
==== Front
Emerg Microbes Infect
Emerg Microbes Infect
Emerging Microbes & Infections
2222-1751
Taylor & Francis

38662785
2348528
10.1080/22221751.2024.2348528
Version of Record
Zika
Research Article
Translocator protein (TSPO) is a biomarker of Zika virus (ZIKV) infection-associated neuroinflammation
Emerging Microbes & Infections
C. B. Victorio et al.
https://orcid.org/0000-0002-1161-5006
Victorio Carla Bianca Luena
Ganasarajah Arun
Novera Wisna
Ong Joanne
https://orcid.org/0000-0002-0094-4285
Msallam Rasha
https://orcid.org/0000-0001-5140-2622
Chacko Ann-Marie
Laboratory for Translational and Molecular Imaging (LTMI), Cancer and Stem Cell Biology Programme, Duke-NUS Medical School, Singapore, Singapore
CONTACT Carla Bianca Luena Victorio carla-bianca.victorio@duke-nus.edu.sg
Ann-Marie Chacko ann-marie.chacko@duke-nus.edu.sg
Supplemental data for this article can be accessed online at https://doi.org/10.1080/22221751.2024.2348528.

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© 2024 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group, on behalf of Shanghai Shangyixun Cultural Communication Co., Ltd
2024
The Author(s)
https://creativecommons.org/licenses/by-nc/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. The terms on which this article has been published allow the posting of the Accepted Manuscript in a repository by the author(s) or with their consent.

ABSTRACT

Zika is a systemic inflammatory disease caused by infection with Zika virus (ZIKV). ZIKV infection in adults is associated with encephalitis marked by elevated expression of pro-inflammatory cytokines and chemokines, as well as increased brain infiltration of immune cells. In this study, we demonstrate that ZIKV encephalitis in a mouse infection model exhibits increased brain TSPO expression. TSPO expression on brain-resident and infiltrating immune cells in ZIKV infection correlates with disease and inflammation status in the brain. Brain TSPO expression can also be sensitively detected ex vivo and in vitro using radioactive small molecule probes that specifically bind to TSPO, such as [3H]PK11195. TSPO expression on brain-resident and infiltrating immune cells is a biomarker of ZIKV neuroinflammation, which can also be a general biomarker of acute viral neuroinflammatory disease.

KEYWORDS

Zika
neuroinflammation
PK11195
translocator protein
TSPO
TSPO neuroinflammation
Singapore’s Ministry of Education Academic 2022-MOET1-0002 National Medical Research Council 10.13039/501100001349 OFLCG19May-0034 This research was supported by Singapore's Ministry of Education Academic Research Fund (AcRF) Tier 1 (2022-MOET1-0002), National Medical Research Council (NMRC) Open Fund Large Collaborative Grant on Integrated Innovations in Infectious Diseases (I3D) (OFLCG19-May-0034) and the Singapore Agency for Science, Technology, and Research (A*STAR) Industry Alignment Pre-positioning Grant for Cancer Immunotherapy Imaging (CITI) Programme (H18/01/a0/018).
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pmcIntroduction

Zika is a systemic inflammatory disease caused by infection with a mosquito-transmitted flavivirus, known as Zika virus (ZIKV). ZIKV is a neurotropic virus prevalent in tropical and subtropical regions. Infection in pregnant women during the first trimester is associated with increased incidence of microcephaly in the developing foetus [1,2]. Microcephaly is caused by ZIKV infection-induced death of neuroprogenitor cells in the developing cerebral cortex [3–5]. Moreover, neuroinflammatory lesions in the brain and spinal cord and elevation of pro-inflammatory cytokines and chemokines – which include IL6, IL9, TNFα, IL-1β, and CXCL12 have been reported in animal infection models [6–8]. Most importantly ZIKV infection in children [9,10] and adults [11–14] can lead to neurologic complications – including encephalitis, transverse myelitis, and Guillain-Barré Syndrome (GBS) – although the exact incidence is unknown. The best prevalence estimates come from prospective studies in northeast Brazil during the 2014–2016 outbreak [15,16]. From 201 recruited patients with symptoms of both recent arbovirus infection and neurological complications, 20.4% (n = 41) was associated with infection with ZIKV alone; the rest of the cases were associated with either single infection with other arboviruses such as Chikungunya and dengue or co-infections with these viruses [15]. In the same cohort, 35.2% (n = 25) of patients diagnosed with primarily a sensorimotor demyelinating subtype of Guillain-Barré Syndrome (GBS) (n = 71) were associated with solo ZIKV infection [16]. These neurological symptoms were proposed to be due to direct ZIKV infection of mature glia [17], which include astrocytes, microglia, and oligodendrocytes, based on evidence from in vitro infection studies [18,19], as well as experimental animal inoculations [20].

