Dual Function of Ccr5 during Langat Encephalitis: Reduction in Neutrophil-Mediated Central Nervous System Inflammation and Increase in T Cell− This information is current as Mediated Viral Clearance of October 1, 2021. Daniela Michlmayr, Susana V. Bardina, Carlos A. Rodriguez, Alexander G. Pletnev and Jean K. Lim J Immunol 2016; 196:4622-4631; Prepublished online 29

April 2016; Downloaded from doi: 10.4049/jimmunol.1502452 http://www.jimmunol.org/content/196/11/4622

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2016 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Dual Function of Ccr5 during Langat Virus Encephalitis: Reduction in Neutrophil-Mediated Central Nervous System Inflammation and Increase in T Cell–Mediated Viral Clearance

Daniela Michlmayr,*,1 Susana V. Bardina,*,1 Carlos A. Rodriguez,* Alexander G. Pletnev,† and Jean K. Lim*

Tick-borne encephalitis virus (TBEV) is a vector-transmitted flavivirus that causes potentially fatal neurologic infection. There are thousands of cases reported annually, and despite the availability of an effective vaccine, the incidence of TBEV is increasing world- wide. Importantly, up to 30% of affected individuals develop long-term neurologic sequelae. We investigated the role of chemokine receptor Ccr5 in a mouse model of TBEV infection using the naturally attenuated tick-borne flavivirus Langat virus (LGTV). Ccr5- Downloaded from deficient mice presented with an increase in viral replication within the CNS and decreased survival during LGTV encephalitis compared with wild-type controls. This enhanced susceptibility was due to the temporal lag in lymphocyte migration into the CNS. Adoptive transfer of wild-type T cells, but not Ccr5-deficient T cells, significantly improved survival outcome in LGTV- infected Ccr5-deficient mice. Concomitantly, a significant increase in neutrophil migration into the CNS in LGTV-infected Ccr52/2 mice was documented at the late stage of infection. Ab-mediated depletion of neutrophils in Ccr52/2 mice resulted in a significant improvement in mortality, a decrease in viral load, and a decrease in overall tissue damage in the CNS compared with http://www.jimmunol.org/ isotype control–treated mice. Ccr5 is crucial in directing T cells toward the LGTV-infected brain, as well as in suppressing neutrophil-mediated inflammation within the CNS. The Journal of Immunology, 2016, 196: 4622–4631.

