Background
Materials and methods
Patient selection and tissue collection
Patient group | Age | Sex | Pathology/COD | Tissue used | Seizure frequency | iEcOG spikes | Anti-epileptic drugs | Engel score (1 yr) |
---|---|---|---|---|---|---|---|---|
1) Control
| 73 | F | subdural hematoma | HC | NA | NA | NA | NA |
2) Control
| 58 | M | unknown. ALS patient | HC & CX | NA | NA | NA | NA |
3) Control
| 62 | M | unknown, non-demented control | HC & CX | NA | NA | NA | NA |
4) Control
| 94 | F | CVA | HC & CX | NA | NA | NA | NA |
5) Control
| 71 | M | pancreas carcinoma | HC & CX | NA | NA | NA | NA |
6) Control
| 64 | F | respiratory failure | HC & CX | NA | NA | NA | NA |
7) Control
| 70 | M | sepsis with broncopneumonia | HC | NA | NA | NA | NA |
8) Control
| 50 | F | metastasized broncocarcinoma | HC & CX | NA | NA | NA | NA |
9) Control
| 48 | M | DMT I induced organ failure | HC | NA | NA | NA | NA |
10) Control
| 74 | M | pulmonary carcinoma | HC & CX | NA | NA | NA | NA |
11) Control
| 62 | F | renal carcinoma (euthanasia) | CX | NA | NA | NA | NA |
12) Control
| 93 | F | heart failure | CX | NA | NA | NA | NA |
13) Control
| 92 | F | cachexia/dehydration | CX | NA | NA | NA | NA |
14) non-HS
| 45 | M | W0, FCD type1 to 2A in cortex | HC | 6 | CX | LTG, PHT | 1A |
15) non-HS
| 46 | F | W0, MCD type 1 in cortex | HC | 12 | CX | CBZ, VPA | 1A |
16) non-HS
| 46 | M | W0, epilepsy after head trauma | HC | 90 | HC & CX | CBZ, VPA, TPR | 1B |
17) non-HS
| 42 | F | W0, DNT WHO grade I | HC & CX | 1 | CX | CBZ, LTG, LEV | 1A |
18) non-HS
| 34 | F | W0, cortical cavernoma | HC | 1.5 | HC & CX | CBZ | 1A |
19) non-HS
| 40 | F | W0, MCD type 1 in cortex | HC | 8 ( C ) | HC & CX | LEV, LTG, CBZ | 1A |
20) non-HS
| 43 | F | W0, therapy resistant epilepsy | HC & CX | 40 ( C ) | HC & CX | PHT, LTG | 1A |
21) non-HS
| 47 | M | W0, therapy resistant epilepsy | HC | 30 | HC | CBZ, VPA, LTG, LEV | 3A |
22) non-HS
| 28 | M | W0, MCD type 1 in cortex | HC | 60 | HC & CX | CBZ, TPR. | 1A |
23) non-HS
| 54 | M | W0, ganglioglioma WHO grade I | HC & CX | 1.5 | HC & CX | OXC, LTG, CLO | 1A |
24) non-HS
| 30 | M | W0, therapy resistant epilepsy | CX | 8 | HC & CX | OXC, LEV, CLO | 1A |
25) non-HS
| 37 | F | W0, therapy resistant epilepsy | CX | 1 | not measured | LTG, CLO | 1A |
26) non-HS
| 61 | M | W0, therapy resistant epilepsy | CX | 5.5 | HC & CX | PHT, CBZ | 2A |
27) non-HS
| 19 | M | W0, ganglioglioma WHO grade II | CX | 1 | None | VPA, LEV | 1A |
28) non-HS
| 27 | F | W0, therapy resistant epilepsy | CX | 16 | HC & CX | LEV | 1A |
29) non-HS
