Resolvin E1 Reduces Proinflammatory Markers in Human Pancreatic Islets in vitro

Abstract

Background: In clinical islet transplantation, inflammatory responses initiated by the transplanted islets and by the host immune system cause acute and chronic graft loss. The resolution of acute inflammation is an active process mediated by specific signals and mediators such as resolvin E1 (RvE1). We investigated the effect of RvE1 on i) the inflammatory status of human pancreatic islets, ii) islet viability and apoptosis, and iii) the instant blood-mediated inflammatory reaction (IBMIR) in vitro.

Methods: Pro-inflammatory cytokines and tissue factor (TF) in isolated human islets were determined by real-time RT-qPCR (mRNA levels), CBA and Gyrolab bioaffy (protein levels) after lipopolysaccaride (LPS) stimulation. Islet viability was measured using insulin secretion in a dynamic model, ADP/ATP ratio and total ATP content. Apoptosis was measured using commercial kits after stimulation with proinflammatory cytokines. To assess effect on IBMIR, human islets were mixed with non-anticoagulated, RvE1 or vehicle pretreated ABO-compatible blood in heparin-coated tubing loops.

Results: Treatment of human islets with RvE1 (500 nM) for 24 h reduced LPS-induced increase in mRNA and protein levels of selected pro-inflammatory markers (IL-8, MCP-1, and TF). RvE1 lowered the ADP/ATP ratio, but had no effect on insulin secretion. RvE1 reduced the apoptotic effect of proinflammatory cytokines. Additionally, RvE1 reduced platelet consumption and TAT complex formation during the first 5 min after islet-blood contact.

Conclusions: RvE1 suppresses proinflammatory markers and lowers the ADP/ATP ratio in human islets in vitro. RvE1 demonstrates anti-apoptotic effects in a proinflammatory milieu. Additionally, RvE1 has modest dampening effects on IBMIR. We conclude that RvE1 may have potential in clinical islet transplantation.

Key words

islet - transplantation - resolvin - cytokine - IBMIR

References

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Supplementary Data Online

[Fig. 6], [7], [Table 2.]

Fig. 6 Dose-response experiments. Dose-response of RvE1 on key inflammatory mediators in human islets, showing best effect at 500 nM IL-8 (A), MCP-1 (B) and TF (C) were used to assess the effect of increasing doses of RvE1 on human islets. Glucocorticoids (MP, methylprednisolone, 1 μg/ml) were used as a positive control, while lipopolysaccharide (LPS, 1 μg/ml) was used as a negative control. Substance exposure was 24 h. Cycle times for the highest-expressing group are shown in the corresponding bar. n=6, purity ranging from 50–95%. *p<0.05, significantly different from vehicle treated. Data represent the mean±SEM.

Fig. 7 Calculation of dynamic insulin secretion (example) Pictures of hand-picked islets was analyzed with Cellimage^® (Andrew Friberg, Uppsala, Sweden), mean islet size calculated, and insulin secretion normalized to mU/ml insulin per 100 islet equivalents (IEQ). This example represent untreated, control islets.

Table 1 Donor and islet preparation characteristics.

Donor pancreas	Age (years)	Sex	Cold ischaemia time (h)	BMI weight (kg/m2)	Pancreas weight (g)	Islet Equivalents	Purity of fractions (%)
1	46	female	12.3	24	93	83 600	50 and 65
2	54	male	3.5	29	133	220 000	85 and 90
3	55	female	13	22	95	388 000	95
4	56	female	6.3	27	122	335 000	90 and 95
5	77	female	12.5	21	90	195 000	50 and 75
6	34	male	11.5	26	105	283 000	55 and 70
7	54	male	7.5	25	110	143 000	70 and 80
8	32	male	6	25	118	94 000	60 and 90
Donor pancreas	Insulin/DNA content (ng)	Insulin Stimulation Index ratio	ADP/ATP (pmol/μg DNA)	TF content	MCP-1(pmol/μg DNA)	IL-8 (pmol/μg DNA)
1	7.1	6.1	0.2	0.025	0.004	0.013
2	6.9	3.4	0.19	0.009	0.011	0.038
3	1.5	9.3	0.07	0.027	0.006	0.014
4	5.4	12	<0.05	0.022	0.002	0.008
5	6.1	7.1	<0.05	0.049	0.012	0.058
6	5.9	1.4	<0.05	0.039	0.011	0.057
7	4.6	1.1	0.11 0	107	–	0.055
8	3.3	4	<0.05	0.04	–	0.017

Effect of RvE1 on human islet gene expression in a TaqMan low density array

The table shows expression levels based on the individual ΔCt-values and is denoted as high, medium and low. No expression is denoted (-). Islets were treated with or without RvE1 (500 nM) for 24 h, with addition of lipopolysaccharide (LPS, 1 μg/ml) after 18 h. The genes tended to be upregulated or downregulated in RvE1 treated islets correspond to a p value below 0.15. n=4, purity ranging from 65–95%. Data represent the mean±SEM. For method, see below.

