Trafficking and Replication Patterns Reveal Splenic. Macrophages As Major Targets of Dengue Virus in Mice

JVI Accepts, published online ahead of print on 29 August 2012 J. Virol. doi:10.1128/jvi.00375-12 Copyright 2012, American Society for Microbiology. All Rights Reserved. 1 2 Trafficking and Replication Patterns Reveal Splenic Macrophages As Major Targets of Dengue Virus in Mice 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 Tyler R. Prestwood, Monica M. May, Emily M. Plummer, Malika M. Morar, Lauren E. Yauch, and Sujan Shresta* Division of Vaccine Discovery, La Jolla Institute for Allergy and Immunology, 9420 Athena Circle, La Jolla, CA 92037, USA * Correspondence and requests for materials should be addressed to S.S.: sujan@liai.org Running Title: Dengue virus infects splenic macrophages in mice Word Count (abstract): 223 Word Count (text): 5,500

22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 ABSTRACT Human post-mortem studies of natural dengue virus (DENV) infection have reported systemically distributed viral antigen. Although it is widely accepted that DENV infects mononuclear phagocytes, the sequence in which specific tissues and cell types are targeted remains uncharacterized. We have previously reported that mice lacking interferon-α/β and -γ receptors permit high levels of DENV replication and show signs of systemic disease (28). Here, we demonstrate that within 6 hours, DENV traffics to and replicates in both CD169 + and SIGN-R1 + macrophages of the splenic marginal zone or draining lymph node, respectively, following intravenous or intrafootpad inoculation. Subsequently, high levels of replication are detected in F4/80 + splenic red pulp macrophages and in the bone marrow, lymph nodes, and Peyer s patches. Intravenously inoculated mice begin to succumb to dengue disease 72 hours after infection, at which time viral replication occurs systemically, except in lymphoid tissues. In particular, high levels of replication occur in CD68 + macrophages of the kidneys, heart, thymus, and gastrointestinal tract. Over the course of infection, proportionately large quantities of DENV traffic to the liver and spleen. However, late during infection, viral trafficking to the spleen decreases, while trafficking to the liver, thymus and kidneys increases. The present study demonstrates that macrophage populations, initially in the spleen and other lymphoid tissues, and later in non-lymphoid tissues, are major targets of DENV infection in vivo. 2

43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 INTRODUCTION The four serotypes of dengue virus (DENV1-4) belong to the Flaviviridae family and cause a mosquito-borne, febrile illness in more than 50 million people per year. In nearly 500,000 cases per year, individuals develop severe hemorrhage and sometimes shock, resulting in more than 25,000 deaths annually (12). The 10.7 kb, positive-stranded RNA genome of DENV encodes a polyprotein that is cleaved into 3 structural proteins, envelope (E), pre-membrane/membrane (prm/m), and capsid (C), and 7 nonstructural (NS) proteins, NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5. During replication, all DENV proteins and both positive- and negative-sense DENV RNA are produced intracellularly, while only positive-sense RNA, the structural proteins, and NS1 are known to be secreted at high levels. Thus, based on current knowledge, NS proteins, with the exception of NS1, are predominantly restricted to cells in which DENV replicates. The cellular tropism of DENV has been investigated in humans (2, 5, 6, 8, 13, 15, 16, 25, 29), and mice (3, 9, 20, 38). In human studies, DENV antigen has been reported in the skin, liver, brain, kidney, spleen, lymph nodes, lungs, stomach, and intestine (2, 5, 6, 8, 13, 15, 16, 25, 29). These studies have mainly relied upon detection of secreted DENV antigens, which may actually localize elsewhere, complicating interpretation of results. Recently, two studies have detected DENV NS antigen in the lymph nodes, spleen, liver, and bone marrow cells of mice (2, 9). With the exception of the study by Fink et al. (9), infected cell types have not been identified by co-staining for specific marker expression. Nevertheless, these studies have consistently reported the major targets of DENV to be 3

66 67 mononuclear phagocytes. The exact cell populations targeted and sequence of infection at the tissue and cellular level, however, remains unclear. 68 69 70 71 72 73 74 75 76 77 78 79 80 81 Previously, we reported that 129/Sv mice lacking type I and II interferon receptors (AG129 mice) are susceptible to lethal disease when challenged with a mouse-passaged DENV2 strain (28, 32, 38). In this experimental setup, the kinetics of DENV dissemination was followed by qrt-pcr (28, 38). Here, we use fluorescent immunohistochemistry (FIHC) to characterize the sequence in which specific tissues and cell types are targeted by analyzing nearly every major tissue for DENV NS3 at several time points over the course of infection and by determining the precise identity of NS3- expressing cells through co-staining with cell type-specific markers. Additionally, we examine viral trafficking using DENV-labeled with the fluorescent lipophilic dye DiD to quantify DENV extravasation at several time points over the course of infection. The present study characterizes the kinetics of DENV infection and trafficking at the tissue and cellular level in vivo. 4

