Macrophage secretory products induce an inflammatory phenotype in hepatocytes

Table 1 Demographic and clinical features of patients with chronic hepatitis C virus infection

No. of patients	n	147
Age (yr)	mean ± SEM	42.3 ± 0.9
Sex	M/F	98/49
Viral genotype	1/ 2/ 3	80/6/61
Stage of fibrosis	0/1/2/3-4	13/65/44/25
Grade of steatosis	0/1/2-3	73/44/29
Necroinflammatory score	1-3/4-5/6-8	56/61/30
BMI (kg/m2)	mean ± SEM	26.2 ± 0.4
BMI	Lean, overweight, obese1	66/51/24
Alcohol - current (g/d)	Median (range)	1 (0-120)
Alcohol - past (g/d)	Median (range)	30 (0-500)
Creatinine (μmol/L)	mean ± SEM	77.8 ± 1.5
Platelets (× 109/L)	mean ± SEM	213 ± 6
Red blood cells (× 1012/L)	mean ± SEM	4.8 ± 0.3
Total WBC (× 109/L)	mean ± SEM	7.2 ± 0.2
Neutrophils (× 109/L)	mean ± SEM	4.0 ± 0.1
Lymphocytes (× 109/L)	mean ± SEM	2.3 ± 0.06
Monocytes (× 109/L)	mean ± SEM	0.6 ± 0.02

¹Lean: ≤ 25 kg/m²; overweight: > 25 and < 30 kg/m²; obese: ≥ 30 kg/m². BMI: Body mass index; WBC: White blood cell.

Table 2 Primer and probe sequences used in real-time polymerase chain reaction assays

E-cadherin	for	5’-ATTGCAAATTCCTGCCATTC-3’
	rev	5’-GCTGGCTCAAGTCAAAGTCC-3’
Vimentin	for	5’-GTTTCCAAGCCTGACCTCAC-3’
	rev	5’-GCTTCAACGGCAAAGTTCTC-3
TGF-β1	for	5’-AAGTGGACATCAACGGGTTC-3’
	rev	5’-TGCGGAAGTCAATGTACAGC-3
MMP-9	for	5’-TTCGACGTGAAGGCGCAGATGG-3’
	rev	5’-AACTCACGCGCCAGTAGAAGCG-3’
CD163	for	5’-CCAACAAGATGCTGGAGTGAC-3’
	rev	5’-TGACAGCACTTCCACATTCAAG-3’
Lipocalin-2	for	5’-TCACCCTCTACGGGAGAACCAAGG-3’
	rev	5’-TGTGCACTCAGCCGTCGATACAC-3’
GAPDH	for	5’-TGCACCACCAACTGCTTAGC-3’
	rev	5’-GGCATGGACTGTGGTCATGAG-3’
	probe	5’-CCTGGCCAAGGTCATCCATGACAACTT-3’
HuPO	for	5’-GCTTCCTGGAGGGTGTCC3-3’
	rev	5’-GGACTCGTTTGTACCCGTTG-3’
	probe	5’-TGCCAGTGTCTGTCTGCAGATTGG-3’
18s	for	5’-GCCCGAAGCGTTTACTTTGA-3’
	rev	5’-TCCATTATTCCTAGCTGCGGTATC-3’
	probe	5’-AAAGCAGGCCCGAGCCGCC-3’

E-cadherin: Epithelial cadherin; TGF-β1: Transforming growth factor-β1; MMP-9: Matrix metalloproteinase-9; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; 18s: 18S ribosomal RNA; HuPO: Human acidic ribosomal protein; for: Forward; rev: Reverse.

Table 3 Selected altered gene ontology, KEGG and BioCarta pathways

	ID	Biological process (no. genes)	Differential expression	P valuea
Gene ontology	0034097	Response to cytokine stimuli (80)	↑	0.00125
	0034341	Response to interferon-γ (11)	↑	0.00165
	0000302	Response to reactive oxygen species (73)	↑	0.00123
	0031663	LPS-mediated signalling pathway (12)	↑	0.00144
	0019882	Antigen processing and presentation (58)	↑	0.00172
	0032393	MHC class I receptor activity (15)	↑	0.00160
	0019217	Regulation of fatty acid metabolic process (48)	↓	0.00127
KEGG	hsa00071	Fatty acid metabolism (51)	↓	0.00001
	hsa00120	Bile acid biosynthesis (41)	↓	0.00001
BioCarta	h_tnfr2Pathway	TNFR2 signaling pathway (17)	↑	0.00130
	h_ctlPathway	CTL mediated immune response against target cells (15)	↑	0.00157
	h_nkcellsPathway	Ras-independent pathway in NK-cell-mediated cytotoxicity (22)	↑	0.00179
	h_cd40Pathway	CD40L signaling pathway (15)	↑	0.00221
	h_fxrPathway	FXR and LXR regulation of cholesterol metabolism (7)	↓	0.00135

^aAverage P-value < 0.005 as measured by least-squares permutation, Kolmogorov-Smirnov permutation, Efron-Tibshirani’s gene set analysis test and Goeman’s global test. LPS: Lipopolysaccharide; TNFR2: Tumour necrosis factor receptor 2; NK: Natural killer; CTL: Cytotoxic lymphocyte; FXR: Farnesoid X receptor; LXR: Liver X receptor.

Table 4 The 21 most differentially expressed genes between control and macrophage-conditioned media-treated HepG2 cells as determined by microarray

Gene		Fold change	Differential expression	P value
Lipocalin-2	Lipocalin-2	17.8	↑	< 1E-07
TIMP1	Metallopeptidase inhibitor 1	11.8	↑	< 1E-07
UBD	Ubiquitin D	10.9	↑	< 1E-07
SERPINA3	Serpin peptidase inhibitor clade A member 3	7.5	↑	< 1E-07
IGFBP1	Insulin-like growth factor binding protein 1	7.5	↑	< 1E-07
S100A3	S100 calcium binding protein A3	6.6	↑	< 1E-07
RASD1	RAS dexamethasone-induced 1	6.4	↑	< 1E-07
CEBPD	CCAAT/enhancer binding protein delta	6.2	↑	< 1E-07
SERPINE1	Serpin peptidase inhibitor clade E member 1	5.9	↑	9.00E-07
NDRG1	N-myc downstream regulated gene 1	4.8	↑	< 1E-07
EMP3	Epithelial membrane protein 3	4.6	↑	< 1E-07
DUSP5	Dual specificity phosphatase 5	4.6	↑	< 1E-07
SOD2	Superoxide dismutase 2, mitochondrial	4.4	↑	< 1E-07
F2RL1	Coagulation factor II receptor-like 1	4.4	↑	< 1E-07
CCL20	Chemokine ligand 20	4.2	↑	< 1E-07
SDC4	Syndecan 4	4.1	↑	6.00E-07
TGM2	Transglutaminase 2	4.1	↑	< 1E-07
NR1H4	Nuclear receptor subfamily 1, group H, member 4	4.2	↓	< 1E-07
LIME1	Lck interacting transmembrane adaptor 1	4.2	↓	1.00E-07
DDC	Dopa decarboxylase	4.3	↓	< 1E-07
ANKRD38	Ankyrin repeat domain 38	5.1	↓	< 1E-07