| 46 | M | W0, cavernoma in uncus | CX | 60 | HC | CBZ, VPA, OXC, LEV, LTG | 1A |
30) non-HS
| 44 | M | W0, therapy resistant epilepsy | CX | 18 | HC & CX | TPR, OXC | 1A |
31) HS
| 41 | M | MTS W4 | HC & CX | 12 | not measured | PHT, CLO, CBZ, LTG | 1A |
32) HS
| 44 | F | MTS W2 | HC | 8 ( C ) | not measured | CBZ, OXC, CLO | 1A |
33) HS
| 41 | M | MTS W4 | HC | 3 | not measured | CBZ | 1A |
34) HS
| 52 | F | MTS W4 | HC & CX | 10 | not measured | CBZ, CLO, DZP | 2D |
35) HS
| 50 | M | MTS W4 | HC | 18 | not measured | CBZ, GBP | 2A |
36) HS
| 36 | F | MTS W4 | HC & CX | 2 | not measured | OXC, LZP | no info |
37) HS
| 42 | M | MTS W4 | HC & CX | 2 | not measured | LEV, LTG | 2A |
38) HS
| 36 | M | MTS W4 | HC & CX | 10 | HC & CX | OXC, PGB | 1B |
39) HS
| 49 | F | MTS W4 | HC | 8 ( C ) | not measured | OXC, CLO | 1A |
40) HS
| 42 | F | MTS W4 | HC | 4.5 | not measured | LEV, LTG, PBT | 1A |
41) HS
| 36 | M | MTS W4 | CX | 5 | not measured | PGB, LTG | 1A |
42) HS
| 34 | F | MTS W4 | CX | 10 ( C ) | HC & CX | LTG, CBZ, VPA,CLO | 2A |
43) HS
| 49 | M | MTS W3 | CX | 1 | None | LTG, CBZ, CLO | 1A |
44) HS
| 45 | M | MTS W4 | CX | 0.5 | not measured | GBP, LTG | 1A |
45) HS
| 48 | M | MTS W4 | CX | 3.5 | not measured | CBZ, LTG, VPA | 1A |
Multiplex bead-based immunoassay analysis
Protein | Hippocampal expression (pg/ml) | Cortical expression (pg/ml) | P-value | ||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Chemokines |
Cntrl
|
mTLE-HS
|
mTLE + HS
|
Cntrl
|
mTLE-HS
|
mTLE + HS
| |||||||
CCL22/MDC
|
0.3
| (0–0.7) |
0.8
| (0.4-1.6) |
0.9
| (0.3-1.4) |
0.2
| (0–0.6) |
0.6
| (0.3-1.2) |
0.9
| (0.7-1.3) |
1.74E-08
|
CCL5/RANTES
|
8.1
| (4–15.8) |
77.4
| (40.9-238.1) |
49.4
| (18.5-167.7) |
10.9
| (1.6-18.5) |
26.0
| (18.5-122.3) |
31.5
| (17.4-38.5) |
1.60E-08
|
CCL4/MIP1β
|
0
| (0–0) |
19.4
| (4–122.3) |
18.9
| (6.2-95.3) |
0
| (0–0) |
0
| (0–7.6) |
0.0
| (0–33.6) |
2.11E-08
|
CXCL9/MIG
|
3.2
| (1.1-11) |
9.0
| (6.7-16.7) |
11.0
| (4.4-17.1) |
5.7
| (2.4-7.7) |
8.3
| (5.7-12) |
10.7
| (7.0-12.3) |
5.99E-07
|
CCL2/MCP1
|
1.1
| (0–4.3) |
11.4
| (2.1-52.8) |
9.3
| (2.1-29.0) |
0.0
| (0–2.9) |
0.7
| (0–4.6) |
0.3
| (0–18.2) |
4.13E-06
|
CCL3/MIP1α
|
44.4
| (11.2-106.2) |
94.5
| (62–177.4) |
94.6
| (62–317.1) |
35.6
| (20.1-57.4) |
62.0
| (35.6-104.9) |
85.1
| (59.0-97.2) |
9.13E-05
|
CCL19/MIP3β
|
0
| (0–0) |
0.5
| (0–1.6) |
0
| (0–1) |
0
| (0–0) |
0
| (0–1.7) |
0.4
| (0–1.3) |
9.90E-05
|
CCL18/PARC | 16.6 | (0.2-151.6) | 24.2 | (1.8-177.4) | 14.0 | (0–29.5) | 13.7 | (0–132.7) | 3.3 | (0–31.9) | 0.7 | (0–37.6) | 0.001966 |
