CSF1R antagonism limits local restimulation of antiviral CD8+ T cells during viral encephalitis
Identifieur interne : 000283 ( Pmc/Corpus ); précédent : 000282; suivant : 000284CSF1R antagonism limits local restimulation of antiviral CD8+ T cells during viral encephalitis
Auteurs : Kristen E. Funk ; Robyn S. KleinSource :
- Journal of Neuroinflammation [ 1742-2094 ] ; 2019.
Abstract
Microglia are resident macrophages of the central nervous system (CNS) locally maintained through colony-stimulating factor 1 receptor (CSF1R) signaling. Microglial depletion via CSF1R inactivation improves cognition in mouse models of neuroinflammation, but limits virologic control in the CNS of mouse models of neurotropic infections by unknown mechanisms. We hypothesize that CSF1R plays a critical role in myeloid cell responses that restrict viral replication and locally restimulate recruited antiviral T cells within the CNS.
The impact of CSF1R signaling during West Nile virus infection was assessed in vivo using a mouse model of neurotropic infection. Pharmacological inactivation of CSF1R was achieved using PLX5622 prior to infection with virulent or attenuated strains of West Nile virus (WNV), an emerging neuropathogen. The subsequent effect of CSF1R antagonism on virologic control was assessed by measuring mortality and viral titers in the CNS and peripheral organs. Immune responses were assessed by flow cytometric-based phenotypic analyses of both peripheral and CNS immune cells.
Mice treated with CSF1R antagonist prior to infection exhibited higher susceptibility to lethal WNV infection and lack of virologic control in both the CNS and periphery. CSFR1 antagonism reduced B7 co-stimulatory signals on peripheral and CNS antigen-presenting cells (APCs) by depleting CNS cellular sources, which limited local reactivation of CNS-infiltrating virus-specific T cells and reduced viral clearance.
Our results demonstrate the impact of CSF1R antagonism on APC activation in the CNS and periphery and the importance of microglia in orchestrating the CNS immune response following neurotropic viral infection. These data will be an important consideration when assessing the benefit of CSF1R antagonism, which has been investigated as a therapeutic for neurodegenerative conditions, in which neuroinflammation is a contributing factor.
The online version of this article (10.1186/s12974-019-1397-4) contains supplementary material, which is available to authorized users.
Url:
DOI: 10.1186/s12974-019-1397-4
PubMed: 30704498
PubMed Central: 6354430
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PMC:6354430Le document en format XML
<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">CSF1R antagonism limits local restimulation of antiviral CD8<sup>+</sup> T cells during viral encephalitis</title><author><name sortKey="Funk, Kristen E" sort="Funk, Kristen E" uniqKey="Funk K" first="Kristen E." last="Funk">Kristen E. Funk</name><affiliation><nlm:aff id="Aff1"><institution-wrap><institution-id institution-id-type="ISNI">0000 0001 2355 7002</institution-id><institution-id institution-id-type="GRID">grid.4367.6</institution-id><institution>Department of Internal Medicine, Division of Infectious Diseases,</institution><institution>Washington University School of Medicine,</institution></institution-wrap>Saint Louis, MO 63110 USA</nlm:aff></affiliation></author><author><name sortKey="Klein, Robyn S" sort="Klein, Robyn S" uniqKey="Klein R" first="Robyn S." last="Klein">Robyn S. Klein</name><affiliation><nlm:aff id="Aff1"><institution-wrap><institution-id institution-id-type="ISNI">0000 0001 2355 7002</institution-id><institution-id institution-id-type="GRID">grid.4367.6</institution-id><institution>Department of Internal Medicine, Division of Infectious Diseases,</institution><institution>Washington University School of Medicine,</institution></institution-wrap>Saint Louis, MO 63110 USA</nlm:aff></affiliation><affiliation><nlm:aff id="Aff2"><institution-wrap><institution-id institution-id-type="ISNI">0000 0001 2355 7002</institution-id><institution-id institution-id-type="GRID">grid.4367.6</institution-id><institution>Department of Pathology and Immunology,</institution><institution>Washington University School of Medicine,</institution></institution-wrap>Saint Louis, MO 63110 USA</nlm:aff></affiliation><affiliation><nlm:aff id="Aff3"><institution-wrap><institution-id institution-id-type="ISNI">0000 0001 2355 7002</institution-id><institution-id institution-id-type="GRID">grid.4367.6</institution-id><institution>Department of Neurosciences,</institution><institution>Washington University School of Medicine,</institution></institution-wrap>Saint Louis, MO 63110 USA</nlm:aff></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">30704498</idno><idno type="pmc">6354430</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6354430</idno><idno type="RBID">PMC:6354430</idno><idno type="doi">10.1186/s12974-019-1397-4</idno><date when="2019">2019</date><idno type="wicri:Area/Pmc/Corpus">000283</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">000283</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">CSF1R antagonism limits local restimulation of antiviral CD8<sup>+</sup> T cells during viral encephalitis</title><author><name sortKey="Funk, Kristen E" sort="Funk, Kristen E" uniqKey="Funk K" first="Kristen E." last="Funk">Kristen E. Funk</name><affiliation><nlm:aff id="Aff1"><institution-wrap><institution-id institution-id-type="ISNI">0000 0001 2355 7002</institution-id><institution-id institution-id-type="GRID">grid.4367.6</institution-id><institution>Department of Internal Medicine, Division of Infectious Diseases,</institution><institution>Washington University School of Medicine,</institution></institution-wrap>Saint Louis, MO 63110 USA</nlm:aff></affiliation></author><author><name sortKey="Klein, Robyn S" sort="Klein, Robyn S" uniqKey="Klein R" first="Robyn S." last="Klein">Robyn S. Klein</name><affiliation><nlm:aff id="Aff1"><institution-wrap><institution-id institution-id-type="ISNI">0000 0001 2355 7002</institution-id><institution-id institution-id-type="GRID">grid.4367.6</institution-id><institution>Department of Internal Medicine, Division of Infectious Diseases,</institution><institution>Washington University School of Medicine,</institution></institution-wrap>Saint Louis, MO 63110 USA</nlm:aff></affiliation><affiliation><nlm:aff id="Aff2"><institution-wrap><institution-id institution-id-type="ISNI">0000 0001 2355 7002</institution-id><institution-id institution-id-type="GRID">grid.4367.6</institution-id><institution>Department of Pathology and Immunology,</institution><institution>Washington University School of Medicine,</institution></institution-wrap>Saint Louis, MO 63110 USA</nlm:aff></affiliation><affiliation><nlm:aff id="Aff3"><institution-wrap><institution-id institution-id-type="ISNI">0000 0001 2355 7002</institution-id><institution-id institution-id-type="GRID">grid.4367.6</institution-id><institution>Department of Neurosciences,</institution><institution>Washington University School of Medicine,</institution></institution-wrap>Saint Louis, MO 63110 USA</nlm:aff></affiliation></author></analytic><series><title level="j">Journal of Neuroinflammation</title><idno type="eISSN">1742-2094</idno><imprint><date when="2019">2019</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><sec><title>Background</title><p id="Par1">Microglia are resident macrophages of the central nervous system (CNS) locally maintained through colony-stimulating factor 1 receptor (CSF1R) signaling. Microglial depletion via CSF1R inactivation improves cognition in mouse models of neuroinflammation, but limits virologic control in the CNS of mouse models of neurotropic infections by unknown mechanisms. We hypothesize that CSF1R plays a critical role in myeloid cell responses that restrict viral replication and locally restimulate recruited antiviral T cells within the CNS.</p></sec><sec><title>Methods</title><p id="Par2">The impact of CSF1R signaling during West Nile virus infection was assessed in vivo using a mouse model of neurotropic infection. Pharmacological inactivation of CSF1R was achieved using PLX5622 prior to infection with virulent or attenuated strains of West Nile virus (WNV), an emerging neuropathogen. The subsequent effect of CSF1R antagonism on virologic control was assessed by measuring mortality and viral titers in the CNS and peripheral organs. Immune responses were assessed by flow cytometric-based phenotypic analyses of both peripheral and CNS immune cells.</p></sec><sec><title>Results</title><p id="Par3">Mice treated with CSF1R antagonist prior to infection exhibited higher susceptibility to lethal WNV infection and lack of virologic control in both the CNS and periphery. CSFR1 antagonism reduced B7 co-stimulatory signals on peripheral and CNS antigen-presenting cells (APCs) by depleting CNS cellular sources, which limited local reactivation of CNS-infiltrating virus-specific T cells and reduced viral clearance.</p></sec><sec><title>Conclusions</title><p id="Par4">Our results demonstrate the impact of CSF1R antagonism on APC activation in the CNS and periphery and the importance of microglia in orchestrating the CNS immune response following neurotropic viral infection. These data will be an important consideration when assessing the benefit of CSF1R antagonism, which has been investigated as a therapeutic for neurodegenerative conditions, in which neuroinflammation is a contributing factor.</p></sec><sec><title>Electronic supplementary material</title><p>The online version of this article (10.1186/s12974-019-1397-4) contains supplementary material, which is available to authorized users.</p></sec></div></front><back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="Petersen, Lr" uniqKey="Petersen L">LR Petersen</name></author><author><name sortKey="Carson, Pj" uniqKey="Carson P">PJ Carson</name></author><author><name sortKey="Biggerstaff, Bj" uniqKey="Biggerstaff B">BJ Biggerstaff</name></author><author><name sortKey="Custer, B" uniqKey="Custer B">B Custer</name></author><author><name sortKey="Borchardt, Sm" uniqKey="Borchardt S">SM Borchardt</name></author><author><name sortKey="Busch, Mp" uniqKey="Busch M">MP 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<front><journal-meta><journal-id journal-id-type="nlm-ta">J Neuroinflammation</journal-id><journal-id journal-id-type="iso-abbrev">J Neuroinflammation</journal-id><journal-title-group><journal-title>Journal of Neuroinflammation</journal-title></journal-title-group><issn pub-type="epub">1742-2094</issn><publisher><publisher-name>BioMed Central</publisher-name><publisher-loc>London</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">30704498</article-id><article-id pub-id-type="pmc">6354430</article-id><article-id pub-id-type="publisher-id">1397</article-id><article-id pub-id-type="doi">10.1186/s12974-019-1397-4</article-id><article-categories><subj-group subj-group-type="heading"><subject>Research</subject></subj-group></article-categories><title-group><article-title>CSF1R antagonism limits local restimulation of antiviral CD8<sup>+</sup> T cells during viral encephalitis</article-title></title-group><contrib-group><contrib contrib-type="author"><name><surname>Funk</surname><given-names>Kristen E.</given-names></name><address><email>kristenfunk@wustl.edu</email></address><xref ref-type="aff" rid="Aff1">1</xref></contrib><contrib contrib-type="author" corresp="yes"><name><surname>Klein</surname><given-names>Robyn S.