Increased IL-23R+ Th Cells Population Exhibits Higher SLEDAI-2K Scores in Systemic Lupus Erythematosus Patients

The IL-23/IL-17 axis plays causative roles in the development and progression of systemic lupus erythematosus (SLE). However, it remains unclear if the IL-17RA+ and IL-23R+ T helper (Th) cells populations are associated with the serum IL-17 and IL-23 levels, or with the immunological parameters and disease activities in SLE patients. Herein, we examined the proportion of IL-17RA+ and IL-23R+ Th cells and serum levels of IL-17 and IL-23 in established SLE patients (n = 50) compared with healthy controls (n = 50). The associations of these interleukins and their receptors with immunological parameters [anti-nuclear antibody (ANA), anti-dsDNA antibody, and C-reactive protein (CRP)] and SLE disease activity (SLEDAI-2K scores) in SLE patients were assessed. CD3+CD4+ Th cells of SLE patients demonstrated significantly elevated IL-17RA+ (p = 1.12 x 10-4) or IL-23R+ (p = 1.98 x 10-29) populations compared with the healthy controls. Serum IL-17 levels were significantly lower in SLE patients compared with the healthy controls (p = 8.32 x 10-5), while no significant difference was observed for the IL-23 serum levels between both groups. IL-23R+ Th cells population was significantly associated with higher SLEDAI-2K scores (p = 0.017). In multivariate analysis, the proportion of IL-23R+ Th cells remained significantly associated with higher SLEDAI-2K scores independent of prednisolone intake (p = 0.027). No associations were observed between the interleukin parameters (i.e., IL-17, IL-23, IL-17RA+ Th cells, and IL-23R+ Th cells) with ANA, anti-dsDNA, and CRP status, suggesting that the IL-17/IL-23 axis acts independently of these immunological parameters. In conclusion, our results support that therapeutic inhibition of the IL-23/IL-17 axis receptors on Th cells, particularly IL-23R, is potentially relevant in SLE patients.


INTRODUCTION
Interleukin (IL) is a group of cytokines crucial in regulating immune responses such as inflammation as well as mediating immune cells growth, differentiation, activation, and migration. Imbalanced T cell subsets have been identified in SLE patients associated with SLE immunopathogenesis (1)(2)(3)(4)(5). Naïve CD4 + T helper (Th) cells differentiate on activation into various cell subsets, namely Th1, Th2, and Th17, with specific profile of cytokines production and different immunopathological implications. IL-17 produced by Th17 cells has been implicated in the pathogenesis of autoimmune diseases including SLE (4). IL-23 is produced by macrophages and dendritic cells to promote the expansion and survival of Th17 cells for the production of IL-17 (6). By stimulating proinflammatory cytokines production, IL-23 is able to activate memory T cells, affect IFN-g production, and trigger the response of Th1 cells, which plays crucial roles in the development of SLE (7)(8)(9)(10). IL-23 can aggravate autoimmune severity by stimulating the activities of pathogenic Th17 cells to continually produce IL-17 (11), and this pathogenic pathway is termed as the IL-23/IL-17 axis in SLE (12,13).
It is thought that cytokine receptors identification is more therapeutically beneficial on account of its stability as well as cytokines pleomorphism. Cytokine receptors activate intracellular signaling which controls a wide range of biological and clinically relevant functions including inflammatory reactions and the expansion of immune cells. The receptor for IL-17 cytokine, IL-17RA, has a cytoplasmic tail with docking sites for various signaling intermediates and has been recognized at elevated levels in specific tissues such as the thymus, bone marrow, spleen tissues, and hematopoietic cells (14). IL-23R, a receptor for IL-23, is expressed by various immune cells including dendritic cells, macrophages, and T cells (15). IL-23 and its receptor IL-23R are necessary for the proliferation of pathogenic IL-17-producing T cells shown to be critical for lupus pathogenesis (16), and IL-23R-deficit prevents the development of lupus nephritis in lupus-prone mice (16).
Multiple studies have reported that elevated IL-17 serum levels in SLE patients is associated with higher SLEDAI scores, a parameter to assess SLE disease activities (17)(18)(19)(20), compared with the controls (21)(22)(23)(24)(25)(26). Independent reports have indicated that Th17 cells can promote the development of SLE (22,27). Although the initial steps of IL-17 differentiation from naïve CD4 + Th cells does not involve IL-23, it has been shown that IL-23 plays a significant role in the expansion and maintenance of Th17 cells, enabling IL-17 to be produced (7,(28)(29)(30). Collectively, the IL-23/IL-17 axis plays key roles in the pathogenesis of SLE. However, the profile of IL-17RA + or IL-23R + Th cells in SLE patients compared with healthy individuals, as well as their associations with relevant parameters in the disease, remain unclear.
In this study, we set out to investigate whether serum IL-17 and IL-23 levels or the proportion of IL-17RA + or IL-23R + Th cells differ between SLE patients and the healthy controls. More importantly, the potential associations between these serum cytokines and Th cell subsets with SLE disease activity measured by the SLEDAI-2K scores were examined. Associations of the SLEDAI-2K scores with the clinicodemographical (e.g., age) and immunological (e.g., ANA, anti-dsDNA) parameters were also investigated, and compared with the IL-23/IL-17 axis parameters using multivariate and false discovery rate (FDR) analyses.

