Inflammatory Phenotype of Intrahepatic Sulfatide-Reactive Type II NKT Cells in Humans With Autoimmune Hepatitis

Abstract

Background: Natural Killer T (NKT) cells are CD1d-restricted innate-like T cells that can rapidly release stored cytokines upon recognition of lipid antigens. In mice, type I NKT cells seem to promote liver inflammation, whereas type II NKT cells seem to restrict hepatitis. Here, we aimed at characterizing the role of human type I and type II NKT in patients with autoimmune hepatitis (AIH). Methods: NKT cells were analyzed by flow cytometry in peripheral blood and liver of AIH patients and control groups. α-galactosylceramide-loaded or sulfatide-loaded tetramers were used to detect type I or II NKT cells, respectively. Hepatic CD1d was stained by in situ-hybridization of liver biopsies. Results and Conclusions: Type II NKT cells were more prevalent in human peripheral blood and liver than type I NKT cells. In AIH patients, the frequency of sulfatide-reactive type II NKT cells was significantly increased in peripheral blood (0.11% of peripheral blood leukocytes) and liver (3.78% of intrahepatic leukocytes) compared to healthy individuals (0.05% and 1.82%) and patients with drug-induced liver injury (0.06% and 2.03%; p < 0.05). Intrahepatic type II NKT cells of AIH patients had a different cytokine profile than healthy subjects with an increased frequency of TNFα (77.8% vs. 59.1%, p < 0.05), decreased IFNγ (32.7% vs. 63.0%, p < 0.05) and a complete lack of IL-4 expressing cells (0% vs. 2.1%, p < 0.05). T cells in portal tracts expressed significantly more CD1d-RNA in AIH livers compared to controls. This study supports that in contrast to their assumed protective role in mice, human intrahepatic, sulfatide-reactive type II NKT cells displayed a proinflammatory cytokine profile in patients with AIH. Infiltrating T cells in portal areas of AIH patients overexpressed CD1d and could thereby activate type II NKT cells.

Keywords: CD1d; alpha-galactosylceramide; lipid antigen; tumor necrosis factor-alpha; unconventional T cells.

Figure 1

Exemplary CD1d tetramer stainings. Mock-loaded human CD1d tetramers served as negative control staining for lipid-loaded human CD1d tetramers. An exemplary peripheral blood (upper row) and intrahepatic (lower row) flow cytometric staining with mock-loaded human CD1d tetramer (left) and sulfatide-loaded human CD1d tetramer (right) from an AIH patient is shown.

Figure 2

Exemplary flow cytometric stainings of type I and type II NKT cells. Human (hu) CD1d-tetramers loaded either with αGalCer or sulfatide were used to stain type I (left) or type II (right) NKT cells in peripheral blood (upper row) or in liver biopsies (lower row). Representative flow cytometric stainings of type I and type II NKT cells in a healthy subject and a patient with AIH are displayed.

Figure 3

Increased frequency of sulfatide-reactive type II NKT cells in patients with autoimmune hepatitis. The frequency of peripheral blood (A) and intrahepatic (B) type II NKT cells and peripheral blood (C) and intrahepatic (D) type I NKT cells in AIH patients in comparison to healthy subjects and patients with DILI is shown.

Figure 4

Exemplary flow cytometric stainings of peripheral and intrahepatic MAIT and γδ T cells. Representative flow cytometric stainings of peripheral blood (upper row) and intrahepatic (lower row) MAIT cells (left) and γδ T cells (right) of a healthy individual are shown.

Figure 5

No increased infrequency of peripheral blood and intrahepatic MAIT cells or γδ T cells in AIH patients. To exclude unspecific elevation of type II NKT in AIH patients, peripheral blood and intrahepatic MAIT cells (A,B) and peripheral blood and intrahepatic γδ T cells (C,D) were also quantified by flow cytometry revealing no increased infrequency of peripheral blood and intrahepatic MAIT cells or γδ T cells in AIH patients compared to healthy subjects or DILI patients.

Figure 6

Exemplary flow cytometric intracellular cytokine stainings for type II NKT cells. Representative intracellular cytokine stainings for TNFα, IFNγ, IL-17 and IL-4 for intrahepatic type II NKT cells of AIH patients (upper row) and healthy subjects (lower row) are shown.

Figure 7

Intrahepatic sulfatide-reactive type II NKT cells in patients with autoimmune hepatitis reveal a different cytokine profile than in healthy subjects. Intrahepatic type II NKT cells of AIH patients have an increased expression of TNFα (A), a lower expression of IFNγ (B) and show a complete lack of IL-4 (C) compared to healthy subjects.

Figure 8

Intrahepatic expression of CD1d is elevated in patients with autoimmune hepatitis. Quantitative PCR analysis of relative RNA expression, normalized on HPRT1, in whole liver tissue samples revealed significantly elevated levels of CD1d RNA in AIH patients in comparison to DILI patients or healthy subjects (A). Due to CD1d-RNA in situ hybridization, the expression of CD1d-RNA was significantly elevated in infiltrating lymphocytes in portal areas of patients AIH in comparison to patients with DILI (B).

Figure 9

Representative in situ stainings of CD1d-RNA in liver biopsies. Representative stainings of CD1d-RNA (purple dots, see arrows) with co-stainings of hepatocytes (A, brown = CK18) and cholangiocytes (B, brown = CK7) and in infiltrating lymphocytes in portal areas (C). Representative analyses of CD1d-RNA expression (see arrows) in infiltrating lymphocytes in portal areas of an AIH patient (D) and a patient with DILI (E).

Figure 10

Co-stainings of CD1d-RNA with CD4 or CD8 in liver biopsies. Representative co-stainings of CD1d-RNA (purple dots) with CD4 (brown) in 20x (A) or 40x (B) magnification are shown. Double positive cells are marked by arrows. Percentage of CD4+CD1d (left bar) or CD8+CD1d (right bar) double positive cells in portal areas of AIH patients are displayed (C).