Visualizing production of interferon - β by astrocytes and microglia in the 1 brain of La Crosse virus - infected mice

26 Interferon (IFN)-β is a major component of innate immunity in mammals, but information on 27 the in vivo source of this cytokine after pathogen infection is still scarce. To identify the cell 28 types responsible for IFN-β production during viral encephalitis, we used reporter mice that 29 express firefly luciferase under control of the IFN-β promoter and stained organ sections with 30 luciferase-specific antibodies. Numerous luciferase-positive cells were detected in regions of 31 La Crosse virus (LACV)-infected mouse brains that contained many infected cells. Double 32 staining experiments with cell-type specific markers revealed that similar numbers of 33 astrocytes and microglia of infected brains were luciferase-positive, whereas virus-infected 34 neurons rarely contained detectable levels of luciferase. Interestingly, if a mutant LACV 35 unable of synthesizing the IFN-antagonistic factor NSs was used for challenge, the vast 36 majority of the IFN-β-producing cells in infected brains were astrocytes rather than microglia. 37 Similar conclusions were reached in a second series of experiments in which conditional 38 reporter mice expressing the luciferase reporter gene solely in defined cell types were infected 39 with wild-type or mutant LACV. Collectively, our data suggest that glial cells rather than 40 infected neurons represent the major source of IFN-β in LACV-infected mouse brains. They 41 further indicate that IFN-β synthesis in astrocytes and microglia is differentially affected by 42 the viral IFN antagonist, presumably due to differences in LACV susceptibility of these two 43 cell types. 44 45 on Jauary 0, 2018 by gest http/jvi.asm .rg/ D ow nladed fom Visualizing of IFN-β in LACV-infected mouse brains 3 Introduction 46 Viruses can trigger pattern recognition receptors of infected hosts which initiate signaling 47 cascades that culminate in transcriptional activation of type I and type III interferon (IFN) 48 genes. Type I and type III IFNs are cytokines that use distinct receptor complexes for 49 signaling and which, thereby, induce an antiviral state in uninfected cells. The family of type I 50 IFN includes more than ten different IFN-α subtypes, IFN-β and minor subtypes such as IFN51 ω or IFN-δ, whereas type III IFN includes IFN-λ1, -λ2 and -λ3 (8, 19). These various IFN 52 genes are typically co-induced in response to virus infection, although the kinetics and the 53 degree of activation of the different IFN genes differ considerably depending on producer cell 54 type and nature of challenge virus (6, 15). In the mouse, IFN-β is the first type I IFN subtype 55 being expressed after viral infection and, together with IFN-α4, is considered to prime cells 56 for the production of other type I IFN family members (2). 57 Pattern recognition receptors which can recognize RNA viruses include cytoplasmic RIG-like 58 helicases and membrane-anchored toll-like receptors (TLR). Cell culture studies indicate that 59 most if not all nucleated mammalian cells can synthesize IFN in response to signals from 60 RIG-like helicases when infected with replication-competent viruses (21). Further, certain 61 immune cells such as macrophages and dendritic cells readily synthesize IFN when receiving 62 signals from TLRs which recognize engulfed virus-derived nucleic acids (14). The situation 63 after infection of an intact organism is much more complex. For viruses that cause viremia, 64 plasmacytoid dendritic cells (pDC) which are mainly present in blood and spleen of mammals 65 are responsible for most of the circulating IFN (1, 7, 24). During influenza virus infection of 66 the lung, pDC seem to play far less important roles (12). Similarly, classical immune cells 67 including pDCs are not present in healthy brains (10, 13), suggesting that other cell types are 68 mainly responsible for IFN synthesis in this organ. However, previous attempts to 69 unambiguously identify these alternative IFN-producing cells did not yield a clear picture. 