Neuron-restricted cytomegalovirus latency in the central nervous system regulated by CD4+ T cells and IFN-γ

doi:10.21203/rs.3.rs-4944489/v1

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Neuron-restricted cytomegalovirus latency in the central nervous system regulated by CD4+ T cells and IFN-γ

https://doi.org/10.21203/rs.3.rs-4944489/v1

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Human cytomegalovirus (HCMV) is the leading cause of congenital viral infections, frequently accompained with long-term neurological sequelae in children. The cell types and mechanisms involved in establishing lifelong CMV latency in brain, from which the virus reactivates intermittently, remain enigmatic. Infection of newborn mice with mouse CMV (MCMV) closely mimicks the pathophysiology of congenital HCMV and was used to unravel the factors involved in CMV infection of the central nervous system (CNS). Here we show that cortex and hippocampus are major sites of productive MCMV infection during the acute phase in newborn mice. Infectious virus was first produced by astrocytes, then microglia, and finally by neurons, which were the major sites of viral replication during the late phase of infection. CD4⁺ T-cells were pivotal in resolving a productive infection in neurons in an interferon-gamma (IFN-γ)-dependent manner. IFN-γ can also suppress HCMV infection of human neuronal cell line and neurons in human fetal brain organotypic tissue culture. Finally, we show that MCMV establishes latency in neurons and that CD4⁺ T-cells are crucial to prevent virus reactivation. This study has important translational potential as it demonstrates that boosting CD4⁺ T-cell mediated immunity could prevent neurological sequelae following congenital CMV infection.

Biological sciences/Microbiology/Virology/Herpes virus

Biological sciences/Microbiology/Virology/Virus–host interactions

Health sciences/Pathogenesis/Infection

Human cytomegalovirus (HCMV) is a highly prevalent herpesvirus, with 40–90% of the population worldwide being seropositive [1]. On average 1% of infants are born with congenital HCMV (cHCMV) infection, making it the most common congenital viral infection [2]. Infected newborns can develop severe disease and permanent neurological sequelae, such as neurodevelopmental disorders, microcephaly, intellectual disability, and sensorineural hearing loss (SNHL) [3]. The underlying mechanisms of how the virus spreads to the brain and causes these neural deficits are still not well understood. Studies of congenital HCMV infection of the brain are mainly limited to observational and histopathological studies on brain specimens of severe cHCMV cases [4].

Like most human and animal herpesviruses, HCMV shows strict species specificity that withholds experimental HCMV infection of experimental animals. However, the pathophysiology of the mouse CMV (MCMV) infection closely mimics that of HCMV infections, including spread and virus-induced neuropathology, underlining its value in deciphering the virus-host interactions [5, 6]. To model cHCMV, newborn mice are experimentally infected by the intraperitoneal (i.p.) route within the first postnatal day to model congenital CMV infection [6, 7]. Following infection of newborn mice, MCMV disseminates to several organs, including the brain, and initiates a local innate and subsequent adaptive immune response [8, 9]. Following the resolution of the acute infection, MCMV establishes a latent infection in the brain, reshaping the immune state of this organ as tissue-resident memory T-cells (T_RM cells) are retained in the brain [10]. Both the cellular sites of CMV latency and the role of T-cells in controlling virus infection in the brain are still unknown.

In this study, we aimed to elucidate viral dissemination and tropism in mouse brain, and immune mechanisms controlling the infection in brain cells. Using recombinant reporter viruses and transgenic mouse lines, we reveal that MCMV disseminates and productively infects principal CNS resident cells with different kinetics and has a remarkable propensity for hippocampus infection. We demonstrate that neurons are the main source of infectious virus during the late phase of acute infection. Finally, we show that IL-12, CD4⁺ T-cells and IFN-γ are essential for the suppression of viral replication, establishment of latency and prevention of reactivation in neurons.

Hippocampus is a major site of productive and latent MCMV infection

During congenital HCMV infection, the virus is detected in different brain regions, with the highest viral load and number of infected cells found in the fetal hippocampus [11]. To correlate these findings to the mouse model, we infected newborn C57BL/6 mice i.p. and harvested brains at multiple time points corresponding to different stages of acute MCMV infection in the brain. MCMV replication in the brain of mice infected as newborns starts around 7 dpi and peaks between 10 and 14 dpi, followed by the resolution of productive infection [10, 12]. The hippocampus, cortex, and cerebellum were isolated from each mouse brain, and viral load was determined (Fig. 1A). At 14, 17 and 21 dpi, the highest MCMV load was detected in the hippocampus, analogous to observations in brains of cHCMV cases [11]. To identify the CNS cell types infected with MCMV, we performed immunohistochemical analysis on brain tissue at the peak of viral replication, 11 days post-infection. Irrespective of the brain region, MCMV was infecting astrocytes (GFAP-positive cells), microglia (IBA-1-positive cells) and neurons (MAP2-positive cells) (Fig. 1B). Again, this data closely mimics the prevalence of HCMV-infected cell types in post-mortem brain specimens of cHCMV cases [13].

The state of latency is defined as the persistence of CMV genomic DNA, without the production of virus progeny [14]. MCMV establishes a latent infection in the brain of mice in the period between one and two months p.i., as evidenced by the lack of infectious virus [10]. Thus, we quantified MCMV DNA in the same brain regions during the latent phase of infection (180 dpi). MCMV genomes were detected in all brain regions, with the highest viral DNA levels again in the hippocampus, followed by the cortex and lastly the cerebellum. Notably, this data correlated with the viral loads of the respective brain regions detected during acute infection (Fig. 1A). Overall, MCMV has a broad cell tropism in the CNS, with a particular propensity to infect and establish latency in the hippocampus.

