Iguratimod represses B cell terminal differentiation linked with the inhibition of PKC/EGR1 axis

Yan Ye,Chunde Bao,Qingran Yan,Mei Liu,Qiong Fu,Longhai Tang,Qiang Guo,Fang Du,Pei Hao,Yuanhua Liu,Xiaoming Zhang

doi:10.1186/s13075-019-1874-2

Abstract

BackgroundThis study aimed to explore the molecular mechanism and clinical relevance of iguratimod in the regulation of human B cell terminal differentiation.MethodsAn in vitro human antibody-secreting cell (ASC) differentiation system was established to test the effect of iguratimod. B cell phenotype and key transcription factors (TFs) relevant to ASC differentiation were analyzed through flow cytometry and qPCR. The COX-2 activity was measured by enzyme immunoassay (EIA). RNA sequencing was used to identify potential targets of iguratimod. We enrolled six treatment-naive rheumatoid arthritis (RA) patients whose blood samples were collected for phenotypic and molecular studies along with 12-week iguratimod monotherapy.ResultsIguratimod inhibited human ASC generation without affecting B cell activation and proliferation. Iguratimod showed only weak COX-2 activity. Gene set enrichment analysis (GSEA) identified that protein kinase C (PKC) pathway was targeted by iguratimod which was confirmed by PKC activity detection. Furthermore, early growth response 1 (EGR1), a target of PKC and a non-redundant TF for ASC differentiation, was found to be the most downregulated gene in iguratimod-treated B cells. Lastly, iguratimod monotherapy decreased peripheral ASCs and was associated with improved disease activity. The expression of major ASC-related TFs, including EGR1, was similarly downregulated in patient blood samples.ConclusionsIguratimod inhibits ASC differentiation both in vitro and in RA patients. Our study suggests that PKC/EGR1 axis, rather than COX-2, is critically involved in the inhibitory effect by iguratimod on human ASC differentiation. Iguratimod could have a broader application to treat B cell-related autoimmune diseases in clinics.

Highlights

This study aimed to explore the molecular mechanism and clinical relevance of iguratimod in the regulation of human B cell terminal differentiation
We have identified that a combination of CpG2006, IL-2, and IL-10 resulted in the highest antibody-secreting cell (ASC) yield after 5-day culture (Additional file 1: Figure S1)
We found that iguratimod at 3 μM inhibited CD27hiCD38hi ASC generation and this effect is obvious at day 5 following CpG/IL-2/IL-10 stimulation (Fig. 1a, b)

Summary

Introduction

This study aimed to explore the molecular mechanism and clinical relevance of iguratimod in the regulation of human B cell terminal differentiation. B cell-targeting therapies have attracted great attentions in recent years. B cell depletion by rituximab has succeeded in trials on rheumatoid arthritis (RA) [3,4,5]; its effects on SLE were variable [6, 7]. Other trials to target B cell-activating factor (BAFF) by belimumab and atacicept have been performed on SLE, while only belimumab has achieved positive endpoints [8, 9]. Attempts to arrest B cell activation such as by Bruton tyrosine kinase (Btk) inhibitor ibrutinib are still at a pre-clinical stage [10]. Targeting plasma cells by proteasome inhibitor bortezomib [11, 12] represents a new strategy to

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