Inflammation is thought to contribute to pulmonary vascular disease and pulmonary arterial hypertension (PAH) on the basis of several lines of epidemiological, biomarker, genetic, animal model, and clinical trial evidence. Epidemiological associations of PAH with systemic inflammatory or infectious disease are reflected in its nosology. PAH associated with connective tissue disease (CTD-PAH), including systemic sclerosis, systemic lupus erythematosus, and rheumatoid arthritis, and PAH associated with HIV infection or schistosomiasis are defined as group 1 PAH by the 6th World Symposium on Pulmonary Hypertension clinical classification. The mechanisms of these associations, however, have yet to be elucidated. In the current issue of Circulation, Yaki et al identify Regnase-1 as a central regulator of inflammation in PAH with important clinical implications.
In the present study, Yaki and colleagues present a set of clinical and translational observations implicating Regnase-1 as a central regulatory node of inflammation in PAH by virtue of its broad dampening effect on a variety of inflammatory cytokines and chemokines. Regnase-1, encoded by ZC3H12A, is a multifunctional protein with RNAse activity that binds specific stem loop motifs within the 3' untranslated region of several inflammatory cytokines, including Il6 and Il12p40, to promote their degradation and thereby prevent the development of autoimmunity.[2,3] Previously validated targets of Regnase-1 have included interleukin (IL)-6, IL-1β, and IL-12B; chemokines such as CXCL1, CXCL2 and CXCL3; and transcription factors, including NFKBID, NFKBIZ, MAFK, and ID1. These observations suggest that Regnase-1 serves as a critical regulatory hub of inflammatory transcriptional programs and immune homeostasis, maintaining the balance between adaptive and maladaptive inflammation. Hypothesizing a role of Regnase-1 in PAH, Yaki et al found diminished ZC3H12A mRNA abundance in peripheral blood mononuclear cells of subjects with idiopathic PAH/heritable PAH or CTD-PAH versus healthy volunteers, with low levels predicting diminished survival among all patients with PAH. Regnase-1 expression correlated with several markers of disease severity, including mean pulmonary arterial pressure and 6-minute walk distance, but only in patients with CTD-PAH and not in those with idiopathic PAH or heritable PAH. These clinical observations of Regnase-1 as a biomarker of CTD-PAH severity and its known role in regulating autoimmunity suggested that Regnase-1 may serve as a link between inflammation and CTD-PAH.
Moreover, these clinical observations were supported by targeted deletion studies of ZC3h12A in various combinations of myeloid and monocytic populations, including alveolar macrophages, using mice expressing CD11c-Cre and LysM-Cre alleles. Deletion of Regnase-1 in these lineages resulted in spontaneous obliterative and inflammatory pulmonary vascular changes in mice, with moderate pulmonary hypertension (PH) and right ventricular hypertrophy. Moreover, the heterozygous loss of ZC3h12A in these lineages sensitized mice to hypoxia-induced PH, albeit with less severity and without right ventricular hypertrophy. The authors then confirmed that selective depletion of Regnase-1 in alveolar macrophages triggers pulmonary vascular remodeling and the development of PH in mice. Transcriptomic analysis of both pulmonary artery and alveolar macrophages from CD11c-Cre+ Zc3h12afl/fl and control mice, ligand-target network, and luciferase reporter analyses yielded several new Regnase-1 targets beyond Il6, Il1b, and Il12b as being sensitive to Regnase-1 RNase activity in alveolar macrophages: Il1a, Il33, Tnfsf10, Pdgfa, and Pdgfb, nearly all of which have previously been described as circulating biomarkers of PAH. Last, the authors define the contributions of IL-6 and platelet-derived growth factor to the PH phenotype in the myelomonocytic Regnase-1–deficient mice by showing that treatment with either an IL-6 neutralizing antibody (MR16-1) or imatinib, a multikinase inhibitor of platelet-derived growth factor receptor β, partially attenuated the PH and vascular obliterative phenotype, whereas the IL1R antagonist anakinra did not have a significant impact. The partial effects of the IL-6– and platelet-derived growth factor–targeted strategies leave open the possibility that intercepting multiple cytokine axes downstream of Regnase-1 regulation might be more successful than more selective strategies not only in this novel PH model but perhaps in clinical PAH as well.
