Background
Systemic lupus erythematosus (SLE) is a chronic autoimmune disease involving multiple systems, organs, and autoantibodies. The incidence rate of SLE in various regions and diverse races is different. The overall incidence rate is 40–80/10 million individuals and the male-to-female ratio is 1:10, with this disease being more prevalent in young women of child-bearing age. At present, interactions among many factors such as heredity, environment, infection, immunity, and hormone levels, are closely related to the pathogenesis of SLE. Immune dysfunction plays an important role in the formation and development of SLE, and especially, the adaptive immune system has been the focus of many studies. Abnormal activation and dysfunction of T cells and B cells results in the production of high levels of autoantibodies, inflammatory cytokines, and circulating immune complexes, as well as their invasion into tissues and organs, causing widespread damage [
1]. As the first barrier, the innate immune system plays an important role in the removal of foreign pathogens and the activation of an effective adaptive immune response. The innate immune system distinguishes pathogen associated molecular patterns (PAMPs) via pattern recognition receptors (PRRs). To date, three types of PRRs have been identified, of which, some NOD-like receptors (NLRs) can be activated by specific endogenous or exogenous stimuli and form a large protein complex referred to as the inflammasome [
2]. Four inflammasome, namely NLRP1, NLRP3, IPAF, and AIM2, have been discovered, among which, the study of NLRP3 has been the most extensive and universal. The NLRP3 inflammasome can be activated by a variety of pathogens such as bacteria, viruses, metabolic toxins, saturated fatty acids, amyloid peptides, adenosine triphosphate, and urate salts, and is related to the development of many diseases [
3]. It consists of three parts, specifically,
NLRP3,
ASC, and
Caspase-1; activation of
NLRP3 further activates the inflammatory caspase protein kinase (
Caspase-1), resulting in production of the active forms of the inflammatory cytokines interleukin-1b (
IL-1b) and interleukin-18 (
IL-18), which plays an important role in inflammation and the immune response.
Studies have found that in mouse models of lupus, the NLRP3-ASC-Caspase-1 signaling pathway is activated, and with a P2X7 receptor blocker (selective potassium channel inhibitor), the activity of the whole pathway was inhibited; thus, specific inhibition of the P2X7 receptor might represent a new direction for SLE therapy [
4]. However, our previous studies found that NLRP3 inflammasome components were expressed at low levels in peripheral blood mononuclear cells (PBMCs) from patients with SLE, which was inversely correlated with disease activities, suggesting that expression of the NLRP3 inflammasome might be a protective factor for SLE patients [
5]. This might be related to the following factors. First, the adaptive immune response system is activated during the pathogenesis of SLE, and T and B lymphocytes are activated in large quantities. It has been reported that upon the activation of adaptive immunity, NLRP3 inflammasomes are directly suppressed by T cells [
6]. In contrast, the low expression of the NLRP3 inflammasome in PBMCs from SLE patients might be related to the high expression of type I interferon (IFN-I), which is common in SLE patients and murine models of lupus. IFN-I has a significant inhibitory effect on the activation of the NLRP3 inflammasome [
7] and can inhibit its activation through the signal transducer and activator of transcription 1 (STAT1) pathway [
8].
Recent research has revealed that NIMA-related kinase 7 (
NEK7), a serine and threonine kinase involved in mitosis, acts as a key mediator of the activation of NLRP3 inflammasome signaling [
9]. In this study, we investigated the potential role of the NEK7- NLRP3 inflammasome in the pathogenesis of SLE and its association with disease activity. We detected expression of the NEK7-NLRP3 inflammasome pathway in Chinese Han SLE patients at the mRNA and protein levels and investigated its clinical significance to determine if it could represent a new target for SLE treatment.
