IL-6 Trans-signaling Controls Liver Regeneration After Partial Hepatectomy

IL-6 Trans-signaling SIL-6R^+/+ Mice Have Drastically Increased SIL-6R Levels

Here we generated transgenic soluble IL-6R^+/+ (sIL-6R^+/+) mice, a strategy to mimic ADAM10/17 hyperactivation for the single target protein IL-6R. sIL-6R^+/+ mice reflect a situation in which only trans-signaling is active, whereas classic signaling is abrogated. sIL-6R^+/+ mice exhibit amplified endogenous IL-6 trans-signaling due to an increased level of sIL-6R, which is not caused by ectodomain shedding but by Cre-mediated deletion of the genetic information coding for the transmembrane and intracellular domain of the IL-6R (Fig. 1A). In detail, exon 9 of the IL-6R gene codes for the transmembrane domain and the first part of the intracellular domain, and the last exon 10 codes for the second part of the intracellular domain followed by the 3′untranslated region (3′UTR). In the targeting vector, the 5,420-base pair (bp)-long intron 9 was deleted, resulting in functional fusion of the 100-bp and 223-bp-long coding regions of exon 9 and exon 10, respectively. Intron 9 was skipped because it would have complicated the construction of the targeting vector. The original IL-6R stop codon located in exon 10 was deleted, and a sequence coding for the 2A-peptide sequence followed by a KDEL-marked GFP was inserted. The 2A peptide from the foot-and-mouth disease virus is a self-processing sequence to achieve expression of at least two separate proteins from a single open reading frame.22 The cleavage of the IL-6R and GFP is thought to happen in a co-translational process. The remaining 2A peptide fragment at the C-terminal end of IL-6R is then cleaved off by the protease furin. In addition, the endoplasmic reticulum retention signal KDEL23 was fused at the C-terminal end of the green fluorescent protein (GFP). The E9-E10-GFP cassette was followed by the flippase recognition target (FRT)-neomycin-resistance-FRT cassette and the original 3′UTR of the IL-6R 3′UTR and ultimately flanked by loxP-sites.

In the founder transgenic mice, the FRT-neomycin-resistance-FRT cassette was deleted by crossing to Flp-recombinase expressing mice, referred to as homozygous sIL-6R^fl/fl mice. We expected that the modified IL-6R protein in IL-6R^fl/fl mice would be produced with comparable efficiency compared with the wild-type IL-6R protein and result in IL-6 classic and trans-signaling comparable to wild-type mice (Fig. 1B). Cre-mediated recombination in the germline resulted in homozygous IL-6R^+/+ mice carrying the deletion of the fused exons 9 and 10 on both alleles (Fig. 1A). As a consequence, the translated IL-6R in Cre-recombined sIL-6R^+/+ mice lack the trans-membrane and intracellular domains and will be directly secreted as soluble IL-6R (Fig. 1C, Supporting Fig. S1A,B). Due to the lack of membrane-bound IL-6R, sIL-6R^+/+ mice will selectively execute only trans-signaling. Introduction of the E9-E10-GFP cassette in sIL-6R^fl/fl mice and deletion of the E9-E10-GFP cassette in sIL-6R^+/+ mice was confirmed by genomic PCR (Fig. 1D). Next, we quantified the IL-6R mRNA level in wild-type, sIL-6R^fl/fl, and sIL-6R^+/+ mice. Whereas the IL-6R mRNA level was comparable in wild-type and sIL-6R^fl/fl mice, a significantly increased sIL-6R mRNA level was detected in sIL-6R^+/+ mice, suggesting that genetic rearrangement in sIL-6R^fl/fl mice did not influence overall stability of the IL-6R mRNA but the deletion of most of the 3′UTR in sIL-6R^+/+ mice enhanced mRNA stability (Fig. 1E,F). Unfortunately, we were not able to detect GFP fluorescence in hepatocytes and immune cells from sIL-6R^fl/fl mice, albeit western blotting of liver and spleen tissue clearly detected GFP proteins in sIL-6R^fl/fl mice, but not in wild-type and sIL-6R^+/+ mice (Fig. 1G). Next, we performed immunohistochemistry of the IL-6R on liver samples from wild-type, IL-6R deficient, and sIL-6R^+/+ mice. We were able to detect IL-6R in cells of wild-type mice but not in IL-6R deficient mice, demonstrating that the staining was specific. Interestingly, staining for sIL-6R in sIL-6R^+/+ mice was also absent, indicating that all membrane-bound IL-6R was converted in sIL-6R in sIL-6R^+/+ mice and rapidly secreted (Fig. 1H). Moreover, staining of the IL-6R in wild-type mice suggests that IL-6R molecules were found primarily in intracellular compartments and not on the cell surface (Fig. 1H). Flow cytometry analysis of IL-6R on CD3⁺ T cells and monocytes demonstrated that wild-type and sIL-6R^fl/fl mice had comparable levels of IL-6R on the cell surface, demonstrating that genetic rearrangement of IL-6R in sIL-6R^fl/fl mice did not interfere with expression (Fig. 1I,J, Supporting Fig. S2). Importantly, IL-6R^-/- and sIL-6R^+/+ mice lacked IL-6R cell surface expression, again supporting our conclusion that all membrane-bound IL-6R was converted into sIL-6R after Cre-recombination in sIL-6R^+/+ mice (Fig. 1I,J, Supporting Fig. S2). Next, determination of the soluble IL-6R levels in the serum by enzyme-linked immunosorbent assay (ELISA) showed that wild-type and sIL-6R^fl/fl mice had almost identical sIL-6R serum levels of approximately 11 ng/mL. Interestingly, sIL-6R^+/+ mice had about 33-fold increased sIL-6R levels of approximately 363 ng/mL (Fig. 1K). The increased sIL-6R level might at least to some degree also be caused by increased IL-6R mRNA levels in sIL-6R^+/+ mice. A previous report demonstrated that serum sIL-6R was originating almost completely from hepatocytes and neutrophils/macrophages, because Alb-Cre-recombined IL-6R^-/- mice had 67.95% and LysMCre-recombined IL-6R^-/- mice displayed 39.95% of the sIL-6R levels of wild-type mice (Supporting Table S1).24 Here, we also crossed our sIL-6R^fl/fl mice with the highly effective Alb-Cre and LysM-Cre lines (Supporting Fig. S3A,B).25-28 Homozygous sIL-6R^+/+ LysM-Cre and sIL-6R^+/+Alb-Cre mice had about 129 ng/mL and 128 ng/mL sIL-6R in the serum, respectively (Fig. 1L). The sum of these sIL-6R serum levels was about 257 ng/mL, representing only about 70.8% of the sIL-6R levels detected in sIL-6R^+/+ mice (Fig. 1L, Supporting Table S1). These data suggest that although the native sIL-6R in wild-type mice were derived primarily from hepatocytes and monocytes/neutrophils, other cells also expressed appreciable amounts of IL-6R, even though they did not contribute to sIL-6R level in wild-type mice.

