Paper

Altered oxidative stress markers in relation to T cells, NK cells & killer immunoglobulin receptors that are associated with disease activity in SLE patients

Lupus 0(0) 1–14 ! The Author(s) 2020 Article reuse guidelines: sagepub.com/journals-permissions DOI: 10.1177/0961203320959441 journals.sagepub.com/home/lup

Ankit Tandon1, Kumari Anupam1, Jyotsana Kaushal1, Preeti Gautam1, Aman Sharma2 and Archana Bhatnagar1

Abstract Systemic Lupus Erythematosus is an autoimmune disease with symptoms pervasive to all organ systems. It affects more females as compared to males (in the ratio 9:1). Oxidative stress plays a major role in the pathogenesis of SLE and other autoimmune diseases. In order to understand the relationship between cell specific oxidative stress and the severity of SLE, this research study involving the estimation of intracellular ROS accumulation in T and NK cell was conducted on SLE patients of North Indian Population. At the same time, to estimate anti-oxidant defense, Keap1 and Nrf2 levels were estimated in these cell types. The relationship between the expression of Killer immunoglobulin receptors i.e., KIR2DL4 & KIR3DL1 and oxidative stress was also evaluated as these receptors are imperative for the function and self-tolerance of NK cells. Oxidative stress was raised along with Keap1 and Nrf2 in T and NK cell subsets in SLE patients. The expression of KIR2DL4 was raised and that of KIR3DL1 was reduced in the NK cells of patients. The intensity of change in expression and its significance varied among the subsets. Nrf2 expression was raised in these species against oxidative stress as the antioxidant defense mechanism pertaining to Keap1-Nrf2 pathway, but the adequacy of response needs to be understood in further studies. The expression of KIR2DL4 and KIR3DL1 varied among the patient and healthy controls and the expression of the latter was found to have a significant positive relationship with plasma Glutathione(reduced) concentration. Keywords Systemic lupus erythematosus, T-cells, natural killer cells, oxidative stress, killer immunoglobulin receptors, Keap1, Nrf2 & Glutathione (reduced) Date received: 25 August 2020; accepted: 11 February 2020

Introduction Systemic Lupus Erythematosus (SLE) is an autoimmune disease that can affect almost all organ systems, be it be the nervous system, sensory system, excretory system, digestive system or circulatory system. The symptoms of SLE depend upon the kind of auto antibodies present in an individual patient. Auto antibodies expressed in SLE are mainly against nuclear bodies like anti-dsDNA, anti-Ro, anti-La, anti-Smith and ANA.1 Oxidative stress is involved in the pathophysiology of autoimmune diseases like SLE. The accumulation of

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Department of Biochemistry, Panjab University, Chandigarh, India Department of Internal Medicine, Post Graduate Institute of Medical Education and Research, Chandigarh, India 2

Corresponding author: Archana Bhatnagar, Department of Biochemistry, Panjab University, Chandigarh 160014, India. Email: [email protected]

2 free radicals such as Reactive Oxygen Species (ROS) in the cells and body leads to unusual chemical reactions with cellular molecules like DNA, Proteins and Lipids; this entails the formation of neo-epitopes or mimic molecules, which stimulate the immune system against self-cells, i.e., auto immunity.2 Cells have their defense system against intracellular oxidative stress, which includes enzymatic scavenger Superoxide Dismutase (SOD), Catalase (CAT), Glutathione Peroxidase (GTPX), Peroxiredoxin (PRX) and Glutathione Transferase (GST) or nonenzymatic ROS scavengers like Glutathione and carotenoids (b-Carotene).3The keap-1-Nrf-2 pathway is a vital signal cascade for antioxidant defense where the Keap-1(Keltch like ECH-associated protein-1), a cytoplasmic protein, binds itself to Nuclear factor erythroid 2-related factor 2(Nrf-2) to retain the latter in the cytosol. Keap-1 is sensitive to oxidative stress, and on being challenged by increased oxidative stress, it releases Nrf2 into the nucleus, while undergoing ubiquitination itself. Nrf-2, which binds to the ARE (antioxidant response element) region on the chromosome, stimulates downstream protective mechanisms against damages due to oxidative stress.4Earlier studies suggested that knockdown of Keap-15 or induction of Nrf-2(by drugs such as dimethyl fumarate)6,7 have alleviated symptoms in various autoimmune diseases. In the current study, we have determined the expression of Keap-1 and Nrf-2 in T cells and NK cells in order to understand the status of the antioxidant defense system in these cells of SLE patients. Aberration in T-cell functions could be responsible for triggering autoimmunity either through release of proinflammatory cytokines, or by helping B-cells produce autoantibodies to expand and also by retaining autoimmune memory or decreased regulatory function. NK cells are large granular cells that form a significant part of innate immunity. There are two types of receptors on the surface of NK cells: the inhibitory receptors (KIR) that bind to MHC class-I receptors and down-regulate activity of NK cells.8The other type, stimulatory receptors, identify stress-induced receptors and amplify the activity of the NK cell.9The balance between inhibitory and activating receptors is required for self-tolerance in NK cells.10 NK cells also attenuate autoimmunity by removing stressed autoreactive T and B cells.11 The present study has been undertaken to understand the roles of an activating receptor of the NK cell, the KIR2DL4 and an inhibiting receptor KIR3DL1 by analyzing their surface expression and mRNA levels. These two receptors are the most commonly expressed in their respective groups. Moreover, a cell-specific study about intracellular oxidative stress, Keap-1 and Nrf-2 expression was carried out in subtypes of T and NK cells to determine