Discussion
Previous studies have found that there are four main pathways of NLRP3 inflammasome activation. First, in the potassium efflux model, metabolites and endotoxin disrupt the integrity of the cell membrane or the binding of ATP to the P2X7 receptor on the cell membrane, leading to the opening of specific potassium channels, intracellular potassium efflux, and a disruption of mitochondrial structure and function, eventually activating the NLRP3 inflammasome [
10,
11]. The second is the direct activation of reactive oxygen species (ROS); here, mitochondrial-derived ROS directly activates the NLRP3 inflammasome and with specific ROS inhibitors, the generation of
IL-1b was found to be markedly reduced [
12]. Third, substances such as crystals or particles entering the cell induce the destruction of corpuscles and the release of cathepsin, thereby activating the NLRP3 inflammasome [
13]. Fourth, metabolites such as fatty acids, peptides, and toxins can also activate the NLRP3 inflammasome by coating microporous structures [
14].In addition to the combined effects of one or more of these pathways, the activation of the NLRP3 inflammasome also requires the synergistic action of nuclear transcription factor kappa B (NF-κB) signaling pathways and the modification of NLRP3 by ubiquitination. In conclusion, activation of the NLRP3 inflammasome is a complex process that is regulated by many factors and pathways [
15]. In 2016, Gabriel et al. [
9] found that
NEK7 plays an important role in the efflux of intracellular potassium ions, and that it is a key protein that activates the NLRP3 inflammasome. The NOD domains and leucine rich repeat (LRR) of NLRP3 components might be the key sites for these interactions. In addition, the lack of
NEK7 also specifically blocks activation of the NLRP3 inflammasome. Studies have also found that murine macrophages deficient in NEK7 exhibit a diminished response to lipopolysaccharide (LPS) stimulation. Moreover, compared to wild-type mice,
NEK7-deficient mice with multiple sclerosis present with fewer
IL-1b-related inflammatory diseases [
16]. Schmid-Burgk et al. [
17] used the clustered regularly interspaced short palindromic repeats (CRISPR/Cas9) system to detect murine macrophage strains, and found that
NEK7-knockout cells show reduced sensitivity to apoptosis induced by nigericin, mediated by low
Caspase-1 and
IL-1b expression. Current studies have also shown that
NEK7 plays a crucial role in the activation of the NLRP3 inflammasome, but most studies have been carried out in mice and in vitro, and there have been no reports of associated disease studies in humans. The purpose of this study was to investigate the expression and clinical significance of NEK7–NLRP3 signaling in Chinese Han SLE patients.
NEK7, as a member of the NIMA-related protein kinase family, concentrates at the poles of the spindle, functions to regulate microtubules, is closely related to the formation of the mitotic spindle and separation of the cytoplasm, and plays an important role in the regulation of cell cycle [
18].
NEK7 and NEK6 are highly homologous; both are not only structurally similar but also serve as a common substrate for NEK9 and are functionally synergistic. NEK6, 7, and 9 comprise an important group of proteins in which all three exert synergistic effects. In its absence or with abnormal expression of
NEK7, cell mitosis is blocked, which results in apoptosis. Presently, research on
NEK7 has been mainly concentrated in the field of malignant tumorigenesis, and this protein is closely related to the occurrence of breast and cervical cancer [
19]. The combination of
NLRP3 and
ASC can lead to mitochondrial structural damage and acetylation of microtubules, and the main function of
NEK7 is to maintain the dynamic stability of microtubule structure. It has also been indirectly shown that
NEK7 is closely related to activation of the NLRP3 inflammasome. The conserved and extensive expression of
NEK7 and its importance in mitosis suggest that it is not a specific NLRP3 inflammasome-activating protein [
17]. Studies have found that during interphase, the NLRP3 inflammasome can be activated by substances such as LPS, ATP, and nigericin, whereas in mitosis, activation of the NLRP3 inflammasome was markedly inhibited under the same stimulation. This shows that the
NEK7-mediated regulation of cell mitosis and NLRP3 inflammasome activation are mutually exclusive events, and that neither can occur simultaneously [
16]. Similarly, compared to that in mitotic cells, the NEK7–NLRP3 complex is highly expressed in interphase cells [
20]. Thus, the expression of this complex in cells is related to growth cycle.
In this study, the mRNA and protein expression of NEK7, NLRP3, and ASC in PBMCs from SLE patients was significantly lower than that in cells from healthy controls, and the difference was statistically significant. Moreover, the mRNA expression of NEK7, NLRP3, and ASC was inversely correlated with the disease activity index of SLE. Interclass correlation analysis showed that the mRNA expression of NEK7, NLRP3, and ASC were positively correlated. These results suggest that NEK7 activates the NLRP3 inflammasome in PBMCs from SLE patients, and that the NEK7–NLRP3 complex is closely related to the immune and inflammatory responses observed in SLE. For patients with SLE, after continuous methylprednisolone treatment (40 mg/day for 2 weeks), SLEDAI scores were reduced, clinical symptoms were relieved, and the mRNA and protein expression levels of NEK7, NLRP3, and ASC in PBMCs from SLE patients were up-regulated, suggesting that the NEK7–NLRP3 complex might act as a protective factor during the pathogenesis of SLE.
The low expression of the NLRP3 inflammasome in PBMCs from SLE patients might be related to the direct inhibitory effect of the adaptive immune system on the activation of T cells and the inhibition NLRP3 by interferon via the STAT1 signaling pathway. It might also be associated with the low expression of NEK7 in patients with SLE. In addition, the mRNA and protein expression levels of NEK7, NLRP3, and ASC in LN patients were significantly lower than those in SLE patients without kidney damage, suggests that this is closely related to the occurrence of LN and could be a protective factor involved in the pathogenesis of LN.
