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TRANSCRIPT
[18F]CB251 PET/MR imaging probe targeting translocator protein (TSPO) independent of its polymorphism in a neuroinflammation model
Kim et al.
LAB/HAB = 37.28
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OCD218F
[18F]fmPBR28-d2
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LAB
HAB
LAB
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HAB LAB HAB LAB
LAB/HAB = 1.14
NN
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18F
[18F]CB251
Ki=0.27 nM
1.0 30.1
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[18F]CB251 PET/MR BLI of immune cells
[18F]CB251 PET/MR imaging probe targeting translocator 1
protein (TSPO) independent of its polymorphism in a 2
neuroinflammation model 3
4 Kyungmin Kim1,2,3, Ha Kim1,3, Sung-Hwan Bae1,3, Seok-Yong Lee1,2,3, Young-Hwa Kim1,3,5, 5
Juri Na1,2,3, Chul-hee Lee1,2,3, Min Sun Lee1, Guen Bae Ko1,8, Kyeong Yun Kim1,8, Sang-Hee 6
Lee4, In Ho Song4,5, Gi Jeong Cheon1,3, Keon Wook Kang1,2,3, Sang Eun Kim4,5,6, June-Key 7
Chung1,9, Euishin Edmund Kim1, Sun-Ha Paek2,7, Jae Sung Lee1,2,8, Byung Chul Lee4,6*, 8
Hyewon Youn1,3* 9 10 1Department of Nuclear Medicine, Seoul National University Hospital, 2Biomedical Sciences, 11 3Laboratory of Molecular Imaging and Therapy, Cancer Research Institute, Seoul National 12
University College of Medicine, 4Department of Nuclear Medicine, Seoul National University 13
Bundang Hospital, 5Department of Transdisciplinary Studies, Graduate School of 14
Convergence Science and Technology, Seoul National University, 6Center for Nanomolecular 15
Imaging and Innovative Drug Development, Advanced Institutes of Convergence Technology, 16
Seoul National University, 7Department of Neurosurgery, Seoul National University Hospital, 17 8Brightonix Imaging Inc., 9Department of Nuclear Medicine, National Cancer Institute, 18
Republic of Korea 19
20
21
*For correspondence 22
Hyewon Youn, Ph.D. 23
Department of Nuclear Medicine, Seoul National University Hospital, 24
#207-4 Samsung Cancer Research Bldg. 25
103 Daehak-No, Chongno-Gu, Seoul, 03080, Korea. 26
Tel: +822-3668-7026; Fax: +822-745-7690; E-mail: [email protected] 27
28
Byung Chul Lee, Ph.D. 29
Department of Nuclear Medicine, Seoul National University Bundang Hospital 30
Goomie-Ro 173, Boondang-Gu, Sungnam, Kyunggi-Do, 13620, Korea 31
Tel: +8231-787-2956; Fax: +8231-787-2957; E-mail: [email protected] 32
Abstract 33
The 18 kDa translocator protein (TSPO) has been proposed as a biomarker for the 34
detection of neuroinflammation. Although various PET probes targeting TSPO have been 35
developed, a highly selective probe for detecting TSPO is still needed because single 36
nucleotide polymorphisms in the human TSPO gene greatly affect the binding affinity of 37
TSPO ligands. Here, we describe the visualization of neuroinflammation with a 38
multimodality imaging system using our recently developed TSPO-targeting radionuclide 39
PET probe [18F]CB251, which is less affected by TSPO polymorphisms. 40
41
Methods: To test the selectivity of [18F]CB251 for TSPO polymorphisms, 293FT cells 42
expressing polymorphic TSPO were generated by introducing the coding sequences of wild-43
type (WT) and mutant (Alanine Threonine at 147th Amino Acid; A147T) forms. 44
Competitive inhibition assay was conducted with [3H]PK11195 and various TSPO ligands 45
using membrane proteins isolated from 293FT cells expressing TSPO WT or mutant-A147T, 46
representing high-affinity binder (HAB) or low-affinity binder (LAB), respectively. IC50 47
values of each ligand to [3H]PK11195 in HAB or LAB were measured and the ratio of IC50 48
values of each ligand to [3H]PK11195 in LAB to HAB was calculated, indicating the 49
sensitivity of TSPO polymorphism. Cellular uptake of [18F]CB251 was measured with 50
different TSPO polymorphisms, and phantom studies of [18F]CB251-PET using 293FT cells 51
were performed. To test TSPO-specific cellular uptake of [18F]CB251, TSPO expression was 52
regulated with pCMV-TSPO (or shTSPO)/eGFP vector. Intracranial lipopolysaccharide (LPS) 53
treatment was used to induce regional inflammation in the mouse brain. Gadolinium (Gd)-54
DOTA MRI was used to monitor the disruption of the blood-brain barrier (BBB) and 55
infiltration by immune cells. Infiltration of peripheral immune cells across the BBB, which 56
exacerbates neuroinflammation to produce higher levels of neurotoxicity, was also monitored 57
with bioluminescence imaging (BLI). Peripheral immune cells isolated from luciferase-58
expressing transgenic mice were transferred to syngeneic inflamed mice. Neuroinflammation 59
was monitored with [18F]CB251-PET/MR and BLI. To evaluate the effects of anti-60
inflammatory agents on intracranial inflammation, an inflammatory cytokine inhibitor, 2-61
cyano-3, 12-dioxooleana-1, 9-dien-28-oic acid methyl ester (CDDO-Me) was administered in 62
intracranial LPS challenged mice. 63
64
Results: The ratio of IC50 values of [18F]CB251 in HAB to LAB indicated similar binding 65
affinity to WT and mutant TSPO and was less affected by TSPO polymorphisms. [18F]CB251 66
was specific for TSPO, and its cellular uptake reflected the amount of TSPO. Higher 67
[18F]CB251 uptake was also observed in activated immune cells. Simultaneous [18F]CB251-68
PET/MRI showed that [18F]CB251 radioactivity was co-registered with the MR signals in the 69
same region of the brain of LPS-injected mice. Luciferase-expressing peripheral immune 70
cells were located at the site of LPS-injected right striatum. Quantitative evaluation of the 71
anti-inflammatory effect of CDDO-Me on neuroinflammation was successfully monitored 72
with TSPO-targeting [18F]CB251-PET/MR and BLI. 73
74
Conclusion: Our results indicate that [18F]CB251-PET has great potential for detecting 75
neuroinflammation with higher TSPO selectivity regardless of polymorphisms. Our 76
multimodal imaging system, [18F]CB251-PET/MRI, tested for evaluating the efficacy of anti-77
inflammatory agents in preclinical studies, might be an effective method to assess the severity 78
and therapeutic response of neuroinflammation. 79
80
Keywords: neuroinflammation, TSPO, polymorphism, PET/MRI, multimodal imaging 81
Introduction 82
Neuroinflammation is the primary defense mechanism against viral and bacterial 83
infections, toxic metabolites, and injury in the central nervous system (CNS). Several studies 84
have shown that prolonged neuroinflammation is linked to the onset and progression of 85
neurodegenerative conditions such as Alzheimer’s and Parkinson’s diseases, and multiple 86
sclerosis [1-3]. Bidirectional interactions between the CNS and peripheral immune systems 87
adversely affect the CNS [4-6] and are considered the main cause of prolonged 88
neuroinflammation and neurodegenerative diseases [5, 6]. These interactions induce not only 89
the activation of microglia and resident immune cells in the brain but also the disruption of 90
the blood-brain barrier (BBB), which results in the migration of peripheral immune cells into 91
the region of CNS neuroinflammation [6-8]. Therefore, imaging the region of 92
neuroinflammation and investigating the interactions between CNS immune cells and 93
infiltrating peripheral immune cells are important for understanding neurodegenerative 94
diseases. 95
Several studies of various imaging techniques have been conducted for identifying 96
the region of neuroinflammation [9-13]. Single-photon emission computed tomography 97
(SPECT) using 99mTc-hexamethylpropyleneamineoxime (HMPAO) or 99mTc-ethyl cysteinate 98
dimer (ECD) has been frequently used to visualize the increased blood flow that occurs in 99
neuroinflammation [14, 15]. Disruption of the BBB and changes in vascular permeability can 100
also be visualized by 99mTc-DTPA SPECT and gadolinium (Gd)-DTPA magnetic resonance 101
imaging (MRI) [16, 17]. Also, [11C]arachidonic acid and [18F]FDG positron emission 102
tomography (PET) have been used for imaging neuroinflammation reflected by changes in 103
incorporation of arachidonic acid and glucose metabolism, respectively, in neurons and 104
inflammatory cells [18]. However, [18F]FDG-PET cannot be used for early diagnosis of 105
neuronal diseases because the basal level of [18F]FDG uptake is too high in the brain to 106
distinguish neuroinflammation from normal brain regions. Therefore, recent studies have 107
focused on identifying better imaging biomarkers to overcome the limitation of [18F] FDG-108
PET for imaging neuroinflammation. 109
The translocator protein 18 kDa (TSPO) has been proposed as an attractive target 110
biomarker of neuroinflammation [19-31]. TSPO, located in the mitochondrial outer 111
membrane, acts as a peripheral benzodiazepine receptor and a cholesterol transporter. 112
Because activated immune cells have been reported to show increased TSPO expression, 113
TSPO-targeting probes such as [11C]PK11195 and [11C]PBR28 have been suggested for PET 114
imaging of inflammation. However, these PET probes have limitations, such as nonspecific 115
binding, a low target-to-background ratio, and different binding sensitivities to TSPO 116
polymorphisms [28-31]. Although radiochemists have been working on developing improved 117
