Engineered extracellular vesicle decoy receptor-mediated modulation of the IL6 trans-signalling pathway in muscle
Introduction
Extracellular vesicles (EVs), such as exosomes and microvesicles, are endogenous nano-sized vesicles that are released by many cell types and mediate intercellular communication under both physiological and pathological conditions [1]. EVs have been extensively studied as delivery vehicles for therapeutics, as they can transport a multitude of molecules between cells (including nucleic acids, proteins, and lipids) [[2], [3], [4], [5], [6]]. Aside from their ability to carry bioactive molecules, EVs derived from specific cell types such as mesenchymal stem cells have been successfully used for clinical applications, as they exhibit other potentially beneficial features (such as low immunogenicity, inherent anti-inflammatory, and pro-regenerative properties [[7], [8], [9], [10], [11], [12]]). It was previously shown that EVs can be engineered to carry biologically active molecules (both targeting ligands and/or therapeutic proteins), by fusing them to EV-associated proteins [3,[13], [14], [15], [16]]. This strategy can be used to express decoy receptors on the surface of EVs, with the capability of binding to specific signalling molecules with high affinity and specificity, and thereby blocking their intercellular signalling cascades [[16], [17], [18]]. This approach presents the opportunity for the modulation of inflammation by targeting key cytokines, such as interleukin 6 (IL6).
IL6 is a multifunctional cytokine, with both pro- and anti-inflammatory properties, that exerts its biological activities primarily through two different mechanisms; the classical- and trans-signalling pathway [19,20]. (Notably, an additional mode of IL6ST activation was recently identified, IL6 trans-presentation by dendritic cells [21]). In the case of the classical signalling pathway, associated with the anti-inflammatory activities of the cytokine, IL6 first binds to the membrane-bound interleukin 6 receptor (IL6R). The complex of IL6 and IL6R further associates with IL6ST (IL6 Signal Transducer, also known as gp130), which then dimerizes and initiates signal transduction via the activation of cytoplasmic tyrosine kinases and STAT3 (signal transducer and activator of transcription 3) [20]. Although the expression of the IL6R is restricted to relatively few cell types (e.g. hepatocytes, neutrophils, and macrophages), IL6 was still found to activate cells that lack membrane-bound IL6R [20], an observation which led to the discovery of the trans-signalling pathway. In this alternative pathway, IL6 first binds to a naturally-occurring, soluble-form of the IL6 receptor (sIL6R, primarily generated by alternative splicing or proteolytic processing of the IL6R protein) in the extracellular space, and subsequently the IL6/IL6R complex binds to and activates IL6ST on the surface of cells that do not express membrane-bound IL6R [22]. The IL6 trans-signalling pathway has been linked to the pro-inflammatory activities of the cytokine, and it is believed to mediate chronic inflammation [[23], [24], [25]]. Since IL6ST is ubiquitously expressed, the activity of the IL6 is greatly expanded relative to the classical pathway [26]. Furthermore, soluble forms of IL6ST were found in human body fluids and these were shown to be able to act as natural inhibitors of the IL6 trans-signalling pathway [27].
Inflammation is a characteristic feature of multiple muscle pathologies, including Duchenne muscular dystrophy (DMD) [28,29]. DMD is caused by mutations which lead to the loss of the dystrophin protein, which is important for maintaining muscle membrane integrity [30]. The lack of this structural protein results in membrane damage, massive infiltration of immune cells, chronic inflammation, impaired regeneration, and severe skeletal muscle degeneration, which are hallmark features of DMD pathology [[31], [32], [33], [34], [35], [36]]. Inflammation is therefore a major target for the treatment of DMD and consequently, almost all DMD patients are treated with corticosteroids (e.g. prednisolone and deflazacort). While chronic treatment with corticosteroids has demonstrated some limited efficacy in terms of delaying the loss of ambulation [37,38], it is also associated with a number of undesirable side effects such as weight gain, cushingoid features, behavioral problems, osteoporosis, and increased risk of infections [38,39]. As such, experimental therapies (such as vamolorone and edasalonexent) which reduce inflammation without the detrimental side effects of corticosteroids, are in late-stage of clinical development [40,41].
