Increased Serum IgG4 Associates with Asthma and Tissue Eosinophilia in Chronic Rhinosinusitis Patients

Chronic Rhinosinusitis (CRS) is a multifactorial disease where microorganisms’ innate and adaptive immunity can play a role. This study assessed the total IgG, IgG subclasses, IgE and IgA levels in serum samples from CRS and non-CRS control patients in relation to the disease severity, phenotype, histopathology and comorbidities. Total serum IgG, IgG1, IgG2, IgG3, IgG4 and IgE was determined from 10 non-CRS controls, 10 CRS without nasal polyp (CRSsNP) and 26 CRS with nasal polyp (CRSwNP) patients using ImmunoCap assays. Tissue lysates were analyzed for IgG levels by ELISA. Immunohistochemical analysis was used to measure the expression of IgE and IgG4 in tissue sections. The presence of anti-nuclear antigens (ANAs) against 12 autoantigens in sera and tissue lysates was determined by immunoblot assays. Total serum IgG/IgG1/IgG2 levels were higher in CRS patients vs. controls (p < 0.001), but were not different between CRSwNP and CRSsNP patients (p = 0.57). Serum IgG4/IgE levels were increased in CRSwNP patients compared to controls (p = 0.006), however, this relationship was attenuated by the inclusion of covariates. Serum IgG4 levels were more strongly associated with asthma (p = 0.038, exact median test) and tissue eosinophilia (Spearman’s rank rho = 0.51, p = 0.016) than IgE levels. No systemic ANAs were detected in any of the subjects tested. There was a polyclonal increase in serum immunoglobulins in CRS patients with elevated IgG4/IgE levels in CRSwNP patients having tissue eosinophilia and asthma.


Preparation of Protein Extract from Tissues
Freshly obtained nasal polyps and mucosal tissues were snap-frozen and stored at −80 °C until needed. Tissues were thawed on ice. Approximately 100 mg of tissue was suspended in 1 mL of Tper tissue protein extraction buffer (product no. 78510, Thermo Fisher Scientific, MA, USA) containing 1% v/v HALT protease inhibitor cocktail (product no. 87786, Thermo Fisher Scientific, MA, US). The samples were then homogenized with Qiagen Tissuelyser (product no. 85220, Qiagen, Hilden, Germany) at 30 hertz for 20 min. The homogenized suspensions were centrifuged at 17,000 g for 10 min at 40 °C and the supernatants were stored at −80 °C until analysis. The protein concentrations of tissue extracts were determined using the Pierce BCA Protein Assay Kit (product no. 23225, Thermo Fisher Scientific, MA, USA).

Histology
Paraffin-embedded tissue samples were cut in 4µ m thickness, stained with Haematoxylin & Eosin (H&E) and scanned using digital whole-slide imaging (WSI) technology (NanoZoomer, Hamamatsu, Japan). Tissue eosinophilia was determined by averaging the number of eosinophils per High Power Field (HPF) (0.035 mm 3 ) from at least 6 HPF's/slide as specified by Ramezanpour et al [3].

Immunohistochemistry Analysis
Slides were deparaffinized and rehydrated. Antigen retrieval was induced at 100 ºC for 10 min in 10 mmol/L sodium citrate buffer, pH 6. Slides were then blocked in 25% normal horse serum blocking buffer for 10 min and incubated with primary antibodies to IgG4 (mouse antihuman IgG4 [A10651] at 1:200 dilution; life technology, OR, USA) or to IgE (rabbit monoclonal [ab195580] at1µ g/ml; Abcam, Cambridge, United Kingdom) overnight at 4 ºC.
Specific binding was detected with the Vectastain Universal Quick kit #PK-7800 and DAB Substrate kit #SK-4100 (Vector Laboratories, Burlingame, CA, USA). Slides were observed with a Nikon Eclipse 90I microscope equipped with NIS-Elements AR3.2 software.

Detection of Autoantibodies
The presence of autoantibodies in serum was tested against 12 Anti-Nuclear Antigen (ANA)associated autoantigens (nRNP, Sm, SS-A (SS-A native and Ro-52), SS-B, Scl-70, Jo-1, CENP B, dsDNA, Nucleosomes, Histones and ribosomal P-Protein) were tested using Euroimmun ANA Profile I Immunoblot kits (catalogue no. DL 1590-6401-8G, Euroimmun, Lübeck, Germany). The tests were performed as per the manufacturer's manual. The blots were evaluated by the EUROLineScan (Euroimmun, Lübeck, Germany) for scanning and analysis. Background cut-off value was ≤ 10 intensity unit. 3 serum samples known to possess ANAs were used as positive controls.

Statistical Analysis and Principal Component Factor Analysis of Serum Immunoglobulin
Principal component factor analysis was performed to both interpret the relationships between serum immunoglobulins and to simplify the analysis of between-group differences. With the exception of total IgG, none of the immunoglobulin measurements were normally distributed, therefore, normalising transformations were applied based on the Tukey ladder of powers. Log transformations were selected for IgE and IgG4, and square root transformations were selected for IgG1, IgG2 and IgG3. Transformed variables were then standardised before principal component factor analysis. Analysis was performed in Stat v15.1 (StataCorp LLC, TX, USA).