Toll-Like Receptor 4 and Myeloid Differentiation Factor 88 Provide Mechanistic Insights Into the Cause and Effects of Interleukin-6 Activation in Mouse Liver Regeneration

INTRODUCTION

Liver regeneration depends on complex interactions between overlapping metabolic, immune, and growth factor-related networks occurring in a timely manner.1 The innate immune system is thought to play important roles in the initiation and modulation of liver regeneration after liver resection or injury, and a hallmark of this involvement is the increase in circulating interleukin-6 (IL-6) that occurs shortly after partial hepatectomy (PH) in humans² and rodents.³ In mice, the increase in IL-6 is thought to originate from liver macrophages⁴ and to depend on tumor necrosis factor (TNF) receptor-1 and nuclear factor kappa B (NF-κB) signaling.³ IL-6 acts on hepatocytes via the IL-6 receptor complex, which activates the signal transducer and activator of transcription 3 (STAT3) and extracellular signal-related kinase 1 and 2 (ERK1/2) pathways, promoting responsiveness to growth factors, hepatoprotection, and survival.^5-7 Indeed, IL-6 regulates up to 40% of immediate-early genes activated in the liver after PH,8 and disruption of Tnfr1,³ Il6,^7,9 or Stat3¹⁰ impairs liver regeneration. Several studies suggest that the effect of IL-6 on hepatocyte proliferation depends on its level and duration. Only subcutaneous (i.e., long-acting), but not intravenous (i.e., short-acting), administration of recombinant IL-6 corrected the phenotype of Il6 null mice after PH.^7,11 In mice without liver resection, a single injection of IL-6 did not promote hepatocyte proliferation, ⁷ whereas sustained IL-6 delivery in nude mice transplanted with human IL-6-producing tumors induced massive liver growth.¹² It is currently unknown, however, whether these observations are physiologically relevant for the variable IL-6 levels observed after PH in individual mice.

The ultimate cause(s) of cytokine activation after PH remains unknown. Cornell’s pioneering work showed transient impairments of liver regeneration after PH in rodents with genetic hyporesponsiveness to lipopolysaccharide (LPS) or those treated with gut flora decontamination strategies,^13,14 suggesting that enteric LPS was responsible for initiating liver regeneration. We and others recently showed that the increase of circulating IL-6 after PH was abrogated in mice lacking Myd88,^15,16 an adaptor protein for the Tolllike receptor (TLR)/IL-1R family of proteins. Interestingly, IL-6 activation was unimpaired in Tlr4 null and Cd14 null mice, which lack genes required for LPS signaling, and in mice deficient in Tlr2 and Tlr9, which, respectively, recognize microbial lipopeptides and unmethylated CpG DNA.^15,16 These results were puzzling, because myeloid differentiation factor 88 (MyD88) is an adaptor protein for most TLRs, and whereas ^Myd88 null mice had profound defects in circulating IL-6, mice deficient in Tlr2, Tlr4, Cd14, and Tlr9 showed normal levels of IL-6 and intact hepatocyte proliferation.^15,16 Redundant signaling among TLR/IL-1R proteins and involvement of different TLR/IL-1R ligands, however, could not be excluded.

The aims of our study were to assess whether levels of circulating IL-6 after PH determine the extent of hepatocyte proliferation, by using a retro-orbital bleed technique that allows mouse survival, and to explore whether redundant signaling among TLR/IL-1R proteins contributes to cytokine activation and liver regeneration after PH, using mice with a triple deficiency of Tlr2, Tlr4 and Tlr9 (Tlr2,4,9), and Tlr2, Tlr4 and Caspase1 (Tlr2,4-Casp1) genes. Because caspase-1 cleaves pro-IL-1 and pro-IL-18 to their active forms, which, in turn, signal via MyD88-associated receptors, the Tlr2,4-Casp1 null strain may reveal whether IL-1 or IL-18 are ligands responsible for the IL-6 increase. As controls, we assessed mice with a single deficiency of Myd88, Tlr2, Tlr4, or Cd14, as well as C3H/HeJ mice, which harbor a missense mutation in Tlr4 that confers LPS hyporesponsiveness.