82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 MATERIALS AND METHODS Mice 129/Sv mice deficient in the IFN-α/β receptor (A129) or the IFN-α/β receptor and the IFN-γ receptor (AG129) were housed under specific pathogen-free conditions. All experiments were approved by the Animal Care Committee at the La Jolla Institute for Allergy and Immunology (LIAI). Mice between 5 and 6 weeks of age were inoculated intravenously (i.v.) in the lateral tail vein with 1.5x10 11 GE (3x10 6 PFU) or 5x10 8 GE (1x10 4 PFU) in 200 µl or intrafootpad (i.f.) with 1.5x10 11 GE in 30 µl of the DENV2 strain E124/128-IC diluted in PBS (Invitrogen) containing 10% fetal bovine serum (FBS, Gemini Bio-Products). For DiD-labeled virus experiments, mice were additionally inoculated i.v. 2 hours before harvesting tissues. Cell cultures and viral stocks C6/36 cells were obtained from the American Type Culture Collection and cultured at 28 C in Leibovitz s L-15 medium (Gibco) supplemented with penicillin, streptomycin, HEPES, and 10% FBS. DENV2 strain E124/128-IC (from pe124/128-ic) was generated as previously described (28). Briefly, an infectious cdna clone of PL046 (ppl046-ic) was previously constructed and site-directed mutagenesis was performed to introduce the two mutations identified in D2S10. The plasmid was linearized with XbaI and gel purified using a QIAquick gel extraction kit (Qiagen). Infectious RNA was generated by in vitro transcription with a RiboMax large-scale RNA production system (T7, Promega) and transfected into BHK-21 cells with Lipofectamine (Invitrogen). The entire genome of the viral stocks was confirmed by sequencing. Virus was amplified twice in C6/36 cells 5

105 106 107 and purified using a sucrose density gradient, as previously described (28). Genomic equivalents (GE) were quantified by real-time reverse transcription-polymerase chain reaction (RT-PCR). 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 DiD-labeled DENV stocks Solid 1,1'-dioctadecyl-3,3,3',3'-tetramethylindodicarbocyanine, 4-chlorobenzenesulfonate salt (DiD, Invitrogen) was reconstituted at 20 mg/ml in DMSO and then diluted 1:10 in 100% ethanol. Twice-amplified stocks of E124/128-IC were used to grow stocks for DiD labeling. The third amplification was performed in CELLine AD 1000 adherent cell chambers (Wilson Wolf) and virus in the supernatant was concentrated by ultracentrifugation, resuspended, and purified over a sucrose density gradient. After dialysis against PBS, virus was incubated with DiD at a 1:38,000 molar ratio of virus:did for 1 hour at room temperature. After incubation, virus was re-purified over a sucrose density gradient, which yielded a distinct band of labeled virus. Virus was dialyzed against PBS, aliquoted, and stored at -80ºC. Infectivity of DiD-labeled virus was reduced to 44% of the unlabeled virus when normalized to GE. Immunohistochemistry Tissues were embedded in O.C.T. compound (Sakura). Sections (6 μm) were cut and stored at -80 C. Frozen sections were thawed and fixed for 10 min in acetone at 25 C, followed by 8 min on ice in 1% paraformaldehyde in 100 mm dibasic sodium phosphate (ph 7.4) containing 60 mm lysine and 7 mm sodium periodate. 6

128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 To stain for DENV replication, tissue sections were blocked with Avidin/Biotin Blocking Kit (Vector), 5% normal goat serum (Caltag Laboratories) and 5% mouse serum in PBS. Sections were stained overnight with affinity purified rabbit polyclonal anti-denv NS3 (a generous gift from the Novartis Institute for Tropical Diseases, Singapore) and purified mouse anti-denv prm/m (clone 2H2) labeled with EZ-Link NHS Biotin (Pierce). After washing, sections were stained with DyLight 649-labeled goat anti-rabbit IgG (Jackson Immunoresearch) and PE-labeled streptavidin (ebioscience). NS3 staining with DiD visualization For DiD and NS3 staining experiments, sections were prepared, fixed, and blocked as stated above and then stained overnight with purified rabbit polyclonal anti-denv NS3. After washing, sections were stained with DyLight 549-labeled goat anti-rabbit IgG (Jackson Immunoresearch). Marginal zone macrophage staining with DiD visualization Sections were prepared, fixed, and blocked as stated above and then stained with rat antimouse CD169 (clone MOMA-1, BMA Biomedicals), biotinylated rat anti-mouse SIGN- R1 (clone ER-TR9, BMA Biomedicals), and rat anti-mouse MARCO (clone ED31, AbD Serotec). After washing, sections were stained with biotinylated goat anti-rat IgG (BD), followed by additional washing and staining of sections with PE-labeled streptavidin (BD). 7

151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 Identification of infected splenocytes Sections were prepared, fixed, and blocked as stated above. For marginal zone macrophage markers, sections were stained overnight with rat anti-mouse CD169 (clone MOMA-1), biotinylated rat anti-mouse SIGN-R1 (clone ER-TR9), and purified rabbit polyclonal anti-denv NS3. After washing, sections were stained with PE-conjugated mouse anti-rat IgG2a (BD), FITC-conjugated streptavidin (BD), and DyLight 649- labeled goat anti-rabbit IgG. Finally, after additional washing, sections were stained with AF488-conjugated goat anti-fitc (Invitrogen). For CD11b and CD11c, sections were stained overnight with FITC-labeled Armenian hamster anti-mouse CD11c (clone HL3, BD), PE-labeled rat anti-mouse CD11b (clone M1/70, BD), and purified rabbit polyclonal anti-denv NS3. After washing, sections were stained with PE-conjugated goat anti-rat IgG, AF488-conjugated goat anti-fitc, and DyLight 649-labeled goat anti-rabbit IgG (Jackson ImmunoResearch Laboratories). Lastly, for pan-macrophage and red pulp macrophage markers, sections were stained overnight with FITC-labeled rat anti-f4/80 (ebioscience), biotinylated rat anti-mouse CD68 (clone FA-11, AbD Serotec), and purified rabbit polyclonal anti-denv NS3. After washing, sections were stained with PE-conjugated streptavidin, AF488-conjugated goat anti-fitc, and DyLight 649-labeled goat anti-rabbit IgG. 8

