Therapeutic perspectives on PDE4B inhibition in adipose tissue dysfunction and chronic liver injury.

INTRODUCTION: Chronic liver disease (CLD) is a complex disease associated with profound dysfunction. Despite an incredible burden, the first and only pharmacotherapy for metabolic-associated steatohepatitis was only approved in March of this year, indicating a gap in the translation of preclinical studies. There is a body of preclinical work on the application of phosphodiesterase 4 inhibitors in CLD, none of these molecules have been successfully translated into clinical use. AREAS COVERED: To design therapies to combat CLD, it is essential to consider the dysregulation of other tissues that contribute to its development and progression. As such, proper therapies must combat this throughout the body rather than focusing only on the liver. To detail this, literature characterizing the pathogenesis of CLD was pulled from PubMed, with a particular focus placed on the role of PDE4 in inflammation and metabolism. Then, the focus is shifted to detailing the available information on existing PDE4 inhibitors. EXPERT OPINION: This review gives a brief overview of some of the pathologies of organ systems that are distinct from the liver but contribute to disease progression. The demonstrated efficacy of PDE4 inhibitors in other human inflammatory diseases should earn them further examination for the treatment of CLD.

Reduction in Obesity-Related Hepatic Fibrosis by SR1664.

Peroxisome-proliferator-activated receptor gamma (PPARγ) is a transcription factor with adipogenic, insulin-sensitizing, and antifibrotic properties. Strong PPARγ activators, such as the thiazolidinediones, can induce unwanted effects such as edema, weight gain, and bone loss, and therefore selective modulators of PPARγ are in development. We previously reported that one selective PPARγ modulator, SR1664, reduced toxin-induced hepatic fibrosis and the activation of hepatic stellate cells (HSCs), the main collagen-producing liver cell in fibrosis. In this study, we used a high fat and high carbohydrate (HFHC) model of hepatic steatosis and fibrosis to determine the effect of SR1664. Mice were placed on a standard chow or HFHC diet for 16 weeks, with SR1664 or control treatment for the final 4 weeks. SR1664 did not alter weight gain or fasting insulin or glucose levels. The size of lipid droplets in the HFHC group was reduced by SR1664, but there was no effect on total liver triglyceride levels. The degree of fibrosis was significantly reduced by SR1664 in mice on the HFHC diet, and this was accompanied by a decrease in activated HSC. In summary, SR1664 improved insulin sensitivity and reduced fibrosis in the HFHC diet, suggesting selective PPARγ modulation is effective in obesity-related liver fibrosis.

Alcohol-induced tubulin post-translational modifications directly alter hepatic protein trafficking.

BACKGROUND: Chronic ethanol exposure leads to enhanced protein acetylation and acetaldehyde adduction. Of the multitude of proteins that are modified on ethanol administration, tubulin is among the best studied. However, an open question is whether these modifications are observed in patient samples. Both modifications have also been implicated in promoting alcohol-induced defects in protein trafficking, but whether they do so directly is also unanswered. METHODS AND RESULTS: We first confirmed that tubulin was hyperacetylated and acetaldehyde-adducted in the livers from ethanol-exposed individuals to a similar extent as observed in the livers from ethanol-fed animals and hepatic cells. Livers from individuals with nonalcohol-associated fatty liver showed modest increases in tubulin acetylation, whereas nonalcohol-associated fibrotic human and mouse livers showed virtually no tubulin modifications. We also asked whether tubulin acetylation or acetaldehyde adduction can directly explain the known alcohol-induced defects in protein trafficking. Acetylation was induced by overexpressing the α-tubulin-specific acetyltransferase, αTAT1, whereas adduction was induced by directly adding acetaldehyde to cells. Both αTAT1 overexpression and acetaldehyde treatment significantly impaired plus-end (secretion) and minus-end (transcytosis)-directed microtubule-dependent trafficking and clathrin-mediated endocytosis. Each modification led to similar levels of impairment as observed in ethanol-treated cells. The levels of impairment by either modification showed no dose dependence or no additive effects suggesting that substoichiometric tubulin modifications lead to altered protein trafficking and that lysines are not selectively modified. CONCLUSIONS: These results not only confirm that enhanced tubulin acetylation is observed in human livers but that it is most relevant to alcohol-induced injury. Because these tubulin modifications are associated with altered protein trafficking that alters proper hepatic function, we propose that changing the cellular acetylation levels or scavenging free aldehydes are feasible strategies for treating alcohol-associated liver disease.