ick-borne encephalitis (TBE) virus (TBEV), a neurotropic rate ranges between 2%, caused by the European subtype, and 40% flavivirus, is predominantly transmitted through the bite of for the Far Eastern subtype (1, 5). Despite the availability of a vac- T an infected tick and can cause TBE (1–3). The outcome of cine, there are ∼15,000 cases of TBE reported annually, with ∼3,000 TBEV infection is dependent on various factors, including the type cases in Europe and the remainder reported in Russia (1, 2, 6, 7). and strain of TBEV, age, immune status, and genetic predisposition. There are no specific antiviral treatments for TBEV, and a better by guest on October 1, 2021 Upon infection, ∼70% of individuals remain asymptomatic, with the understanding of pathogenesis is required for the development of remaining individuals developing a febrile illness that can potentially targeted therapies. progress to meningitis and/or encephalitis and death (2–4). Thirty A hallmark of is the accumulation of leukocytes percent of patients who develop TBE suffer from long-term neuro- in the infected CNS, which is essential for viral clearance but can also logic sequelae and often require prolonged rehabilitation. The fatality lead to neuroimmunopathology (8, 9). Leukocyte migration into infected tissues is guided, in part, by chemokines produced during inflammation, with their receptors found on circulating leukocytes. *Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY 10029; and †Laboratory of Infectious Diseases, National Institute of Allergy and In the context of numerous inflammatory conditions, the chemokines Infectious Diseases, National Institutes of Health, Bethesda, MD 20892 Ccl3, Ccl4, and Ccl5 are induced both early and to high levels in 1D.M. and S.V.B. contributed equally to this work and are cofirst authors. the periphery and within the CNS. The common receptor for these Received for publication November 19, 2015. Accepted for publication March 29, ligands, chemokine receptor Ccr5, is expressed by a wide range of 2016. leukocytes, including activated T cells, NK cells, and monocytes/ This work was supported by National Institute of Allergy and Infectious Diseases macrophages, and it was shown to have a critical role in neuroprotec- Grant R01AI108715 and in part by the Division of Intramural Research, National tion against Cryptococcus neoformans, HSV-2, and, most recently, Institute of Allergy and Infectious Diseases, National Institutes of Health. S.V.B. was supported in part by National Institutes of Health Research Training Award neurotropic flaviviruses (10–16). In a murine model of West Nile T32AI007647 and National Institute of Allergy and Infectious Diseases Fellowship virus (WNV) infection, Ccr5 was shown to be a critical antiviral F31AI110071. and survival determinant; in Ccr5-deficient mice, viral replication D.M., S.V.B., and J.K.L. conceived and designed the experiments and wrote the was uncontrolled within the CNS, which was concomitant with manuscript. D.M., S.V.B., C.A.R., and J.K.L. conducted the experiments, assembled and analyzed the data, and interpreted the results. A.G.P. provided technical assis- the global loss of infiltrating peripheral leukocytes into the in- tance and provided key reagents. All authors reviewed and commented on the man- fected CNS (17). The protective role of Ccr5 in mice has been uscript and approved the final version. translated in cohorts of WNV-infected individuals in whom Address correspondence and reprint requests to Dr. Jean K. Lim, Department of CCR5D32 homozygosity, which renders individuals CCR5 defi- Microbiology, Icahn School of Medicine at Mount Sinai, One Gustave L. Levy Place, Box 1124, New York, NY 10029. E-mail address: [email protected] cient, was associated with an increased risk for developing Abbreviations used in this article: FFU, focus-forming unit; JEV, Japanese enceph- symptomatic WNV disease; conversely, CCR5-deficient individ- alitis virus; LGTV, Langat virus; MPO, myeloperoxidase; RT, room temperature; uals were absent from asymptomatic WNV-infected blood donors TBE, tick-borne encephalitis; TBEV, TBE virus; WNV, ; WT, (11, 18, 19). In a related mouse model of wild-type. virus (JEV) infection, Larena et. al (13) found that Ccr5-deficient Copyright Ó 2016 by The American Association of Immunologists, Inc. 0022-1767/16/$30.00 mice exhibited increased viral titers in the CNS and enhanced www.jimmunol.org/cgi/doi/10.4049/jimmunol.1502452 The Journal of Immunology 4623 mortality compared with wild-type (WT) mice; this was due to staining. The following Abs were used: CD3-FITC (17A2), CD8-PE impaired trafficking and reduced functional activity of NK cells (53.6.7), CD4-PerCp5.5 (GK1.5), Ly6C-FITC (HK1.4), Ly6G-PE (1A8), and CD8+ T cells in the CNS. Because of the relative absence of CD11b-allophycocyanin (M1/70), CD45–allophycocyanin–Cy7 (30-F11), D NK1.1-PE (PK136), and CD19-allophycocyanin (eBio1D3) (all from the CCR5 32 allele in the human population in Asian countries, eBioscience). Data were collected using an LSR II flow cytometer (Becton where the majority of JEV cases occur, the ability to test the role Dickinson) and analyzed using FlowJo software 8.5.3 (TreeStar). Cell of CCR5 in human genetic susceptibility to JEV is limited (20). numbers were quantified using counting beads (Spherotech). WNV and JEV are mosquito-transmitted neurotropic flaviviruses Protein quantification that are related genetically and serologically. It is unclear whether the protective function of CCR5 extends to more distantly related, Cytokines and chemokines were measured using a multiplex ELISA-based assay, as previously described (27, 28). Briefly, Abs and cytokine standards serologically distinct neurotropic flaviviruses, such as the tick- were purchased from R&D Systems or PeproTech. Individual Luminex borne flaviviruses. bead regions were coupled to capture Abs for each cytokine or chemokine In this study, we sought to understand the role of Ccr5 during measured. Biotinylated detection Abs were used at twice the recommended TBEV infection. To address this, we used Langat virus (LGTV), concentration. Approximately 1200 beads were used for each cytokine or which shares 78–88% amino acid identity among the structural chemokine sample. The plates were read on a Luminex MAGPIX system, with $50 beads collected for each region/sample. The median fluorescence enveloped proteins of the tick-borne flaviviruses and was once intensity was determined with Milliplex software using a five-parameter considered a live attenuated vaccine strain (2, 21–26). We found regression algorithm. that efficient effector lymphocyte migration into the CNS was Immunohistochemistry and TUNEL staining dependent on Ccr5; in the absence of this receptor, uncontrolled viral replication resulted in a dysregulation of neutrophil migra- Perfused brains were immediately fixed in 10% neutral-buffered formalin m Downloaded from tion into the CNS, leading to enhanced apoptosis. Our findings (VWR) prior to paraffin embedding. Sections (5–6 m) were rehydrated prior to Ag retrieval with citrate buffer, as previously described (29). Slides suggest that Ccr5 is a critical host response gene that contributes were incubated with the primary Ab rat anti-mouse B220 (1:500; Bio- to maintaining a balance between the antiviral response and im- Legend) and mouse anti-TBEV (1:1000; American Type Culture Collec- munopathology within the CNS. tion) at 4˚C overnight. The sections were incubated with 2.5 mg/ml biotinylated anti-rat or anti-mouse IgG Abs for 30 min at room tempera- ture (RT) and incubated with VECTASTAIN Elite ABC-peroxidase re- agent (all from Vector Laboratories). Staining for CD3 (1:100; Vector Materials and Methods http://www.jimmunol.org/ Laboratories) and myeloperoxidase (1:1000; DAKO) was used in con- Virus and virus quantification junction with the rabbit EnVision/HRP Kit (DAKO), according to the WT LGTV strain TP21 (GenBank accession no. AF253419; http://www. manufacturer’s guidelines. All sections were visualized by incubating 9 ncbi.nlm.nih.gov/nuccore/AF253419) stock was generated in Vero cells slides with 3,3 diaminobenzidine HRP substrate for 1–2 min at RT and (World Health Organization, passage 143) from a plaque-purified LGTV counterstaining with Gill’s hematoxylin (Vector Laboratories) for 2 min. TP21 virus preparation, as described previously (24). To measure infec- Sections were dehydrated in a series of increasing ethanol concentrations tious virus, Vero cell monolayers in 24-well plates were infected with 100 and mounted with DPX Mountant (Sigma). A TUNEL assay was used to ml supernatants for 1 h at 37˚C. Cells were overlaid with 1 ml Opti-MEM visualize apoptotic cells. For this, sections were stained using the DeadEnd (Invitrogen) containing 0.8% methylcellulose (Fisher Scientific), 2% FBS, Colorimetric TUNEL Systems Kit (Promega), counterstained with Gill’s and 50 mg/ml gentamicin sulfate. After a 3-d incubation, plates were fixed hematoxylin for 2 min, and mounted with 100% glycerol (Sigma). The with 100% methanol. Ag was detected with TBEV-specific Abs (1:2500; positive control slide was treated with DNase for 10 min at RT to introduce by guest on October 1, 2021 American Type Culture Collection), followed by staining with goat anti-mouse nicks in the DNA. All slides were analyzed using an Axio Imager Z2 IgG Abs (1:500; Invitrogen) conjugated with HRP. LGTV focus-forming units microscope and Zen 2012 software (both from Zeiss). 