Neurons are the main source of infectious virus during the late phase of acute infection

To determine which cells produce infectious virus within the CNS, we used a recombinant reporter virus MCMV-flox that encodes the enhanced green fluorescent protein (EGFP) preceded by a floxed stop codon. Thus, EGFP expression is activated by the Cre recombinase. By infecting transgenic mice expressing Cre under the control of a brain cell type-specific promoter, the virus undergoes Cre-mediated recombination only in cells expressing Cre and further disseminates as EGFP-positive (EGFP⁺) virus (Fig. 2A) [15]. We have i.p. infected newborn mice in which Cre is selectively expressed in astrocytes (GFAP-Cre^+/− mice), microglia (Sall1-CreERT2^+/− mice), neurons (BAF53b-Cre^+/− mice), as well as respective Cre-negative littermate controls. The CreERT2 transgenic line received tamoxifen (TAM) treatment on three subsequent days within the first postnatal week (Suppl. Figure 1A). High efficiency of TAM-induced recombination was confirmed by crossing Sall1-Cre^+/−/ERT2 mice with reporter Rosa26-loxP-tdTomato (R26^tdTomato) mice (Suppl. Figure 1B).

Brain and peripheral organs were harvested at 7, 10, 14, and 17 dpi. The number of reporter-tagged and untagged infectious viruses in organ homogenates of transgenic mice and littermate controls was determined by plaque assays and confocal microscopy (Fig. 2B). The results obtained by infecting GFAP-Cre^+/− transgenic mice revealed that astrocytes are the major brain cell type producing infectious virus, given that reporter virus conversion occurs as early as 7 dpi. Furthermore, less than 60% of virus replicating in the brain has originated from astrocytes at any time point of infection, suggesting that virus can enter the brain without infecting blood-brain barrier astrocytes (Fig. 2B). Microglia-derived virus was detected on 10 dpi, but not on other times points, suggesting that microglia-derived virus does not readily spread to other cells. Infection of BAF53b-Cre^+/− mice showed that mature neurons are not the initial targets during the acute phase of MCMV infection in the brain, as no and < 20% of viruses were neuron-derived at 7 and 10 dpi, respectively. Notably, neurons became the major cell type producing infectious viruses at later time points (Fig. 2B). This observation was validated by histological quantification of MCMV-infected cells (IE1⁺ cells) in the brains of C57BL/6 mice infected with wild-type MCMV. MCMV-infected cells were exclusively MAP2⁺ neurons on 17 dpi (Fig. 2C-D), hereby providing independent confirmation of the data obtained using the reporter system (Fig. 2B). We did not observe EGFP⁺ plaques in peripheral organs of GFAP-Cre^+/− and BAF53b-Cre^+/− mouse lines, suggesting that the virus does not spread from the CNS back to the periphery. We observed some conversion in the peripheral organs of Sall1CreERT2^+/− mice, which was in line with the known expression of Sall1 in several macrophage populations [16]. Importantly, we did not observe EGFP⁺ plaques in MCMV-flox-infected Cre-negative littermate controls, indicating that no conversion of MCMV-flox occurred in the absence of Cre (Suppl. Figure 1C). Altogether, our findings demonstrate that MCMV productively infects multiple brain-resident cell types, of which neurons are the dominant source of MCMV during the late phase of acute infection.

CD4⁺ T-cells are required to resolve productive infection in cortical and hippocampal neurons

During acute MCMV infection, CD8⁺ and CD4⁺ T-cells infiltrate the brain and contribute to virus control [9, 17]. Since T-cells infiltrate the brain only at about 10 dpi [9], our data suggest that the T-cell-mediated control of virus infection in neurons is delayed compared to other cell types (Fig. 2B-D). To determine the T-cell subset involved in resolving the productive infection in neurons, we infected newborn wild-type C57BL/6, CD4- (CD4^−/−) and CD8-deficient (CD8^−/−) mice by the i.p. route with MCMV. Brain and peripheral organs were harvested at 14, 30, and 70 dpi. We did not observe significant differences in both body weight and survival of MCMV-infected mice that lack CD4⁺ or CD8⁺ T-cells compared to wild-type C57BL/6 mice (Suppl. Figure 2A-B). Notably, mice lacking CD4⁺ T-cells had significantly higher viral titers at 14 dpi onwards, and could not resolve productive infection in the brain (Fig. 3A). Even at 120 dpi, replicating MCMV was still detectable in brains of CD4^−/− mice (Fig. 3B). In contrast, CD8⁺ T-cells were not essential for the resolution of productive MCMV infection in the brain (Fig. 3A). In agreement with MCMV infection in adult mice [18], CD4⁺ T-cells were not essential for the resolution of productive infection in other peripheral organs, including the spleen and lungs, but were required in salivary glands (Suppl. Figure 2C-E).

To define if CD4⁺ T-cells are pivotal in controlling MCMV infection in neurons, brains from CD4^−/− mice were collected at 37 dpi for immunohistochemical detection of MCMV-infected cells (IE1⁺ cells). The viral IE1 protein was mainly detected in the cortex and hippocampus (Fig. 3C), where MAP2⁺ cells (neurons) were the predominant MCMV-infected cell type (Fig. 3D-E). Therefore, our results strongly suggest that CD4⁺ T-cells are essential to control MCMV infection in neurons. Accordingly, we detected CD4⁺ T-cells in the parenchyma of infected animals (Fig. 3F). Altogether, we show that CD4⁺ T-cells, and not CD8⁺ T cells, are essential to terminate productive MCMV infection and the subsequent establishment of latency in the brain.

