Vascular-Parenchymal Cross-Talk Promotes Lung Fibrosis through BMPR2 Signaling.

Rationale: Idiopathic pulmonary fibrosis (IPF) is a fatal lung disease characterized by progressive lung scarring. IPF-related pulmonary vascular remodeling and pulmonary hypertension (PH) result in a particularly poor prognosis. Objectives: To study the pathogenesis of vascular remodeling in fibrotic lungs and its contribution to progression of fibrosis. Methods: We used an experimental model of lung fibrosis associated with PH by transient overexpression of active TGF-ß1 (transforming growth factor-ß1). Samples from patients with fibrotic lung diseases were analyzed in depth using immunostaining, gene expression, and gene mutations. Measurements and Main Results: We found a reduction in endothelial cells (ECs) and activation of vascular smooth muscle cells (VSMCs) in fibrotic lungs. Coculturing fibroblasts with VSMCs or ECs from fibrotic lungs induced fibrotic phenotypes in fibroblasts. IPF fibroblasts induced EC death and activation of VSMCs in coculture systems. Decreased concentrations of BMPR2 (bone morphogenic protein receptor 2) and its signaling were observed in ECs and VSMCs from fibrotic lungs in both rats and humans. On fibroblasts treated with media from VSMCs, BMPR2 suppression in VSMCs led to fibrogenic effects. Tacrolimus activated BMPR2 signaling and attenuated fibrosis and PH in rodent lungs. Whole-exome sequencing revealed rare mutations in PH-related genes, including BMPR2, in patients with IPF undergoing transplantation. A unique missense BMPR2 mutation (p.Q721R) was discovered to have dysfunctional effects on BMPR2 signaling. Conclusions: Endothelial dysfunction and vascular remodeling in PH secondary to pulmonary fibrosis enhance fibrogenesis through impaired BMPR2 signaling. Tacrolimus may have value as a treatment of advanced IPF and concomitant PH. Genetic abnormalities may determine the development of PH in advanced IPF.

MicroRNA-326 negatively regulates CD155 expression in lung adenocarcinoma.

Treatment with immune checkpoint inhibitors induces a durable response in some patients with non-small-cell lung cancer, but eventually gives rise to drug resistance. Upregulation of CD155 expression is implicated as one mechanism of resistance to programmed death receptor-1 (PD-1)/PD-1 ligand (PD-L1) inhibitors, and it is therefore important to characterize the mechanisms underlying regulation of CD155 expression in tumor cells. The aim of this study was to identify microRNAs (miRNAs) that might regulate CD155 expression at the posttranscriptional level in lung cancer. Comprehensive miRNA screening with target prediction programs and a dual-luciferase reporter assay identified miR-346, miR-328-3p, miR-326, and miR-330-5p as miRNAs that bind to the 3'-UTR of CD155 mRNA. Forced expression of these miRNAs suppressed CD155 expression in lung cancer cell lines. Immunohistochemical staining of CD155 in tissue specimens from 57 patients with lung adenocarcinoma revealed the median tumor proportion score for CD155 to be 68%. The abundance of miR-326 in these specimens with a low level of CD155 expression was significantly greater than in specimens with a high level (p < 0.005). Our results thus suggest that miR-326 negatively regulates CD155 expression in lung adenocarcinoma and might therefore play a role in the development of resistance to PD-1/PD-L1 inhibitors.

Intrinsic BMP inhibitor Gremlin regulates alveolar epithelial type II cell proliferation and differentiation.

