Interstitial macrophage phenotypes in Schistosoma-induced pulmonary hypertension.

Background: Schistosomiasis is a common cause of pulmonary hypertension (PH) worldwide. Type 2 inflammation contributes to the development of Schistosoma-induced PH. Specifically, interstitial macrophages (IMs) derived from monocytes play a pivotal role by producing thrombospondin-1 (TSP-1), which in turn activates TGF-ß, thereby driving the pathology of PH. Resident and recruited IM subpopulations have recently been identified. We hypothesized that in Schistosoma-PH, one IM subpopulation expresses monocyte recruitment factors, whereas recruited monocytes become a separate IM subpopulation that expresses TSP-1. Methods: Mice were intraperitoneally sensitized and then intravenously challenged with S. mansoni eggs. Flow cytometry on lungs and blood was performed on wildtype and reporter mice to identify IM subpopulations and protein expression. Single-cell RNA sequencing (scRNAseq) was performed on flow-sorted IMs from unexposed and at day 1, 3 and 7 following Schistosoma exposure to complement flow cytometry based IM characterization and identify gene expression. Results: Flow cytometry and scRNAseq both identified 3 IM subpopulations, characterized by CCR2, MHCII, and FOLR2 expression. Following Schistosoma exposure, the CCR2+ IM subpopulation expanded, suggestive of circulating monocyte recruitment. Schistosoma exposure caused increased monocyte-recruitment ligand CCL2 expression in the resident FOLR2+ IM subpopulation. In contrast, the vascular pathology-driving protein TSP-1 was greatest in the CCR2+ IM subpopulation. Conclusion: Schistosoma-induced PH involves crosstalk between IM subpopulations, with increased expression of monocyte recruitment ligands by resident FOLR2+ IMs, and the recruitment of CCR2+ IMs which express TSP-1 that activates TGF-ß and causes PH.

VEGF-C/VEGFR-3 signalling in macrophages ameliorates acute lung injury.

BACKGROUND: Successful recovery from acute lung injury requires inhibition of neutrophil influx and clearance of apoptotic neutrophils. However, the mechanisms underlying recovery remain unclear. We investigated the ameliorative effects of vascular endothelial growth factor (VEGF)-C/VEGF receptor 3 (VEGFR-3) signalling in macrophages in lipopolysaccharide (LPS)-induced lung injury. METHODS: LPS was intranasally injected into wild-type and transgenic mice. Gain and loss of VEGF-C/VEGFR-3 signalling function experiments employed adenovirus-mediated intranasal delivery of VEGF-C (Ad-VEGF-C vector) and soluble VEGFR-3 (sVEGFR-3) or anti-VEGFR-3 blocking antibodies and mice with a deletion of VEGFR-3 in myeloid cells. RESULTS: The early phase of lung injury was significantly alleviated by the overexpression of VEGF-C with increased levels of bronchoalveolar lavage (BAL) fluid interleukin-10 (IL-10), but worsened in the later phase by VEGFR-3 inhibition upon administration of Ad-sVEGFR-3 vector. Injection of anti-VEGFR-3 antibodies to mice in the resolution phase inhibited recovery from lung injury. The VEGFR-3-deleted mice had a shorter survival time than littermates and more severe lung injury in the resolution phase. Alveolar macrophages in the resolution phase digested most of the extrinsic apoptotic neutrophils and VEGF-C/VEGFR-3 signalling increased efferocytosis via upregulation of integrin αv in the macrophages. We also found that incubation with BAL fluid from acute respiratory distress syndrome (ARDS) patients, but not from controls, decreased VEGFR-3 expression and the efficiency of IL-10 expression and efferocytosis in human monocyte-derived macrophages. CONCLUSIONS: VEGF-C/VEGFR-3 signalling in macrophages ameliorates experimental lung injury. This mechanism may also provide an explanation for ARDS resolution.

Endothelial cell PHD2-HIF1α-PFKFB3 contributes to right ventricle vascular adaptation in pulmonary hypertension.

