Multiple sclerosis (MS) is a chronic autoimmune disease of the central nervous system (CNS), characterized by demyelination and neurodegeneration [1
], which represents the most frequent neuroinflammatory disease in young adults, with a mean age of diagnosis of about 30 years and a worldwide prevalence rate of 30.1 cases per 100,000 population in 2016 [2
We and others have shown that proinflammatory cytokines such as macrophage migration inhibitory factor (MIF), interleukin (IL)12, and IL18 can be implicated in progressive forms of the disease [3
] and that an altered balance between proinflammatory Th1 and Th17 cytokines and anti-inflammatory Th2 and Th3 cytokines may represent important pathogenetic events contributing to the onset and clinical course of relapsing remitting forms of the disease, as well as its therapeutic response. Indeed, endogenous anti-inflammatory mediators such as IL1-receptor antagonist (IL1-Ra, encoded by the gene IL1RN) [9
], soluble IL1-receptor [10
], transforming growth factor (TGF)-β [11
], and IL4 can be augmented by disease-modifying drugs such as interferon (IFN)-β or glatiramer acetate [12
]. Both glatiramer acetate and IFN-β reduce IL17A [13
]. We have also shown that corticosteroids that are used during relapses of the disease increase the circulating levels of soluble IL1 type II receptor and IL1-Ra [10
Hence, a tightly regulated endogenous network composed of proinflammatory and anti-inflammatory cytokines and other cellular and soluble mediators may control the onset and the progression of the disease and may also be implicated in its therapeutic response [14
IL37 is a cytokine belonging to the IL1 family that was originally discovered by in silico research [15
]. IL37 can be found in the cytoplasm and nucleus [16
] and is detectable in many human cells, including monocytes; natural killer (NK) cells; stimulated B cells; and tissues such as thymus, bone marrow, lymph nodes, colon, lung, and uterus [17
]. The human IL37 gene is located on chromosome 2 and undergoes alternative splicing. There are five splice variants from IL37a to IL37e. IL37b, the longest sequence in the five isoforms of IL37, contains 218 amino acids and represents the cytokine expressed at maturity [18
]. To exert its biological function, IL37 binds to a receptor complex composed of single Ig IL-1-related receptor (SIGIRR), also known as IL1-R8 or Toll interleukin-1 receptor 8 (TIR8) and IL18-Rα congregated on the surface of peripheral blood mononuclear cells (PBMCs) upon pro-inflammatory stimulation [19
]. IL37 may also interact with low affinity and without influencing the biological function of IL18 with the α receptor subunit of IL18 receptor (IL18Rα or IL18R1). However, the inhibition of IL18 proinflammatory activity results from the binding of IL37 with the IL18 binding protein (IL18BP) and with IL18-Rβ, which results in an inactive complex that reduces the production of IFN-γ levels [16
]. Once bound to the receptor, IL37 is translocated to the nucleus, where it interacts with Smad3 before activating specific gene transcription [20
In addition, it was recently demonstrated that human IL37 has a caspase-1 cleavage site and that nuclear translocation of IL37 is inhibited by caspase-1 inhibition [21
Several preclinical studies support the concept that IL37 may potently downregulate immune-inflammmatory responses. Among others, these include the anti-inflammatory function of IL37 in human coronary artery endothelial cells [22
], as well as the augmented production of proinflammatory cytokines in human peripheral blood mononuclear cells secondary to IL37 knockdown [20
]. Moreover, treatment with recombinant human IL37 suppresses lipopolysaccharide (LPS)-induced cytokines IL1-β, IL6, and tumor necrosis factor (TNF)-α; inhibits p38 mitogen-activated protein kinase (MAPK) activation in M1-differentiated human blood macrophages; and reduces innate inflammation markers in mice subjected to endotoxemia [23
]. In rodent models of autoimmune diseases, the intra-articular injection of an adenovirus vector expressing IL37 into the knee joints of mice with type II collagen-induced arthritis (CIA) markedly down-regulated the clinical and histological signs of arthritis [24
Other studies have shown that IL37 exerts beneficial anti-inflammatory effects in animal models of colitis and myocardial ischemia-reperfusion injury and concanavalin A (ConA)-induced immunoinflammatory hepatitis [25
]. In the clinical setting, it has concordantly been shown that circulating and/or PBMC levels of IL37 are increased in several autoimmune diseases including systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), inflammatory bowel disease, ankylosing spondylitis, psoriasis, and Graves’ disease [25
]. These increased levels often occur during exacerbations of the diseases and correlate with biomarkers of disease activity, suggesting that the expression of IL37 corresponds to the disease activity of RA, ankylosing spondylitis (AS), Graves’ disease (GD), and SLE [25
