Herpes simplex virus infection induces necroptosis of neurons and astrocytes in human fetal organotypic brain slice cultures

doi:10.21203/rs.3.rs-3231988/v1

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Research Article

Herpes simplex virus infection induces necroptosis of neurons and astrocytes in human fetal organotypic brain slice cultures

https://doi.org/10.21203/rs.3.rs-3231988/v1

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Journal Publication

published 01 Feb, 2024

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Background

Herpes simplex virus (HSV) encephalitis (HSE) is a serious and potentially life-threatening disease, affecting both adults and newborns. Progress in understanding the virus and host factors involved in neonatal HSE has been hampered by the limitations of current brain models that do not fully recapitulate the tissue structure and cell composition of the developing human brain in health and disease. Here, we developed a human fetal organotypic brain slice culture (hfOBSC) and determined its value in mimicking the neuropathology of HSE in vitro .

Methods

Cell viability and tissues integrity were determined by lactate dehydrogenase release in supernatant and detailed immunohistological (IHC) analyses. Brain slices were infected with green fluorescent protein (GFP-) expressing HSV-1 and HSV-2. Virus replication and spread were determined by confocal microscopy, PCR and virus culture. Cell tropism and HSV-induced neuropathology was determined by IHC analysis. Finally, the in situ data of HSV-infected hfOBSC were compared to the neuropathology detected in human HSE brain sections.

Results

Slicing and serum-free culture conditions were optimized to maintain the viability and tissue architecture of ex vivo human fetal brain slices for at least 14 days at 37^oC in a CO₂ incubator. The hfOBSC supported productive HSV-1 and HSV-2 infection, involving predominantly infection of neurons and astrocytes. Both viruses induced programmed cell death – especially necroptosis – in infected brain slices at later time points after infection. The virus spread, cell tropism and role of programmed cell death in HSV-induced cell death closely mimicked the neuropathology of HSE.

Conclusions

We developed a novel human brain model in which the viability of the major brain-resident cells – including neurons, microglia, astrocytes and oligodendrocytes – and the tissue architecture is maintained for at least 2 weeks in vitro under serum-free conditions. The close resemblance of cell tropism, spread and neurovirulence of HSV-1 and HSV-2 in the hfOBSC model with the neuropathological features of human HSE cases underscores its potential to detail the pathophysiology of other neurotropic viruses and as preclinical model to test novel therapeutic interventions.

Herpes simplex virus

encephalitis

human brain

organotypic cultures

neurotropism

neurovirulence

programmed cell death & necroptosis

Various pathogens grouped under the acronym ‘TORCH’ [Toxoplasma gondii, Others (e.g. parvovirus B and Zikavirus and Rubella virus), Cytomegalovirus and Herpes simplex virus (HSV)], remain a substantial threat to newborns and fetuses worldwide [1]. Next to rubella virus, the neurotropic HSV are the second most prevalent TORCH pathogens during pregnancy that can cause neonatal herpes associated with serious morbidity (e.g. neurological and disseminated disease) and mortality [2]. The global incidence rate of neonatal HSV infection is about 10 cases per 100,000 livebirths and 0.82 deaths per 1000,000 births in industrialized countries due to either HSV-1 or HSV-2 [2, 3]. While the majority of neonatal HSV infections are acquired at birth, congenital HSV due to intrauterine HSV infection can occur leading to severe abnormalities affecting multiple organs including the brain [4, 5].