9 (FFU) were visualized by the addition of 1 ml 3,3 diaminobenzidine Adoptive-transfer studies tetrahydrochloride hydrate HRP substrate (Sigma). Viral RNA was iso- lated from mouse plasma using a QIAamp Viral RNA Mini Kit and On day 8 post-LGTV infection, T cells from spleens of Ccr52/2 and WT converted into cDNA using a QuantiTect Reverse Transcription Kit (both donor mice were enriched using the Pan T Cell Isolation Kit II and the from QIAGEN), according to the manufacturer’s protocol. The following AutoMACS Magnetic Cell Sorter (both from Miltenyi Biotec), according primers were used for quantitative real-time PCR: forward 59-CAGTG- to the manufacturer’s guidelines. Enrichment resulted in ∼85% CD45+ GACACAGAGCGAATG-39 and reverse 59-ACAGTCAGGTTTGCCT- CD3+ T cell purity. On day 5 postinfection, Ccr52/2 recipient mice were CACC-39. The samples were run for 40 cycles using the Roche LightCycler injected i.v. with 106 T cells from WT or Ccr52/2 donor mice in 100 ml 480 Real Time PCR System. The absolute copy number was calculated using PBS and monitored for survival for 18 d. a standard curve generated using an LGTV NS5–containing plasmid DNA. In vivo depletion of neutrophils LGTV infection model Neutrophils were depleted using anti-Ly6G Ab clone 1A8 (Bio X Cell); rat Mouse studies were carried out in an animal biosafety level two facility IgG2a isotype was used as a control. Briefly, 250 mg anti-Ly6G or isotype under a protocol approved by the Icahn School of Medicine at Mount Sinai control Ab in 100 ml PBS was injected i.p. into each mouse on days 8, 10, 2/2 Animal Care and Use Committee. Ccr5 and WT C57BL/6J mice were and 12 postinfection. purchased from The Jackson Laboratory. All experiments were initiated using female mice at 4 wk of age. Mice were infected s.c. in the scruff of Statistical analysis the neck with 106 FFU LGTV suspended in 50 ml PBS. Mice were monitored for morbidity and mortality for up to 18 d. All data were analyzed using Prism Version 5 software (GraphPad); a Pearson Omnibus K2 and Shapiro–Wilk test was performed to test whether Immune cell analysis data were distributed normally. In brief, a Student unpaired t test or Mann– Whitney U test was performed. For all survival analyses, a Kaplan–Meier Mice were deeply anesthetized with ketamine/xylazine prior to cardiac survival curve was generated, and statistical significance was determined perfusion with ice-cold PBS. Brains were removed aseptically, collected in using a log-rank test. 7 ml FACS buffer (PBS + 2% FBS), and homogenized using a dounce homogenizer. After the addition of 3 ml 100% isotonic Percoll (GE Healthcare), the homogenate was underlain with 1 ml 70% isotonic Percoll. Results After centrifugation at 2470 rpm for 30 min at 4˚C, cells at the interphase were washed in FACS buffer prior to Ab staining. Anticoagulated blood Ccr5 deficiency increases susceptibility to LGTV infection was incubated with Pharm Lyse buffer (BD Biosciences) for 2 min on ice, To understand the role of Ccr5 in host defense during LGTV in- according to the manufacturer’s protocol. An Ultraviolet LIVE/DEAD cell 2 2 fection, we infected WT and Ccr5 / mice s.c. with 106 FFU of stain kit (Invitrogen) was used to assess cell viability, and cells were 2/2 blocked for nonspecific Fc-mediated interactions with 0.5 mg purified anti- LGTV strain TP21. Although both WT and Ccr5 mice exhibited mouse CD16/CD32 (BD Biosciences) for 20 min at 4˚C prior to Ab weight loss, lethargy, hunchback posture, and fur ruffling starting on 4624 CCR5 IS CRITICAL FOR LGTV-INDUCED ENCEPHALITIS day 7 postinfection, only Ccr52/2 mice displayed signs of neurologic LGTV-infected WT and Ccr52/2 mice was in the levels of Ccl4 disease, including unilateral or bilateral limb paralysis, severe dis- and Ccl5 (Fig. 2B). Significantly higher levels of Ccl5 were orientation, ataxia, and seizures. Consistent with these observations, measured in the steady-state in Ccr5-deficient mice; following Ccr52/2 mice exhibited decreased survival (Fig. 1A) compared with infection, Ccl4 and Ccl5 levels were 3–6-fold higher than WT WT mice (48% versus 90%, p = 0.0008). Daily evaluation of LGTV- levels. This is consistent with previous studies showing that the infected Ccr52/2 mice showed a transient and significantly increased loss of Ccr5 results in an inability to bind and degrade these li- weight loss on days 8 and 9 postinfection compared with WT mice gands (15, 30, 31). Because all leukocyte numbers, as well as (Fig. 1B). cytokine and chemokine levels (with the exception of the cognate Given the increased morbidity and mortality in Ccr52/2 mice, ligands) were unaltered, the increased susceptibility observed in we next examined viral burden in the periphery and CNS. Viremia Ccr5-deficient mice is not a direct result of a deficit in leukocyte on days 1, 3, 5, and 7 was similar in WT and Ccr52/2 mice numbers in the periphery or differences in the initial host cytokine (Fig. 1C). Similarly, no differences in viral clearance were ob- response to infection. served within the spleen (Fig. 1D), demonstrating that viral Ccr5 results in impaired T and NK cell trafficking to the CNS clearance in the periphery was nearly identical in the two strains. In the CNS, initial viral loads were similar in WT and Ccr52/2 Because no difference in peripheral viral loads, leukocyte numbers, mice in the brain and spinal cord; however, viral burden remained or cytokine production was observed following LGTV infection, high in the brain of Ccr52/2 mice compared with WT mice on day we next assessed whether the accumulation of lymphocytes into the 12 postinfection (p = 0.045) and in the spinal cord on day 10 CNS was altered given the role of Ccr5 in leukocyte migration. postinfection (p = 0.034) (Fig. 1E, 1F), which is consistent with Leukocytes were isolated from brains of WT and Ccr5-deficient the observed symptom onset. These data point to a protective, mice on days 8 and 12 postinfection and analyzed by flow Downloaded from antiviral role for Ccr5 within the CNS. cytometry. On day 8 postinfection, after virus has entered the CNS, we found a significant reduction in CD4+ T cells (∼46%), CD8+ Ccr5 does not alter the cellular or inflammatory response in T cells (∼49%), and NK cells (∼53%) in the brains of Ccr52/2 the periphery during LGTV infection mice compared with WT mice (Fig. 3A–F). This delay coincided Because depressed effector cell function can hinder viral clearance with weight loss (Fig. 1B) and disease symptom onset and was within the CNS, we investigated whether Ccr5 may be involved in transient because differences were no longer apparent by day http://www.jimmunol.org/ the initiation of the cellular response to LGTV. To investigate this, 12 postinfection, with CD4+ Tcell,CD8+ T cell, and NK cell we examined the role of Ccr5 on lymphocyte numbers in the numbers returning to those nearly identical to WT mice. To periphery of WT and Ccr52/2 mice. In the steady-state, as well as further evaluate the nature of the differences in T cell migra- following infection on days 1, 3, and 5, the number of inflam- tion into the CNS, we conducted immunohistochemical anal- matory monocytes, neutrophils, NK cells, CD4+ T cells, CD8+ yses on the brain tissue of WT and Ccr5-deficient mice. As T cells, and B cells present in the blood was unaffected in the shown in Fig. 3G, the number of CD3+ T cells was greatly absence of Ccr5 (Fig. 2A). Because viral infections induce innate reduced in the brains of Ccr5-deficient mice on day 8 postin- immune mediators indicative of the antiviral response, we next fection, which was observed in all regions in the brain; repre- measured several proinflammatory factors known to be produced sentative images are shown for the cerebellum. These differences by guest on October 1, 2021 following infection, such as IFN-stimulated proteins (Ccl2, Cxcl9, were no longer observed on day 12; similar staining for CD3+ and Cxcl10), immunosuppressive cytokine IL-10, and the Ccr5 T cells was noted between the strains, confirming the flow cytom- ligands (Ccl4 and Ccl5). The only differences observed between etry results.