CXCL8/IL-8 | 0.8 | (0–13.8) | 2.7 | (0–26) | 1.0 | (0.3-20.1) | 0.6 | (0–1.6) | 0.5 | (0–10.3) | 0.9 | (0–3.6) | 0.02352 |
CCL11/Eotaxin | 0 | 0 | 0 | 0 | 0 | 0 | b.d. | ||||||
CCL17/Tarc | 0 | 0 | 0 | 0 | 0 | 0 | b.d. | ||||||
CXCL10/IP-10 | 0 | 0 | 0 | 0 | 0 | 0 | b.d. | ||||||
Cytokines
| |||||||||||||
IL-7
|
0
| (0–5.6) |
8.3
| (3.8-9.6) |
7.3
| (0.6-12.2) |
0.9
| (0.0-4.6) |
8.7
| (4.6-14.5) |
11.0
| (8–12.7) |
5.07E-15
|
IL-13
|
2.6
| (0.9-6.8) |
13.4
| 7.9-20.5) |
11.2
| (8.1-17.4) |
6.4
| (2.6-12.5) |
12.7
| (10.3-17.4) |
15.4
| (10.7-16.5) |
7.60E-13
|
IL-22
|
92.5
| (58.3-212.2) |
351.2
| (236.6-540.6) |
304.1
| (244.8-449.9) |
212.2
| (122.8-359.4) |
380.0
| (293.8-548.8) |
425.2
| (261.1-499.3) |
1.65E-12
|
IL-5
|
0
| (0–1.2) |
2.8
| (1.5-7.4) |
2.8
| (0.3-5.5) |
0
| (0–1) |
2.3
| (1–6.7) |
5.0
| (2–6.1) |
2.06E-07
|
IL-25/IL-17E
|
726.9
| (273.4-1780) |
868.3
| (348.3-1616) |
1976.4
| (258.5-2499) |
303.3
| (109.9-757.4) |
673.5
| (483.8-1234.5) |
922.1
| (468.7-1327.5) |
1.93E-05
|
IL-1RA
|
13.5
| (6.7-26.8) |
19.0
| (13.5-33.4) |
20.7
| (12.3-29) |
15.7
| (13.5-31.2) |
29.0
| (13.5-165.2) |
53.1
| (26.8-92.2) |
2.96E-05
|
IL-1a
|
0
| (0–0.2) |
0.3
| (0–3) |
0.3
| (0–1.3) |
0
| (0–0) |
0
| (0–0.9) |
0.3
| (0–1.6) |
5.06E-05
|
IL-27
|
40.9
| (0–153.8) |
33.7
| (0–133.4) |
132.1
| (0–370.5) |
0
| (0–42.3) |
28.6
| (0–89.7) |
49.4
| (29.4-78.7) |
8.93E-05
|
IL-10
|
2.2
| (0–5.9) |
1.4
| (0–3.4) |
7.8
| 0-12.4) |
0
| (0–2.2) |
0.5
| (0–1.9) |
0.9
| (0–3.4) |
1.02E-04
|
IFNα
|
22.5
| (12.8-41.5) |
31.5
| (22.7-58.5) |
66.3
| (10.1-114.3) |
27.0
| (4.1-73.1) |
41.3
| (26.9-51.5) |
38.5
| (30.7-59) |
3.40E-04
|
MIF | 11135.4 | (7054–13156) | 8200.7 | (6406–19843) | 6907.3 | (4910–9650) | 11062.2 | (4645–17980) | 7112.0 | (4645–9872) | 7180.8 | (5547–9470) | 0.0024 |
IL-1β | 0.2 | (0–0.9) | 0.8 | (0.2-1.7) | 0.9 | (0–1.7) | 0.2 | (0–0.9) | 0.5 | (0–1.5) | 0.9 | (0–1.7) | 0.0063 |
IL-23 | 298.9 | (190.4-568.7) | 575.1 | (473.2-675) | 556.2 | (311.2-691) | 308.1 | (238.2-717) | 500.2 | (286.7-633) | 543.2 | (372.9-710) | 0.0063 |
IL-4 | 0 | (0–0.1) | 0.1 | (0–0.2) | 0 | (0–0.2) | 0 | (0–0) | 0 | (0–0.2) | 0.1 | (0–0.2) | 0.0234 |
IL-6 | 0 | (0–11.7) | 0 | (0–3.9) | 0 | (0–1.1) | 0 | (0–2.1) | 0 | (0–0) | 0.0 | (0–0) | 0.0428 |
IL-21 | 336.5 | (117.7-944.2) | 510.6 | (0–944.2) | 831.0 | (55.8-1270.4) | 431.2 | (0–1009.1) | 415.4 | (0–847.1) | 654.1 | (179.9-1534) | 0.0848 |
TNFα | 10.4 | (8.6-12.7) | 11.5 | (7.0-13.2) | 11.2 | (8.2-13.2) | 9.3 | (6.3-12.9) | 10.6 | (6.9-12.3) | 10.5 | (9.2-13.2) | 0.1341 |
IL-18 | 3.7 | (1.7-14.2) | 3.2 | (1.5-5.2) | 2.1 | (1.1-23.8) | 5.0 | (1.6-163.1) | 2.9 | (1.7-12.4) | 2.9 | (2.2-4.8) | 0.1481 |
