Interleukin 6 plays an important role in host defense against environmental stress such as infection and injury. Dysregulated IL-6 production has been implicated in the development of various autoimmune diseases and chronic inflammatory diseases. IL-6 is a prototypical four-helix bundle cytokine that is a member of the neuropoietins, which includes IL-6, IL-11, IL-27, IL-31, leukemia inhibitory factor, oncostatin M, cardiotrophin-1, neuropoietin, and neurotrophin-1 . These cytokines are structurally related and bind to Class I cytokine receptors. With the exception of IL-31, all IL-6 type cytokines share the membrane glycoprotein gp130 as a common beta receptor and signal transducer subunit [24, 25].
IL-6 signaling occurs through two cellular pathways: the classical and trans-pathway. In the classical signaling pathway, IL-6 binds to membrane-bound type I receptor complex consisting of the ligand-binding glycoprotein, IL-6α. The expression of this receptor is mostly restricted to leukocytes and hepatocytes. The IL-6/IL-6α complex subsequently associates with gp130 leading to gp130-homodimer formation . In trans-pathway, IL-6 provides signaling to cells lacking IL-6R via binding to soluble IL-6R (sIL-6R), which is generated by alternative splicing or ectodomain shedding of the membrane-bound IL-6 receptor (Fig. 1) . Both classical and trans-signaling pathways are gp130-mediated and activate the same intracellular pathways.
After the formation of gp130 homodimer, IL-6 initiates the intracellular signaling by activating the Janus kinase family tyrosine kinases (JAKs) . Activation of these kinases leads to phosphorylation and activation of signal transducers and activators of transcription 3 (STAT3) and the SH2-domain containing protein tyrosine phosphatase-2 (SHP2) [29, 30]. Phosphorylated STAT3 translocates to the nucleus and regulates transcription of various genes. SHP2 activates SOS/Ras-Raf-MEK-MAP kinase pathway to regulate genes .
It is important to note that the activation of STAT3 in turn induces the suppressor of cytokine signaling 1 (SOCS1) and SOCS3, which bind tyrosine-phosphorylated JAK and gp130 respectively, to stop IL-6 signaling by means of a negative feedback loop [32, 33].
There is counter-regulation by a soluble form of gp130 (sgp130), present at high concentrations in serum of healthy individuals. As part of the physiological IL-6 buffer in the blood, this natural inhibitor forms a complex with IL-6/sIL-6R, preventing the binding of IL-6/sIL-6R to membrane-bound gp130. This ensures that IL-6/sIL-6R trans-signaling is tightly regulated .
Various studies have shown that classic signaling via the membrane-bound receptor is regenerative and protects from bacterial infections, whereas trans-signaling via the soluble receptor is proinflammatory . Therefore, it may make sense to block only the IL-6 trans-signaling alone, which would maintain the regenerative function of IL-6 and specifically suppress only inflammatory arm mediating the disease process (Fig. 2) .
IL-6 and autoimmunity
As mentioned earlier, IL-6 is a pleiotropic cytokine that plays important roles in hematopoiesis, immune defense, and oncogenesis . Historically, IL-6 molecule had been studied under many different names such as B cell stimulatory factor-2 (BSF-2), IFN-β2, Hybridoma/plasmacytoma growth factor, hepatocyte-stimulating factor (HSF), until advancements in molecular testing. In the following sections, we will discuss the role of IL-6 in immune cascades and defense mechanisms, pathological significance of IL-6 signaling in inflammatory autoimmune systemic and ocular diseases, as well as therapeutic implications of IL-6 targeted therapy.
Immunomodulatory role of IL-6
IL-6 was originally identified and named as B cell stimulatory factor 2 because it promotes the differentiation of activated B cells into plasma cells which are responsible for antibody production . Interactions between T and B cells during antibody production were first reported in 1968 and it was theorized that certain molecules were released from T cells which stimulate B cells to produce antibodies . B cells can produce antibodies, but not without having T cells producing those growth and differentiation factors. IL-6 produced by plasmacytoid dendritic cells is critical for this process . IL-6 also promotes T follicular helper cell differentiation as well as production of IL-21, which also promotes B cell differentiation and increase immunoglobulin synthesis [39, 40].
Moreover, IL-6 may promote the survival of the plasma blasts that secrete immunoglobulin or pathological autoantibodies, e.g., anti-aquaporin 4 in patients with neuromyelitis optica (NMO) . IL-6 may act as an autocrine growth factor in some types of multiple myelomas while some others are themselves able to produce IL-6 .
