Our study was designed to investigate the role of IRF3 transgene expression in microglial inflammatory activation. Our data in primary human microglial cultures show that adenovirus-mediated IRF3 transgene expression changes the microglial cytokine profile from a proinflammatory phenotype to an anti-inflammatory or immunoregulatory phenotype. Specifically, the expression of IL-1ra, IL-10 and IFNβ was markedly induced, while the expression of many proinflammatory cytokines such as IL-1 was suppressed consistently and significantly. Additional suppressed proinflammatory genes included TNFα, IL-6 and IL-8 and CXCL1.
We refer to the microglial cytokine expression profile changes described here as "M1-like" or "M2-like", following the general scheme of M1 and M2 activation phenotypes developed in mouse macrophages and subsequently adopted to describe microglial activation phenotypes [6
]. There are a number of differences between human microglia and murine microglia. For example, although iNOS is a prototypic marker of M1-activated murine microglia, it is not expressed by human microglia [37
]. In addition, human microglia do not express certain Th1 or Th2 cytokines such as IFNγ or IL-4. There might also be additional differences between macrophages and microglia. For these and other reasons, we refer to the microglial phenotypes described here as "M1-like" or "M2-like".
Importantly, we note these changes regardless of the types of immunological stimuli applied (TLR3/4 ligands as well as IL-1/IFNγ, to mimic non-infectious neuroinflammatory conditions). The observed effects of IRF3 transgene in the suppression of proinflammatory cytokine genes is novel and points to a mechanism by which IRF3 influences other signaling pathways. In addition, we have obtained novel findings that indicate that the PI3K pathway plays a predominantly anti-inflammatory role in microglial activation. It played a particularly potent role in the induction of anti-inflammatory and immunoregulatory cytokines such as IL-10, IL-1ra and IFNβ. These results together suggest that activation of the PI3K/Akt pathway (via Ad-IRF3, for example) in microglia can lead to the resolution of inflammation and promotion of repair under neuroinflammatory conditions [38
The PI3K/Akt pathway is unique for its multitudes of roles in transcriptional regulation of cytokine genes. Employing a pharmacological inhibitor, we show that the PI3K/Akt pathway is involved in both the suppression (of "M1-like") and the enhancement (of "M2-like") of cytokine genes in IRF3-transduced microglia. One might speculate that the impressive amounts of suppression of proinflammatory genes in Ad-IRF3-transduced cells are at least in part secondary to the induction of anti-inflammatory and immunoregulatory genes, as IL-1ra, IL-10 and IFNβ each can function as a suppressor of proinflammatory cytokine expression. For example, we have previously shown that recombinant IFNβ suppresses IL-1 and increases IL-1ra production in human microglia [41
]. IFNβ also induces certain chemokines [42
]. Microarray analysis of human peripheral blood mononuclear cells (PBMCs) exposed to IFNβ demonstrated that distinct sets of genes are upregulated or downregulated by IFNβ, the latter including IL-1β, CXCL1, and IL-8 [43
]. Therefore, IFNβ most certainly played a role as an intermediary cytokine that mediated the effect of Ad-IRF3 in our system. Additional cytokines that might have played a role in our system include IFNα, as well as type III IFNs. Type III IFNs are newly discovered interferons that share a number of similarities with type I IFNs including their mechanism of induction and their biological activities [30
]. One might also speculate that the opposite effects of LY294002 on the two groups of genes can be best (and most simply) explained by the prominent role played by PI3K/Akt on microglial "M2-like" cytokine induction. Furthermore, we show that PI3K/Akt might play a different role in proinflammatory gene expression (without Ad-IRF3) depending on the stimulus applied, as that induced by IL-1/IFNγ was suppressed by PI3K/Akt, while little changes were noted in PIC-stimulated microglia, and PIC-induced IL-1β production was even increased. We also note that although IL-1 expression was consistently and potently suppressed by Ad-IRF3 transduction in microglia, its expression appeared to be least affected by the PI3K inhibitor. Therefore, multiple mechanisms must exist that mediate the effects of Ad-IRF3 on microglial cytokine expression. Additionally, the adenoviral vector may have evoked some elements of inflammatory activation in microglia and that this may have created conditions that contributed to the effects seen 48 h after adenovirus infection. Our results with LY294002 are reminiscent of those obtained in mouse macrophages deficient in phosphatase and tensin homologue (PTEN), a negative regulator of Akt, which showed similar differential regulation of cytokines, i.e., decrease in TNFα/IL-6 and increase in IL-10 [44
] supporting the dual role played by PI3K/Akt in Ad-IRF3-transduced microglial cytokine expression. Our results demonstrating a pivotal role of pAkt in IFNβ production is also in line with another study of murine macrophages which demonstrated a critical role of pAkt in TLR-induced IRF3 activation and IFNβ expression downstream of TRIF signaling [36
]. The anti-inflammatory role of Akt in mouse macrophages has been most convincingly demonstrated in a study in which Akt1-deficient mice injected with LPS showed increases in proinflammatory cytokine production compared to wildtype mice [45
]. In the latter study, the effect of Akt1 was attributed in part to its suppression of microRNA-155 (miR-155) expression. miR-155 is a proinflammatory microRNA that increases cytokine production by targeting specific mRNAs such as suppressor of cytokine signaling (SOCS1) mRNA [29
]. These results are interesting, since miR-155 was significantly elevated by IL-1/IFNγ in human microglia (data not shown), suggesting that suppression of miR-155 may be the mechanism by which Akt modulated "M1-like" cytokines in IL-1/IFNγ-stimulated microglia (Figure ).
