Allergy Asthma Immunol Res.  2018 Nov;10(6):698-715. 10.4168/aair.2018.10.6.698.

Differential Hrd1 Expression and B-Cell Accumulation in Eosinophilic and Non-eosinophilic Chronic Rhinosinusitis With Nasal Polyps

• Chen K
• Han M
• Tang M
• Xie Y
• Lai Y
• Hu X
• Zhang J
• Yang J
• Li H

• KMID: 2441818
• DOI: http://doi.org/10.4168/aair.2018.10.6.698

Abstract

PURPOSE
Hrd1 has recently emerged as a critical regulator of B-cells in autoimmune diseases. However, its role in the pathogenesis of chronic rhinosinusitis with nasal polyps (CRSwNP) remains largely unexplored. This study aimed to examine Hrd1 expression and B-cell accumulation and their possible roles in CRSwNP.
METHODS
Quantitative real-time polymerase chain reaction, immunohistochemistry, enzyme-linked immunosorbent assay and Western blotting were used to assess gene and protein expression in nasal tissue extracts. Cells isolated from nasal tissues and peripheral blood mononuclear cells were characterized by flow cytometry. Local antibody production was measured in tissue extracts with a Bio-Plex assay. Additionally, changes in Hrd1 expression in response to specific inflammatory stimuli were measured in cultured dispersed polyp cells.
RESULTS
Nasal polyps (NPs) from patients with eosinophilic CRSwNP (ECRS) had increased levels of Hrd1, B-cells and plasma cells compared with NPs from patients with non-eosinophilic CRSwNP (non-ECRS) or other control subjects (P < 0.05). The average Hrd1 levels in B-cells in NPs from ECRS patients were significantly higher than those from non-ECRS patients and control subjects (P < 0.05). NPs also contained significantly increased levels of several antibody isotypes compared with normal controls (P < 0.05). Interestingly, Hrd1 expression in cultured polyp cells from ECRS patients, but not non-ECRS patients, was significantly increased by interleukin-1β, lipopolysaccharide and Poly(I:C) stimulation, and inhibited by dexamethasone treatment (P < 0.05).
CONCLUSIONS
Differential Hrd1 expression and B-cell accumulation between the ECRS and non-ECRS subsets suggests that they can exhibit distinct pathogenic mechanisms and play important roles in NP.

Keyword

Hrd1; chronic rhinosinusitis; B-cells; nasal polyps; innate immunity

MeSH Terms

Antibody Formation
Autoimmune Diseases
B-Lymphocytes*
Blotting, Western
Dexamethasone
Enzyme-Linked Immunosorbent Assay
Eosinophils*
Flow Cytometry
Humans
Immunity, Innate
Immunohistochemistry
Nasal Polyps*
Plasma Cells
Polyps
Real-Time Polymerase Chain Reaction
Tissue Extracts
Dexamethasone
Tissue Extracts

Figure

• 

  Fig. 1 Hrd1 mRNA expression in nasal tissues. Hrd1 (A), CD19 (B), CD20 (C), CD138 (D) and BAFF (E) mRNA levels were significantly enhanced in polyp tissues from patients with ECRS compared with values seen in the other subgroups. Results are expressed as medians (interquartile ranges). BAFF, B-cell activating factor; ECRS, eosinophilic chronic rhinosinusitis with nasal polyp; non-ECRS, non-eosinophilic chronic rhinosinusitis with nasal polyp; CRSsNP, chronic rhinosinusitis without nasal polyps.

• 

  Fig. 2 Increased Hrd1 protein levels and accumulation of B cells and plasma cells in NPs. Representative IHC staining of Hrd1 (A), CD19 (C), CD20 (E), and CD138 (G) (magnification, × 400) in nasal tissues. Relative positive cells of Hrd1 (B), CD19 (D), CD20 (F), and CD138 (H) were significantly increased in polyp tissues from patients with ECRS relative to those in other subgroups. Representative Western blotting results (I), densitometric analysis (J). Positive correlation between relative Hrd1 protein levels in the polyp tissues and Lund-Mackay CT scores of the matched patients (K). ELISA of BAFF in nasal tissues (L). Positive correlation between BAFF and Hrd1 protein levels in the matched NPs (M). Bars = 20 μm. NP, nasal polyp; ECRS, eosinophilic chronic rhinosinusitis with nasal polyp; non-ECRS, non-eosinophilic chronic rhinosinusitis with nasal polyp; CRSsNP, chronic rhinosinusitis without nasal polyps; HPF, high-power field; CT, computed tomography; ELISA, enzyme-linked immunosorbent assay; BAFF, B-cell activating factor.

• 

  Fig. 3 Enhanced Hrd1 expression in B-cells from polyp tissues and matched PBMCs. Gating strategy and representative flow plots for nasal tissues (A). MFI levels of Hrd1 in CD19+ B-cells were significantly increased in polyp tissues from patients with ECRS compared with those in in polyps from patients with non-ECRS, middle turbinates from control subjects (B). Gating strategy and representative flow plots for PBMCs (C). MFI levels of Hrd1 in CD19+ B-cells were significantly increased in PBMCs from patients with CRSwNP compared with those in PBMCs from healthy controls (D). PBMC, peripheral blood mononuclear cell; MFI, mean fluorescence intensity; ECRS, eosinophilic chronic rhinosinusitis with nasal polyp; non-ECRS, non-eosinophilic chronic rhinosinusitis with nasal polyp; CRSwNP, chronic rhinosinusitis with nasal polyps.

• 

  Fig. 4 Increased local immunoglobulin levels in polyp tissues. Protein levels of IgA, IgE, IgM, IgG and IgG subclasses in tissue homogenates from ECRS, non-ECRS, CRSsNP, and control tissue are detected by using the Bio-Plex assay. Positive correlations are found between relative Hrd1 protein levels and IgM, IgG, IgG1, and IgG2. Ig, immunoglobulin; ECRS, eosinophilic chronic rhinosinusitis with nasal polyp; non-ECRS, non-eosinophilic chronic rhinosinusitis with nasal polyp; CRSsNP, chronic rhinosinusitis without nasal polyps.

• 

  Fig. 5 Hrd1 mRNA and protein levels in cultured DPCs in response to IL-1β, LPS, Poly(I:C) and Dex. mRNA expression and representative western blot results of DPCs lysates from ECRS patients after 20-hour stimulation (A-C). mRNA expression and representative western blot results of DPCs lysates from non-ECRS patients after 20-hour stimulation (D-F). Results represent mean values from 3 independent experiments. Data are expressed as means (standard error of the means). DPC, dispersed polyp cell; IL, interleukin; LPS, lipopolysaccharide; Poly(I:C), polyinosinic-polycytidylic acid; Dex, dexamethasone; ECRS, eosinophilic chronic rhinosinusitis with nasal polyp;non-ECRS, non-eosinophilic chronic rhinosinusitis with nasal polyp. *P < 0.05; †P < 0.01; ‡P < 0.001.

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