As mentioned, IL-6 was first identified as a B cell function and differentiation factor; however, T cell differentiation and activation is another major action of IL-6 . IL-6 signaling has been found to control proliferation of resting T cells and reinforcing their resistance against apoptosis by inducing IL-2 production and STAT-3 activation . IL-6 has also been identified as major regulator between regulatory T cells (Treg) and effector Th17 cells. In combination with transforming growth factor (TGF)-β, IL-6 brings about differentiation into Th17 cells, but inhibits TGF-β-induced Treg development [44, 45]. This results in an increase of Th17 cell population over Treg cells which may have a role in altered immunological tolerance and resulting in the development of autoimmune inflammatory diseases .
IL-6 also modulates Th1 and Th2 balance. It enhances the production of Th2 cells by promoting IL-4 and IL-13 production . On the other hand, it inhibits Th1 cell differentiation and interferon-gamma (IFN-γ) production .
IL-6 and acute-phase response
IL-6 is a major cytokine in the initiation process of acute-phase responses . In the serum of healthy individuals, the IL-6 level is less than 5 pg/ml; however, IL-6 concentration increases dramatically during infectious and non-infectious events . IL-6 acts as an important factor in the synthesis of acute-phase proteins by the liver, such as C-reactive protein (CRP), serum amyloid A (SAA), fibrinogen, hepcidin, and α1-antichymotrypsin . Administration of IL-6 inhibitors completely normalizes the serum levels of CRP and SAA, indicating that their synthesis depends primarily on IL-6 . These major acute-phase reactants act as an inducer of systemic inflammatory and infectious response. Elevated levels of CRP have been reported in serum of patients with various autoinflammatory diseases such as rheumatoid arthritis, systemic lupus erythematosus, among others, as well as bacterial and viral infections . When the emergent stimuli are completely removed from the host, the IL-6-mediated signal transduction cascade is terminated, leading to normalization of the CRP level in serum (Fig. 3) .
Due to the rapid plasma clearance, IL-6 levels are largely regulated at the transcriptional and post-transcriptional gene expression level . The molecular aspects of IL-6 regulation comprise of complex interactions between proteins, miRNAs, and IL-6 gene expression, which is beyond the scope of this review.
IL-6 in various systemic autoimmune diseases
IL-6 was first associated with disease development in a case of cardiac myxoma, a benign heart tumor . Subsequently, excessive IL-6 expression patterns were detected in several other autoimmune inflammatory diseases including chronic rheumatoid arthritis, juvenile idiopathic arthritis, systemic lupus erythematosus, Adamantiades-Behcet’s disease, and systemic sclerosis [30, 50, 55, 56]. It is also known that the extent of elevation of serum IL-6 level depends on the type and severity of the disease . On this basis, IL-6 inhibition strategy is currently being pursued to develop novel therapies for inflammatory pathologic conditions. The following sections will focus on IL-6 inhibitors and their usage in systemic and ocular inflammatory diseases.
Experimental autoimmune uveitis—animal model
Several experimental autoimmune uveitis (EAU) studies demonstrated the importance of IL-6 in non-infectious uveitis (NIU). In an animal model of T cell-mediated uveitis (interphotoreceptor retinoid binding protein (IRBP) immunization model), Yoshimura et al. showed that IL-6-deficient mice could not induce Th17 cells and the EAU score was found to be decreased in those mice in the entire time course . On the other hand, systemic administration of anti-IL-6 receptor antibody reduced uveitic inflammation. This effect in EAU appears to occur via the suppression of both Th1 and Th17 differentiation, both of which are important in this animal model of uveitis. Haruta et al. induced EAU in wild-type (WT) mice and in mice lacking IL-6, IL-17, and IFN-γ and also in IL-6-lacking mice treated with anti-CD 25 monoclonal antibody (mAb) to deplete Treg cells . IL-6 deficiency resulted in the inhibition of the antigen-specific Th1 response and enhanced the generation of antigen-specific Treg cells. Authors concluded that blockade of IL-6 signaling suppresses not only Th17 but also IRBP-specific Th1 by promoting regulatory T cells in EAU.
IL-6 in ocular pathologies and non-infectious uveitis
IL-6 is a key cytokine which is strongly upregulated during infection/inflammation and associated with variety of systemic autoimmune diseases. Elevated levels of IL-6 have been detected in many ocular diseases such as glaucoma, central vein occlusion, dry eye disease, chemical burn injuries, corneal infections, allergic eye diseases, and ocular inflammatory diseases [56, 60].
