INTRODUCTION

Interleukin 6 (IL-6) was initially described as a B-cell growth factor [1], but more recent studies identified IL-6 as one of the key pro-inflammatory cytokines that plays an important immunoregulatory role associated with cell proliferation and differentiation. The range of its functions in the body is not restricted to the immune system [2, 3]. Defects in the IL-6 signaling cascade lead to systemic inflammation and lymphoproliferative disorders [4]. Approaches to systemic and selective inhibition of IL-6 and its downstream signaling cascade have intensely developed over recent years and have found application in medicine [5].

IL-6 belongs to a family of cytokines that, apart from their structural homology, are similar in utilizing a common receptor subunit, gp130, in signal transduction [6, 7]. The signal is transmitted via a classical pathway through a membrane-associated complex of IL-6 with its receptor (IL-6R), IL-6/IL-6R/gp130, on the cell surface (Fig. 1a) or a trans-signaling pathway wherein the soluble IL-6/sIL-6R complex interacts with gp130 on other cells (Fig. 1b). A third pathway was recently described and termed “trans-presentation”, or IL-6 cluster signaling. In trans-presentation, the IL-6/IL-6R forms intracellularly and is presented on the surface of one cell type to induce gp130 dimerization and intracellular signal transduction in cells of another type (Fig. 1c). In particular, this signaling mechanism is characteristic of a dendritic cell population that induce the formation of pathogenic Th17 cells in experimental autoimmune encephalomyelitis (EAE) [8].

Fig. 1.
figure 1

Scheme of IL-6 signaling pathways. (a) In the classical pathway, IL-6 target cells express the IL-6 receptor (IL-6Rα). (b) In the trans-signaling pathway, certain cells (e.g., hepatocytes, leukocytes, and epithelial cells) express a membrane IL-6Rα form, which can be processed to yield a soluble form (sIL-6Rα). The interaction of sIL-6Rα with IL-6 results in a signaling complex, which binds to gp130 to induce signal transduction on any gp130-expressing cells. (c) In trans-presentation, dendritic cells express IL-6 and IL-6Rα, which presumably form a signaling complex within the cell. The complex finds its way on the cell surface and may induce signaling through gp130 present on the surface of naïve T cells. The images used in the figure are available at https://smart.servier.com.

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IL-6 performs many functions in normal and pathological conditions. Although IL-6 was initially identified as a B-cell differentiation factor [1], it is clear now that IL-6 plays an important role in regulating T-cell immunity as well. For example, IL-6 is necessary for differentiation of several CD4+ T-cell subpopulations, such as Th17, and regulates the switching between induction of regulatory T cells (Foxp3+) and Th17 cells [9–12]. In addition, IL-6 produced by follicular dendritic cells is of importance for T-follicular helpers (Tfh) because the cytokine regulates Bcl-6 expression [13–15]. In turn, Tfh are specialized in maintenance of germinal center B-cells [16, 17]. It is known that IL-6 controls glucose metabolism and, in particular, is responsible for hepatocyte sensitivity to insulin [2]. Recent studies implicated IL-6 in regulating muscle tissue metabolism, and neutralization or lack of IL-6 in mice may decrease their endurance in physiological tests [3]. Finally, IL-6 plays an important role in epithelial homeostasis, including tissue regeneration after damage. Mice with a complete IL-6 knockout show a substantial delay in full-thickness skin wound healing [18, 19], and IL-6-dependent inflammation is necessary for maintaining the regenerative potential of intestinal epithelial cells [20, 21]. It is of immense importance for medicine to find a means to selectively inhibit the pathogenic effects of IL-6 without affecting its functions in homeostasis.

The signal transduction mechanisms that are triggered by IL-6 binding with the receptor complex were elucidated mostly using reverse genetics approach, and earlier data on IL-6-mediated signaling were refined with improvements in reverse genetics techniques. Current views of the mechanisms of IL-6 signal transduction are most likely incomplete and will be supplemented and expanded in the future.

