Noninvasive approach to indicate risk factors of nonalcoholic steatohepatitis overlapping autoimmune hepatitis based on peripheral lymphocyte pattern

Abstract

Background Nonalcoholic fatty liver disease (NAFLD)/nonalcoholic steatohepatitis (NASH) clinically includes autoimmunity as indicated by antinuclear antibody (ANA) positivity and overlap of autoimmune hepatitis (AIH). Discriminating AIH-overlap NASH from NAFLD/NASH is required for proper treatment, and typically involves pathological diagnosis by invasive liver biopsy. Differential patterns of peripheral lymphocytes in NAFLD and AIH were assessed to noninvasively indicate risk factors of AIH-overlap NASH by flow cytometry (FCM). Methods We assessed the differential frequencies of peripheral lymphocytes in 115 patients: 70 NASH (ANA negative:positive:AIH-overlap = 36:20:14), 18 NAFL, and 27 AIH (acute:chronic = 12:15) patients diagnosed by FCM. We focused on the following populations of lymphocytes: T cells, B cells, natural killer (NK) cells, NKT cells, helper T cell (Th) subsets (Th1, Th2, and Th17), and regulatory T cells; we also examined programmed cell death (PD) 1 and cytotoxic T-lymphocyte antigen levels. Results Several significant differences in laboratory parameters and peripheral lymphocyte frequencies were found among the NAFLD and AIH subgroups. In univariate and multivariate analyses, hyaluronic acid level, liver stiffness, and the frequencies of Th17 and CD8+ PD1+ T cells were independent risk factors of NASH in NAFLD. Regarding overlap of AIH, only the frequency of CD8+ PD1+ T cells (odds ratio, 0.01; 95% CI 0.00–38.9, p = 0.004) was an independent risk factor in NASH and significantly decreased in AIH. Conclusions The decreased frequency of peripheral CD8+ PD1+ T cells is an independent risk factor of NASH overlapping with AIH in the present cohort. Our findings will facilitate development of a new noninvasive FCM method for indicating risk factors of NASH, including autoimmunity.

Differential peripheral memory T cell subsets sensitively indicate the severity of nonalcoholic fatty liver disease

“…The PBMC samples were separated from the blood using a Ficoll kit as previously described. 9,18 This study was approved by the Ethics Committee of the University of Tokyo and carried out in accordance with the Helsinki Declaration and Japanese government policies.…”

“…Peripheral blood mononuclear cells were processed for surface staining of human peripheral lymphocytes based on an 11-color antibody cocktail as previously reported (Table S1). 18 They were processed for FCM using FACSAria (BD Biosciences). Flow cytometry data were exported and analyzed using FlowJo version 10.8.1 (Treestar).…”

“…Briefly, single and viable peripheral lymphocytes were selected as previously described. 18 Flow cytometry plots and representative gating strategies were used to identify specific lymphocytes (Figure 1). Subsequently, CD3 þ (whole T cells) were selected from peripheral lymphocytes, followed by CD4 þ CD8 − (Th) cells and CD4…”

“…We previously reported several predictors of NAFLD/NASH, including autoimmunity with peripheral lymphocyte patterns, using FCM 18 ; several patterns of peripheral memory T lymphocytes could 526 predict NAFLD severity. In this study, we aimed to elucidate the association between differential patterns of these T cell subsets and NAFLD severity and develop a new noninvasive method to sensitively determine the disease severity in addition to existing biomarkers.…”

“…In the univariate analysis, no association was observed between ballooning and age (Table 4); ballooning may be associated with immunosenescence without being influenced by aging, unlike liver FIB. Nonalcoholic steatohepatitis is diagnosed by the presence of ballooning in NAFLD according to the Matteoni classification, and peripheral CTL are closely associated with NAFLD/ NASH pathology 18,47 ; therefore, peripheral immunosenescence in CTL may also be more sensitive to ballooning in NAFLD/NASH.…”