Murray et al. was the first to demonstrate elevated aqueous humor levels of IL-6 in 24 human subjects with uveitis, including, Fuchs’ heterochromic iridocyclitis and toxoplasma uveitis . Several groups also found IL-6 to be elevated in ocular fluids including vitreous in patients with active or chronic NIU supporting its mainstream role in ocular inflammatory process [7, 62, 63].
All these evidences, since the first report on the discovery of IL-6 in 1973 by Kishimoto and Ishizaka, have led to a pursuit of new IL-6 inhibitory drugs, for the management of pathologic inflammatory conditions including several types of NIU .
Vascular endothelial growth factor (VEGF) plays an important role in the pathogenesis of macular edema in central retinal vein occlusion (CRVO) and diabetic retinopathy by enhancing vascular permeability and altering retinal endothelial tight junctions. In the presence of hypoxia, IL-6 plays a major role in VEGF induction. Vitreous levels of IL-6 and VEGF correlate with the severity of ischemia and are found to be significantly elevated in patients with ischemic CRVO and correlated with the severity of disease [65, 66].
Elevated levels of IL-6 with other cytokines have been shown in proliferative diabetic retinopathy (PDR) wherein the progression may lead to tractional or combined mechanism retinal detachment . IL-6 has also been implicated in the pathogenesis of proliferative vitreo-retinopathy (PVR) after rhegmatogenous retinal detachment (RRD). IL-6 stimulated the production of matrix metalloproteinases (MMPs) which play a major role in the development of PVR .
IL-6 has also been found to have a role in various other ocular diseases such as allergic conjunctivitis and dry eye disease, infectious keratitis, ocular neovascularization, and posterior capsular opacification [69,70,71,72,73].
Non-specific anti-IL-6 drugs
The mechanism of action of corticosteroids on IL-6 inhibition is not fully understood; however, they are known to inhibit IL-6 production at the transcriptional level [57, 74]. This process may involve suppression of gene upregulation by nuclear factor-κβ (NF-κβ) or by the occlusion of promotor elements in the IL-6 promotor . Although corticosteroids are the mainstay treatment in many types of inflammatory diseases and IL-6 inhibition is one of their pleiotropic mechanisms of action, their dose-dependent side effects limit long-term therapeutic usage.
These effects are mediated by its specific inhibition of IkB kinase-b, which prevents activation of nuclear factor -kB (NF-kB)
Tetracyclines are broad-spectrum antibiotics that can act against a wide range of microorganisms via inhibition of protein synthesis . The immunomodulatory and anti-inflammatory properties of tetracyclines suggested that this drug might be effective in the treatment of autoimmune disorders . Wide spectrum anti-inflammatory effects of these drugs are thought to be partially due to suppression of IL-6 by the blockage of NF-κβ signaling .
Targeted biological anti-IL-6 drugs
Tocilizumab (Actemra®, Roche AG, Basel, Switzerland) is a humanized mouse monoclonal antibody inhibitor of IL-6 receptor. Tocilizumab (TCZ) prevents the binding of IL-6 with its membrane and soluble receptors and antagonizes its action [4, 6]. It is currently approved for the treatment of rheumatoid arthritis (RA), juvenile idiopathic arthritis (JIA), and giant cell arteritis (GCA) by the FDA [37, 78]. Apart from these, many retrospective open-label studies have shown efficacy of TCZ in inflammatory and/or autoimmune diseases refractory to conventional therapy and/or other biologics which included series of other large-vessel vasculitis (Takayasu’s arteritis), Adamantiades-Behçet’s disease (ABD), adult onset Still’s disease, multicentric Castleman disease (approved in Japan), relapsing polychondritis, Cogan’s disease, inflammatory myositis, and lupus .
Large randomized controlled trials (RCTs) on TCZ therapy for RA paved way for its usage in other systemic and ocular inflammatory conditions, since it provided valuable information on the efficacy and side effect profile of the drug. TCZ demonstrated therapeutic potential in moderate to severe active RA patients with inadequate response to methotrexate (MTX), or other conventional disease-modifying antirheumatic drugs (cDMARDs) or biological disease-modifying antirheumatic drugs (bDMARDs) like TNF antagonists [36, 37, 49, 50]. Also, TCZ was found to be better than MTX monotherapy, with rapid improvement in signs and symptoms in patients with active RA, for whom previous treatment with MTX/biological agents had not failed . On the other hand, two phase 3 studies showed that immediate initiation of TCZ with or without MTX in early progressive RA was also more effective and associated with sustained remission and low disease activity, but with a similar safety profile compared with initiation of MTX alone [80, 81]. The results of the phase 4 ADACTA study comparing the efficacy of TCZ 8 mg/kg monotherapy versus anti-TNF-α agent adalimumab monotherapy were also in favor of TCZ in terms of reduction of signs and symptoms of RA, in patients for whom MTX was deemed inappropriate .
