Smoking enhances proliferation, inflammatory markers, and immunoglobulins in peripheral blood mononuclear cells from Graves’ patients

in Endocrine Connections
Authors:
Bushra Shahida Department of Clinical Sciences, Genomics, Diabetes and Endocrinology, Lund University, Malmö, Sweden
Department of Diabetes & Endocrinology, Skåne University Hospital, Malmö, Sweden

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Tereza Planck Department of Clinical Sciences, Genomics, Diabetes and Endocrinology, Lund University, Malmö, Sweden
Department of Diabetes & Endocrinology, Skåne University Hospital, Malmö, Sweden

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Tania Singh Department of Clinical Sciences, Genomics, Diabetes and Endocrinology, Lund University, Malmö, Sweden

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Peter Åsman Department of Clinical Sciences Malmö, Ophthalmology, Lund University, Malmö, Sweden
Department of Ophthalmology, Skåne University Hospital, Malmö, Sweden

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Mikael Lantz Department of Clinical Sciences, Genomics, Diabetes and Endocrinology, Lund University, Malmö, Sweden
Department of Diabetes & Endocrinology, Skåne University Hospital, Malmö, Sweden

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Correspondence should be addressed to B Shahida: Bushra.shahida@med.lu.se
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Graves’ disease (GD) and Graves’ ophthalmopathy (GO) are complex autoimmune diseases. This study delved into the impact of cigarette smoke extract (CSE), simvastatin, and/or diclofenac on peripheral blood mononuclear cells (PBMCs). Specifically, we explored alterations in IL-1B, IL-6, PTGS2 expression, B- and T-lymphocyte proliferation, and Immunoglobulin G (IgG) production. We also assessed IGF1’s influence on B- and T-lymphocyte proliferation. PBMCs from Graves’ patients were exposed to CSE with/without simvastatin and/or diclofenac. Gene and protein expression was compared with untreated PBMCs. B- and T-lymphocyte proliferation was assessed following IGF1 treatment. PBMCs exposed to CSE exhibited increased expression of IL-1B (6-fold), IL-6 (10-fold), and PTGS2 (5.6-fold), and protein levels of IL-1B (4-fold), IL-6 (16-fold) and PGE2 (3.7-fold) compared with untreated PBMCs. Simvastatin and/or diclofenac downregulated the expression of PTGS2 (0.5-fold), IL-6 (0.4-fold), and IL-1B (0.6-fold), and the protein levels of IL-1B (0.6-fold), IL-6 (0.6-fold), and PGE2 (0.6-fold) compared with untreated PBMCs. CSE exposure in PBMCs increased the proliferation of B and T lymphocytes by 1.3-fold and 1.4-fold, respectively, compared with untreated. CSE exposure increased IgG (1.5-fold) in supernatant from PBMCs isolated from Graves’ patients. IGF1 treatment increased the proliferation of B and T lymphocytes by 1.6-fold. Simvastatin downregulated the proliferation of B and T lymphocytes by 0.7-fold. Our study shows that CSE significantly upregulated the expression and release of the inflammatory markers PTGS2, IL-6 and IL-1B,the IgG levels, and the proliferation of B and T lymphocytes. Additionally, IGF1 increased the proliferation of B and T lymphocytes. Finally, these effects were decreased by diclofenac and/or simvastatin treatment.

Abstract

Graves’ disease (GD) and Graves’ ophthalmopathy (GO) are complex autoimmune diseases. This study delved into the impact of cigarette smoke extract (CSE), simvastatin, and/or diclofenac on peripheral blood mononuclear cells (PBMCs). Specifically, we explored alterations in IL-1B, IL-6, PTGS2 expression, B- and T-lymphocyte proliferation, and Immunoglobulin G (IgG) production. We also assessed IGF1’s influence on B- and T-lymphocyte proliferation. PBMCs from Graves’ patients were exposed to CSE with/without simvastatin and/or diclofenac. Gene and protein expression was compared with untreated PBMCs. B- and T-lymphocyte proliferation was assessed following IGF1 treatment. PBMCs exposed to CSE exhibited increased expression of IL-1B (6-fold), IL-6 (10-fold), and PTGS2 (5.6-fold), and protein levels of IL-1B (4-fold), IL-6 (16-fold) and PGE2 (3.7-fold) compared with untreated PBMCs. Simvastatin and/or diclofenac downregulated the expression of PTGS2 (0.5-fold), IL-6 (0.4-fold), and IL-1B (0.6-fold), and the protein levels of IL-1B (0.6-fold), IL-6 (0.6-fold), and PGE2 (0.6-fold) compared with untreated PBMCs. CSE exposure in PBMCs increased the proliferation of B and T lymphocytes by 1.3-fold and 1.4-fold, respectively, compared with untreated. CSE exposure increased IgG (1.5-fold) in supernatant from PBMCs isolated from Graves’ patients. IGF1 treatment increased the proliferation of B and T lymphocytes by 1.6-fold. Simvastatin downregulated the proliferation of B and T lymphocytes by 0.7-fold. Our study shows that CSE significantly upregulated the expression and release of the inflammatory markers PTGS2, IL-6 and IL-1B,the IgG levels, and the proliferation of B and T lymphocytes. Additionally, IGF1 increased the proliferation of B and T lymphocytes. Finally, these effects were decreased by diclofenac and/or simvastatin treatment.

