1. IntroductionRabies is caused by RABV, a negative-sense, single-stranded RNA virus belonging to the family Mononegavirales [1]. RABV belongs to the genus Lyssavirus, family Rhabdoviridae, and presents an RNA genome 12 kb in length: the viral genome encodes nucleoprotein (N-protein), phosphoprotein (P-protein), matrix protein (M-protein), glycoprotein (G-protein), and the large protein (L-protein). RABV, classically believed to present a neuron-specific tropism, reaches the CNS via retrograde transport along the neural network, where it induces fatal encephalomyelitis in mammals, including humans [2,3]. Once inside the CNS, RABV successfully hides inside the neural network from glial surveillance [4]. To date, we do not fully understand the exact mechanisms underlying viral-mediated immune evasion of glial cells.Cellular tropism relies on two major determinants: the expression of entry receptors, which enables viral entry, and the cellular immune response, which allows or restricts productive viral replication [5]. In detail, the innate immune system represents the first line of defense against viral invaders. It senses viruses via germline-encoded pattern recognition receptors (PRRs), including toll-like receptors (TLRs), retinoic acid-inducible gene I (RIG-I) like helicases (RLRs), and nucleotide-binding oligomerization domain-like receptors (NOD-like receptors). TLR3 and RLRs in the host cells recognize virus-derived RNAs, leading to the activation of transcription factors, more specifically interferon regulatory factor 3 (IRF-3) and nuclear factor κB (NF-κB), which establish antiviral responses via the production of type I IFNs and proinflammatory cytokines [6]. Subsequently, type I IFNs bind type I IFN receptors (IFNAR) and activate the Janus kinase (JAK) and signal transducer and activator of the transcription protein 1 (STAT1) signaling pathway, elevating the expression of interferon-stimulated genes (ISGs) with antiviral activity.RABV uses several receptors to enter cells via clathrin-mediated endocytosis [7,8,9]: nicotinic acetylcholine receptor (nAChR) [10], neuronal cell adhesion molecule (NCAM) [11], low-affinity p75 neurotrophin receptor (p75NTR) [12], and metabotropic glutamate receptor subtype 2 (mGluR2) [13]. However, those broadly expressed receptors are not essential for RABV entry per se [14] but lead to an acceleration of RABV infection [15]. In contrast to its profound neuron-specific tropism in vivo [16,17], most cell types are susceptible to RABV infection in vitro [18,19,20]. Whereas most of the research is focussed on the discovery of RABV receptors [10,11,12,13] and their interaction with the RABV G-protein [14], less research focusses on how cellular host immune responses shape RABV tropism and how distinct neural immune responses in the CNS could collectively lead to the establishment of an antiviral response. Recently, several publications reported infection of different glial cells in vivo, particularly astrocytes [21,22] and Schwann cells [23], depending on the viral strain and the infection route used [24]. Nevertheless, we are far from understanding the molecular pathways underlying susceptibility to RABV infection, although it remains crucial to determine infection outcome.Here, we investigated susceptibilities and cellular immune responses of different CNS cell types towards infection with two canine RABV strains (Tha and Th2P-4M) in vitro. Tha is a cell culture-adapted virus isolate [25] that shares the same genetic background with Th2P-4M, except for mutations introduced into P- (W265G andM287V) and M-proteins (R77K, D100A, A104S, and M110L), consequently inhibiting viral evasion of the NF-κB and JAK-STAT pathways [26,27,28,29]. We provide evidence that virulent Tha and less virulent Th2P-4M successfully replicate in hiNeurons and, to a lesser extent, in hiAstrocytes but not in hiMicros. Further, Tha strongly represses innate immune gene expression and the secretion of inflammatory proteins in neurons in contrast to glial cell types. Whereas successful infection might highly depend on the concentration of RABV receptors on the cellular surface, we suggest that cell type-specific innate immune responses are critical for the success of RABV replication and spread in the distinct CNS cell types. Hence, our study emphasizes the need to study RABV in relevant CNS culture models to understand the underlying pathways shaping RABV tropism as well as to elucidate the role of glial cells in the RABV-infected brain. 2. Materials and Methods 2.1. VirusesThailand virus, referred to as 8764THA (Genbank No. EU293111) is a field strain of RABV isolated from the brain of a Thai patient who died of rabies after being bitten by a rabid dog [25]. This virus was further adapted to cell culture on BSR cells (a BHK-21 clone, kindly provided by Monique Lafon, Institute Pasteur, Paris) [30], consequently called 8743THA (Genbank No. EU293121, EVAg collection, Ref-SKU: 014V-02106) [25] and in this manuscript referred to as Tha. Sequence comparison between the cell culture-adapted RABV strain 8743THA (GenBank No. EU293121) and the original field isolate 8764THA (GenBank No. EU293111) demonstrated 98.56% identity (52 mutations) and 99.36% similarity between concatenated protein sequences. The recombinant Th2P-4M virus harbours the same genetic background as Tha apart from bearing two mutations in the viral P-protein (W265G and M287V) and four mutations in the viral M-protein (R77K, D100A, A104S, and M110L), which were previously shown to inhibit evasion of the NF-κB and JAK-STAT pathways [26,31,32]. To monitor viral infection, recombinant viral Tha-eGFP and Th2P-4M-eGFP constructs were used, which were generated by cloning sequences of eGFP (recovered from pEGFP-C1 plasmid, Promega, Charbonnières-les-Bains, France) into the genetic sequence of Tha [33] and Th2P-4M, respectively. Viral strains were sequenced before being used for infection experiments. Viral titres were determined by virus titration on BSR cells, more specifically via staining of the viral nucleoprotein (5100, Sigma-Aldrich, Saint-Quentin-Fallavier Cedex, France). No significant difference was observed in the number of viral particles detected in the supernatant of Tha and Th2P-4M-infected BSR-T7 cells (a BHK-21 clone) [26]. Further, the impact of introduced mutations into viral P- and M-proteins was shown to have no impact on viral replication (Figure S1). 2.2. Cell Culture and Infection