Polymeric Chloroquine as an Effective Antimigration Agent in the Treatment of Pancreatic Cancer.

Hydroxychloroquine (HCQ) has been the subject of multiple recent preclinical and clinical studies for its beneficial use in the combination treatments of different types of cancers. Polymeric HCQ (PCQ), a macromolecular multivalent version of HCQ, has been shown to be effective in various cancer models both in vitro and in vivo as an inhibitor of cancer cell migration and experimental lung metastasis. Here, we present detailed in vitro studies that show that low concentrations of PCQ can efficiently inhibit cancer cell migration and colony formation orders of magnitude more effectively compared to HCQ. After intraperitoneal administration of PCQ in vivo, high levels of tumor accumulation and penetration are observed, combined with strong antimetastatic activity in an orthotopic pancreatic cancer model. These studies support the idea that PCQ may be effectively used at low doses as an adjuvant in the therapy of pancreatic cancer. In conjunction with previously published literature, these studies further undergird the potential of PCQ as an anticancer agent.

Dually Active Polycation/miRNA Nanoparticles for the Treatment of Fibrosis in Alcohol-Associated Liver Disease.

Alcohol-associated liver disease (AALD) is a major cause of liver disorders worldwide. Current treatment options are limited, especially for AALD-associated fibrosis. Promising approaches include RNA interference for miR-155 overexpression in Kupffer cells (KCs), as well as the use of CXCR4 antagonists that inhibit the activation of hepatic stellate cells (HSCs) through the CXCL12/CXCR4 axis. The development of dual-functioning nanoparticles for the effective delivery of antifibrotic RNA together with a CXCR4 inhibitor thus promises to improve the treatment of AALD fibrosis. In this study, cholesterol-modified polymeric CXCR4 inhibitor (Chol-PCX) was synthesized and used to encapsulate anti-miR-155 or non-coding (NC) miRNA in the form of Chol-PCX/miRNA nanoparticles. The results indicate that the nanoparticles induce a significant miR-155 silencing effect both in vitro and in vivo. Treatment with the Chol-PCX/anti-miR-155 particles in a model of moderate alcohol consumption with secondary liver insult resulted in a significant reduction in aminotransferase enzymes as well as collagen content in the liver parenchyma. Overall, our data support the use of Chol-PCX as a carrier for anti-miR-155 for the combined therapeutic inhibition of CXCR4 and miR-155 expression as a way to improve fibrotic damage in the liver.

Relaxin as an anti-fibrotic treatment: Perspectives, challenges and future directions.

Fibrosis refers to the scarring and hardening of tissues, which results from a failed immune system-coordinated wound healing response to chronic organ injury and which manifests from the aberrant accumulation of various extracellular matrix components (ECM), primarily collagen. Despite being a hallmark of prolonged tissue damage and related dysfunction, and commonly associated with high morbidity and mortality, there are currently no effective cures for its regression. An emerging therapy that meets several criteria of an effective anti-fibrotic treatment, is the recombinant drug-based form of the human hormone, relaxin (also referred to as serelaxin, which is bioactive in several other species). This review outlines the broad anti-fibrotic and related organ-protective roles of relaxin, mainly from studies conducted in preclinical models of ageing and fibrotic disease, including its ability to ameliorate several aspects of fibrosis progression and maturation, from immune cell infiltration, pro-inflammatory and pro-fibrotic cytokine secretion, oxidative stress, organ hypertrophy, cell apoptosis, myofibroblast differentiation and ECM production, to its ability to facilitate established ECM degradation. Studies that have compared and/or combined these therapeutic effects of relaxin with current standard of care medication have also been discussed, along with the main challenges that have hindered the translation of the anti-fibrotic efficacy of relaxin to the clinic. The review then outlines the future directions as to where scientists and several pharmaceutical companies that have recognized the therapeutic potential of relaxin are working towards, to progress its development as a treatment for human patients suffering from various fibrotic diseases.

A Selective PPARγ Modulator Reduces Hepatic Fibrosis.

