Mir-155-5p targets TP53INP1 to promote proliferative phenotype in hypersensitivity pneumonitis lung fibroblasts.

Background: Hypersensitivity pneumonitis (HP) is an inflammatory disorder affecting lung parenchyma and often evolves into fibrosis (fHP). The altered regulation of genes involved in the pathogenesis of the disease is not well comprehended, while the role of microRNAs in lung fibroblasts remains unexplored. Methods: We used integrated bulk RNA-Seq and enrichment pathway bioinformatic analyses to identify differentially expressed (DE)-miRNAs and genes (DEGs) associated with HP lungs. In vitro, we evaluated the expression and potential role of miR-155-5p in the phenotype of fHP lung fibroblasts. Loss and gain assays were used to demonstrate the impact of miR-155-5p on fibroblast functions. In addition, mir-155-5p and its target TP53INP1 were analyzed after treatment with TGF-ß, IL-4, and IL-17A. Results: We found around 50 DEGs shared by several databases that differentiate HP from control and IPF lungs, constituting a unique HP lung transcriptional signature. Additionally, we reveal 18 DE-miRNAs that may regulate these DEGs. Among the candidates likely associated with HP pathogenesis was miR-155-5p. Our findings indicate that increased miR-155-5p in fHP fibroblasts coincides with reduced TP53INP1 expression, high proliferative capacity, and a lack of senescence markers compared to IPF fibroblasts. Induced overexpression of miR-155-5p in normal fibroblasts remarkably increases the proliferation rate and decreases TP53INP1 expression. Conversely, miR-155-5p inhibition reduces proliferation and increases senescence markers. TGF-ß, IL-4, and IL-17A stimulated miR-155-5p overexpression in HP lung fibroblasts. Conclusion: Our findings suggest a distinctive signature of 53 DEGs in HP, including CLDN18, EEF2, CXCL9, PLA2G2D, and ZNF683, as potential targets for future studies. Likewise, 18 miRNAs, including miR-155-5p, could be helpful to establish differences between these two pathologies. The overexpression of miR-155-5p and downregulation of TP53INP1 in fHP lung fibroblasts may be involved in his proliferative and profibrotic phenotype. These findings may help differentiate and characterize their pathogenic features and understand their role in the disease.

Lack of ZNF365 Drives Senescence and Exacerbates Experimental Lung Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is characterized by aberrant activation of the alveolar epithelium, the expansion of the fibroblast population, and the accumulation of extracellular matrix. Global gene expression of human lung fibroblasts stimulated with TGFß-1, a strong fibrotic mediator revealed the overexpression of ZNF365, a zinc finger protein implicated in cell cycle control and telomere stabilization. We evaluated the expression and localization of ZNF365 in IPF lungs and in the fibrotic response induced by bleomycin in WT and deficient mice of the orthologous gene Zfp365. In IPF, ZNF365 was overexpressed and localized in fibroblasts/myofibroblasts and alveolar epithelium. Bleomycin-induced lung fibrosis showed an upregulation of Zfp365 localized in lung epithelium and stromal cell populations. Zfp365 KO mice developed a significantly higher fibrotic response compared with WT mice by morphology and hydroxyproline content. Silencing ZNF365 in human lung fibroblasts and alveolar epithelial cells induced a significant reduction of growth rate and increased senescence markers, including Senescence Associated ß Galactosidase activity, p53, p21, and the histone variant γH2AX. Our findings demonstrate that ZNF365 is upregulated in IPF and experimental lung fibrosis and suggest a protective role since its absence increases experimental lung fibrosis mechanistically associated with the induction of cell senescence.

Single Nucleotide Polymorphisms (SNP) and SNP-SNP Interactions of the Surfactant Protein Genes Are Associated With Idiopathic Pulmonary Fibrosis in a Mexican Study Group; Comparison With Hypersensitivity Pneumonitis.

