1. Introduction
Under the circumstances of host pathology and defense, the cells comprising the immune system produce an assortment of biological products that enable them to communicate and mount specific and effective immune responses. Among these biological products, cytokines are perceived as crucial mediators, which are integral to the progression and outcome of numerous infections, auto-reactive disorders, allergic illnesses as well as neoplasms in which a range of cytokines are produced to modulate subsequent cellular responses of the host towards the invading pathogen or noxious stimuli. [
1].
Essentially, cytokines represent an integrated network of cellular mediators capable of eliciting diverse biological effects influenced by the prevailing state of the organism [
2]. They represent a distinct, heterogeneous array of proteins which are fundamental in conveying signals amidst various cells of the immune system and consequently oversee gene expression in these cells; operate the scope and extent of an inflammatory response, in addition to controlling cellular differentiation, expansion, antibody secretion, inflammatory immune responses as well as immune pathology [
3,
4]. Basically, cytokines mediate the turnout of an effective immune response and serve as an interface between the two arms (i.e., innate and adaptive elements) of an otherwise complex immune system [
5].
Usually categorized as being either pro-inflammatory or anti-inflammatory, cytokines are secreted by diverse cell populations upon stimulation [
6]. Notably, both pro-inflammatory and anti-inflammatory types elicit distinct responses to immunogens at different stages of an infection. Moreover, cytokine response profiles generated towards the same stimuli have been shown to differ between different individuals, among species and depend on the nature of a disease, either chronic or acute, pathogenic or autoreactive in etiology [
7]. Certain cytokines have pleiotropic functions and hence exhibit functional redundancy [
8]. Cytokine repertoires influence the make-up of cellular infiltrate(s), cellular activation and intrinsic responses to inflammation. Furthermore, their actions may be “autocrine,” whereby they exert their biological function on the cell(s) that secrete them or “pancrine” in which case they evoke actions on cells other than those from which they are secreted [
1].
Although cytokines are beneficial in the initiation and coordination of immune responses, unregulated cytokine signaling inadvertently constitutes principal determinants of immunopathology, giving rise to autoimmune disorders such as rheumatoid arthritis as well as hypersensitivity reactions [
9,
10]. Cytokines are of central importance in the development of immunopathological symptoms in consort with several diseases owing more or less to the fact that their signaling is complex and tightly regulated and they are capable of acting synergistically, hence, minor disturbances in their homeostasis may result in immunopathology.
Figure 1 depicts the consequences of unregulated cytokine activation which will eventually leads to immunopathological events seen in various diseases.
Contemporary advancements in understanding cytokine biology have brought to light the benefits of focusing on this class of proteins as druggable targets for the development of therapeutic biological macromolecules that are efficacious in disease amelioration and highly selective with fewer incidences of ambiguous side effects [
1]. Remarkable success has been achieved in the areas of targeting (blocking or enhancing) cytokine activities for therapeutic purposes [
9,
11]. Over the past decade, IL-35 and IL-37 have been the focus of immunoregulatory cytokine research in a fair share of disease models. Intense investigations into the possible implications of their immune suppressing properties as well as their potential for therapeutic application as cutting-edge biological macromolecule is being explored in diseases ranging from autoimmune diseases, various types of cancer as well as infectious diseases [
12].
2. Immunobiological Significance of IL-35 in Disease Pathogenesis
The IL-12 cytokine family to which IL-35 belongs (as the most recently identified member) is quite unique in that the members comprise of two distinct units; an α-subunit (p19, p28 or p35) along with a β-subunit (p40 or Ebi3). This family of cytokine exists as heterodimeric molecules [
12,
13], viz IL-12 (p35/p40), IL-23 (p19/p40), IL-27 (p28/Ebi3), IL-35 (p35/Ebi3), IL-39 (p19/Ebi3) as well as IL-Y (p28/p40). Due to similarities in chain pairing, family members exhibit structural homology and feature similar receptors (signaling pathways) for the execution of their respective contrasting biological functions [
14]. This family of cytokines exerts multiple functions to produce diverse phenotypic traits. Egwuagu et al. [
15] demonstrated their profound actions in shaping and regulating host immunity. These cytokines operate a diverse multiplicity of immune responses, from inflammation promoting T
H1, T
H2 and T
H17 type responses to retroactive anti-inflammatory immune defenses mediated primarily via regulatory T cell (T
regs) proliferation and expansion of IL-10 expressing T
regs [
15].
Figure 2 depicts the IL-12 family members and their associated subunits together with their roles as pro-inflammatory and anti-inflammatory cytokines.
Contrasting in expression pattern and biological activity, the serendipitous finding of IL-35 occurred when the α-subunit of IL-12 (p35) was co-incubated with Ebi3 (which is itself a subunit shared by the IL-6 cytokine super family as well as the IL-12 cytokine family) in the pursuit to discover probable catenation pairs for Ebi3 [
16]. The ensuing novel heterodimer formed (Ebi3/p35) was later discovered to be capable of exerting potent immunoregulatory actions [
16,
17]. Both α and β-subunits of IL-35 are largely derived from regulatory Foxp3
+ T
regs distinctive from most IL-12 cytokine family members which are chiefly expressed on the surface of activated dendritic cells, B-cells, monocytes and macrophages viz antigen presenting cells via which they drive T
H1 responses solely or synergistically in addition to promoting interferon-γ (IFN-γ) generation by T cells [
16,
18]. Contrariwise, a study by Li et al. [
19] involving gene-expression profiles of
IL-35 revealed a broader tissue distribution than was earlier portrayed i.e., confined to T
regs [
19]. The p35/p40 heterodimer (i.e., IL-12) along with p19/p40 (viz IL-23) constitute the strictly pro-inflammatory members of this enigmatic cytokine family, whereas IL-27 on the other hand is described as capable of exerting pleomorphic actions, specifically driving T
H1 responses similar to IL-12 on one hand and promoting IL-10 dependent immunosuppression on the other [
13,
20,
21].