</given-names></name><address><email>rklein@wustl.edu</email></address><xref ref-type="aff" rid="Aff1">1</xref><xref ref-type="aff" rid="Aff2">2</xref><xref ref-type="aff" rid="Aff3">3</xref></contrib><aff id="Aff1"><label>1</label><institution-wrap><institution-id institution-id-type="ISNI">0000 0001 2355 7002</institution-id><institution-id institution-id-type="GRID">grid.4367.6</institution-id><institution>Department of Internal Medicine, Division of Infectious Diseases,</institution><institution>Washington University School of Medicine,</institution></institution-wrap>Saint Louis, MO 63110 USA</aff><aff id="Aff2"><label>2</label><institution-wrap><institution-id institution-id-type="ISNI">0000 0001 2355 7002</institution-id><institution-id institution-id-type="GRID">grid.4367.6</institution-id><institution>Department of Pathology and Immunology,</institution><institution>Washington University School of Medicine,</institution></institution-wrap>Saint Louis, MO 63110 USA</aff><aff id="Aff3"><label>3</label><institution-wrap><institution-id institution-id-type="ISNI">0000 0001 2355 7002</institution-id><institution-id institution-id-type="GRID">grid.4367.6</institution-id><institution>Department of Neurosciences,</institution><institution>Washington University School of Medicine,</institution></institution-wrap>Saint Louis, MO 63110 USA</aff></contrib-group><pub-date pub-type="epub"><day>31</day><month>1</month><year>2019</year></pub-date><pub-date pub-type="pmc-release"><day>31</day><month>1</month><year>2019</year></pub-date><pub-date pub-type="collection"><year>2019</year></pub-date><volume>16</volume><elocation-id>22</elocation-id><history><date date-type="received"><day>24</day><month>9</month><year>2018</year></date><date date-type="accepted"><day>2</day><month>1</month><year>2019</year></date></history><permissions><copyright-statement>© The Author(s). 2019</copyright-statement><license license-type="OpenAccess"><license-p><bold>Open Access</bold>This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (<ext-link ext-link-type="uri" xlink:href="http://creativecommons.org/licenses/by/4.0/">http://creativecommons.org/licenses/by/4.0/</ext-link>), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (<ext-link ext-link-type="uri" xlink:href="http://creativecommons.org/publicdomain/zero/1.0/">http://creativecommons.org/publicdomain/zero/1.0/</ext-link>) applies to the data made available in this article, unless otherwise stated.</license-p></license></permissions><abstract id="Abs1"><sec><title>Background</title><p id="Par1">Microglia are resident macrophages of the central nervous system (CNS) locally maintained through colony-stimulating factor 1 receptor (CSF1R) signaling. Microglial depletion via CSF1R inactivation improves cognition in mouse models of neuroinflammation, but limits virologic control in the CNS of mouse models of neurotropic infections by unknown mechanisms. We hypothesize that CSF1R plays a critical role in myeloid cell responses that restrict viral replication and locally restimulate recruited antiviral T cells within the CNS.</p></sec><sec><title>Methods</title><p id="Par2">The impact of CSF1R signaling during West Nile virus infection was assessed in vivo using a mouse model of neurotropic infection. Pharmacological inactivation of CSF1R was achieved using PLX5622 prior to infection with virulent or attenuated strains of West Nile virus (WNV), an emerging neuropathogen. The subsequent effect of CSF1R antagonism on virologic control was assessed by measuring mortality and viral titers in the CNS and peripheral organs. Immune responses were assessed by flow cytometric-based phenotypic analyses of both peripheral and CNS immune cells.</p></sec><sec><title>Results</title><p id="Par3">Mice treated with CSF1R antagonist prior to infection exhibited higher susceptibility to lethal WNV infection and lack of virologic control in both the CNS and periphery. CSFR1 antagonism reduced B7 co-stimulatory signals on peripheral and CNS antigen-presenting cells (APCs) by depleting CNS cellular sources, which limited local reactivation of CNS-infiltrating virus-specific T cells and reduced viral clearance.</p></sec><sec><title>Conclusions</title><p id="Par4">Our results demonstrate the impact of CSF1R antagonism on APC activation in the CNS and periphery and the importance of microglia in orchestrating the CNS immune response following neurotropic viral infection. These data will be an important consideration when assessing the benefit of CSF1R antagonism, which has been investigated as a therapeutic for neurodegenerative conditions, in which neuroinflammation is a contributing factor.</p></sec><sec><title>Electronic supplementary material</title><p>The online version of this article (10.1186/s12974-019-1397-4) contains supplementary material, which is available to authorized users.</p></sec></abstract><kwd-group xml:lang="en"><title>Keywords</title><kwd>West Nile virus</kwd><kwd>Microglia</kwd><kwd>Antiviral T cells</kwd><kwd>Viral encephalitis</kwd><kwd>Colony-stimulating factor 1 receptor</kwd><kwd>PLX5622</kwd></kwd-group><funding-group><award-group><funding-source><institution-wrap><institution-id institution-id-type="FundRef">http://dx.doi.org/10.13039/100000060</institution-id><institution>National Institute of Allergy and Infectious Diseases</institution></institution-wrap></funding-source><award-id>R01 NS052632</award-id></award-group></funding-group><funding-group><award-group><funding-source><institution-wrap><institution-id institution-id-type="FundRef">http://dx.doi.org/10.13039/100000002</institution-id><institution>National Institutes of Health</institution></institution-wrap></funding-source><award-id>R01 AI101400</award-id><award-id>K99 AG053412</award-id><principal-award-recipient><name><surname>Funk</surname><given-names>Kristen E.</given-names></name></principal-award-recipient></award-group></funding-group><funding-group><award-group><funding-source><institution-wrap><institution-id institution-id-type="FundRef">http://dx.doi.org/10.13039/100000124</institution-id><institution>Office of Research on Women's Health</institution></institution-wrap></funding-source><award-id>U19 AI083019</award-id></award-group></funding-group><custom-meta-group><custom-meta><meta-name>issue-copyright-statement</meta-name><meta-value>© The Author(s) 2019</meta-value></custom-meta></custom-meta-group></article-meta></front><body><sec id="Sec1"><title>Background</title><p id="Par16">West Nile virus (WNV) is an emerging human pathogen that causes seasonal outbreaks through the Western hemisphere since its introduction in 1999 [<xref ref-type="bibr" rid="CR1">1</xref>]. A member of the <italic>Flavivirus</italic> genus, WNV is an enveloped, single-stranded positive sense RNA virus that cycles between birds and mosquitos. Symptoms of WNV infection can range from a relatively mild flu-like illness, West Nile fever, to severe neuroinvasive disease. After peripheral infection, WNV replicates within lymphoid tissues before entering the CNS, where it targets neurons in the cerebellum, brainstem, and cerebral cortex [<xref ref-type="bibr" rid="CR2">2</xref>, <xref ref-type="bibr" rid="CR3">3</xref>]. In 2017, 2002 symptomatic WNV cases were reported to the CDC, and of those, 1339 (67%) were classified as neuroinvasive [<xref ref-type="bibr" rid="CR4">4</xref>]. Prolonged neurological deficits, which include motor functions, verbal learning and memory, and executive functions, are a significant problem for survivors of WNV infection and appear to manifest even in cases where neuroinvasion is absent [<xref ref-type="bibr" rid="CR5">5</xref>–<xref ref-type="bibr" rid="CR7">7</xref>]. Recent work indicates that persistent phagocytic microglia contribute to prolonged cognitive dysfunction following WNV encephalitis [<xref ref-type="bibr" rid="CR8">8</xref>], and thus, limiting microglial activation may alleviate the associated neurological sequelae.</p><p id="Par17">Microglia are the resident immune cells in the CNS, originating from yolk sac progenitor cells that seed the brain early in development [<xref ref-type="bibr" rid="CR9">9</xref>]. In addition to a multitude of homeostatic functions throughout development and healthy aging [<xref ref-type="bibr" rid="CR10">10</xref>, <xref ref-type="bibr" rid="CR11">11</xref>], microglia participate in both innate and adaptive immune responses through recognition of pathogen-associated molecular patterns and damage-associated molecular patterns [<xref ref-type="bibr" rid="CR12">12</xref>], which stimulate morphological changes [<xref ref-type="bibr" rid="CR13">13</xref>, <xref ref-type="bibr" rid="CR14">14</xref>] and increase production of reactive oxidative species [<xref ref-type="bibr" rid="CR15">15</xref>, <xref ref-type="bibr" rid="CR16">16</xref>] and cytokines/chemokines [<xref ref-type="bibr" rid="CR17">17</xref>, <xref ref-type="bibr" rid="CR18">18</xref>]. During neurotropic virus infection, virally infected cells secrete chemokines including CCL2, CCL5, and CXCL10, which facilitate immune cells crossing the blood brain barrier (BBB) [<xref ref-type="bibr" rid="CR19">19</xref>–<xref ref-type="bibr" rid="CR21">21</xref>]. Mononuclear cell infiltration into the CNS is a hallmark of WNV encephalitis, and monocytes and T cells are critical for CNS virologic control. Elimination of monocytes using a liposome-encapsulated drug dichloromethylene diphosphonate or by genetic deletion of <italic>CCR2</italic> increases mortality in mice infected with a neurotropic strain of WNV [<xref ref-type="bibr" rid="CR22">22</xref>, <xref ref-type="bibr" rid="CR23">23</xref>]. However, monocytes may also contribute to dissemination of virus to the CNS [<xref ref-type="bibr" rid="CR24">24</xref>, <xref ref-type="bibr" rid="CR25">25</xref>], and inhibition of monocyte infiltration may also protect against nitric oxide-mediated immunopathology [<xref ref-type="bibr" rid="CR26">26</xref>]. CD4<sup>+</sup> T cells produce antiviral molecules, such as interferon γ (IFNγ) and IL-2, which promotes antiviral humoral immunity and sustains WNV-specific CD8<sup>+</sup> T cell responses, enabling viral clearance in all organs [<xref ref-type="bibr" rid="CR27">27</xref>, <xref ref-type="bibr" rid="CR28">28</xref>]. CD8<sup>+</sup> T cells contribute to viral clearance and recovery from WNV infection via both cytopathic and noncytopathic mechanisms [<xref ref-type="bibr" rid="CR29">29</xref>–<xref ref-type="bibr" rid="CR32">32</xref>]. Mice lacking either CD8<sup>+</sup> T cells or MHC-I molecules have higher CNS viral burdens, increased mortality after infection, and prolonged viral persistence, suggesting that cell-mediated immunity controls virus within the CNS [<xref ref-type="bibr" rid="CR32">32</xref>].</p><p id="Par18">CSF1 receptor (CSF1R) signaling is essential for the development of many mononuclear phagocytes including microglia. Tissue-resident macrophages, such as microglia, fail to develop in <italic>Csf1r</italic><sup>−/−</sup> mice [<xref ref-type="bibr" rid="CR9">9</xref>], but in contrast to many tissue macrophages, adult microglia can still form in <italic>Csf1</italic><sup><italic>op/op</italic></sup> mice, which carry a natural null mutation in the <italic>Csf1</italic> gene [<xref ref-type="bibr" rid="CR33">33</xref>]. This is due to an alternative CSF1R ligand, IL-34, which is highly expressed by neurons [<xref ref-type="bibr" rid="CR34">34</xref>, <xref ref-type="bibr" rid="CR35">35</xref>]. Similar to <italic>Csf1</italic><sup><italic>op/op</italic></sup> mice, <italic>IL-34</italic><sup><italic>−/−</italic></sup> mice develop fewer microglia but do not lack peripheral macrophages [<xref ref-type="bibr" rid="CR36">36</xref>]. These mice are more susceptible to lethal neurotropic WNV infection, which is associated with increased neuronal apoptosis without increased viral replication [<xref ref-type="bibr" rid="CR36">36</xref>]. In order to temporally control microglial depletion, pharmacologic agents that antagonize CSF1R have been developed, including antagonist PLX5622, which depletes microglia in as little as 3 days and may be sustained for at least 6 weeks [<xref ref-type="bibr" rid="CR37">37</xref>]. These agents are being investigated as therapeutics in models of neurodegenerative diseases in which microglial activation is considered a contributing factor, such as traumatic brain injury [<xref ref-type="bibr" rid="CR38">38</xref>, <xref ref-type="bibr" rid="CR39">39</xref>], experimental autoimmune encephalomyelitis [<xref ref-type="bibr" rid="CR40">40</xref>], and Alzheimer’s disease [<xref ref-type="bibr" rid="CR41">41</xref>]. Here, we used PLX5622 in a model of WNV encephalitis to examine the effect of CSF1R antagonism in the setting of neurotropic viral infection. Our results show that mice treated with PLX5622 are more susceptible to lethal WNV infection, which is associated with increased CNS viral burden and neuronal apoptosis. PLX5622 also reduced expression of proinflammatory cytokines and B7 co-stimulatory molecules within the infected CNS by depleting cellular sources, including resident microglia and infiltrating macrophages, as well as decreasing expression on peripheral antigen-presenting cells (APCs). The loss of APCs in the CNS resulted in limited local reactivation of recruited antiviral CD8<sup>+</sup> T cells, which are required for virologic control in the CNS. Together, these data demonstrate the impact of CSF1R antagonism on APC activation during viral infection in both the CNS and periphery and highlight the importance of microglia in orchestrating cell-mediated antiviral immunity during neurotropic infection.