Elevated Interleukin-18 Receptor Accessory Protein Mediates Enhancement in Reactive Oxygen Species Production in Neutrophils of Systemic Lupus Erythematosus Patients

Interleukin-18 receptor accessory protein (IL18RAP) is an indispensable subunit for the IL-18 receptor (IL-18R) complex’s ability to mediate high-affinity IL-18 binding and signalling transduction. Interest in IL-18 in systemic lupus erythematosus (SLE) has been mostly focused on its role as a type 1 T helper cell-driving cytokine. The functional significance of IL18RAP in mediating the IL-18-driven response in myeloid cells in SLE remains largely unexplored. This study aimed to investigate the expression and function significance of IL18RAP in neutrophils of SLE patients. By qRT-PCR and Western blot analyses, elevated expressions of IL18RAP mRNA and protein were observed in neutrophils from SLE patients—particularly those with a history of nephritis. IL18RAP expression correlated negatively with complement 3 level and positively with disease activity, with higher expression in patients exhibiting renal and immunological manifestations. The increased IL18RAP expression in SLE neutrophils could be attributed to elevated type I interferon level in sera. Functionally, neutrophils from SLE patients showed higher IL-18-mediated enhancement in reactive oxygen species (ROS) generation, which showed positive correlation with IL18RAP expression and could be neutralized by anti-IL18RAP blocking antibodies. Taken together, our findings suggest that IL-18 could contribute to SLE pathogenesis through mediation of neutrophil dysfunction via the upregulation of IL18RAP expression.


Introduction
Systemic lupus erythematosus (SLE) is a female-biased, chronic systemic autoimmune disorder that affects multiple organ systems, including renal, nervous, musculoskeletal, haematological, respiratory, and cardiovascular systems [1,2]. Renal involvement is one of the most common clinical features of SLE, with approximately 50-60% of Asian SLE patients developing lupus nephritis (LN) during the disease course. It is also a major cause of morbidity and mortality in SLE patients [3,4]. SLE pathogenesis is highly complex; the intricate interplay of environmental, genetic, and hormonal factors lead to disturbances in both the innate and adaptive immune compartments and the loss of self-tolerance [5]. Cytokine dysregulation is a typical characteristic of SLE. Imbalanced proinflammatory and anti-inflammatory cytokine production, together with abnormal responsiveness of immune cells to various cytokines, exacerbate immune dysregulation and contribute to tissue inflammation and organ damage in lupus [6].

Human Subjects
SLE patients fulfilling both the 1997 American College of Rheumatology (ACR) and the 2012 Systemic Lupus International Collaborating Clinics (SLICC) criteria were recruited from the Queen Mary Hospital, Hong Kong. LN patients were SLE patients with biopsyconfirmed nephritis. Non-nephritic lupus (NLN) patients were SLE patients with no history of renal manifestation for at least ten years after disease onset. Age-and sex-matched healthy control (HC) samples were collected from buffy coats obtained from healthy blood donors from the Hong Kong Red Cross. All participants gave their informed consent for inclusion in the study. The study was approved by the Institutional Review Board of the University of Hong Kong/Hospital Authority Hong Kong West Cluster (study approval number: UW 16-286).

Isolation and Stimulation of Human Neutrophils
Neutrophils were isolated from buffy coats or fresh EDTA-anticoagulated blood. Reagents used in the isolation and culture process were tested to contain <0.5EU/mL endotoxin level using a Pierce Chromogenic Endotoxin Quant Kit (Thermo Fisher Scientific, Rockford, IL, USA). Briefly, neutrophils were separated using Ficoll-Paque Plus (GE Healthcare Life Science, Uppsala, Sweden) gradient centrifugation, followed by erythrocyte sedimentation in 6% dextran (Sigma-Aldrich, Oslo, Norway). Remaining erythrocytes were lysed with deionized water for 30 s, followed by neutralization with PBS. Neutrophils were cultured in RPMI 1640 medium supplemented with 20 mM GlutaMAX, 1 mM sodium pyruvate, nonessential amino acids, 100 U/mL penicillin, 100 µg/mL streptomycin, and 10% foetal bovine serum (all from Gibco, Gaithersburg, MD, USA). For stimulation, purified neutrophils were treated with or without 1000 IU/mL recombinant human interferon-alpha (IFN-α) (Abcam, Cambridge, UK) at 37 • C for 6 h in specified assays. In separate experiments, neutrophils were incubated with 10% human sera derived from healthy controls or from SLE patients with low or high interferon (IFN) activity. Type I IFN activity in individual serum samples was determined using HEK-Blue IFNα/β cells and a QUANTI-Blue assay system (InvivoGen, San Diego, CA, USA). SLE sera with high levels of IFN (IFN hi ) were pooled from sera with more than 500 IU/mL, whereas IFN lo sera were pooled from sera with less than 100 IU/mL.

