Interleukin-22 in acute-on-chronic liver failure: A matter of ineffective levels, receptor dysregulation or defective signalling?

Interleukin-22 ameliorates acute-on-chronic liver failure by reprogramming impaired regeneration pathways in miceJournal of HepatologyVol. 72Issue 4PreviewAcute-on-chronic liver failure (ACLF) is a clinical syndrome defined by liver failure on pre-existing chronic liver disease. It is often associated with bacterial infection and high short-term mortality. Experimental models that fully reproduce ACLF are lacking, so too are effective pharmacological therapies for this condition. Full-Text PDF Reply to: “Interleukin-22 in acute-on-chronic liver failure: A matter of ineffective levels, receptor dysregulation or defective signalling?”: The search for an optimal mouse modelJournal of HepatologyVol. 73Issue 4PreviewWe greatly appreciate Schwarzkopf et al.'s comments on our work. The first question raised relates to the role of endogenous interleukin (IL)-22 and IL-22 binding protein (IL-22BP) in acute-on-chronic liver failure (ACLF). Serum levels of IL-22 are elevated in patients with ACLF1 and in mouse models of ACLF induced by infection of adenovirus-CYP4502D6 plus injection of cecal slurry (CS) or chronic CCl4 plus 2 binges of ethanol.2 Actually, we also found that serum IL-22 levels were mildly but significantly elevated in our ACLF model, which was illustrated in Fig. S6B in our paper. Full-Text PDF Xiang et al. published interesting insights about the role of interleukin (IL)-22 in acute-on-chronic liver failure (ACLF).[1]Xiang X. Feng D. Hwang S. Ren T. Wang X. Trojnar E. et al.Interleukin-22 ameliorates acute-on-chronic liver failure by reprogramming impaired regeneration pathways in mice.J Hepatol. 2020; 72: 736-745Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar The authors elegantly analysed the effects of IL-22 in a mouse model of ACLF after chronic carbon tetrachloride (CCl4)-treatment (0.2 ml/kg, 2×/week, i.p.) and acute precipitating injury using 2 injections of 0.4 ml/kg CCl4 in combination with an i.p. challenge with Klebsiella pneumonia (K.P.), mimicking a bacterial infection. The model seems to induce a shift in the hepatic IL-22 signalling from the pro-regenerative IL-6/STAT3 towards the anti-regenerative IFNγ/STAT1 pathway paralleled by suppression of liver regeneration. Interestingly, treatment with IL-22Fc fusion protein reduces bacterial load, improves microcirculation (hepatic/renal) and survival rate in mice with ACLF. The authors suggest that an impaired production of IL-6 from “tolerant” Kupffer cells in the fibrotic liver might be responsible for reduced liver regeneration in ACLF. They demonstrated that high-dose treatment with IL-22Fc restores downstream STAT1/STAT3 signalling imbalances. Using IL-22Fc to ameliorate ACLF is an elegant approach that can be translated into clinical trials using the IL-22 agonist F-652.[2]Tang K.-Y. Lickliter J. Huang Z.-H. Xian Z.-S. Chen H.-Y. Huang C. et al.Safety, pharmacokinetics, and biomarkers of F-652, a recombinant human interleukin-22 dimer, in healthy subjects.Cell Mol Immunol. 2019; 16: 473-482Crossref PubMed Scopus (46) Google Scholar,[3]Arab J.P. Sehrawat T.S. Simonetto D.A. Verma V.K. Feng D. Tang T. et al.An open label, dose escalation study to assess the safety and efficacy of IL-22 agonist F-652 in patients with alcoholic hepatitis.Hepatology. 2019; https://doi.org/10.1002/hep.31046Crossref PubMed Scopus (72) Google Scholar However, there are significant discrepancies between the data reported by Xiang et al. and data reported earlier in human ACLF. We recently demonstrated that circulating IL-22 levels are significantly elevated and strongly associated with mortality in these patients.[4]Schwarzkopf K. Rüschenbaum S. Barat S. Cai C. Mücke M.M. Fitting D. et al.IL-22 and IL-22-binding protein are associated with development of and mortality from acute-on-chronic liver failure.Hepatol Commun. 2019; 3: 392-405Crossref PubMed Scopus (25) Google Scholar Circulating levels of the endogenous IL-22, however, were not significantly elevated in the study by Xiang et al. unlike the human experience. To further assess the role of IL-22 in ACLF, we have developed different animal models. In a C57BL/6J mouse model of ACLF, we combined chronic liver disease (CLD) (CCl4/ethanol[5]Brol M.J. Rösch F. Schierwagen R. Magdaleno F. Uschner F.E. Manekeller S. et al.Combination of CCl4 with alcoholic and metabolic injuries mimics human liver fibrosis.Am J Physiol Gastrointest Liver Physiol. 