Mechanobiology and Resolution of Lung Fibrosis.

The Lung Macrophage: A Jack of All Trades

Idiopathic pulmonary fibrosis (IPF) is a progressive disease thought to result from impaired lung repair following injury and is strongly associated with aging. While vascular alterations have been associated with IPF previously, the contribution of lung vasculature during injury resolution and fibrosis is not well understood. To compare the role of endothelial cells (ECs) in resolving and non‐resolving models of lung fibrosis, we applied bleomycin intratracheally to young and aged mice. We found that injury in aged mice elicited capillary rarefaction, while injury in young mice resulted in increased capillary density. ECs from the lungs of injured aged mice relative to young mice demonstrated elevated pro‐fibrotic and reduced vascular homeostasis gene expression. Among the latter, Nos3 (encoding the enzyme endothelial nitric oxide synthase, eNOS) was transiently upregulated in lung ECs from young but not aged mice following injury. Young mice deficient in eNOS recapitulated the non‐resolving lung fibrosis observed in aged animals following injury, suggesting that eNOS directly participates in lung fibrosis resolution. Activation of the NO receptor soluble guanylate cyclase in human lung fibroblasts reduced TGFβ‐induced pro‐fibrotic gene and protein expression. Additionally, loss of eNOS in human lung ECs reduced the suppression of TGFβ‐induced lung fibroblast activation in 2D and 3D co‐cultures. Altogether, our results demonstrate that persistent lung fibrosis in aged mice is accompanied by capillary rarefaction, loss of EC identity, and impaired eNOS expression. Targeting vascular function may thus be critical to promote lung repair and fibrosis resolution in aging and IPF.

Matrix metalloprotease 13 (MMP13) deficiency in pulmonary fibrosis has described contradictory phenotypes on inflammatory and fibrotic responses after lung injury, and its role during lung fibrosis resolution is still undefined. MMP13 has been considered the main collagenase in rodents, and the remodeling of fibrillar collagen is widely attributed to the action of this enzyme. In this study we aimed to explore the role of MMP13 during lung fibrosis progression and resolution. Lung fibrosis was induced by intratracheal instillation, and inflammatory, fibrotic, and resolution stages were evaluated in Mmp13-null and wild-type (WT) mice. Bronchoalveolar lavage fluid was taken for cytokine array analysis and activity of gelatinases. Our results showed that MMP13 is upregulated mainly during two stages after lung injury, inflammation and resolution of fibrosis, and it is mainly expressed by alveolar and interstitial macrophages. Mmp13-null mice exhibited more extensive inflammation at 7 days after bleomycin treatment, and it was characterized by increased macrophage infiltration and significant alterations in proinflammatory cytokines. We also documented that Mmp13-deficient mice experienced more severe and prolonged lung fibrosis compared with WT mice. Delayed resolution in Mmp13-deficient lungs was characterized by a decreased overall collagenolytic activity and persistent fibrotic foci associated with emphysema-like areas. Together, our findings indicate that MMP13 plays an antifibrotic role and its activity is crucial in lung repair and restoration of tissue integrity during fibrosis resolution.

Idiopathic pulmonary fibrosis is a devastating lung disease with limited treatment options. The signaling molecule adenosine is produced in response to injury and serves a protective role in early stages of injury and is detrimental during chronic stages of disease such as seen in lung conditions such as pulmonary fibrosis. Understanding the association of extracellular adenosine levels and the progression of pulmonary fibrosis is critical for designing adenosine based approaches to treat pulmonary fibrosis. The goal of this study was to use various models of experimental lung fibrosis to understand when adenosine levels are elevated during pulmonary fibrosis and whether these elevations were associated with disease progression and severity. To accomplish this, extracellular adenosine levels, defined as adenosine levels found in bronchioalveolar lavage fluid, were determined in mouse models of resolvable and progressive pulmonary fibrosis. We found that relative bronchioalveolar lavage fluid adenosine levels are progressively elevated in association with pulmonary fibrosis and that adenosine levels diminish in association with the resolution of lung fibrosis. In addition, treatment of these models with dipyridamole, an inhibitor of nucleoside transporters that potentiates extracellular adenosine levels, demonstrated that the resolution of lung fibrosis is blocked by the failure of adenosine levels to subside. Furthermore, exacerbating adenosine levels led to worse fibrosis in a progressive fibrosis model. Increased adenosine levels were associated with elevation of IL-6 and IL-17, which are important inflammatory cytokines in pulmonary fibrosis. These results demonstrate that extracellular adenosine levels are closely associated with the progression of experimental pulmonary fibrosis and that this signaling pathway may mediate fibrosis by regulating IL-6 and IL-17 production.