173 174 175 176 177 178 179 180 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 Identification of infected cells in lymph nodes Sections were prepared, fixed, and blocked as stated above. For MARCO and SIGN-R1 staining, sections were stained overnight with purified rat anti-marco (clone ED31, AbD Serotec), biotinylated rat anti-mouse SIGN-R1 (clone ER-TR9), and purified rabbit polyclonal anti-denv NS3. After washing, sections were stained with PE-conjugated streptavidin (BD), FITC-conjugated mouse anti-rat IgG1 (BD), and DyLight 649-labeled goat anti-rabbit IgG (Jackson ImmunoResearch Laboratories). For CD68 and CD169 staining, sections were stained overnight with biotinylated rat antimouse CD68 (clone FA-11, AbD Serotec), purified rat anti-mouse CD169 (clone MOMA-1), and purified rabbit polyclonal anti-denv NS3. After washing, sections were stained PE-conjugated mouse anti-rat IgG2a (BD), FITC-conjugated streptavidin (BD), and DyLight 649-labeled goat anti-rabbit IgG (Jackson ImmunoResearch Laboratories). Comparison of infected spleen cells in A129 and AG129 mice Tissues sections were prepared, fixed, and blocked as stated above. For marginal zone macrophage markers, sections were stained overnight with rat anti-mouse CD169 (clone MOMA-1), biotinylated rat anti-mouse SIGN-R1 (clone ER-TR9), and purified rabbit polyclonal anti-denv NS3. After washing, sections were stained with PE-conjugated mouse anti-rat IgG2a and FITC-conjugated goat anti-rabbit IgG. Finally, after additional washing, sections were stained with AF488-conjugated goat anti-fitc and APC-labeled streptavidin (ebioscience). 9

196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 For pan-macrophage and red pulp macrophage markers, sections were stained overnight with PE-labeled rat anti-f4/80 (clone BM8, Biolegend), biotinylated rat anti-mouse CD68 (clone FA-11, AbD Serotec), and purified rabbit polyclonal anti-denv NS3. After washing, sections were stained with FITC-conjugated goat anti-rabbit IgG. Finally, after additional washing, sections were stained with AF488-conjugated goat anti-fitc and APC-labeled streptavidin. Identification of infected cells in the kidney, heart and thymus For cell type identification, sections were prepared, fixed, and blocked as stated above. Sections were stained overnight with FITC-labeled rat anti-mouse CD11b (clone M1/70), biotinylated rat anti-mouse CD68 (clone FA-11), and purified rabbit polyclonal anti- DENV NS3. After washing, sections were stained with PE-conjugated streptavidin, AF488-conjugated goat anti-fitc, and DyLight 649-labeled goat anti-rabbit IgG. Image acquisition and preparation Images were recorded using a Marianas deconvolution fluorescence microscope (3i) or DM6000B upright laser scanning confocal microscope (Leica). Final preparation of all images was performed using ImageJ software (NIH), Adobe Photoshop, and Adobe Illustrator (Adobe Systems). Quantification of DiD For quantitative analysis, images were processed with the noise reduction despeckle function in ImageJ. DiD intensity was then quantified using the Batch>Measure function 10

219 220 221 for 6-24 non-overlapping low-power images from 2 tissues sections for each of the 5 mice per group per time point. Data were graphed and analyzed with Prism 5.0 (GraphPad). Statistical significance was determined using the unpaired t-test. 222 11

223 224 225 226 227 228 229 230 231 232 233 234 235 236 237 238 239 240 241 242 243 244 245 RESULTS Early DENV replication occurs in lymphoid tissues We previously reported that serum levels of DENV virions decrease rapidly following intravenous (i.v.) inoculation of AG129 mice (28). However, levels of DENV RNA in the serum begin to increase between 5 and 7 hours after infection, suggesting that initial viral replication in vivo occurs in less than 7 hours. To screen for DENV replication patterns characteristic of lethal infection and to coordinate with our previous RNA kinetics study (28), we utilized a high dose of DENV. AG129 mice were inoculated i.v. with 1.5x10 11 genomic equivalents (GE) of the DENV2 strain E124/128-IC. At 0 (naïve mice), 6, 12, 24, 48, and 72 hours after infection, the following tissues were collected: liver, heart, lung, thymus, spleen, kidney, stomach, small intestine (including Peyer s patches), large intestine, mesenteric lymph nodes (MLN), cutaneous lymph nodes (CLN: brachial, axillary, inguinal LNs were pooled because of equivalent trends), skin, blood, spinal cord, brain, and bone marrow. For analysis of viral replication, 6 µm frozen tissue sections were cut, mounted, and subsequently stained with rabbit polyclonal antibodies raised against purified NS3, in combination with biotinylated mouse anti-denv prm/m monoclonal antibody (clone 2H2). By 6 hours after infection, DENV replication was detected in the spleen (Figure 1), at which time both NS3 and prm/m were present in the marginal zone encircling the white pulp. At 12 hours after infection, NS3 expression was still present in the marginal zone of the spleen, although the circular staining pattern had extended into the red pulp and become more diffuse. At 24 hours after infection, DENV NS3 was distributed 12