MATERIALS AND METHODS

RESULTS

DISCUSSION

In the present study, we addressed two unresolved questions concerning early cytokine activation after PH, in particular whether the physiologic increases of circulating IL-6 determine the extent of hepatocyte proliferation and whether LPS and TLR/IL-1R proteins are responsible for triggering IL-6 activation. Using the retro-orbital bleed technique, we showed that LPS/TLR-4 signaling contributes, in a nonredundant fashion, to IL-6 activation after PH, although the TLR-4-independent component of IL-6 increase is sufficient to maintain intact downstream signaling in the regenerating liver. These data are consistent with the poor correlation between circulating IL-6 levels and extent of hepatocyte proliferation found in individual mice after PH. A striking observation was the accelerated start of hepatocyte proliferation after PH in Myd88 null mice, despite the profound abrogation of IL-6 increase. This IL-6 abrogation was accompanied by opposite changes in two signaling pathways downstream of IL-6 (i.e., STAT3-Socs3 and Ras/ERK), suggesting a mechanism for the Myd88 null phenotype and highlighting the potential relevance of an antiproliferative arm of IL-6/ STAT3 signaling, possibly via the suppressor of cytokine signaling 3 (SOCS3), in the liver-regenerative process.

Experiments using exogenous delivery of IL-6 suggested that the magnitude and duration of circulating IL-6 determine its effects on hepatocyte proliferation. ^7,11,12,22 To assess whether this circumstance is relevant for the physiologic increases in IL-6 noted after PH, we used the retro-orbital bleed technique, which allows mouse survival and correlation of IL-6 levels early after PH with downstream events in individual animals. Importantly, this technique uncovered serious artifacts in IL-6 measurements after PH, when blood is collected by cardiac puncture. Circulating IL- 6 levels measured in retro-orbital blood strongly relate to the amount of liver tissue resected in mice undergoing one-third versus two-thirds PH, but our data do not support a direct correlation between IL-6 levels and extent of hepatocyte proliferation. First, levels of circulating IL-6 early after PH are not correlated with extent of hepatocyte proliferation in individual mice. Second, neither the attenuation nor abrogation of IL-6 increase, respectively, in Tlr4-deficient and Myd88 null mice translate into reduced hepatocyte proliferation. The intact hepatic expression of phospho-STAT3 protein and Socs3 mRNA in Tlr4 null mice, despite attenuated IL-6 levels, suggest the existence of a certain threshold of circulating IL-6, above which downstream signaling pathways remain intact in the regenerating liver.

Identifying the factors ultimately responsible for triggering cytokine activation after PH has long been evasive. Recently, cytokine activation after PH was shown to require the adaptor protein, MyD88, but the specific receptors implicated were not identified.^15,16 Here, we reveal a global attenuation (~40-60%) of increase in IL-6 after PH in all mouse strains harboring TLR-4 signaling defects (namely, C3H/HeJ, Tlr4 null, Cd14 null, Tlr2,4,9 null, and Tlr2,4-Casp1 null mice), supporting a role for LPS in triggering IL-6 activation after PH. Because of artifacts in the cardiacpuncture technique, the contribution of LPS/TLR-4 signaling may have passed unnoticed in prior studies (including ours) that assessed Tlr4 and Cd14 null strains.^15,16 Despite attenuated IL-6 production, Tlr4 null mice did not display noticeable impairments of downstream signaling (e.g., STAT3 phosphorylation, Socs3 induction) in the liver, and no compensatory changes were noted in other circulating cytokines (e.g., TNF-α, IL-1β, IL-12, and IFN-ɣ). Furthermore, we did not observe defective hepatocyte proliferation in any Tlr4-deficient strain, including the C3H/HeJ strain, in which Cornell reported a transient impairment. ¹⁴ Notably, liver regeneration was preserved in Tlr2,4,9 null and Tlr2,4-Casp1 null mice, reasonably excluding redundancy among these receptors as an explanation for the preservation of liver regeneration in mice with single genetic defects. We believe that differences in the strain chosen as a control and health status of the mice explain the discrepancy with Cornell’s observation in C3H/HeJ mice.¹⁴ The use of C3H/HeJ mice for modeling defective TLR-4 signaling is problematic, as C3H substrains present significant allelic differences that affect results.²³ Whereas Cornell used C3H/HeN mice as a control strain (from Harlan Sprague-Dawley), we used C3H/HeOuJ mice, which are genetically more similar to C3H/HeJ and bred by the same vendor (The Jackson Laboratory). At the time of Cornell’s studies, C3H mice harbored mouse mammary tumnor virus (MMTV), being rederived in 1999 to eliminate the viral contamination (http://jaxmice.jax.org/strain/000659.html). Although our results demonstrate a role of LPS in triggering IL-6 activation after PH, they also show that the contribution of LPS is dispensable for normal liver regeneration.^13,14

Our study shows that early cytokine activation after PH is mediated by at least two distinct MyD88- dependent pathways, only one of which involves TLR- 4. We excluded IL-1, IL-18, the specific ligands of TLR-2 and TLR-9, and redundancy among TLR-2, TLR-4, and TLR-9 receptors as potential MyD88- related factors leading to IL-6 increase. Other factors that affect the production of IL-6 after PH are the complement system,²⁴ the lymphotoxin β receptor,²⁵ TNF receptor 1,³ TLR-3,²⁶ and the intercellular adhesion molecule 1,²⁷ but none of these signal via MyD88 TLR-4-independent pathways causing IL-6 production after PH, therefore, still need to be identified.