Neuroinflammation is a complex response to altered homeostasis in the central nervous system (CNS) caused by either internal or external insults. This response is orchestrated by brain-resident (e.g. microglia) and infiltrating immune cells (e.g. monocytes and granulocytes) and often result in elevated CNS expression of translocator protein (TSPO) [21]. TSPO is mitochondrial protein widely accepted as a general biomarker of neuroinflammation. Chronic inflammation in neurodegenerative diseases is associated with increased brain TSPO expression in astrocytes, glia, and neurons [22–24]. In contrast, acute neuroinflammation associated with viral encephalitis is marked by increased brain infiltration of immune cells that express TSPO [25]. Elevated immune cell TSPO expression has been described as a hallmark of inflammation in a variety of viral infections, including Ebola virus (EboV) [26], human immunodeficiency virus (HIV) [27,28], and simian immunodeficiency virus (SIV) [29]. Elevated TSPO expression was also monitored in brains of experimental animals inoculated with encephalitic viruses, such as Venezuela Equine Encephalitis Virus (VEEV) [30], Herpes simplex virus (HSV) [31] or SARS-CoV-2 [32] using Positron Emission Tomography (PET) imaging with TSPO-targeted small molecules labelled with radioactive isotopes (i.e. radioligands). Recently, elevated TSPO expression in ZIKV-infected mouse brains using both interferon-deficient [25] and fully immunocompetent mice [33,34] has also been non-invasively detected by PET imaging.

Here, we describe the modulation of TSPO expression in encephalitic ZIKV-infected mouse brains contributed by brain-resident and infiltrating immune cells. Encephalitis was validated against traditional measures of neuroinflammation. We also report a sensitive method for the ex vivo and in vitro detection of ZIKV neuroinflammation using the first-generation commercially available TSPO radioligand [3H]PK11195. We propose that TSPO expression is an immune-driven biomarker of ZIKV neurological infection and inflammation that can be used as proxy for dynamic changes in disease progression and treatment response.

Materials and methods

Materials

[3H]PK11195 (NET885250UC, 9.25 MBq, 2912 GBq/mmol) was purchased from Perkin-Elmer (now Revvity), Singapore. Nonradioactive “cold” PK11195 (Cat. no. 85532-75-8) was purchased from Sigma Aldrich, Singapore. The ZIKV virus strain Paraiba01/2015 (Genbank Accession No. KX, 280026.1) was a kind gift from Dr. Pedro Vasconcelos (Instituto Evandro Chagas, Brazil). Adult AG129 male mice deficient in interferon (IFN)-α,β,γ receptor expression were obtained from in-house breeding colony maintained at the Duke-NUS Animal Facility.

Animal infection

All animal experiments were conducted with approval from the Institutional Animal Care and Use Committee (IACUC) of Duke-NUS Medical School and SingHealth (IACUC approval no. 2020/SHS/1607) and conformed to the National Institutes of Health (NIH) guidelines and public law. The animals were housed in individually-ventilated cages and provided with food pellets and water ad libitum.

The ZIKV model used in this study was established as described [25,35]. Briefly, male AG129 were (8–11 weeks old) infected with 106 plaque-forming units (pfu) virus by intraperitoneal (i.p.) injection. Mock-infected controls were similarly inoculated with an equal volume of sterile phosphate buffered saline (PBS, pH 7.4). In total, twenty (20) animals were allocated into three groups used in this study: Mock infection, mid-disease, and late disease groups.

