Defining the mechanism of galectin-3–mediated TGF-β1 activation and its role in lung fibrosis

Integrin-mediated activation of the profibrotic mediator transforming growth factor-β1 (TGF-β1), 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-β1 activation and/or signaling in the lung to promote fibrogenesis. We show that galectin-3 induces TGF-β1 activation in human lung fibroblasts (HLFs) and specifically that extracellular galectin-3 promotes oleoyl-L-α-lysophosphatidic acid sodium salt–induced integrin-mediated TGF-β1 activation. Surface plasmon resonance analysis confirmed that galectin-3 binds to αv integrins, αvβ1, αvβ5, and αvβ6, and to the TGFβRII 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 β1 integrin was validated in vitro by coimmunoprecipitation in HLFs. Proximity ligation assays indicated that galectin-3 and β1 integrin colocalize closely (≤40 nm) on the cell surface and that colocalization is increased by TGF-β1 treatment and blocked by galectin-3 inhibitors. In the absence of TGF-β1 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-β type I receptor inhibitor. These data suggest that galectin-3 promotes TGF-β1 signaling and may induce fibrogenesis by interacting directly with components of the TGF-β1 signaling cascade.

Proximity ligation assays indicated that galectin-3 and b1 integrin colocalize closely (≤40 nm) on the cell surface and that colocalization is increased by TGF-b1 treatment and blocked by galectin-3 inhibitors.In the absence of TGF-b1 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-b type I receptor inhibitor.These data suggest that galectin-3 promotes TGF-b1 signaling and may induce fibrogenesis by interacting directly with components of the TGF-b1 signaling cascade.
Idiopathic pulmonary fibrosis (IPF) is a chronic and progressive interstitial lung disease (1, 2).It is the most common interstitial lung disease subtype with over 32,000 patients living with the disease in the United Kingdom and more than 6000 newly diagnosed cases annually (3).Older adults and males are primarily affected, with a median survival of 2.5 to 3.5 years from time of diagnosis (1,4,5).Repeated injury to the distal lung parenchyma of a genetically predisposed individual promotes apoptotic, necrotic, and senescent pathways, causing alveolar collapse and exposure of the underlying basement membrane to damage (6)(7)(8)(9).Compromised re-epithelialization following injury enables interstitial cells to proliferate and induces downstream signaling pathways, promoting progressive lung fibrosis (10).Currently, there is no cure for IPF and only two antifibrotic drugs, pirfenidone and nintedanib, are licensed for IPF management (11)(12)(13).
Therefore, alternative strategies to inhibit TGF-b1 activation and signaling are required.
Galectin-3 is a chimeric and self-associating, profibrotic betagalactoside-binding protein localized both intracellularly and extracellularly that is believed to contribute to the pathological wound healing seen in IPF individuals (24)(25)(26)(27).Usual interstitial pneumonia patient lung sections immunostained for galectin-3 show elevated galectin-3 expression within fibroblastic foci that is temporospatial associated with fibrosis (28).Galectin-3 levels are also higher in the serum and bronchoalveolar lavage fluid of IPF patients than controls (28,29).Following irradiationinduced lung injury in vivo, galectin-3 is predominately secreted by alveolar macrophages and its expression markedly increased in fibrotic lung tissue (30).Additionally, the distribution of galectin-3 is spatially related to fibrotic regions of the lung following administration of adenoviral TGF-b1 or intratracheal bleomycin in vivo and its mitogenic activity on cultured human lung fibroblasts (HLFs) has also been demonstrated (28,31).Galectin-3 significantly induces fibroblast migration and increases fibroblast collagen synthesis in vitro further suggesting a role for galectin-3 in fibrogenesis (29).In murine models of pulmonary fibrosis, loss of galectin-3 is protective, with galectin-3−/− mice demonstrating decreased collagen staining and lower total lung collagen compared with WT mice (28).Adding to this, the novel small-molecule inhaled galectin-3 inhibitor, GB0139 (formerly TD139), designed to modulate the fibrogenic response to tissue injury has been shown to decrease galectin-3 expression in the lung and significantly reduce bleomycin-induced fibrosis as assessed by total lung collagen content (28).In a phase 1/2a clinical trial (ID: NCT02257177), inhalation of GB0139 was shown highly suitable for dosing IPF patients and was associated with reductions in plasma biomarkers linked to IPF pathobiology, including platelet-derived growth factor-beta, plasminogen activator inhibitor-1, galectin-3, chemokine (C-C motif) ligand 18, and chitinase-3-like protein 1 (YKL-40) (32).GB0139 has since progressed into a European Union and US phase 2b clinical trial in IPF subjects "GALACTIC-1" (ID: NCT03832946) with the full readout yet to the published.
Galectin-3 preferentially binds to N-glycan residues, which are covalently linked to the TGF-b1 receptor during posttranslation processing.Galectin-3 has been proposed to mediate cell surface retention of the TGF-b1 receptor by binding to its poly-N-acetyllactosamine residues, potentially sustaining profibrotic signaling (28,33).Similar to the TGF-b1 receptor, the literature demonstrates that galectin-3 is also capable of regulating integrin activity by interacting with their surface glycans (34)(35)(36)(37).Therefore, we sought to define the mechanism through which galectin-3 may enhance TGF-b1 activation and contribute to the development of IPF.