Surfactant proteins (SPs) are important for normal lung function and innate immunity of the lungs and their genes have been identified with significant genetic variability. Changes in quantity or quality of SPs due to genetic mutations or natural genetic variability may alter their functions and contribute to the host susceptibility for particular diseases. Alternatively, SP single nucleotide polymorphisms (SNPs) can serve as markers to identify disease risk or response to therapies, as shown for other genes in a number of other studies. In the current study, we evaluated associations of SFTP SNPs with idiopathic pulmonary fibrosis (IPF) by studying novel computational models where the epistatic effects (dominant, additive, recessive) of SNP-SNP interactions could be evaluated, and then compared the results with a previously published hypersensitivity pneumonitis (HP) study where the same novel models were used. Mexican Hispanic patients (IPF=84 & HP=75) and 194 healthy control individuals were evaluated. The goal was to identify SP SNPs and SNP-SNP interactions that associate with IPF as well as SNPs and interactions that may be unique to each of these interstitial diseases or common between them. We observed: 1) in terms of IPF, i) three single SFTPA1 SNPs to associate with decreased IPF risk, ii) three SFTPA1 haplotypes to associate with increased IPF risk, and iii) a number of three-SNP interactions to associate with IPF susceptibility. 2) Comparison of IPF and HP, i) three SFTPA1 and one SFTPB SNP associated with decreased risk in IPF but increased risk in HP, and one SFTPA1 SNP associated with decreased risk in both IPF and HP, ii) a number of three-SNP interactions with the same or different effect pattern associated with IPF and/or HP susceptibility, iii) one of the three-SNP interactions that involved SNPs of SFTPA1, SFTPA2, and SFTPD, with the same effect pattern, was associated with a disease-specific outcome, a decreased and increased risk in HP and IPF, respectively. This is the first study that compares the SP gene variants in these two phenotypically similar diseases. Our findings indicate that SNPs of all SFTPs may play an important role in the genetic susceptibility to IPF and HP. Importantly, IPF and HP share some SP genetic variants, suggesting common pathophysiological mechanisms and pathways regarding surfactant biogenesis, but also some differences, highlighting the diverse underlying pathogenic mechanisms between an inflammatory-driven fibrosis (HP) and an epithelial-driven fibrosis (IPF). Alternatively, the significant SNPs identified here, along with SNPs of other genes, could serve as markers to distinguish these two devastating diseases.

Fibroblasts From Idiopathic Pulmonary Fibrosis Induce Apoptosis and Reduce the Migration Capacity of T Lymphocytes.

Idiopathic pulmonary fibrosis (IPF) is a progressive and irreversible lung disease of unknown etiology. Myofibroblasts are organized in peculiar subepithelial fibroblasts foci (FF), where they abnormally persist and exclude lymphocytes by unclear mechanisms. FF are the source of an excessive extracellular matrix, which results in progressive stiffening and destruction of the lung architecture. We hypothesized that the absence of T cells inside the FF could be related, at least partially, to an inefficient function of lymphocytes induced by IPF fibroblasts. Here, we evaluated the effect of a supernatant from IPF fibroblasts on T-cell apoptosis and migration capacity. Data showed that IPF fibroblasts secrete pro-apoptotic molecules (both from extrinsic and intrinsic pathways), generating a microenvironment that induces apoptosis of T cells at 3 h of culture, despite a weak anti-apoptotic profile exhibited by these T cells. At 24 h of culture, the supernatants from both IPF and control fibroblasts provoked T-cell death. However, at this time of culture, IPF fibroblasts caused a marked decrease in T-cell migration; in contrast, control lung fibroblasts induced an increase of T-cell migration. The reduction of T-cell migratory capacity provoked by IPF fibroblasts was associated with a negative regulation of RHOA and ROCK, two essential GTPases for migration, and was independent of the expression of chemokine receptors. In conclusion, our findings demonstrate that IPF fibroblasts/myofibroblasts induce apoptosis and affect T-cell migration, revealing a mechanism involved in the virtual absence of T lymphocytes inside the FF.