The human neuroblastoma cell line SK-N-SH (ATCC® HTB-11™), the human astrocyte-like cell line SVGp12 (ATCC® CRL8621™), and the human microglial cell line HMC3 (ATCC® CRL-3304™) were cultured in Dulbecco’s Modified Eagle Medium (10566016, Thermo Scientific, Illkirch-Graffenstaden, France) supplemented with 10% heat-inactivated foetal bovine serum (FCS, S182H-500, Eurobio Scientific, Les Ulis, France) at 37 °C and 5% CO2.

Human neuronal progenitor cells (EnStem-A™ SCC003, Merck Millipore, Molsheim, France) were cultured on plates previously coated with Geltrex (A1413201, Invitrogen, Waltham, MA, USA) in complete neural stem cell (CNSC) medium containing Knock-out D-MEM/F-12 (A1370801, Thermo Scientific), 2 mM Glutamax (35050038, Thermo Scientific), 2% StemPro Neural Supplement (A1050801, Thermo Scientific), 20 ng/mL FGF (PHG0026, Thermo Scientific), and 20 ng/mL EGF (PHG0311, Thermo Scientific) for amplification purposes at 37 °C and 5% CO2.

Human foetal pAstrocytes (N7805100, Thermo Scientific) were cultured on Geltrex-coated plates (A1413201, Thermo Scientific) in complete astrocyte medium containing D-MEM (10566016, Thermo Scientific), 2% foetal bovine serum (S182H-500, Eurobio), and N-2 Supplement (17502048, Thermo Scientific) at 37 °C and 5% CO2.

Human iPSCs (XCL-1, IP-001-1V, XCell Science) were cultured on Matrigel-coated plates (354277, Corning, Amsterdam, The Netherlands) in mTeSR1 medium (85850, STEMCELL Technology, Saint-Égrève, France). The medium was changed daily.