Galectin-3 induces TGF-b1 activation in fibroblasts but not epithelial cells
To investigate whether galectin-3 was able to induce TGF-b1 activation, primary HLFs and an immortalized human bronchial epithelial cell line (iHBECs) were treated with exogenous galectin-3 protein and Smad2 phosphorylation was assessed by Western blotting.Although treatment with galectin-3 induced Smad2 phosphorylation in HLFs (Fig. 1A), it was unable to promote phosphorylation of Smad2 in the epithelial cell line (Fig. 1B).In contrast, direct stimulation with TGF-b1 was shown to induce Smad2 phosphorylation in both cell types (Fig. 1, A and B).Galectin-3-induced Smad2 phosphorylation in HLFs was completely inhibited by pretreatment either with the high affinity galectin-3 inhibitor, GB0139, or with the selective inhibitor of the TGF-b type I receptor, activin receptor-like kinase 5 (ALK5) SB-431542 (Fig. 1A).

LPA-induced integrin-mediated TGF-b1 activation requires galectin-3
To investigate the potential involvement of galectin-3 in promoting integrin-mediated TGF-b1 activation in fibroblasts using galectin-3 inhibitors with varying K d values and cell permeability (Table S1), HLFs were stimulated with oleoyl-La-lysophosphatidic acid sodium salt (LPA), a G-protein agonist, which promotes reorganization of the cytoskeleton and integrin activation in a variety of cell types.LPA-induced TGF-b1 activation in HLFs as measured by Smad2 phosphorylation was inhibited in a concentration-dependent manner by pretreatment of HLFs with the b1 integrin small molecule inhibitor, NOTT199SS (Fig. 2A).LPA-induced Smad2 phosphorylation in HLFs was also inhibited by pretreatment both with the inhaled galectin-3 inhibitor, GB0139 (Fig. 2B), and by the orally active galectin-3 inhibitors, GB1107 and GB1211 (Fig. 2C).Pretreatment with the cell-impermeable extracellular galectin-3 inhibitor, GB0149 (38) also inhibited phosphorylation of Smad2 in a concentration-dependent manner (Fig. 2D), suggesting that galectin-3 might be mediating its effects on TGF-b1 activation extracellularly.

Galectin-3 interacts physically with integrins avb1, avb5, and avb6 and the TGFbRII subunit
To determine how galectin-3 promotes integrin-mediated TGF-b1 activation, protein interaction studies were performed by surface plasmon resonance (SPR).Galectin-3 bound to recombinant human integrins avb1 (Fig. 3A), avb5 (Fig. 3B), and avb6 (Fig. 3C) in a concentration-dependent manner.Binding responses were 2-fold higher with avb1 and avb5 than avb6 (Fig. 3, A-C).The highest binding responses recorded for all three integrins were considerably higher than the theoretical maximum response (approximately 135 Rmax) for analyte-ligand binding 1:1.It was not possible to saturate the immobilized integrins at the galectin-3 concentrations tested, as evidenced by the lack of a plateau.As it was not possible to yield an accurate K d value or Bmax for these binding interactions, the minimum number of individual galectin-3 proteins involved in the binding interactions were calculated: 16, 16, and 8 for avb1, avb5, and avb6, respectively.
The binding of galectin-3 to all three integrins (red lines) was seen to be glycosylation-dependent, as enzymatic removal of all N-linked and common O-linked glycans with protein deglycosylation mix II completely inhibited these binding interactions (black lines) (Fig. 3, A-C).Treatment with PNGase F digestion to remove N-linked glycans alone resulted in only a partial decrease in integrin-galectin-3 binding (blue lines) (Fig. 3, A-C).
Galectin-3 also bound to the extracellular domain of the recombinant human TGFbRII protein in a concentrationdependent manner (Fig. 3D).As with the av integrins, the highest binding response recorded was higher than the theoretical maximum response (approximately 251 Rmax) for analyte-ligand binding 1:1.As a higher analyte concentration was used for the TGFbRII experiments than for the av integrins, saturation of the TGFbRII subunit was reached, as evidenced by the plateauing of the curve.Subsequent nonlinear regression analysis yielded a K d value of 5288 nM and a Bmax of 1473 response units (RU), and it was calculated that a minimum of six individual galectin-3 proteins were involved in the binding interaction.Galectin-3 binding to TGFbRII (red line) was also shown to be glycosylation-dependent with enzymatic removal of all N-linked and common O-linked glycans inhibiting the binding interaction (black line) (Fig. 3D).However, a similar decrease in the level of binding was observed after the removal of only N-linked glycans (blue line) (Fig. 3D), suggesting that the galectin-3-TGFbRII interaction is predominantly N-linked glycan-dependent.

