Decoding the link between inflammation and pulmonary arterial hypertension

Inflammation is thought to contribute to pulmonary vascular disease and pulmonary arterial hypertension (PAH), based on several lines of epidemiologic, biomarker, genetic, animal model and clinical trial evidence. Epidemiologic 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, as well as PAH associated with human immunodeficiency virus infection or schistosomiasis are defined as Group 1 PAH by the 6^th World Symposium on Pulmonary Hypertension clinical classification. The mechanisms of these associations, however, have yet to be elucidated. In the current issue of Circulation, Yaku et al. identify Regnase-1 as a central regulator of inflammation in PAH with important clinical implications.

In the current study Yaku 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.¹ Regnase-1, encoded by ZC3H12A, is a multifunctional protein with RNAse activity that binds specific stem loop (SL) motifs within the 3’ untranslated region (UTR) 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 IL-6, IL1-β, and IL-12B, chemokines such as CXCL1, CXCL2 and CXCL3, and transcription factors including NFKBID, NFKBIZ, MAFK and ID1.³ These observations suggest 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, Yaku et al. found diminished ZC3H12A mRNA abundance in peripheral blood mononuclear cells of subjects with IPAH/HPAH or CTD-PAH vs. healthy volunteers, with low levels predicting diminished survival among all PAH patients.¹ Regnase-1 expression correlated with several markers of disease severity including mean pulmonary arterial pressure (mPAP) and 6-min walk distance (6MWD), but only in CTD-PAH and not idiopathic (IPAH) or heritable (HPAH) PAH patients. 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 PAH associated with connective tissue disease.

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 and RV hypertrophy. Moreover, the heterozygous loss of ZC3h12A in these lineages sensitized mice to hypoxia-induced pulmonary hypertension (PH), albeit with less severity and without RV 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⁺Zc3h12a^fl/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 been previously described as circulating biomarkers of PAH. Finally, the authors define the contributions of IL-6 and PDGF 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 multi-kinase inhibitor of PDGFRβ, both partially attenuated the PH and vascular obliterative phenotype, while IL1R antagonist anakinra did not exert a significant impact. The partial effects of the IL-6- and PDGF-targeted strategies leaves 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 clinical PAH as well.

These novel findings may provide mechanistic understanding for prior clinical data regarding inflammation in PAH. Numerous biomarker studies have demonstrated that inflammatory cytokines are broadly elevated in PAH patients. A study of 60 IPAH or HPAH patients and 21 healthy volunteers revealed elevated serum interleukin (IL)-1β, −2, −4, −6, −8, −10, and −12bp70, and tumor necrosis factor (TNF)-α, with levels of IL-6, −8, and −12p70 predicting survival.⁴ A study of CTD-PAH revealed elevated circulating IL-6 and C-X-C motif chemokines CXCL9 and CXCL13,⁵ while a recent machine learning proteomic approach identified a distinct cluster of PAH patients (predominantly IPAH) with a circulating cytokine profile that included IL-4, −6, −8, PDGFβ, and CCL11 associated with an intermediate prognosis, and a cluster of PAH patients expressing elevated Tnfsf10/TRAIL marked by a poor prognosis.⁶ A pro-inflammatory phenotype associated with HPAH 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 (LPS).⁷ 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 via the Clara cell promoter CC10 developed perivascular inflammation and severe hypoxia-induced pulmonary hypertension and vascular remodeling via pro-proliferative and anti-apoptotic mechanisms.⁸ The recent success of the CANTOS trial in demonstrating that an anti-inflammatory therapy targeting IL-1β reduces atherosclerotic disease risk⁹ has rekindled interest in applying anti-inflammatory therapy more broadly to PAH and other vascular diseases. Yet despite abundant epidemiologic, 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 unproven if treating underlying auto-immunity is helpful for CTD-PAH. A randomized controlled trial testing bardoxolone methyl, a small molecule NRF2 activator and inhibitor of NFκB signaling, in a diverse set of Group 1 PAH and Group 3 and 5 PH patients (LARIAT, NCT02036970) failed to achieve its primary endpoint of improved 6-minute walk distance at 16 weeks. An open-label pilot study of IL-1 antagonist anakinra in PAH (NCT01479010) did not meet its primary endpoint of improving peak oxygen consumption and ventilatory efficiency by cardiopulmonary exercise testing, nor improvements in NT-pro-BNP or right ventricular (RV) function by echocardiography, despite a reduction in high sensitivity C-reactive protein and strong trend towards decreased IL-6 levels.¹⁰ A small open-label clinical study using IL-6 receptor antagonist tocilizumab in PAH (TRANSFORM-UK, NCT02676947) did not meet its primary endpoint of improved pulmonary vascular resistance (PVR) at 6 months in the overall study population, but demonstrated a possible efficacy signal among enrollees with CTD-PAH as part of a pre-specified sub-analysis.¹¹ These PAH clinical studies demonstrate dysregulation of almost all of the cytokines and chemokines regulated by Regnase-1, and suggest IL-6 is a common denominator, yet 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 current study opens up several possible avenues for future research. Regnase-1 is known to be stimulated by chemokine MCP-1, TNFα, LPS, and IL-1β, microbial infections, and various chemical and mechanical stimuli,¹² 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 (IKKs) phosphorylate Regnase-1 in response to TLR ligands or IL-1β stimulation, resulting in rapid degradation of Regnase-1¹³. In response to TCR stimulation, paracaspase Malt1 cleaves Regnase-1 at R111 to inactivate Regnase-1.¹⁴ 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 mRNAs¹⁵. The fact that transgenic mice overexpressing IL6 had only mild PH under normoxia,⁸ 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 current studies build upon a growing understanding of Regnase-1 as an essential inflammation-sensing and dampening hub, offering compelling biomarker and experimental biology evidence of its role in regulating pulmonary vascular disease. Revisiting parallel conceptual advances in pulmonary and systemic vascular disease, the potent phenotypes associated with Regnase-1 suggest its therapeutic modulation could impact 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 current work may be that whether we act on central regulators like 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.