Whereas progress have been made in the early diagnosis and antiviral treatment of neonatal HSE, still about 60% of patients will experience lifelong neurological sequela including cognitive impairment and epilepsy [2–4]. Thus, besides development of better prevention strategies, including effective HSV vaccines and prevention of maternal infection during pregnancy, there is an unmet need to identify the detrimental virus and host parameters involved in the pathogenesis of neonatal HSE as potential targets of therapeutic intervention [3, 6]. Various in vitro and in vivo models have been developed to address this issue, but most if not all fail to mimic HSV neurovirulence in the developing human brain [7–9]. Human 2D brain cell models lack the cell complexity of brain tissue, experimental rodent models are evidently lacking the human component and the more recently use of human brain organoid models lack microglia and vasculature [7, 10–12]. Organotypic brain slice cultures (OBSC), which largely fulfill the shortcomings of the aforementioned models, have been successfully applied for rodents to both unravel the pathogenesis of various neurological diseases as well as provide the unique opportunity to determine the efficacy and safety of various therapeutic interventions in parallel using a limited number of animals [13–15]. In comparison, studies on human OBSC are limited not only by the scarce availability of clinical brain specimens, but also due to technical drawbacks including limited cell viability and aberrant tissue structure caused by long-term culture in the presence of serum in culture media [7, 12, 16]. To overcome these limitations, the use of serum-free medium combined with various growth factors is preferred to create a more optimal and reproducible environment while minimizing glial reactivity [17–19].

The aim of the current study was twofold. First, to develop a novel human fetal OBSC (hfOBSC) platform that recapitulates the complexity of the developing human brain and that can be successfully cultured in serum-free medium for several weeks in vitro. Second, to study the cell tropism, virus spread and neurovirulence of HSV-1 and HSV-2 in hfOBSC and to compare with archived brain specimens of neonatal HSV cases by in situ analysis.

Archived human brain specimens

Formalin-fixed and paraffin-embedded (FFPE) brain samples were obtained for diagnostic purposes from two fatal HSE patients, including one adult HSV-1- and one neonatal HSV-2-associated HSE case. The brain samples were provided by the BioBank of the Amsterdam University Medical Center (Amsterdam, The Netherlands). According to the institutional “Opt-Out” system, which is defined by the National “Code of Good Conduct” [Dutch: Code Goed Gebruik, May 2011], these surplus human brain tissues were made available for the current study.

Human fetal organotypic brain slice cultures

Human fetal brain tissue from legally terminated second trimester pregnancies (17–20 weeks) was obtained by the HIS-Mouse Facility of Academic Medical Center (AMC; Amsterdam, The Netherlands), after written informed consent of the mother for the tissue’s use in research and with approval of the Medical Ethical Review Board of the AMC (MEC: 03/038) and Erasmus MC (MEC-2017-009). Study procedures were performed according to the Declaration of Helsinki, and in compliance with relevant Dutch laws and institutional guidelines. The tissues obtained were anonymized and non-traceable to the donor. On request by the researchers, only gender and gestational age is provided. Upon removal of exterior blood vessels and meninges, brain tissue fragments (~ 0.5 x 0.5 cm) were cut into 350 µm-thick slices using a vibratome (Leica; type VT1200S) in artificial cerebrospinal fluid (aCSF) under constant oxygenation (95% O₂, 5% CO₂) as described previously [20]. Slices were transferred to 12 mm transwell plates with polyester membrane inserts (4 µm pore size; Corning) to recuperate the tissue in recovery medium composed of a 7:3 (v/v) mixture of Neurobasal media and advanced DMEM/F12 culturing medium (both Life Technologies) supplemented with 20% heat-inactivated fetal bovine serum (FBS) and antibiotics. After 1 hr incubation in a CO₂ incubator at 37°C, the recovery medium was replaced with optimized hfOBSC serum-free culture medium containing a 7:3 (v/v) mixture of Neurobasal media and advanced DMEM/F12 culturing medium, B27 (2% v/v), N2 (1% v/v), glutaMAX 100x (1% v/v, all from Life Technologies), primocin (1:500, Invitrogen), TGF-β2 human (2 ng/mL, ProspecBio), cholesterol (1.5 µg/mL, Sigma Aldrich), human recombinant m-CSF (100 ng/mL, Peprotech), BDNF (50 µg/mL,), NT-3 (10 ng/mL, Peprotech), FGF2 (10 ng/mL, R&D Systems) and EGF (10 ng/mL, R&D Systems). To insure fluidic flow in the transwell system, 750 µl was added to the basolateral compartment and 50 µl added to the apical compartment. The culture medium of the human fetal organotypic brain slice cultures (hfOBSC) was refreshed every 48 hrs.