Galectin-3 inhibitors prevent galectin-3 binding to av integrins and the TGFbRII subunit
To further understand the binding of galectin-3 to integrins and the TGFbRII subunit and the potential to disrupt this interaction with galectin-3 inhibitors, solution competition binding assays were performed.Target inhibition of the galectin-3 carbohydrate recognition domain (CRD) with GB0139 (blue line) or GB1107 (black line) inhibited the binding of galectin-3 to av integrins: avb1 (Fig. 4A), avb5 (Fig. 4B), and avb6 (Fig. 4C) and the TGFbRII subunit (Fig. 4D) in a concentration-dependent manner.These data confirm that the interaction of galectin-3 with both the integrins and TGFbRII are CRD-dependent.

Galectin-3 coimmunoprecipitates with b1 integrin in HLFs
In order to confirm the key SPR binding data in a cell-based system, coimmunoprecipitation (Co-IP) experiments were performed to determine whether galectin-3 is a b1 integrinbinding partner in HLFs (Fig. 5).Galectin-3 (30 kDa) was successfully coimmunoprecipitated with the b1 integrin, following pulldown (immunoprecipitation [IP]) with a b1 integrin antibody and immunoblot detection with a galectin-3-specific antibody (upper panel).No galectin-3 band was detectable in the sample immunoprecipitated with an IgG control antibody (upper panel).As expected, galectin-3 protein  pre-treated with (A) NOTT199SS b1 inhibitor (0.1-100 nM) or (B-D) galectin-3 inhibitors GB0139, GB1107, and GB1211 (1 mM) or GB0149 (0.1-10 mM) for 20 min prior to stimulation with 2 ng/ml TGF-b1 (2-h) or 50 mM LPA (4-h).Cells pretreated with 50 mM SB-431542 (ALK5 inhibitor) were included as a control demonstrating maximal inhibition of pSmad2 signaling.Western blot bands were quantified using densitometry analysis and presented as a ratio of pSmad2/tSmad2.ALK, activin receptor-like kinase; HLF, human lung fibroblast; iHBEC, immortalized human bronchial epithelial cell line; IPF, idiopathic pulmonary fibrosis; LPA, lysophosphatidic acid; TGF-b1, transforming growth factor-b1.Mechanism of galectin-3-mediated TGF-b1 activation was detected in all input and flow through (FT) control samples (upper panel).For further confirmation of the galectin-3 and b1 integrin interaction, the Co-IP experiment was also performed in the opposite direction (lower panel).The b1 integrin (130 kDa) was successfully coimmunoprecipitated following pulldown with a galectin-3 antibody, although a low level of nonspecific binding was detected in the IgG control IP (lower panel).The b1 integrin was detected in all input and FT controls (lower panel).

Galectin-3 and b1 integrin are in close proximity in IPF HLFs
To further confirm that galectin-3 and the b1 integrin colocalize closely on the cell surface of HLFs, a proximity ligation assay (PLA) was performed in both non-IPF (Fig. 6A) and IPF (Fig. 6B) HLFs.In these experiments, the punctate red fluorescent signal visible in the z-stack images is a measure of colocalization events in which galectin-3 and the b1 integrin protein were found within 40 nm of each other within the same cellular compartment.The use of fixed and unpermeabilized cells is consistent with detection of PLA signal only at the cell surface, as it is the effect of treatment with the small molecule GB0139 which shows low cell permeability at the time points used in these experiments.No PLA signal was observed in untreated non-IPF HLFs (panel A1), while stimulation of the non-IPF HLFs with TGF-b1 for 24 h resulted in the appearance of a low-level red fluorescence signal (panel A2).In HLFs isolated from fibrotic lung tissue, higher levels of PLA signals were readily apparent.This was observed in untreated IPF HLFs (panel B1) and was augmented further by stimulation with TGF-b1 (panel B2).
To confirm the previous SPR and Co-IP data suggesting that compounds which target the galectin-3 CRD could prevent the interaction of galectin-3 with integrins, PLA studies were also performed in the presence of GB0139 (0.1-10 mM).
Pretreatment of the unstimulated and TGF-b1 stimulated IPF HLFs with GB0139 reduced the level of galectin-3 and b1 integrin colocalization in a concentration-dependent manner (panels B1, B3, B5, B7 for unstimulated cells and panels B2, B4, B6, B8 for cells stimulated with TGF-b1).There was abrogation of PLA signal at 1 mM and 10 mM compared to the vehicle control in both stimulated and unstimulated cells.
This was also apparent in the unstimulated HLFs at 0.1 mM, but in the stimulated cells, it was not clearly appreciable at this inhibitor concentration, relative to the high expression levels in the vehicle control.