70 Experiments were either performed with isolated brain cells or were focused on certain cell 71 types without addressing the question of the contribution of such cells to the overall IFN72 response in the central nervous system (10, 22, 23, 26). One difficulty with these experimental 73 approaches was that IFNs are quickly secreted and are not accumulating to high intracellular 74 levels in producer cells, thus complicating their detection in tissue slices by IFN-specific 75 antibodies. 76 La Crosse virus (LACV) is a mosquito-borne pathogen that infects up to 300,000 people in 77 the United States and can cause encephalitis in children and young adults (3). LACV belongs 78 to the genus Orthobunyavirus, family Bunyaviridae. These viruses have a tri-segmented 79 on Jauary 0, 2018 by gest http/jvi.asm .rg/ D ow nladed fom Visualizing of IFN-β in LACV-infected mouse brains 4 single-stranded RNA genome of negative polarity and replicate in the cytoplasm of infected 80 cells. The smallest genome segment of LACV codes for the viral nucleoprotein and a non81 structural protein, termed NSs, which efficiently inhibits the IFN system of infected 82 mammalian hosts. NSs of LACV induces degradation of cellular RNA polymerase II which, 83 in turn, results in reduced transcription of many cellular genes, including the genes for type I 84 and type III IFN (27). A mutant of LACV lacking a functional NSs gene (LACV-ΔNSs) was 85 generated (5). As expected if NSs served as IFN antagonist, LACV-ΔNSs induced 86 significantly more IFN in cultured cells and brains of infected mice than wild-type LACV (4, 87 18). Further, the LACV-ΔNSs mutant was less virulent than wild-type LACV in mice, 88 although both viruses cause encephalitis and death after 5-10 days if administered 89 intraperitoneally into juvenile mice (4). Using conventional in situ hybridization and 90 immunostaining techniques, we previously identified cells with macrophage and ependymal 91 markers as major sources of IFN-α and -β in the brain of mice with acute LACV encephalitis, 92 and we observed that neurons represent a minor but substantial source of IFN during viral 93 encephalitis (10). 94 Transgenic mice in which reporter genes were inserted into the coding regions of the IFN-α or 95 -β genes are promising new tools for studying virus-induced expression of IFN genes in vivo. 96 A reporter mouse which expresses green-florescent protein (GFP) in place of IFN-α6 was 97 successfully used to demonstrate that alveolar macrophages contribute to IFN synthesis in 98 virus-infected lungs (17). Cells from a reporter mouse with a modified IFN-β locus encoding 99 GFP were used to demonstrate the stochastic nature of type I IFN gene expression (28). We 100 recently employed another reporter mouse in which the IFN-β coding region is replaced by 101 luciferase to visualize IFN synthesis in virus-infected animals by in vivo imaging (18). We 102 now used the same luciferase reporter mouse to analyze the contribution of various brain cell 103 types to IFN-β synthesis in mice with LACV encephalitis. We visualized IFN-β-producing 104 cells by staining brain sections with antibodies that simultaneously recognize luciferase and 105 marker proteins of neurons, astrocytes and microglia, respectively. We further took advantage 106 of the possibility that the loxP-flanked luciferase gene in this reporter mouse can be 107 rearranged in defined cell types by crossing these animals with mice that express Cre 108 recombinase in a cell type-specific manner. Using these two approaches we identified 109 astrocytes and microglia as the main IFN-β producers in LACV-infected brains. We further 110 observed that the LACV-encoded IFN-antagonistic factor NSs strongly impairs IFN 111 production by astrocytes but not microglia. 112 113 on Jauary 0, 2018 by gest http/jvi.asm .rg/ D ow nladed fom Visualizing of IFN-β in LACV-infected mouse brains 5 Materials and Methods 114 Mice: Mice were bred in the animal facility of the Department of Virology at the University 115 of Freiburg. All mice used in this study were on the C57BL/6 background, or backcrossed 116 onto C57BL/6 for at least 5 generations. IFN-β and conditional reporter IFN-β 117 mice have been described previously (18, 25). IFN-β mice were crossed to 118 LysM-Cre (25), Thy1-Cre (11) (Jackson Lab, Stock Number 006143) and Synapsin1-Cre 119 mice (29) to generate mice that express the reporter gene either in microglia and 120 macrophages, astrocytes and neurons, and neurons, respectively. 