2019; 317: G182-G194Crossref PubMed Scopus (26) Google Scholar or CYP2D6-linked adenovirus (ADV)-induced autoimmune hepatitis[6]Holdener M. Hintermann E. Bayer M. Rhode A. Rodrigo E. Hintereder G. et al.Breaking tolerance to the natural human liver autoantigen cytochrome P450 2D6 by virus infection.J Exp Med. 2008; 205: 1409-1422Crossref PubMed Scopus (162) Google Scholar) with different precipitating events (2 binges of ethanol or i.p. polymicrobial infection) (Fig. S1A+D). After 7 weeks CCl4/ethanol we induced ACLF with 2 binges of alcohol (alcohol gavage with 31.5% Vol.) with an interval of 3 days in between the binges. Mice showed mortality (Fig. S1B), as well as systemic inflammation (Fig. 1D) and significant elevation of serum alanine aminotransferase (ALT) levels (Fig. S1C) alongside other ACLF features. In mice with chronic autoimmune hepatitis we induced polymicrobial infection according to an adapted protocol by Wynn et al.[7]Wynn J.L. Scumpia P.O. Delano M.J. O'Malley K.A. Ungaro R. Abouhamze A. et al.Increased mortality and altered immunity in neonatal sepsis produced by generalized peritonitis.Shock. 2007; 28: 675-683Crossref PubMed Scopus (113) Google Scholar to achieve dose-dependent bacterial infection. Cecal slurry of a naïve donor C57BL/6J mouse was used to prepare a stool suspension (concentration 50 mg cecal slurry/1 ml sodium chloride solution) and 1 mg cecal slurry per g of bodyweight was injected.[8]Schwarzkopf K. Rüschenbaum S. Mücke M.M. Fuchs S. Messmer M. Bayer M. et al.THU-051 establishment of a new animal model for the acute-on-chronic liver failure.J Hepatol. 2019; 70: e183Abstract Full Text PDF Google Scholar After injection of cecal slurry mice develop a serious systemic infection (Fig. 1D), mortality (Fig. S1E) and elevation of ALT serum levels (Fig. S1F). In our opinion polymicrobial sepsis is closer to human infection-triggered ACLF than the K.P. injection used in the study by Xiang et al. This may be the reason why the authors tried to induce ACLF with cecal ligation and puncture surgery. Yet, this method may have drawbacks, since it is not easy to control the amount of cecal contents spilled into the peritoneal cavity, additionally the mice are exposed to a surgical procedure, which influences outcome as well. In our polymicrobial infection model, we could find a method to control the severity of infection with a specific amount of cecal slurry (Fig. S1). For both precipitating events, IL-22 serum concentrations in ACLF were elevated when compared to CLD alone. Of note, in infections, IL-22 levels are elevated regardless of the presence of CLD (Fig. 1A), suggesting that the mouse model used in the study by Xiang and co-workers is potentially not a suitable model to study mechanistic effects of IL-22, specifically in ACLF. Based on the paper by Xiang and co-workers, one may speculate that the increased IL-22 levels might be an endogenous attempt to improve organ function. Yet, the clinical course of disease shows no benefit from elevated IL-22 levels, which cannot be explained by the data published. A possible explanation is that IL-22 might be scavenged by the IL-22 binding protein (IL-22BP), which is highly upregulated in this condition as we recently showed in patients with ACLF[4]Schwarzkopf K. Rüschenbaum S. Barat S. Cai C. Mücke M.M. Fitting D. et al.IL-22 and IL-22-binding protein are associated with development of and mortality from acute-on-chronic liver failure.Hepatol Commun. 2019; 3: 392-405Crossref PubMed Scopus (25) Google Scholar and Xiang et al. also show in their mouse model. Correspondingly, using a rat model of ACLF, IL-22BP levels are elevated in peripheral blood mononuclear cells in ACLF (lipopolysaccharide 6.25 μg day 21 and day 25, n = 4) compared to acute decompensation (AD, lipopolysaccharide 6.25 μg day 21, n = 5) (Fig. 1B). The novel experimental data are in line with our previous findings on ACLF and IL-22 in humans, showing that a significant elevation of IL-22BP is strongly associated with mortality in patients with ACLF and in vitro inhibition of hepatocellular IL-22 signalling in human hepatoma cells.