C-type lectin receptor DCIR contributes to hippocampal injury in acute neurotropic virus infection

Neurotropic viruses target the brain and contribute to neurologic diseases. C-type lectin receptors (CLRs) are pattern recognition receptors that recognize carbohydrate structures on endogenous molecules and pathogens. The myeloid CLR dendritic cell immunoreceptor (DCIR) is expressed by antigen presenting cells and mediates inhibitory intracellular signalling. To investigate the effect of DCIR on neurotropic virus infection, mice were infected experimentally with Theiler’s murine encephalomyelitis virus (TMEV). Brain tissue of TMEV-infected C57BL/6 mice and DCIR−/− mice were analysed by histology, immunohistochemistry and RT-qPCR, and spleen tissue by flow cytometry. To determine the impact of DCIR deficiency on T cell responses upon TMEV infection in vitro, antigen presentation assays were utilised. Genetic DCIR ablation in C57BL/6 mice was associated with an ameliorated hippocampal integrity together with reduced cerebral cytokine responses and reduced TMEV loads in the brain. Additionally, absence of DCIR favoured increased peripheral cytotoxic CD8+ T cell responses following TMEV infection. Co-culture experiments revealed that DCIR deficiency enhances the activation of antigen-specific CD8+ T cells by virus-exposed dendritic cells (DCs), indicated by increased release of interleukin-2 and interferon-γ. Results suggest that DCIR deficiency has a supportive influence on antiviral immune mechanisms, facilitating virus control in the brain and ameliorates neuropathology during acute neurotropic virus infection.


Neurotropic viruses target the brain and contribute to neurologic diseases. C-type lectin receptors (CLRs) are pattern recognition receptors that recognize carbohydrate structures on endogenous molecules and pathogens. The myeloid CLR dendritic cell immunoreceptor (DCIR) is expressed by antigen presenting cells and mediates inhibitory intracellular signalling. To investigate the effect of DCIR on neurotropic virus infection, mice were infected experimentally with Theiler's murine encephalomyelitis virus (TMEV). Brain tissue of TMEV-infected C57BL/6 mice and DCIR −/− mice were analysed by histology, immunohistochemistry and RT-qPCR, and spleen tissue by flow cytometry.
To determine the impact of DCIR deficiency on T cell responses upon TMEV infection in vitro, antigen presentation assays were utilised. Genetic DCIR ablation in C57BL/6 mice was associated with an ameliorated hippocampal integrity together with reduced cerebral cytokine responses and reduced TMEV loads in the brain. Additionally, absence of DCIR favoured increased peripheral cytotoxic CD8 + T cell responses following TMEV infection. Co-culture experiments revealed that DCIR deficiency enhances the activation of antigen-specific CD8 + T cells by virus-exposed dendritic cells (DCs), indicated by increased release of interleukin-2 and interferon-γ. Results suggest that DCIR deficiency has a supportive influence on antiviral immune mechanisms, facilitating virus control in the brain and ameliorates neuropathology during acute neurotropic virus infection.
Neurotropic viruses target the brain and can cause asymptomatic or acute and fatal diseases 1,2 . Moreover, cognitive deficits and memory impairment, suggestive of hippocampal dysfunction, as well as an increased risk of developing epilepsy are often observed in patients surviving acute viral encephalitis [3][4][5] .

Results
DCIR −/− mice show preservation of hippocampal integrity and reduced viral load in the brain. The effect of DCIR deficiency on hippocampal and neuronal integrity following TMEV infection was determined (Fig. 1). Two-way ANOVA yielded a significant effect of DCIR deficiency on neuronal integrity determined by histology (HE score; p = 0.0008) and immunohistochemistry (NeuN + area/mm 2 , p = 0.0006), as well as on TMEV load (TMEV + cells/mm 2 , p = 0.0257). Subsequent Mann-Whitney U tests at different time points post infection revealed a diminished hippocampal damage of infected DCIR −/− mice with a significant difference compared to WT mice at 14 dpi (p = 0.005, Fig. 1a-c). Similarly, a significantly reduced loss of NeuN + neurons in the hippocampus of DCIR −/− animals compared to WT controls was found at 14 dpi (p = 0.002, Fig. 1d-f). Although increased numbers of β-APP + axons (damaged axons) were found in hippocampal regions with severe neuronal damage and loss mainly in TMEV-infected WT mice, group differences did not reach the level of significance ( Supplementary Fig. S1a). Likewise, an elevation of GFAP + astrocytes (astrogliosis) was present within the hippocampus of WT animals compared to DCIR −/− mice at 14 dpi, but differences did not reach the level of significance ( Supplementary Fig. S1b). No hippocampal inflammation and damage were found in non-infected, age-matched WT and DCIR −/− animals. In addition, non-infected groups showed a similar amount of NeuN + neurons and GFAP + astrocytes in the hippocampus ( Supplementary Fig. S5).