Brain cell viability assays

The viability of the hfOBSC was determined by lactate dehydrogenase (LDH) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays. The LDH assays were performed on conditioned medium of hfOBSC cultures using the CyQUANT™ LDH Cytotoxicity Assay kit (Invitrogen) according to the manufacturer’s protocol. LDH levels of brain slices incubated in lysis buffer according to the manufacturer’s instructions were included as positive control. To assess metabolic activity, brain slice cultures were treated with MTT (0.5 g/L, Abcam) and incubated for 45 minutes at 37°C before imaging by using Zeiss LSM 700 confocal microscope (Zeiss).

HSV infection of human fetal organotypic brain slice cultures

Recombinant HSV-1 expressing green fluorescent protein (GFP) N-terminally tagged to VP16 (HSV-1-GFP) and HSV-2 (333-ZAG) containing a GFP expression cassette under the control of a human cytomegalovirus promoter inserted in an intergenic region between UL3 and UL4, was propagated on human retinal pigment epithelial (ARPE-19) cells to obtain cell-free virus stocks, as previously described [21, 22]. The hfOBSC were infected with 100 plaque-forming units (PFU) or 10⁵ PFU of cell-free HSV-1-GFP or HSV-2-GFP. After 1 hr incubation at 37^oC, the inoculum was removed, and the brain slices were washed with PBS and subsequently maintained in culture medium for 72 to 96 hours post-infection (hpi) at 37°C in a CO₂ incubator. Infection was monitored by collecting supernatants every 24 hrs for quantitative real-time PCR (qPCR) and assessing GFP expression using a fluorescent microscope (Zeiss AXIO inverted microscope). At the indicated time points, the hfOBSC were fixed in PBS containing 4% paraformaldehyde and embedded in paraffin for histological analyses.

Quantitative real-time PCR

HSV-1 and HSV-2 DNA loads in supernatants were quantified by Taqman quantitative real-time PCR (qPCR) as described previously (PMID: 24671719). In brief, assays were performed in Taqman fast Advanced mastermix (Thermo Fisher scientific) using primers/probes specific for the following targets: HSV-1 gene US4 (glycoprotein G) and HSV-2 gene US6 (glycoprotein D) [23]. The qPCR was performed on a 7500 Real time PCR system (Applied Biosystems) and the PCR cycling program used consisted of 5 minutes at 50°C and 2 minutes at 95°C, followed by 40 cycles of 3 seconds at 95°C and 30 seconds at 60°C.

In situ analysis of brain slices and tissues

The FFPE brain slices and archived brain sections of two HSE cases were serially sectioned at 4 µm thickness. Heat-induced antigen retrieval was performed using citric acid buffer (pH 6.0). Consecutive sections from three different levels of the brain slices (i.e. apical, middle and basal level) were immunohistochemically stained (IHC) with the following primary antibodies: polyclonal rabbit anti-Iba1 (microglia marker; Wako), anti-GFAP (glial fibrillary acidic protein, astrocyte marker; Dako), monoclonal rabbit anti-Olig2 (oligodendrocyte transcription factor 2, oligodendrocyte marker; clone EPR2673, Abcam) or polyclonal guinea pig anti-MAP2 (microtubule-associated protein 2, neuron marker; Synaptic Systems). Next, sections were washed and incubated with the appropriate secondary antibody including rabbit anti-mouse Ig biotinylated (Dako), goat anti-rabbit Ig biotinylated (Dako), or rabbit anti-guinea pig IgG (H + L) horseradish peroxidase (HRP)-conjugated (Invitrogen), respectively. Horseradish peroxidase-labeled streptavidin (Dako) was applied to sections with biotinylated antibodies, followed by 3-amino-9-ethylcarbazole substrate. Sections were counterstained with hematoxylin, mounted with Kaiser’s glycerol, and scanned using the Hamamatsu NanoZoomer 2.0 HT (Hamamatsu).