Introduction

Graves’ disease (GD) and Graves’ ophthalmopathy (GO) are both complex autoimmune diseases that are caused by an interplay of environmental factors and genetic susceptibility (1). Risk factors such as smoking, emotional stress, and dietary iodine are associated with the development of GD (2). GD stands as a multifaceted autoimmune disorder, with thyroid gland inflammation marked by the infiltration of mononuclear cells. This infiltration notably comprises T lymphocytes and antigen-presenting cells including dendritic cells, monocytes, and B lymphocytes. Upon encountering antigen-presenting cells, T lymphocytes undergo proliferation, differentiating into distinct phenotypes. While some evolve into effector T lymphocytes, others assume the role of regulatory T lymphocytes, capable of mitigating immune reactivity (2). The autoimmune response observed in GD originates from the presence of circulating immunoglobulin G (IgG) antibodies targeting the thyroid-stimulating hormone receptor (TSHR), commonly referred to as TRAb. These antibodies, generated by B-cell clones, prompt the production of thyroid hormones. TRAb specifically binds to TSHR, initiating the activation of the G-protein-coupled receptor (GPCR) pathway, and subsequently activating adenylate cyclase. This activation leads to the generation of cyclic adenosine monophosphate (cAMP). Elevated levels of cAMP induce the proliferation of thyrocytes, thereby fostering thyroid growth and the secretion of thyroid hormones T3 and T4 (3).

Not only more than 90% of GD patients exhibit morphological changes in the retrobulbar space at the time of diagnosis but only one-third of the patients develop clinical ophthalmopathy. Smoking is the strongest risk factor for developing GO (4, 5, 6). GO is a complex autoimmune disease characterized by inflammation in orbital tissue and increased adipogenesis (7). Inflammation of the orbital tissue includes elevated synthesis of hyaluronic acid by orbital fibroblasts, which can differentiate into adipocytes or myofibroblasts and contribute to tissue expansion (8). Fibroblasts and adipocytes are activated by the TSHR, which is expressed on preadipocytes and orbital fibroblasts (9). It is thought that the activation of TSHR induces cross-talk with insulin-like growth factor 1 receptor (IGF1R) (10), which results in elevated levels of hyaluronic acid, leading to the disruption of the extraocular muscles (11). T cells bind to CD40 on orbital fibroblasts and induce further T-cell infiltration. Orbital fibroblasts are activated by CD40 ligation, which induces the production of proinflammatory cytokines and prostaglandin E2 (PGE2). TSHR autoantibodies are then produced by B cells, which also interact with CD4+ T cells (12).

We have previously shown that prostaglandin-endoperoxide synthase 2 (COX-2/PTGS2), which is an immediate early gene (IEG) and is expressed in the first 30–60 min of adipogenesis in response to a mitogen (13), was overexpressed in intraorbital adipose/connective tissues in GO patients with optic nerve dysfunction compared to that in healthy controls (14) and in smokers with active, severe GO compared to nonsmokers (13). In another study, we showed that cigarette smoke extract (CSE) induced the expression of the inflammatory markers PTGS2, IL-1B, and IL-6 in orbital fibroblasts from patients with GO. Furthermore, the expression of these genes and adipogenesis of orbital fibroblasts was decreased by the treatment with simvastatin (4).

Based on our previous results, we have now investigated the effect of CSE on peripheral mononuclear blood cells (PBMCs) from patients with GD and whether this effect can be modulated by simvastatin and/or diclofenac treatment. Furthermore, the effects of simvastatin on the proliferation of PBMCs were examined.

Materials and methods

Blood samples were obtained from 12 newly diagnosed GD patients (Table 1) at the Endocrinology Clinic, Skånes University Hospital, Malmo. Ten Thyroid healthy control samples (Table 1) were obtained from personnel at Endocrinology Clinic, Skånes University Hospital, Malmo. The inclusion criteria for the subjects in the GD group were that they were newly diagnosed and, consequently, had not undergone any treatment for GD or were treated with any immunosuppressive medication, which also applied to the control subjects. Furthermore, the control subjects were selected based on the absence of any autoimmune diseases. Written informed consent was acquired from all patients and the project was approved by the Swedish Ethical Committee.

Table 1

Clinical data of controls and newly diagnosed Graves’ disease patients.

Patients, n (%) Controls, n (%)
n 12 (100) 10 (100)
Female 7 (58) 6 (60)
Male 5 (42) 4 (40)
Age 45 ± 18 41 ± 9
GD treatment No No
Immune suppressive drugs No No
Other autoimmune disease No No
Smokers 4 (33) 3 (30)

Heparinized blood was obtained, and PBMCs were isolated using density centrifugation by Ficoll-Paque Plus (VWR/GE Healthcare, Stockholm, Sweden). The PBMCs isolated from whole blood were washed twice in RPMI 1640 medium and resuspended in complete RPMI 1640 culture medium (ATCC modification) containing 2 mM L-glutamine, 10 mM HEPES, 1 mM sodium pyruvate, 25 mM D-glucose, 17 mM sodium bicarbonate, 10% heat-inactivated fetal bovine serum (Gibco), and 1% Pen-Strep (Gibco).

Activation of simvastatin

Simvastatin (Sigma-Aldrich) was activated as previously described (2). Briefly, 25 mg simvastatin was dissolved in 0.2 mL ethanol (95–100%) followed by the addition of 0.3 mL NaOH. After heating at 50°C for 2 h, the solution was neutralized with 1 N HCl to pH 7.2 and adjusted to a 1 mL volume with normal saline.

Cigarette smoke extract

CSE was produced as described by Cawood et al. and Shahida et al. (4, 15) with a validated peristaltic pump system. Four cigarettes were smoked through 30 mL RPMI 1640 medium. Each cigarette was smoked for 10 puffs, each puff involving 35 mL smoke, every 30 s, which used approximately 75% of the cigarette. The media was sterile filtered, and the density of the media was measured at 450 nm.

Cytotoxicity assay

Cell viability was investigated using a Cell Proliferation Kit I (MTT assay, Sigma). PBMCs were treated with phytohemagglutinin (PHA) and exposed to 5% or 10% CSE for 1 h, 3 h, and 24 h, 3 days, 4 days, and 5 days in the presence or absence of diclofenac and/or simvastatin (5 µM or 10 µM). Then, cell viability was tested using the MTT assay. The absorbance of the solution was measured at 490 nm.