Viral quantification within the brain was performed by RT-qPCR and TMEV-specific immunohistochemistry. At 7 dpi, TMEV RNA concentration was significantly decreased in DCIR −/− mice compared to WT mice (p = 0.047, Fig. 2a). Immunohistochemistry revealed a preferential infection of hippocampal neurons of infected  www.nature.com/scientificreports/ mice in both groups at 7 dpi. Similar to the diminished viral RNA load, reduced numbers of TMEV-infected cells were observed in the brain of DCIR −/− mice at 7 dpi, but differences did not reach level of significance (p = 0.12, Fig. 2b). Both, WT and DCIR −/− mice, showed reduced viral RNA levels and TMEV antigen at 14 dpi, indicating viral elimination (Fig. 2a,b). TMEV RNA concentration in the brain did not differ significantly between both groups at 14 dpi (p = 0.87, Fig. 2b). However, the number of TMEV + cells within the hippocampus was significantly reduced at 14 dpi in DCIR −/− mice compared to WT mice, indicating a reduced residual infection following acute infection phase in DCIR deficient animals (p = 0.005, Fig. 2b-d). No TMEV was detected in non-infected WT mice and DCIR −/− mice by immunohistochemistry and RT-qPCR (data not shown). Data show that preserved hippocampal morphology in mice lacking DCIR is associated with an enhanced early virus elimination from the brain, indicating a refined induction of protective responses in DCIR −/− mice following TMEV infection. Weekly clinical examination, body weight recordings, Racine score evaluation and RotaRod performance test revealed no symptoms in both groups, indicating a subclinical acute infection ( Supplementary Fig. S4) 39 .
DCIR deficiency leads to diminished brain sequestration of effector immune cells. Immunohistochemistry revealed a reduced infiltration of CD3 + T cells (p = 0.007, Fig. 3a) and CD45R + B cells (p = 0.005, Fig. 3b) in the hippocampus of DCIR −/− mice compared to WT controls at 14 dpi. At both time points, the hippocampus of DCIR −/− mice contained similar numbers of activated CD107b + macrophages/microglia in comparison to WT controls (Fig. 3c).
Brain-infiltrating GrB + effector cells decreased in DCIR −/− mice at 14 dpi (p = 0.05, Fig. 3d). Moreover, reduced numbers of CD4 + and CD8 + T cells were found in the hippocampus of DCIR −/− mice at 14 dpi (CD4 + T cells: p = 0.002, Fig. 3e, CD8 + T cells: p = 0.011, Fig. 3f), likely related to decreased virus-trigged immune responses in receptor deficient animals. Comparison of CD4 + and CD8 + T cell proportions revealed a slightly reduced ratio of CD4 + to CD8 + T cells in the brain of DCIR −/− mice at 14 dpi (p = 0.00045, Fig. 3g), showing a relative increase of cytotoxic T cells in animals lacking DCIR. Statistical analyses (Pearson's correlation coefficient R) revealed negative correlations between neuronal integrity of the hippocampus (NeuN + area/mm 2 ) and the amount of www.nature.com/scientificreports/ CD107b + , arginase 1 + and CD45R + cells at 7 and 14 dpi. Foxp3 + cells and GFAP + astrocytes were negatively correlated with neuronal integrity at 14 dpi (Supplemental Table S6). Non-infected control animals of both groups showed no leukocyte infiltrations in the hippocampus. Collectively, these findings suggest that the reduced viral brain load in DCIR −/− mice leads to an accelerated termination of brain inflammatory responses.
Reduced pro-inflammatory cytokine expression in the brain at the later stage of acute polioencephalitis (14 dpi) in DCIR −/− mice is likely a direct consequence of reduced viral burden and accelerated termination of neuroinflammation in comparison to WT mice.