Humans and animals with pulmonary hypertension (PH) show right ventricular (RV) capillary growth, which positively correlates with overall RV hypertrophy. However, molecular drivers of RV vascular augmentation in PH are unknown. Prolyl hydroxylase (PHD2) is a regulator of hypoxia-inducible factors (HIFs), which transcriptionally activates several proangiogenic genes, including the glycolytic enzyme 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3). We hypothesized that a signaling axis of PHD2-HIF1α-PFKFB3 contributes to adaptive coupling between the RV vasculature and tissue volume to maintain appropriate vascular density in PH. We used design-based stereology to analyze endothelial cell (EC) proliferation and the absolute length of the vascular network in the RV free wall, relative to the tissue volume in mice challenged with hypoxic PH. We observed increased RV EC proliferation starting after 6 h of hypoxia challenge. Using parabiotic mice, we found no evidence for a contribution of circulating EC precursors to the RV vascular network. Mice with transgenic deletion or pharmacological inhibition of PHD2, HIF1α, or PFKFB3 all had evidence of impaired RV vascular adaptation following hypoxia PH challenge. PHD2-HIF1α-PFKFB3 contributes to structural coupling between the RV vascular length and tissue volume in hypoxic mice, consistent with homeostatic mechanisms that maintain appropriate vascular density. Activating this pathway could help augment the RV vasculature and preserve RV substrate delivery in PH, as an approach to promote RV function.

Targeted Phage Display-based Pulmonary Vaccination in Mice and Non-human Primates.

BACKGROUND: The extensive alveolar capillary network of the lungs is an attractive route for administration of several agents. One key functional attribute is the rapid onset of systemic action due to the absence of first-pass metabolism. METHODS: Here we applied a combinatorial approach for ligand-directed pulmonary delivery as a unique route for systemic targeting in vaccination. FINDINGS: We screened a phage display random peptide library in vivo to select, identify, and validate a ligand (CAKSMGDIVC) that specifically targets and is internalized through its receptor, α3ß1 integrin, on the surface of cells lining the lung airways and alveoli and mediates CAKSMGDIVC-displaying phage binding and systemic delivery without compromising lung homeostasis. As a proof-of-concept, we show that the pulmonary delivery of targeted CAKSMGDIVC-displaying phage particles in mice and non-human primates elicit a systemic and specific humoral response. CONCLUSIONS: This broad methodology blueprint represents a robust and versatile platform tool enabling new ligand-receptor discovery with many potential translational applications. FUNDING: Cancer Center Support Grants to the University of Texas M.D. Anderson Cancer Center (CA016672), University of New Mexico Comprehensive Cancer Center (CA118100), Rutgers Cancer Institute of New Jersey (CA072720), research awards from the Gillson Longenbaugh Foundation, and National Institutes of Health (NIH) grant no. 1R01CA226537.

Persistent, Progressive Pulmonary Fibrosis and Epithelial Remodeling in Mice.

Idiopathic pulmonary fibrosis (IPF) is a progressive fibrotic interstitial lung disease with underlying mechanisms that have been primarily investigated in mice after intratracheal instillation of a single dose of bleomycin. However, the model has significant limitations, including transient fibrosis that spontaneously resolves and its failure to fully recapitulate the epithelial remodeling in the lungs of patients with IPF. Thus, there remains an unmet need for a preclinical model with features that more closely resemble the human disease. Repetitive intratracheal instillation of bleomycin has previously been shown to recapitulate some of these features, but the instillation procedure is complex, and the long-term consequences on epithelial remodeling and fibrosis persistence and progression remain poorly understood. Here, we developed a simplified repetitive bleomycin instillation strategy consisting of three bi-weekly instillations that leads to persistent and progressive pulmonary fibrosis. Lung histology demonstrates increased collagen deposition, fibroblast accumulation, loss of type I and type II alveolar epithelial cells within fibrotic areas, bronchiolization of the lung parenchyma with CCSP+ cells, remodeling of the distal lung into cysts reminiscent of simple honeycombing, and accumulation of hyperplastic transitional KRT8+ epithelial cells. Micro-computed tomographic imaging demonstrated significant traction bronchiectasis and subpleural fibrosis. Thus, the simplified repetitive bleomycin instillation strategy leads to progressive fibrosis and recapitulates the histological and radiographic characteristics of IPF. Compared with the single bleomycin instillation model, we suggest that the simplified repetitive instillation model may be better suited to address mechanistic questions about IPF pathogenesis and preclinical studies of antifibrotic drug candidates.