Gene expression studies

PBMCs were counted and plated at a density of 2 × 105 in a 96-well plate and treated with 1.5% PHA (Thermo Fisher), 10% CSE with or without 10 µM simvastatin and/or 10 µM diclofenac, followed by incubation for 1, 2, 3, 4, 24, and 72 h (37°C and 5% CO2). PBMCs were cultured in triplicates for each treatment at every time point. RNA was isolated using the RNeasy Plus Mini Kit (Qiagen) according to the manufacturer’s instructions, and the purity and concentration were determined spectrophotometrically using a NanoDrop ND-1000. The integrity of the RNA was assessed utilizing Agilent 2100 Bioanalyzer (Agilent Technologies, Inc. 2016) (details are given in Supplementary S1, see section on supplementary materials given at the end of this article). In total, 0.2 µg of total RNA was reverse transcribed using a QuantiTect Reverse Transcription Kit (Qiagen) according to the manufacturer’s instructions. RT-PCR was performed using the QuantStudio 7 Flex sequence detection system (Applied Biosystems) according to the manufacturer’s instructions, employing TaqMan mRNA Expression Master Mix (Applied Biosystems). The following assays (Applied Biosystems) were used to assess the mRNA expression in human PBMCs: PTGS2/Hs00153133_m1/, IL-6/Hs00174131_m1/, and IL-1B/Hs01555410_m1/. PHA activates T lymphocytes and was used as a positive control. All the samples were run in duplicate, and the data were analyzed with the standard curve method using cyclophilin A as an endogenous control. The results were confirmed in at least three biological replicates.

Enzyme-linked immunosorbent assay

Cell culture supernatants were harvested, and the protein levels of PGE2, IL-1B, IL-6, and IgG were analyzed with commercially available ELISA kits (R & D Systems) according to the manufacturer’s guidelines.

Cell culture for flow cytometry

PBMCs were stained with CellTrace™ CFSE (Thermo Fisher), plated at a density of 2 × 105 cells in 96-well plates, treated with the T-lymphocyte activator PHA (1.5%; Thermo Fisher), recombinant human CD40-Ligand, Cross-Linking Antibody, IL-4 (B-cell expansion kit, Miltenyi, Stockholm, Sweden), IGF1 or 10 µM simvastatin and/or 10 µM diclofenac (Sigma-Aldrich), with or without 10% CSE, and incubated for 72 h. After 72 h of incubation, IL-2 (5 U/mL, Miltenyi Biotec, Lund, Sweden) was added to the wells and incubated for an additional 48 hat 37°C in 5% CO2. PBMCs were harvested and resuspended in 1× PBS with 0.5% bovine serum albumin (BSA) (Sigma-Aldrich) and 2 mM EDTA followed by staining with fixable viability dye (FVD eFluor 780, APC-750) and the following monoclonal antibodies (all from Miltenyi): CD3 (PE-Vio 770, PC7), CD14 (VioBlue, BV421), and CD19 (APC).

PHA and B-cell expansion kits were used together as a proliferation cocktail (PC).

7-AAD (7-amino-actinomycin D) was used to exclude nonviable cells. Cell analysis was carried out on CytoFLEX (Beckman Coulter; FACS facility center Clinical Research Center, Malmö, Sweden). Gates were set on viable lymphocytes according to forward (FSC) and side scatter (SSC). Listmode data were generated using CytExpert acquisition software on 50,000 events. For viability assays, all the lymphocytes were gated to assess the number of nonviable 7-AAD-positive cells. For proliferation, nonviable 7AAD cells were excluded. Proliferation index = total number of divisions divided by the number of cells that initiated division calculated using FlowJo™ Version v10.7.

Statistical analysis

For all continuous variables among multiple groups, the analysis of variance test (one-way ANOVA) was used. When the overall test was found to be significant, pairwise comparisons using Tukey’s multiple comparisons test were applied to control the familywise error rate.

The data were analyzed using GraphPad Prism Software 9.0.

Results

Expression of PTGS2 in PBMCs after exposure to CSE and the effects of simvastatin and/or diclofenac on PTGS2, IL-6, and IL-1B expression

After 3 h of treatment, CSE and PHA increased the expression of PTGS2 in the PBMCs from both healthy control subjects (Fig. 1A) and GD patients (Fig. 1B) by 1.8-fold and 1.5-fold (P ≤ 0.001), respectively, compared to that in PHA-treated PBMCs. CSE exposure alone for 3 h increased the expression of PTGS2 similar to PHA in GD patients (Fig. 1B). The expression of PTGS2 at 3 h was increased in PBMCs from healthy control subjects and GD patients (Fig. 1) exposed to CSE alone compared to untreated PBMCs, with fold changes of 3.3 and 5.6, respectively (P ≤ 0.001). Furthermore, we investigated the effects of simvastatin and/or diclofenac on PTGS2 expression in PBMCs after 72 h of CSE exposure by measuring mRNA levels (Fig. 2); it was shown that CSE exposure increased the expression of PTGS2 in both healthy control subjects and GD patients by 13-fold (P ≤ 0.0001) (Fig. 2A) and 7-fold (P ≤ 0.0001) (Fig. 2B), respectively, when compared to control treatment. Moreover, compared to PHA treatment alone, combined treatment with PHA and CSE further elevated the mRNA expression of PTGS2 in healthy control subjects and GD patients by 1.3-fold (P ≤ 0.05) and 1.2-fold (P ≤ 0.05), respectively (Fig. 2), and treatment with either simvastatin or diclofenac downregulated PTGS2 expression by 0.4-fold (P ≤ 0.0001) and 0.8-fold (P ≤ 0.05), respectively, in healthy control subjects (Fig. 2A) and 0.5-fold (P ≤ 0.0001) and 0.7-fold (P ≤ 0.001), respectively, in GD patients (Fig. 2B). Furthermore, the expression of PTGS2 decreased by 0.3-fold (P ≤ 0.0001) (Fig. 2) when PBMCs from controls and GD patients were treated with diclofenac, simvastatin, and PHA and exposed to CSE compared to treatment with PHA and exposure to CSE.