SIL-6R in SIL-6R^+/+ Mice Mediated IL-6 Signals Through Trans-signaling

Injection of recombinant IL-6 in wild-type mice showed that the colon, and to a lesser extent also the liver and lung, are targeted by IL-6 trans-signaling.11 Our sIL-6R^+/+ mice have a 33-fold higher sIL-6R serum level compared with wild-type mice (Fig. 1K), suggesting that sIL-6R^+/+ mice efficiently and specifically execute trans-signaling. To test this, sIL-6R^+/+ mice were injected with 5 µg recombinant IL-6, and phosphorylation of signal transducer and activator of transcription 3 (pSTAT3) was analyzed in the colon, liver, and lung. IL-6 induced sustained STAT3 phosphorylation in these organs as demonstrated by immunohistochemical staining and western blotting compared with control mice (Fig. 2A-L, Supporting Fig. S4A-C). Moreover, mice were co-injected with 50 µg sgp130Fc or 250 µg neutralizing IL-6 monoclonal antibody (mAb) to block IL-6 signaling. Co-injection of IL-6 with sgp130Fc or IL-6 mAb significantly blocked pSTAT3 in colon, liver, and lung (Fig. 2A-L, Supporting Fig. S4A-C). To analyze whether the acute phase response differs between wild-type and sIL-6R^+/+ trans-signaling mice, we injected per mouse 5 µg IL-6 and analyzed the expression of the acute phase protein serum amyloid A2 (Saa2) by quantitative real-time PCR. As shown in Fig. 2M, induction of Saa2 expression in wild-type and sIL-6R^+/+ trans-signaling mice was almost identical, suggesting that at least in the liver, IL-6 trans-signaling can completely compensate for the loss of IL-6 classic signaling. Our results demonstrate that the sIL-6R in sIL-6R^+/+ mice is biologically active and transmits IL-6 signals through trans-signaling, thereby indicating that forced IL-6 trans-signaling can activate all cells of the organism. Thus, endogenous IL-6 trans-signaling is a very efficient cellular activation mode, which is specifically triggered during pathophysiological conditions.

