healthmedicinet

TsSP and reagents

Soluble products of Trichuris suis (TsSP) were prepared as described previously 9], 10]. A limulus amebocyte lysate assay (Thermo scientific, USA) was used to determine
endotoxin levels in TsSP, which appeared to be similar to background levels, thereby
excluding LPS contamination. Cleavage of TsSP peptide chains (using ?-chymotrypsin
(CT, Sigma, USA) and oxidation of glycan moieties (using sodium periodate (PI; 10 mM,
Sigma) was performed as described previously 9]. We used blocking antibodies for the human mannose receptor (MR, CD206, BD Pharmingen,
USA) or Dectin-2 (Clone 545943, RD Systems, USA). Recombinant human TNF-? was obtained
from Invitrogen (Carlsbad, CA, USA). To study the involvement of PKC or Rho GTPases,
we used the panPKC inhibitor Bisindolylmaleimide I (GF109203, Enzo Life Sciences,
the Netherlands) as well as the Rac1-GEF inhibitor NSC-23766 (Bio-Techne, Abingdon,
UK) and the Rho kinase inhibitor Y27632 (Sigma-Aldrich, Steinheim, Germany).

Monocyte isolation

Peripheral blood mononuclear cells (PBMCs) were isolated using Ficoll density gradient
(Lymphoprepâ„¢, Axis- Shield, Oslo, Norway) from buffy coats obtained from healthy donors
(Sanquin Blood Bank, Amsterdam, the Netherlands) or patients who signed informed consent
based on principles outlined in the Declaration of Helsinki. The studies were approved
by the local ethics committee of Sanquin Blood Supply, Medical University of Vienna,
Vienna, Austria and Comitè de Protection des Personnes Sud-EST IV, France. Patients’
characteristics including details about PRKCD gene mutations have been described before 21], 22]. Monocyte isolation was performed by gradient centrifugation on Percoll (Amersham
Biosciences, USA) according to manufacturer’s protocol. To increase monocyte purity,
we used anti-CD14 magnetic beads (Miltenyi Biotec, Germany) with a MACS® MultiStand
and LS Column by passing 3 ml of MACS buffer (2 mM EDTA and 0.1 % FCS in PBS) according
to the manufacturers’ protocol. Monocyte purity (based on CD68 expression) was 90 %
as assessed by flow cytometry performed (FACSCaliburâ„¢ using CELLQuestâ„¢ software (BD
Biosciences) and monocytes were cultured in RPMI 1640 medium supplemented with 10 %
heat-inactivated fetal calf serum (FCS), 2 mM L-glutamine, 100 U/ml penicillin and
100 ?g/ml streptomycin (all obtained from Gibco-BRL Life Technologies, Breda, the
Netherlands).

Flow cytometry

Freshly isolated monocytes were cultured in the presence or absence of TsSP (40 ?g/ml)
for 16 h and subsequently harvested. Next, monocytes were washed twice with PBS and
labelled for 1 h at 4 °C with primary antibody (Table 1) diluted in PBS containing 0.1 % BSA. Cells were washed twice in PBS and incubated
for 1 h (4 °C) with fluorescent labelled secondary antibodies diluted in PBS/0,5 %
BSA (FACS buffer), washed twice and resuspended in FACS buffer prior to FACS analysis.
We used either four or eight colour flow cytometry. Four colour flow cytometry (FACSCalibur,
Becton Dickinson, Belgium) was used in combination with Cell Quest software (Becton
Dickinson) and FlowJo software version 9.4.0 for Microsoft (Tree Star, San Carlos,
CA) to analyse expression of markers. For eight colour flow cytometry, we used a Cyan
ADP High Performance Research Flow Cytometer (Beckman Coulter) and results were analyzed
with Summit Software v4.3. Single stained cells were used to compensate for spectral
overlap. Fluorescence Minus One (FMO) stained cells were used to set boundaries between
positively and negatively stained cells. Sytox Blue dead cell stain (Molecular Probes,
the Netherlands) was used to discriminate between live and dead cells.

Table 1. Antibodies used for FACS analysis

Cytokine assays

The production of pro- and anti-inflammatory cytokines was assessed by enzyme-linked
immunosorbent assay (ELISA) in cell-free supernatant samples using the Human Inflammatory
5-Plex Panel (Invitrogen, USA). This multiplex bead assay was performed according
to the user manual supplied by Invitrogen. Samples were measured by Luminex® 200TM
(Bio-Rad, California, USA) and analysed using Bio-plex ManagerTM 6.0 software.