Figure 1
Figure 1

The effect of cigarette smoke extract on PBMCs isolated from healthy control subjects and Graves’ disease patients. PBMCs were isolated from healthy control subjects (A) and Graves’ disease patients (B) followed by treatment with 10% cigarette smoke extract (CSE) and/or phytohemagglutinin (PHA). PBMCs were harvested after treatment for 1 h, 2 h, 3 h, or 4 h followed by analysis of PTGS2 mRNA expression. The values are presented as the mean ± SD of three independent experiments. ***P ≤ 0.0001.

Citation: Endocrine Connections 13, 6; 10.1530/EC-23-0374

Figure 2
Figure 2

PBMCs from healthy control subjects and Graves’ disease patients treated with simvastatin and/or diclofenac. PBMCs were isolated from healthy control subjects (A) and Graves’ disease patients (B) and then cultured with cigarette smoke extract (CSE) and/or phytohemagglutinin (PHA) in the presence of absence of diclofenac and/or simvastatin. After 72 h of treatment, the cells were harvested, and the mRNA expression of PTGS2 was quantified. The values are presented as the mean ± s.d. of three independent experiments. *P ≤0.05, ***P ≤ 0.001, ****P ≤ 0.0001.

Citation: Endocrine Connections 13, 6; 10.1530/EC-23-0374

The expression of IL-6 and IL-1B was measured after 72 h of CSE exposure and treatment with simvastatin and/or diclofenac (Fig. 3). Exposure to CSE alone upregulated the expression of IL-6 by 10-fold (P ≤ 0.05) (Fig. 3A) when compared to untreated PBMCs. Furthermore, when CSE was added in combination with PHA, the expression increased by 2.4-fold (P ≤ 0.05) when compared to the treatment with PHA alone. Simvastatin downregulated the expression of IL-6 by 0.4-fold (P ≤ 0.05), and diclofenac combined with simvastatin treatment decreased IL-6 expression by 0.3-fold (P ≤ 0.01).

Figure 3
Figure 3

Effects of simvastatin and/or diclofenac on PBMCs from Graves’ disease patients activated by cigarette smoke extract (CSE). PBMCs were isolated from Graves’ disease patients and cultured with CSE and/or phytohemagglutinin (PHA) in the presence or absence of diclofenac and/or simvastatin for 72 h. The cells were harvested, and mRNA expression of IL-6 (A) and IL-1B (B) was quantified. The values are presented as the mean ± s.d. of three independent experiments. *P ≤ 0.05, **P ≤ 0.01.

Citation: Endocrine Connections 13, 6; 10.1530/EC-23-0374

The expression of IL-1B increased by six-fold (P ≤ 0.05) when the cells were exposed to CSE alone compared to the untreated control (Fig. 3B), and the exposure to CSE combined with treatment with PHA increased the expression of IL-1B by two-fold (P ≤ 0.01) compared to treatment with PHA alone. Exposure to CSE combined with simvastatin and diclofenac downregulated the expression by 0.6-fold (P ≤ 0.05). The combination of simvastatin and diclofenac downregulated IL-6 and IL-1B expression in PBMCs from healthy controls by 0.5-fold (P ≤ 0.01) compared to exposure to CSE and PHA (data not shown).

There were no cytotoxic effects of simvastatin, diclofenac, or CSE at any incubation time or concentration, as determined by a cytotoxicity assay as described in the Materials and methods section (data not shown).

The mean and SD underlying Figs. 13 can be found in Supplementary S2.

Effects of CSE in the presence or absence of simvastatin and/or diclofenac on the release of PGE2, IL-6, and IL-1B

CSE and PHA increased the release of PGE2 by 1.5-fold at 4 h in both groups (P ≤ 0.01, P ≤ 0.001) (Fig. 4A and B) compared to PHA alone. The elevated level of PGE2 in the supernatant of PBMCs treated with 10% CSE and PHA peaked at 24 h and was elevated by 1.3-fold (Fig. 4A) and 1.5-fold (Fig. 4B) (P ≤ 0.0001) in both groups compared to that in the PHA-treated groups. Exposure to CSE increased the release of PGE2 in both groups compared to the untreated control, peaking at 24 h with a fold-change of 3.7 (P ≤ 0.0001) (Fig. 4A) and 4.6 (P ≤ 0.0001) (Fig. 4B).

Figure 4
Figure 4

Protein levels of PGE2 in the supernatants of PBMCs isolated from healthy subjects and Graves’ disease patients. The protein levels of PGE2 in the supernatants of PBMCs isolated from control subjects (A) and Graves’ disease patients (B) were measured after exposure to 10% cigarette smoke extract for 1 h, 2 h, 3 h, 4 h, and 24 h. The PBMCs isolated from control subjects (C) and Graves’ disease patients (D) were exposed to 10% CSE in the presence or absence of simvastatin and/or diclofenac for 72 h. The protein levels of PGE2 in the supernatants were measured using ELISA. The values are presented as the mean ± s.d. of three independent experiments. **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001.

