key: cord-0824510-n1yjzk3j authors: Grist, Jonathan J.; Marro, Brett; Lane, Thomas E. title: Neutrophils and viral-induced neurologic disease date: 2018-04-01 journal: Clinical Immunology DOI: 10.1016/j.clim.2016.05.009 sha: 9e0f1a931626ce9554b73e38693d47de81893dbb doc_id: 824510 cord_uid: n1yjzk3j Infection of the central nervous system (CNS) by neurotropic viruses represents an increasing worldwide problem in terms of morbidity and mortality for people of all ages. Although unique structural features of the blood-brain-barrier (BBB) provide a physical and physiological barrier, a number of neurotropic viruses are able to enter the CNS resulting in a variety of pathological outcomes. Nonetheless, antigen-specific lymphocytes are ultimately able to accumulate within the CNS and contribute to defense by reducing or eliminating the invading viral pathogen. Alternatively, infiltration of activated cells of the immune system may be detrimental, as these cells can contribute to neuropathology that may result in long-term cellular damage or death. More recently, myeloid cells e.g. neutrophils have been implicated in contributing to both host defense and disease in response to viral infection of the CNS. This review highlights recent studies using coronavirus-induced neurologic disease as a model to determine how neutrophils affect effective control of viral replication as well as demyelination. Intracranial infection of susceptible mice with the JHM strain of mouse hepatitis virus (JHMV) causes an acute encephalomyelitis followed by a chronic demyelinating disease similar to the human demyelinating disease multiple sclerosis (MS). Early following JHMV infection of the CNS, the virus targets ependymal cells lining the ventricles, replicates, and rapidly disseminates into the brain parenchyma at which point the virus infects and replicates within astrocytes, oligodendroglia, and microglia throughout the brain and spinal cord [45] . In response to viral infection of glial cells, a rapid and orchestrated expression of chemokines occurs that contribute to attracting inflammatory cells into the CNS. In terms of host defense, secretion of chemokines derived from the CNS, including CXCL10 and CCL5, promote the migration and accumulation of virus-specific CD4+ and CD8+ T cells that control viral replication via secretion of IFN-γ and cytolytic activity. While inflammatory T cells are effective in eliminating virus, sterile immunity is not achieved; viral protein and/or RNA persist within astrocytes and oligodendroglia resulting in chronic expression of chemokine genes leading to chronic neuroinflammation and demyelination. Histological features associated with viral persistence include the development of an immune-mediated demyelinating disease similar to the human demyelinating disease MS in that both T cells and macrophages are critical mediators of disease severity and contribute to myelin damage [5, 32] . Through the course of both acute and chronic JHMV-induced neurologic infection, there is a coordinated expression of chemokines and chemokine receptors that regulate inflammation contributing to both host defense and disease exacerbation. Among the chemokines expressed during infection are members of the ELR(+) chemokine family CXCL1, CXCL2, and CXCL5. CXCL1 and CXCL2 are potent chemoattractants for neutrophils via binding and signaling through the receptor CXCR2 [28, 39, 48] . Moreover, PMNs have been shown to enhance CNS inflammation by disrupting blood brain barrier (BBB) integrity in various animal models of chronic neuroinflammation including spinal cord injury (SCI) [9, 44] and autoimmune demyelination [4] while blocking or silencing of CXCR2 signaling mutes inflammation and tissue damage in mouse models in which PMN infiltration is critical to disease initiation [2, 4, 9, 18, 24, 25, 43, 47] . Neutrophils represent a component of the innate immune response and provide an essential role in killing invading pathogens through an arsenal of defense mechanisms including release of microbicidal granules and release of reactive oxygen/nitrogen species [3, 30] . While a clear role for neutrophils in combating bacterial pathogens is documented [3, 30] , how these cells contribute to host defense and disease in response to CNS viral infection is less well characterized. McGavern et al. [19] employed two-photon microscopy to elegantly demonstrate that neutrophils, along with monocytes, were responsible for vascular leakage and acute lethality following lymphocytic choriomeningitis virus (LCMV) infection of the CNS. Human immunodeficiency virus-1 (HIV-1) infection of monocyte-derived macrophages increases expression of CXCL5 that serves to attract neutrophils that may augment neuropathology by contributing to neuron death [10] . Experimental Clinical Immunology j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / y c l i m infection of mice with West Nile virus (WNV) in which neutrophil trafficking to the CNS is impaired results in increased protection from WNV encephalitis by limiting immune cell access to the CNS thus diminishing neuropathology [46] . With regards to JHMV-induced encephalomyelitis, early work by Stohlman et