FIGURE 1. Ccr5 is critical for survival and viral clearance in the CNS during LGTV infection. (A) Kaplan–Meier survival analysis of LGTV-infected WT and Ccr52/2 mice. (B) Body weight was measured for LGTV-infected WT and Ccr52/2 mice. All data (A and B) are pooled from three independent experiments (31 WT mice and 32 Ccr52/2 mice). Viral titers were quantified in the blood (C) and spleen (D) by quantitative real-time PCR, as well as in the brain (E) and spinal cord (F) by FFU assay. Dashed lines indicate the limit of detection. All data (C–F) are mean 6 SD for three to eight mice/genotype and time point from two independent experiments. *p , 0.05, ***p , 0.001. The Journal of Immunology 4625 Downloaded from http://www.jimmunol.org/ by guest on October 1, 2021

FIGURE 2. Peripheral leukocytes and inflammatory cytokine production following LGTV infection. (A) Total cell numbers of monocytes (CD45hiLy6Chi CD11b+Ly6G2), neutrophils (CD45hiLy6CintCD11b+Ly6G+), NK cells (CD45hiNK1.1+CD32), CD4+ T cells (CD45hiCD3+CD4+), CD8+ T cells (CD45hi CD3+CD8+), and B cells (CD45hiCD19+) were assessed by flow cytometry from the blood of WT and Ccr52/2 mice on days 0, 1, 3, and 5 postinfection. Cells were gated on live singlet CD45hi cells. (B) Levels of Ccl2, Cxcl9, Cxcl10, IL-10, Ccl4, and Ccl5 in the blood of WT and Ccr52/2 mice were measured by multiplex ELISA. The dashed line represents the assay limit of detection. All data are shown as mean 6 SD for 4–11 mice/genotype and time point from three independent experiments. *p , 0.05, **p , 0.01, ***p , 0.001.

