated dermatophyte species (T. violaceum, P-value: 0.029), and with the age of younger than Cyclic nucleotide phosphodiesterase 16 years old (P-value: 0.005), while there was no association with gender (P-value: 0.672). A small number of household contacts accepted to proceed for screening. ASC was found in nearly all screened household contacts and was associated with T. violaceum and younger age. The low number of household contacts that accepted screening may reflect the ignorance of the general population about the possibility of ASC among household contacts in case of a patient with TC. “
“Biofilm formation is one of the most important attributes for virulence in Candida species and contributes to increased resistance to antifungal drugs and host immune mechanisms.

[40] This may reflect model-dependent differences in inflammatory pathophysiology. An alternative explanation is that although cysts are macroscopically small at week 3, other pathophysiological processes (such as cell proliferation) may already be established at this stage, and stimulate macrophage infiltration. The phenotypes of macrophages in PKD may also provide a clue to the role of inflammation. Karihaloo et al. investigated macrophages in two murine models of ADPKD, the Pkd1fl/fl;Pkhd1-Cre and Pkd2WS25/− mouse.[19] In both mouse strains there were increased numbers of F4/80-positive

macrophages compared with disease controls. In Pkd1fl/fl;Pkhd1-Cre mice, Ly6Clow cells comprised the predominant population of macrophages,[19] a phenotype characteristic of alternatively activated macrophages.[12] Clodronate-induced depletion of macrophages SB203580 purchase in Pkd1fl/fl;Pkhd1-Cre mice resulted in a decrease in circulating monocyte numbers, Ki67-positive cell proliferation, cystic R788 index and blood urea nitrogen (BUN) compared with vehicle-treated controls.[19] These findings suggest that macrophage depletion delays disease progression, which seems inconsistent with the authors’ previous observation of a predominantly Ly6Clow macrophage population which should theoretically

have restorative roles in disease. The proportions of Ly6Clow and Ly6Chigh macrophages were not significantly changed following clodronate treatment, indicating that the improved disease outcomes were not due to selective depletion of one

macrophage subtype.[19] Tyrosine-protein kinase BLK This implies that in PKD, alternatively activated macrophages may have detrimental rather than restorative roles. Indeed, alternatively activated macrophages can induce cell proliferation.[41] Although cell proliferation facilitates the repair of damaged renal parenchyma in other types of renal injury such as IRI,[41] proliferation, particularly of the CEC, promotes cyst expansion in PKD.[7] Since Karihaloo et al. observed a concomitant decrease in cell proliferation with macrophage depletion,[19] it is plausible that inflammatory cells such as macrophages exacerbate cyst growth in this disease. Although macrophages are the most well-studied infiltrating cell type in PKD, other cells have also been observed (see Table 3). CD45-positive lymphocytes[11] and CD4-positive lymphocytes[10] have been identified in the renal interstitium of ADPKD patients. Lymphocytes were also reported in kidneys of kat2J/kat2J mice,[30] DBA/2FG-pcy mice,[26] and Han:SPRD rats,[36] although lymphocyte-specific markers were not employed in any of these studies. In other inflammatory renal states such as IRI, lymphocytes produce chemokines (e.g. TNF-α and interferon-γ),[71] and may therefore instigate similar inflammatory effects in PKD. McPherson et al. identified interstitial mast cells surrounded by chymase in ADPKD kidney tissue.

TGR5 is expressed in several tissues, with the highest levels detected in the gall bladder, followed by the ileum and colon. TGR5 expression is not detectable in primary hepatocytes.8,19 In contrast, FXR is highly expressed in the liver, intestine, kidney and adrenal glands.8–10,13,24–27 FXR expression in immune cells, such as CD14+ monocytes, has also been reported, but its expression in these cells is relatively low compared with the expression of other nuclear receptors such as LXRα (Liver X Receptor alpha).3

In addition, MK-2206 manufacturer we could not detect expression of BA transporter mRNA in monocytes. These findings are consistent with our demonstration that the FXR agonist did not influence DC differentiation in our experiments. In the present study,

we found expression of TGR5 on CD14+ peripheral blood monocytes. Furthermore, the presence of the TGR5-specific agonist promoted the differentiation of IL-12 hypo-producing DC in a similar manner to that seen in the presence of BA. Taken together, these results suggest that BAs can regulate the DC differentiation process through TGR5 expressed on primary peripheral blood monocytes. Expression of TGR5 was rapidly down-regulated during DC differentiation from monocytes, and differentiated DCs did not express detectable levels of cell surface TGR5. Although the mechanisms of TGR5 gene transcription regulation have not been identified, our study of mRNA transcription revealed that the Rucaparib supplier amount of TGR5 mRNA transcript was dramatically reduced following GM-CSF and IL-4 stimulation. In addition, it has been reported that ligand stimulation causes ZD1839 datasheet cellular internalization of TGR5.8 These findings suggest that the binding of the BA to TGR5 on monocytes at the initial phase of differentiation is crucial if differentiation outcomes are to be influenced by the BA. Activation of TGR5 leads

to intracellular cAMP accumulation, which activates CREB.8,18 The CREB then transactivates target genes by binding to the cAMP response element in the promoter region of these genes.8,20,22,23 In our studies, stimulation of monocytes by BA or a TGR5-specific agonist led to up-regulated intracellular cAMP concentrations. It has been reported that intracellular cAMP concentration is an important modulator of pro-inflammatory cytokine transcription.28 Consistent with these observations, treatment of monocytes with cAMP also promoted cellular differentiation into IL-12 hypo-producing DC. The cAMP promotes the differentiation of CD14+ monocytes into CD1alow CD209+ DCs.29 We observed BA-DCs and TGR5-DCs, but not cAMP-DCs, expressing low levels of CD1a (Fig. 1), although all three DC types displayed a similarly low capacity to produce IL-12. Interestingly, FXR-DCs also showed a CD1a-positive DC phenotype, but FXR-DCs did not display an IL-12 hypo-producing phenotype.