Statistical Analyses
Data were analysed using GraphPad Prism 8 software (GraphPad Software Inc., San Diego, CA, USA). Normal distribution of the data was verified by the Shapiro-Wilk normality test prior to statistical analysis. Statistical significance between two groups was calculated using the Mann-Whitney U test. A paired t test was used to compare two different treatments that were applied to the same samples. Correlation analysis was performed by Spearman's rank-order correlation test. All p-values were considered significant at p < 0.05.

Neutrophils of SLE Patients Show Elevated IL18RAP Expression, Which Correlates Positively with Disease Activity and Renal Involvement
Results from our pilot screening assay on SLE peripheral leukocytes showed highly correlated expression between IL18RAP and neutrophil-associated genes, and the former was significantly elevated in patients with nephritis development ( Figure S1). We therefore hypothesized that IL18RAP in neutrophils could be dysregulated in expression and function in lupus patients. To investigate this, we first compared the expression of IL18RAP in neutrophils of HC (n = 30) and SLE patients (n = 95). qRT-PCR analysis indeed showed higher mRNA expression of IL18RAP in SLE patients ( Figure 1A). At the protein level, an independent set of HC (n = 11) and SLE (n = 11) samples were found to express the canonical isoform of IL18RAP protein at the expected molecular weight of around 68 kDa by western blotting ( Figure 1B). However, in some samples, significant immunoreactivity was also detected at a lower molecular weight, which likely corresponds to the shorter IL18RAP isoform with approximately 52 kDa (UniProtKB identifier: O95256-2). Nevertheless, an increased expression of IL18RAP protein was observed in neutrophils from SLE patients ( Figure 1B,C). These results indicate that IL18RAP expression is dysregulated in SLE neutrophils.
We then examined if the elevated expression of IL18RAP had any association with clinical manifestations in these 95 SLE patients. Basic demographics and selected clinical characteristics of the study participants were shown in Table 1. As IL-18 has been implicated in nephritis development, we specifically compared SLE patients with biopsy-confirmed nephritis (LN group, n = 55) with patients without any renal manifestations for at least 10 years (non-LN, NLN group, n = 40). Reinforcing our pilot data, LN patients indeed expressed significantly higher IL18RAP when compared with NLN patients (Figure 2A). Also, the expression level of IL18RAP correlated negatively with serum complement 3 (C3) level, and positively with SLE disease activity index 2000 (SLEDAI-2K) score ( Figure  2B,C). No significant correlation was observed with other serological parameters such as anti-dsDNA antibody, C4, urea, creatinine, albumin, or globulin levels. We further investigated if the dysregulated IL18RAP expression was associated with specific organ or system manifestations. As shown in Figure 2D,E, higher IL18RAP expression was observed in SLE patients exhibiting active manifestations of the renal system (presence of urinary casts, haematuria, proteinuria, or pyuria-as defined by the descriptors in the SLEDAI-2K scoring system) and the immunological system (low complement or increased DNA binding). tigated if the dysregulated IL18RAP expression was associated with specific organ or system manifestations. As shown in Figure 2D,E, higher IL18RAP expression was observed in SLE patients exhibiting active manifestations of the renal system (presence of urinary casts, haematuria, proteinuria, or pyuria-as defined by the descriptors in the SLEDAI-2K scoring system) and the immunological system (low complement or increased DNA binding).       (for A,D,E). Results were shown as Tukey's boxes. RQ, relative quantity.

