Killing of Escherichia coli by Crohn’s disease monocyte-derived macrophages and its enhancement by hydroxychloroquine and Vitamin D. Paul K. Flanagan, Direkrit Chiewchengchol, Helen Wright, Steven W. Edwards, Abdullah Alswied, Jack Satsangi, Sreedhar Subramanian, Jonathan M. Rhodes and Barry J. Campbell.
Supplementary file S1
Table S1 – Inclusion and exclusion criteria for recruitment to provide blood for monocyte isolation, quantification of serum Vitamin D and hsCRP levels, and genomic analysis.
Inclusion/Exclusion criteria
Inclusion 1) Patient or control is willing to participate in the study and has provided informed consent 2) Patients or controls aged 16 or over
3) Patients’ with Crohn’s disease diagnosed by conventional clinical, radiological and histological criteria. The disease can be either active or inactive
4) Controls: Patients with a diagnosis of Irritable bowel syndrome diagnosed by standard means, or normal volunteers with no GI disorder and no personal or family history of inflammatory bowel disease
Exclusion 1) Patients under 16 or unable to give informed consent.
2) Patients receiving anti-tumour necrosis factor (anti-TNF) medications
3) Patients receiving corticosteroids
4) Patients on immunosuppressants
5) Patients who are pregnant, breastfeeding or <3 months post-partum
Killing of Escherichia coli by Crohn’s disease monocyte-derived macrophages and its enhancement by hydroxychloroquine and Vitamin D. Paul K. Flanagan, Direkrit Chiewchengchol, Helen Wright, Steven W. Edwards, Abdullah Alswied, Jack Satsangi, Sreedhar Subramanian, Jonathan M. Rhodes and Barry J. Campbell.
Table S2: Comparison of baseline characteristics for healthy controls and Crohn’s disease patients.
Healthy controls (N=10)
Crohn’s disease
(N=10) P value
Male (%) 50% 20% 0.177
Age (y) 51.7 ± 3.8 44.8 ± 5.2 0.297
Smoker (%) 10% 30% 0.290
Harvey Bradshaw Index n/a 5.0 ± 1.7 n/a
Serum Vitamin D3 (nmol/L) 30.8 ± 4.7 26.0 ± 3.7 0.433
hsCRP (mg/L) 2.2 ± 0.6 7.8 ± 2.0 0.03
NOD2/CARD15 homozygote wt 90% 80% 0.556
NOD2/CARD15 heterozygote 10% 20% 0.556
ATG16L1 homozygote wt 40% 40% 0.999
ATG16L1 heterozygote 60% 50% 0.677
ATG16L1 homozygote variant 0% 10% 0.343
IRGM homozygote wt 80% 70% 0.628
IRGM heterozygote 20% 30% 0.628
Data presented as % population affected or mean ± SEM as relevant.
Specific SNPs analyzed; NOD2/CARD15 (rs2066844, rs2066845 and rs2066847), ATG16L1 (rs2241880) and IRGM (rs13361189) hsCRP, high-sensitivity C-reactive protein; n/a, not applicable; wt, wild-type
P values as shown obtained by Mann-Whitney U.
Killing of Escherichia coli by Crohn’s disease monocyte-derived macrophages and its enhancement by hydroxychloroquine and Vitamin D. Paul K. Flanagan, Direkrit Chiewchengchol, Helen Wright, Steven W. Edwards, Abdullah Alswied, Jack Satsangi, Sreedhar Subramanian, Jonathan M. Rhodes and Barry J. Campbell.
Table S3: Individual baseline characteristics with results of genomic analysis.