DHR assay

Reactive oxygen species (ROS) production of monocytes was measured using dihydrorhodamine
(DHR), which reacts with ROS in a peroxidase-like reaction to yield fluorescent rhodamine
123 23]. After culturing of monocytes in the presence or absence of TsSP (40 ?g/ml) for 16 h,
cells were rinsed twice with RPMI, incubated for 30 min at 37 ° C with 0.5 ?M DHR
(Sigma Aldrich, Germany) in RPMI medium. After that, cells were rinsed twice with
PBS/0.1 % BSA, transferred to FACS tubes and analysed on a FACSCalibur (see above)
with excitation at 488 nm and the emitted fluorescence collected at 525 nm.

RNA isolation and quantitative real-time PCR

Messenger RNA (mRNA) was isolated using a mRNA Capture kit (Roche, Switzerland), and
subsequently transcribed into cDNA using the Reverse Transcription System kit (Promega,
USA), as described previously 24]. Quantitative real-time PCR was performed with the SYBR Green method as previously
described 24]. Oligonucleotides were designed using Primer Express 2.0 (Applied Biosystems, USA)
computer software. All primer sequences are listed in Table 2 and expression levels of transcripts obtained with real-time PCR were normalized
to GAPDH expression levels.

Table 2. Primers used for RT-PCR

Western blotting

Freshly isolated monocytes were cultured in the presence or absence of TsSP (40 ?g/ml)
for 30 min. Cell lysates were prepared using NP-40 lysis buffer (1 % Nonidet P-40,
10 % glycerol, 100 mM NaCl
₂
, 10 mM MgCl
₂
, 50 mM Tris, pH 7.4) mixed with protease and phosphatase inhibitors (Halt protease
and phosphatase inhibitor cocktail, Thermo Fisher Scientific, Rockford, IL, USA).
Prior to immunoblotting, lysates were boiled 10 min in Laemmli sample buffer containing
1 % ?-mercaptoethanol. Equal sample volumes were subjected to 10 % sodium dodecyl
sulfate–PAGE and transferred to nitrocellulose membranes (Schleicher Schuell, Dassel,
Germany). Membranes were washed with TSM/0,05 % Tween and blocked in TSM/0,05 % Tween/10 %
roti-block (Techmate, Milton Keynes, UK, #A151.4). The following primary antibodies
(in TSM/0,05 % Tween/5 % BSA) were used: PhosphoPKC (Cell signaling, Beverly, MA,
USA, #9371S) and GAPDH (Santa Cruz Biotechnology, Heidelberg, Germany, #sc-32233)
as a loading control. Followed by incubation with polyclonal goat anti-rabbit HRP
(Dako, Heverlee, Belgium, #p0448) and polyclonal rabbit anti-mouse HRP (Dako, Heverlee,
Belgium, #p0161). Proteins were detected using SuperSignal West Pico Chemiluminescent
Substrate (Thermo Scientific, USA), in an EpiChemi II Darkroom (UPV Laboratory Products).
Bands were quantified using Image J.

Live cell imaging for assessment of monocyte motility

Real-time video recordings of monocytes were performed with an inverted phase-contrast
microscope (Olympus, IX81-ZDC, Suffolk, U.K.) housed in a humidified, 5 % CO
₂
gassed, temperature-controlled (37 °C) chamber. For this, 3?×?10
⁵
freshly isolated monocytes were applied in each well of an IBIDI slide in the presence
or absence of TsSP (40 ?g/ml), and randomly selected fields were recorded for 240 min.
Pictures were taken every 3 min with an Olympus ColorView II camera (Olympus Nederland
BV). Recordings were analyzed using CELL F trackIT software (Olympus Soft Imaging
Solutions, Münster, Germany).

Monocyte adhesion and migration assays

The immortalized human brain endothelial cell line hCMEC/D3, which preserves the key
features of brain endothelium, was cultured as described 25]. Monocyte migration of primary human monocytes across confluent monolayers of hCMEC/D3
cells was studied with time-lapse video microscopy as described previously 26]. Freshly isolated monocytes were cultured in the presence or absence of TsSP (40 ?g/ml)
for 16 h and subsequently washed and added (7.5?×?10
⁵
cells/ml) to hCMEC/D3 cells, and the number of migrated monocytes were counted after
4 h. For monocyte adhesion experiments, the TsSP-treated or untreated monocytes were
fluorescently labelled with 0.5 ?M BCECF-AM (Molecular Probes) for 15 min at 37 °C.
Labelled monocytes (1?×?10
⁶
cells/ml) were added to confluent monolayers of hCMEC/D3 cells and allowed to adhere
for 30 min at 37 °C and 5 % CO
₂
. Nonadherent cells were removed by gentle washing with prewarmed medium. Adhered
cells were lysed with 0.1 M NaOH, and fluorescence intensity was determined (Fluostar
32, BMG; excitation 485 nm, emission 535 nm). The number of adherent monocytes was
calculated using a calibration curve.

Statistical analysis

All data were analyzed statistically by means of analysis of variance (ANOVA) and
Student-t test. Statistical significance was defined as *p??0.05, ** p??0.01, *** p??0.001.