Mitochondrial Dysfunction and Alterations in Mitochondrial Permeability Transition Pore (mPTP) Contribute to Apoptosis Resistance in Idiopathic Pulmonary Fibrosis Fibroblasts.

Idiopathic pulmonary fibrosis (IPF) is a devastating disease characterized by increased activation of fibroblasts/myofibroblasts. Previous reports have shown that IPF fibroblasts are resistant to apoptosis, but the mechanisms remain unclear. Since inhibition of the mitochondrial permeability transition pore (mPTP) has been implicated in the resistance to apoptosis, in this study, we analyzed the role of mitochondrial function and the mPTP on the apoptosis resistance of IPF fibroblasts under basal conditions and after mitomycin C-induced apoptosis. We measured the release of cytochrome c, mPTP opening, mitochondrial calcium release, oxygen consumption, mitochondrial membrane potential, ADP/ATP ratio, ATP concentration, and mitochondrial morphology. We found that IPF fibroblasts were resistant to mitomycin C-induced apoptosis and that calcium, a well-established activator of mPTP, is decreased as well as the release of pro-apoptotic proteins such as cytochrome c. Likewise, IPF fibroblasts showed decreased mitochondrial function, while mPTP was less sensitive to ionomycin-induced opening. Although IPF fibroblasts did not present changes in the mitochondrial membrane potential, we found a fragmented mitochondrial network with scarce, thinned, and disordered mitochondria with reduced ATP levels. Our findings demonstrate that IPF fibroblasts are resistant to mitomycin C-induced apoptosis and that altered mPTP opening contributes to this resistance. In addition, IPF fibroblasts show mitochondrial dysfunction evidenced by a decrease in respiratory parameters.

Development of a Diagnostic Biosensor Method of Hypersensitivity Pneumonitis towards a Point-of-Care Biosensor.

In spite of a current increasing trend in the development of miniaturized, standalone point-of-care (PoC) biosensing platforms in the literature, the actual implementation of such systems in the field is far from being a reality although deeply needed. In the particular case of the population screenings for local or regional diseases related to specific pathogens, the diagnosis of the presence of specific antibodies could drastically modify therapies and even the organization of public policies. The aim of this work was to develop a fast, cost-effective detection method based on the manipulation of functionalized magnetic beads for an efficient diagnosis of hypersensitivity pneumonitis (HP), looking for the presence of anti-pigeon antigen antibodies (APAA) in a patient's serum. We presented a Diagnostic Biosensor Method (DBM) in detail, with validation by comparison with a traditional high-throughput platform (ELISA assay). We also demonstrated that it was compatible with a microfluidic chip that could be eventually incorporated into a PoC for easy and broad deployment using portable optical detectors. After standardization of the different reaction steps, we constructed and validated a plastic chip that could easily be scaled to high-volume manufacturing in the future. The solution proved comparable to conventional ELISA assays traditionally performed by the clinicians in their laboratory and should be compatible with other antibody detection directly from patient samples.

From pulmonary fibrosis to progressive pulmonary fibrosis: a lethal pathobiological jump.

The month of September is Pulmonary Fibrosis Awareness Month. In this context, we would like to highlight the concept of progressive pulmonary fibrosis, a common denominator/phenotype of many interstitial lung diseases other than idiopathic pulmonary fibrosis, leading to clinical deterioration, decreased quality of life, and high mortality.

Loss of MT1-MMP in Alveolar Epithelial Cells Exacerbates Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a lethal age-related lung disease whose pathogenesis involves an aberrant response of alveolar epithelial cells (AEC). Activated epithelial cells secrete mediators that participate in the activation of fibroblasts and the excessive deposition of extracellular matrix proteins. Previous studies indicate that matrix metalloproteinase 14 (MMP14) is increased in the lung epithelium in patients with IPF, however, the role of this membrane-type matrix metalloproteinase has not been elucidated. In this study, the role of Mmp14 was explored in experimental lung fibrosis induced with bleomycin in a conditional mouse model of lung epithelial MMP14-specific genetic deletion. Our results show that epithelial Mmp14 deficiency in mice increases the severity and extension of fibrotic injury and affects the resolution of the lesions. Gain-and loss-of-function experiments with human epithelial cell line A549 demonstrated that cells with a deficiency of MMP14 exhibited increased senescence-associated markers. Moreover, conditioned medium from these cells increased fibroblast expression of fibrotic molecules. These findings suggest a new anti-fibrotic mechanism of MMP14 associated with anti-senescent activity, and consequently, its absence results in impaired lung repair. Increased MMP14 in IPF may represent an anti-fibrotic mechanism that is overwhelmed by the strong profibrotic microenvironment that characterizes this disease.

