Defining the mechanism of galectin-3-mediated TGF-ß1 activation and its role in lung fibrosis.

Integrin-mediated activation of the profibrotic mediator transforming growth factor-ß1 (TGF-ß1), plays a critical role in idiopathic pulmonary fibrosis (IPF) pathogenesis. Galectin-3 is believed to contribute to the pathological wound healing seen in IPF, although its mechanism of action is not precisely defined. We hypothesized that galectin-3 potentiates TGF-ß1 activation and/or signaling in the lung to promote fibrogenesis. We show that galectin-3 induces TGF-ß1 activation in human lung fibroblasts (HLFs) and specifically that extracellular galectin-3 promotes oleoyl-L-α-lysophosphatidic acid sodium salt-induced integrin-mediated TGF-ß1 activation. Surface plasmon resonance analysis confirmed that galectin-3 binds to αv integrins, αvß1, αvß5, and αvß6, and to the TGFßRII subunit in a glycosylation-dependent manner. This binding is heterogeneous and not a 1:1 binding stoichiometry. Binding interactions were blocked by small molecule inhibitors of galectin-3, which target the carbohydrate recognition domain. Galectin-3 binding to ß1 integrin was validated in vitro by coimmunoprecipitation in HLFs. Proximity ligation assays indicated that galectin-3 and ß1 integrin colocalize closely (≤40 nm) on the cell surface and that colocalization is increased by TGF-ß1 treatment and blocked by galectin-3 inhibitors. In the absence of TGF-ß1 stimulation, colocalization was detectable only in HLFs from IPF patients, suggesting the proteins are inherently more closely associated in the disease state. Galectin-3 inhibitor treatment of precision cut lung slices from IPF patients' reduced Col1a1, TIMP1, and hyaluronan secretion to a similar degree as TGF-ß type I receptor inhibitor. These data suggest that galectin-3 promotes TGF-ß1 signaling and may induce fibrogenesis by interacting directly with components of the TGF-ß1 signaling cascade.

An Inhaled Galectin-3 Inhibitor in COVID-19 Pneumonitis: A Phase Ib/IIa Randomized Controlled Clinical Trial (DEFINE).

Rationale: High circulating galectin-3 is associated with poor outcomes in patients with coronavirus disease (COVID-19). We hypothesized that GB0139, a potent inhaled thiodigalactoside galectin-3 inhibitor with antiinflammatory and antifibrotic actions, would be safely and effectively delivered in COVID-19 pneumonitis. Objectives: Primary outcomes were safety and tolerability of inhaled GB0139 as an add-on therapy for patients hospitalized with COVID-19 pneumonitis. Methods: We present the findings of two arms of a phase Ib/IIa randomized controlled platform trial in hospitalized patients with confirmed COVID-19 pneumonitis. Patients received standard of care (SoC) or SoC plus 10 mg inhaled GB0139 twice daily for 48 hours, then once daily for up to 14 days or discharge. Measurements and Main Results: Data are reported from 41 patients, 20 of which were assigned randomly to receive GB0139. Primary outcomes: the GB0139 group experienced no treatment-related serious adverse events. Incidences of adverse events were similar between treatment arms (40 with GB0139 + SoC vs. 35 with SoC). Secondary outcomes: plasma GB0139 was measurable in all patients after inhaled exposure and demonstrated target engagement with decreased circulating galectin (overall treatment effect post-hoc analysis of covariance [ANCOVA] over days 2-7; P = 0.0099 vs. SoC). Plasma biomarkers associated with inflammation, fibrosis, coagulopathy, and major organ function were evaluated. Conclusions: In COVID-19 pneumonitis, inhaled GB0139 was well-tolerated and achieved clinically relevant plasma concentrations with target engagement. The data support larger clinical trials to determine clinical efficacy. Clinical trial registered with ClinicalTrials.gov (NCT04473053) and EudraCT (2020-002230-32).

Target inhibition of galectin-3 by inhaled TD139 in patients with idiopathic pulmonary fibrosis.