121 122 Viruses and infection of mice: Wild-type LACV and mutant LACV-ΔNSs that cannot express 123 the IFN antagonistic factor NSs were previously described (5). Virus stocks were generated in 124 Vero cells. Juvenile mice (12-15 days old) were infected intraperitonally with 10 plaque 125 forming units (PFU) of wild-type or mutant LACV diluted in 100 μl of phosphate buffer 126 saline (PBS). Animals were checked for neurological symptoms at 8 h intervals and sacrificed 127 when first signs of ataxia were noted. Brains were either collected without fixation for virus 128 titration and measuring luciferase activity or were perfusion fixed for immunohistochemical 129 analyses as described below. 130 131 Virus titrations: Plaque assays were performed in Vero cells using 6-well plates. Serial 132 dilutions of lysates were applied for 1 h. Supernatants were then removed and replaced by a 133 1:1 mixture of 3% Avicel-cellulose (FMC BioPolymer) and double concentrated DMEM 134 (Gibco). Vero cells were incubated for 72 h at 37 °C and 5% CO2 before supernatants were 135 removed. Cells were fixed with 4% paraformaldehyde, and plaques were visualized by 136 staining with 0.5% crystal violet. 137 138 Ex vivo luciferase measurement: Brains homogenized in 800 μl of PBS using the FastPrep-24 139 equipment and Lysing Matrix A (MP Biomedicals). Samples (200 μl) were treated with 50 μl 140 of 5x Cell Culture Lysis Buffer (Promega), and luciferase activity was measured in a Sirius 141 Tube Luminometer (Berthold Technologies) using the single Luciferase Assay System 142 (Promega) according to the manufacturer’s protocol. 143 144 Immunohistochemistry: Animals were sacrificed with a mixture of ketamine (3.7%), xylazine 145 (0.2%) and acepromacine (0.02%) and transcardially perfused with 0.9% NaCl followed by 146 4% buffered paraformaldehyde in PBS. Brains were postfixed in the same solution for 6 more 147 on Jauary 0, 2018 by gest http/jvi.asm .rg/ D ow nladed fom Visualizing of IFN-β in LACV-infected mouse brains 6 hours. Fixed brains were cut horizontally into 50-μm-thick sections on a Leica Vibratome. 148 Free-floating tissue sections were blocked and permeabilized in PBS containing 5% normal 149 donkey serum and 0.1% Triton X-100 for 30 min. Sections were then incubated with rabbit 150 anti-luciferase antibody (Fitzgerald, 70C-CR2020RAP), mouse anti-NeuN (Millipore, 151 MAB377), rat anti-F4/80 (AbD Serotec, MCA497R), mouse anti-GFAP (SIGMA-ALDRICH, 152 G3893) or mouse anti-LACV-G2 (QED Bioscience, 18752) in PBS containing 3% normal 153 donkey serum at 4 °C overnight. For detection of luciferase, signal amplification with the 154 TSA Fluorescein System (PerkinElmer) was performed according to the manufacturer's 155 instructions using a biotin-conjugated donkey anti-rabbit antibody (Jackson 156 ImmunoResearch). For cellular markers, appropriate DyLight488-, DyLight549-, Cy3-, or 157 Cy2-conjugated secondary antibodies (Jackson ImmunoResearch) were used. Slides were 158 mounted in DAPI-containing IS Mounting Medium (Dianova). Digital images were taken 159 with an ApoTome fluorescence microscope (Zeiss) using AxioVision software. 160 161 162 163 Results 164 Identification of IFN-β-producing cells in LACV-infected brains 165 To visualize IFN-β-producing cells in the brain of LACV-infected heterozygous reporter 166 mice, we stained tissue slices from animals exhibiting clinically apparent encephalitis with 167 luciferase-specific antibodies. Because luciferase expression levels were expected to be low 168 and paraffinor cryo-embedding might affect epitope recognition, we decided to work 169 exclusively with paraformaldehyde-fixed free floating slices prepared by vibratome 170 sectioning. We observed that luciferase signals were very faint if standard histological 171 staining techniques were applied. Consequently, signal amplification was routinely employed 172 for better visualization of IFN-β-producing cells. 