It is expected that blocking specific mediators of the inflammatory response, such as the IL6 trans-signalling pathway, might provide another alternative to steroidal drugs, with increased efficacy and decreased side effects. Multiple lines of evidence suggest that targeting the IL6 pathway in muscle-related pathologies might be beneficial; (i) chronic elevation of IL6 promotes skeletal muscle wasting [[42], [43], [44]], (ii) transient inhibition of the STAT3 signalling pathway stimulates muscle regeneration in aged and dystrophic mice [45,46], (iii) elevated levels of IL6 results in an increase in myostatin expression in muscle and consequent loss of muscle mass [47], (iv) serum levels of IL6 are increased in DMD patients and dystrophic mouse models [[48], [49], [50]], (v) the use of corticosteroids decreases the levels of IL6 in DMD patients and a mouse model of DMD, suggesting that the benefits of targeting the IL6 pathway are partially responsible for the positive effects of corticosteroids in DMD [49,51], (vi) targeting the IL6 pathway using IL6R neutralizing antibodies attenuates muscular dystrophy in mouse models of DMD [49,52] and (vii) overexpression of IL6 in a DMD mouse model exacerbates the dystrophic muscle phenotype and can more faithfully recapitulate the disease features observed in patients [50].
Here, we first explored the relevance of the IL6 trans-signalling pathway in myogenic cultures and mouse models of DMD, which led to the identification of this pathway as the dominant IL6 signalling pathway in muscle cells. We then engineered EVs to express signalling incompetent IL6ST decoy receptors, which could specifically block the IL6 trans-signalling pathway. This study demonstrates the activity of IL6ST decoy receptor EVs in reporter cell lines, muscle cell cultures, and in vivo muscle.
Section snippets
Cell culture
C2C12, RAW264.7, HEK293T (all from American Type Culture Collection, Manassas, VA, USA), immortalized human bone marrow derived mesenchymal stromal cells (MSCs) [53], HEK-Blue IL6 (InvivoGen, San Diego, CA, USA), and STAT3 Luciferase HeLa cells (Signosis, Santa Clara, CA, USA) were grown at 37 °C in 5% CO2. C2C12 cells were maintained in growth media (GM) Dulbecco's Modified Eagle's Media supplemented with 15% fetal bovine serum (FBS) and 1 × antibiotic-antimycotic (Ab-Am, all Life
The IL6 trans-signalling pathway as a therapeutic target for muscle pathology
Cellular responsiveness towards IL6 or IL6/IL6R complexes is dependent on the ratio between IL6R and IL6ST protein levels in cells [59]. We therefore first evaluated the cell surface expression of these receptors in C2C12 murine skeletal muscle cells (both myoblasts and differentiated myotubes). Flow cytometry data showed that 99.6% of the myoblasts expressed IL6ST while only 6.4% were double-positive for both IL6R and IL6ST (Fig. 1A). For myotubes, approximately 86.2% expressed IL6ST, with
Discussion
Chronic inflammation, in which there is prolonged release of pro-inflammatory cytokines, such as IL6, plays a critical role in the pathogenesis of many muscle pathologies and contributes to muscle atrophy and wasting [[42], [43], [44],74]. In this study, we identified the IL6 trans-signalling pathway as a therapeutic target for muscle-related pathologies, and specifically for DMD. Analysis of IL6ST and IL6R levels, STAT3 phosphorylation status, and changes in expression of IL6-regulated genes
CRediT authorship contribution statement
Mariana Conceição: Conceptualization, Methodology, Investigation, Formal analysis, Data curation, Visualization, Writing - original draft, Writing - review & editing, Funding acquisition. Laura Forcina: Investigation. Oscar P.B. Wiklander: Conceptualization, Methodology, Validation. Dhanu Gupta: Conceptualization, Methodology, Validation. Joel Z. Nordin: Conceptualization, Methodology, Validation. Besarte Vrellaku: Investigation. Graham McClorey: Investigation. Imre Mäger: Conceptualization.