</p></sec><sec id="Sec2"><title>Methods</title><sec id="Sec3"><title>Ethics statement</title><p id="Par19">All experiments were performed in strict compliance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health and according to the international Guiding Principles for Biomedical Research Involving Animals. The protocol was approved by the Washington University School of Medicine in St Louis Animal Safety Committee (#20170064).</p></sec><sec id="Sec4"><title>Viruses</title><p id="Par20">WNV-NY is strain 3000.0259, isolated in New York in 2000 [<xref ref-type="bibr" rid="CR42">42</xref>] and passaged twice in C6/36 <italic>Aedes albopictus</italic> cells to generate an insect cell-derived stock for f.p. inoculations. For i.c. inoculations of WNV-NY, virus strain 3000.0259 was passaged once in Vero cells to generate mammalian cell-derived stock. Attenuated WNV-NS5-E218A was constructed from WNV 3356 strain as described previously [<xref ref-type="bibr" rid="CR43">43</xref>, <xref ref-type="bibr" rid="CR44">44</xref>] and was passaged once in Vero cells, as described previously [<xref ref-type="bibr" rid="CR45">45</xref>]. Stock titers for all viruses were determined using BHK21 cells for viral plaque assay as previously described [<xref ref-type="bibr" rid="CR46">46</xref>].</p></sec><sec id="Sec5"><title>Mouse experiments</title><p id="Par21">All mice used in these experiments were male C57BL/6 inbred mice obtained commercially (The Jackson Laboratory). All mice were housed in pathogen-free facilities at the Washington University School of Medicine. PLX5622 was provided by Plexxikon Inc. and formulated in AIN-76A rodent diet at a dose of 1200 mg/kg by Research Diets. Mice were provided PLX5622 or control AIN-76A chow starting at 6 weeks of age for 2 weeks. For viral inoculations, mice were anesthesthetized with a cocktail of ketamine/xylazine/acepromazine, then inoculated subcutaneously via footpad injection (f.p., 50 μl) or intracranially (i.c., 10 μl) with a guided 29G needle into the brain’s third ventricle.</p></sec><sec id="Sec6"><title>Viral tissue burden and viremia quantification</title><p id="Par22">For in vivo virus replication experiments, mice were infected with WNV and euthanized at specific days post-infection, as indicated. For tissue collection, mice were deeply anesthetized with ketamine/xylazine/acepromazine, blood collected in serum separator tubes, then transcardially perfused with sterile Dulbecco’s phosphate-buffered saline (dPBS; Gibco). The spleen and kidneys were collected, then the brain and spinal cord removed and microdissected. All organs were snap frozen, weighed, and then homogenized in dPBS. Virus was titered by standard plaque assay with BHK21 cells, as described previously [<xref ref-type="bibr" rid="CR46">46</xref>]. Serum viremia was measured using TaqMan quantitative RT-PCR (qRT-PCR) primers and probe listed in Table <xref rid="Tab1" ref-type="table">1</xref>, as described previously [<xref ref-type="bibr" rid="CR47">47</xref>].<table-wrap id="Tab1"><label>Table 1</label><caption><p>Primer and probe sequences used for qRT-PCR</p></caption><table frame="hsides" rules="groups"><thead><tr><th>Target</th><th>Primer sequence (5′➔3′)</th></tr></thead><tbody><tr><td colspan="2"><italic>GAPDH</italic></td></tr><tr><td> Fwd</td><td>GGC AAA TTC AAC GGC ACA GT</td></tr><tr><td> Rev</td><td>AGA TGG TGA TGG GCT TCC C</td></tr><tr><td colspan="2"><italic>IFNα</italic></td></tr><tr><td> Fwd</td><td>CTT CCA CAG GAT CAC TGT GTA CCT</td></tr><tr><td> Rev</td><td>TTC TGC TCT GAC CAC CTC CC</td></tr><tr><td colspan="2"><italic>IFNβ</italic></td></tr><tr><td> Fwd</td><td>CTG GAG CAG CTG AAT GGA AAG</td></tr><tr><td> Rev</td><td>CTT CTC CGT CAT CTC CAT AGG G</td></tr><tr><td colspan="2"><italic>IFNγ</italic></td></tr><tr><td> Fwd</td><td>AAC GCT ACA CAC TGC ATC TTG G</td></tr><tr><td> Rev</td><td>GCC GTG GCA GTA ACA GCC</td></tr><tr><td colspan="2"><italic>TNFα</italic></td></tr><tr><td> Fwd</td><td>GCA CAG AAA GCA TGA TCC G</td></tr><tr><td> Rev</td><td>GCC CCC CAT CTT TTG GG</td></tr><tr><td colspan="2"><italic>iNOS</italic></td></tr><tr><td> Fwd</td><td>GGA GCC TTT AGA CCT CAA CAG A</td></tr><tr><td> Rev</td><td>TGA ACG AGG AGG GTG GTG</td></tr><tr><td colspan="2"><italic>CD86</italic></td></tr><tr><td> Fwd</td><td>ACG ATG GAC CCC AGA TGC ACC A</td></tr><tr><td> Rev</td><td>GCG TCT CCA CGG AAA CAG CA</td></tr><tr><td colspan="2"><italic>CD68</italic></td></tr><tr><td> Fwd</td><td>CCA CAG GCA GCA CAG TGG ACA</td></tr><tr><td> Rev</td><td>TCC ACA GCA GAA GCT TTG GCC C</td></tr><tr><td colspan="2"><italic>Arg1</italic></td></tr><tr><td> Fwd</td><td>TTA GGC CAA GGT GCT TGC TGC C</td></tr><tr><td> Rev</td><td>TAC CAT GGC CCT GAG GAG GTT C</td></tr><tr><td colspan="2"><italic>TGFβ</italic></td></tr><tr><td> Fwd</td><td>TAC TAT GCT AAA GAG GTC ACC C</td></tr><tr><td> Rev</td><td>CTT CCC GAA TGT CTG ACG TAT TG</td></tr><tr><td colspan="2"><italic>IL10</italic></td></tr><tr><td> Fwd</td><td>TTG GAA TTC CCT GGG TGA GAA</td></tr><tr><td> Rev</td><td>GGA GAA ATC GAT GAC AGC GC</td></tr><tr><td colspan="2"><italic>WNV</italic></td></tr><tr><td> Fwd</td><td>TCA GCG ATC TCT CCA CCA AAG</td></tr><tr><td> Rev</td><td>GGG TCA GCA CGT TTG TCA TTG</td></tr><tr><td> Probe</td><td>/56-FAM/TGC CCG ACC /ZEN/ ATG GGA GAA GCT C/3IABkFQ/</td></tr></tbody></table></table-wrap></p></sec><sec id="Sec7"><title>Leukocyte isolation and flow cytometry</title><p id="Par23">For flow cytometry experiments, mice were deeply anesthetized with ketamine/xylazine/acepromazine, then blood was collected in 5 μM EDTA in dPBS (Gibco). Mice were transcardially perfused with dPBS, and then, the spleen, brain, popliteal LNs, and femur were removed. Bone marrow was collected from the femur by centrifuging the bone in a 0.65-ml microtube punctured with an 18G needle nested inside a 1.7-ml tube. Brain tissue was minced and digested in a HBSS (Gibco) containing 0.05% collagenase D (Sigma), 0.1 μg/ml TLCK trypsin inhibitor (Sigma), 10 μg/ml DNase I (Sigma), and 10 mM Hepes pH 7.4 (Gibco) for 1 h at 22 °C with shaking. Brain, spleen, and LN tissues were pushed through a 70-μm strainer and centrifuged at 500<italic>g</italic> for 10 min. Brain cell pellets were resuspended in 37% isotonic Percoll (GE healthcare) and centrifuged at 1200<italic>g</italic> for 30 min to remove myelin debris, and pellet was resuspended in dPBS. Red blood cells were lysed in blood, spleen, and bone marrow samples with ACK lysing buffer (Gibco) for 5 min, then centrifuged at 500<italic>g</italic> for 10 min and resuspended in dPBS. Prior to immunostaining, all cells were blocked with 1:50 TruStain FcX anti-mouse CD16/32 (Clone 93, Biolegend, Cat 101320) for 5 min. Cells were stained with antibodies, as indicated for 15 min at 22 °C, then washed thrice with dPBS, and fixed with 2% paraformaldehyde (PFA). Data were collected with a BD LSR Fortessa X-20 flow cytometer and analyzed with FlowJo software.</p></sec><sec id="Sec8"><title>Flow cytometry antibodies</title><p id="Par24">All used at 1:200: CD11b (Clone M1/70, Biolegend, Cat 10137), CD45 (Clone 30-F11, eBioscience, Cat 56-0451-82), MHCII (Clone M5/114.15.2, Biolegend, Cat 107626), CD86 (Clone GL1, BD Biosciences, 553691), CD80 (Clone 16-10A1, Biolegend, 104706), CD11c (Clone N418, Biolegend, Cat 117335), CD103 (Clone 2E7, eBioscience, Cat 48-1031-80), CD8a (Clone 53-6.7, Biolegend, Cat 100712), CD4 (Clone RM4-5, BD Biosciences, Cat 550954), CD69 (Clone H1.2F3, eBiosciene, Cat 12-0691-81), Ly6C (Clone HK1.4, Biolegend, Cat 128003), Ly6G (Clone 1A8, Biolegend, Cat 127616), CD160 (Clone 7H1, Biolegend, Cat 143007), Rat-anti-P2RY12 (Clone S16007D, Biolegend, Cat 848002), and Goat-anti-Rat-AlexaFluor 350 (Invitrogen, Cat A21093). WNV-specific CD8+ T cells were identified with fluorescent-labeled immunodominant <italic>D</italic><sup><italic>b</italic></sup>-restricted NS4B peptide.</p></sec><sec id="Sec9"><title>Quantitative RT-PCR</title><p id="Par25">RNA was isolated from tissue homogenates using Qiagen RNeasy kit according to the manufacturer’s instructions. RNA was treated with DNAse, then cDNA was synthesized using random primers and MultiScribe reverse transcriptase (Applied Biosystems). A single reverse-transcriptase master mix was used to reverse-transcribe all samples in order to minimize differences in reverse-transcription efficiency using the following conditions: 25 °C for 10 min, 48 °C for 30 min, and 95 °C for 5 min. For all primers except WNV, qRT-PCR was performed using Power SYBR Green PCR mastermix, and data are reported as ΔCt (Ct<sub>target</sub> − Ct<sub>GAPDH</sub>). Viral RNA was isolated from serum using Qiagen Viral RNA kit. A standard curve of viral genome with known PFU and serum samples were quantified using TaqMan reagents and the primers and probe listed in Table <xref rid="Tab1" ref-type="table">1</xref>.</p></sec><sec id="Sec10"><title>Tunel stain, immunohistochemistry, and confocal microscopy</title><p id="Par26">Following perfusion with ice cold PBS and 4% paraformaldehyde (PFA), brains were immersion-fixed overnight in 4% PFA, followed by cryoprotection in two exchanges of 30% sucrose for 48 h, then frozen in OCT (Fisher). Ten-micrometer sagittal sections were cut, then washed with PBS, and permeabilized with 0.1% Triton X-100/0.1% sodium citrate. Tunel reaction was prepared according to the manufacturer’s directions (Roche in situ cell death kit, TMR Red, Cat 12-156-792-910). Sections were immunostained for NeuN or Iba1 by blocking sections with 5% goat serum/0.1% Tween 20/PBS, then incubating overnight at 4 °C with rabbit-anti-NeuN (1:1000, Clone D3S3I, Cell Signaling Technology Cat 12943S) or rabbit-anti-Iba1 (1:1000, Wako Cat 019-19741). Sections were washed with 0.1% Tween 20/PBS, then incubated with goat-anti-rabbit-AF488 (4 μg/ml, Invitrogen, Cat A11008), counterstained with 1 μg/ml DAPI, and coverslipped with Prolong Gold Antifade Mountant (ThermoFisher, Cat P36930). Z-stack images (seven images separated by 1 μm) were captured using a Zeiss LSM 510 laser-scanning confocal microscope at × 40 objective magnification then compressed into a maximum intensity projection with accompanying software. For each region of each mouse, three to six images were taken from two different sagittal sections spaced at least 50 μm apart. The number of DAPI-positive nuclei also positive for NeuN and Tunel were counted by an individual blinded to the conditions.</p></sec><sec id="Sec11"><title>Statistical analysis</title><p id="Par27">Statistical analyses were performed using Prism 7.0 (GraphPad Software). All data were analyzed using an unpaired <italic>t</italic> test or two-way ANOVA with Sidak’s post-test to correct for multiple comparisons, as indicated in the corresponding figure legends. A <italic>P</italic> value < 0.05 was considered significant.