IL18RAP Expression in Neutrophils Is Regulated by Type I Interferon
Next, we asked whether the inflammatory milieu in serum could have an impact on IL18RAP expression in SLE patients. SLE peripheral leukocytes remarkably express a spectrum of IFN signature genes as a result of perturbed production and signalling of type I IFN [26]. We therefore hypothesized and tested a causal relationship between type I IFN and IL18RAP using separate sets of HC and SLE samples. Firstly, a positive expression correlation between IL18RAP and IFIT1, one of the interferon-stimulated genes (ISGs), was observed ( Figure 3A), suggesting that IL18RAP expression in SLE neutrophils might be regulated by IFN in circulating blood. Next, sera from HC and SLE patients were tested for the capacity to modulate IL18RAP expression in neutrophils. As shown in Figure 3B, only SLE sera with high level of type I IFN, but not those with low IFN, could upregulate IL18RAP expression when compared with cells cultured in HC sera. Furthermore, an increase in IL18RAP expression could be readily induced upon recombinant IFN-α stimulation in vitro ( Figure 3C). Taken together, it is likely that type I IFN in circulating blood could cause, at least in part, the upregulated IL18RAP expression in neutrophils of SLE patients.

IL-18 Enhances fMLP-Mediated ROS Generation in SLE Neutrophils
We then assessed the functional impact of the elevated IL18RAP expression in SLE neutrophils. The inflammatory and antimicrobial action of neutrophils is highly associated with the induction of a strong oxidative burst, generating almost the whole spectrum of ROS including superoxide, hydrogen peroxide, and hypochlorous acid [27]. N-formylmethionyl-leucyl-phenylalanine (fMLP), a bacterial analogue, is one of the most common agents used to study ROS generation in research. We therefore used fMLP to evaluate . Linear regression line is shown; r = correlation coefficient; * p < 0.05 by Spearman's rank-order test. (B) IL18RAP expression in healthy neutrophils upon culture with sera from HC and SLE patients was evaluated. Samples from healthy individuals (n = 12) were treated with HC sera and SLE sera with high IFN activity (IFN hi ) (left panel). Six individuals (n = 6) were treated with HC sera and SLE sera with low IFN activity (IFN lo ) (right panel). A paired t test was used to compare data from the same samples treated with HC sera and SLE sera. (C) IL18RAP expression in healthy neutrophils (n = 8) was compared between those cultured in medium alone (untreated group, UT) and those cultured in the presence of recombinant human IFN-α (1000 IU/mL for 6 h). ** p < 0.01 by paired t test. RQ, relative quantity. Data are shown as mean + SEM.

IL-18 Enhances fMLP-Mediated ROS Generation in SLE Neutrophils
We then assessed the functional impact of the elevated IL18RAP expression in SLE neutrophils. The inflammatory and antimicrobial action of neutrophils is highly associated with the induction of a strong oxidative burst, generating almost the whole spectrum of ROS including superoxide, hydrogen peroxide, and hypochlorous acid [27]. N-formylmethionyl-leucyl-phenylalanine (fMLP), a bacterial analogue, is one of the most common agents used to study ROS generation in research. We therefore used fMLP to evaluate neutrophil functional response. Our results showed that IL-18 stimulation alone had no effect on the induction of ROS generation in neutrophils from both HC and SLE patients, however, it could augment fMLP-mediated ROS generation in vitro ( Figure 4A,B). Notably, the level of IL-18-mediated ROS enhancement was significantly higher in neutrophils from SLE patients than in those from HC ( Figure 4C). Interestingly, this enhancement positively correlated with the expression level of IL18RAP ( Figure 4D). Although neutrophils from HC and SLE were obtained from samples of slightly different preparation-i.e., buffy coat and fresh blood, respectively-it was anticipated to have minimal impact on the functional response of neutrophils, including oxidative burst, phagocytosis, migration, and formation of neutrophil extracellular traps (NETs) [28,29]. Nevertheless, we compared HC neutrophils from fresh blood and buffy coat, and observed that they indeed had similar ability to produce ROS in vitro ( Figure S2). We also evaluated the expression of the IL-18Rα subunit in neutrophils and found no significant difference between HC and SLE patients ( Figure S3). Thus, it is likely that the augmentation in IL-18-mediated ROS enhancement in SLE neutrophils is attributable to the dysregulation of IL18RAP expression. In support of this argument, IL-18-mediated enhancement in ROS generation in SLE neutrophils could indeed be efficiently neutralized by anti-IL18RAP antibody ( Figure 4E,F). Finally, we further tested whether IL-18-mediated ROS enhancement in neutrophils could be observed using an SLE-relevant stimulus-the anti-dsDNA immune complexes. Similar to fMLP stimulation, immune complex-induced ROS was also increased in response to IL-18 pretreatment, especially in neutrophils from SLE patients ( Figure S4). Taken together, our data suggests that heightened IL18RAP expression may contribute to the dysregulated function of neutrophils in SLE patients. 4E,F). Finally, we further tested whether IL-18-mediated ROS enhancement in neutrophils could be observed using an SLE-relevant stimulus-the anti-dsDNA immune complexes. Similar to fMLP stimulation, immune complex-induced ROS was also increased in response to IL-18 pretreatment, especially in neutrophils from SLE patients ( Figure S4). Taken together, our data suggests that heightened IL18RAP expression may contribute to the dysregulated function of neutrophils in SLE patients.