imaging ligands for targeting TSPO, a highly selectable probe for detecting TSPO is still 118
needed. 119
Our group recently developed a new imidazopyridine TSPO ligand, [18F]CB251, 120
which showed promise as a TSPO radiotracer [26]. We introduced fluorine-18 because 18F-121
labelled compounds with a longer half-life (109.8 min for fluorine-18 and 20.3 min for 122
carbon-11) are preferable to 11C-labelled compounds for clinical applications. We reported on 123
the synthesis of [18F]CB251, which showed a 5.1-times higher TSPO binding affinity than 124
[11C]PK11195 [26, 31]. 125
In this study, we evaluated the selectivity of the [18F]CB251 probe for human TSPO 126
harboring genetic variants, and its ability to reveal activated immune cells in regions of 127
neuroinflammation. We established a mouse model of intracranial lipopolysaccharide (LPS)-128
induced regional inflammation to visualize neuroinflammation using multimodality imaging. 129
Both CNS and peripheral immune cells infiltrating across the BBB play an important role in 130
neuroinflammation. We isolated peripheral immune cells from spleens of luciferase-131
expressing transgenic mice and systemically injected them into mice with intracranial LPS-132
induced regional neuroinflammation, and visualized with bioluminescence imaging (BLI). By 133
comparing TSPO-targeting [18F]CB251 PET/MR and peripheral immune cells-targeting BLI, 134
we attempted to determine whether the [18F]CB251 probe can quantitatively assess the 135
severity of neuroinflammation and therapeutic response to anti-inflammatory agents. 136
Results 137
In vitro evaluation of [18F] CB251 as a probe for targeting TSPO to image activated 138
immune cells 139
As we reported previously [26], an 18F-labeled alpidem analog, [18F]CB251, was 140
synthesized from 2-phenyl-imidazo [1,2-a] pyridine, and used as a specific probe for TSPO. 141
We also reported that the binding affinity of CB251 was higher than that of the widely used 142
TSPO ligands, PK11195 [26, 32] , PBR28 [26, 32] , fluoremethyl-PBR28-d2 (fmPBR28-d2), 143
and GE-180 [33] (Figure 1A). 144
For TSPO ligand development, TSPO selectability regardless of TSPO 145
polymorphism (rs6971; A147T) is important because TSPO polymorphism is known to affect 146
both in vitro and in vivo radio-ligand bindings. TSPO ligands show substantial differences in 147
affinity between subjects classified as a high-affinity binder (HAB; TSPO wild-type (WT)) 148
and low-affinity binder (LAB; TSPO A147T mutant (Mut). TSPO genotypes and 149
classification is showed in Figure S1A. PBR28 was reported to have a different binding 150
affinity to TSPO polymorphism compared to PK11195 [28-31]. 151
To test the ligand-binding affinity and cellular uptake, we generated 293FT cells 152
expressing polymorphic TSPOs, including the most abundant type (WT) and mutant TSPO 153
(C to T point mutation generating Alanine to Threonine at 147th amino acid of TSPO; Mut-154
A147T). Figure S1B shows the mutagenesis site and the map of expression vectors. 155
To compare ligand binding affinity to the TSPO polymorphism, we conducted 156
competitive inhibition assay with [3H]PK11195 and various TSPO ligands such as PK11195, 157
fluoromethyl-PBR28-d2 (fmPBR28-d2) and CB251 using membrane proteins isolated from 158
293FT cells expressing TSPO polymorphism (TSPO WT and TSPO Mut-A147T) (Figure 159
1B). The ratio of IC50 values (IC50 of LAB/ IC50 of HAB) for PK11195 was 0.83 and similar 160
to earlier reports [34, 35]. Unlike fmPBR28-d2 (IC50 of LAB/ IC50 of HAB=37.28), CB251 161
showed a similar ratio (IC50 of LAB/ IC50 of HAB =1.14), which was close to 1. These results 162
indicated that fmPBR28-d2 ligand had a difference in binding affinity according to the TSPO 163
polymorphism, whereas CB251 showed ratios similar to PK11195. 164
The higher affinity of ligand binding to the target protein is an important factor. 165
However, clinical applications also require consideration of biochemical, cellular, and 166
physiological factors, such as lipophilicity of the ligand and its non-specific insertion in the 167
membrane, cellular metabolism of the ligand, endogenous cellular TSPO levels, and in vivo 168
kinetics. Also, the higher affinity of a ligand to the target proteins does not automatically 169
reflect target selectivity. Therefore, we performed cellular uptake of various radiolabeled 170
ligands targeting TSPO because it reflects the entire cellular process from infiltration through 171
the cell membrane to its binding to TSPO on the mitochondrial outer membrane. As expected, 172
our results demonstrated that [18F]fmPBR28-d2 was sensitive to the A147T mutation. GE-173
180, another well-known TSPO ligand, showed less sensitivity to A147T mutation. However, 174
[11C]PK1195 and [18F]CB251 were much less sensitive to TSPO polymorphism, and 175
[18F]CB251 showed similar cellular uptake levels between the polymorphic TSPOs (Figure 176
1C). 177
We also observed differences in PET signals from the cells with different 178
polymorphic TSPOs using phantoms. [18F]fmPBR28-d2 was used as a sensitive probe for 179
TSPO polymorphism. Similar to the cellular uptake test results, [18F]CB251 was less 180
sensitive to TSPO polymorphism (Figure 1D, 1E), indicating that it could be a good PET 181
probe with better selectivity for targeting TSPO. 182
In a previous study, the specificity of [18F CB251 ligand for TSPO was not directly 183
addressed in a biological system [26]; therefore, in this study, the specificity of [18F]CB251 184
was tested by regulating TSPO expression in cell culture. [18F]CB251 uptake was measured 185
in the cells with different levels of TSPO expression, which was regulated by transfection 186
with pCMV-TSPO/eGFP for overexpression and pCMV-shTSPO/eGFP for reduced 187
expression. 293FT cells expressing low levels of TSPO were used for TSPO overexpression 188
(Figure 2A), and MDA-MD-231 cells expressing high levels of TSPO were used for reduced 189
expression (Figure 2C). Fluorescence microscopic images showed increased eGFP signals, 190
reflecting the successful transfection of each vector in the target cells (Figure 2A-C). 191
Increased TSPO expression was observed by both immunoblotting and fluorescence 192
microscopy of pCMV-TSPO/eGFP transfected cells (Figure 2A), and [18F]CB251 uptake was 193
increased in TSPO-overexpressing cells (Figure 2B). In contrast, decreased TSPO expression 194
was observed in pCMV-shTSPO/eGFP-transfected cells (Figure 2C), and [18F]CB251 uptake 195
was decreased in cells with reduced TSPO expression (Figure 2D). These data demonstrated 196
that [18F]CB251 uptake was directly reflected by changes in TSPO expression, indicating that 197
[18F]CB251 is specific for TSPO at the cellular level. 198
Next, we monitored TSPO expression as a marker for activated immune cells and 199
tested whether [18F]CB251 could be used as a specific imaging probe for activated immune 200
cells. The mouse macrophage cell line, RAW264.7, and primary white blood cells obtained 201
from mouse spleens (splenocytes) were treated with LPS and IFN-γ for 24 h to activate 202
immune cells. Following activation, immunoblotting in RAW264.7 cells and mouse 203
splenocytes showed increased expression of TSPO as well as iNOS, a marker of activated 204
macrophages (Figure 2E). Increased [18F]CB251 uptake was also observed in activated 205
immune cells with increased TSPO expression (Figure 2F). These data indicated that 206
[18F]CB251 directly reflected the activation of immune cells by targeting TSPO. 207
208
BLI of peripheral immune cells in mice receiving an intracranial injection of LPS 209
In addition to activation of CNS immune cells, infiltration of peripheral immune cells 210
through the BBB into the inflammatory area is considered to be critical for the 211
neuroinflammatory process [34-36]. Therefore, imaging depicting the infiltration of 212
peripheral immune cells across the BBB provides useful information for the evaluation of the 213
inflammatory process in the brain. Since BLI has been frequently used to visualize the 214
distribution and proliferation of immune cells in vivo [37, 38] , we employed this technique 215
to observe peripheral immune cell infiltration into the neuroinflammatory region after 216
systemic administration of luciferase-expressing peripheral immune cells. Splenocytes from 217
luciferase-expressing transgenic mice (C57BL/6.LucTg) were used as a source of peripheral 218
immune cells [38]. We previously reported that the composition and function of immune cells 219
in the C57BL/6.LucTg mice were similar to those of the normal C57BL/6 mice [38]. 220
Flow cytometry analysis showed that splenocytes from reporter transgenic mice 221
(C57BL/6.LucTg) used in this study had immune cell populations similar to those of normal 222
(C57BL/6) mice (Figure 3A). The BLI signals of splenocytes isolated from luciferase-223
expressing transgenic mice increased in a cell-number-dependent manner with a high 224
correlation value (r2 = 0.9991), which could quantitatively assess the number of splenocytes 225
that exhibited BLI signals (Figure 3B). To assess peripheral immune cell biodistribution by 226