Resolution of productive MCMV infection in neurons is dependent on IFN-γ and IL-12

CD4⁺ T-cells possess many antiviral functions, from regulating antiviral immunity mediated by other cell types to directly suppressing viral replication in infected cells [19]. To elucidate the mechanism of CD4⁺ T-cell-mediated control of MCMV infection in the brain, we infected knockout mice that lack critical elements of T-cell effector functions. First, the role of cytotoxic mechanisms was determined by infecting newborn mice that lack the cytolytic T cell proteins perforin (Prf1^−/−) or granzymes A and B (Gzma^−/−Gzmb^−/−). Compared to wild-type C57BL/6 controls, no significant differences were observed in viral titers of Prf1^−/− or Gzma^−/−Gzmb^−/− mice, suggesting that non-cytolytic mechanism controls MCMV infection in neurons (Fig. 4A-B). As expected, perforin and granzymes A and B were necessary to control MCMV in the lungs and salivary glands at 14 and 30 dpi (Suppl. Figure 3A-B) [20, 21]. Next, we tested the outcome of MCMV infection in Tumor Necrosis Factor Receptor p55 (TNFRp55) (TNFRp55^−/−) and IFN-γ (IFN-γ^−/−) knockout mice. The TNFRp55^−/− mice had modest but significantly increased viral titers in the brain compared to WT control mice at 30 dpi, while the majority of animals resolved productive infection in the brain by 70 dpi (Fig. 4C). The same was observed in the lungs, as TNFRp55^−/− mice cleared MCMV infection with a delayed kinetic compared to C57BL/6 controls, while no difference was observed in the spleen and salivary glands (Supp. Figure 3C). In contrast, IFN-γ^−/− mice displayed increased virus titers of nearly two orders of magnitude and were not able to resolve brain infection even by 70 dpi (Fig. 4D). A similar effect was observed in salivary glands, previously described to require IFN-γ to control productive infection (Supp. Figure 3D) [22]. Histological analysis of brains from IFN-γ-deficient animals at 37 dpi showed the same pattern of MCMV infection (Fig. 4E), as observed in CD4⁺ T-cell deficient animals (Fig. 3C). Again, MCMV-infected cells were predominantly found in the cortex and hippocampus (Fig. 4E, Supp. Figure 3E), and MAP2⁺ neurons were the main infected population of cells in IFN-γ^−/− mice (Fig. 4F-G).

Interleukin-12 (IL-12) is a pro-inflammatory cytokine important for T helper 1 (Th1) cell differentiation and the production of IFN-γ [23]. Thus, we infected IL-12 receptor subunit beta 2 deficient (Il12rb2^−/−) newborn mice. Analogous to IFN-γ^−/− mice, Il12rb2^−/− mice could not resolve MCMV infection in the brain (Fig. 4H). MCMV was detected predominantly in the murine cortex and hippocampus (Fig. 4I, Supp. Figure 3F), and exclusively within MAP2⁺ neurons (Fig. 4J-K). In conclusion, these findings show the crucial role of IFN-γ and IL-12 signaling, but not perforin and granzyme-mediated cytotoxicity, to control productive MCMV infection of neurons.

IFN-γ inhibits HCMV infection of human neurons

Since we have observed that IFN-γ is critical for controlling MCMV infection in mouse neurons, we determined if the same applies to HCMV infection in human neurons. First, we used SH-SY5Y cells, a human neuroblastoma cell line [24]. Treatment with recombinant IFN-γ either prior (Fig. 5A-B, Suppl. Figure 4A) or following (Fig. 5C-D, Suppl. Figure 4B) HCMV infection significantly decreased HCMV infection of SH-SY5Y cells in a dose-dependent manner. Next, to model the neuropathology of congenital HCMV infection, we infected human fetal organotypic brain slice cultures (hfOBSCs) with HCMV, similar to the recently reported model for herpes simplex virus (HSV) infection [25]. Notably, HCMV efficiently infected cells and spread in the hfOBSCs (Fig. 5E), and IFN-γ decreased the number of HCMV-infected NeuN⁺ neurons, demonstrating the neuroprotective role of IFN-γ against HCMV infection (Fig. 5F-G, Suppl. Figure 4C). Overall, these data show that IFN-γ inhibits HMCV infection in human neurons.

Neurons are sites of MCMV latency and reactivation

Despite the resolution of productive infection, MCMV still establishes life-long latency in the brain [10]. However, the cellular sites of MCMV latency in the brain remain unclear. To identify which cell types harbor the latent virus, we used a recombinant virus MCMV-GFP_Cre that codes for simultaneous co-expression of GFP and Cre-recombinase under HCMV major immediate-early promoter (MIEP) (Suppl. Figure 5A). We infected newborn Rosa26-loxP-tdTomato (R26^tdTomato) reporter mice, which have a loxP-flanked STOP cassette preventing expression of tdTomato fluorescent protein. Thus, infection of cells with MCMV-GFP_Cre enables tdTomato expression following Cre-mediated recombination. Brains of latently MCMV-GFP_Cre-infected mice were collected at 45 dpi and analyzed for the expression of tdTomato reporter protein by confocal microscopy. Interestingly, tdTomato-positive cells displayed a neuronal morphology, which strengthens the notion that neurons are the major cell type infected during the late phase of productive MCMV infection (Fig. 6A). By using the neuronal marker NeuN, we confirmed that tdTomato is expressed exclusively in neurons but not in astrocytes or microglia (Fig. 6B), suggesting that MCMV established a latent infection in neurons. Accordingly, we could not detect MCMV genomes in sorted astrocytes and microglia by qPCR (Suppl. Figure 5B). As shown in Fig. 1B, the hippocampus is the dominant site of MCMV latency in the brain. To test if MCMV can reactivate in the hippocampus and other brain regions, we isolated different brain regions from latently infected mice and cultured these specimens as ex-vivo explants for 6-weeks. In accordance with latent genome loads (Fig. 1B), the hippocampus was the prime site of MCMV reactivation in tissue explants (Fig. 6C). To exclude that there was no active viral replication in mice from which we collected brain tissues, no infectious virus was detected in the animals’ salivary glands (Suppl. Figure 5C).