Immunofluorescent (IF) stainings were performed on consecutive sections treated with TrueBlack® (Biotium) after antigen retrieval to decrease autofluorescence, according to the manufacturer’s protocol. The following primary antibodies were used for IF: polyclonal chicken anti-GFAP (Abcam), polyclonal rabbit anti-Iba1 (Wako), monoclonal rabbit anti-Olig2 (clone EPR2673, Abcam), polyclonal guinea pig anti-MAP2 (Synaptic Systems), and FITC polyclonal goat anti-GFP (Abcam). The primary antibodies were labeled with the appropriate secondary antibodies from Invitrogen: Rabbit anti-GFP, AlexaFluor™ 647 (AF647)- and AF555-conjugated goat anti-guinea pig or chicken IgG or donkey anti-Rabbit IgG, respectively. Nuclei were stained with Hoechst 33342 Solution. Images were taken using a Leica Stellaris 5 Low Incidence Angle confocal microscope.

Terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) assay

TUNEL assay were performed using the ApopTag® Plus in situ apoptosis fluorescein S7111 detection kit (Sigma) according to the manufacturer’s protocol, following instructions for a combined IF staining. Sections were treated with TrueBlack® (Biotium) after antigen retrieval to decrease autofluorescence, followed by staining with the following primary antibodies: polyclonal guinea pig anti-MAP2 (Synaptic Systems), polyclonal chicken anti-GFAP (Abcam), polyclonal rabbit anti-CC3 (cleaved caspase 3 protein; Cell Signalling Technology), polyclonal rabbit anti-pMLKL (phosphorylation of mixed lineage kinase domain-like protein; Abcam) and monoclonal mouse anti-GSDMD (gasdermin D protein; Abnova). Subsequently, slides were incubated with the following secondary antibodies (Invitrogen): AF555-conjugated donkey anti-rabbit IgG, AF647-conjugated goat anti-guinea pig IgG, AF647-conjugated goat anti-chicken IgG or AF647 goat anti-mouse IgG2a, respectively.

Image processing

Following confocal imaging, the acquired images were subjected to quantitative analysis using ImageJ software (version 1.53t, National Institutes of Health, Bethesda, MD) and a customized script. Individual channels were analyzed after splitting the images. For Hoechst staining, a Gaussian blur filter of 1 was applied to remove noise, and a ‘Moments dark’ threshold was utilized for image segmentation. Particle quantification was performed exclusively on particles larger than 5 µm. TUNEL staining was processed by subtracting the median of 6 to eliminate background noise, followed by the application of a Gaussian blur of 1 and a ‘Yen dark’ threshold for accurate image segmentation. Quantification was limited to particles positive for both Hoechst and TUNEL staining. In the case of CC3, pMLKL, and GSDMD staining, background noise was efficiently removed by applying a median of 16 and a Gaussian blur of 0.5. A ‘Yen dark’ threshold was then utilized to identify particles larger than 20 µm. The percentage of positive cells was calculated by determining the ratio of positive cells to the total number of Hoechst-positive cells. Finally, all processed images were converted to mask, and the watershed method was employed to accurately separate particles for comprehensive analysis. Phenotypes of infected cells were identified and quantified using the classifications function on the QuPath 0.3.2 software [24].

Statistical analysis

Data were analyzed using Prism 8.0.2 (GraphPad). A two-tailed Student’s t test was used to test for statistical significance as indicated in the figure legends. The p values were assigned: p > 0.05 (ns, not significant); * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001.

Development of a human fetal organotypic brain slice culture platform

The aim of this study was to develop a human fetal OBSC (hfOBSC) platform that recapitulates the complexity of the developing human brain and that can be successfully cultured in serum-free medium for several weeks. Hereto, human fetal brain tissue, obtained at 17 to 20 weeks of gestation, was cut with a vibratome into 350 µm thick slices of 25 mm² in size. Brain slice were recuperated in aCSF under constant oxygenation to reduce cell damage due to hypoxia [25, 26] (Fig. 1A). The slices were cultured in the upper compartment of a transwell system in a CO₂ incubator at 37^oC, a well-established method to culture organotypic tissue slices [15, 27, 28]. Addition of a minimal amount of media to this compartment allowed optimal oxygenation of the tissue, whereas the transwell membrane ensured diffusion of nutrients from the large volume of media from the lower compartment into the tissue. To assess the overall viability of the hfOBSC, LDH concentrations measured in conditioned medium showed negligible cellular cytotoxicity throughout 2 week of culture (Fig. 1B). In line with this, the MTT assay, which measures cellular metabolic activity, showed no signs of reduction in cellular metabolism during 14 days of culture (Fig. S1).