173 Luciferase-positive cells were typically observed in distinct clusters which were present in all 174 parts of virus-infected brains. Double-staining experiments revealed that the luciferase175 positive cell clusters exclusively mapped to brain regions in which virus-infected cells were 176 highly abundant (Fig. 1). As expected from the fact that LACV encodes the IFN-antagonist 177 factor NSs, the number of luciferase-positive cells was at least 10-fold higher in brains of 178 diseased mice infected with the LACV-ΔNSs mutant (Fig. 1A-D) compared to brains infected 179 with LACV-wt (Fig. 1E-H). Detailed inspection revealed that although mostly found in close 180 proximity to virus-infected cells, luciferase-positive cells were usually not positive for viral 181 on Jauary 0, 2018 by gest http/jvi.asm .rg/ D ow nladed fom Visualizing of IFN-β in LACV-infected mouse brains 7 antigen (Fig. 1B, 1F, 1D and 1H). It was shown previously that LACV predominantly infects 182 neurons (16). Shape and distribution of luciferase-positive cells indicated that they might be 183 mostly astrocytes. This was particularly obvious in the cerebellum, where luciferase-positive 184 Bergmann glia cells were typically observed in immediate vicinity of virus-infected Purkinje 185 cell somata (Fig. 1C and 1D). 186 187 Majority of IFN-β-producing cells in LACV-infected brains are astrocytes and microglia 188 To assess the extent to which the various cell types in LACV-infected brains might contribute 189 to IFN-β synthesis, we determined which fractions of luciferase-positive cells could 190 unambiguously be classified as astrocytes, neurons or microglia by double-staining with 191 antibodies that recognize cell type-specific markers. This analysis was performed with both 192 wild-type LACV and the NSs-deficient mutant virus to address the question of whether the 193 IFN-antagonistic factor NSs might act predominantly in certain cell types. In agreement with 194 previous results (4), we found that luciferase levels in brains of mice infected with the ΔNSs 195 virus were about 6-fold enhanced, although the mutant virus replicated substantially less well 196 than wild-type virus with about 30-fold reduced peak brain titers (Fig. 2). A large number of 197 luciferase-positive cells in brains of mice infected with wild-type or NSs-deficient LACV 198 expressed GFAP, indicating that they represent astrocytes (Fig. 3A and 3B). Another fraction 199 of luciferase-positive cells expressed the macrophage/microglia marker protein F4/80 (Fig. 200 3C and 3D). A very small proportion of luciferase-expressing cells stained positive for the 201 neuron marker NeuN (Fig. 3E). Interestingly, luciferase-positive neurons were detected at low 202 frequency in brains of mice infected with LACV-ΔNSs (Fig. 3E) but not LACV-wt (Fig. 3F). 203 To make this analysis more quantitative, we compiled the results from a detailed inspection of 204 tissue slices from three or more severely diseased animals per virus strain. At least 300 205 luciferase-positive cells were evaluated individually for each double-staining experiment 206 listed in table 1. In brains of mice infected with the LACV-ΔNSs mutant, the vast majority 207 (89%) of luciferase-positive cells expressed GFAP, suggesting that they represent astrocytes. 208 The F4/80 marker which is present on microglia and infiltrating macrophages was expressed 209 by approximately 5% of the luciferase-positive cells in brains of mice infected with LACV210 ΔNSs. The number of luciferase-positive cells expressing NeuN was approximately 1%, 211 suggesting that only very few neurons can synthesize large amounts of IFN-β. A strikingly 212 different picture emerged when the staining data from wild-type LACV-infected mice were 213 compiled. In this case, the frequency of luciferase-positive astrocytes was only 35%, whereas 214 the frequency of luciferase-positive microglia/macrophages was 62% (Table 1). No 215 on Jauary 0, 2018 by gest http/jvi.asm .rg/ D ow nladed fom Visualizing of IFN-β in LACV-infected mouse brains 8 luciferase-positive cells expressing NeuN were detected in brains which were infected with 216 wild-type LACV. 217 218 LACV replicates predominantly in neurons 219 As the majority of IFN-producing cells were seemingly uninfected astrocytes and microglia, 220 we carefully analyzed if LACV can establish productive infections in those cell types. To do 221 this, we subjected brains infected with LACV-wt or LACV-ΔNSs to simultaneous staining for 222 viral antigen and specific markers for neurons, astrocytes or microglia. In line with previous 223 data (16), we found that LACV-wt and LACV-ΔNSs replicated almost exclusively in neurons 224 (Fig 4A and 4B). We could not detect any virus-positive microglia (data not shown), and less 225 than 1% of the virus-positive cells in the infected brains were astrocytes (Fig. 4C and 4D). 