Chronic consumption of ethanol is tightly linked to liver inflammation and steatosis in human disease as well as in experimental models. Whereas activation of TLR4-dependent pathways by gut-derived LPS and induction of inflammatory cytokines has been traditionally attributed to bone marrow-derived Kupffer cells (22
), the role of crosstalk between parenchymal and nonparenchymal (bone marrow-derived immune cells) in ALD remains elusive. Here we demonstrate that liver response to insults is a multistep process: IRF3 in parenchymal cells drives Type I IFN induction in the liver and parenchymal cell-derived Type I IFN leads to a modulation of inflammatory cytokines in non-parenchymal BM-derived cells (). Our novel findings outline a link between parenchymal and liver immune cells in modulation of innate immune signaling in ALD.
Proposed mechanism of parenchymal cell-mediated control of inflammatory responses in alcohol-induced liver injury
BM-derived cells are considered to be the main targets of pathogen-derived products in the liver due to their strategical position to encounter pathogens in the portal system and broad-range expression of TLRs. Our study provides novel lines of evidence that parenchymal cells are the main producers of Type I IFNs in response to alcohol/LPS exposure, and that IRF3 is a dominant signaling molecule inducing Type I IFN in alcoholic liver disease.
First, chimeric mice containing IRF3-deficient liver parenchymal cells and wild-type bone marrow-derived cells show a similar reduction in baseline and ethanol-induced expression of Type I IFNs as mice with global IRF3 deficiency. Second, no decrease in liver expression of Type I IFNs was observed in mice with selective deficiency of IRF3 in bone marrow-derived cells. Third, ex vivo stimulation of wild-type primary mouse hepatocyte isolates with LPS resulted in phosphorylation of IRF3 and in a significant upregulation of Type I IFNs, in contrast to hepatocyte isolates from IRF3KO mice that failed to induce Type I IFNs. In addition, phenotypic analysis of hepatocyte isolates employed in our study indicated that the IRF3-dependent Type I IFN induction indeed originates from hepatocytes, while the role of other cell types remains negligible.
Our study defines induction of Type I IFNs via IRF3 in hepatocytes and downregulation of inflammatory cytokines in BM–derived cells as two complementary, yet independent mechanisms by which TLR4 controls the extent of alcohol-induced liver inflammation and injury. Kupffer cells stimulated via TLR4 are a main source of inflammatory cytokines in the liver and promote tissue inflammation, injury and fibrosis (22
). Thus, TLR4 seems to activate IRF3 in both parenchymal and non-parenchymal liver cells: here we demonstrate that while the signaling pathways are shared, we observed a cell-specific response to LPS, with a distinct outcome.
Studies by Zhao et al. (21
) suggested that IRF3, activated by TLR4/TRIF and ethanol, induces inflammatory cytokines in macrophages, thereby playing a proinflammatory role. We observed no induction of inflammatory cytokines in mice with bone marrow-specific deficiency of IRF3; however, our novel data show that this effect was not sufficient to prevent alcohol-induced liver injury. These findings suggest that both myeloid- and parenchymal-cell specific IRF3 contribute to ALD, i.e. that the solo contribution IRF3 in bone marrow-derived cells is not sufficient for the development of ALD. The type of signal in IRF3 deficient bone-marrow derived cells that improves ALD in the global IRF3 knockouts, and the reason why this signal requires the absence of IRF3 in parenchymal cells remains to be further investigated.
Recently, Klein et al. (23
) and Kennedy et al. (24
) reported that in chimeric mice, 2 populations of liver macrophages co-exist: radioresistant macrophages which show tolerogenic properties, and radiosensitive macrophages with are immunogenic; the latter macrophages are rapidly replaced by BM transplantation and expected to be the dominant subtype that participates in the immunoinflammatory reactions in the liver post-transplant. Thus, the lack of protection from ALD in mice with bone-marrow specific deficiency of IRF3 could be attributable, at least in part, to an incomplete substitution of the host with BM-derived cells and/or differential host/donor macrophage ratio (24
), either of which could influence the nature of the response to LPS and/or alcohol.
The differential role of IRF3 in ALD seems to be dominated by its parenchymal cell-specific protective effect. Our data demonstrate that IRF3 in parenchymal cells dampens TLR4-induced inflammatory response by indirect (paracrine) mechanism mediated by Type I IFNs. The importance of this cell-specific activation of IRF3 and Type I IFNs is emphasized by our finding that aggravated liver inflammation and injury was observed in mice chimeras lacking IRF3 in parenchymal cells, and was further associated with a significantly decreased expression of IL-10, a major anti-inflammatory cytokine, in the liver.
Our finding of Type I IFN-dependent induction of the anti-inflammatory state in the liver is supported by the fact that the IL-10 promoter contains a Type I IFN-dependent responsive element (25
) which makes this cytokine a Type I IFN-dependent anti-inflammatory mediator. We found that, ex vivo
, LPS-stimulated liver mononuclear cells synthesized significantly more IL-10 when co-cultured with primary hepatocytes that produced significant amounts of Type I IFNs. This synergism was completely absent in co-cultures of WT hepatocytes with IFNAR1-deficient LMNCs, but only partially abolished in co-cultures containing LMNCs that lacked IRF3, suggesting that it is the parenchymal-cell derived Type I IFN that acts synergistically with LPS on LMNCs to produce IL-10, rather than IRF3 in LMNCs per se
. The existence of a hepatocyte/immune cell regulation loop is further supported by our finding that the facilitation of LPS-induced production of IL-10 by hepatocyte-specific Type I IFNs in liver mononuclear cells was abrogated in cells lacking Type I IFN receptor. Furthermore, our data show that administration of IL-10 to mouse macrophages or human PBMCs stimulated with LPS significantly suppresses inflammatory cytokines, and therefore support the critical role of IL-10 in determining the pro- and anti-inflammatory balance in the pathogenesis of ALD (19
). Taken together, these findings demonstrate that full expression of anti-inflamatory factors in BM-derived cells is dependent on Type I IFN signaling from parenchymal cells, which is regulated by IRF3.
TLRs fulfill a variety of functions in the liver, and inhibition of TLR4 signaling may alter biological processes related to liver inflammation, injury and fibrosis (27
). TLR4 also promotes disease progression in alcoholic and nonalcoholic steatohepatitis (13
), primary sclerosing cholangitis (32
) and ischemia-reperfusion injury (33
), and therefore represents a potential therapeutic target. Indeed, use of probiotics, antifibrotics or anti-inflammatory agents are proposed as potential therapeutic options for these diseases (34
). However, excessive TLR signaling triggers not only harmful responses, but also beneficial responses, such as clearance of microorganisms (35
), tissue regeneration (36
), and, as we demonstrate in this study, indirect induction of anti-inflammatory loop via induction of protective Type I IFNs in hepatocytes and anti-inflammatory IL-10 in macrophages in an IRF3-dependent manner. Therefore, it seems plausible that fine tuning, in contrast to approaches that would completely abrogate TLR signaling, may have a future in efforts to translate TLR pathophysiology into clinical practice in human liver diseases.