There was a significant main effect of the factor Object, F(1, 53

There was a significant main effect of the factor Object, F(1, 53) = 13.551, p = .001, η² = 0.203. The previously uncued objects were fixated significantly longer (mean of 0.414, standard error of 0.015) compared with the previously cued objects (mean of 0.328, standard error of 0.013). No effects were found for Cue Condition and Location. No interaction effects were found,

indicating that head and eye gaze cues yielded similar effects. The same infants that were tested in the eye-tracking experiment were also tested in a subsequent ERP experiment. The final sample Selleckchem R788 consisted of 46 infants (26 females) with an average age of 4 months and 16 days (age range: 4 months and 0–29 days; 23 infants for the eye gaze condition, 23 infants for the head condition). Forty-seven infants were excluded because of technical problems (N = 4), fussiness (N = 11) or poor data quality due to movement artifacts, and/or high impedances (N = 28). In the eye gaze condition, infants were presented with the same footage

PD0325901 of the person as in the eye-tracking experiment. However, only one object was presented next to the head; therefore, the object was either cued or uncued by the person’s eye gaze. The person looked straight ahead for 1000 ms, then shifted gaze to the side (1000 ms). The last frame was held for 1000 ms. After a brief blank screen period (400–600 ms, on average 500 ms), only the object was presented again at the center of the screen (test phase, 1000 ms; see Figure 1 for an example of a trial). Different objects (N = 80) were used than in the eye-tracking experiment, but the same stimulus descriptions apply. ERPs for the previously cued objects are based on averaging 10–29 Atazanavir trials (mean of 16 trials), for the previously uncued object on 10–28 trials (mean of 16 trials). The same procedure was used in the head condition. As in the eye-tracking experiment, the person turned her head toward one of the objects while constantly keeping her eyes gazing toward the infant. ERPs are based on 10–30 trials (mean of 16 trials)

for the previously cued objects and on 10–27 trials (mean of 16 trials) for the previously uncued objects. A total of 160 trials were presented on a computer screen using the software Presentation (Neurobehavioural Systems, Inc., Albany, CA). EEG was recorded at 32 channels with a sample rate of 250 Hz using a BrainAmp amplifier (Brain Products GmbH, Munich, Germany). Signals were rereferenced to the linked mastoids, and a bandpass filter of 0.3–30 Hz was applied offline. Electrooculogram (EOG) was recorded bipolarly. ERPs were time-locked to the test phase and were assessed based on 1700 ms long EEG segments (including a 200 ms prestimulus baseline). Automatic artifact detection methods were applied using ERPLAB Software (http://erpinfo.org).

The percent time each mouse spent in the central and peripheral z

The percent time each mouse spent in the central and peripheral zones of the arena was quantified by an EthoVision automated tracking system (Noldus Information Technology, Wageningen, The Netherlands) and an anxiety index was calculated by dividing the time spent in peri-pheral zones by the time spent in the central zone. The arena was cleaned with 70% ethanol and thoroughly dried between sessions. Mice were individually placed in a Plus Maze apparatus elevated 40 cm above the ground. This apparatus consisted

of four arms (each 35 cm long and 5 cm wide), two of Abiraterone which enclosed by 15 cm high walls (“closed arms”) and two without walls (“open arms”). A mouse was allowed to freely explore for 5 min, during which the total number of entries into the open and closed arms, as well as the time spent in each arm, was recorded by the experimenter. An anxiety index ranging from 0 (low anxiety) to 100 (high anxiety) was calculated based on the following formula: Individual body weight was measured weekly throughout the experimental period. Individual spleen weight was measured following the 24-day experimental period and immediately after killing the mouse. To avoid stressing mice in the nonstressed group, CORT Epigenetics inhibitor levels were determined in urine (rather than by

drawing blood) by gently massaging the urinary bladder to induce urination. Urine was collected daily at 9:00 a.m. and prior to applying the stressor. For mice in which EAE was induced, urine was also collected during the development