226 Thus, astrocytes are susceptible for LACV, although infection of such cells might mostly be 227 non-productive. 228 229 Luciferase production in virus-infected conditional reporter mice 230 To confirm the IFN-β expression patterns observed by immunohistochemistry, we created 231 reporter mice in which luciferase is expressed exclusively in predetermined cell types. Such 232 animals may be generated by breeding reporter mice which contain strategically positioned 233 loxP sites with mice that selectively express Cre recombinase in defined cell types (25). For 234 the current study we used Synapsin1-Cre mice to generate animals in which expression of 235 luciferase is restricted to neurons (29). Further, we used Thy1-Cre mice to produce reporter 236 mice which express luciferase in both neurons and astrocytes (11). Finally, we employed 237 LysM-Cre mice to generate reporter mice in which luciferase expression is restricted to 238 microglia and macrophages (9, 20). 239 To verify the predicted luciferase gene expression patterns in our conditional reporter mice we 240 performed double-staining experiments of brain sections from LACV-ΔNSs-infected LysM241 Cre, Thy1-Cre or Synapsin-Cre reporter mice with antibodies recognizing luciferase and the 242 corresponding markers F4/80, GFAP and NeuN. As expected if the reporter mice expressed 243 the luciferase gene with desired cell type specificity, we found that luciferase-positive cells in 244 brains of infected LysM-Cre mice expressed F4/80, an antigen of microglia and macrophages 245 (Fig. 5A), but not the astrocyte marker GFAP (data not shown). Similarly, the luciferase246 positive cells observed in brains of Thy1-Cre mice expressed GFAP (Fig. 5B), but not F4/80 247 (data not shown). Further, the few luciferase-positive cells that we detected in Synapsin-Cre 248 reporter mice expressed the neuron marker NeuN (Fig. 5C). Thus, the conditional reporter 249 on Jauary 0, 2018 by gest http/jvi.asm .rg/ D ow nladed fom Visualizing of IFN-β in LACV-infected mouse brains 9 mice used here represent suitable tools for the assessment of the relative contributions of the 250 various cell types to overall IFN synthesis in LACV-infected brains. 251 Since luciferase expression is driven by the virus-inducible IFN-β promoter in our mice, a 252 direct comparison of reporter gene activity in individual animals will only yield meaningful 253 quantitative data if virus replication in brains is similar. Although the different conditional 254 reporter mice showed only slight differences in onset of symptoms or course of disease (Fig. 255 6), we tried to minimize errors resulting from such variation by restricting this comparison to 256 animals containing matching viral brain titers (~8x10 pfu in the case of LACV-ΔNSs and 257 ~1x10 pfu in the case of LACV-wt). Under these experimental conditions, “global” Δβ-luc 258 mice infected with LACV-ΔNSs contained, at average, 5x10 RLU of luciferase activity per 259 μl of brain extract (Fig. 7A). Luciferase activity in brain extracts from Thy1-Cre mice 260 infected with LACV-ΔNSs was only slightly reduced, suggesting that at least 71.4% of the 261 luciferase signal originates from virus-mediated stimulation of astrocytes and neurons (Fig. 262 7B). Luciferase activity in extracts from brains of Synapsin1-Cre mice was 9.4% of “global” 263 mice, whereas luciferase activity in extracts of brains from LysM-Cre mice infected with 264 LACV-ΔNSs was comparatively low. If compared to “global” mice, the signal in LysM-Cre 265 mice was only about 1.7% at average (Fig. 7B). Thus, astrocytes, neurons and microglia 266 together accounted for ~75% of luciferase activity of “global” reporter mice. The cellular 267 origin of the missing ~25% of activity (white sector in Fig. 7B) remains unclear. It most likely 268 indicates incomplete Cre-mediated recombination of the lox-P-tagged target gene in our mice. 269 A different picture emerged when LACV-wt was used for the infection study. First, as 270 discussed above, luciferase activity in brain extracts of “global” mice infected with LAVC-wt 271 was about 6-fold lower than in “global” mice infected with LACV-ΔNSs and, at