Assessing the expression of immunosuppressive cytokines in the newly diagnosed systemic lupus Erythematosus patients: a focus on B cells

The systemic lupus erythematosus (SLE) is a multifactorial chronic autoimmune disease that is more common in women [1]. Evidence showed that genetic and environmental factors, including cigarette smoking, drugs, ultraviolet (UV) light, chemical substances, gut microbiota, and viral infections, could be involved in SLE onset [1, 2]. Regarding the existence of an imbalance between apoptosis and removal of apoptotic substances in SLE patients, nuclear antigens such as histones, centromere proteins, single- and double-stranded deoxyribonucleic acid (ss- and ds-DNA), nucleosome, Smith antigen (Sm Ag), Ro and La proteins, as well as ribonucleoproteins (RNPs) become exposed to the immune system cells and components [3]. These autoantigens are able to stimulate B-cells to produce auto-antibodies such as anti-nuclear antibody (ANA) and anti-double-stranded DNA (ds-DNA) antibody as well as other inflammatory mediators [4].

Previous studies revealed that regulatory immune cells, such as regulatory T cells (Tregs) and B regs with immunosuppressive properties, are involved in the homeostasis processes [5]. Recent experimental and human studies have further indicated that Bregs can suppress inflammatory responses via production and secretion of anti-inflammatory cytokines like IL-10, IL-35, and TGF-β as well as expression of inhibitory molecules [6, 7]. Besides, Bregs come up from common progenitor of transitional 2-marginal zone precursor (T2-MZP) B cells capable of being autoreactive following interaction with pathogens and even activating release anti-inflammatory mediators [8]. Some investigations in this respect have correspondingly suggested that Bregs are impaired in inflammatory autoimmune disorders such as SLE, rheumatoid arthritis (RA), and Graves’ disease [9]. However, signals necessary for differentiation of Bregs have remained poorly understood, and previous studies have revealed that, under normal conditions, plasmacytoid dendritic cells (pDCs) would stimulate differentiation of CD19 + CD24^hiCD38^hi immature B-cells into the CD24⁺CD38^hi mature Bregs, which could produce IL-10 through release of interferon-alpha (IFN-α) and the cluster of differentiation 40 (CD40) engagement. Conversely, the mentioned Bregs can have an inhibitory effect on the generation of IFN-α through pDCs by releasing IL-10 [10]. In SLE, the cross-talk between pDCs and Bregs has defected, and pDCs fail to trigger differentiating CD19⁺CD24^hiCD38^hi B-cells into IL-10-producing CD24⁺CD38^hi Bregs [10]. In addition, Bregs can suppress CD4⁺ T-cell proliferation, induce Foxp3⁺ regulatory T and Tr1 cells, and suppress T helper (Th) 1 (Th1). Bregs also can stimulate Th17 and CD8⁺ effector T-cells differentiation through cell-cell interactions and release of the anti-inflammatory cytokine, including TGF-β, IL-35, and IL-10. As well, TGF-β and IL-10, which are secreted by Bregs, can have an inhibitory effect on antigen-presenting function, cytokine secretion by DCs, neutrophils, natural killer (NK) cells, M1 macrophages, and conversely, induction of M2 macrophage differentiation.

Moreover, FasL (or CD178) or PD-L1 (or CD274) that are expressed on the Bregs surface are involved in the apoptosis of effector T cells following ligation with PD-1 (CD279) and Fas (CD95) on the surface of mentioned T-cells [11]. Since changes in immune cells phenotype, as well as their number and function, are closely related to the disease activity, measuring the produced mediators and inhibitory molecules by regulatory immune cells before treatment based on diseases activity status could be critical in further understanding the role of the modulatory mechanisms of the immune system in the pathogenesis of SLE. Therefore, this study aimed to measure inhibitory molecules’ expression on B cells’ surface and anti-inflammatory cytokines produced by B cells in newly diagnosed SLE patients compared to healthy subjects.