of the disease. To determine the fraction of free CORT in urine and blood of male and female C57BL/6 mice, samples were centrifuged in centrifree micropartition tubes (Ultracel YM-T cellulose membrane with a 30,000 MW cut-off) purchased from Millipore (Co. Cork, IRL). CORT levels were determined by CORT ELISA kit (Endocrine Technologies Inc, CA) according to manufacturer’s instructions. For peripheral Farnesyltransferase blood analysis, 50 μL of fresh blood were drawn into heparinized tubes and incubated with 100 μL of ACK lysis buffer at 37°C for 10 min to eliminate red blood cells. For splenocyte analysis, spleens were removed, weighed and dissociated in DMEM medium containing 10% fetal calf serum, 10 mM HEPES, 1 mM sodium pyruvate, 10 mM nonessential amino acids, 1% Pen/Strep, and 50 μM β-mercaptoethanol. ACK lysis buffer was added for 1 min to eliminate red blood cells. Viable mononuclear cells were counted in a haemocytometer using trypan blue and adjusted to 5 × 105 cells/mL in medium containing PBS supplemented with 2% fetal bovine serum. Cell surface staining was performed was performed using anti-CD4 (FITC or PERCP), anti-CD25 (PE), and anti-CD127 (allophycocyanin) antibodies, all purchased from BioLegend (San Diego, CA). To detect intracellular FoxP3 we used anti-FoxP3 (FITC or allophycocyanin) antibodies according to manufacturer’s instructions (BioLegend) or used transgenic mice expressing enhanced green florescent protein under the control of the mouse FoxP3 promoter.

The

The FK506 datasheet present data reveal a possible role that IL-9+IL-10+ T cells may attract Mϕ to the local tissue and the latter contribute further to inflammation. The data support the hypothesis; a portion of Mo is F4/80+ Mϕ. Our results are in line with other investigations reported previously that also observed that the levels of MIP1, together with other proinflammatory cytokines, were elevated in patients with chronic allergic asthma [15], chronic atopic dermatitis [16] or animal studies [17]. The present results reveal that in allergic reactions, a portion of IL-9+IL-10+ T cells extravasate into

local tissue such as the intestine. As MIP1 plays an important role in inflammation, the source of MIP1 is of significance to be understood. Our results indicate that, upon antigen-induced TCR activation, IL-9+IL-10+ T cells produce MIP1 that has the capacity to attract Mϕ; the latter may be responsible for further

pathological changes in local tissue. It is well documented that Mos extravasate in allergic hypersensitivity reactions [18,19]. The present data are in line with these published data by revealing abundant Mos in the intestine PCI-32765 clinical trial after antigen challenge, as shown by flow cytometry and histology studies. Furthermore, we have shown that these Mos express high levels of MIP2γ, indicating that they have the capacity to attract neutrophils to local tissue. Meanwhile, we also observed an increase in neutrophils in the intestine during LPR. A link between the extravasation of Mos and neutrophils has been noted in the present study. Thus, we may envisage a scenario that TCR activation induces IL-9+IL-10+ T cells to express MIP1; MIP1 attracts Mϕ to local tissue; Mϕ-derived MIP2γ attracts neutrophils to extravasate in the intestine to release proinflammatory molecules, such as MPO (Fig. 3),

that may damage intestinal tissue and induce inflammation, as shown by the present study as well as by other investigators [9]. Allergic hypersensitivity Epothilone B (EPO906, Patupilone) plays an important role in the induction of pathological changes in chronic allergic inflammation [20]. A skewed cellular response is proposed to play a major role in the inflammatory process [21]. These results are in line with previous reports [19,21] showing that the cellular elements in local tissue (the intestine) include eosinophils, mast cells, Mos and neutrophils. The data demonstrate that the extravasation of eosinophils and mast cells occurs mainly in early allergic responses; the frequency of these cells declines gradually after antigen challenge. At 48 h after antigen challenge, neutrophil becomes the major inflammatory cellular element together with a portion of Mo; the latter has been reduced markedly compared to cell counts at 2 h after antigen challenge. Neutrophil contains several enzymes, such as MPO, that essentially function to fight against invaded microbes as well as to damage local tissue and to cause inflammation such as inflammatory bowel disease.