Recruitment of SLE Patients and Healthy Controls
According to published flow cytometry and/or ELISA casecontrol studies utilizing peripheral blood samples to investigate IL-17 or IL-23 parameters in SLE patients compared with controls (31)(32)(33)(34)(35), sample size calculation was performed according to the difference of means between two independent groups using the software G*Power (version 3.1.9.3). A two-tailed hypothesis, a-error probability of 0.05, power (1-b error probability) of 0.80, effect size of 0.6, allocation ratio (N2/N1) of 1, and dropout rate of 10% were adopted. This yielded a total sample size of 100 participants divided equally between the healthy controls and SLE patients group (n = 50 per group). Thus, in this cross-sectional case-control study, 100 participants were recruited consisting of 50 Table 1). The selection of SLE patients and healthy controls for this study was conducted on the basis of random sampling. The clinicodemographic data of the patients were obtained from the Records Unit of Hospital Universiti Sains Malaysia (HUSM). SLEDAI-2K score for each patient was assigned by the attending clinician at the time of this study (i.e., on the same day when the samples were taken) according to the SLEDAI-2K score calculator (https://qxmd.com/calculate/calculator_335/sledai-2k) (38). Data on the current treatments received (i.e., prednisolone, immunosuppressants, or anticonvulsant) were obtained at the time of the study. Recruitment of the subjects and retrieval of their data were conducted from November 2018 to May 2019. The study procedures were approved by the Human Research Ethics Committee of Universiti Sains Malaysia (JEPeM) (approved ethics code: USM/JEPeM/17120680). All procedures carried out in this study involving peripheral blood samples were in accordance with the 1964 Declaration of Helsinki and its later updates, and with the institutional ethical standards. All participants were briefed about the study during the recruitment, and both verbal and written informed consents were obtained from each participant after detailed explanations of the study. The documented written informed consent permitted the investigators to conduct the recruitment, draw the blood of the participants for the experiments, and publish the results. All samples were labeled anonymously, and all data were recorded, stored, and analyzed anonymously to ensure that none of the private information such as the name of participants were disclosed. All experimental protocols were conducted according to the institutional relevant guidelines and regulations.