We showed that CD4⁺ T-cells are critical to suppress productive infection in the brain (Fig. 3). To analyze the distribution of CD4⁺ T-cells in the latently infected brain regions, we performed flow cytometry analysis. The hippocampus harbored the highest frequency of CD4⁺ T-cells (Fig. 6D), correlating with the number of latent genomes (Fig. 1B) and potential to reactivate the virus (Fig. 6C). To assess the role of CD4⁺ T cells in the control of latent MCMV in neurons, we depleted CD4⁺ T-cells in latently infected R26^tdTomato reporter mice infected with MCMV-GFP_Cre. Depletion of CD4⁺ T cells resulted in a significant increase in number of tdTomato-positive neurons, suggesting viral reactivation and further spread among neurons following loss of CD4⁺ T-cell-mediated control (Fig. 6E). Finally, we depleted CD4⁺ T-cells in latently infected C57BL/6 mice and isolated brain regions. Depletion efficiency of CD4⁺ T-cells in the brain was confirmed by flow cytometry (Suppl. Figure 5D). In the control, non-depleted group, we did not detect reactivating virus. The highest frequency of MCMV reactivation was observed in the hippocampus, suggesting that the hippocampus is also the primary site of MCMV reactivation upon loss of CD4⁺ T-cell-mediated control (Fig. 6F). MCMV reactivation was detected exclusively in neurons following depletion of CD4⁺ T-cells (Fig. 6G-H). Overall, these data suggest that neurons are sites of MCMV latency and that CD4⁺ T-cells are required to prevent reactivation.

HCMV is a leading infectious cause of congenital abnormalities with limited treatment options [5], warranting a better understanding of the disease pathogenesis. Here, we investigated MCMV dissemination, immune response, and resolution of productive infection in the brain in an experimental mouse model of cHCMV infection. MCMV preferentially localized in the hippocampus during both acute infection and latency. While initially infecting diverse cell types in the CNS, neurons become the primary reservoir of MCMV during the late productive and the subsequent latent phase of infection. Mechanistically, we show that IL-12, CD4⁺ T-cells and IFN-γ are crucial at two different stages of MCMV infection of the brain. First, they all contributed to the control of productive MCMV infection in neurons, and later, they prevented the reactivation of latent virus in cortical and hippocampal neurons.

The highest HCMV load in the brain has been reported in the hippocampus of congenitally infected fetuses [11]. Similarly, we show that the hippocampus is the main region targeted by MCMV. However, it remains to be determined what is the underlying mechanistic reason for the preferential CMV infection of the hippocampus. Findings from animal studies demonstrate notable variations in the brain regions susceptible to different neurotropic viruses [26]. Preference for hippocampal infection has been reported for other viruses, including DNA viruses (eg herpesviruses) and RNA viruses (eg flaviviruses) [27, 28]. In the case of HSV-1, the preference for hippocampal infection has been related to the high expression of viral receptors, lower levels of antiviral cytokines, and a neurogenic niche [29]. Furthermore, neuronal populations differ between brain regions, which can determine their susceptibility to infection [30–32]. Moreover, microglia display regional heterogeneity in the brain, which could play a significant role in antiviral responses [33]. Finally, the hippocampus blood-brain barrier is more fragile to insults than in other brain regions, potentially enabling more efficient hematogenous virus spread [34].

Like HCMV [35], MCMV has a broad cell tropism in acutely infected brains. By following MCMV dissemination in the acutely infected brain, we have shown that astrocytes are initial targets, but also a substantial cellular source of infectious viruses. Astrocytes are often targets of neurotropic viral infections, including cHCMV infection, due to their anatomical location and morphological properties that support productive virus infection [13, 36]. Microglia were also infected with MCMV. However, the microglia-derived virus does not readily spread to other cells, suggesting an efficient microglial mechanism for virus containment. Our observation fits with data for the infection of other tissue-resident macrophages, which are often infected with CMV, but are not major virus producers [37, 38]. For example, MCMV infects subcapsular sinus macrophages (SSMs) in the lymph nodes, which restrict viral spread and thereby protect other cells from infection [37]. At later time points of acute infection, the virus is cleared from microglia and astrocytes, and neurons become the primary source of infectious virus, demonstrating slower immune control of the virus in neurons.