Ex vivo [3H]PK11195 tissue biodistribution

At pre-infection (day 0) and late ZIKV (day 8 post-infection), mice were injected intravenously (i.v.) with [3H]PK11195 (5 µCi) and sacrificed 1 h post tracer injection. In another group of animals, 55 min post-administration of [3H]PK11195, mice were injected i.v. with a blocking dose of “cold” PK11195 (5 mg/kg) to assess specific binding of the tracer. Organs were harvested, and brains were micro-dissected as previously described [25,36]. Tissues were completely dissolved in Soluene® 350 (PerkinElmer, USA) for 2–7 days and subsequently mixed with Ultima Gold liquid scintillant (PerkinElmer, USA) prior to detection of radioactivity with TriCarb 4810TR liquid scintillation counter (PerkinElmer, USA). The distribution of tracer in tissues was reported as % injected dose per gram tissue (%ID/g).

In a separate cohort of animals, mouse brains were harvested at 60 min post-tracer injection, sliced in the midline, and embedded in OCT medium. Cryosections (20 µm) were air-dried and subjected to autoradiographic imaging for 72 h.

In vitro radioligand binding and autoradiography

In separate cohorts of mice, sagittal cryosections of mouse brains (20 µm) harvested from either sham-infected or ZIKV-infected mice were subjected to in vitro radioligand binding assay as previously described [25]. Tissues were incubated with 10 nM (∼ 0.87 MBq) [3H]PK11195 for 45 min at room temperature (RT). Some tissue sections were incubated with 10 nM [3H]PK11195, mixed with 10 µM PK11195, for blocking experiments. Tissue sections were washed in 0.17M Tris-Cl, pH 7.4 several times prior to air-drying. Autoradiography was done by image acquisition in BeaQuant autoradiograph (Ai4R, France) for 24 h. Images were analyzed using Beamage software (Ai4R, France).

Immunohistopathology

Brain tissue cryosections (10 µm) were subjected to histochemical staining with hematoxlyin & eosin (H&E) as described [37]. Tissues were also subjected to immunohistochemical staining or immunofluorescence staining for TSPO and ZIKV envelope protein expression using TSPO antibody (Invitrogen, Cat. no. MA5-31966) and 4G2 (Novus Biologicals, Cat. no. NBP2-52709) monoclonal antibodies, respectively.

Brain infection and inflammation assessments

Brains were harvested as described previously [37] and frozen immediately. Frozen brains were homogenized to extract total protein, which were processed for detection of IL-6 and TNF-α expression using the Ready-Set-GO! Kit following the manufacturer’s protocol (eBioscience, USA). Total RNA was extracted from frozen brains using RNEasy RNA extraction kit (Qiagen, Germany) according to manufacturer’s instructions. Real-time RT-PCR was carried out with primers targeting the E gene of ZIKV genome (ZK_F: 5′- CCGCTGCCCAACACAAG– 3′; ZK_R: 5′- CCACTAACGTTCTTTTGCAGACAT- 3′) and viral RNA standards for absolute viral RNA quantitation as previously described [25,35].

Brain immune cell profiling

Brain tissue immune profiling was conducted as previously described [25]. Briefly, mechanically disaggregated brains were incubated in digestion buffer containing DNaseI and collagenase, and single cell suspensions were collected by passing the suspension through a nylon mesh strainer (70 μm). Brains homogenates were fat depleted by centrifugation in Percoll. Cells were labelled with fluorescent antibody cocktails and immune cell subsets identified by flow cytometry (Fortessa, BD Biosciences, USA) and analyzed using FlowJo V10.8.0 (BD Biosciences, USA). Detected events were normalized to absolute cell number using CountBright Absolute Counting Beads (ThermoFisher, USA) [38]. Cells were gated using immune cells (CD45+) and translocator protein (TSPO) expression. TSPO expression was expressed as Mean Fluorescence Intensity (MFI) from samples tagged with anti-mouse TSPO-AlexaFluor 488 (ab199779; Abcam, USA). The following markers were used to identify the immune landscape: total immune cells (CD45+); granulocytes (Ly6G + Ly6C + CD11b+); microglia (CD11b + F4/80 + Ly6c−Ly6G−CD3−); monocyte-derived macrophages or MDM (CD11b + F4/80 + Ly6c + Ly6G−CD3−); monocytes (CD11b + CD115+); dendritic cells (CD11c + MHCII+); total T cells (CD19 − CD49b− B220 − LY6G−CD3+); cytotoxic T cells (CD19 − CD49b−B220 − LY6G−CD3 + CD8+); and helper T cells (CD19 − CD49b−B220 − LY6G−CD3 + CD4+).

