TLR9-dependent liver damage is involved in the pathogenesis of several liver diseases, such as alcoholic liver disease,5
primary biliary cirrhosis,6
primary sclerosing cholangitis7
and acetaminophen-induced liver injury,8
as well as in pathological processes such as liver fibrosis,10
and liver graft rejection.13
Thus, TLR9 holds the common link between different processes which lead to liver diseases; therefore, unraveling the pathogenesis of TLR9-induced liver injury may aid in identification of novel, efficient pathogenesis-based management or cure. Here we report the novel finding that Type I IFNs have an important regulatory role in TLR9-induced liver injury by limiting inflammation and liver injury. Further, we demonstrate that Type I IFN-induced IL-1ra could serve as a potential therapeutic target in liver injury.
In the current liver injury model, three TLR agonists are being used. Our recent and previous 15, 16
data show that liver inflammation and injury induced in this model are primarily dependent on TLR9; liver damage elicited by CpG is aggravated by co-stimulation with TLR2 and a secondary stimulation with TLR4. Of these TLRs, TLR2, 4 and 9 activate the MyD88-dependent pathway, while IRF3 activation is exclusive to TLR4. Type I IFN and IRF7 induction can occur via both TLR9 and TLR4. 18
We first demonstrated that Type I IFNs, IFNα and IFNs, were induced at the mRNA level in the TLR9-associated liver injury. This Type I IFN was biologically active as suggested by the increased expression of the IFN-inducible genes, ISG15 and IP-10 in the liver. Our novel observation of the increased liver inflammatory infiltrate and exacerbated liver injury in IRF7 and IFNAR1-deficient mice suggested a protective role for the Type I IFN induction in the liver. Indeed, previous studies suggested that Type-I IFNs can mediate anti-inflammatory effects (summarized in 28
Compared to IFNAR1-deficient mice, where the protective Type I IFN signaling was fully inhibited, IRF7-deficient mice could still produce TLR4-induced Type I IFNs (preferentially IFNβ) via the TLR4-IRF3 pathway. In our study, TLR9-associated liver injury was associated with induction of IFNα, IFNβ and interferon-stimulated genes in wild-type mice. Notably, IFNAR1-deficiency abrogated the induction of Type I IFN response to much greater extent than IRF7 deficiency. Also, IFNAR1-deficient mice showed the most severe TLR9-associated histological liver damage, which exceeded the extent present in IRF7-deficient mice. The different observation between IRF7- and IFNAR1-deficient mice could be explained by differential composition of inflammatory liver infiltrate, or by the fact that Type I IFNs are potentially inducible by multiple pathways, including IRF1, IRF3 and IRF7.23, 29
Therefore, protective Type I IFNs could still be induced in IRF7-deficient mice by TLR9 in an IRF7- independent, IRF3-dependent manner, while the downstream protective effect of Type I IFNs is completely abolished in IFNAR1-deficient mice which cannot respond to type I IFNs.
We found that deficient Type I IFN signaling was associated with decreased liver recruitment of dendritic cells and with decreased expression of CCL-21and CCR7, a key chemokine-receptor pair involved in dendritic cell trafficking.30
Our novel findings indicating a role of Type I IFNs in recruitment of dendritic cells and in expression of CCL-21, together with data showing that TLR9-activated dendritic cells are key producers of Type I IFNs,31
suggest a novel self-sustaining mechanism for dendritic cell recruitment into the liver. It has been reported that dendritic cells are essential for Th1 response in the liver induced by TLR9 + TLR2 ligands 32
and contribute to liver fibrosis,9
Consistent with earlier observations15
TLR9-induced inflammatory infiltrates, that require dendritic cell recruitment to the liver,32
amplified pro-inflammatory cytokine induction by LPS. Our novel data suggest a dual role for dendritic cells in the liver. First, dendritic cells are necessary for TLR9-induced inflammatory cell infiltrates and sensitization to TLR4 ligands. Second, dendritic cells provide anti-inflammatory signals mediated by Type I IFNs.
We observed a synergistic effect of TLR9 + TLR2 and TLR4 ligands in upregulation of IL-1ra in wild-type animals; both IRF7-deficient and IFNAR1-deficient mice showed decreased induction of IL-1ra, compared to WT. One can speculate that this synergistic effect of TLR ligands on IL-1ra induction is mediated by Type I IFNs and IRF7. This notion is consistent with our data showing a synergistic effect of TLR9+2 and TLR4 ligands on Type I IFN expression in wild-type mice. We observed that Type I IFN induces IL-1ra in hepatocytes and liver mononuclear cells, and that IL-1ra protects hepatocytes against the sensitizing effect of IL-1β towards cytotoxicity induced by TNF-α. Our findings support previous reports that in order to become susceptible to the cytotoxic effect of TNF-α, hepatocytes require priming with ligands that interfere with their proliferation.27
Indeed, IL-1β has been shown to inhibit DNA synthesis in rat hepatocytes.33
We further showed that administration of recombinant pegIFNα2 induced IL-1ra in vivo
. This finding was in agreement with our in
vitro data from hepatocytes and liver mononuclear cells, with in vitro
data that IL-1RA
promoter region contains Type I IFN-inducible elements,34
and with data demonstrating increased in vivo
production of IL-1ra upon treatment with recombinant IFN in humans.35
In addition, we observed that in vivo
administration of pegIFNα2 significantly ameliorated TLR9-associated survival, liver injury and inflammation.
Our novel data demonstrated that pretreatment with recombinant IL-1ra significantly ameliorated liver injury and inflammation in WT mice. These findings confirm the anti-inflammatory effect of IL-1ra in liver injury and are complementary to study of Iizasa et al.36
who showed that deficiency of IL-1ra resulted in exacerbated liver injury and inflammation induced by Propionibacterium Acnes
, which activates TLR9 and TLR2 receptors.15, 16
Our results show for the first time that Type I IFNs potentially protect from liver injury by inducing anti-inflammatory IL-1ra.
IL-1ra acts as a natural antagonist of IL-1β,26
and we show that in the liver IL-1ra is produced by both hepatocytes and liver mononuclear cells. Our data also demonstrates the protective effect of IL-1ra in TLR9-associated liver injury and survival. In previous studies, Imaeda et al.8
showed that in acetaminophen hepatotoxicity, inflammatory response is triggered by apoptotic mammalian DNA that increases transcription of IL-1β, and that inflammatory response is ameliorated in mice deficient for TLR9 and in mice treated with anti-IL-1β antibody. Importantly, IL-1ra has been reported for the treatment of hepatic failure in rats using a bioartificial liver device.37
These studies support our finding that the balance between IL-1β/IL-1ra is of crucial importance in TLR9-induced liver damage (Suppl. Fig. 4
In conclusion, our findings suggest that the endogenous anti-inflammatory signaling induced by Type I IFNs and mediated by IL-1ra regulates the extent of TLR9-induced liver damage, and support the indispensable role of Type I interferon signaling in immune mediated liver injury. Finally, we suggest the potential role of IL-1ra in therapy of TLR9-associated liver diseases.