NOD2- and disease-specific gene expression profiles of peripheral blood mononuclear cells from Crohn’s disease patients

Table 1 Primer for NOD2-genotyping

SNP	Primer
8	Forward: 5’CCTCTTCAATTGTGGCAGGC-3’
	Reverse: 5’-CTCCTGCATCTCGTACAGGC-3’
12	Forward: 5’-ATGGAGGCAGGTCCACTTTG-3’
	Reverse: 5’-TTACCTGAGCCACCTCAAGC-3’
13	Forward: 5’-GATGGTACTGAGCCTTTGTTGA-3’
	Reverse: 5’-CAGACTTCCAGGATGGTGTCAT -3’

Table 2 Numbers and maximum changes of up- and downregulated genes in peripheral blood mononuclear cells from Crohn’s disease patient vs identically treated controls

Treatment of PBMCs	Upregulated genes	Downregulated genes
Untreated	85 (59-fold)	39 (39-fold)
Vitamin D3	25 (21-fold)	12 (9-fold)
LPS/PGN	54 (6-fold)	15 (5-fold)
Vitamin D3 + LPS/PGN	29 (15-fold)	8 (5-fold)

P < 0.05; Threshold: ≥ 2-fold change. PBMCs: Peripheral blood mononuclear cells; LPS: Lipopolysaccharide; PGN: Peptidoglycan.

Table 3 Genes selected for real-time PCR studies

Transcript	Fold changes: patients vs controls				Details on function/ reasons to study
	Basal	+D, -L/P	-D, +L/P	+D, +L/P
MSR1	9.56	13.19		14.72	macrophage scavenger receptor[49]; differentially expressed in 3 of 4 groups
CD101	2.66				expressed on various immune cells; inhibits expansion of colitogenic T cells[30]
CLEC5A		2.82		3.11	C-type lectin member 5A, pattern recognition receptor; involved in antibacterial/antiviral defense[43]
CLEC7A	4.53			3.94	C-type lectin member 7A, pattern recognition receptor; control of fungal infections[50]
CLEC12A	6.04	4.52			C-type lectin member 12A, pattern recognition receptor, inhibits cell death-induced inflammation[51]
ITGAM	2.18				CD11b; integrin αM; expressed by many immune cells; polymorphisms linked to autoimmunity[52]
LYZ			3.51	2.03	antimicrobial enzyme; essential role in innate immunity; increased production linked to CD[31]
PECAM1	3.15				CD31; implicated in transendothelial leukocyte migration in experimental colitis[32]
CCL20	-2.33		-5.08		chemokine expressed by neutrophils, enterocytes, Bcells and dendritic cell; IBD predilection gene[33]
CXCL5	-38.89		-5.32		regulates neutrophil homeostasis and chemotaxis; increased serum levels in IBD patients reported[34]
IL-24			-3.49	-4.82	Involved in host defence against bacteria and fungi; increased expression in patients with active IBD[35]
TREM1					amplifier of antimicrobial immune responses and inflammation in experimental colitis and IBD[36]

P < 0.05; Threshold: ≥ 2-fold change. Positive values refer to genes upregulated and negative values to genes downregulated in CD patients. LPS: Lipopolysaccharide; PGN: Peptidoglycan; PBMCs: Peripheral blood mononuclear cells. CD: Crohn’s disease; IBD: Inflammatory bowel diseases.

Table 4 Characteristics of the patients -nucleotide-binding oligomerization domain 2 status, classification and activity of the disease, C-reactive protein and vitamin D levels, and medication at the time of the study

No.	Sex	Age	NOD2	Montreal classification	CDAI	CRP (mg/L)	Vitamin D (nmol/L)	Prednisolon (> 10 mg/d)	Azathio-prine	Anti-TNF-α
1	M	24	WT1	A2 L3 L4 B1	121	2.58	104.0	Yes	No	Yes
2	M	28	WT1	A2 L3 B3p	46	< 1.0	62.9	No	No	Yes
3	M	64	WT1	A3 L3 B2p	111	4.58	27.0	Yes	No	Yes
4	F	60	WT	A2 L3 B2	100	2.27	72.5	No	No	Yes
5	F	62	WT	A3 L3 B3p	82	< 1.0	58.2	Yes	Yes	Yes
6	M	46	WT	A2 L3 L4 B2p	110	4.97	32.9	No	No	Yes
7	M	38	HO	A2 L3 B3p	92	13.30	40.2	No	Yes	No
8	M	64	HO	A3 L3 B1	34	< 1.0	53.0	No	No	Yes
9	F	48	HO2	A2 L3 B2	54	< 1.0	67.6	No	No	Yes
10	M	54	HO	A2 L3 B2p	103	1.63	51.9	No	Yes	No
11	M	26	HO	A1 L3 B2p	120	7.86	31.0	No	Yes	Yes
12	M	27	HT	A2 L3 B1	106	1.41	47.2	No	No	Yes
13	M	37	HT	A2 L3 B2p	166	4.56	37.9	No	Yes	No
14	F	48	HT	A2 L1 B3p	115	1.01	55.2	No	Yes	Yes
15	M	57	HT	A1 L3 B2p	132	12.30	52.3	No	No	Yes
16	M	47	HT	A2 L3 B3	60	2.62	92.0	No	Yes	No

¹Patients who were also included into microarray analysis;

²SNP8 mutation. All other HO/HT mutations refer to SNP13. NOD2: Nucleotide-binding oligomerization domain 2; CDAI: Crohn’s disease activity index; CRP: C-reactive protein; TNF : Tumor necrosis factor; WT: Wild-type; HO: Homozygous mutation; HT: Heterozygous mutation.

Table 5 differentially expressed transcripts in vitamin D-pretreated peripheral blood mononuclear cells from Crohn’s disease patients and controls (with and without additional stimulation with lipopolysaccharide and peptidoglycan, respectively, age adjusted)

Gene	P value	Upregulated in
CLEC5A	0.030	CD patients
LYZ	0.047	CD patients
TREM1	0.023	CD patients

CD: Crohn’s disease.

Table 6 Transcripts with a NOD2-dependent expression pattern

Gene	P value	Highest levels
CD101	0.002	Heterozygotes
CLEC5A	0.020	Heterozygotes
CXCL5	0.009	Heterozygotes
IL-24	0.044	Heterozygotes
ITGB2	0.041	Heterozygotes
LYZ	0.042	Heterozygotes