Next, we determined whether these culture conditions preserved the four main CNS cell types (i.e., neurons, astrocytes, oligodendrocytes, and microglia) and overall brain tissue architecture in cultured hfOBSC. Immunohistochemical staining was performed on sections derived from three different levels (i.e., apical, middle and basal layer) of the hfOBSC to identify the presence and spatial orientation of GFAP⁺ astrocytes, MAP2⁺ neurons, Iba1⁺ microglia and Olig2⁺ oligodendrocytes. All four major cell types were detected throughout the hfOBSC and their morphology did not change during 14 days of culture. The microglia population contained both ramified and amoeboid-like Iba1⁺ cells, but no astrogliosis nor microgliosis was observed at later times (Fig. 1C). In conclusion, the slicing and serum-free culture conditions developed maintained the viability and overall integrity of ex vivo human fetal brain slices for at least 14 days in culture.

HSV-1 and HSV-2 predominantly infect neurons and astrocytes in hfOBSC

The second aim of our study was to determine the cell tropism, virus spread and neurovirulence of HSV-1 and HSV-2 in hfOBSC and to compare these with archived brain specimens of human HSE cases by in situ analysis. Recombinant HSV strains encoding GFP were used to determine whether the cultured brain slices were permissive to productive HSV-1 and HSV-2 infection. The use of GFP reporter viruses facilitated both the identification of infected cells and to monitor virus spread longitudinally by fluorescence microscopy. The introduction of the GFP cassette does not affect viral replication in vitro [21, 22]. Infections were initiated at day 3 of culture (DIV3) to allow the brain slices to equilibrate following the slicing procedure. Following inoculation of the brain slices with 100 PFU HSV-1-GFP or HSV-2-GFP, several foci of infected GFP-expressing cells were observed at 24 hpi, from which infection spread throughout the tissue until 72 hpi (Fig. 2A). Quantitative PCR analysis showed an exponential increase in viral DNA in the supernatant of hfOBSC over time, which demonstrated productive HSV-1 and HSV-2 infection of hfOBSC (Fig. 2B).

To investigate whether this in vivo cell tropism was recapitulated in the hfOBSC model developed, IF stainings were performed on hfOBSC sections to identify and quantify the cell types infected. At 72 hpi, HSV-1 and HSV-2 mainly infected MAP2⁺ neurons and GFAP⁺ astrocytes, while only minor proportions (< 10%) of Iba1⁺ microglia and Olig2⁺ oligodendrocytes were infected by both viruses (Fig. 3A, B). Analogous to human HSE brain tissues, no obvious differences in cell tropism were observed between HSV-1 and HSV-2 [29–33]. In conclusion, these results indicated that hfOBSC were permissive to productive HSV-1 and HSV-2 infection and spread throughout the tissue. Furthermore, the in vivo tropism of HSV-1 and HSV-2 observed in neonatal HSE patients is recapitulated in our hfOBSC platform.

HSV-1 and HSV-2 infection induces cell death in astrocytes and neurons

Intrinsic and innate immune responses of CNS recent cells including neurons and especially microglia prevent HSV spread within the CNS. Indeed, genetic studies of patients with viral brain infections as well as experimental cell and animal models have provided evidence of the pivotal role of these immune mechanisms, which mediate a first line of antiviral control without significantly triggering inflammatory activities [34, 35]. However, in rare cases, this first line of defence fails to control HSV infection leading to profound inflammation and extensive death of neurons and astrocytes, as well as lower numbers of oligodendrocytes and microglia in HSE [8, 36, 37]. Cell death in particular is thought to provide a significant contribution to virus-induced encephalitis and HSE pathogenesis in particular [38–40].

