An ex vivo RT-qPCR-based assay for human peripheral leukocyte responsiveness to glucocorticoids in surgically induced inflammation

Figures & data

Figure 1 Gene expression of GC-regulated genes in peripheral leukocytes in response to 1 µM dexamethasone (DEX) in healthy blood donors (BD1, n=21, A) and in surgical patients (PAT1, n=9, B) using the endogenous control G6PD.

Notes: *P<0.05, **P<0.01, and ***P<0.0001, vs unstimulated sample. Data are presented as box plots showing median and IQR with whiskers at quartiles ±1.5 IQR.

Abbreviations: FKBP5, FK506 binding protein 5; G6PD, glucose-6-phosphate dehydrogenase; GILZ, glucocorticoid-induced leucine zipper; GRα, glucocorticoid receptor alpha; HLA-DR, human leukocyte antigen DR; IL1R2, interleukin 1 receptor II; IQR, interquartile range.

Table 1 Characteristics of patients, procedures, and outcomes

Patient cohort 1 (PAT1)	n	Median (IQR)	Patient cohort 2 (PAT2)	n	Median (IQR)
Type of surgery
Pancreas	1		Pancreas	5
Liver	5		Liver	13
Esophagus	2		Spleen	2
Gastric	2		Other	1
Total	10		Total	21
Sex
Female	3			9
Male	7			12
Age (years)		62.90 (56.60–73.39)			66.29 (59.84–72.63)
ASA class
1	4
2	5			12
3	1			9
Duration of operation (minutes)		295 (227–420)*			210 (165–322)
Blood loss (mL)		1,000 (800–1,600)**			500 (350–1,000)
Clavien–Dindo grade
I	6			7
II	4			9
IIIa				1
IIIb				1
IV				1
V				2
ICU-LOS (days)		3.5 (3–6)			2 (0–3)
Length of stay in the hospital (days)		12 (10–29)			10 (8–14)

Notes:

* Missing data for three patients;

** missing data for one patient.

Abbreviations: ICU-LOS, length of stay in the intensive care unit; IQR, interquartile range; ASA class, American Society of Anesthesiologists class.

Figure 2 Correlation between preoperative gene expression of GILZ (P<0.05, A) and GRα (P<0.05, B) in response to 10 nM DEX in peripheral leukocytes from the patient cohort 2 (PAT2) (surgical patients, n=21) and length of stay (days) intensive care unit (ICU).

Note: Scatterplot with fitted regression lines with 95% confidence interval of predicted means.

Abbreviations: GILZ, glucocorticoid-induced leucine zipper; GRα, glucocorticoid receptor alpha.

Table 2 Cortisol and markers of inflammation and metabolism in patient cohort 2 (PAT2)

	Preoperative		Postoperative day 1		P value
	n	Median (IQR)	n	Median (IQR)
Cortisol (nmol/L)	20	396 (227–514)	21	434 (169–697)	0.654
C-reactive protein (mg/L)	20	2 (1–8)	18	52 (26–76)	<0.0001
Interleukin 6 (ng/L)	20	16.3 (10.9–26.2)	18	56.2 (24.2–86.2)	0.009
White blood cells (10^9/L)	21	5.1 (3.9–6.5)	21	10.4 (8.2–14.4)	<0.0001
Neutrophils (10^9/L)	21	2.8 (1.7–4)	21	8.6 (6.3–11)	<0.0001
Lymphocytes (10^9/L)	21	1.4 (1.2–2.0)	21	1.0 (0.6–1.3)	0.010
Monocytes (10^9/L)	21	0.5 (0.3–0.8)	21	0.8 (0.6–0.9)	0.004
Adiponectin (mg/L)	21	6.7 (4.7–9.2)	21	4.4 (3.8–6.3)	<0.0001
Resistin (mg/L)	21	2.5 (2.3–3.9)	21	5 (3.8–6.8)	<0.0001
Fasting glucose (mmol/L)	21	5.9 (5.4–6.8)	21	8.3 (7.4–9)	<0.0001
Insulin (pmol/L)	21	55.2 (43.4–78.8)	21	97.4 (60.8–209)	0.017
C-peptide (nmol/L)	21	0.86 (0.81–1.1)	21	1.3 (0.82–1.85)	0.058
Non-esterified fatty acids (mmol/L)	21	0.47 (0.35–0.66)	21	0.46 (0.31–0.57)	0.192

Abbreviation: IQR, interquartile range.