</p></sec></sec><sec id="Sec12"><title>Results</title><sec id="Sec13"><title>CSF1R antagonism depletes myeloid populations within peripheral blood and CNS</title><p id="Par28">CSF1R signaling is essential for the development of mononuclear phagocytes including microglia, but pharmacological antagonism has been reported to selectively deplete microglia [<xref ref-type="bibr" rid="CR48">48</xref>]. To examine the impact of CSF1R antagonism on both peripheral and CNS mononuclear cells, we used PLX5622, which reportedly depletes microglia in as little as 3 days and may be sustained for at least 6 weeks [<xref ref-type="bibr" rid="CR37">37</xref>, <xref ref-type="bibr" rid="CR41">41</xref>]. Mice were provided either control chow or PLX5622-embedded chow for 2 weeks, after which microglia numbers were assessed by flow cytometry using the microglia-specific marker P2RY12 (Additional file <xref rid="MOESM1" ref-type="media">1</xref>) and by immunohistochemistry using Iba1 (Additional file <xref rid="MOESM2" ref-type="media">2</xref>). Quantification shows that 2 weeks of PLX5622 treatment reduced microglia in the CNS by 90–95% (Additional file <xref rid="MOESM1" ref-type="media">1</xref> A–F, P, Q). Specificity of P2RY12 for CNS-resident microglia is demonstrated by lack of staining in leukocytes isolated from peripheral immune compartments (Additional file <xref rid="MOESM1" ref-type="media">1</xref> G–O). Within the CNS, PLX5622 treatment significantly depleted microglia across hippocampal, cortical, and cerebellar brain regions (Additional file <xref rid="MOESM2" ref-type="media">2</xref>). To determine whether PLX5622 treatment depleted mononuclear cells in peripheral immune compartments, we assessed numbers of antigen-presenting cells (APCs, Fig. <xref rid="Fig1" ref-type="fig">1</xref>) and T cells (Additional file <xref rid="MOESM3" ref-type="media">3</xref>) in the spleen and blood of uninfected mice in control versus PLX5622-treated mice. PLX5622 treatment significantly reduced circulating CD11c<sup>+</sup> (Fig. <xref rid="Fig1" ref-type="fig">1</xref>a–c), MHCII<sup>+</sup>CD11c<sup>+</sup> (Fig. <xref rid="Fig1" ref-type="fig">1</xref>d–f), and CD11b<sup>neg</sup>CD11c<sup>+</sup> (Fig. <xref rid="Fig1" ref-type="fig">1</xref>g–i) cells in the blood. In the spleen, however, there was no significant difference in populations of APCs with PLX5622 treatment (Fig. <xref rid="Fig1" ref-type="fig">1</xref>j–r). In addition, no significant difference in populations of T cells was detected in uninfected mice in either compartment (Additional file <xref rid="MOESM3" ref-type="media">3</xref>). Because of the sensitivity of microglia and APCs to CSF1R antagonism, we assessed whether CSF1R antagonism depleted CD11b<sup>+</sup>, Ly6C<sup>+</sup>, and Ly6G<sup>+</sup> cells in the blood, spleen, and bone marrow of PLX5622-treated versus control-treated uninfected mice. Flow cytometry analysis revealed no significant differences in populations of these cells in uninfected mice (Additional file <xref rid="MOESM4" ref-type="media">4</xref>). Together, these data indicate that circulating APCs, but not bone marrow or splenic mononuclear cells, are decreased in the context of CSF1R antagonism.<fig id="Fig1"><label>Fig. 1</label><caption><p>CSF1R antagonism reduces circulating APC populations in uninfected mice. Mice were fed PLX5622 chow or control chow for 2 weeks, and APCs were assessed in blood (<bold>a</bold>–<bold>i</bold>) and spleen (<bold>j</bold>–<bold>r</bold>). <bold>a</bold>, <bold>j</bold> Representative flow cytometry plots of CD11c expression on CD45<sup>+</sup> cells. <bold>b</bold>, <bold>k</bold> Quantification of percentages and <bold>c</bold>, <bold>l</bold> total numbers of CD45<sup>+</sup>CD11c<sup>+</sup> cells. <bold>d</bold>, <bold>m</bold> Representative flow cytometry plots of MHCII expression on CD11c<sup>+</sup>CD45<sup>+</sup>. <bold>e</bold>, <bold>n</bold> Quantification of percentages and <bold>f</bold>, <bold>o</bold> total numbers of MHCII<sup>+</sup>CD11c<sup>+</sup>CD45<sup>+</sup> cells. <bold>g</bold>, <bold>p</bold> Representative flow cytometry plots of CD103 vs CD11b expression on CD11c<sup>+</sup>CD45<sup>+</sup> cells. <bold>h</bold>, <bold>q</bold> Quantification of percentages and <bold>i</bold>, <bold>r</bold> total numbers of (I) CD103<sup>+</sup>CD11b<sup>neg</sup>CD11c<sup>+</sup>CD45<sup>+</sup>, (II) CD103<sup>+</sup>CD11b<sup>+</sup>CD11c<sup>+</sup>CD45<sup>+</sup>, (III) CD103<sup>neg</sup>CD11b<sup>+</sup>CD11c<sup>+</sup>CD45<sup>+</sup>, and (IV) CD103<sup>neg</sup>CD11b<sup>neg</sup>CD11c<sup>+</sup>CD45<sup>+</sup> cells. For quantification panels, each symbol represents an individual control (black) or PLX5622-treated (red) mouse, and bars indicate mean ± SEM. Data shown represent analysis from one experiment with three to five mice per group, repeated in three independent experiments. Statistical significance was calculated using two-way ANOVA with Sidak’s multiple comparisons test. For all data: ns, not significant at <italic>P</italic> < 0.05; *<italic>P</italic> < 0.05; **<italic>P</italic> < 0.01; ***<italic>P</italic> < 0.001.</p><p> CTRL: Control; PLX: PLX5622</p></caption><graphic xlink:href="12974_2019_1397_Fig1_HTML" id="MO1"></graphic></fig></p></sec><sec id="Sec14"><title>CSF1R antagonism increases susceptibility of mice to fatal WNV infection</title><p id="Par29">To determine whether CSF1R antagonism impacts survival from WNV infection, mice were fed PLX5622-embedded chow for 2 weeks, then infected with WNV-NY [<xref ref-type="bibr" rid="CR42">42</xref>] at either 10<sup>2</sup> or 10<sup>4</sup> PFU via footpad (f.p.) inoculation. Consistent with published data [<xref ref-type="bibr" rid="CR8">8</xref>, <xref ref-type="bibr" rid="CR49">49</xref>], 30% of control-treated mice survived infection with 10<sup>4</sup> PFU and 90% of control-treated mice survived infection with 10<sup>2</sup> PFU; however, mice treated with PLX5622 universally succumbed to infection with either inoculum titer (Fig. <xref rid="Fig2" ref-type="fig">2</xref>a). This was associated with significantly increased weight loss (Fig. <xref rid="Fig2" ref-type="fig">2</xref>b) and more severe clinical score (Fig. <xref rid="Fig2" ref-type="fig">2</xref>c, d) in PLX5622-treated compared with control-treated mice infected with 10<sup>2</sup> PFU WNV-NY. To determine whether increased mortality caused by PLX5622 treatment is associated with increased viral replication in peripheral or CNS tissues, tissue viral loads were assessed by plaque assay, and serum viral loads were determined by quantitative RT-PCR (qRT-PCR) at 2, 4, 6, and 8 days post-infection (dpi). In the CNS, virus was first detected in the olfactory bulb (Fig. <xref rid="Fig2" ref-type="fig">2</xref>e), then sequentially in more caudal regions including the cortex (Fig. <xref rid="Fig2" ref-type="fig">2</xref>f), cerebellum (Fig. <xref rid="Fig2" ref-type="fig">2</xref>g), brainstem (Fig. <xref rid="Fig2" ref-type="fig">2</xref>h), and spinal cord (Fig. <xref rid="Fig2" ref-type="fig">2</xref>i). Compared with control-treated animals, PLX5622-treated mice exhibited higher viral titers in the olfactory bulb and cortex at 6 and 8 dpi and in the cerebellum, brainstem, and spinal cord at 8 dpi; however, viral titers were also significantly higher in peripheral tissues of PLX5622-treated mice including the spleen at 4 dpi (Fig. <xref rid="Fig2" ref-type="fig">2</xref>j), kidney at 4 and 6 dpi (Fig. <xref rid="Fig2" ref-type="fig">2</xref>k), and serum at 2, 4, 6, and 8 dpi (Fig. <xref rid="Fig2" ref-type="fig">2</xref>l). PLX5622 treatment did not cause increased BBB permeability in infected mice (Additional file <xref rid="MOESM5" ref-type="media">5</xref>). Once WNV-NY enters the CNS via intracranial (i.c.) inoculation, PLX5622 treatment did not affect neuronal permissivity to infection (Additional file <xref rid="MOESM6" ref-type="media">6</xref>). Together, these data indicate a loss of immune-mediated virologic control in both peripheral and CNS tissues in CSF1R antagonist-treated mice, consistent with prior data suggesting that CSF1R signaling plays important roles in the function of both peripheral and CNS myeloid cells [<xref ref-type="bibr" rid="CR50">50</xref>–<xref ref-type="bibr" rid="CR52">52</xref>].<fig id="Fig2"><label>Fig. 2</label><caption><p>PLX5622-treated mice exhibit increased mortality, increased encephalitis score, and impaired virologic control compared with control mice following peripheral infection with WNV-NY. <bold>a</bold>–<bold>d</bold> Mice were fed chow containing PLX5622 or control chow for 2 weeks, then infected with 10<sup>2</sup> or 10<sup>4</sup> PFU via footpad infection, then monitored for (<bold>a</bold>) mortality, (<bold>b</bold>) weight loss, and (<bold>c</bold>, <bold>d</bold>) encephalitis score for up to 25 dpi. <bold>a</bold> Survival curves show a significant increase in mortality in mice treated with PLX5622 compared with control-treated mice infected with either viral inoculum dose, as calculated by log-rank (Mantel-Cox) test. <bold>b</bold> Weight was measured daily during acute illness as a measure of illness. After death, the last measured weight was carried through to the end of the experiment. Significantly greater weight loss was measured in PLX5622-treated mice compared with controls infected with 10<sup>2</sup> PFU as calculated by two-way ANOVA with matched values comparing group means without multiple comparisons. <bold>c</bold> Clinical scores of PLX5622 or control-treated mice infected with 10<sup>2</sup> PFU or <bold>d</bold> 10<sup>4</sup> PFU as indicated during acute illness. 0 = subclinical, 1 = hunched/ruffled fur, 2 = altered gate/slow movement, 3 = no movement, but responsive to stimuli, 4 = moribund, 5 = dead. <bold>e</bold>–<bold>k</bold> Tissue viral loads as measured by plaque assay at 2, 4, 6, and 8 dpi following infection with 10<sup>2</sup> PFU in control (black) or PLX5622-treated (red) mice. <bold>l</bold> Serum viral loads as measured by qRT-PCR. <bold>e</bold>–<bold>l</bold> Data are presented as scatter plots with each mouse represented by a dot with the SEM indicated by a line. Dotted lines in <bold>e</bold>–<bold>l</bold> indicate assay limit of detection. All data presented are the compilation of two independent experiments with 10 mice per group. Statistical significance was calculated using two-way ANOVA with Sidak’s multiple comparisons test. For all data: ns, not significant at <italic>P</italic> < 0.05; *<italic>P</italic> < 0.05; **<italic>P</italic> < 0.01; ****<italic>P</italic> < 0.0001</p></caption><graphic xlink:href="12974_2019_1397_Fig2_HTML" id="MO2"></graphic></fig></p><p id="Par30">To determine whether microglia depletion impacts innate immune mechanisms of virologic control specifically within the CNS, mice were fed control or PLX5622-embedded chow for 2 weeks, then i.c. infected with an attenuated strain of WNV (WNV-NS5-E218A) at 10<sup>4</sup> PFU. The NS5-E218A point mutation abolishes 2′-<italic>O</italic>-methyltransferase activity, which functions to methylate the 5′ cap of viral RNA and facilitate escape of IFN-induced proteins with tetratricopeptide repeats (IFIT)-mediated suppression; thus, this attenuation limits viral replication by increasing sensitivity of the virus to type I IFN-mediated innate immune mechanisms [<xref ref-type="bibr" rid="CR45">45</xref>, <xref ref-type="bibr" rid="CR49">49</xref>]. Despite this attenuation, mice treated with PLX5622 were significantly more susceptible to lethal WNV-NS5-E218A infection than those receiving control chow (Fig. <xref rid="Fig3" ref-type="fig">3</xref>a). Weight loss was also significantly increased in PLX5622-treated mice (Fig. <xref rid="Fig3" ref-type="fig">3</xref>b). Consistent with this, viral titers measured by plaque assay at 2, 4, 6, and 8 dpi in the olfactory bulb (Fig. <xref rid="Fig3" ref-type="fig">3</xref>d), cortex (Fig. <xref rid="Fig3" ref-type="fig">3</xref>e), cerebellum (Fig. <xref rid="Fig3" ref-type="fig">3</xref>f), brainstem (Fig. <xref rid="Fig3" ref-type="fig">3</xref>g), and spinal cord (Fig. <xref rid="Fig3" ref-type="fig">3</xref>h) were significantly higher in PLX5622-treated compared in control-treated mice infected with WNV-NS5-E218A. As virus was inoculated into the cortex, it was detected there at 2 dpi, followed by spread to the olfactory bulb by 4 dpi, and cerebellum, brainstem, and spinal cord by 6 dpi. PLX5622 treatment also led to decreased proinflammatory cytokine expression in the cortex at 8 dpi (Fig. <xref rid="Fig3" ref-type="fig">3</xref>c) and increased neuronal apoptosis in the hippocampus and cerebellum at 6 dpi (Fig. <xref rid="Fig3" ref-type="fig">3</xref>l–p) after i.c. infection with WNV-NS5-E218A.