al. [50] highlighted a previously unrecognized role for neutrophils in effectively controlling viral replication within the CNS. The underlying mechanisms by which neutrophils contribute to an effective host defense are related to neutrophil-mediated permeabilization of the blood-brain-barrier (BBB) through release of matrix metalloproteinase 9 (MMP-9) [50] although other factors independent of MMP-9 may also be involved [37] . In addition, monocytes can also enhance T cell accumulation within the CNS of JHMV-infected mice through the glia limitans [37] . Neutrophils are rapidly mobilized from the bone-marrow and into the blood in response to CNS infection by JHMV and this most likely reflects the precipitous increase in expression of the neutrophil chemoattractants CXCL1, CXCL2, and CXCL5 that all bind to their cognate receptor CXCR2 with high binding [12] . Indeed, treatment of JHMV-infected mice with a blocking antibody specific for CXCR2 resulted impaired migration of CXCR2-bearing neutrophils to the CNS and this resulted in increased mortality that was associated with impaired ability to control viral replication within the CNS [12] . Blocking neutrophil accumulation within the CNS resulted in reduced expression of MMP-9, limited permeabilization of the BBB, and diminished infiltration of virus-specific T cells [12] . Collectively, these findings illustrate that neutrophils are an important component of an effective host defense following CNS infection with a neurotropic virus. Neutrophil infiltration into the CNS has been associated with neurologic disease in pre-clinical animal models [6, 14, 34, 38, 40] . Herz et al. [11] have recently demonstrated that CXCR2 antagonization reduced neurological deficits and infarct volumes following middle cerebral artery occlusion and this was associated with reduced neutrophil infiltration into the CNS. Similarly, depletion of neutrophils following subarachnoid hemorrhage was found to improve memory in a model of aneurysmal subarachnoid hemorrhage (SAH) [33] . Additionally, Zenaro et al. [49] have demonstrated a role for the adhesion molecule lymphocyte function-associated antigen 1 (LFA-1) in promoting neutrophil accumulation within the CNS and amplifying AD-like pathology in transgenic models of Alzheimer's disease (AD). Depletion of neutrophils and/or a deficiency in LFA-1 resulted in protection from cognitive decline and reduced gliosis arguing that blocking neutrophil trafficking may be beneficial in AD [49] . Within models of spinal cord injury/ trauma, neutrophils are among the first cells to accumulate within the site of injury and a number of studies argue for a pathogenic role for these cells through limiting tissue sparing and motor recovery while increasing expression of pro-inflammatory cytokines [1, 36] . Collectively, these studies demonstrate that in animal models of chronic neuroinflammation/neurodegeneration neutrophils can amplify the severity histologic disease and argue that blocking entry into the CNS may limit the severity of neurologic disease. A role for neutrophils in immune-mediated demyelination remains to be well characterized. Ransohoff et al. [22] , have shown that CXCR2-positive neutrophils are essential for cuprizone-induced demyelination and potentially contribute to oligodendrocyte cell loss. Questions remain regarding the importance of neutrophils in the pathogenesis of MS given the paucity of these cells in active lesions; however, elevated neutrophil numbers within the cerebrospinal fluid (CSF) of MS patients have been correlated with clinical relapse [20] . Administration of granulocyte-colony-stimulating factor (G-CSF), a neutrophil activating molecule, to MS and neuromyelitis optica (NMO) patients resulted in disease exacerbation arguing for a role for these cells in amplifying disease severity [16, 31] . Additionally, neutrophils have been reported to be more numerous and exhibit a more primed state in MS patients [29] . Recent studies [15, 35] highlight the importance of CXCL1 as well as other myeloid-chemoattractant molecules as having a possible role in potentiating disease in patients with either relapsing-remitting or progressive forms of MS, suggesting that soluble factors that attract neutrophils and/or neutrophil-related molecules may be important therapeutic targets for MS patients. Support for this notion is derived from studies employing experimental autoimmune encephalomyelitis (EAE) as a model for MS in which disease onset is mute when neutrophil trafficking to the CNS is disrupted [4, 27] . More recently, Stoolman et al. [42] have expanded on these findings to show that enriched expression of CXCL2 within the brainstem attracts neutrophils that substantially contribute to atypical EAE. Similarly, mice in which neutrophils lack suppressor of cytokine signaling 3 (SOCS3) exhibit an increase in susceptibility to the atypical EAE and this correlates with preferential recruitment of neutrophils into the cerebellum and brainstem [23] . The site of neutrophil recruitment may be critical in terms of amplifying histopathology as neutrophil accumulation within the brain, but to a limited extent in the spinal cord, contribute to tissue injury [41] . Collectively, these findings indicate that neutrophils can affect the severity of clinical disease and neuroinflammation in EAE. In attempt to better understand how neutrophils influence both host defense and disease following CNS viral infection, we have recently engineered transgenic mice to utilize the tetracycline-controlled transcriptional activation system in which the human glial fibrillary acidic protein (hGFAP) promoter drives expression of a modified version of the reverse tetracycline transactivator protein (rtTA*M2) [26] (Fig. 1A) . Astrocytes were chosen for targeted expression of CXCL1 as previous studies [7, 8, 17] have shown that JHMV-infected astrocytes express CXCL1 [13, 21] . In the presence of doxycycline (Dox), transcription initiates at a tet-operon and leads to production of recombinant CXCL1 mRNA transcripts. Double transgenic (tg) mice (pBI-CXCL1-rtTA) and single tg mice (pBI-CXCL1) were generated; characterization of double tg mice revealed Dox-dependent expression of CXCL1 from cultured astrocytes as determined by ELISA (Fig. 1B) [26] . I.c. infection of Doxtreated double tg mice with JHMV resulted in a selective increased expression of CXCL1 mRNA transcripts and protein within the brain and spinal cords when compared to Dox-treated single tg mice infected with JHMV (Fig. 1C) [26] . Dox-induced overexpression of CXCL1 did not enhance control of viral replication within the CNS as both infected double and single tg mice exhibited similar viral titers at defined times post-infection (p.i.) nor were there differences in either frequency or numbers of virus-specific CD4+ and CD8+ T cells within the CNS of double tg mice compared to single tg mice [26] . However, Doxtreatment of JHMV-infected double tg mice resulted in increased clinical disease and mortality when compared to infected single tg mice [26] . In conjunction with increased expression of CXCL1 initiated within the CNS of Dox-treated double tg mice infected with JHMV, there was a rapid increase in CXCL1 protein levels in serum [26] . Correspondingly, there is a rapid increase in neutrophils within the blood at days 4 (p b 0.05) and 7 (p b 0.001) in double tg mice compared to infected single tg controls [26] . Dox-induced CXCL1 production in JHMV-infected double tg mice also resulted in an increase in neutrophil frequency within the brain at days 4 and 7 p.i. [26] . Similarly, there was an increase in neutrophil frequency within spinal cords of double tg mice at days 4 (p b 0.01) and 7 (p b 0.05) p.i. compared to single tg mice [26] . Immunofluorescence staining for neutrophils (Ly-6B.2) supported the flow cytometric data and revealed increased numbers of neutrophils accumulating within the meninges of double tg mice at day 7 p.i. [26] . The increased presence of neutrophils within the CNS of double tg mice suggested that there would also be a corresponding increase in blood-brain-barrier (BBB) permeability. Surprisingly, no differences were observed in BBB permeability within the brain or spinal cord at day 4 p.i. as measured by sodium fluorescein (NaF) uptake [26] . Examination of spinal cords from JHMV-infected Dox-treated double tg mice revealed an overall increase (p b 0.05) in the severity of demyelination when compared to infected single tg animals ( Fig. 2A ) [26] . The increase in demyelination in double tg mice was associated with a significant (p b 0.05) loss of mature oligodendrocytes (as determined by expression of GST-π) within the spinal cords and increased numbers of microglia in Dox-treated JHMV-infected double tg mice compared to infected single tg mice [26] . Flow cytometric data indicated that neutrophil frequencies within the spinal cords of infected double tg were significantly increased (p b 0.01) as well as their total numbers (p b 0.001) at day 12 p.i. compared to single tg mice (Fig. 2B) [26] . Additionally, neutrophils were detected within the spinal cord parenchyma of double tg mice compared to single tg mice (Fig. 2B) [26] . Elimination of neutrophils via administration of anti-Ly6g monoclonal antibody injection into JHMV-infected double tg mice treated with Dox resulted in a reduction in the severity of demyelination when compared to mice treated with isotype control antibody (Fig. 2C ) thus demonstrating that neutrophils are capable of augmenting the severity of white matter damage [26] . Although a role for neutrophils in host defense following infection with bacterial pathogens has been appreciated for a number of years, how neutrophils affect host defense in response to viral infection of the CNS has not been as well studied. However, it is now clear that neutrophils are capable of enhancing control of viral replication within the CNS through increasing the permeabilization of the BBB thereby allowing antigen-specific lymphocytes access to sites of infection. Equally interesting is how neutrophil infiltration into the CNS contributes to neuropathology e.g. demyelination. Compelling new information derived from clinical studies from MS patients as well as preclinical animal models of MS have emphasized a potential role for these cells in amplifying white matter damage opening the possibility of targeting