Twenty-three patients suffering from SLE and thirty normal-age and gender-matched subjects were enrolled in this study. The demographic features of SLE patients and healthy subjects are shown in Table 1. All the patients were classified as individuals having an active disease (SLEDAI ≥4). The mean ± SD of SLEDAI in patients was 9.73 ± 6.01and we divided patients into three groups (SLEDAI I; 4 to 7, SLEDAI II; 7 to10, SLEDAI III more than 10) based on SLEDAI scores (min:5, max: 34, and median: 8). At the time of the blood collection, newly diagnosed SLE patients were not under treatment. Clinical and laboratory data of studied SLE patients are shown in Table 2.

Evidence revealed that SLE is a multiple-organ autoimmune disease described by autoantibodies’ increased production against autoantigens and enhanced immune complex deposition. Both T- and B-cells with different phenotypes are also involved in SLE’s pathogenesis [12]. Previous studies on a subset of B cells with modulatory properties in patients with RA, primary Sjogren’s syndrome (SjS), and SLE have demonstrated a hyperfunction in these B cells anti-inflammatory cytokines like IL-10 production and eventually homeostasis [9, 13, 14]. Several investigations have shown that depletion of pan B cell could help SLE patients, while phase III trials of the pan-B cell depletion were unsuccessful [15]. This problem can be due to the depletion of both the effector and regulatory B cell subsets [6]. These findings could accordingly confirm the immune-modulatory properties of Bregs and their involvement in the homeostasis mechanisms [16]. The first clinical investigation of peripheral blood B cells in SLE patients revealed that the proportion of CD5⁺ B cells producing IL-10 was significantly higher than healthy subjects [17]. Although there are several contradictions in this field, some studies have shown that the number of Bregs in autoimmune diseases such as RA and SLE decreased [13]; on the other hand, some experimental and human studies have demonstrated that the number of Bregs was increased in SLE which can ultimately depend on various factors such as treatment protocol and disease activity. In this study, the results show that apart from the number, a group of B cells might be hyperactivated to cause homeostasis in patients with lower SLEDAI scores [17‐21]. In line with our findings, it was shown that in humans, IL-10⁺ B cell frequency in SLE patients was higher than the healthy control group [17, 20]. These findings also confirm the possibility of hyperactive Bregs involvement in balancing inflammatory and anti-inflammatory responses. Although mechanisms that mediate development and induction of the Bregs have thus far remained unclear, a study suggested that the increased IL-10⁺ Bregs may be secondary to expanded T follicular helper (Tfh) cells provide help to effector B cells and promote autoimmunity. Tfh cell and IL-10⁺ Bregs are increased in SLE and Tfh cell-derived IL-21 induced IL-10 [22]. In most human studies that have been done so far, SLE patients were under different treatment protocols, affecting the outcomes [13]. For instance, methotrexate can increase the number and possibly, the activity of B cells [23]. Another study on murine collagen-induced arthritis showed that treating animals with methotrexate, alone or together with cyclophosphamide, can reduce Bregs and DCs in lymph nodes and spleen [24].

In contrast, another investigation reported no associations between azathioprine, methotrexate, mycophenolate mofetil, and hydroxychloroquine with the expression of IL-10. However, the mentioned study showed that the serum level of IL-10 was twice more in SLE patients with Asian ethnicity than non-Asians [25]. The results of this study show that patients’ ethnicity can also affect the level of anti-inflammatory cytokines. Recently it has been shown that systemic treatment with methotrexate may cause dysregulation of anti-inflammatory cytokines [26]. According to the mentioned studies, it seems that treatment with immunomodulatory drugs can change the cytokine profile and negatively affect the results. In our study, the results showed that in newly diagnosed SLE patients with mild or moderate disease activity who were not under any treatment, anti-inflammatory cytokines as well as inhibitory molecules (as suppressive indicators of B cells) expression increased by isolated B cells in compared to healthy subjects and by increasing the SLEDAI score, serum levels of IL-10 and TGF-β decreased remarkably. However, the balance between regulatory and effector functions is a finely regulated immunological process that is not fully understood.