Ex vivo human IPF precision cut lung slice studies
The antifibrotic effect of blocking the interactions of galectin-3 with integrins and TGFbRII with GB0139 was investigated ex vivo by measurement of soluble fibrogenesis markers (Co1a1, TIMP-1, MMP-7, galectin-3, hyaluronan in precision cut lung slices (PCLuSs) derived from IPF lung explants.Treatment with GB0139 caused a concentration-dependent reduction in secreted galectin-3 compared to vehicle, yet no effect on galectin-3 was observed with control drugs SB-525334 (TGF-b type I receptor inhibitor), pirfenidone, or nintedanib (Fig. 7).The reduction in galectin-3 levels was associated with a reduction in markers of fibrosis including Co1a1, TIMP-1, and hyaluronan but not on MMP7 and at the highest concentration of GB0139 tested (10 mM) effects were comparable to that observed with SB-525334 and with currently approved IPF therapies, pirfenidone, and nintedanib (Fig. 7).In addition, following treatment of PCLuS with 10 mM GB0139 for 4 days, proteomic analysis confirmed that several signaling pathways were downregulated and these included proteins associated with canonical TGF-b1 Smad signaling (transcriptional activity of Smad2/Smad3:Smad4) in each of the IPF patient-derived PCLuS indicating that galectin-3 inhibition reduces downstream signaling from the TGFbRI (Table S2).