IL-6R | 1.4 | (0–10) | 1.4 | (0–5.6) | 4.5 | (1.4-8.9) | 3.4 | (0–17) | 3.4 | (0–10) | 4.5 | (0–7.8) | 0.4944 |
IL-2 | 0 | 0 | 0 | 0 | 0 | 0.0 | b.d. | ||||||
IL-12p70 | 0 | 0 | 0 | 0 | 0 | 0.0 | b.d. | ||||||
Other
| |||||||||||||
VCAM1
|
219.7
| (33.9-828.3) |
985.5
| (548–1722.6) |
1181.3
| (364.5-1649) |
210.3
| 67.3-527.2) |
817.5
| (444.9-1287) |
1239.9
| (740.4-1551) |
2.71E-11
|
VEGF
|
10.0
| (0.4-28.8) |
30.3
| (18.6-47) |
31.1
| (7.2-59.3) |
4.5
| (0–12.8) |
22.9
| (12.8-39.3) |
32.6
| (17.1-36.3) |
3.70E-10
|
HGF
|
33.0
| (26.7-54.5) |
105.2
| (65.8-157.4) |
90.3
| (34.6-156.4) |
24.0
| (19–50.3) |
71.3
| (37–207.7) |
52.4
| (32.2-83.6) |
1.38E-07
|
ICAM1
|
9099.6
| (3364–17901) |
5400.2
| (3174–9985) |
16011.1
| (5781–26620) |
5039.5
| (1532–16739) |
3440.4
| (1532–10754) |
4698.9
| (1861–8725) |
9.13E-05
|
Cathepsin S | 463.3 | (284.2-1010) | 311.9 | (129.1-525.7) | 218.0 | (160.4-425.5) | 350.6 | (198.2-993.6) | 303.8 | (198.2-478.7) | 327.2 | (190.4-413) | 0.0025 |
TIMP-1 | 101.0 | (36.2-2243.7) | 124.3 | (81.6-382.5) | 103.2 | (50.7-204) | 81.6 | (53.7-137.7) | 97.7 | (64.5-324.4) | 112.5 | (83.5-305.6) | 0.2672 |
Adiponectin | 415.0 | (14–1412.5) | 727.8 | (193.3-1643) | 480.3 | (221–1787.4) | 722.4 | (22.4-1979.9) | 490.0 | (22.4-940.7) | 349.3 | (0–1751) | 0.4253 |
Antigen | Protein source | Antibody set |
---|---|---|
CCL4 | R&D | R&D |
CCL19 | R&D | R&D |
IL-1RA | R&D | Bioledgend |
IL-6R | R&D | Sanquin |
IL-7 | BD | BD |
IL-21 | Abnova | eBioscience |
IL-22 | Peprotech | Peprotech |
IL-23 | R&D | eBioscience |
IL-25 | R&D | R&D |
IL-27 | R&D | R&D |
IFNa | eBioscience | eBioscience |
VEGF | R&D | R&D |
HGF | R&D | R&D |
Cathepsin S | R&D | R&D |
TIMP1 | R&D | R&D |
Adiponectin | R&D | R&D |
Immunohistochemistry and immunofluorescence
Statistical analyses
Parameter | HC | HC | HC-CX | HC | HC-CX | CX | CX |
---|---|---|---|---|---|---|---|
+HS vs – HS | +HS vs Cntrl | +HS vs + HS | -HS vs Cntrl | -HS vs –HS | +HS vs Cntrl | -HS vs Cntrl | |
IL1 RA | 0.64497528 | 0.116 |
0.0008
| 0.137 | 0.108 |
0.0005
| 0.027 |
IL1a | 0.24252404 |
0.0007
| 0.687 |
0.003
| 0.0722 |
0.0021
| 0.171 |
IL-5 | 0.706 |
0.0002
| 0.0261 |
0.001
| 0.705 |
1.32E-06
|
0.001
|
IL-7 | 0.853 |
6.65E-05
| 0.007 |
3.28E-07
| 0.469 |
1.78E-10
|
7.37E-06
|
IL-10 |
4.1619E-06
|
0.004
|
2E-06
| 0.138 | 0.079 | 0.068 | 0.305 |
IL-13 | 0.25819279 |
2.78E-06
| 0.0449 |
7.35E-07
| 0.518 |
1.16E-06
|
4.90E-05
|
IL-22 | 0.491 |
4.86E-07
| 0.0149 |
9.13E-07
| 0.478 |
1.23E-05
|
0.00023
|
IL-25 |
1.8845E-05
|
0.001
|
1E-07
| 0.470 | 0.0865 |