Upon 80% confluency, cells were used for infection experiments. The cell medium was aspirated, and cells were washed once with PBS (10010023, Thermo Scientific) prior to infection. Adjacently, Tha and Th2P-4M were diluted in culture medium according to the appropriate multiplicity of infection (MOI). Cells were incubated with viral suspension for 2 h at 37 °C and 5% CO2. After two hours, the viral suspension was removed, and the appropriate culture medium was added. Twenty-four hours post infection, cells were treated with 2500 U/mL of IFN-α (I4401-100KU, Sigma-Aldrich) and incubated for 24 h at 37 °C and 5% CO2 prior to cell lysis.

2.3. Differentiation of hNSC to hiNeurons

To induce neural differentiation, EnStem-A cells were cultured at a density of 5 × 104 cells/cm2 on plates previously coated with Geltrex (A1413302, Thermo Scientific) according to the manufacturer’s instructions. One day after seeding, CNSC was changed to a neural differentiation medium (NDM) consisting of neurobasal medium (10880022, Thermo Scientific), B-27 Serum Free Supplement (17504044, Thermo Scientific), 2 mM GlutaMAX (35050038, Thermo Scientific), CultureOne Supplement (A33202-01, Thermo Scientific), and 200 μM ascorbic acid (A4403, Sigma-Aldrich). After 21 days of differentiation, differentiated neurons were used for downstream experiments.

2.4. Differentiation of hiPSCs to hiMacsHuman-induced macrophage-like cells (hiMacs) were generated from iPSCs (IP-001-1V, XCell Science, Novato, CA, USA) according to a published method described by Takata et al. [34]. In short, 5 ng/mL BMP-4 (314-BP-050, R&D Systems, Minneapolis, MN, USA), 50 ng/mL VEGF (293-VE-500, R&D Systems), and 2 µM CHIR99021 (4423, TOCRIS, Bristol, United Kingdom) induced mesoderm specification of human XCL-1 iPSC colonies and hemangioblast-like cell formation [35]. Replacement of CHIR99021 with 20 ng/mL FGF-2 (223-FB-500, R&D Systems), followed by maintenance of 15 ng/mL VEGF and 5 ng/mL FGF-2, induced differentiation into the hematopoietic lineage according to a modified protocol from Grigoriadis et al. [36]. Wnt signalling was inhibited and hematopoietic stem cells were matured by incubation with 50 ng/mL SCF (255-SC-01M), 10 ng/mL FGF-2, 20 ng/mL IL-3 (203-IL-050, R&D Systems), 10 ng/mL IL-6 (206-IL-050, R&D Systems), 10 ng/mL VEGF, and 30 ng/mL DKK-1(5439-DK-500) for 6 days and with 50 ng/mL SCF, 10 ng/mL FGF-2, 20 ng/mL IL-3, and 10 ng/mL IL-6 for 4 days. To promote terminal differentiation of hiMacs, cells were cultured with 50 ng/mL CSF-1 (216-MC-01M, R&D Systems) for 14 days [37,38,39]. 2.5. Differentiation of hiMacs to hiMicros

After hiMac generation, hiMacs were positively selected by fluorescence-activated cell sorting (FACS, MoFlo Astrios, Beckman Coulter, Villepinte, France) using CD45-BV605 (304042, Biolegend, Paris, France, 1:300), CD11b-BV421 (301324, Biolegend, 1:300), CD14-FITC (982502, Biolegend, 1:300), CD163-APC (333610, Biolegend, 1:300), and CX3CR1-PE (341604, Biolegend, 1:300). FACS-sorted cells were co-cultured with 70–80% confluent three-week-old (day 0 = induction of neural induction) hiNeurons for three weeks. After three weeks of co-culture, cells were used for imaging. To obtain a pure culture of hiMicros for downstream qPCR analysis, FACS-sorted hiMacs were co-cultured for three weeks on 24-well-plates in inserts (353495, Falcon, pore size 0.4 µm) with confluent three-week-old hiNeurons. During co-culture, 50 ng/mL recombinant human CSF-1 (216-MC-010, R & D Systems) and 50 ng/mL human IL-34 (5265-IL-010, R & D Systems) were added every third day to the culture medium.

