IL-37 inhibits the production of inflammatory cytokines in peripheral blood mononuclear cells of patients with systemic lupus erythematosus: its correlation with disease activity

The interleukin (IL)-1 family of cytokines possesses a variety of immunoregulatory properties in response to inflammation and autoimmune diseases [1]. As the members of the IL-1 family, seven cytokines (IL-1α, IL-1β, IL-18, IL-33, IL-36α, IL-36β and IL-36γ) act as agonists, and two are classified as naturally occurring receptor antagonists (IL-1Ra and IL-36Ra) [2, 3]. IL-37, the most recently identified IL-1 member, originally defined as IL-1 family member 7 (IL-1F7), is a fundamental inhibitor of innate immunity [4].

Recent studies demonstrated that anti-inflammatory cytokines TGF-β, IL-10, several toll-like receptor (TLR) ligands and pro-inflammatory cytokines such as IL-1β, TNF-α, IFN-γ and IL-18 induce IL-37 production in peripheral blood mononuclear cells (PBMCs) [4, 5]. However, over-expressed human IL-37 suppressed the TLR-induced pro-inflammatory cytokines in a mouse macrophage RAW cell line, in human monocyte cell line THP-1 and in alveolar epithelial A549 cells [1, 4]. In vivo, mice transgenic for IL-37 (IL-37tg) exhibited markedly reduced manifestations of DSS colitis, ischemia–reperfusion injury and obesity-induced inflammation [6‐8]. Compared to health subjects, IL-37 was constitutively expressed in tissues from patients with rheumatoid arthritis [4]. Similar studies have found that IL-37 was not detected in the normal colonic mucosa, but in the inflamed mucosa of IBD patients [9]. These findings tend to imply that IL-37 mediates a negative feedback mechanism to suppress excessive inflammation.

Systemic lupus erythematosus (SLE) is an autoimmune and inflammatory disease characterized by the activation of T and polyclonal B lymphocytes. The activation of B cells produces numerous auto-antibodies and form immune complexes with variety antigens, which result in tissue and organ damage [10]. Cytokines collectively play key roles in the regulation of systemic inflammation, local tissue damage and immunoreactions [11]. Abnormal release various cytokines have been identified in SLE patients and animal models both in vitro and in vivo[12]. Abundant studies have demonstrated that pro- and anti-inflammatory cytokines, including TNF-α, IL-1β, IL-6 and IL-10, play crucial pathogenic roles in the disease of SLE [12, 13]. Although the serum IL-37 levels have been found to be elevated in active patients with SLE [14], its causal relationship with disease activity and their clinical association and disease manifestations in SLE is still unclear, especially the regulations of cytokines expression by IL-37 in SLE PBMCs remain to be studied.

In present study, we compared expressions of IL-37 mRNAs in PBMCs and serum IL-37 protein levels in SLE patients with healthy controls. In addition, we determined the correlation of serum IL-37 levels with disease activity and clinical manifestations in SLE, investigated the effect of IL-37 on the expressions of cytokine TNF-α, IL-1β, IL-6 and IL-10.

Although IL-37 mRNA expression and serum protein level has been demonstrated to be higher in active SLE patients [14], but information with regard to its clinical association and disease manifestation is lacking. Our results provide a detailed analysis of IL-37 expression in SLE patients (SLEDAI ≥ 6, active SLE and SLEDAI < 6, inactive SLE) and healthy controls. We demonstrated that IL-37 mRNAs expressions and serum protein levels were higher in 66 patients with SLE than in 41 healthy controls (Figure 1). Further investigation revealed that IL-37 mRNAs and protein levels were significantly higher in 36 patients with active diseases than in 30 patients with inactive diseases and in 41 healthy controls. However, there is no difference between inactive diseases and healthy controls (Figure 2). In order to further proved the relationship between IL-37 expression and SLE laboratory values, we made efforts to analyze the correlation between serum IL-37 protein levels and several laboratory values. Our novel data suggested that serum IL-37 levels were negatively correlated with C3 and C4 (Figure 3), but it lacked association with other laboratory values (anti-dsDNA, smith, AnuA, rRNP antibody, IgG, IgM, IgA and ESR) (data not shown). Thus, our findings implied that the expression of IL-37 is correlated with activity of SLE, and IL-37 probably involved in the mediation of the disease activity. In patients with SLE, skin involvement, arthritis and renal disorder are very common manifestations. We showed that no significant differences in serum IL-37 protein levels between the patients who had malar rash, serositis, arthritis, fever, neurological disorder, anemia, leucopenia and thrombocytopenia and the patients who absented the above-mentioned clinical manifestations (data not shown). Surprisingly, serum IL-37 levels were significantly higher in patients with renal disease compared with patients without renal involvement together with healthy controls (Figure 4). In summary, our experiments demonstrated that the expression levels of IL-37 correlated with the disease activity, laboratory values and clinical manifestations of SLE.

