Meanwhile, blood urea nitrogen

level, serum creatinine, proteinuria, blood routine tests and immunological parameters including serum C3, C4, immunoglobulins, CRP and autoantibodies (anti-dsDNA, AnuA and anti-Sm) levels were also analysed. For the control group, 43 age- and sex-matched normal individuals were included as healthy controls (HC, 41 women, two men; age of 33.6 ± 5.5). The study protocol was designed in compliance with Helsinki Declaration and approved by the Ethics check details Board of Provincial Hospital Affiliated to Shandong University. Each participant signed an informed consent for participating in this study. Assay for sRAGE.  Plasma was collected using EDTA as an anticoagulant, aliquoted and stored at −80 °C. The level of sRAGE was detected using an ELISA kit (R&D systems, Minneapolis, MN, USA) according to the manufacturer’s protocol. ELISA plates coated with monoclonal antibody specific for RAGE (extracellular domain) were used for quantitative analysis of sRAGE in plasma. The minimum detectable level of sRAGE was 4 pg/ml. As indicated in the datasheet, no significant cross-reactivities to EN-RAGE, AG-014699 manufacturer HMGB1, S100A10 or S100B were observed. Assays for autoantibodies. 

Antinuclear autoantibodies (ANA) were detected by ANA mosaic indirect immuno-fluorescence assay kit (Euroimmun Medizinische Labordiagnostika AG, Lübeck, Germany). Antibodies of the IgG class against dsDNA, Sm and nucleosome were detected by 17-DMAG (Alvespimycin) HCl ELISA kits from EUROIMMUN

according to the manufacturer’s instructions. The upper limit for anti-dsDNA recommended by EUROIMMUN was 100 International Units (IU)/ml and ≥100 IU/ml is regarded to be positive, while the upper limit for anti-Sm and AnuA was 20 Relative Units (RU)/ml. Measurement of C3, C4, IgA, IgG, IgM and CRP. Blood C3, C4, IgA, IgG, IgM and CRP were detected by nephelometric assay kits from Dade Behring Marburg GmbH (Germany) according to the manufacturer’s instructions. Quantification of proteinuria and urinalysis.  Proteinuria was quantified by Olympus AU5400 (Olympus, Japan). Urinalysis was performed by Urisys 2400 Urinalysis System from Roche Diagnostics (USA). Statistical analysis.  Data were expressed as the Mean ± SEM. Comparisons between patients with SLE and HC were analysed by the Student’s t-test, One-way anova. Correlation analysis was performed by Spearman’s rank correlation test. All analyses were performed by spss (version 17.0, SPSS Inc., Chicago, Illinois, USA). A two-tailed P-value <0.05 was considered as statistically significant. Characteristics of patients with SLE and HC are shown in Tables 1 and 2. The average level of plasma sRAGE in patients with SLE (842.7 ± 50.6 pg/ml) was significantly lower than that in HC (1129.3 ± 80.1 pg/ml) (P = 0.003, Fig. 1A).

Future work should examine whether NF-κB and JAK-STAT directly mediate disease progression in vivo, and identify specific genes

downregulated by NF-κB inhibition to select the most crucial targets for directed therapy. MT was supported by the Michael Stern Polycystic Kidney Disease Research Fellowship, and p38 MAPK inhibitor review an Australian Postgraduate Award (University of Sydney). Research work of the authors cited in this review was supported by the NHMRC (Grants no. 632647 and 457575). “
“Aim:  Spot urine measurement of albumin is now the most commonly accepted approach to screening for proteinuria. Exertion prior to the collection may potentially influence the result of spot urine albumin estimation. We aim to evaluate the effect of exercise on albuminuria in subjects at various stages of diabetic nephropathy in comparison with healthy control volunteers. Methods:  Thirty-five people with diabetes (19 with normoalbuminuria (NA), nine with microalbuminuria (MA) and seven with overt proteinuria (OP)) and nine control subjects were assessed. A 1 km treadmill walk was performed. Four spot urine specimens were collected: first morning void, immediately prior to exercise, and 1 h and 2 h after exercise. A random Cobimetinib ic50 effects linear regression mixed model was used

