William G. Glass

CCR5 deficiency increases risk of symptomatic West Nile virus infection

West Nile virus (WNV) is a reemerging pathogen that causes fatal encephalitis in several species, including mouse and human. Recently, we showed that the chemokine receptor CCR5 is critical for survival of mice infected with WNV, acting at the level of leukocyte trafficking to the brain. To test whether this receptor is also protective in man, we determined the frequency of CCR5Δ32, a defective CCR5 allele found predominantly in Caucasians, in two independent cohorts of patients, one from Arizona and the other from Colorado, who had laboratory-confirmed, symptomatic WNV infection. The distribution of CCR5Δ32 in a control population of healthy United States Caucasian random blood donors was in Hardy-Weinberg equilibrium and CCR5Δ32 homozygotes represented 1.0% of the total group (n = 1,318). In contrast, CCR5Δ32 homozygotes represented 4.2% of Caucasians in the Arizona cohort (odds ratios [OR] = 4.4 [95% confidence interval [CI], 1.6–11.8], P = 0.0013) and 8.3% of Caucasians in the Colorado cohort (OR = 9.1 [95% CI, 3.4–24.8], P < 0.0001). CCR5Δ32 homozygosity was significantly associated with fatal outcome in the Arizona cohort (OR = 13.2 [95% CI, 1.9–89.9], P = 0.03). We conclude that CCR5 mediates resistance to symptomatic WNV infection. Because CCR5 is also the major HIV coreceptor, these findings have important implications for the safety of CCR5-blocking agents under development for HIV/AIDS.

Chemokine receptor CCR5 promotes leukocyte trafficking to the brain and survival in West Nile virus infection

The molecular immunopathogenesis of West Nile virus (WNV) infection is poorly understood. Here, we characterize a mouse model for WNV using a subcutaneous route of infection and delineate leukocyte subsets and immunoregulatory factors present in the brains of infected mice. Central nervous system (CNS) expression of the chemokine receptor CCR5 and its ligand CCL5 was prominently up-regulated by WNV, and this was associated with CNS infiltration of CD4+ and CD8+ T cells, NK1.1+ cells and macrophages expressing the receptor. The significance of CCR5 in pathogenesis was established by mortality studies in which infection of CCR5−/− mice was rapidly and uniformly fatal. In the brain, WNV-infected CCR5−/− mice had increased viral burden but markedly reduced NK1.1+ cells, macrophages, and CD4+ and CD8+ T cells compared with WNV-infected CCR5+/+ mice. Adoptive transfer of splenocytes from WNV-infected CCR5+/+ mice into infected CCR5−/− mice increased leukocyte accumulation in the CNS compared with transfer of splenocytes from infected CCR5−/− mice into infected CCR5−/− mice, and increased survival to 60%, the same as in infected CCR5+/+ control mice. We conclude that CCR5 is a critical antiviral and survival determinant in WNV infection of mice that acts by regulating trafficking of leukocytes to the infected brain.

Mechanisms of Host Defense following Severe Acute Respiratory Syndrome-Coronavirus (SARS-CoV) Pulmonary Infection of Mice

We describe a model of severe acute respiratory syndrome-coronavirus (SARS-CoV) infection in C57BL/6 mice. A clinical isolate of the virus introduced intranasally replicated transiently to high levels in the lungs of these mice, with a peak on day 3 and clearance by day 9 postinfection. Viral RNA localized to bronchial and bronchiolar epithelium. Expression of mRNA for angiotensin converting enzyme 2, the SARS-CoV receptor, was detected in the lung following infection. The virus induced production in the lung of the proinflammatory chemokines CCL2, CCL3, CCL5, CXCL9, and CXCL10 with differential kinetics. The receptors for these chemokines were also detected. Most impressively, mRNA for CXCR3, the receptor for CXCL9 and CXCL10, was massively up-regulated in the lungs of SARS-CoV-infected mice. Surprisingly Th1 (and Th2) cytokines were not detectable, and there was little local accumulation of leukocytes and no obvious clinical signs of pulmonary dysfunction. Moreover, beige, CD1−/−, and RAG1−/− mice cleared the virus normally. Infection spread to the brain as it was cleared from the lung, again without leukocyte accumulation. Infected mice had a relative failure to thrive, gaining weight significantly more slowly than uninfected mice. These data indicate that C57BL/6 mice support transient nonfatal systemic infection with SARS-CoV in the lung, which is able to disseminate to brain. In this species, proinflammatory chemokines may coordinate a rapid and highly effective innate antiviral response in the lung, but NK cells and adaptive cellular immunity are not required for viral clearance.