Type 1 alveolar epithelial cells (AT1s) and type 2 alveolar epithelial cells (AT2s) regulate the structural integrity and function of alveoli. AT1s mediate gas exchange, whereas AT2s serve multiple functions, including surfactant secretion and alveolar repair through proliferation and differentiation into AT1s as progenitors. However, mechanisms regulating AT2 proliferation and differentiation remain unclear. Here we demonstrate that Gremlin, an intrinsic inhibitor of bone morphogenetic protein (BMP), induces AT2 proliferation and differentiation. Transient overexpression of Gremlin in rat lungs by adenovirus vector delivery suppressed BMP signaling, induced proliferation of AT2s and the production of Bmp2, which in turn led to the recovery of BMP signaling and induced AT2 differentiation into AT1s. Bleomycin-induced lung injury upregulated Gremlin and showed a similar time course of biomarker expression comparable to the adenovirus model. TGF-ß and IL-1ß induced Gremlin expression in fibroblasts. Taken together, our findings implicate that Gremlin expression during lung injury leads to precisely timed inhibition of BMP signaling and activates AT2s, leading to alveolar repair.

Connective-Tissue Growth Factor Contributes to TGF-ß1-induced Lung Fibrosis.

Idiopathic pulmonary fibrosis is a fatal lung disease characterized by progressive and excessive accumulation of myofibroblasts and in the lung. Connective-tissue growth factor (CTGF) exacerbates pulmonary fibrosis in radiation-induced lung fibrosis, and in this study, we demonstrate upregulation of CTGF in a rat lung fibrosis model induced by an adenovirus vector encoding active TGF-ß1 (AdTGF-ß1). We show that CTGF is also upregulated in patients with idiopathic pulmonary fibrosis. Expression of CTGF was upregulated in vascular smooth muscle cells cultured from fibrotic lungs on Days 7 and 14 as well as endothelial cells sorted from fibrotic lungs on Days 14 and 28. These findings suggest contributions of different cells in maintaining the fibrotic phenotype during fibrogenesis. Treatment of fibroblasts with recombinant CTGF along with TGF-ß increases profibrotic markers in fibroblasts, confirming the synergistic effect of recombinant CTGF with TGF-ß in inducing pulmonary fibrosis. Also, the fibrotic extracellular matrix upregulated CTGF expression, compared with the normal extracellular matrix, suggesting that not only profibrotic mediators but also a profibrotic environment contributes to fibrogenesis. We also showed that pamrevlumab, a CTGF inhibitory antibody, partially attenuates fibrosis in the model. These results suggest that pamrevlumab could be an option for treatment of pulmonary fibrosis.

Fibrotic extracellular matrix induces release of extracellular vesicles with pro-fibrotic miRNA from fibrocytes.

RATIONALE: Extracellular vesicles (EVs) are small lipid vesicles, and EV-coupled microRNAs (miRNAs) are important modulators of biological processes. Fibrocytes are circulating bone marrow-derived cells that migrate into the injured lungs and contribute to fibrogenesis. The question of whether EV-coupled miRNAs derived from fibrocytes are able to regulate pulmonary fibrosis has not been addressed yet. METHODS: Pulmonary fibrosis was induced in rats by intratracheal administration of an adenoviral gene vector encoding active transforming growth factor-ß1 (TGF-ß1) or control vector. Primary fibrocytes and fibroblasts were cultured from rat lungs and were sorted by anti-CD45 magnetic beads. Human circulating fibrocytes and fibrocytes in bronchoalveolar lavage fluid (BALF) were isolated by fibronectin-coated dishes. Fibrocytes were cultured on different stiffness plates or decellularised lung scaffolds. We also determined the effects of extracellular matrix (ECM) and recombinant TGF-ß1 on the cellular and EV-coupled miRNA expression of fibrocytes. RESULTS: The EVs of fibrocytes derived from fibrotic lungs significantly upregulated the expression of col1a1 of fibroblasts. Culturing on rigid plates or fibrotic decellularised lung scaffolds increased miR-21-5 p expression compared with soft plates or normal lung scaffolds. Dissolved ECM collected from fibrotic lungs and recombinant TGF-ß1 increased miR-21-5 p expression on fibrocytes, and these effects were attenuated on soft plates. Fibrocytes from BALF collected from fibrotic interstitial pneumonia patients showed higher miR-21-5 p expression than those from other patients. CONCLUSIONS: Our results indicate that ECM contributes to fibrogenesis through biomechanical and biochemical effects on miRNA expression in fibrocytes.