Serum and PBMCs Isolation
A total of 10 mL of peripheral blood was withdrawn from the healthy controls (n = 50) and SLE patients (n = 50). Of this, 6 mL were allocated in a clot activator tube for serum collection and 4 mL was placed in a BD Vacutainer ® EDTA tube (Becton Dickinson, USA) for peripheral blood mononuclear cells (PBMCs) isolation. The collected blood samples were left in an upright position for 30 minutes at room temperature (RT) prior to centrifugation at 1,000 x g at 4°C for 15 min to remove the blood clot. The serum obtained from centrifugation was aliquoted immediately into 1.5 ml microcentrifuge tubes which were labeled with the identification number of the subject and the intended tests (e.g., ANA, anti-dsDNA, CRP, IL-17, IL-23), and stored at -20°C. On the other hand, PBMCs were isolated from peripheral blood by standard Histopaque-1077 (Sigma-Aldrich, USA) density centrifugation. The collected peripheral blood was inverted and left in an upright position for 30 min at RT. Four milliliters of Histopaque-1077 was transferred into a new falcon tube followed by 4 mL of peripheral blood from the EDTA tube layered carefully on top of the Histopaque-1077 with a 1:1 ratio. The blood mixture was then centrifuged at 400 x g (30 min at RT, brakes off). The PBMCs layer obtained was transferred carefully using a Pasteur pipette into a new falcon tube containing 2 mL of PBS. The mixture was then added with PBS to a total of 10 mL, mixed gently, and resuspended. The supernatant obtained after being centrifuged at 200 x g for 10 min was discarded and the pellet was resuspended using 5 mL of PBS and centrifuged. The washing process was then repeated with 500 ml of PBS before cell counting using a hemocytometer.
Flow Cytometry Analysis for IL-17RA + and IL-23R + Th Cells A total of 5 mL of isolated PBMCs were centrifuged at 1,000 rpm for 10 min. The cells were washed with 1 ml of cold FBS followed by centrifuging at 1,000 rpm for 15 min. Then, cells were resuspended with cold FBS to a final concentration of 2 x 10 6 cells/ml. The cell mixtures were distributed and 50 ml of cell suspension was aliquoted into a microcentrifuge tube. Fluorochrome-labeled antibodies for CD3 (APC-Cy7; cat no. 560176), CD4 (PE-Cy7; cat no. 557852), IL-17RA (BB700/ PerCP-Cy5.5; cat no. 747946), IL-23R (FITC; cat no. FAB14001F), and isotype controls (APC-Cy7; cat no. 560167, PE-Cy7; cat no. 557872, BB700/PerCP-Cy5.5; cat no. 566404, FITC; cat no. GZ-IC0041F) (Beckton Dickinson, USA) were used at 5 ml per test whereby the antibody concentration for each test was 0.0182 mg/ml in a final volume of 55 ml, corresponding to 1 mg of antibody per test according to the protocols of the manufacturer. Each antibody was added accordingly into the cells mixture and incubated on ice protected from light for 30 min. Washing steps were conducted twice with 1 ml of FBS, the tube inverted, and supernatant blotted from the cell pellets. The cell pellets were resuspended in 500 ml of FBS. The stained cells were then transferred into an FACS tube and analyzed using the FACS CANTO II (Beckton Dickinson). The results were acquired with the FACS Diva software (Beckton Dickinson) and analyzed with FlowJo (v10; TreeStar, USA).
ELISA for Serum IL-17 and IL-23 Levels ELISA tests were conducted according to manufacturer's instructions (BioLegend, USA). Briefly, monoclonal antibodies specific for human IL-17 (cat no. 433917) and IL-23 (cat no. 437607) were pre-coated onto the provided 96-well strip microplate. Standards and samples for both IL-17 and IL-23 levels detection were run in duplicate, and the standard curve was obtained for each assay. All reagents were brought to RT prior to use, and 50 ml of 20X wash buffer was diluted in 950 ml of sterilized dH 2 O to prepare a 1X wash buffer. Each microplate used was labeled accordingly, and the washing process to remove unbound materials was conducted with 300 ml of 1X wash buffer per well, and the residual buffer was blotted by tapping firmly the microplate upside down on a clean absorbent paper. The final washing process was conducted for 30 seconds to 1 min for each wash to minimize the background. Absorbance values were recorded at 450 nm and 570 nm by the ELISA microplate reader (Tecan, Switzerland).