<fig id="Fig3"><label>Fig. 3</label><caption><p>CSF1R antagonism increases mortality and CNS viral burdens during i.c. infection with WNV-NS5-E218A. Mice were fed chow containing PLX5622 or control chow for 2 weeks, infected i.c. with WNV-NS5-E218A at 10<sup>4</sup> PFU, then monitored for <bold>a</bold> mortality and <bold>b</bold> weight loss for up to 25 dpi. <bold>a</bold> Survival curves show a significant increase in mortality in mice treated with PLX5622 compared with control as calculated by log-rank (Mantel-Cox) test. <bold>b</bold> Weight was measured daily during acute illness as a measure of illness. After death, the last measured weight was carried through to the end of the experiment. Significantly greater weight loss was measured in PLX5622-treated mice compared with controls as calculated by two-way ANOVA with matched values comparing group means without multiple comparisons. For <bold>a</bold> and <bold>b</bold>, data represent the compilation of two independent experiments with 10 mice per group. <bold>c</bold> At 8 dpi, cortical brain tissue was extracted and relative transcript levels in tissue homogenates were measured by SYBR qRT-PCR for indicated cytokines. Data for individual mice were normalized to <italic>Gapdh.</italic> Data are presented as mean ± SEM of three to five mice from one experiment. Statistical significance between treatment groups for each cytokine was calculated by <italic>t</italic> test. <bold>d</bold>–<bold>j</bold> Tissue viral loads were measured by plaque assay at 2, 4, 6, and 8 dpi in control (black) or PLX5622-treated (red) mice. <bold>k</bold> Serum viral loads as measured by qRT-PCR. <bold>d</bold>–<bold>k</bold> Data are presented as scatter plots with each mouse represented by a dot with the mean indicated by a line. For <bold>d</bold>–<bold>k</bold>, data represent the compilation of two-independent experiments with 10 mice per group. Dotted lines in <bold>d</bold>–<bold>k</bold> indicate assay limit of detection. <bold>l</bold>–<bold>o</bold> Representative confocal microscopic images of Tunel (red), NeuN (green), and DAPI (blue) of hippocampus (<bold>l</bold>, <bold>m</bold>) and cerebellum (<bold>n</bold>, <bold>o</bold>) of control- (<bold>l</bold>, <bold>n</bold>) or PLX5622-treated (<bold>m</bold>, <bold>o</bold>) mice infected i.c. with WNV-NS5-E218A at 6 dpi. Arrow heads point to DAPI-positive nuclei positive for both Tunel and NeuN. Asterisks denote DAPI- and Tunel-positive but NeuN-negative nuclei. <bold>p</bold> Quantification of confocal microscopic images for number of DAPI-positive nuclei positive for Tunel and NeuN in the hippocampus (Hpc), cerebellum (Cb), and brainstem (Bs). The number of Tunel+ NeuN+ nuclei per high-power field (HPF) was quantified from three to six images captured at × 40 per brain region across two separate brain sections for each of five independent mice collected in one experiment. Each symbol represents the average number for an individual mouse, with bar indicating mean ± SEM. Statistical significance was calculated using two-way ANOVA with Sidak’s multiple comparisons test. For all data: ns, not significant at <italic>P</italic> < 0.05; *<italic>P</italic> < 0.05; **<italic>P</italic> < 0.01; ****<italic>P</italic> < 0.0001</p></caption><graphic xlink:href="12974_2019_1397_Fig3_HTML" id="MO3"></graphic></fig></p><p id="Par31">As expected, WNV-NS5-E218A was barely detected in the kidney (Fig. <xref rid="Fig3" ref-type="fig">3</xref>j), and the virus was steadily cleared from the serum (Fig. <xref rid="Fig3" ref-type="fig">3</xref>k) in both control- and PLX5622-treated mice. However, significantly higher levels of virus were detected in the spleen of PLX5622- versus control-treated, WNV-NS5-E218A-infected mice at 4 dpi (Fig. <xref rid="Fig3" ref-type="fig">3</xref>i), which was consistent with data obtained during f.p. infection with WNV-NY. Overall, CSF1R antagonism leads to enhanced mortality and loss of virologic control in both the periphery and CNS regardless of the WNV strain utilized, suggesting a critical role for this molecule in antiviral immunity.</p></sec><sec id="Sec15"><title>CSF1R signaling is required for activation of APC co-stimulatory function</title><p id="Par32">To determine whether alterations in APC populations impact antiviral immune responses, control- and PLX5622-treated mice were infected via f.p. with WNV-NY (100 PFU). After f.p. infection, WNV-infected cells traffic to draining popliteal lymph nodes (pLN) and stimulate an antiviral immune response [<xref ref-type="bibr" rid="CR53">53</xref>]. At 4 dpi, a time-point of high serum viral loads (Fig. <xref rid="Fig2" ref-type="fig">2</xref>l), flow cytometric assessment revealed a trend towards decreased CD11c<sup>+</sup>CD45<sup>+</sup> cells in the blood (Fig. <xref rid="Fig4" ref-type="fig">4</xref>a–d) and significantly reduced percentages and total numbers of CD11c<sup>+</sup>CD45<sup>+</sup> cells in the pLN of PLX5622-treated compared with control-treated mice (Fig. <xref rid="Fig4" ref-type="fig">4</xref>k–n). There was no difference in numbers or percentages of MHCII<sup>+</sup>CD11c<sup>+</sup>CD45<sup>+</sup> cells in either blood or pLN (Fig. <xref rid="Fig4" ref-type="fig">4</xref>e–g, o–q); however, the percentages of CD11c<sup>+</sup>CD45<sup>+</sup> cells in the blood positive for co-stimulatory molecule CD80<sup>+</sup> was significantly decreased in PLX5622- versus control-treated mice (Fig. <xref rid="Fig4" ref-type="fig">4</xref>h–j). In the pLN, the numbers and percentages of CD11c<sup>+</sup>CD45<sup>+</sup> cells positive for co-stimulatory molecule CD86 were significantly reduced in PLX5622-treated mice compared with control mice (Fig. <xref rid="Fig4" ref-type="fig">4</xref>r–t). The impact of CSF1R antagonism on CD80 and CD86 expression appears to be tissue-dependent as the blood showed no difference in APCs expressing CD86 and the pLN showed no difference in APCs expressing CD80 (Additional file <xref rid="MOESM7" ref-type="media">7</xref>). Together, these data indicate that CSF1R antagonism decreases the expression of co-stimulatory B7 molecules on peripheral APC populations.<fig id="Fig4"><label>Fig. 4</label><caption><p>CSF1R antagonism reduces response of APCs in peripheral immune compartments following peripheral infection with WNV-NY. Mice were fed PLX5622 or control chow for 2 weeks, then infected via f.p. with WNV-NY (100 PFU). At 4 dpi, leukocytes were isolated from blood (<bold>a</bold>–<bold>j</bold>) and draining popliteal LNs (k–t). <bold>a</bold>, <bold>k</bold> Representative flow cytometry plots of CD11c expression on CD45<sup>+</sup>-gated cells and <bold>b</bold>, <bold>l</bold> fluorescence minus one (FMO) gating controls. <bold>c</bold>, <bold>m</bold> Quantification of percentages and <bold>d</bold>, <bold>n</bold> total numbers of CD11c<sup>+</sup> CD45<sup>+</sup> cells. <bold>e</bold>, <bold>o</bold> Representative flow cytometry plots of MHCII<sup>+</sup> expression on CD11c<sup>+</sup>CD45<sup>+</sup> cells. <bold>f</bold>, <bold>p</bold> Quantification of percentages and <bold>g</bold>, <bold>q</bold> total numbers of MHCII<sup>+</sup>CD11c<sup>+</sup>CD45<sup>+</sup> cells. <bold>h</bold> Representative flow cytometry plots of CD86<sup>+</sup> expression on CD11c<sup>+</sup>CD45<sup>+</sup> cells. <bold>i</bold> Quantification of percentages and <bold>j</bold> total numbers of CD86<sup>+</sup>CD11c<sup>+</sup>CD45<sup>+</sup> cells. <bold>r</bold> Representative flow cytometry plots of CD80<sup>+</sup> expression on CD11c<sup>+</sup>CD45<sup>+</sup> cells. <bold>s</bold> Quantification of percentages and <bold>t</bold> total numbers of CD80<sup>+</sup>CD11c<sup>+</sup>CD45<sup>+</sup> cells. For quantification panels, each symbol represents an individual control- (black) or PLX5622-treated (red) mouse, and bars indicate mean ± SEM. Data shown represent analysis from one experiment with three to five mice per group. Statistical significance was calculated using unpaired <italic>t</italic> test in all panels except <bold>d</bold> and <bold>n</bold>, which used two-way ANOVA with Sidak’s multiple comparisons test. For all data: ns, not significant at <italic>P</italic> < 0.05; *<italic>P</italic> < 0.05; **<italic>P</italic> < 0.01</p></caption><graphic xlink:href="12974_2019_1397_Fig4_HTML" id="MO4"></graphic></fig></p><p id="Par33">Because infectious virus was detected in the spleens of mice infected i.c. with WNV-NS5-E218A (Fig. <xref rid="Fig3" ref-type="fig">3</xref>i), splenic APCs were analyzed using flow cytometry at 8 dpi. Results indicated no significant difference in the numbers or percentages of MHCII<sup>+</sup>CD11c<sup>+</sup>CD45<sup>+</sup> cells in the spleens of PLX5622-treated mice compared with control-treated mice (Fig. <xref rid="Fig5" ref-type="fig">5</xref>a–c). However, splenic APCs collected from PLX5622-treated mice exhibited significantly reduced populations of cells expressing co-stimulatory molecules CD80 (Fig. <xref rid="Fig5" ref-type="fig">5</xref>d–f) and CD86 (Fig. <xref rid="Fig5" ref-type="fig">5</xref>g–i) compared with control-treated mice. Furthermore, splenic MHCII<sup>+</sup>CD11c<sup>+</sup>CD45<sup>+</sup> cells showed significant reduction in the expression level of CD86 (Fig. <xref rid="Fig5" ref-type="fig">5</xref>l, m) and non-significant reduction of CD80 (Fig. <xref rid="Fig5" ref-type="fig">5</xref>j, k) in PLX5622-treated compared with control-treated mice. This correlated with a small, but significant increase in the numbers of CD4<sup>+</sup> and CD8<sup>+</sup> T cells, but no difference in numbers or percentages of WNV-specific NS4B tetramer staining or CD69 positivity in spleens of PLX5622-treated mice compared with control-treated mice infected i.c. with WNV-NS5-E218A (Additional file <xref rid="MOESM8" ref-type="media">8</xref>). Together, these data indicate that CSF1R antagonism reduces the activation of inflammatory responses in peripheral APCs even without significant depletion.