IL-35 represents a relatively newcomer to the already established class of extensively characterized suppressive cytokines comprising of IL-10, transforming growth factor-β (TGF-β) as well as the pleomorphic co-IL-12 cytokine family member IL-27 [
12]. These suppressive cytokines comprise critical immunomodulatory networks and tolerance-promoting channels essential for adequate functioning of the immune system [
12]. Collison et al. [
16] characterized IL-35 as a strictly immunosuppressive cytokine possessing the capacity to abolish T
eff cell expansion while simultaneously mediating the conversion of naive T cells toward a robust population of induced T
regs (iTr) that function exclusively via IL-35 [
16,
22].
Resting/non-stimulated mouse T
regs have been well-established to be capable of secreting IL-35 whereas, non-stimulated human T
regs are not capable of doing so [
16]. This insinuates that IL-35 is inducible and not fundamentally expressed by humans. This was further substantiated by Li et al. [
19] who demonstrated the presence of
IL-35 mRNA in monocytes, smooth muscle cells as well as endothelial cells only after pyrogenic perturbation with lipopolysaccharide (LPS) or inflammatory cytokine(s). Taken together, this data delineates the role of IL-35 as a responsive cytokine which is only secreted in reaction to inflammatory stimuli [
19]. Contrarily a later study by Mao et al. established that IL-35 was intrinsically expressed in human placental trophoblast cells [
23].
Notably, endothelial cells, activated B cells, dendritic cells, monocytes, smooth muscle cells as well as macrophages have been reported to secrete IL-35 although to a lesser extent [
24,
25]. Secreted in response to interferon-γ (IFN-γ), IL-35 acts as an agonist on toll-like receptors (TLR) 3 and 4. The immunoregulatory function of IL-35 occurs principally via the inhibition of CD4
+ effector cell expansion (T
H1 and T
H17 included), along with the enhancement of T
reg cell proliferation and IL-10 production [
24]. IL-35 functions by expanding T
reg cell population effectively inhibiting T
eff cells thereby forestalling immune injury in the event of an ensuing chronic infection and inhibition of T
H17 cell differentiation away from unbridled autoimmune reaction [
24].
Although described as an initiator and effector of anti-inflammatory signaling, the principal actions of IL-35 in living systems are yet to be clearly elucidated [
26]. It is however evident that in the presence of an antigen presenting cell-free culture, IL-35 directly suppresses the proliferation of T
eff cells. Additionally, IL-35 deficient T
reg cells have been observed to display greatly diminished suppressive ability in vivo resulting in a failure to hinder the homeostatic expansion of T
eff cells and predisposition to subsequent exuberant autoimmune sequelae, evidenced by the impaired in vivo ability of IL-35 deficient T
regs to alleviate colitis [
16].
The IL-12 receptor (IL-12R) family comprises of five main receptor subunits. It is presumed that a combination of receptor types is used by IL-35 for signal transduction. Similarly, various unique receptor chain combinations are employed by other family members for signal transduction including interleukin-12 receptor (IL-12Rβ1 and IL-12Rβ2), interleukin-23 receptor (IL-23R), interleukin 27 receptor (WSX-1) and glycoprotein 130 receptor (gp130) as illustrated in
Figure 3 [
27].
Interestingly, IL-35 does not comply with the quintessential high affinity and lower affinity receptor complex paradigm signaling peculiar to a majority of cytokines. The signaling pathway for IL-35 appears to overlap with that of IL-12 as well as IL-27. This is evidenced by the deployment of signals via its non-preferential binding to homodimers (“IL-12Rβ2: IL-12Rβ2” and “gp130:gp130”) or heterodimers of IL-12Rβ2 as well as gp130 which comprise of IL-12R and IL-27 receptor (viz “gp130: IL-12Rβ2” and “IL-12Rβ2: WSX-1”) respectively [
28]. gp130 receptor is ubiquitous in addition to being an integral moiety of receptors for numerous cytokines including; IL-6, IL-11 and IL-27. The IL-12Rβ2 on the other hand is strictly an IL-12 cytokine family receptor [
29]. Signal transduction of IL-35 is initiated upon its binding to either homodimers or heterodimers comprising of gp130 and/or IL-12Rβ2. Following receptor engagement, signal transduction takes place via the janus kinase and signal transducer and activator of transcription (JAK-STAT) pathway [
28]. Phosphorylation of signal transducer and activator of transcription STAT1 was demonstrated to occur upon binding of IL-35 to gp130 receptor whereas STAT4 phosphorylation occurs upon binding of IL-35 to IL-12Rβ2 [
28]. Co-incubation of IL-35 with T cells deficient in either STAT1 or STAT4 revealed a marked reduction in their suppressive capacity, hence, the coordinated role of STAT1 and STAT4 are essential for IL-35 mediated suppression [
28].