Galectin (Gal)-3 is a profibrotic ß-galactoside-binding lectin that plays a key role in the pathogenesis of idiopathic pulmonary fibrosis (IPF) and IPF exacerbations. TD139 is a novel and potent small-molecule inhibitor of Gal-3.A randomised, double-blind, multicentre, placebo-controlled, phase 1/2a study was conducted to assess the safety, tolerability, pharmacokinetics and pharmacodynamics of inhaled TD139 in 36 healthy subjects and 24 patients with IPF. Six dose cohorts of six healthy subjects were evaluated (4:2 TD139:placebo ratio) with single doses of TD139 (0.15-50 mg) and three dose cohorts of eight patients with IPF (5:3 TD139:placebo ratio) with once-daily doses of TD139 (0.3-10 mg) for 14 days.Inhaled TD139 was well tolerated with no significant treatment-related side-effects. TD139 was rapidly absorbed, with mean time taken to reach maximum plasma concentration (C max) values ranging from 0.6 to 3 h and a plasma half-life (T 1/2) of 8 h. The concentration of TD139 in the lung was >567-fold higher than in the blood, with systemic exposure predicting exposure in the target compartment. Gal-3 expression on alveolar macrophages was reduced in the 3 and 10 mg dose groups compared with placebo, with a concentration-dependent inhibition demonstrated. Inhibition of Gal-3 expression in the lung was associated with reductions in plasma biomarkers centrally relevant to IPF pathobiology (platelet-derived growth factor-BB, plasminogen activator inhibitor-1, Gal-3, CCL18 and YKL-40).TD139 is safe and well tolerated in healthy subjects and IPF patients. It was shown to suppress Gal-3 expression on bronchoalveolar lavage macrophages and, in a concerted fashion, decrease plasma biomarkers associated with IPF progression.

Alternatively activated macrophages promote resolution of necrosis following acute liver injury.

BACKGROUND & AIM: Following acetaminophen (APAP) overdose, acute liver injury (ALI) can occur in patients that present too late for N-acetylcysteine treatment, potentially leading to acute liver failure, systemic inflammation, and death. Macrophages influence the progression and resolution of ALI due to their innate immunological function and paracrine activity. Syngeneic primary bone marrow-derived macrophages (BMDMs) were tested as a cell-based therapy in a mouse model of APAP-induced ALI (APAP-ALI). METHODS: Several phenotypically distinct BMDM populations were delivered intravenously to APAP-ALI mice when hepatic necrosis was established, and then evaluated based on their effects on injury, inflammation, immunity, and regeneration. In vivo phagocytosis assays were used to interrogate the phenotype and function of alternatively activated BMDMs (AAMs) post-injection. Finally, primary human AAMs sourced from healthy volunteers were evaluated in immunocompetent APAP-ALI mice. RESULTS: BMDMs rapidly localised to the liver and spleen within 4 h of administration. Injection of AAMs specifically reduced hepatocellular necrosis, HMGB1 translocation, and infiltrating neutrophils following APAP-ALI. AAM delivery also stimulated proliferation in hepatocytes and endothelium, and reduced levels of several circulating proinflammatory cytokines within 24 h. AAMs displayed a high phagocytic activity both in vitro and in injured liver tissue post-injection. Crosstalk with the host innate immune system was demonstrated by reduced infiltrating host Ly6Chi macrophages in AAM-treated mice. Importantly, therapeutic efficacy was partially recapitulated using clinical-grade primary human AAMs in immunocompetent APAP-ALI mice, underscoring the translational potential of these findings. CONCLUSION: We identify that AAMs have value as a cell-based therapy in an experimental model of APAP-ALI. Human AAMs warrant further evaluation as a potential cell-based therapy for APAP overdose patients with established liver injury. LAY SUMMARY: After an overdose of acetaminophen (paracetamol), some patients present to hospital too late for the current antidote (N-acetylcysteine) to be effective. We tested whether macrophages, an injury-responsive leukocyte that can scavenge dead/dying cells, could serve as a cell-based therapy in an experimental model of acetaminophen overdose. Injection of alternatively activated macrophages rapidly reduced liver injury and reduced several mediators of inflammation. Macrophages show promise to serve as a potential cell-based therapy for acute liver injury.

Cerebral Concussion Primes the Lungs for Subsequent Neutrophil-Mediated Injury.