To assess the extent of cell death during HSV infection in vivo as well as in our hfOBSC platform, TUNEL assays were performed on hfOBSC sections (Fig. 4A). This assay facilitates the histological identification of dead cells by staining for fragmented DNA [41]. First, brain sections of the two human HSV-1 and HSV-2 encephalitis cases were analyzed, which showed high numbers of TUNEL-positive cells in HSE lesions (Suppl Fig. 2A). Next, the induction of cell death was investigated in mock- and HSV-infected hfOBSC. Inoculation of human brain slices with 10⁵ PFU HSV-1-GFP or HSV-2-GFP led to a significant increase in the amount of TUNEL⁺ cells relative to the mock control at 72 hpi (Fig. 4B). In line with these results, LDH release steadily increased between 24 and 96 hpi following HSV-1 or HSV-2 infection, whereas it remained low in the mock-infected hfOBSC (Fig. 4C).

To determine which cell types were undergoing cell death the TUNEL assay was combined with IF staining for the four major CNS cell types. In both HSV-1 and HSV-2 infected hfOBSC, the majority of TUNEL⁺ cells were either GFAP⁺ astrocytes or MAP2⁺ neurons (Fig. 4D). These data resembled the preferential death of astrocytes and neurons in brain tissues of both HSE patients (Suppl Fig. 2B). In conclusion, the data demonstrate that both HSV-1 and HSV-2 infection induce high level of cell death in the hfOBSC model, predominantly in neurons and astrocytes, which is in line with in situ analysis of human HSE brain specimens reported here and others previously [29, 30, 39, 42].

HSV-1 and HSV-2 induce necroptosis in hfOBSC

Besides the direct cytopathic effect of HSV on brain cells, the virus may also induce cell damage indirectly by programmed cell death (PCD) [43]. PCD is a tightly regulated process that plays pivotal roles in tissue homeostasis as well as antiviral defense to control infection [44]. Various PCD pathways can be induced by HSV, which include non-inflammatory apoptosis and autophagy and inflammation inducing PCD including pyroptosis and necroptosis [30, 40, 43, 44]. Since we observed robust induction of cell death in HSV-infected hfOBSC, we determined whether this involved specific PCD pathways. We inoculated hfOBSC with 10⁵ PFU HSV-1-GFP and HSV-2-GFP and determined three HSV-induced PCD pathways (i.e. apoptosis, pyroptosis and necroptosis) by in situ analysis of brain slice sections at 3 dpi (Fig. 5A). Expression of the following markers were used to differentiate between the three PCD pathways: cleaved caspase 3 (CC3) for apoptosis, gasdermin D (GSDMD) for pyroptosis and phosphorylation of mixed lineage kinase domain-like protein (pMLKL) as a marker for necroptosis [39, 43]. Whereas neither HSV subtype induced expression of GSMD, HSV-2 infection showed a trend to increased CC3 expression. Notably, productive infection with both HSV-1 and HSV-2 significantly induced expression of pMLKL (Fig. 6B). These results demonstrate that HSV-2 infection led to increased levels of apoptosis, while both viruses induced necroptotic cell death in hfOBSC.

The highly prevalent HSV-1 and HSV-2 are the most common causes of infectious encephalitis worldwide, with a disparate burden in individuals older than 50 or less than 1 year old [45, 46]. The cellular and molecular events that occur within the HSV-infected brain that lead to HSE are largely unknown.