BLI, we isolated splenocytes from reporter mice and grafted them into inflamed mice (Figure 227
3C). 228
BLI signals produced by grafted luciferase-expressing splenocytes were observed in 229
peripheral immune cell-targeting organs such as the lung, heart, liver, spleen, and intestine. 230
However, BLI signals from grafted splenocytes were observed only in the brain of inflamed 231
mice 4 days after intracranial LPS injection (Figure 3D). Most of the immune cell-homing 232
organs showed bioluminescent signals in both saline-injected control mice and LPS-injected 233
inflamed mice. However, the brain showed higher signals in mice receiving intracranial LPS 234
injection than the saline injection. Since the mouse brain is not a homing organ of peripheral 235
immune cells, our results indicated that grafted peripheral immune cells infiltrated the 236
inflamed brain of mice. 237
238
MRI using Gd-DOTA to monitor BBB disruption, immune cell infiltration, and 239
activation 240
Our observations on peripheral immune cell infiltration of the LPS-injected mouse 241
brains led us to monitor BBB disruption induced by mechanical stress. Our mouse model was 242
created by injecting saline (control agent) or LPS (inflammation-inducing agent) into the 243
mouse brain using a robotic stereotaxic device with a Hamilton syringe. We, therefore, had to 244
investigate the possibility of peripheral immune cell infiltration through the injection hole 245
rather than BBB. For this, closure of the injection hole and no BBB disruption in control mice 246
had to be verified. Previously, Gd-DOTA (Dotarem), a gadolinium-based MRI contrast agent 247
with a macrocyclic structure, has been used as an efficient paramagnetic contrast agent to 248
increase MR sensitivity and specificity for the identification of BBB defects [39-41]. 249
Generally, T1-weighted MRI has been used to visualize the presence of gadolinium 250
penetration through disrupted BBB. On the other hand, T2-weighted MRI usually provides 251
information about water content or vasogenic edema of brain lesions induced by LPS 252
injection. Therefore, we performed MRI using Gd-DOTA as a contrast agent to assess the 253
level of BBB disruption and vasogenic edema in mouse models. 254
We injected saline or LPS into the right striatum of the mouse brain to create 255
intracranial inflammation (Figire 4A). The MR signals in the right striatum were higher in the 256
LPS-injected mice than in the saline-injected mice in both the T1- (Figure 4B) and T2- 257
weighted images (Figure 4C). Representative MR images demonstrated that BBB disruption 258
caused by mechanical stress (injection hole) was recovered on day 1, as shown in the saline-259
injected group, while in the LPS-induced group, signal peaks indicating BBB disruption were 260
still visible on day 3 (Figure 4B; N=3). As shown in Figure 4C, the T2 signal representing 261
vasogenic edema due to LPS injection was observed. Peak T2 signal in LPS-treated mice, 262
representing tissue damage resulting from inflammation, was observed on day 3. On day 4, 263
T2 signals were reduced in the LPS-treated mice. Since T2-weighted signal enhancement was 264
reported to be produced by gadolinium-based chelates trapped in viable immune cells [41], 265
high T2 signals are thought to indicate the presence of viable immune cells in the region. Our 266
results showed that tracer uptake on day 4 in T1- and T2-weighted MRIs with Gd-DOTA was 267
due to the upregulation of neuroinflammation induced by LPS and not due to BBB disruption 268
induced by mechanical stress. Based on these results, we used only T2-weighted MRI with 269
Gd-DOTA to monitor the severity of neuroinflammation for further investigation. 270
271
Simultaneous [18F]CB251 PET/MRI and BLI for visualization of neuroinflammation 272
It has been reported that simultaneous PET/MRI provides information on anatomical 273
and functional changes with precise co-registration of signals and accurate correction of 274
attenuation [42, 43]. Herein, we performed simultaneous [18F]CB251 PET/MRI and BLI to 275
visualize neuroinflammation indicative of immune cell activation and peripheral immune cell 276
infiltration in brain regions in the intracranial LPS mouse model (Figure 5A). Although T1- 277
and T2-weighted MR signals peaked on day 3 after LPS injection, neuroinflammation was 278
still observed on day 4. Imaging studies at this time point could provide additional useful 279
information about peripheral immune cell infiltration without interference due to severe 280
tissue damage. Therefore, we performed [18F]CB251 PET/MRI and BLIs on day 4. 281
Radiation signals from [18F]CB251-targeting TSPO proteins reflected activation of 282
immune cells, such as resident microglia and infiltrated peripheral immune cells. T2-283
weighted MR signals reflected localization of all viable immune cells, and the BLI signals 284
reflected only luciferase-expressing peripheral immune cells. On day 4, LPS-injected mice 285
showed higher signals from each imaging modality in the inflamed region of the right 286
striatum than in the saline-injected mice (Figure 5B-C). T2-weighted MR signals from LPS-287
injected mice appeared to emanate from a slightly wider area of immune cells (Figure 5B 288
left) than the [18F]CB251 PET signals (Figure 5B middle), but the critical region of T2-289
weighted MR signal was co-localized with the region of increased [18F]CB251 PET signals. 290
In the BLI, luciferase-expressing splenocytes representing peripheral immune cells were 291
visualized in the right striatum of the LPS-injected mice (Figure 5B right). The degree of 292
neuroinflammation could be observed using T2-weighted MR reflecting localization of all 293
viable immune cells, including residential and peripheral immune cells. However, we used 294
MR images of simultaneous PET/MR to obtain anatomical information for locating 295
inflammation and BBB disruption, since the observation of inflammatory cell activation 296
using quantitative PET is more sensitive. 297
Next, we compared the differences between the signal intensity ratio of the right 298
(saline or LPS injection) to the left (no injection) striatum in the saline- (N=6) and LPS-299
injected (N=7) mice (Figure 5C). [18F] CB251 PET signal was expressed as standard uptake 300
value (SUVmax), and BLI optical signal was expressed as total flux (photon/sec/cm2/sr). In 301
LPS-injected mice, [18F]CB251 radioactivity and BLI optical signals were significantly 302
increased compared to saline-injected mice (p=0.023 for [18F]CB251; p=0.011 for BLI ). 303
When we compared the difference between the non-injected left striatum and the saline-304
injected right striatum, [18F]CB251 PET signal increased slightly in the saline-treated right 305
striatum (p=0.012). However, the BLI signal did not change significantly in the saline-treated 306
right striatum. Since [18F]CB251 PET reflects residential microglial activity as well as 307
peripheral immune cell activity, these results suggested that [18F]CB251 PET was more 308
sensitive to mechanical stress due to intracranial injection than BLI, which only reflected 309
peripheral immune cells. Considering the change in the strength of BLI signals after LPS 310
injection, BLI appeared to be more sensitive to the development of inflammation than PET 311
MRI using the TSPO-targeting probe [18F]CB251. However, the BLI signals were not 312
detectable through the black skin of mice, which had to be removed to detect and assess the 313
BLI signals. Taken together, the simultaneous analysis by [18F]CB251 PET targeting TSPO 314
and BLI targeting grafted peripheral immune cells revealed resident and peripheral immune 315
cell activation due to inflammatory processes, as well as the location of infiltrating peripheral 316
immune cells. 317
318
Identification of immune cells in inflamed regions of the mouse brain 319
We performed immunohistochemical (IHC) staining to identify the presence of 320
different types of immune cells in the brain region of neuroinflammation (Figure 5D). 321
Antibodies were used for staining TSPO-expressing cells (anti-TSPO) and immune cells in 322
the CNS parenchyma including perivascular macrophages, monocytes, microglia (anti-CD68, 323
anti-Iba-1), antigen-presenting cells (anti-CD86), and T cells (anti CD4, CD8). Cells from 324
monocyte lineages such as circulating macrophages and resident microglia expressing CD68 325
were clearly increased and were located with TSPO expressing cells at the site of 326
inflammation (right striatum). Also, the antigen-presenting cells expressing CD89, which are 327
expressed only in peripheral immune cells and critical for T cell activation, increased 328
significantly in the right striatum of LPS-injected mice. Since macrophages and dendritic 329
cells from bone marrow generally exhibit higher antigen-presenting ability than microglial 330
cells, we confirmed that infiltrated peripheral immune cells were localized at the site of 331
inflammation. However, helper T cells (anti-CD4) and cytotoxic T cells (anti-CD8) appeared 332
widely distributed on the cross-sections of mouse brains. 333