Diminished induction of immunomodulatory responses in DCIR −/− mice following neurotropic virus infection. The suppressive cytokine interleukin-10, secreted by regulatory T cells (Treg) and M2-type macrophages/microglia, is thought to exhibit neuroprotective effects in infectious disorders 40 . On the other hand, Treg and M2-type myeloid cells may dampen effective antiviral responses and thus promote deleterious effects on tissue integrity [41][42][43] . In order to test whether neuronal preservation in the hippocampus in DCIR −/− mice is accompanied by reduced virus load or attributed to immunomodulatory mechanisms, Foxp3 + Treg, arginase 1 + M2-type macrophages/microglia and the key immunomodulatory cytokine IL-10 were quantified. At 14 dpi, numbers of Foxp3 + regulatory T cells (p = 0.018, Fig. 5a) and Foxp3 mRNA copy numbers (p = 0.009, Fig. 5b), determined by immunohistochemistry and RT-qPCR, respectively, were significantly increased in brain samples of mice with intact DCIR signalling (WT mice) compared to DCIR −/− mice upon infection. Similarly, an increase of arginase 1 + cells was found in the hippocampus of WT mice compared to DCIR −/− mice at both investigated time points with significant differences at 14 dpi (p = 0.006, Fig. 5c). Moreover, significantly increased www.nature.com/scientificreports/ transcription of IL-10 was detected in WT controls compared to DCIR −/− mice at 14 dpi (p = 0.034, Fig. 5d). In non-infected WT and DCIR −/− mice cerebral IL-10 mRNA expression was not detectable. Results suggest that TMEV infection elicits compensatory immune pathways, mediated by Treg and M2-type macrophages/microglia in the brain of DCIR intact C57BL/6 mice. Consequently, the observed neuroprotective effect of DCIR deficiency is likely not mediated by classical immunomodulatory mechanisms at the infection site, but rather due to improved virus elimination and timely onset of peripheral protective immune responses. In addition, diminished induction of immunomodulatory and suppressive mechanisms, including decreased numbers of arginase 1 + M2-type macrophages/microglia and Treg, may promote virus control in DCIR deficient animals.

DCIR deficiency enhances peripheral T cell responses following neurotropic virus infection.
The accelerated TMEV elimination observed in the brain of DCIR −/− mice suggests an enhanced antiviral immune response. Priming of naïve T cells in lymphoid organs during the early phase of TME was shown to be crucial for robust antiviral responses 44,45 . Therefore, splenic cytokine mRNA expression was quantified by RT-qPCR and splenic T cell responses were analysed by flow cytometry.
Flow cytometric analysis of TMEV-infected groups revealed an increased fraction of CD8 + T cells in the spleen of DCIR −/− mice compared to WT group at 7 dpi (p = 0.016, Fig. 6f), while CD4 + T cell frequency remained unchanged at this time point (Fig. 6e). Accordingly, a significant shift of the CD4 + /CD8 + T cell ratio with dominance of cytotoxic T cells was observed at 7 dpi (p = 0.009, Fig. 6g) as well as at 14 dpi (p = 0.028, Fig. 6g). Subsequently, the portion of splenic CD4 + T cells decreased in DCIR −/− mice during the disease course, resulting in a significant difference between the groups at 14 dpi (p = 0.028, Fig. 6e).
Further characterization of splenic T cell subsets revealed a significantly higher proportion of activated CD4 + T cells, displayed by higher fractions of CD4 + CD62L low T cells in DCIR −/− mice compared to WT mice at 7 dpi (p = 0.016) and 14 dpi (p = 0.047, Fig. 6d,j). Moreover, the level of activated CD4 + CD44 + T cells was elevated at both time points in DCIR −/− animals compared to WT mice with statistically significant difference between the groups at 7 dpi (p = 0.047, Fig. 6i). Similarly, CD8 + CD44 + cell populations were significantly increased in www.nature.com/scientificreports/ DCIR −/− animals compared to WT mice at 7 dpi (p = 0.009) and 14 dpi (p = 0.016, Fig. 6l). Splenic CD4 + -and CD8 + T cell subpopulations expressing CD25 as well as CD8 + CD62L low T lymphocytes did not significantly differ between groups at any time point (Fig. 6h,k,m). Flow cytometry of non-infected control mice showed a slight difference for the portion of CD8 + T cells and the CD4 + /CD8 + T cell ratio comparing the spleens of WT and DCIR −/− mice. However, no major differences in surface expression of T cell activation markers were observed between both non-infected control groups ( Supplementary Fig. S6). Thus, flow cytometry and cytokine expression analysis revealed an enhanced peripheral T cell activation and an early proportional shift towards cytotoxic CD8 + T cell responses in DCIR −/− mice during viral encephalitis.