OBJECTIVES: Mild traumatic brain injury in the form of concussion is extremely common, and the potential effects on pulmonary priming have been underestimated. The aim of this study was to characterize the pulmonary response following mild traumatic brain injury and assess the pulmonary susceptibility to lung injury after a subsequent innocuous pulmonary insult. DESIGN: Experimental in vivo study. SETTING: University research laboratory. SUBJECTS: Male CD1 mice. INTERVENTIONS: We developed a model of concussive traumatic brain injury in mice followed by pulmonary acid microaspiration. To assess the dependent role of neutrophils in mediating pulmonary injury, we specifically depleted neutrophils. MEASUREMENTS AND MAIN RESULTS: Lateral fluid percussion to the brain resulted in neuronal damage and neutrophil infiltration as well as extensive pulmonary interstitial neutrophil accumulation but no alveolar injury. Following subsequent innocuous acid microaspiration, augmented alveolar neutrophil influx led to the development of pulmonary hemorrhage that was reduced following neutrophil depletion. CONCLUSIONS: This model shows for the first time that innocuous acid microaspiration is sufficient to induce neutrophil-mediated lung injury following mild concussion and that the extracranial effects of mild traumatic brain injury have been underestimated.

CSF1 Restores Innate Immunity After Liver Injury in Mice and Serum Levels Indicate Outcomes of Patients With Acute Liver Failure.

BACKGROUND & AIMS: Liver regeneration requires functional liver macrophages, which provide an immune barrier that is compromised after liver injury. The numbers of liver macrophages are controlled by macrophage colony-stimulating factor (CSF1). We examined the prognostic significance of the serum level of CSF1 in patients with acute liver injury and studied its effects in mice. METHODS: We measured levels of CSF1 in serum samples collected from 55 patients who underwent partial hepatectomy at the Royal Infirmary Edinburgh between December 2012 and October 2013, as well as from 78 patients with acetaminophen-induced acute liver failure admitted to the Royal Infirmary Edinburgh or the University of Kansas Medical Centre. We studied the effects of increased levels of CSF1 in uninjured mice that express wild-type CSF1 receptor or a constitutive or inducible CSF1-receptor reporter, as well as in chemokine receptor 2 (Ccr2)-/- mice; we performed fate-tracing experiments using bone marrow chimeras. We administered CSF1-Fc (fragment, crystallizable) to mice after partial hepatectomy and acetaminophen intoxication, and measured regenerative parameters and innate immunity by clearance of fluorescent microbeads and bacterial particles. RESULTS: Serum levels of CSF1 increased in patients undergoing liver surgery in proportion to the extent of liver resected. In patients with acetaminophen-induced acute liver failure, a low serum level of CSF1 was associated with increased mortality. In mice, administration of CSF1-Fc promoted hepatic macrophage accumulation via proliferation of resident macrophages and recruitment of monocytes. CSF1-Fc also promoted transdifferentiation of infiltrating monocytes into cells with a hepatic macrophage phenotype. CSF1-Fc increased innate immunity in mice after partial hepatectomy or acetaminophen-induced injury, with resident hepatic macrophage as the main effector cells. CONCLUSIONS: Serum CSF1 appears to be a prognostic marker for patients with acute liver injury. CSF1 might be developed as a therapeutic agent to restore innate immune function after liver injury.

Galectin-3-Binding Glycomimetics that Strongly Reduce Bleomycin-Induced Lung Fibrosis and Modulate Intracellular Glycan Recognition.

Discovery of glycan-competitive galectin-3-binding compounds that attenuate lung fibrosis in a murine model and that block intracellular galectin-3 accumulation at damaged vesicles, hence revealing galectin-3-glycan interactions involved in fibrosis progression and in intracellular galectin-3 activities, is reported. 3,3'-Bis-(4-aryltriazol-1-yl)thiodigalactosides were synthesized and evaluated as antagonists of galectin-1, -2, -3, and -4 N-terminal, -4 C-terminal, -7 and -8 N-terminal, -9 N-terminal, and -9 C-terminal domains. Compounds displaying low-nanomolar affinities for galectins-1 and -3 were identified in a competitive fluorescence anisotropy assay. X-ray structural analysis of selected compounds in complex with galectin-3, together with galectin-3 mutant binding experiments, revealed that both the aryltriazolyl moieties and fluoro substituents on the compounds are involved in key interactions responsible for exceptional affinities towards galectin-3. The most potent galectin-3 antagonist was demonstrated to act in an assay monitoring galectin-3 accumulation upon amitriptyline-induced vesicle damage, visualizing a biochemically/medically relevant intracellular lectin-carbohydrate binding event and that it can be blocked by a small molecule. The same antagonist administered intratracheally attenuated bleomycin-induced pulmonary fibrosis in a mouse model with a dose/response profile comparing favorably with that of oral administration of the marketed antifibrotic compound pirfenidone.

