Effect of Add-on Hydroxychloroquine Therapy on Serum Proinammatory Factor Levels in Patients with Systemic Lupus Erythematosus With or Without Lupus Nephritis

Background: We investigated the effects of add-on hydroxychloroquine (HCQ) therapy on the expression of proinammatory cytokines and other factors in systemic lupus erythematosus (SLE) patients with low disease activity. Methods: Patients who had low disease activity of at least 3 months duration were included. Patients with a history of lupus nephritis (LN+) must have been in remission for at least 3 months prior to enrollment. Serum levels of interferon interferon-α, S100A8, S100A9, tumor necrosis factor(TNF) -α, interleukin(IL)-2, IL-6, IL-8, vascular endothelial growth factor (VEGF)-A, Monocyte Chemotactic Protein-1, macrophage inammatory protein-1α, IL-1β, Interleukin 1 receptor antagonist(IL-1ra), and Granulocyte Colony Stimulating Factor were measured immediately before and 3 months after treatment with oral HCQ treatment. Results: Of the 42 patients enrolled in the study (4 males, 38 females, mean age ± standard deviation age 41.4±13.3 years), 19 patients had a history of lupus nephritis but were currently in remission (LN+), and the remaining 23 patients had no history of LN (LN−). Serum levels of IL-1ra, S100A8, and S100A9 at baseline were signicantly higher in the LN+ group compared with the LN− group (p=0.0092, p=0.012, and p=0.0043, respectively). In the full cohort, HCQ treatment led to signicantly reduced serum levels of TNF-α, IL-6, VEGF-A, IL-1ra, IL-2, S100A8, and S100A9, and to decreased, albeit not signicantly, levels of IL-8 and MIP-1α. The HCQ-induced changes in serum IL-8, IL-1ra, S100A8, and S100A9 levels were greater for patients in the LN+ group than those in the LN−group (p=0.0039, p=0.0011, p=0.0201, and p=0.0092, respectively).


Introduction
Systemic lupus erythematosus (SLE) is a chronic systemic autoimmune disease in which multiple organ systems can be damaged by autoantibodies, immune complexes, and in ammation [1]. The pathogenesis of SLE results from the complex interplay of immunological, genetic, and environmental factors [2]. Several studies have identi ed associations between levels of various proin ammatory cytokines and SLE disease activity with speci c clinical manifestations. In particular, dysregulation of interferons (IFNs) has been suggested to be involved in the pathogenesis of SLE. Accordingly, transcriptome analysis has con rmed that numerous IFN-stimulated genes are upregulated in the peripheral blood mononuclear cells of patients with SLE, and serum IFN-α levels are elevated in SLE patients in a manner that correlates with disease activity and severity [3][4][5]. Bauer et al. reported that levels of IFN and IFN-inducible chemokines, such as macrophage in ammatory protein-1 (MIP-1), monocyte chemotactic protein-1 (MCP-1) and interferon-inducible protein-10, correlate with disease activity, as measured by various disease activity indices such as erythrocyte sedimentation rate and anti-dsDNA antibody titers [6,7]. Other proin ammatory cytokines that have been shown to correlate with SLE disease activity include tumor necrosis factor-α (TNF-α), interleukin (IL)-6, IL-8, IL-10, and vascular endothelial growth factor (VEGF) [8][9][10][11][12]. Recent work has reported that myeloid-related proteins (MRPs), also known as damage-associated molecular patterns, may be involved in the pathogenesis of multiple autoimmune diseases [13,14]. In SLE patients, serum levels of these proteins, which include MRP8, MRP14, and S100 proteins, are positively correlated with disease activity, especially lupus nephritis (LN) [15]. Histological analyses have also shown these proteins to be expressed in renal tissues in SLE patients, with levels proportional to the severity of LN [16,17].
Despite these studies, the associations between serum levels of proin ammatory cytokines and disease characteristics remain to be fully elucidated. Moreover, there is a need to identify biomarkers to aid in the diagnosis and monitoring of disease activity in patients with SLE, especially those with involvement of the kidneys, the major organs involved in SLE. However, the effects of many SLE treatments on proin ammatory cytokine levels remain unclear.
Hydroxychloroquine (HCQ) is a common add-on treatment for SLE patients with low disease activity (LDA), although the European League Against Rheumatism recommended HCQ for all patients with SLE in 2019 [18]. HCQ was approved for the treatment of SLE in Japan in July 2015, and since then, it has been prescribed as an add-on treatment for many SLE patients on immunosuppressants, especially women of child-bearing age. Although the mechanisms may be unclear, there is evidence for multiple bene cial effects of HCQ in SLE [19]. Willis et al. showed that HCQ therapy resulted in signi cant clinical improvement in a manner that strongly correlated with reductions in IFN-α levels [20]. Our previous study suggested that HCQ modulated the expression of S100 proteins in SLE patients with LDA [21]. However, little is known about the effects of HCQ on biomarkers in SLE patients with LDA. In the present study, we investigated the effect of add-on HCQ on the expression of proin ammatory cytokines and other serum factors in SLE patients with LDA who were receiving immunosuppressants.

