Interleukin-6 (IL-6)-type cytokines not only have key immunomodulatory functions that affect the pathogenesis of diseases such as autoimmune diseases, chronic inflammatory conditions, and cancer, but ...also fulfill important homeostatic tasks. Even though the pro-inflammatory arm has hindered the development of therapeutics based on natural-like IL-6-type cytokines to date, current synthetic trends might pave the way to overcome these limitations and eventually lead to immune-inert designer cytokines to aid type 2 diabetes and brain injuries. Those synthetic biology approaches include mutations, fusion proteins, and inter-cytokine swapping, and resulted in IL-6-type cytokines with altered receptor affinities, extended target cell profiles, and targeting of non-natural cytokine receptor complexes. Here, we survey synthetic cytokine developments within the IL-6-type cytokine family and discuss potential clinical applications.
Cytokine-induced signal transduction is executed by natural biological switches, which among many others control immune-related processes. Here, we show that synthetic cytokine receptors (SyCyRs) can ...induce cytokine signaling using non-physiological ligands. High-affinity GFP- and mCherry-nanobodies were fused to transmembrane and intracellular domains of the IL-6/IL-11 and IL-23 cytokine receptors gp130 and IL-12Rβ1/IL-23R, respectively. Homo- and heterodimeric GFP:mCherry fusion proteins as synthetic cytokine-like ligands were able to induce canonical signaling in vitro and in vivo. Using SyCyR ligands, we show that IL-23 receptor homodimerization results in its activation and IL-23-like signal transduction. Moreover, trimeric receptor assembly induces trans-phosphorylation among cytokine receptors with associated Janus kinases. The SyCyR technology allows biochemical analyses of transmembrane receptor signaling in vitro and in vivo, cell-specific activation through SyCyR ligands using transgenic animals and possible therapeutic regimes involving non-physiological targets during immunotherapy.
Highlights • Interleukin (IL-)23 is a central cytokine controlling TH 17 development. • Overshooting IL-23 signaling contribute to autoimmune diseases. • IL-23 is a member of the IL-12-type cytokine ...family and consists of IL-23p19 and p40. • IL-23 triggers heterodimerization of IL-12Rβ1 and the IL-23R.
Interleukin‐6 (IL‐6) is critically involved in liver regeneration after partial hepatectomy (PHX). Previous reports suggest that IL‐6 trans‐signaling through the soluble IL‐6/IL‐6R complex is ...involved in this process. However, the long‐term contribution of IL‐6 trans‐signaling for liver regeneration after PHX is unknown. PHX‐induced generation of the soluble IL‐6R by ADAM (a disintegrin and metallo) proteases enables IL‐6 trans‐signaling, in which IL‐6 forms an agonistic complex with the soluble IL‐6 receptor (sIL‐6R) to activate all cells expressing the signal‐transducing receptor chain glycoprotein 130 (gp130). In contrast, without activation of ADAM proteases, IL‐6 in complex with membrane‐bound IL‐6R and gp130 activates classic signaling. Here, we describe the generation of IL‐6 trans‐signaling mice, which exhibit boosted IL‐6 trans‐signaling and abrogated classic signaling by genetic conversion of all membrane‐bound IL‐6R into sIL‐6R proteins phenocopying hyperactivation of ADAM‐mediated shedding of IL‐6R as single substrate. Importantly, although IL‐6R deficient mice were strongly affected by PHX, survival and regeneration of IL‐6 trans‐signaling mice was indistinguishable from control mice, demonstrating that IL‐6 trans‐signaling fully compensates for disabled classic signaling in liver regeneration after PHX. Moreover, we monitored the long‐term consequences of global IL‐6 signaling inhibition versus IL‐6 trans‐signaling selective blockade after PHX by IL‐6 monoclonal antibodies and soluble glycoprotein 130 as fragment crystallizable fusion, respectively. Both global IL‐6 blockade and selective inhibition of IL‐6 trans‐signaling results in a strong decrease of overall survival after PHX, accompanied by decreased signal transducer and activator of transcription 3 phosphorylation and proliferation of hepatocytes. Mechanistically, IL‐6 trans‐signaling induces hepatocyte growth factor production by hepatic stellate cells. Conclusion: IL‐6 trans‐signaling, but not classic signaling, controls liver regeneration following PHX.