Figure S1 Correlation between preoperative gene expression of IL1R2 (P=0.06) in response to 10 nM dexamethasone (DEX) in peripheral leukocytes from the PAT2 cohort and length of stay (days) in the intensive care unit (ICU).

Note: Scatterplot with fitted regression lines with 95% confidence interval of predicted means.

Figure S2 Correlation between preoperative gene expression of IL1R2 (P=0.051) in response to 10 nM dexamethasone (DEX) in peripheral leukocytes from the PAT2 cohort and Clavien–Dindo grade of complications.

Note: Scatterplot with fitted regression lines with 95% confidence interval of predicted means.

Table S1 Primer and probe sequences for investigated genes and endogenous control genes

Gene	Forward primer	Reverse primer	Probe (FAM-TAMRA)
IL1R2	CCCTCAAGACCATATCAGCTTCTC	TGTGCCGGTTCCCAGAAA	TCAAGACTGACAATCCCGTGTAAGGT
FKBP5	GGAGCAGGCTGCCATTGT	ACCGCCTGCATGTATTTGC	AGAGAAGGGAACCGTATACTTCAAGGGA
GILZ	CGTGGTGGCCATAGACAACA	CCTCTCTCACAGCATACATCAGATG	CGAACAGGCCATGGATCTGGTGAAG
HLA-DR	ACTGGGAGTTTGATGCTCCAA	CCCACCAGACCCACAGTCA	ACTACAGAGAACGTGGTGTGTGCCCTGG
GRα	CATTGTCAAGAGGGAAGGAAACTC	TTAAGGAGATTTTCAACCACTTCATG	CAGAACTGGCAGCGGTTTTATCAACTGAC
B2m	GAGTATGCCTGCCGTGTG	AATCCAAATGCGGCATCT	CCTCCATGATGCTGCTTACATGTCTC
Cyclophilin	ACGGCGAGCCCTTGG	TTTCTGCTGTCTTTGGGACCT	CGCGTCTCCTTTGAGCTGTTTGCA
RPLP0	CTTGTCTGTGGAGACGGATTACAC	CCACAAAGGCAGATGGATCA	CCCACTTGCTGAAAAGGTCAAGGCC
G6PD	TGCCCCCGACCGTCTAC	ATGCGGTTCCAGCCTATCTG	ACTCGTGAATGTTCTTGGTGACGGCC
ABL	TGGAGATAACACTCTAAGCATAACTAAAGGT	GATGTAGTTGCTTGGGACCCA	CCATTTTTGGTTTGGGCTTCACACCATT

Table S2 Gene expression of candidate endogenous control genes in healthy blood donors (n=9)

	Unstimulated	10 nM DEX	1 μM DEX
ABL
Median	26.56	26.37	24.85
IQR	25.39–26.77	25.65–26.91	24.17–27.26
B2M
Median	17.00	16.84	16.64
IQR	16.45–17.40	16.43–17.83	16.24–18.76
Cyclophilin
Median	21.16	21.34	20.86
IQR	20.30–21.44	20.40–21.67	20.07–22.36
G6PD
Median	24.66	23.89	23.42
IQR	23.98–25.12	22.73–24.71	22.07–24.85
RPLP0
Median	20.05	20.23	19.66
IQR	19.47–20.79	19.40–20.72	19.08–21.33

Note: Gene expression is displayed as medians and interquartile range (IQR) of crude CT values.

Abbreviations: DEX, dexamethasone; ABL, Abelson protein; B2M, Beta-2-microglobulin; G6PD, glucose-6-phosphate dehydrogenase; RPLP0, Ribosomal protein, large, P0.

Table S3 Gene expression of endogenous control genes in surgical patients (n=21)

	Preoperative	Postoperative
B2M
Median	27.14	26.56
IQR	26.04–27.56	24.83–27.21
Cyclophilin
Median	28.85	27.71
IQR	27.34–29.23	26.09–28.67
RPLP0
Median	29.40	28.93
IQR	28.97–29.9	27.92–29.45

Note: Gene expression is displayed as medians and interquartile range (IQR) of crude CT values.

Abbreviations: B2M, Beta-2-microglobulin; RPLP0, Ribosomal protein, large, P0.