Anti-Nuclear Antibodies Indirect Immunofluorescence Assay (IIFA)
The IIFA for the AESKUSLIDES ANA-HEp-2 test was performed according to the instructions of the manufacturer (AESKU DIAGNOSTICS, Germany). All components of AESKUSLIDES-ANA-Hep-2 were allowed to reach RT before usage. Serum samples were diluted following the ratio of 1:80 with PBS (20 ml of serum + 1,580 ml of PBS), and 25 ml of each diluted serum and controls (ready-to-use format) were pipetted into the appropriate wells. Slides were incubated in a humidified chamber for 30 min at RT. The slides were subsequently rinsed with the 1X wash buffer using a squeeze wash bottle followed by 10 min of washing in a glass staining dish containing fresh wash buffer. The slides were then transferred to the humidified chamber and excess wash buffer was carefully removed by filter paper. Subsequent incubation with secondary antibody conjugated with FITC was conducted in the humidified chamber for 30 min at RT in the dark. Washing steps were then repeated before a coverslip was mounted on each slide. The specific green fluorescent staining of the antigen-antibody complex was observed and interpreted with an Olympus BX-60 fluorescence microscope (Olympus, Tokyo, Japan). Interpretation of the ANA staining patterns were conducted by immunologists (KKW, CMCH) according to the International Consensus on ANA Patterns (ICAP; http://www.anapatterns. org/) nomenclature and definitions of 29 HEp-2 IIFA patterns, where each pattern is ascribed with an alphanumeric code (AC-1 to AC-29) (39).

Anti-dsDNA ELISA Test
ImmuLisa ™ Anti-dsDNA Antibody Enhanced ELISA tests were conducted according to the instructions of the manufacturer (Immco Diagnostics, USA). Anti-dsDNA IgG antibodies were detected by the test. Firstly, the microwells were coated with purified dsDNA antigen followed by blocking steps to reduce non-specific binding. Controls, calibrators, and patient sera were incubated in the antigen-coated wells, allowing the specific antibody in the serum to bind to the dsDNA antigens. Bound antibodies were detected by adding the anti-human IgG conjugate enzyme to the microwells and the presence of the antibodies were detected by a color change produced after a specific enzyme substrate (TMB) were added. After the washing process, to remove the unbound antibody and conjugate, the reaction was stopped and the intensity of the color change was detected by a spectrometer at 450 nm. Linear measurement was recorded and the results were reported as anti-dsDNA antibody positive or negative.

C-Reactive Protein Immunoturbidimetric Test
QuikRead go CRP test (Orion Diagnostica, Finland) is an immunoturbidimetric test based on microparticles coated with anti-human CRP F(ab) 2 fragments, and the test was carried out according to manufacturer's instructions. The CRP present in the serum reacts with the microparticles resulting in the turbidity of the solution as quantitatively measured by the QuikRead go instrument.

Statistical Analysis
The categorical and numerical data for the clinicodemographical and immunological characteristics of SLE patients and healthy controls were described using frequency (%) and median (interquartile rage; IQR), respectively. Normality of data was tested using the Shapiro-Wilk test. For comparison of numerical variables between two groups, unpaired t-test or Mann-Whitney test was used for normally and not normally distributed variables, respectively. Kruskal-Wallis test and the subsequent Dunn's multiple comparisons post hoc test were used to compare numerical variables in more than two groups. For correlation analysis between two sets of numerical variables, Pearson and Spearman correlation coefficient was used for normally and not normally distributed variables, respectively. For categorical variables, their associations were compared using the Chi-square test and Fisher's exact test (used when >20% of cells have expected frequencies below five) (40). Statistical analysis was performed using the GraphPad Prism (v9; GraphPad Software Inc., USA). Multivariate analysis of IL-23R + Th cells combined with age or prednisolone intake versus SLEDAI-2K score groups (i.e., SLEDAI-2K score 0 or ≥1) was conducted using multiple logistic regression (IBM SPSS v22; SPSS Inc., Chicago, IL, USA). For all tests, two-tailed p<0.05 was considered statistically significant.