]. Of additional interest was the observation that in vitro-added IL37 decreased production of proinflammatory cytokines from PBMCs of AS, GD, and SLE patients, but not of healthy controls [25
]. These augmented levels of IL37 in inactive phases of autoimmune diseases have been interpreted as a compensatory attempt that is physiologically activated by the body to counteract excessive ongoing immunoinflammatory responses. Nonetheless, even though it is inconsistent with the potent anti-inflammatory action of IL37 in experimental studies, the hypothesis can neither be ruled out that the augmented levels of IL37 may play an unexpected pathogenetic role in these autoimmune diseases by activating unknown inflammatory pathways that have so far not been recognized to be modulated by IL37. Cytokines exerting primarily anti-inflammatory effects have subsequently been shown to be also capable of upregulating immunoinflammatory responses, as has been shown with endogenous IL4 in models of autoimmune hepatitis [29
] and exogenously administered IL10 in models of orchitis [30
]. Vice versa, anti-inflammatory effects of prototypical Th1 cytokines such as IFN-γ and also TNF-α have been reported in models of type 1 diabetes [31
In spite of these data of IL37 in autoimmune diseases, only a limited number of studies have evaluated the possible role of this cytokine in MS [33
]. Farrokhi et al. showed that IL37 serum levels were higher both in patients suffering from relapsing-remitting MS (RR-MS) and those with neuromyelitis optica (NMO) as compared to healthy controls. In addition, they noticed a positive correlation between the serum levels of IL37 and Expanded Disability Status Scale (EDSS) of patients [33
]. These findings were subsequently confirmed in another independent study that demonstrated that serum levels of IL37 were higher in patients with MS and correlated with disease activity as well as with the levels of IL33 and soluble vascular endothelial growth factor (VEGF) receptor 2 [34
Taken together, these papers seem to indicate that endogenous IL37 is produced in response to ongoing immunoinflammatory events during the course of MS with the possible, and yet unsuccessful, attempt to downregulate the progression of the disease. Along this line of research, recent data have shown that the beneficial effects of hypoxia-preconditioned human periodontal ligament cell secretome in myelin oligodendrocyte glycoprotein peptide (MOG)-induced experimental autoimmune encephalomyelitis (EAE) were associated with marked expression of IL37 [35
These initial data propelled us to further study the involvement of IL37 in MS. To do so, we evaluated in silico expression and circulating levels of IL37 in patients with different forms of the disease and also upon treatment with different disease modifying drugs [37
]. The combined interpretation of the resulting data strengthens and extends the current emerging concept that endogenous IL37 plays an important role in determining onset and progression of MS. In addition, the present demonstration that fingolimod, but not other disease-modifying therapies (DMTs), augmented the levels of IL37 in a significant manner indicates that this drug may at least partially exert its beneficial effects in MS by upregulating the production of IL37.
In the present study, we first analyzed the expression levels of IL37, SIGIRR, and IL18R1 in circulating immune cells from MS patients. In particular, we performed a DNA microarray analysis that represents a useful in silico tool for the better understanding of pathogenic pathways and the possible prediction of novel diagnostic therapeutic strategies, as it has been shown in a variety of clinical settings, such as autoimmune and immunoinflammatory diseases [38
] and cancer [45
], leading to the identification of novel therapeutic targets [52
Following activation, CD4+ T cells from MS patients showed lower levels of expression of IL37 than helper T cells from healthy controls. Opposite results were observed for the IL37 receptors. Indeed, in stimulated CD4+ T cells, either in healthy control or in MS patients, we observed a downregulation of SIGIRR and an upregulation of IL18R1. Conversely, in unstimulated CD4+ T cells, both in healthy control or in SM patients, SIGIRR was upregulated and IL18R1 was downregulated.
Moreover, we found a reduction of IL37 levels in PBMCs from MS patients undergoing a relapse compared to stable patients. We also investigated the level expression of IL18R1 and SIGIRR, which showed only a moderate or no modulation between patients with stable or relapsing disease.
Of particular interest, in the in silico analysis had the observation that higher levels of IL37 levels in PBMC entailed a significant protection to the exacerbation of the disease.
