Effects of Curcumin on Pro-Inflammatory Cytokines in Sjögren's Syndrome

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https://doi.org/10.1007/s00296-021-04859-7 GENES AND DISEASE

In vitro effects of curcumin on proinflammatory cytokines

and expression of their genes in minor salivary gland tissue of patients with Sjogren’s syndrome

Jayakanthan Kabeerdoss¹ · Pulukool Sandhya^1,2 · Biji T. Kurien^3,4,5 · Robert Hal Scofield^3,4,5 · Debashish Danda¹

Received: 14 February 2021 / Accepted: 5 April 2021 / Published online: 16 April 2021

Abstract

Curcumin reduces disease severity and ameliorates lupus-like/Sjögren’s Syndrome-like disease in mice model. The immunological basis of these effects is largely unknown. This study examined the effects of curcumin on pro-inflammatory cytokines secreted by minor salivary glands in patients with primary Sjögren’s syndrome (pSS). Minor salivary gland (MSG) tissue samples were collected from patients undergoing biopsy for suspected pSS. The tissues were treated with phytohemagglutinin (PHA) alone as well as PHA with curcumin (30 μM) and cultured in RPMI 1640 medium for 48 h at 37 °C in CO₂ incubator. After the incubation period, culture supernatant and tissues were stored in the freezer (−80 °C). IL-6 levels were measured in supernatant by ELISA kit. Gene expressions of pro-inflammatory cytokines, namely IL-6, IL-8, TNF-α, IL-1β, IL-4, IL-10, IL-17, IL-21, and IFN-γ, were measured by qPCR. IL-6 secretion levels and gene expressions were compared statistically between groups by Student’s t test. Forty-seven patients were screened. Eight patients satisfied ACR/EULAR criteria for pSS. Seven patients with absent glan- dular inflammation and negative serology constituted sicca controls. These 15 subjects were included in final analysis. In pSS group, but not in controls, median IL-6 levels in supernatant were less in curcumin-treated as compared to PHA-alone subset [5.5 (0.7–13.34) vs 18.3 (12–32) ng/ml; p = 0.0156]. mRNA expression levels of IL-6 were also lower in curcumin-treated samples as compared to PHA alone, when cases and controls were analyzed together as well as in cases alone (p = 0.0009 and p = 0.0078, respectively); however, mRNA expression of IL-1β was lower in curcumin-treated samples as compared to PHA alone, only when cases and controls were analyzed together (p = 0.0215). There was no difference in other cytokine gene expression levels between the subsets under the in-vitro experimental conditions. In conclusion, curcumin reduced mRNA expression as well as secretion of IL-6 levels by salivary gland tissue of patients with pSS. Curcumin also suppressed PHA-induced mRNA expression levels of IL-6 and IL-1β in MSG tissue of patients with pSS and controls when analyzed together as a combined group.

Keywords Curcumin · Cytokines · Sjogren’s syndrome · Labial salivary gland

Introduction

Primary Sjogren’s syndrome (pSS) is a chronic systemic inflammatory autoimmune disease, affecting approxi- mately 0.6% of the general population [1]. pSS is mainly

Rheumatology

INTERNATIONAL

This study was presented as oral presentation in 35th Annual Conference of The Indian Rheumatology Association held inPuducherry, from 5th to 8th December 2019 and abstract was published in Indian J Rheumatol 2019;14, Suppl S2:99–249. In addition, an abstract representing the work was also published inThe Journal of Immunology, May 1, 2020, 204 (1 Supplement) 238.14.

* Debashish Danda

[email protected]

1 Department of Clinical Immunology and Rheumatology, Christian Medical College and Hospital, Vellore, Tamil Nadu 632004, India

2 Present Address: Department of Rheumatology, St Stephen’s Hospital, Tis Hazari, Delhi 110054, India

3 Arthritis and Clinical Immunology Research Program, Oklahoma Medical Research Foundation, Oklahoma City, OK, USA

4 Department of Medicine, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA

5 Department of Veterans Affairs Medical Center, Oklahoma City, OK, USA

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characterized by exocrinopathy with lymphocytic infiltration in salivary and lacrimal glands, eventually leading to dry- ness of mouth and eyes. However, the disease can virtually affect other organs in the body leading to musculoskeletal, pulmonary, renal, hematological, dermatological, vascular, and neurological manifestations.