Collison et al. [
28] illustrated that signaling via homodimer receptor pairs is moderated by STAT1 and STAT4 with a resultant partial loss of IL-35’s suppressive activity when compared to its signaling via the wholly functional IL-12Rβ2-gp130 heterodimeric receptor complex [
30]. Signal transduction via homodimer receptors culminates in the negation of T
eff cell expansion while failing to induce their conversion to iTr35, reason being that only one arm of signal transduction pathway (comprising the homodimer) is “switched on”. Contrastingly, Wu et al. [
31], reported that signal transduction via the homodimer receptor pair comprising IL-12Rβ2:gp130 is capable of mediating both iTr35 induction as well as T
eff cell suppression. Evidence also shows that IL-12Rβ2 can be expressed by both B cells and dendritic cells invariably influencing the bioactivity of IL-35 in the immune system [
32]. Research by Shen et al. [
33] illustrated that signal transduction of IL-35 in B-cells is moderated via a heterodimer receptor complex comprising of IL-12Rβ2: WSX-1.
2.1. IL-35 Generates a Lineage of Functionally Suppressive IL-35 Expressing T-cells (iTr35) in Human and Murine Subjects
The proliferation of induced T
regs cells (iTr) in the periphery from naive CD4
+ T cells constitutes a substantial research interest in the context of their probable therapeutic application as tolerance-promoting immunobiologicals [
34]. This is further supported by the fact that iTr cells can be produced in copious numbers hence making them ideal therapeutic targets. In addition to the potent inhibitory actions of iTr aimed at specific antigens, these cells have been proven to be beneficial in restoring regulatory networks upon application in conditions where T
regs are exhausted or inherently defective [
34].
The immunosuppressive attribute of IL-35 is not simply limited to the subversion of T
eff cell proliferation along with their ensuing cellular responses; IL-35 can in turn stimulate induction of T
reg cells invariably prompting tolerance effect in an infectious state. Earlier research demonstrated that co-incubation of supernatant obtained from IL-35 transfected human embryonic kidney cell line (HEK293T) on naïve mouse or human T
regs induced a population of T cells denoted iTr35 cells that were themselves capable of exerting suppressive effects strictly mediated by IL-35, not IL-10 nor TGF-β and in turn these cells were able to enhance immunosuppression by further secreting additional IL-35 [
22,
35].
Figure 4 depicts the schematic representation of IL-35 mediated suppression.
2.2. Function(s) of IL-35 in Autoimmune Disorders
Autoimmune disorders typically hold significance as a leading non-communicable disease worldwide. Despite limited knowledge pertaining to their causative mechanisms, researchers have revealed that these disorders are not strictly governed by a single immunologic anomaly (e.g., generation of bad clones or autoantibodies) but rather span across multiple factors and a range of mechanisms including; a breakdown in tolerance and subsequent auto-antibody release at different sites in addition to generation and selection of bad clones amongst other factors [
36]. These disorders are now known to stem from the intricate interplay between diverse cell populations in the immune system (T cells, B cells, classical antigen presenting cells, etc.) culminating in auto-aggressive responses to self viz loss of tolerance [
37]. Cytokines represent key elements that operate the ensuing interplay between cells of the immune system and have been documented in some instances to support the recruitment, protraction and multiplication of auto reactive immune cells [
36].
Several experimental models have demonstrated the functional profile and regulatory mechanisms of inflammation mitigating cytokines (IL-10, TGF-β) including IL-35 in a range of autoimmune diseases. In broad terms, the suppressive capacity of T
reg cells in (
IL-35 deficient)
p35−/− or
Ebi3−/− murine subjects (achieved via targeted deletion of either of the two genes) is significantly reduced in contrast to their wild-type counterparts [
24].
2.2.1. Rheumatoid Arthritis
Typically, rheumatoid arthritis (RA) patients elaborate markedly diminished IL-35 and T
reg titers, which by extension hinders their ability to effectively suppress pro-inflammatory cytokine release by T
eff cells [
6,
35]. Niedbala et al. [
35] demonstrated a delay in normal disease progression as well as amelioration of disease severity in a rodent model of collagen induced arthritis (CIA), following systematic administration of IL-35 [
35,
38]. Murine arthritic subjects receiving exogenous IL-35 therapy demonstrated considerable reduction in critical clinicopathological features associated with arthritis (including arthritic paw count, adjacent cartilage and bone erosion, synovial hyperplasia) as well as decreased histological articular damage when compared to their positive control counterparts receiving only phosphate buffered saline [
35]. The observed amelioration of CIA by IL-35 was attributed to an IL-35 mediated enhancement of T
regs cell proliferation in addition to its restriction of T
eff cell expansion and differentiation towards a T
H1 and T
H17 phenotype [
39]. Consequently, IL-35 augmented mice elaborated a marked decrease in T
H1 as well as T
H17 cytokine titers and high circulating levels of IL-10 in their serum. A study involving human arthritic patients detected lower circulating indices of IL-35 in sera of arthritic patients in comparison to controls [
6].
