Alcoholic hepatitis is associated with a high incidence of infection, a major driver of short-term mortality. While several pathways have been investigated in decompensated cirrhosis9xGustot, T., Fernandez, J., Szabo, G., Albillos, A., Louvet, A., Jalan, R. et al. Sepsis in alcohol-related liver disease. J Hepatol. 2017;
Abstract | Full Text | Full Text PDF | PubMed | Scopus (27) | Google ScholarSee all References there are fewer data in alcoholic hepatitis. This study shows that the IL-33/ST2 pathway is altered in PMNs from patients with SAH and that this defect can be restored, at least in part, by IL-33. Indeed, the levels of circulating sST2, a biomarker indicative of IL-33/ST2 pathway activity, are associated with short-term mortality, development of infection and response to steroids. We also observed that different downstream effectors of the IL33/ST2 pathway were altered in SAH. Indeed, SAH is associated with an increased expression of GRK2 and decreased expression of CXCR2, which can be restored by challenging PMNs with IL-33. Decreased CXCR2 expression can be linked to a reduced migration capacity induced by IL-8, which can be improved by challenge with IL-33. The main study findings are shown in Fig. 6Fig. 6.
The issue of controls is critical in translational research for alcoholic hepatitis. Indeed, most patients admitted with SAH (i.e. with a Maddrey discriminant function >32) have underlying cirrhosis. Thus, we used 2 different control groups (i.e. patients with cirrhosis and healthy controls) to prove that changes in the IL-33/ST2 pathway in SAH are related to alcoholic hepatitis rather than decompensated cirrhosis. Comparison between cirrhosis and controls also shows the defect in this pathway during liver decompensation without any inflammatory process.
Endotoxemia was quantified using the HPLC-MS/MS method to determine serum concentrations of 3-HM. We chose this technique because HPLC-MS/MS can more accurately identify patients with low-grade endotoxemia, compared to the classical LAL (limulus amebocyte lysate) test, due to its high specificity and low limits of detection and quantification. Moreover, the measurement is not affected by the lipid content of samples, thus avoiding bias due to lipid-rich particles observed with the LAL test.23xPais de Barros, J.-P., Gautier, T., Sali, W., Adrie, C., Choubley, H., Charron, E. et al. Quantitative lipopolysaccharide analysis using HPLC/MS/MS and its combination with the limulus amebocyte lysate assay. J Lipid Res. 2015;
Crossref | PubMed | Scopus (33) | Google ScholarSee all References Such a technique has been validated in patients with cirrhosis.22xWeil, D., Pais de Barros, J.-P., Mourey, G., Laheurte, C., Cypriani, B., Badet, N. et al. Circulating levels of 3-hydroxymyristate, a direct quantification of endotoxaemia in noninfected cirrhotic patients. Liver Int. 2019;
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Herein, we confirm that patients with SAH have higher endotoxemia than patients with cirrhosis and controls.30xMichelena, J., Altamirano, J., Abraldes, J.G., Affò, S., Morales-Ibanez, O., Sancho-Bru, P. et al. Systemic inflammatory response and serum lipopolysaccharide levels predict multiple organ failure and death in alcoholic hepatitis. Hepatology. 2015;
Crossref | PubMed | Scopus (112) | Google ScholarSee all References The weak although significant correlation between sST2 levels and MELD score or endotoxemia suggests that liver failure and latent infection do not play an exclusive role to drive sST2 up in patients with SAH. Compared to the study by Alves-Filho et al.20xAlves-Filho, J.C., Sônego, F., Souto, F.O., Freitas, A., Verri, W.A., Auxiliadora-Martins, M. et al. Interleukin-33 attenuates sepsis by enhancing neutrophil influx to the site of infection. Nat Med. 2010;
Crossref | PubMed | Scopus (289) | Google ScholarSee all References we think that it is not only infection or liver insufficiency that inhibits the IL33-ST2 pathway but rather that this defect is a characteristic of SAH. Future studies are required to determine if systemic inflammation associated with SAH plays a role in the defect in the IL-33/ST2 pathway.