Declaration of competing interest
The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: MJAW and SELA are founders of, and consultants for, Evox Therapeutics. IM, AG, and DG are consultants for Evox Therapeutics. MJAW, SELA, JZN, OW, AG, and DG are shareholders in Evox Therapeutics. PL is a founder, employee, and shareholder of Evox Therapeutics. The remaining authors declare no conflicts of interest.
Acknowledgements
This work was supported by grants from Oxford University Press John Fell Fund (awarded to TCR and IM), Evox Therapeutics (to MJAW and SELA), The University Challenge Seed Fund (UCSF) and MRC Confidence in Concept (to MJAW and SELA), Muscular Dystrophy Association (to MJAW and MC), Muscular Dystrophy UK (to MJAW, MC, and IM), Ricerca Finalizzata (to AM), Ateneo Sapienza (to AM), Swedish Medical Research Council (to SELA), and Swedish Strategic Foundation of Medical Research - SSF-IRC, FormulaEx
References (85)
- et al.
Reduction of myocardial infarct size by human mesenchymal stem cell conditioned medium
Stem Cell Res.
(2008) - et al.
Exosome secreted by MSC reduces myocardial ischemia/reperfusion injury
Stem Cell Res.
(2010) - et al.
Engineered exosomes as vehicles for biologically active proteins
Mol. Ther.
(2017) - et al.
Effects of exosome-mediated delivery of myostatin propeptide on functional recovery of mdx mice
Biomaterials
(2020) - et al.
The pro- and anti-inflammatory properties of the cytokine interleukin-6
Biochim. Biophys. Acta Mol. Cell Res.
(2011) - et al.
Interleukin-6: from basic biology to selective blockade of pro-inflammatory activities
Semin. Immunol.
(2014) - et al.
The physiopathologic interplay between stem cells and tissue niche in muscle regeneration and the role of IL-6 on muscle homeostasis and diseases
Cytokine Growth Factor Rev.
(2018) - et al.
Immune-mediated mechanisms potentially regulate the disease time-course of duchenne muscular dystrophy and provide targets for therapeutic intervention
Pharm. Manag. PM R
(2009) - et al.
Dystrophin: the protein product of the duchenne muscular dystrophy locus
Cell
(1987) The muscular dystrophies
Lancet
(2002)
Muscular dystrophies involving the dystrophin–glycoprotein complex: an overview of current mouse models
Curr. Opin. Genet. Dev.
Stakeholder cooperation to overcome challenges in orphan medicine development: the example of Duchenne muscular dystrophy
Lancet Neurol.
Phase IIa trial in Duchenne muscular dystrophy shows vamorolone is a first-in-class dissociative steroidal anti-inflammatory drug
Pharmacol. Res.
Muscle undergoes atrophy in association with increase of lysosomal cathepsin activity in interleukin-6 transgenic mouse
Biochem. Biophys. Res. Commun.
Stat3 activation links a C/EBPδ to myostatin pathway to stimulate loss of muscle mass
Cell Metabol.
Functional and morphological improvement of dystrophic muscle by interleukin 6 receptor blockade
EBioMedicine
Ultrafiltration with size-exclusion liquid chromatography for high yield isolation of extracellular vesicles preserving intact biophysical and functional properties
Nanomed. Nanotechnol. Biol. Med.
Interleukin-6 and soluble interleukin-6 receptor: direct stimulation of gp130 and hematopoiesis
Blood
Differential activation of acute phase response factor/stat3 and Stat 1 via the cytoplasmic domain of the interleukin 6 signal transducer gp130: II. Src homology SH2 domains define the specificity OF STAT factor activation
J. Biol. Chem.
Utrophin-dystrophin-Deficient mice as a model for duchenne muscular dystrophy
Cell
Age-related changes in replication of myogenic cells in mdx mice: quantitative autoradiographic studies
J. Neurol. Sci.
Mdx respiratory impairment following fibrosis of the diaphragm
Neuromuscul. Disord.