Genome editing methods (genetic knockout, knockin, and transgenesis) experimentally identify the consequences of gene inactivation or overexpression and report primarily the non-redundant functions of a gene or its product. Shared redundant functions are possibly missed, while such functions are almost always present given that IL-6, like any other cytokine, belongs to a family of cytokines similar in structure and signal transduction mechanisms. However, constructing model organisms with gene inactivation in a particular cell type or restricted overexpression of the same gene in the same cells is still a promising field of reverse genetics. Moreover, an inducible deletion or overexpression of a gene is is routinely used in model organisms or cell lines, allowing to switch the gene on or off at a particular stage of development or pathogenesis. Based mostly on reverse genetics approach, a pathogenic role of IL-6 produced by particular cell type was demonstrated in the context of many experimental disorders to justify a selective blockade of the pathogenic signal, which is possible to abolish, for example, by neutralizing the soluble receptor sIL-6R or blocking IL-6 in a tissue-specific manner [22].

Neutralizing monoclonal antibodies are broadly used now to treat many autoimmune and inflammatory disorders associated with defects in cytokine signaling cascades. Antibodies provided a breakthrough in therapy for many obstinate chronic conditions. However, preclinical evaluations of the efficacy and safety of therapeutic monoclonal antibodies are often difficult to perform in experimental animal models because the majority of such antibodies show extremely low cross-reactivity with orthologous molecules of animals other than primates (which are the closest phylogenetically to humans). Models in smaller experimental animals, such as mice and rats, are therefore poorly suitable in spite of their apparent advantages, requiring additional modification of the animal genome. This circumstance explains the broad use of methods to humanize the genes for cytokines and their receptors along with reverse genetics approach, which are designed to study the results of complete or partial loss of gene function in vivo. Two methods are widely used to humanize a gene. One is constructing transgenic mice so that the transgene function is artificially enhanced by using a potent promoter to allow high-level transgene expression in all cells or under control of a tissue-specific promoter [23, 24]. Another method is more laborious, but more physiological and is known as a knock-in; the gist is replacing a mouse gene with the orthologous human gene while preserving the genome regions responsible for endogenous gene expression [25]. It should be noted that double humanization of both the cytokine and receptor genes is necessary in the case of the IL-6/ IL-6R system because mouse IL-6 does not interact with the human IL-6 receptor [26, 27]. Interestingly, both anti-IL-6 and anti-IL-6R neutralizing antibodies proved effective in treating autoimmune disorders; it is therefore necessary to study their pharmacokinetics, pharmacodynamics, and efficacy in vivo in the context of available experimental mouse models of human diseases.

CONDITIONAL INACTIVATION OF IL-6 DIRECTS TOWARDS THE CELL SOURCE OF THE PATHOGENIC CYTOKINE IN EXPERIMENTAL MODELS OF DISEASES

IL-6 is a pro-inflammatory cytokine, and a mouse model of multiple sclerosis is intensely used to study its role. Earlier studies showed that mice with genetic or drug-induced inactivation of IL-6 or IL-6R are fully resistant to experimental autoimmune encephalomyelitis (EAE) [28, 29]. Further investigation of the respective signaling pathway made it possible to establish the principal mechanism whereby IL-6 facilitates disease development. The IL-6 signaling pathway was found to play a role in regulating the balance between pathogenic Th17 cells, which are the main effectors in EAE, and regulatory T cells that suppress autoimmune reactions. Acting together with other pro-inflammatory cytokines (IL-1, IL-23, IL-21, and TGF-β), IL-6 can induce de novo development of Th17 cells from naive T cells. Although IL-6 inhibited FoxP3 and prevented the development of regulatory T cells to aggravate the pathological condition in many experimental models, the development of regulatory T cells, which are necessary for maintaining homeostasis and controlling barrier tissues, was found to depend on the STAT3 signaling pathway, which is triggered, in particular, by IL-6 [30, 31]. Thus, IL-6 plays both pathogenic and regulatory roles in the context of.