Immunological Characteristics of SLE Patients
The patients had their SLE diagnosis established previously where all patients were positive for ANA, and 36 out of 43 (83.7%; anti-dsDNA test was not initially conducted for 7 patients) were positive for anti-dsDNA antibody leading to their SLE diagnosis according to the established criteria. In the current study, ANA and anti-dsDNA tests were conducted anew along with the CRP test. The proportion of SLE patients according to their ANA (n = 45/50 ANA-positive; 90%), anti-dsDNA (n = 36/50 anti-dsDNA-positive; 72%), or CRP status is presented in Figure 1A. The proportion of SLE patients positive for ANA (n = 45/50; 90%) was higher than those negative for ANA (n = 5/50; 10%, i.e., ANA seroconversion to ANA-negative post-treatment). The ANA and anti-dsDNA test results of SLE patients prior to disease diagnosis and current ANA and anti-dsDNA results as well as CRP levels are summarized in Supplementary Table 2.

Determination of IL-17RA + and IL-23R + Th Cells Populations by Flow Cytometry
The peak of flow cytometry histogram for unstained lymphocytes were fixed at 1 for all fluorochromes (PE-Cy7, APC-Cy7, PerCP, and FITC). To observe a distinct peak between the negative and positive cells populations, the histograms of isotype control (red shaded) and fluorochrome-stained (blue shaded) were overlapped for each fluorochrome to distinguish the negative and positive cells populations (Supplementary Figure 3). The gating strategy for CD3 + CD4 + Th cells and subsequent determination of IL-17RA + or IL-23R + Th cells subset is summarized in Supplementary Figure 4.

Serum IL-17 and IL-23 Levels in SLE Patients and Healthy Controls
Serum IL-17 levels were significantly lower in SLE patients compared with the healthy controls (4.63 vs 5.16 pg/ml; p = 8.32 x 10 -5 ). The serum levels of IL-23 did not differ significantly between the SLE patients and healthy controls (22.85 vs 25.23 pg/ml; p = 0.728) (Figure 3). Higher serum IL-17 levels were significantly and positively associated with the proportion of IL-17RA + Th cells in SLE patients (r = 0.294; p = 0.038). However, these associations were not observed in the healthy controls. In addition, serum IL-17 and IL-23 levels were not associated with the proportion of IL-23R + Th cells in both the healthy controls and SLE patients ( Figure 4).
In addition, we also examined the association between serum IL-17 and IL-23 levels, and IL-17RA + or IL-23R + Th cells population with the immunological characteristics in SLE patients. No significant association was observed in any of the comparisons made between serum IL-17 or IL-23 levels, and IL-17RA + or IL-23R + Th cells proportion with ANA, anti-dsDNA, or CRP status (Supplementary Table 5).

Multivariate and False Discovery Rate Analyses of the Associations With SLEDAI-2K Scores
As younger age, prednisolone intake, and the proportion of IL-23R + Th cells were associated with higher SLEDAI-2K scores, the univariate association of IL-23R + Th cells proportion with age or prednisolone intake was first assessed.  Higher IL-23R + Th cells proportion was significantly associated with a younger age (p = 0.025) ( Figure 5A), and no significant association was observed for prednisolone intake ( Figure 5B). Multivariate analysis of IL-23R + Th cells and age or prednisolone intake versus SLEDAI-2K scores demonstrated that IL-23R + Th cells proportion was not significantly associated with higher SLEDAI-2K scores when age was taken into account (OR: 1.10, 95% CI: 0.97-1.25; p = 0.146). However, IL-23R + Th cells proportion remained significantly associated with higher SLEDAI-2K scores when prednisolone intake was taken into account (OR: 1.18, 95% CI: 1.02-1.36; p = 0.027) ( Figure 5C). A total of 36 associations were tested for a potential significance in this study where 6 of the associations demonstrated significant p-values. To account for multiple hypothesis testing, the p-values of these associations were corrected using the Benjamini-Hochberg (BH) FDR procedure. The threshold to use for significance for the generated q-values (BH-corrected p-values) was 0.25 (41,42). Two of the six   Table 6). Finally, the complete data of the clinico-demographical and immunological characteristics, as well as the serum interleukin and their receptors data for each individual healthy control and SLE patient involved in this study, are presented in Supplementary Table 7.