Discussion
We show that galectin-3 can activate TGF-b1 and that inhibiting galectin-3 can prevent agonist-induced integrin-  mediated TGF-b1 activation in fibroblasts.These data demonstrate that galectin-3 binds integrins and the TGF-b1 receptor in a glycosylation-dependent manner.Galectin-3 binding responses were higher for avb1 and avb5 than for avb6, and this may partially explain the lack of galectin-3mediated TGF-b1 activation detected in iHBECs despite the expression of high levels of avb6 integrin in epithelial cells (39).Although we were only able to detect this mechanism in fibroblasts, we cannot exclude the possibility it may also occur in epithelial cells but that higher concentrations of galectin-3 may be required at the cell surface to initiate the response.We demonstrate that galectin-3 inhibition prevents binding of galectin-3 to integrins, which in turn inhibits TGF-b1 activation in fibroblasts.Given the requirement for the close physical association of the integrin, the latent TGF-b1 complex and TGF-b1 receptor for agonist-induced integrin-mediated TGF-b1 activation (39), we hypothesise that galectin-3 promotes integrin-mediated TGF-b1 activation in lung fibroblasts by facilitating clustering of the b1 integrin and TGF-b1 receptor on their respective cell surfaces (Fig. 8).
Traction-dependent TGF-b1 activation is mediated by the integrins avb1, avb3, avb5, and avb6, which directly bind to the latency-associated peptide portion of the latent TGF-b1 complex (19,21,(40)(41)(42), and the avb1 integrin is suggested to be the principal integrin responsible for activation of latent TGF-b1 in fibroblasts (43,44).The phospholipid LPA has previously been shown to bind its G protein-coupled receptor LPA 2 to induce avb6-mediated TGF-b1 activation in epithelial cells via both Ras homolog family member A (RhoA) and Rhoassociated protein kinase (45).Although LPA has been shown to mediate primary lung fibroblast chemotaxis and proliferation via LPA 1 following lung injury, the potential contribution of LPA signaling to integrin-mediated TGF-b1 activation in fibroblasts is unexplored (46,47).
As with the b1 integrin, these findings show endogenous galectin-3 as being essential for LPA-induced TGF-b1 activation.More specifically, we confirm that it is the extracellular galectin-3 that is responsible for this mechanism as the cellimpermeable galectin-3 inhibitor (GB0149) (38) also reduced Smad2 phosphorylation in a concentration-dependent manner.This role of extracellular galectin-3 was confirmed in experiments with GB0139 in which the drug completely inhibited TGF-b1 activation as denoted by Smad2 phosphorylation despite being in use for a short incubation time (20 min).During this short time frame, target engagement of intracellular galectin-3 could not occur in response to GB0139 due to poor cell permeability (38), therefore highlighting that the effects on TGF-b1 activation are mediated by extracellular galectin-3.
Galectin-integrin binding by SPR was glycan-dependent, thus supporting the view that extracellular galectin interactions are commonly carbohydrate-dependent (48)(49)(50).Enzymatic removal of all integrin N-linked and common Olinked glycans inhibited the galectin-integrin binding interaction, while only a partial reduction in binding was observed following the removal of N-linked glycans alone.From these experiments, it was not possible to determine whether the partial binding observed following PNGase F treatment was due to O-linked glycan interactions or residual N-linked glycans.Although the efficiency of PNGase F treatment was visually confirmed by a shift in protein mobility by SDS-PAGE, this does not verify 100% N-glycan removal and therefore, some N-glycans may potentially remain on the integrin surface for galectin binding.Alternatively, the O-linked glycans may partially compensate for the higher affinity N-linked glycan interactions, when they are no longer available or there is cooperative binding between the glycan subtypes.These findings are in agreement with the published literature, whereby the carbohydrate dependency of galectin-integrin binding has been demonstrated using a number of techniques other than SPR (35,37).
The galectin-3 binding responses recorded across all three integrins were considerably higher than the theoretical maximum response for analyte-ligand binding 1:1, demonstrating that multiple individual galectins are involved in the binding interaction.As SPR measures a change in mass, it is not possible to accurately conclude if the total estimated number of galectin-3 protein is directly interacting with the immobilized ligand or if this number is a result of galectin-3 oligomerization.As galectin-3 can oligomerize through either the N-terminal or C-terminal domain, this may have resulted in multivalency of carbohydrate-binding activity, where multivalent galectin-3 binds to one or more cell surface glycans causing high avidity binding (24,25,(51)(52)(53).Alternatively, each galectin-3 protein could be binding to a single binding site on the integrin as a number of glycosylation sites have been reported or predicated from the NXS/T consensus sequence on the av (13 sites), b1 (12 sites), b5 (8 sites), and b6 subunits (9 sites) (54,55).There is currently no consensus sequence motif to enable the prediction and identification of O-glycans.However, it is less likely that each galectin protein is binding to a single glycosylation site given the estimated number of proteins and the size of galectin-3 when compared to the integrin receptor extracellular domain.It is therefore most probable that galectin-3 binds to one or more binding sites on the integrin causing subsequent selfassociation and lattice formation.Supporting this conclusion, galectin-3 has previously demonstrated positive cooperativity upon binding by SPR (56).
Galectin-3 also bound to the TGFbRII extracellular domain in a glycan-dependent manner.In contrast with integrin- following binding to TGF-b1 receptors on neighboring cells.In the absence of galectin-3 (A), the avb1 integrin and TGF-b1 receptors may not always be found in close proximity on adjacent cells, limiting the degree of TGF-b1 receptor mediated cell signaling initiated.B, in the presence of galectin-3, the galectin-3 carbohydrate-binding domain binds to the glycosylation sites on both the av integrins and the TGF-b1 receptor forming a galectin lattice at the cell surface.This galectin lattice facilitates receptor clustering, bringing the integrin and TGF-b1 receptor into close proximity, and ensuring that the active TGFb1 molecule can interact with its receptor potentiating TGF-b1 downstream signaling and phosphorylation of Smad2 and Smad3.Galectin inhibitors including GB0139 bind to the galectin-3 carbohydrate recognition domain, block the protein-glycan interactions and prevent galectin lattice-induced clustering.TGF-b1, transforming growth factor-b1.
galectin-3 interactions, PNGase F treatment of TGFbRII prevented TGFbRII-galectin-3 binding to a similar level observed with total glycan removal.This suggests that the N-linked oligosaccharides on the extracellular domain of TGFbRII predominantly mediate the galectin-3 binding interaction.This finding is consistent with a previous study from Partridge et al (2004) reporting alpha-1,6-mannosylglycoprotein 6-beta-N-acetylglucosaminyltransferase-dependent binding of galectin-3 to the TGFbRII subunit by co-IP (33).In their study, the half-life of the TGF-b1 receptor and subsequent cytokine signaling was reduced in alpha-1,6-mannosylglycoprotein 6-beta-N-acetylglucosaminyltransferase−/− cells, leading to decreased Smad2/3 nuclear translocation in response to TGF-b1 (33).This suggests that N-linked glycans and their subsequent interactions are necessary for TGF-b1 canonical signaling, following TGF-b1 ligand binding.Although it has been confirmed that TGFbRII has two N-linked glycan sites and a third is predicted by sequence analysis (57), as with the integrins, it is not possible to conclude whether galectin-3 binds to all three sites or oligomerizes upon binding.However, our experiments do confirm that protein-glycan binding events occur at the galectin-3 C terminus as small molecule inhibitors of galectin-3 (GB0139 and GB1107), both of which act via the galectin CRD, prevented galectin-3 binding to all three integrins and TGFbRII in a concentration-dependent manner.
Using a combination of co-IP and PLAs, we validated our positive SPR data generated with recombinant proteins and confirmed firstly that endogenous galectin-3 binds to the b1 integrin in primary lung fibroblasts and secondly that colocalization of the two proteins (≤40 nm) occurs on the cell surface.In the absence of TGF-b1 stimulation, such colocalization was detectable by PLA in IPF HLFs but not non-IPF cells, suggesting that the proteins are inherently more closely associated in the disease state.The failure to detect a PLA signal in the untreated non-IPF fibroblasts above the limit of detection for this assay may result from a lower level of avb1 integrin expression and/or galectin-3 at the cell surface in these cells.Fibroblasts from IPF patients are consistently shown to have increased avb1 mRNA expression compared to non-IPF controls (58).Similarly avb1 protein levels are increased in IPF lung (59) and galectin-3 expression is known to be increased in fibroblasts within fibrotic foci (28).Exposure of lung fibroblasts to a fibrotic environment, for example, stimulation with TGFb for 24 h may potentiate both avb1 expression and galectin-3 release from on fibroblasts in vitro increasing the likelihood that sufficient stochastic galectin-3-avb1 interactions are detected by PLA to visualize the signal.