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•Constitutive-active cytokine receptors are directly linked to human diseases.•Gain-of-function cytokine receptors are promising targets to modulate immunotherapeutic ...strategies.•Synthetic biology approaches resulted in gain-of-function cytokine receptors which might be useful for therapeutics.•This review gives insight into the field of naturally occurring and synthetic constitutive-active cytokine receptors.
Cytokines control immune related events and are critically involved in a plethora of patho-physiological processes including autoimmunity and cancer development. Mutations which cause ligand-independent, constitutive activation of cytokine receptors are quite frequently found in diseases. Many constitutive-active cytokine receptor variants have been directly connected to disease development and mechanistically analyzed. Nature’s solutions to generate constitutive cytokine receptors has been recently adopted by synthetic cytokine receptor biology, with the goal to optimize immune therapeutics. Here, CAR T cell immmunotherapy represents the first example to combine synthetic biology with genetic engineering during therapy. Hence, constitutive-active cytokine receptors are therapeutic targets, but also emerging tools to improve or modulate immunotherapeutic strategies. This review gives a comprehensive insight into the field of naturally occurring and synthetic constitutive-active cytokine receptors.
Cytokines of the IL-12 family show structural similarities but have distinct functions in the immune system. Prominent members of this cytokine family are the pro-inflammatory cytokines IL-12 and ...IL-23. These two cytokines share cytokine subunits and receptor chains but have different functions in autoimmune diseases, cancer and infections. Accordingly, structural knowledge about receptor complex formation is essential for the development of new therapeutic strategies preventing and/or inhibiting cytokine:receptor interaction. In addition, intracellular signaling cascades can be targeted to inhibit cytokine-mediated effects. Single nucleotide polymorphisms can lead to alteration in the amino acid sequence and thereby influencing protein functions or protein–protein interactions. To understand the biology of IL-12 and IL-23 and to establish efficient targeting strategies structural knowledge about cytokines and respective receptors is crucial. A highly efficient therapy might be a combination of different drugs targeting extracellular cytokine:receptor assembly and intracellular signaling pathways.
Cytokines control immune-related events and are critically involved in a plethora of physiological and pathophysiological processes including autoimmunity and cancer development. Accordingly, ...modulation of natural cytokine signaling by antibodies and small molecules has improved therapeutic regimens. Synthetic biology sets out to optimize immunotherapeutics, with chimeric antigen receptor (CAR) T cell immmunotherapy being the first example to combine synthetic biology with genetic engineering during therapy. Hence, synthetic cytokines and cytokine receptors, as well as constitutively active cytokine receptor variants, are emerging as tools to improve or modulate immunotherapeutic strategies. This review focuses on recent developments in the growing field of synthetic cytokine signaling, providing an outlook for developing applications that involve physiological targets of immunotherapy.
Synthetic cytokine biology deconstructs and reassembles cytokines and their receptors to design biological devices as therapeutics.
Synthetic cytokines, including neoleukins and synthekines, can activate selective or nonnatural receptor combinations and subsequently induce unique signaling patterns.
Cytokines coupled to nanoparticles, peptides, or antibodies and fusokines can allow cell-targeted therapies.
Naturally occurring and synthetic constitutively active cytokine receptors have been described for all cytokine receptor classes and might improve immunotherapy approaches.
Fully synthetic cytokine signaling systems can allow precisely orchestrated cellular responses. They have been shown to modulate immune responses and presumably, might support immunotherapeutics.