Rationale: Idiopathic pulmonary fibrosis (IPF) is a chronic lung disease characterized by tissue stiffening and loss of organ function. Chronic alveolar injury and failure to repair are intrinsic to IPF pathogenesis. IPF lungs exhibit vascular abnormalities, including perivascular inflammation and vascular leak, suggesting that aberrant vascular repair contributes to IPF progression. We found that lung general capillary (gCap) endothelial cells (ECs) acquire an activated state in response to bleomycin injury that was characterized by tropomyosin receptor kinase B (TrkB) expression. TrkB+ capillary ECs were also detected in IPF lungs, in regions characterized by tissue remodeling. TrkB and its ligand BDNF have established roles in neurite outgrowth and angiogenesis, however their contribution to lung vascular repair and fibrosis is poorly understood. Here, we investigate the role of BDNF/TrkB pathway in lung gCap EC repair and fibrosis after injury. Methods: TrkB expression was assessed by IHC in mouse lungs exposed to a single dose of bleomycin (self-resolving model) or repetitive doses of bleomycin (persistent fibrosis). The TrkB antagonist ANA-12 and agonist (7,8-DHF) were administered to mice receiving a single dose of bleomycin to assess whether manipulation of TrkB pathway interfere with the lung fibrogenesis. TrkB was overexpressed in human lung microvascular ECs using lentiviral transduction followed by BDNF stimulation and Western blotting to assess TrkB pathway activation. Results: IHC analysis demonstrated that TrkB was highly expressed in mouse lungs receiving multiple bleomycin doses compared to those receiving a single bleomycin dose. Interestingly, TrkB was expressed in lung areas with abundant collagen deposition. Inhibition of TrkB pathway using ANA-12 delayed lung fibrosis resolution and worsened vascular leak following a single dose of bleomycin. In contrast, activation of TrkB pathway by DHF accelerated lung fibrosis resolution and attenuated vascular leak. Mechanistically, we found that TrkB phosphorylates and inhibits YAP1, a member of the Hippo pathway implicated lung fibrosis, angiogenesis, and vascular permeability. BDNF treatment of IPF-derived ECs promoted YAP phosphorylation, reduced nuclear YAP, and attenuated vascular permeability. Conclusion: This study demonstrates that lung capillary BDNF/TrkB pathway is essential for restoring capillary homeostasis after injury. TrkB expression in lung gCap ECs correlates with fibrotic regions and collagen deposition, with higher levels observed in progressive fibrosis models. Pharmacological inhibition of TrkB delayed fibrosis resolution and exacerbated vascular leak, while TrkB activation accelerated repair and reduced vascular permeability. These findings underscore the therapeutic potential of targeting the BDNF/TrkB pathway to promote vascular repair and mitigate IPF.

Two-Way Conversion between Lipogenic and Myogenic Fibroblastic Phenotypes Marks the Progression and Resolution of Lung Fibrosis

Recent studies have presented compelling evidence that it is not tissue-resident, but rather monocyte-derived alveolar macrophages (TR-AMs and Mo-AMs, respectively) that are essential to development of experimental lung fibrosis. However, whether apolipoprotein E (ApoE), which is produced abundantly by Mo-AMs in the lung, plays a role in the pathogenesis is unclear. In this study, we found that pulmonary ApoE was almost exclusively produced by Mo-AMs in mice with bleomycin-induced lung fibrosis. We showed that, although ApoE was not necessary for developing maximal fibrosis in bleomycin-injured lung, it was required for the resolution of this pathology. We found that ApoE directly bound to Collagen I and mediated Collagen I phagocytosis in vitro and in vivo, and this process was dependent on low-density lipoprotein receptor-related protein 1 (LPR1). Furthermore, interference of ApoE/LRP1 interaction impaired the resolution of lung fibrosis in bleomycin-treated WT mice. In contrast, supplementation of ApoE promoted this process in ApoE-/- animals. In conclusion, Mo-AM-derived ApoE is beneficial to the resolution of lung fibrosis, supporting the notion that Mo-AMs may have distinct functions in different phases of lung fibrogenesis. The findings also suggest a potentially novel therapeutic target for treating lung fibrosis, to which effective remedies remain scarce.