Animals were sacrificed 4 days after LPS injections, and only the initial response 334
consisting of activated resident immune cells and recruited antigen-presenting cells could be 335
observed at the sites of inflammation. Our multimodality imaging data and IHC data 336
colocalizing TSPO-, CD68- and CD86-expressing cells in the inflamed region of the brain, 337
clearly demonstrated that immune cells in the LPS-injected region were activated, and 338
recruitment of peripheral immune cells occurred after intracranial LPS challenges. 339
340
Comparative analysis of neuroinflammation by [18F]CB251 PET and BLI after anti-341
inflammatory treatment 342
We compared the usefulness of [18F]CB251 PET with BLI to evaluate the therapeutic 343
efficacy of an anti-inflammatory agent in neuroinflammation. We used 2-cyano-3, 12-344
dioxooleana-1, 9-dien-28-oic acid methyl ester (CDDO-Me), which is known to inhibit 345
inflammatory cytokines such as IL-6, IL-10, and IL-12 [44-48]. We administered CDDO-Me 346
daily for 3 days after intracranial LPS-injection in the right striatum (Figure 6A). As shown in 347
the previous experiments, the [18F]CB251 uptake ratio (SUVmax, right/left striatum) of the 348
inflamed region in LPS-injected mice was significantly increased compared to saline-injected 349
control mice (p=0.002).On the other hand, the [18F]CB251 uptake ratio of CDDO-Me-treated 350
mice after LPS injection significantly decreased compared to LPS-injected mice (p=0.0323, 351
Figure 6B). A similar pattern was also confirmed by the ratios of SUVmean values (data not 352
shown). 353
In LPS-injected mice, the BLI signal ratio from the brain (expressed as the ratio of 354
the total photon flux of the right and the left striatum) was 3.93-fold higher than that from 355
saline-injected control mice (p=0.0009). The BLI signal ratio from the brain of mice treated 356
with anti-inflammatory CDDO-Me after LPS injection decreased dramatically compared to 357
LPS-injected mice (p=0.0025, Figure 6C). These data demonstrated that CDDO-Me 358
successfully inhibited peripheral immune cell infiltration and reduced their recruitment 359
leading to neuronal damage. Our results clearly showed that both modalities, TSPO-targeting 360
[18F]CB251 PET and peripheral immune cell-targeting BLI, revealed the degree of neuronal 361
inflammation and reduced activation of immune cells. 362
363
Discussion 364
In a previous report [26], we described synthesis, binding, and competition studies 365
with [18F]CB251 and its future applications. In this study, we attempted to test [18F]CB251, 366
targeting TSPO as a clinically applicable PET probe for imaging neuroinflammation. Our 367
results indicated that [18F]CB251 targeted TSPO with higher selectivity in human TSPO 368
polymorphisms (Figure 1), was specific for TSPO protein, and taken up by activated immune 369
cells with high TSPO expression (Figure 2), These observations suggested that [18F]CB251 is 370
a suitable candidate for imaging of TSPO-related neuroinflammation. We also established an 371
intracranial LPS-induced neuroinflammation mouse model, and designed experiments with a 372
multimodal imaging approach using [18F]CB251 PET/MR and BLI technologies to 373
understand the interaction between CNS and the peripheral immune system during 374
neuroinflammation. Furthermore, we tested whether [18F]CB251 PET could be used to 375
evaluate the therapeutic effect of anti-inflammatory drugs in this model. 376
The higher affinity of a ligand for the target protein does not automatically reflect the 377
target selectivity. Especially, in vitro and in vivo discrepancies of targeting ability of TSPO 378
ligands have been reported in many human brain studies [20-25, 27, 28, 33, 49, 50]. While 379
developing TSPO targeting ligands, we noticed that human rs6971 polymorphism was 380
important, and TSPO ligand binding was influenced by A147T mutation (Figure S1A). 381
Generally, TSPO with Ala at 147th amino acid is the abundant phenotype and high-affinity 382
ligand binder (HAB). Single point mutation of C to T in the TSPO gene induces amino acid 383
change (Ala to Thr at 147th amino acid). This phenotype is a low-affinity ligand binder 384
(LAB). PBR28 is a well-known TSPO ligand that shows a different binding ability to the 385
TSPO rs6971 polymorphism. 386
Although testing the sensitivity of the rs6971 polymorphism (A147T) for developing 387
clinically applicable TSPO targeting ligands is important, we could not find enough donors 388
with the rs6971 polymorphism to test the affinity for [18F]CB251 in Korea. About 30% of 389
Caucasian and 3% of Chinese/Japanese are reported as LABs (“TSPO polymorphism by 390
ethnic differences” in the Hapmap database http://hapmap.ncbi.nih.gov or 391
http://asia.ensembl.org/Homo_sapiens/Variation/Explore?r=22:43162420-392
43163420;v=rs6971;vdb=variation;vf=210028659). The LAB percentage of Koreans has not 393
been reported, but less than 3% of Koreans are expected to have LAB considering the genetic 394
similarity between the Chinese and Japanese. 395
Thus, we established cells that ectopically express polymorphic TSPOs (WT for 396
HAB or Mut-A147T for LAB). In vitro site mutagenesis was introduced in the coding 397
sequence of 147th Ala in TSPO (Figure S1B). To test the sensitivity of the rs6971 398
polymorphism, competitive inhibition assays of CB251 and other ligands with [3H]PK11195 399
were performed (Figure 1B). We calculated the IC50 value of each ligand to [3H]PK11195 400
from % inhibition in HAB or LAB. The ratios of IC50 LAB/HAB for each ligand were also 401
calculated. The ratio of IC50 values of PK11195 was consistent with previous reports (0.83 to 402
0.85 for PK11195 [34, 35]). Similar to PBR28, the ratios of IC50 LAB/HAB for fm-PBR28-d2 403
was 37.28 (55 for PBR28 [34, 35]), indicating lower binding to LAB. The ratios of IC50 404
LAB/HAB were 1.14 for CB251 and 3.96 for GE-180 [33]. After comparing the Ki 405
(generated from recombinant TSPO protein) and the ratio of IC50 LAB/HAB (generated from 406
membrane TSPO from cells), we concluded that CB251 had a higher binding affinity 407
(Ki=0.27 nM) and selectivity (ratio of IC50 LAB/HAB=1.14) than other TSPO ligands 408
regardless of TSPO polymorphism. 409
To test in vitro and in vivo selectivity for rs6971 polymorphism of [18F]CB251 as a 410
PET probe, [18F]CB251 cell uptake PET scans were performed. Our results showed that 411
[18F]CB251 had a higher binding affinity (Figure 1A) with favorable selectivity (Figure 1B- 412
E) in vitro and in vivo. However, there were limitations because cellular uptake of TSPO 413
ligands in humans can be influenced by factors such as cellular metabolism, endogenous 414
TSPO level, and in vivo kinetics. [11C]PK11195 and [11C]ER176 were both reported to be 415
significantly insensitive to TSPO polymorphism in vitro, however, in human studies, 416
[11C]PK11195 showed the different uptake of lung and heart in binders and non-binders [49]. 417
In addition, [11C]ER176 represented different radioactivity according to each genotype in the 418
human brain [50]. Therefore, it is difficult to conclude that in vitro uptake completely reflects 419
human kinetics. Unfortunately, we could not perform human study because it is difficult to 420
find Korean volunteers to express rs6971 A147T polymorphism. Evaluation of the ligand in 421
humans should be performed as a further study. 422
In this study, we also tested the specificity of [18F]CB251 as a TSPO-targeting ligand 423
for imaging activated immune cells (Figure 2). By modulating the TSPO expression levels in 424
the cells and immune cell activation, we found that [18F]CB251 was specific for TSPO 425
protein and its uptake was specifically observed in activated immune cells with high TSPO 426
expression. 427
The role of resident immune cells in the brain and peripheral immune cells that 428
infiltrate across the BBB in neuroinflammation has been studied and emphasized [34-36] as 429
the main target of neuroinflammation imaging. The resident CNS immune system usually 430
consists of innate immune cells and operates under homeostatic conditions. However, when 431
peripheral innate and adaptive immune cells enter the CNS, they execute distinct cell-432
mediated effects. Infiltration of peripheral immune cells across the BBB exacerbates 433
neuroinflammation resulting in higher levels of neurotoxicity [4, 8]. Therefore, it is important 434
to visualize peripheral immune cell infiltration to evaluate neuroinflammatory damages. 435
Herein, we tested whether [18F]CB251 PET signal reflects neuroinflammation compared to 436
other conventional methods such as MRI and BLI. 437
We adoptively transferred splenocytes from luciferase-expressing transgenic mice 438
into an intracranial LPS-injected mouse model to visualize the distribution of infiltrating 439
peripheral immune cells in the brain by BLI. The total population of splenocytes was used 440
because immune responses are initiated by the co-stimulation of different populations of 441
immune cells. Despite the limitations of optical imaging, the grafted splenocytes were found 442
in the inflamed region of the mouse brain (Figure 3). 443