Mesenchymal-Epithelial Transition in Fibroblasts of Human Normal Lungs and Interstitial Lung Diseases.

. In passages above ten and growing very actively, we observed that some human lung fibroblasts cultured under standard conditions were transformed into a lineage of epithelial-like cells (ELC). To systematically evaluate the possible mesenchymal-epithelial transition (MET) occurrence, fibroblasts were obtained from normal lungs and also from lungs affected by idiopathic interstitial diseases. When an unusual epithelial-like phenotypic change was observed, cultured cells were characterized by confocal immunofluorescence microscopy, immunoblotting, immunocytochemistry, cytofluorometry, gelatin zymography, RT-qPCR, and hybridization in a whole-transcript human microarray. Additionally, microvesicles fraction (MVs) from ELC and fibroblasts were used to induce MET, while the microRNAs (miRNAs) contained in the MVs were identified. Pattern-gene expression of the original fibroblasts and the derived ELC revealed profound changes, upregulating characteristic epithelial-cell genes and downregulating mesenchymal genes, with a marked increase of E-cadherin, cytokeratin, and ZO-1, and the loss of expression of α-SMA, collagen type I, and Thy-1 cell surface antigen (CD90). Fibroblasts, exposed to culture media or MVs from the ELC, acquired ELC phenotype. The miRNAs in MVs shown six expressed exclusively in fibroblasts, and three only in ELC; moreover, twelve miRNAs were differentially expressed between fibroblasts and ELC, all of them but one was overexpressed in fibroblasts. These findings suggest that the MET-like process can occur in human lung fibroblasts, either from normal or diseased lungs. However, the biological implication is unclear.

When things go wrong: exploring possible mechanisms driving the progressive fibrosis phenotype in interstitial lung diseases.

Interstitial lung diseases (ILDs) comprise a large and heterogeneous group of disorders of known and unknown aetiology characterised by diffuse damage of the lung parenchyma. In recent years it has become evident that patients with different types of ILD are at risk of developing progressive pulmonary fibrosis, known as progressive fibrosing ILD (PF-ILD). This is a phenotype that behaves similar to idiopathic pulmonary fibrosis, the archetypical example of progressive fibrosis. PF-ILD is not a distinct clinical entity but describes a group of ILDs with similar clinical behaviour. This phenotype may occur in diseases displaying distinct aetiologies and different biopathology during their initiation and development. Importantly, these entities may have the potential for improvement or stabilisation prior to entering the progressive fibrosing phase. The crucial questions are: 1) why does a subset of patients develop a progressive and irreversible fibrotic phenotype even with appropriate treatment? and 2) what are the possible pathogenic mechanisms driving progression? Here, we provide a framework highlighting putative mechanisms underlying progression, including genetic susceptibility, ageing, epigenetics, structural fibrotic distortion, aberrant composition and stiffness of the extracellular matrix, and the emergence of distinct pro-fibrotic cell subsets. Understanding the cellular and molecular mechanisms behind PF-ILD will provide the basis for identifying risk factors and appropriate therapeutic strategies.

Risk factors associated with the development of interstitial lung abnormalities.