Curcumin is an anti-inflammatory agent and an antioxi- dant. Chemically, curcumin is diferuloylmethane (1,7-bis[4- hydroxy-3-methoxyphenyl]-1,6- heptadiene-3,5-dione) obtained from the rhizome of Curcuma longa (turmeric) [2]. Oral administration of heat/pressure-solubilized curcumin in MRL-lpr/lpr mice models of SS and systemic lupus erythematosus (SLE) leads to amelioration of disease features by reduction of lymphoproliferation and suppression of autoantibody formation. Curcumin induces apoptosis of lymphocytes and thereby reduces lymphocyte infiltration in the salivary gland of these mice [3]. Curcumin has also been shown to be protective against development of SS in mice model; however, molecular mechanisms are not well known.

Increased activation of Th1, Th17, Th2 and follicular T cells has also been well described in minor salivary glands (MSG) of patients with primary Sjögren’s Syndrome (pSS) [4, 5]. There is, however, paucity of data on the role of curcumin in altering cytokine responses in pSS.

Hypothesis: based on these lacunae in literature, we hypothesized that curcumin would directly reduce expression of pro-inflammatory cytokines in MSG of patients with pSS.Novelty: our present study is the first study proposed to examine effect of curcumin on cytokine gene expression in minor salivary gland tissues of patients with primary Sjogren syndrome, and, therefore, it is a novel study.

In this ‘In vitro’ culture experiment, we aimed to evaluate the role of curcumin on expression of cytokines originating from different T helper cell subsets in minor salivary gland biopsy tissue samples of patients with primary Sjögren’s syndrome.

Methods

Minor salivary gland biopsies

The study was done between June 2017 and August 2018.

Minor salivary gland (MSG) biopsy tissue samples were collected from adult patients undergoing the procedure as a part of evaluation for the diagnosis of pSS after obtaining informed consent. pSS patients fulfilling 2016 ACR-EULAR Classification Criteria constituted the cases [6]. Patients who tested negative for serologies [ANA, RF, Anti-Ro (or SSA;

henceforth will be referred as Anti-Ro for historical reasons throughout the manuscript)] as well as lacked histopathologic criteria of pSS and those who did not fulfil criteria for any

systemic autoimmune disease constituted the sicca controls.

Ethical Approval for the study was granted by the Institutional Review Board, Christian Medical College, Vellore, India.

Tissue samples were collected and transported to the laboratory in Roswell Park Memorial Institute (RPMI) medium 1640 (Sigma, St. Louis, MO, USA). Laboratory bench staff were blinded to the samples and were unaware of clinical data as well as the source of the tissue, i.e. case or control till full analysis of data. MSG tissue from each subject was divided into two parts, one part was treated with 100 ng/ml of phytohemagglutinin [(PHA), Sigma, St. Louis, MO,USA] alone and the other part of the tissue was treated with both 100 ng/ml PHA and 30 µM of curcumin (Sigma, St. Louis, MO,USA) [7]. Curcumin used in this experiment was heat- and pressure-solubilized in water as described previously [3]. Briefly, 5 mg/ml of curcumin was heated in a conical flask for 15 min followed by autoclaving for 30 min, and finally filtered by Whatman filter paper. Biopsy tissue samples cultured in RPMI 1640 medium were sup- plemented with 10% fetal bovine serum (GIBCO, Paisley, UK), penicillin/streptomycin and gentamicin (Sigma, St.

Louis, MO, USA) for 48 h at 37 °C in a CO₂ incubator.

After the incubation period, the culture supernatant and tissues were separated and stored in the freezer (-80º C) till further processing.

ELISA for IL‑6

Supernatants from the tissue cultures were subjected to ELISA for IL-6 following manufacturer’s instructions (R&D systems, Minneapolis, MN, USA). After appropriate stand- ardisation experiment, each diluted sample was run in dupli- cate against the appropriate standard and optical densities read out by microplate reader (iMark reader, BioRad, Hemel Hempstead, UK).

Real‑time PCR for gene expression

Cultured tissues were subjected to homogenization by handheld homogenizer (Arlington Heights, IL, USA). RNA extraction was done using TRI reagent solution (Sigma, St. Louis, MO, USA) as per manufacturer recommended protocol. RNA was quantified by NanoDrop 2000 (Thermo fisher scientific, Wilmington, DE, U.S.A.). One microgram of RNA was used for cDNA conversion using ProtoScript^® First-Strand cDNA Synthesis Kit (Catalog No E6560, New England Biolabs, Ipswich, MA, USA).

We have chosen the following cytokine genes:

Interferon-γ (IFN-γ), Interleukin-4 (IL-4), IL-10, IL-17 and IL-21 as representatives for each of the T helper subsets, namely Th1, Th2, Treg, Th17 and follicular T helper cell (TFH), respectively. In addition, we have included cytokine genes involved in innate immune responses, namely IL-6

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Tumor necrosis factor-α (TNF-α), IL-8 and IL-1β. Gene expressions were quantified using specific primers with SYBR green on the real-time PCR system. Briefly, PCR reactions were performed using Power SYBR Green qPCR master mix (Catalog No 1804574, Life Technologies, War- rington, UK), with 250 nM of both forward and reverse primers as well as five-time diluted cDNA. All cDNA samples were then amplified for the genes of interest along with beta-actin as a housekeeping gene. All samples were run in duplicates for each gene expression assay.