We also visualized BBB disruption and immune cell localization by MRI with the 444
contrast agent, Gd-DOTA. When Gd-DOTA crosses the damaged BBB into the extracellular 445
space of brain parenchyma, its paramagnetic property is changed [41, 51]. T1-weighted MRI 446
contrast enhancement shows the rapid diffusion of gadolinium-based chelates from non-447
viable cells, while T2-weighted MRI contrast enhancement shows slow diffusion of 448
gadolinium-based chelates from viable immune cells [39]. We used T1-weighted MRI to 449
visualize gadolinium penetration through disrupted BBB. T2-weighted MRI was used to 450
monitor the vasogenic edema and localization of viable immune cells, including residential 451
microglia and peripheral immune cells due to LPS induced-neuroinflammation (Figure 4). In 452
our mouse model of neuroinflammation induced by intracranial injection of LPS, we opted to 453
use T2-weighted imaging. However, since the sensitivity to detect BBB disruption is limited, 454
there is a limitation that only MRI signal is not sufficient to evaluate BBB disruption. 455
Therefore, it is difficult to exclude the possibility of micro-disruptions of BBB. 456
To validate the usefulness of [18F]CB251 as a PET probe for imaging 457
neuroinflammation, we performed simultaneous PET/MRI and BLI (Figure 5), which 458
provided information on anatomical and functional changes with precise co-registration of 459
signals and accurate correction of attenuation [42, 43]. IHC staining showed increased TSPO 460
expression at the site of neuroinflammation. It also demonstrated that inflammatory 461
monocytes, macrophages, and antigen-presenting cells were present with resident immune 462
cells (i.e., microglia) at the site of inflammation, suggesting cooperation between the 463
peripheral and CNS immune system in the region of neuroinflammation [34-36]. BLI 464
revealed peripheral immune cell infiltration only in the region of the inflamed brain, but the 465
radioactive signal from [18F]CB251 showed the effects of both infiltrating peripheral immune 466
cells and resident microglia. 467
Furthermore, simultaneous [18F]CB251 PET/MR and comparative analysis of 468
[18F]CB251 PET and BLI were performed to evaluate the efficacy of an anti-inflammatory 469
agent and verify the usefulness of [18F]CB251 PET. CDDO-Me was used as an anti-470
inflammatory drug, which was reported to induce antioxidant proteins that prevent oxidative 471
damage from inflammation [44-48]. In our LPS-induced neuroinflammation model, treatment 472
with CDDO-Me reduced [18F]CB251 PET and BLI signals, indicating a decrease in 473
inflammatory response and recruitment of peripheral immune cells (Figure 6). Despite the 474
higher sensitivity of BLI, its signals were obscured by the skin and black fur of the mice in 475
our animal model. Because of the limitations of optical imaging, BLI can be used only for 476
preclinical studies. However, [18F]CB251 PET is a noninvasive imaging procedure that is 477
directly applicable to clinical investigations that provide diagnostic and therapeutic 478
information on neuroinflammation. 479
We demonstrated that [18F]CB251 is specific to TSPO-expressing inflammatory cells 480
with better selectivity for TSPO polymorphism. TSPO-targeting [18F]CB251 PET identified 481
the regions of neuroinflammation and also allowed us to evaluate the therapeutic effect of 482
anti-inflammatory treatments. In preclinical studies, our multimodal imaging approach using 483
PET/MR and BLI is expected to enable further understanding of the interactions between 484
CNS and the peripheral immune system during neuropathogenesis. Furthermore, [18F]CB251 485
PET/MRI, which provides information on anatomical and functional changes with precise co-486
registration of signals, can be an efficient tool for testing the efficacy of various anti-487
inflammatory drugs and treatment planning for both preclinical and clinical application. 488
Materials & Methods 489
Cell culture and reagents 490
RAW264.7, a mouse macrophage cell line and mouse splenocytes were used to evaluate 491
TSPO expression. LPS (50 ng/mL, Sigma-Aldrich, St.Louis, MO, USA) and IFN-γ (20 ng/mL, 492
Sigma-Aldrich) were used to activate immune cells. The 293FT (Invitrogen, Carlsbad, CA, 493
USA) and MDA-MB-231 (ATCC, Manassas, VA, USA) cell lines were used for various TSPO 494
expression levels. Cells were maintained in complete DMEM (for Raw264.7 and MDA-MB-495
231) or MEM (for 293FT) with 10% FBS and 1% antibiotics (penicillin-streptomycin). All 496
media and reagents for cell culture, including FBS and antibiotics were purchased from Gibco 497
(Grand Island, NY, USA). 498
499
TSPO expression in Cells 500
To express the genetic variants of TSPO proteins (Wild-type, WT; Mutant with 501
alanine-threonine at 147 amino acid, A147T), we generated different TSPO expression 502
vectors from the PBR (TSPO) (NM_000714) human tagged ORF clone RG220107 (Origene, 503
Rockville, MD, USA) by site mutagenesis (Figure S1B). We changed the nucleotide 504
sequences encoding alanine at amino acid 147 to threonine (A147T). We then transfected 505
these vectors into 293FT cells and performed an in vitro uptake test of these cells at 24 hours 506
after transfection. 507
For overexpression of the TSPO protein, the 293FT cell line was transiently 508
transfected with vectors (pCMV-eGFP/TSPO) (AddGene, Cambridge, MA, USA) harboring 509
the eGFP and TSPO coding sequences. MDA-MB-231, which highly expresses TSPO, was 510
used for the reduction of TSPO expression by shRNA (pCMV-eGFP/shTSPO; AddGene). 511
Each vector used for various levels of TSPO expression was transfected by Lipofectamine 512
2000 (Invitrogen, Waltham, MA, USA) at a total concentration of 2.5 µg/mL. Fluorescent 513
images were acquired by a confocal laser-scanning microscope (Leica TCS SP8; Wetzlar, 514
Hesse, Germany), and Western blotting was performed for verification. 515
516
Western blotting 517
Cell lysates were extracted by RIPA lysis buffer (Sigma-Aldrich), with a protease 518
inhibitor (Roche, Branchburg, NJ, USA) cocktail. Protein concentrations were measured by 519
the BCA protein assay kit (Pierce Endogen, Rockford, IL, USA), and samples containing 20 520
µg of proteins were separated on 10% polyacrylamide gels. Proteins were then transferred to 521
PVDF membranes (Millipore, Watford, Herts, UK), which were blocked by 5% skim milk in 522
Tris-buffered saline (20 mM Tris, 138 mM NaCl, and pH 7.4) with Tween-20 (0.1%) for 1 h. 523
Membranes were then incubated with primary antibodies followed by incubation with 524
secondary antibodies. The following antibodies were used: anti-TSPO (Abcam, Cambridge, 525
Cambs, UK), anti-iNOS (Santa-Cruz Biotechnology, Santa-Cruz, CA, USA), anti-β-actin 526
(Sigma-Aldrich) were anti-rabbit IgG (Cell Signaling Technology, Danvers, MA, USA), and 527
anti-mouse IgG (Cell Signaling Technology) for β-actin. 528
529
Radiochemical synthesis of radioactive TSPO-targeting ligands 530
The [11C]PK11195 [21], deuterium-substituted fluoromethyl-PBR28 ([18F]fmPBR28-531
d2 [52]), and [18F]GE-180 [33] were synthesized according to previous studies. The 532
[18F]CB251 probe was also synthesized following a protocol described in a previous study 533
[26]. 534
535
Membrane protein extraction 536
Membrane protein extraction was conducted by protocols from the membrane protein 537
extraction kit (Mem-PER plus kit, Thermo Fisher, Waltham, MA, USA). 293FT cells (5 x 538
106/100 Ø) were seeded for transient transfection and transfected with TSPO WT or A147T 539
vector for 24 hours. Cells were harvested using a scraper and washed twice with cell wash 540
solution. After centrifugation (300 x g, 5 min), the cell pellet was permeabilized for 10 min at 541
4°C and centrifuged (16000 x g, 15 min, 4°C). The supernatant containing cytosolic proteins 542
was removed, and the pellet was solubilized with the buffer from the Mem-PER plus kit for 543
30 min at 4°C. The solubilized pellet was centrifuged again (16000 x g, 15 min, 4°C), and the 544
supernatant containing membrane-associated proteins was used for competitive binding 545
assay. 546
547
Competitive inhibition assay 548
To verify the binding affinity of TSPO-targeting ligand for the TSPO polymorphism 549
rs6971, we performed competitive inhibition assay [34, 35] with [3H]PK11195 550
(NET885250UC, 9.25 MBq, Perkin Elmer, Boston, MA, USA) and 3 different TSPO-551
targeting ligands (PK11195, fmPBR28-d2, and CB251). Membrane proteins (100 µg/100 µL) 552
isolated from 293FT cells expressing TSPO WT (HABs) or A147T (LABs) were incubated 553
with [3H]PK11195 (0.019 MBq/100 µL) at different concentrations of each ligand (from 0.1 554
nM to 1000 µM) in assay buffer (50 mM Tris base, 140 mM NaCl, 1.5 mM MgCl2, 5 mM 555
KCl, 1.5 mM CaCl2, pH 7.4) for 1 hour at 37°C. Subsequently, the mixtures were filtrated 556
through Whatman filter paper under vacuum and washed three times with the wash buffer (50 557
mM Tris base, 1 mM MgCl2, pH7.4). Filters were harvested, and the radioactivity of each 558
filter in scintillation buffer (Perkin Elmer) was counted using β-counter (Liquid scintillation 559