Enhanced neutrophil recruitment in the absence of Ccr5 leads 4H). To further characterize neutrophil infiltration into the CNS, to neuropathology immunohistochemistry was performed on brain slices from WT We next evaluated monocytes and neutrophils within the CNS and Ccr5-deficient mice on days 8 and 12 postinfection. Staining because these cell populations were shown to accumulate in the for myeloperoxidase (MPO), an enzyme abundantly expressed by CNS during WNVand JEV infection (13, 28, 32). In the absence of neutrophils, was similar on day 8 postinfection in the two strains, Ccr5, the number of inflammatory monocytes was similar in WT but it was markedly increased in the brains of Ccr5-deficient mice and Ccr5-deficient mice (Fig. 4A, 4B). Consistent with this result, on day 12, including the meninges and the cortex (Fig. 5A). Taken no significant differences were observed in the protein levels of together, these data confirm that neutrophil infiltration into the Ccl2 and Ccl7, known chemoattractants for inflammatory mono- brain is increased in the absence of Ccr5 during the late phase of cytes (Fig. 4C, 4D) (33). Evaluation of neutrophils in the infected LGTV infection. CNS revealed similar numbers on day 8 postinfection, but by day Previous studies showed that flaviviruses, including LGTV, can 12 postinfection, there was a 2–3-fold increase in the total number induce apoptosis of infected cells in vitro, suggesting a mechanism 2 2 of neutrophils in the brains of Ccr5 / mice (Fig. 4E, 4F). by which damage to neurons within the CNS can occur (34–37). Evaluation of the neutrophil-attracting chemokines Cxcl1 and Given that Ccr52/2 mice have increased viral replication within Cxcl2 within the CNS also showed significantly elevated levels in the CNS, we hypothesized that this may lead to a significant increase the absence of Ccr5, specifically on day 12 postinfection (Fig. 4G, in apoptotic cells. To test this, we evaluated WT and Ccr52/2 CNS 4626 CCR5 IS CRITICAL FOR LGTV-INDUCED ENCEPHALITIS