In most viral brain infections studied so far, CD8⁺ T cells were shown to critically contribute to virus control [39]. Here we have shown that CD4⁺ T-cells are required to resolve productive MCMV infection in the brain. CD8⁺ T-cells play a pivotal role in protecting against MCMV in most organs, except salivary glands, where the virus is controlled by CD4⁺ T-cells [18]. In addition, CD4⁺ T-cells provide compensatory protective activity in adult mice depleted of CD8⁺ T-cells [40]. In cases of cHCMV infection, T-cells infiltrate the infected fetal brains [41, 42]. Histological analysis correlated higher numbers of CD8⁺ T-cells in the brain with increased disease severity [42]. Conversely, cHCMV cases that present more severe sequelae at birth have significantly lower counts of CD4⁺ T-cells [43]. In contrast to infected adults, infants with cHCMV have long-term impaired CD4⁺ T-cell response, while CD8⁺ T-cell response is not altered, demonstrating the importance of early-life CD4⁺ T-cell-mediated immunity to HCMV [44, 45]. Adult patients with human immunodeficiency virus (HIV) and HCMV co-infection present with neurotropic HCMV infections when the number of CD4⁺ T-cells is particularly low [46, 47]. The protective role of CD4⁺ T-cells has also been reported in the rhesus macaque model of congenital rhesus CMV (rhCMV) infection [48]. Thus, the importance of CD4⁺ T-cells in the context of congenital CMV infection in both humans and nonhuman primates aligns with our findings obtained in the mouse model of congenital infection.

The resolution of productive MCMV infection in neurons does not depend on cytolytic mechanisms. This is unsurprising, as cytolytic mechanisms may cause neuronal death [49]. Cytolytic mechanisms are more important for the clearance of viral infections from glial cells [26]. This is in accordance with our data, which demonstrated faster clearance of infection from astrocytes and microglia. We show that IFN-γ is critical for controlling MCMV infection and limits the establishment of viral latency in neurons. Previous studies have shown that IFN-γ is necessary in the control of several other virus infections in the brain [50–54]. During HSV-1 infection, CD8⁺ T-cells control HSV-1 latency in trigeminal ganglia by blocking viral reactivation via IFN-γ [55]. CD4⁺ T-cells are not directly involved in the control of HSV-1 latency in trigeminal ganglia, but rather mediate early CD8⁺ T-cell priming to generate an efficient local effector memory CD8⁺ T-cell population [56]. However, we show here that CD8⁺ T-cells are not critical for the control of MCMV infection in the brain. This is in accordance with the control of MCMV infection in salivary glands, where the resolution of productive infection does not relay on CD8⁺ T-cells. To support the data on Th1-dependent infection control in neurons, we have shown that animals lacking IL-12 receptors cannot control MCMV infection in neurons. To relate our findings to cHCMV infection, we have developed a new model of human fetal organotypic brain slice cultures that mimic the complexity of the human brain and maintain microglia [25]. We used this system to demonstrate that IFN-γ treatment protected human neurons from HCMV infection. Thus, IFN-γ can protect both human and mouse neurons from CMV infection.

The state of latency is defined as the presence of viral genomes without the production of new infectious virions [57]. While the viral gene expression is generally silenced during latency, it is suggested that stochastic transcription of CMV genes occurs during latency [58, 59]. Herpesviruses can replicate and establish latency in many cell types [60]. HCMV establishes latency in hematopoietic stem cells, myeloid progenitors, and monocytes, while monocytes, endothelial cells, and fibroblasts were shown to be sites of MCMV latency [61, 62]. The sites of CMV latency in the CNS remained elusive until now. The data presented in this study indicate that neurons are the main site of MCMV latency in the mouse brain, a state from which MCMV can reactivate upon loss of CD4⁺ T-cells. HCMV can infect neurons and neural stem and precursor cells [35]. Interestingly, some studies suggest that primitive neural stem cells are infected nonproductively, with HCMV replication restricted at the IE gene level, while HCMV reactivates upon complete neuronal differentiation ex vivo [63], thus supporting our notion of the ability of CMV to reactivate in neurons. The exact mechanism of CD4⁺ T-cell mediated prevention of MCMV reactivation remains unknown. Unlike antigen-presenting cells, neurons lack major histocompatibility complex (MHC II) molecules, preventing them from directly triggering CD4⁺ T-cells in an antigen-specific manner [64]. Thus, CD4⁺ T-cells might control CMV reactivation in neurons through intermediate cells, such as MHC II expressing microglia, which can cross-present antigens to T-cells [65]. It was shown that uninfected microglia can effectively cross-present vesicular stomatitis virus(VSV) antigens acquired from neighboring neurons to CD8⁺ T-cells to secure control of VSV in neurons [66], and it is at least plausible that this might contribute to MCMV control. Finally, a TCR-independent mechanism of direct interaction between CD4⁺ T-cells and neurons could be involved [67].

This study provides novel insights into the virus-host interactions involved in controlling CMV infection of the developing brain. We show that MCMV can establish latency in neurons, and that IFN-γ producing CD4⁺ T-cells are pivotal in the control of both productive and latent brain infection. This result echoes the weak CD4⁺ T-cell responses that were observed to correlate with aggravated disease outcomes in infants born with congenital CMV infection. HCMV remains a major clinical problem, as there is no approved vaccine against HCMV, and pharmacotherapy during pregnancy is constrained by concerns on drug toxicity and teratogenicity [68]. HCMV is also linked to declines in neurocognitive and neuropsychiatric health and can be found in patients with glioblastoma multiforme [69, 70]. Therefore, a better understanding of the immune response to CMV infection in neuronal tissue remains a pressing need. Our findings suggest that exploiting CD4⁺ T-cell responses in preventive and therapeutic strategies might be beneficial in thwarting CMV-associated neurological complications.