2.6. Transfer of Conditioned Medium

Cell lines (SK-N-SH, HMC3, and SVGp12) were seeded in 6-well-plates at a density of 1 × 105 cells/cm2. Twenty-four hours after seeding, cells were infected with Tha or Th2P-4M at an MOI of 5. After two hours, the viral suspension was removed, and 1 mL of culture medium was added per well. SK-N-SH cells were seeded into 96-well-plates (655086, Greiner Bio-One, Les Ulis, France) at a density of 1.8 × 104 cells/cm2. After 24 h, SK-N-SHs previously seeded in 96-well-plates (655086, Greiner Bio) were infected with Tha-eGFP or Th2P-4M-eGFP at an MOI of 0.5. After two hours the viral suspension was removed and replaced via 100 µL of the supernatant taken from previously infected cells (SK-N-SH, HMC3, and SVGp12, at 24 h post-infection) filtered through a 100 kilodalton membrane (28-9322-58, Dominique Dutscher, Bernolsheim, France). After 24 h, the medium was removed, and cells were treated with 2500 U/mL of IFN-α (I4401-100KU, Sigma-Aldrich). Cells were imaged at 48 h post-infection using the Opera Phenix™ High Content Screening System (Perkin Elmer, Villebon-sur-Yvette, France).

2.7. Treatment of hiNeurons with Human Recombinant IL-1β, IL-6, and LIF

Human NSCs were seeded at a density of 1.6 × 105 cells/cm2 into 96-well-plates (655086, Greiner Bio) and differentiated to hiNeurons as described above. Twenty-one days after differentiation, hiNeurons were infected with Tha-eGFP or Th2P-4M-eGFP at an MOI of 0.5. Two hours after infection, the viral suspension was removed and replaced via 100 µL of NDM or NDM supplemented with 100 ng/mL of recombinant human IL-1β (200-01B, Peprotech, Neuilly-sur-Seine, France), IL-6 (200-06, Peprotech), or LIF (300-05, Peprotech). Cells were imaged at 48 h post-infection using the Opera Phenix™ High Content Screening System (Perkin Elmer).

2.8. Opera Phenix™ High Content Screening AssayCell lines were seeded at a density of 1.8 × 104 cells/cm2. In the case of co-cultures, different cell lines were seeded in equal quantities within wells. Twenty-four hours after seeding, cells were infected with Tha-eGFP and Th2P-4M-eGFP. Twenty-four hours post-infection, cells were treated with 2500 U/mL of IFN-α (I4401-100KU, Sigma-Aldrich) and incubated for 24 h at 37 °C and 5% CO2 prior to fixation. Cells were fixed using 4% PFA (Thermo Scientific, J61984) for 15 min at room temperature, washed with PBS (10010023, Thermo Scientific) and permeabilized using 0.5% triton X-100 (648463, Millipore) for 10 min. Cells were stained with primary and secondary antibodies listed according to the manufacturer’s instructions (Table S1). Apoptotic cells were quantified by the in situ cell death detection kit (12156792910, Roche, Meylan, France) according to the manufacturer’s instructions. Dead cells were detected with the ReadyProbes® Cell Viability Imaging Kit (R37610, Thermo Scientific). Images were acquired via the Opera Phenix™ High Content Screening System (Perkin Elmer) using the parameters mentioned in Table S2. To identify eGFP+ cells, we used the software Columbus 2.9.1 (Perkin Elmer), which automatically detects nuclei and the cellular cytoplasm. Intensity thresholds to distinguish eGFP+ from eGFP− cells were based on the autofluorescence level of non-infected cells (Table S2). 2.9. RNA Isolation and cDNA Synthesis

Total RNA was isolated using the RNeasy Mini Kit (74104, Qiagen, Courtaboeuf, France) by the following procedure: EzDNase (11766051, Thermo Scientific) was used to eliminate genomic DNA. Adjacently, 500 ng or 1 μg of purified RNA was converted into first-strand cDNA using the SuperScript™ VILO™ IV enzyme (11756050, Thermo Scientific) according to the manufacturer’s instructions. For real-time qPCR experiments, cDNA was diluted 1/50 or 1/100, respectively.