Platelet activation contributes to hypoxia-induced inflammation.

Inflammation is central to the pathogenesis of pulmonary vascular remodeling and pulmonary hypertension (PH). Inflammation precedes remodeling in preclinical models, thus supporting the concept that changes in immunity drive remodeling in PH. Platelets are recognized as mediators of inflammation, but whether platelets contribute to hypoxia-driven inflammation has not been studied. We utilized a murine hypoxia model to test the hypothesis that platelets drive hypoxia-induced inflammation. We evaluated male and female 9-wk-old normoxic and hypoxic mice and in selected experiments included hypoxic thrombocytopenic mice. Thrombocytopenic mice were generated with an anti-GP1bα rat IgG antibody. We also performed immunostaining of lung sections from failed donor controls and patients with idiopathic pulmonary arterial hypertension. We found that platelets are increased in the lungs of hypoxic mice and hypoxia induces platelet activation. Platelet depletion prevents hypoxia-driven increases in the proinflammatory chemokines CXCL4 and CCL5 and attenuates hypoxia-induced increase in plasma CSF-2. Pulmonary interstitial macrophages are increased in the lungs of hypoxic mice; this increase is prevented in thrombocytopenic mice. To determine the potential relevance to human disease, lung sections from donors and patients with advanced idiopathic pulmonary arterial hypertension (iPAH) were immunostained for the platelet-specific protein CD41. We observed iPAH lungs had a two-fold increase in CD41, compared with controls. Our data provide evidence that the platelet count is increased in the lungs and activated in mice with hypoxia-induced inflammation and provides rationale for the further study of the potential contribution of platelets to inflammatory mediated vascular remodeling and PH.

Endothelial HIF-2α as a Key Endogenous Mediator Preventing Emphysema.

Rationale: Endothelial injury may provoke emphysema, but molecular pathways of disease development require further discernment. Emphysematous lungs exhibit decreased expression of HIF-2α (hypoxia-inducible factor-2α)-regulated genes, and tobacco smoke decreases pulmonary HIF-2α concentrations. These findings suggest that decreased HIF-2α expression is important in the development of emphysema.Objectives: The objective of this study was to evaluate the roles of endothelial-cell (EC) HIF-2α in the pathogenesis of emphysema in mice.Methods: Mouse lungs were examined for emphysema after either the loss or the overexpression of EC Hif-2α. In addition, SU5416, a VEGFR2 inhibitor, was used to induce emphysema. Lungs were evaluated for HGF (hepatocyte growth factor), a protein involved in alveolar development and homeostasis. Lungs from patients with emphysema were measured for endothelial HIF-2α expression.Measurements and Main Results: EC Hif-2α deletion resulted in emphysema in association with fewer ECs and pericytes. After SU5416 exposure, EC Hif-2α-knockout mice developed more severe emphysema, whereas EC Hif-2α-overexpressing mice were protected. EC Hif-2α-knockout mice demonstrated lower levels of HGF. Human emphysema lung samples exhibited reduced EC HIF-2α expression.Conclusions: Here, we demonstrate a unique protective role for pulmonary endothelial HIF-2α and how decreased expression of this endogenous factor causes emphysema; its pivotal protective function is suggested by its ability to overcome VEGF antagonism. HIF-2α may maintain alveolar architecture by promoting vascular survival and associated HGF production. In summary, HIF-2α may be a key endogenous factor that prevents the development of emphysema, and its upregulation has the potential to foster lung health in at-risk patients.

Perspective: pathobiological paradigms in pulmonary hypertension, time for reappraisal.

