Changes in natural killer cells and exhausted memory regulatory T Cells with corticosteroid therapy in acute autoimmune hepatitis

Autoimmune hepatitis (AIH) is an immune-mediated liver disease currently treated by immunosuppressive medications with significant side effects. Thus, novel mechanistic treatments are greatly needed. We performed prospective deep immunophenotyping of blood immune cells in patients with acute AIH before and after corticosteroid therapy. Blood samples from 26 patients with acute AIH (United Kingdom-AIH Consortium) were phenotyped by flow cytometry at baseline and 4 months after starting corticosteroids. Pretreatment liver tissues were stained for forkhead box P3-positive (FOXP3POS) regulatory T cells (Tregs), clusters of differentiation (CD)56POS natural killer (NK) cells, and chemokine (C-X-C motif) ligand 10. Chemokine secretion by cultured primary hepatocyte and biliary epithelial cells was measured by enzyme-linked immunosorbent assay. Functional coculture assays with stimulated NK cells and Tregs were performed. CD161 ligand, lectin-like transcript-1 expression by intrahepatic immune cells was demonstrated with flow cytometry. Frequencies of NKbright cells declined with therapy (P < 0.001) and correlated with levels of alanine aminotransferase (P = 0.023). The Treg:NKbright ratio was lower pretreatment, and Tregs had an activated memory phenotype with high levels of CD39, cytotoxic T lymphocyte antigen 4, and FOXP3 but also high programmed death ligand 1, indicating exhaustion. Coculture experiments suggested the Tregs could not efficiently suppress interferon-γ secretion by NK cells. Both Tregs and NK cells had high expression of liver infiltration and T helper 17 plasticity-associated marker CD161 (P = 0.04). Pretreatment and CD161pos NK cells expressed high levels of perforin and granzyme B, consistent with an activated effector phenotype (P < 0.05). Lectin-like transcript 1, a ligand for CD161, is expressed on intrahepatic B cells, monocytes, and neutrophils. Conclusion: Activated effector NK cells, which correlate with biochemical measurements of hepatitis, and exhausted memory Tregs are increased in the blood of patients with treatment-naive AIH and decline with corticosteroid therapy. Inadequate regulation of NK cells by exhausted FOXP3pos Tregs may play a role in AIH pathogenesis and contribute to liver injury. (Hepatology Communications 2018;2:421-436)

Human intrahepatic ILC2 are IL-13^*positive Amphiregulin^*positive and their frequency correlates with Model of End stage Liver Disease score

Innate lymphoid cells (ILC) have been implicated in the initiation of inflammation and fibrosis in mice. However, ILC have not been characterized in inflamed human liver tissue.Human intrahepatic lymphocytes were isolated by mechanical digestion and phenotyped by flow cytometry. Conditioned medium from cultures of primary human biliary epithelial cells, stellate cells, fibroblasts and inflamed human liver tissue was used to model the effects of the inflammatory liver environment of ILC phenotype and function.All three ILC subsets were present in the human liver, with the ILC1 (CRTH2negCD117neg) subset constituting around 70% of intrahepatic ILCs. Both NCRpos (NKp44+) and NCRneg ILC3 (CRTH2negCD117pos) subsets were also detected. ILC2 (CRTH2pos) frequency correlated with disease severity measured by model of end stage liver disease (MELD) scoring leading us to study this subset in more detail. ILC2 displayed a tissue resident CD69+ CD161++ phenotype and expressed chemokine receptor CCR6 allowing them to respond to CCL20 secreted by cholangiocytes and stellate cells. ILC2 expressed integrins VLA-5 and VLA-6 and the IL-2 and IL-7 cytokine receptors CD25 and CD127 although IL-2 and IL-7 were barely detectable in inflamed liver tissue. Although biliary epithelial cells secrete IL-33, intrahepatic ILC2 had low expression of the ST2 receptor. Intrahepatic ILC2 secreted the immunoregulatory and repair cytokines IL-13 and amphiregulin.Intrahepatic ILC2 express receptors allowing them to be recruited to bile ducts in inflamed portal tracts. Their frequencies increased with worsening liver function. Their secretion of IL-13 and amphiregulin suggests they may be recruited to promote resolution and repair and thereby they may contribute to ongoing fibrogenesis in liver disease

CD127^pos ILC2 express a high level of CD25, but inflamed human liver supernatant contains minimal IL-2 and IL-7.

<p>(A) The CD45^pos CD3^neg lineage^neg CD127^pos CRTH2^pos ILC2 subset was gated and expression of the IL-2 receptor α-chain, CD25, was analysed. CD25 representative overlay and summary data in normal and diseased livers is shown. (** = <i>p</i><0.01 by Mann Whitney test). In the histogram overlay the dotted line represents isotype staining and the shaded histogram reports CD25 staining. (B) Human inflamed liver supernatant was analysed for IL-2, IL-7, IL-9 and IFN-γ. The secretion of cytokines by normal and inflamed human liver tissue was analysed by luminex of 24-hour liver tissue supernatants prepared for 1g of tissue/1ml culture medium. (C) IL-33 production by human liver tissue (Normal and diseased) and (D) IL-33 production by Primary human biliary epithelial (BEC). IL-33 in 24-hour supernatants generated by 1g liver tissue/1ml medium or BEC cells unstimulated, stimulated with IFN-γ and TNF-α or with lipopolysaccharide (LPS) was analysed by ELISA. Summary data are median ± Interquartile range.</p

Biliary epithelial cells secrete IL-33 and intrahepatic ILC2 express IL-13 and amphiregulin.