Discussion
IL18RAP is an indispensable subunit of the IL-18R complex for the mediation of highaffinity IL-18 binding and subsequent signalling transduction [8]. Its extracellular region contains two immunoglobulin-like domains for ligand interaction, and the cytoplasmic tail possesses a TIR domain which is also found in IL-1R and many toll-like receptors (TLRs). In mice, IL18RAP deficiency led to pronounced impairment in IL-18-induced responses in multiple cell types, including IFN-γ production in Th-1 cells, NK cell cytotoxicity, as well as neutrophil activation [30]. Also, responses to IL-1 and TLR ligand stimulation remained normal in these mice, indicating that this receptor is specific for mediating the IL-18 signalling cascade. The majority of studies on IL18RAP focus on genetic association analyses that encompass a diverse spectrum of conditions including cancer, cardiovascular disease, autoimmunity, and infections. Pertaining to inflammatory disorders, the rs917997 single nucleotide polymorphism (SNP) in IL18RAP was found to have a divergent role, conferring risk for celiac disease but protection for type I diabetes [31]. A follow-up study further demonstrated that the risk-associated allele of this SNP could yield a higher IFN-γ production in peripheral blood mononuclear cells (PBMCs) upon IL-12 and IL-18 costimulation [32]. Using combined genome-wide association studies (GWAS) and expression quantitative trait loci (eQTL) analyses, Andiappan et al. revealed another functional IL18RAP SNP-rs2058660-of which the CC/TT variants could mediate differential IL-18 responses by altering IL18RAP expression levels in neutrophils [33]. Intriguingly, this SNP also affects leprosy and Crohn's disease susceptibility in opposite directions. Not only affecting disease susceptibility, IL18RAP expression in synovial tissues was shown to associate with treatment response in rheumatoid arthritis patients [34]. It is worth noting that gene overexpression does not always endow functional enhancement. In systemiconset juvenile idiopathic arthritis (SJIA), an overexpression of IL18RAP in neutrophils was observed in patients with active disease [35]. However, NK cells of SJIA patients were also shown to have impaired IL-18-mediated cytotoxicity due to defective IL18RAP phosphorylation [36]. This could be one of the reasons to account for the divergent role of IL18RAP in different clinical conditions. Nevertheless, dysregulated IL18RAP expression has been observed in a number of inflammatory diseases.
In SLE research to date, there is only one published study on IL18RAP with implicated functional relevance. Neumann et al. reported an elevated IL18RAP expression in lymphocytes of MRL/lpr mice, which in turn was associated with heightened IL-18-induced lymphocyte proliferation and IFN-γ production [21]. Here, we demonstrate that IL18RAP expression in SLE patients-both at mRNA and protein levels-was significantly higher in circulating neutrophils when compared with healthy controls (Figure 1). Interestingly, our results suggest that a shorter isoform of IL18RAP may be expressed by neutrophils. The 52-kDa IL18RAP short isoform (UniProtKB identifier: O95256-2) has 142 amino acids missing at the N-terminal in the extracellular domain. The functional impact of this isoform is not clear. Structural and biochemical analyses showed that this region is not involved in ligand binding of the IL-18/IL-18Rα/IL18RAP ternary complex [7]. Thus, it is likely that the absence of this region would have minimal impact on IL-18 signalling. Nevertheless, the overall IL18RAP protein levels were higher in SLE neutrophils. We did not perform IL18RAP SNP analysis and whether this overexpression is related to genetic predisposition is yet to be tested. However, our data clearly show that the inflammatory milieu in patient sera can, at least in part, contribute to the IL18RAP expression upregulation. We further demonstrate that type I IFN is a potent mediator for the upregulation of IL18RAP expression in neutrophils (Figure 3). Type I IFN is known to play a central pathologic role in driving SLE development and disease progression [37]. Peripheral leukocytes of SLE patients often exhibit a highly expressed ISG signature in correlation with disease activity [26,38]. Additionally, upregulation of granulocyte-associated genes were also reported, suggesting that neutrophils also play functional roles in disease development [39]. In fact, type I IFN has been shown to prime neutrophils to undergo NETosis, a cell death pathway with the formation of NETs, which release cellular proteins and DNA that further fuel the inflammatory process in SLE patients [40]. In line with this, our findings also demonstrate another functional impact of type I IFN on neutrophil function in SLE, as highlighted by the heightened IL-18-mediated ROS production enhancement in neutrophils of SLE patients in relation to the elevated IL18RAP expression (Figure 4). On a side note, the priming or enhancement effect of IL-18 on ROS generation in neutrophils has also been shown in settings of bacterial infections, anti-neutrophil cytoplasm autoantibody (ANCA)-associated vasculitis, and rheumatoid arthritis [25,41,42].
With increasing evidence to support its pathological involvement, neutrophils from SLE patients indeed display multiple aberrations, including dysregulated cytokine and chemokine expression, defective phagocytic and antimicrobial capacity, and enhanced oxidative stress responses [43]. Notably, studies on ROS production capacity in SLE neutrophils have conflicting findings. Perozzio et al. showed that, when compared with healthy controls, neutrophils from SLE patients exhibited a higher ROS production at basal level as well as under Staphylococcus aureus or Pseudomonas aeruginosa stimulation [44]. Conversely, SLE neutrophils were shown to have decreased ROS generation after phorbol-myristate acetate (PMA) or Escherichia coli stimulation in an independent study [45]. The reason for such discrepancy is not clear but it could be due to intrinsic variations in different patient cohorts and stimuli. Nevertheless, a relatively recent study unravelled an essential role of mitochondrial (mt) ROS in mediating ribonucleoprotein immune complex (RNP-IC)-induced NETosis, particularly in the unique subpopulation of low density-gradient granulocytes (LDGs) that are found in SLE patients [46]. In vivo ablation of mt ROS in MRL/lpr mice could attenuate lupus-like symptoms, including anti-dsDNA level and renal manifestations with a concurrent reduction in NETosis [46]. Moreover, there exists increasing evidence to link nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-derived ROS (e.g., induced by fMLP stimulation) with NETs formation [47]. All these studies suggest that ROS production by neutrophils could have significant pathological contribution to lupus development.
Consistent with this reasoning, our data indeed show a positive correlation between IL18RAP expression and SLE disease activity. A higher expression was also observed in patients with a history of nephritis and in patients exhibiting renal manifestations at the time of investigation (Figure 2). IL-18 has long been implicated in promoting nephritis development in SLE in the capacity of a prominent Th-1 response driver [22]. Elevated levels of IL-18 have been found in serum and urine, as well as kidney biopsies, of LN patients [13,48,49]-this could have functional impact on both circulating and kidney-infiltrating neutrophils. Potentially, higher IL18RAP expression in the infiltrating neutrophils could enhance the generation of ROS, which is known to play a major role in glomerulonephritis [50]. Taken together, our findings provide evidence to support an additional pathogenic role of IL-18 in that it may promote nephritis through the mediation of neutrophil dysfunction via the upregulated expression of IL18RAP.