Statistical analysis

Statistical analyses and graphing were performed with Prism v9.5 (GraphPad Software, USA). Mann-Whitney test and Kruskal-Wallis tests with Dunn’s post-hoc correction were used to compare medians among different groups. Spearman’s coefficient (ρ) was used to assess the correlation between two parameters. Results were considered statistically significant at p < 0.05. Correlation heatmaps were generated in RStudio (version 2023.09.0 + 463 “Desert Sunflower”).

Results

Evidence of neuroinflammation in ZIKV murine infection model

ZIKV infection in AG129 mice led to acute lethal disease with median overall survival (mOS) of 10 days post-infection (Figure 1(A)). The progression of Zika disease was accompanied by severe wasting and body weight loss (Figure 1(B)), as well as neurological impairment, including ataxia and limb paresis or paralysis (Figure 1(C)). Infected mouse brains harvested on days 0 (pre-infection), 4 (mid disease), and 8 (late disease) post-infection revealed increased ZIKV replication, where viral RNA copies in infected brains increased by >3-log values relative to pre-infection (Figure 1(D)). ZIKV-infected brains also exhibited elevated IL6 expression on days 4 and 8, with 2.2-fold and 1.5-fold higher IL6 production than pre-infection, respectively (Figure 1(E)). Most importantly, ZIKV-infected brains exhibited increased infiltration of immune cells (CD45+) at late disease as shown by flow cytometry (FC) (Figure 1(F)) and immunohistopathological (i.e. hematoxylin and eosin; H&E) staining of brain tissue (Figure 1(G)). Other histopathological findings include perivascular cuffing and gliosis (Figure 1(G)). These results confirmed that the adult AG129 mouse model of ZIKV infection exhibited classical signs of neuroinflammation. Figure 1. Disease and neuroinflammation in ZIKV-infected mice. (A) Kaplan-Meier survival curves, (B) Body weight loss, and (C) Neurological scores of AG129 mice infected i.p. with 106 pfu ZIKV Paraiba01/Brazil strain. (D) Viral replication, (E) IL6 expression, and (F) infiltration of CD45+ immune cells in ZIKV-infected mouse brains. (G) Representative images of mock-infected vs. ZIKV-infected hind brain tissue sections subjected to hematoxylin & eosin staining. Highlighted areas revealing gliosis and perivascular cuffing in ZIKV-infected brains, as well as a comparable region in Mock-infected brain, are enlarged. Data are shown as mean ± SD, and means were compared by Kruskal-Wallis test with Dunn’s post-hoc correction. Scale bars in 1G represent 1 mm unless stated otherwise.

