This study was designed to investigate the therapeutic potential of IRF3 overexpression during inflammation. Data in primary human astrocyte and mixed neuronal and glial cultures showed that adenovirus-mediated overexpression of IRF3 changes the cytokine production profile from proinflammatory (A1) to anti-inflammatory (A2), associated with neuroprotection. Since neurons were not transduced with adenovirus in these cultures, the neurotrophic effect of IRF3 was strictly mediated by glial (mostly astrocytic) cells. Ad-IRF3-upregulated genes included IFNβ, IFN-induced protein with tetratricopeptide repeats 1 (IFIT1, aka ISG56, often the highest induced) and IP-10, all known IRF3 target genes (Grandvaux et al, 2002
), the transcription factor IRF7 which synergizes with IRF3 in the induction of IFNα and ISGs, and the Th2 cytokine IL-13 ().
Unexpectedly, the expression of many proinflammatory genes was suppressed by IRF3 and these included iNOS, TNFα, IL-1 receptor (IL-1RI), IL-8, CXCL1 (GROα), and A20. iNOS and TNFα induction in human astrocytes requires stimulation with IL-1, with IFNγ providing synergistic effects due to the presence of IFNγ-activated sequence (GAS) in their promoters (Hua et al, 2002
; Hua and Lee, 2000
; McManus et al, 2000
). We have shown previously that IFNβ suppresses these genes by preventing STAT1 binding to GAS sequences (Hua et al, 2002
). However, Ad-IRF3-suppressed astrocyte genes also included chemokine genes such as IL-8 and GROα that bear no known GAS or IFN-stimulated response element (ISRE). In addition, A20, an NF-κB-dependent gene involved in feedback inhibition of macrophage innate immunity (Turer et al, 2008
; Lin et al, 2006
), was also suppressed by Ad-IRF3. A20 mRNA suppression in IRF3-overexpressing human cell lines has been previously observed, in direct (inverse) proportionality to the amount of cellular IRF3 expression (Elco et al, 2005
). Furthermore, the IL-1 receptor (IL-1RI) expression was also downregulated by Ad-IRF3, suggesting that receptor downregulation may also participate in the suppression of IL-1 (NF-κB) signaling by IRF3. These results together suggest that the mechanism by which Ad-IRF3 suppresses proinflammatory genes in astrocytes is probably multifaceted and not simply explained by over-production of anti-inflammatory cytokines such as IFNβ.
We also find that IRF3 overexpression is associated with a change in balance in M1 and M2 cytokines in microglia (for example, IL-1 receptor antagonist > IL-1)1
. This is highly significant since IL-1 is a major proinflammatory cytokine expressed in several neurodegenerative disorders, and also is a prime inflammatory activator of astrocytes that acts through the MyD88 pathway (Lee, 2010
; Burger et al, 2009
; Simi et al, 2007
). IL-1 and TLRs share the same receptor component (the toll/IL-1 receptor “TIR” domain) that signals through the MyD88 pathway or the non-MyD88 (TRIF) pathway. The TRIF pathway is triggered exclusively by TLR3 or TLR4 ligation and converges on the activation of IRF3. Although IL-1 is capable of activating IRF3 in astrocytes (Rivieccio et al, 2005
), a direct comparison with PIC in this study shows that IL-1/IFNγ induces very little IFNβ expression (). Consistent with these findings, our previous studies have shown that human astrocytes activated with PIC conferred effective antiviral immunity against HIV and HCMV in an IRF3-dependent manner, while IL-1 did not (Suh et al, 2007
). Importantly, we observe robust increase in IFNβ production by IRF3 transduction (+ IL-1/IFNγ), resembling PIC-activated astrocytes. These results suggest that while cytokines alone do not elicit significant IRF3-dependent gene expression, they do so in the presence of increased amounts of IRF3 protein, as can be induced therapeutically by viral vector-mediated gene transfer.
MicroRNAs (miRNAs) are small non-coding RNAs important in regulation of gene expression and immune responses. Among these, miR-155 has emerged as a multifunctional miRNA involved in the regulation of inflammation and antiviral responses in macrophages (Baltimore et al, 2008
; O’Connell et al, 2007
). In addition, miR-155 has been shown to be highly expressed in reactive astrocytes in multiple sclerosis lesions (Junker et al, 2009
), and furthermore, miR-155-deficient mice are resistant to the development of experimental autoimmune encephalitis, an animal model for multiple sclerosis (O’Connell et al, 2010
). The positive role of miR-155 in autoimmunity has been largely attributed to its ability to drive Th17 differentiation of T cells (O’Connell et al, 2010
), and its role in endogenous CNS cells such as astrocytes have not been considered. Our microarray profiling of IL-1/IFNγ-activated astrocytes demonstrates that several miRNAs are significantly upregulated, confirming previous results in cytokine-activated human astrocytes (Junker et al, 2009
). These include miR-155, miR-147, miR-147b and miR-146a, miRNAs that are shown to be induced in activated macrophages and involved in immune responses (Taganov et al, 2006
; Baltimore et al, 2008
). Our study using a specific miR-155 inhibitor oligonucleotide showed that miR-155 is involved in astrocyte proinflammatory gene expression. Interestingly, we find that the star-form partner miR-155* is the most highly induced miRNA in cytokine-activated astrocytes. The star-form partner miRNAs are derived from the same precursor as a passing strand but their roles have not been systemically studied (Yang et al, 2011
). Although a recent study reported opposite roles that miR-155 and miR-155* play in dendritic cell cytokine production (Zhou et al, 2010
), our own study of astrocytes show that miR-155 and miR-155* are co-regulated by cytokines and TLR ligand, and that they have the same proinflammatory function.
Our results in astrocytes agree with the proinflammatory role of miR-155 generally reported in TLR-activated macrophages (Androulidaki et al, 2009
). We show that miR-155 plays an “M1-like” role in astrocytes (which we propose to be termed “A1” for astrocytes), and that the immune modulatory effect of IRF3 transgene may in part be mediated through inhibition of miR-155 transcription, thereby suppressing proinflammatory cytokine production, while preserving anti-inflammatory cytokine production (this astrocyte phenotype we propose to be termed “A2”). One of the many discovered targets of miR-155 is SOCS1. SOCS1 is an important negative regulator of cytokine and TLR signaling (Baker et al, 2009
). The classic function of SOCS1 is to inhibit IFN signaling through interaction with p-JAK, thereby limiting activation of STAT proteins. SOCS1 can target additional signaling components such as NF-κB p65 (ibid
). In astrocytes, we find that SOCS1 is induced by IL-1/IFNγ and this is further increased by anti-miR155 inhibitor. Furthermore, Ad-IRF3 increases SOCS1 expression, while suppressing miR-155 (and miR-155*). Together, these results demonstrate that IRF3 transgene reduces the “A1” gene expression by suppressing miR-155, which, in turn, increases the expression of miR-155 target genes such as SOCS1, a negative regulator of cytokine signaling ().
The serendipitous discovery that overexpression of IRF3 suppresses some of the key proinflammatory molecules is particularly important to our understanding of glial biology. We believe IRF3 gene transfer will predispose glial cells to become an “A2” (and M2) phenotype by coordinately modulating the expression of various gene groups upon exposure to proinflammatory stimuli (such as IL-1/IFNγ). Since transduced IRF3 protein is dormant, there will be fewer undesirable effects originating from the transgene expression per se. The activating signals could be provided by intercurrent systemic infections or stress, conditions known to trigger CNS inflammation These results provide rationale for IRF3 gene therapy for CNS diseases.