It is noteworthy that our in silico data show how IL37 and IL1Ra are directly correlated. In PBMCs from MS patients undergoing a relapse, the mutual levels increase of IL1Ra and IL37 was observed. This finding adds IL37 to the IL1 family of endogenous anti-inflammatory networks composed from IL1Ra-soluble IL1 receptor type II that we and others have previously studied in MS [56
]. The above data suggest that the elevated levels of endogenous IL1Ra and IL37 may indicate an attempt to promote anti-inflammatory responses, possibly inhibiting immuno-inflammatory events induced by IL1.
In addition to the data elaborated from the in silico study, we also generated new data in an in vivo study by analyzing IL37 serum level in MS patients with different phenotype conditions and different therapeutic responses. We studied IL37 levels in patients with PP-, SP-, and RR-MS in stable disease or during relapses. It has been generally accepted that inflammatory processes within the CNS in MS are more exaggerated in the younger population [57
], and therefore our findings that higher IL37 levels were associated with younger age (p
= 0.047) are in line with this notion. Additionally, we demonstrated that higher IL37 levels were associated with lower MSSS (p
= 0.039), thus supporting the previously mentioned hypothesis that IL37 produced in response to ongoing immunoinflammatory events in MS might downregulate the progression of the disease. Therefore, the activation of IL37 and IL1Ra could be crucial in limiting the harmful effects of IL1 during the recovery phase in MS.
Furthermore, our data demonstrate that the serum levels of IL37 were significantly augmented during relapses of the disease, as well as in patients with stable disease upon treatment with highly effective DMT. In particular, the greatest percentage of MS patients with stable disease exhibiting dosable levels of IL37 was found in the MS patients treated with fingolimod, with six out of nine having detectable levels of IL37 in their circulation. It seems therefore possible that one of the immunopharmacological mode of action by which fingolimod ameliorates RR-MS may rely on the induced production of IL37. In vitro and in vivo studies are necessary to more precisely dismantle the mode of action of fingolimod on IL37 production. Previous studies on the immunopharmacological mode of action of this drug in RR-MS have shown that it increases in a non-specific fashion both pro- and anti-inflammatory cytokine-producing T helper subsets (IFN-γ, TNF-α, IL4, and IL10-producing CD4+ T cells) [58
]. We also observed an increasing trend of IL37 level expression after steroid therapy. These data could indicate that IL37 and IL1Ra are closely related, demonstrated by the fact that glucocorticoids and transforming growth factor (TGF)-β are among the agents that reduce the production of proinflammatory cytokines of the IL1 family [59
The combined comparison of in silico analysis and in vivo data appear in conflict with regard to the finding that a significant reduction in IL37 expression was observed in PBMCs from MS patients undergoing exacerbation of the disease in the in silico analysis whereas augmented levels of IL37 were observed in the circulation of MS patients suffering an exacerbation. These apparently conflicting results may be explained by the fact that in the in silico analysis only the production of IL37 from PBMC was evaluated, whereas measurement of the circulating levels of IL37 also takes into account the production of IL37 from other non-immune sources including uterus and gastrointestinal cells for example [17
]. Although our study is the first to demonstrate increased levels of IL37 during relapses of MS, two previous studies found augmented blood levels of IL37 in Iranian patients suffering from RR-MS and NMO. It is very difficult to compare the studies and dismantle reasons for the possible differences. In particular, in the two studies in the Iranian population, it was not specified whether the patients were sampled during stable phases of MS or during exacerbations. Information on the treatment of the patients was not provided, yet this information is clearly essential as DMT modifies the course of the disease and the counter-inflammatory responses that could evoke IL37 production.
Our results thus indicate that IL-1Ra activation and expression of IL37 could be one of the defense mechanisms leading to remissions in MS and lower disease severity as measured by MSSS. Further studies are warranted to understand whether the dysregulated balance between pro-inflammatory and anti-inflammatory cytokines, their receptors, and endogenous inhibitors might play a role in designing novel diagnostic tools and therapeutic approaches for MS. Indeed, our present work represents only an observational study and cross sectional analysis for the treatment arm on the effects of DMTs on IL-37 levels, and no direct or causal relation between the observed level of IL37 and the clinical parameters can be presently inferred. Nonetheless, our study adds new important pieces of new information to the potential role of IL37 in MS and its eventual contribution to mediate the action of fingolimod on the course of the disease.