abscessus (4–6) One of them, M abscessus Group II strains, was

abscessus (4–6). One of them, M. abscessus Group II strains, was reported as M. massiliense and M. bolletii (7). As a genetic identification method to differentiate M. massiliense from M. abscessus and other species recently became available, human infections caused by M. massiliense have been continuously

reported (8–12). Nearly half of the RGM isolates initially identified as M. abscessus, which is the species of RGM that is most frequently Selleckchem Napabucasin isolated in Korea, are actually M. massiliense (7). So far, differentiation between M. abscessus and M. massiliense depended on sequence analysis of housekeeping genes (e.g. rpoB and hsp65) (7, 9). However, additional housekeeping genes were analyzed because of the discordant results between rpoB and hsp65 gene analysis (7, 13). Clarithromycin is a 14-membered ring macrolide that binds

to the large ribosomal subunit in the vicinity of the peptidyltransferase center and inhibits protein synthesis, which results in the arrest of bacterial growth (14). Clarithromycin is given orally, and is highly active against many species of NTM. Although M. massiliense shares many traits with M. abscessus and M. bolletii, M. massiliense can be differentiated by marked susceptibility to clarithromycin (2, 7, 11). Moreover, patterns of clarithromycin resistance differed between M. massiliense and M. abscessus (7), which led us to investigate another mechanism, involvement of erm. This is because the erm gene is frequently involved in macrolide resistance in human pathogens as with the 23 rRNA gene mutation. GSK1120212 research buy The erm gene encodes N6-mono or N6, N6-dimethyltransferases that cause specific methylation of nucleotide A2058 and/or neighboring nucleotides (A2057 and A2059; based on Escherichia coli numbering) in the 23S rRNA, which Sitaxentan results in resistance to macrolide. Because Mycobacterium species possess only one or two rrn operons, alteration of this specific site is critical to the development of resistance (25). Among the 33 erm genes that have

been reported and numbered to date, five innate erm genes [erm(37), erm(38), erm(39), erm(40) and erm(41)] have been identified within the genus Mycobacterium (15). Recently, three types of erm(41) of M. abscessus were reported. One M. massiliense clinical isolate was confirmed to have short erm(41) by PCR and was reported as one of the three erm(41) types without sequence analysis (16). Because quite different responses of M. massiliense compared to M. abscessus against clarithromycin were observed in our previous report (7), exact information on erm(41) of more clinical M. massiliense isolates, and their relevance to the susceptibility pattern of clarithromycin was needed. In the present study, the erm(41) sequences of M. massiliense, M. abscessus and M. bolletii isolates were investigated in relation with MIC to clarithromycin, and a simple erm(41) PCR to differentiate M. massiliense from closely related M. abscessus and M.

High IL-22 expression in skin lesions and serum levels of patient

High IL-22 expression in skin lesions and serum levels of patients with active psoriasis suggests deleterious effects of this cytokine on tissue inflammation 22, 23. Indeed, recent biologic therapies for psoriatic patients include anti-IL-23 treatment, a cytokine directly involved in the expansion of IL-17- and IL-22-secreting CD4+ T www.selleckchem.com/products/chir-99021-ct99021-hcl.html cells 24, 25. In contrast, although IL-22 transcripts are also elevated in inflamed lesions of patients with Crohn’s disease 26, studies using mouse models of ulcerative colitis show that IL-22, produced by CD4+ T cells and a subset of NK cells, had a protective

effect 27. Altogether, it is at present uncertain whether IL-22 exerts predominantly regulatory or pro-inflammatory effects. The present study was undertaken in an attempt to clarify the phenotypic and functional plasticity of putative inflammation-inducing human CD4+ T-cell subsets. Our goal was also to investigate the potential ontogenic relationships between these subsets, and other T-cell subsets, including induced Tregs. Our results argue for the existence Fulvestrant concentration of a highly polyfunctional IL-22-producing T-cell population, distinct from IL-17 “only”-producing T cells. Despite

the pronounced functional differences, we found extensive TCRαβ sharing across all the effector and regulatory subsets defined. Our data therefore underscore the fact that one T-cell precursor is able to adopt multiple Th-subset profiles irrespective of antigen specificity. To explore phenotypic and functional differences