to assess the effect of exercise on albumin/creatinine ratio (uACR). Results are presented separately for male and female subjects with diabetes due to a Nabilone significant exercise/gender interaction (P < 0.05). Results:  No significant effect of exercise on uACR was seen in control subjects. In NA males with diabetes no effect of exercise was seen, while in females uACR 1 h after exercise was significantly higher than the early morning sample (3.55 mg/mmol (96% confidence interval 0.27–6.83). Both female and male diabetes subjects with MA have increase in uACR 1 h after exercise (87.8, −24.3–199.4 and

6.7, 2.1–11.3). For both males and females with OP, uACR was significantly increased 1 h post exercise (67.5, 22–113 and 21.6, 8.4–34.8, respectively). In all groups uACR at 2 h after exercise was not significantly different to the early morning sample. Conclusions:  Exercise increased uACR estimation in normoalbuminuric subjects with diabetes with a larger effect in females. Whether exercise unmasks early diabetic nephropathy in NA subjects requires further study. “
“To evaluate the reliability of contrast-enhanced ultrasonography (CEUS) for the detection of renal microvascular blood perfusion in a type 2 diabetic Goto-Kakizaki (GK) rat model. Male GK and Wistar rats at the age of 4, 12 and 20 weeks (n = 10, respectively) were used for the study. Real-time and haemodynamic imaging of the renal cortex was performed using CEUS with SonoVue.

After three washes, goat anti-mouse IgG1-HPR (1 : 10 000, Southern Biotech, Birmingham, AL, USA) or goat anti-mouse IgG2a-HPR (1 : 10 000; Southern Biotech) was added and incubated for

2 h at 37°C. After four washes, plates were incubated for 30 min at 37°C with peroxidase substrate system (KPL, ABTS®) as substrate. Reactions were stopped with 1% sodium dodecyl sulphate (SDS), and the absorbance was measured at 405 nm. Alectinib Three mice from each group were sacrificed before and also 4 and 8 weeks after challenge and spleens were homogenized. After lysis using ACK lysis buffer (0·15 m NH4Cl, 10 mm KHCO3 and 0·1 mm Na2EDTA), splenocytes were washed and resuspended in complete RPMI medium (RPMI-1640 supplemented with 5% FCS, 1% L-glutamine, 1% HEPES, 0·1% 2ME, 0·1% gentamicin). Cells were then seeded at a density of 3·5 × 106 cells/mL in the presence of rA2 (10 μg/mL), rCPA (10 μg/mL) and rCPB (10 μg/mL), or L. infantum F/T (25 μg/mL), or medium alone. Concanavalin A (Con A; 5 μg/mL) was also used in all experiments as the positive control. Plates were incubated for 24 h for IL-2 measurement and 5 days for IFN-γ and IL-10 measurements and also for nitric oxide assay at 37°C in 5% CO2-humidified atmosphere. The IL-2, IFN-γ and IL-10 production in supernatants of splenocyte cultures was measured by sandwich ELISA kits (R&D, Minneapolis, MN, USA),

according to the manufacturer’s instructions. Nitrite release was determined at 8 weeks after challenge by mixing 5-day-incubated splenocyte supernatant with an equal volume of Griess

reagent Birinapant mw Bay 11-7085 [0·1N (1-naphthyl)ethylenediamine dihydrochloride and 1% sulphanil amide in 5% H3PO4] and incubated 10 min at room temperature. Absorbance of the coloured complex was determined at 550 nm. The nitric oxide concentration of each corresponding sample was extrapolated from the standard curve plotted with sodium nitrite serial dilution in culture medium. All experiments were run in duplicates. Two mice from each group were sacrificed at 2, 4, 8 and 12 weeks after challenge, and parasite burdens were determined as follows. A piece of spleen and liver were excised, weighed and then homogenized with a tissue grinder in 2 mL of Schneider’s Drosophila medium supplemented with 20% heat-inactivated foetal calf serum and gentamicin (0·1%). Under sterile conditions, serial dilutions ranging from 1 to 10−20 were prepared in wells of 96-well microtitration plates. After 7 and 14 days of incubation at 26°C, plates were examined with an inverted microscope at a magnification of 40×. The presence or absence of mobile promastigotes was recorded in each well. The final titre was the last dilution for which the well contained at least one parasite. The number of parasites per gram was calculated in the following way: parasite burden = −log10 (parasite dilution/tissue weight) [25, 26].