Hepatic fibrosis is the accumulation of excess collagen as a result of chronic liver injury. If left unabated, hepatic fibrosis can lead to the disruption of the liver architecture, portal hypertension, and increased risk of progression to cirrhosis and hepatocellular carcinoma. The thiazolidinedione class of antidiabetic drugs, through their target peroxisome proliferator-activated receptor γ (PPARγ), have protective effects against liver fibrosis, and can inhibit the profibrotic activity of hepatic stellate cells, the major collagen-producing liver cells. However, these drugs have been ineffective in the treatment of established fibrosis, possibly due to side effects such as increased weight and adiposity. Recently, selective PPARγ modulators that lack these side effects have been identified, but their role in treating fibrosis has not been studied. In this study, we tested the effectiveness of one of these selective modulators, SR1664, in the mouse carbon tetrachloride model of established hepatic fibrosis. Treatment with SR1664 reduced the total and type 1 collagen content without increasing body weight. The abundance of activated hepatic stellate cells was also significantly decreased. Finally, SR1664 inhibited the profibrotic phenotype of hepatic stellate cells. In summary, a selective PPARγ modulator was effective in the reduction of established hepatic fibrosis and the activated phenotype of hepatic stellate cells. This may represent a new treatment approach for hepatic fibrosis.

Evidence for a Spatially Modulated Superfluid Phase of

In superfluid ^{3}He-B confined in a slab geometry, domain walls between regions of different order parameter orientation are predicted to be energetically stable. Formation of the spatially modulated superfluid stripe phase has been proposed. We confined ^{3}He in a 1.1 µm high microfluidic cavity and cooled it into the B phase at low pressure, where the stripe phase is predicted. We measured the surface-induced order parameter distortion with NMR, sensitive to the formation of domains. The results rule out the stripe phase, but are consistent with 2D modulated superfluid order.

Relaxin and fibrosis: Emerging targets, challenges, and future directions.

The peptide hormone relaxin is well-known for its anti-fibrotic actions in several organs, particularly from numerous studies conducted in animals. Acting through its cognate G protein-coupled receptor, relaxin family peptide receptor 1 (RXFP1), serelaxin (recombinant human relaxin) has been shown to consistently inhibit the excessive extracellular matrix production (fibrosis) that results from the aberrant wound-healing response to tissue injury and/or chronic inflammation, and at multiple levels. Furthermore, it can reduce established scarring by promoting the degradation of aberrant extracellular matrix components. Following on from the review that describes the mechanisms and signaling pathways associated with the extracellular matrix remodeling effects of serelaxin (Ng et al., 2019), this review focuses on newly identified tissue targets of serelaxin therapy in fibrosis, and the limitations associated with (se)relaxin research.

Relaxin and extracellular matrix remodeling: Mechanisms and signaling pathways.

Fibrosis is associated with accumulation of excess fibrillar collagen, leading to tissue dysfunction. Numerous processes, including inflammation, myofibroblast activation, and endothelial-to-mesenchymal transition, play a role in the establishment and progression of fibrosis. Relaxin is a peptide hormone with well-known antifibrotic properties that result from its action on numerous cellular targets to reduce fibrosis. Relaxin activates multiple signal transduction pathways as a mechanism to suppress inflammation and myofibroblast activation in fibrosis. In this review, the general mechanisms underlying fibrotic diseases are described, along with the current state of knowledge regarding cellular targets of relaxin. Finally, an overview is presented summarizing the signaling pathways activated by relaxin and other relaxin family peptide receptor agonists to suppress fibrosis.

Targeting the Relaxin Pathway for Liver Disease Treatment.

Hepatic fibrosis is a progressive disease with few treatment options outside of transplantation. Relaxin is a member of the insulin/relaxin superfamily of peptide hormones. Originally known for its roles in pregnancy, relaxin promotes reproductive tissue remodelling and regulates vascular changes, including increased arterial compliance and reduced vascular resistance. Outside of pregnancy, relaxin plays a major role in the protection of organs from excess extracellular matrix accumulation, as demonstrated by the relaxin-null mouse, which develops widespread fibrosis with ageing. Relaxin reduces scarring due to excess collagen deposition by inhibiting collagen production while simultaneously promoting its degradation and can reduce established fibrosis in several animal models of extracellular matrix-associated disease, including liver fibrosis. Treatment with relaxin reduces the myofibroblastic phenotype of activated hepatic stellate cells, the major hepatic collagen-producing cell in fibrosis and cirrhosis. Relaxin also has haemodynamic effects, including vasodilation, and can reduce portal hypertension associated with cirrhosis. In this review, a brief overview of hepatic fibrosis and the role of the hepatic stellate cell will be presented, followed by an introduction to relaxin and its actions. The use of relaxin to treat preclinical models of fibrotic diseases, including liver diseases, will also be discussed. Finally, the completed, current, and ongoing clinical trials of relaxin in human disease will be described, followed by the limitations and future directions for the use of relaxin for disease treatment.