246 247 248 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 throughout the red pulp. By 48 and 72 hours after infection, levels of NS3 in the spleen had decreased substantially, but low levels continue to be detectable in the red pulp at the 72 hour time point. Staining with the anti-denv prm/m antibody 2H2 showed near complete colocalization with anti-ns3 staining, confirming specificity to sites of DENV replication. The kinetics of DENV infection in other lymphoid tissues was similar to that observed in the spleen (Figure 1). In the bone marrow, NS3 expression became detectable at 12 hours and peaked at 24 hours after infection. In the lymph nodes and Peyer s patches, infection was consistently observed 24 hours after inoculation and peaked at 48 hours. As in the spleen, infection in the bone marrow, lymph nodes, and Peyer s patches decreased substantially and became almost undetectable by 72 hours. Collectively, these results demonstrate that systemic lymphoid tissues are targeted by DENV before nonlymphoid tissues in this model. Late DENV replication occurs in non-lymphoid tissues We additionally examined DENV replication in non-lymphoid tissues including the kidney, heart, thymus, adrenal gland, liver, lung, forestomach, stomach, jejunum, duodenum, ileum, cecum, colon, and rectum (Figure 2A). In contrast to replication in lymphoid tissues, replication was undetectable in non-lymphoid tissues early after infection, but became detectable between 24 and 48 hours and continued to increase until 72 hours (Figure 2A). Notably, a high level of DENV NS3 expression was detected in the heart, thymus, and kidneys. Several of the non-lymphoid tissues analyzed, including the 13

269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 brain, spinal cord, esophagus, blood, bladder, and skin, showed little or no viral replication, even 72 hours after infection (data not shown). Based on these data, a model of DENV dissemination was developed (Figure 3). These findings are in agreement with our previous kinetic study measuring DENV RNA levels in tissues (28). DENV replication occurs in the draining popliteal lymph node following intrafootpad inoculation To validate our results using a more relevant route of infection than the i.v. route (34), AG129 mice were inoculated intrafootpad with 1.5x10 11 GE of E124/128-IC. The kinetics of systemic DENV dissemination was assessed by monitoring viral RNA levels in the serum at 6, 12, 24, 48, 72 and 96 hours after infection. Levels of DENV RNA were almost undetectable at 6 hours but increased steadily, peaking at 72 hours and decreasing by 96 hours after infection (data not shown). Based on these kinetics, the spleen, draining popliteal lymph node (PLN), contralateral PLN, cutaneous lymph nodes (CLN), and kidneys were harvested 6, 24, and 72 hours after infection. Tissues were stained for the presence of DENV NS3. At 6 hours after infection, NS3 expression was detected only in the draining PLN (Figure 2B). By 24 hours, NS3 expression was observed at a reduced level in the draining PLN, while it became detectable in the marginal zone of the spleen. By 72 hours, NS3 expression was detected in the contralateral PLN as well as distant CLNs, splenic red pulp, and kidneys, but not the draining PLN. These results show that the pattern of DENV replication (as determined by staining for NS3 expression) following intrafootpad inoculation mirrors results obtained with intravenous inoculation: early replication in the draining lymphoid tissue 14

292 293 followed by systemic dissemination to other lymphoid tissues, and lastly, replication in non-lymphoid tissues. 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 Intravenous DENV particles traffic to macrophages in the marginal zone of the spleen Based on the pattern of initial replication in the splenic marginal zone and subsequent dissemination to the red pulp, we hypothesized that the ability of the splenic marginal zone to absorb blood-borne DENV particles diminishes over the course of infection, resulting in leakage of viral particles into the red pulp. To investigate this hypothesis, mice were inoculated i.v. with 1.5x10 11 GE E124/128-IC, as in previous experiments, and administered an additional 5x10 10 GE of E124/128-IC labeled with DiD, a lipophilic farred fluorescent dye, 2 hours before tissue harvest at 0 (naïve mice), 6, 12, 18, 24, 36, 48, and 72 hours after the initial inoculation (Figure 4A). When large amounts of DiD are incorporated into the lipid bilayer of the virion, it undergoes autoquenching and emits only low levels of fluorescence. However, once the lipid bilayer fuses with the host cell membrane, the probe is diluted and renders target cells highly fluorescent. In naïve mice and mice 6 hours after DENV infection, DiD signal in the spleen was restricted to the marginal zone (Figure 4A). DiD distribution became more diffuse in spleens of mice 12 and 18 hours after infection. After 24 hours, dissemination of DENV particles was readily observed throughout the red pulp. However, at all time points after infection, the majority of the DiD accumulated in rings within the marginal zone of the spleen, implying that DENV particles primarily trafficked here throughout infection, even at time points when most of the NS3 signal was located in the red pulp (Figure 4A, 24 48 hours). 15

315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 Because macrophages of the marginal zone often serve to filter blood-borne virus, we speculated that these cells were the initial targets of intravenous DENV. To investigate this, AG129 and WT129 mice were inoculated with 5x10 10 GE of DiD-labeled E124/128- IC, and spleens were harvested 6 hours later. Tissues were sectioned and stained with antibodies against MARCO, CD169 and SIGN-R1, which label the heterogenous pool of macrophages in the marginal zone. Nearly all of the DiD signal in both AG129 and wildtype 129/SV (WT129) mice (Figure 4B) localized to cells expressing marginal zone macrophage markers, implicating these cells as the initial targets of DENV in this model, regardless of host immune status. DENV trafficking to the liver, kidneys and thymus increases over the course of infection, while trafficking to the spleen decreases To determine how viral trafficking and fusion change with time in the major target tissues, the spleen, liver, thymus, and kidneys were analyzed for the presence of DiD and DENV NS3 (Figure 4A). DiD signal intensity was quantified on 6 24 images of the spleen, liver, kidneys, and thymus for 5 mice at 0 (naïve mice), 12, 36, and 72 hours after infection. These results are summarized in Figure 4C. Large amounts of DENV trafficked to the spleen and liver, with the amounts decreasing in the spleen and increasing in the liver over the course of infection. Similar to the liver, the kidneys and thymus had increasing levels of DiD at late time points after infection, albeit less virus trafficked to these tissues than to the spleen and liver. These data show that the bloodborne DENV trafficking pattern changes during infection, favoring early dissemination to lymphoid tissues and late dissemination to non-lymphoid tissues. 16