Patients
This was a single-center prospective study. We enrolled subjects who were diagnosed with SLE using the American College of Rheumatology criteria [22] or the Systemic Lupus Collaborating Clinics criteria [23] and began HCQ treatment for the rst time between September 2015 and March 2019. All patients had a ≥ 3-month history of LDA prior to enrollment, de ned as (i) a SELENA-SLEDAI score of ≤ 8 with no activity in major organ systems, such as renal involvement, neuropsychiatric SLE, cardiopulmonary involvement, and vasculitis; (ii) current prednisolone or equivalent dose of ≤ 10 mg per day; and (iii) welltolerated maintenance doses of other immunosuppressants. Pregnant women and patients who were not currently in complete renal remission [24], regardless of LN history, were excluded. Patients who began anti-thrombotic therapy or add-on immunosuppressants after starting the HCQ treatment were excluded from the study. Informed consent was obtained from all participants. The study was approved by the ethical committee of Kagawa University (Heisei30-047).

Treatment and outcomes
Patients were administered oral HCQ sulfate (Plaquenil; Sano -Winthrop, Paris, France) continuously for at least 3 months. HCQ was administered at a dose based on ideal body weight (IBW) calculated using the modi ed Broca's method: 200 mg daily for patients with IBW < 46 kg; 200 mg and 400 mg on alternate days for IBW ≥ 46 kg and < 62 kg; and 400 mg daily for IBW ≥ 62 kg. Clinical parameters (age, gender, HCQ dose, immunological biomarkers, disease activity indices, and skin scores) were recorded before and after HCQ treatment. Disease activity was evaluated using the SELENA-SLEDAI 2011 criteria [25]. Cutaneous disease activity was evaluated using the Cutaneous Lupus Erythematous Disease Area and Severity Index (CLASI) [26]. In accord with the CLASI improvement criteria of Klein et al. [27], the principal investigator designated CLASI as improved, unchanged, or worse compared with the previous visit.
Patients classi ed as improved were de ned as "CLASI responders" and those classi ed as unchanged or worse were de ned as "CLASI non-responders". Immunological activity was determined by measuring serum levels of complement factors (C3, C4, CH50), anti-double stranded DNA (dsDNA) antibodies, and total white blood cell counts, lymphocyte counts, and platelet counts. Speci c ELISA kits were used to measure serum S100A8, S100A9 (CircuLex ELISA Kits

Statistical analysis
Data are presented as the mean ± standard deviation (SD) unless otherwise noted. Immunological biomarkers and proin ammatory cytokine levels were compared using Student's t test for continuous variables or the Wilcoxon signed-rank test for non-normally distributed data. Comparisons between groups were performed using the Wilcoxon rank sum test. All p values were two-sided, and a p value < 0.05 was considered signi cant. Data were analyzed using JMP® 13 software (SAS Institute, Cary, NC, USA).

Baseline characteristics
Forty-two SLE patients (38 women, 4 men) with sustained LDA of at least 3 months duration were enrolled in this study. Their baseline characteristics are shown in Table 1. The mean (± SD) age was 41.4 ± 13.3 years, the mean disease duration was 14.8 ± 11.8 years, the mean SELENA-SLEDAI score was 3.7

Baseline serum levels of proin ammatory factors
Serum levels of IL-1ra, S100A8, and S100A9 at baseline were signi cantly higher in SLE patients with a history of LN (LN+) compared with those without a history of LN (LN−) (p = 0.0092, p = 0.012, and p = 0.0043, respectively; Fig. 1). There were no signi cant differences between the LN + and LN − groups in the baseline levels of other proin ammatory cytokines (Fig. 1). Some proin ammatory cytokines were not detected in all SLE patients; for example, serum IFN-α and IL-1β were detected in only 6 and 15 of the 42 patients, respectively (Fig. 1).