FIGURE 3. T and NK cell infiltration into the CNS is impaired in the absence of Ccr5 during LGTV infection. The total number of CD4+ T cells (CD45hiCD3+ CD4+)(A), CD8+ T cells (CD45hiCD3+ CD8+)(C), and NK cells (CD45hiNK1.1+ CD32)(E) was assessed by flow cytom- etry in the brains of WT and Ccr52/2 mice. Data are mean 6 SD for three to nine mice/genotype and time point Downloaded from from two independent experiments. Rep- resentative flow cytometry plots on day 8 postinfection for CD4+ T cells (B), CD8+ T cells (D), and NK cells (F). Cells were gated on live singlet CD45hi cells. (G) Paraffin-embedded brain sections from http://www.jimmunol.org/ WT and Ccr52/2 mice were stained for CD3. Positive cells (brown cells, indi- cated by arrows) are shown within the cerebellum of LGTV-infected mice on days 8 and 12 postinfection for three to five mice/genotype from two inde- pendent experiments (original magni- fication 320, including for the insets). *p , 0.05, ***p , 0.001. by guest on October 1, 2021

tissue for apoptosis using TUNEL staining. On day 8 postinfection, mortality observed in Ccr52/2 mice (38–41). To determine the when viral loads are similar in WT and Ccr52/2 mice, the number of relative contribution of T cells to survival, we adoptively trans- apoptotic cells was nearly identical in both strains. By day 12 post- ferred T cells isolated from the spleens of LGTV-infected WT or infection, there was a significant increase in the number of apoptotic Ccr52/2 donors into LGTV-infected Ccr5-/- recipients 5 d postin- cells in the CNS of Ccr5-deficient mice, which we observed in var- fection and evaluated survival (Fig. 6A). LGTV-infected Ccr52/2 ious regions of the CNS, including the cortex and meninges (Fig. 5B). recipients receiving WT T cells showed a significant improvement in survival compared with Ccr52/2 recipients receiving Ccr52/2 Survival during LGTV encephalitis involves promoting T cells Tcells(p , 0.05, Fig. 6B). This increase in survival was accom- and reducing neutrophils in the CNS panied by an increase in CD3+ T cells accumulating in the CNS of Thus far, our data suggest that Ccr5 may regulate the efficient Ccr52/2 recipients receiving WT T cells in comparison with Ccr52/2 migration of effector lymphocytes (T and NK cells) into the CNS. recipients receiving Ccr52/2 T cells (Fig. 6C). Although CCR5- In the absence of Ccr5, the delayed migration of these cells likely expressing T cells appeared to provide a survival benefit, protection results in increased viral replication that coincides with aberrant was not completely restored to WT levels, suggesting that other production of neutrophil-associated chemokines, leading to in- factors are important for Ccr5-mediated survival. creased neutrophil influx and associated immunopathology. As a We next evaluated the impact of neutrophil migration into the result of the known antiviral role of T cells in neuroprotection and CNS. Because neutrophil numbers in the CNS were nearly identical the known role of neutrophils in neuropathology, we hypothesized in WT and Ccr52/2 mice on day 8 postinfection, and they differed that T cells and neutrophils may be involved in the increased only on day 12 postinfection, we depleted neutrophils starting on The Journal of Immunology 4627

FIGURE 4. Neutrophil numbers are increased in the CNS in the absence of Ccr5. The total number of monocytes (CD45hiCD11b+Ly6ChiLy6G2)(A)and neutrophils (CD45hiCD11b+Ly6Cint Ly6G+)(E) in the brain of uninfected and LGTV-infected WT and Ccr52/2 mice on days 8 and 12 postinfection were assessed by flow cytometry. Rep- Downloaded from resentative flow cytometry plots from day 12 postinfection for monocytes (B) and neutrophils (F). Cells were gated on live singlet CD45hi cells. Neutrophils were additionally gated on CD11b+ and Ly6Cint cells. The protein levels of monocyte chemo- http://www.jimmunol.org/ attractants Ccl2 (C) and Ccl7 (D)and neutrophil chemoattractants Cxcl1 (G) and Cxcl2 (H) were measured by multiplex ELISA. All data are mean 6 SD with 4–10 mice/time point and genotype and were pooled from two independent experiments. *p , 0.05, Student t test. by guest on October 1, 2021