Mice

Mice were strictly age-matched within experiments and handled in accordance with institutional and national guidelines. Newborn mice of both sexes were included in all experiments. All mice were housed and bred under specific pathogen-free conditions at the animal facility of the Faculty of Medicine, University of Rijeka where they were maintained at 22°C in a 12-h light-dark cycle, and relative humidity (40–50%). Wild-type C57BL/6J (strain #000664), GFAP-Cre line 77.6 (#024098), Sall1CreERT2 (provided by Ryuchi Nishinakamura at Kumamoto University, [71, 72]), BAF53b-Cre (#027826), CD4^−/− (#002663), CD8^−/− (#002665), Prf1^−/− (#002407), Gzma^−/−Gzmb^−/− (#010608), TNFRp55^−/− (#002818), Ifng^−/− (#002287), and Il12rb2^−/− (#003248) and Rosa26-loxP-tdTomato (#007914) mice were obtained from The Jackson Laboratory. The Animal Welfare Committee at the University of Rijeka, Faculty of Medicine, and The National Ethics Committee for the Protection of Animals Used for Scientific Purposes (Ministry of Agriculture) approved all animal experiments (UP/I-322-01/19 − 01/58, UP/I-322-01/21 − 01/51, UP/I-322-01/23 − 01/33).

Viruses and cell lines

Tissue culture-derived wilde-type (WT) MCMV reconstituted from BAC pSM3fr-MCK-2fl was used in the majority of experiments [73]. MCMV-Δm157-flox-egfp (MCMV-flox) was previously described[74]. MCMV-GFP_Cre (ΔIE2) expressing EGFP and Cre recombinase under the control of the endogenous MIEP promoter was generated by replacing the entire IE2 ORF with a construct encoding Cre recombinase in MCMV^IE−GFP [75], in which a construct encoding EGFP plus a distal P2A-encoding sequence is inserted in the start codon of IE1/3. The backbone of MCMV-GFP_Cre (ΔIE2) is the Mck2-repaired BAC-encoded MCMV strain, clone pSM3fr-MCK-2fl 3.3 [73], and genetic modifications were done using en passant mutagenesis [76].

Virus stocks and organ homogenates were titrated on mouse embryonic fibroblasts (MEFs) using standard plaque assay procedures [7]. Newborn pups were infected i.p. 24–48 h postnatally with either 200 PFU WT MCMV, 1500 PFU MCMV-flox, or 1000 PFU MCMV-GFP_Cre. For HCMV infection experiments RV-TB40-BACKL7-SE-EGFP (KL7-EGFP) virus was used, a kind gift from Christian Sinzger [77].

SH-SY5Y cells were infected with KL7-EGFP at a multiplicity of infection (MOI) of 10 diluted in 500 µl of culture medium. For treatment experiments, cells were treated with 100, 1000, or 2500 U/ml of recombinant human IFN-γ (Peprotech, #300-02) diluted in 500 µl of culture medium.

In-vivo treatments

To induce site-specific recombination in CreERT2 transgenic mice, TAM (Sigma-Aldrich, #T5648) was dissolved in corn oil (Sigma-Aldrich, #C8267) to prepare 10 mg/ml stock solutions [78]. On 1–3 dpi, 50 µg of TAM in a volume of 50 µL was administered daily in by intragastric (i.g.) injection using a 30G needle (BD Microlance, #304000).

Histology

Brains were fixed in 4% paraformaldehyde and paraffin-embedded. Double immunohistochemical staining was performed on mouse brains for the detection of viral antigen and the detection of CNS cell types. Antigen retrieval was performed in a citrate buffer. Endogenous peroxidase activity was blocked with peroxidase and alkaline phosphatase blocking reagent (Dako, #S200380) for 10 min. Nonspecific binding was blocked with 3% BSA (Roth, #8076.1). Viral antigen IE1 was detected using biotinylated (Thermofisher Scientific, #A39256) anti-IE1 antibody (clone IE1.01; Center for Proteomics, Faculty of Medicine, University of Rijeka, Rijeka, Croatia, #HR-MCMV-12). Antibody binding was visualized with streptavidin alkaline phosphatase conjugate (1:1000, Roche, #11089153001), followed by liquid permanent red as a chromogen (Dako, #K064030). CNS cells were detected with rabbit anti-Iba-1 (1:1000, Fujifilm; Wako Chemicals, #019-19741) for microglia, rabbit anti-GFAP (1:200, Cell signaling, #80788) for astrocytes and rabbit anti-MAP2 (1:1000, Abcam, #ab183830) antibody for neurons. Antibody binding was visualized with peroxidase-conjugated goat anti-rabbit IgG antibody (1:500, Abcam, #ab6721), followed by 3,39-diaminobenzidine (DAB) as a chromogen (Dako, #GC80611-2). Tissues were counterstained with hematoxylin (Shandon, #12687926). Images were acquired with Olympus BX51, 100x magnification (oil).

Detection of reporter protein tdTomato was performed using frozen brain tissue. Mice were perfused with PBS and brains were fixed in 4% paraformaldehyde. Following fixation, brains were submerged in 30% sucrose-PBS. The tissue was frozen on dry ice in OCT (Sakura, #4583) embedding media and stored at -80°C until cutting. Before staining, tissue slides were blocked with 3% BSA. CNS cells were detected with rabbit anti-Iba-1 (1:1000, Fujifilm; Wako Chemicals, #019-19741) for microglia, rabbit anti-GFAP (1:200, Cell signaling, #80788) for astrocytes and rabbit anti-NeuN (1:250, Cell signaling, #12943) for neurons. Primary antibodies were labeled with goat anti-rabbit IgG antibody conjugated to Alexa Fluor (AF) 555 (1:250, Cell signaling, #4413). Nuclei were stained with 4',6-Diamidino-2-Phenylindole (DAPI) (Biolegend, #422801).