Idiopathic pulmonary fibrosis (IPF) is a progressive and fatal lung disease of unknown etiology with no cure. A better understanding of the disease processes and identification of druggable targets will benefit the development of effective therapies for IPF. We previously reported that MDM4 promoted lung fibrosis through the MDM4-p53-dependent pathway. However, it remained unclear whether targeting this pathway would have any therapeutic potential. In this study, we evaluated the efficacy of XI-011, a small molecular inhibitor of MDM4, for treating lung fibrosis. We found that XI-011 significantly reduced MDM4 expression and increased the expression of total and acetylated p53 in primary human myofibroblasts and a murine fibrotic model. XI-011 treatment resulted in the resolution of lung fibrosis in mice with no notable impact on normal fibroblast death or the morphology of healthy lungs. Based on these findings, we propose that XI-011 might be a promising therapeutic drug candidate for treating pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a progressive and fatal disease with undefined etiology and minimally effective therapies. The greatest risk factor for developing IPF is aging. The central paradigm to developing antifibrotic drugs for the last half century has focused on directly targeting proliferative lung fibroblasts. However, recent high-resolution analyses of IPF patient lungs suggests disease unique populations of resident lung cells are enriched for markers of senescence. Published work by our group and others further supports that senescent cells are key drivers of fibrosis and may provide an opportunity to develop an effective antifibrotic drug. Multiple naturally derived flavonoids can selectively induce apoptosis in senescent cells (senolytic) and improve end points in models of lung fibrosis; however, these natural phytochemicals are not structurally optimized to maximize their translational potential. Inspired by this opportunity we have performed hit-to-lead studies and medicinal chemistry optimization to generate a novel synthetic flavanoid (F-4N) with ∼ 50× greater senolytic potency in vitro- compared to fisetin or quercetin, two naturally derived senolytic flavonols. Furthermore, in bleomycin injury models of lung fibrosis we have shown treatment with F-4N (10 mg/kg-30 mg/kg, daily) promotes reduced senescence burden, resolution of chronic lung fibrosis, and markers of enhanced alveolar epithelial repair.

To date, phenotyping and disease course prediction in idiopathic pulmonary fibrosis (IPF) primarily relies on lung function measures. Blood biomarkers were recently proposed for diagnostic and outcome prediction in IPF, yet their correlation with lung function and histology remains unclear. Here, we comprehensively assessed biomarkers in liquid biopsies and correlated their abundance with lung function and histology during the onset, progression, and resolution of lung fibrosis, with the aim to more precisely evaluate disease progression in the preclinical model of bleomycin-induced pulmonary fibrosis in vivo. Importantly, the strongest correlation of lung function with histological extent of fibrosis was observed at day 14, whereas lung function was unchanged at days 28 and 56, even when histological assessment showed marked fibrotic lesions. Although matrix metalloproteinase-7 (MMP-7), MMP-9, and PAI-1 were significantly elevated in broncheoalveolar lavage of fibrotic mice, only soluble ICAM-1 (sICAM-1) was elevated in the peripheral blood of fibrotic mice and was strongly correlated with the extent of fibrosis. Importantly, tissue-bound ICAM-1 was also elevated in lung homogenates, with prominent staining in hyperplastic type II alveolar epithelial and endothelial cells. In summary, we show that lung function decline is not a prerequisite for histologically evident fibrosis, particularly during the onset or resolution thereof. Plasma levels of sICAM-1 strongly correlate with the extent of lung fibrosis, and may thus be considered for the assessment of intraindividual therapeutic studies in preclinical studies of pulmonary fibrosis.