Studying the functions of IL-6 produced by a particular cell type and the consequences of IL-6 signaling in target cells is essential for understanding the molecular mechanisms of immunoregulation, pharmacological stimulation of which is an important part of drug development. In particular, an Il6 knockout in dendritic cells was shown to confer EAE resistance in mice [8]. A new type of IL-6 presentation was identified in mice with a conditional knockout in Il6 and Il6ra in dendritic cells. IL-6 and IL-6R were implicated in formation of an intracellular complex in dendritic cells. The complex is then exposed on the dendritic cell surface and interacts with gp130 on the T‑cell surface, leading eventually to differentiation of pathogenic Th17 cells [8].

We also studied the role of IL-6 in EAE. To induce EAE, mice were immunized with a myelin oligodendrocyte glycoprotein peptide (MOG35–55) combined with complete Freund’s adjuvant and then injected twice with pertussis toxin to increase the permeability of the blood–brain barrier (Fig. 2a). Clinical signs of EAE became detectable 9 days after the disease induction. One group of mice was injected with anti-IL-6 neutralizing antibodies (clone MP5-20F3) as soon as they developed the first signs of the disease. Figure 2b shows the development of clinical signs of EAE in wild-type mice, mice with a conditional Il6 knockout in monocytes and macrophages (Mlys-Cre IL-6fl/fl), and mice with a pharmacological blockade of IL-6 (@IL-6). A pharmacological blockade of IL-6 was found to decelerate the EAE development and to decrease the clinical signs of the disease (Fig. 2). The finding is in agreement with published data on a genetic or pharmacological ablation of the IL-6 receptor [28, 29]. It is of interest that an Il6 knockout in myeloid cells only did not affect the development and clinical signs of EAE (Fig. 2), although myeloid cells are the cell source that produces proinflammatory cytokines involved in the pathogenesis of other autoimmune disorders.

Fig. 2.
figure 2

Pharmacological blockade of IL-6 protects mice from experimental autoimmune encephalitis (EAE), while genetic inactivation of Il6 in monocytes and macrophages only does not affect the EAE development. (a) To induce EAE, mice were immunized with a myelin oligodendrocyte glycoprotein (MOG) peptide in complete Freund’s adjuvant and injected with anti-IL-6 neutralizing antibodies (@IL-6). (b) Clinical signs of EAE were evaluated in wild-type mice (WT), mice with a conditional Il6 knockout in monocytes and macrophages (Mlys-Cre IL-6fl/fl), and mice with a pharmacological block of IL-6 (@IL-6). Clinical signs were scored daily on the following scale: 0, no sign of disease; 0.5, partial loss of tail tone; 1, complete loss of tail tone; 2, muscle weakness of the hind limbs; 2.5, altered gait; 3, paresis of the hind limbs. (c) Time of EAE sign development in days after disease induction. (d) Maximal EAE scores in moue groups.

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As previously established, IL-6 is involved in airway inflammation because an increase in IL-6 expression is observed in exacerbations of allergic asthma. IL-6 was shown to play a key role in the development of allergic inflammation induced via adoptive transfer of activated dendritic cells [32]. However, our studies in a mouse model of allergic asthma induced with a house dust mite (HDM) extract [33] showed that, apart from dendritic cells, cells of the macrophage–monocyte lineage are an important source of pathogenic IL-6 in mice with a tissue-specific Il6 knockout in myeloid cells (Fig. 3a). For example, both the proportion (Fig. 3b) and absolute count (Fig. 3c) of IL-4+IL-13+ lymphocytes are decreased in the bronchoalveolar fluid and lung-draining lymph nodes of mice with a total Il6 knockout or an Il6 knockout in macrophages. Expression of IL-4 and IL-13 is characteristic of a Th2 response and is necessary for B-cell antibody isotype switching in atopic asthma. Our findings give grounds to assume that IL-6 produced by macrophages induces expansion of IL-4+IL-13+ lymphocytes to cause a stronger Th2 response, which is characteristic of allergic pulmonary inflammation, thus acting as a pathogenic factor in the disease.