average, 272 reached values of only 9x10 RLU per μl of brain extract (Fig. 7C). Second, compared to 273 infection with LACV-ΔNSs, luciferase activity in brains of wild-type LACV-infected LysM274 Cre mice was significantly increased. At average, it accounted for 41.4% of the signal 275 observed in “global” mice infected with wild-type LACV (Fig. 7D). Third, the contribution of 276 neurons to overall luciferase activity did not differ substantially in mice infected with either 277 wild-type or mutant LACV, whereas the contribution of astrocytes was clearly less prominent 278 in wild-type LACV-infected mice compared to mice infected with the ΔNSs mutant virus 279 (Fig. 7D). Thus, our experiments with the conditional reporter mice could confirm the 280 conclusions from our double-staining experiments with the “global” reporter mice which 281 suggested that the two virus variants induce a strikingly different cellular IFN-β expression 282 pattern. 283 on Jauary 0, 2018 by gest http/jvi.asm .rg/ D ow nladed fom Visualizing of IFN-β in LACV-infected mouse brains 10 Discussion 284 Employing transgenic mice that express a luciferase reporter gene under control of the IFN-β 285 promoter we established a staining protocol that can identify single IFN-β-producing cells in 286 virus-infected brain tissue. To quantify the relative contribution of specific cell types to 287 overall IFN-β synthesis in the brain, we took advantage of the Cre-Lox system and generated 288 reporter mice that express the luciferase transgene either in astrocytes and neurons, neurons 289 only, or microglia and macrophages. When using La Crosse virus as a model for viral 290 encephalitis, we found that astroyctes and microglia were the main producers of IFN-β in the 291 infected brain, whereas the contribution of infected neurons was relatively small. 292 Interestingly, when a mutant virus was used for challenge that cannot synthesize the IFN293 antagonistic factor NSs, the balance was shifted and astrocytes became the dominant IFN-β 294 producers. Our work demonstrates that besides infected cells, seemingly uninfected cells also 295 contribute massively to IFN synthesis in the central nervous system. Our work further 296 demonstrates that virus-encoded antagonistic factors can affect IFN production by acting 297 selectively on distinct cell types. 298 Previous attempts to characterize the production of IFN during viral encephalitis by in situ 299 hybridization technology had already indicated that various cell types, including neurons, 300 contain detectable levels of type I IFN (10). These authors had used specific antisera to 301 visualize IFN-αand IFN-β-producing cells in virus-infected brains. However, signals were 302 weak, and quantitative analyses were depended on visual interpretation of histological data. 303 This difficulty presumably originates from the fact that type I IFN is quickly secreted from 304 producer cells and fails to accumulate to high intracellular levels, thus complicating detection 305 by immunostaining. The reporter mice that we used in this study overcome this problem as 306 the luciferase molecule lacks export signals and thus accumulates in the cytoplasm of the 307 producer cells. Nevertheless, staining of tissue for luciferase in virus-infected reporter mice 308 was challenging as standard histological protocols failed to produce detectable signals. We 309 could eventually overcome these problems by using mild conditions for sectioning and 310 staining, and by including a signal amplification step. 311 Our immunostaining approach is backed-up well by results from experiments with conditional 312 reporter mice that express the IFN-β-promoter-regulated luciferase gene exclusively in 313 defined cell types. We stringently evaluated the specificity of Cre-mediated recombination in 314 these mice and excluded the possibility that reporter gene expression by undesired cell types 315 clouded the picture. As predicted, LysM promoter-driven expression of Cre recombinase 316 seemed to rearrange the IFN-β locus exclusively in cells that were positive for the 317 on Jauary 0, 2018 by gest http/jvi.asm .rg/ D ow nladed fom Visualizing of IFN-β in LACV-infected mouse brains 11 microglia/macrophage marker F4/80. Similarly, Thy1 promoter-driven expression of Cre 318 recombinase seemed to activate the loxP-tagged reporter gene only in astrocytes and 319 presumably neurons. Synapsin1-driven expression of Cre recombinase resulted in selective 320 rearrangement of the loxP-tagged IFN-β locus in neurons. 