Novel Anti-fibrotic Therapies.

Fibrosis is a major player in cardiovascular disease, both as a contributor to the development of disease, as well as a post-injury response that drives progression. Despite the identification of many mechanisms responsible for cardiovascular fibrosis, to date no treatments have emerged that have effectively reduced the excess deposition of extracellular matrix associated with fibrotic conditions. Novel treatments have recently been identified that hold promise as potential therapeutic agents for cardiovascular diseases associated with fibrosis, as well as other fibrotic conditions. The purpose of this review is to provide an overview of emerging antifibrotic agents that have shown encouraging results in preclinical or early clinical studies, but have not yet been approved for use in human disease. One of these agents is bone morphogenetic protein-7 (BMP7), which has beneficial effects in multiple models of fibrotic disease. Another approach discussed involves altering the levels of micro-RNA (miR) species, including miR-29 and miR-101, which regulate the expression of fibrosis-related gene targets. Further, the antifibrotic potential of agonists of the peroxisome proliferator-activated receptors will be discussed. Finally, evidence will be reviewed in support of the polypeptide hormone relaxin. Relaxin is long known for its extracellular remodeling properties in pregnancy, and is rapidly emerging as an effective antifibrotic agent in a number of organ systems. Moreover, relaxin has potent vascular and renal effects that make it a particularly attractive approach for the treatment of cardiovascular diseases. In each case, the mechanism of action and the applicability to various fibrotic diseases will be discussed.

Serelaxin increases the antifibrotic action of rosiglitazone in a model of hepatic fibrosis.

AIM: To determine the effect of combined serelaxin and rosiglitazone treatment on established hepatic fibrosis. METHODS: Hepatic fibrosis was induced in mice by carbon tetrachloride administration for 6 wk, or vehicle alone (nonfibrotic mice). For the final 2 wk, mice were treated with rosiglitazone, serelaxin, or both rosiglitazone and serelaxin. Serum liver enzymes and relaxin levels were determined by standard methods. The degree of liver collagen content was determined by histology and immunohistochemistry. Expression of type I collagen was determined by quantitative PCR. Activation of hepatic stellate cells was assessed by alpha-smooth muscle actin (SMA) levels. Liver peroxisome proliferator activated receptor-gamma coactivator 1 alpha (PGC1α) was determined by Western blotting. RESULTS: Treatment of mice with CCl4 resulted in hepatic fibrosis as evidenced by increased liver enzyme levels (ALT and AST), and increased liver collagen and SMA. Monotherapy with either serelaxin or rosiglitazone for 2 wk was generally without effect. In contrast, the combination of serelaxin and rosiglitazone resulted in significantly improved ALT levels (P < 0.05). Total liver collagen content as determined by Sirius red staining revealed that only combination treatment was effective in reducing total liver collagen (P < 0.05). These results were supported by immunohistochemistry for type I collagen, in which only combination treatment reduced fibrillar collagen levels (P < 0.05). The level of hepatic stellate cell activation was modestly, but significantly, reduced by serelaxin treatment alone, but combination treatment resulted in significantly lower SMA levels. Finally, while hepatic fibrosis reduced liver PGC1α levels, the combination of serelaxin and rosiglitazone resulted in restoration of PGC1α protein levels. CONCLUSION: The combination of serelaxin and rosiglitazone treatment for 2 wk was effective in significantly reducing established hepatic fibrosis, providing a potential new treatment strategy.

Lack of hepcidin expression attenuates steatosis and causes fibrosis in the liver.