Resveratrol mitigates miR-212-3p mediated progression of diesel exhaust-induced pulmonary fibrosis by regulating SIRT1/FoxO3

<b>Rationale:</b> Idiopathic Pulmonary Fibrosis (IPF) is an age-related lung disease. Our group and others had identified the accumulation of senescent fibroblasts and epithelial cells in the lungs of IPF patients. Activated Natural killer lymphocytes (NK) cells have been implicated in the clearance of senescent cells. <b>Methods:</b> The proportion of NK cells and their senescent status was evaluated by flow cytometry. The effect of the lung microenvironment was evaluated using lung fibroblast conditioned media. The NK migration capacity was determine by the expression of chemokine receptors, presence of plasma chemokines and transwell migration assays. A mouse model of NK depletion and bleomycin inducing lung fibrosis was used to asses the relation between the NK cells and the senescent cell clearance. <b>Results:</b> The proportion of NK cells in the IPF lung is reduced and have an impaired gene expression. IPF lung NK cells have increased immunosenescence with IL-6 production. The IPF lung fibroblast conditioned media increases the percent of senescent cells and reduces NK cytotoxic activity. The presence of IL-15 was able to revert the detrimental effect of the IPF CM. Plasma cytokines showed a decreased in NK lung recruitment chemokines and lung NK cells have a reduction in the expression of CCR2. This impaired recruitment may be inducing their accumulation in the blood. In vivo lung fibrosis mouse model with NK depletion showed the importance of NK cells in the resolution of lung fibrosis and clearance of senescent cells. <b>Conclusions:</b> We propose that defects in NK activity could be one of the mechanisms responsible for the perpetration of the accumulation of senescent cells in IPF lungs.

ABSTRACTLung development is a tightly regulated process that progresses through five distinct stages: embryonic, pseudoglandular, canalicular, saccular, and alveolar. Each stage is characterized by coordinated cellular interactions and structural changes driven by signals from epithelial, endothelial, and mesenchymal cells. The extracellular matrix (ECM) plays a pivotal role in this process, acting as both a structural scaffold and a dynamic regulator of cell behavior. Key ECM components, including collagens, elastin, proteoglycans, and glycosaminoglycans, provide the framework for tissue integrity while modulating critical signaling pathways essential for lung branching morphogenesis, alveolar formation, and vascular development. Disruptions in ECM dynamics are associated with pathological conditions such as bronchopulmonary dysplasia, chronic obstructive pulmonary disease, and pulmonary fibrosis. In these diseases, aberrant ECM remodeling leads to tissue stiffening, scarring, and impaired lung function. The ECM plays a critical role in storing and regulating the activity of growth factors, such as FGFs and VEGFs, thereby shaping cellular proliferation, differentiation, and repair processes. Understanding the complex interplay between ECM components and cellular signaling during normal and aberrant lung development provides valuable insights into therapeutic strategies aimed at restoring lung function. This review explores the multifaceted roles of proteoglycans and hyaluronan in lung development and disease, emphasizing its impact on tissue architecture, signaling environments, and repair processes.