Fig. 3.
figure 3

Mice with a complete Il6 knockout are protected from a Th2-mediated response in allergic asthma; genetic inactivation of Il6 in dendritic cells (CD11c-Cre) does not affect the count and proportion of pathogenic IL-4+IL-13+ cells in the bronchoalveolar lavage fluid (BALF) and mediastinal lymph node (MLN); and genetic inactivation of Il6 in macrophages partly protects from an allergy-induced Th2 response. (a) Scheme of the experiment where acute asthma was induced via intranasal administration of a house dust mite (HDM) extract in wild-type, IL-6–/–, Mlys-Cre IL-6fl/fl, and CD11c-Cre IL-6fl/fl mice. (b) Percentages of IL-4+IL13+CD45+ cells in the BALF and MLN in wild-type (WT), IL-6–/–, Mlys-Cre IL-6fl/fl, and CD11c-Cre IL-6fl/fl mice. (c) IL-4+IL13+CD45+ cell counts in the BALF and MLN in wild-type (WT), IL-6–/–, Mlys-Cre IL-6fl/fl, and CD11c-Cre IL-6fl/fl mice. Differences were significant at (*) p < 0.05, (**) p < 0.01, (***) p < 0.001, or (****) p < 0.0001.

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Thus, IL-6 produced by myeloid cells with the active Mlys promoter (which was used to control Cre-dependent genetic inactivation of Il6 in our experimental system) acted differently in two different models. The difference is presumably explained by the fact that myeloid cells are a highly heterogeneous population and may utilize different mechanisms to play their role in the pathogenesis in the two experimental models of allergic airway inflammation.

CONDITIONAL INACTIVATION OF IL-6R AND gp130 AS A MEANS TO DESIGN NEW APPROACHES TO SELECTIVE INHIBITION OF IL-6

The IL-6 receptor consists of a ligand-binding α subunit and a signal-transducing β subunit (gp130). The transmembrane protein gp130 is found in all cell types, while IL-6R is expressed predominantly in cells of the immune system, hepatocytes, and microglial cells. It is noteworthy that IL-6R can be from a membrane-associated to a soluble (sIL-6R) form via proteolytic cleavage by metalloproteinases ADAM10 and ADAM17 [34] and, to a lesser extent, alternative splicing of the IL-6R mRNA [35].

Two main pathways mediate signal transduction from IL-6R. A classical signaling pathway is triggered by the binding of soluble IL-6 (sIL-6) with IL-6R and gp130 on the surface of a target cell. An alternative signaling pathway includes two modalities, trans-signaling, where the complex of IL-6 with sIL-6R forms extracellularly and then binds with gp130 on a target cell, and cluster signaling (or trans-presentation), where the IL-6/IL-6R complex is assembled within the producer cell, transferred onto the cell surface, and then binds with gp130 on a target cell (Fig. 1) [8].

A complete genetic knockout in Gp130 leads to embryonic lethality presumably because the gp130 signaling molecule is involved in other cytokine signaling cascades, such as LIF. The role of gp130 molecules expressed on different cell types is possible to study using the conditional knockout approach and has been previously described [36, 37]. IL-6 is known to facilitate differentiation of naïve T cells into Th17 cells, which are involved in the pathogenesis of various autoimmune disorders [37]. In vitro experiments showed that a Gp130 deletion in T cells prevents them from acquiring the Th17 phenotype when cultured in the presence of IL-6 and TGFβ [38].

In vivo studies with a mouse model of multiple sclerosis showed that a Gp130 deletion in T cells fully protects mice from EAE by shifting differentiation of naïve T cells toward regulatory T cells [30]. A conditional Gp130 knockout in GFAP+ astrocytes substantially increases the signs of EAE, and the increase correlates with a higher astrocyte mortality in demyelinated sites; a decreased count of regulatory T cells; and a higher production of proinflammatory cytokines, such as IL-17, IFN-γ, and TNF [39]. Genetic inactivation of Gp130 in monocytes and neutrophils in the EAE model leads to the development of clinical signs, while an Il6ra deletion in monocytes and neutrophils does not affect the disease course [40]. It is possible that the protective role in the EAE model is played by other cytokines that are secreted by myeloid cells and utilize gp130 as a receptor subunit.