AIM: To investigate the role of key iron-regulatory protein, hepcidin in non-alcoholic fatty liver disease (NAFLD). METHODS: Hepcidin (Hamp1) knockout and floxed control mice were administered a high fat and high sucrose (HFS) or a regular control diet for 3 or 7 mo. Steatosis, triglycerides, fibrosis, protein and gene expression in mice livers were determined by histological and biochemical techniques, western blotting and real-time polymerase chain reaction. RESULTS: Knockout mice exhibited hepatic iron accumulation. Despite similar weight gains, HFS feeding induced hepatomegaly in floxed, but not knockout, mice. The livers of floxed mice exhibited higher levels of steatosis, triglycerides and c-Jun N-terminal kinase (JNK) phosphorylation than knockout mice. In contrast, a significant increase in fibrosis was observed in knockout mice livers within 3 mo of HFS administration. The hepatic gene expression levels of sterol regulatory element-binding protein-1c and fat-specific protein-27, but not peroxisome proliferator-activated receptor-alpha or microsomal triglyceride transfer protein, were attenuated in HFS-fed knockout mice. Knockout mice fed with regular diet displayed increased carnitine palmitoyltransferase-1a and phosphoenolpyruvate carboxykinase-1 but decreased glucose-6-phosphatase expression in the liver. In summary, attenuated steatosis correlated with decreased expression of lipogenic and lipid storage genes, and JNK phosphorylation. Deletion of Hamp1 alleles per se modulated hepatic expression of beta-oxidation and gluconeogenic genes. CONCLUSION: Lack of hepcidin expression inhibits hepatic lipid accumulation and induces early development of fibrosis following high fat intake. Hepcidin and iron may play a role in the regulation of metabolic pathways in the liver, which has implications for NAFLD pathogenesis.

Relaxin activates peroxisome proliferator-activated receptor γ (PPARγ) through a pathway involving PPARγ coactivator 1α (PGC1α).

Relaxin activation of its receptor RXFP1 triggers multiple signaling pathways. Previously, we have shown that relaxin activates PPARγ transcriptional activity in a ligand-independent manner, but the mechanism for this effect was unknown. In this study, we examined the signaling pathways of downstream of RXFP1 leading to PPARγ activation. Using cells stably expressing RXFP1, we found that relaxin regulation of PPARγ activity requires accumulation of cAMP and subsequent activation of cAMP-dependent protein kinase (PKA). The activated PKA subsequently phosphorylated cAMP response element-binding protein (CREB) at Ser-133 to activate it directly, as well as indirectly through mitogen activated protein kinase p38 MAPK. Activated CREB was required for relaxin stimulation of PPARγ activity, while there was no evidence for a role of the nitric oxide or ERK MAPK pathways. Relaxin increased the mRNA and protein levels of the coactivator protein PGC1α, and this effect was dependent on PKA, and was completely abrogated by a dominant-negative form of CREB. This mechanism was confirmed in a hepatic stellate cell line stably that endogenously expresses RXFP1. Reduction of PGC1α levels using siRNA diminished the regulation of PPARγ by relaxin. These results suggest that relaxin activates the cAMP/PKA and p38 MAPK pathways to phosphorylate CREB, resulting in increased PGC1α levels. This provides a mechanism for the ligand-independent activation of PPARγ in response to relaxin.

Relaxin decreases the severity of established hepatic fibrosis in mice.

BACKGROUND & AIMS: Hepatic fibrosis is characterized by excess collagen deposition, decreased extracellular matrix degradation and activation of the hepatic stellate cells. The hormone relaxin has shown promise in the treatment of fibrosis in a number of tissues, but the effect of relaxin on established hepatic fibrosis is unknown. The aim of this study was to determine the effect of relaxin on an in vivo model after establishing hepatic fibrosis METHODS: Male mice were made fibrotic by carbon tetrachloride treatment for 4 weeks, followed by treatment with two doses of relaxin (25 or 75 µg/kg/day) or vehicle for 4 weeks, with continued administration of carbon tetrachloride. RESULTS: Relaxin significantly decreased total hepatic collagen and smooth muscle actin content at both doses, and suppressed collagen I expression at the higher dose. Relaxin increased the expression of the matrix metalloproteinases MMP13 and MMP3, decreased the expression of MMP2 and tissue inhibitor of metalloproteinase 2 (TIMP2) and increased the overall level of collagen-degrading activity. Relaxin decreased TGFß-induced Smad2 nuclear localization in mouse hepatic stellate cells. CONCLUSIONS: The results suggest that relaxin reduced collagen deposition and HSC activation in established hepatic fibrosis despite the presence of continued hepatic insult. This reduced fibrosis was associated with increased expression of the fibrillar collagen-degrading enzyme MMP13, decreased expression of TIMP2, and enhanced collagen-degrading activity, and impaired TGFß signalling, consistent with relaxin's effects on activated fibroblastic cells. The results suggest that relaxin may be an effective treatment for the treatment of established hepatic fibrosis.

Tacrolimus and sirolimus have distinct effects on insulin signaling in male and female rats.