SIGIRR has been suggested to be a downregulator of the Toll-like receptor pathways implicated in sepsis in murine models, especially in neutrophils, with conflicting results in terms of functional consequences.31xUeno-Shuto, K., Kato, K., Tasaki, Y., Sato, M., Sato, K., Uchida, Y. et al. Lipopolysaccharide decreases single immunoglobulin interleukin-1 receptor-related molecule (SIGIRR) expression by suppressing specificity protein 1 (Sp1) via the Toll-like receptor 4 (TLR4)-p38 pathway in monocytes and neutrophils. J Biol Chem. 2014;
Crossref | PubMed | Scopus (21) | Google ScholarSee all References,32xBlok, D.C., van Lieshout, M.H., Hoogendijk, A.J., Florquin, S., de Boer, O.J., Garlanda, C. et al. Single immunoglobulin interleukin-1 receptor-related molecule impairs host defense during pneumonia and sepsis caused by Streptococcus pneumoniae. J Innate Immun. 2014;
Crossref | PubMed | Scopus (13) | Google ScholarSee all References In the present study, we did not observe any significant difference in SIGIRR expression between controls, patients with cirrhosis and those with SAH, in the presence or the absence of IL-33.
Because the incidence of infection is a major driver of mortality in SAH,5xThursz, M.R., Richardson, P., Allison, M., Austin, A., Bowers, M., Day, C.P. et al. Prednisolone or pentoxifylline for alcoholic hepatitis. N Engl J Med. 2015;
Crossref | PubMed | Scopus (255) | Google ScholarSee all References,8xLouvet, A., Wartel, F., Castel, H., Dharancy, S., Hollebecque, A., Canva-Delcambre, V. et al. Infection in patients with severe alcoholic hepatitis treated with steroids: early response to therapy is the key factor. Gastroenterology. 2009;
Abstract | Full Text | Full Text PDF | PubMed | Scopus (195) | Google ScholarSee all References,33xVergis, N., Atkinson, S.R., Knapp, S., Maurice, J., Allison, M., Austin, A. et al. In patients with severe alcoholic hepatitis, prednisolone increases susceptibility to infection and infection-related mortality, and is associated with high circulating levels of bacterial DNA. Gastroenterology. 2017;
Abstract | Full Text | Full Text PDF | PubMed | Scopus (40) | Google ScholarSee all References there is an urgent need for reliable markers able to identify patients at a greater risk of becoming infected. Although baseline severity scores have been combined with scores evaluating medical management response, the percentage of well-classified patients is still around 80%. Thus, the outcome of some patients is not properly predicted in close to 20% of cases. Moreover, in patients initially classified as having good prognosis and who respond to therapy, the occurrence of an infection is responsible for a major drop in survival.8xLouvet, A., Wartel, F., Castel, H., Dharancy, S., Hollebecque, A., Canva-Delcambre, V. et al. Infection in patients with severe alcoholic hepatitis treated with steroids: early response to therapy is the key factor. Gastroenterology. 2009;
Abstract | Full Text | Full Text PDF | PubMed | Scopus (195) | Google ScholarSee all References Circulating sST2 has been proposed as a biomarker in several diseases34xCoronado, M.J., Bruno, K.A., Blauwet, L.A., Tschöpe, C., Cunningham, M.W., Pankuweit, S. et al. Elevated sera sST 2 is associated with heart failure in men ≤50 years old with myocarditis. J Am Heart Assoc. 2019;
Crossref | PubMed | Scopus (9) | Google ScholarSee all References,35xAlam, M.L., Katz, R., Bellovich, K.A., Bhat, Z.Y., Brosius, F.C., de Boer, I.H. et al. Soluble ST2 and Galectin-3 and progression of CKD. Kidney Int Rep. 2019;
Abstract | Full Text | Full Text PDF | PubMed | Scopus (9) | Google ScholarSee all References,36xWatanabe, M., Nakamoto, K., Inui, T., Sada, M., Honda, K., Tamura, M. et al. Serum sST2 levels predict severe exacerbation of asthma. Respir Res. 2018;
Crossref | PubMed | Scopus (6) | Google ScholarSee all References and adding this marker to available scoring systems could optimize the prediction of clinical events in SAH. We show here using a large sample size and a robust methodology that sST2 not only predicts mortality but also the probability of getting infected in SAH and in cirrhosis, even though circulating levels of sST2 are different between the 2 conditions. Such results are promising because there is a large unmet need for biomarkers to predict the development of infection in both SAH and cirrhosis. Since elevated baseline sST2 values are associated with the risk of development of infection after treatment, patients with high sST2 might be candidates for trials testing strategies against infection. We observed higher levels of sST2 after 7 days of prednisolone exposure in both responders and non-responders to corticosteroids. It has already been reported that sST2 levels are increased by steroid treatment in ulcerative colitis and in systemic lupus erythematosus37xDíaz-Jiménez, D., Núñez, L., De la Fuente, M., Dubois-Camacho, K., Sepúlveda, H., Montecino, M. et al. A functional IL1RL1 variant regulates corticosteroid-induced sST2 expression in ulcerative colitis. Sci Rep. 2017;
Crossref | PubMed | Scopus (5) | Google ScholarSee all References,38xDíaz-Jiménez, D., Núñez, L.E., Beltrán, C.J., Candia, E., Suazo, C., Alvarez-Lobos, M. et al. Soluble ST2: a new and promising activity marker in ulcerative colitis. World J Gastroenterol. 2011;
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Crossref | PubMed | Scopus (96) | Google ScholarSee all References and we feel that its evolution under prednisolone is not indicative of the global activity of the IL-33/ST2 pathway.
