Background
MicroRNAs (miRNAs) are a new class of evolutionarily conserved gene-regulatory molecules that function to repress key target genes, primarily at the post-transcriptional level through specific mRNA 3′ untranslated region (3′ UTR) interactions [
1]. Because miRNAs commonly target critical signaling proteins and transcription factors with potent regulatory impacts on the immune system [
2,
3], it is accepted that miRNAs have an important effect on immune system activation and cellular differentiation. Recent work by our group and others has determined that miR-155 is an important regulator of immune cell development and function. Following its original identification as an oncogene in chicken lymphomas [
4], miR-155 was discovered to be overexpressed in mammalian hematopoietic cancers and shortly thereafter established as an immunomodulatory noncoding RNA in macrophages and B lymphocytes [
5‐
9]. It is now clear that miR-155 is expressed by and functions within a variety of activated immune cell types that include various T cell populations, NK cells, and dendritic cells [
6,
7,
10‐
12]. In addition, miR-155 represses a variety of immunoregulatory proteins that include signaling molecules such as Ship1 [
13] and Socs1 [
14], as well as transcriptional regulators such as Jarid2 [
15], Ets1 [
16,
17], PU.1 [
18], and Fosl2 [
19].
Consistent with its known roles in regulating immune factors, multiple studies have demonstrated that miR-155 is important in shaping the immune responses that govern viral pathogenesis [
20]. Genetic silencing of miR-155 results in increased sensitivity to experimental infection with lymphocytic choriomeningitis virus (LCMV) [
21,
22], influenza virus [
23], and herpes simplex virus (HSV) [
24,
25]. While miR-155 had previously been shown to help tailor CD4
+ T cell responses in models of autoimmunity, viral studies have since illustrated the importance of miR-155 in strengthening CD8
+ T cell responses. Recent reports showed that miR-155 is required for optimal CD8
+ T cell function following experimental infection with LCMV in terms of CTL activity, cytokine secretion, and proliferation [
21,
22]. With regard to viral-induced encephalitis, miR-155 is important in controlling neuroinflammation, presumably by regulating T cell responses [
24,
26]. These reports have emphasized the importance of miR-155 in augmenting host defense following viral infection; however, there have been few rigorous studies examining how miR-155 influences immune cell responses in a model of viral-induced encephalomyelitis.
Inoculation of the neurotropic JHM strain of mouse hepatitis virus (JHMV) into the CNS of susceptible strains of mice provides an excellent model for examining host response mechanisms responsible for controlling viral replication and modulating neuroinflammation within distinct cell lineages present in the brain [
27,
28]. During acute disease, control of viral replication is mediated by infiltrating CD4
+ and CD8
+ T cells [
29‐
31]; however, clearance of virus is not complete, and animals that survive the acute disease develop an immune-mediated demyelinating disease with both T cells and macrophages amplifying disease severity by contributing to myelin damage [
32‐
38]. Our findings demonstrated that miR-155 was necessary for optimal T cell accumulation, cytolytic activity, cytokine secretion, and trafficking to the CNS after JHMV infection. Macrophage migration and accumulation within the CNS was not impaired in the absence of miR-155 during the time period studied, and there were no differences in the severity of demyelination at 14 days pi, when peak disease severity generally occurs. These results demonstrate that miR-155 has an important role in regulating antiviral T cell responses following viral-induced neuroinflammation.
Discussion
In this report, we have examined the mechanisms by which miR-155 affects both host defense and disease progression following JHMV infection of the CNS. Our findings revealed that miR-155 expression is associated with susceptibility to JHMV-induced neurologic disease. Expression of miR-155 is necessary for effective antiviral T cell responses as ablation of miR-155 resulted in increased morbidity/mortality that was associated with elevated viral titers within the CNS. Increased disease severity most likely reflects dampened CD8
+ T cell responses, as reflected by reduced CNS accumulation of virus-specific CD8
+ T cells. Furthermore, cytolytic activity by CD8
+ T cells, as well as secretion of IFN-γ, was reduced in
miR-155
−/−
CD8
+ T cells, highlighting a role for this molecule in configuring effective responses by virus-specific CD8
+ T cells. While expansion of virus-specific CD4
+ T cells was not affected in the absence of miR-155, IFN-γ secretion by CD4
+ T cells was diminished. Importantly, the ability of T cells to migrate to the CNS was dramatically reduced in the absence of miR-155 expression, and this was associated with increased susceptibility to JHMV-induced neurologic disease. Whether increased susceptibility to JHMV-induced neurologic disease reflects a T cell intrinsic problem or whether our findings reflect an extrinsic effect via other immune cells, e.g., dendritic cells, is not known at this time. While this is an important question, we believe that muted antiviral T cell responses in
miR-155
−/−
mice following JHMV infection reflects an intrinsic problem in that (i) adoptive transfer of
miR-155
−/−
virus-specific T cells into JHMV-infected mice was unable to effectively reduce CNS viral titers and (ii) recent studies employing experimental infection of
miR-155
−/−
mice with lymphocytic choriomeningitis virus (LCMV) indicate that impaired T cell responses are due to specific deficiencies in T cells [
21].