321 The results of our immunostaining experiments and those of our Cre-loxP approach showed a 322 very good correlation in the case of astrocytes and microglia, but no clear correlation in the 323 case of neurons. This discrepancy can easily be explained by the high detection threshold of 324 the immunostaining technique. Most likely, the IFN-β-promoter-driven luciferase gene got 325 activated to a low extent in LACV-infected neurons, but luciferase levels in individual cells 326 remained too low for detection by antibody staining. Since a large percentage of neurons got 327 productively infected with LACV in our mice, it is likely that the small contribution of 328 individual neurons did add up considerably. These considerations may explain why 329 experiments with our Synapsin1-Cre reporter mice indicated a more substantial contribution 330 of neurons to luciferase activity in LACV-infected brains than the histological analysis. 331 A remarkable finding of our study was that astrocytes contribute substantially to IFN 332 synthesis in the virus-infected brain. This result was not expected, as we (Fig. 4 A and 4B) 333 and others (4, 10, 16) showed that productive replication of LACV is largely restricted to 334 neurons. Since the contribution of astrocytes was much more pronounced if mutant LACV 335 was used for infection that cannot synthesize the IFN-antagonistic factor NSs, we assume that 336 a high number of brain astrocytes actually got infected by LACV, although such infections 337 usually remained non-productive. In line with this hypothesis, our immunofluorescence 338 analysis (Fig. 4C and 4D) demonstrated that LACV can indeed establish productive infection 339 of astrocytes, although this may occur only rarely. 340 We noted that the luciferase signal in microglia was not negatively regulated by NSs. In fact, 341 IFN production by microglia was even higher if wild-type virus was administered instead of 342 LACV-ΔNSs. Therefore, IFN production by microglia correlated directly with the virus load 343 in the brain, irrespective of whether the challenge virus coded for NSs or not. Since the two 344 viruses used here differ slightly with regard to kinetics of disease induction, it remains 345 possible that the kinetics of virus-induced cytokine gene activation in astrocytes and micoglia 346 also differed somewhat and thus contributed to the difference which we observed between 347 LACV-wt and LACV-ΔNSs. To minimize variations of this sort, we restricted our analysis to 348 animals which showed similar signs of neurological disease. Taking all these caveats into 349 account, the most coherent explanation of the various observations reported here seems that, 350 in contrast to astrocytes, IFN production by microglia is not triggered by intracellular virus 351 on Jauary 0, 2018 by gest http/jvi.asm .rg/ D ow nladed fom Visualizing of IFN-β in LACV-infected mouse brains 12 sensors such as Rig-I but rather by alternative sensors such as TLR which can detect viral 352 components in the extracellular space. Interestingly, recent LACV infection experiments with 353 TLR7-deficient mice failed to reveal a prominent role of this virus sensor in our system 354 (unpublished data), suggesting that several virus sensors might get activated simultaneously in 355 the LACV-infected mouse brain. 356 Taken together, our study demonstrates that three cell types are responsible for the bulk of 357 IFN synthesis during acute encephalitis after LACV infection, namely productively infected 358 neurons, abortively infected astrocytes and uninfected microglia. Interestingly, if not inhibited 359 by the viral IFN-antagonistic factor NSs, the amount of IFN-β expressed by neurons exceeds 360 the quantity of IFN-β produced by microglia and macrophages. 