There has been no significant improvement in the pharmaceutical management of SAH in the past few years. Based on the knowledge that infection is associated with a high short-term probability of death, results in trials testing antibiotic prophylaxis are expected (Rifaximin: NCT02116556; Ciprofloxacin: NCT02326103; Amoxicillin and Clavulanate: NCT02281929). While strategies with antibiotics are promising, they do not restore the immune defect associated with SAH. Stimulating PMN migration to the site of infection with IL-33 might represent an appealing option. Indeed, alterations in migration capacity have been associated with an increased risk of infection in humans.40xEgger, G., Aigner, R., Glasner, A., Hofer, H.P., Mitterhammer, H., and Zelzer, S. Blood polymorphonuclear leukocyte migration as a predictive marker for infections in severe trauma: comparison with various inflammation parameters. Intensive Care Med. 2004;
Crossref | PubMed | Scopus (29) | Google ScholarSee all References Interestingly an increased migration capacity of circulating PMNs with IL-33 was found in both responders and non-responders to treatment in patients with SAH. We previously showed that non-responders to steroids are more likely to develop infection than responders.8xLouvet, A., Wartel, F., Castel, H., Dharancy, S., Hollebecque, A., Canva-Delcambre, V. et al. Infection in patients with severe alcoholic hepatitis treated with steroids: early response to therapy is the key factor. Gastroenterology. 2009;
Abstract | Full Text | Full Text PDF | PubMed | Scopus (195) | Google ScholarSee all References The present study shows that PMNs from non-responders have a lower migration capacity following a challenge with IL-8 than those from responders and that IL-33 leads to an improvement in migration capacity in both groups. Although PMNs from non-responders are less responsive to IL-33, this cytokine improves migration capacity. This is particularly important because of the high rate of infection in these patients. Furthermore, PMNs from non-responders are less responsive to IL-33 at D0, suggesting that the defect in migration in these patients is already present in part before steroid exposure. The defect in the IL-33/ST2 pathway in the neutrophils of non-responders to corticosteroids seems to be related to higher levels of the decoy receptor sST2 but also to a less good response to IL-33 than responders that cannot be improved with higher doses of IL-33.
While neutrophils were treated with a dose of 50 ng/ml in the ex vivo experiments, the detected dose in the serum was around 100 pg/ml. Such a difference can be viewed as a supraphysiological stimulus. Other authors have also used similar doses.20xAlves-Filho, J.C., Sônego, F., Souto, F.O., Freitas, A., Verri, W.A., Auxiliadora-Martins, M. et al. Interleukin-33 attenuates sepsis by enhancing neutrophil influx to the site of infection. Nat Med. 2010;
Crossref | PubMed | Scopus (289) | Google ScholarSee all References,41xBandara, G., Beaven, M.A., Olivera, A., Gilfillan, A.M., and Metcalfe, D.D. Activated mast cells synthesize and release soluble ST2-a decoy receptor for IL-33. Eur J Immunol. 2015;
Crossref | PubMed | Scopus (38) | Google ScholarSee all References,42xHayakawa, H., Hayakawa, M., Kume, A., and Tominaga, S. Soluble ST2 blocks interleukin-33 signaling in allergic airway inflammation. J Biol Chem. 2007;
Crossref | PubMed | Scopus (365) | Google ScholarSee all References,43xLe, H.T., Tran, V.G., Kim, W., Kim, J., Cho, H.R., and Kwon, B. IL-33 priming regulates multiple steps of the neutrophil-mediated anti-Candida albicans response by modulating TLR and dectin-1 signals. J Immunol. 2012;
Crossref | PubMed | Scopus (47) | Google ScholarSee all References,44xLan, F., Yuan, B., Liu, T., Luo, X., Huang, P., Liu, Y. et al. Interleukin-33 facilitates neutrophil recruitment and bacterial clearance in S. aureus-caused peritonitis. Mol Immunol. 2016;
Crossref | PubMed | Scopus (22) | Google ScholarSee all References In addition, we cannot exclude that the detection of IL-33 in the serum of patients lacks sensitivity. Based on our results, we propose that in patients with SAH, restoration of the IL-33/ST2 pathway may help prevent and treat infection in both responders and non-responders.