Our observations are consistent with nonpeer reviewed results
showing that although colocalization of galectin-3 and the b1 integrin by PLA was also not detected in untreated lung ex vivo human lung tissue (60), it was detectable in TGF-b1-treated lung tissue samples created using a validated model of lung fibrogenesis (61).Stimulation with TGF-b1 increased the signal in both IPF and non-IPF fibroblasts, although more markedly in the IPF tissue-derived cells, and this response could be entirely abrogated by antagonism of galectin-3 glycan binding using the small molecule inhibitor GB0139.Together these findings support a mechanistic link between TGF-b1 signaling, enhanced complexation of galectin-3 with the b1 integrin via CRD:glycan interactions and fibrogenesis in disease.These data are also encouraging given that the concentration of compound used here covers the concentration range used in the phase 1/2a study and is therefore translatable to the clinic (ID: NCT02257177) (32).
Collectively, this work shows that CRD-dependent galectin-3 binding interactions are implicated in TGF-b1 signaling in human lung fibroblasts and that the inhibition of these interactions can modulate TGFb-dependent signaling and release of fibrogenic mediators in PCLuS from IPF patients.We have performed ex vivo validation studies of biophysical data, which confirm that galectin-3 interacts with the b1 integrin in HLFs.We have shown extracellular galectin-3 to be involved in LPA-induced integrin-mediated TGF-b1 activation and alluded to a role for galectin-3 in TGF-b1 signaling at the receptor level.We hypothesize that upon binding, galectin-3 self-associates to form a lattice between the avb1 integrin and TGF-b1 receptor on adjacent cells to facilitate receptor clustering.Subsequently, when TGF-b1 is activated via traction-dependent mechanisms, it is in close enough proximity to the TGF-b1 receptor to bind and signal, causing fibrogenesis.Understanding the precise role of galectin-3 in IPF pathogenesis may be critical for the continued development of more effective and selective treatments for IPF patients.

Culture of primary cells
IPF and non-IPF HLFs were obtained from explanted human lung tissue post lung biopsy following informed, written consent, and ethical review (Ethical approval numbers: 08/ H0407/1, 20/SC/0142, 10/H0402/12).Briefly, the tissue was washed with PBS and then cut into 1 mm 2 sections.Two sections were placed per well of a 6-well plate and left to adhere to the plastic for 5 to 10 min before adding culture media.Cells were cultured in Dulbecco's Modified Eagle medium high glucose (Sigma-Aldrich) supplemented with 10% fetal bovine serum (FBS) (Gibco), 4 mM L-glutamine (Sigma-Aldrich), 100 U/ml penicillin, and 100 mg/ml streptomycin (Sigma-Aldrich).Cells were grown in a humidified incubator set to 37 C with 5% CO 2 .Primary cells were tested for mycoplasma and confirmed to be mycoplasma free prior to experimentation.
Culture of immortalized cell lines iHBECs (gifted from Professor Jerry Shay, University of Texas) were cultured in keratinocyte serum-free media with L-glutamine (Gibco) supplemented with 0.2 ng/ml human recombinant epidermal growth factor (Gibco), 25 mg/ml bovine pituitary extract (Gibco), 25 mg/ml G418 disulphate (VWR), and 250 ng/ml puromycin dihydrochloride (Sigma-Aldrich).Cells were grown in a humidified incubator set to 37 C with 5% CO 2 .Immortalized cell lines were tested for mycoplasma and confirmed to be mycoplasma free prior to experimentation.

Treatment of cells
Cells were growth arrested in serum-free media for 24 h prior to stimulation in the presence or absence of inhibitors.
When used, inhibitors were applied in serum-free media for 20 min prior to stimulation.All of the small molecule glycomimetics utilized in this study, GB0139 (formerly TD139), GB1107, GB1211, or GB0149 were synthesized by the Galecto Biotech AB Medicinal Chemistry Department (Table S1) and used at a concentration range of 0.1 to 10 mM.
The ALK5/TGFbRI inhibitor SB-525334 (Sigma-Aldrich) (62,63) was used at a concentration of 50 mM.The b1 integrin inhibitor NOTT199SS (gifted) was used at a concentration range of 0.1 to 100 nM.All inhibitors were dissolved in 100% dimethyl sulfoxide (DMSO), and all cells, including untreated controls, were exposed to a DMSO concentration equivalent to that used in the highest inhibitor concentration in each experiment.