between IL-17A+IL-22+, IL-17A+IL-22− and IL-17A−IL-22+ CD4+ T cells, Aprepitant co-expression of IFN-γ, TNF-α, IL-2, CD161 and CCR6 was analyzed on circulating CD4+ T cells using multiparametric flow cytometry (Fig. 1A and Supporting Information Fig. S1A). Circulating cytokine-secreting cells were present at similar proportions and absolute numbers in psoriasis patients and in controls (Supporting Information Fig. S1B). Also, the three combinations of IL-17A- and IL-22-secreting CD4+ T cells were present with similar frequencies and absolute numbers in controls and psoriasis patients, although IL-17A+IL-22+ CD4+ T cells were moderately, albeit non-significantly, increased in the latter (Fig. 1B). The killer cell lectin-like receptor CD161 was recently reported to be preferentially expressed on Th17 precursor cells as well as on gut 10 and skin 28 homing Th17 cells, but the CD161 status of ex vivo IL-22-secretors is not known. CD161 expression (Supporting Information Fig. S2A) was found to be more pronounced on IL-17A-secreting CD4+ T cells, as compared with cells producing IL-22 (p=0.0086 and p=0.0102 in healthy controls and psoriasis patients respectively) (Supporting Information Fig. S2B). Of note, CD161 expression is retained on IL-17A+IL-22+ cells (Supporting Information Fig. S2C).

Mice were on C57BL/6J genetic background (at least 10 back-crosse

Mice were on C57BL/6J genetic background (at least 10 back-crosses) and WT C57BL/6J mice were used as control. For experiments, 7/11-week-old mice were kept in filtered-cages in a P2 animal facility. All animal experimental protocols complied with the French ethical and animal experiments regulations and were approved by the Ethics Committee for Animal Experimentation of CNRS Campus Orleans (N° CLE CCO 2011–028 to V.Q., UMR7355). Plasmodium berghei ANKA (PbA) 15cy1 line constitutively expressing GFP under EF1α-promoter control, was obtained from Dr. A. Waters [23]. Mice were infected intraperitoneally with GSK1120212 in vitro 105 parasitized erythrocytes

as described [41]. Alternatively, mice were infected intravenously with 1000 motile sporozoites obtained from salivary gland homogenate of day-21-PbA-infected females Anopheles stephensi. Mice were observed daily and scored for ECM neurological signs, namely ataxia, paralysis, and coma. Parasitaemia was assessed with EGFP-PbA as described [41] and fluorescent cells analyzed by BD CANTO II flow cytometer. Data were acquired by using DIVA software (BD Bioscience, Rungis, France) and analyzed with FlowJO software (Treestar, Ashland, USA). Blood was drawn under Isofluorane anesthesia (CSP, Fontenay-sous-Bois,

France) into tubes containing Ethylenediaminetetraacetic BGJ398 in vivo (EDTA), (Vacutainer; Becton, Grenoble, France) and hematological parameters determined using 5-part-differential-hematology Uroporphyrinogen III synthase analyzer MS9.5 (Melet Schloesing Laboratoires, France). Histological analysis was performed as described [41]. Briefly, mice were euthanized and perfused with intracardiac PBS/2 mM-EDTA. Organs were fixed in PBS/3.6%-formaldehyde for 72

h. Brain and lung microvascular obstruction was quantified on H&E stained sections, using a semiquantitative score with increasing severity of changes (0–5) by two independent observers, including a trained pathologist (B.R.). MRI and MRA measurements of cerebral vascular blood flow were performed using a horizontal 7 T/16 Bruker Biospec MR system (Bruker Biospin, Wissembourg, France), as described [8]. A homogeneous coil with inner diameter of 23 mm and length 55 mm was used to achieve uniform excitation and reception. A custom-built stereotaxic head holder was used to fix the animal into the birdcage coil (see below). The mice were anaesthetized with isoflurane (1.5%) and O2 (0.5 L/min) applied with a face mask allowing free breathing. Respiration was monitored using a balloon taped to the abdomen and connected to a pressure transducer (SA Instruments, Inc., Stony Brook, NY, USA). Body temperature was kept at 37 ± 0.5°C throughout the experiment, using warm water circulation. Brain lesions and global changes in tissue structure were accessed by T2 weighted (T2w) MR images using a MSME sequence, in both axial and sagittal planes, with the following parameters: RARE factor = 8, TR/TEeff.