The role of the PI3K/Akt pathway in cytokine production is also cell-type specific. In human astrocytes, we see that LY294002 suppresses both "M1-like" (TNFα and IL-8, for example) and "M2-like" cytokine (IFNβ) expression induced by PIC or IL-1/IFNγ (Figure ). These results suggest that in astrocytes, Akt is activated upstream of NF-κB (and MAPK) following activation of TLR3 or IL-1R. In addition, LY294002 suppresses miR-155 expression in astrocytes, indicating a positive role for PI3K/Akt in miR-155 expression in astrocytes (Figure ). These results demonstrate that the PI3K/Akt pathway plays a fundamentally different role in the inflammatory activation of the two glial cell types (astrocytes vs. microglia). It is also possible that microglia and astrocytes express different combinations of Akt isoforms, with each isoform having distinct immune regulatory functions. These are some of the topics that need to be explored in future studies.
Our results suggest that in Ad-IRF3-transduced microglia, a positive feed forward loop between Akt and IRF3 might be established resulting in downmodulation of inflammatory activation. For example, evidence supports that signaling through TRIF (TLR3 or TLR4) or MyD88 (TLR4 or IL-1R) activates Akt [21
] that is critical in the activation of IRF3 [28
]. Furthermore, Ad-IRF3 increases the level of pAkt, likely contributing to increased activation of IRF3, in addition to increase in total IRF3 (Figures and ). It is unclear how Ad-IRF3 increases pAkt in microglia. We do not believe this was mediated by IFNβ because we do not see measurable IFNβ in cultures treated with Ad-IRF3 alone (not shown). In addition, our previous studies showed that while IFNβ activates microglial NF-κB and MAP kinases (ERK) immediately, IFNβ (or IFNγ) does not activate Akt until later time points (6 h), indicating an indirect mechanism of activation [21
The major change that we see in IRF3-transduced microglia is downmodulation of the IL-1 axis. IL-1 is a non-redundant cytokine expressed primarily by microglia and macrophages but also by T cells. Microglial IL-1 is induced early after CNS insult and is capable of activating downstream cytokine cascades, as well as auto-amplification cascades [41
]. In vitro
, microglial IL-1 is induced by diverse types of stimuli [4
] and serves as a potent neurotoxin [53
]. IL-1 is also crucial in the Th17 differentiation of human T cells [55
]. The amount of IL-1 signal transduction is primarily determined by the relative abundance of the agonists (IL-1α or IL-1β) and the antagonist (IL-1ra). The importance of IL-1ra in human biology has been elucidated in recent discovery of an inflammatory disease caused by homozygous deletion/mutations of the IL1RN
]. A term DIRA (deficiency of the IL-1ra) has been proposed to denote this life-threatening autoinflammatory disease caused by unopposed action of IL-1. Of interest, IFNβ and glatiramer acetate, disease-modifying treatments for multiple sclerosis, are both known to exert opposing effects on IL-1α/β and IL-1ra [41
]. Therefore, the combined effects of IL-1 receptor antagonism and the robust increase in IL-10 and IFNβ production in Ad-IRF3-transduced microglia could significantly alter the neuroimmune environment in favor of resolution of inflammation and promotion of repair. The data obtained in this study should be useful in future development of therapeutic strategies aiming at neuroinflammation.