PTPα promotes fibroproliferative responses after acute lung injury.

The acute respiratory distress syndrome (ARDS) is a major healthcare problem, accounting for significant mortality and long-term disability. Approximately 25% of patients with ARDS will develop an overexuberant fibrotic response, termed fibroproliferative ARDS (FP-ARDS) that portends a poor prognosis and increased mortality. The cellular pathological processes that drive FP-ARDS remain incompletely understood. We have previously shown that the transmembrane receptor-type tyrosine phosphatase protein tyrosine phosphatase-α (PTPα) promotes pulmonary fibrosis in preclinical murine models through regulation of transforming growth factor-ß (TGF-ß) signaling. In this study, we examine the role of PTPα in the pathogenesis of FP-ARDS in a preclinical murine model of acid (HCl)-induced acute lung injury. We demonstrate that although mice genetically deficient in PTPα (Ptpra-/-) are susceptible to early HCl-induced lung injury, they exhibit markedly attenuated fibroproliferative responses. In addition, early profibrotic gene expression is reduced in lung tissue after acute lung injury in Ptpra-/- mice, and stimulation of naïve lung fibroblasts with the BAL fluid from these mice results in attenuated fibrotic outcomes compared with wild-type littermate controls. Transcriptomic analyses demonstrate reduced extracellular matrix (ECM) deposition and remodeling in mice genetically deficient in PTPα. Importantly, human lung fibroblasts modified with a CRISPR-targeted deletion of PTPRA exhibit reduced expression of profibrotic genes in response to TGF-ß stimulation, demonstrating the importance of PTPα in human lung fibroblasts. Together, these findings demonstrate that PTPα is a key regulator of fibroproliferative processes following acute lung injury and could serve as a therapeutic target for patients at risk for poor long-term outcomes in ARDS.

Influenza virus infection increases ACE2 expression and shedding in human small airway epithelial cells.

BACKGROUND: Patients with coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) demonstrate high rates of co-infection with respiratory viruses, including influenza A (IAV), suggesting pathogenic interactions. METHODS: We investigated how IAV may increase the risk of COVID-19 lung disease, focusing on the receptor angiotensin-converting enzyme (ACE)2 and the protease TMPRSS2, which cooperate in the intracellular uptake of SARS-CoV-2. RESULTS: We found, using single-cell RNA sequencing of distal human nondiseased lung homogenates, that at baseline, ACE2 is minimally expressed in basal, goblet, ciliated and secretory epithelial cells populating small airways. We focused on human small airway epithelial cells (SAECs), central to the pathogenesis of lung injury following viral infections. Primary SAECs from nondiseased donor lungs apically infected (at the air-liquid interface) with IAV (up to 3×105 pfu; ∼1 multiplicity of infection) markedly (eight-fold) boosted the expression of ACE2, paralleling that of STAT1, a transcription factor activated by viruses. IAV increased the apparent electrophoretic mobility of intracellular ACE2 and generated an ACE2 fragment (90 kDa) in apical secretions, suggesting cleavage of this receptor. In addition, IAV increased the expression of two proteases known to cleave ACE2, sheddase ADAM17 (TACE) and TMPRSS2 and increased the TMPRSS2 zymogen and its mature fragments, implicating proteolytic autoactivation. CONCLUSION: These results indicate that IAV amplifies the expression of molecules necessary for SARS-CoV-2 infection of the distal lung. Furthermore, post-translational changes in ACE2 by IAV may increase vulnerability to lung injury such as acute respiratory distress syndrome during viral co-infections. These findings support efforts in the prevention and treatment of influenza infections during the COVID-19 pandemic.

Protein tyrosine phosphatase-α amplifies transforming growth factor-ß-dependent profibrotic signaling in lung fibroblasts.