For the past 120 years, there has been a progressive evolution of the pathobiological concepts underlying pulmonary hypertension. Conceptual frameworks, build around the paradigms of excessive vasoconstriction (vs. vasodilation) and, more recently, of the cancer-like hypothesis of pulmonary hypertension, have served to consolidate key discoveries; moreover, they have and continue contributing to innovative advances that have been translated into either successful or potential new therapies. However, those frameworks do not fully address the complexity and challenges facing pulmonary hypertension, particularly those involving the marked heterogeneity of disease presentation and the dynamic changes occurring over time in affected tissues and cells. This is particularly relevant in regards to the molecular pathways of pulmonary hypertension; the ever growing understanding of molecular and cellular pathways requires clarification if they drive distinctive pulmonary vascular lesions in a given lung and disease patients with the same group pulmonary hypertension. Novel methodologies and approaches can start dissecting this key challenge in the field as it is critical to address the key angle of heterogeneity of the disease and reappraisal of disease-modifying therapies.

Localization of Macrophages in the Human Lung via Design-based Stereology.

Rationale: Interstitial macrophages (IMs) and airspace macrophages (AMs) play critical roles in lung homeostasis and host defense, and are central to the pathogenesis of a number of lung diseases. However, the absolute numbers of macrophages and the precise anatomic locations they occupy in the healthy human lung have not been quantified.Objectives: To determine the precise number and anatomic location of human pulmonary macrophages in nondiseased lungs and to quantify how this is altered in chronic cigarette smokers.Methods: Whole right upper lobes from 12 human donors without pulmonary disease (6 smokers and 6 nonsmokers) were evaluated using design-based stereology. CD206 (cluster of differentiation 206)-positive/CD43+ AMs and CD206+/CD43- IMs were counted in five distinct anatomical locations using the optical disector probe.Measurements and Main Results: An average of 2.1 × 109 IMs and 1.4 × 109 AMs were estimated per right upper lobe. Of the AMs, 95% were contained in diffusing airspaces and 5% in airways. Of the IMs, 78% were located within the alveolar septa, 14% around small vessels, and 7% around the airways. The local density of IMs was greater in the alveolar septa than in the connective tissue surrounding the airways or vessels. The total number and density of IMs was 36% to 56% greater in the lungs of cigarette smokers versus nonsmokers.Conclusions: The precise locations occupied by pulmonary macrophages were defined in nondiseased human lungs from smokers and nonsmokers. IM density was greatest in the alveolar septa. Lungs from chronic smokers had increased IM numbers and overall density, supporting a role for IMs in smoking-related disease.

Interstitial macrophage-derived thrombospondin-1 contributes to hypoxia-induced pulmonary hypertension.

AIMS: Transforming growth factor-ß (TGF-ß) signalling is required for chronic hypoxia-induced pulmonary hypertension (PH). The activation of TGF-ß by thrombospondin-1 (TSP-1) contributes to the pathogenesis of hypoxia-induced PH. However, neither the cellular source of pathologic TSP-1 nor the downstream signalling pathway that link activated TGF-ß to PH have been determined. In this study, we hypothesized that circulating monocytes, which are recruited to become interstitial macrophages (IMs), are the major source of TSP-1 in hypoxia-exposed mice, and TSP-1 activates TGF-ß with increased Rho-kinase signalling, causing vasoconstriction. METHODS AND RESULTS: Flow cytometry revealed that a specific subset of IMs is the major source of pathologic TSP-1 in hypoxia. Intravenous depletion and parabiosis experiments demonstrated that these cells are circulating prior to recruitment into the interstitium. Rho-kinase-mediated vasoconstriction was a major downstream target of active TGF-ß. Thbs1 deficient bone marrow (BM) protected against hypoxic-PH by blocking TGF-ß activation and Rho-kinase-mediated vasoconstriction. CONCLUSION: In hypoxia-challenged mice, BM derived and circulating monocytes are recruited to become IMs which express TSP-1, resulting in TGF-ß activation and Rho-kinase-mediated vasoconstriction.

Immunoglobulin-driven Complement Activation Regulates Proinflammatory Remodeling in Pulmonary Hypertension.