analyzer Tri-Carb3100 TR, Perkin Elmer, Waltham, MA, USA). IC50 values of each ligand to 560
[3H]PK11195 in HAB or LAB were measured. The ratio of IC50 values of each ligand to 561
[3H]PK11195 in the LAB to HAB was also calculated, representing the sensitivity of TSPO 562
polymorphism. A value close to 1 indicates lower sensitivity to TSPO polymorphism. 563
564
Measurement of cell uptake of radioactive ligands 565
Cells were seeded in 6-well plates (Sigma-Aldrich) until they reached approximately 566
80% confluency. Before the uptake experiments, the cells were preincubated with glucose-567
free RPMI-1640 medium (Gibco) for 4 h. The cells were then trypsinized (Gibco), counted, 568
and 1×105 cells transferred into a 5 mL test tube containing warmed Hank’s balanced salt 569
solution (HBSS, Sigma-Aldrich) with 0.5% (w/v) bovine serum albumin (Sigma-Aldrich). 570
Subsequently, approximately 0.185 MBq of radioactive ligands were added to the tubes, and 571
the cells were incubated in a humidified incubator with 5% CO2 for 1 h at 37°C. The cells 572
were then washed 3 times with cold HBSS and lysed for 5 min in 200 μL of 1% sodium 573
dodecyl sulfate (SDS, Sigma-Aldrich). Cell lysates were collected, and radioactivity was 574
measured by a Cobra II gamma counter (Canberra Packard; Vaughan, Ontario, Canada). 575
Radioactivity was normalized according to the amount of total protein at the time of assay. 576
Total protein levels were quantified by the BCA protein assay kit (Pierce, Rockford, IL, 577
USA). All experiments were performed in triplicate. 578
579
Preparation of splenocytes from B6. LucTg mice 580
Luciferase-expressing cells were isolated from the dissected spleens of transgenic 581
C57BL/6lucTg mice and centrifuged at 1300 rpm for 5 min at 4 °C). The red blood cell lysis 582
buffer (1 mL/spleen, Sigma-Aldrich) was added to the cell pellet, and the mixture was 583
incubated at room temperature for 3 min to remove red cells. After a double volume of RPMI 584
medium was added to the cells, 2-3 volumes of red blood cell lysis buffer were added until 585
complete lysis of red blood cells. Subsequently, the cells were re-suspended, ensuring a 586
suspension of single cells, and filtered through a cell strainer (40 µm nylon, Falcon, Oneonta, 587
NY). Cells (3.175 × 105 to 5 × 106) were then plated in 24-well plates, and bioluminescent 588
images were acquired after the addition of D-luciferin (300 µg/mL, Caliper Life Science, 589
Hopkinton, MA) to measure luciferase activity. Bioluminescence intensity was measured by 590
an IVIS 100 system (Perkin Elmer, Waltham, MA). Cells (2 × 107) were adoptively 591
transferred by intravenous injection into recipient C57BL/6 mice to monitor the in vivo 592
distribution of luciferase-expressing immune cells. All experimental designs and procedures 593
for animal experiments were approved by the Institutional Animal Care and Use Committee 594
of Seoul National University and in accordance with the ethical standards of Seoul National 595
University Hospital guidelines of Seoul National University Hospital. 596
597
Intracranial inflammation mouse model by LPS administration 598
C57BL/6 mice (male, 6 weeks old) were used for generating the intracranial 599
inflammation model. LPS (Sigma-Aldrich, 5 µg/2 µL dissolved in saline) was used to induce 600
local inflammation in the right striatum of the mouse brain (anteroposterior, +1.8; 601
mediolateral, +2.0; dorsoventral, -3.0) via intracranial injection by a robotic stereotaxic 602
device (Stoelting, Wood Dale, IL) equipped with a Hamilton syringe at a rate of 0.25 µL/min 603
[52-54]. Saline was injected as a control. The mouse was placed in the isoflurane induction 604
box (5% isoflurane, 95% air, 250 cc/min) until the animal was fully anesthetized, and the 605
isoflurane percentage was adjusted to 2 % at a flow rate of 250 cc/min during the process. 606
607
[18F]CB251 PET/MR imaging 608
To visualize neuroinflammation, PET/MR scans were acquired by a simultaneous 609
small animal PET insert (SimPET, Brightonic imaging, Seoul, Korea) combined with an M7 610
1.0 T MRI system (Aspect Imaging, Jerusalem, Israel). [18F]CB251 (9.25 to 11.1 MBq per 611
mouse) was injected intravenously, and images were acquired 4 days after intracranial 612
injection of LPS. Mice were anesthetized with 1.5% isoflurane during imaging. 613
To evaluate breach of the BBB, gadolinium (Gd-DOTA, 0.5 mmol/kg) was injected 614
intravenously, and T1- and T2-weighted MR scans were acquired [26]. The T1-weighted fast-615
spin echo sequence consisted of a repetition time of 200 msec and an echo time of 10 msec. 616
The T2-weighted fast spin echo sequence consisted of a repetition time of 3000 msec and an 617
echo time of 63.84 msec. 618
A PET/MR scan was performed simultaneously for 40 min with a 30 x 30 mm field-619
of-view (FOV) at 20 min post-[18F]CB251 injection. PET images were reconstructed using 620
3D OSEM algorithm (12 subsets and 3 iterations). An ellipsoidal region-of-interest was 621
drawn around the peak PET activity on the right striatum in a coronal PET/MRI slice, and the 622
region-of-interest was copied to the left striatum using AMIDE program (ver 0.9.0, 623
http://amide.sourceforge.net). PET and MR images were reconstructed and analyzed by the 624
AMIDE program (ver 0.9.0, http://amide.sourceforge.net). The ratio of maximum PET SUV 625
in the right and left striatum was then calculated. 626
627
Bioluminescence imaging 628
An in vivo IVIS 100 bioluminescence/optical imaging system (Xenogen, Alameda, 629
CA, USA) was used to follow luciferase-expressing peripheral immune cells. For the ex vivo 630
imaging of luciferase-expressing peripheral immune cells in the brain, D-luciferin (3 631
mg/mouse) was intravenously injected into the tail vein at 10 min before sacrificing the 632
animal in a CO2 chamber. The bioluminescent signal was acquired and analyzed by Living 633
Image ver.2.50.2 software (Xenogen). 634
635
Immunohistochemistry 636
Brain tissues from mice were fixed in 4% paraformaldehyde, embedded in paraffin, 637
and sectioned into 4 µm-thick segments. After heat-induced antigen epitope retrieval, slides 638
were blocked with PBS containing 3% normal serum and incubated overnight at 4 °C with 639
primary antibodies as follows: anti-CD68, anti-CD86 (Santa Cruz Biotechnology, Santa Cruz, 640
CA, USA), anti-CD4, anti-CD8, anti-Iba-1, and anti-TSPO (Abcam, Cambridge, UK). The 641
slides were then washed with PBS and incubated with biotinylated secondary antibodies 642
(anti-goat for CD68; anti-rabbit for CD4, CD8, and Iba-1; all from Santa Cruz 643
Biotechnology). The slides were then treated with avidin-biotin solution (Vector Laboratories, 644
Burlingame, CA, USA) for 1 h. The antigenic signal was developed using DAB (3, 3 –645
diaminobenzidine) substrate (Vector Laboratories, Burlingame, CA, USA), according to the 646
manufacturer’s instructions. Finally, the slides were counterstained with hematoxylin and 647
mounted with the Permount Mounting Medium (Thermo Fisher Scientific, Fair Lawn, NJ, 648
USA). 649
650
CDDO-methyl ester (CDDO-Me) treatment 651
2-Cyano-3,12-dioxo-oleana-1,9(11)-dien-28-oic acid methyl ester (CDDO-methyl 652
ester or CDDO-Me, Sigma-Aldrich) was dissolved in DMSO to produce a 10 mM stock 653
solution. The stock solution was further diluted with 7.5% PBST (1.5 ml Tween-20 dissolved 654
in 20 ml PBS) to obtain a 200 nM CDDO-ME solution. Mice received 100 µL of CDDO-ME 655
solution via intraperitoneal injection administered once daily for 3 days beginning 1 day after 656
intracranial administration of LPS. 657
658
Statistical analysis 659
All results were calculated as means ± standard deviation (SD). Statistically 660
significant differences were analyzed by a paired 2-sample Student t-test. Statistical 661
significance was considered to be p<0.05. GraphPad Prism 5 software (San Diego, CA) was 662
used for statistical analysis.663
Acknowledgments 664
This study was supported by grants the from the National Research Foundation (NRF), 665
funded by the Ministry of Science and ICT (MSIT) of Korea (No. 20110030001, 666
2020R1A2C2011695 to H.Y.), grants from the Korea Health Industry Development Institute 667
(KHIDI), and by the Ministry of Health & Welfare of Korea (HI16C0947 to S.E.K. and 668
B.C.L.). 669
670
Author Contributions 671
K. K., Y-H. K., J. N., and C-H. L. performed transfections, Western blots, 672
immunohistochemistry, confocal microscopy, FACS analysis, animal imaging, and 673
interpretation of data. S-Y L. generated the cells that expressed TSPO polymorphisms 674
(genetic variants) and performed the evaluation of the in vitro cell uptake of radio-labeled 675
compounds. H.K. and S-H. B. maintained transgenic animals and isolated immune cells from 676
transgenic mice. M.S.L., G.B.K., and J.S.L. analyzed and interpreted simultaneous PET/MR 677
imaging. S.H.L., K.Y.K, J.H.J., I.H.S, and B.C.L. synthesized the described compound, 678
performed radiosynthesis of the described radiotracers, and participated in scientific 679
discussions. J.C.P., G.J.C., K.W.K., S.E.K., J-K C., E.E.K., and S-H P. also participated in 680