BACKGROUND: Around 8-10% of individuals over 50 years of age present interstitial lung abnormalities (ILAs), but their risk factors are uncertain. METHODS: From 817 individuals recruited in our lung ageing programme at the Mexican National Institute of Respiratory Diseases, 80 (9.7%) showed ILAs and were compared with 564 individuals of the same cohort with normal high-resolution computed tomography to evaluate demographic and functional differences, and with 80 individuals randomly selected from the same cohort for biomarkers. We evaluated MUC5B variant rs35705950, telomere length, and serum levels of matrix metalloproteinase (MMP)-1, MMP-2, MMP-3, MMP-7, MMP-8, MMP-9, MMP-12, MMP-13, interleukin (IL)-6, surfactant protein (SP)-D, α-Klotho and resistin. RESULTS: Individuals with ILAs were usually males (p<0.005), older than controls (p<0.0001), smokers (p=0.01), with a greater frequency of MUC5B rs35705950 (OR 3.5, 95% CI 1.3-9.4; p=0.01), and reduced diffusing capacity of the lung for carbon monoxide and oxygen saturation. Resistin, IL-6, SP-D, MMP-1, MMP-7 and MMP-13 were significantly increased in individuals with ILAs. Resistin (12±5 versus 9±4 ng·mL-1; p=0.0005) and MMP-13 (357±143 versus 298±116 pg·mL-1; p=0.004) were the most increased biomarkers. On follow-up (24±18 months), 18 individuals showed progression which was associated with gastro-oesophageal reflux disease (OR 4.1, 95% CI 1.2-12.9; p=0.02) and in females with diabetes mellitus (OR 5.3, 95% CI 1.0-27.4; p=0.01). CONCLUSIONS: Around 10% of respiratory asymptomatic individuals enrolled in our lung ageing programme show ILAs. Increased serum concentrations of pro-inflammatory molecules and MMPs are associated with ILAs.

Determination of the phenotypic age in residents of Mexico City: effect of accelerated ageing on lung function and structure.

INTRODUCTION: Phenotypic age better represents age-related biological dysregulation than chronological age. Recently, a multisystem-based ageing measure, which integrates chronological age and nine biomarkers, was proposed. METHODS: Phenotypic age was determined in 774 residents of Mexico City over 60 years old and without respiratory problems. We arbitrarily classified as "accelerated" ageing, those showing >4 years compared with their chronological age, and "slowed" ageing, those with <4 years compared with chronological age. Demographic risk factors were analysed with structured questionnaires. Lung structure was evaluated by high-resolution computed tomography and functional competence was analysed by forced vital capacity (FVC), forced expiratory volume in 1 s (FEV1), diffusion capacity of carbon monoxide (D LCO), and the 6-minute walk test (6MWT). RESULTS: Overall, 13% of this cohort showed accelerated ageing, which was corroborated with four independent biomarkers of ageing, 42% had normal ageing and 46% had slowed ageing. Risk factors associated with accelerated ageing were male sex (OR 4.4, 95% CI 2.4-7.9; p<0.01), diabetes mellitus (OR 9.7, 95% CI 5.5-17.2; p<0.01), and long-term sleep duration (OR 2.9 95% CI 1.34-6.35, p<0.01). Among smokers, there was a slight but significant association with the number of pack-years. Subjects with accelerated ageing showed decreased FVC (p<0.0001), FEV1 (p<0.0001), D LCO (p<0.02) and walking distance in the 6MWT (p=0.0001). Slowed-ageing subjects presented less frequently with emphysematous lesions compared with those with accelerated ageing. CONCLUSIONS: A small but significant proportion of residents of Mexico City age rapidly, which is associated with male sex, diabetes, and long-term sleep duration. They exhibit lower levels of lung function and develop emphysema more frequently.

Identification of Autophagy-related Proteins in Lungs From Hypersensitivity Pneumonitis Patients.