day 8 postinfection, after the onset of weight loss and symptoms. with MPO showed no reactivity in 1A8-treated mouse brains in To deplete neutrophils, we treated LGTV-infected Ccr52/2 mice comparison with isotype control–treated mice, which exhibited with the mAb Ly6G (clone 1A8) or isotype control on days 8, 10, prominent neutrophil staining (Fig. 6G). To determine the extent and 12 postinfection. An ∼99% depletion of neutrophils was of neuropathology in the brain, a TUNEL assay was performed. achieved that lasted 48 h, as shown previously(42–44). Although Staining revealed that the number of apoptotic cells within the weight loss was observed in both groups of mice starting on day brain was lower in neutrophil-depleted mice compared with con- 7 postinfection, it was significantly less pronounced in the trols, suggesting that the presence of neutrophils in the CNS is neutrophil-depleted mice on days 10–12 postinfection compared damaging (Fig. 6H–J, left panels). Apoptotic cells were observed with isotype control–treated mice (Fig. 6D). Accordingly, 1A8- in various regions within the CNS, including the meninges treated mice exhibited fewer signs of illness and showed improved (Fig. 6H), hippocampus (Fig. 6I), and cortex (Fig. 6J). We next survival outcome, with a survival rate of 75% versus 35% for the determined whether regions of apoptosis were also regions of vi- isotype control–treated mice (Fig. 6E, p , 0.05). Interestingly, ral replication. To test this, serial brain sections of isotype evaluation of the brain tissue showed an ∼12-fold decrease in viral control– and 1A8-treated mice were also stained for LGTV. We titers in neutropenic mice (p = 0.034) compared with isotype found that LGTV-infected cells were in close proximity to control–treated mice (Fig. 6F). TUNEL+ cells in various parts of the CNS (Fig. 6H–J, right To further characterize this phenotype, we conducted immu- panels). There were fewer LGTV-infected cells in neutrophil- nohistochemical analysis of brains from 1A8- or isotype control– depleted mice, consistent with the decrease in viral titers, which treated mice. Consistent with the flow cytometry data, staining correlated with an overall decrease in apoptosis within the tissue. 4628 CCR5 IS CRITICAL FOR LGTV-INDUCED ENCEPHALITIS

models of WNVor JEV. Given these results in mice, our data have implications for individuals deficient for CCR5, who are likely at higher risk for developing symptomatic TBEV infection. CCR5- deficient cohorts include individuals with genetically altered CCR5 function, including CCR5D32 homozygotes found in ∼1% of individuals of European decent (45), as well as individuals prescribed the U.S. Food and Drug Administration–approved CCR5 antagonist maraviroc for the treatment of HIV infection and other chronic inflammatory conditions (46, 47). In fact, several human cohort studies investigated the association of CCR5D32 and TBEV. Two studies showed that the frequency of CCR5D32 homozygotes was elevated among TBE patients, whereas a third study was unable to find an association (48–50). Our studies with LGTV in mice support a protective role for CCR5 during tick- borne flavivirus infections. Because Ccr5 is expressed primarily on activated subsets of T cells, a large percentage of the infiltrating, virus-specific T cells within the CNS is likely to express this receptor. A previous study showed that a small percentage of T cells expresses Ccr5 in the steady-state and that expression increases 10-fold following in- Downloaded from flammation (10, 51). In humans, CCR5 expression was shown to increase significantly following infection (51). Functionally, the infiltration of activated CD4+ and CD8+ T cells into the CNS was shown to be critical for controlling viral repli- cation in the context of WNV and JEV. In these studies, CD8+ T cells strongly correlated with increased survival through http://www.jimmunol.org/ perforin- and Fas-mediated mechanisms (40, 52). Similarly, CD4+ T cells are critical for survival because the absence of these cells in mice resulted in persistent infection in the CNS, ultimately leading to uniform mortality (53). CCR5 is expressed on a large proportion of T cells found in the CSF of TBEV-infected indi- viduals (54). However, the role of T cells during TBEV infection is unclear; in the context of infection with the Central European subtype, T cells appear to be immunopathogenic because CD8- by guest on October 1, 2021 deficient mice or SCID mice have a significant survival advantage (41). The data generated in this study do not support a pathogenic role for T cells; rather, they suggest that T cells within the CNS FIGURE 5. Enhanced neutrophil accumulation in the brain is accom- promote survival, consistent with data generated in WNV and JEV panied by increased apoptosis in Ccr52/2 mice during LGTV-induced infection models. encephalitis. Paraffin-embedded brain sections of LGTV-infected mice on The role of neutrophils during flavivirus infections is poorly days 8 and 12 postinfection were stained for MPO (A) and apoptosis (B) defined; however, the presence of neutrophils into the CNS was using TUNEL assay for three to five mice/genotype and condition from demonstrated in numerous models of flavivirus infections, in- two independent experiments. Arrows indicate the positive cells (brown cluding Murray Valley encephalitis virus, TBEV, and WNV (28, 3 staining) within the cortex and meninges (original magnification 20, 55, 56). Our data suggest that neutrophils promote viral replication including for the insets). and pathology by shuttling virus into the CNS. Support for this model comes from our data showing that increased neutrophils in the CNS correlate with greater viral replication and apoptosis in Together, our data suggest that neutrophils promote viral repli- the brain tissue, a phenotype that is reversed following neutrophil cation within the CNS, drive apoptosis, and negatively impact depletion. This model is supported by another published report survival. showing that neutrophils are cellular targets for infection with TBEV and also undergo apoptosis following infection (35). Discussion Similarly, neutrophils are readily infected ex vivo by the related One of the hallmarks of viral encephalitis is the influx of leukocytes virus WNV and may serve as an important reservoir of virus into the CNS to clear virus and aid recovery. This process can also replication in vivo (57). Furthermore, a recent study showed cause collateral damage, particularly in delicate tissues, such as the that neutrophils have a detrimental role in an ocular model of brain, by causing the destruction of irreparable neurons and glial HSV-1 infection, where neutrophils serve as a conduit for virus cells. In this study, we demonstrate that Ccr5 regulates this balance replication (58). Thus, our data align with the idea that neu- by coordinating the efficient migration of effector T cells and NK trophils contribute to pathology through their function as a cells into the CNS. In the absence of Ccr5, virus is inefficiently reservoir for and presumably through immune-mediated controlled at the early time points because of a lag in lymphocyte mechanisms. migration. This tips the balance toward greater virus replication, One limitation of our study is that we do not know which cells causing an excess production of chemokines, the subsequent in- within the CNS are undergoing apoptosis. Within the CNS, neurons filtration of neutrophils, and induction of neuroimmunopathology, are the primary target for infection by TBEVand undergo apoptosis an unanticipated phenotype that has not been described to occur in (59–62). Although astrocytes can also become infected, these cells The Journal of Immunology 4629 Downloaded from http://www.jimmunol.org/