Citation: Endocrine Connections 13, 6; 10.1530/EC-23-0374

Simvastatin and diclofenac decreased the release of PGE2 compared to CSE exposure and PHA treatment. Simvastatin decreased the release of PGE2 by 0.5-fold (P ≤ 0.0001) from the cells from healthy controls (Fig. 4C) and by 0.6-fold (P ≤ 0.0001) from the cells from GD patients (Fig. 4D). Diclofenac decreased the release by 0.6-fold (P ≤ 0.001) from the cells from control subjects and 0.7-fold (P ≤ 0.0001) from the cells from GD patients (Fig. 4C and D). The inhibition of the release of PGE2 was even stronger when simvastatin and diclofenac treatment was combined, and this combined treatment decreased this secretion by 0.3-fold (P ≤ 0.0001).

CSE alone increased the release of IL-6 and IL-1B from PBMCs from GD patients by 16-fold (Fig. 5A) (P ≤ 0.0001) and 4-fold (Fig. 5B) (P ≤ 0.0001), respectively, compared to that from untreated PBMCs. When CSE was added in combination with PHA, the release of IL-6 and IL-1B increased by 1.7-fold (P ≤ 0.0001) and 1.2-fold (P ≤ 0.0001), respectively, compared with PHA alone. IL-6 release was downregulated by 0.6-fold (P ≤ 0.0001) when PBMCs were treated with either simvastatin or diclofenac and 0.4-fold (P ≤ 0.0001) when both treatments were combined compared to PBMCs that were treated with CSE and PHA. Simvastatin and diclofenac decreased the release of IL-1B by 0.6-fold (P ≤ 0.0001) compared with exposure to CSE and PHA.

Figure 5
Figure 5

Protein levels of IL-6 and IL-1B in the supernatants of PBMCs isolated from Graves’ disease patients after 72 h of exposure to 10% CSE in the presence or absence of simvastatin and/or diclofenac. PBMCs isolated from Graves’ disease patients were cultured and exposed to 10% cigarette smoke extract in the presence or absence of simvastatin and/or diclofenac for 72 h. The protein levels of IL-6 (A) and IL-1B (B) in the supernatants were measured using ELISA. The values are presented as the mean ± s.d. of three independent experiments. ***P ≤ 0.001, ****P ≤ 0.0001.

Citation: Endocrine Connections 13, 6; 10.1530/EC-23-0374

The mean and SD underlying Figs. 4 and 5 can be found in Supplementary S2.

CSE exposure effects on the production of IgG from B lymphocytes isolated from Graves’ disease patients and healthy controls

PBMCs were exposed to CSE in combination with the PC. We investigated the production of IgG in response to CSE exposure and found that IgG levels in the supernatants from Graves’ disease patients were increased at day 5 by 1.5-fold (Fig. 6) (*P ≤ 0.01) compared to that of healthy controls.

Figure 6
Figure 6

IgG levels in the supernatants of PBMCs isolated from Graves’ disease patients and healthy controls after 1–5 days of exposure to 10% CSE. PBMCs isolated from Graves’ disease patients were treated with phytohemagglutinin (PHA), recombinant human CD40 ligand, cross-linking antibody and IL-4 (proliferation cocktail (PC)) in the presence of 10% cigarette smoke extract (CSE) for 5 days. The IgG levels in the supernatants were measured using ELISA. The values are presented as the mean ± s.d. of three independent experiments. *P ≤ 0.05, **P ≤ 0.01.

Citation: Endocrine Connections 13, 6; 10.1530/EC-23-0374

The mean and SD underlying Fig. 6 can be found in Supplementary S2.

Effects of CSE, simvastatin, or IGF1 treatment on the proliferation of B lymphocytes and T lymphocytes from GD patients

We exposed lymphocytes to CSE in combination with the PC and investigated the proliferation of lymphocytes treated with PC alone. Exposure to CSE increased the proliferation of both B lymphocytes (Fig. 7A) and T lymphocytes (Fig. 7B). PC is indicated in red, and CSE-exposed lymphocytes are indicated in blue. The proliferation index of lymphocytes exposed to CSE was elevated by 1.3-fold (P ≤ 0.01) for B lymphocytes and 1.4-fold (P ≤ 0.0001) for T lymphocytes (Fig. 7C) compared to that of lymphocytes treated with PC.

Figure 7
Figure 7

Effects of cigarette smoke extract exposure and simvastatin treatment on the proliferation of B cells and T cells isolated from Graves’ disease patients. PBMCs isolated from Graves’ disease patients were treated with phytohemagglutinin (PHA), recombinant human CD40 ligand, cross-linking antibody and IL-4 (proliferation cocktail (PC)) in the presence of 10% cigarette smoke extract (CSE) or 10 µM simvastatin for 5 days. The effects of CSE exposure and simvastatin treatment (indicated in blue) on the proliferation of B lymphocytes (A and D) and T lymphocytes (B and E) were measured and compared with the control (indicated in red). The proliferation index data of B and T lymphocytes exposed to CSE or simvastatin compared with PC-treated cells (C and F) are presented as the median ± range pooled from three independent experiments **P ≤ 0.01 and ****P ≤ 0.0001. Proliferation index = total number of divisions divided by the number of cells that initiated division, and this value was calculated using FlowJo™ Version v10.7.

Citation: Endocrine Connections 13, 6; 10.1530/EC-23-0374

Simvastatin treatment (Fig. 7D and E marked in blue) decreased the proliferation index of both B and T lymphocytes by 0.7-fold (P ≤ 0.01; Fig. 7F) compared to PC treatment (indicated in red, Fig. 7D and E).

IGF1 treatment increased the proliferation index of B lymphocytes (Fig. 8A) by 1.7-fold (P ≤ 0.001) (Fig. 8C) compared to PC treatment. The proliferation index of T lymphocytes treated with IGF1 compared to PC-treated T lymphocytes (Fig. 8B) was increased by 1.6-fold (P ≤ 0.001) (Fig. 8C). The proliferation index of T lymphocytes treated with IGF1 compared to PC-treated T lymphocytes (Fig. 8B) was increased by 1.6-fold (P ≤ 0.001) (Fig. 8C).