Rationale: Pulmonary hypertension (PH) is a life-threatening cardiopulmonary disorder in which inflammation and immunity have emerged as critical early pathogenic elements. Although proinflammatory processes in PH and pulmonary arterial hypertension (PAH) are the focus of extensive investigation, the initiating mechanisms remain elusive.Objectives: We tested whether activation of the complement cascade is critical in regulating proinflammatory and pro-proliferative processes in the initiation of experimental hypoxic PH and can serve as a prognostic biomarker of outcome in human PAH.Methods: We used immunostaining of lung tissues from experimental PH models and patients with PAH, analyses of genetic murine models lacking specific complement components or circulating immunoglobulins, cultured human pulmonary adventitial fibroblasts, and network medicine analysis of a biomarker risk panel from plasma of patients with PAH.Measurements and Main Results: Pulmonary perivascular-specific activation of the complement cascade was identified as a consistent critical determinant of PH and PAH in experimental animal models and humans. In experimental hypoxic PH, proinflammatory and pro-proliferative responses were dependent on complement (alternative pathway and component 5), and immunoglobulins, particularly IgG, were critical for activation of the complement cascade. We identified Csf2/GM-CSF as a primary complement-dependent inflammatory mediator. Furthermore, using network medicine analysis of a biomarker risk panel from plasma of patients with PAH, we demonstrated that complement signaling can serve as a prognostic factor for clinical outcome in PAH.Conclusions: This study establishes immunoglobulin-driven dysregulated complement activation as a critical pathobiological mechanism regulating proinflammatory and pro-proliferative processes in the initiation of experimental hypoxic PH and demonstrates complement signaling as a critical determinant of clinical outcome in PAH.

GDF15 is an epithelial-derived biomarker of idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is the most common and devastating of the interstitial lung diseases. Epithelial dysfunction is thought to play a prominent role in disease pathology, and we sought to characterize secreted signals that may contribute to disease pathology. Transcriptional profiling of senescent type II alveolar epithelial cells from mice with epithelial-specific telomere dysfunction identified the transforming growth factor-ß family member, growth and differentiation factor 15 (Gdf15), as the most significantly upregulated secreted protein. Gdf15 expression is induced in response to telomere dysfunction and bleomycin challenge in mice. Gdf15 mRNA is expressed by lung epithelial cells, and protein can be detected in peripheral blood and bronchoalveolar lavage following bleomycin challenge in mice. In patients with IPF, GDF15 mRNA expression in lung tissue is significantly increased and correlates with pulmonary function. Single-cell RNA sequencing of human lungs identifies epithelial cells as the primary source of GDF15, and circulating concentrations of GDF15 are markedly elevated and correlate with disease severity and survival in multiple independent cohorts. Our findings suggest that GDF15 is an epithelial-derived secreted protein that may be a useful biomarker of epithelial stress and identifies IPF patients with poor outcomes.

Pseudomonas aeruginosa stimulates nuclear sphingosine-1-phosphate generation and epigenetic regulation of lung inflammatory injury.

INTRODUCTION: Dysregulated sphingolipid metabolism has been implicated in the pathogenesis of various pulmonary disorders. Nuclear sphingosine-1-phosphate (S1P) has been shown to regulate histone acetylation, and therefore could mediate pro-inflammatory genes expression. METHODS: Profile of sphingolipid species in bronchoalveolar lavage fluids and lung tissue of mice challenged with Pseudomonas aeruginosa (PA) was investigated. The role of nuclear sphingosine kinase (SPHK)2 and S1P in lung inflammatory injury by PA using genetically engineered mice was determined. RESULTS: Genetic deletion of Sphk2, but not Sphk1, in mice conferred protection from PA-mediated lung inflammation. PA infection stimulated phosphorylation of SPHK2 and its localisation in epithelial cell nucleus, which was mediated by protein kinase C (PKC) δ. Inhibition of PKC δ or SPHK2 activity reduced PA-mediated acetylation of histone H3 and H4, which was necessary for the secretion of pro-inflammatory cytokines, interleukin-6 and tumour necrosis factor-α. The clinical significance of the findings is supported by enhanced nuclear localisation of p-SPHK2 in the epithelium of lung specimens from patients with cystic fibrosis (CF). CONCLUSIONS: Our studies define a critical role for nuclear SPHK2/S1P signalling in epigenetic regulation of bacterial-mediated inflammatory lung injury. Targeting SPHK2 may represent a potential strategy to reduce lung inflammatory pulmonary disorders such as pneumonia and CF.