Successful use of tocilizumab with an acceptable safety profile has been reported in patients with NIU who did not respond to several other treatments. In 2011, Muselier et al. reported the first two cases of refractory uveitis (birdshot chorioretinopathy (BSC) and idiopathic panuveitis) treated by tocilizumab with favorable results and acceptable short-term safety profile . After that report, several case series have demonstrated efficacy of tocilizumab in the treatment of uveitis refractory to anti-TNF agents [8, 84, 85]. Papo et al. in their study with eight consecutive unselected patients with severe and refractory non-infectious uveitis including BD, BSC, and idiopathic cases concluded that tocilizumab was safe and promising . After 8 months of median follow-up, six out of eight patients improved in terms of inflammation control. Mesquida et al. reported on the long-term efficacy and safety of tocilizumab for refractory uveitis-associated macular edema . All their patients were refractory to cDMARD and at least one bDMARD prior to initiation of tocilizumab. Uveitis diagnoses were BSC (n = 3), JIA-associated uveitis (n = 3), and idiopathic panuveitis (n = 1). After a 15-month follow up, no serious adverse events were observed. Mean central foveal thickness improved significantly from 550 to 274 μm at month 12. Visual acuity also significantly improved from 0.67 to 0.4 at month 12. The same group published 24 months results of quiescent uveitis patients with recalcitrant uveitic macular edema (ME), treated with TCZ . Diagnoses included patients with BSC, JIA-associated uveitis, idiopathic panuveitis, sympathetic ophthalmia, and ankylosing spondylitis. Sustained inflammatory remission was maintained in all 12 patients. However, an attempt to withdraw TCZ could only be made in five of them because of systemic disease and perceived high risk of visual loss. All five patients in whom TCZ therapy was withdrawn, ME relapsed within 1 to 3 months after cessation. A re-challenge with TCZ infusions in those patients induced recovery. In the study, tocilizumab was generally well-tolerated except one case of dose-dependent neutropenia and another case of pneumonia .
In addition to these encouraging results in small case series and retrospective studies with relatively small number of patients, the first prospective randomized clinical trial STOP-Uveitis was conducted to assess the safety and efficacy of tocilizumab in NIU . STOP-Uveitis was a 6-month study of 37 patients treated with one of two intravenous doses (either 4 or 8 mg per kg) of tocilizumab for posterior NIU. The majority of the cases were of idiopathic origin (28/37 patients) but subjects who had uveitis secondary to Vogt-Koyanagi-Harada syndrome, sarcoidosis, punctate inner choroiditis, and ABD were also included. Only 20–25% of the study population had history of immunomodulatory therapy usage. Two patients developed low absolute neutrophil counts (ANC) after receiving the first infusion of tocilizumab; one normalized before the second infusion while the other subject exited the study. No ocular adverse events related to the study drug were observed. The STOP-Uveitis study demonstrated that the therapy was well-tolerated and associated with a reduction in vitreous haze and cystoid macular edema at both doses (Fig. 4) .
Tocilizumab: dose and route of administration
The approved starting dose of TCZ differs in various regions of the world. TCZ also has two approved systemic modes of administration: intravenous (IV) and subcutaneous (SC). In the USA, induction therapy employs 4 mg/kg monthly IV regimen followed by step up to 8 mg/kg monthly based on therapeutic response. However, in Europe, the approved initiating and maintenance dose is 8 mg/kg IV monthly, titrated to 4 mg/kg if side effects occur.
SC route of administration has important advantages such as providing more convenient route of administration, including self-application at home, negating the requirement for and associated health care costs of intravenous access, and frequent clinic visits. The efficacy of the SC route over IV route was evaluated in several studies. Two randomized, double-masked, 24-week comparative, phase 3 studies demonstrated the non-inferiority of TCZ-SC to TCZ-IV: SUMMACTA in Europe and MUSASHI in Japan [87,88,89]. SUMMACTA compared TCZ-SC 162 mg every week to TCZ-IV 8 mg/kg every 4 weeks, and MUSASHI compared TCZ-SC 162 mg every 2 weeks or TCZ-IV 8 mg/kg every 4 weeks, showed safety and non-inferior efficacy of TCZ-SC. BREVACTA, another randomized, double-masked, 24-week comparative trial in the USA, evaluated 162 mg TCZ-SC every 2 weeks versus placebo . BREVACTA showed that TCZ-SC was superior to placebo. These studies led to the approval of the subcutaneous formulation and the dosing recommendations in the European Union and the USA. Consistent with the respective intravenous labels, in the EU, the approved starting dose for TCZ-SC is 162 mg every week, with a possible decrease in dosage frequency to every 2 weeks. In the USA, the starting dose for TCZ-SC is 162 mg every 2 weeks, with a possible increase in dosing to every week, based on clinical response approved . In summary, TCZ-SC appears to be as effective as TCZ-IV with comparable safety.