Modulation of TSPO expression in ZIKV-infected brains

We next evaluated how TSPO expression levels changed in the brain during ZIKV infection. Immunohistochemical (IHC) staining of ZIKV-infected mouse brain tissues revealed increased numbers of TSPO-expressing cells compared to pre-infection in all brain regions evaluated (Figure 2(A)). Whereas immunoblotting analysis of bulk brain tissues did not reveal differences in TSPO expression following infection (Figure S1), FC analysis of infected whole brains revealed an increase in the number of brain cells expressing TSPO (Figure 2(B)). The number of TSPO+ brain cells at late disease was 5.40 ± 3.90-fold (p = 0.006) and 5.17 ± 2.94-fold (p = 0.002) higher than either pre-infection or mid disease, respectively (Figure 2(B)). Further gating of live cells using the CD45 marker revealed that majority of brain cells exhibiting elevated TSPO expression are immune cells (CD45+) and not brain-resident (non-immune) cells (CD45-) (Figure 2(C and D)). This relationship was recapitulated in the immune cell population: 15.30 ± 3.50-fold change (p < 0.001) on late disease vs. pre-infection; and 12.36 ± 2.83-fold change (p = 0.008) on late- vs. mid-disease (Figure 2(D)), but not in the non-immune cell subset of the brain (Figure 2(C)). Similarly, mean TSPO expression normalized to all live cells in the brain did not vary between pre-infection vs. ZIKV disease (Figure 2(E)). However, cells gated for CD45 expression revealed increased TSPO expression in immune cells but not in non-immune cells in the bulk brain (Figure 2(F and G)). Indeed, immune cells in mid and late disease brains exhibited 1.17 ± 0.22-fold (p < 0.001) and 1.21 ± 0.27-fold (p < 0.001) higher TSPO mean fluorescence intensity (MFI) relative to pre-infection, respectively (Figure 2(G)). Lastly, whole brain (WB) TSPO expression directly correlated with other markers of neuroinflammation – specifically immune cell infiltration (r = 0.96; p < 0.001) and IL6 expression (r = 0.52; p = 0.027) – as well as viral load in the brain (r = 0.57; p = 0.013) (Figure 2(H)). All these data indicate that elevated TSPO expression in the brain is a hallmark of ZIKV brain infection and associated neuroinflammation. Figure 2. Elevated translocator protein (TSPO) expression in ZIKV-infected mouse brains. (A) Representative images of mock-infected vs. ZIKV-infected hind brain tissue sections subjected to immunohistochemical staining for TSPO expression. Highlighted areas revealing intense TSPO staining in ZIKV-infected brain, as well as a comparable region from Mock-infected brain, are enlarged. (B–D) Total counts of TSPO+ dissociated cells from ungated total brain cells (B), CD45¯ gated non-immune cells (C), and CD45+ gated immune cells (D) as determined by Flow Cytometry. (E-G) TSPO expression on dissociated cells reported as Mean Fluorescence Intensity (MFI) from the unsorted total brain cells (E), CD45¯ gated non-immune cells (F), and CD45+ gated immune cells (G) as determined by Flow Cytometry. (H) Heatmap of Spearman correlations between TSPO expression, viral load, IL6 expression, and infiltration of CD45+ immune cells into ZIKV-infected mouse brains. Asterisks indicate p­-values. * p < 0.05. ** p < 0.005. *** p < 0.001. ns, not significant. Data in B-G are shown as mean ± SD, and means were compared by Kruskal-Wallis test with Dunn’s post-hoc correction. Scale bars in 2A represent 1 mm unless stated otherwise.

Detection of ZIKV-associated neuroinflammation with [3H]PK11195 TSPO radioligand

To sensitively measure in vivo changes in mouse brain TSPO expression, mice were systemically injected with the TSPO probe [3H]PK11195 and sacrificed 1 h later. The radioactivity taken up by various tissues was measured on a liquid scintillation counter. Of the tissues evaluated, only the heart (2.17-fold change; p = 0.004), gonad (2.08-fold change; p = 0.004), and brain (1.81-fold change; p = 0.004) exhibited higher probe uptake at late disease relative to pre-infection (Figure 3(A)). Within micro-dissected brain regions (Figure 3(B)), only the hippocampal formation (HPF; 1.40 ± 0.55-fold change; p = 0.06) and cerebellar cortex (CBX; 1.66 ± 0.62-fold change; p = 0.06) did not exhibit higher probe uptake at late disease vs. pre-infection (Figure 3(C)). In vitro [3H]PK11195 binding onto brain tissue sections and subsequent autoradiographic imaging confirmed the ex vivo findings (Figure 3(D and E)). The amount of probe bound to ZIKV-infected brains increased globally relative to pre-infected brains, except in the CBX (1.66 fold-change; p = 0.06) (Figure 3(D)). Radioligand binding to late ZIKV diseased brain tissue sections was 1.67 ± 0.23-fold higher than pre-infection (p = 0.03) and 1.37 ± 0.70-fold higher (p = 0.04) than in brains pre-incubated with cold PK11195 blocker (Figure 3(F)). Brain uptake of [3H]PK11195 exhibited strong linear correlation with TSPO expression (r = 0.76; p = 0.006), viral load (r = 0.75; p = 0.007), IL6 expression (r = 0.76; p = 0.006), and immune cell infiltration (r = 0.76; p = 0.007) in the brain (Figure 3(G)). Lastly, brain regions with higher TSPO expression also coincide with foci of increased probe binding (Figure 3(H)). These data confirm that the modulation of TSPO brain expression in ZIKV-associated neuroinflammation can be sensitively detected ex vivo and in vitro using the TSPO radioligand [3H]PK11195. Figure 3. Detection of translocator protein (TSPO) expression in ZIKV-infected mouse brains using [3H]PK11195 radioligand. (A) Ex vivo tissue biodistribution of [3H]PK11195 in mice. LN, lymph nodes. (B) Schematic diagram of mouse brain sagittal sections revealing the various brain regions. Illustration created using Biorender©. CTX, cerebral cortex. HPF, hippocampal formation. TH + HY, diencephalon. CBX, cerebellum. MY, medulla. P, pons. (C) Ex vivo tissue biodistribution of radioligand in micro-dissected mouse brains: WB, whole brain. (D–E) Representative images of mouse brain sagittal sections subjected to (D) in vitro binding with [3H]PK11195 and detected by autoradiography (DAR), and (E) hematoxylin & eosin (H&E) staining. Regions of interest (ROIs) in D are enclosed in dashed white boxes. (F) Quantification of [3H]PK11195 binding to brain tissue sections detected by autoradiography. (G) Heatmap of Spearman correlations between [3H]PK11195 binding to brain tissue sections, viral load, and expression of TSPO, IL6 and TNFα in ZIKV-infected mouse brains. (H) Immunofluorescence (I.F.) staining of brain tissue sections for translocator protein (TSPO) and ZIKV envelope protein. Corresponding ROIs within dashed white boxes in D and H that show increased immune cell TSPO expression are marked with yellow # and $ symbols. Areas with increased TSPO expression contributed by perivascular macrophages are shown in white # and $ symbols. Asterisks indicate p­-values. * p < 0.05. ** p < 0.005. *** p < 0.001. Data in A, C, F are shown as mean ± SD, and means were compared by Kruskal-Wallis test with Dunn’s post-hoc correction. ns, not significant. Scale bars in 3D, 3E, and 3H represent 1 mm unless stated otherwise.