Galectin-3 regulates hepatic progenitor cell expansion during liver injury.

OBJECTIVE: Following chronic liver injury or when hepatocyte proliferation is impaired, ductular reactions containing hepatic progenitor cells (HPCs) appear in the periportal regions and can regenerate the liver parenchyma. HPCs exist in a niche composed of myofibroblasts, macrophages and laminin matrix. Galectin-3 (Gal-3) is a ß-galactoside-binding lectin that binds to laminin and is expressed in injured liver in mice and humans. DESIGN: We examined the role of Gal-3 in HPC activation. HPC activation was studied following dietary induced hepatocellular (choline-deficient ethionine-supplemented diet) and biliary (3,5-diethoxycarbonyl-1,4-dihydrocollidine supplemented diet) injury in wild type and Gal-3(-/-) mice. RESULTS: HPC proliferation was significantly reduced in Gal-3(-/-) mice. Gal-3(-/-) mice failed to form a HPC niche, with reduced laminin formation. HPCs isolated from wild type mice secrete Gal-3 which enhanced adhesion and proliferation of HPCs on laminin in an undifferentiated form. These effects were attenuated in Gal3(-/-) HPCs and in wild type HPCs treated with the Gal-3 inhibitor lactose. Gal-3(-/-) HPCs in vitro showed increased hepatocyte function and prematurely upregulated both biliary and hepatocyte differentiation markers and regulated cell cycle genes leading to arrest in G0/G1. CONCLUSIONS: We conclude that Gal-3 is required for the undifferentiated expansion of HPCs in their niche in injured liver.

Polysialic acid/neural cell adhesion molecule modulates the formation of ductular reactions in liver injury.

UNLABELLED: In severe liver injury, ductular reactions (DRs) containing bipotential hepatic progenitor cells (HPCs) branch from the portal tract. Neural cell adhesion molecule (NCAM) marks bile ducts and DRs, but not mature hepatocytes. NCAM mediates interactions between cells and surrounding matrix; however, its role in liver development and regeneration is undefined. Polysialic acid (polySia), a unique posttranslational modifier of NCAM, is produced by the enzymes, ST8SiaII and ST8SiaIV, and weakens NCAM interactions. The role of polySia with NCAM synthesizing enzymes ST8SiaII and ST8SiaIV were examined in HPCs in vivo using the choline-deficient ethionine-supplemented and 3,5-diethoxycarbonyl-1,4-dihydrocollidine diet models of liver injury and regeneration, in vitro using models of proliferation, differentiation, and migration, and by use of mouse models with gene defects in the polysialyltransferases (St8sia 2+/-4+/-, and St8sia2-/-4-/-). We show that, during liver development, polySia is required for the correct formation of bile ducts because gene defects in both the polysialyltransferases (St8sia2+/-4+/- and St8sia2-/-4-/- mice) caused abnormal bile duct development. In normal liver, there is minimal polySia production and few ductular NCAM+ cells. Subsequent to injury, NCAM+ cells expand and polySia is produced by DRs/HPCs through ST8SiaIV. PolySia weakens cell-cell and cell-matrix interactions, facilitating HGF-induced migration. Differentiation of HPCs to hepatocytes in vitro results in both transcriptional down-regulation of polySia and cleavage of polySia-NCAM. Cleavage of polySia by endosialidase (endoN) during liver regeneration reduces migration of DRs into parenchyma. CONCLUSION: PolySia modification of NCAM+ ductules weakens cell-cell and cell-matrix interactions, allowing DRs/HPCs to migrate for normal development and regeneration. Modulation of polySia levels may provide a therapeutic option in liver regeneration.

Phenotypic and functional characterization of macrophages with therapeutic potential generated from human cirrhotic monocytes in a cohort study.