Immune-checkpoint profiles for T cells in bronchoalveolar lavage fluid of patients with immune-checkpoint inhibitor-related interstitial lung disease.

Immune-checkpoint inhibitors (ICIs) have improved clinical outcomes and are becoming a standard treatment for many cancer types. However, these drugs also induce immune-related adverse events, among which interstitial lung disease (ILD) is potentially fatal. The underlying mechanism of ILD induction by ICIs is largely unknown. With the use of flow cytometry, we determined the expression levels of the immune-checkpoint proteins PD-1, TIM-3, TIGIT, LAG-3 and PD-L1 in T cells of bronchoalveolar lavage fluid (BALF) from patients with ICI-related ILD and compared them with those for patients with sarcoidosis or with ILD related to connective tissue disease or cytotoxic drug use. The proportions of CD8+ T cells positive for both PD-1 and TIM-3 or for TIGIT in BALF were significantly higher for ICI-related ILD patients than for those with other types of ILD. A prominent increase in the proportion of PD-1+PD-L1+ cells among CD8+ T cells was also apparent in BALF of a patient with a fatal case of ICI-related ILD, and the proportion of such cells was positively correlated with the grade of ICI-related ILD. Our data reveal the immune-checkpoint profiles of T cells in ICI-related ILD and may provide mechanistic insight into the development of this adverse event.

A measuring method for occupancy of immune checkpoint inhibitors in the cell surface.

Monoclonal antibodies, including immune-checkpoint inhibitors, are becoming popular in treatments of many cancers and connective tissue diseases. However, little is known about how long the antibodies combine with antigens on targeted cells or how this duration of binding associates with therapeutic efficacy or potential adverse events. Here, we show the principle and the results of a feasible method for measuring the antibodies' occupancy on the targeted cells using two different detecting antibodies in conjunction with different fluorochromes. Nivolumab occupancy was measured using two detecting antibodies, MIH4 and EH12.2, which are commercially available in vitro (programmed cell death-1 [PD-1] expressing the cell line MIT9 and human T cells) and in T cells from patients treated with nivolumab. Our method has potential for use as a simple and feasible monitoring system in the clinical setting.

Insights into pathogenesis and clinical implications in myositis-associated interstitial lung diseases.

PURPOSE OF REVIEW: Interstitial lung diseases (ILDs) have been reported to be associated with myositis (including polymyositis and dermatomyositis). These myositis-associated ILDs carry significant morbidity and mortality. This review summarizes recent findings on myositis-associated ILD with a focus on pathogenesis and emerging treatment. RECENT FINDINGS: Recent advances in genetics have revealed 22 myositis-associated genome-wide loci, which were significantly enriched in regulatory regions in immune cells. An analysis of such disease-associated loci elucidated potential drug targets (e.g., TYK2 targeted by tofacitinib). In another study, an intronic variant in WDFY4 in association with clinically amyopathic dermatomyositis (CADM) had an effect for higher expression of a truncated WDFY4 isoform. Truncated WDFY4 markedly enhanced the MDA5-mediated NF-κB activation and cell apoptosis, indicating the dysregulated WDFY4-MDA5 pathway as a novel pathogenesis of CADM. As a novel strategy, tofacitinib treatment showed a promising improvement in survival and clinical features of CADM-associated ILD. SUMMARY: The genetic differences in the myositis-susceptible loci may explain the heterogeneous phenotypes and treatment responses in myositis-associated ILD. The understanding of pathogenesis with the genetic background as well as autoantibodies will enable the practice of personalized treatment in the management of the disease.

Mechanical stress-induced mast cell degranulation activates TGF-ß1 signalling pathway in pulmonary fibrosis.