It is known that IL-6 is involved in differentiation of CD4+ T cells and is necessary for naive T cells to acquire the Th17 and Tfh phenotypes [41]. Nish et al. [42] showed that, in T cells with a conditional Il6ra knockout, the IL-6 signaling pathway is necessary for their function, but does not affect the development of Tfh cells, whose main function is to help B cells in subsequent secretion of high-affinity antibodies and generation of memory B cells. In addition, activation of the IL-6/IL-6R pathway is required for effector T cells to inhibit the suppressor effects of regulatory T cells.

As already mentioned, IL-6 acts not only in the immune system and, in particular, controls certain metabolic processes [2]. For example, mice with an Il6ra knockout in hepatocytes develop systemic insulin resistance associated with defects in glucose metabolism [43]. In mice with Il6ra inactivation in myeloid cells, high-fat diet (HFD) leads not only to insulin resistance, but also to an increase in systemic inflammation and the proinflammatory M1 phenotype of macrophages. In this case, the anti-inflammatory effect of IL-6 signaling in myeloid cells is mediated by STAT3 phosphorylation, which increases Il4ra expression and causes macrophage polarization to anti-inflammatory M2 cells [44]. Thus, activation of the IL-6 signaling pathway in hepatocytes and myeloid cells plays an important anti-inflammatory effect when insulin resistance is induced or metabolic syndrome develops.

The role of IL-6R was studied in detail in vivo in experimental models of autoimmune disorders, where a pathogenic function is attributed to the Th17 cell subpopulation and a protective function, by regulatory T cells. A colitis model was obtained via adoptive transfer of CD4+ T cells to RAG2-deficient mice, and lack of IL-6R expression on T cells protected mice from developing the disease [42]. In this case, a complete Il6ra deletion or a tissue-specific Il6ra knockout in myeloid cells does not alleviate the clinical signs of experimental colitis [45]. In addition, Il6ra was genetically inactivated in intestinal epithelial cells in a chemically induced colitis model, and IL-6-mediated signal transduction via the classical pathway (Fig. 1a) was not found to play a considerable role in epithelial regeneration in inflammatory diseases of the intestine [46]. In the EAE model, lack of Il6ra in CD11с+ dendritic cells fully protected mice from the disease, the effect being due to the absence of IL-6 trans-presentation by antigen-specific dendritic cells and subsequent differentiation of pathogenic Th17 cells [8]. A milder disease developed in mice with a tissue-specific Il6ra knockout in T cells as compared with wild-type mice, while depletion of regulatory T cells substantially increased the disease signs in mice with a conditional Il6ra knockout in T cells [42]. Thus, activation of the IL-6/IL-6R signaling pathway in T cells plays an important nonredundant role in autoimmune disorders by regulating Th17 cell differentiation. In addition, IL-6 produced by dendritic cells is of critical relevance in terms of cluster signaling during disease development in the EAE model.

HUMANIZATION OF THE IL-6/IL-6R SYSTEM FOR COMPARATIVE EVALUATION OF ANTICYTOKINE DRUGS IN MOUSE MODELS

In order to desigh effective therapeutics, the role of the IL-6/IL-6R signaling pathways in the pathogenesis of various disorders needs to be established.

Tocilizumab, a humanized anti-IL-6R monoclonal antibody, is currently approved as a drug for autoimmune disorders, in particular, rheumatoid arthritis [47]. When the drug was still under development, mice with humanized IL-6R were used to evaluate the IL-6R inhibitors in preclinical studies and to demonstrate the efficacy of tocilizumab in a model of Castleman’s disease, which is a genetic lymphoproliferative syndrome [26]. More recently, tocilizumab has been approved for rheumatoid arthritis. A main advantage of tocilizumab over TNF blockers is that IL-6R is not involved in granuloma assembly and, therefore, a blockade of IL-6R-mediated signaling does not induce complications due to reactivation of mycobacterial infections, including tuberculosis. Siltuximab is a chimeric monoclonal antibody against human IL-6 and another drug of the kind approved for medical use (Table 1). Clinical studies of various phases are currently performed to evaluate several other inhibitors, including sgp130Fc, which is a soluble form of the gp130 receptor [22]. It should be noted that a systemic block of IL-6 or IL-6R is still associated with higher risk of reactivation of infections, and this circumstance is important to consider when developing inhibitors of the IL-6/IL-6R signaling cascade.

