The role of interleukin-1 beta in the pathophysiology of Schnitzler’s syndrome

Patients and patient samples

The study was approved by the local medical ethical committee of the Radboud university
medical center, as the patients and controls were recruited there. Eight patients
with SchS, either classical or variant type, and seventeen healthy controls that were
age- and sex-matched as much as possible provided written informed consent. Patients
stopped anakinra in order to enter the canakinumab trial and multiple blood samples
were collected 13]. Polymorphonuclear cells (PMNs) and peripheral blood mononuclear cells (PBMCs) were
isolated during anakinra treatment, during disease relapse after discontinuation of
anakinra (symptomatic episode), 14 days and 6 months after the first monthly canakinumab
injection, and upon disease relapse after discontinuation of canakinumab. At each
time point, blood samples from a matched healthy donor control were collected too.
B cells and T cells were isolated from blood samples collected during anakinra, canakinumab
and during the symptomatic phase. Serum samples were also taken on those occasions,
as well as at 3 and 7 days and then monthly after the first canakinumab injection.

PBMC and polymorphonuclear cells (PMNs) processing

PBMCs were isolated from EDTA-blood using Ficoll-paque Plus (GE Healthcare, Eindhoven,
The Netherlands) separation, and PMNs were isolated from the pellet by lysing erythrocytes
with a hypotonic 155mM NH
₄
Cl, 10 mM KHCO
₃
lysis buffer. For RNA isolation, 5 million cells of each sample were dissolved in
1 ml Trizol (Invitrogen, Bleiswijk, The Netherlands) and stored until further processing.
For protein analysis, 6 million cells were lysed with a lysisbuffer (50 mM Tris (pH
7.4), 150 mM NaCl, 2 mM EDTA, 2 mM ethylene glycol tetraacetic acid (EGTA), 10 % glycerol,
1 % Triton X-100, 40 mM ?-glycerophosphate, 50 mM sodium fluoride, and 200 mM sodium
vanadate, supplemented with protease inhibitor cocktail (Roche, Mannheim, Germany))
and stored at ?80 °C until measurement.

PBMC culture

PBMCs isolated at the five indicated time points were also used for in vitro experiments.
Cells from patients (N = 8) and controls (N = 17) were stimulated for 24 hours with
LPS (TLR4 ligand, 0.1, 1, 10 ng/ml) (Sigma, St Louis, MO, USA; Escherichia coli serotype 055:B5, purified in our own laboratory), Pam3Cys (TLR2 ligand, 10 ?g/ml)
(EMC Microcollections, Tubingen, Germany), poly:IC (TLR3 ligand, 5 ?g/ml) (InvivoGen,
Toulouse, France), or no ligand. For IL-17 assays, cells were stimulated for 7 days
with heat-killed (homemade) Candida albicans (10^6/ml). Supernatants were collected and stored at ?80 °C. Cytokine concentrations
in serum, supernatants and lysates were measured by means of enzyme-linked immunosorbent
assay (ELISA): IL-1? (RD, DY 201E, Minneapolis, MN, USA), IL-6 (Sanquin, M9316, Nijmegen,
The Netherlands), TNF-? (RD, DY210E), IL-17 (RD, DY317E), MRP8/14 and S100A12 as
previously described 18], 19].

RNA extraction and quantitative real-time polymerase chain reaction (qPCR)

mRNA was extracted and first-strand cDNA was generated and amplified by means of qPCR
as previously described 20]. Specific qPCR primers were designed with Primer Express 1.0 Software (Applied Biosystems)
and purchased from Biolegio (Nijmegen, The Netherlands). By means of the comparative
delta-DCt method, relative mRNA expression levels of all examined genes were calculated
21].