SIL-6R^+/+ Mice Fully Compensated Disabled IL-6 Classic Signaling by IL-6 Trans-signaling During Liver Regeneration After PHX

PHX was performed in wild-type, sIL-6R^fl/fl, sIL-6R^+/+, and IL-6R^-/- mice to analyze whether IL-6 trans-signaling is sufficient for normal liver regeneration. Overall survival rate of wild-type and sIL-6R^fl/fl mice 12 days after PHX was about 80%, whereas IL-6R deficient mice had an overall survival rate of only 20%. Survival of sIL-6R^+/+ trans-signaling mice after PHX was identical to wild-type mice and sIL-6R^fl/fl mice (Fig. 3A). Moreover, large necrotic areas were only found in the liver of IL-6R^-/- mice but not in wild-type, sIL-6R^fl/fl, and sIL-6R^+/+ mice (Fig. 3B). Consequently, the necrotic score was increased in IL-6R^-/- mice in comparison to wild-type, sIL-6R^fl/fl, and sIL-6R^+/+ mice (Fig. 3C). There was, however, only a trend for increased alanine aminotransferase (ALT)/aspartate aminotransferase (AST) levels 12 and 24 hours following PHX in IL-6R^-/- mice compared with wild-type, sIL-6R^fl/fl, and sIL-6R^+/+ mice (Supporting Fig. S5A,B). Liver weight to body weight ratio 12, 24, and 168 hours after PHX was not different between wild-type and sIL-6R^+/+ mice (Supporting Fig. S6A). Next, we analyzed sIL-6R levels following PHX in wild-type, IL-6R^-/-, and sIL-6R^+/+ mice. As reported previously14 and supported by our data, sIL-6R level in wild-type mice increased 1.5 fold from 11 ng/mL to 15 ng/mL 24 hours after PHX (Fig. 3D), whereas no sIL-6R was detectable in IL-6R^-/- mice at any time after PHX (Fig. 3E). Importantly, sIL-6R^+/+ mice also showed a 3.2-fold increase of sIL-6R from 372 ng/mL to 1,200 and 620 ng/mL 12 hours and 24 hours after PHX (Fig. 3F). As shown previously, the sIL-6R in sIL-6R^+/+ mice was not generated by ADAM-mediated shedding, but by direct secretion of sIL-6R. Therefore, we analyzed mRNA levels of IL-6R following PHX in wild-type and sIL-6R^+/+ mice in liver and spleen by quantitative real-time PCR. IL-6R mRNA levels were significantly increased in livers of wild-type mice and sIL-6R^+/+ mice by a factor of 6.6 and 5.5, respectively (Fig. 3G), suggesting that transcriptional activation of IL-6R is the main driving force of sIL-6R generation in sIL-6R^+/+ mice following PHX. Increased transcription likely contributes to increased sIL-6R level in wild-type mice following PHX; however, ectodomain shedding is the final trigger for sIL-6R generation. IL-6R mRNA levels were not increased in the spleen of wild-type mice and in sIL-6R^+/+ mice only after 24 hours (Fig. 3H). Because in wild-type mice the membrane-bound IL-6R is converted into sIL-6R by ectodomain shedding, production of sIL-6R will be delayed in comparison to direct secretion of sIL-6R in sIL-6R^+/+ mice, which might be the reason for the faster increase of the sIL-6R protein level in sIL-6R^+/+ mice compared with wild-type mice (Fig. 3D,F).

Next, we analyzed STAT3 phosphorylation in the liver of wild-type, IL-6R^-/-, and sIL-6R^+/+ 12 hours and 24 hours following PHX. STAT3 phosphorylation was comparable in wild-type and sIL-6R^+/+ mice as determined by immunohistochemistry (Fig. 4A). Importantly, IL-6R^-/- mice had significantly reduced pSTAT3 levels in the liver 12 hours and 24 hours following PHX (Fig. 4A-D, Supporting Fig. S7). Proliferating cell nuclear antigen (PCNA) is required for DNA synthesis during replication and hepatocyte proliferation.28 Immunohistochemical staining of PCNA in liver sections 24 hours after PHX revealed a reduction of about 76.4% of PCNA-positive cells in IL-6R^-/- mice compared with wild-type mice, whereas PCNA levels of wild-type and IL-6R^+/+ mice were comparable (Fig. 4E,F).

Taken together, our data demonstrate that IL-6 trans-signaling can fully compensate for the loss of classic signaling to ensure liver regeneration following PHX.

Selective Inhibition of IL-6 Trans-signaling Prevents Liver Regeneration Following PHX