Lupus 0(0) their antioxidant capacity, alterations in their proportions and relationship of these parameters with the severity of the disease.

Materials and methods Materials 1. 1.Anti-human antibodies for Keap-1, Nrf-2 and goat anti-mouse IgG for secondary labeling (Santacruz, USA) 2. 2.Antihuman antibodies for CD3, CD4, CD8, CD56, CD25, KIR2DL4 and KIR3DL1 (BiolegendTM) 3. 20 -70 -Dichlorodihydrofluorescein diacetate (DCFHDA) (Sigma-Aldrich, USA) 4. HiSepTM LSM1077 Ficoll (Himedia) 5. TRIzolTM reagent (Thermofischer scientific) 6. RevertaidTM kit (Thermofischer scientific) for cDNA synthesis 7. Isolation Kits for CD4þ, CD8þ and CD56þ cells (Stemcell technologies, Canada) 8. 5, 5’-dithio-bis-2-nitrobenzoic acid (DTNB) (ThermofischerScientific) 9. Fixation/permeabilization kit (BD biosciences)

Patient recruitment SLE patients (n ¼ 33) were enrolled from the OutPatient Department (OPD) at the Rheumatology Clinic, the Post Graduate Institute of Medical Education and Research (PGIMER), Chandigarh. Age and Sex matched healthy volunteers (n ¼ 20) were also enrolled. For the study, the score of disease activity was evaluated by Rheumatologist Prof.Aman Sharma according to SLE disease activity index (SLEDAI-2K).12 All samples were collected upon receiving informed written consent for every case and ethical clearance for the study was obtained from the Ethics committee of PGIMER, Chandigarh (IEC/ 2015/103).

Procedure The isolation of peripheral blood mononuclear cells (PBMC). The PBMC were isolated from venous using HiSepTM LSM1077 Ficoll (Himedia).13 The isolation of CD4þ, CD8þ and CD56þ cells from PBMC using magnet associated cell sorting (MACS). Isolation of CD4þ cells, CD8þ cells and CD56þ cells from PBMC were carried out by protocols provided by the manufacturer (Stem Cell Technologies, Canada). The purity of isolated cells was determined by flowcytometric analysis (FACScan, Becton Dickinson, USA) and found to be more than 90% in each case.

Tandon et al. Flowcytometry analysis and ROS estimation. Immunophenotyping was done on PBMCs by using monoclonal antibodies for studying various parameters. For intracellular ROS analysis, PBMC were also incubated with a cell-permeable DCFDA dye, an esterified precursor of fluorescent molecule DCF, that fluoresces at 530 nm when excited at 485 nm in the presence of H2O2, Intensity of fluorescence is directly proportional to the amount of H2O2 present. The gating strategy used is shown in Supplementary Figure 1. Estimation of reduced glutathione concentration in plasma. Levels of reduced Glutathione were measured by allowing the development of a yellow-colored complex, 2nitro-5-mercaptobenzoic acid due to the reduction of 5, 5’-dithio-bis-2-nitrobenzoic acid (DTNB) with a free-SH group of GSH. Glutathione concentration is directly proportional to the absorption maxima measured at 412 nm.14 Quantitative real-time polymerase chain reaction. Total RNA from the T and NK cells was extracted using TRIzolTM reagents as per manufacturer’s protocol, followed by cDNA synthesis. Quantitative PCR for various genes was performed on step-one PCR(Applied Biosystem, USA). The expression of genes Keap-1, Nrf-2, FOXP3, HMOX-1, KIR2DL4, KIR3DL1 and GAPDH were normalized by the expression of housekeeping gene Glyceraldehyde 3-phosphate dehydrogenase (GAPDH).