A striking observation in our study is the accelerated initiation of hepatocyte proliferation in Myd88 null mice after PH, corroborated by Ser¹⁰ phosphorylation of histone H3 and expression of cyclins E and A. Although we previously reported intact liver regeneration in Myd88 null mice (15),¹⁵ Seki et al. reported impaired hepatocyte proliferation after PH in this strain.¹⁶ In our previous study,¹⁵ rederivation of Myd88 null mice to exclude confounding effects of Helicobacter infection could not reproduce Seki et al.’s findings. Noteworthy, Myd88 null mice in Seki et al.’s study displayed normal cyclin D1 induction, and full recovery of initial liver mass was not delayed.¹⁶ Different anesthetics, surgical techniques, and housing conditions likely explain the differing results. In any case, our present finding of earlier initiation of hepatocyte proliferation in Myd88 null mice is puzzling, given the concomitant abrogation of circulating IL- 6 and blunted STAT3 activation—features considered important for initiation of liver regeneration.

Do our findings preclude a role for IL-6 as an essential promitogenic stimulus after PH? How do they fit into the current knowledge? Our study adds to the body of conflicting information concerning the role of IL-6 in liver regeneration and calls for a new integrative perspective. Previous studies in Il6 null mice are particularly conflicting, because the degrees of impaired hepatocyte proliferation, liver injury, and mortality after PH differ among them from severe to nonexistent,^7,9,11 but it is unquestionable that a subset of Il6 null mice regenerate their livers normally.¹¹ Technical issues and the role of IL-6 as inducer of the acute phase response may underlie contradictory data, but a major difficulty in reconciling all observations lies in the assumption that the effect of IL-6 after PH is purely proproliferative. Our study suggests that a parallel antiproliferative effect of IL-6 after PH needs to be included in the equation. IL-6 hyperstimulation in mice after PH has been reported to induce the cellcycle inhibitory protein, p21, and to delay hepatocyte proliferation,²² an observation also reproduced in vitro.²⁸ Recent work, including our own, suggests that an antiproliferative effect of IL-6 via Socs3 also occurs after PH, in the absence of exogenous IL-6 administration. ^19,21 The hepatic expression of SOCS3 is induced shortly after PH in a manner dependent on the IL-6- gp130-JAK-STAT3 signaling pathway.^6,19,29 Once expressed, SOCS3 interacts with activated gp130 receptors and inhibits further JAK catalytic activity.³⁰ In mice with hepatocyte-specific deletion of Socs3,²¹ the absence of this negative feedback loop led to enhanced hepatocyte proliferation after PH in association with increased ERK1/2 activation, a signaling pathway that drives cell-cycle progression in hepatocytes. ³¹ Partially mimicking this scenario, the blunted Socs3 induction in Myd88 null mice shown here was also associated with enhanced, prolonged phosphorylation of ERK1/2 and accelerated cell-cycle entry. Multiple cytokines and growth factors relevant for hepatocyte proliferation, including the epidermal growth factor receptor (EGFR), hepatocyte growth factor, and its receptor c-Met, insulin, and others are sensitive to the inhibitory actions of SOCS3^21,32,33 and could contribute to these changes in ERK1/2. An interaction with the EGFR is particularly conceivable, given that SOCS3 is capable of interacting with EGFR, and that Socs3 null hepatocytes display increased ERK1/2 activation and hepatocyte proliferation when exposed to EGF.^21,34 Our finding of accelerated initiation of hepatocyte proliferation in Myd88 null mice points to SOCS3 as a critical element in the interplay between the innate immune system, IL-6, and liver regeneration. From our perspective (Fig. 6), the effects of IL-6 signaling after PH may be viewed as a double-edged sword, with both pro- and antiproliferative arms. Studies focused on the regulation of SOCS3 and the identification of its major targets should provide valuable information for testing this hypothesis and understanding the effects of IL-6 in liver regeneration.

Supplementary Material