Detection of West Nile virus sequences in cerebrospinal fluid

We have established a sensitive and specific real-time PCR method for detection of West Nile virus. Analysis of specimens obtained during the 1999 New York outbreak indicated the presence of viral sequences In cerebrospinal fluid of all of four Individuals with fatal outcomes, and in only one of four who survived.

Antibody Targeting of the CC Chemokine Ligand 5 Results in Diminished Leukocyte Infiltration into the Central Nervous System and Reduced Neurologic Disease in a Viral Model of Multiple Sclerosis

Intracerebral infection of mice with mouse hepatitis virus, a member of the Coronaviridae family, reproducibly results in an acute encephalomyelitis that progresses to a chronic demyelinating disease. The ensuing neuropathology during the chronic stage of disease is primarily immune mediated and similar to that of the human demyelinating disease multiple sclerosis. Secretion of chemokines within the CNS signals the infiltration of leukocytes, which results in destruction of white matter and neurological impairment. The CC chemokine ligand (CCL)5 is localized in white matter tracts undergoing demyelination, suggesting that this chemokine participates in the pathogenesis of disease by attracting inflammatory cells into the CNS. In this study, we administer a mAb directed against CCL5 to mice with established mouse hepatitis virus-induced demyelination and impaired motor skills. Anti-CCL5 treatment decreased T cell accumulation within the CNS based, in part, on viral Ag specificity, indicating the ability to differentially target select populations of T cells. In addition, administration of anti-CCL5 improved neurological function and significantly (p ≤ 0.005) reduced the severity of demyelination and macrophage accumulation within the CNS. These results demonstrate that the severity of CNS disease can be reduced through the use of a neutralizing mAb directed against CCL5 in a viral model of demyelination.

Reduced macrophage infiltration and demyelination in mice lacking the chemokine receptor CCR5 following infection with a neurotropic coronavirus

Abstract Studies were performed to investigate the contributions of the CC chemokine receptor CCR5 in host defense and disease development following intracranial infection with mouse hepatitis virus (MHV). T cell recruitment was impaired in MHV-infected CCR5−/− mice at day 7 postinfection (pi), which correlated with increased (P ≤ 0.03) titers within the brain. However, by day 12 pi, T cell infiltration into the CNS of infected CCR5−/− and CCR5+/+ mice was similar and both strains exhibited comparable viral titers, indicating that CCR5 expression is not essential for host defense. Following MHV infection of CCR5+/+ mice, greater than 50% of cells expressing CCR5 antigen were activated macrophage/microglia (determined by F4/80 antigen expression). In addition, infected CCR5−/− mice exhibited reduced (P ≤ 0.02) macrophage (CD45highF4/80+) infiltration, which correlated with a significant reduction (P ≤ 0.001) in the severity of demyelination compared to CCR5+/+ mice. These data indicate that CCR5 contributes to MHV-induced demyelination by allowing macrophages to traffic into the CNS.

Mouse hepatitis virus infection of the central nervous system: chemokine-mediated regulation of host defense and disease.

Infection of the central nervous system (CNS) of susceptible mice with mouse hepatitis virus (MHV), a positive-strand RNA virus that is a member of the Coronaviridae family, reproducibly results in an acute encephalomyelitis followed by a demyelinating disease similar to the human demyelinating disease multiple sclerosis (MS). MHV infection triggers a robust cell-mediated response in which both CD4+ and CD8+ T cells are essential in controlling viral replication and spread. However, viral clearance is incomplete and viral RNA and protein can persist within white matter tracts, areas of viral persistence are often associated with demyelinating lesions, and recent studies have indicated an important role for both T cells and macrophages in contributing to myelin destruction. The molecular mechanisms governing leukocyte trafficking and accumulation within the CNS of MHV-infected mice are just now being understood and recent studies indicate that chemokines and chemokine receptors have an important role in this process. This article will provide an overview on how these molecules regulate T cell and macrophage trafficking into the CNS of MHV-infected mice and illustrate the delicate balance that exists with regards to expression of chemokines and their receptors as it relates to both host defense and disease development.