Table 1.   Characteristics of IL-6 and IL-6R inhibitors approved for medical use*
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To allow comparative evaluations of inhibitors targeting human IL-6, we constructed new humanized mice with tamoxifen-dependent induction of a human IL-6-coding transgene in myeloid cells. Constitutive expression of the transgene in myeloid cells led to neonatal lethality, which was presumably due to systemic inflammation and anemia. The IL-6 production was extremely high in primary macrophage cultures obtained from bone marrow of newborn transgenic mice; data on reporter EGFP expression supported the finding [24]. When transgene expression was induced in mice by a course of tamoxifen administration (Fig. 4a), the serum IL-6 concentration similarly increased to a pathological level and then decreased as the myeloid cell pool was renewed in the blood, but did not reach its baseline value. The serum IL-6 content did not decrease to the baseline level possibly because resident CX3CR1 macrophages (in particular, microglial and other long-lived resident myeloid cells) continued expressing the transgene (Fig. 4b). The observation was in agreement with data on reporter EGFP expression in blood cells (Fig. 4c). Mice with tamoxifen-dependent transgene activation are viable and can be used to study the pathologies accompanied by higher IL-6 expression and to evaluate the efficacy of anticytokine therapy (Table 1).

Fig. 4.
figure 4

Characterization of Cx3cr1CreERhIL-6 Tg mice with tamoxifen-inducible overexpression of the hIL-6 transgene in CX3CR1+ myeloid cells. (a) Construction of the Cx3cr1CreERhIL-6 Tg mouse strain. Overexpression of the hIL-6 transgene is induced in response to intraperitoneal administration of tamoxifen (75 μg/g) for 5 days. (b) Representative distributions of VD–CD11b+ peripheral blood mononuclear cell populations with respect to the CX3CR1 markers and EGFP reporter. (c) Dynamics of EGFP reporter expression in VD–CD11b+CX3CR1+ peripheral blood mononuclear cells after a course of tamoxifen administration. (d) Serum concentration of hIL-6 prior to (day –5), during (day –2), and after (days 0, 12, and 24) a course of tamoxifen administration.

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It should be emphasized in conclusion that reverse genetics approach, especially conditional or inducible inhibition or activation of genes, made it possible to identify at the molecular level and to separate the protective and pathogenic functions of IL-6 and IL-6-dependent complex signaling cascades. The result of studies in the field give grounds to expect that selective anticytokine drugs will be designed to avoid the side effects that are inevitable in systemic anti-IL-6 therapy. An additional modality involves construction of mouse models with a humanized cytokine–receptor system, offering broad opportunities for comparative evaluation of inhibitors targeting human IL-6 and its receptor in experimental and preclinical studies.

ACKNOWLEDGMENTS

We are grateful to O.A. Namakanova and A.S. Zhdanova for help with some experiments, E.A. Gorshkova for discussion of the results, K.V. Korneev and D.V. Kuprash for their contribution to the design of the transgene construct, and A.V. Deikin for construction of the hIL-6 Tg mouse strain at the Collective Access Center of the Institute of Gene Biology. Other mouse strains mentioned above were from the Collection of Laboratory Animals for Basic, Biomedical, and Pharmacological Research of the Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry. The collection was supported by the Program of Bioresource Collection of the Federal Agency of Scientific Organizations of Russia.

This work was supported by the Russian Science Foundation (project no. 14-25-00160) and the Program of Basic Research at the State Academies of Sciences from 2013 to 2020 (project no. 01201363822).

COMPLIANCE WITH ETHICAL STANDARDS

The authors declare that they have no conflict of interest. This article does not contain any studies involving animals or human participants performed by any of the authors.