BACKGROUND AIMS: Macrophages have complex roles in the liver. The aim of this study was to compare profiles of human monocyte-derived macrophages between controls and cirrhotic patients, to determine whether chronic inflammation affects precursor number or the phenotype, with the eventual aim to develop a cell therapy for cirrhosis. METHODS: Infusion of human macrophages in a murine liver fibrosis model demonstrated a decrease in markers of liver injury (alanine transaminase, bilirubin, aspartate transaminase) and fibrosis (transforming growth factor-ß, α-smooth muscle actin, phosphatidylserine receptor) and an increase in markers of liver regeneration (matrix metalloproteinases [MMP]-9, MMP-12 and TNF-related weak inducer of apoptosis). CD14+ monocytes were then isolated from controls. Monocytes were matured into macrophages for 7 days using a Good Manufacturing Practice-compatible technique. RESULTS: There was no significant difference between the mean number of CD14+ monocytes isolated from cirrhotic patients (n = 9) and controls (n = 10); 2.8 ± SEM 0.54 × 10(8) and 2.5 ± 0.56 × 10(8), respectively. The mean yield of mature macrophages cultured was also not significantly different between cirrhotic patients and controls (0.9 × 10(8) ± 0.38 × 10(8), with more than 90% viability and 0.65 × 10(8) ± 0.16 × 10(8), respectively. Maturation to macrophages resulted in up-regulation of a number of genes (MMP-9, CCL2, interleukin [IL]-10 and TNF-related weak inducer of apoptosis). A cytokine and chemokine polymerase chain reaction array, comparing the control and cirrhotic macrophages, revealed no statistically significant differences. CONCLUSIONS: Macrophages can be differentiated from cirrhotic patients' apheresis-derived CD14 monocytes and develop the same pro-resolution phenotype as control macrophages, indicating their suitability for clinical therapy.

Bone marrow contributions to fibrosis.

Bone marrow transplant experiments in mice using labelled donor bone marrow have indicated that following injury bone marrow derived cells can circulate and home to the injured organs. In particular fibrocytes and myofibroblasts are capable of contributing to the wound healing response, including collagen deposition. In chronic injury this can lead to a pathological degree of fibrosis. Experiments have shown that this can be a relatively insignificant contribution to the scar forming population in certain organs and that the majority of the scar forming cells are intrinsic to the organ. Conversely, in certain circumstances, the circulating cells become major players in the organs fibrotic response. Whilst cell tracking experiments are relatively simple to perform, to actually determine a functional contribution to a fibrotic response more sophisticated approaches are required. This can include the use of bone marrow transplantation from recipients with collagen reporter systems which gives a read out of bone marrow derived cells that are transcriptional active for collagen production in a damaged organ. Another technique is to use bone marrow transplants from donors that have a mutation in the collagen to demonstrate a functional difference in fibrosis when bone marrow transplants performed. Recent reports have identified factors mediating recruitment of circulating fibrocytes to injured organs, such as CXCL12 and CXCL16 and shown that blocking these factors reduced fibrocyte recruitment and subsequent fibrosis. The identification of such factors may enable the development of novel therapies to block further fibrocyte engraftment and fibrosis in situations of pathological scarring. This article is part of a Special Issue entitled: Fibrosis: Translation of basic research to human disease.

Temporal expression of galectin-3 following myocardial infarction.

OBJECTIVE: Galectin-3 is a pro-inflammatory, pro-fibrotic molecule implicated in the pathogenesis of heart failure, and associated with poor prognostic outcome. When measured following ST-elevation myocardial infarction (STEMI), a high plasma galectin-3 predicts greater 30-day morbidity and mortality, and increased heart failure incidence at a median of 2 years. This study aims to elucidate the temporal aspects of galectin-3 expression immediately post-STEMI and how expression relates to severity of myocardial injury. METHODS: Plasma galectin-3 levels were compared in 53 STEMI patients and 23 control patients with stable angina. Consecutive plasma galectin-3 levels, measured at a mean of 30 hours (sample A) and 54 hours (sample B) post pain, and analysis of galectin-3 vs time since onset of pain/time since reperfusion allowed assessment of temporal expression in STEMI patients. Myocardial injury markers included troponin and left ventricular ejection fraction (LVEF) at primary percutaneous coronary intervention (PCI). RESULTS: Circulating galectin-3 levels were significantly higher in STEMI patients than control patients when measured at a mean of 30 hours post pain (t = 2.72, df = 66, P = 0.008). However, levels had significantly decreased when measured 24 hours later (t = 2.13, df = 47, P = 0.039), with a negative linear relationship apparent between plasma galectin-3 levels and time since reperfusion on univariate analysis (OR = 0.871, 95% CI = 0.779-0.975, P = 0.021). A significantly lower circulating galectin-3 concentration was also found for sample A in those reperfused within 3 hours post-onset of pain (OR 0.045, 95% CI 0.003-0.669, P = 0.029). CONCLUSIONS: Plasma galectin-3 levels vary significantly following a STEMI over a short time period, in relation to timing of reperfusion.