Identification of potential influencing factors for hippocampal damage using regression analyses.
Regression analyses were performed to identify factors that correlate with hippocampal damage following TMEV infection. Simple regression models confirmed that neuronal integrity of the hippocampus (NeuN + area/mm 2 ) was significantly associated with the TMEV load. In addition, hippocampal damage was significantly associated with the amount of CD107b + , CD3 + , CD45R + Foxp3 + , arginase 1 + , GFAP + , and granzyme B + cells in the hippocampus, as well as with IFN-β mRNA expression in the brain. The amount of TMEV + cells in the hippocampus remains the only significant parameter in the multiple regression model, pointing at a high collinearity among explanatory variables (Supplementary Table S5).  To determine the impact of DCIR deficiency on early T cell responses upon TMEV infection in vitro, antigen presentation assays using WT and DCIR −/− MEGs or BMDCs were performed. T cells were isolated from OT-I TCR-transgenic mice, which specifically recognise the OVA-peptide presented via the MHC-I molecule H2-K b . T cells were co-cultured with MEGs or BMDCs, previously exposed to TMEV-OVA [47][48][49] . BMDCs were used for the in vitro stimulation of OT-I T cells since BMDCs from WT and DCIR −/− mice had been previously compared in a global and unbiased manner through genome-wide transcriptome analysis and thus represent a well characterized source of APCs 36 . To analyse CD8 + T cell activation, cytokine release was measured by ELISA, and expression of the early T cell activation marker CD69 was measured by flow cytometry. Microglia, as part of the glial cell mixtures, represent the CNS' local APC population. Incubating MEGs with TMEV-OVA, however, did not result in a difference between WT or DCIR −/− microglia-mediated CD8 + T cell response (Fig. 8a). Combined with the marginal levels of released proinflammatory cytokines (data not shown), these findings suggest that the potential of microglia to process and present antigens is limited. As an additional source of APCs, BMDCs were used in a co-culture system to stimulate antigen-specific CD8 + T cells. To avoid alterations in TMEV antigenicity and modification of (potential) DCIR ligands, live virus was used for incubation with BMDCs. However, to exclude a productive infection of BMDCs leading to classical antigen presentation via MHC-I molecules, viral RNA loads in TMEV DA-exposed BMDCs as well as viral titers in the supernatant were determined (Supplementary Fig. S7). While an initial increase in viral RNA load in BMDCs between 2 and 6 h was observed ( Supplementary Fig. S7a), viral titers in the supernatant decreased continuously from 2 to 22 h after TMEV DA incubation ( Supplementary Fig. 7b). Thus, the initial increase of TMEV RNA in BMDCs may be mediated by initial TMEV replication, but it most likely does not reflect a productive infection of BMDCs, but rather an increased TMEV internalisation. In addition, incubation with live TMEV did not lead to a significant decrease in MEG and BMDC viability compared to OVA-or mock-stimulated samples and the vast majority of the cells remained viable ( Supplementary Fig. S8), further supporting that BMDCs present viral antigens to CD8 + T cells. Upon TMEV DA incubation, BMDCs were activated, but no difference between WT and DCIR −/− BMDCs was detected ( Supplementary Fig. S9). Simi- www.nature.com/scientificreports/ larly, the activation status of BMDCs did not differ between WT and DCIR −/− BMDCs following co-cultivation with OT-I T cells (Supplementary Fig. S10). However, TMEV-OVA stimulation of DCIR −/− BMDCs led to an increased expression of CD69 by CD8 + T cells compared to WT BMDCs (Fig. 8b). Further, the release of IL-2, IFN-γ and GrB by CD8 + T cells was elevated if co-cultured with DCIR −/− BMDCs (Fig. 8c-e). These results indicate that DCIR deficiency in BMDCs impacts subsequent CD8 + T cell activation and T cell effector functions in this BMDC/T cell co-culture system. Possibly, DCIR in DCs may balance type I and II IFN signaling directly influencing T cell priming 36,50 . Additionally, cross-talk of DCIR with other immune receptors, such as Toll-like receptors, is conceivable, which can affect the quality of induced T cell responses even without alterations in the expression of co-stimulatory markers CD80 and CD86, as it was shown for human DCIR [51][52][53] . However, the mechanism by which the differential CD8 + T cell activation by DCIR −/− BMDCs shown here is mediated, remains to be determined in future studies.