neutrophil migration into the CNS as a therapeutic strategy to limit CNS damage. The systemic inflammatory response after spinal cord injury in the rat is decreased by alpha4beta1 integrin blockade CXCR2/CXCR2 ligand biology during lung transplant ischemia-reperfusion injury Neutrophils, from marrow to microbes The Th17-ELR+ CXC chemokine pathway is essential for the development of central nervous system autoimmune disease A murine virus (JHM) causing disseminated encephalomyelitis with extensive destruction of myelin Mast cell activation and neutrophil recruitment promotes early and robust inflammation in the meninges in EAE Chemokine expression in GKO mice (lacking interferon-gamma) with experimental autoimmune encephalomyelitis Expression of chemokines RANTES, MIP-1alpha and GRO-alpha correlates with inflammation in acute experimental autoimmune encephalomyelitis Reparixin, an inhibitor of CXCR2 function, attenuates inflammatory responses and promotes recovery of function after traumatic lesion to the spinal cord Transcriptional regulation of CXCL5 in HIV-1-infected macrophages and its functional consequences on CNS pathology Role of neutrophils in exacerbation of brain injury after focal cerebral ischemia in hyperlipidemic mice A protective role for ELR+ chemokines during acute viral encephalomyelitis CXCR2 signaling protects oligodendrocytes and restricts demyelination in a mouse model of viral-induced demyelination Inflammatory monocytes damage the hippocampus during acute picornavirus infection of the brain Dysregulation of the IL-23/IL-17 axis and myeloid factors in secondary progressive MS Detrimental role of granulocyte-colony stimulating factor in neuromyelitis optica: clinical case and histological evidence Act1 mediates IL-17-induced EAE pathogenesis selectively in NG2+ glial cells CXC chemokine receptor-2 ligands are required for neutrophil-mediated host defense in experimental brain abscesses Myelomonocytic cell recruitment causes fatal CNS vascular injury during acute viral meningitis Colic, IL-17 and glutamate excitotoxicity in the pathogenesis of multiple sclerosis Dynamic regulation of alpha-and beta-chemokine expression in the central nervous system during mouse hepatitis virus-induced demyelinating disease CXCR2-positive neutrophils are essential for cuprizone-induced demyelination: relevance to multiple sclerosis Preferential recruitment of neutrophils into the cerebellum and brainstem contributes to the atypical experimental autoimmune encephalomyelitis phenotype CXCR2 is critical for dsRNA-induced lung injury: relevance to viral lung infection CXCR2/ CXCR2 ligand biological axis impairs alveologenesis during dsRNA-induced lung inflammation in mice Inducible expression of CXCL1 within the central nervous system amplifies viral-induced demyelination Treatment with anti-granulocyte antibodies inhibits the effector phase of experimental autoimmune encephalomyelitis Neutrophil-activating properties of the melanoma growth-stimulatory activity Neutrophils in multiple sclerosis are characterized by a primed phenotype Neutrophils and immunity: challenges and opportunities Multiple sclerosis flares associated with recombinant granulocyte colony-stimulating factor Coronaviruses: hepatitis, peritonitis, and central nervous system disease Neutrophil depletion after subarachnoid hemorrhage improves memory via NMDA receptors Innate immunity in the central nervous system Neutrophil-related factors as biomarkers in EAE and MS The LTB4-BLT1 axis mediates neutrophil infiltration and secondary injury in experimental spinal cord injury Monocytes regulate T cell migration through the glia limitans during acute viral encephalitis Meningeal mast cells affect early T cell central nervous system infiltration and blood-brain barrier integrity through TNF: a role for neutrophil recruitment? High-and low-affinity binding of GRO alpha and neutrophil-activating peptide 2 to interleukin 8 receptors on human neutrophils Complement C3 and C5 play critical roles in traumatic brain cryoinjury: blocking effects on neutrophil extravasation by C5a receptor antagonist Cytokine-regulated neutrophil recruitment is required for brain but not spinal cord inflammation during experimental autoimmune encephalomyelitis Site-specific chemokine expression regulates central nervous system inflammation and determines clinical phenotype in autoimmune encephalomyelitis The role of CXCR2/CXCR2 ligands in acute lung injury A neutrophil elastase inhibitor (ONO-5046) reduces neurologic damage after spinal cord injury in rats Sequential infection of glial cells by the murine hepatitis virus JHM strain (MHV-4) leads to a characteristic distribution of demyelination CXCR2 is required for neutrophil recruitment to the lung during influenza virus infection, but is not essential for viral clearance Identification and characterization of macrophage inflammatory protein 2 Neutrophils promote Alzheimer's disease-like pathology and cognitive decline via LFA-1 integrin Neutrophils promote mononuclear cell infiltration during viral-induced encephalitis This work was supported by NIH grant R01NS041249 to T.E.L. B.S.M. was supported by NIH T32 Training Grant 5T32A1007319.