Vascular endothelial growth factor (VEGF) and angiopoietins (Angs) represent key factors which act synergistically to foster synovial angiogenesis and inflammation in RA [
40]. The suppressive capacity of varying concentrations of IL-35 on VEGF/Ang2 pro-angiogenic pathway (translated in terms of decreased endothelial cell adhesion, migration and tube formation) was demonstrated in in vitro cultures of human umbilical vein endothelial cells (HUVECs) and an ex vivo RA synovial tissue explant set up [
41]. IL-35 was observed to antagonize intrinsic as well as VEGF-induced tube formation in vitro and in vivo in addition to the observed inhibition of HUVECs migration and adhesion in vitro. The observed antiangiogenic property of IL-35 was surmised to occur as a result of its direct inhibitory action on Ang2 expression in addition to an IL-35 mediated interruption of signal transduction via the angiopoietin receptor (Tie2) pathway. Furthermore, the enhanced secretion of vascularization/inflammation response modulating genes
matrix metalloproteinase 2 (
MMP-2) and
matrix metalloproteinase 9 (
MMP-9) in addition to increased IL-6 and IL-8 secretion upon exogenous VEGF and/or Ang2 application to HUVECs and RA synovial explants was potently abrogated by IL-35 [
41].
2.2.2. Experimental Autoimmune Encephalomyelitis (EAE)
Experimental autoimmune encephalomyelitis (EAE) represents a murine (experimental) archetype for the human condition multiple sclerosis [
42]. Conventionally, EAE is initiated by the generation of pathogenic T helper cells in the central nervous system (CNS) via immunization of myelin oligodendrocyte glycoprotein (MOG) to susceptible mice causing CNS demyelination (evidenced by neuroinflammation and limb paralysis). Susceptible mice revealed a complete protection from CNS symptoms upon iTr35 treatment as compared to
Ebi3−/
− mice treated with iTr35 in which disease progression was indistinguishable from their saline treated counterparts. IL-35
+ B
regs generated from IL-35 releasing B lymphocytes also accounted for disease amelioration [
33]. Shen et al. [
33] demonstrated that IL-10 is required by B
reg cells in addition to IL-35 for the effective resolution of EAE, reason being that a deficiency in either one of the two cytokines abrogated completely the regulatory potential of B cells. Experimental autoimmune uveitis (EAU) serves as a template for preclinical research on human uveitis (or eye-specific antigens). EAU is potently suppressed by IL-35, this is evidenced by reduction in ocular inflammation and associated disease severity upon recombinant IL-35 therapy (rIL-35). Such potent disease suppression is thought to be mediated by the inhibitory actions of IL-35 on T
eff cell proliferation in addition to its suppressive actions on T
H17 cell expansion as well as the enhancement of IL-35
+ B
regs and T
reg cell expansion [
32].
2.2.3. Hashimoto’s Thyroiditis
A role was delineated for IL-35 in Hashimoto’s thyroiditis—an autoimmune thyroid disorder (AITD) that targets the thyroid gland, characterized by defective (self)-tolerance to thyroid antigens. Clinical symptoms develop as a result of the attacks on the thyroid gland by auto antibodies released defectively by a malfunctioning immune system. The disease features decreased thyroid follicular cell viability resulting from inept expression of apoptosis pathway molecules and genes,
Fas or B-cell lymphoma-2 gene (
Fas or
Bcl-2) respectively, lymphocytic thyroid infiltration and thyroid hyperplasia. Disease initiation and progression has been attributed to defective T
regs cell function and expansion, in addition to a concomitant increase in follicular helper T cell activation [
43]. Yilmaz et al. [
44], illustrated that IL-35 serum concentrations correlated inversely with the levels of thyroid stimulating hormone (TSH) and anti-thyroid peroxidase antibodies (TPOAb). They proposed that lower circulating levels of IL-35 (which hinders T
reg proliferation and function) promotes a loss of tolerance owing to a decrease or functional impairment of T
regs invariably setting up a favorable environment for the development and exacerbation of autoimmune disorders including Hashimoto’s thyroiditis [
44]. Owing to the previously cited suppressive action of IL-35 on mouse B
regs, decreased IL-35 titers were surmised to be a predisposing factor to a B
reg-mediated increase in TPOAb expression [
32]. Consequently, it was deduced that optimal IL-35 titers are essential for protection against thyroid immunopathology and this action of IL-35 is mediated through its action on T
reg cell expansion rather than its direct abrogation of pro-inflammatory cytokine function [
44].
2.2.4. Multiple Low Dose Streptozotocin (MLDSTZ)
Multiple low dose streptozotocin (MLDSTZ) represents a murine model of human diabetes. Susceptible mice, pre-treated with rIL-35 and subsequently subjected to MLDSTZ treatments, failed to develop hyperglycemia and stayed normoglycaemic in contrast to their saline-treated age and sex-matched controls, where insulitis and hyperglycemia were manifested [
45]. The proffered mechanism of disease attenuation was described as an IL-35 mediated build-up in anti-inflammatory signaling particularly, IL-10 and IL-35 [
45]. Additionally, IL-35 was proffered to effectively reverse the phenotypic shift of (functionally impaired) T
regs restoring their suppressive capacity [
45].