Figure 8
Figure 8

Effects of IGF1 treatment on the proliferation of B cells and T cells isolated from Graves’ disease patients compared with controls. PBMCs isolated from Graves’ disease patients were treated with phytohemagglutinin (PHA), recombinant human CD40 ligand, cross-linking antibody and IL-4 (proliferation cocktail (PC)) with or without IGF1 for 5 days. The effects of IGF1 treatment (indicated in blue) on the proliferation of B lymphocytes (A) and T lymphocytes (B) compared with PC treatment (indicated red). The proliferation index data of B and T lymphocytes treated with IGF1 compared with PC-treated cells (C) are presented as the median ± range pooled from three independent experiments. ***P ≤ 0.0001. Proliferation index = total number of divisions divided by the number of cells that initiated division, and this value was calculated using FlowJo™ Version v10.7.

Citation: Endocrine Connections 13, 6; 10.1530/EC-23-0374

The mean and SD underlying Fig. 7C and F and 8C can be found in Supplementary S2.

Differences on the expression of PTGS2, release of PGE2, IL-6, and IL-1B between PBMCs from patient group and control group

We investigated PTGS2 expression, release of PGE2, IL-6, and IL-1B in response to each treatment between the patient and control groups. No statistical differences were observed in the expression of PTGS2, release of PGE2, IL-6, or IL-1B in PBMCs from GD patients compared to healthy controls (data not shown).

Discussion

Smoking cigarettes is strongly linked to the development of Graves’ disease (GD) and significantly increases the risk of developing Graves’ Ophthalmopathy (GO), with the risk escalating in correlation with the number of cigarettes smoked per day (16). In this study, we used an in vitro model to investigate the effect of CSE on PBMCs isolated from GD patients to investigate the role of smoking in the pathogenesis of GD and GO.

We investigated the expression of PTGS2, IL-1B, and IL-6 in PBMCs exposed to CSE. Expression of the inflammatory genes PTGS2, IL-6, and IL-1B was significantly upregulated by CSE exposure. CSE exposure combined with treatment with PHA, a common in vitro T-cell activator, enhanced the expression of PTGS2, IL-6, and IL-1B. Treatment with the lipid-lowering drug simvastatin alone or in combination with the NSAID drug diclofenac normalized the upregulated expression of these inflammatory genes. Furthermore, we found that CSE and IGF1 enhanced the proliferation of B and T lymphocytes from GD patients and that simvastatin downregulated proliferation.

In an earlier study, we showed that CSE exposure increased the expression of PTGS2, IL-6, and IL-1B in orbital fibroblasts from GO patients, and this expression was downregulated by simvastatin (4). The pathogenesis of GO includes both increased adipogenesis and inflammation due to infiltration of mononuclear cells in the orbital tissue (8). We have previously shown that CSE exposure to orbital fibroblasts isolated from GO patients increased the expression of PTGS2 and IL-6 (4).

It has been shown that among GD patients, thyroid TSH receptor autoantibodies (TRAbs) persist at higher levels in smokers than in nonsmokers during treatment with methimazole (17). Furthermore, it is known that cigarette smoking is associated with increased expression of proinflammatory markers, such as IL-1 and IL-6, and increased numbers of granulocyte macrophages (18). These effects of cigarette smoking are all very important for the pathogenesis of both GD and GO, and we found the above-mentioned cytokines to be upregulated by CSE in PBMCs compared to untreated PBMCs.

IL-6 plays an important role in the activation of B lymphocytes and the development of antibody-producing plasma cells (19). These processes are both important for the development of GO and GD.

A study by Lania et al. found a relationship between thyrotoxicosis and higher serum IL-6 levels in patients who were hospitalized for COVID-19 infection. These authors found that higher levels of IL-6 were associated with thyrotoxicosis, and they suggested that the inflammation might be triggered and sustained by the cytokine storm associated with COVID-19 infection, mimicking thyroid disorders (20). In another study, the authors reported that patients with active steroid-resistant GO had significantly reduced TRAb levels and that the condition of GO was improved by treatment with tocilizumab, an anti-IL-6 receptor monoclonal antibody (19). These results suggest the importance of IL-6 in both acute and chronic inflammation processes; if IL-6 levels are decreased, the level of inflammation might be decreased. We have shown that IL-6 gene expression and release from PBMCs was downregulated by diclofenac and simvastatin.

To the best of our knowledge, no in vitro study has reported the effect of CSE on the proliferation of B and T lymphocytes. However, Szydelko et al. reported that GO patients who currently smoke displayed elevated white blood cell and neutrophil counts compared with former smokers (21). These observations are consistent with our study, in which we found that CSE enhanced the proliferation of both B and T lymphocytes from GD patients compared to PC-treated lymphocytes. This suggests that CSE influences the overall cytokine release leading to the proliferation of lymphocytes.

In our study, exposure to CSE increased the release of PGE2 by PBMCs isolated from GD patients and control subjects. PGE2 is a proinflammatory protein and is encoded by the PTGS2 gene, which is expressed in response to various stimuli, including mitogens and cytokines (22). PGE2 is produced by orbital fibroblasts in GO patients (23). PGE2 plays a role in the maturation of B lymphocytes, stimulates the production of IL-6 by orbital fibroblasts, affects the differentiation of T lymphocytes and activates the degranulation of mast cells (24, 25). Hence, PGE2 is involved in many processes leading to inflammation in GO. PTGS2 (COX-2) is overexpressed in thyroiditis and benign and malignant thyroid lesions but not in normal thyroid tissue, suggesting that the expression of COX-2 (26) is associated with not only autoimmune disease but also thyroid cancer. It has been shown that PGE2 expression is decreased in response to NSAIDs (27). In a previous study, we showed that the expression of COX-2 in adipocytes was decreased to approximately 50% in response to the NSAID diclofenac. In the present study, the expression of PTGS2/COX-2 and the protein levels of PGE2 were both downregulated by diclofenac and even further decreased by the combined treatment of diclofenac and simvastatin.