TaqMan low density array

In the current study, we applied a TaqMan LDA card to examine the expression of 48 genes in human islets after exposure to RvE1 (500 nM) for 24 h and compared the results to vehicle treated islets (controls). Aliquots (n=2) of islets from 2 independent donors in quadruplicates where used. The 48 genes belonged to several pathways involved in inflammation, coagulation and apoptosis. 1 μg of RNA was reverse transcribed using the High Capacity Archive Kit (Applied Biosystems) in a final volume of 50 μL. For real-time PCR analysis we created a TaqMan LDA card based on a 7900 HT Micro Fluidic card (Applied Biosystems, Foster City, CA). Each gene was present as duplicates in the analysis. GAPDH was used as a reference gene. The total volume of each PCR reaction was 1 μL containing an equivalent of 2ng RNA. The data acquired were analyzed with the Sequence Detector software (version 1.6.3, Applied Biosystems) and the relative gene expression levels were calculated using the 2-ΔΔCT method (Livak & Schmittgen 2001). To provide a mRNA expression profile of RvE1 exposed islets and controls, we displayed the expression levels as high, medium and low, based on ΔCt values (Ct (target gene)-Ct (GAPDH)) ([Table 2] ). The groups were made by dividing the range between the lowest and highest ΔCt into three groups for high, medium and low expression. Genes with a ΔCt above 15 cycles were defined as absent. For the calculation of differential expression in RvE1 exposed islets compared to controls, the ΔCt-value for RvE1 was divided with ΔCt-value for controls (values >1: downregulation, and <1: upregulation). Trends obtained were used for targeted real-time qRT-PCR analysis.

Reference

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Table 2 Expression of genes on the Taqman^®Low Density Array.

Gene	Assay ID	Mean expression level	ΔCt RvE1/ΔCt Control
			Mean	p-value
NFKB1	Hs00765730_m1	medium	1.30	0.52
CCL2	Hs00234140_m1	medium	1.04	0.39
SMAD3	Hs00232222_m1	medium	1.00	0.87
DIABLO	Hs00219876_m1	medium	1.04	0.27
SLC30A10	Hs00218883_m1	low	0.98	0.12
CCR2	Hs00174150_m1	–	–	–
INS	Hs02741908_m1	high	0.98	0.22
CD40	Hs00386848_m1	medium	1.02	0.65
CASP8	Hs00154256_m1	low	0.98	0.33
BAD	Hs00188930_m1	low	1.05	0.27
TNF	Hs00174128_m1	low	1.04	0.08
VEGFA	Hs00900054_m1	medium	0.98	0.38
TLR4	Hs00152939_m1	medium	1.00	1.00
IL15	Hs00542562_m1	low	1.09	0.44
CCL3	Hs00234142_m1	low	1.02	0.39
FOXP3	Hs00203958_m1	low	0.99	0.86
JAK1	Hs00233820_m1	low	1.03	0.67
CXCL10	Hs00171042_m1	low	1.07	0.20
ANGPT1	Hs00181613_m1	low	0.96	0.41
IL8	Hs00174103_m1	high	1.23	0.10
STAT1	Hs00234829_m1	medium	1.00	0.99
CCR5	Hs00152917_m1	low	1.09	0.04
BAX	Hs00180269_m1	medium	1.04	0.51
EGFR	Hs00193306_m1	medium	1.08	0.35
SSTR5	Hs00265647_s1	low	1.02	0.62
CASP3	Hs00234385_m1	low	1.03	0.13
CCL5	Hs00174575_m1	medium	1.04	0.10
CXCL12	Hs00930455_m1	low	1.00	0.92
IL1B	Hs00174097_m1	medium	1.02	0.31
GCG	Hs00174967_m1	high	0.98	0.39
INSR	Hs00169631_m1	medium	1.04	0.35
C5	Hs00156197_m1	low	0.98	0.48
IL4	Hs00174122_m1	–	–	–
IL18	Hs00155517_m1	low	1.05	0.03
IFNG	Hs00174143_m1	–	–	–
TXNIP	Hs00197750_m1	medium	1.03	0.21
ACTB	Hs99999903_m1	medium	0.82	0.28
IL6	Hs00174131_m1	low	1.00	0.83
FAS	Hs00163653_m1	low	1.01	0.81
TGFB1	Hs99999918_m1	medium	0.95	0.03
SLC39A1	Hs00205358_m1	medium	0.98	0.64
MAPK1	Hs00177066_m1	medium	0.99	0.78
CASP9	Hs00154261_m1	low	1.03	0.08
CCL4	Hs99999148_m1	medium	1.04	0.29
BCL2	Hs00153350_m1	low	0.97	0.61
IL10	Hs00174086_m1	low	1.16	0.53
ICAM1	Hs00164932_m1	medium	1.02	0.33

Correspondence