<fig id="Fig5"><label>Fig. 5</label><caption><p>CSF1R antagonism reduces co-stimulatory signal 2 on splenic APCs during i.c. infection with WNV-NS5-E218A. Mice were fed PLX5622 chow or control chow for 2 weeks, then infected i.c. with WNV-NS5-E218A (10<sup>4</sup> PFU). At 8 dpi, splenic leukocytes were analyzed. <bold>a</bold> Representative flow cytometry contour plots of CD11c and MHCII expression on CD45<sup>+</sup>-gated cells and <bold>b</bold> quantification of percentages and <bold>c</bold> total numbers of CD11c<sup>+</sup>MHCII<sup>+</sup>CD45<sup>+</sup> cells. <bold>d</bold> Representative flow cytometry plots of CD80 expression on CD11c<sup>+</sup>MHCII<sup>+</sup>CD45<sup>+</sup> cells. <bold>e</bold> Quantification of percentages and <bold>f</bold> total numbers of CD80<sup>+</sup>CD11c<sup>+</sup>MHCII<sup>+</sup>CD45<sup>+</sup> cells. <bold>g</bold> Representative flow cytometry plots of CD86 expression on CD11c<sup>+</sup>MHCII<sup>+</sup>CD45<sup>+</sup> cells. <bold>h</bold> Quantification of percentages and <bold>i</bold> total numbers of CD86<sup>+</sup>CD11c<sup>+</sup>MHCII<sup>+</sup>CD45<sup>+</sup> cells. <bold>j</bold> Representative flow cytometry histograms of CD80 expression on CD11c<sup>+</sup>MHCII<sup>+</sup>CD45<sup>+</sup> and <bold>k</bold> quantification of MFI. <bold>l</bold> Representative flow cytometry histograms of CD86 expression on CD11c<sup>+</sup>MHCII<sup>+</sup>CD45<sup>+</sup> and <bold>m</bold> quantification of MFI. AU, arbitrary units. For quantification panels, each symbol represents an individual control- (black) or PLX5622-treated (red) mouse, and bars indicate mean ± SEM. Data represent analysis from one experiment with three to four mice per group. Statistical significance was calculated using unpaired two-way <italic>t</italic> tests. For all data: ns, not significant at <italic>P</italic> < 0.05; *<italic>P</italic> < 0.05; **<italic>P</italic> < 0.01</p></caption><graphic xlink:href="12974_2019_1397_Fig5_HTML" id="MO5"></graphic></fig></p></sec><sec id="Sec16"><title>Loss of cellular sources of co-stimulatory signals in the CNS results in decreased T cell reactivation</title><p id="Par34">Microglia are hypothesized to act as immediate responders to CNS pathogens as resident APCs that coordinate intra-parenchymal adaptive immune response [<xref ref-type="bibr" rid="CR54">54</xref>]. Previous studies indicated that clearance of WNV in the CNS requires antiviral CD8<sup>+</sup> T cells [<xref ref-type="bibr" rid="CR19">19</xref>, <xref ref-type="bibr" rid="CR32">32</xref>, <xref ref-type="bibr" rid="CR55">55</xref>], and that microglia, astrocytes, and neurons express chemokines that attract mononuclear cells to the CNS [<xref ref-type="bibr" rid="CR21">21</xref>, <xref ref-type="bibr" rid="CR56">56</xref>, <xref ref-type="bibr" rid="CR57">57</xref>]. To determine the impact of CSF1R antagonism on recruitment of these cells to the CNS, leukocytes isolated from the cortices of WNV-NS5-E218A-infected mice were assessed by flow cytometry at 8 dpi. As expected, CD45<sup>lo</sup>CD11b<sup>+</sup> (P1) population, which is primarily microglia [<xref ref-type="bibr" rid="CR58">58</xref>], remained significantly depleted in the PLX5622-treated compared with control-treated mice (Fig. <xref rid="Fig6" ref-type="fig">6</xref>a–c). The population of CD45<sup>hi</sup>CD11b<sup>+</sup> (P2), which is primarily infiltrating monocytes/macrophages [<xref ref-type="bibr" rid="CR58">58</xref>], were non-significantly reduced in the PLX5622-treated compared with control-treated mice. However, the number of CD45<sup>hi</sup>CD11b<sup>neg</sup> (P3) lymphocyte population [<xref ref-type="bibr" rid="CR58">58</xref>] were non-significantly elevated in PLX5622-treated compared with control mice, though the percentage of this population was significantly elevated (Fig. <xref rid="Fig6" ref-type="fig">6</xref>a–c). This increase was largely due to elevated numbers of CD8<sup>+</sup> T cell infiltration in PLX5622-treated mice (Fig. <xref rid="Fig6" ref-type="fig">6</xref>d–f). Though there was no significant difference in the percentage of CD8<sup>+</sup> T cells specific for the WNV immunodominant peptide between PLX5622 and control-treated mice, there were significantly more WNV-specific CD8<sup>+</sup> T cells isolated from PLX5622 compared with control-treated cortex (Fig. <xref rid="Fig6" ref-type="fig">6</xref>g–i). Despite the increased infiltration of WNV-specific CD8<sup>+</sup> T cells in PLX5622-treated mice, the percentages of cells expressing early activation marker CD69 and their level of expression were significantly reduced in PLX5622-treated compared with control-treated mice (Fig. <xref rid="Fig6" ref-type="fig">6</xref>j–m). Additionally, the percentage of WNV-specific CD8<sup>+</sup> T cells positive for CD160 as well as the level of CD160 expression, which is specifically expressed on highly activated CD8<sup>+</sup> T cells [<xref ref-type="bibr" rid="CR59">59</xref>], were reduced in PLX5622-treated compared with control-treated mice. However, the numbers of NS4B+CD8+ cells positive for either CD69 or CD160 were non-significantly elevated in PLX5622-treated compared with control-treated cortices (Fig. <xref rid="Fig6" ref-type="fig">6</xref>l, p) due to the increased numbers of NS4B+CD8+ cells.<fig id="Fig6"><label>Fig. 6</label><caption><p>CSF1R antagonism does not hinder antiviral T cell recruitment to the CNS, but recruited T cells lack full activation during i.c. infection with WNV-NS5-E218A. Mice were fed PLX5622 chow or control chow for 2 weeks, then infected i.c. with WNV-NS5-E218A (10<sup>4</sup> PFU). At 8 dpi, leukocytes were isolated from the cortex and analyzed by flow cytometry. <bold>a</bold> Representative flow cytometry plots of CD45<sup>+</sup>-gated cells analyzed by CD11b vs CD45 expression, identifying three populations of cells: CD11b<sup>+</sup>CD45<sup>lo</sup> (P1), CD11b<sup>+</sup>CD45<sup>hi</sup> (P2), and CD11b<sup>neg</sup>CD45<sup>hi</sup> (P3). <bold>b</bold> Quantification of percentages and <bold>c</bold> number of cells in each gate. <bold>d</bold> Representative flow cytometry plots of CD4 vs CD8 expression and <bold>e</bold> quantification of percentages and <bold>f</bold> total numbers of CD4<sup>+</sup> vs CD8<sup>+</sup> cells within P3 gate. <bold>g</bold> Representative flow cytometry plots of NS4B<sup>+</sup>WNV-specific tetramer staining and <bold>h</bold> quantification of percentages and <bold>i</bold> total number of NS4B<sup>+</sup>CD8<sup>+</sup>CD45<sup>+</sup> cells. <bold>j</bold> Representative flow cytometry plots of CD69 expression on NS4B<sup>+</sup>CD8<sup>+</sup>CD45<sup>+</sup> cells and <bold>k</bold> quantification of percentages, <bold>l</bold> total numbers, and <bold>m</bold> MFI of CD69<sup>+</sup>NS4B<sup>+</sup>CD8<sup>+</sup>CD45<sup>+</sup> cells. <bold>n</bold> Representative flow cytometry plots of CD160 expression on NS4B<sup>+</sup>CD8<sup>+</sup>CD45<sup>+</sup> cells and <bold>o</bold> quantification of percentages, <bold>p</bold> total numbers, and <bold>q</bold> MFI of CD160<sup>+</sup>NS4B<sup>+</sup>CD8<sup>+</sup>CD45<sup>+</sup> cells. MFI quantifications are shown as arbitrary units (AU) by normalizing each sample to the MFI of FMO negative control value [<xref ref-type="bibr" rid="CR77">77</xref>]. For quantification panels, each symbol represents an individual control- (black) or PLX5622-treated (red) mouse, and bars indicate mean ± SEM. Data presented are the compilation of two independent experiments with seven to nine mice per group. Statistical significance was calculated using unpaired <italic>t</italic> tests with Welch’s correction. For all data: ns, not significant at <italic>P</italic> < 0.05; *<italic>P</italic> < 0.05; **<italic>P</italic> < 0.01; ***<italic>P</italic> < 0.001; ****<italic>P</italic> < 0.0001</p></caption><graphic xlink:href="12974_2019_1397_Fig6_HTML" id="MO6"></graphic></fig></p><p id="Par35">Because numbers of infiltrating WNV-specific CD8<sup>+</sup> T cells derived from the CNS of PLX5622-treated animals were increased, we hypothesized that their lack of local reactivation may be the result of loss of co-stimulatory signals and/or inflammatory cytokines (Fig. <xref rid="Fig3" ref-type="fig">3</xref>c) in the CNS. In order to distinguish resident microglia from infiltrating monocytes/macrophages, CNS myeloid cells were examined for expression of the microglia-specific marker P2RY12 (Fig. <xref rid="Fig7" ref-type="fig">7</xref>a). Infiltrating monocytes/macrophages were then identified via gating of P2RY12<sup>neg</sup>CD11b<sup>+</sup> cells (Fig. <xref rid="Fig7" ref-type="fig">7</xref>b). Populations of both CNS-derived P2RY12<sup>+</sup>CD45<sup>+</sup> microglia (P1, Fig. <xref rid="Fig7" ref-type="fig">7</xref>a) and CD11b<sup>+</sup>P2RY12<sup>neg</sup>CD45<sup>+</sup> monocytes/macrophages (P2, Fig. <xref rid="Fig7" ref-type="fig">7</xref>b) were reduced in PLX5622-treated compared with control-treated mice (Fig. <xref rid="Fig7" ref-type="fig">7</xref>i, j). While no differences in the overall number of cells expressing MHCII were observed in P2RY12<sup>+</sup>CD45<sup>+</sup>, a significant reduction of MHCII<sup>+</sup>CD11b<sup>+</sup>P2RY12<sup>neg</sup>CD45<sup>+</sup> was seen (Fig. <xref rid="Fig7" ref-type="fig">7</xref>c, d, l). This corresponded with a relative increase in the percentage of MHCII<sup>+</sup> cells in both P2RY12<sup>+</sup>CD45<sup>+</sup> and CD11b<sup>+</sup>P2RY12<sup>neg</sup>CD45<sup>+</sup> populations due to the decreased numbers of total P2RY12<sup>+</sup>CD45<sup>+</sup> and CD11b<sup>+</sup>P2RY12<sup>neg</sup>CD45<sup>+</sup> cells in the cortices of PLX5622-treated mice compared with control-treated mice (Fig. <xref rid="Fig7" ref-type="fig">7</xref>k). However, the numbers of P2RY12<sup>+</sup>CD45<sup>+</sup> cells expressing B7 co-stimulatory signals CD80 and CD86 were significantly reduced in the CNS of PLX5622-treated mice compared with control-treated animals (Fig. <xref rid="Fig7" ref-type="fig">7</xref>e–h, m–p). The percentage of remaining P2RY12<sup>+</sup>CD45<sup>+</sup> cells expressing CD80 or CD86 were not reduced (Fig. <xref rid="Fig7" ref-type="fig">7</xref>m, o); however, the percentage of remaining CD11b<sup>+</sup>P2RY12<sup>neg</sup>CD45<sup>+</sup> cells expressing CD86 was significantly reduced (Fig. <xref rid="Fig7" ref-type="fig">7</xref>o), similar to results seen in peripheral APCs (Figs. <xref rid="Fig4" ref-type="fig">4</xref>, <xref rid="Fig5" ref-type="fig">5</xref>) Together, these data suggest that resident microglia and infiltrating macrophages are important sources of CD80 and CD86 in the CNS, and PLX5622 treatment depletes these cellular sources of co-stimulatory molecules.<fig id="Fig7"><label>Fig. 7</label><caption><p>Microglia supply co-stimulatory signal 2 for T cell activation in the WNV-NS5-E218A-infected CNS. Mice were fed PLX5622 chow or control chow for 2 weeks, then i.c. infected with WNV-NS5-E218A (10<sup>4</sup> PFU). At 8 dpi, leukocytes were analyzed from the cortex. <bold>a</bold> Representative flow cytometry contour plots of P2RY12 expression on CD45<sup>+</sup>-gated cells to identify resident microglia (P1). <bold>b</bold> Representative flow cytometry plots of CD11b expression on P2RY12<sup>neg</sup>CD45<sup>+</sup> cells to identify infiltrating macrophages (P2). <bold>c</bold>, <bold>d</bold> Representative flow cytometry plots of MHCII expression on <bold>c</bold> P1 microglia and <bold>d</bold> P2 macrophages. <bold>e</bold>, <bold>f</bold> Representative flow cytometry plots of CD80 expression on <bold>e</bold> P1 microglia and <bold>f</bold> P2 macrophages. <bold>g</bold>, <bold>h</bold> Representative flow cytometry dot plots of CD86 expression on <bold>g</bold> P1 microglia and <bold>h</bold> P2 macrophages. <bold>i</bold>, <bold>j</bold> Quantification of flow cytometry analysis of <bold>i</bold> percentages and <bold>j</bold> total numbers of P1- vs P2-gated cells. <bold>k</bold> Quantification of percentages and <bold>l</bold> total numbers of MHCII<sup>+</sup>CD45<sup>+</sup> cells within each P1 and P2 population. <bold>m</bold> Quantification of percentages and <bold>n</bold> total number of CD80<sup>+</sup>CD45<sup>+</sup> cells within each P1 and P2 population. <bold>o</bold> Quantification of percentages and <bold>p</bold> total numbers of CD86<sup>+</sup>CD45<sup>+</sup> cells within each P1 and P2 population. For quantification panels, each symbol represents an individual control- (black) or PLX5622-treated (red) mouse, and bars indicate mean ± SEM. Data presented are the compilation of two independent experiments with seven to nine mice per group. Statistical significance was calculated using multiple <italic>t</italic> tests with Welch’s correction. For all data: ns, not significant at <italic>P</italic> < 0.05; *<italic>P</italic> < 0.05; **<italic>P</italic> < 0.01; ***<italic>P</italic> < 0.001; ****<italic>P</italic> < 0.0001</p></caption><graphic xlink:href="12974_2019_1397_Fig7_HTML" id="MO7"></graphic></fig></p></sec></sec><sec id="Sec17"><title>Discussion</title><p id="Par36">Recent studies have reported increased viral titers in the CNS of mice treated with CSF1R antagonists [<xref ref-type="bibr" rid="CR36">36</xref>, <xref