The mRNA and protein expression of Caspase-1 in PBMCs from SLE patients was significantly higher than that in healthy controls, and was positively correlated with SLE disease activities. After treatment, the expression of Caspase-1 decreased significantly. In addition, the mRNA and protein expression of Caspase-1 in the LN group was significantly higher than that in SLE patients without kidney damage. This reveals that high Caspase-1 expression is associated with the pathogenesis of SLE and LN and is positively related to disease activity. These results were in conflict with the observed low expression of NEK7, NLRP3, and ASC, and suggests that the expression of Caspase-1 in PBMCs from SLE patients is not only affected by NLRP3 inflammasome signaling, but also by other signaling pathways.
Consistent with our experimental results, Zhang et al. [
21] reported that compared to those in healthy controls, the expression levels of
Caspase-1 and
IL-1b in patients with SLE were significantly increased and positively correlated with disease activity. Moreover, they further confirmed that in patients with SLE, ds-DNA interacts with Toll like receptor 4 (TLR4) and induces the mitochondrial production of ROS in monocytes and macrophages, eventually leading to high
Caspase-1 expression. As is known,
Caspase-1 is widely involved in cell growth, differentiation, injury, repair, and apoptosis, and it is differentially expressed in diverse contexts [
22]. Furthermore, a large number of apoptotic bodies and abnormal apoptosis have been observed in PBMCs from SLE patients, which might be related to the high expression of
Caspase-1 [
23]. Experiments in mouse models of lupus have shown that Caspase-1P20 is activated, and that inhibition of this activation can significantly reduce infection and improve disease severity [
24]. In a mouse model of LN, the severity of clinical signs was markedly improved by specific
Caspase-1 inhibitors [
25]. However, in our previous study,
Caspase-1 was found to be poorly expressed in SLE patients, and the results were inconsistent. Possible reasons for this are as follows. First, in the early stage of disease,
Caspase-1 is highly expressed and induces the maturation and release of downstream factors; as a risk factor, it is involved in immune and inflammatory reactions in the organism. When the disease develops,
IL-1b and
IL-18 cluster together to activate programmed cell apoptosis, which is dependent on the
Caspase-1 pathway via feedback. Comparing these studies, the two groups had different disease periods, which might be the main reason for these inconsistent results. Second, the two groups had different degrees of disease activity. In our previous study, the mean SLEDAI score of patients with SLE was 15 (2–26), and in this study, it was 8.4 (0–18); thus, there were significant differences in disease activity between the two groups. Differences might also be related to individual differences, experimental errors, and other factors.
In our study, the mRNA and protein expression of
IL-1b and
IL-18 in PBMCs from SLE patients was significantly higher than that in healthy controls, and this was positively correlated with disease activity. Further, after treatment, the expression of these markers decreased obviously. Similarly, serum levels of
IL-1b and
IL-18 in patients with SLE were also high and positively correlated with the evaluation index of SLE disease activities; these levels were also significantly decreased after drug treatment. The mRNA and protein expression of
IL-1b and
IL-18 in the LN group was significantly higher than that in the SLE group without kidney damage, suggesting that high expression of these markers is closely related to the incidence of SLE and LN, because high activation of
Caspase-1 eventually leads to high expression of
IL-1b and
IL-18. Wang et al. [
26] found that in patients with SLE,
IL-1b was highly expressed and involved in multiple organ damage during SLE, and was especially associated with the occurrence of neuropsychiatric lupus. The fact that it is difficult to induce lupus in
IL-1b-deficient mice indirectly suggests that
IL-1b has an irreplaceable role in the pathogenesis of SLE [
27]. The possible role of
IL-1b in the pathogenesis of SLE might also be related to the activation of B cells and the production of immunoglobulin IgG and autoantibodies such as anti-ds-DNA [
28]. In addition, anakinra, an IL-1 receptor antagonist, can effectively reduce clinical symptoms and disease severity in patients with SLE [
29,
30]. One study found that compared to that in healthy controls, the expression of
IL-18 in the serum of SLE patients increased significantly. In patients with LN, this increase was more pronounced, and the serum levels of
IL-18 were positively correlated with SLEDAI score and urinary proteins [
31,
32]. In addition,
IL-18 gene polymorphisms were found to be significantly associated with the occurrence of arthritis symptoms in SLE patients [
33]. The role of
IL-18 in the development of SLE might be related to the induction of other proinflammatory cytokines such as TNF-α, IFN-γ, and IL-1. This suggests that the activation and release of
IL-1b and
IL-18, mediated by excessive activation of
Caspase-1 in PBMCs from SLE patients, contribute to the pathogenesis of SLE and LN.