MCMV reactivation assays

To induce MCMV reactivation in vivo, mice were depleted of CD4⁺ T-cells by administering 150 µg of anti-CD4 antibody (GK1.5, BioXCell, #BE0003-1) i.p. every 4–5 days for 30 days. Brains were collected, and the tissue was homogenized and centrifuged for 1 min at 400 g. The supernatant was collected, mixed with 5 ml of 3% DMEM medium, and distributed on a 24-well plate seeded with MEF. Plates were left to incubate for 5–6 days. Afterward, supernatants were transferred to new 24-well plates with MEF and analyzed for plaque formation after 5–6 days of incubation.

Ex-vivo reactivation was performed using a previously published procedure [79]. Brains were collected, hemispheres were separated sagitally, and specific regions were isolated from each hemisphere. Tissues were cultured in 1ml of 3% DMEM media in individual wells of 24-plates for 6 weeks. The supernatant was collected weekly, culture media was replaced, and infectious virus was detected by the plaque assay.

Flow cytometry and cell sorting

Mice were sacrificed and brains were collected in RPMI 1640 with 3% FCS and mechanically dissociated. Single-cell suspensions of brains were prepared according to standard protocols [9]. In brief, 30% Percoll (Cytivia, #17089101) and brain homogenate suspension was underlaid with 70% Percoll in PBS and then centrifuged at 1,800 rpm for 25 min. Cells in the interphase were collected for further analysis of microglia and T-cell populations. For efficient isolation of astrocytes, microglia and oligodendrocytes from the brain, Adult Brain Dissociation Kit (Miltenyi Biotec, # 130-107-677) was used. After tissue dissociation based on enzymatic digestion (enzyme papain), debris, and red blood cell removal, myelin was additionally removed using magnetic beads (Myelin Removal Beads, Miltenyi Biotec, #130-096-433). Flow cytometric analysis were performed using following anti-mouse antibodies: CD45.2 (104), CD11b (M1/70), CD8a (53 − 6.7), CD4 (RM4-5), CD69 (H1.2F3), CD103 (2E7), F4/80 (BM8), CD3e (145-2C11), CD19 (eBio1D3), O1 (O1) purchased from ThermoFisher and ACSA-2 (IH3-18A3) were purchased from Miltenyi Biotec. All data were acquired using a FACSAria (BD Biosciences). Microglia and astrocytes were sorted by FACS. Microglia were sorted as CD45.2^intCD11b⁺ population, while astrocytes were sorted as CD45.2⁻CD11b⁻O1⁻ACSA-2⁺ population. For sorting of splenic stromal cells or red pulp macrophages, the cell suspension was subjected to immunomagnetic depletion of CD45⁺ cells or positive selection of VCAM-1⁺ cells using MACS (Miltenyi Biotech).

qPCR detection of latent MCMV genomes

Total DNA was extracted from brain tissue or sorted cells by using AllPrep DNA/RNA Micro Kit (Qiagen, #80204). MCMV and mouse genome copies were quantified as described previously [62, 80]. Viral gB and mouse Pthrp sequences were assayed in technical duplicates using 9 µl of 200 ng sample DNA per reaction. Serial dilutions of 10¹-10⁶ copies per reaction of the pDrive_gB_PTHrP_Tdy plasmid were used to generate standard curves for both qPCR reactions. Quantitative PCR was performed using Fast Plus EvaGreen qPCR master mix (Biotium) in a 7500 Fast Real Time PCR (Applied Biosystems). Cycling conditions were as follows: enzyme activation, 2 minutes (min) at 95°C, followed by 50 cycles of denaturation for 10 seconds (sec) at 95°C, annealing for 20 sec at 56°C and extension for 30 sec at 72°C. The specificity of amplified sequences was validated for all samples in each run by inspecting the respective melting curve profiles. Primer sequences that were used: Pthrp forward 5’-ggtatctgccctcatcgtctg-3’ and reverse 5’-cgtttcttcctccaccatctg-3’, gB forward 5’-gcagtctagtcgctttctgc-3’ and reverse 5’-aaggcgtggactagcgataa-3’.