339 340 341 342 343 344 345 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360 DENV initially infects splenic macrophages in the marginal zone, and later spreads to red pulp macrophages In order to identify the DENV-permissive cells in the spleen of AG129 mice, we sought to distinguish between macrophages, monocytes and dendritic cells (DCs), all of which have been reported to be targeted by DENV in humans. Macrophages in the spleen exist as several heterogeneous, overlapping populations (23). Sialoadhesin, or CD169, is expressed by marginal metallophilic macrophages (MMMs), which reside closest to the white pulp in the innermost part of the marginal zone. SIGN-R1 (a murine homolog of DC-SIGN) and MARCO are expressed by two highly overlapping populations of macrophages in the outer part of the marginal zone, collectively termed marginal zone macrophages (MZMs). We have observed that some of the outer MARCO and SIGN- R1 expressing MZMs also express F4/80. Additionally, we have observed a marked upregulation of MARCO expression on F4/80 + red pulp macrophages between 12 and 24 hours after infection (data not shown). MARCO was therefore not used to identify splenocytes in this study. Lastly, splenic red pulp macrophages (RPMs) can be distinguished based on their high level of F4/80 expression and lack of MZM and MMM markers. Thus, to differentiate the mononuclear phagocytes potentially permissive to DENV infection, we utilized CD11c (dendritic cell marker), CD11b (present at high levels on monocytes and neutrophils), CD169 and SIGN-R1 (present on macrophages of the marginal zone), F4/80 (present on macrophages of the red pulp), and CD68 (expressed at high levels in macrophages). 17

361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377 378 379 380 381 382 383 Using a sublethal dose of virus (5x10 8 GE of E124/128-IC) to more accurately model a natural human infection, AG129 mice were inoculated intravenously and spleens were harvested 12 and 48 hours after infection. At this dose, replication was observed selectively in the marginal zone at 12 hours. DENV NS3-expressing cells were distinct from cells expressing high levels of CD11b or CD11c (Figure 5A, top row). Instead, NS3 localized to cells expressing CD68, some of which also expressed F4/80 (Figure 5A, middle row) and most of which were also found to express CD169 and/or SIGN-R1, identifying them as MMMs and MZMs, respectively, (Figure 5A, bottom row). At 48 hours, DENV NS3-expressing cells did not express high levels of CD11b, CD11c, CD169 or SIGN-R1 (Figure 5B, top and bottom rows), but instead expressed high levels of CD68 and F4/80 (Figure 5B, middle row). This cell marker expression and cell localization is consistent with RPMs. Thus, in this model of primary DENV infection, virus replicates early in the spleen in the MZMs and MMMs, and later in RPMs. Additionally, we did not observe DENV replication in CD11c high-expressing dendritic cells or CD11b high-expressing monocytes or neutrophils. Interferon-γ signaling restricts DENV replication in macrophages of the red pulp, but not in macrophages of the marginal zone To validate our tropism findings in less immunocompromised mice than AG129, we next sought to determine the pattern of DENV replication in the spleens of A129 mice, which lack the interferon-α/β receptor, but have functional IFN-γ signaling. A129 and AG129 mice were infected i.v. with 5x10 8 GE of E124/128-IC, and spleens were harvested at 12, 24, 48, and 72 hours after infection. Frozen sections of the spleen were stained for 18

384 385 DENV NS3 together with either F4/80 and CD68 (Figure 6A), or CD169 and SIGN-R1 (Figure 6B). At 12 and 24 hours post-inoculation, A129 and AG129 mice showed an 386 approximately equivalent infection occurring predominantly in CD169 + MMMs and 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 SIGN-R1 + MZMs. At 48 hours, however, infection in F4/80 + RPMs was strikingly reduced in A129 mice compared to AG129 (Figure 6A). Thus, in this experimental setup, IFN-γ receptor signaling does not affect initial DENV replication in MMMs or MZMs, but is able to reduce replication in RPMs. Additionally, DENV cellular tropism in this model is unaffected by IFN-γ receptor signaling. DENV systemically infects CD68 + macrophages late in infection We have previously reported that in AG129 mice, DENV replicates in CD68 + lamina propria macrophages of the small intestine at late time points after infection (38). To determine whether macrophages are the cells infected at late time points in other tissues, AG129 mice were inoculated i.v. with 1.5x10 11 GE of E124/128-IC, and 72 hours later the kidney, heart and thymus were harvested and stained for CD11b and CD68. In the kidney, heart, and thymus, most of the DENV NS3 signal was present in cells expressing CD68 and, additionally but less often, CD11b (Figure 7A). These results confirm that macrophages in many non-lymphoid tissues are late targets of DENV infection. 19