Discussion
This study highlights the role of DCIR in neuropathology of C57BL/6 mice following acute TMEV infection. Genetic ablation of DCIR appears to exert a supporting effect on viral clearance from the CNS and ameliorates hippocampal damage following virus infection. While susceptible mouse strains (e.g. SJL mice) show an inefficient antiviral immunity and persistent TMEV infection in the CNS, C57BL/6 mice develop vigorous TMEV-specific responses during acute infection 54 . The ability of C57BL/6 mice to eliminate TMEV is caused by robust MHC class I-restricted antiviral CD8 + T cell responses 39,40,46,[55][56][57] . As shown in the present study, the lack of DCIR contributes to a more effective priming of peripheral T cells with increased CD44 and reduced CD62L expression by CD4 + T cells together with an increased IFN-γ expression in the spleen during the early phase of TMEV infection. In general, DCIR −/− mice show an age-related increase of CD4 + CD44 high and CD4 + CD62 low T cells by expanding DC populations in lymphoid organs, demonstrating that DCIR deficiency predisposes to effector-memory T cell development. CD4 + T cells are required for protective immunity in TMEV infection, since CD4 deficiency has been shown to cause virus persistence in C57BL/6 mice. CD4 + helper T cells support antiviral CD8 + T cell responses by cytokine release (e.g. IL-2) and by improving the ability of DCs to prime cytotoxic T cell responses (DC licensing) 33,58,59 . An increased frequency of splenic CD8 + T cells together with an upregulation of the activation marker CD44 was found in infected DCIR −/− mice, suggesting an enhancement of cytotoxic CD8 + T cell responses. The skewed ratio of CD4 + to CD8 + T cells observed in DCIR −/− mice indicates an early dominance of peripheral cytotoxic responses. Increased frequencies of circulating CD8 + T cells were shown to improve antiviral immunity and account for TMEV elimination in C57BL/6 mice 39,60 . In agreement with the present findings, enhanced T cell responses in DCIR −/− mice control experimental mycobacteria infection better than WT controls. Of note, DCs of DCIR −/− mice exhibit several transcriptional changes that promote Th1 immunity also under non-infectious conditions 36 .
Noteworthy, besides protecting from viral infection, T cell immunity has the ability to contribute to acute brain pathology following TMEV infection. Virus-specific CD8 + T cells target infected neurons of the hippocampus in acutely infected C57BL/6 mice. MHC class I-restricted cytotoxicity towards TMEV epitopes contributes to neuronal loss and brain atrophy 57,61,62 . Moreover, cytotoxicity boosted by TMEV peptides leads to fatal CNS inflammation in infected C57BL/6 mice, demonstrating the difficulty of balancing immune responses in neurotropic virus infection 62,63 . As observed in experimental autoimmune encephalomyelitis, rheumatoid arthritis models, and experimental colitis, DCIR −/− mice are prone to develop autoimmunity and T cell-mediated immunopathology, respectively 33,64,65 . Strikingly, despite enhanced peripheral cytotoxic responses in the present study, no exacerbated brain injury was observed in DCIR −/− mice, but on the contrary, a reduced hippocampal damage following TMEV infection. DCIR deficiency seems to fine-tune protective immune responses without evoking additional virus-mediated immunopathology in the TME model. The underlying mechanisms remain speculative, but might be associated with diminished pro-inflammatory cytokine responses found in the brain of DCIR −/− mice. Reduced expression of IFN-β and TNF-α in the brain of DCIR −/− mice during the early phase of polioencephalitis (7 dpi) indicates a diminished cytokine response at the infection site. Particularly, increased IFN-β mRNA levels were significantly associated with hippocampal damage in TMEV-infected mice as determined by correlation analyses. IFN-β (type I interferon) expression in the brain is driven by TMEV infection and involved in the induction of innate and adaptive immune responses 66 . Robust antiviral immunity trigged by type I interferons accounts for viral elimination in C57BL/6 mice but also elicit neuronal damage following TMEV infection 10 . Thus, reduced IFN-β expression might have contributed to diminished T cell sequestration in the brain and decreased hippocampal damage in DCIR −/− mice during advanced infection (14 dpi). Similarly, TNF-α is a cytokine produced by activated microglia and macrophages, which initiate protective responses against certain viral infections, including TMEV infection 67,68 . However, TNF-α also displays cytotoxic effects and contributes to hippocampal damage in C57BL/6 mice following TMEV infection 7,10,20 . In addition, TNF-α has been shown to cause excitotoxicity and neuronal damage in murine HIV encephalitis models 69 . Thus, in addition to the accelerated virus elimination, the alleviated brain cytokine response at the infection site might also contribute to the neuroprotective effect observed in DCIR −/− mice. Despite differences of hippocampal integrity and cytokine expression profiles, no obvious clinical changes were observed between DCIR −/− mice and WT mice (subclinical infection). More targeted diagnostic methods such as video/EEG monitoring and behavioral tests (e.g. Morris water maze) are needed to detect subtle clinical changes and fully discover the functional relevance CNS alteration in receptor deficient animals in future studies.