How CD23 on B cells modulates active systemic anaphylaxis needs f

How CD23 on B cells modulates active systemic anaphylaxis needs further Autophagy activator studies. A direct effect of CD23 on effector cells or a proposed negative regulatory function of CD23 on B cells could be involved [23, 31]. Because the CD23−/− IgE knock-in mice displayed increased anaphylaxis, we reasoned that they would be better targets to test a potential protective effect of basophil depletion on active anaphylaxis. Therefore, we treated sensitized mice with Ba103 Ab (anti-CD200R3), which depletes basophils, but not mast cells [32] to examine the effect on IgE or IgG1 dominated anaphylaxis.

In WT, heterozygous and homozygous IgEki mice 65, 80, and 85% of basophils (CD49b+-IgE+) in peripheral blood were depleted, respectively (Supporting Information Fig. 2). The depletion of basophils resulted in reduction of body temperature drop in all three genotypes. This effect was most prominent in IgE knock-in mice in the late phase of anaphylaxis, between 60–90 min. At the endpoint after 90 min the body temperature was 3–4°C lower in untreated mice as compared with that of basophil-depleted mice. In line with this observation, the mortality rate dropped to zero in treated mice. However, at the peak of anaphylaxis around 30–40 min past challenge, basophil-depleted IgE knock-in mice also reacted with substantial anaphylaxis,

although they recovered faster than the untreated mice (Fig. 4B and C, panels 3 and 4). Due to genetic differences between BALB/c mice (where the knock-in was made) and C57BL/6 mice (used for backcrosses) the IgEki/ki mice express the IgG2a isotype, whereas the WT littermates express IgG2c [33]. This feature of the genetic see more manipulation is not due to insufficient backcrossing, but results from the close linkage of the IgG isotypes in the immunoglobulin locus. A contribution of differentially expressed antigen-specific IgG2a versus IgG2c (Fig. 3B and C) to the anaphylaxis phenotype, MTMR9 or the moderately increased IgG2b in CD23-competent IgE knock-in (Fig. 3B) is unlikely, because in mice immunized with alum as adjuvant, specific IgG1 is the dominating IgG isotype, resulting in reduced antigen-specific IgG in the IgE knock-in mice

[34]. In summary, IgE-sensitized basophils are most likely responsible for the severe body temperature drop in the late phase of anaphylaxis and contribute to death due to anaphylaxis. However, in the early phase of anaphylaxis, sensitized mast cells do have an important contribution in IgE-dominated systemic anaphylaxis. This is supported by the detection of significantly increased mouse mast cell protease 1 (Mmcp1) in IgEwt/ki mice, but not in IgEki/ki mice (Fig. 4D). Mmcp1 has been identified as a marker that distinguishes IgE- from IgG-mediated anaphylaxis [7]. As basophils do not express this protease, whereas mast cells do – albeit weakly – [35], this suggests that mast-cell degranulation via IgE may partially contribute to the anaphylaxis phenotype.

These data point to IL-2 signaling as another target for zinc in

These data point to IL-2 signaling as another target for zinc in T cells, in addition to TCR signaling. Upon stimulation, the IL-2-receptor (IL-2R) activates signal transduction pathways, including STAT5 and ERK1/2. The β- and γ-chains of the IL-2R are associated with

JAK1 and 3, which transphosphorylate each other and subsequently the β receptor-chain at the key positions Tyr338, Tyr393, and Tyr51010. Phosphorylation of these tyrosines forms binding sites for the SH2-domain of STAT5, which becomes activated by JAK via phosphorylation selleck chemical of Tyr694, leading to dimerization to a transcription factor that promotes transcription of genes such as c-myc, bcl-2, CD25, and bcl-x 11. Additional feedback regulation of this pathway occurs via SOCS and cytokine