403 404 405 406 407 408 409 410 411 412 413 414 415 DENV replicates in macrophages of draining lymph nodes following intrafootpad inoculation Initial screening studies for DENV replication following intrafootpad inoculation revealed early replication in the draining PLNs of AG129 mice. To identify the cells in the lymph node targeted by DENV, AG129 mice were inoculated i.f. with 1.5x10 11 GE of E124/128-IC, and PLNs were harvested 6 hours after infection. Frozen sections of the PLNs were stained for DENV NS3 together with either MARCO and SIGN-R1 (Figure 7B, top row) or CD169 and CD68 (Figure 7B, bottom row). As in the spleen, cells expressing these markers were overlapping, and cells expressing each or combinations were found to permit DENV replication. These results show that initial DENV infection occurs in macrophages of draining PLNs within 6 hours following intrafootpad inoculation. Downloaded from http://jvi.asm.org/ on October 2, 2018 by guest 20

416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 DISCUSSION In this study, we sought to characterize the tissues and cells targeted by DENV in vivo and the sequence in which they are infected. Following i.v. inoculation, DENV initially traffics to the spleen where it infects macrophages of the marginal zone, including MZMs and MMMs. Following i.f. inoculation, DENV initially targets macrophages of the draining popliteal LN before entering the blood and subsequently infecting the splenic marginal zone. Although blood-borne DENV continues to traffic to the marginal zone throughout the course of infection, replication in the marginal zone decreases and bloodborne DENV later disseminates into the red pulp, where it infects RPMs. The initial wave of replication in the spleen is followed by replication in the bone marrow and then lymph nodes and Peyer s patches. Finally, late during infection, when mice are becoming overtly ill, replication decreases in the lymphoid tissues and increases in macrophages of non-lymphoid tissues, most notably in the kidneys, heart, thymus, and GI tract. In humans, a broad array of tissues and cell types have been reported to harbor DENV: perivascular mononuclear cells of the skin; hepatocytes, endothelial cells, and Kupffer cells of the liver; cortical neurons, astrocytes, microglia, Purkinje s cells, and choroid plexus epithelial cells of the brain; immunoblasts, mononuclear cells, and macrophages of the kidney; immunoblasts, lymphocytes, plasma cells, and macrophages of the spleen, lymph nodes, and kidneys; endothelial cells of the spleen; alveolar macrophages of the lung; mucosal lymphocytes and lamina propria plasma cells of the stomach; and plasma and mononuclear cells of the small and large intestine (2, 5, 6, 8, 13, 15, 16, 25, 29). Importantly, because these studies detected components of mature viral particles and 21

439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 identified cells based on cellular morphology alone or with non-specific markers in serial sections, it is unclear whether these studies have accurately identified sites of viral replication. Despite these caveats, the published studies with human tissues have set a framework for understanding DENV behavior in vivo. Due to logistical complications of studies with human tissues, small animal studies can provide critical information to elucidate viral behavior in vivo. For example, the present study investigated the kinetics of DENV dissemination by analyzing several time points and tissues after infection at a consistent viral dose, which is nearly impossible in human studies. However, the use of non-natural mammalian hosts, particularly mice lacking IFN receptors, indicates potential caveats for interpretation of our results. Both type I and type II IFN have been reported to restrict the cellular and tissue tropism of flaviviruses in mice (30, 31), which may result in findings that do not represent what occurs in natural human infections. However, DENV, like other flaviviruses, possesses mechanisms to overcome or circumvent IFN responses in humans and non-human primates (7, 17, 18, 22, 24), and this ability appears to be species-specific for a number of mosquito-borne viruses (1, 4, 24). As we are not currently able to validate our findings with human samples, it remains unclear whether other differences between mice and humans could be confounding our results. For example, there may be cell populations targeted by DENV in humans that are either not present or not infected in mice. Thus, although small animal studies are invaluable for deducing the nature of virus replication and virus-host interactions in vivo, it is crucial that findings are evaluated in focused human studies, even if the same rigor is not possible. 22

462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 Identification of the precise cellular targets of DENV in vivo has been hindered by the heterogeneity of mononuclear phagocytes present in different tissues and even within a given tissue. Macrophages of the spleen in particular are highly heterogeneous and, therefore, difficult to distinguish based on cell markers (23). Certain markers, such as CD11b, are expressed at variable levels on multiple cell populations. Moreover, the populations of cells present in a tissue, as well as cell marker expression, can change under inflammatory conditions such as viral infections. This may help explain the inconsistent reports in the literature of individual markers on specific cell populations. For example, RPMs have been variably reported to lack or express CD11b (11, 19, 23, 37). FIHC is thus particularly useful in identifying cell populations, including monocytes/neutrophils (CD11b high ), MZMs (SIGN-R1 + ), MMMs (CD169 + ), and RPMs (F4/80 + ), because it is based on anatomic location, cellular morphology, and expression of cell population-specific markers. Tissue and cellular tropism of DENV has been investigated previously in AG129 mice, both by our group and others (2, 3, 9, 20, 38). It has been reported that in the spleen, DENV infection occurs in CD11c + DCs and in CD11b high-expressing, F4/80 + macrophages (2, 20). In contrast to these publications, we did not find evidence that CD11c + DCs in the spleen are major targets of DENV, nor did we find evidence of notable CD11b expression on DENV-infected F4/80 + red pulp macrophages. Instead, our findings demonstrated that MMMs, MZMs, and RPMs, but not cells expressing high levels of CD11b or CD11c, were highly permissive to DENV infection. In agreement with the present study, Balsitis et al. identified the red pulp and marginal zone as the 23

