RNASE2 Mediates Age-Associated B Cell Expansion Through Monocyte Derived IL-10 in Patients With Systemic Lupus Erythematosus

Yantong Zhu, Xiaojun Tang, Yang Xu, Si Wu, Weilin Liu, Linyu Geng, Xiaolei Ma, Betty P Tsao, Xuebing Feng, Lingyun Sun, Yantong Zhu, Xiaojun Tang, Yang Xu, Si Wu, Weilin Liu, Linyu Geng, Xiaolei Ma, Betty P Tsao, Xuebing Feng, Lingyun Sun

Abstract

Systemic lupus erythematosus (SLE) is characterized by the production of pathogenic autoantibodies. Ribonuclease A family member 2 (RNase2) is known to have antiviral activity and immunomodulatory function. Although RNASE2 level has been reported to be elevated in SLE patients based on mRNA microarray detection, its pathologic mechanism remains unclear. Here, we confirmed that RNASE2 was highly expressed in PBMCs from SLE patients and associated with the proportion of CD11c+T-bet+ B cells, a class of autoreactive B cells also known as age-associated B cells (ABCs). We showed that reduction of RNASE2 expression by small interfering RNA led to the decrease of ABCs in vitro, accompanied by total IgG and IL-10 reduction. In addition, we demonstrated that both RNASE2 and IL-10 in peripheral blood of lupus patients were mainly derived from monocytes. RNASE2 silencing in monocytes down-regulated IL-10 production and consequently reduced ABCs numbers in monocyte-B cell co-cultures, which could be restored by the addition of recombinant IL-10. Based on above findings, we concluded that RNASE2 might induce the production of ABCs via IL-10 secreted from monocytes, thus contributing to the pathogenesis of SLE.

Keywords: age-associated B cells; interleukin 10; monocytes; ribonuclease A family member 2; systemic lupus erythematosus.

Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2022 Zhu, Tang, Xu, Wu, Liu, Geng, Ma, Tsao, Feng and Sun.

Figures

Figure 1
Figure 1
Increased RNASE2 mRNA expression in PBMCs from SLE patients. (A) Detection of RNASE2 expression by real-time PCR in PBMCs from 37 healthy controls (HC), 60 SLE patients, 20 patients with rheumatoid arthritis (RA) and 20 patients with primary Sjögren’s syndrome (SS). (B–E) Associations of RNASE2 mRNA levels with SLE disease activity index (SLEDAI) score, the amount of 24hour proteinuria as well as the levels of serum creatinine and uric acid by the Pearson or Spearman correlation test. (F–I) RNASE2 mRNA expression in SLE patients with or without positive anti-SSA, anti-Sm, anti-dsDNA or anti-SSB antibodies. Data are presented as mean ± SEM, *p < 0.05, **p < 0.01, ****p < 0.0001.
Figure 2
Figure 2
Association of age-associated B cells subset (ABCs) with RNASE2 expression in SLE patients. (A) PBMCs from 24 HC, 26 SLE, 5 RA and 6 SS patients was collected and analyzed for the proportion of CD11c+T-bet+ cells in CD19+ B cells by flow cytometry. (B, C) The percentage of CD11c+T-bet+ B cells was related to SLE disease activity score (SLEDAI and BILAG) (by Spearman correlation test). (D) The percentage of CD11c+T-bet+ B cells was associated with the amount of 24hour proteinuria (by Spearman correlation test). (E) The proportion of CD11c+T-bet+ B cells was increased in SLE patients with positive anti-Sm antibody. (F) The proportion of CD11c+T-bet+ B cells was closely related to RNASE2 mRNA levels in SLE patients (n=14). Data are presented as mean ± SEM, *p < 0.05.
Figure 3
Figure 3
RNASE2 participated in the regulation of ABCs. (A) Nearly 80% reduction in the gene expression 3 days after the treatment of RNASE2 siRNA in vitro (n=5). (B) Decreased percentage and absolute number of CD11c+T-bet+ B cells after RNASE2 silencing by flow cytometry (n=13). (C) Down-regulated expression of total immunoglobulin (Ig) G in culture supernatants from RNASE2 silencing group (n=9), as determined by enzyme-linked immunosorbent assay (ELISA). (D) The difference of IgG level between silence group and control group was positively correlated to the alteration of ABCs proportion (n= 6). Data are presented as mean ± SEM, *p < 0.05, **p < 0.01.
Figure 4
Figure 4
IL-10 was a major effector of RNASE2. (A–G) The expression of multiple cytokines (IL-2, IL-4, IL-6, IL-10, IL-12p70, IL-21 and TNF-α) in cultured supernatant was determined by using luminex liquid phase chip, among which only IL-10 level was significantly decreased in RNASE2 silencing group (n=11). The lower limit of detection (LLOD) for each cytokine was 2.96 pg/ml, 5.61 pg/ml, 0.72 pg/ml, 0.74 pg/ml, 1.26 pg/ml, 2.69 pg/ml and 0.98 pg/ml respectively, and levels lower than LLOD were regarded as having the minimum detection value. (H) Independent sample validation by ELISA confirmed the reduction of IL-10 level in cultured supernatants after RNASE2 silencing (n=8) (LLOD 0.05pg/ml). (I)In vivo study showed the level of plasma IL-10 was increased in SLE patients (n=16) compared with healthy controls (n=14). (J) Plasma IL-10 levels in SLE patients were closely linked to peripheral RNASE2 mRNA expression (n=10). Data are presented as mean ± SEM, *p < 0.05, **p < 0.01.
Figure 5
Figure 5
RNASE2 promoted the secretion of IL-10 from lupus monocytes. (A) Flow cytometry sorting technology was used to separate lupus CD14+ monocytes, CD19+ B cells, CD4+ T cells and the remaining CD14-CD19-CD4- cells. (B) mRNA expression of IL-10 in each cell subgroup was detected by real-time PCR, and the highest expression was seen in monocyte subgroup (n=5), especially those from SLE patients (n=8). (C, D) mRNA expression of RNASE2 was also prominent in monocyte subgroup from lupus patients, which was correlated with monocyte IL-10 mRNA level (by Spearman correlation test, n=8). (E, F) RNASE2 silencing down-regulated IL-10 mRNA expression in lupus monocytes and IL-10 protein levels in cultured supernatants (n=7). Data are presented as mean ± SEM, *p < 0.05, **p < 0.01, ns, no significant statistical difference.
Figure 6
Figure 6
IL-10 regulated the production of ABCs in SLE patients. (A) Lupus B cells were co-cultured with monocytes with the presence of either RNASE2 siRNA (RNASE2si) or RNASE2 siRNA plus 50ng/ml human recombinant IL-10 (RNASE2si+IL-10) (n=6). The proportion of CD11c+T-bet+ B cells was restored after the replenishment of IL-10 in RNASE2 silencing group. (B) High dose anti-IL-10 (5ug/ml) but not low dose anti-IL-10 (1ug/ml) blocked the production of CD11c+T-bet+ B cells in B cell and monocyte co-cultures (n=5). (C) The effect of recombinant IL-10 on the proportion of CD11c+T-bet+ B cells in B-cell plus monocyte co-cultures (n=8). (D) The effect of recombinant IL-10 on the proportion of CD11c+T-bet+ B cells in CD19+ B cell culture (n=5). (E) Hypothesis schema of RNASE2 pathogenetic role involved in SLE patients (picture material was from http://smart.servier.com). Data are presented as mean ± SEM, paired t test, *p < 0.05, **p < 0.01.

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