scientific discussions. K.K. interpreted the data and wrote the initial draft of the manuscript. 681
H.Y. supervised projects, contributed to the conception of the study, interpretation of data, 682
and wrote the final manuscript. This manuscript has been seen and approved by all authors, 683
who contributed to preparing the manuscript, and no person other than the authors listed has 684
contributed significantly to its preparation. 685
686
Competing Interests 687
Guen Bae Ko, Kyeong Yun Kim, and Jae Sung Lee declare that they are employees of 688
Brightonix Imaging Incorporation (for PET/MR). Other authors have nothing to declare. 689
Figure Legends 690
Figure 1. [18F]CB251 as a TSPO-targeting radioactive ligand. (A) Various TSPO ligands 691
and the structure of TSPO-targeting radioactive ligands. CB251 is shown compared with 692
known TSPO ligands, such as PK11195, PBR28, fm-PBR-d2, and GE-180. [18F]fmPBR28-693
d2 is fluoromethyl-PBR28-d2 (ref. 24, 30). Ki of ligands for TSPO from refs. 47a, 50b, 31c, 694
and 24d. (B) Competitive inhibition assay of PK11195, fmPBR28-d2, and CB251 with 695
[3H]PK11195 on membrane proteins . HAB indicates high affinity ligand binding phenotype 696
(wild type TSPO); LAB indicates low affinity ligand binding phenotype (A147T mutant) 697
isolated from 293FT cells expressing polymorphic TSPOs. CPM indicates count per minute. 698
The ratio of IC50 LAB/HAB represents the sensitivity of TSPO polymorphism; a value close 699
to 1 indicates less sensitive to TSPO polymorphism. (C) In vitro cellular uptake of CB251 in 700
293FT cells expressing polymorphic TSPO was compared with TSPO ligands. (D) PET scan 701
images of 293FT cells expressing polymorphic TSPOs (E) Representative PET/MR images 702
of mice bearing 293FT cells with polymorphic TSPOs (N=2 for each treatment) 703
Figure 2. [18F]CB251 as a TSPO-targeting ligand for imaging activated immune cells. 704
(A) TSPO expression in 293FT cells was increased by transfection with pCMV-TSPO/eGFP. 705
(B) [18F]CB251 uptake was increased in TSPO-overexpressing cells. (C) TSPO expression in 706
MDA-MB-231 cells was reduced by transfection with pCMV-shTSPO/eGFP. (D) 707
[18F]CB251 uptake was decreased in cells with reduced TSPO expression. (E) Western blots 708
of TSPO and iNOS (activated immune cell marker) in a mouse macrophage cell line 709
(RAW264.7) and mouse splenocytes. (F) [18F]CB251 uptake was increased in activated cells 710
(both in Raw264.7 and mouse splenocytes). All experiments were replicated at least three 711
times. 712
Figure 3. Bioluminescence imaging of peripheral immune cells in a mouse 713
neuroinflammation model using intracranial LPS injection. (A) Flow cytometry analysis 714
of immune cells from splenocytes of C57BL/6 (wild type) and C57BL/6.LucTg (reporter 715
transgenic) mice. (B) Semiquantitative analysis of luciferase-expressing mouse splenocytes 716
from C57BL/6.LucTg. The intensity of luciferase signal and the number of cells were highly 717
correlated (R2=0.9991) (C) Schematic representation of experimental design using luciferase-718
expressing splenocytes to monitor peripheral immune cell infiltration in the brain of a mouse 719
with neuroinflammation induced by intracranial LPS injection. (D) Representative 720
biodistribution of luciferase-expressing splenocytes in mice 4 days after intracranial injection 721
of saline or LPS (N=2 for each treatment). 722
Figure 4. Representative MR scans using gadolinium-DOTA to monitor the Blood-Brain 723
Barrier (BBB) disruption and immune cell activation. (A) Intracranial LPS injection 724
model (saline as a control). (B) T1-weighted MR scans and (C) T2-weighted MR scans on 725
day 1, 3, and 4 after intracranial injection (N=3). T1-weighted MRI was used to visualize the 726
presence of gadolinium penetration through disrupted BBB, and T2-weighted MRI was used 727
to monitor the vasogenic edema and localization of viable immune cells due to LPS induced-728
neuroinflammation. 729
Figure 5. Visualization of the intracranial LPS-induced neuro-inflammatory region 730
using multi-modal imaging. (A) Experimental scheme of simultaneous PET/MR using 731
[18F]CB251 and BLI. (B) Representative images of [18F]CB251 PET/MR and 732
bioluminescence (BLI). The red circle indicates a region of interest (ROI). (C) Signal 733
intensity ratio of the right striatum (injected with saline or LPS) to the left striatum (no 734
injection) in the saline-injected (N=6) and LPS-injected (N=7) mice. PET signal was 735
expressed as standard uptake value (SUVmax) and the BLI signal was expressed as total flux 736
(photon/sec/cm2/sr). (D) IHC staining of intracranial LPS injected mouse brain tissues. Iba-1 737
(macrophages/microglia marker), CD68 (macrophages/monocytes marker), CD86 (antigen-738
presenting cells), CD4 (helper T cells), and CD8 (cytotoxic T cells) were used as markers for 739
each cell type. 740
Figure 6. Evaluation of the therapeutic effect of the anti-inflammatory drug (CDDO-741
Me) using [18F]CB251 PET and BLI. (A) Experimental design of treatments and imaging 742
schedules, including simultaneous [18F]CB251 PET/MRs and BLIs. (B) Representative 743
[18F]CB251 PET/MR scans and SUVmax ratios were plotted. (C) Representative BLI image 744
and total flux ratio were plotted. Signal ratios from each modality (SUVmax ratio for PET; 745
Total flux ratio for BLI) were calculated from the region of interest (ROI) of right 746
(intracranially injected with saline, LPS, or LPS plus anti-inflammatory agent CDDO-Me) 747
and left striatum (non-injected) in the mouse brain. The values are mean ± SD. (N=8 for 748
saline-treated; N=12 for LPS-treated; N=5 for LPS plus CDDO-Me). 749
Figure S1. TSPO genotypes and classfication. (A) TSPO ligand binding classification. Single 750
nucleotide polymorphism rs6971 TSPO (CT point mutation; rs6971 A147T; Ala to Thr at 751
amino acid 147 of TSPO) influences the binding affinity of TSPO ligands. TSPO with Thr/Thr 752
at amino acid 147 shows a lower binding affinity to its ligand. TSPO ligand binding phenotypes 753
are HAB or LAB, which indicate high- or low-affinity binding of ligands, respectively 754
*Adapted from ref 32, 33. (B) Expression vector design for TSPO mutagenesis 755
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916
D E
NN
O
11CH3
Cl
[11C]PK11195
N
NOO
18F
[18F]GE180
NN
Cl
ClO
N
O
18F
[18F]CB251
N N
O
O CH3
OCD218F
[18F]fmPBR28-d2
Compound Ki (nM)PK11195 1.4a
PBR28 6.1a
fmPBR28-d2 3.6b
GE-180 0.87c
CB251 0.27d
ATSPO ligands
3.30.2
coronal
sagittal
[18F]CB251[18F]fmPBR28-d2
WT Mut
0.1 2.1
WT
Mut
WT
Mut
[18F]CB251[18F]fmPBR28-d2
1.0 30.1
WT
Mut
WT
Mut
FIGURE 1. [18F]CB251 as a TSPO-targeting radioactive ligand (A) Various TSPO ligands and the structure of TSPO-targeting radioactive ligands. CB251 is shown compared with known TSPO ligands, such as PK11195, PBR28, fm-PBR-d2, and GE-180. [18F]fmPBR28-d2 is fluoromethyl-PBR28-d2 (ref. 24, 30). Ki of ligands for TSPO from refs. 47a, 50b, 31c, and 24d. (B) Competitive inhibition assay of PK11195, fmPBR28-d2, and CB251 with [3H]PK11195 on membrane proteins . HAB indicates high affinity ligand binding phenotype (wild type TSPO); LAB indicates low affinity ligand binding phenotype (A147T mutant) isolated from 293FT cells expressing polymorphic TSPOs. CPM indicates count per minute. The ratio of IC50 LAB/HAB represents the sensitivity of TSPO polymorphism; a value close to 1 indicates less sensitive to TSPO polymorphism. (C) In vitro cellular uptake of CB251 in 293FT cells expressing polymorphic TSPO was compared with TSPO ligands. (D) PET scan images of 293FT cells expressing polymorphic TSPOs (E) Representative PET/MR images of mice bearing 293FT cells with polymorphic TSPOs (N=2 for each treatment)
C100806040200
% u
ptak
e
[11C]PK11195 [18F]fmPBR28-d2 [18F]GE-180 [18F]CB251
Ctr WT Mut(A147T)
Ctr WT Mut(A147T)
Ctr WT Mut(A147T)
Ctr WT Mut(A147T)
B
-11 -10 -9 -8 -7 -6 -5 -40
50
100
150
Log10 (M)%
CP
M-11 -10 -9 -8 -7 -6 -5 -4
0
50
100
150
Log10 (M)
% C
PM
LAB (Mut) IC50=7.61 µMHAB (WT) IC50=0.204 µM
LAB (Mut) IC50=0.305 µMHAB (WT) IC50=0.267 µM
-11 -10 -9 -8 -7 -6 -5 -40
50
100
150
200
Log10 (M)
% C
PM
LAB (Mut) IC50=5.01 µMHAB (WT) IC50=6.05 µM
[11C]PK11195 [18F]fmPBR28-d2 [18F]CB251
LAB/HAB of IC50= 0.83 LAB/HAB of IC50= 37.28 LAB/HAB of IC50= 1.14
%C
PM
%C
PM
200
150
100
50
0%
CPM
150
100
50
0
150
100
50
0-11 -10 -9 -8 -7 -6 -5 -4 Log10(M)-11 -10 -9 -8 -7 -6 -5 -4 Log10(M)-11 -10 -9 -8 -7 -6 -5 -4 Log10(M)
20
15
10
5
0
[18F]
CB
251
upta
ke
( 106
cpm
/mg)
ctr TSPO/eGFP(+)
P <0.01
E F
C D
A B
FIGURE 2. [18F]CB251 as a TSPO-targeting ligand for imaging activated immune cells. (A) TSPO expression in 293FT cells was increased by transfection with pCMV-TSPO/eGFP. (B) [18F]CB251 uptake was increased in TSPO-overexpressing cells. (C) TSPO expression in MDA-MB-231 cells was reduced by transfection with pCMV-shTSPO/eGFP. (D) [18F]CB251 uptake was decreased in cells with reduced TSPO expression. (E) Western blots of TSPO and iNOS (activated immune cell marker) in a mouse macrophage cell line (RAW264.7) and mouse splenocytes. (F) [18F]CB251 uptake was increased in activated cells (both in Raw264.7 and mouse splenocytes). All experiments were replicated at least three times.