Li et al. [
46] demonstrated that sera from patients with active stage irritable bowel disease (IBD) had notably lower levels of IL-35 compared to healthy controls. Previous studies report a decrease in T
reg cell count to be associated with ulcerative colitis. Suffice to say that T
regs being a primary source of IL-35, such decrease translates to a corresponding decrease in peripheral circulating levels of IL-35 invariably offsetting an imbalance in the mitigating counter-inflammatory response required to effectively curb mucosal release of inflammatory mediators consequently exacerbating symptoms in patients with IBD [
46]. Conversely elevations in the level of IL-35 was detected in T
reg and B
reg cells of IBD patients further supporting the immunoregulatory function of IL-35 [
47].
2.3. Role of IL-35 in Respiratory Disorders
Earlier research accorded primacy to polymorphisms in cytokine genes and their signaling pathways in the pathogenesis of (allergic) airway disorders such as asthma [
48]. Both T
H2 and T
H17 cells have been implicated as the principal effector cells involved in asthma pathogenesis. T
H2 have been reported to mediate local (airway) recruitment of inflammatory cell subsets (mast cells, eosinophils, etc.) while T
H17 cells represent the principal source of neutrophil attracting cytokines in the lung parenchyma [
48].
Kanai et al. [
49], ascribed a central role to IL-35 in an experimental model of asthma. Excessive airway eosinophilia observed in susceptible
Ebi3−/
− mice previously sensitized and subsequently exposed (intratracheally) to ovalbumin (OVA) or LPS was potently suppressed upon rIL-35 administration. Furthermore, the associated increased recruitment of eosinophil-attracting chemokines, eotaxin-1 and eotaxin-2 (CCL11 and CCL24) as well as ensuing airway eosinophilia were potently antagonized by IL-35. Similarly, in vitro studies involving perturbation of human bronchial epithelial cells (BEAS-2B cells) with tumor necrosis factor alpha (TNF-α) as well as IL-1β revealed a regulatory role for IL-35 modulated by its suppressive action on STAT1 and STAT3 phosphorylation. The latter of which has been described for its role in regulating CCL11 expression [
50]. Treatment with rIL-35 was also reported to result in an increase in BEAS-2B associated suppressor of cytokine signaling 3 (SOCS3) expression thereby enhancing the inhibition of gp130-mediated IL-6 signaling [
49].
A significant alleviation in symptoms of allergic rhinitis (nasal rubbing gestures as well as total number of sneezes) was observed upon rIL35 administration in a murine model of allergic rhinitis [
51]. Intranasal IL-35 administration was reported to negate T
eff cell responses as well as the pathological production of IL-4 and IL-5 (T
H2 cytokines) both implicated in the exacerbation of allergic rhinitis. Furthermore, an IL-35 mediated increase in circulating IL-10 expression was observed, which in turn promotes T
reg survival [
51].
An earlier study by Chen et al. [
52] delineated CD8
+ T cells as the principal mediators driving the lung and airway infiltrations (i.e., neutrophilia) peculiar to individuals suffering from chronic obstructive pulmonary disease (COPD). Investigation in to the dynamics of IL-35 secretion in asthma and COPD revealed lower IL-35 titers in COPD patients compared to the asthmatic and control group. The observed difference in IL-35 titers (between COPD sufferers and asthmatic patients) was attributed to the disparate cellular sources that promote each airway pathology namely; CD4
+ T cells in asthma and CD8
+ T cells in COPD.Foxp3
+ T
regs cells are a principal source of IL-35, hence the higher expression levels in asthma. Moreover, the higher levels of IL-35 observed in disease-free control groups was attributed to inherent (effective) anti-allergy mechanism mounted towards sensitization from exposure to typical everyday environmental allergens. This implies that maladaptive anti-allergy mechanisms may contribute to the lower IL-35 titers observed in respiratory disorders [
52].
2.4. A Role for IL-35 in Cardiovascular Diseases
Atherosclerosis is a consequence of the interaction between immune effector molecules and metabolic susceptibility which trigger lesions (atheromata) in arterial vasculature [
53]. Compelling evidence has delineated the key role of T
H1 cytokines (particularly IL-1β and IL-18) in aberrant immune responses. These aberrant immune responses foster the propagation and activation of arterial lesions resulting in the development of notoriously unstable atherosclerotic plaques and thrombi. Rupture of (unstable) plaques or arterial tree occlusion by a thrombus leads to cardiovascular events with varying (potentially fatal) clinical outcomes including myocardial infarction, cerebrovascular accident (stroke) as well as coronary artery disease [
54].
Sanchez et al. [
54], demonstrated the involvement of
IL-35 gene polymorphisms in atherosclerosis and its associated clinical ramifications (e.g., coronary artery disease—CAD). Two polymorphisms in the
IL-35 gene namely;
Ebi3 rs428253 and
IL-12A rs2243115 were found to be associated with a decreased risk of premature CAD development. The same polymorphisms were found to be associated with reduced risk of type-2-diabetes mellitus and metabolic syndrome respectively, both of which are known cardiovascular disease risk factors. Surprisingly, no association was observed between the two polymorphisms and IL-35 levels, probably due to the fact that IL-35 levels were measured in systemic circulation and not localized to the site of sclerotic lesions [
54].