A study by Henrotin et al. showed that diclofenac significantly decreased IL-1B-stimulated IL-6 production by human chondrocytes (28). In our study, we showed that both IL-1B and IL-6 gene expression and release by PBMCs were downregulated by diclofenac.

Simvastatin is known for its immunomodulatory and anti-inflammatory effects; however, the precise molecular mechanisms underlying these effects are not fully known (29).

Statins are used as lipid-lowering drugs, but recently, they have been suggested to exert apoptotic and anti-inflammatory effects as well (30).

In a cohort study by Stein et al. participants with newly diagnosed GD treated with simvastatin for more than 60 days had a 40% decreased risk of developing GO compared to those who had been treated for shorter time periods (31). Gullu et al. found that simvastatin treatment resulted in a change in the subtypes of lymphocytes and increased thyroid function in patients with Hashimoto’s thyroiditis and that simvastatin treatment in vitro decreased the number of lymphocyte subtypes through apoptosis (32). In our study, we showed that simvastatin downregulates the proliferation of B and T lymphocytes in PBMCs activated by PHA and CD-40. CD-40-dependent activation of B lymphocytes is interesting, as it is known to be enhanced in orbital fibroblasts from GO patients (33) and expressed on thyroid epithelial cells, suggesting their potential to directly interact with antigen-specific T-lymphocytes in GD (2).

It has been shown that B lymphocytes acquire strong Ag-presenting abilities after the crosslinking of CD-40, and activation of B lymphocytes via the CD-40 ligand may be the strongest stimulus for the induction of B-lymphocyte-mediated Ag presentation and cytokine production (34). In a study by Vornhagen et al., it was described that B-lymphocyte activation through CD-40 resulted in an upregulation of enzymes in the mevalonate pathway, which is involved in cholesterol biosynthesis and hence a target of simvastatin. The researchers blocked the pathway by simvastatin treatment and inhibited the activation of B lymphocytes through CD-40 activation (35, 36), which might in part explain the decreased proliferation of B lymphocytes in our study.

Pritchard et al. reported that IgG molecules circulating in GD patients are directed against IGF1R and detected these IgG in almost all patients but in very few control donors. Additionally, they found that orbital fibroblasts from GD patients, when treated with IGF1 or IgG, resulted in very high levels of IL-16 and RANTES when compared to controls. Both IL-16 and RANTES are very strong T-lymphocyte chemoattractants (37, 38). In another study, Douglas et al. reported that B lymphocytes from patients with GD were more skewed towards the CD19 IGF1R phenotype, leading to enhanced B-lymphocyte expansion (39). Smith et al. demonstrated that IGF1R is a target for GO treatment, as the IGF1 pathway plays a crucial role in the pathogenesis of GO through autoantibody-activated signaling and autocrine/paracrine mechanisms (40). Furthermore, these authors have shown that Tepezza, a human monoclonal antibody with absolute specificity for IGF1, is a promising treatment for GO. In placebo-controlled Phase III clinical trials, they reported that Tepezza exhibited a rapid reduction in proptosis and clinical activity scores compared to placebo for moderate-to-severe GO (41). In this study, we demonstrate that PBMCs activated by PHA and CD-40 and treated with IGF1 enhance B- and T-lymphocyte expansion, suggesting that IGF1 may have an impact on the proliferation of B and T lymphocytes in GD and GO patients.

We observed that PBMCs derived from GD patients, which were exposed to CSE, exhibited significantly higher levels of IgG compared to those from healthy controls. This intriguing finding is noteworthy, considering that previous studies have demonstrated that smoking typically leads to reduced IgG production when compared to non-smokers (42), and smoking is generally associated with immunosuppressive effects (43). To the best of our knowledge, there have been no prior investigations on the impact of CSE, specifically on the production of IgG from B lymphocytes isolated from GD patients.

Our discovery suggests that the behavior of B lymphocytes and their response to CSE in GD patients may diverge significantly from that of healthy individuals. However, to comprehend the underlying mechanisms more comprehensively, further studies are required. Examining epigenetic changes in B lymphocytes from both GD and associated ophthalmopathy patients could shed light on the unique nature of B lymphocytes in the context of GD.

Conducting such studies could offer valuable insights into the complex interactions between CSE exposure, B lymphocytes, and the pathogenesis of GD. Ultimately, a deeper understanding of these mechanisms may pave the way for more targeted therapeutic interventions and management strategies for patients with GD and related conditions.

We are aware that the conclusions drawn from our study are limited to an in vitro model. However, background research by other groups supports our results, and we suggest that in future studies, the levels of IL-6, IL-1B, and PGE2 in the serum of GO patients should be investigated. Moreover, we recommend a more comprehensive study of B lymphocytes from individuals with GD and/or GO, with a particular emphasis on investigating epigenetic alterations.

In conclusion, this study shows that CSE significantly upregulates the expression and release of the inflammatory markers PTGS2, IL-6, and IL-1B,the IgG levels and the proliferation of B and T lymphocytes. These findings suggest that CSE has an impact on inflammation both by recruiting mononuclear cells to the site of inflammation and by systemically elevating the numbers of B- and T-lymphocyte populations; these results explain the severe state of GO in smokers and may in part explain the association of smoking with GD. Additionally, CSE increases the production of IgG by PBMCs from GD patients compared to PBMCs from healthy controls. Furthermore, IGF1 also increased the proliferation of B and T lymphocytes in our in vitro model. Finally, these effects were decreased by diclofenac and/or simvastatin treatment, strongly supporting the need to investigate simvastatin and diclofenac as treatments for GD and GO in a clinical trial.