Participants were 48 infants, 24 6- to 7-month-olds (12 females) and 24 9- to 10-month-olds (12 females). For the 6- to 7-month-olds, mean age of the females was 193.83 days, SD = 16.99, and mean age of the males

was 186.08 days, SD = 12.56, a difference that was not SCH727965 nmr significant, t(22) = 1.27, p > .20, two-tailed. Likewise, for the 9- to 10-month-olds, mean age of the females was 280.58 days, SD = 13.03, and mean age of the males was 277.25 days, SD = 8.74, a difference that was again not reliable, t(22) = 0.73, p > .20, two-tailed. Three additional 6- to 7-month-olds were tested (one female), but one did not complete the procedure due to fussiness and two were excluded from analyses because of failure to compare the test stimuli. Two additional 9- to 10-month-olds were tested (both female), but one did not complete

the procedure due to fussiness, and the other was excluded from analyses because of side preference. Familiarization included seven 15-s familiarization trials, Obeticholic Acid chemical structure each presenting the number 1 (or its mirror image) in a different degree of rotation. Two identical copies of each stimulus were presented on each trial. The seven values of rotation and their order of presentation were randomly chosen for each female and a corresponding male participant. There were two 10-s preference test trials, each of which paired the rotation of the number 1 (or its mirror image) not experienced during familiarization with its mirror image. Left-right positioning of the two test stimuli was counterbalanced across both females and males on the first test trial and reversed on the second test trial. Interobserver agreement was calculated for the preference test trials of six infants (three female) in each

age group. Average level of agreement was 98.48% (SD = 0.71) for the 6- to 7-month-olds, Methane monooxygenase and 97.60% (SD = 2.19) for the 9- to 10-month-olds. As in Experiment 1, preliminary analyses indicated that left versus right orientation of the familiar stimulus (i.e., number 1 versus mirror image) did not impact looking time during familiarization or novelty preference for either gender. Individual looking times were summed over left and right copies of the stimulus presented on each trial and then averaged across the first three trials and last three trials. Mean looking times are shown in Table 2. An analysis of variance (ANOVA), Sex of Participant (female versus male), Age of Participant (6–7 months versus 9–10 months) × Trial Block (1–3 versus 5–7), performed on the looking times revealed only a significant effect of trial block, F(1, 44) = 4.96, p < .03. The trial block effect indicates that infants displayed a reliable decrement in looking time from the first to last half of familiarization that is consistent with the presence of habituation (Cohen & Gelber, 1975). Each infant’s looking time to the mirror image stimulus was divided by looking time to both test stimuli and converted to a percentage score.

As aforementioned, CCL3 and CCL4 are two structurally and functionally related CC chemokines. CCL3 and CCL4 were both discovered in 1988, when Wolpe et al. purified a protein doublet from the supernatant of lipopolysaccharide (LPS)-stimulated murine macrophages [57]. Because of its inflammatory properties in vitro as well as in vivo, the protein mixture was called macrophage inflammatory protein-1 (MIP-1). Further biochemical separation and characterization of the protein doublet yielded

two distinct, but highly related proteins, MIP-1α and MIP-1β[58]. From 1988 to Lenvatinib ic50 1991, several groups reported independently the isolation of the human homologues of MIP-1α and MIP-1β[59–61]. As Metformin a consequence, alternate designations were used for MIP-1α (LD78α, AT464·1, GOS19-1) and MIP-1β (ACT-2, AT744·1), similar to other members of chemokine superfamily. In an attempt to clarify the confusing nomenclature associated with chemokines and their receptors, a new nomenclature was introduced by Zlotnik and Yoshie in 2000 [37]. MIP-1α and MIP-1β were renamed as CCL3 and CCL4. The non-allelic