BACKGROUND: The role of mast cells accumulating in idiopathic pulmonary fibrosis (IPF) lungs is unknown. OBJECTIVES: We investigated the effect of fibrotic extracellular matrix (ECM) on mast cells in experimental and human pulmonary fibrosis. RESULTS: In IPF lungs, mast cell numbers were increased and correlated with disease severity (control vs 60%90% vs 60%90% vs FVC<60%, mean difference=-268.6, 95% CI of difference -441.0 to -96.17, p=0.0007). Plasma tryptase levels were increased in IPF and negatively correlated with FVC (control vs FVC<60%, mean difference=-17.12, 95% CI of difference -30.02 to -4.22, p=0.006: correlation curves R=-0.045, p=0.025). In a transforming growth factor (TGF)-ß1-induced pulmonary fibrosis model, chymase-positive and tryptase-positive mast cells accumulated in fibrotic lung. Lung tissue was decellularised and reseeded with bone marrow or peritoneum-derived mast cells; cells on fibrotic ECM released more TGF-ß1 compared with normal ECM (active TGF-ß1: bone marrow-derived mast cell (BMMC)-DL vs BMMC-TGF-ß1 p=0.0005, peritoneal mast cell (PMC)-DL vs PMC-TGF-ß1 p=0.0003, total TGF-ß1: BMMC-DL vs BMMC-TGF-ß1 p=0.013, PMC-DL vs PMC-TGF-ß1 p=0.001). Mechanical stretch of lungs caused mast cell degranulation; mast cell stabilisers inhibited degranulation (histamine: cont vs doxantrazole p=0.004, ß-hexosaminidase: cont vs doxantrazole, mean difference=1.007, 95% CI of difference 0.2700 to 1.744, p=0.007) and TGF-ß1 activation (pSmad2/Smad2: cont vs dox p=0.006). Cromoglycate attenuated pulmonary fibrosis in rats (collagen: phosphate-buffered saline (PBS) vs cromoglycate p=0.036, fibrotic area: PBS vs cromoglycate p=0.031). CONCLUSION: This study suggests that mast cells may contribute to the progression of pulmonary fibrosis.

Macitentan reduces progression of TGF-ß1-induced pulmonary fibrosis and pulmonary hypertension.

Idiopathic pulmonary fibrosis (IPF) is a progressive disease with an unknown cause. Two drugs, nintedanib and pirfenidone, have been shown to slow, but not stop, disease progression. Pulmonary hypertension (PH) is a frequent complication in IPF patients and is associated with poor prognosis. Macitentan is a dual endothelin receptor antagonist that is approved for pulmonary arterial hypertension treatment. We hypothesised that using macitentan to treat animals with pulmonary fibrosis induced by adenoviral vector encoding biologically active transforming growth factor-ß1 (AdTGF-ß1) would improve the PH caused by chronic lung disease and would limit the progression of fibrosis.Rats (Sprague Dawley) which received AdTGF-ß1 were treated by daily gavage of macitentan (100 mg·kg-1·day-1), pirfenidone (0.5% food admix) or a combination from day 14 to day 28. Pulmonary artery pressure (PAP) was measured before the rats were killed, and fibrosis was subsequently evaluated by morphometric measurements and hydroxyproline analysis.AdTGF-ß1 induced pulmonary fibrosis associated with significant PH. Macitentan reduced the increase in PAP and both macitentan and pirfenidone stopped fibrosis progression from day 14 to day 28. Macitentan protected endothelial cells from myofibroblast differentiation and apoptosis whereas pirfenidone only protected against fibroblast-to-myofibroblast differentiation. Both drugs induced apoptosis of differentiated myofibroblasts in vitro and in vivoOur results demonstrate that dual endothelin receptor antagonism was effective in both PH and lung fibrosis whereas pirfenidone only affected fibrosis.

