Restoring balance with recombinant interleukin-37: hope for halting chronic inflammation in calcific aortic valve disease

This editorial refers to ‘Interleukin-37 attenuates aortic valve lesions by inhibiting N6-methyladenosine-mediated interleukin-1 receptor-associated kinase M degradation’, by G. Xian et al., https://doi.org/10.1093/cvr/cvaf012. Once considered a degenerative process, calcific aortic valve disease (CAVD) is now recognized as a condition of chronic inflammation. While healthy aortic valve leaflets contain few immune cells, infiltrates of predominantly macrophages and T lymphocytes are observed in both the early and late stages of CAVD.1 A unique population of pluripotent mesenchyme-derived aortic valvular interstitial cells (AVICs) differentiate into myofibroblast-like and osteoblast-like phenotypes under the influence of pro-inflammatory mediators and are the primary effectors of extracellular matrix remodelling and tissue calcification in CAVD. This process is thought to begin with an initial injury to the aortic valve such as transient infection, deposition of oxidized lipids, or the altered haemodynamic stress of congenital bicuspid valves and is then perpetuated by chronic inflammation.2 In this edition of Cardiovascular Research, Xian et al.3 tackle one of the crucial, unanswered questions in CAVD: what mechanisms perpetuate the chronic inflammation that drives CAVD? The authors isolated and cultured human AVICs from heart transplant recipients without valve disease and from patients with CAVD undergoing valve replacement. Toll-like receptor (TLR)-2, -3, and -4 are known to be expressed by healthy AVICs and mediate interleukin (IL)-6 and IL-8 secretion and osteogenic differentiation in response to lipopolysaccharide (LPS) stimulation.4 The authors performed LPS stimulation of isolated AVICs, which resulted in increased nuclear factor kappa-B (NF-κB) phosphorylation and up-regulation of osteoblastic markers, with concurrent increased expression of IL-1 receptor-associated kinase M (IRAK-M). Unlike other IRAK family members that propagate TLR signalling, IRAK-M acts as a negative regulator of TLR signalling, inhibiting NF-κB activation and downstream pro-inflammatory cytokine production. The authors further tested the ability of IRAK-M to inhibit CAVD disease processes by performing knockdown and adenoviral vector IRAK-M overexpression studies to inhibit osteogenic AVIC differentiation and calcification and by demonstrating aortic valve thickening and calcification in IRAK-M−/− mice on a high-fat diet. These findings illustrate a pathway of AVIC inflammation in response to insults such as LPS, pathogen-associated molecular patterns (PAMPs), or disease-associated molecular patterns (DAMPs), with a TLR4:IRAK-M feedback axis that mediates timely resolution of inflammation (Figure 1). Potential roles of IRAK-M as a counter-regulator of TLR-4-mediated inflammation and rIL-37 as novel therapeutic approach in CAVD. Diverse initiating insults result in stimulation of TLR-4 by LPS, PAMPs, or DAMPs, with subsequent activation of the MyD88-IRAK complex and nuclear translocation of NF-κB, resulting in increased IL-6 and IL-8 production and downstream osteogenic transformation of AVICs. Resolution of inflammation occurs when the NF-κB p65 subunits bind to the IRAK-M promoter, resulting in increased IRAK-M expression, which in turn inhibits IRAK activity, providing a negative feedback mechanism on pro-inflammatory TLR-4 signalling. Dysregulation of the TLR-4:IRAK-M axis occurs after prolonged TLR-4 stimulation, via P65 subunit binding to the METTL3 promoter, resulting in increased m6A modification and inactivation of IRAK-M with subsequent unchecked pro-inflammatory signalling. Potential therapy with rIL-37 inhibits m6A-mediated IRAK-M degradation, thereby restoring the anti-inflammatory TLR-4/IRAK-M feedback loop (figure created with BioRender.com). Post-transcriptional modifications, such as RNA methylation, play a critical role in regulating gene expression by modulating RNA stability, splicing, translation, and degradation. After observing that the counter-regulatory IRAK-M expression in LPS-stimulated AVICs was attenuated after 48 h, the authors went on to identify N6-methyladenosine (m6A) modification of IRAK-M as a potential disruptor of the TLR-4:IRAK-M feedback loop. In the pro-inflammatory context, m6A modifications are key modulators of immune signalling pathways, including those mediated by TLRs. Using the LPS-stimulated AVIC model, the authors demonstrated that as a by-product of TLR-4 activation, methyltransferase-like 3 (METTL3) activity and subsequent m6A modification of IRAK-M mRNA were increased. This epigenetic inactivation of IRAK-M resulted in the interruption of the protective counter-regulatory TLR-4:IRAK-M feedback axis, introducing a pathway of sustained inflammation, which could further promote the pathological remodelling observed in CAVD. IL-37 is a member of the IL-1 cytokine family produced by innate immune cells, regulatory T cells, and endo/epithelial cells in pro-inflammatory conditions. When bound to IL-1R8 and IL-18Rα on the surface of macrophages, T cells, NK cells, or epithelial cells, IL-37 suppresses NF-κB and other pro-inflammatory pathways, making recombinant IL-37 (rIL-37) an attractive potential therapy in autoimmune diseases. IL-37 has been previously shown to inhibit NF-κB activation and osteogenic differentiation in human AVICs, and IL-37 expression is reduced in human CAVD leaflets.5 In this investigation, the authors established that the anti-osteogenic effect of rIL-37 in both isolated human AVICs and transgenic mice is mediated by inhibition of m6A-mediated IRAK-M degradation, resulting in increased IRAK-M expression and thereby restoring the anti-inflammatory TLR-4:IRAK-M feedback axis. Despite being a common disorder easily diagnosed in its early stages, no effective drugs are available to slow or halt the progression of CAVD. As the incidence of CAVD in many countries is projected to double by 2040 and triple by 2060, the search for therapeutic alternatives to costly and invasive valve replacement is an area of research priority.6 Xian et al.3 have not only expanded existing knowledge of TLR signalling in AVICs but also have crucially identified uncoupling of the TLR-4:IRAK-M negative feedback axis by m6A modification as a potential mechanism for unchecked chronic inflammation and proposed rIL-37 as a potential medical therapy to restore protective IRAK-M activity. rIL-37 has shown promise as a potential anti-inflammatory drug in several disease models; however, efficacy as well as safety of rIL-37 therapy in humans remains untested. Several other factors contribute to inflammation in CAVD, including haemodynamic stress, which in a vicious cycle activates both circulating cells such as monocytes and neutrophils and dissociates proteins such as pentameric C-reactive protein into highly pro-inflammatory monomeric C-reactive protein, thereby amplifying the inflammatory response and calcification observed in aortic valve stenosis.7–9 To date, however, no medical treatment has been developed or successfully tested that can reverse or prevent further progression of CAVD. Therefore, the potential therapeutic approach based on rIL-37 seems highly attractive and worthwhile to be further tested. One of the major difficulties in CAVD research is the lack of pre-clinical models that reflect human CAVD pathophysiology. As AVICs are highly sensitive to substrate stiffness and mechanical stress, organ-on-a-chip models may provide an alternative opportunity to identify and test therapeutic targets by recreating the intricate mechanical, cellular, and molecular environment of the aortic valve within a controlled micro-scale system.10 Incorporating organ-on-a-chip technology may provide an important link between in vitro and in vivo studies of inflammation and calcification pathways in CAVD, aiding in the identification of novel pharmacological therapies to fill the significant unmet need for medical therapies in CAVD.