copies of CCL3 and CCL4 were designated as CCL3L (previously LD78β, AT 464·2, GOS19-2) and CCL4L (previously LAG-1, AT744·2). CCL3 and CCL4 precursors and mature proteins share 58% and 68% identical amino acids, respectively (Fig. 2). Both chemokines are expressed upon stimulation by monocytes/macrophages, T and B lymphocytes and dendritic cells (although they are inducible in most mature haematopoietic cells). Functionally, CCL3 and CCL4 are potent chemoattractants of monocytes, T lymphocytes, dendritic cells and natural killer cells [47]. Despite these similarities, CCL3 and CCL4 differ in the recruitment of specific T cell subsets: CCL3 preferentially Carnitine dehydrogenase attracts CD8 T cells

while CCL4 preferentially attracts CD4 T cells [62]. Interestingly, Bystry and co-workers demonstrated that B cells and professional antigen-presenting cells (APCs) recruit CD4+CD25+ regulatory T cells via CCL4 [63]. This role of CCL4 in immune regulation was reinforced later by Joosten et al. [64], who identified a human CD8+ regulatory T cell subset that mediates suppression through CCL4 but not CCL3. CCL3 and CCL4 also differ in their effect on stem cell proliferation: CCL3 suppresses proliferation of haematopoietic progenitor cells [65]. CCL4 has no suppressive or enhancing activity on stem cells or early myeloid progenitor cells by itself, but has the capacity to block the suppressive actions of CCL3 [66]. A different receptor usage may help to explain, at least in part, why these molecules have overlapping, but not identical, bioactivity profiles: CCL3 signals through the chemokine receptors CCR1 and CCR5.

For functional assays, mouse anti-human CD3 (HIT3a) and anti-human CD28 (CD28.2) were purchased from BD Biosciences. For ELISPOT assays, mouse anti-human IFN-γ capture mAbs and a biotinylated anti-human Selisistat IFN-γ mAbs were purchased from Fisher Scientific (Pierce Biotechnology, Rockford, IL); mouse anti-human IL-2 capture mAbs, biotinylated anti-human IL-2 mAbs and recombinant IL-2 were purchased from

R&D Systems. Pooled human AB serum was purchased from Pel Freeze Biologicals (Rogers, AR). Rapamycin was gifted to the laboratory by Wyeth-Ayest Research (Princeton, NJ) and CsA was purchased from Novartis Pharmaceuticals (East Hanover, NJ). Human peripheral blood was obtained from healthy volunteers consented in accordance with IRB approval by Children’s Hospital Boston. CD4+ T cells were isolated from PMBCs using magnetic beads (Dynal CD4 Positive Isolation Kit, Invitrogen, click here Carlsbad, CA) according

to the manufacturer’s instructions. The purity of isolated CD4+ cells was found to be >97% by FACS. For depletion studies, purified CD4+ T cells were incubated for 20 min with 1 μg per 106 target cells of anti-CXCR3 mAbs (1C6, BD Biosciences) or anti-CD25 mAbs (M-A251, BD Biosciences) at 4°C, and were washed in PBS/0.5% BSA. The cells were subsequently incubated with Pan mouse IgG magnetic beads (Dynal Cellection Kit, Invitrogen) and CXCR3+ or CD25+ cells were removed