Synergistic role of HSP90α and HSP90ß to promote myofibroblast persistence in lung fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a progressive disease of the lung parenchyma, causing significant morbidity through worsening dyspnoea and overall functional decline. IPF is characterised by apoptosis-resistant myofibroblasts, which are a major source for the excessive production of extracellular matrix (ECM) overtaking normal lung tissue. We sought to study the role of heat shock protein (HSP) isoforms HSP90α and HSP90ß, whose distinct roles in lung fibrogenesis remain elusive.We determined the level of circulating HSP90α in IPF patients (n=31) and age-matched healthy controls (n=9) by ELISA. The release of HSP90α and HSP90ß was evaluated in vitro in primary IPF and control lung fibroblasts and ex vivo after mechanical stretch on fibrotic lung slices from rats receiving adenovector-mediated transforming growth factor-ß1.We demonstrate that circulating HSP90α is upregulated in IPF patients in correlation with disease severity. The release of HSP90α is enhanced by the increase in mechanical stress of the fibrotic ECM. This increase in extracellular HSP90α signals through low-density lipoprotein receptor-related protein 1 (LRP1) to promote myofibroblast differentiation and persistence. In parallel, we demonstrate that the intracellular form of HSP90ß stabilises LRP1, thus amplifying HSP90α extracellular action.We believe that the specific inhibition of extracellular HSP90α is a promising therapeutic strategy to reduce pro-fibrotic signalling in IPF.

Thymic epithelial cell-specific deletion of Jmjd6 reduces Aire protein expression and exacerbates disease development in a mouse model of autoimmune diabetes.

Thymic epithelial cells (TECs) establish spatially distinct microenvironments in which developing T cells are selected to mature or die. A unique property of medullary TECs is their expression of thousands of tissue-restricted self-antigens that is largely under the control of the transcriptional regulator Aire. We previously showed that Jmjd6, a lysyl hydroxylase for splicing regulatory proteins, is important for Aire protein expression and that transplantation of Jmjd6-deficient thymic stroma into athymic nude mice resulted in multiorgan autoimmunity. Here we report that TEC-specific deletion of Jmjd6 exacerbates development of autoimmune diabetes in a mouse model, which express both ovalbumin (OVA) under the control of the rat insulin gene promoter and OT-I T cell receptor specific for OVA peptide bound to major histocompatibility complex class I Kb molecules. We found that Aire protein expression in mTECs was reduced in the absence of Jmjd6, with retention of intron 2 in Aire transcripts. Our results thus demonstrate the importance of Jmjd6 in establishment of immunological tolerance in a more physiological setting.

Treatment with a programmed cell death-1-specific antibody has little effect on afatinib- and naphthalene-induced acute pneumonitis in mice.

Although several antibodies developed to target programmed cell death-1 (PD-1) and its ligand (PD-L1) have demonstrated great promise for the treatment of non-small cell lung cancer (NSCLC), and other malignancies, these therapeutic antibodies can cause pneumonitis. Furthermore, epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI)-induced pneumonitis was reported after treatment with anti PD-1 antibodies. We previously demonstrated that mice with naphthalene-induced airway epithelial injury developed severe gefitinib-induced pneumonitis through a neutrophil-dependent mechanism. The present study aimed to investigate whether treatment with afatinib, an EGFR-TKI that effectively targets EGFR mutation-positive NSCLC, and anti PD-1 antibodies induces pneumonitis in mice. C57BL/6J mice were treated intraperitoneally with naphthalene (200 mg/kg) on day 0. Afatinib (20 mg/kg) was administered orally on days -1 to 13. An anti-PD-1 antibody (0.2 mg/mice) was also administered intraperitoneally every 3 days from day 1 until day 13. The bronchoalveolar lavage fluid (BALF) and lung tissues were sampled on day 14. As observed previously with gefitinib, afatinib significantly increased the severity of histopathologic findings and the level of protein in BALF on day 14, compared to treatment with naphthalene alone. A combined anti-PD-1 antibody and afatinib treatment after naphthalene administration had yielded the same histopathological grade of lung inflammation as did afatinib treatment alone. Our results suggest that anti-PD-1 antibody treatment has little effect on afatinib-induced lung injury.

Amphiregulin suppresses epithelial cell apoptosis in lipopolysaccharide-induced lung injury in mice.