Hypomorphic ADAM17ex/ex mice showed defects in mucosal regeneration due to inefficient enhanced GFR shedding. ADAM17 is the main sheddase of interleukin-6 receptor (IL-6R) to induce IL-6 ...trans-signaling. However, serum levels of soluble murine IL-6R were not reduced in ADAM17ex/ex mice, and murine ADAM17 was not the major sheddase of murine IL-6R. Shedding of murine IL-6R by murine ADAM17 was rescued in chimeric murine IL-6R proteins containing any extracellular domain but not the transmembrane and intracellular domain of human IL-6R. Apoptosis is a physiological stimulus of ADAM17-mediated shedding of human IL-6R. Even though apoptosis induced IL-6R shedding in mice, the responsible protease was identified as ADAM10. ADAM10 also was identified as protease responsible for ionomycin-induced shedding of murine and human IL-6R. However, in ADAM10-deficient murine embryonic fibroblasts, compensatory shedding of human IL-6R was mediated by ADAM17, but loss of ADAM10-mediated shedding of murine IL-6R was compensated by an as-yet-unidentified protease. Finally, we identified physiological purinergic P2X7 receptor stimulation as a novel inducer of murine and human IL-6R shedding solely mediated by ADAM10. In conclusion, we describe an unexpected species specificity of ADAM10 and ADAM17 and identified ADAM10 as novel inducible sheddase of IL-6R in mice and humans, which might have consequences for the interpretation of phenotypes from ADAM17- and ADAM10-deficient mice.
Synthetic biology has emerged as a useful technology for studying cytokine signal transduction. Recently, we described fully synthetic cytokine receptors to phenocopy trimeric receptors such as the ...death receptor Fas/CD95. Using a nanobody as an extracellular-binding domain for mCherry fused to the natural receptor’s transmembrane and intracellular domain, trimeric mCherry ligands were able to induce cell death. Among the 17,889 single nucleotide variants in the SNP database for Fas, 337 represent missense mutations that functionally remained largely uncharacterized. Here, we developed a workflow for the Fas synthetic cytokine receptor system to functionally characterize missense SNPs within the transmembrane and intracellular domain of Fas. To validate our system, we selected five functionally assigned loss-of-function (LOF) polymorphisms and included 15 additional unassigned SNPs. Moreover, based on structural data, 15 gain-of-function or LOF candidate mutations were additionally selected. All 35 nucleotide variants were functionally investigated through cellular proliferation, apoptosis and caspases 3 and 7 cleavage assays. Collectively, our results showed that 30 variants resulted in partial or complete LOF, while five lead to a gain-of-function. In conclusion, we demonstrated that synthetic cytokine receptors are a suitable tool for functional SNPs/mutations characterization in a structured workflow.
•Algorithm to directly align dbSNP information to DNA and protein sequence.•SNP landscape was generated for all tall cytokine receptors of the IL-6/IL-12 family.•Structural insights of ...disease-causing SNPs in cytokine/cytokine receptor interfaces.•The algorithm can be easily applied to other cytokine receptor networks.
Cytokines control immune related events and are critically involved in a plethora of patho-physiological processes including autoimmunity and cancer development. In rare cases, single nucleotide polymorphisms (SNPs) or single nucleotide variations (SNVs) in cytokine receptors eventually cause detrimental ligand-independent, constitutive activation of signal transduction. Most SNPs have, however, no or only marginal influences on gene expression, protein stability, localization and function and thereby only slightly affecting pathogenesis probability. The SNP database (dbSNP) is an archive for a broad collection of polymorphisms in which SNPs are categorized and marked with a locus accession number “reference SNP” (rs). Here, we engineered an algorithm to directly align dbSNP information to DNA and protein sequence information to clearly illustrate a genetic SNP landscape exemplified for all tall cytokine receptors of the IL-6/IL-12 family, including IL-23R, IL-12Rβ1, IL-12Rβ2, gp130, LIFR, OSMR and WSX-1. This information was complemented by a comprehensive literature summary and structural insights of relevant disease-causing SNPs in cytokine/cytokine receptor interfaces. In summary, we present a general strategy with potential to apply to other cytokine receptor networks.
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