FIGURE 6. Early recruitment of T cells and the absence of neutrophils in the CNS of LGTV-infected Ccr5-deficient mice result in decreased neuropathology. (A) Schematic diagram of the experimental layout for the T cell–adoptive transfer experiment. Donor mice (WT or Ccr52/2)were infected with LGTV, and T cells were isolated from spleens harvested on day 8 postinfection. A total of 1 3 106 T cells was transferred i.v. into LGTV-infected recipient mice (Ccr52/2 mice) on day 5 postinfection, and mice were evaluated for survival for 18 d. (B) Kaplan–Meier analysis of recipient mice (18–20 Ccr52/2 mice/group). WT→Ccr52/2 mice received T cells from WT mice, and Ccr52/2→Ccr52/2 mice received T cells from Ccr52/2 mice. (C)ThetotalnumberofCD3+ T cells (CD45hiCD3+) was assessed by flow cytometry on day 8 postinfection in the by guest on October 1, 2021 brains of Ccr52/2 mice receiving WT or Ccr52/2 CD3+ T cells. Data are mean 6 SDforfourtosixmicepergroup.(D) Body weight was measured daily for isotype-treated and anti-Ly6G (clone 1A8)-treated LGTV-infected mice for 12 d postinfection (16–17 mice/group pooled from two independent experiments). (E) Ccr52/2 mice were injected with isotype or 1A8 Ab, and survival was assessed by a Kaplan–Meier analysis through day 18 postinfection (16–17 mice/group pooled from two independent experiments). The arrows in (D)and(E) indicate the days on which the Ab was administered. (F) The viral load was quantified in the brains of isotype- or 1A8 Ab–treated mice on day 12 postinfection. Data are mean 6 SD from seven or eight mice/group on day 12 postinfection from two independent experiments. (G) Immunohistochemical analysis of MPO was performed on paraffin-embedded brain sections from mice treated with isotype or 1A8 Ab on day 12 postinfection after staining for apoptosis (TUNEL stain, left panels)orLGTV(right panels) in the meninges (H), hippocampus (I), and deep cortical layers (J) for three to five mice/genotype and condition from two independent experiments (original magnification 320, including for the insets). *p , 0.05.

appear to be primarily involved in cytokine production and do that neutrophil depletion during symptom onset would promote not undergo morphological changes or apoptosis (63, 64). We recovery following TBEV infection in humans and, in fact, may showed that LGTV Ag colocalized to areas of apoptotic cells; be beneficial because neutrophils in the CNS, viral load, and based on the literature, these infected cells may primarily be damage are positively correlated and intimately coupled. Be- neurons and/or neutrophils (35, 59–62). Our study also did not cause no specific antivirals or immunomodulatory therapies address the mechanism by which neutrophils function within the exist for the treatment of individuals infected with any neuro- CNS. Although the CNS injury observed in the current study tropic flavivirus, our findings provide novel directions with may be caused directly by viral infection, neuronal destruction regard to immunomodulators of the host response, which are was attributed to an indirect effect of infiltrating immune cells greatly needed. and production of inflammatory cytokines (4, 59, 65). Therefore neutrophil-mediated indirect destruction of cells in the brain cannot be excluded. Recent studies showed that neutrophils Acknowledgments promote Alzheimer’s disease–like pathology in the brain We thank the Flow Cytometry Shared Resource Facility, the Center for through the release of neutrophil extracellular traps (66). Comparative Medicine and Surgery, and the Microscopy CORE at Icahn Therefore, neutrophils may be further mediating brain damage School of Medicine at Mount Sinai for technical assistance. through the release of neutrophil extracellular traps and/or re- active oxygen/nitrogen intermediates during tick-borne flavivi- Disclosures rus infection. From a translational standpoint, our data suggest The authors have no financial conflicts of interest. 4630 CCR5 IS CRITICAL FOR LGTV-INDUCED ENCEPHALITIS

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