ref-type="bibr" rid="CR60">60</xref>, <xref ref-type="bibr" rid="CR61">61</xref>]; however, the mechanisms underlying this loss of virologic control and the effects on peripheral immune responses have not been well characterized. We sought to determine the role of CSF1R signaling in viral clearance of both systemic and neurotropic infection. Our results indicate that antagonism of CSF1R signaling limits virologic control within the CNS via reduction of critical APCs that express co-stimulatory B7 molecules, CD80 and CD86, necessary for local reactivation of antiviral CD8<sup>+</sup> T cells. Our data also indicate that CSF1R antagonism reduces expression of co-stimulatory B7 molecules on peripheral APCs, contributing to loss of virologic control in the periphery and dissemination of virus to the CNS. As previously reported, WNV-NY inoculated into mice via f.p. is detected first within the olfactory bulb then spreads caudally through the cerebral cortex to the brain stem with significant virus not detected in the cerebellum until 8 dpi [<xref ref-type="bibr" rid="CR20">20</xref>]. CSF1R antagonism with PLX5622 leads to increased viral replication within each of these CNS regions and also in the peripheral organs: the spleen, kidney, and serum. In contrast to CNS titers, the virus is cleared from the spleen and serum by 8 dpi in WNV-infected, control, and PLX5622-treated mice. Notably, we detected expansion of viral tropism with significant viral replication in the kidney of PLX5622-treated mice at 4 and 6 dpi compared with control-treated animals. These data suggest that renal macrophages may be sensitive to CSF1R antagonism, which is consistent with a study utilizing a model of recovery from acute kidney injury in which CSF1R inhibition with GW2580 reportedly decreased both kidney macrophage proliferation and their polarization towards wound-healing phenotypes [<xref ref-type="bibr" rid="CR62">62</xref>]. In order to better isolate the role of microglia during neurotropic infection, we examined the effect of PLX5622 in an established murine model of CNS infection using an attenuated strain of WNV (WNV-NS5-E218A), which lacks 2′-<italic>O</italic>-methyltransferase activity, thus increasing sensitivity to IFIT-mediated suppression and limiting virulence in peripheral organs with intact immunity [<xref ref-type="bibr" rid="CR8">8</xref>, <xref ref-type="bibr" rid="CR45">45</xref>, <xref ref-type="bibr" rid="CR49">49</xref>]. Mice infected i.c. with WNV-NS5-E218A exhibited increased viral titers in the CNS and increased lethality with PLX5622 treatment compared with control treatment. These data suggest that loss of CNS immunity increases lethality of WNV infection.</p><p id="Par37">Clearance of WNV within the CNS requires local reactivation of antiviral CD8<sup>+</sup> T cells for efficient T cell-mediated adaptive immune responses [<xref ref-type="bibr" rid="CR55">55</xref>, <xref ref-type="bibr" rid="CR63">63</xref>]. Under homeostatic conditions, microglia express very low levels of co-stimulatory B7 molecules (CD80/CD86); however, microglia upregulate expression of these molecules in response to inflammatory cytokines [<xref ref-type="bibr" rid="CR54">54</xref>, <xref ref-type="bibr" rid="CR64">64</xref>]. Our results show PLX5622 treatment decreased expression of these molecules by depleting their cellular sources in the CNS, which include both resident microglia and infiltrating monocytes/macrophages. In the periphery, PLX5622 also reduced cellular expression of B7 molecules on splenic, blood, and pLN APCs. Under certain conditions, other co-stimulatory molecules are able to compensate for the loss of B7 signals in T cell reactivation. For example, expansion of lymphocytic choriomeningitis virus-specific antiviral T cells can be driven by either alternative co-stimulatory TNFR superfamily members or by enhanced expression of antiviral cytokines, such as high levels of type I IFNs [<xref ref-type="bibr" rid="CR65">65</xref>]. However, during WNV encephalitis, PLX5622 treatment also significantly decreased transcriptional expression of both TNF and IFNβ. Together, our results indicate that CSF1R antagonism contributes impaired local reactivation of antiviral T cells within the CNS by depleting co-stimulatory signals including both B7 molecules and inflammatory cytokines.</p><p id="Par38">In addition to augmenting antiviral T cell activation, many cytokines have essential protective roles during WNV infection. Infected human monocyte-derived macrophages release IL-8, IFNα, IFNβ, and TNF [<xref ref-type="bibr" rid="CR66">66</xref>, <xref ref-type="bibr" rid="CR67">67</xref>]. Microglia become activated in response to neurotropic WNV infection and upregulate expression of CXCL10, CXCL1, CCL5, CCL3, CCL2, TNF, and IL-6 [<xref ref-type="bibr" rid="CR18">18</xref>]. Neurons and astrocytes in WNV-infected brains also release proinflammatory mediators including CCL2, CCL5, CXCL10, IL1β, IL-6, IL-8, and TNF, which promote lymphocyte trafficking to the CNS [<xref ref-type="bibr" rid="CR19">19</xref>–<xref ref-type="bibr" rid="CR21">21</xref>]. In fact, results from a recent study demonstrated that peripheral infection with WNV increased CNS expression of chemokines important for lymphocyte trafficking, including CCL2, CCL7, CXCL9, and CXCL10, in PLX5622-treated mice compared with control-treated mice [<xref ref-type="bibr" rid="CR61">61</xref>]. Type I IFN response is widely accepted as the most immediate antiviral host response, essential for controlling viral replication during the initial infection. Mice lacking type I IFN signaling are highly vulnerable to uncontrolled WNV replication, exhibiting 100% mortality [<xref ref-type="bibr" rid="CR68">68</xref>]. Clearance of viral infections, however, requires additional immune response including type II IFN, i.e., IFNγ, which is produced by activated CD8<sup>+</sup> T cells. Mice deficient in IFNγ show higher peripheral viral load, increased CNS infection, and increased lethality [<xref ref-type="bibr" rid="CR69">69</xref>]. Data described here show significantly decreased cytokine expression in PLX5622-treated mice, which may also contribute to the loss of virologic control via the above-described mechanisms.</p><p id="Par39">The exact role of microglia during viral infection has been difficult to define because infiltrating monocytes can mask the impact of resident microglia. In a previous report, <italic>IL34</italic><sup><italic>−/−</italic></sup> mice, which have significantly reduced development of microglia, are more susceptible to lethal infection after WNV-NS5-E218A i.c. inoculation [<xref ref-type="bibr" rid="CR36">36</xref>]. Similar to results reported here, <italic>IL34</italic><sup><italic>−/−</italic></sup> mice exhibited increased neuronal death compared with wildtype controls. In contrast with PLX5622-treated mice, <italic>IL34</italic><sup><italic>−/−</italic></sup> mice exhibited similar levels of viral burden compared with wildtype controls in the brain at 3 and 6 dpi. Numbers of infiltrating monocytes/macrophages and WNV-specific T cells were also similar between <italic>IL34</italic><sup><italic>−/−</italic></sup> and wildtype controls; however, the authors did not investigate antiviral T cell activation in this model [<xref ref-type="bibr" rid="CR36">36</xref>]. In another model of flavivirus encephalitis, in which suckling mice were infected with dengue virus, depletion of microglia with liposome-encapsulated clodronate resulted in increased viral replication and reduced infiltration of IFNγ<sup>+</sup>CD8<sup>+</sup> T cells [<xref ref-type="bibr" rid="CR70">70</xref>]. Here, we report no reduction in CD8<sup>+</sup> T cell infiltration; however, this difference may be due to the age of mice, virus, and/or method of microglial depletion. Another recent study reported use of PLX5622 in a model of neuroattenuated murine corona virus, mouse hepatitis virus (MHV). Similar to our results, PLX5622 treatment resulted in increased viral replication and lethality. In contrast to our results, PLX5622 treatment reduced expression of MHCII on microglia and macrophages and reduced CD4<sup>+</sup> T cell response [<xref ref-type="bibr" rid="CR60">60</xref>]. While CD4<sup>+</sup> T cells can improve CD8<sup>+</sup> T cell response, the dependence of CD8<sup>+</sup> T cells on CD4<sup>+</sup> T cells varies by virus, antigen exposure, tissue environment, and effector sites [<xref ref-type="bibr" rid="CR71">71</xref>]. In models of MHV encephalitis, control of viral replication requires a collaborative effort between CD4<sup>+</sup> and CD8<sup>+</sup> T cells [<xref ref-type="bibr" rid="CR71">71</xref>]. However, when virus replication induces activation of APCs, activation of CD8<sup>+</sup> T cells is relatively CD4<sup>+</sup> T cell-independent [<xref ref-type="bibr" rid="CR71">71</xref>].</p><p id="Par40">Recently, chronic activation of innate immune response by microglia is believed to be a major contributor to neurodegenerative conditions [<xref ref-type="bibr" rid="CR72">72</xref>]. Genetic studies have confirmed the relevance of inflammatory response in common neurodegenerative diseases, such as Alzheimer’s disease and multiple sclerosis, indicating that neuroinflammation is likely implicated in the primary pathogenesis, rather than a secondary response [<xref ref-type="bibr" rid="CR73">73</xref>]. We and others have reported that microglia impact neurocognitive function by executing complement-mediated synapse elimination [<xref ref-type="bibr" rid="CR8">8</xref>, <xref ref-type="bibr" rid="CR74">74</xref>, <xref ref-type="bibr" rid="CR75">75</xref>]. Because of the neurological consequences of microglial activation, researchers have attempted to use CSF1R antagonism to deplete microglia in a variety of models to improve neurological function, including traumatic brain injury [<xref ref-type="bibr" rid="CR38">38</xref>, <xref ref-type="bibr" rid="CR39">39</xref>], brain irradiation [<xref ref-type="bibr" rid="CR50">50</xref>], myelin-induced catatonia [<xref ref-type="bibr" rid="CR76">76</xref>], experimental autoimmune encephalomyelitis [<xref ref-type="bibr" rid="CR40">40</xref>], and Alzheimer’s disease [<xref ref-type="bibr" rid="CR41">41</xref>]. However, our results indicate that CSF1R antagonism may ultimately be detrimental to brain health due to loss of innate and adaptive immune response in the CNS, as well as decreased peripheral APC activation. Thus, in assessing the benefit of CSF1R antagonism, it will be important to consider the diminished immune response in both the CNS and peripheral immune compartments.</p></sec><sec id="Sec18"><title>Conclusions</title><p id="Par41">This study provides evidence that CSF1R signaling is critically important for cellular immunity during WNV neurotropic infection. Pharmacologic antagonism of CSF1R signaling limits expression of co-stimulatory signals through depletion of CNS cellular sources and decreased expression on peripheral APCS. This reduces local restimulation of antiviral CD8<sup>+</sup> T cells within the CNS and results in loss of virologic control and increased mortality. These data raise important considerations regarding the use of CSF1R antagonists for the treatment of neurological disorders associated with neuroinflammation.