2.10. Quantitative PCRQuantitative PCR, based on the detection of the SYBR Green dye, was performed by using 2.5 µL of the synthesized and diluted cDNA in the presence of 5 µL QuantiTect SYBR Green (204143, Qiagen) and 1 mM specific primers (Table S3) in a final volume of 10 µL. Oligonucleotides were used for PCR at a concentration of 10 pmol/µL. All the samples were measured in triplicate. Gene expression levels were normalized to the endogenous expression of the housekeeping gene 18S (Eurofins) and the respective non-infected cells (mock). Quantitative PCR was performed on the 7500 Real-Time PCR System (Thermo Scientific, 7500 Software v2.3) using the following: initial denaturation step (1× repetition, 10 min, 95 °C), amplification step (40 repetitions, 15 s at 95 °C, 1 min at 60 °C), and melting curve determination step (1× repetition, 15 s at 95 °C, 1 min at 60 °C, 15 s at 95 °C, 15 s at 60 °C). Gene expression was normalized to expression of housekeeping gene 18S (ΔCT), and the difference in gene expression was calculated as the difference between infected and non-infected samples (ΔΔCT) [40]. 2.11. Protein Quantification

Cells were seeded at a density of 1 × 105 cells/cm2 and infected with Tha or Th2P-4M at an MOI of 5. Forty-eight hours post-infection, cells were lysed using Procartaplex™ cell lysis buffer (EPX-99999-000, Thermo Scientific) according to the manufacturer’s instructions to quantify protein expression in the cellular cytoplasm. Intracellular protein concentrations of human IFN-β, IFN-γ, IL-1β, IL-6, IL-15, CXCL10, LIF, CCL5, and TNF-α were quantified using a 9-plex Procartaplex assay (PPX-09, Thermo Scientific). In short, DropArray 96-well-plates (96-CC-BD-05, Clinisciences, Nanterre, France) were blocked using 1% BSA for 30 min. After blocking, 20 µL of cell lysate was added per well. According to the protocol, plates were stepwise incubated with 5 µL detection antibody, 10 µL streptavidin-PE, and 10 µL reading buffer per well before being read by the Luminex 200™ instrument (Thermo Scientific).

2.12. Statistical Analysis

Percentages and means ± standard deviation (SD) were calculated with Prism version 9 (GraphPad, San Diego, CA, USA) and R version 4.0.4 (R Foundation, Vienna, Austria). Standard deviation and statistical significance were only calculated when three independent experiments were conducted. Multiple comparisons of data were performed using either Prism version 9 or the lme4 package within R version 4.1.1 (R Foundation, Vienna, Austria), with the test being indicated in the figure legend. If multiple comparisons were performed, significance thresholds were lowered to account for multiple testing. Principal component analysis and hierarchical clustering on gene expression values were performed using R (version 4.0.4). Heatmaps represent normalized gene expression values that were clustered after centring gene expression values on their mean. Figures were generated using Illustrator CC 2019 (Adobe, San Jose, CA, USA).

The analysis of the proportion of eGFP+ cells in co-cultures (mixed in equal ratios) performed in Figure S3 was based on the comparison of confidence intervals in each co-culture to a linear combination (with equal weights because the mixture ratios are the same) of the corresponding monocultures. More precisely, the first step of this analysis is the computation of the expected confidence intervals (from monocultures) on the percentages of eGFP+ cells for co-cultures (1) and triple cultures (2).