Functional Expression of Chemokine Receptor CCR5 on CD4+ T Cells during Virus-Induced Central Nervous System Disease

ABSTRACT Intracranial infection of C57BL/6 mice with mouse hepatitis virus (MHV) results in an acute encephalomyelitis followed by a demyelinating disease similar in pathology to the human disease multiple sclerosis (MS). CD4+ T cells are important in amplifying demyelination by attracting macrophages into the central nervous system (CNS) following viral infection; however, the mechanisms governing the entry of these cells into the CNS are poorly understood. The role of chemokine receptor CCR5 in trafficking of virus-specific CD4+ T cells into the CNS of MHV-infected mice was investigated. CD4+ T cells from immunized CCR5+/+ and CCR5−/− mice were expanded in the presence of the immunodominant epitope present in the MHV transmembrane (M) protein encompassing amino acids 133 to 147 (M133-147). Adoptive transfer of CCR5+/+-derived CD4+ T cells to MHV-infected RAG1−/− mice resulted in CD4+-T-cell entry into the CNS and clearance of virus from the brain. These mice also displayed robust demyelination correlating with macrophage accumulation within the CNS. Conversely, CD4+ T cells from CCR5−/− mice displayed an impaired ability to traffic into the CNS of MHV-infected RAG1−/− recipients, which correlated with increased viral titers, diminished macrophage accumulation, and limited demyelination. Analysis of chemokine receptor mRNA expression by M133-147-expanded CCR5−/−-derived CD4+ T cells revealed reduced expression of CCR1, CCR2, and CXCR3, indicating that CCR5 signaling is important in increased expression of these receptors, which aid in trafficking of CD4+ T cells into the CNS. Collectively these results demonstrate that CCR5 signaling is important to migration of CD4+ T cells to the CNS following MHV infection.

Functional analysis of the CC chemokine receptor 5 (CCR5) on virus-specific CD8+ T cells following coronavirus infection of the central nervous system

Abstract Intracranial infection of C57BL/6 mice with mouse hepatitis virus (MHV) results in an acute encephalomyelitis followed by a demyelinating disease similar in pathology to the human disease multiple sclerosis (MS). T cells participate in both defense and disease progression following MHV infection. Expression of chemokine receptors on activated T cells is important in allowing these cells to traffic into and accumulate within the central nervous system (CNS) of MHV-infected mice. The present study evaluated the contributions of CCR5 to the activation and trafficking of virus-specific CD8+ T cells into the MHV-infected CNS mice. Comparable numbers of virus-specific CD8+ T cells derived from immunized CCR5+/+ or CCR5−/− mice were present within the CNS of MHV-infected RAG1−/− mice following adoptive transfer, indicating that CCR5 is not required for trafficking of these cells into the CNS. RAG1−/− recipients of CCR5−/−-derived CD8+ T cells exhibited a modest, yet significant (P ≤ 0.05), reduction in viral burden within the brain which correlated with increased CTL activity and IFN-γ expression. Histological analysis of RAG1−/− recipients of either CCR5+/+or CCR5−/−-derived CD8+ T cells revealed only focal areas of demyelination with no significant differences in white matter destruction. These data indicate that CCR5 signaling on CD8+ T cells modulates antiviral activities but is not essential for entry into the CNS.

Generation of a Protective T-Cell Response Following Coronavirus Infection of the Central Nervous System Is Not Dependent on IL-12/23 Signaling

The functional role of IL-12 and IL-23 in host defense and disease following viral infection of the CNS was determined. Instillation of mouse hepatitis virus (MHV, a positive-strand RNA virus) into the CNS of mice results in acute encephalitis followed by a chronic immune-mediated demyelinating disease. Antibody-mediated blocking of either IL-23 (anti-IL-23p19) or IL-12 and IL-23 (anti-IL-12/23p40) signaling did not mute T-cell trafficking into the CNS or antiviral effector responses and mice were able to control viral replication within the brain. Therapeutic administration of either anti-IL-23p19 or anti-IL-12/23p40 to mice with viral-induced demyelination did not attenuate T-cell or macrophage infiltration into the CNS nor improve clinical disease or diminish white matter damage. In contrast, treatment of mice with anti-IL-12/23p40 or anti-IL-23p19 resulted in inhibition of the autoimmune model of demyelination, experimental autoimmune encephalomyelitis (EAE). These data indicate that (1) IL-12 and IL-23 signaling are dispensable in generating a protective T-cell response following CNS infection with MHV, and (2) IL-12 and IL-23 do not contribute to demyelination in a model independent of autoimmune T-cell-mediated pathology. Therefore, therapeutic targeting of IL-12 and/or IL-23 for the treatment of autoimmune diseases may offer unique advantages by reducing disease severity without muting protective responses following viral infection.