Table_1_High L-Carnitine Levels Impede Viral Control in Chronic Hepatitis B Virus Infection.xlsx

Persistent antigen exposure during chronic hepatitis B infection leads to exhausted immune responses, thus impeding viral control. In recent years, immunometabolism opens new therapeutic possibilities for the modulation of immune responses. Herein, we investigated the immunomodulatory effect of L-carnitine (L-Cn) on immune cells in chronic HBV infection. In this study, 141 treatment-naïve patients with chronic HBV infection, 38 patients who achieved HBsAg loss following antiviral treatment, and 47 patients who suffered from HBV-related HCC from real-life clinical practice were recruited. The plasma L-Cn levels were measured by ELISA. RNA sequencing was conducted to define the transcriptional profiles of peripheral blood mononuclear cells after L-Cn stimulation. In vitro assays were performed to assess the effect of L-Cn on immune cells; the frequencies and function of immune cells were analyzed by flow cytometry. We found that compared with patients with HBsAg loss, patients with HBsAg positivity and patients who suffered from HBV-related HCC had higher levels of L-Cn, and the plasma levels of L-Cn in the HBeAg-positive chronic hepatitis patients who had elevated ALT were significantly higher than that of HBeAg-negative chronic infection and HBsAg loss groups. Moreover, a positive correlation between plasma levels of L-Cn and HBsAg levels was found. Additionally, RNA sequencing analysis demonstrated that L-Cn altered the transcriptional profiles related to immune response. In vitro assays revealed that L-Cn suppressed the proliferation of and IFN-γ production by CD4+ and CD8+ T cells. It also down-regulated the proliferation and IgG production of B cells. Notably, L-Cn enhanced IL-10 secretion from regulatory T cells and up-regulated the expression of inhibitory receptors on T cells. Moreover, a variant in CPT2 (rs1799821) was confirmed to be associated with L-Cn levels as well as complete response in CHB patients following Peg-IFNα antiviral therapy. Taken together, the immunosuppressive properties of L-Cn may hinder the control of HBV in chronic HBV infection, implicating that L-Cn manipulation might influence the prognosis of patients with HBV infection.

慢性乙型肝炎（chronic hepatitis B, CHB）感染过程中，持续的抗原暴露会引发免疫耗竭，进而阻碍病毒的有效控制。近年来，免疫代谢学（immunometabolism）为免疫应答的调控开辟了全新的治疗途径。本研究旨在探讨左旋肉碱（L-carnitine, L-Cn）对慢性乙型肝炎病毒（hepatitis B virus, HBV）感染患者免疫细胞的免疫调节作用。本研究从真实世界临床实践中招募了141例初治慢性HBV感染患者、38例经抗病毒治疗后实现乙型肝炎表面抗原（hepatitis B surface antigen, HBsAg）血清学清除的患者，以及47例HBV相关肝细胞癌（hepatocellular carcinoma, HCC）患者。采用酶联免疫吸附试验（enzyme-linked immunosorbent assay, ELISA）检测受试者血浆L-Cn水平；通过RNA测序（RNA sequencing）分析L-Cn刺激后人外周血单个核细胞（peripheral blood mononuclear cells, PBMC）的转录组特征；并开展体外实验评估L-Cn对免疫细胞的作用，通过流式细胞术（flow cytometry）分析免疫细胞的比例与功能。结果显示，与实现HBsAg清除的患者相比，HBsAg阳性患者及HBV相关HCC患者的血浆L-Cn水平更高；其中，丙氨酸氨基转移酶（alanine aminotransferase, ALT）升高的乙型肝炎e抗原（hepatitis B e-antigen, HBeAg）阳性慢性乙型肝炎患者的血浆L-Cn水平，显著高于HBeAg阴性慢性感染者与HBsAg清除组患者。此外，血浆L-Cn水平与HBsAg滴度呈正相关。进一步的RNA测序分析显示，L-Cn可调控与免疫应答相关的转录组特征。体外实验结果表明，L-Cn可抑制CD4+T细胞与CD8+T细胞的增殖及γ干扰素（interferon-γ, IFN-γ）的分泌，同时下调B细胞的增殖与免疫球蛋白G（immunoglobulin G, IgG）的产生。值得注意的是，L-Cn可促进调节性T细胞（regulatory T cells, Treg）分泌白细胞介素10（interleukin-10, IL-10），并上调T细胞表面抑制性受体的表达。此外，肉碱棕榈酰转移酶2（carnitine palmitoyltransferase 2, CPT2）位点rs1799821的基因变异，与血浆L-Cn水平及聚乙二醇干扰素α（peginterferon α, Peg-IFNα）抗病毒治疗后慢性乙型肝炎患者的完全应答显著相关。综上，L-Cn的免疫抑制特性可能会阻碍慢性HBV感染患者的病毒控制，提示靶向调控L-Cn或可改善HBV感染患者的预后。