Statistics

Quantification of gene expression was expressed as relative expression calculated from quantification cycle [(Cq), for- merly known as cycle of threshold i.e. Ct] values, which were obtained from quantifying target gene expression relative to the beta-actin gene expression using the 2−△Cq method [8]. The relative difference in each gene expression levels was expressed as mean with standard error of the mean, and the significance of differences between two groups was assessed using paired t test or Wilcoxon matched pair test based on normality of distribution. Non-parametric Spearman correlation was used to assess correlation between gene expression levels and secretion levels for IL-6. P values less than 0.05 were considered as statistically significant.

Statistical analysis was done using GraphPad prism version 5 (GraphPad Software, Inc. La Jolla, CA USA).

Results

Demography

Forty-seven patients were screened for the study. At the end of the study period, eight patients fulfilled the 2016 ACR- EULAR Classification Criteria for diagnosis of pSS. Seven patients were classified as sicca controls. The remaining 32

patients who neither fulfilled classification criteria for pSS nor considered as controls were eliminated from the study.

Totally 15 subjects only were included in final analysis.

Demographic details for patients with pSS and controls are described in Table 1.

IL‑6 levels in supernatant and tissues

Values of IL-6 levels were numerically lower in tissues treated with curcumin + PHA as compared to those treated with PHA alone, when pSS and controls were analysed as a single group; however, when each individual group is analysed separately, PHA-induced IL-6 production in the supernatant was significantly lower in the curcumin treated samples from patients with pSS. No difference was observed in the control group (Fig. 1 bottom row). A similar but more significant trend was observed for IL-6 gene expression levels in tissues (Fig. 1 top row). IL-6 gene expression levels were lower in curcumin + PHA-treated tissues compared to PHA alone in combined group and pSS, but not in the control group (Fig. 1). Significant positive correlation was also observed between secreted IL-6 levels and gene expression levels in both PHA and curcumin-treated experiments (Fig. 2).

Tissue cytokine gene expression levels

IFN-γ gene expression levels were not different between PHA- and curcumin-treated samples in combined groups, i.e. pSS as well as control groups (Fig. 3). Gene expressions of IL-4, IL-10, IL-17 and IL-21 were below detectable lim- its. Hence, quantitative levels of these cytokine genes were not available.

IL-1β gene expression levels were significantly lower in curcumin-treated groups compared to only PHA-treated samples, when cases and controls were analysed together.

However, statistical significance was not seen when the cases

Table 1 Clinical demography of study participants

a For historical reasons, we preferred to use Anti-La, rather than SSB (similar to the replacement done for SSA by Anti-Ro)

Parameters pSS (n = 8) Sicca controls (n = 7)

Age [median (range)] 39.5 (33–64) 46 (35–57)

Sex ratio (F:M) 7:1 5:2

Dry eye 6 7

Dry mouth 5 7

Biopsy grade > 3 7 0

Anti-Ro Ab positivity 8 0

Anti-La^a Ab positivity 4 0

Antinuclear antibodies (ANA) positivity 6 0

Rheumatoid factor positivity 6 0

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and controls were analysed individually. IL-8 and TNF-α gene expression levels were not different between PHA- and curcumin-treated samples in combined groups, or individually in pSS or control groups (Fig. 4).

Discussion

Our study demonstrated that IL-6 and IL-1β cytokine genes are highly expressed in minor salivary gland tissue upon mitogen stimulation; and curcumin significantly reduced these levels.

Fig. 1 IL-6 gene expression levels and secretion levels by MSG tissues of patients with pSS and sicca controls following experimental treatments with PHA and PHA + curcumin

Fig. 2 Correlation between secreted IL-6 levels and IL-6 gene expression levels by MSG tissues of patients with pSS and sicca controls following experimental treatments with PHA and PHA + Curcumin

Fig. 3 IFN-γ gene expression levels by MSG tissues of patients with pSS and sicca controls following experimental treatments with PHA and PHA + Curcumin

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These findings are relevant in the light of the role of pro- inflammatory cytokines especially IL-6 and IL-1β in the pathogenesis of pSS. High salivary IL-6 levels were noted in pSS and it correlated with mononuclear cell infiltration in salivary gland tissues of these patients [9]. Epithelial cells from the salivary gland were the primary cellular sources for increased IL-6 secretion in these patients [10].