In addition to an enhanced CD8 + T cell activation in the periphery during the early infection phase, an altered immune environment at the site of infection, including reduced infiltrations of Foxp3 + Treg and arginase 1 + M2-type cells in DCIR −/− animals at 14  www.nature.com/scientificreports/ a decreased expression of genes specific for M2-type cells (including arginase 1) can be found in DCIR −/− mice following mycobacteria infection 36 . Moreover, arginase 1 + myeloid cells have been shown to exert suppressive effects on antiviral immunity 42 . For instance, ablation of arginase 1 in macrophages reduces the viral load and ameliorates tissue integrity after experimental Ross River virus infection of mice 43 . Similarly, Treg are able to dampen antiviral responses during TMEV infection 39 .
The interplay between innate and adaptive immunity is mediated by APCs, such as macrophages, microglia and DCs, which have the ability to recognize pathogens and induce effector T cell responses 51,70,71 . Microglia are CNS-resident APCs and play an important role in TMEV-mediated hippocampal damage and seizure development 17 . However, within the present study, in vitro TMEV exposure of DCIR −/− and WT MEGs did not show differences in CD8 + T cell activation. Although there was a slight OVA-and TMEV-OVA-mediated increase of CD69 observed in the MEG/T cell co-cultivation assay, cytokine levels were not elevated. Thus, in comparison to DCs, the in vitro potential of adult microglia to perform APC function and present specific antigens is apparently limited, as previously shown 72,73 .
DCIR is expressed on all DC subsets and exerts mainly inhibitory effects on immune responses via its intracellular ITIM 28,30,33,50,74,75 . The present study shows an enhanced activation of CD8 + T cells when DCIR −/− BMDCs were used to prime CD8 + T cells. In addition, the release of IL-2 by activated T cells was elevated upon co-cultivation with DCIR −/− BMDCs. DCIR deficiency results also in an increased production of IFN-γ by lymphocytes which was also observed in the present study 50 . Likewise, Chikungunya virus infection of DCIR −/− mice causes an elevation of IFN-γ in vivo 37 . However, in contrast to the TME model, intact DCIR signalling in experimental Chikungunya virus infection contributes to protection against virus-induced pathology of the joint, demonstrating that the effect of DCIR signalling on disease progression is clearly context dependent and differs between pathogens and the primarily affected organ in infectious disorders 37 .
Conclusively, DCIR deficiency seems to support antiviral immune responses of C57BL/6 mice during the initial phase of TMEV infection and to reduce virus-induced neuropathology. Previous studies highlight the potential of DCIR for cell specific targeting and immune modulation 51,52,76 . Thus, this CLR represents a potential target for intervention strategies to selectively enhance protective immunity in neurotropic virus infection.  [47][48][49] . Virus strains were cultivated and passaged in BHK-21 cells and plaque assays were performed using L-cells for virus titration [79][80][81] .Virus isolation was performed by freezing and thawing. Plaque assays were performed as independent duplicates. Virus titres were determined by calculating the plaque forming units per ml (PFU/ml) as previously described 82,83 . Experimental design. Five-week old female DCIR −/− and WT mice were anaesthetised with medetomidine (1 mg/kg, Domitor) and ketamine (100 mg/kg) and inoculated into the right cerebral hemisphere with TMEV DA in a total volume of 20 µl DMEM (Biochrom GmbH, Berlin, Germany) supplemented with 2% FCS (PAA Laboratories GmbH, Pasching, Austria) and 50 µg/kg gentamicin (Sigma Aldrich Chemie GmbH, Taufkirchen, Germany) as described 39 . Weekly clinical examination included body weight recordings as well as clinical scorings with evaluation of "posture and outer appearance", "behaviour and activity" and "gait" 39 . Additionally, a 5 point scale scoring system according to Racine (Racine score) was applied for recording motor seizures 84 . A RotaRod (TSE Systems GmbH, Bad Homburg, Germany) performance test for motor function and coordination was carried out weekly 85 .