3.81E-05
|
0.001
|
CCL2 | 0.376 |
0.0009
| 0.0185 |
0.0006
|
0.0001
| 0.372 | 0.227 |
CCL3 | 0.521 |
0.015
| 0.106 |
0.002
| 0.0132 |
1.34E-06
|
0.002
|
CCL5 | 0.250 |
0.006
| 0.0661 |
0.001
| 0.0166 |
5.78E-07
|
0.0002
|
CCL19 | 0.258 | 0.045 | 0.203 |
0.0015
| 0.2107 |
0.002
| 0.126 |
CCL22 | 0.99 |
0.00029
| 0.5204 |
0.0002178
| 0.1207 |
2.00E-07
|
0.001
|
CXCL9 | 0.168 |
0.00020
| 0.0958 |
0.001
| 0.249 |
1.70E-05
|
0.003
|
HGF | 0.327 |
0.001
|
0.0021
|
9.61E-08
| 0.142 |
0.001
|
0.006
|
sICAM1 |
5.9897E-05
|
0.004
|
2E-05
| 0.052 | 0.281 | 0.136 | 0.147 |
sVCAM1 | 0.326 |
3.01E-05
| 0.9371 |
7.48E-05
| 0.1186 |
6.88E-09
|
0.00011
|
VEGF | 0.714 |
0.001
| 0.5836 |
8.71E-05
| 0.0925 |
3.09E-10
|
3.07E-05
|
IFNa |
0.00177
|
0.002
|
0.014
| 0.036 | 0.1402 | 0.327 | 0.318 |
IL-27 |
0.0012
|
0.008
|
0.0051
| 0.933 | 0.4785 |
3.62E-06
| 0.022 |
CCL4 | 0.795 |
0.0000108
| 0.022 |
0.00001
|
0.00004
| 0.49 | 0.27 |
Results
Protein expression profiling of the immune system in human mTLE
Inflammatory mediators show distinct patterns of upregulation across patient groups and tissues
Cellular localization of IL25 and CCL4
Principle component analyses revealed pathology-associated immunological profiles in mTLE
A | |||||||
---|---|---|---|---|---|---|---|
Component-HC | |||||||
1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 |
37.7% | 13.4% | 11.1% | 7.5% | 6.7% | 5.3% | 3.7% | 3.2% |
IL-13 | CCL4 | CCL3 | IL-6R | TIMP1 | CCL19 | TNFα | IL-23 |
IL-5 | CCL2 | IL-10 | IL-18 | IL-6 | CCL5* | IL-1RA | CCL18 |
IL-22 | IL-1α | ICAM1 | Adiponectin | Cathepsin-S | IL-1β * | IL-4 | |
CCL19 | IL-8 | IL-27 | IL-1β | MIF | |||
VEGF | CCL3 | IL-25 | |||||
CCL22 | CCL5 | IL-21 | |||||
CXCL9 | IFNα | ||||||
VCAM1 | IL-6R | ||||||
IL-7 | |||||||
HGF | |||||||
IL-23 | |||||||
mTLE Δ | mTLE Δ | mTLE + HS Δ | ND | ND | mTLE + HS Δ | ND | ND |
control | control | -HS & control | & control | ||||
B
| |||||||
Component-CX
| |||||||
1
|
2
|
3
|
4
|
5
|
6
|
7
|
8
|
40.1%
|
10%
|
8.4%
|
8%
|
5.8%
|
5%
|
4%
|
3.3%
|
IL-5 | IL-18 | CCL2 | Adiponectin | CXCL9 | TIMP1 | IL-1β | IL-25 |
VEGF | IL-6R | CCL4 | ICAM1 | CCL5 | CXCL8.IL-8 | IL-6 | IL-21 |
VCAM1 | MIF | Adiponectin | Cathepsin-S | HGF | IL-1α | IFNα | IL-4 |
CCL3 | IL-1β | CXCL8.IL-8 | CCL18 | TNFα | IL-23 | IL-10 | |
CCL19 | TNFα | IL-4 | |||||
IL-1RA | |||||||
IL-27 | |||||||
IL-22 | |||||||
IL-13 | |||||||
IL-25 | |||||||
CCL22 | |||||||
IL-7 | |||||||
CXCL9 | |||||||
IL-1α | |||||||
IL-23 | |||||||
TNFα | |||||||
mTLE Δ | ND | ND | ND | mTLE Δ | mTLE + HS Δ | ND | mTLE + HS Δ |
control | control | & control | -HS & control |