In another study, IL-1β along with IFN-γ reduced salivary amylase secretion in the parotid gland, thereby implicating these pro-inflammatory cytokines in causation of exocrine dysfunction in patients with pSS [11]. Further, enhanced activation of NLRP3 inflammasome was also reported in the macrophages infiltrating salivary glands as well as peripheral blood mononuclear cells of patients with pSS; and this is in conformation with our finding of high expression of IL-1β gene in minor salivary gland tissue of patients with pSS [12, 13].

Other inflammatory cytokines have also been reported to be overexpressed in pSS. Fox et al. showed that mRNA

expression of IL-1 alpha, IL-6, IFN-γ and TNF-α was 40-fold higher in salivary glands of pSS as compared to normal healthy individuals. Levels of these cytokines were also higher in saliva of pSS patients [14]. We were able to measure gene expressions of IFN-γ and TNF-α, but not IL-4, IL-17, IL-21 and IL-10 in MSG tissue. A prior study using immunohistochemistry, however, had reported low expression of Th2 cytokine IL-4 in MSG tissue of pSS patients [15].

There are data in literature on the ameliorating effect of curcumin on inflamed tissues including salivary gland.

Curcumin prevented secretion of IL-6 by submandibular glands (SMG) in patients with IgG4-related disease, thereby reducing SMG fibrosis [16]. Similarly, in the present study, curcumin effectively reduced mitogen- induced IL-6 secretion and IL-6 gene expression in MSG of patients with pSS. A meta-analysis study of randomised controlled trials showed that curcumin effectively low- ered circulating IL-6 levels, thereby preventing systemic

Fig. 4 IL-1β, IL-8 and TNF- α gene expression levels by MSG tissues of patients with pSS and sicca controls following experimental treatments with PHA and PHA + Curcumin

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inflammation [17]. Earlier study had also showed that reduction in IL-6 expression by curcumin was mediated through inhibition of mitogen-activated protein kinase (MAPK) and nuclear factor-κB (NF-κB) [7, 18]. Further, curcumin inhibited formation of the NLRP3 Inflamma- some and attenuated IL-1β secretion [19].

In the present study, curcumin did not reduce mitogen- induced TNF-α expression. This may not be of much con- cern considering the fact that TNF-α blockade was inef- fective in mice models of SS as well as human subjects with pSS [20].

Major limitations of this study include the small sample size. During 14 months of study period, we were able to screen 47 subjects; of them, only eight patients satisfied the 2016 ACR-EULAR Classification Criteria for diagnosis of primary SS (pSS) including 7 of them fulfilling biopsy criteria. pSS is a relatively rare disease and usu- ally a good number of patients are not willing to undergo biopsy. Hence, the majority of them are diagnosed by serology and the objective clinical criteria.

Treatment of MSG tissue with curcumin and the mitogen has been done for 48 h only in our study. This time period might be enough for induction of innate immune cytokines, but expression of T helper cytokines probably requires a longer duration. Further studies would be required to study the effect of curcumin on adaptive immune response-related cytokines too in patients with pSS. Also, we had divided the tiny biopsy tissues by visually apparent equal half portions and then treated with either PHA or Curcumin plus PHA.

Could the unequal cell numbers between the divided tissue portions may have influenced the degree of gene expression and secretion of cytokines, in addition to apoptosis of cells induced by curcumin as reported previously? [3] This is unlikely, as both cases and controls were subjected to the same experimental conditions and the laboratory staff han- dling the samples was blinded to the samples till the data were analysed. In addition, expression values were repre- sented by relative differences; also, values were normalised by housekeeping genes which would have minimised the impact of weight changes between the divided tissues also.

The results of the current study can, therefore, be considered as preliminary and further studies with larger sample size is warranted to validate the findings of this study.

In conclusion, curcumin reduces gene expression of IL-6 and IL-1β as well as secretion of IL-6 by MSG tissue of our patients with pSS.

Acknowledgements We are thankful to Mr. Karthi for transporting samples, Ms. Hindhumathi for technical help and Ms. Tunny Sebastian for sample size calculation. Finally, we appreciate all participants who were generous in providing the samples and written consents.

Author contributions JK was responsible for laboratory analyses. SP and DD were responsible for clinical data collection. All authors were

involved in design of the study and write-up of the manuscript. JK and SP drafted the initial version of the manuscript. BTK, RHS and DD were involved in editing and approving the final manuscript.

Funding This work was funded by an intramural grant (IRB Min No:

10395 dated 30.11.2016) from Christian Medical College, Vellore.

Declarations

Confict of interest The author(s) declare no competing interests

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