Gene expression changes in human islets exposed to type 1 diabetic serum

Figures & data

Figure 1. Complement deposition on islets exposed to type 1 diabetic serum. Isolated islets were fixed and cryosectioned at 6 μm thickness and consecutive sections were used per condition. Islet sections were incubated in a 1:2 dilution of autologous serum (A–D), allogeneic serum (E–H) or type 1 diabetic serum (I–L) at 37°C for 2 h. Sections were washed and labeled with rabbit polyclonal anti-C3-FITC (green) or mouse monoclonal anti-insulin conjugated to AF647 (red). Only type 1 diabetic serum displayed signs of complement deposition (K), which corresponded to an islet cell as evidenced by positive insulin staining (J). DAPI was used for counterstaining (blue).

Figure 2. Complement activation initiated by auto-antibodies in type 1 diabetic serum induce a specific profile of gene expression. Islets exposed to complement in diabetic serum or control sera (allogenic or autologous) were compared using Illumina micro-array analysis. The resulting data was analyzed using the GeneSpring Software. Supervised clustering revealed 679 differentially expressed genes in diabetic serum when compared with autologous serum. Data from one representative experiment out of five independent experiments is shown.

Figure 3. class prediction analysis of islets exposed to type 1 diabetic serum. One representative experiment was chosen for class prediction analysis to establish a list of genes that could differentiate the serum conditions based on gene expression changes. Samples were normalized to the mean of the autologous condition. Yellow reflects unchanged expression, blue reflects downregulation and red reflects upregulation.

Table 1. Average signal intensity for class predictor genes in T1DM serum-treated islets

Gene ID	Average	p	Gene ID	Average	p
RRAD*	343.17±62.37	0.0008	ARL4	131.02±11.24	0.0083
GMNN	284.59±40.54	<0.0001	ZNF644	130.73±10.43	0.0016
ANKRD1	279.99±45.87	0.0003	ZNF658	129.53±20.99	0.2211
CCL4L1	266.89±57.03	0.1038	CCL3	122.87±18.69	0.2104
1L-1B*	255.66±31.31	<0.0001	CCL3L3	120.05±18.04	0.2372
IL-23A	251.37±35.85	0.0002	CNNM2	119.42±15.84	0.347
MMP9*	243.37±35.13	0.0001	RND1	117.11±14.88	0.3477
1L11*	220.12±45.46	0.0135	KYNU	116.29±16.86	0.3803
ENC1	219.3±15.27	<0.0001	CCL3L1	112.77±16.41	0.462
SDS	210.56±26.46	0.0004	SERPINB2	109.23±17.98	0.5174
1780523	192.66±32.69	0.01	SLC25A36	101.29±11.33	0.9154
HLA-E	187.16±32.35	0.2664	NR1D2	97.71±10.52	0.3283
STAT5A	187.03±35.81	0.2182	SFRS7	96.49±10.16	0.7461
FAM43A	184.9±37.17	0.0263	TLCD1	85.71±7.8	0.0828
ZNF659	183.94±30.03	0.0049	NR5A2	83.99±14.84	0.2859
GBP1	181.43±16.84	<0.0001	GCNT3	79.71±13.39	0.115
NEXN	179.03±15.65	<0.0001	ARRDC3	72.9±11.45	0.0576
EPC1	175.61±16.43	0.0001	CXCL2	68.26±11.37	0.6288
C1QR1	158.63±36.78	0.7955	SDCBP2	66.73±8.78	0.004
VNN1	148.68±108.07	0.2311	HSPC065	64.27±10.42	0.0243
CXCL1	146.62±19.66	0.0256	IL-1RN*	64±6.02	0.0049
NCOA7	145.57±19.01	0.0216	CCL20	61.86±8.36	0.0008
NEDD9	137.78±18.92	0.0161	ANGPTL4	48.1±7.76	<0.0001
IL-18R1	136.37±30.01	0.8777	SPRY1	37.13±6.24	<0.0001
SLC25A3	133.19±15.01	0.0434	MT1E	34.48±10.71	<0.0001
IL-12A*	132.94±13.7	0.0578	LAMB3	33.93±18.56	0.3128

* Indicates gene expression corroborated with Q-PCR

Figure 4. Real time quantitative PCR analysis confirms micro-array data in representative genes. Representative genes were chosen for corroboration by using relative quantification real-time PCR. TaqMan probes specific for MMP-9 (A), IL-1β (B), IL-12A (C), IL-11 (D), RAD (E), and IL-1RN (F) were used to produce cDNA from representative mRNA samples from each experiment. Real-time PCR detected significant gene expression changes of 7.50 ± 3.17 (p = 0.07), 2.23 ± 0.33 (p = 0.005), 3.19 ± 0.66 (p = 0.01), 1.92 ± 0.53 (p = 0.12), 6.13 ± 1.87 (p = 0.03), 0.42 ± 0.14 (p = 0.003) respectively. Data expressed as mean ± SEM.