Chemokines (chemotactic cytokines) are involved in a wide variety of biological processes. Following microbial infection, there is often robust chemokine signaling elicited from infected cells, which contributes to both innate and adaptive immune responses that control growth of the invading pathogen. Infection of the central nervous system (CNS) by the neuroadapted John Howard Mueller (JHM) strain of mouse hepatitis virus (JHMV) provides an excellent example of how chemokines aid in host defense as well as contribute to disease. Intracranial inoculation of the CNS of susceptible mice with JHMV results in an acute encephalomyelitis characterized by widespread dissemination of virus throughout the parenchyma. Virus-specific T cells are recruited to the CNS, and control viral replication through release of antiviral cytokines and cytolytic activity. Sterile immunity is not acquired, and virus will persist primarily in white matter tracts leading to chronic neuroinflammation and demyelination. Chemokines are expressed and contribute to defense as well as chronic disease by attracting targeted populations of leukocytes to the CNS. The T cell chemoattractant chemokine CXCL10 (interferon-inducible protein 10 kDa, IP-10) is prominently expressed in both stages of disease, and serves to attract activated T and B lymphocytes expressing CXC chemokine receptor 3 (CXCR3), the receptor for CXCL10. Functional studies that have blocked expression of either CXCL10 or CXCR3 illuminate the important role of this signaling pathway in host defense and neurodegeneration in a model of viral-induced neurologic disease.
The Chemokine CXCL10 and JHMV-Induced Acute Encephalomyelitis
The human CXCL10/IP-10 (interferon-inducible protein 10 kDa) was originally cloned and characterized following IFN-γ treatment of the human monocyte-like U937 in 1985 by Luster et al. The mouse ortholog, originally dubbed cytokine response gene-2 (crg-2), was subsequently cloned and characterized in 1990 by Vanguri and Farber. The molecular and biochemical characterization of CXCL10 are outside the scope of this review, yet there are numerous articles detailing these specific biological aspects of this chemokine related to apoptosis, cell growth, and proliferation, as well as regulating angiostasis.
CXCL10 is a member of the non-ELR CXC chemokine along with CXCL9 and CXCL11, and these three chemokine ligands all bind to the surface receptor CXC chemokine receptor 3 (CXCR3) that is expressed on numerous different cell types. Binding of CXCL10 to CXCR3 expressed by cells of the immune system has been shown to influence migration/homing of macrophages, dendritic cells, NK cells, and activated T cell subsets to areas of inflammation. Initially described as potentially important in attracting T cells to psoriatic plaques, CXCL10 has subsequently been shown to be expressed in numerous human inflammatory diseases. In addition, CXCL10 is expressed in response to microbial infection, and is important in attracting targeted CXCR3-positive leukocytes to sites of infection that help control/eliminate the invading pathogen.
We became interested in host factors governing neuroinflammation in response to JHMV infection of the CNS. Previously, numerous cytokines had been shown to be increased in response to CNS infection, yet it was unclear whether chemokines were expressed. Using a RNAse protection assay (RPA) targeting chemokines, we demonstrated that transcripts encoding a number of different chemokines are rapidly synthesized in response to JHMV infection of the CNS . Of these, the chemokine CXCL10 is the predominant transcript detected at both acute and chronic stages of disease arguing for a potentially important role in both host defense and disease. In situ hybridization of CXCL10 transcripts revealed strict colocalization of CXCL10 messenger RNA (mRNA) transcripts with viral trans, arguing that soluble factors released from infected cells, for example, type I interferons may enhance CXCL10 expression.
We have determined that resident glial cells including astrocytes as well as inflammatory macrophage/microglia express CXCL10 within the CNS of JHMV-infected mice. The early and dominant expression of CXCL10 following CNS infection by JHMV argued for a potential role as a key sentinel molecule in host defense. In support of this notion, treatment of infected mice with an anti-CXCL10-neutralizing antibody resulted in increased mortality and impaired ability to control JHMV replication that correlated with reduced levels of IFN-γ-producing T cells within the CNS . Therefore, these results argued that early expression of CXCL10 aided in host defense by attracting CXCR3-positive virus-specific T cells. These findings were further supported by subsequent studies employing JHMV infection of germline CXCL10−/− mice that resulted in decreased entry of IFN-γ-positive T cells into the CNS and reduced ability for JHMV replication. These findings indicated that blocking CXCL10 signaling, through use of either neutralizing antibody or genetic ablation, reduced activated virus-specific T cell entry into the CNS.
Interestingly, we demonstrated through flow cytometry for staining of CXCR3 and intracellular IFN-γ following stimulation with virus-specific peptides that >90% of these virus-specific T cells expressed the CXCL10 receptor . However, CXCL10 neutralization selectively reduced accumulation and/or retention of virus-specific CD4+ T cells to the CNS, yet exhibited a milder effect on virus-specific CD8+ T cells (79). Furthermore, administration of anti-CXCR3 antibody to JHMV-infected mice reduced CD4+ T cell infiltration, while CD8+ T cell trafficking was not dramatically affected (78). The selective effect of anti-CXCR3 treatment on CD4+ T cells was not the result of either reduced proliferation or modulation in chemokine receptor gene expression. Therefore, CXCR3 signaling has a nonredundant role in T cell subset trafficking in response to viral infection, and argue that differential signals are required for trafficking and retention of virus-specific CD4+ and CD8+ T cells in response to JHMV CNS infection.
As an additional method to assess the importance of CXCL10 in host defense against JHMV-induced neurologic disease, we generated a recombinant virus strain of MHV capable of expressing CXCL10. The CXCL10-expressing recombinant of MHV (MHV-CXCL10) was generated through targeted recombination using a reverse genetic approach. In addition, an isogenic wild-type control virus was constructed in the same manner. For both viruses, the exogenous gene was inserted into open reading frame (ORF)4 of the MHV-A59 parental virus. Notably, the A59 strain of MHV is capable of replicating in both the CNS and the liver following i.c. inoculation, allowing us the opportunity to explore whether the protective effects of CXCL10 are restricted to the CNS. Importantly, MHV ORF4 encodes for a nonstructural protein that is not essential for growth in tissue culture or within the mouse CNS.
Studies over the past 20 years from our laboratory and others have helped shape our understanding of the functional role of CXCL10 in host defense and disease in response to JHMV infection of the CNS. Using either antibody targeting or genetic silencing of CXCL10, it has been determined that early expression of CXCL10 is beneficial as it serves to attract CXCR3-positive T cells into the CNS that subsequently aid in controlling viral replication. Equally important is the demonstration that ASCs respond to CXCL10 expression in the CNS to enter the parenchyma and suppress viral replication through secretion of virus-specific antibody. Conversely, sustained expression of CXCL10 also contributes to JHMV-induced demyelination through attraction of CD4+ T cells that amplifies neuroinflammation through IFN-γ-mediated expression of other chemokines.
Importantly, subsequent studies by other investigators have demonstrated that CXCL10 is important in host defense against other neurotropic viruses, including Herpes Simplex Virus-1 (HSV-1) and West Nile Virus (WNV). Although much is known about CXCL10 and how it shapes inflammation in acute and chronic diseases following viral infection of the CNS, there are undoubtedly a number of additional questions that need to be addressed with regard to how the CXCL10:CXCR3 signaling pathway influences glial biology and repair in response to viral-induced neurologic disease.
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