Discovery of the Selective and Orally Available Galectin-1 Inhibitor GB1908 as a Potential Treatment for Lung Cancer.

We have previously described a new series of selective and orally available galectin-1 inhibitors resulting in the thiazole-containing glycomimetic GB1490. Here, we show that the introduction of polar substituents to the thiazole ring results in galectin-1-specific compounds with low nM affinities. X-ray structural analysis of a new ligand-galectin-1 complex shows changes in the binding mode and ligand-protein hydrogen bond interactions compared to the GB1490-galectin-1 complex. These new high affinity ligands were further optimized with respect to affinity and ADME properties resulting in the galectin-1-selective GB1908 (Kd galectin-1/3 0.057/6.0 µM). In vitro GB1908 inhibited galectin-1-induced apoptosis in Jurkat cells (IC50 = 850 nM). Pharmacokinetic experiments in mice revealed that a dose of 30 mg/kg b.i.d. results in free levels of GB1908 in plasma over galectin-1 Kd for 24 h. GB1908 dosed with this regimen reduced the growth of primary lung tumor LL/2 in a syngeneic mouse model.

Inhibition of galectin-3 reduces atherosclerosis in apolipoprotein E-deficient mice.

Atherosclerosis is a major risk factor for cardiovascular disease (CVD) and stroke. Galectin-3 is a carbohydrate-binding lectin implicated in the pathophysiology of CVD and is highly expressed within atherosclerotic lesions in mice and humans. The object of this present study was to use genetic deletion and pharmacological inhibition in a well-characterized mouse model of atherosclerosis to determine the role of galectin-3 in plaque development. Apolipoprotein-E/galectin-3 knockout mice were generated and fed a high-cholesterol "western" diet. Galectin-3 deletion had no consistent effect on the serum lipid profile but halved atherosclerotic lesion formation in the thoracic aorta (57% reduction), the aortic arch (50% reduction) and the brachiocephalic arteries. The aortic plaques were smaller, with reduced lipid core and less collagen. In apolipoprotein E-deficient (ApoE(-/-)) mice, there was a switch from high inducible nitric oxide expression in early lesions (6 weeks) to arginase-1 expression in later lesions (20 weeks), which was reversed in ApoE(-/-)/gal-3(-/-) mice. Administration of modified citrus pectin, an inhibitor of galectin-3, during the latter stage of the disease reduced plaque volume. We conclude that inhibiting galectin-3 causes decreased atherosclerosis. Strategies to inhibit galectin-3 function may reduce plaque progression and potentially represent a novel therapeutic strategy in the treatment of atherosclerotic disease.

Regulation of transforming growth factor-ß1-driven lung fibrosis by galectin-3.

RATIONALE: Idiopathic pulmonary fibrosis (IPF) is a chronic dysregulated response to alveolar epithelial injury with differentiation of epithelial cells and fibroblasts into matrix-secreting myofibroblasts resulting in lung scaring. The prognosis is poor and there are no effective therapies or reliable biomarkers. Galectin-3 is a ß-galactoside binding lectin that is highly expressed in fibrotic tissue of diverse etiologies. OBJECTIVES: To examine the role of galectin-3 in pulmonary fibrosis. METHODS: We used genetic deletion and pharmacologic inhibition in well-characterized murine models of lung fibrosis. Further mechanistic studies were performed in vitro and on samples from patients with IPF. MEASUREMENTS AND MAIN RESULTS: Transforming growth factor (TGF)-ß and bleomycin-induced lung fibrosis was dramatically reduced in mice deficient in galectin-3, manifest by reduced TGF-ß1-induced EMT and myofibroblast activation and collagen production. Galectin-3 reduced phosphorylation and nuclear translocation of ß-catenin but had no effect on Smad2/3 phosphorylation. A novel inhibitor of galectin-3, TD139, blocked TGF-ß-induced ß-catenin activation in vitro and in vivo and attenuated the late-stage progression of lung fibrosis after bleomycin. There was increased expression of galectin-3 in the bronchoalveolar lavage fluid and serum from patients with stable IPF compared with nonspecific interstitial pneumonitis and controls, which rose sharply during an acute exacerbation suggesting that galectin-3 may be a marker of active fibrosis in IPF and that strategies that block galectin-3 may be effective in treating acute fibrotic exacerbations of IPF. CONCLUSIONS: This study identifies galectin-3 as an important regulator of lung fibrosis and provides a proof of principle for galectin-3 inhibition as a potential novel therapeutic strategy for IPF.