Animals. DCIR
At 7 and 14 days post infection (dpi) mice were anaesthetised as described above and euthanised with an overdose of medetomidine (1 mg/kg) and ketamine (200 mg/kg). The rostral part of the left cerebrum (contralateral to injection site) was formalin fixed and paraffin embedded (FFPE), and caudal part of the left cerebrum was snap frozen and stored at − 80 °C 39,86 . Spleens were taken for flow cytometry and parts of splenic tissue were snap frozen and stored at − 80 °C. Serial sections (2-3 µm thickness) of FFPE coronal brain sections at the hippocampal level (Bregma − 1.46 to − 1.82) were used for histology (hematoxylin and eosin staining) and www.nature.com/scientificreports/ immunohistochemistry, respectively 39,87 . In addition, non-infected age matched controls were used to determine baseline differences of splenic and hippocampal immune cell compositions as well as cerebral cytokine and transcription factor expression profiles between DCIR −/− and WT mice.

Isolation of an adult microglia-enriched glial cell mixture (MEG).
To isolate MEGs, a previously used method was modified 98 . Brains of WT and DCIR −/− mice were dissected and stored temporarily in HBSS (Sigma Aldrich, St. Louis, MO, USA) containing 15 mM HEPES (Carl Roth, Karlsruhe, Germany) and 0.5% glucose (Carl Roth, Karlsruhe, Germany). For dissociation, brains were squashed with the top end of a syringe in a 6-well plate containing a digestion cocktail (HBSS, 1 mg/ml collagenase D, 5 U/ml DNase I; Roche, Basel, Switzerland). After 10 min of incubation at 37 °C , brains were gently dissociated manually. Afterwards, a 40% Percoll centrifugation (10 min, 350×g, 18 °C; GE Healthcare, Chicago, IL, USA) and erythrocyte lysis were performed. To check the percentage of microglia within the glial cell mixture, cells were blocked with anti-mouse CD16/32, stained with anti-mouse CD11b-PE and anti-mouse CD45-APC and fixed in 1% PFA. Flow cytometry was performed using an Attune NxT Flow Cytometer. Data analysis was conducted with FlowJo software 97 . The purity of microglia (CD11b + /CD45 low+ ) within MEG used for co-culture experiments ranged between 40 to 60% for both WT and DCIR −/− cell suspensions.

Bone marrow-derived dendritic cells/T cell co-cultivation.
To generate BMDCs, bone marrow cells were isolated from femurs and tibias of DCIR −/− and C57BL/6 control mice and differentiated into BMDCs by cultivation with differentiation medium (culture medium + 10% X63-GM-CSF supernatant) at 37 °C for 8 to 10 days. Following generation and differentiation, BMDCs were seeded with 2 × 10 5 cells/ml in culture medium in a 96-well U-bottom plate and co-cultivation was performed as described above.
Statistical analysis. Statistical analyses were performed using SPSS for Windows (version 21, SPSS Inc., IBM Corp.) 99 applying multiple Mann-Whitney U tests (Supplementary Table S4) and the statistics software R (version 4.0.4) 100 for nonparametric two-way analysis of variance (ANOVA). Moreover, statistics software R was used for applying simple and multiple regression models to study the influence of infiltrating immune cell composition, virus load and cytokine profile on hippocampal neuronal integrity. First independent parameters were preselected by single regression models. Surviving parameters were subjected to multiple regression models, which were further reduced by automatic backwards variable selection. Due to lower sample sizes at individual time points, regression models were avoided at time-specific subgroup analyses. Instead, correlation analyses using Pearson's correlation coefficient R regarding specific analyses at time points 7 dpi and 14 dpi were performed. Graphs were designed using GraphPad Prism software (version 8, GraphPad Software Inc., San Diego, CA, USA) 101 . Statistical tests were performed with a significance level of α = 5%.

Data availability
The data presented in this study are available on request from the corresponding author.