[3H]PK11195 brain uptake and brain immune cell infiltration

To determine what immune cell subsets are enriched in the brain following ZIKV-induced neuroinflammation, we performed multiplex immunostaining of single cells isolated from whole brains followed by flow cytometry (FC). The gating strategy used identified both myeloid and lymphoid lineages of immune cells (Figure 4(A)). CD45+ immune cells were found enriched at late disease compared to pre-infection (10.74 ± 8.10-fold change) and to mid disease (7.47 ± 5.63-fold change) (Figure 4(B)). Similar trends of post-infection enrichment were also observed in myeloid-lineage immune cells – particularly granulocytes, monocyte-derived macrophages (MDM) and dendritic cells (DC); (Figure 4(C)) and lymphoid-lineage immune cells: total T cells, CD4+ T (helper) cells, and CD8+ T (cytotoxic) cells (Figure 4(D)). More importantly, total cellular TSPO expression at late disease was elevated on all CD45+ immune cells and myeloid-lineage cells: monocytes and microglia (Figure 4(E)). Probe brain uptake assayed by ex vivo gamma counting (Figure 3(A)) exhibited strong direct correlation with both TSPO expression and absolute counts of most myeloid lineage immune cells (Figure 4(F)). These data confirmed that [3H]PK11195 binding in the brain is due to the increased presence of TSPO – both by upregulated expression on microglia and monocytes, as well as increased infiltration of immune cells into the brain. Figure 4. Immune cell profile and TSPO expression in ZIKV-infected mouse brains. (A) Gating strategy for analysing live CD45+ immune cell subsets from flow cytometry data. (B–D) Absolute cell counts of total CD45+ immune cells (B), myeloid lineage immune cells (C) and lymphoid lineage immune cells (D) in mouse brains. (E) TSPO expression of various immune cells in the brain. (F) Heatmap of linear correlations between [3H]PK11195 brain uptake vs. either TSPO expression or absolute counts of various myeloid lineage immune cells in the brain. Asterisks indicate p­-values. * p < 0.05. ** p < 0.005. *** p < 0.001. ns, not significant. Data in B-E are shown as mean ± SD, and means were compared by Kruskal-Wallis test with Dunn’s post-hoc correction (B-D) or Mann-Whitney test (E). ns, not significant. MDM, monocyte-derived macrophages. DC, conventional dendritic cells.