Discussion
Distinct expression patterns of upregulated inflammatory mediators
“Pro-epileptogenic” | “Anti-epileptogenic” | ||||
---|---|---|---|---|---|
Protein
|
Function in CNS
|
reference
|
Protein
|
Function in CNS
|
reference
|
CCL2
| - contributes to immune-cell recruitment across the BBB | [49] |
IL-1ra
| - An anti-infllammatory protein with seizure inhibiting properties in experimental TLE | |
CCL3
| - Capable of inducing Ca2+ transients in neuronal and microglial cultures. |
IL-7
| Neurotrophic actions in embryonic brain cultures | ||
- Inhibition of systemic receptor leads to decrease in seizure activity | |||||
CCL4
| - Capable of inducing Ca2+ transients in neuronal and microglial cultures. |
IL-10
| - Inhibits development of seizures in FS and a hypoxia model for epilepsy. | ||
- SNPs that result in increased IL-10 are decreased in FS patients. | |||||
- Inhibition of systemic receptor leads to decrease in seizure activity | |||||
-Can give trophic support to neurons | |||||
CCL5
| - Capable of inducing Ca2+ transients in neuronal and microglial cultures |
IL-13
| - Protects BBB integrity. | [62] | |
- Glut release from CNS cells in hypothalamus | |||||
- contributes to immune-cell recruitment across the BBB | |||||
- Inhibition of systemic receptor leads to decrease in seizure activity | |||||
CCL19
| immune-cell recruitment across the BBB. |
IL-25
| - Protects BBB integrity | ||
CCL22
| -Immune-cell recruitment across the BBB. |
IL-27
| Marked anti-inflammatory actions in EAE. | ||
CXCL9
| -Immune-cell recruitment across the BBB. |
IFNα
| - Suppression of hippocampal CA1 neurons & LTP | ||
IL-1α
| Polymorphisms that lead to increased transcription are associated with TLE |
HGF
| - Enhances neuronal survival | ||
IL-1β
| - Increased NMDA R subunit NR2B phosphorylation leading to increased Ca2+ influx |
VEGF
| - neuroprotective after experimental seizures | ||
- reduces hippocampal excitability | |||||
- Higher levels correlate with increased SRS after eFS | |||||
IL-5
| - Implicated in BBB disruption in CNS tumor bloodvessels |
IL-5
| - Anti-inflammatory actions in the periphery as a Th2 cytokine | ||
- Induces microglial proliferation | |||||
- Induces microglial activation | |||||
IL-7
| Induces neuronal apoptosis in human NT2 cells | [90] | |||
IL-22
| - BBB disruption through down-regulation of occludin. | [91] | |||
VEGF
| BBB disruption → reduction in tight junction proteins in kainate model for TLE | ||||
ICAM-1
| - implicated in BBB disruption | [20] | |||
VCAM-1
| - Immune-cell recruitment across the BBB | [20] |