Monocytes control second-phase neutrophil emigration in established lipopolysaccharide-induced murine lung injury.

RATIONALE: Acute lung injury (ALI) is an important cause of morbidity and mortality, with no currently effective pharmacological therapies. Neutrophils have been specifically implicated in the pathogenesis of ALI, and there has been significant research into the mechanisms of early neutrophil recruitment, but those controlling the later phases of neutrophil emigration that characterize disease are poorly understood. OBJECTIVES: To determine the influence of peripheral blood monocytes (PBMs) in established ALI. METHODS: In a murine model of LPS-induced ALI, three separate models of conditional monocyte ablation were used: systemic liposomal clodronate (sLC), inducible depletion using CD11b diphtheria toxin receptor (CD11b DTR) transgenic mice, and antibody-dependent ablation of CCR2(hi) monocytes. MEASUREMENTS AND MAIN RESULTS: PBMs play a critical role in regulating neutrophil emigration in established murine LPS-induced lung injury. Gr1(hi) and Gr1(lo) PBM subpopulations contribute to this process. PBM depletion is associated with a significant reduction in measures of lung injury. The specificity of PBM depletion was demonstrated by replenishment studies in which the effects were reversed by systemic PBM infusion but not by systemic or local pulmonary infusion of mature macrophages or lymphocytes. CONCLUSIONS: These results suggest that PBMs, or the mechanisms by which they influence pulmonary neutrophil emigration, could represent therapeutic targets in established ALI.

Galectin-3: therapeutic targeting in liver disease.

INTRODUCTION: The rising incidence of liver diseases is a worldwide healthcare concern. However, the therapeutic options to manage chronic inflammation and fibrosis, the processes at the basis of morbidity and mortality of liver diseases, are very limited. Galectin 3 (Gal-3) is a protein implicated in fibrosis in multiple organs. Several Gal-3 inhibitors are currently in clinical development. AREAS COVERED: This review describes our current understanding of the role of Gal-3 in chronic liver diseases, with special emphasis on fibrosis. Also, we review therapeutic advances based on Gal-3 inhibition, describing drug properties and their current status in clinical research. EXPERT OPINION: Currently, the known effects of Gal-3 point to a direct activation of the NLRP3 inflammasome leading to its activation in liver macrophages and activated macrophages play a key role in tissue fibrogenesis. However, more research is needed to elucidate the role of Gal-3 in the different activation pathways, dissecting the intracellular and extracellular mechanisms of Gal-3, and its role in pathogenesis. Gal-3 could be a target for early therapy of numerous hepatic diseases and, given the lack of therapeutic options for liver fibrosis, there is a strong pharmacologic potential for Gal-3-based therapies.

Resistance to anti-PD-1/anti-PD-L1: galectin-3 inhibition with GB1211 reverses galectin-3-induced blockade of pembrolizumab and atezolizumab binding to PD-1/PD-L1.

Background: Galectin-3 (Gal-3) is a ß-galactoside-binding lectin that is highly expressed within the tumor microenvironment of aggressive cancers and has been suggested to predict a poor response to immune checkpoint therapy with the anti-PD-1 monoclonal antibody pembrolizumab. We aimed to assess if the effect of Gal-3 was a result of direct interaction with the immune checkpoint receptor. Methods: The ability of Gal-3 to interact with the PD-1/PD-L1 complex in the absence and presence of blocking antibodies was assessed in in vitro biochemical and cellular assays as well as in an in vivo syngeneic mouse cancer model. Results: Gal-3 reduced the binding of the checkpoint inhibitors pembrolizumab (anti-PD-1) and atezolizumab (anti-PD-L1), by potentiating the interaction between the PD-1/PD-L1 complex. In the presence of a highly selective Gal-3 small molecule inhibitor (GB1211) the binding of the anti-PD-1/anti-PD-L1 therapeutics was restored to control levels. This was observed in both a surface plasmon resonance assay measuring protein-protein interactions and via flow cytometry. Combination therapy with GB1211 and an anti-PD-L1 blocking antibody reduced tumor growth in an in vivo syngeneic model and increased the percentage of tumor infiltrating T lymphocytes. Conclusion: Our study suggests that Gal-3 can potentiate the PD-1/PD-L1 immune axis and potentially contribute to the immunosuppressive signalling mechanisms within the tumor microenvironment. In addition, Gal-3 prevents atezolizumab and pembrolizumab target engagement with their respective immune checkpoint receptors. Reversal of this effect with the clinical candidate GB1211 offers a potential enhancing combination therapeutic with anti-PD-1 and -PD-L1 blocking antibodies.