Microarray

A microarray using the Illumina Direct Hybridization Assay (performed by ServiceXs,
Leiden, The Netherlands) was performed on purified whole blood RNA samples from two
patients with classical SchS and 1 IgG variant case with myeloid-restricted NLRP3 mosaicism. Integrity of the RNA samples was confirmed by Eukaryote Total RNA Nano
Bioanalyzer analysis. The microarray data were further analyzed by loading the log
expression values into Partek Genomics Suite Software (Version 6.4; Partek, Inc.,
St Louis, MO, USA). Quantile normalization was performed including all arrays, to
correct for large overall expression differences between the arrays. In addition,
to adjust for the baseline expression level of each of the individual patients in
the different subgroups, a correction for the factor ‘individual’ was performed, as
one would do for batch correction, using the batch remove option from the software.
On 1 August 2015, the data will be released online in the Gene Expression Omnibus
[GSE70019].

Flow cytometric analysis

T-lymphocyte subsets

To detect intracellular expression of the transcription factors Foxp3, ROR?t and Tbet
in CD3+CD4+ T cells, Ficoll-isolated PBMCs were first labeled with CD3(UCHT1)-ECD
and CD4(SFCI12T4D11)-PC7 (Beckman Coulter), and subsequently fixed and permeabilized
using Fix/Perm buffer (eBioscience), labeled with FoxP3(PCH101)-FITC (eBioscience),
ROR?t(AFKJS-9)-APC and T-bet(4B10)-PE (Santa Cruz Biotechnology, Santa Cruz, CA) and
measured by five-color flow cytometry (FC500, Beckman Coulter). Data were analyzed
using CXP software (Beckman Coulter). Isotype controls were used for gate settings.

B-lymphocyte subsets

Cells from heparinized blood were phenotypically analyzed in a 10-color MoAb conjugate
combination using a Naviosâ„¢ instrument with 10-color PMTs and three solid-state lasers
(Beckman Coulter, Fullerton, FL). The list mode data files were further analyzed using
Kaluzaâ„¢ software (Beckman Coulter). In order to guarantee that the optics, laser,
fluidics and fluorescence intensity were stable during all measurements calibration
was performed using Flow Check Pro Fluorospheres (Beckman Coulter) and Cyto-Cal Multifluor
+ Violet Fluorescence Alignment Beads (Thermo Scientific, Fremont, CA, USA). After
erythrocyte lysis (BD Pharm-Lyse, BectonDickinson) cells were washed with PBS with
1 % bovine serum albumin before being labeled with fluorochrome-conjugated mAbs. After
incubation for 30 minutes at 4 °C in the dark, cells were washed twice to remove unbound
antibodies and analyzed. For cell surface staining, the following mAbs were used:
IgD-FITC, IgM-PE (both Dako, Denmark) and CD3-ECD, CD4-PECy5.5, CD27-PECy7, CD20-PacB,
CD45-KromeOrange, CD56-APC, CD8-APC-Alexa Fluor700 and CD19-APC-Alexa Fluor750 (all
Beckman Coulter, Marseille, France). Subsequently, the various lymphocyte subpopulations
were analyzed on the flow cytometer using CD45/SSC to gate the lymphocyte population.

M-protein and free light chain analysis

To detect and quantify the presence of an M-protein, agarose gel electrophoresis and
immunofixation were performed on the Hydrasys (Sebia, Evry, France) according to the
manufacturer’s protocol. Serum free light-chain analysis was performed on a BNII analyzer
(Siemens, Marburg, Germany) using Freelite reagents (The Binding Site Ltd, Birmingham,
UK) according to the manufacturer’s protocol.

Statistical analysis

Repeated-measures analysis of variance (ANOVA) using SPSS v16.0 (SPSS Inc) was performed
on the delta-cycle threshold (dCt) values of the qPCR data corrected for primer efficiency.
dCt is the difference between the Ct of the target gene and the reference gene (RPLP0). The ELISA data were analyzed by one-way ANOVA with Bonferroni post hoc testing
for the models. Fold changes and p values of the microarray data for the contrast, patients vs. controls, were calculated
by conducting multifactorial ANOVA on the factor ‘treatment’.