Conclusions
In summary, we have revealed an overexpression of IL18RAP in neutrophils which is associated with disease activity and renal involvement in SLE patients. Type I IFN in the inflammatory milieu could cause this upregulation, which in turn leads to IL-18-mediated enhancement in ROS generation. These findings provide new insights into the role of IL-18 in contributing to the dysregulation of neutrophil function, which is now known to play a crucial role in SLE and LN development.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/ 10.3390/cells10050964/s1, Figure S1: Expression correlation of IL18RAP and neutrophil-associated genes in peripheral leukocytes of SLE patients, Figure S2: Neutrophils from healthy fresh blood and buffy coat have similar ability to produce ROS, Figure S3: No difference in IL-18Rα expression in neutrophils between HC and SLE patients, Figure S4: IL-18 enhances immune complex-mediated ROS production in neutrophils.  Informed Consent Statement: Informed consent was obtained from all subjects involved in the study.

Data Availability Statement:
No new data outside those presented in this study were created or analyzed. Data sharing is not applicable to this article.

Acknowledgments:
We thank the Hong Kong Red Cross for providing buffy coats of healthy blood donors, Ivy Lo and Albert Chan for collecting and processing clinical samples, and Stephen Au for organizing clinical data.

Conflicts of Interest:
The authors declare no conflict of interest.