<p><b>(</b>A) Intrahepatic ILC were freshly isolated and stimulated with PMA and Ionomycin for 4 hours and both CD45^pos CD3^neg lineage^neg CD127^pos CRTH2^pos ILC2 and CD45^pos CD3^neg lineage^neg CD127^pos CRTH2^neg populations were analysed for expressions of IL-4, IL-5, IL-13 and IFN-γ. Representative dot plots and summary data are shown. (B) Immunohistochemistry for amphiregulin in CD3 positive and negative intrahepatic immune cells in human liver. CD3 (Vector Red, red color); Amphiregulin (DAB, Brown color). (C) Amphiregulin expression by intrahepatic ILC2. Amphiregulin representative overlay and summary data in normal and diseased livers are shown. In the histogram overlay the dotted line represents isotype staining and the shaded histogram reports amphiregulin staining.</p

ILC2 subset correlates with MELD score.

<p>Correlation between MELD score and frequency of intrahepatic ILC subsets. Correlation of ILC subset frequency with MELD score was assessed by Spearman’s rank correlation test. End-stage livers of all types of inflammatory liver disease (autoimmune, and non-autoimmune) were included in this analysis. Model of End-stage Liver Disease (MELD).</p

Intrahepatic ILC2 highly express biliary tropic chemokine receptor CCR6 and integrins for fibronectin and laminin.

<p>(A) The CD45^pos CD3^neg lineage^neg CD127^posi CRTH2^pos ILC2 subset was gated and expressions of CXCR3, CCR6, Very Late Antigen-5 (VLA-5) and Very Late Antigen-6 (VLA-6) were analysed. Representative overlays and summary data in normal and diseased livers are shown (normal livers = open squares; diseased livers = filled squares). Summary data are median ± Interquartile range. In histogram overlays, dotted lines represent isotype staining and shaded histograms show the marker expression. (B) Primary human biliary epithelial cells (BEC), stellate cells and fibroblasts were isolated and stimulated with IFN-γ and TNF-α. Interferon gamma-induced Protein-10 (IP-10) secretion was analysed by ELISA. (* = p<0.05, ** = <0.01 by Paired <i>t-</i>test). Summary data are mean ± SEM. (C) Expressions of CXCR3 and CCR6 by the peripheral blood ILC subsets of normal donors and autoimmune liver disease patients with the condition autoimmune hepatitis (AIH). Summary data are median ± interquartile range. (* = p<0.05 by Mann-Whitney test).</p

Summary graphical diagram.

<p>Intrahepatic ILC2 are characterized by expression of CD127 and CRTH2, which binds PDG2. They express tissue residence maker CD69; CD161; the IL-2 cytokine receptor CD25; liver and tissue homing chemokine receptors CXCR3 and CCR6 and cytokine receptor ST2, which bind respectively to IP-10, CCL20 and IL-33 produced by biliary epithelial cells upon stimulation with inflammatory cytokines or bacteria. ILC2 secrete IL-13 and amphiregulin, which may contribute to bile duct regeneration. In addition, they express the integrins VLA-5 and VLA-6, which attach to the extracellular matrix proteins fibronectin and laminin found in the fibrous stroma.</p

Intrahepatic human ILC2 display a tissue resident phenotype, express CD161 and PGD2 is present in human inflamed livers.

<p>(A) The CD45^pos CD3^neg lineage^neg CD127^pos CRTH2^pos ILC2 subset was gated and CD161 and CD69 expressions were analysed. CD161 and CD69 representative overlays and summary data are shown. (B) PGD2 production by human liver. The secretion of PGD2 by normal and inflamed human liver tissue was analysed by ELISA on 24-hour liver tissue supernatants prepared for 1g-tissue/1ml culture medium. Summary data are median ± Interquartile range. (C) Expression of the IL-33 receptor, ST2 was analysed on ILC2. Representative overlay and summary data are shown. In histogram overlays, dotted lines are isotype staining and shaded histograms marker expression.</p

All three ILC subsets are present in inflamed human livers.

<p>(A) ILC gating strategy. Intrahepatic lymphocytes were freshly isolated and gated on the CD45^pos CD3^neg population. The total ILC population was defined as lineage^neg and CD127^pos. Of these, ILC1 were defined as CD117^negCRTH2^neg, ILC2 as CD117^pos/negCRTH2^pos and ILC3 as CD117^pos CRTH2^neg. (B) Frequency of the total ILC population among the CD3^neg CD45^pos population. (C) Frequencies of the three major ILC subsets in the total intrahepatic ILC in different inflammatory liver diseases. AILD: Autoimmune Liver Diseases (PSC, PBC, AIH); NASH: Nonalcoholic Steatohepatitis; ALD: Alcoholic Liver Disease. (* = <i>p</i><0.05 by Kruskal Wallis test). (D) Distribution of NKp44 expression among intrahepatic ILC. ILC3 were NKp44^pos (NCR^pos) or NKp44^neg (NCR^neg). (E) Frequencies of NKp44 expression by intrahepatic ILC3 in normal and diseased livers. Summary data are median ± Interquartile range.</p