485 dominant compartments for DENV infection in the spleen (2) and Fink et al. reported 486 infection of CD169 + subcapsular sinus macrophages (SCSMs) of the draining LN 487 following subcutaneous administration of DENV (9). In the present study, SCSMs and 488 the equivalent CD169 + cells in the marginal zone of the spleen, respectively, were 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 infected following i.f. and i.v. routes of administration, and all of the lymph nodes and Peyer s patches analyzed 48 hours after infection contained high levels of DENV NS3. These particular macrophage populations of the splenic marginal zone and subcapsular sinus of lymph nodes have been shown to be important for clearance of high molecular mass antigens including viral particles from blood and lymph (21, 26, 33). These cells are believed to permit low levels of viral replication in order to amplify antigen and facilitate generation of high affinity antibodies (14). Additionally, many viruses, including DENV, bind to DC-SIGN (36), which is the human homolog of murine SIGN- R1. This molecule is expressed at high levels on macrophages of the marginal zone and subcapsular sinus and it acts as a pattern recognition receptor for sampling antigens in the surrounding fluid (10). These characteristics all support results that these macrophage populations, which possess molecules thought to be responsible for permitting DENV infection, are initial interaction partners and important initial targets of blood- and lymphborne DENV. To our knowledge, this is the first use of fluorescence-tagged viral particles for tracking the temporal and spatial patterns of DENV trafficking in vivo. The far-red fluorescent, lipophilic dye DiD has previously been used for single DENV particle tracking to 24

508 509 510 511 512 513 514 515 516 517 518 519 520 521 522 523 524 525 526 527 528 529 530 investigate cell entry in vitro (35). Here, DiD-labeled DENV particles were utilized in vivo to visualize the fate of the injected viral particles. In contrast to tissue section staining for DENV NS3, DiD-labeled DENV particles can be used to probe the outcome of blood-borne DENV in order to visualize trafficking at any time during infection. Our results show that high levels of DENV traffic to cells of the liver and the spleen, initially in the marginal zone and later also in the red pulp. The amount of DiD accumulating in the spleen decreases over time, while it increases in the liver, kidneys and thymus. Although kinetically not identical, this trend mirrors levels of viral replication within these organs over time, suggesting that extravasation of viral particles may dictate the sequence of cells and tissues targeted by DENV in vivo. Our results contribute to the understanding of DENV tropism in vivo over the course of infection. We show that DENV inoculated via a peripheral route initially targets draining lymph nodes. Once DENV enters the blood, it first targets the spleen and bone marrow and later spreads to the lymph nodes and Peyer s patches, and finally to non-lymphoid tissues throughout the mouse. These findings augment our published studies and show that DENV largely infects macrophages in a number of tissues of AG129 mice: the kidneys, heart, thymus, and gastrointestinal tract, marginal zone and red pulp of the spleen, subcapsular sinus of the lymph nodes, as well as sinusoidal endothelial cells of the liver (9, 38). By elucidating the pattern of DENV replication in mice, our study provides an overall framework for DENV behavior in vivo to guide subsequent investigations of DENV tropism in naturally infected humans. 25

531 532 533 534 535 536 537 538 539 540 541 542 543 544 FIGURE LEGENDS Figure 1 Dengue virus first infects the spleen, followed by bone marrow, and then lymph nodes, and Peyer s patches after intravenous inoculation. Representative immunohistochemical staining of tissue sections from naïve AG129 mice, and at 6, 12, 24, 48, and 72 hours after infection (1.5x10 11 GE E124/128-IC i.v. on day 0). Spleens, cutaneous lymph nodes (CLN: axillary, brachial, and inguinal lymph nodes), mesenteric lymph nodes (MLN), Peyer s patches (PP) and bone marrow (BM) were sectioned and stained for DENV NS3 (white when alone, green when shown with prm/m) and DENV prm/m (antibody clone 2H2, shown in red). Results are representative of 3 mice. WP labels the white pulp. LN labels the center of the lymph nodes. PP labels the center of the Peyer s patches. 26

545 546 547 548 549 550 551 552 553 554 555 556 557 Figure 2 Dengue virus infects systemic non-lymphoid tissues late during infection. Representative immunohistochemical staining of tissue sections from AG129 mice. A) Tissues were harvested from naïve mice and mice 48 and 72 hours after infection (1.5x10 11 GE E124/128-IC i.v. on day 0) and stained for DENV NS3 (white) in the kidney, heart, thymus, adrenal gland, liver, lung, forestomach, stomach, duodenum, jejunum, ileum, cecum, colon, and rectum. Results are representative of 3 mice. B) Tissues were harvested from mice 6, 24, and 72 hours after infection (1.5x10 11 GE E124/128-IC i.f. on day 0) and stained for DENV NS3 (white) in the spleen, draining popliteal lymph node (drpln), contralateral popliteal lymph node (clpln), cutaneous lymph nodes (CLN: axillary, brachial, and inguinal lymph nodes), and kidney. Results are representative of 3 mice. Downloaded from http://jvi.asm.org/ on October 2, 2018 by guest 27

558 559 560 Figure 3 A schematic depicting the kinetics of DENV tissue tropism over the course of infection in mice. 561 562 563 564 565 566 Model of DENV infection. DENV first infects the spleen, followed by the bone marrow, and then the lymph nodes and Peyer s patches (LN, PP). Infection decreases in these tissues while it increases systemically in non-lymphoid tissues at which point mice become moribund. Downloaded from http://jvi.asm.org/ on October 2, 2018 by guest 28