Serum levels of proin ammatory factor after HCQ treatment
We analyzed serum levels of proin ammatory cytokines at 3 months after initiation of HCQ treatment. As shown in Fig. 2, TNF-α, IL-6, VEGF-A, IL-1ra, IL-2, S100A8, and S100A9 decreased signi cantly after HCQ treatment. Although IL-8, MIP-1α, and IL-1β levels were also decreased, the difference from baseline was not signi cant. In the patients with detectable levels of MCP-1 (n = 42), G-CSF (n = 37), and IFN-α (n = 6) at baseline, no change after add-on HCQ treatment was detected (Fig. 2). The HCQ-induced changes in IL-8, IL-1ra, S100A8, and S100A9 levels were signi cantly greater in the LN + group compared with the LN − group (Fig. 3). As for association with proin ammatory factors, changes in IL-8, IL-1β, IL-1ra and IL-2 levels correlated with those of serum S100 protein ( Table 2). No associations were detected between changes in serum proin ammatory cytokine levels and immunological biomarkers or disease activity scores (Supplemental Material Table).

Discussion
Previous studies have demonstrated signi cant changes in many proin ammatory cytokines, especially IL-1, IL-6, TNF-α, and IFNs, in SLE patients [28,29]. However, serum levels of most of the proin ammatory cytokines measured here were not associated with disease activity indices such as, SLEDAI and CLASI, or with serum immunological biomarkers, most likely because our study only included SLE patients with LDA.
We detected decreases in serum levels of TNF-α, IL-6, VEGF-A, IL-1ra, IL-2, MIP-1α, IL-8, IL-1β, S100A8, and S100A9 after add-on HCQ treatment. Our results differ from those of Monzavi et al., who reported no change in serum IL-8 levels after treatment of newly diagnosed SLE patients with HCQ [30]. This difference, together with the observed lack of response of other factors, such as MCP-1, to HCQ treatment, is likely to be due to the inclusion of only patients with LDA. Plasma and urinary IL-8 levels are reportedly associated with LN activity [8,[31][32][33], and serum IL-6, IL-8 and IL-18 levels have been proposed to be useful predictors of relapse in SLE [34]. We found that HCQ had a greater effect on serum IL-8 levels in patients with a history of renal involvement compared with patients with no history, suggesting that HCQ may help improve the prognosis and prevent relapse of LN. Of note, it is possible that even renal lesions considered to be in remission may express elevated levels of proin ammatory cytokines.
Despite its common use, the mechanism of action of HCQ in autoimmune diseases is unclear. HCQ is a weak base and is known to raise the pH of acidic intracellular vesicles and interfere with their physiological functions, including autophagy and antigen processing [35]. In addition, HCQ interferes with intracellular signaling, which may suppress the response to engagement of the innate Toll-like receptors (TLRs), thereby inhibiting the production and release of cytokines and promoting apoptosis in lymphocytes and endothelial cells [36,37]. Thus, both acidi cation of endosomal vesicles and increased lymphocyte apoptosis following HCQ treatment may contribute to the decreased production of proin ammatory cytokines. In addition, HCQ-mediated inhibition of TLR activation suppresses the activity of plasmacytoid dendritic cells and autoreactive B cells in SLE patients [37], leading to a reduction in in ammation. Indeed, Sacre et al. demonstrated that HCQ treatment of SLE patients reduced the ability of plasmacytoid dendritic cells to produce IFN-α and TNF-α in response to TLR-9 and TLR-7 stimulation in vivo [38]. We believe that the mechanism of cytokine reduction by HCQ in the present study may be mediated by these effects.
We previously reported that HCQ modulates serum levels of S100A8 and S100A9 in SLE patients with LDA [21]. S100 proteins are components of neutrophil extracellular traps (NETs), which play an important role in the pathogenesis of SLE [39] [40]. In addition, chloroquine and HCQ have been reported to inhibit NETs in vivo and in vitro [41,42], which may suggest a mechanism for the regulation of S100 proteins. S100A8 and S100A9 proteins upregulate the expression of proin ammatory cytokines such as IL-6 and IL-8 [43,44]. Therefore, HCQ-mediated modulation of S100 proteins may also be involved in the suppression of proin ammatory cytokine expression in SLE patients.
There are several limitations to this study. First, we did not monitor HCQ adherence by measuring blood HCQ levels. Second, whether the change in proin ammatory cytokine levels were a direct result of add-on HCQ treatment is di cult to determine unequivocally because some patients were receiving other immunosuppressants. Third, the sample size was small due to the strict inclusion and exclusion criteria. Finally, serum IFN-α levels were un detectable in most patients. Nevertheless, our study has merit because it is the rst to demonstrate the effect of add-on HCQ treatment on proin ammatory cytokines in SLE patients with LDA.

Conclusions
We found that add-on HCQ treatment decreased the levels of several proin ammatory cytokines in SLE patients with LDA, especially those with LN. Our results suggest that HCQ treatment may reduce IL-8 expression in patients with LN in remission, which could improve the renal prognosis. The study was approved by the ethical committee of Kagawa University (Heisei30-047) and and prospectively registered. All participants gave their written informed consent prior to entering the study.

List Of Abbreviations
The study was conducted in accordance with the Declaration of Helsinki.