Human fetal organotypic brain slice cultures

Human fetal organotypic brain slice cultures (hfOBSCs) are described in more detail [25]. In brief, 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. At the request by the researchers, only gender and gestational age are provided. Abortions were not performed for medical indications and fetuses did not have any major anatomical deformities (checked by ultrasound prior to the procedure) or trisomy. The procedure was performed by in utero dissection and removal of fetal tissue specimens did not enable recovery of intact organs. Brain tissue fragments (approximately 0.5 × 0.5 cm in size) were cut into 350 µm thick slices using a vibratome (Leica, #VT1200S) in artificial cerebrospinal fluid (aCSF) (prepared in house) under constant oxygenation (95% O2, 5% CO2). Brain slices were transferred to 12-mm Transwells with polyester membrane inserts (0.4 µm pore size; Corning, #3470) and recuperated for 1 h in recovery medium that was 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; Sigma, cat: F7524, lot: 0001644044) and antibiotics. After 1-h incubation in a CO2 incubator at 37°C, the recovery medium was replaced with optimized hfOBSC serum-free culture medium consisting of a 7:3 (v/v) mixture of Neurobasal media (Gibco, #21103049) and advanced DMEM/F12 culturing medium (Gibco, #12634010), B27 (2% v/v, Gibco, #17504044), N2 (1% v/v, Gibco, #17502048), glutaMAX (1% v/v, Gibco, #35050061), primocin, 1:500, Invivogen, #ant-pm-05), TGF-β2 human (2 ng/mL, ProspecBio, #CYT-441), cholesterol (1.5 µg/mL, Sigma Aldrich, #C3045), human recombinant m-CSF (100 ng/mL, Peprotech, #300 − 25), BDNF (50 µg/mL, Peprotech, #450-02), NT-3 (10 ng/mL, Peprotech, #450-03), FGF2 (10 ng/mL, R&D Systems, #233-FB) and EGF (10 ng/mL, R&D Systems, #236-EG). The culture medium was refreshed every 48 h. The hfOBSCs were infected with 10⁷ PFU of cell-free KL7-EGFP. After 1-h incubation at 37°C, the inoculum was removed, and the brain slices were washed with PBS and subsequently maintained in the culture medium for 48 h post-infection (hpi) at 37°C in a CO2 incubator. Additionally, one group of brain slices was treated with 1,000 U/ml of recombinant human IFN-γ (Peprotech, #300-02) for 48 h. At the indicated time point, the hfOBSCs were fixed in PBS containing 4% paraformaldehyde and embedded in paraffin for histological analyses. For immunofluorescent staining, brain slices were treated with TrueBlack (Biotium, #23007) after citrate buffer antigen retrieval to decrease autofluorescence. The following primary antibodies were used for IF: FITC goat anti-GFP (1:250, Abcam, #ab6662) and rabbit anti-NeuN (1:500, Abcam, #ab177487). Unconjugated primary antibodies were labelled with the appropriate secondary antibody from donkey anti-rabbit IgG conjugated to AF555 (1:250, Invitrogen, #A-31572). Nuclei were stained with Hoechst 33342 Solution (Thermo Scientific, #62249). Images were taken using a Leica Stellaris 5 Low Incidence Angle confocal microscope.

Statistical analyses

Statistical analyses were performed with Prism 5 software (GraphPad Software Inc.). Appropriate statistical tests were employed depending on the number of animals per group and data distribution (Student’s t-test, One-way ANOVA test, two-way ANOVA test). p values above < 0.05 were considered to be statistically significant;. *,p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p ≤ 0.0001.

Acknowledgments

This work has been fully supported by “Research Cooperability“ program of the Croatian Science Foundation funded by the European Union from the European Social Fund under the “Operational Programme Efficient Human Resources 2014 2020“ (PZS-2019-02-7879, I.B. ), the grant “Strengthening the capacity of CerVirVac for research in virus immunology and vaccinology” (KK.01.1.1.01.0006) granted to the Scientific Centre of Excellence for Virus Immunology and Vaccines and co-financed by the European Regional Development Fund (S.J.), the Croatian Science Foundation under the project numbers IP-2022-10-3371 and DOK-2020-01-5362 to (I.B.), and University of Rijeka (uniri-iskusni-biomed-23-231, I.B.), the German Scientific Foundation (DFG) through the grants EXC 2155 “RESIST”—Project ID 39087428 and FOR2830 (project 5) to LCS, the European Union’s Horizon 2020 research and innovation program under Grant Agreement 793858 (to K.M.S.). The study was in part supported by The Lundbeck Foundation (R359-2020-2287) (author GMGMV). We thank Mijo Golemac, Dijana Rumora, Ante Miše, Mihaela Gašparević, Cristina Paulović, and Antonija Šarlija for the excellent technical and administrative support. We thank Vanda Juranić Lisnić for the critical reading of the manuscript. Schematic representations are created with BioRender.com.

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Neuron-restricted cytomegalovirus latency in the central nervous system regulated by CD4+ T cells and IFN-γ

Status:

Version 1

Abstract

Figures

Introduction

Results

Hippocampus is a major site of productive and latent MCMV infection

Neurons are the main source of infectious virus during the late phase of acute infection

CD4⁺ T-cells are required to resolve productive infection in cortical and hippocampal neurons

Resolution of productive MCMV infection in neurons is dependent on IFN-γ and IL-12

IFN-γ inhibits HCMV infection of human neurons

Neurons are sites of MCMV latency and reactivation

Discussion

Material and methods

Mice

Viruses and cell lines

Histology

MCMV reactivation assays

Flow cytometry and cell sorting

qPCR detection of latent MCMV genomes

Human fetal organotypic brain slice cultures

Statistical analyses

Declarations

Acknowledgments

References

Additional Declarations

Supplementary Files

Status:

Version 1

Neuron-restricted cytomegalovirus latency in the central nervous system regulated by CD4+ T cells and IFN-γ

Status:

Version 1

Abstract

Figures

Introduction

Results

Hippocampus is a major site of productive and latent MCMV infection

Neurons are the main source of infectious virus during the late phase of acute infection

CD4+ T-cells are required to resolve productive infection in cortical and hippocampal neurons

Resolution of productive MCMV infection in neurons is dependent on IFN-γ and IL-12

IFN-γ inhibits HCMV infection of human neurons

Neurons are sites of MCMV latency and reactivation

Discussion

Material and methods

Mice

Viruses and cell lines

Histology

MCMV reactivation assays

Flow cytometry and cell sorting

qPCR detection of latent MCMV genomes

Human fetal organotypic brain slice cultures

Statistical analyses

Declarations

Acknowledgments

References

Additional Declarations

Supplementary Files

Status:

Version 1

CD4⁺ T-cells are required to resolve productive infection in cortical and hippocampal neurons