Idiopathic pulmonary fibrosis (IPF) is a progressive, often fatal, fibrosing lung disease for which treatment remains suboptimal. Fibrogenic cytokines, including transforming growth factor-ß (TGF-ß), are central to its pathogenesis. Protein tyrosine phosphatase-α (PTPα) has emerged as a key regulator of fibrogenic signaling in fibroblasts. We have reported that mice globally deficient in PTPα (Ptpra-/-) were protected from experimental pulmonary fibrosis, in part via alterations in TGF-ß signaling. The goal of this study was to determine the lung cell types and mechanisms by which PTPα controls fibrogenic pathways and whether these pathways are relevant to human disease. Immunohistochemical analysis of lungs from patients with IPF revealed that PTPα was highly expressed by mesenchymal cells in fibroblastic foci and by airway and alveolar epithelial cells. To determine whether PTPα promotes profibrotic signaling pathways in lung fibroblasts and/or epithelial cells, we generated mice with conditional (floxed) Ptpra alleles (Ptpraf/f). These mice were crossed with Dermo1-Cre or with Sftpc-CreERT2 mice to delete Ptpra in mesenchymal cells and alveolar type II cells, respectively. Dermo1-Cre/Ptpraf/f mice were protected from bleomycin-induced pulmonary fibrosis, whereas Sftpc-CreERT2/Ptpraf/f mice developed pulmonary fibrosis equivalent to controls. Both canonical and noncanonical TGF-ß signaling and downstream TGF-ß-induced fibrogenic responses were attenuated in isolated Ptpra-/- compared with wild-type fibroblasts. Furthermore, TGF-ß-induced tyrosine phosphorylation of TGF-ß type II receptor and of PTPα were attenuated in Ptpra-/- compared with wild-type fibroblasts. The phenotype of cells genetically deficient in PTPα was recapitulated with the use of a Src inhibitor. These findings suggest that PTPα amplifies profibrotic TGF-ß-dependent pathway signaling in lung fibroblasts.

Transitional human alveolar type II epithelial cells suppress extracellular matrix and growth factor gene expression in lung fibroblasts.

Epithelial-fibroblast interactions are thought to be very important in the adult lung in response to injury, but the specifics of these interactions are not well defined. We developed coculture systems to define the interactions of adult human alveolar epithelial cells with lung fibroblasts. Alveolar type II cells cultured on floating collagen gels reduced the expression of type 1 collagen (COL1A1) and α-smooth muscle actin (ACTA2) in fibroblasts. They also reduced fibroblast expression of hepatocyte growth factor (HGF), fibroblast growth factor 7 (FGF7, KGF), and FGF10. When type II cells were cultured at an air-liquid interface to maintain high levels of surfactant protein expression, this inhibitory activity was lost. When type II cells were cultured on collagen-coated tissue culture wells to reduce surfactant protein expression further and increase the expression of some type I cell markers, the epithelial cells suppressed transforming growth factor-ß (TGF-ß)-stimulated ACTA2 and connective tissue growth factor (CTGF) expression in lung fibroblasts. Our results suggest that transitional alveolar type II cells and likely type I cells but not fully differentiated type II cells inhibit matrix and growth factor expression in fibroblasts. These cells express markers of both type II cells and type I cells. This is probably a normal homeostatic mechanism to inhibit the fibrotic response in the resolution phase of wound healing. Defining how transitional type II cells convert activated fibroblasts into a quiescent state and inhibit the effects of TGF-ß may provide another approach to limiting the development of fibrosis after alveolar injury.

Impaired non-homologous end joining in human primary alveolar type II cells in emphysema.

Emphysema is characterized by alveolar wall destruction induced mainly by cigarette smoke. Oxidative damage of DNA may contribute to the pathophysiology of this disease. We studied the impairment of the non-homologous end joining (NHEJ) repair pathway and DNA damage in alveolar type II (ATII) cells and emphysema development. We isolated primary ATII cells from control smokers, nonsmokers, and patients with emphysema to determine DNA damage and repair. We found higher reactive oxygen species generation and DNA damage in ATII cells obtained from individuals with this disease  in comparison with controls. We also observed low phosphorylation of H2AX, which activates DSBs repair signaling, in emphysema. Our results indicate the impairement  of NHEJ, as detected by low XLF expression. We also analyzed the role of DJ-1, which has a cytoprotective activity. We detected DJ-1 and  XLF interaction in ATII cells in emphysema, which suggests the impairment of their function. Moreover, we found that DJ-1 KO mice are more susceptible to DNA damage induced by cigarette smoke. Our results suggest that oxidative DNA damage and ineffective the DSBs repair via the impaired NHEJ may contribute to ATII cell death in emphysema.

TGF beta inhibits HGF, FGF7, and FGF10 expression in normal and IPF lung fibroblasts.

TGF beta is a multifunctional cytokine that is important in the pathogenesis of pulmonary fibrosis. The ability of TGF beta to stimulate smooth muscle actin and extracellular matrix gene expression in fibroblasts is well established. In this report, we evaluated the effect of TGF beta on the expression of HGF, FGF7 (KGF), and FGF10, important growth and survival factors for the alveolar epithelium. These growth factors are important for maintaining type II cells and for restoration of the epithelium after lung injury. Under conditions of normal serum supplementation or serum withdrawal TGF beta inhibited fibroblast expression of HGF, FGF7, and FGF10. We confirmed these observations with genome wide RNA sequencing of the response of control and IPF fibroblasts to TGF beta. In general, gene expression in IPF fibroblasts was similar to control fibroblasts. Reduced expression of HGF, FGF7, and FGF10 is another means whereby TGF beta impairs epithelial healing and promotes fibrosis after lung injury.