by magnetic separation. The purity of the depleted populations was >92% as assessed by flow cytometry. For migration assays, CD4+CD25+CD127dim/− cells were isolated from PBMCs using magnetic beads (Miltenyi Biotec) and were FACS-sorted (using 1C6, BD Biosciences) into CXCR3+ and CXCR3neg populations. Cell culture was performed at 37°C in 5% CO2 in RPMI 1640 media (Cambrex, Charles City, IA) containing 10% human AB serum, 2 mM L-glutamine, 100 U/mL penicillin/streptomycin (Gibco-Invitrogen), 1% sodium bicarbonate and 1% sodium pyruvate (Cambrex) in Diflunisal 96-well, round-bottom plates (Corning Life Sciences, Lowell, MA). Mitogen-dependent assays were performed in 96-well round bottom cell culture plates in triplicate wells (1×105 T cells/well) in a final volume of 200 μL. The cells were stimulated with either immobilized anti-CD3 mAbs (5 μg/mL) alone or with immobilized anti-CD3 mAbs in combination with soluble anti-CD28 mAbw (1 μg/mL) for 3 days. Mixed lymphocyte reactions were performed using 2×105 responders and γ-irradiated PBMCs (1700 rad) as stimulators in a ratio of 1:1. Cells were cultured in triplicate wells using either allogeneic or autologous stimulators. Proliferation was assessed after 5 days by 3H–Thymidine (Perkin Elmer, Boston, MA; 1 μCi/well) incorporation for the final 18 h of culture, and data were analyzed using suppression ratios.

Indeed, treatment with rhIL-10 significantly reduced both CD8+ and CD4+ T-cell proliferation (Fig. 7C), thus proving a central role of IL-10 in the regulation of the T-cell response to allogenic monocytes. In this study, we demonstrated the role of the IRAK4 kinase as a differential switch between TLR-induced pro-inflammatory and anti-inflammatory cytokine production. This observation is of interest as to date IRAK4 is mainly being viewed as a central executor of the MyD88 pathway that unselectively transduces all signals downstream of MyD88. As previously described in IRAK4-deficient mice [26], stimulation of IRAK4 knockdown monocytes with TLR4 and TLR2 ligands resulted in markedly reduced pro-inflammatory cytokine

secretion such as TNF and IL-12 (Fig. 2A–C) and this website concomitant reduction of the NF-κB subunits p50 and p65 responsible for the transcription of these cytokines (Fig. 2D). The results obtained are further in accordance CP-868596 cell line with observations made in cells of IRAK4- and MyD88-deficient patients. There, TLR stimulation fails to activate the NF-κB pathway and cells display an impaired cytokine response after stimulation with agonists for TLR-1, TLR-2, TLR-4, TLR-5, TLR-7, and TLR-8 [17-20]. Similarly, siRNA-mediated silencing of MyD88 caused a significant reduction in TNF and IL-12 cytokine production in human monocytes (Fig. 4C,D), highlighting the cooperative interaction

of MyD88 and IRAK4 in TLR-induced synthesis of pro-inflammatory cytokines such as TNF and IL-12. IRAK4-deficient patients are more susceptible to infections with pyogenic bacteria, especially Gram-positive species such as S. pneumoniae and/or S. aureus [18]. Consistently, the predisposition to S. aureus infections was also reported in IRAK4-deficient mice [26]. Until now, little is known about the role of monocytes in response to these pathogens albeit these cells belong to those first to encounter bacterial pathogens in blood stream infections.

In this study, we tested the role of IRAK4 in the TLR-mediated response of human monocytes to bacterial infections. In particular, our results showed diminished IL-12 www.selleck.co.jp/products/Pomalidomide(CC-4047).html secretion and elevated TNF and IL-10 levels after treatment with live S. aureus and S. pneumoniae (Fig. 1C). To further assess these findings we conducted MyD88 knockdown experiments, obtaining concomitantly reduced IL-12 and IL-10 secretion. However, TNF levels were only slightly diminished (Fig. 4E). This strongly suggests that IL-12 and IL-10 secretion evoked by bacterial infections is MyD88-dependent, whereas TNF production is also regulated in a MyD88-independent fashion, possibly triggered via TRIF or cytosolic PRR-induced IFN-I [27]. In general, bacteria represent complexes of multiple ligands that can be sensed by membrane-bound as well as cytosolic PRR. The most prominent difference between stimulation with bacteria and single TLR ligands was an increase in TNF release under IRAK4-silencing conditions (Fig. 1C) and under rapamycin treatment (Fig. 5B).