Dorsomorphin ic50 inducible SH2-containing protein (CIS) 12. MAPK transduce extracellular signals from hormones, growth factors, cytokines, and environmental stress, thereby regulating a variety of cellular responses including cell proliferation, migration, differentiation, and apoptosis 13. Phosphorylation of Tyr338 of the IL-2R β-chain leads to the assembly of adaptor proteins SHC, Grb2, and SOS1. This triggers a MAPK-cascade consisting of the dual-specific kinases RAF and MEK, which activates ERK via phosphorylation of conserved tyrosine and threonine residues in its catalytic domain 10. Upon activation, ERK phosphorylates several other kinases and activates Vasopressin Receptor transcription factors, such as c-fos, c-jun, elk-1, and c-myc 14. Negative regulation of the ERK pathway is mediated by various phosphatases, including several dual specificity Thr/Tyr protein phosphatases (DUSP) and protein phosphatase 2A (PP2A) 13. Here, we demonstrate that IL-2 induces a zinc signal, i.e. a

translocation of zinc ions from lysosomes into the cytosol. This signal is required for inhibition of ERK dephosphorylation and IL-2-induced T-cell proliferation, but has no effect on STAT5 signaling. Upon staining of the murine cytotoxic T-cell line CTLL-2 with the zinc-selective fluorescent probes Zinquin and FluoZin-3, we found differential intracellular localization of the probes (Fig. 1A). Zinquin showed a relatively uniform staining throughout the entire cell, whereas FluoZin-3 exclusively labeled vesicular structures. These so-called zincosomes sequester high amounts of zinc 15. After stimulation with IL-2, intracellular translocation of zinc occurred (Fig. 1B and C; Supporting Information Fig. 1A). Vesicular FluoZin-3 fluorescence decreased in response to IL-2. In contrast, an increase of the cytoplasmic zinc-dependent fluorescence was measured with Zinquin. There were no major differences in the intracellular localization of the fluorescence before and after treatment with IL-2, indicating that IL-2 affects the intensity of zinc staining in the different compartments, rather then the distribution of the fluorescent dyes (Supporting Information Fig. 2).

More importantly, T-cell-specific genes encoding proteins such as

More importantly, T-cell-specific genes encoding proteins such as CD3 and CD4 were absent from the FDC data sets. The comparison with the gene expression profiles

of macrophages showed an overlap in 167/575 genes. Again, the expression of genes diagnostic for macrophages such as Cd11b, Cd68 or Emr1 (F4/80) was absent or low (Signal<100) in FDC. These findings suggest that the number of follicular T cells and macrophages in the FDC network is too low to significantly distort the FDC gene expression profile. For the genes Cxcl13, Serpina1, Cilp, Lrat, Enpp2, Ltbp3, 9130213B05Rik (prostatic androgen-repressed message-1), Coch and Postn-specific expression in FDC was controlled by in situ hybridization (Fig. 2A). Staining of consecutive splenic tissue sections of BALB/c mice showed that the expression of the genes Enpp2, Serpina1, Cilp, Postn, find more Lbp3 and Lrat was restricted

to the area of CXCL13 expressing FDC. By contrast, the gene Coch showed, in addition, expression in reticular cells of the red pulp and the gene 9130213B05Rik was also expressed in reticular cells of the T-cell zone (Fig. 2A). Expression of Postn and Coch was upregulated in FDC of secondary follicles (Fig. 2B). Staining of consecutive sections with peanut agglutinin (PNA), which labels GC B cells and M2, an FDC-specific Ab, demonstrated that the upregulation of Postn and Coch is restricted to FDC in GC. The gene expression profile obtained for FDC overlapped to a large extent with that of mesenchymal cells (NCBI GEOS data base). Thus, the comparison showed that selleck 342 of the 575 genes expressed in FDC are also expressed in myoblasts and a similar close relationship was found with the transcriptome of fibroblasts (337/575). To