Correlation of [3H]PK11195 uptake with immune cell counts and TSPO expression in various brain regions

Whole brains were micro-dissected to isolate the cerebral cortex (CTX), hippocampal formation (HPF), midbrain (MB), cerebellar cortex (CBX), and hindbrain (HB). Isolated single cells were subjected to flow cytometry to identify specific immune cell subsets and to determine intracellular TSPO expression. Consistent with trends in the whole brain, TSPO expression increased on total immune cells, monocytes, and microglia in most of the brain regions evaluated (Figure 5(A)). Elevated TSPO expression on microglia and monocytes is most prominent in CBX, and HPF. Interestingly, TSPO expression in cytotoxic (CD8+) T cells declined in ZIKV-infected whole brains and brain regions (Figure 5(A)). On the other hand, absolute counts of various immune cells – except monocytes and microglia – increased in ZIKV-infected brains and brain regions (Figure 5(B)). The greatest degree of immune cell infiltration in various brain regions is contributed by CD8+ T cells. These data indicate massive infiltration of both myeloid and lymphoid immune cells in the entire brain, consistent with global encephalitis at late ZIKV infection. Figure 5. Correlation of immune cell TSPO expression and immune cell counts with [3H]PK11195 uptake in various brain regions. (A–B) Heatmap of fold-change (FC) in (A) TSPO expression and (B) immune cell counts in different brain regions: whole brain (WB), cerebellar cortex (CBX), cerebral cortex (CTX), hippocampal formation (HPF), midbrain (MB), and hindbrain (HB). Blue-coloured cells denote reduced expression or counts, while red-coloured cells denote increased expression or counts. Mean values between day 8 and day 0 post-ZIKV infection were compared by Mann-Whitney test. (C–D) Spearman correlation (ρ) between [3H]PK11195 uptake and (C) TSPO expression of various immune cells or (D) absolute counts of various immune cells. Green-coloured cells denote negative correlation, while orange-coloured cells denote positive correlation. Circle sizes denote the degree of statistical significance. MDM, monocyte-derived macrophages. DC, conventional dendritic cells.

Correlating immune cell TSPO expression with [3H]PK11195 uptake in various brain regions, we found that myeloid cells – especially microglia and monocytes – exhibited the strongest linear correlation with radioligand binding in various brain regions (Figure 5(C)). TSPO expression on lymphoid immune cells, particularly cytotoxic T cells, did not correlate with radioligand binding (Figure 5(C)). On the other hand, absolute counts of the different immune cells, except for microglia and monocytes, strongly correlated with radioligand binding in various brain regions (Figure 5(D)). These data indicate that the increased TSPO expression in the different brain regions was due to increased TSPO expression on myeloid-derived immune cells and increased infiltration of both myeloid and lymphoid immune cells due to ZIKV brain infection.

Discussion

In this study, we showed that ZIKV infection in mice lacking type-I/II interferon receptors (AG129 strain) induced lethal disease exhibiting neurological infection and neuroinflammation. ZIKV replicated in the brains and resulted in elevated IL6 expression and infiltration of immune cells (encephalitis) in the brain, which are consistent with prior observations in ZIKV mouse infection models [39,40]. The progression of ZIKV-induced neuroinflammation also manifested in mild neurological deficits – such as limb paresis, paralysis, and ataxia, in the affected animals and resulted in increased TSPO expression in the brain at late disease. Brain TSPO expression correlated with other indicators of brain infection and inflammation, specifically viral replication, pro-inflammatory cytokine expression, and immune cell infiltration in the brain. Hence, brain TSPO expression is a biomarker of ZIKV infection and inflammation in mouse brains.

Brain TSPO expression during ZIKV infection was mainly contributed by immune cells that constitute a minority population in the brain. In our studies, CD45+ cells constitute < 10% of live cells isolated from whole brains (data not shown). Consequently, changes in TSPO were undetectable on bulk tissue lysate immunoblots primarily because this method was not sufficiently sensitive to detect expression changes on a small number of cells.

