Liver fibrosis (scarring) and its end stage, cirrhosis, are a major cause of morbidity and mortality worldwide, but there are no licensed antifibrotic therapies and the availability of liver transplantation is limited. In recent years, there has been increasing interest in the possible role of the naturally occurring peptide hormone human relaxin-2 (RLX) as a therapeutic agent. Now, writing in Nature Nanotechnology, Hu et al. show that hepatic targeting of plasmid DNA encoding RLX (pRLX), represents a potential treatment strategy for liver fibrosis¹.

RLX, acting through its cognate G protein-coupled receptor relaxin family peptide receptor 1 (RXFP1), is vasodilatory, promotes tissue remodelling and is antifibrotic across most major organs. Indeed, mice lacking RLX develop spontaneous fibrotic diseases with ageing, which are completely reversed by treatment with recombinant RLX². In rodent models, RLX treatment attenuated the fibrogenic properties of activated hepatic stellate cells (HSC; the major scar-producing cells in liver fibrosis)³, reduced histological features of non-alcoholic fatty liver disease⁴ and reversed established fibrosis by inhibiting collagen production while simultaneously promoting its degradation by upregulating expression of matrix metalloproteinases⁵. However, RLX in its native form poses challenges for therapeutic development, in particular production complexity and a very short half-life with intravenous dosing (necessitating continuous infusion). Accordingly, several alternative approaches have recently been investigated such as discovery of a small molecule RXFP1 agonist compound⁶, adenovirus-mediated RLX delivery⁷ and RLX conjugated to PEGylated superparamagnetic iron oxide nanoparticles⁸. These divergent strategies were all shown to promote antifibrotic effects in vivo. Hu et al. have used lipid nanoparticles conjugated with aminoethyl anisamide, a potent ligand for the sigma-1 receptor that is highly expressed on activated HSC, to enable hepatic targeting of pRLX in mice, resulting in a specific increase in tissue RLX levels and a striking inhibition of fibrosis following chronic liver injury¹.

Importantly, in addition to confirming the antifibrotic efficacy of pRLX administration in pathologically-distinct mouse models of liver fibrosis (carbon tetrachloride toxicity and two dietary models of non-alcoholic steatohepatitis (NASH)), Hu et al. have provided critical mechanistic insights into the possible mode of action of RLX-based therapies. Notably, they identified potent immunomodulatory effects of pRLX administration, resulting in the expansion of hepatic restorative Ly6C^lo macrophages and a consequent reduction in fibrosis¹. The finding of macrophages as a ‘central hub’ for RLX-mediated effects on liver fibrosis contrasts sharply with previous studies that reported direct modulation of RXFP1-expressing activated HSC^2,3,5,7. Macrophages and monocytes, their precursors in the circulation, have long been known to be key regulators of hepatic fibrosis and represent an attractive pharmacological target. Indeed, inhibition of monocyte recruitment using the CCR2/CCR5 dual antagonist cenicriviroc is currently being investigated in Phase 3 clinical trials for patients with NASH-induced liver fibrosis. However, liver monocyte-derived macrophages are highly heterogeneous and plastic cells, capable of promoting both fibrogenesis and fibrosis resolution⁹. Therefore, global inhibition of monocyte recruitment in patients with established liver fibrosis may in fact hinder matrix remodelling. Hence, a therapeutic strategy aimed at modulating macrophage phenotype in situ to inhibit pro-fibrotic properties and enhance restorative functions is extremely appealing, although has so far remained elusive. The data presented by Hu et al. showed that pRLX gene therapy can act via RXFP1 on fibrogenic macrophages, triggering a phenotypic switch and promoting the scar-resolving properties. Whilst the potential for RLX to modulate macrophage phenotype in the context of fibrosis has been described previously¹⁰, the identification of Nur77 as a key transcriptional driver of pro-resolution macrophage properties and the demonstration that restorative macrophages promote the deactivation of activated HSC via the release of exosomes containing microRNA miR-30a-5p, provide important new insights for the field (Fig. 1)¹.

In response to liver injury, circulating inflammatory monocytes are recruited into the liver and differentiate into pro-fibrotic Ly6C^hi macrophages. These cells produce a number of soluble mediators that promote hepatic stellate cell (HSC) activation, proliferation and survival, resulting in deposition of scar proteins (extracellular matrix (ECM)) and production of tissue inhibitor of matrix metalloproteinases 1 (TIMP1), which protects ECM from degradation. Pro-fibrotic Ly6C^hi macrophages can then undergo a phenotypic switch in situ to form pro-resolution Ly6C^lo macrophages that promote fibrosis regression via a number of mechanisms. Following HSC-targeted relaxin (RLX) gene therapy, RLX acts via the RXFP1 receptor on pro-fibrotic Ly6C^hi macrophages, promoting NUR77 expression and enhancing the macrophage phenotypic switch. Pro-resolution Ly6C^lo macrophages produce exosomes containing miR-30a-5p, which signal to activated HSC resulting in their deactivation. Deactivated HSCs demonstrate increased matrix metalloproteinase (MMP) expression and reduced TIMP1 expression, which favours ECM degradation and fibrosis resolution. CCL2, C–C motif chemokine ligand 2; CCR2, C–C chemokine receptor type 2; TRAIL, tumor necrosis factor-related apoptosis-inducing ligand. New findings from Hu et al.¹ are highlighted in red. Figure adapted with permission from ref. ¹², Springer Nature Ltd.

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One of the challenges in translating pre-clinical data from rodent models into tractable treatments for patients is the lack of comparative analyses in relevant human cells and models. Therefore, the demonstration that pRLX treatment induced a similar modulation of primary human monocyte-derived macrophages is pertinent. However, recent single-cell RNA-sequencing studies have highlighted the heterogeneity of human hepatic macrophages in health and disease, with specific subpopulations responsible for pro-fibrogenic effects¹¹. Hence, more detailed interrogation of human RXFP1 and NUR77 expression patterns, human liver macrophage exosome production and effects on human HSC activation across a spectrum of liver disease etiologies, is essential to better define the potential utility of targeted pRLX gene therapy as an antifibrotic strategy in patients. Furthermore, monocyte-derived macrophages also have a key pathogenic role in fibrosis in organs such as the lung, heart and kidney, so the applicability of pRLX treatment in other fibrosis contexts also merits more detailed study.

This article is also of broader interest as there are multiple other potential development opportunities, beyond fibrosis, for RLX-based therapeutics including acute kidney injury/hepatorenal syndrome, musculoskeletal conditions (for example, adhesive capsulitis), ischemia-reperfusion injury (for example, kidney transplantation, coronary angioplasty) and cardiovascular diseases (for example, pulmonary hypertension, heart failure with preserved ejection fraction). Plausibly, the nanomedicine described by Hu et al. could be engineered to incorporate targeting moieties selective for different cell types. The key advantage of tissue-specific RLX expression is that it mitigates potential off-target effects of systemic RLX in other RXFP1 expressing tissues. Obviously, questions around the durability of effect, scalability and safety of pRLX gene therapy for liver fibrosis will need to be addressed before clinical application can be contemplated. Nevertheless, this study provides new impetus to exploit the intrinsic tissue remodelling properties of RLX that have tantalized researchers since its discovery by Frederick Hisaw in 1926.