Discrimination between classic and trans-signaling is also possible by comparing the functional outcome of classic and trans-signaling blockade by IL-6 and/or IL-6R antibodies with the specific blockade of trans-signaling by sgp130Fc. Therefore, we injected a high dose of monoclonal antibodies directed against IL-6 (250 µg/mouse) and a low dose of sgp130Fc (50 µg/mouse) into wild-type mice, to independently characterize the contribution of classic and trans-signaling to liver regeneration. A total of 50 µg/mouse (1.25 mg/kg) sgp130Fc was chosen because previous studies showed selective inhibition of trans-signaling for this dosage, whereas lower amounts had no effect.29, 30 Abrogation of IL-6 classic and trans-signaling in wild-type mice treated with neutralizing IL-6 mAb (250 µg/mouse) every 2 days following PHX resulted in an overall survival of 10% after 12 days (Fig. 5A), which is in good agreement with the survival of IL-6R^-/- mice after PHX (Fig. 3A). Importantly, selective inhibition of IL-6 trans-signaling by repetitive intraperitoneal injection of a low dose of 50 µg/mouse sgp130Fc (1.25 mg/kg) resulted in a comparably low survival rate as observed for mice treated with 250 µg/mouse IL-6 mAb (6.25 mg/kg) after PHX (Fig. 5A). In contrast, an even lower dose of 10 µg/mouse sgp130Fc (0.25 mg/kg) had no effect on survival (Supporting Fig. S8). Histological examination of the overall liver damage 12 hours and 24 hours after PHX showed larger necrotic areas in mice treated with IL-6 mAb and sgp130Fc (1.25 mg/kg) compared with control mice (Fig. 5B). Consequently, the necrotic score was increased in IL-6 mAb and sgp130Fc treated mice in comparison to control mice (Fig. 5C). There was, however, just a trend for increased ALT/AST level 12 hours and 24 hours after PHX in IL-6 mAb treated mice compared with wild-type and sgp130Fc treated mice (Supporting Fig. S5A,B). Liver weight to body weight ratio 12 hours and 24 hours after PHX was not different between experimental groups (Supporting Fig. S6B). Due to the low survival rate of IL-6 mAb-treated and sgp130Fc-treated mice liver weight to body weight ratio at a later time point was not determined. Next, we analyzed STAT3 phosphorylation in the liver following PHX in mice treated with IL-6 mAb, sgp130Fc, or left untreated. STAT3 phosphorylation in the liver was decreased after global blockade of IL-6 signaling for 12 hours and 24 hours compared with control mice, as determined by immunohistochemistry (Fig. 6A,B) and western blotting (Fig. 6C-F, Supporting Fig. S9A,B). Importantly, selective inhibition of IL-6 trans-signaling also prevented phosphorylation of STAT3 in the liver 12 hours and 24 hours after PHX (Fig. 6A-F, Supporting Fig. S9A,B). Immunohistochemical staining of PCNA in liver sections 24 hours following PHX demonstrated a 94.1% reduction of PCNA-positive cells after global blockade of IL-6 signaling and a 85.4% reduction after selective inhibition of IL-6 trans-signaling compared with control mice (Fig. 6G,H). Taken together, our data showed that IL-6 trans-signaling is the main pathway controlling liver regeneration following PHX.

IL-6 Trans-signaling Induces HGF Expression in Mice Following PHX and in Hepatic Stellate Cells

HGF is important for induction of hepatocyte proliferation26 and was up-regulated following PHX in wild-type mice in the liver at the mRNA level (Fig. 7A). Importantly, inhibition of classic and trans-signaling by IL-6 mAb or trans-signaling by sgp130Fc prevented up-regulation of Hgf on mRNA level in wild-type mice following PHX (Fig. 7A). Up-regulation of Hgf was, however, abrogated in IL-6R^-/- mice (Fig. 7A,B), but not in sIL-6R^+/+ mice (Fig. 7A,B). Next, we determined HGF level in the liver lysate of mice following PHX. Whereas wild-type and sIL-6R^+/+ mice had significantly increased HGF-level 12 hours and 24 hours following PHX, the IL-6 mAb-treated, sgp130Fc-treated, and IL-6R^-/- mice failed to show up-regulation of HGF (Fig. 7C). These data indicate that HGF up-regulation following PHX is dependent on IL-6 trans-signaling.

Hepatic stellate cells (HSCs) are a known source of HGF following PHX.31 To answer the question of whether IL-6 classic and/or trans-signaling trigger expression of HGF, rat HSCs were analyzed. Interestingly, HSCs express IL-6R and Gp130 on the mRNA level as determined by quantitative real-time PCR (Fig. 8A). Expression of IL-6R protein was verified by immunohistochemical staining (Fig. 8B), and again IL-6R was found primarily in intracellular compartments and only sparingly on the cell membrane. Despite the expression of IL-6R, only the stimulation of HSCs with hyper-IL-6 but not with IL-6 or sgp130Fc, resulted in increased of Hgf mRNA level (Fig. 8C). Even though increased HGF protein level found only in lysates of hyper-IL-6 stimulated HSCs (Fig. 8D), release of HGF was not stimulated by IL-6 trans-signaling in this in vitro setting (Fig. 8E). Our data show that IL-6 trans-signaling induced HGF production in HSCs, but suggested that in addition to IL-6 trans-signaling, additional factors might be needed to induce release of HGF from HSCs.