DISCUSSION
In this study, we demonstrated that CD3 + CD4 + Th cells were significantly higher in SLE patients compared with the healthy controls. In terms of IL-23/IL-17 axis receptors, we observed that IL-17RA + and IL-23R + Th cells were significantly increased in SLE patients than in the healthy controls. This is comparable with another study where it was reported that the percentages of CD4 + IL-23R + cells from PBMCs was significantly higher in SLE patients (35). The production of IL-17 is driven by IL-23 (11,43) and IL-23R is expressed on activated Th17 cells (44). Furthermore, the increased IL-17RA + and IL-23R + Th cells populations in SLE patients may be attributable to their shared molecular signaling pathway as the binding of IL-23 with its receptor complex activates STAT3 signaling in Th17 cells leading to IL-17 production through differentiation of Th17 cells (45,46). Along with our observation of increased IL-23R + Th cells in SLE patients associated with higher SLEDAI-2K scores, IL-23R + Th cells may play crucial roles in the progression and aggressive disease course of SLE patients. Multiple studies have reported significantly elevated serum IL-23 levels in SLE patients compared with healthy controls (12,33,47). Specifically, serum IL-23 levels were significantly higher in active SLE patients compared with inactive and healthy controls group (32,48). However, our study demonstrated that serum IL-23 levels did not differ significantly between SLE patients and healthy controls, suggesting that increased IL-23R signaling might be achieved via higher surface levels of IL-23R instead of serum IL-23 levels. On the other hand, serum IL-17 was lower in SLE patients but IL-17RA + Th cells population was higher in the patients, suggesting that a higher consumption of serum IL-17 by increased IL-17RA may occur in SLE patients. This is also supported by our observation that serum IL-17 levels had a positive relationship with IL-17RA + Th cells in SLE patients but not in the healthy controls. However, these hypotheses require further validation by functional and mechanistic studies such as gene knockdown or knockout methodologies.
In addition, significantly downregulated serum IL-17 levels in our cohort of SLE patients is in contrast with the findings of elevated serum IL-17 levels in SLE patients as reported previously (22)(23)(24)26). This may also be due to the use of hydroxychloroquine, an immunosuppressant routinely used to treat SLE patients (27,(49)(50)(51)(52), by the majority of our cohort of SLE patients (n = 45/50; 90%). It has been reported that hydroxychloroquine use resulted in a significant reduction of disease activities in SLE patients where most of the cytokines and pro-inflammatory markers were reduced following the treatment (51)(52)(53)(54). Hydroxychloroquine has been shown to inhibit Th17 cells proliferation and differentiation along with reduced IL-17 production by Th17 cells in vitro (27). Essentially, SLE patients (n = 6) demonstrated lower levels of serum IL-17 and reduced proportion of CD3 + CD8 -IL-17 + Th17 cells after receiving 4 weeks of treatment with hydroxychloroquine and prednisone (27). Furthermore, in PBMCs derived from a cohort of 18 SLE patients, stimulation of these PBMCs with phorbol 12-myristate 13-acetate and ionomycin displayed reduced IL-17 production after treatment with hydroxychloroquine (55). Elevated serum levels of IL-17 correlated positively with the SLEDAI-2K scores in SLE patients as reported in previous studies (26,56). In contrast, several studies demonstrated that there were no associations observed between IL-17 (21,57) or IL-23 (58) with the SLEDAI-2K scores, similar with the findings in our study.
In our multivariate analysis, IL-23R + Th cells and SLEDAI-2K association was dependent on age in our SLE cohort, tallying with the univariate comparison where younger SLE patients had a significantly higher proportion of IL-23R + Th cells. These observations are comparable with independent studies in which younger SLE patients are more likely to present with nephritis and greater organ damage (59)(60)(61)(62), and more likely to be hospitalized with SLE as a diagnosis (59). Whether an increased proportion IL-23R + Th cells plays a direct, causative role in exacerbating disease activities in younger SLE patients remains to be elucidated. On the other hand, IL-23R + Th cells proportion remained significantly predictive of worse SLEDAI-2K scores when prednisolone intake was taken into account in the multivariate analysis, suggesting that prednisolone did not affect IL-23R + Th cells proportion in SLE patients. This also suggests that alternative therapeutics specifically targeting IL-23R or its downstream signaling may be beneficial for patients who fail to respond to prednisolone.