ctr TSPO/eGFP(+)
β-actin
TSPO
ctr TSPO/eGFP (+)
ctr shTSPO/eGFP(-)
ctr shTSPO/eGFP(-)
β-actin
TSPO
[18F]
CB
251
upta
ke
( 106
cpm
/mg)
2
1.5
1
0.5
0
ctr shTSPO/eGFP(-)
P <0.05
RAW264.7 Splenocytesβ-actin
TSPO
iNOS
ctractivatedLPS+INFγctr
activatedLPS+INFγ
[18F]
CB
251
upta
ke
( 106
cpm
/mg)
5
4
3
2
1
0 ctr activated ctr activatedRAW264.7 Splenocytes
P <0.005P <0.01
Figure 3. Bioluminescence imaging of peripheral immune cells in a mouse neuroinflammation model using intracranial LPS injection. (A) Flow cytometry analysis of immune cells from splenocytes of C57BL/6 (wild type) and C57BL/6.LucTg (reporter transgenic) mice. (B) Semiquantitative analysis of luciferase-expressing mouse splenocytes from C57BL/6.LucTg. The intensity of luciferase signal and the number of cells were highly correlated (R2=0.9991) (C) Schematic representation of experimental design using luciferase-expressing splenocytes to monitor peripheral immune cell infiltration in the brain of a mouse with neuroinflammation induced by intracranial LPS injection. (D) Representative biodistribution of luciferase-expressing splenocytes in mice 4 days after intracranial injection of saline or LPS (N=2 for each treatment).
A B
C D
C57BL/6.LucTg C57BL/6
Saline or
LPS
Luciferase-expressingsplenocytes
BLI
Splenocytes
CD8 CD4 Mac-1
CD
4
C
D4
CD8 CD4 Mac-1
B22
0
B22
0
Gr-
1
G
r-1
B cells
B cellsT cells
T cells
Macrophages
Macrophages
Granulocytes
Granulocytes
WT
LucTg
R2=0.9991
3
2.5
2
1.5
1
0.5
0
Tota
l Flu
x (x
106
phot
on/s
ec/c
m2 /s
r)
0 2 4 6 (106 cells)
0.3175 0.625 1.25 2.5 5 (106 cells/well)
Saline LPS
Brain Brain
Heart
Lung
Kidney
Liver
StomachSpleen
Intestine Intestine
SpleenStomach
Liver
Kidney
Lung
Heart
A
B
C
Figure 4. Representative MR scans using gadolinium-DOTA to monitor the Blood-Brain Barrier (BBB) disruption and immune cell activation. (A) Intracranial LPS injection model (saline as a control). (B) T1-weighted MR scans and (C) T2-weighted MR scans on day 1, 3, and 4 after intracranial injection (N=3). T1-weighted MRI was used to visualize the presence of gadolinium penetration through disrupted BBB, and T2-weighted MRI was used to monitor the vasogenic edema and localization of viable immune cells due to LPS induced-neuroinflammation.
Saline / LPSSaline / LPS
T2- weighted MRDay 1 Day 3 Day 4
T2- weighted MRDay 1 Day 3 Day 4
Saline
LPS
Saline
LPS
Day 1 Day 3 Day 4 T1-weighted MR T1- weighted MR
Day 1 Day 3 Day 4
Figure 5. Visualization of the intracranial LPS-induced neuro-inflammatory region using multi-modal imaging. (A) Experimental scheme of simultaneous PET/MR using [18F]CB251 and BLI. (B) Representative images of [18F]CB251 PET/MR and bioluminescence (BLI). The red circle indicates a region of interest (ROI). (C) Signal intensity ratio of the right striatum (injected with saline or LPS) to the left striatum (no injection) in the saline-injected (N=6) and LPS-injected (N=7) mice. PET signal was expressed as standard uptake value (SUVmax) and the BLI signal was expressed as total flux (photon/sec/cm2/sr). (D) IHC staining of intracranial LPS injected mouse brain tissues. Iba-1 (macrophages/microglia marker), CD68 (macrophages/monocytes marker), CD86 (antigen-presenting cells), CD4 (helper T cells), and CD8 (cytotoxic T cells) were used as markers for each cell type.
A
B
C
D
Day 0 Day 1 Day 4
Intracranial injection with LPS
i.v. injectionLuciferse-expressing splenocytes
[18F]CB251 PET/MRBLI
Saline
LPS
MRI (T2) [18F]CB251 PET/MR BLI
CD4 CD8
CD68
CD86
TSPO Iba-1
Left Right Rightno injection Saline LPS
PET (SUVmax) p=0.023
p=0.012
Left Right Rightno injection Saline LPS
p=0.011
n.s.
BLI
SUVm
axra
tio(R
ight
/Lef
t stri
atum
)
Tota
l Flu
x ra
tio(R
ight
/Lef
t stri
atum
)1.8
1.6
1.4
1.2
1.0
0
6
4
2
0
[18F]CB251 PET/MR
Saline LPS LPS + CDDO-Me
A
B
CBLI
Saline LPS LPS + CDDO-Me
Day 0 Day 1 Day 2 Day3 Day 4
Intracranial injection with LPS
i.v. injectionLuciferse-expressing splenocytes [18F]CB251 PET
T2-MRIBLI i.p. injection 200 nM CDDO-Me
0.7 2.0
PET
***p=0.002 **
p= 0.0323
n.s.
Saline LPS LPSCDDO-Me
SUVm
axra
tio(R
ight
/Lef
t stri
atum
)
1.8
1.6
1.4
1.2
1.0
0
BLI
*** p=0.0009
**p= 0.0025
*p= 0.042
Saline LPS LPSCDDO-Me
Tota
l Flu
x ra
tio(R
ight
/Lef
t stri
atum
)
8
6
4
2
0
Figure 6. Evaluation of the therapeutic effect of the anti-inflammatory drug (CDDO-Me) using [18F]CB251 PET and BLI. (A) Experimental design of treatments and imaging schedules, including simultaneous [18F]CB251 PET/MRs and BLIs. (B) Representative [18F]CB251 PET/MR scans and SUVmax ratios were plotted. (C) Representative BLI image and total flux ratio were plotted. Signal ratios from each modality (SUVmax ratio for PET; Total flux ratio for BLI) were calculated from the region of interest (ROI) of right (intracraniallyinjected with saline, LPS, or LPS plus anti-inflammatory agent CDDO-Me) and left striatum (non-injected) in the mouse brain. The values are mean ± SD. (N=8 for saline-treated; N=12 for LPS-treated; N=5 for LPS plus CDDO-Me).
Vectors for TSPO Mutagenesis
pTSPO-WT or –Mut(7.1kb)
TSPO genotype LigandBinding
PhenotypeDNA polymorphism rs6971
Protein position 147
Wild type C/C Ala/Ala HAB*
Mutant T/T Thr/Thr LAB*
TSPO ligand binding classification
HAB High Affinity Binder ; LAB Low Affinity Binder
B
A
FIGURE S1. TSPO genotypes and classfication. (A) TSPO ligand binding classification. Single nucleotide polymorphism rs6971 TSPO (CT point mutation; rs6971 A147T; Ala to Thr at amino acid 147 of TSPO) influences the binding affinity of TSPO ligands. TSPO with Thr/Thr at amino acid 147 shows a lower binding affinity to its ligand. TSPO ligand binding phenotypes are HAB or LAB, which indicate high- or low-affinity binding of ligands, respectively *Adapted from ref 32, 33. (B) Expression vector design for TSPO mutagenesis.