Shedding of endogenous interleukin-6 receptor (IL-6R) is governed by A Disintegrin and metalloproteinase (ADAM) proteases while a full-length IL-6R isoform localizes to circulating microvesicles
J. Biol. Chem.
The GCN4 basic region leucine zipper binds DNA as a dimer of uninterrupted alpha helices: crystal structure of the protein-DNA complex
Cell
ExoCarta: a web-based compendium of exosomal cargo
J. Mol. Biol.
Monoclonal antibodies and fusion proteins in medicine
J. Allergy Clin. Immunol.
Decoy receptors: a strategy to regulate inflammatory cytokines and chemokines
Trends Immunol.
Receptor-Fc fusion therapeutics, traps, and MIMETIBODY™ technology
Curr. Opin. Biotechnol.
Effective dystrophin restoration by a novel muscle-homing peptide–morpholino conjugate in dystrophin-deficient mdx mice
Mol. Ther.
Shedding light on the cell biology of extracellular vesicles
Nat. Rev. Mol. Cell Biol.
Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells
Nat. Cell Biol.
Delivery of siRNA to the mouse brain by systemic injection of targeted exosomes
Nat. Biotechnol.
Extracellular vesicles: biology and emerging therapeutic opportunities
Nat. Rev. Drug Discov.
Advances in therapeutic applications of extracellular vesicles
Sci. Transl. Med.
The biology, function, and biomedical applications of exosomes
Science
Therapeutic potential of multipotent mesenchymal stromal cells and their extracellular vesicles
Hum. Gene Ther.
Clinical potential of mesenchymal stem/stromal cell-derived extracellular vesicles
Stem Cell Invest.
Preclinical translation of exosomes derived from mesenchymal stem/stromal cells
Stem Cell.
Human embryonic mesenchymal stem cell-derived conditioned medium rescues kidney function in rats with established chronic kidney disease
PloS One
Exosome-mediated delivery of siRNA in vitro and in vivo
Nat. Protoc.
Extracellular vesicles as a platform for membrane-associated therapeutic protein delivery
J. Extracell. Vesicles
Decoy exosomes as a novel biologic reagent to antagonize inflammation
Int. J. Nanomed.
Cited by (24)
Recent advances of exosomes in soft tissue injuries in sports medicine: A critical review on biological and biomaterial applications
2023, Journal of Controlled ReleaseFibrinogen on extracellular vesicles derived from polyhexamethylene guanidine phosphate-exposed mice induces inflammatory effects via integrin β
2023, Ecotoxicology and Environmental SafetyThe hormetic and hermetic role of IL-6
2022, Ageing Research ReviewsCitation Excerpt :It has been also demonstrated that glycosylation is an important regulatory mechanism in terms of proteolysis and sIL-6R generation, whereas it is not dispensable for trafficking, stabilization, and signalling of the IL-6R (Riethmueller et al., 2017). Although in human, but not in mice, sIL-6R also derive from alternative mRNA splicing, the shedding of receptor is through to be responsible for the bulk of serum sIL-6R (Conceição et al., 2021; Dimitrov et al., 2006; Honda et al., 1992; Jones et al., 2001; Riethmueller et al., 2017; Schumacher et al., 2015). Contrasting data have been reported about the prevalent involvement of one or the other mechanism (Chalaris et al., 2010; Ferreira et al., 2013; Garbers et al., 2014; Reich et al., 2007; Schumacher et al., 2016).
Neutralization of SARS-CoV-2 pseudovirus using ACE2-engineered extracellular vesicles
2022, Acta Pharmaceutica Sinica BCitation Excerpt :Till July 2021, more than 200 clinical trials involving EVs-related treatments and diagnoses of different diseases have been registered at https://clinicaltrials.gov/. EVs bearing decoy receptors that competitively bind with the target receptors as a potential treatment has been proposed in skeletal muscle pathophysiology42. It was proposed that ACE2-expressing EVs that bind with SARS-Cov-2 could be a possible therapy43, and subsequently, it was demonstrated by in vitro tests44.
- 1
equal contribution.