Understanding the pathogenesis of HSE and other neurotropic virus infections requires the development of accurate and relevant model systems. Traditional two-dimensional (2D) cell cultures fail to capture the complex cellular interactions and three-dimensional architecture of human brain tissue, limiting their ability to reproduce the intricacies of brain development and diseases like HSE [47, 48]. Animal models, while valuable in elucidating HSE pathogenesis, exhibit variations in immune responses, cellular composition, and developmental stages among different species [48, 49]. As a result, translating results from animal models to the human context, especially for human-specific pathogens like HSV, has proven challenging [50, 51]. More sophisticated cell culture model systems such as brain organoids, although they exhibit self-organization and partial recapitulation of specific brain structures, fall short in replicating the precise cellular composition, cellular diversity, and complex structural organization observed in in vivo human brain tissue [52, 53]. To address these limitations, we have developed the hfOBSC model system presented here. This system offers a promising alternative with preserved three-dimensional architecture, cellular interactions, and native human brain physiology, enabling comprehensive investigations into the cellular mechanisms underlying HSE. This advanced model has allowed us to investigate the interplay between virus infection and PCD, showing that both HSV-1 and HSV-2 induce necroptosis in human brain tissue, offering leads that can be explored for targeted therapeutic interventions in HSE and related conditions.

The data demonstrate that the hfOBSC model recapitulates the cell tropism and neuropathology of HSV-1 and HSV-2 that is observed in affected brain tissue of human HSE cases [29, 30, 39, 42]. This highlights the relevance and translational value of our hfOBSC model in studying HSE pathogenesis. Moreover, since we used human fetal tissue, this system also provides a valuable platform to study the neurovirulence of other TORCH pathogens as well as emerging neurotropic viruses that pose an increasing risk to human health worldwide [1, 54]. Indeed, our preliminary investigations on infections of hfOBSC with human cytomegalovirus and zika virus demonstrate efficient viral replication and clinically relevant neurovirulence (data not shown).

In previous studies, apoptosis was identified as the primary mode of PCD observed across different HSE models [29, 39, 55–57]. Apoptosis involves controlled cellular dismantling, and its activation is often associated with the host's attempt to limit viral spread and inflammation [58]. While these studies have shed light on the apoptotic pathways triggered during HSE, the involvement of necroptosis, a regulated form of necrosis controlled by specific signaling pathways, is largely unknown. In contrast to previous studies on human (neonatal) HSE cases, which also reported apoptotic cell death as major neuropathological feature [39, 42, 55], our hfOBSC model shows only a trend towards HSV-induced apoptosis alongside a significant induction of HSV-induced necroptosis. Notably, recent studies have highlighted the pivotal role of the cytoplasmic protein RIPK3, a key player in necroptosis, in modulating the severity of HSE [59, 60]. Future studies are warranted to detail the different PCD pathways in HSE during the development of disease upon intracerebral HSV infection..

This study has several limitations. First, the brain specimens were obtained following second-trimester surgical abortions, at which stage it is difficult to determine the anatomical orientation of the brain tissue fragments. This may contribute to variability in the results. Second, the abortions were performed for various reasons, including pregnancy termination due to fetal anomalies. Due to complete anonymization we cannot rule out the presence of underlying diseases or genetic defects that may have influenced the obtained results. Lastly, while hfOBSC provides a more physiologically relevant environment compared to traditional in vitro models, our system lacks the dynamic in vivo interactions with blood-derived immune cells that limit the full translation of the findings to the clinical setting.

In conclusion, we developed a novel human fetal organotypic brain slice model in which the viability of the major CNS resident cells, including microglia, and the tissue architecture is maintained for at least 14 days under serum-free conditions in culture. The close resemblance in cell tropism, spread and neurovirulence of HSV-1 and HSV-2 in the hfOBSC model with the neuropathological features of HSE. These data emphasize its potential to study the pathophysiology - including the role of PCD - of human brain infections with HSV and other neurotropic viruses. Furthermore, the hfOBSC model may also be used as preclinical model to test the efficacy and safety of novel therapeutic intervention strategies that specifically target viral replication or the detrimental host responses in human brain tissue.

aCSF: artificial cerebrospinal fluid

CC3: cleaved caspase 3

CNS: central nervous system

FFPE: formalin-fixed paraffin embedded

GFAP: glial fibrillary acidic protein

GFP: green fluorescent protein

GSDMD: gasdermin D

hpi: hours post-infection

HSE: herpes simplex encephalitis

HSV-1: herpes simplex virus type 1

hfOBSC: human fetal organotypic brain slice culture

IF: immunofluorescence

IHC: immunohistochemistry

LDH: lactate dehydrogenase

MAP2: microtubule-associated protein 2

MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

Olig2: oligodendrocyte transcription factor 2

PCD: programmed cell death

PFU: plaque-forming units

pMLKL: phosphorylation of mixed lineage kinase domain-like protein

RT-qPCR: real-time quantitative PCR

TUNEL: Terminal deoxynucleotidyl transferase mediated dUTP nick end labeling

Ethics approval and consent to participate.