Alternative TSPO-detection methods with improved detection sensitivity include TSPO-binding small molecule ligands attached to a radioisotope, such as [3H]PK11195. Tritium [3H] is a radioactive isotope of hydrogen [1H] that releases low-energy β-particles (19 keV) in its decay process. This released energy is converted to light when the radioactive sample is mixed with liquid scintillant, and the emitted light can be detected in the scintillation counter. Similarly, this released energy can also be directly quantified in a digital autoradiography imager. Radioactivity detection methods are highly sensitive and facilitate efficient monitoring of minute changes in the released energy. Thus, [3H]PK11195 binding for detecting TSPO expression is far more sensitive than classical light-based techniques. PK11195 is an isoquinoline carboxamide and most widely used TSPO radioligand in the literature. It has high affinity to TSPO (KD = 1.028 nM), and [3H]PK11195 has been used to detect and monitor TSPO expression in a variety of inflammatory models since 1983 [41].

Indeed, systemic injection of 1 μCi (12.7 nmol) [3H]PK11195 was sufficient to detect changes in TSPO expression in testes during ZIKV infection by ex vivo liquid scintillation. The increased radioligand uptake in the testes was notable due to previous reports of testicular inflammation and tissue destruction caused by ZIKV infection in mouse models [42–45]. TSPO expression in the gonads could be further examined as a potential biomarker for Zika testicular disease and would be relevant in studies exploring viral persistence and infection-induced tissue destruction within the gonads [46–49].

Similarly, ex vivo detection of [3H]PK11195 uptake in whole brains and micro-dissected brain regions was significantly higher at late disease (day 8) relative to pre-infection. In vitro [3H]PK11195 binding to brain tissue sections also confirmed these findings. Brain [3H]PK11195 uptake exhibited strong positive correlation with viral replication, IL6 expression, and infiltration of TSPO-expressing myeloid and lymphoid immune cells into the brain. More importantly, radioligand binding strongly correlated with the number of myeloid and lymphoid cells infiltrating the different brain regions, which suggest that the degree of [3H]PK11195 uptake could also predict encephalitis severity. These results also concurred with previous reports of global elevated brain TSPO expression and increased uptake of TSPO radioligands in ZIKV-infected brains [25,33,50]. Thus, binding of [3H]PK11195 to ZIKV-infected brains – either ex vivo or in vitro – could be used as proxy for disease and inflammation.

TSPO radioligands with varying specificity for TSPO have been reported in the literature [51]. Of these, radioligands tagged with the [18F] radioisotope, such as [18F]FEPPA [52] and [18F]DPA-714 [53], have been used to non-invasively monitor immune cell infiltration and modulation of TSPO expression in ZIKV brain infection using Positron Emission Tomography (PET) imaging [25,33]. However, PET imaging systems are not readily available within the research setting, especially in emerging countries. Thus, we present here an alternative method for in vitro and ex vivo detection of low-expression tissue biomarkers – i.e. TSPO – through liquid scintillation and autoradiography paired with a commercially available radioligand [3H]PK11195. These assays are obtainable at a fraction of the cost of nuclear imaging systems, require significantly less specialized training to perform, and more easily adopted in infectious disease-focused laboratories.

In addition to ZIKV, [18F]DPA-714 has been used to monitor SARS-CoV-2 neuroinflammation [32] and pulmonary inflammation [54]; and active HIV neuroinflammation [28,55]. Moreover, [18F]FEPPA has been used to detect lung inflammation in mouse models of malaria [56], SARS-CoV-2, (unpublished data), and rhinovirus (unpublished data) infections. These suggest that TSPO expression on immune cells is a versatile biomarker that can be applied to other viral encephalitis as well as peripheral inflammatory diseases. Future studies can also evaluate how TSPO expression in the brain (or other tissue inflammatory site) is modulated in the presence of therapeutic and prophylactic interventions, and whether TSPO expression could also be a surrogate early predictor for response to these therapeutics.

Supplementary Material

Figure_S1

Acknowledgements

We would like to thank Marie Reolo, Agnieszka Dorota Sekula, Haziq bin Razeli, Dr. Chaw Suyin, and Dr. Jing Guo (Duke-NUS) for assisting in experiments, data analysis, and data visualization. We are grateful to Dr. Pedro Vasconcelos at Instituto Evandro Chagas (Brazil) for providing the Paraiba01/Brazil (ZIKVBR) strain and to Prof. Subhash G. Vasudevan (Duke-NUS) for providing resources and mentorship in this project.

Disclosure statement

No potential conflict of interest was reported by the author(s).
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