Significantly decreased amounts of IL-35, IL-10 as well as TGF-β were observed in plasma of patients with coronary artery disease (CAD) with a concomitant elevation in plasma IL-12 and IL-27 levels. IL-35 showed a positive correlation with left ventricular ejection fraction in the CAD patients studied hinting at the involvement of IL-35 in CAD and its potential relevance as a prognostic marker for CAD [
55].
2.5. IL-35 Mediates Inflammation in Sepsis
Systemic inflammation in conjunction with unfettered release of large amounts of pro-inflammatory mediators results in the systemic inflammatory response syndrome (SIRS) peculiar to sepsis [
56]. A potentially fatal illness in which signs and symptoms are often ambiguous, sepsis normally arises from aberrant host immune response to severe infection. Sepsis is responsible for a high percentage of mortality in adults and neonates [
57]. The physiology of cytokines has been extensively evaluated for their likely relevance as prognostic indicators in patients suffering from sepsis. Previous research utilizing IL-10 and IL-27 (an IL-12 cytokine family member) in murine models of sepsis have yielded promising outcomes directed towards the understanding of cytokine dynamics in septic shock. Hence, IL-35 which is also a member of the IL-12 cytokine family was therefore examined for its role in the pathogenesis of sepsis [
57].
A study investigating the dynamics of IL-35 in both adult and pediatric septic patients revealed higher circulating levels of IL-35 which were strikingly consistent with the degree of disease severity [
58]. IL-35 levels were observed to display a positive correlation with the levels of other pro-inflammatory mediators released in sepsis (including; IL-6, IL-8, IL-27, TNF-α and IL-10). An inquisition on the effect of neutralizing IL-35 via the administration of mouse anti-IL-35p35 antibody, demonstrated an increase in peritoneal neutrophil recruitment as evidenced by their increased numbers in peritoneal lavage fluid of IL-35 neutralized mice in contrast to placebo treated healthy mice. Likewise, higher titers of pro-inflammatory mediators (viz TNF-α etc.) at an early time point (6 h) were observed in anti-IL-35p35 treated mice which implies a preservation of vital early inflammatory cytokine response necessary for typical immune activation and pathogen clearance. Quantitation by polymerase chain reaction (PCR) of organ lysates obtained from septic rodents revealed high levels of IL-35 subunits (p35 and
Ebi3 as early as six hours post disease initiation) particularly in the lungs and spleen. As an added advantage, Du et al. [
58] revealed IL-35 was able to predict the likelihood of sepsis onset in neonates in a capacity which was highly comparable (and potentially superior) to procalcitonin a known predictive marker for sepsis. Certain immune cells (dendritic cells, monocytes, macrophages, etc.) have been documented to contribute to the cytokine repertoire in sepsis upon activation. Hence, human and murine elevations in IL-35 concentration with ongoing sepsis were ascribed to the stimulation of immune cells at the onset of infection. Detailed reappraisal of the study outcome implicated IL-35 in the pathogenesis of sepsis as well as its probable beneficial application as a predictive (prognostic) marker for disease outcome in septic patients [
58].
2.6. Role for IL-35 in Connective Tissue Disorders
2.6.1. Systemic Sclerosis (Scleroderma)
Atypical TGF-β activation of dermal and tissue fibroblasts has been proposed to be the primary insult driving the clinical manifestations seen in systemic sclerosis. This multi system connective tissue disorder is characterized by exaggerated build-up of collagen and extracellular matrix around vital organs ultimately resulting in varying degrees of tissue fibrosis and diverse organ malfunction. Tomcik et al. [
59] implicated IL-35 in the pathogenesis of systemic sclerosis. He investigated lesional IL-35 expression in sclerotic dermal sections and reported an upregulation of serum IL-35 levels in systemic sclerosis patients as compared to their control counterparts. Moreover, overexpression of both subunits comprising IL-35 (i.e., p35 and Ebi3) was observed in sclerotic dermal lesions from either localized or disseminated sclerosis patients particularly in inflammatory cells and fibroblasts. Sustained elevations of IL-35 levels were observed upon culture of fibroblasts previously acquired from sclerotic skin lesions. Likewise, TGF-β application to cultured (normal) fibroblasts induced IL-35 expression which in turn promoted a switch of resting fibroblasts to an activated state thereby promoting the release of IL-35 and collagen, inadvertently enhancing abnormal TGF-β signaling via a positive feedback loop [
59]. T
regs are the main source of IL-35 and their numbers are concurrently increased with increasing IL-35 concentration in systemic sclerotic patients. Studies have reported a positive correlation between T
reg expansion and clinicopathological features of systemic sclerosis as well as disease progression owing to a characteristic loss of their suppressive function (phenotypic shift) with a resultant CD4
+ CD25
+ Foxp3
low CD45RA
− (non-suppressive) phenotype and an associated propensity to secrete IL-17 [
59].
According to Tomcik et al. [
59], capillaroscopic assessments in early stage sclerotic patients demonstrated a negative correlation with IL-35. Early stage sclerotic patients demonstrated higher IL-35 levels when compared to patients with a more chronic (protracted) disease duration. IL-35 is known to signal via STAT1 and STAT4, consequently, the molecular mechanisms of IL-35 in systemic sclerosis were adduced to IL-35 mediated influence of pro-fibrotic STAT4 clones on T cell activation, expansion as well as patterns of cytokine release [
59]. Hence, Dantas et al. [
60] suggested exploiting the dynamics of IL-35 in systemic sclerosis as a serological marker/prognostic indicator.