Within the context of neuroinflammatory diseases, miR-155 was initially shown to be critical in the induction of myelin-reactive Th17 cells in EAE, the prototypic model of the human demyelinating disease multiple sclerosis (MS) [
67,
68]. In addition, miR-155 expression by endothelial cells of the blood-brain barrier (BBB) has been shown to regulate BBB function and affect neuroinflammation during EAE [
69]. More recently, a role for miR-155 has been implicated in contributing to neuroinflammation in models of Parkinson’s disease [
70], Alzheimer’s disease [
71], alcohol-induced neuroinflammation [
72], and amyotrophic lateral sclerosis (ALS) [
73]. Although the mechanisms by which miR-155 affects neuroinflammation have not been firmly established, an emerging concept is that expression of miR-155 by microglia is important in regulating expression of proinflammatory genes that subsequently influence neuroinflammation [
74‐
77]. We are currently further investigating the mechanisms by which miR-155 affects host defense and disease progression in models of viral encephalitis. Results from the current study are congruent with recent reports by Rouse and colleagues [
24] demonstrating that miR-155 affects susceptibility to HSV-1-induced encephalitis as a result of impaired antiviral T cell responses as well as homing to the CNS. Similarly, the severity of neuroinflammation is reduced following experimental infection with Japanese encephalitis virus (JEV) in the absence of miR-155, and this is associated with dampened expression of proinflammatory cytokines [
26]. These findings emphasize an important role for miR-155 in augmenting host defense in response to CNS infection by neurotropic viruses through different mechanisms, including regulating gene expression by resident glia and tailoring T cell responses. With regard to the former, negative regulation of Ship1 by miR-155 was found to affect expression of proinflammatory cytokines and modulate neuroinflammation during JEV infection [
26]. A number of different mechanisms by which miR-155 controls T cell responses following viral infection have been proposed. In the absence of miR-155, virus-specific CD8
+ T cells have enhanced type-I interferon signaling, leading to increased susceptibility to interferon’s anti-proliferative effect [
23]. Impaired antiviral CD8
+ T cell responses have also been associated with reduced activation of the prosurvival Akt pathway, arguing that miR-155 promotes T cell survival/function in response to viral infection [
21]. Targeting of Socs1 by miR-155 has also been shown to disrupt T cell function in response to viral infection, and these studies emphasized the importance of both cell type and context in determining how miR-155 affects lymphocyte function [
22]. Whether these miR-155-related pathways and/or targets are affected in response to JHMV infection of the CNS remains to be determined and is the focus of ongoing studies by our group.
Previous work from our lab and others has implicated chemokines as important in regulating lymphocyte migration to the CNS in response to viral infection [
78]. Specifically, we have shown that expression of both CXCR3 and CCR5 promote migration of virus-specific T cells into the CNS of JHMV-infected mice [
42,
43,
45,
79]. Our findings that impaired migration of miR-155-deficient, virus-specific CD8+ T cells to the CNS of JHMV-infected mice correlated with reduced expression of CXCR3, but not CCR5, are interesting and argue that expression of chemokine homing receptors may be modulated by miR-155. In our hands, this effect was restricted to CD8
+ T cells, as neither CXCR3 nor CCR5 expression was affected in miR-155-deficient CD4
+ T cells. Nonetheless, homing to the CNS by CD4
+ cells was reduced, arguing that the absence of miR-155 may affect the ability of these cells to efficiently migrate to sites of infection. This theory was further supported by adoptive transfer experiments demonstrating that in
RAG-1
−/−
mice that received miR-155-deficient CD4
+ or CD8
+ cells, there was a dramatic deficiency in CNS accumulation of CD4
+ or CD8
+ T cells, respectively. In addition, there was an impaired ability to control viral replication compared to recipients of WT cells. Recent work has demonstrated that miRNAs, including miR-155, may influence chemokine receptor expression on circulating lymphocytes [
80‐
82], suggesting that sufficient expression of these homing receptors is intrinsically influenced by miRNAs.
Mice persistently infected with JHMV develop an immune-mediated demyelinating disease in which chronic infiltration of virus-specific T cells and macrophages amplifies the severity of demyelination. The profile of clinical symptoms and accompanying histopathology associated with JHMV persistence has been employed as a pre-clinical animal model of the human demyelinating disease multiple sclerosis (MS) [
28,
83,
84]. Previous studies have demonstrated that genetic silencing of miR-155 ameliorates the severity of EAE and this was associated with a reduction in the severity of neuroinflammation and demyelination, highlighting that miR-155 has a functional role in pre-clinical MS models [
67,
68]. Clinical studies in MS patients have suggested that microRNAs may be used as novel diagnostic and predictive biomarkers, as well as affect disease progression [
85‐
87]. Evidence demonstrating a potentially important role for miR-155 in MS includes demonstration that miR-155 expression is increased in peripheral blood mononuclear cells [
88] as well as in brain lesions [
89] of MS patients. In addition, glatiramer acetate treatment resulted in normalization of deregulated miRNAs, including miR-155, in peripheral blood mononuclear cells in patients with relapsing-remitting MS, arguing that miR-155 has a role in the regulation of immune responses in MS patients [
90]. Other potential roles for miR-155 in controlling disease progression include regulation of proinflammatory responses in blood-derived and CNS-resident myeloid cells [
91]. Furthermore, microRNAs may represent novel regulators of oligodendrocyte differentiation via control of transcriptional networks that influence myelin gene expression and cell cycle transitions [
92,
93]. Our findings indicate that in the JHMV model, miR-155 does not affect demyelination per se, as there were similar levels of myelin damage in JHMV-infected WT and
miR-155
−/−
mice at the peak of disease. Whether these results reflect the use of the JHM strain of MHV is not known at this time. The A59 strain of MHV has been shown to induce demyelination in the absence of the adaptive immune suggesting that macrophage/microglia may be sufficient to initiate white matter damage [
94]. We are currently investigating whether miR-155 influences processes governing demyelination and/or remyelination at later stages of JHMV through control of oligodendrocyte progenitor maturation. In addition, we are examining whether the absence of miR-155 affects proinflammatory gene expression by resident glia, e.g., astrocytes and microglia.