These novel findings may provide mechanistic understanding for previous clinical data on inflammation in PAH. Numerous biomarker studies have demonstrated that inflammatory cytokines are broadly elevated in patients with PAH. A study of 60 patients with idiopathic PAH or heritable PAH and 21 healthy volunteers revealed elevated serum IL-1β, -2, -4, -6, -8, -10, and -12bp70 and tumor necrosis factor-α, with levels of IL-6, -8, and -12p70 predicting survival. A study of CTD-PAH revealed elevated circulating IL-6 and C-X-C motif chemokines CXCL9 and CXCL13, whereas a recent machine learning proteomic approach identified a distinct cluster of patients with PAH (predominantly idiopathic PAH) with a circulating cytokine profile that included IL-4, -6, -8, platelet-derived growth factor-β, and CCL11 associated with an intermediate prognosis, and a cluster of patients with PAH expressing elevated Tnfsf10/TRAIL marked by a poor prognosis. A proinflammatory phenotype associated with heritable PAH is supported also by the finding that mice deficient in BMPR2, the most common locus affected in congenital PAH, demonstrate higher levels of lung and circulating IL-6 and IL-8 analog KC when exposed to lipopolysaccharide. The concept that IL-6 may be a critical driver of inflammation-mediated pulmonary vascular remodeling is supported by the observation that transgenic mice overexpressing IL-6 in the lungs through the Clara cell promoter CC10 developed perivascular inflammation and severe hypoxia-induced PH and vascular remodeling through proproliferative and antiapoptotic mechanisms. The recent success of the CANTOS trial (Canakinumab Anti-Inflammatory Thrombosis Outcome Study) in demonstrating that an anti-inflammatory therapy targeting IL-1β reduces atherosclerotic disease risk has rekindled interest in applying anti-inflammatory therapy more broadly to PAH and other vascular diseases. However, despite abundant epidemiological, biomarker, and animal model data supporting a role of inflammation in PAH, the goal of translating these insights into therapy has remained elusive, much as it remains unproved whether treating underlying autoimmunity is helpful for CTD-PAH. A randomized controlled trial testing bardoxolone methyl, a small-molecule NRF2 activator and inhibitor of nuclear factor-κB signaling, in a diverse set of patients with group 1 PAH and group 3 and 5 PH (LARIAT [Bardoxolone Methyl Evaluation in Patients with Pulmonary Hypertension (PH)], NCT02036970) failed to achieve its primary end point of improved 6-minute walk distance at 16 weeks. An open-label pilot study of the IL-1 antagonist anakinra in PAH (NCT01479010) did not meet its primary end point of improving peak oxygen consumption and ventilatory efficiency by cardiopulmonary exercise testing or improvements in NT-proBNP (N-terminal pro-B-type natriuretic peptide) or right ventricular function by echocardiography, despite a reduction in high-sensitivity C-reactive protein and a strong trend toward decreased IL-6 levels. A small open-label clinical study using the IL-6 receptor antagonist tocilizumab in PAH (TRANSFORM-UK [A Therapeutic Open Label Study of Tocilizumab in the Treatment of Pulmonary Arterial Hypertension], NCT02676947) did not meet its primary end point of improved pulmonary vascular resistance at 6 months in the overall study population but demonstrated a possible efficacy signal among enrollees with CTD-PAH as part of a prespecified subanalysis. These PAH clinical studies demonstrate dysregulation of almost all of the cytokines and chemokines regulated by Regnase-1 and suggest that IL-6 is a common denominator, yet they leave unanswered whether any single cytokine axis constitutes a sufficient therapeutic strategy. The broad immune dysregulation in PAH raises questions of whether there might be a hierarchy or a central regulator of these diverse inflammatory contributions to PAH that could be targeted to improve the success of anti-inflammatory strategies in PAH. The finding of Regnase as a central PAH node may just be that factor, having the potential to integrate many piecemeal clinical observations in PAH under the umbrella of diminished Regnase-1 function.
The present study opens up several possible avenues for future research. Regnase-1 is known to be stimulated by the chemokine monocyte chemoattractant protein-1, tumor necrosis factor-α, lipopolysaccharide, IL-1β, microbial infections, and various chemical and mechanical stimuli, suggesting an important immune feedback inhibition function. Less is known, however, about factors that may suppress Regnase-1 activity or diminish its expression in CTD-PAH, questions relevant for restoring its function. The authors previously showed that IκB kinases phosphorylate Regnase-1 in response to toll like receptor ligands or IL-1β stimulation, resulting in rapid degradation of Regnase-1. In response to T cell receptor stimulation, paracaspase Malt1 cleaves Regnase-1 at R111 to inactivate Regnase-1. A recent study from this group documented an interaction of 14–3-3 proteins with Regnase-1 induced by inflammation, resulting in cytoplasmic sequestration of Regnase-1 to limit its degradation of inflammatory mRNAs. The fact that transgenic mice overexpressing IL-6 had only mild PH under normoxia whereas loss of Regnase-1 in alveolar macrophages led to spontaneous obliterative remodeling and PH seems consistent with much broader anti-inflammatory effects of Regnase-1 while implicating alveolar monocytes as reservoirs for these effects. The present studies build on a growing understanding of Regnase-1 as an essential inflammation-sensing and dampening hub, offering compelling biomarker and experimental biological evidence of its role in regulating pulmonary vascular disease. Revisiting parallel conceptual advances in pulmonary and systemic vascular disease indicates that the potent phenotypes associated with Regnase-1 suggest that its therapeutic modulation could affect systemic vascular conditions, including atherosclerosis. Augmenting or restoring Regnase-1 function might be easier to accomplish therapeutically with a deeper understanding of its upstream regulation; delineating the function of this critical hub and its pleiotropic downstream effects and target cells will help answer persistent questions about the link between inflammation and PAH. A more prosaic implication of the present work may be that whether we act on central regulators such as Regnase-1 or its downstream mediators, anti-inflammatory strategies for PAH may need to target a plurality of cytokine and chemokine axes to achieve efficacy.
Circulation. 2022;146(13):1023-1025. © 2022 American Heart Association, Inc.