Here, we discuss how miRNAs regulate TLRs, particularly in macrophages, a process likely to occur in the resolution phase of inflammation and speculate on the importance of miRNAs in diseases, which feature dysregulated innate immunity. We discuss three particular miRNAs – miR-155, miR-146a, and miR-21 – since these miRNAs have been strongly implicated in the regulation of TLRs in a number of cells including macrophages 3. Interestingly, miR-155 and miR-146 are specifically present in LPS-induced macrophages, as compared with

similarly activated polymorphonuclear neutrophils (PMNs), NVP-LDE225 in vivo suggesting a particular role for these miRNAs in macrophages 4. We also speculate on the potential novel therapies that target miRNAs

in infection and inflammation that could be developed. The gene-encoding miR-155 is located on chromosome 21 in the B-cell integration cluster (BIC) 5. BIC is highly conserved between humans and mice and is highly expressed in lymphoid organs. miR-155 expression is strongly induced in response to LPS or type I interferons, in both monocytes and macrophages of human or mouse origin, demonstrating that this miRNA participates in the innate immune response to both bacterial and viral infection 6, 7. Furthermore, miR-155 is highly expressed in activated B and T cells and has been shown to play a role in regulating cytokine expression in the germinal center 8. miR-155 is induced by either the MyD88 or the TRIF pathways through LPS or poly I:C stimulation 7. Unlike the miRNAs discussed later in this MLN0128 concentration Viewpoint, the evidence so far presented on miR-155 function indicates that it is likely

to be pro- rather than anti-inflammatory. This is because one of the roles of miR-155 in macrophages is to allow the translation of tumor necrosis factor (TNF), a key pro-inflammatory cytokine click here 6, 9. In resting macrophages, the 3′ UTR of TNF induces a self-repression, which is released upon LPS stimulation via the binding of miR-155. This has been shown in macrophages, where miR-155 overexpression results in increased TNF production and miR-155 deficiency results in lower levels of TNF 9. Targeting miR-155 in macrophages would therefore limit TNF production and would be useful therapeutically in TNF-mediated disorders. An in vivo study has shown that B cells that overexpress miR-155 transgenically produce more TNF and the corresponding transgenic mice have an elevated susceptibility to LPS-induced septic shock 8. miR-155-deficient B cells, on the other hand, fail to produce TNF 8. As shown in Fig. 1, in macrophages, miR-155 is negatively regulated by IL-10, an anti-inflammatory cytokine 10. Inhibition of miR-155 by IL-10 increases expression of Src homology2 (SH2) domain-containing inositol 5′-phosphatase 1 (SHIP1), a known target of miR-155 11, 12. Previously, SHIP1 has been shown to function as a negative regulator of TLR-induced responses 13–15.

Macaque and human pDC were shown to have similar TLR expression profiles [25], which is in agreement with the response patterns observed by us. Also TLR-7, TLR-9 and myeloid differentiation primary response gene 88 (MYD88) SCH727965 concentration sequences were shown to be identical, whereas there were important differences for interferon regulatory factor 7 (IRF-7) [26]. Other regulatory pathways still need to be explored [37]. Beside TLRs, the C-type lectin receptor (CLR) family plays an important role in the modulation of innate immune responses [38, 39]. Human pDC express the CLRs blood dendritic cell antigen 2 (BDCA2) and dendritic cell immunoreceptor (DCIR) [40]. Cross-linking of DCIR was shown to result in reduced IFN-α induction upon

TLR-9 stimulation [40], and similar inhibitory effects were reported following incubation with the CLR ligand mannan [41]. Interestingly, BDCA2 [our unpublished observation and documented at the NIH non-human primate reagent resource portal (http://nhpreagents.bidmc.harvard.edu/NHP)] and DCIR [42] were shown to be absent on pDC in rhesus macaques. Although not investigated here, a difference in the balance between activating TLRs and inhibitory CLRs could lead to different levels of pDC activation, possibly translating into a difference in cytokine production pattern. A direct comparison between the absolute numbers of pDC, mDC and monocytes in rhesus versus human blood showed that rhesus

macaques had a lower number of pDC, while Selleck Obeticholic Acid there was no difference in the abundance of the other subsets. The number

