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. 2021 Apr 21;10(5):964.
doi: 10.3390/cells10050964.

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

Jie Ma  1 , Ian Kar Yin Lam  1 , Chak-Sing Lau  1 , Vera Sau Fong Chan  1
Affiliations

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

Jie Ma et al. Cells. .

Abstract

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.

Keywords: SLE; cellular function; interleukin-18 receptor accessory protein; type I interferon.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Elevated expression of IL18RAP in neutrophils from SLE patients. (A) qRT-PCR analysis on IL18RAP expression in neutrophils from healthy controls (HC, n = 30) and SLE patients (n = 95). RQ, relative quantity; **** p < 0.0001 by Mann–Whitney U test. (B) Western blotting on protein expression of IL18RAP in neutrophils was performed using an independent set of samples. Representative blot of three independent experiments showing four samples in HC and SLE patients. Arrows indicate the position of molecular weight markers. (C) Summary chat showing relative IL18RAP protein expression in neutrophils between HC (n = 11) and SLE patients (n = 11), as measured by normalized densitometry signal intensity. A.U., arbitrary unit; * p < 0.05 by Mann–Whitney U test. Data were shown as the median (horizontal line) with interquartile range (IQR, box), lower and upper whiskers (data within 1.5 × IQR), and outliers (points) (Tukey’s box).
Figure 2
Figure 2
Correlation analyses between IL18RAP expression and clinical and laboratory parameters in 95 SLE patients. (A) IL18RAP expression was compared between non-nephritic lupus patients (NLN, n = 40) and lupus nephritis patients (LN, n = 55). ** p < 0.01 by Mann–Whitney U test. Data were expressed as Tukey’s boxes. Scatter plots showing IL18RAP expression correlation with (B) C3 levels, and (C) SLEDAI-2K scores in SLE patients (n = 95). Linear regression line is shown; r = correlation coefficient; ** p < 0.01 by Spearman’s rank-order test. Stratified analyses on IL18RAP expression comparison between SLE patients (D) with (+, n = 12) and without (−, n = 83) renal involvement; and between SLE patients (E) with (+, n = 64) and without (−, n = 31) immunological involvement at the time of blood sample collection. * p < 0.05; ** p < 0.01 by Mann–Whitney U test (for A,D,E). Results were shown as Tukey’s boxes. RQ, relative quantity.
Figure 3
Figure 3
Regulation of IL18RAP expression by type I interferon (IFN) in neutrophils. (A) Expression correlation between IL18RAP and IFIT1 in neutrophils of SLE patients (n = 21). 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 (IFNhi) (left panel). Six individuals (n = 6) were treated with HC sera and SLE sera with low IFN activity (IFNlo) (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.
Figure 4
Figure 4
IL-18 enhances fMLP-mediated ROS generation in neutrophils via IL18RAP. Representative plots showing kinetics of ROS generation in neutrophils from (A) HC and (B) SLE patients upon treatment with or without rhIL-18 (100 ng/mL) and fMLP (100 nM) stimulation. (C) Summary chart showing IL-18-enhanced ROS generation in neutrophils at 30 min poststimulation in HC (n = 14) and SLE patients (n = 14). ** p < 0.01 by Mann–Whitney U test. (D) Correlation analysis between IL18RAP expression and IL-18-enhanced ROS generation using an independent set of HC and SLE neutrophil samples (n = 15). * p < 0.05 by Spearman’s rank-order correlation test; r = correlation coefficient and the linear regression line is shown. (E) Representative plot showing fMLP-mediated ROS generation in SLE neutrophils with anti-IL18RAP antibody (Ab) or goat IgG isotype control (10 μg/mL) pretreatment prior to IL-18 (100 ng/mL) stimulation. (F) Summary chart showing IL-18-enhanced ROS generation in SLE neutrophils (n = 3) upon pretreatment with goat IgG isotype antibody or anti-L18RAP antibody at 30 min poststimulation. ** p < 0.05 by paired t test. Data shown as mean + SEM.
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