Statistical analysis Quantitative data presented as mean  standard error mean (SEM) were analyzed using unpaired student’s Ttests for parametric quantitative data and MannWhitney U test for non-parametric data. Likewise, the correlational study of parametric data was done with Pearson’s correlation and for non-parametric data Spearman’s rank correlation analysis. The software Graph pad prism version5.1 was used for analysis.

Results Demographics details of SLE patients and healthy controls In the study, SLE(n ¼ 33) patients and age and sex matched healthy control(n ¼ 20) were enrolled. Out of thirty-three patients, twenty-six had the Anti-Nuclear Antibody (ANA), sixteen had anti-dsDNA antibodies out of total patients, there were single cases positive for anti-Ro and anti-La antibody each. Ten patients out of the total number were diagnosed with a low C3

3 complement factor and eight had a low C4 complement factor. Alopecia was observed in twelve patients and four patients had thrombocytopenia, eight had myalgia, thirteen were observed with new rash, sixteen had Proteinuria, nine had arthritis, two had mucosal ulcers, six patients had nephritis while a single case out of the total was observed for symptoms like Lupus headache, fever, cardiac and ocular manifestations. Data is shown in the Supplementary Table 1.

Alteration in proportions of subsets of T and NK cells Proportions of Helper T- cells (Supplementary Figure 2 (a)), Tregs (Supplementary Figure 2(c)), and of CD3-CD56dim cells (Supplementary Figure 2(d)) were significantly diminished in SLE patients. However, the proportion of cytotoxic T-cells did not vary significantly. In contrast, the proportion of CD3-CD56bright cells (Supplementary Figure 2(e)) tends to increase, though not significantly.

Alteration of ROS The upsurge in intracellular oxidative stress was observed in all subsets of T and NK cells, i.e., Helper T cells (Figure 1(a)), Cytotoxic T-cells (Figure 1(b)) and Treg cells (Figure 1(c)), CD3-CD56dim (Figure 1 (d)) and CD3-CD56bright cells (Figure 1(e)) of SLE patients. Glutathione concentration was reduced significantly in patients as compared to the healthy controls. (Supplementary Figure 3) Glutathione (reduced) oxidizes to the GS-SG (oxidized) form, to neutralize reactive oxidative ions. Glutathione concentration in plasma was reduced significantly with an increase in SLEDAI score. (Figure 6(h))

Alteration in Keap-1/Nrf-2 expression Protein expressions of Keap-1and Nrf-2 were significantly increased in CD3þCD8þ cells (Figures 2(b) and 3(b)) and CD3-CD56dim cells (Figure 2(d) and (d)) of SLE patients as compared to healthy controls. While only the protein expression of Keap-1was elevated significantly in CD4þCD25hi cells (Figures 2(c) and 3(c)) of SLE patients. Expression of neither Keap-1 nor Nrf-2 did vary significantly in CD3þCD4þ (Figures 2 (a) and 3(a)) and CD3-CD56bright cells (Figures 2(e) and 3(e)) in patients from the healthy controls. mRNA expression of Keap-1 did not vary significantly in either CD4þ( Figure 2(f)), CD8þ(Figure 2 (g)) cells or CD56þ cells (Figure 2(h)) in SLE patients while mRNA expression of Nrf-2 was significantly increased only in CD8þ cells (Figure 3(g)) of SLE patients.

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Figure 1. Analysis of intracellular oxidative stress in T and NK subsets by flowcytometry using DCFDA dye in samples from healthy controls and SLE patients. Representative overlay histogram (i) and scatter plot (ii) describing DCFDA MFI as following (a) CD3þCD4þ cells: Healthy (8624  1375); SLE patient (13860  1645) (b) CD3þCD8þ cells: Healthy (8604  1346); SLE patient (15680  2034), (c) CD4þCD25hi cells: Healthy(56590  14280); SLE patient(55400  12960),(d) CD3-CD56dim cells: Healthy (4465  531.6); SLE patients (7600  633.9) (e) CD3-CD56bright cells: Healthy(4027  196.8); SLE patient (9789 558) . *p-value