Although the contribution of the immunosuppressants tacrolimus (TAC) and sirolimus (SIR) to the development of posttransplant diabetes mellitus (PTDM) are being increasingly recognized, the mechanisms of immunosuppressant-induced hyperglycemia are unclear. SIR induces insulin resistance predominantly, but is associated with ß-cell dysfunction in rodents. TAC affects islet function but is associated with worsening insulin sensitivity in a few, and improvement in some, clinical studies. We sought to clarify the contributions of TAC and SIR to insulin resistance and islet function. Four groups of male and female Sprague-Dawley rats received TAC, SIR, TAC and SIR, or control for 2 weeks. All rats were administered an oral glucose challenge at the end of treatment. Half the groups were sacrificed 10 minutes after administration of regular insulin whereas the other half did not receive insulin before sacrifice. Liver, pancreas, fat, and muscle were harvested subsequently. Quantification of Western blots revealed that SIR and TAC plus SIR suppressed the phospho-Akt (pAkt)-to-Akt ratios in liver, muscle, and fat compared with control, regardless of sex. TAC alone did not impair the pAkt-to-Akt ratios in any of the tissues in male and female rats. ß-Cell mass was reduced significantly after TAC treatment in male rats. SIR did not affect ß-cell mass, regardless of sex. Our study demonstrated very clearly that SIR impairs insulin signaling, without any effect on ß-cell mass, and TAC does not impair insulin signaling but reduces ß-cell mass. Our efforts are key to understanding the mechanisms of immunosuppressant-induced hyperglycemia and to tailoring treatments for PTDM.

Dominant-negative and knockdown approaches to studying PPAR activity.

Manipulation of PPAR activity is often a valuable approach toward elucidation of the cellular effects of PPARs. The activity of specific PPARs can be decreased using chemical inhibitors, but these approaches can be affected by nonspecific interactions or cell toxicity. Alternative approaches include targeting PPAR gene expression or activity through molecular biology strategies. Here, we describe the targeting of PPARγ through dominant-negative and siRNA-mediated knockdown constructs.

Metformin improves immunosuppressant induced hyperglycemia and exocrine apoptosis in rats.

BACKGROUND: Immunosuppressants are an important cause of posttransplantation diabetes mellitus. We have shown that tacrolimus and sirolimus induce hyperglycemia and hyperinsulinemia in normal rats. We hypothesized that metformin, given concurrently with tacrolimus and/or sirolimus, prevents disturbances in glucose and insulin metabolism. METHODS: Eight groups (n=6) of normal Sprague-Dawley rats were studied: four groups received tacrolimus, sirolimus, tacrolimus/sirolimus, or control for 14 days, and four more groups received similar treatments along with metformin. Daily glucoses were measured. All rats were administered an oral glucose challenge before sacrifice. Pancreata were analyzed by terminal deoxynucleotide tranferase-mediated dUTP nick-end labeling staining and immunohistochemistry. RESULTS: Tacrolimus, sirolimus, and tacrolimus/sirolimus impaired glucose tolerance compared to control. Sirolimus and tacrolimus/sirolimus also increased random blood glucose levels. Sirolimus alone resulted in hyperinsulinemia after oral glucose challenge compared to control. In the sirolimus/metformin and tacrolimus/sirolimus/metformin groups, mean daily random glucose was no longer increased, although the response to glucose challenge was still impaired. Metformin decreased pancreatic exocrine and trended to decrease endocrine apoptosis in tacrolimus/sirolimus group and reduced islet insulin content in sirolimus group. CONCLUSIONS: This is the first study to show that metformin can improve immunosuppressant-induced hyperglycemia, when administered concurrently, and reduces exocrine apoptosis (reducing the impact on potential islet progenitor cells).

Surface-induced order parameter distortion in superfluid ³He-B measured by nonlinear NMR.

The B phase of superfluid 3He is a three-dimensional time-reversal invariant topological superfluid, predicted to support gapless Majorana surface states. We confine superfluid 3He into a thin nanofluidic slab geometry. In the presence of a weak symmetry-breaking magnetic field, we have observed two possible states of the confined 3He-B phase manifold, through the small tipping angle NMR response. Large tipping angle nonlinear NMR has allowed the identification of the order parameter of these states and enabled a measurement of the surface-induced gap distortion. The results for two different quasiparticle surface scattering boundary conditions are compared with the predictions of weak-coupling quasiclassical theory. We identify a textural domain wall between the two B phase states, the edge of which at the cavity surface is predicted to host gapless states, protected in the magnetic field.