Exploring The Pathogenic Mechanisms Of Plasmacytoid Dendritic Cells In IgG4-related Disease Based On High-throughput Sequencing

BackgroundThe IgG4-related disease(IgG4-RD)is an immune-mediated disorder with fibrotic manifestations.However,the transcriptional profiles of immune cell subsets at the single-cell level are unknown.Herein,single-cell sequencing was used to assess the specific cell subpopulations and pathways in peripheral blood mononuclear cells(PBMCs)of IgG4-RD.MethodsSingle-cell sequencing was performed using the PBMCs from four patients with IgG4RD and three healthy controls(HCs).Functional enrichment and cell analysis were performed through re-clustering of PBMCs to assess functional pathways and intercellular communication networks in IgG4-RD.Western blot and flow cytometry were used to verify sequencing and functional enrichment results.ResultsFour major cell types and 21 subtypes were identified.Further sub-clustering demonstrated that plasma B-cell proportions increased with increasing glycolysis/gluconeogenesis activity in IgG4-RD.Re-clustering of myeloid cells showed that CD14+monocytes(CD 14+Mono)were significantly increased in IgG4-RD patients and dendritic cells(DC)were significantly decreased.EGR1 and CD36 expressions were significantly increased in CD14+monocytes of IgG4-RD,as validated by Western blot analysis.Moreover,tumor necrosis factor(TNF)production pathways were positively regulated in CD 14+monocytes of IgG4-RD.In vitro stimulation showed that CD 14+monocytes of IgG4-RD could secrete higher levels of TNF-a.Notably,the proportions of CD8 central memory T(TCM)and TIGIT+CD8 cytotoxic T(CTL)increased in patients with IgG4-RD compared with HCs.Further interaction analysis showed that B cell activation factor(BAFF)signaling pathways were enriched from myeloid cell(CD 14+Mono and dendritic cells)subsets to B cells.ConclusionThis study enhances the understanding of the cellular heterogeneity and transcriptional features involved in the pathogenesis of IgG4-RD,myeloid cells(CD 14+Mono and DC),B lymphocytes,and their interactions playing a crucial role in the pathogenesis of IgG4-RD.This study provides key clinical implications.BackgroundTo further validate the immunophenotype and function of plasmacytoid dendritic cells(pDCs)in patients with IgG4-related disease(IgG4-RD)based on single-cell sequencing.MethodsFlow cytometry was used to detect the percentage of plasmacytoid dendritic cells in the peripheral blood mononuclear cells and the fluorescence intensity of their surface activation markers and functional molecules in IgG4-RD patients and matched healthy controls.Immunohistochemistry and immunofluorescence were used to detect the infiltration of pDC and co-localization with other lymphocytes in the affected tissues of IgG4-RD patients and disease controls,respectively.Apoptosis Detection Kit was used to detect the level of apoptosis of pDC in direct isolation and after plasma stimulation from IgG4-RD patients and healthy controls.Plasmacytoid dendritic cells were co-cultured with B cells and Naive T cells in vitro to verify the effect of pDC on B and Na?ve T lymphocyte activation,proliferation,and differentiation.ResultsThe percentage of plasmacytoid dendritic cells was significantly decreased in the peripheral blood mononuclear cells of IgG4-RD patients(IgG4-RD 0.14±0.13%vs HC0.30±0.20%),which rebounded significantly in IgG4-RD patients after treatment to achieve remission.And there was a significant negative correlation between the decreased level of pDCs and patients’ inflammatory status and disease activity.Apoptosis of pDCs in peripheral blood of IgG4-RD patients was not significantly different compared to healthy controls but showed abnormal activation with significantly enhanced mean fluorescence intensity of HLA-DR(35332.07±10710.27 vs 27909.80±9600.15),CD86(582.28±173.11 vs 478.93±108.08),and TLR7(16153.14±6242.71 vs 9482.33±906.04).The higher expression of CCR5 on the surface of IgG4-RD peripheral blood pDC and the significant increase of pDC cell infiltration in the affected tissues of IgG4-RD patients suggested that peripheral blood pDC might infiltrate into the affected tissues through chemotaxis.In addition,immunofluorescence co-localization suggested the presence of pDC in the affected tissues of IgG4-RD patients in an adjacent location to CD 19+B lymphocytes as well as CD4+T lymphocytes.Further co-culture experiments confirmed that activated pDC of IgG4RD promoted the activation,proliferation,and differentiation of B cells to plasma cells and the production of IgG and IgG4 antibodies more significantly than healthy controls;they also promoted the proliferation of T lymphocytes and their differentiation to Treg cells.ConclusionThe abnormal activation of peripheral blood pDCs in IgG4-RD patients and their ability to chemotactic to the affected tissues may promote the development of tissue fibrosis by interacting with lymphocytes,which is a possible therapeutic target for the disease.