Multicytokine-producing CD4+ T cells characterize the livers of patients with NASH
Anna Woestemeier, Pasquale Scognamiglio, Yu Zhao, Jonas Wagner, Franziska Muscate, Christian Casar, Francesco Siracusa, Filippo Cortesi, Theodora Agalioti, Simone Müller, Adrian Sagebiel, Leonie Konczalla, Ramez Wahib, Karl-Frederick Karstens, Anastasios D. Giannou, Anna Duprée, Stefan Wolter, Milagros N. Wong, Anne K. Mühlig, Agata A. Bielecka, Vikas Bansal, Tianran Zhang, Oliver Mann, Victor G. Puelles, Tobias B. Huber, Ansgar W. Lohse, Jakob R. Izbicki, Noah W. Palm, Stefan Bonn, Samuel Huber, Nicola Gagliani
Anna Woestemeier, Pasquale Scognamiglio, Yu Zhao, Jonas Wagner, Franziska Muscate, Christian Casar, Francesco Siracusa, Filippo Cortesi, Theodora Agalioti, Simone Müller, Adrian Sagebiel, Leonie Konczalla, Ramez Wahib, Karl-Frederick Karstens, Anastasios D. Giannou, Anna Duprée, Stefan Wolter, Milagros N. Wong, Anne K. Mühlig, Agata A. Bielecka, Vikas Bansal, Tianran Zhang, Oliver Mann, Victor G. Puelles, Tobias B. Huber, Ansgar W. Lohse, Jakob R. Izbicki, Noah W. Palm, Stefan Bonn, Samuel Huber, Nicola Gagliani
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Resource and Technical Advance Hepatology Immunology

Multicytokine-producing CD4+ T cells characterize the livers of patients with NASH

Abstract

A role of CD4+ T cells during the progression from nonalcoholic fatty liver disease (NAFLD) to nonalcoholic steatohepatitis (NASH) has been suggested, but which polarization state of these cells characterizes this progression and the development of fibrosis remain unclear. In addition, a gut-liver axis has been suggested to play a role in NASH, but the role of CD4+ T cells in this axis has just begun to be investigated. Combining single-cell RNA sequencing and multiple-parameter flow cytometry, we provide the first cell atlas to our knowledge focused on liver-infiltrating CD4+ T cells in patients with NAFLD and NASH, showing that NASH is characterized by a population of multicytokine-producing CD4+ T cells. Among these cells, only those with a Th17 polarization state were enriched in patients with advanced fibrosis. In parallel, we observed that Bacteroides appeared to be enriched in the intestine of NASH patients and to correlate with the frequency of multicytokine-producing CD4+ T cells. In short, we deliver a CD4+ T cell atlas of NAFLD and NASH, providing the rationale to target CD4+ T cells with a Th17 polarization state to block fibrosis development. Finally, our data offer an early indication to test whether multicytokine-producing CD4+ T cells are part of the gut-liver axis characterizing NASH.

Authors

Anna Woestemeier, Pasquale Scognamiglio, Yu Zhao, Jonas Wagner, Franziska Muscate, Christian Casar, Francesco Siracusa, Filippo Cortesi, Theodora Agalioti, Simone Müller, Adrian Sagebiel, Leonie Konczalla, Ramez Wahib, Karl-Frederick Karstens, Anastasios D. Giannou, Anna Duprée, Stefan Wolter, Milagros N. Wong, Anne K. Mühlig, Agata A. Bielecka, Vikas Bansal, Tianran Zhang, Oliver Mann, Victor G. Puelles, Tobias B. Huber, Ansgar W. Lohse, Jakob R. Izbicki, Noah W. Palm, Stefan Bonn, Samuel Huber, Nicola Gagliani

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Figure 5

IL-17A–producing CD4+ T cells were enriched in the liver of NASH patients with fibrosis.

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IL-17A–producing CD4+ T cells were enriched in the liver of NASH patient...
(A) Comparison of the FACS analysis of the frequencies of the indicated multicytokine-producing CD4+ T cells isolated from the NASH patients with and without fibrosis (same cohort as in Figure 4). Each dot represents 1 patient. Data are presented as mean ± SEM. P values were determined by Mann-Whitney U test. NS, not significant (P > 0.05). (B–D) Correlations between fibrosis score and (B) frequencies of liver IL-17A–producing CD4+ T cells obtained by FACS, (C) IL17A expression obtained form the scRNA-seq data set, and (D) COL1A1 expression obtained from real-time PCR of total liver tissue of NASH patients undergoing bariatric surgery. The COL1A1 expression is normalized to HPRT expression. P values were calculated with Pearson’s correlation coefficient. (E) Immunofluorescent staining of the livers of patients with fibrosis (patients 1, 2, and 3) and without fibrosis (control 1 and 2). αSMA in cyan, CCR6 in orange, and DRAQ5 in far red for nuclei. (F) Frequency of CCR6+ cells per field of view (FOV). Each dot represents the number of CCR6+ cells per FOV. Data are presented as mean ± SEM. P value was determined by unpaired t test with Welch’s correction. (G) Real-time PCR of ACTA2 in fibroblasts either left untreated or stimulated in vitro with TGF-β (10 ng/mL) and IL-17A (150 ng/mL) for 24 hours. Data are a pool of 2 independent experiments and each dot represents the ACTA2 expression obtained from each culture well. Data are presented as mean ± SEM. P values were determined by unpaired 1-way ANOVA test. (H) Real-time PCR of CD74 in liver tissues of NASH patients with and without fibrosis. The CD74 expression is normalized to HPRT expression. Each dot represents 1 patient. Data are presented as mean ± SEM. P value was determined by Mann-Whitney U test.