Hypersensitivity pneumonitis (HP) is a complex immune-mediated interstitial lung disease triggered by repeated or persistent inhalation of a variety of inhaled antigens in genetically predisposed individuals. The disease encompasses a wide clinical spectrum and may present as predominantly inflammatory or fibrotic forms, the latter associated with irreversible architectural distortion and progressive loss of pulmonary function. Although mechanisms involving dysregulated T-cell activation, aberrant antigen processing and maladaptive tissue-repair pathways are recognised as central contributors to HP pathogenesis, the precise mechanisms and the sequence of immunologic events that occur during inflammation and mainly those that drive the transition from inflammation to fibrosis remains unclear. Animal models, particularly murine systems, have helped in dissecting these mechanisms, yet they continue to fall short in fully recapitulating the heterogeneity and chronicity observed in human HP. In this review, we provide an in-depth and critical examination of existing wild-type and genetically modified mouse models used to investigate HP, with emphasis on antigen sources, exposure paradigms and the strengths and limitations of each approach. We synthesise current insights into how specific genes, signalling pathways and immune cell subsets, including T-helper cell polarisation, regulatory T-cell function, dendritic cell activation and immune complexes contribute to disease initiation, amplification and progression.

ABSTRACT Autophagy has been implicated in several lung diseases, either protecting tissues or driving pathology. Hypersensitivity pneumonitis (HP) is a complex inflammatory lung disease, and autophagy is heavily involved in regulating inflammation. The role of autophagy in HP remains unclear. The aim of our study was to understand the role of autophagy in HP pathogenesis. GFP-LC3 transgenic mice were exposed intranasally to Saccharopolyspora rectivirgula (SR) to induce HP and follow autophagy activation in the lung. Then, we take advantage of our Atg4b-deficient mouse model to assess how autophagy disruption impacts lung inflammation in response to SR antigen challenge. Increased autophagy activation was observed in epithelial and inflammatory cells after SR antigen exposure in GFP-LC3 transgenic lungs. GFP-LC3 puncta colocalized with ATG4B and ATG5 in epithelial and inflammatory cells after antigen exposure. Autophagy impairment limits the inflammatory response after SR antigen exposure in the lungs from the Atg4b -deficient mice when compared to WT mice. To evaluate whether lipopolysaccharide (LPS) exacerbates the inflammatory response in the Atg4b-deficient, a SR+LPS combined treatment was developed and we discovered that LPS aggravates the SR-induced HP in WT but not in Atg4b-deficient mice. Reduced HP severity in Atg4b -deficient mice was associated with decreased expression of NFkB, CCL1, CCL25, CXCL1, TNFR1, IL-13, and IL-17A, diminished CD4+ T cell recruitment and expansion, reduced M2-like macrophages, and decreased granuloma and iBALT development. Our findings highlight autophagy as a critical driver in HP pathogenesis and as a potetial target for novel theraphy development.

Rationale: Hypersensitivity pneumonitis (HP) is classified as an interstitial lung disease (ILD) caused by the chronic inhalation of a wide variety of antigens in susceptible and sensitized individuals, that develop an exaggerated immune response. Usually, the HP is associated with an occupational setting and is categorized as fibrotic or non-fibrotic. We have previously shown evidence that proteins from the autophagy pathway are highly expressed in the lungs of HP patients. However, the role of autophagy in HP has not been studied yet.Method: To reduce the multivariable challenge in human disease that impacts autophagy flux, we developed a HP mouse model in GFP-LC3B transgenic, atg4b+/+, and atg4b-/- mice, which were instilled intranasally with 50µg of Saccharopolyspora rectivirgula (SR) in combination with 5µg of LPS three times a week for three weeks and mice were euthanized three days after the last exposure.In this study, we explored the autophagic flux and the cellular localization of autophagy markers such as LC3B, p62, ATG4B, and ATG5 by immunofluorescence, and the protein level by western blot in lungs from GFP-LC3B transgenic and WT SR+LPS-exposed mice compared to control mice.Results: SR+LPS exposed GFP-LC3 transgenic mice showed an increase in the number of autophagosomes/cells compared with controls. By immunofluorescence, we colocalized LC3B with ATG4B and p62 mainly in macrophages and epithelial cells. In addition, by immunoblot, we observed an increased LC3B, p62, ATG4B and ATG5 protein level in SR+LPS-exposed atg4b+/+ lungs compared with controls. These data suggested that autophagy could be activated in the lung after SR exposure and induced inflammation. We also found LC3B colocalization with SPC, CD68, BLA. Then we evaluated the inflammatory response, lung damage and the cytokine profile in the bronchoalveolar lavage from atg4b-/- and atg4b+/+ SR+LPS treated mice after 3 weeks post-exposure. We found the protein level of CXCL1, CCL1 and CCl25 proinflammatory cytokines were significantly lower in the BALF from atg4b-/- compared to atg4b+/+. Conclusion: this study showed that autophagy could have a pathogenic role in HP.