of pDC observed, i.e. 3020 ± 1357 cells/μl, is in agreement with several reports on rhesus macaques [16, 18, 24, 25, 43] and considerably less Methane monooxygenase than in humans [44]. In contrast, two other studies, where a direct head-to-head comparison was made, showed no difference in pDC number [17, 28], although it must be noted that in those studies the quantification was either performed on PBMC or cynomolgus monkeys imported from Mauritius were used, which have a more limited genetic diversity and might differ from rhesus macaques. The strong IL-12p40 expression in rhesus pDC may have implications for preclinical evaluation of vaccines in this model. For instance, TLR-7/8 containing adjuvants might trigger different responses in macaques than in humans and involve pDC as IL-12 producing cells. Also TLR-9 agonists could be expected to induce an IL-12 response in rhesus macaques, in contrast to humans. Simultaneous production of IFN-α and the inflammatory cytokines TNF-α and T helper type 1 (Th1)-skewing cytokine IL-12 might also lead to a slightly different response pattern to bacterial and viral infection and have consequences for the induction of CD8 responses [45, 46]. We would like to thank Dr F. Verreck for critical reading of the manuscript, Dr S.B. Geutskens for organizing the collection of the human blood samples and H. van Westbroek for preparing the figures.

) were used, when necessary, for stimulation. For evaluation Z-IETD-FMK in vitro of cytokine secretion, supernatants from ML-stimulated monocytes were harvested after 1 day of culture and stored at −20 °C until future use. For live or dead bacteria detection, the LIVE/DEAD® BacLight™ Bacterial Viability Kits were used according to the manufacturer’s

instructions (Invitrogen Corporation). To block endogenous IL-10, the neutralizing anti-IL-10 rat anti-human or isotype control—IgG1 at a final concentration of 1 μg/mL (BD PharMingen, San Diego, CA, USA) was added to the monocytic culture. The neutralizing antibody was added to the culture 30 min before ML stimulation. After 24 h, the percentage of CD163+ was evaluated by flow cytometry (AccuriTM, Ann Arbor,

MI, USA) and IDO activity was evaluated in the supernatants. To detect IDO activity, supernatants from ML-stimulated monocytic cultures were collected and frozen in −20°C until HPLC analysis. When necessary, IDO activity was evaluated in rIL-10 (10 ng/mL)- or anti-IL-10 (1 μg/mL)-stimulated cell supernatants. Tryptophan (Trp) and Kynurenine (Kyn) concentrations were measured by HPLC, as previously described [6]. Monocytes were pretreated with RM3/1 CD163 antibody or its isotype control—Mouse IgG1 (20 μg/mL, Santa Cruz Biotechnology®) for 30 min on ice. Prior to bacterial interaction assays, ML was stained with PKH26 Red Fluorescence cell linker Kit (Sigma) according to the manufacturer’s instructions. Adherent CDK phosphorylation monocytes were infected with PKH 26-labeled ML (MOI 5: 1) and after 2, 16, and 24 h postinfection, the percentage of eukaryotic cells with bacterial association was measured using an AccuriTM flow cytometry. The index of bacterial association is expressed as percentage of cells taking up PKH26-ML. To determine bacterial internalization, ML was labeled with PKH67 Green Fluorescence cell linker Kit

(Sigma) prior to infection and the fluorescent signal of extracellular bacteria after incubation time was quenched with trypan blue, as previously described [39]. The percentage of ML phagocytosis was measured by PKH-67 and oxyclozanide measured at the FL1 channel via flow cytometry. Alternatively, ML association and internalization were evaluated at 2 and 16 h using the human embryonic kidney cell line 293 (HEK293) cells transfected with CD163 mRNA (splice variant AC1) as previously described [40]. In parallel, microscopy images were obtained from cells pretreated with the PKH 67 Green Fluorescence cell linker Kit (Sigma) (green) to visualize the eukaryotic cell membrane, prior saturation with the antibodies, and PKH 26-labeled ML (red) infection, as described below regarding the cytometry assay. Cells were also labeled with the DAPI nuclear stain. Preparations were examined using Microscope Axio Observer Z1 (Carl Zeiss) via Axiovision 4.7 software.