ABSTRACT
The maintenance of homeostasis in the central nervous system (CNS) during neurotropic viral infections is critical
for host survival. Inflammation of the CNS is characterized by the recruitment of leukocytes to the brain and the
activation of resident CNS cells, including astrocytes. The pivotal in vivo function of astrocytes during viral
encephalitis is poorly defined. Toll like receptor 3 (TLR3) is an interferon-inducing double stranded RNA sensor
that senses viral infections. Although it is well known that astrocytes express functional TLR3, its role in response
to viral RNA infections of the CNS remains poorly understood. The interplay between transcription factors and
microRNAs, and their upstream pattern recognition receptors during viral encephalitis has not been extensively
studied. MicroRNA-155 (miR-155) has a key role of in the fine-tuning of TLR responses, and exerts both positive
and negative regulation during inflammation. We and others have identified miR-155 as one of the most over-
expressed miRNAs after stimulation of TLR3 with the synthetic dsRNA agonist Poly (I:C) in macrophages. In the
CNS, miR-155 is also expressed in glial cells. However, a CNS-intrinsic role of miR-155 has yet to be defined in
vivo. Signal transducers and activators of transcription (STAT) proteins and their negative regulators, the
suppressors of cytokine signaling (SOCS) protein family play central roles in regulating many cytokines and
growth factors. Recently, two STAT3 binding sites have been identified in the promoter of miR-155. Furthermore,
miR-155 targets SOCS1 and SOCS3 suppressing their negative regulatory effect on JAK/STAT signaling
pathway. Coronaviruses (CoVs) are enveloped, positive-sense, single-stranded, polyadenylated RNA viruses.
Mouse hepatitis virus JHM (MHV-JHM) is a CNS-tropic strain that induces fatal encephalitis, associated with
non-protective, enhanced macrophage and neutrophil infiltration that correlates with exacerbated chemokine
and Th1 response and no Th17 involvement. We have identified a TLR3-dependent, miR-155 /STAT3 regulatory
loop that promotes exacerbated, detrimental neuroinflammation and blood brain barrier (BBB) disruption in
response to MHV-JHM fatal infection. Intriguingly, TRIF, the only TLR3 adaptor known, is not required for TLR3-
dependent activation of STAT3 and induction of miR-155 both in vitro and in vivo. We have identified astrocytes
and inflammatory monocytes infiltrating the brain as the cell sources driving exacerbated STAT3 activation. Our
findings prompted us to investigate the following hypotheses: AIM 1 MHV-JHM infection in astrocytes and
macrophages induces STAT3 activation through a TRIF-independent, TLR3-dependent non-canonical axis that
is negatively regulated by its adaptor TRIF; AIM 2 Cell type specific genetic ablation of STAT3 and miR-155
expression in astrocytes or myeloid cells will reduce detrimental neuroinflammation and increase host survival
after fatal MHV-JHM infection; and AIM 3 TLR3 contributes to BBB disruption through early and sustained
upregulation of miR-155 expression in astrocytes, which deliver exosome-miR155 to brain endothelial cells
affecting the expression of (to be identified in AIM 3) AJPs, TJPs, and/or MMPs.