Several types of cytotoxic insults disrupt endoplasmic reticulum (ER) homeostasis, cause ER stress, and activate the unfolded protein response (UPR). The role of ER stress and UPR activation in hypersensitivity pneumonitis (HP) has not been described. HP is an immune-mediated interstitial lung disease that develops following repeated inhalation of various antigens in susceptible and sensitized individuals. The aim of this study was to investigate the lung expression and localization of the key effectors of the UPR, BiP/GRP78, CHOP, and sXBP1 in HP patients compared with control subjects. Furthermore, we developed a mouse model of HP to determine whether ER stress and UPR pathway are induced during this pathogenesis. In human control lungs, we observed weak positive staining for BiP in some epithelial cells and macrophages, while sXBP1 and CHOP were negative. Conversely, strong BiP, sXBP1- and CHOP-positive alveolar and bronchial epithelial, and inflammatory cells were identified in HP lungs. We also found apoptosis and autophagy markers colocalization with UPR proteins in HP lungs. Similar results were obtained in lungs from an HP mouse model. Our findings suggest that the UPR pathway is associated with the pathogenesis of HP.

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:

The nuclear architecture of mammalian cells can be altered as a consequence of anomalous accumulation of nuclear proteins or genomic alterations. Most of the knowledge about nuclear dynamics comes from studies on cancerous cells. How normal healthy cells maintain genome stability, avoiding accumulation of nuclear damaged material, is less understood. Here, we describe that primary mouse embryonic fibroblasts develop a basal level of nuclear buds and micronuclei, which increase after etoposide-induced DNA double-stranded breaks. Both basal and induced nuclear buds and micronuclei colocalize with the autophagic proteins BECN1 and LC3B (also known as MAP1LC3B) and with acidic vesicles, suggesting their clearance by nucleophagy. Some of the nuclear alterations also contain autophagic proteins and type II DNA topoisomerases (TOP2A and TOP2B), or the nucleolar protein fibrillarin, implying they are also targets of nucleophagy. We propose that basal nucleophagy contributes to genome and nuclear stability, as well as in response to DNA damage.

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.

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.

Idiopathic pulmonary fibrosis (IPF) is a chronic and progressive aging-associated disease of unknown etiology. A growing body of evidence indicates that aberrant activated alveolar epithelial cells induce the expansion and activation of the fibroblast population, leading to the destruction of the lung architecture. Some matrix metalloproteinases (MMPs) are upregulated in IPF, indicating that they may be important in the pathogenesis and/or progression of IPF. In the present study, we examined the expression of MMP28 in this disease and evaluated its functional effects in two alveolar epithelial cell lines and in human primary bronchial epithelial cells. We found that the enzyme is expressed in bronchial (apical and cytoplasmic localization) and alveolar (cytoplasmic and nuclear localization) epithelial cells in two different groups of patients with IPF. In vitro MMP28 epithelial silencing decreased the proliferation rate and delayed wound closing, whereas overexpression showed opposite effects, protecting from apoptosis and enhanced epithelial-mesenchymal transition. Our findings demonstrate that MMP28 is upregulated in epithelial cells from IPF lungs, where it may play a role in increasing the proliferative and migratory phenotype in a catalysis-dependent manner.

RESUMEN.La autofagia es un proceso fundamental de degradación intracelular de organelos y proteínas dañadas.La autofagia es también un mecanismo esencial para la adaptación al estrés, la supervivencia y la homeostasis celular.Paradójicamente, la autofagia también puede promover la muerte celular, pero cuándo y cómo la autofagia puede tener funciones pro o antiapoptóticas, es aún desconocido.La autofagia involucra el reclutamiento y la degradación de organelos dañados, agregados de proteínas, proteínas de larga vida y patógenos en vesículas de doble membrana llamadas autofagosomas, que luego se fusionan con los lisosomas para formar los autofagolisosomas (o autolisosomas).Alteraciones en la autofagia han sido implicadas en una amplia gama de trastornos, incluyendo enfermedades pulmonares.En esta revisión, se discute lo que al presente se sabe sobre el papel de la autofagia en el inicio y la progresión de diferentes trastornos pulmonares.Algunos estudios indican que la autofagia podría tener un papel deletéreo en la patogénesis de la enfermedad pulmonar obstructiva crónica y del asma.En otros trastornos como la fi brosis pulmonar idiopática y la fi brosis quística, la inhibición de la autofagia puede contribuir a la patogénesis.

Idiopathic pulmonary fibrosis (IPF) is a progressive and devastating lung disorder of unknown origin, with very poor prognosis and no effective treatment. The disease is characterized by abnormal activation of alveolar epithelial cells, which secrete numerous mediators involved in the expansion of the fibroblast population, its differentiation to myofibroblasts, and in the exaggerated accumulation of extracellular matrix provoking the loss of lung architecture. Among the excessively produced mediators are several matrix metalloproteases (MMPs) which may contribute to modify the lung microenvironment by various mechanisms. Thus, these enzymes can not only degrade all the components of the extracellular matrix, but they are also able to release, cleave and activate a wide range of growth factors, cytokines, chemokines and cell surface receptors affecting numerous cell functions including adhesion, proliferation, differentiation, recruiting and transmigration, and apoptosis. Therefore, dysregulated expression of MMPs may have profound impact on the biopathological mechanisms implicated in the development of IPF. This review focuses on the current and emerging evidence regarding the role of MMPs on the fibrotic processes in IPF as well as in mouse models of lung fibrosis.