Tocilizumab: safety and pre-administration evaluation
The safety of TCZ in patients with RA has been evaluated in several phase 3 and 4 RCTs [37, 50, 53, 80, 82, 87, 88]. Infections such as nasopharyngitis, upper respiratory tract infections, pneumonia, and cellulitis were the most common. Other major adverse events were gastrointestinal perforations (GIPs), neutropenia, and malignancies. Laboratory test abnormalities were also reported with TCZ therapy, including elevated liver enzyme levels, decreased neutrophil counts, and change in lipid levels.
The real-world studies, in line with the data from RCTs, also found higher incidence of GIPs in patients treated with TCZ than in those treated with other biologics or cDMARDs [91, 92]. For every 1000 patients treated with TCZ per year, between one and two additional GIPs might be expected to occur compared with those treated with other TNF inhibitors . In particular, the risk for lower-intestinal perforations (LIPs) seems to be higher in patients with a history of diverticulitis [91, 92].
Malignancy and neutropenia are two additional concerns for patients receiving TCZ. The risk for malignancy is potentially greater in patients with RA who use immunosuppressive agents. The current hypothesis is that inflammatory activity associated with RA drives the increased lymphoma risk. Additionally, some evidence suggests that patients with RA treated with biologics are at increased risk for malignancy, specifically non-melanoma skin cancers, compared with the general population . Despite these inferences, the analysis of the data from phase 3 trials and long-term extension studies did not demonstrate increased risk for overall or site-specific malignancies above the risk expected in patients with RA. The overall malignancy rate observed in tocilizumab, all-exposure population was higher than that in the general population but was consistent with that expected in the RA population and in the geographic regions studied .
Data for neutropenia incidence with TCZ are divergent. De Benedetti et al. reported neutropenia incidences as high as 57% in a cohort of 112 patients with JIA . A dose-dependent effect on neutropenia-onset was observed in RCTs, where the incidence of neutropenia at 8 mg/kg was almost twice as observed at 4 mg/kg [37, 49]. In a retrospective cohort study, TCZ was associated with a higher incidence of neutropenia compared with abatacept and infliximab. However, the increased incidence of neutropenia did not result in a higher risk for severe infections . Also, data from these trials confirm that grade 4 neutropenia (< 0.5 ANC × 109/l) is extremely uncommon.
Adverse events reported in RCTs with TCZ in pediatric age group, treated for JIA, were similar to the adult group and included infections, neutropenia, and abnormalities in liver function test results [95, 97]. Notably, no or very few cases of macrophage activation syndrome (MAS), which resolved, were reported in those studies. On the other hand, a patient registry post-marketing surveillance (PMS) in Japan, which was conducted to investigate the safety and effectiveness of TCZ in real-world clinical settings, reported slightly higher adverse event rates than those reported in clinical trials of TCZ for systemic JIA . This dissimilarity was attributed to the fact that RCTs had excluded patients with concurrent medical or surgical conditions; or concomitant diseases of the nervous, renal, endocrine, or hepatic systems.
All this data from RCTs and cohort studies highlights the need for careful patient selection when treating with anti-IL-6 agents (i.e., exclusion of individuals with previous diverticulitis, exclusion of infections such as tuberculosis, fungal, etc.). Patients should be closely monitored for the development of signs and symptoms of infection during and after treatment. In order to ensure safe use of TCZ in daily practice, physicians and patients should also be aware that, under TCZ, conditions such as GIP, MAS (in pediatrics) may occur with mild symptoms only and careful examination and testing are crucial.
Newer IL-6 inhibitors
TCZ was launched as the first biologic drug targeting IL-6 in 2010, which provided a strong alternative to anti-TNF-α agents. Clinical success of TCZ was encouraging, which led pharmaceutical industry to undertake research in discovering other IL-6 blocking pathways. Currently, several IL-6/IL-6R inhibitors are under investigation in various phases of different studies (Table 1). These molecules can be broadly divided into two categories:
Targeting IL-6 with Sirukumab, Siltuximab, Olokizumab, Clazakizumab, and EBI-031