</p></sec><sec sec-type="supplementary-material"><title>Additional files</title><sec id="Sec19"><p><supplementary-material content-type="local-data" id="MOESM1"><media xlink:href="12974_2019_1397_MOESM1_ESM.tif"><label>Additional file 1:</label><caption><p>Example gating strategies and P2RY12 staining specificity. Mice were fed chow containing PLX5622 or control chow for 2 weeks, then P2RY12 microglia staining was assessed by flow cytometry in the (A–C) forebrain, (D–F) hindbrain, (G–I) blood, (J–L) spleen, and (M–O) bone marrow of uninfected mice. (A, D, G, J, M) Representative flow cytometry dot plots are shown for each tissue type to demonstrate gating strategy to identify CD45<sup>+</sup> cells. (B, C, E, F, H, I, K, L, N, O) Representative flow cytometry dot plots of P2RY12 expression on CD45<sup>+</sup> cells in (B, E, H, K, N) control- and (C, F, I, L, O) PLX5622-treated mice in each tissue. (P) Quantification of percentages and (Q) numbers of P2RY12<sup>+</sup>CD45<sup>+</sup> cells in the forebrain (FB), hindbrain (HB), blood (Bl), spleen (Sp), and bone marrow (BM). For quantification panels, each symbol represents an individual control (black) or PLX5622 (red)-treated mouse, and bars indicate mean ± SEM. Data shown represent analysis from one experiment with 5 mice per group, repeated in two independent experiments. Statistical significance was calculated using two-way ANOVA with Sidak’s multiple comparisons test. *<italic>P</italic> < 0.05; **<italic>P</italic> < 0.01; ****<italic>P</italic> < 0.0001. (TIF 14813 kb)</p></caption></media></supplementary-material><supplementary-material content-type="local-data" id="MOESM2"><media xlink:href="12974_2019_1397_MOESM2_ESM.tif"><label>Additional file 2:</label><caption><p>PLX5622 broadly depletes Iba1+ cells. Mice were fed chow containing PLX5622 or control chow for 2 weeks, then microglia depletion was assessed by Iba1 immunohistochemical staining in the (A) hippocampus, (B) cortex, and (C) cerebellum. (D) Quantification of confocal microscopic images for number of Iba1-positive DAPI-positive cells per high-power field (HPF) in the hippocampus (Hpc), cortex (Ctx), and cerebellum (Cb). Three to six images were captured at × 40 per brain region across two separate brain sections for each of four to five independent mice collected in one experiment. Each symbol represents the average number for an individual mouse, with bars indicating mean ± SEM. Statistical significance was calculated using two-way ANOVA with Sidak’s multiple comparisons test, <italic>P</italic> < 0.05; *<italic>P</italic> < 0.05; **<italic>P</italic> < 0.01. (TIF 11593 kb)</p></caption></media></supplementary-material><supplementary-material content-type="local-data" id="MOESM3"><media xlink:href="12974_2019_1397_MOESM3_ESM.tif"><label>Additional file 3:</label><caption><p>PLX5622 treatment does not impact T cell populations in peripheral immune compartments of uninfected mice. Mice were fed PLX5622 or control chow for 2 weeks, then T cell populations were assessed in (A, B, E, F) blood and (C, D, G, H) spleen of uninfected mice. (A, C) Representative flow cytometry plots and example gating strategy to identify CD45<sup>+</sup> cells. (B, D) Representative flow cytometry plots of CD4 vs CD8 expression on CD45<sup>+</sup> cells. (E, G) Quantification of percentages and (F, H) total numbers of CD4<sup>+</sup>CD45<sup>+</sup> vs CD8<sup>+</sup>CD45<sup>+</sup> cells in uninfected mice. For quantification panels, each symbol represents an individual control (black) or PLX5622 (red)-treated mouse, and bars indicate mean ± SEM. Data shown represent analysis from one experiment with three to five mice per group, repeated in three independent experiments. Multiple unpaired <italic>t</italic> test analyses indicate no significant difference among any of these populations. (TIF 8042 kb)</p></caption></media></supplementary-material><supplementary-material content-type="local-data" id="MOESM4"><media xlink:href="12974_2019_1397_MOESM4_ESM.tif"><label>Additional file 4:</label><caption><p>PLX5622 treatment does not impact macrophage/monocyte population in peripheral immune compartments of uninfected mice. Mice were fed PLX5622 chow or control chow for 2 weeks, then monocytes/macrophages were assessed in (A–F) blood, (G–L) spleen, and (M–R) bone marrow of uninfected mice. (A, G, M) Representative flow cytometry plots of CD11b expression on CD45<sup>+</sup>-gated cells. (B, H, N) Quantification of percentages and (C, I, O) total numbers of CD11b<sup>+</sup>CD45<sup>+</sup> cells. (D, J, P) Representative flow cytometry plots of Ly6G vs Ly6C expression on CD11b<sup>+</sup>CD45<sup>+</sup> cells. (E, K, Q) Quantification of percentages and (F, L, R) total numbers of Ly6G<sup>+</sup>CD45<sup>+</sup> vs Ly6C<sup>+</sup>CD45<sup>+</sup> cells. For quantification panels, each symbol represents an individual control (black) or PLX5622 (red)-treated mouse, and bars indicate mean ± SEM. Data shown represent analysis from one experiment with five mice per group, repeated in three independent experiments. Multiple unpaired <italic>t</italic> test analyses indicate no significant difference among any of these populations. (TIF 11595 kb)</p></caption></media></supplementary-material><supplementary-material content-type="local-data" id="MOESM5"><media xlink:href="12974_2019_1397_MOESM5_ESM.tif"><label>Additional file 5:</label><caption><p>PLX5622 treatment does not enhance BBB permeability. Mice were fed PLX5622 chow or control chow for 2 weeks, then infected via footpad with WNV-NY (10<sup>2</sup> PFU). BBB permeability was measured by detection of sodium fluorescein accumulation in brain tissue homogenates derived from (A) olfactory bulb, (B) cortex, (C) cerebellum, (D) brainstem, and (E) spinal cord. Data are represented as mean ± SEM of individual mouse values normalized to serum sodium fluorescein concentration. Group means were then normalized to the mean values for uninfected controls. Statistical significance was calculated using two-way ANOVA with Sidak’s multiple comparisons test, indicating significantly different curves, but no significant difference at any 1 day. *<italic>P</italic> < 0.05; **<italic>P</italic> < 0.01; (TIF 3314 kb)</p></caption></media></supplementary-material><supplementary-material content-type="local-data" id="MOESM6"><media xlink:href="12974_2019_1397_MOESM6_ESM.tif"><label>Additional file 6:</label><caption><p>Control and PLX5622-treated CNS tissue are equally permissive to WNV-NY infection. Mice were fed chow containing PLX5622 or control chow for 2 weeks, infected i.c. with 10 PFU WNV-NY, and then monitored for (A) mortality and (B) weight loss. (A) Mice universally die from i.c. infection with WNV-NY regardless of PLX5622 treatment, with no difference in length of survival as calculated by log-rank (Mantel-Cox) test. (B) Weight was measured daily during acute illness as a measure of illness. After death, the last measured weight was carried through to the end of the experiment. Statistically significant greater weight loss was measured in control mice compared with PLX5622-treated mice as calculated by two-way ANOVA with matched values comparing group means without multiple comparisons. (C-I) Tissue viral loads were measured by plaque assay at 2 and 4 dpi in control (black) or PLX5622-treated (red) mice. (J) Serum viral loads as measured by qRT-PCR. For viral titers, each symbol represents an individual control (black) or PLX5622 (red)-treated mouse and bars indicate mean. Dotted line indicates assay limit of detection. Data shown represent analysis from one experiment with five mice per group. Statistical significance was calculated using two-way ANOVA with Sidak’s multiple comparisons test. ns, not significant at <italic>P</italic> < 0.05; *<italic>P</italic> < 0.05; ****<italic>P</italic> < 0.0001. (TIF 7958 kb)</p></caption></media></supplementary-material><supplementary-material content-type="local-data" id="MOESM7"><media xlink:href="12974_2019_1397_MOESM7_ESM.tif"><label>Additional file 7:</label><caption><p>Impact of CSF1R antagonism on APC co-stimulatory expression is tissue-dependent. Mice were fed PLX5622 or control chow for 2 weeks, then infected via f.p. with WNV-NY (100 PFU). At 4 dpi, leukocytes were isolated from blood (A–C) and draining popliteal LNs (D–F). (A) Representative flow cytometry plots of CD86 expression on CD11c<sup>+</sup>CD45<sup>+</sup>-gated cells in the blood. (B) Quantification of percentages and (C) total numbers of CD86<sup>+</sup>CD11c<sup>+</sup>CD45<sup>+</sup> cells in the blood. (D) Representative flow cytometry plots of CD80 expression on CD11c<sup>+</sup>CD45<sup>+</sup>-gated cells in the pLN. (E) Quantification of percentages and (F) total numbers of CD80<sup>+</sup>CD11c<sup>+</sup>CD45<sup>+</sup> cells in the blood. For quantification panels, each symbol represents an individual control (black) or PLX5622 (red)-treated mouse, and bars indicate mean ± SEM. Data shown represent analysis from one experiment with four to five mice per group. Statistical significance was calculated using unpaired <italic>t</italic> test. For all data: ns, not significant at <italic>P</italic> < 0.05; *<italic>P</italic> < 0.05; **<italic>P</italic> < 0.01. (TIF 4465 kb)</p></caption></media></supplementary-material><supplementary-material content-type="local-data" id="MOESM8"><media xlink:href="12974_2019_1397_MOESM8_ESM.tif"><label>Additional file 8:</label><caption><p>PLX5622 treatment does not impact T cell activation in spleens of WNV-NS5-E218A infected mice. Mice were fed PLX5622 or control chow for 2 weeks, then infected i.c. with WNV-NS5-E218A (10<sup>4</sup> PFU). At 8 dpi, splenic leukocytes were isolated and analyzed by flow cytometric analysis. (A) Representative flow cytometry gating strategy for CD45<sup>+</sup> cells. (B) Representative flow cytometry contour plots of CD4 vs CD8 expression on CD45<sup>+</sup>-gated cells. (C) Quantification of percentages and (D) total numbers of CD4<sup>+</sup>CD45<sup>+</sup> vs CD8<sup>+</sup>CD45<sup>+</sup> cells. (E) Representative flow cytometry plots of WNV-specific NS4B<sup>+</sup> tetramer staining of CD8<sup>+</sup>CD45<sup>+</sup> cells. (F) Quantification of percentages and (G) total numbers of NS4B<sup>+</sup>CD8<sup>+</sup>CD45<sup>+</sup> cells. (H) Representative flow cytometry plots of CD69 expression on NS4B<sup>+</sup>CD8<sup>+</sup>CD45<sup>+</sup> cells. (I) Quantification of percentages and (G) total numbers of CD69<sup>+</sup>NS4B<sup>+</sup>CD8<sup>+</sup>CD45<sup>+</sup> cells. For quantification panels, each symbol represents an individual control (black) or PLX5622 (red)-treated mouse, and bars indicate mean ± SEM. Data shown represent analysis from one experiment with three to five mice per group. Multiple unpaired <italic>t</italic> test analyses indicate no significant difference among any of these populations. (TIF 7349 kb)</p></caption></media></supplementary-material></p></sec></sec></body><back><glossary><title>Abbreviations</title><def-list><def-item><term>APC</term><def><p id="Par5">Antigen-presenting cell</p></def></def-item><def-item><term>BBB</term><def><p id="Par6">Blood brain barrier</p></def></def-item><def-item><term>CNS</term><def><p id="Par7">Central nervous system</p></def></def-item><def-item><term>CSF1R</term><def><p id="Par8">CSF1 receptor</p></def></def-item><def-item><term>DPI</term><def><p id="Par10">Days post-infection</p></def></def-item><def-item><term>FMO</term><def><p id="Par11">Fluorescence minus one</p></def></def-item><def-item><term>PFA</term><def><p id="Par12">Paraformaldehyde</p></def></def-item><def-item><term>qRT-PCR</term><def><p id="Par14">Quantitative RT-PCR</p></def></def-item><def-item><term>WNV</term><def><p id="Par15">West Nile virus</p></def></def-item></def-list></glossary><ack><title>Acknowledgements</title><p>We would like to thank Plexxikon Inc. for supplying PLX5622 compound and Dr. Michael Diamond for access to reagents.</p><sec id="FPar1"><title>Funding</title><p id="Par42">This work was supported by National Institutes of Health grants K99 AG053412 to KEF and U19 AI083019, R01 NS052632, and R01 AI101400 to RSK.</p></sec><sec id="FPar2" sec-type="data-availability"><title>Availability of data and materials</title><p id="Par43">Data sharing is not applicable to this article as no datasets were generated or analyzed during the current study.</p></sec></ack><notes notes-type="author-contribution"><title>Authors’ contributions</title><p>KEF and RSK designed the experiments. KEF performed experiments and compiled data. KEF and RSK prepared figures, analyzed data, and wrote the manuscript. 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