Supplementary materials

This is linked to the online version of the paper at https://doi.org/10.1530/EC-23-0374.

Declaration of interest

There is no conflict of interest that could be perceived as prejudicing the impartiality of study reported.

Funding

This work was supported by the Agreement for Medical Education and Research (ALF 2023 230101).

Acknowledgements

We gratefully acknowledge Ylva Wessman for her assistance in handling blood samples and Perparim Cerri for helping with the RNA extractions.

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Supplementary Materials

 

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  • Figure 1

    The effect of cigarette smoke extract on PBMCs isolated from healthy control subjects and Graves’ disease patients. PBMCs were isolated from healthy control subjects (A) and Graves’ disease patients (B) followed by treatment with 10% cigarette smoke extract (CSE) and/or phytohemagglutinin (PHA). PBMCs were harvested after treatment for 1 h, 2 h, 3 h, or 4 h followed by analysis of PTGS2 mRNA expression. The values are presented as the mean ± SD of three independent experiments. ***P ≤ 0.0001.

  • Figure 2

    PBMCs from healthy control subjects and Graves’ disease patients treated with simvastatin and/or diclofenac. PBMCs were isolated from healthy control subjects (A) and Graves’ disease patients (B) and then cultured with cigarette smoke extract (CSE) and/or phytohemagglutinin (PHA) in the presence of absence of diclofenac and/or simvastatin. After 72 h of treatment, the cells were harvested, and the mRNA expression of PTGS2 was quantified. The values are presented as the mean ± s.d. of three independent experiments. *P ≤0.05, ***P ≤ 0.001, ****P ≤ 0.0001.

  • Figure 3

    Effects of simvastatin and/or diclofenac on PBMCs from Graves’ disease patients activated by cigarette smoke extract (CSE). PBMCs were isolated from Graves’ disease patients and cultured with CSE and/or phytohemagglutinin (PHA) in the presence or absence of diclofenac and/or simvastatin for 72 h. The cells were harvested, and mRNA expression of IL-6 (A) and IL-1B (B) was quantified. The values are presented as the mean ± s.d. of three independent experiments. *P ≤ 0.05, **P ≤ 0.01.

  • Figure 4

    Protein levels of PGE2 in the supernatants of PBMCs isolated from healthy subjects and Graves’ disease patients. The protein levels of PGE2 in the supernatants of PBMCs isolated from control subjects (A) and Graves’ disease patients (B) were measured after exposure to 10% cigarette smoke extract for 1 h, 2 h, 3 h, 4 h, and 24 h. The PBMCs isolated from control subjects (C) and Graves’ disease patients (D) were exposed to 10% CSE in the presence or absence of simvastatin and/or diclofenac for 72 h. The protein levels of PGE2 in the supernatants were measured using ELISA. The values are presented as the mean ± s.d. of three independent experiments. **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001.

  • Figure 5

    Protein levels of IL-6 and IL-1B in the supernatants of PBMCs isolated from Graves’ disease patients after 72 h of exposure to 10% CSE in the presence or absence of simvastatin and/or diclofenac. PBMCs isolated from Graves’ disease patients were cultured and exposed to 10% cigarette smoke extract in the presence or absence of simvastatin and/or diclofenac for 72 h. The protein levels of IL-6 (A) and IL-1B (B) in the supernatants were measured using ELISA. The values are presented as the mean ± s.d. of three independent experiments. ***P ≤ 0.001, ****P ≤ 0.0001.

  • Figure 6

    IgG levels in the supernatants of PBMCs isolated from Graves’ disease patients and healthy controls after 1–5 days of exposure to 10% CSE. PBMCs isolated from Graves’ disease patients were treated with phytohemagglutinin (PHA), recombinant human CD40 ligand, cross-linking antibody and IL-4 (proliferation cocktail (PC)) in the presence of 10% cigarette smoke extract (CSE) for 5 days. The IgG levels in the supernatants were measured using ELISA. The values are presented as the mean ± s.d. of three independent experiments. *P ≤ 0.05, **P ≤ 0.01.

  • Figure 7

    Effects of cigarette smoke extract exposure and simvastatin treatment on the proliferation of B cells and T cells isolated from Graves’ disease patients. PBMCs isolated from Graves’ disease patients were treated with phytohemagglutinin (PHA), recombinant human CD40 ligand, cross-linking antibody and IL-4 (proliferation cocktail (PC)) in the presence of 10% cigarette smoke extract (CSE) or 10 µM simvastatin for 5 days. The effects of CSE exposure and simvastatin treatment (indicated in blue) on the proliferation of B lymphocytes (A and D) and T lymphocytes (B and E) were measured and compared with the control (indicated in red). The proliferation index data of B and T lymphocytes exposed to CSE or simvastatin compared with PC-treated cells (C and F) are presented as the median ± range pooled from three independent experiments **P ≤ 0.01 and ****P ≤ 0.0001. Proliferation index = total number of divisions divided by the number of cells that initiated division, and this value was calculated using FlowJo™ Version v10.7.

  • Figure 8

    Effects of IGF1 treatment on the proliferation of B cells and T cells isolated from Graves’ disease patients compared with controls. PBMCs isolated from Graves’ disease patients were treated with phytohemagglutinin (PHA), recombinant human CD40 ligand, cross-linking antibody and IL-4 (proliferation cocktail (PC)) with or without IGF1 for 5 days. The effects of IGF1 treatment (indicated in blue) on the proliferation of B lymphocytes (A) and T lymphocytes (B) compared with PC treatment (indicated red). The proliferation index data of B and T lymphocytes treated with IGF1 compared with PC-treated cells (C) are presented as the median ± range pooled from three independent experiments. ***P ≤ 0.0001. Proliferation index = total number of divisions divided by the number of cells that initiated division, and this value was calculated using FlowJo™ Version v10.7.

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