Sex Age Current smoker
Montreal Harvey Bradshaw
Index
Vitamin D3 (nM)
hsCRP (mg/L)
No. of altered alleles NOD2 (rs2066844)
No. of altered alleles NOD2 (rs2066845)
No. of altered alleles NOD2 (rs2066847)
No. of altered alleles ATG16L (rs2241880)
No. of altered alleles IRGM (rs13361189)
Fold change in intra- macrophage
HM605
Fold change in intra- macrophage
K12
Fold change in intra- macrophage
Staph
Relative MDM induced Neutrophil chemotaxis
HC 1 M 36 No n/a n/a 45 0.4 0 0 0 1 0 1.56 1.80 0.58 n/d
HC 2 F 59 Yes n/a n/a 20 3.07 0 0 0 1 0 1.39 1.75 0.55 2.95
HC 3 F 56 No n/a n/a 42 5.66 0 0 0 0 1 0.39 0.31 0.54 2.34
HC 4 M 51 No n/a n/a 46 0.57 0 0 0 0 0 3.00 0.41 0.61 2.49
HC 5 M 74 No n/a n/a 22 2.65 0 0 0 0 0 2.00 0.18 0.26 2.46
HC 6 F 34 No n/a n/a 10 0.35 0 0 0 1 0 1.00 0.65 0.54 2.70
HC 7 M 57 No n/a n/a 41 4.62 0 0 0 1 0 1.00 1.04 0.55 3.07
HC 8 F 43 No n/a n/a 29 3.37 0 0 0 1 0 1.56 1.24 0.41 2.56
HC 9 M 59 No n/a n/a 8 1.12 0 0 0 0 0 1.57 0.58 0.25 n/d
HC 10 F 48 No n/a n/a 45 0.63 1 0 0 1 1 n/d 0.65 0.49 n/d
CD 1 F 38 No A2, L1,
B3p
0 31 2.75 1 0 0
0 0 0.00 0.00 0.04
3.00
CD 2 F 64 Yes A3, L1, B2 5 12 14.15 0 0 1 1 0 1.42 0.11 0.42 3.19
CD 3 F 45 No A2, L1, B2 0 23 1.46 0 0 0 1 1 0.15 0.37 0.18 2.43
CD 4 F 21 No A1, L3, B1 2 13 1.04 0 0 0 0 1 1.28 1.22 1.09 2.31
CD 5 F 48 No A2, L1, B2 7 43 18.3 0 0 0 2 0 3.43 0.84 0.38 2.24
CD 6 M 39 No A2, L1, B2 10 16 10.14 0 0 0 0 0 2.40 1.12 0.61 1.94
CD 7 F 73 No A3, L2, B1 9 38 14.91 0 0 0 0 0 0.23 0.17 0.17 2.27
CD 8 M 24 No A1, L1,
B3p
1 36 3.95 0 0 0
1 1 0.38 0.18 0.20
2.87
CD 9 F 40 Yes A2, L1, B2 16 33 4.84 0 0 0 1 0 1.20 1.16 0.31 2.80
CD10 F 56 Yes A2, L3,
B2p
0 15 6.95 0 0 0
1 0 0.36 0.27 0.25
2.48 Sex Age Current
smoker
Montreal Harvey Bradshaw
Index
Vitamin D3 (nM)
hsCRP (mg/L)
No. of altered alleles NOD2 (rs2066844)
No. of altered alleles NOD2 (rs2066845)
No. of altered alleles NOD2 (rs2066847)
No. of altered alleles ATG16L (rs2241880)
No. of altered alleles IRGM (rs13361189)
Fold change in intra- macrophage
HM605
Fold change in intra- macrophage
K12
Fold change in intra- macrophage
Staph
Relative MDM induced Neutrophil chemotaxis
HC 1 M 36 No n/a n/a 45 0.4 0 0 0 1 0 1.56 1.80 0.58 n/d
HC 2 F 59 Yes n/a n/a 20 3.07 0 0 0 1 0 1.39 1.75 0.55 2.95
HC 3 F 56 No n/a n/a 42 5.66 0 0 0 0 1 0.39 0.31 0.54 2.34
HC 4 M 51 No n/a n/a 46 0.57 0 0 0 0 0 3.00 0.41 0.61 2.49
HC 5 M 74 No n/a n/a 22 2.65 0 0 0 0 0 2.00 0.18 0.26 2.46
HC 6 F 34 No n/a n/a 10 0.35 0 0 0 1 0 1.00 0.65 0.54 2.70
HC 7 M 57 No n/a n/a 41 4.62 0 0 0 1 0 1.00 1.04 0.55 3.07
HC 8 F 43 No n/a n/a 29 3.37 0 0 0 1 0 1.56 1.24 0.41 2.56
HC 9 M 59 No n/a n/a 8 1.12 0 0 0 0 0 1.57 0.58 0.25 n/d
HC 10 F 48 No n/a n/a 45 0.63 1 0 0 1 1 n/d 0.65 0.49 n/d
CD 1 F 38 No A2, L1,
B3p
0 31 2.75 1 0 0
0 0 0.00 0.00 0.04
3.00
CD 2 F 64 Yes A3, L1, B2 5 12 14.15 0 0 1 1 0 1.42 0.11 0.42 3.19
CD 3 F 45 No A2, L1, B2 0 23 1.46 0 0 0 1 1 0.15 0.37 0.18 2.43
CD 4 F 21 No A1, L3, B1 2 13 1.04 0 0 0 0 1 1.28 1.22 1.09 2.31
CD 5 F 48 No A2, L1, B2 7 43 18.3 0 0 0 2 0 3.43 0.84 0.38 2.24
CD 6 M 39 No A2, L1, B2 10 16 10.14 0 0 0 0 0 2.40 1.12 0.61 1.94
CD 7 F 73 No A3, L2, B1 9 38 14.91 0 0 0 0 0 0.23 0.17 0.17 2.27
CD 8 M 24 No A1, L1,
B3p
1 36 3.95 0 0 0
1 1 0.38 0.18 0.20
2.87
CD 9 F 40 Yes A2, L1, B2 16 33 4.84 0 0 0 1 0 1.20 1.16 0.31 2.80
CD10 F 56 Yes A2, L3,
B2p
0 15 6.95 0 0 0
1 0 0.36 0.27 0.25
2.48