TGF beta inhibits expression of SP-A, SP-B, SP-C, but not SP-D in human alveolar type II cells.

TGF beta is a multifunctional cytokine that regulates alveolar epithelial cells as well as immune cells and fibroblasts. TGF beta inhibits surfactant protein A, B and C expression in fetal human lung and can inhibit type II cell proliferation induced by FGF7 (KGF). However, little is known about direct effects of TGF beta on adult human type II cells. We cultured alveolar type II cells under air/liquid interface conditions to maintain their state of differentiation with or without TGF beta. TGF beta markedly decreased expression of SP-A, SP-B, SP-C, fatty acid synthase, and the phospholipid transporter ABCA3. However, TGF beta increased protein levels of SP-D with little change in mRNA levels, indicating that it is regulated independently from other components of surfactant. TGF beta is a negative regulator of both the protein and the phospholipid components of surfactant. TGF beta did not induce EMT changes in highly differentiated human type II cells. SP-D is an important host defense molecule and regulated independently from the other surfactant proteins. Taken together these data are the first report of the effect of TGF beta on highly differentiated adult human type II cells. The effects on the surfactant system are likely important in the development of fibrotic lung diseases.

The lung response to ozone is determined by age and is partially dependent on toll-Like receptor 4.

BACKGROUND: Ozone pollution has adverse effects on respiratory health in children and adults. This study was carried out in the mouse model to investigate the influence of age and to define the role of toll-like receptor four (TLR4) in the lung response to ozone exposure during postnatal development. METHODS: Female mice (1 to 6 weeks of age) were exposed for 3 h to ozone (1 part per million) or filtered air. Analyses were carried out at six and 24 h after completion of exposure, to assess the effects on lung permeability, airway neutrophilia, expression of antioxidants and chemokines, and mucus production. The role of TLR4 was defined by examining TLR4 expression in the lung during development, and by investigating the response to ozone in tlr4-deficient mice. RESULTS: Metallothionein-1, calcitonin gene-related product, and chemokine C-X-C ligand (CXCL) five were consistent markers induced by ozone throughout development. Compared with adults, neonates expressed lower levels of pulmonary TLR4 and responded with increased mucus production, and developed an attenuated response to ozone characterized by reduced albumin leakage and neutrophil influx into the airways, and lower expression of CXCL1 and CXCL2 chemokines. Examination of the responses in tlr4-deficient mice indicated that ozone-mediated airway neutrophilia, but not albumin leakage or mucus production were dependent on TLR4. CONCLUSIONS: Collectively, the data demonstrate that the response to ozone is determined by age and is partially dependent on TLR4 signaling. The reduced responsiveness of the neonatal lung to ozone may be due at least in part to insufficient pulmonary TLR4 expression.

Transcriptome profiling of the newborn mouse lung response to acute ozone exposure.

Ozone pollution is associated with adverse effects on respiratory health in adults and children but its effects on the neonatal lung remain unknown. This study was carried out to define the effect of acute ozone exposure on the neonatal lung and to profile the transcriptome response. Newborn mice were exposed to ozone or filtered air for 3h. Total RNA was isolated from lung tissues at 6 and 24h after exposure and was subjected to microarray gene expression analysis. Compared to filtered air-exposed littermates, ozone-exposed newborn mice developed a small but significant neutrophilic airway response associated with increased CXCL1 and CXCL5 expression in the lung. Transcriptome analysis indicated that 455 genes were down-regulated and 166 genes were up-regulated by at least 1.5-fold at 6h post-ozone exposure (t-test, p < .05). At 24h, 543 genes were down-regulated and 323 genes were up-regulated in the lungs of ozone-exposed, compared to filtered air-exposed, newborn mice (t-test, p < .05). After controlling for false discovery rate, 50 genes were identified as significantly down-regulated and only a few (RORC, GRP, VREB3, and CYP2B6) were up-regulated at 24h post-ozone exposure (q < .05). Gene ontology enrichment analysis revealed that cell cycle-associated functions including cell division/proliferation were the most impacted pathways, which were negatively regulated by ozone exposure, an adverse effect that was associated with reduced bromo-deoxyuridine incorporation. These results demonstrate that acute ozone exposure alters cell proliferation in the developing neonatal lung through a global suppression of cell cycle function.