2.6.2. Actions of IL-35 in a Murine Model of Systemic Lupus Erythematosus (SLE)
Loss of peripheral and central immune homeostasis (tolerance-promoting B and T cells) in addition to subsequent auto-antibody generation and ensuing organ damage are key features of systemic lupus erythematosus (SLE). A study was undertaken by Cai et al. [
61] to characterize the immune-regulatory profile of IL-35 as well as its soluble receptors (gp130 and IL-12Rβ2) from peripheral blood mononuclear cells (PBMC) of SLE patients with varying disease activity indices (SLEDAI). Significantly elevated titers of IL-35 were observed in SLE patients unlike their control counterparts. The authors delineated an inverse correlation between the proportion of peripheral circulating T
regs cells and disease activity in SLE patients. Additionally, an increased T
eff cell to T
reg cells ratio (T
eff; T
reg) was observed in patients suffering from severe SLE [
61]. This observation is in line with reports from previous studies describing a reduced Foxp3
+ expression by T
regs of SLE patients in conjunction with an intrinsic dysfunction of their suppressive capacity. Taken together the authors [
61] surmised that the flawed suppressive capacity of T
regs from SLE patients may represent the primary element underpinning the immunopathogenesis of SLE. They further interpreted the induction of IL-35 production as a direct result of stimulation by the inflammatory milieu following T
eff cell stimulation and adduced inflammatory pathogenesis of SLE to diminished expression and functional capacity of T
regs cells. Despite the elevated levels of IL-35 seen in SLE patients, Cai et al. [
61] speculated that the expressed IL-35 was unable to sufficiently induce a robust population of T
regs cells owing to the decreased expression of gp130 receptor on the surface of CD4
+ T-helper cells. Suffice to say, a higher expression of gp130 receptor is pertinent for the actualization of suppressive action by IL-35 [
61].
In a murine model of SLE, daily administration of recombinant IL-35 (800 ng rIL-35/mouse) was observed to suppress SLE. Cai et al. [
61] opined that rIL-35 restored the (otherwise absent) delicate balance between autoimmune tolerance mediators and inflammatory immune response in SLE murine subjects and ascribed this action to effects of IL-35 on T
eff cells (viz abridged expansion and effector function). Recombinant IL-35 brought about a reduced T
H17 effector cell differentiation from CD4
+ T
H cells, which culminated in propagation of immune tolerance in SLE mice. Additionally, significantly decreased SLE-specific plasma auto-antibody concentrations were observed. The action of IL-35 on SLE-specific auto-antibodies suggests that other regulatory cell populations (e.g., B
reg cells) may contribute to the actualization of IL-35 mediated immunosuppression. Tolerance-promoting attribute of IL-35 was linked in part to its expansion of peripheral, thymic and splenic IL-10 producing B
regs cells [
61].
2.7. Actions of IL-35 in Hepatitis Infection
Independent researchers have illustrated the immunomodulatory role of IL-35 in hepatitis C virus infection. Liu et al. [
62] observed a direct correlation between hepatitis C viral load and IL-35 concentration. The role of IL-35 was described as being somewhat ambiguously centered around its influence on T cell activity (in a specific and non-specific manner). Although failing to show a correlation with the levels of alanine amino transferase (a liver enzyme usually elevated in hepatic dysfunctions) and liver inflammation, IL-35 mediated a reduction in the levels of HCV-induced upregulation of STAT1 as well as STAT3 signaling. Additionally, IL-35 mediated suppression of pro-inflammatory cytokine production was adduced to an inhibition of STAT signaling by IL-35 in both hepatitis C virus infected cells as well as the hepatocellular carcinoma cell line (Huh. 7.5). There was however no evidence of any direct action of IL-35 on viral replication [
62].
A study by Shi et al. [
63] aimed to characterize the dynamics of IL-35 expression in patients suffering from chronic severe hepatitis B (CSHB), chronic hepatitis B (CHB), liver cirrhosis (LC) and asymptomatic carriers (ASC). IL-35 titers were elevated in sera of patients with HBV infected compared to normal controls. Essentially a progressive increase in IL-35 serum levels was observed with increasing disease severity (i.e., CSHB > CHB > LC > ASC). In CHB patients aside from an elevation of serum IL-35 titers, a positive correlation was also observed between IL-35 levels and partial liver indices, severe liver inflammation and incidence of necrosis [
63]. IL-35 was proffered to function in an immune modulating manner by ensuring a homeostatic balance between the hepatitis B virus and T cells in an effort to curtail liver injury. Hence, no direct influence on hepatitis B viral load in the peripheral circulation was ascribed to IL-35 neither was a role proposed for IL-35-mediated direct inhibition of viral replication. The study proposed that the homeostatic balance between HBV and CD4
+ CD25
+ T
reg cells create an immunotolerant environment which translates to the chronic disease course known to occur in Hepatitis B virus infection. Thus, on the one hand, IL-35 forestalls liver injury provoked by the hepatitis B virus while inadvertently promoting immune tolerance to the HBV via immunosuppressive T
reg cells [
63].