Formalin-fixed and paraffin-embedded (FFPE) brain samples were obtained for diagnostic purposes from two fatal HSE patients, including one adult HSV-1- and one neonatal HSV-2-associated HSE case. The brain samples were provided by the BioBank of the Amsterdam University Medical Center (Amsterdam, The Netherlands). According to the institutional “Opt‐Out” system, which is defined by the National “Code of Good Conduct” [Dutch: Code Goed Gebruik, May 2011], these surplus human brain tissues were made available for the current study.

Human fetal brain tissue was obtained from legally terminated second trimester pregnancies (17–20 weeks) by the HIS-Mouse Facility of Academic Medical Center (AMC; Amsterdam, The Netherlands), after written informed consent of the mother for the tissue’s use in research and with approval of the Medical Ethical Review Board of the AMC (MEC-2003-038) and the Erasmus MC (MEC-2017-009). Study procedures were performed according to the Declaration of Helsinki, and in compliance with relevant Dutch laws and institutional guidelines. The fetal brain tissues obtained were anonymized and non-traceable to the donor. On request by the researchers, only gender and gestational age was provided.

Consent for publication.

Not applicable.

Availability of data and materials.

The datasets and scripts used and/or analyzed during the current study are available from the corresponding author upon reasonable request.

Competing interests.

The authors declare that they have no competing interests.

Funding.

The study was in part supported by The Lundbeck Foundation (R359-2020-2287) (author ASR).

Authors' contributions.

Conceptualization: GMGMV; Methodology: ASR & DNR; Formal analysis: ASR, DNT & CRP; Investigation: ASR, DNR & CRP; Writing Original Draft: ASR & DNT; Writing, Review & Editing: ASR, DNT, MvG, WJDO & GMGMV; Visualization: ASR, DNT & CRP; Supervision: MvG, WJDO & GMGMV; Project Administration and Funding Acquisition: GMGMV.

Acknowledgements

The study was in part supported by The Lundbeck Foundation (R359-2020-2287) (ASR).

AUTHOR CONTRIBUTIONS

Conceptualization: GMGMV; Methodology: ASR & DNR; Formal analysis: ASR, DNT & CRP; Investigation: ASR, DNR & CRP; Writing – Original Draft: ASR & DNT; Writing – Review & Editing: ASR, DNT, MvG, WJDO & GMGMV; Visualization: ASR, DNT & CRP; Supervision: MvG, WJDO & GMGMV; Project Administration and Funding Acquisition: GMGMV.

DECLARATION OF INTERESTS

The authors declare no competing interests.

DATA AVAILABILITY

The datasets and scripts used and/or analyzed during the current study are available from the corresponding author upon reasonable request.

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Herpes simplex virus infection induces necroptosis of neurons and astrocytes in human fetal organotypic brain slice cultures

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Journal Publication

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Abstract

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INTRODUCTION

MATERIALS AND METHODS

Archived human brain specimens

Human fetal organotypic brain slice cultures

Brain cell viability assays

HSV infection of human fetal organotypic brain slice cultures

Quantitative real-time PCR

Terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) assay

Image processing

Statistical analysis

RESULTS

Development of a human fetal organotypic brain slice culture platform

HSV-1 and HSV-2 predominantly infect neurons and astrocytes in hfOBSC

HSV-1 and HSV-2 infection induces cell death in astrocytes and neurons

HSV-1 and HSV-2 induce necroptosis in hfOBSC

DISCUSSION

Abbreviations

Declarations

References

Additional Declarations

Supplementary Files

Status:

Journal Publication

Version 1