analyze the lineage relationship between why mature FDC and mesenchymal stromal cells, we made use of the fact that FDC do not develop in SCID mice. In the SCID mouse, the BP3 Ab labels reticular cells, which define the area in which lymphocyte-positive mice give rise to the B-cell compartment 19. To analyze the developmental relationship between FDC and reticular cells, BP3hi cells were micro-dissected from the spleen of SCID mice and their transcriptome examined. Since the FDC transcriptome was determined by subtraction of the B-cell signature, which includes all of the housekeeping genes (see above), we carried out the same procedure on the transcriptome of the BP3hi cells (Fig. 1A). Subtraction resulted in a set of 541 genes with significant expression in BP3hi cells. In the next step, the gene expression profile of primary FDC was compared with that of BP3hi reticular cells of the SCID mouse. This analysis yielded a set of 690 genes expressed in either one or both cell populations (Fig. 3). There was a striking similarity in the gene expression patterns of BP3hi reticular cells from SCID mice and FDC from wild-type BALB/c. In total, 85.

The Ct value of target gene in each sample was normalized to that

The Ct value of target gene in each sample was normalized to that of reference gene, giving ΔCt. Then the ΔCt values of treated macrophages were compared with Saracatinib purchase that of untreated ones, giving ΔΔCt. The logarithm was used to calculate the relative expression of the target gene.

The macrophages were pre-treated with recombinant mouse IL-17A for 24 hr before BCG infection at a multiplicity of infection of 1. After 3 hr of BCG infection, infected macrophages were washed with PBS and replenished with fresh medium containing 1 μg/ml actinomycin D (Sigma-Aldrich). At the indicated time-points, total RNA from infected macrophages was extracted by using TRIzol reagent and reverse transcribed to complementary DNA. The relative expression level of iNOS mRNA was determined by qPCR. After 2 hr (phagocytosis assay) or 48 hr (bacteria survival assay) of BCG infection, the intracellular bacteria were recovered based on the methods described previously.[21] Briefly, the infected macrophages were washed thrice with PBS. The cells were then lysed by lysis buffer (PBS, 0·5% Triton X-100) to recover intracellular bacteria. The cell lysates were appropriately diluted in learn more PBS containing 0·05% Tween-80 and were plated onto Middlebrook 7H10 agar (BD Biosciences). The agar plates were incubated at 37° supplemented with 5% CO2. Colony-forming units (CFU) were enumerated after 3 weeks of incubation. To collect

whole cell lysates, the macrophages were washed once with PBS and lysed by ice-cold whole cell lysis buffer (10 mm Tris–HCl, pH 7·4, 50 mm NaCl, 50 mm NaF, 10 mm β-glycerophosphate, 0·1 mm EDTA, 10% glycerol, 1% Triton X-100, 2 μg/ml aprotinin, 1 mm sodium orthovanadate, 2 μg/ml leupeptin, 2 μg/ml pepstatin and 1 mm PMSF). Soluble proteins were harvested after centrifugation at 16 000 g for 5 min. The protein concentrations in the whole cell lysates were quantified by bicinchoninic acid (BCA) protein assay kit (Thermo Fisher Scientific, Waltham, MA) according to the manufacturer’s instructions. The extraction of cytoplasmic proteins and also nuclear proteins was based on the methods described previously.[22]

Briefly, the macrophages were washed twice with cold 1 × PBS, followed by incubation with buffer A (10 mm HEPES, pH 7·9, 10 mm KCl, 0·1 mm EDTA, 0·1 mm EGTA, 1 mm dithiothreitol, 2 μg/ml aprotinin, 1 mm sodium orthovanadate, 2 μg/ml leupeptin, 2 μg/ml pepstatin and 1 mm PMSF) on ice for 15 min. The cells were lysed by adding nonidet P-40 to a final concentration of 0·625%. The lysates were centrifuged at 16 000 g for 5 min at 4°. The supernatant containing cytoplasmic proteins was harvested. The pellets were washed once with buffer A and then lysed in buffer C (20 mm HEPES, pH 7·9, 0·4 mm NaCl, 50 mm NaF, 1 mm EDTA, 0·1 mm EGTA, 1 mm dithiothreitol, 2 μg/ml aprotinin, 1 mm sodium orthovanadate, 2 μg/ml leupeptin, 2 μg/ml pepstatin and 1 mm PMSF). The lysates were centrifuged at 16 000 g for 5 min at 4°.