Low Interleukin-22 Binding Protein Is Associated With High Mortality in Alcoholic Hepatitis and Modulates Interleukin-22 Receptor Expression

Abstract

Introduction: In alcoholic hepatitis (AH), high interleukin (IL)-22 production is associated with disease improvement, purportedly through enhanced infection resistance and liver regeneration. IL-22 binding protein (BP) binds and antagonizes IL-22 bioactivity, but data on IL-22BP in liver disease suggest a complex interplay. Despite the scarcity of human data, IL-22 is in clinical trial as treatment of AH. We, therefore, in patients with AH, described the IL-22 system focusing on IL-22BP and associations with disease course, and mechanistically pursued the human associations in vitro.

Methods: We prospectively studied 41 consecutive patients with AH at diagnosis, days 7 and 90, and followed them for up to 1 year. We measured IL-22 pathway proteins in liver biopsies and blood and investigated IL-22BP effects on IL-22 in hepatocyte cultures.

Results: IL-22BP was produced in the gut and was identifiable in the patients with AH' livers. Plasma IL-22BP was only 50% of controls and the IL-22/IL-22BP ratio thus elevated. Consistently, IL-22-inducible genes were upregulated in AH livers at diagnosis. Low plasma IL-22BP was closely associated with high 1-year mortality. In vitro, IL-22 stimulation reduced IL-22 receptor (R) expression, but coincubation with IL-22BP sustained IL-22R expression. In the AH livers, IL-22R mRNA expression was similar to healthy livers, although IL-22R liver protein was higher at diagnosis.

Discussion: Plasma IL-22BP was associated with an adverse disease course, possibly because its low level reduces IL-22R expression so that IL-22 bioactivity was reduced. This suggests the IL-BP interplay to be central in AH pathogenesis, and in future treatment trials (see Visual abstract, Supplementary Digital Content 5, http://links.lww.com/CTG/A338).

Figure 1.

IL-22BP is present in the liver and produced in the gut, and plasma levels are markedly reduced. (a) Immunohistochemical staining for IL-22BP in paraffin-embedded sections of liver from patients with alcoholic hepatitis and healthy liver and colon. Representative sections displayed. (b) Plasma IL-22BP by ELISA from the portal vein and peripheral blood in a cohort of patients with cirrhosis and in (c–e) peripheral plasma from patients with alcoholic hepatitis (AH) and controls; nonalcoholic fatty liver (NAFL) and steatohepatitis (NASH) and stable alcoholic (Alc) cirrhosis (analysis of variance, T-test, *P < 0.05,**P < 0.01, ***P < 0.001, bars represent means and SDs).

Figure 2.

Low IL-22BP at diagnosis is associated with 1-year mortality. Plasma IL-22BP by ELISA. (a) Kaplan-Meier survival curve of all patients with alcoholic hepatitis stratified by plasma IL-22BP at diagnosis above or below 225 ng/mL (Log-rank test: P = 0.037). (b) The plasma IL-22BP at day 7 was compared between alcoholic hepatitis patients, who had died by day 90 and those still alive (T-test, bars display mean and SD, *P < 0.05). BP, binding protein; IL, interleukin.

Figure 3.

Upregulation of lipocalin-2 in liver and blood. (a and b) Immunohistochemical staining for lipocalin-2 in livers of patients with alcoholic hepatitis (AH) compared with healthy controls (Fisher exact test, P < 0.001). (c) Blood lipocalin-2 by ELISA in patients with alcoholic hepatitis, healthy controls, nonalcoholic fatty liver (NAFL) and steatohepatitis (NASH) and from stable alcoholic (Alc) cirrhosis patients (T-test, bars represent means and SDs, *P < 0.05, **P < 0.01).

Figure 4.

Presence of IL-22BP sustains IL-22 receptor expression. (a) HepG2 cells stimulated with 50 ng/mL recombinant IL-22 alone or in combination with varying concentrations of recombinant IL-22BP for 4 hours. (b and c) primary human hepatocytes and HepG2 cells stimulated with 50 ng/mL recombinant IL-22 alone or in combination with 250 ng/mL recombinant IL-22BP for 24 h and followed by repeat IL-22 stimulation for 4 hours. IL-22 receptor (R) expression by quantitative polymerase chain reaction relative to GAPDH and an unstimulated control. (d) IL-22R expression by RNA sequencing and (e and f) immunohistochemistry of livers from alcoholic hepatitis patients compared with healthy liver. BP, binding protien; GAPDH, Glyceraldehyde 3-phospahte dehydrogenase; IL, Interleukin.