Western blot
Protein expression levels of phospho-Smad2 (pSmad2), total Smad2 (tSmad2), and GAPDH were determined by Western blot as previously described (64).Briefly, cells were lysed in protein lysis buffer and protein concentrations determined by Pierce bicinchoninic acid assay (Thermo Fisher Scientific).
Prior to commencing experiments the instrument was successfully primed and underwent routine normalization to ensure a uniform signal during the assay.Commercially available recombinant human integrins avb1, avb5, and avb6 (R&D Systems) or TGF beta receptor II protein (Abcam) were diluted in 10 mM sodium acetate pH 4 immobilization buffer and immobilized onto a Series S Sensor Chip CM5 (Cytiva) via the standard amine coupling protocol.Serial dilutions (2-fold) of galectin-3 in running buffer were injected at a 30 ml/min flow rate, 20 C, contact time: 120 s and dissociation time: 1200 s (integrins) or 300 s (TGFbRII) into flow cells containing approximately 1000 RU of integrin (equating to approximately 135 Rmax) or approximately 400 RU TGFbRII protein (equating to approximately 251 Rmax).The sensor chip surface was regenerated with 5 mM EDTA for integrin-galectin-3 experiments.All sensorgrams and kinetic plots were baselinecorrected using a flow cell with blank immobilization prior to data analysis (Fig. S1).Binding data was analyzed in GraphPad Prism, if steady state was reached then the K d value (M) and Bmax were determined by nonlinear regression (binding saturation) one-site specific binding.If steady state was not reached, then the minimum number of binding sites was estimated from the response at the highest analyte concentration tested (5000 nM).This was calculated using the following formula (56):

Minimum number of binding sites ¼ Maximum binding response Rmax
To determine the effects of deglycosylation on galectin-3 binding to integrins or TGFbRII, all glycans were removed from recombinant proteins using protein deglycosylation mix II (New England BioLabs) or N-linked glycans alone removed via PNGase F digestion (New England BioLabs) under nondenaturing conditions.Successful deglycosylation was confirmed by SDS-PAGE and deglycosylated proteins immobilized as described above.The same galectin-3 serial dilution was injected across all flow paths at a 30 ml/min flow rate, 20 C, contact time: 120 s and dissociation time: 600 s (integrins) or 300 s (TGFbRII).
SPR was used to assess the effects of small molecule galectin-3 inhibitors (GB0139 and GB1107) on galectin-3 binding to integrins or TGFbRII.Glycosylated recombinant proteins were immobilized as described above.Galectin-3 inhibitors were serially diluted (2-fold) in running buffer containing 625 nM galectin-3 across the dilution series (stocks of compound in 100% DMSO diluted to a 1.5% final concentration).The serial dilution of compound (+galectin-3) was injected across all flow paths at a 30 ml/min flow rate, 20 C, contact time: 120 s and dissociation time: 600 s (integrins) or 300 s (TGFbRII).The response values were normalized with respect to the highest RU response.Competition binding curves were analyzed in GraphPad Prism and IC 50 values determined by nonlinear regression analysis (binding saturation), and specific binding was determined with hill slope.

Coimmunoprecipitation
Co-IP was performed using the Pierce Crosslink IP Kit (Thermo Fisher Scientific).Cells were harvested by scraping in 1× PBS, pellets were lysed in ice cold IP lysis/wash buffer (300 ml/50 mg wet pellet), and protein concentrations were determined by Pierce bicinchoninic acid assay (Thermo Fisher Scientific).Lysates (650 mg/Co-IP reaction) were precleared using control agarose resin slurry (52 ml/Co-IP reaction) for 1 h at 4 C.The precleared lysates were then incubated at 4 C overnight with protein A/G plus agarose coupled and crosslinked to 10 mg of pulldown antibody: mouse anti-integrin beta 1/CD29 mAb (R&D Systems, Clone No. P5D2), rabbit antigalectin-3 mAb (Abcam, Clone No. EPR19244), mouse IgG1 isotype control mAb (R&D Systems, Clone No. 11711), or rabbit IgG isotype control mAb (Abcam, Clone No. EPR25A).FTs and wash steps were collected until confirmation of successful IP.Elution buffer was added to spin columns and eluates analyzed by Western blot for the presence of antigen.Samples were subjected to SDS-PAGE on a 4 to 12% Bis-Tris gel (Invitrogen) and transferred onto a polyvinylidene fluoride membrane (Invitrogen).After blocking for 2 h; 5% nonfat milk in PBS plus 0.05% Tween 20, the membrane was incubated overnight at 4 C with primary antibody: rabbit anti-galectin-3 mAb (Abcam, Clone No. EPR19244; 1:1000) or mouse antiintegrin beta 1 mAb (Abcam, Clone No. 12G10; 1:1000).After washes (PBS plus 0.05% Tween 20), the membrane was incubated for 1 h at room temperature with a HRP-conjugated secondary antibody: goat anti-rabbit immunoglobulins/HRP pAb (Dako; 1:3000) or goat anti-mouse IgG/HRP pAb (Invitrogen; 1:5000).All primary and secondary antibodies used were diluted in blocking buffer.The membrane was then incubated with ECL Western blot detection reagent and visualized using chemiluminescent detection.