Cutting edge: FasL+ immune cells promote resolution of fibrosis

Pulmonary fibrosis is characterized by progressive scarring of the lung parenchyma surrounding the alveoli that leads to respiratory failure and ultimately death. The idiopathic form of pulmonary fibrosis (or IPF), while rare in incidence, affects over 100,000 people in the US alone, and 30,000–40,000 new cases arise worldwide every year.1 Prognosis for IPF remains poor with a median survival of less than 5 years after initial diagnosis. Currently there is no cure, although, pharmacologic treatments (Pirfenidone/Nintedanib) have been shown to slow down disease progression by blocking myofibroblast activation, collagen synthesis and the transforming growth factor-beta (TGF-β) pathway.2 TGF-β signaling is well documented to serve a central role in mediating fibrogenesis; however, TGF-β is critical in biological development and specifically targeting this pathway may lead to undesirable consequences. In addition, IPF has multiple etiologies; therefore, developing therapeutic targets has remained a challenge (Figure 1). Some non- pharmacologic approaches have focused on cellular therapy, or mesenchymal stem cell (MSC) infusion, as a treatment to regulate lung injury and repair,3 but current and approved treatment strategies are mainly symptomatic and include oxygen therapy, exercise for pulmonary rehabilitation, and lung transplantation.2 Aging factors such as fibroblast growth factor 23 (FGF23) and its co-receptor/anti-aging hormone, klotho (KL), have also been suggested as possible targets to promote anti-fibrotic and anti-inflammatory responses.4 Pharmacologic approaches, on the other hand, have focused on inhibiting pro-fibrotic mediators, one of which is galectin-3, a beta-galactoside binding protein that interacts with multiple growth factor receptors to activate profibrotic signaling pathways. TD139 (currently known as GB0139), a small molecule galectin-3 inhibitor, was shown to be safe and well tolerated in IPF subjects, who showed improvements in plasma biomarkers of inflammation.5 In addition, pharmacologic approaches focusing on pentraxin supplementation have also been reported in IPF. PTX2, a member of the PTX superfamily, has been targeted as a novel therapeutic option to treat IPF. PTX2 has been shown to modulate wound healing and fibrotic remodeling of injured tissue through binding to cellular debris, enhancing phagocytosis by leukocytes, and inhibiting the production of TGF-β.6 PRM-151 (Zinpentraxin Alfa), a recombinant human PTX2 protein, has been proposed for pharmacologic treatment of treat IPF and is currently in a phase 3 trial with an estimated completion date of March 2023. In the Journal of Clinical and Translational Medicine, Chi et al. evaluated the pentraxin 3 (PTX3)/CD44 axis as an effective strategy to treat lung injury-induced fibrosis. PTX3 is an acute phase protein that participates in resistance against inflammation and microorganisms. CD44 is a transmembrane receptor that interacts with various ligands and growth factor receptors to facilitate cellular processes. They note that PTX3 is a novel biomarker of inflammation and elevated serum PTX3 levels are associated with extracellular matrix (ECM) formation thus providing the impetus for their present study. The authors show that PTX3 binding to its receptor, CD44, activates downstream signaling pathways NF-κB, PI3K/AKT, and MAPKs promoting lung fibroblast-mediated fibrogenesis. Blocking PTX3 and CD44 interaction in vivo and in vitro was suggested to effectively reduce myofibroblast activation, ECM formation, and an overall reduction in lung injury. Compared to the current treatment regimen, such as pirfenidone and nintedanib, their αPTX3i treatment showed similar effectiveness in lung injury resolution, pulmonary function, and overall survival in their in vivo experiments. These findings suggest that PTX3/CD44 may be a potential option for fibrosis resolution. However, the role of PTX3/CD44 in fibrogenesis and/or how its blockade contributes to resolution still remain unclear. It is known that PTX3 is produced by a wide variety of cell types and the PTX3/CD44 axis has been explored in other diseases. In influenza infection, PTX3 responds to pro-inflammatory stimuli and binds to a variety of viral strains to mediate host anti-viral responses.7 In cancer, PTX3 antibodies and synthetic peptides can disrupt the PTX3/CD44 interaction and attenuate/restrict the stemness and metastasis/invasion of tripe-negative breast cancers.8 These studies detail the importance of the PTX3/CD44 axis and its contribution to a wide array of inflammatory- and non-inflammatory-mediated processes, which may impact fibrosis and fibrosis resolution. Does PTX3/CD44 blockade, which leads to fibrosis resolution in animal models, translate to humans? Are there limitations to the pre-clinical models used? Bleomycin-induced pulmonary fibrosis in young mice has been shown to resolve over time whereas a non-resolving aged mouse or a repetitive bleomycin instillation model would better recapitulate human pulmonary fibrosis.9, 10 What are the side-effects of PTX3/CD44 blockade? As stated above, PTX3 is produced in multiple cell types, and blockade may cause unwarranted side effects. PTX3 is also involved in the innate immunity response, and blocking its function may lead to immune-wide complications. Is there a connection between PTX2 and PTX3 in IPF? PTX2 is downregulated in IPF, but PTX3 is upregulated; therefore, both have differing therapeutic intervention strategies. PTX2 therapy centers on intravenous infusion of exogenous proteins to delay fibrosis whereas Chi et al. focuses on blocking PTX3 function to prevent or resolve fibrosis. Overall, Chi et al. characterize PTX3 as a novel and attractive target for anti-fibrotic therapy and its underlying mechanism, but more studies are needed to understand the role of the PTX3/CD44 axis in prevention and resolution of pulmonary fibrosis as well as its potential as a therapeutic target in IPF. The authors declare no conflict of interest.