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Genes Dev 15:859-876.456457458onJauary0,2018bygesthttp/jvi.asm.rg/Downladedfom Visualizing of IFN-β in LACV-infected mouse brains15 Figure Legends460461Figure 1: IFN-β-producing cells are found near LACV-infected cells in various brain regions.462Brain sections from cortex (A, B, E, F) and cerebellum (C, D, G, H) of diseased IFN-β463mice infected with LACV-ΔNSs (A, B, C, D) or LACV-wt (E, F, G, H) were simultaneously464stained for luciferase (green), viral antigen (LACV-G2, red) and cell nuclei (DAPI, blue).465Note that although in close proximity to infected cells, luciferase-positive cells did usually not466stain for viral antigen. Size bars: 50 μm.467468Figure 2: LACV-ΔNSs activates the IFN-β reporter gene more efficiently than LACV-wt,469although it replicates less well in the brain. Brains of infected IFN-β mice showing signs470of neurological symptoms were collected and homogenized. Viral titers (A) and luciferase471 activity (B) in brain homogenates were determined. *** p< 0.0005472 473Figure 3: Astrocytes, microglia and neurons of LACV-infected mouse brains express IFN-β474 reporter gene. Double-staining of brain slices for luciferase (luc) and astroycte marker GFAP475(A, B), microglia/macrophage marker F4/80 (C, D) and neuron marker NeuN (E, F).476 Luciferase positive neurons were detectable only in LACV-ΔNSs (E) but not in LACV477infected brains (F). Cells were counterstained with DAPI. Single channel and merged pictures478of the same frames are shown. Size bars: 10 μm.479480481 Figure 4: LACV replicates predominantly in neurons. Brains of mice infected with LACV-482ΔNSs (A, C) or LACV-wt (B, D) were stained simultaneously for viral antigen (LACV-G2,483green) and either the neuron marker NeuN (red in panels A and B) or the astrocyte marker484GFAP (red in panels C and D). Size bars: 20μm.485486Figure 5: Conditional reporter mice express the luciferase reporter gene in the predicted cell487types. (A)LysM-Cre-IFN-β mice were infected with LACV-ΔNSs and brains of488diseased mice were simultaneously stained for luciferase (green) and the489microglia/macrophage marker F4/80 (red). (B)Thy1-Cre-IFN-β mice were infected490with LACV-ΔNSs, and brains of diseased mice were simultaneously stained for luciferase491(green) and the astrocyte marker GFAP (red). (C)Synapsin1-Cre-IFN-β mice492onJauary0,2018bygesthttp/jvi.asm.rg/Downladedfom Visualizing of IFN-β in LACV-infected mouse brains16 infected with LACV-ΔNSs and brain sections of diseased mice were simultaneously stained493for luciferase (green) and the neuron marker NeuN (red). Size bars: 50 μm.494495Figure 6: Conditional reporter mice show no significant differences in susceptibility to496LACV. “Global” reporter mice (Δβ-luc) and conditionalThy1-Cre-IFN-β (thy),497LysM-Cre-IFN-β(lys) and Synapsin1-Cre-IFN-β (syn) reporter mice498infected with LACV-ΔNSs (A) or LACV-wt (B) were monitored for neurological symptoms.499 Diseased animals were killed and brain titers were determined. No significant differences500were observed between the different mouse strains.501502Figure 7: IFN-β synthesis by astrocytes and neurons but not microglia is repressed by LACV-503encoded IFN-antagonistic factor NSs. Reporter mice in which the luciferase gene is expressed504 exclusively in astroytes and neurons (thy), neurons only (syn) or microglia/macrophages505(lysM) were infected with LACV-ΔNSs (A, B) or LACV-wt (C, D). “Global” IFN-β506reporter mice (Δluc) served as reference. Brains of diseased animals with very similar virus507 load were selected for further analysis. Mean luciferase activities (with standard deviation) in508brain samples from the various mouse strains infected with either LACV-ΔNSs (A) or LACV-509wt (C) are shown. The average contributions of different cell types to luciferase activity in510 brains of mice infected with LACV-ΔNSs (B) or LACV-wt (D) are shown as pie charts, in511which the activity of “global” IFN-β reporter mice was set to 100%.512onJauary0,2018bygesthttp/jvi.asm.rg/Downladedfom Table 1: Frequency of luciferase-positive astrocytes, microglia/macrophages and neurons in mouse brains infected with wild-type or mutant LACV Virus Astrocytes a) Microglia/Macrophages b) Neurons c)LACV-ΔNSs 89% (536/603) 5% (29/561) 1% (4/424)LACV-wt 35% (134/379) 62% (189/306) 0% (0/337) d)a) Astrocytes are defined here as GFAP-positive cellsb) The microglia/macrophage population is defined here as F4/80-positive cellsc) Neurons are defined here as NeuN-positive cellsd) Cells positive for corresponding marker/number of luciferase-positive cells analyzed onJauary0,2018bygesthttp/jvi.asm.rg/Downladedfom