Proximity ligation assay
Cells were growth arrested in serum-free media for 24 h prior to experiments.After serum starvation, cells were stimulated with 2 ng/ml recombinant human TGF-b1 protein (R&D Systems) for 24 h and then pretreated with 0.1 to 10 mM galectin-3 inhibitor (GB0139) for 20 min.PLA was performed using the Duolink In Situ Red Kit Mouse/Rabbit (Sigma-Aldrich).Prior to PLA, cells were fixed with 4% paraformaldehyde for 10 min and incubated with blocking solution (1× PBS pH 7.4, 10% FBS) for 2 h at 37 C. Slides were then incubated overnight at 4 C with primary antibody (5 mg/ml): Mechanism of galectin-3-mediated TGF-b1 activation buffer (1:80) and added to slides for 2 h at 37 C after wash steps (wash buffer A).After washes (wash buffer B), slides were then stained with 4 0 ,6-diamidino-2-phenylindole solution and mounted with fluoroshield for imaging (Leica LSM 980 Airyscan2).
Serial z-stack images of individual cells were collected by confocal imaging and data presented as the z-stack with maximum intensity projection.
Ex vivo human IPF precision cut lung slice studies IPF PCLuS experiments were completed as previously described (66).Briefly, human fibrotic lung tissue was sourced ethically from human explants from three patients with IPF undergoing lung transplantation.Tissue was obtained in accordance with local human biological sample management procedures, which were approved by regional ethics approval (11/NE/0291).All patients were male and had their IPF diagnosis confirmed based on medical history and evaluation of their explanted lung tissue by a board-certified respiratory pathologist (IPF donor demographics: age 62, 64, and 65; FEV1 1.85, 2.37, and 1.53; for donors 1, 2, and 3, respectively).Tissue was inflated with 2 to 3% low boiling point agarose and allowed to set at 4 C. PCLuS were then cut at 400 mm on a vibrating microtome and cultured in small airway epithelial cell growth medium containing bovine pituitary extract (0.004 ml/ml), recombinant human insulin (5 mg/ml), hydrocortisone (0.5 mg/ml), epinephrine (0.5 mg/ml), triiodo-L-thyronine (6.7 ng/ml), recombinant human transferrin (10 mg/ml), retinoic acid (0.1 ng/ml), bovine serum albumin fatty acid free (2.5 mg/ml).PCLuS were rested for 48 h prior to treatment with inhibitors (SB-525334 (ALK5 inhibitor) [Sigma], pirfenidone [Cambridge Biosciences], nintedanib [Cambridge Biosciences], and GB0139 for 4 days with media replaced every 24 h.Soluble mediators released were measured daily using R&D Duoset ELISA kits according to the manufacturer's protocol.In addition, analysis of experimental proteomics data derived from mass spectrometry testing on the PCLuS at the end of the study (day 6) were completed as previously described (see Supplemental methods).Functional pathway enrichment analysis was performed using the analysis tools at Reactome (https://reactome.org),where upregulated and downregulated proteins from all donors were evaluated for pathway enrichment (see Supplemental methods).

Statistical analysis
Data analyzed in GraphPad Prism and presented as mean with individual replicates shown.

Figure 4 .
Figure 4. Galectin-3 inhibitors block av integrin and TGFbRII binding to galectin-3.Solution competition binding assays performed with the galectin-3 inhibitor GB0139 (blue) or GB1107 (black) for av integrins: (A) avb1, (B) avb5, and (C) avb6 or (D) TGFbRII in the presence of galectin-3 at 625 nM.Response values are normalized with respect to the highest binding response (DMSO control) and competitive inhibition graphs plotted in GraphPad Prism.IC 50 values were calculated by nonlinear regression analysis (binding saturation), and specific binding was calculated with hill slope.DMSO, dimethyl sulfoxide; TGF, transforming growth factor.

Figure 5 .
Figure 5. Binding of galectin-3 to b1 integrin is detected in lung fibroblasts.Representative Western blots showing coimmunoprecipitation of galectin-3 and the b1 integrin.Whole-cell protein lysates (650 mg/IP reaction) from untreated non-IPF HLFs p6 (N = 3) were immunoprecipitated with an anti-b1 integrin antibody (10 mg/IP reaction) and immunoblotted for galectin-3 (upper panel) or immunoprecipitated with an anti-galectin-3 antibody (10 mg/IP reaction) and immunoblotted for the b1 integrin (lower panel).Co-IP input, FT, and wash steps loaded as controls.Proteins separated by reducing SDS-PAGE and target protein size estimated from the marker migration pattern.Co-IP, coimmunoprecipitation; FT, flow through; IP, immunoprecipitation; IPF, idiopathic pulmonary fibrosis; HLF, human lung fibroblast.

Figure 7 .
Figure 7. Galectin-3 inhibitors reduce secretion of fibrotic markers in IPF lung tissue.The daily average change in the level of fibrotic markers secreted by IPF patient-derived PCLuS was measured in response to treatment with inhibitor.PCLuS were treated with galectin-3 inhibitor GB0139 (0.3-10 mM), 10 mM SB-525334, 2.5 mM pirfenidone, or 2.5 mM nintedanib for 4 days with media replaced every 24 h.Levels of secreted galectin-3, Col1a1 (A), TIMP1 (B), hyaluronan (C), and MMP7 (D) were measured daily, and the average value for each mediator (N = 3 donors) is presented as % change in drug treated samples compared with vehicle control.Comparative levels of galectin-3 are shown in each graph.IPF, idiopathic pulmonary fibrosis; PCLuS, precision cut lung slice.