Baines et al.
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ONLINE DEPOSITORY
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Differential gene expression and cytokine production from neutrophils in asthma
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phenotypes
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Katherine J Baines * PhD BBiomedSci(Hons) 1,2
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Jodie L Simpson PhD BSc (Hons) 1,2,
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Nikola A Bowden PhD BBiomedSci(Hons)3
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Rodney J Scott PhD, DSc, FRCPath, FHGSA 3
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Peter G Gibson MBBS FRACP 1,2,
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Newcastle, Callaghan NSW 2308 AUSTRALIA
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John Hunter Hospital, New Lambton NSW 2305 AUSTRALIA
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Institute, The University of Newcastle, Callaghan NSW 2308 AUSTRALIA
Priority Research Centre for Asthma and Respiratory Diseases, The University of
Department of Respiratory and Sleep Medicine, Hunter Medical Research Institute
Priority Research Center for Information Based Medicine, Hunter Medical Research
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*Address for correspondence:
Dr Katherine Baines
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Level 3, HMRI, John Hunter Hospital
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Locked Bag 1, Hunter Region Mail Centre
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Newcastle, NSW 2310, AUSTRALIA
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Email: katherine.baines@newcastle.edu.au
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Phone: 61 2 49855766 Fax: 61 2 49855850
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Methods
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Airway Neutrophil Isolation
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CD16 positive cells were isolated from the remainder of the sputum sample using magnetic
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cell separation. The cell pellet obtained was resuspended in phosphate-buffered saline (PBS)
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supplemented with 0.1% bovine serum albumin (BSA) and 2mM EDTA (MACS buffer), and
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incubated with CD16 microbeads (60uL per 50 x 106cells, Miltenyi Biotec, Gladbach,
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Germany) for 30 minutes. The cells were applied to a LS MACS column surrounded by a
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magnetic field (VarioMACS System, Miltenyi Biotec Gladbach, Germany), and the CD16-
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cell fraction was eluted. The LS column was removed from the magnetic field and the CD16+
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neutrophil enriched cell fraction was eluted. Cytospins were prepared for each fraction,
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stained (May-Grunwald Giemsa) and a differential cell count obtained from 400 cells, to
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assess cell purity. Viability of the sputum neutrophil enriched fraction was assessed by trypan
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blue and found to be 67%.
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Peripheral Blood Neutrophil Isolation
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Peripheral blood was collected in sodium citrate tubes. After centrifugation at 200g for 20
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minutes the platelet rich plasma was removed. The remaining pellet was diluted and layered
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over Percoll (GE Healthcare, Uppsala, Sweden) and centrifuged to deplete mononuclear cells.
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After red blood cells were lysed, granulocytes were washed. The granulocyte cell pellet
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obtained was resuspended in phosphate-buffered saline (PBS) supplemented with 0.1%
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bovine serum albumin (BSA) and 2mM EDTA (MACS buffer), and incubated with CD16
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microbeads (60uL per 50 x 106cells, Miltenyi Biotec, Gladbach, Germany). Neutrophils were
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isolated from peripheral blood granulocytes via magnetic cell separation as per manufacturers
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instructions (VarioMACS System, Miltenyi Biotec Gladbach, Germany). Cytospins were
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prepared for each fraction, stained (May-Grunwald Giemsa) and a differential cell count
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obtained from 400 cells, to assess cell purity. Neutrophil viability was assessed by trypan blue
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exclusion and found to be 98%.
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Neutrophil Culture and Stimulation
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Highly pure blood neutrophils [100% (96-100%)] or the sputum neutrophil enriched cell
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fraction [59% (30-78%) neutrophils; 35% (22-57%) macrophages] were resuspended in
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phenol red free RPMI 1640 (Gibco Invitrogen, Mount Waverley, Australia) with 10mM
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HEPES, 1% heat inactivated fetal calf serum and antibiotics (Penicillin/Streptomycin). The
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cells were cultured at 1 x 105 (sputum) 1 x 106 (blood) cells/ml with or without
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lipopolysaccharide (100ng/ml E.coli LPS, Sigma, Sydney, Australia) at 37˚C (5% CO2). Cell
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cultures were collected at 24hrs. Neutrophil viability at 24 hours was assessed by trypan blue
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exclusion and found to be 95% for resting neutrophils and 96% for LPS stimulated
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neutrophils. Cell free supernatants were prepared and cell pellets were and stored in buffer
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RLT (Qiagen, Hilden, Germany) at -80˚C.
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Results
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Table E1: Clinical characteristics of participants with eosinophilic and non-eosinophilic
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asthma selected for microarray analysis.