2.8. Function(s) of IL-35 in Cancer
Different assorted yet ambiguous roles in the tumor microenvironment ranging from tumor-promoting to anti-tumor effects have been attributed IL-35 in various types of cancers. A study by Turnis et al. [
64] demonstrated a reduction in tumor enlargement in several murine cancer models as a consequence of T
reg specific IL-35 deletion in addition to neutralization with an antibody against IL-35. The authors postulated that T
reg cell-derived IL-35 fosters tumor inception accomplished via T
reg cell exhaustion which in turn restrains anti-tumor immunity [
64].
Ordinarily, immune surveillance limits tumor (cancer) development, such that oftentimes, tumors which thrive are poorly immunogenic. Decades of research have revealed that tumors have an uncanny ability to survive via the subversion of host immune anti-tumor responses. Contributory to tumor survival is the genomic instability of precancerous cells resulting in clones of varying (often poor) immunogenicity with an uncanny ability to bypass recognition by immune cells (T cells), thereby impeding tumor-directed immune response. Additionally, conversion of naïve T cells to adaptive Treg cells and their subsequent proliferation represents yet another tumor-mediated mechanism adopted to bypass immune surveillance. Suffice to say that tumors create a suppressive environment via the recruitment of immune subverting factors and suppressive cell populations.
Nishikawa and Sakaguchi [
65] demonstrated enhanced tumor rejection and improved disease outcomes upon T cell depletion which was negated upon T cell augmentation. Peripheral and localized cancer tissues (from B16 melanoma and prostate cancer sufferers) reveal enhanced population of immune suppressing T
reg cells in tumor microenvironment as well as IL-35 [
65]. Researchers recently revealed an enrichment of IL-35 in tumor-recruited T
reg cells where it is adduced to partake in immune evasion. T cell exhaustion which is a hallmark of most cancers has been linked to T
regs and their associated inhibitory cytokines. The phenomenon (T cell exhaustion) was reversed by simultaneous blockade of T
regs, Programmed cell death protein-1, IL-10 or TGF-β signal transduction [
65].
An earlier study by Wang et al. [
66] delineated tumor-promoting property of IL-35 in two different murine models of cancer, namely plasmacytoma J558 and B16.F10 melanoma. In these cancer models, IL-35 was observed to considerably increase tumorigenesis in both immunocompetent mice and their immune deficient counterparts [
66]. Cao et al. [
67] reported that the observed tumor promoting property associated with IL-35 is mediated via the inhibition of anti-tumor cytotoxic T lymphocyte (CTL) responses which is driven by CD4
+ CD25
+ T
regs recruitment at malignant sites in addition to the build-up of myeloid derived suppressor cells (MDSC) in the tumor microenvironment [
66,
67]. Additionally, cancer cell resistance to chemotherapy is shown to be mediated by signaling of IL-35 through its gp130 homodimer receptor which in the interim is one of the receptors upregulated in tumor cells [
68]. Thus, it is highly probable that IL-35 signaling via gp130 homodimer receptor promotes cancer cell resistance to CTL destruction [
28].
A later study undertaken by Zeng et al. [
69] to elucidate the action(s) of IL-35 in human colorectal cancer (CRC) patients revealed elevated levels of IL-35 in sera and cancer cell lysates of CRC sufferers. The levels of IL-35 showed high correlation with the clinical stage of tumor as well as the degree (extent) of malignancy. In addition, appreciable reduction of serum IL-35 was observed in patients after surgical resection of tumors, revealing that poor prognosis in CRC is linked to elevated IL-35 titers. Furthermore, negative expression (depletion) of
Ebi3 gene a subunit of IL-35 correlated with a better prognosis and increased overall survival and disease-free survival in a study undertaken to evaluate IL-35 expression patterns in nasopharyngeal carcinoma cells [
70,
71].
Contrarily, Long et al. [
72] cited that disease progression in hepatocellular cancer patients is associated with a decrease in IL-35 expression by tumor tissues. A recent study by Zhang et al. [
73] illustrated a beneficial role for IL-35 in inflammation related tumors. He provided evidence that IL-35 production is reduced in colon cancer in comparison to healthy controls and that applying various concentrations of IL-35 to human colon adenocarcinoma cell line (DLD1) and human colorectal adenocarcinoma cell line (HT-29) suppressed their proliferation (invasiveness and migration) in vitro. Additionally, IL-35 was shown to decrease the expression of β-catenin (a molecule known to be promote colon cancer development) invariably hindering colon cancer progression as well as sensitizing colon cancer cells to chemotherapeutic agents [
73]. Increased IL-35 titers in a cohort of patients suffering from papillary thyroid carcinoma (PTC) with concurrent Hashimoto’s thyroiditis (HT) was surmised to be beneficial due to the observed improved prognosis in this group of patients (PTC + HT) compared to their counterparts with either PTC or nodular goiter [
74].
The deployment of small interfering RNA (siRNA) directed towards
Ebi3 in lung cancer cells was shown to impede lung cancer cell proliferation albeit stable
Ebi3 gene expression confers growth promoting activity in vitro and is associated with poor prognosis [
70]. Taken together, numerous human cancer tissues produce ample amounts of IL-35 which hints at the possibility of its application as an independent prognostic biomarker for various types of cancers. On the other hand, anti-IL-35 may serve as a potential anti-tumor therapeutic biomolecule in various malignancies [
75]. The overall involvement of IL-35 in disease pathogenesis is summarized in
Table 1.