[4]Schwarzkopf K. Rüschenbaum S. Barat S. Cai C. Mücke M.M. Fitting D. et al.IL-22 and IL-22-binding protein are associated with development of and mortality from acute-on-chronic liver failure.Hepatol Commun. 2019; 3: 392-405Crossref PubMed Scopus (25) Google Scholar Hence, the lack of a hepatoprotective effect of IL-22 in ACLF could potentially be due to reduced circulating levels of this cytokine in its free/unbound form. Alternatively, based on differential expression levels of the receptor subunits IL-22R1 and IL-10R2, we infer that IL-22 signalling could be modified at the receptor level, possibly by impaired receptor assembly (Fig. 1C). Such a mechanism could cause ineffective signalling or potentially modulate the downstream signalling cascade as suggested by Xiang et al. It seems likely that both mechanisms, IL-22BP scavenging and modified IL-22 receptor assembly, are not mutually exclusive but rather could exist in parallel. However, mechanisms on the IL-22 receptor level remain largely unexplained. In addition, Xiang and co-workers suggest that an increase in hepatic IL-6 might be beneficial in ACLF and could lead to enhanced liver regeneration. This conclusion seems puzzling given that in human ACLF high IL-6 levels were associated with worse outcomes in several independent studies.[9]Trebicka J. Amoros A. Pitarch C. Titos E. Alcaraz-Quiles J. Schierwagen R. et al.Addressing profiles of systemic inflammation across the different clinical phenotypes of acutely decompensated cirrhosis.Front Immunol. 2019; 10: 476Crossref PubMed Scopus (92) Google Scholar,[10]Clària J. Stauber R.E. Coenraad M.J. Moreau R. Jalan R. Pavesi M. et al.Systemic inflammation in decompensated cirrhosis: characterization and role in acute-on-chronic liver failure.Hepatology. 2016; 64: 1249-1264Crossref PubMed Scopus (430) Google Scholar Systemic inflammation including an increase in IL-6 precedes ACLF in humans and is, even in the absence of infection, strongly associated with the development of ACLF and death.[9]Trebicka J. Amoros A. Pitarch C. Titos E. Alcaraz-Quiles J. Schierwagen R. et al.Addressing profiles of systemic inflammation across the different clinical phenotypes of acutely decompensated cirrhosis.Front Immunol. 2019; 10: 476Crossref PubMed Scopus (92) Google Scholar,[10]Clària J. Stauber R.E. Coenraad M.J. Moreau R. Jalan R. Pavesi M. et al.Systemic inflammation in decompensated cirrhosis: characterization and role in acute-on-chronic liver failure.Hepatology. 2016; 64: 1249-1264Crossref PubMed Scopus (430) Google Scholar In our mouse models, IL-6 is elevated in ethanol- as well as in infection-triggered ACLF (Fig. 1D). One may speculate that high levels of IL-6 reflect dysfunction of the immune system and lead to failure in the regeneration process.[11]Wasmuth H.E. Kunz D. Yagmur E. Timmer-Stranghöner A. Vidacek D. Siewert E. et al.Patients with acute on chronic liver failure display “sepsis-like” immune paralysis.J Hepatol. 2005; 42: 195-201Abstract Full Text Full Text PDF PubMed Scopus (384) Google Scholar Given the different lines of evidence and lack of mechanistic detail, caution is required in the interpretation of the results and further studies on IL-22 are needed to fully understand its impact on the course of liver disease and ACLF, as well as its therapeutic or preventive potential. Deutsche Forschungsgemeinschaft (SFB TRR57 P18 & CRC1382) H2020 European Institute of Innovation and Technology (668031 & 825694) H2020 Societal Challenges (731875) and CELLEX Foundation (PREDICT). The funders had no influence on study design, data collection and analysis, decision to publish or preparation of the manuscript. Dr. Katharina Maria Schwarzkopf was funded by Nachwuchsförderung of Goethe University, Frankfurt, Germany. KMS designed and conducted the experimental studies and wrote the paper; LE, FEU, SK, RS, MMM conducted some experimental studies, performed data analysis and edited the paper; LS, JC, SZ, EH, UC, CML helped with experimental design and edited the paper; JT and CW designed the experimental studies, supervised the whole project and wrote the paper. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. 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