CI95 (expected) = 0.5 × CI95 (cell type A) + 0.5 × CI95 (cell type B)

(1)

CI95 (expected) = 0.33 × CI95 (cell type A) + 0.33 × CI95 (cell type B) + 0.33 × CI95 (cell type C)

(2)

The second step is to compute an observed confidence interval on the actual values measured in the co-cultures. If the observed confidence intervals overlapped with the expected confidence intervals, we concluded that the proportion of eGFP+ cells in co-cultures was not a consequence of an interaction between culture cell types.

4. DiscussionIn this section, we describe different factors that determine RABV tropism. The first factor relies on the divergent susceptibilities of CNS cell types towards Tha infection. We report that the two RABV strains (Tha-eGFP and its attenuated variant Tha2P-4M-eGFP) used in this study infect hiNeurons and hiAstrocytes, whereas hiMicros are not susceptible to RABV infection in vitro (Figure 3A and Figure S4). Although human CNS cell lines did not fully corroborate these results (Figure 1), Tha-eGFP and Th2P-4M-eGFP still displayed a higher neuron-specific tropism in CNS cell lines (Figure 1C,D). The main difference, however, is the low susceptibility of microglial cell line HMC3 (Figure 1) compared to the absence of RABV infection recorded in iPSC-derived human microglia (Figure 2 and Figure S4). In short, our results question the ability of canine RABV strains to successfully enter and replicate in human microglia (Figure 2). Previously, Ray and colleagues reported that the tissue culture-adapted ERA strain and mouse-adapted bat SRV strain successfully replicate in primary adult human microglia in vitro [19]. Despite these dissimilarities, we suggest that the different nature of RABV strains, their culture adaptation, or insufficient cell purification or differentiation might account for different susceptibilities towards RABV infection observed between these studies. Microscopic analysis of post mortem human brain tissues of rabid patients revealed enhanced activation of microglia surrounding degenerated neurons [44], which are referred to as Babes nodules [45] and observed in other viral encephalitis and infectious disorders [46]. Given that microglia phagocytes degenerate neurons and thereby take up RABV components [44], viral transcripts that are detected in microglia [47] do not necessarily mean that microglia actively support RABV infection. Nevertheless, more research is needed to elucidate the susceptibility and function of microglia in human rabies infection, particularly by using more sophisticated models that reflect the complexity of the human CNS. The second factor determining RABV tropism relies on the type and extent of the immune response induced by RABV infection in the different CNS cell types. A limited comparative transcriptomic analysis between Tha- and Th2P-4M-infected cells focusing on innate immune gene expression implied in RABV pathogenesis (Figure 3) revealed that Tha specifically evades neuronal immune responses. Further, we confirm that the evasion of the NF-κB and JAK-STAT pathways is mediated by the specific domains of P- and M-proteins as shown by the transcriptome comparison between Tha and Th2P-4M (Figure 3). Although antiviral signalling via IFNs was originally considered as a universal mechanism to control viral infections, recent evidence suggests that neurons lack robust innate immune signalling pathways to minimize the detrimental effects of viral infection on this non-renewable cell population [48,49]. Further, the neuronal ability to respond to IFN stimulation seems limited given the low gene expression of IFNAR1 and IFNAR2 (Figure S8) and the moderate induction of innate immune gene expression following IFN stimulation compared to cells of glial origin (Figure S5B). In contrast to neuronal impairment to respond to Tha infection via IFN induction, we show that glial cells induce strong innate immune responses upon Tha and Th2P-4M infection (Figure 3). Consequently, we suggest that the pronounced neuron-specific tropism of Th2P-4M in cultures consisting of hiNeurons and hiAstrocytes results from its limited or delayed capacity to evade strong immune responses compared to Tha (Figure 2B) [49].Apart from the crucial role of neurons during RABV infection, our results indicate that astrocytes may also play an important role during infection: in our study, astrocytes strongly induce the transcription of cytokines and adaptor molecules (IFIH1, TLR7, IFNB1, CCL5, CXCL10, IL1B, and