567 568 Figure 4 Blood-borne DENV particles traffic mainly to the spleen and liver. 569 570 571 572 573 574 575 576 577 578 579 580 581 582 583 584 585 586 587 (A) Representative immunohistochemical staining of spleen, liver, thymus, and kidney from naïve AG129 mice, and 6, 12, 18, 24, 36, 48, and 72 hours after infection (1.5x10 11 GE E124/128-IC i.v. on day 0 and 5x10 10 GE DiD-labeled E124/128-IC i.v. 2 hours before harvest). Sections were stained for DENV NS3 (white, top row of each panel) and DiD in the tissue was imaged (white, middle row of each panel). Merged images are shown on the bottom row of each panel (NS3 is shown as green and DiD is shown as red). B) Representative immunohistochemical staining of spleen from AG129 (top) or WT129 (bottom) mice 6 hours after infection (5x10 10 GE DiD-labeled E124/128-IC i.v.). Sections were stained for CD169, SIGN-R1, and MARCO (white, left) and DiD in the tissue was imaged (white, middle). The merged image is shown on the right (DiD is shown as green and CD169, SIGN-R1, and MARCO are shown as red). C) Summary of results from panels (A). DiD intensity was quantified from 6-24 images per mouse with 5 mice per time point. Symbols and error bars represent means ± SEM. P- values from two-tailed unpaired t-test, CI 95%: * P 0.05, ** P 0.01, *** P 0.001. 29

588 589 590 Figure 5 In the spleen, DENV first infects macrophages of the marginal zone, and later infects macrophages of the red pulp. 591 592 593 594 595 596 597 598 599 600 601 602 603 604 A) Representative immunohistochemical staining of spleen sections from AG129 mice 12 hours after infection (5x10 8 GE E124/128-IC i.v. on day 0). Tissue sections were stained for DENV NS3 (green) and CD11b and CD11c (red and blue, respectively, top row), CD68 and F4/80 (red and blue, respectively, middle row), and CD169 and SIGN-R1 (red and blue, respectively, bottom row). Low magnification (5x) images are shown on the left with DENV NS3 alone, while each individual channel and the merged image are shown on the right. Grey boxes on the DENV NS3 panels depict areas of enlargement shown on the right. Results are representative of 3 mice. B) Representative immunohistochemical staining of spleen sections from AG129 mice 48 hours after infection (5x10 8 GE E124/128-IC i.v. on day 0). Sections are stained as in A. Results are representative of 3 mice. 30

605 606 Figure 6 Interferon-γ-receptor signaling limits DENV replication in splenic red pulp macrophages. 607 608 609 610 611 612 613 614 615 616 617 618 619 620 621 622 623 624 A) Representative immunohistochemical staining of spleen sections from AG129 or A129 mice 12, 24, 48, and 72 after infection (5x10 8 GE E124/128-IC i.v. on day 0). Tissue sections were stained for F4/80 (red), DENV NS3 (green), and CD68 (blue). Low magnification (5x) merged images are shown on the left as merged images only, medium magnification (20x) images are shown in the middle as merged images only, and high magnification (33x) images are shown on the right as the merged image followed by each individual channel (F4/80, DENV NS3, and CD68). Grey boxes on the left depict the area of enlargement shown on the immediate right. Two enlarged areas from each middle magnification image are shown. Results are representative of 3 mice. B) Representative immunohistochemical staining of spleen sections from AG129 or A129 mice 12, 24, 48, and 72 after infection (5x10 8 GE E124/128-IC i.v. on day 0), with each time point represented by a row. Images are arranged as in A, except tissue sections were stained for CD169 (red), DENV NS3 (green), and SIGN-R1 (blue). Results are representative of 3 mice. 31

625 626 Figure 7 DENV infects macrophages of the kidney, heart, thymus and lymph nodes. 627 628 629 630 631 632 633 634 635 636 637 638 639 640 A) Representative immunohistochemical staining of kidney, heart, and thymus sections from AG129 mice 72 hours after infection (1.5x10 11 GE E124/128-IC i.v. on day 0). Tissue sections were stained for DENV NS3 (green), CD68 (red), and CD11b (blue). 5x magnification of all channels is shown on left and 20x magnification images on the right with each channel shown separately in white. Results are representative of 3 mice. B) Representative immunohistochemical staining of draining popliteal lymph node sections from AG129 mice 6 hours after infection (1.5x10 11 GE E124/128-IC i.f.). Tissue sections were stained for DENV NS3 (green), SIGN-R1 (red), and MARCO (blue) in the upper panel and DENV NS3 (green), CD169 (red), and CD68 (blue) in the lower panel. 5x magnification of all channels is shown on left and 20x magnification images on the right with each channel shown separately in white. Results are representative of 3 mice. 32

641 642 643 644 645 646 647 648 649 ACKNOWLEDGEMENTS We thank Drs. Chris Benedict, Raphaël M. Zellweger, and Stuart T. Perry at LIAI for experimental advice. We thank Dr. Olga Turovskaya, Sarala Joshi, Steven Lada, and Dr. Satoshi Fukuyama for technical help. This work was supported by NIH grants U01 AI082185 to S.S. and U54 AI057157 from Southeast Regional Center of Excellence for Emerging Infections and Biodefense to P.F. Sparling. The authors declare they have no competing financial interests. Downloaded from http://jvi.asm.org/ on October 2, 2018 by guest 33

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