Autophagy has been involved in the pathogenesis of various lung diseases. However, it is not yet known whether autophagy plays a role in hypersensitivity pneumonitis (HP). HP is an interstitial lung disease resulting from exposure to a wide variety of antigens that provoke an exaggerated immune response in susceptible individuals. The aim of this study was to explore the localization of autophagy key proteins in lungs from HP patients and controls by immunohistochemistry and analyze their expression levels by immunoblot. Macrophages and epithelial cells were strongly positive for the autophagosome biomarker LC3B (microtubule-associated protein light chain 3 beta) in HP lungs compared with controls. A similar pattern was found for the autophagy receptor p62 and the enzyme ATG4B. Unexpectedly, nuclear p62 signal was also noticed in macrophages from HP lungs. Regarding ATG5 and ATG7 localization, we observed positive staining in neutrophils, vascular smooth muscle cells, and endothelial cells. Our findings provide for the first time evidence that proteins from the autophagy machinery are highly expressed in the lungs of HP patients and describe the specific cellular and subcellular localization of LC3B, p62, ATG4B, ATG5, and ATG7 in HP lungs.

SNP and Haplotype Interaction Models Reveal Association of Surfactant Protein Gene Polymorphisms With Hypersensitivity Pneumonitis of Mexican Population.

Background: Hypersensitivity pneumonitis (HP) is an interstitial lung disease caused by inhalation of common environmental organic particles. Surfactant proteins (SPs) play a role in innate immunity and surfactant function. We hypothesized that single nucleotide polymorphisms (SNPs) or haplotypes of the SP genes associate with HP. Methods: Seventy-five HP patients caused by avian antigen and 258 controls, asymptomatic antigen exposed and non-exposed were enrolled. SNP association was performed using logistic regression analysis and SNP-SNP interaction models. Results: Based on odds ratio, regression analyses showed association of (a) rs7316_G, 1A3 (protective) compared to antigen exposed; (b) male sex, smoking, rs721917_T and rs1130866_T (protective) compared to non-exposed controls with HP; (c) compared to antigen exposed, 25 interactions associated with HP in a three-SNP model; (d) compared to non-exposed, (i) rs1136451 associated with increased, whereas rs1136450 and rs1130866 associated with lower HP risk, (ii) 97 interactions associated with HP in a three-SNP model. The majority of SNP-SNP interactions associated with increased HP risk involved SNPs of the hydrophilic SPs, whereas, the majority of interactions associated with lower HP risk involved SNPs of both hydrophilic and hydrophobic SPs; (e) haplotypes of SP genes associated with HP risk. Conclusions: The complexity of SNPs interactions of the SFTP genes observed indicate that the lung inflammatory response to avian antigens is modulated by a complex gene interplay rather than by single SNPs.

Transforming growth factor beta 1 induces methylation changes in lung fibroblasts.

Idiopathic pulmonary fibrosis is a complex disease of unknown etiology. Environmental factors can affect disease susceptibility via epigenetic effects. Few studies explore global DNA methylation in lung fibroblasts, but none have focused on transforming growth factor beta-1 (TGF-ß1) as a potential modifier of the DNA methylome. Here we analyzed changes in methylation and gene transcription in normal and IPF fibroblasts following TGF-ß1 treatment. We analyzed the effects of TGF-ß1 on primary fibroblasts derived from normal or IPF lungs treated for 24 hours and 5 days using the Illumina 450k Human Methylation array and the Prime View Human Gene Expression Array. TGF-ß1 induced an increased number of gene expression changes after short term treatment in normal fibroblasts, whereas greater methylation changes were observed following long term stimulation mainly in IPF fibroblasts. DNA methyltransferase 3 alpha (DMNT3a) and tet methylcytosine dioxygenase 3 (TET3) were upregulated after 5-days TGF-ß1 treatment in both cell types, whereas DNMT3a was upregulated after 24h only in IPF fibroblasts. Our findings demonstrate that TGF-ß1 induced the upregulation of DNMT3a and TET3 expression and profound changes in the DNA methylation pattern of fibroblasts, mainly in those derived from IPF lungs.

Lower levels of α-Klotho in serum are associated with decreased lung function in individuals with interstitial lung abnormalities.