LIF) upon Tha and Th2P-4M infection in vitro (Figure 3A). We assume that astrocytes sense Tha via RLRs (IFIH1, DDX58, and DHX58) and TLRs (TLR3 and TLR7), in turn inducing the expression of type I IFNs (IFNB1), chemokines (CCL5 and CXCL10), and interleukins (IL1B). Both IFN-β and IL-1β are known to increase BBB permeability, to activate monocytes, microglia, and astrocytes, and to induce the production of neuroprotective mediators [50]. Previously, astrocytic expression of CCL5 and CXCL10 chemokines was shown to induce the recruitment of macrophages, dendritic cells, lymphocytes, and neutrophils, and to regulate microglial activity as well as astrocyte survival [50]. Further, it has been shown that murine astrocytes strongly respond to infection with a recombinant RABV carrying the G-protein of the CVS strain (SAD-GCVS) via RLR- and TLRs-induced expression of IFN-β in vivo. As a consequence, the IFN response was shown to abort RABV infection of astrocytes in mice [21]. In contrast to attenuated RABV strains, a recent quantitative analysis of RABV tropism in rats revealed a strong tropism for astrocytes by RABV field strains (8–27%). In accordance with these data, we showed that Tha is able to infect human astrocytes in vitro (Figure 2) despite the induction of a strong astrocyte-mediated immune response upon infection (Figure 3). Although human astrocytes constitutively express modest levels of LIF, IL-1β, and IL-6 (Figure 4), none of those factors actively restricted RABV replication in hNSC-derived CNS cultures (Figure S9). Still, the fact that the antiviral activity of LIF [51,52], IL-1β [53], and IL-6 [54,55] has been described previously in different in vitro and in vivo models for various human pathogens urges further research to understand the pleiotropic nature of these interleukins during viral infections, particularly during RABV-mediated encephalitis.Neurons and astrocytes are not the only cell types playing important roles during RABV infection: here, we identified the upregulation of IFN-inducible genes with antiviral responses (CXCL10, ISG15, and MX1) in hiMicros (Figure 3) as well as a modest increase in IL-6 protein expression in microglial-like cells upon Tha and Th2P-4M infection (Figure 4). Microglia are already known to induce CXCL10 [56,57] and ISG15 [47,57,58] expression during viral infection. Generally, activated microglia are well known to release pro-inflammatory cytokines in pathological conditions such as IL-6, resulting in cytotoxicity, immune activation, neuronal excitotoxicity, and apoptosis [59]. Here, we show that IL-6 does not directly restrict RABV replication in either hiNeurons or in hiAstrocytes (Figure S9), nor does it reduce the percentage of dead cells in Tha-eGFP- or Th2P-4M-infected co-cultures (Figure S10). Despite the lack of IL-6-mediated antiviral or anti-apoptotic activity in our model, IL-6 might potentially modulate other biological processes in the CNS, such as microglia and T-cell activation, BBB permeability [60], or synaptic function [61]. The third factor determining RABV tropism relies on the interplay between the different CNS cell types. Although we were unable to characterize this factor in differentiated CNS cell types, our results show the importance of the communication between the different CNS cell types during RABV infection. Apart from the simple presence of glial cells in co-cultures, the transfer of supernatant from glial cells protected neuron-like cells from Tha infection (Figure 5). Possible underlying mechanisms comprise glial-mediated signalling directly via cell-to-cell contacts (Figure 5), the induction of inflammatory genes (Figure 3), as well as the expression of cell type-specific inflammatory proteins (Figure 4) that can induce secondary signalling cascades in surrounding neuronal cells. Even though we could not identify the precise factors restricting Tha infection in glial cells (Figure S9), we provide evidence that glial immune responses partly shape RABV tropism. This has already been shown for poliovirus, where the type I IFN system induces the expression of ISGs, particularly PKR and OAS in atrophic tissues [62]. Further, the IFN system dictates viral tropism for VSV [63], West Nile Virus [64], and neurotropic coronavirus [65]. Thus, we urge further research to characterize the interplay between CNS cell types during RABV infection by studying more complex CNS models such as brain organoids or human brain sections.