BACKGROUND AND OBJECTIVE: As a member of the epidermal growth factor family, amphiregulin contributes to the regulation of cell proliferation. Amphiregulin was reported to be upregulated in damaged lung tissues in patients with chronic obstructive pulmonary disease and asthma and in lung epithelial cells in a ventilator-associated lung injury model. In this study, we investigated the effect of amphiregulin on lipopolysaccharide (LPS)-induced acute lung injury in mice. METHODS: Acute lung injury was induced by intranasal instillation of LPS in female C57BL/6 mice, and the mice were given intraperitoneal injections of recombinant amphiregulin or phosphate-buffered saline 6 and 0.5 h before and 3 h after LPS instillation. The effect of amphiregulin on apoptosis and apoptotic pathways in a murine lung alveolar type II epithelial cell line (LA-4 cells) were examined using flow cytometry and western blotting, respectively. RESULTS: Recombinant amphiregulin suppressed epithelial cell apoptosis in LPS-induced lung injury in mice. Western blotting revealed that amphiregulin suppressed epithelial cell apoptosis by inhibiting caspase-8 activity. CONCLUSION: Amphiregulin signaling may be a therapeutic target for LPS-induced lung injury treatment through its prevention of epithelial cell apoptosis.

Neutrophil elastase inhibitor sivelestat ameliorates gefitinib-naphthalene-induced acute pneumonitis in mice.

Gefitinib, an epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI), is an effective therapeutic agent for non-small cell lung cancer with EGFR mutations. It can cause severe acute pneumonitis in some patients. We previously demonstrated that mice with naphthalene-induced airway epithelial injury developed severe gefitinib-induced pneumonitis and that neutrophils played important roles in the development of the disease. This study aimed to investigate the effects of the neutrophil elastase inhibitor sivelestat on gefitinib-induced pneumonitis in mice. C57BL/6J mice received naphthalene (200 mg/kg) intraperitoneally on day 0. Gefitinib (250 or 300 mg/kg) was orally administered to mice from day -1 until day 13. Sivelestat (150 mg/kg) was administered intraperitoneally from day 1 until day 13. Bronchoalveolar lavage fluid (BALF) and lung tissues were sampled on day 14. Sivelestat treatment significantly reduced the protein level, neutrophil count, neutrophil elastase activity in BALF, and severity of histopathologic findings on day 14 for mice administered with 250 mg/kg of gefitinib. Moreover, sivelestat treatment significantly improved the survival of mice administered with 300 mg/kg of gefitinib. These results indicate that sivelestat is a promising therapeutic agent for severe acute pneumonitis caused by gefitinib.

DOCK2 and DOCK5 act additively in neutrophils to regulate chemotaxis, superoxide production, and extracellular trap formation.

Neutrophils are highly motile leukocytes that play important roles in the innate immune response to invading pathogens. Neutrophils rapidly migrate to the site of infections and kill pathogens by producing reactive oxygen species (ROS). Neutrophil chemotaxis and ROS production require activation of Rac small GTPase. DOCK2, an atypical guanine nucleotide exchange factor (GEF), is one of the major regulators of Rac in neutrophils. However, because DOCK2 deficiency does not completely abolish fMLF-induced Rac activation, other Rac GEFs may also participate in this process. In this study, we show that DOCK5 acts with DOCK2 in neutrophils to regulate multiple cellular functions. We found that fMLF- and PMA-induced Rac activation were almost completely lost in mouse neutrophils lacking both DOCK2 and DOCK5. Although ß2 integrin-mediated adhesion occurred normally even in the absence of DOCK2 and DOCK5, mouse neutrophils lacking DOCK2 and DOCK5 exhibited a severe defect in chemotaxis and ROS production. Similar results were obtained when human neutrophils were treated with CPYPP, a small-molecule inhibitor of these DOCK GEFs. Additionally, we found that DOCK2 and DOCK5 regulate formation of neutrophil extracellular traps (NETs). Because NETs are involved in vascular inflammation and autoimmune responses, DOCK2 and DOCK5 would be a therapeutic target for controlling NET-mediated inflammatory disorders.