In terms of immunological parameters, 10% of the established SLE patients in our cohort demonstrated ANA seroconversion. Loss of ANA over time in established SLE patients has been reported in up to approximately 30% of the patients (63)(64)(65)(66)(67). ANA seroconversion may reflect responses to therapies or naturally-occurring exhaustion of autoimmune clones (66), and whether our population of SLE patients displayed a reduced retention of autoimmune clones post-therapy is subject to future investigations. We observed that the routine biomarker anti-dsDNA did not correlate with the SLEDAI-2K scores in SLE patients. This is in agreement with multiple independent reports whereby anti-dsDNA status does not show significant relationship with SLE disease activities (68)(69)(70). As anti-dsDNA antibodies do not always correlate with SLE disease activities, it is thought that anti-dsDNA encompasses a large spectrum of different chromatin targets that may explain their heterogeneity in lupus activities (70,71). This proposal appears to tally with past observation in which IgA, but not IgG or IgM, anti-dsDNA was significantly elevated in SLE patients with higher disease activities (72). This represents fertile grounds for further investigation and validation. In terms of CRP, it has been reported that IL-17 correlates with CRP levels and other immunology diagnostic parameters including IgG and IgM (57). However, our study demonstrated that there were no associations between the interleukin parameters (i.e., IL-17, IL-23, IL-17RA + Th cells, and IL-23R + Th cells) with the immunological parameters investigated in this study, suggesting that the IL-17/ IL-23 axis acts independently of ANA, anti-dsDNA, and CRP. We acknowledge the limitations of the study as follows: 1) Established SLE patients were recruited whereby all patients had been on treatment, hence, a proportion of the patients demonstrated ANA seroconversion and lower disease activities; 2) A confirmatory cohort of SLE patients especially of SLE patients recruited from multiple centers is recommended to validate and expand on the findings of the current study; and 3) We did not investigate which Th cell subsets expressing IL-17RA or IL-23R were altered such as Th1 and Th17 populations. Nonetheless, we recommend this as an avenue for future investigations particularly for IL-23R.
In conclusion, our study demonstrated that increased populations of IL-17RA + and IL-23R + Th cells occurred in SLE patients, and IL-23R + Th cells population was associated with higher SLEDAI-2K scores. Our studies support future research either in the production or clinical trials of therapeutic antibodies targeting IL-23/IL-17 axis receptors in SLE, particularly IL-23R, and the inhibition of IL-23/IL-17 receptors is a promising therapeutic strategy in this autoimmune disorder.

DATA AVAILABILITY STATEMENT
The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article's/ Supplementary Material.

ETHICS STATEMENT
The studies involving human participants were reviewed and approved by Human Research Ethics Committee of Universiti Sains Malaysia (JEPeM) (approved ethics code: USM/JEPeM/ 17120680). All patients and participants provided their written informed consent to participate in this study.

AUTHOR CONTRIBUTIONS
CMCH and KKW conceived the study and recruited research grants. AFI, NDMS, WSWG and CMCH recruited the samples and retrieved the clinico-demographical data of the subjects. AFI and NDMS conducted the experiments, and KKW supervised the experiments. AFI, CMCH and KKW designed the study. AFI and KKW performed data analysis, generated the figures and tables, conducted literature searches, and wrote the manuscript. KKW revised the manuscript. All authors contributed to the article and approved the submitted version.

ACKNOWLEDGMENTS
We would like to thank the staff members at the Department of Immunology, School of Medical Sciences, USM and nurses at the Rheumatology Clinic, HUSM for their technical assistance.