Safety and pharmacokinetics of GB1211, an oral galectin-3 inhibitor: a single- and multiple-dose first-in-human study in healthy participants.

PURPOSE: Galectin-3, a ß-galactoside-binding lectin, plays a key role in several cellular pathways involved in chronic inflammation, heart disease and cancer. GB1211 is an orally bioavailable galectin-3 inhibitor, developed to be systemically active. We report safety and pharmacokinetics (PK) of GB1211 in healthy participants. METHODS: This phase 1, double-blind, placebo-controlled, first-in-human study (NCT03809052) included a single ascending-dose phase (with a food-effect cohort) where participants across seven sequential cohorts were randomized 3:1 to receive oral GB1211 (5, 20, 50, 100, 200 or 400 mg) or placebo. In the multiple ascending-dose phase, participants received 50 or 100 mg GB1211 or placebo twice daily for 10 days. All doses were administered in the fasted state except in the food-effect cohort where doses were given 30 min after a high-fat meal. RESULTS: All 78 participants received at least one GB1211 dose (n = 58) or placebo (n = 20) and completed the study. No safety concerns were identified. Following single and multiple oral doses under fasted conditions, maximum GB1211 plasma concentrations were reached at 1.75-4 h (median) post-dose; mean half-life was 11-16 h. There was a ~ twofold GB1211 accumulation in plasma with multiple dosing, with steady-state reached within 3 days; 30% of the administered dose was excreted in urine as unchanged drug. Absorption in the fed state was delayed by 2 h but systemic exposure was unaffected. CONCLUSION: GB1211 was well tolerated, rapidly absorbed, and displayed favorable PK, indicating a potential to treat multiple disease types. These findings support further clinical development of GB1211. CLINICAL TRIAL REGISTRATION: The study was registered with ClinicalTrials.gov (identifier: NCT03809052).

Small cell lung cancer tumour cells induce regulatory T lymphocytes, and patient survival correlates negatively with FOXP3+ cells in tumour infiltrate.

Small cell lung cancer (SCLC) kills at least one person every 2 hr in the United Kingdom. Some patients do relatively well but most have rapidly progressive disease. There is no effective treatment and overall 2-year survival is less than 5%. Patients with SCLC have poorly understood local and systemic immune defects and can be immunocompromised. As CD4(+) T lymphocytes coordinate and regulate immunity, a better understanding of interactions between SCLC tumour cells and CD4(+) T cells may lead to effective molecular immunotherapy. We show that some, but not all, SCLC tumour cell lines secrete molecules that induce IL-10 secretion by and de novo differentiation of functional CD4(+)CD25(+)FOXP3(+)CD127(lo)Helios(-) regulatory T (Treg) cells in healthy blood lymphocytes. FOXP3(+) T cells were found in SCLC tumour biopsies, and patients with higher ratios of FOXP3(+) cells in tumour infiltrates have a worse survival rate. The inhibitory effect of SCLC tumour cells was not affected by blocking IL-10 receptor or TGF-ß signalling but was partially reversed by blocking IL-15, which is reported to be involved in human Treg cells induction. IL-15 was secreted by SCLC cells that inhibited CD4(+) T-cell proliferation and was present in SCLC biopsy tumour cells. These novel findings demonstrate that SCLC tumour cells can induce CD4(+) T-cell-mediated immunosuppression. This gives a potential mechanism by which SCLC tumour cells may downregulate local and systemic immune responses and contribute to poor patient survival. Our data suggest that IL-15 and Treg cells are potential new therapeutic targets to improve immune response and patient survival in SCLC.