In the present study, the IL-33/ST2 pathway did not modulate the phagocytic capacity of circulating PMNs suggesting that this pathway only improves PMNs influx at the site of infection. We would like to underline that impairment in neutrophil function during ALD is not restricted to decreased phagocytosis but also includes defects in microbial killing with several pathways involved such as myeloperoxidase, TIM-3, PD-1, etc.9xGustot, T., Fernandez, J., Szabo, G., Albillos, A., Louvet, A., Jalan, R. et al. Sepsis in alcohol-related liver disease. J Hepatol. 2017;
Abstract | Full Text | Full Text PDF | PubMed | Scopus (27) | Google ScholarSee all References Thus, future studies targeting pathways involved in phagocytic capacity in circulating PMNs are needed.
Alcoholic hepatitis is a complex disease and the role of PMNs is not fully understood. Indeed, there is a paradox between the liver infiltration by neutrophils (suggesting that activation of these cells is harmful to the liver) and a defect in neutrophil migration capacity (suggesting that a defect in PMN function is harmful to infection). In addition, IL-8 is the most upregulated cytokine in patients with acute drinking/alcoholic hepatitis vs. patients without SAH in the CANONIC study evaluating the profile of patients with acute-on-chronic liver failure.45xClària, 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;
Crossref | PubMed | Scopus (173) | Google ScholarSee all References IL-8 is also elevated in the blood and in the liver of patients with SAH in the study by Taïeb et al.46xTaïeb, J., Mathurin, P., Elbim, C., Cluzel, P., Arce-Vicioso, M., Bernard, B. et al. Blood neutrophil functions and cytokine release in severe alcoholic hepatitis: effect of corticosteroids. J Hepatol. 2000;
Abstract | Full Text | Full Text PDF | PubMed | Scopus (92) | Google ScholarSee all References Based on our results on neutrophil migration upon IL-8 and IL-33, we cannot exclude that promoting the IL-33/ST2 pathway in patients with ALD could result in a higher recruitment of neutrophils in the liver. Although the present study does not address this issue, it can be hypothesized that neutrophil migration triggers are different for the liver and the site of infection. The discrepancies between migration in the liver and to the site of infection could be related to different patterns of chemokine expression. However, IL-8 is not the only signal leading to neutrophil recruitment, as reviewed by Gustot et al.9xGustot, T., Fernandez, J., Szabo, G., Albillos, A., Louvet, A., Jalan, R. et al. Sepsis in alcohol-related liver disease. J Hepatol. 2017;
Abstract | Full Text | Full Text PDF | PubMed | Scopus (27) | Google ScholarSee all References Specific studies testing chemotaxis targets are needed for alcoholic hepatitis.
In conclusion, this study shows that circulating neutrophil migration is altered in SAH and that this defect is reversible by restoring the IL-33/ST2 pathway. Circulating sST2 levels are associated with survival and the probability of infection. Our results suggest that treatment with IL-33 represents a new potential therapeutic strategy to decrease the risk of infection in SAH.
3-HM, 3-hydroxymyristate; ALD, alcohol-related liver disease; AH, alcoholic hepatitis; Cirrh, cirrhosis; Ctrls, controls; DAMP, damage-associated molecular pattern; HR, hazard ratio; IFN, interferon; LPS, lipopolysaccharide; MELD, model for end-stage liver disease; MFI, mean fluorescent intensity; MIRU, mean intensity with relative units; PMN, polymorphonuclear neutrophils; SAH, severe alcoholic hepatitis; sST2, soluble ST2.