In this study, it was observed that IL-35 levels significantly elevated in SLE patients compared to healthy subjects. In this regard, an investigation reported that IL-35 could induce B cells and stimulate their differentiation into a regulatory subset producing IL-35 and IL-10. These findings similarly proposed that IL-35 is a potential inducer of the autologous and IL-35⁺ B cells in treating inflammatory and autoimmune diseases [27]. It had been further confirmed that circulatory IL-10⁺ Bregs had significantly increased in SLE patients, accompanied by fluctuation such that the number and activity of the Bregs had elevated during SLE flares and reduced subsequent remission of the disease [22]. Our findings also showed that as the SLEDAI score increased, the levels of IL-10 and TGF-β significantly decreased in studied SLE patients, although no decrease was observed in the serum level of IL-35. These findings could confirm the role of disease activity in the fluctuation of the B cells’ number and activity in SLE patients.

It should be noted that myeloid-derived suppressor cells (MDSCs) as an immune suppressor cell can also induce the development of Bregs through inducible nitric oxide synthase (iNOS) and relieve self-immune responses in a lupus animal model [28]. Moreover, Bregs are not the only source of immunosuppressive cytokines because other cells might produce and release IL-10, TGF-β, and IL-35 in response to inflammatory and pathological conditions to modulate the immune response. Evidence showed that the regulatory T cells (Tregs) and Bregs involvement in different phases of autoimmune diseases could be different. For instance, a study had shown that IL-10⁺ Bregs could mainly regulate the initiation phase of the disease, while Tregs could cooperatively inhibit late-phase via covering IL-10⁺ Bregs in an experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis (MS) disease [29].

Berthelot et al. had reported that induction of apoptosis by Bregs through PD-1, FasL, or TNF-related apoptosis-inducing ligand (TRAIL) differed with the nature of the target T cell. T-cell subsets’ sensitivity probably shifted from Th1 and Th2 to Th17, resulting in a reduction in Tregs [30]. The outputs of this study also demonstrated that the expressions of the PDL-1 and FasL gene had significantly elevated in isolated B-cells of the patients with SLE compared to healthy subjects. It could be another mechanism of B cell for modulating the immune response by eliminating effector immune cells.

On the other hand, some studies found that regulatory immune cells’ function to inhibit effector T cells in inflammatory and autoimmune illnesses like lupus was defective. However, in most of these investigations, serum levels of IL-35, IL-10, and TGF-β cytokines along with PDL-1 and FasL had not been measured in B cells. The wide range of disease activity in SLE patients and treatment protocols might lead to these discrepancies in the studies’ findings.

Our findings showed a negative association between serum level of TGF-β and SLEDAI; IL-10 and EBI3 genes may be due to reduced Bregs function in patients with higher disease activity scores. However, a positive and significant correlation was observed between the PDL-1 gene and serum level of IL-35, as well as IL-10 and TGF-β serum levels [7]. Furthermore, regarding the studied cytokines’ extracted data at mRNA and protein levels (components 1 and 2), clustering showed that it is possible to differentiate patients from healthy subjects using the information in components 1 and 2. Additionally, statistical analysis demonstrated that with increasing the SLEDAI score, component 2 was significantly decreased. Therefore, with increasing disease activity, the level of immunomodulatory cytokines produced by B cells in patients with SLE might significantly be decreased.

One of the strengths of this study was that all the patients enrolled in the study were newly diagnosed cases, and blood collection was done before treatment onset because, as discussed before, routine treatment for lupus patients could affect the number and activity of B cells and outcome of the studies [17]. The inaccessibility of the materials for fluorescence-activated cell sorting (FACS) technique to specific isolation and evaluation cytokine levels in Bregs was also declared one of the major limitations of this study.

Taken together, the results of this study indicated that a group of SLE patients B cells might modulate immune responses in mild to moderate disease activity by producing anti-inflammatory cytokines and expressing inhibitory molecules. The findings can also clarify SLE disease activity’s effect on the fluctuation and expression of IL-10, TGF-β serum levels. However, the source of the studied cytokines might be other regulatory immune cells. Future studies are needed to fully elucidate the source of released anti-inflammatory cytokines and evaluate the producer regulatory cells’ number and activity to a well understanding of the immunomodulatory mechanisms in SLE.