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Eosinophilic
Non-Eosinophilic
Asthma
Asthma
n
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Age years
63 (55-69)
70 (64-75)
0.33
Gender M | F
3|2
2|2
1.00
Atopy n (%)
4 (80)
3 (75)
1.00
FEV1 % predicted
66 (15)
53 (15)
0.21
FEV1/FVC %
80 (7)
71 (9)
0.15
ICS ‡‡ dose (μg) median (IQR)
1000 (500-2000)
2000 (1200-2500)
0.30
‡‡
p
ICS dose is calculated 1μg of beclomethasone = 1μg of budesonide = 0.5μg of fluticasone.
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Table E2: Relative levels of cytokine gene expression in resting and LPS stimulated peripheral blood neutrophils isolated from participants with
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eosinophilic (n=8) and non-eosinophilic asthma (n=9) and healthy controls (n=11).
Resting
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LPS Stimulated
Healthy Controls
Eosinophilic
Asthma
Non-Eosinophilic
Asthma
p
Healthy Controls
Eosinophilic
Asthma
Non-Eosinophilic
Asthma
p
IL-8
2.24 (1.59-3.57)
1.07 (0.71-3.63)
3.07 (2.4-3.66)
0.16
54.2 (40.6-95.3)
26.4 (10.9-56.4)
44.6 (29.7-84.4)
0.23
IL-1β
0.07 (0.03-0.19)
0.01 (0.01-0.04)
0.03 (0.02-0.04)
0.07
1.4 (0.6-2.0)
0.4 (0.1-0.75)*
0.7 (0.2-1.3)
0.03
TNF-α
0.02 (0.01-0.06)
0.02 (0.01-0.03)
0.08 (0.02-0.1)
0.23
1.2 (0.6-1.7)
1.5 (0.4-1.9)
1.0 (0.6-1.5)
0.83
OSM
0.18 (0.07-0.24)
0.1 (0.07-0.15)
0.15 (0.13-0.18)
0.29
0.66 (0.35-0.84)
0.64 (0.26-1.09)
0.56 (0.46-0.72)
0.87
TLR2
0.6 (0.3-0.9)
0.14 (0.06-0.4)*
0.2 (0.2-0.3)
0.02
3.9 (2.5-8.4)
2.1 (1.6-3.4)
2.3 (1.1-2.9)*
0.02
TLR4
0.5 (0.2-0.8)
0.2 (0.2-0.5)
0.4 (0.3-0.4)
0.48
0.9 (0.5-1.0)
1.0 (0.5-1.3)
0.6 (0.5-1.0)
0.66
*p<0.008 versus healthy controls as determined by Kruskal-Wallis non parametric test for significance
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Table E3: Mean fold changes in gene expression of selected targets identified to be
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differentially expressed by microarrays and confirmed by relative real-time PCR.
Gene Symbol
Array Fold
Change
Real-Time PCR
Fold Change
PCR t test p
value
Altered by LPS stimulation (n=9)
TLR2
+ 11.7
+ 20.6
<0.01
OSM
+ 3.2
+ 14.3
<0.01
IL-8
+ 9.2
+ 23.2
<0.01
Altered in resting neutrophils in NEA (n=4) versus EA (n=5)
GADD45B
+3.1
+1.6
0.01
TNFRSF14
+2.2
+1.6
0.05
Altered in LPS stimulated neutrophils in NEA (n=4) versus EA (n=5)
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PLAU
+3.4
+1.1
0.55
CCL23
-5.9
-7.1
0.01
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Table E4: Spearman Rank Correlation Analysis between the expression of evaluated upregulated genes in noneosinophilic asthma and lung function (FEV1% predicted; n=9).
FEV1% predicted
GADD45B
IRAK3
HM74
GADD45B
IRAK3
HM74
MAIL
PI3
STX4A
HLAE
HDAC5
TNFRSF14
-0.70b
-0.77b
-0.72b
-0.78b
-0.83c
-0.67c
-0.67b
-0.77a
-0.60a
0.68b
0.77b
0.63a
0.72b
0.80c
0.73b
0.95c
0.92c
0.82c
0.97c
0.75b
0.80c
0.70b
0.53
0.60a
0.77b
0.70b
0.80c
0.92c
0.63a
0.83c
0.80c
0.75b
0.65a
0.45
0.60a
0.72b
0.53
0.62a
0.70b
0.77b
0.78b
0.72b
0.63a
0.77b
MAIL
PI3
STX4A
HLAE
HDAC5
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Data shows the Spearman rank correlation coefficient (r).
p<0.10, b p<0.05 c p<0.01
a
0.80c