Imbalances between pro- and anti-inflammatory cytokines are hallmarks of the pathogenesis of SLE [19, 20]. It has been demonstrated that pro-inflammatory cytokines TNF-α, IL-1β, IL-6 and cytokine IL-10 levels are significantly elevated in the serum of SLE patients and correlate with disease activity [13, 21, 22]. In particular, the expressions of TNF-α, IL-6 and IL-10 are markedly elevated in SLE patients with lupus nephritis [20‐22]. Published data have shown that blocking these cytokines significantly improved the development of SLE [23‐26]. Uppal SS et al showed that nine SLE patients treated with anti-TNF-α monoclonal antibody, five patients showed improvement in disease activity (SLEDAI) [24]. Two very small uncontrolled studies of three and four SLE patients suggested that blocking IL-1 signaling might have beneficial effects on lupus-related manifestations [24]. In addition, SLE patients with tocilizumab (anti-IL-6 receptor antibody) treatment resulted in significant clinical improvement, especially in SLE patients with arthritis [25]. Another study found that administration of anti-IL-10 monoclonal antibody (21 consecutive days) to six patients with moderate lupus (an open study) resulted in healing of arthritis and cutaneous lesions [26].

In our present study, the expressions of TNF-α, IL-1β, IL-6 and IL-10 were significantly increased in PBMCs of SLE patients than in PBMCs from healthy controls. Interestingly, our result also showed that the expression of IL-37 was correlated with the activity of disease in SLE. What are the functions of a fundamental innate immunity inhibitor IL-37 [4] in SLE? To answer the question, recombinant purified IL-37 was used to stimulate PBMCs from SLE patients and health controls. Our study is the first to reveal that IL-37 could effectively decrease the productions of pro-inflammatory cytokines TNF-α, IL-6 and IL-1β (Figure 5A-F), whereas the productions of cytokine IL-10 were unaffected in PBMCs of SLE patients (Figure 5G-H). Researcher showed that IL-37 can be up-regulated by cytokines IL-1β, TNF-α and IL-10 [4, 5], indicating these cytokines act as positive feedback loops for up-regulation of IL-37 production. Therefore, it is reasonable to explain the correlation between higher activity of disease and the higher expression levels of IL-37 in SLE. Recently, IL-37 has been shown to inhibit the production of pro-inflammatory cytokines TNF-α, IL-1β and IL-6 in PBMCs in vitro[4]. Together with this result, the down regulation of pro-inflammatory cytokines in our experiments imply that IL-37 may play an important role in the inhibition of inflammatory response in SLE, the up regulation of IL-37 by SLE immune reaction may be the result of inflammatory diseases self-limiting. It has been shown that the expression of IL-10 in PBMCs was unaffected by treatment with siIL-37 [4], which was consistent with our finding. These data demonstrated that IL-37 acts as an inhibitor of inflammatory responses in SLE disease, but this function does not extend to cytokines IL-10.

Interestingly, the pro-inflammatory cytokine productions in PBMCs of healthy controls and inactive disease with SLE patients were unaffected by treatment with IL-37 (Figure 5), suggesting that anti-inflammatory actions of IL-37 are only in the inflammatory phase and active disease conditions of SLE. Nold et al. showed that IL-37 suppresses the phosphorylation of p38 MAPK [4], which are known to be involved in the pathogenesis of SLE and contributes to several pro-inflammatory signaling cascades [27]. It has been demonstrated that inhibition of p38 MAPK activation reduced serum TNF-a, IL-1β and IL-6 levels and attenuated SLE renal injury [28, 29]. Thus, it is plausible that down-regulation of several kinase activity by IL-37, such as p38 MAPK, might result in reduced expression of pro-inflammatory cytokines in PBMC during inflammatory responses, which could lead to suppression of excessive immune response, and protection of tissue damage in SLE.

In summary, our study showed that the expressions of IL-37 correlated with the disease activity of SLE. Meanwhile, it was likely to mediate a negative feedback mechanism to suppress excessive inflammation in SLE, especially in renal disease of SLE. Furthermore, we demonstrated that the expressions of pro-inflammatory cytokines TNF-α, IL-6 and IL-1β were suppressed by IL-37 in active SLE patients. These observations implicate that IL-37 probably played an important role in the inhibition of pathogenesis of SLE. Future efforts need to define the regulatory mechanisms of IL-37 in the mediation of immune reaction of SLE.