Interstitial lung abnormalities (ILA) represent aging-associated bilateral interstitial abnormalities in nondependent areas of the lung. However, the aging mechanisms associated with ILA remain uncertain. α-Klotho is an anti-aging molecule that decreases progressively with age, and abnormally low circulating levels of this protein have been revealed in several chronic-degenerative diseases. In this study, we evaluated α-Klotho serum concentrations in individuals with ILA, and examined whether its levels were associated with pulmonary function decline. α-Klotho was measured by ELISA in 50 respiratory asymptomatic adults with ILA and 150 healthy individuals over 60 years. Compared with controls, ILA subjects were predominantly older males, and showed lower lung diffusing capacity (DLCO), higher desaturation after exercise, and higher concentrations of serum matrix metalloprotease-7 (6.24 ± 4.1 versus 4.3 ± 1.7 ng/ml; p = 0.002). No differences were found in serum concentrations of α-Klotho. However, lower levels of this protein in ILA significantly correlated with lower values of forced vital capacity (Rho = 0.39; p = 0.005), forced expiratory volume in one second (Rho = 0.39; p = 0.005), and DLCO (Rho = 0.29, p = 0.04). These findings suggest that decreased concentrations of α-Klotho may be a predictive biomarker of accelerated decline of lung function in individuals with ILA.

Delayed resolution of bleomycin-induced pulmonary fibrosis in absence of MMP13 (collagenase 3).

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.

Accelerated aging induced by deficiency of Zmpste24 protects old mice to develop bleomycin-induced pulmonary fibrosis.

Idiopathic pulmonary fibrosis is a devastating aging-associated disease of unknown etiology. Despite that aging is a major risk factor, the mechanisms linking aging with this disease are uncertain, and experimental models to explore them in lung fibrosis are scanty. We examined the fibrotic response to bleomycin-induced lung injury in Zmpste24-deficient mice, which exhibit nuclear lamina defects developing accelerated aging. We found that young WT and Zmpste24(-/-) mice developed a similar fibrotic response to bleomycin. Unexpectedly, while old WT mice developed severe lung fibrosis, accelerated aged Zmpste24-/- mice were protected showing scant lung damage. To investigate possible mechanisms associated with this resistance to fibrosis, we compared the transcriptome signature of the lungs and found that Zmpste24(-/-) mice showed downregulation of several core and associated matrisome genes compared with WT mice. Interestingly, some microRNAs that target extracellular matrix molecules such as miR23a, miR27a, miR29a, miR29b-1, miR145a, and miR491 were dysregulated resulting in downregulation of profibrotic pathways such as TGF-ß/SMAD3/NF-κB and Wnt3a/ß-catenin signaling axis. These results indicate that the absence of Zmpste24 in aging mice results in impaired lung fibrotic response after injury, which is likely associated to the dysregulation of fibrosis-related miRNAs.

Impaired autophagic activity and ATG4B deficiency are associated with increased endoplasmic reticulum stress-induced lung injury.

Aging is the main risk factor for the development of idiopathic pulmonary fibrosis (IPF), a progressive and usually lethal lung disorder. Although the pathogenic mechanisms are uncertain, endoplasmic reticulum (ER) stress and impaired proteostasis that have been linked with aging are strongly associated with the pathogenesis of IPF. Using the Atg4b-deficient mice as a model, that partially reproduces the autophagy deficient conditions reported in aging and IPF lungs, we show for the first time how autophagy impairment and ER stress induction, contribute simultaneously to development of lung fibrosis in vivo. Increased expression of ER stress markers, inflammation and apoptosis of alveolar epithelial cells were observed in Atg4b-deficient mice compared to WT mice, when treated with the ER stress inducer tunicamycin. After tunicamycin treatment, Atg4b null lungs showed accumulation of its substrate LC3-I, demonstrating that these mice failed to induce autophagy despite the ER stress conditions. We also showed that compromised autophagy in lungs from Atg4b null mice is associated with exacerbated lung damage, epithelial apoptosis and the development of lung fibrosis at 21 days after tunicamycin treatment. Our findings indicate that ATG4B protein and autophagy are essential to mitigate ER stress and to prevent tunicamycin-induced epithelial apoptosis and lung fibrosis.