A Review of Neuropathological Features of Familial and Adult Hemophagocytic Lymphohistiocytosis.

Hemophagocytic lymphohistiocytosis (HLH) is a hematological disorder that can be due to genetic (primary HLH) causes or excessive activation of the immune system in association with infection, malignancy, rheumatologic disorders, or immune suppression (secondary HLH). Hemophagocytic lymphohistiocytosis remains an under-recognized condition among neuropathologists, especially the secondary forms, where it may be diagnosed only at brain biopsy or autopsy due to confounding comorbidities. The CNS is frequently affected, but neuropathological features are underappreciated. We place our own experience with HLH in context with review of neuropathological features from the literature. A 10-year database search for cases from our pediatric and adult hospitals with re-review of neuropathological features revealed 1 biopsy and 5 autopsies. Literature that reported neuropathological features was tabulated and 8 adult and 12 pediatric cases were identified. Children had predominantly secondary HLH: 5/12 co-associated with Epstein Barr (or dual) viral infections, 3/12 with malignancy. One biopsy showed florid lymphohistiocytic infiltrates and hemophagocytosis and served as first diagnosis; 2/5 CNS autopsies had originally been reported as negative for HLH, but on re-review had subtle lymphohistiocytic infiltrates with hemophagocytosis confined to leptomeninges. In conclusion, the neuropathological features are highly variable in HLH; features such as focal erythrophagocytosis may be histologically subtle in early phases, but should be sought.

Paclitaxel blocks Th2-mediated TGF-ß activation in Schistosoma mansoni-induced pulmonary hypertension.

Schistosomiasis is a leading cause of pulmonary hypertension (PH) worldwide. Recent studies reveal that the type-2 immune cytokines IL-4 and IL-13, as well as consequent activation of TGF-ß, are key factors in the pathogenesis of Schistosoma-PH. Paclitaxel has been reported to act as an adjuvant for Th2 inflammation while downregulating TGF-ß activation. Moreover, paclitaxel blocks PH in monocrotaline and SU5416-hypoxia models. We hypothesized that paclitaxel would augment Th2 inflammation while blocking TGF-ß activation and PH after schistosomiasis exposure. Wild-type mice (C57BL6/J; 6/group) were intraperitoneally (IP) sensitized and then intravenously (IV) challenged with Schistosoma mansoni eggs. One day after IV egg challenge, the mice were treated with a single IP dose of 25 mg/kg paclitaxel or vehicle. Right ventricular (RV) catheterization was performed and granuloma volumes and vascular remodeling were quantified. Lung cytokines were quantified by ELISA and reverse transcription polymerase chain reaction, and the quantity of active TGF-ß was determined using a cell reporter line. We also investigated hypoxia-induced PH. Paclitaxel treatment significantly protected mice from Schistosoma-PH, with decreased RV systolic pressure ( P = 0.005) and pulmonary vascular media thickness. Inflammation was significantly suppressed, contrary to our hypothesis, with decreased IL-4 and IL-13 levels, smaller granulomas, and less active TGF-ß following paclitaxel treatment. There was no change in IFN-γ or FoxO1 or FoxO3 expression. Paclitaxel did not suppress chronic hypoxia-induced PH, which is also TGF-ß-driven but independent of type-2 immunity. Paclitaxel protects against Schistosoma-induced PH in mice, although by blocking proximate Th2 inflammation rather than suppressing distal TGF-ß activation.

S100A8 Protects Human Primary Alveolar Type II Cells against Injury and Emphysema.

Pulmonary emphysema is characterized by alveolar wall destruction, and cigarette smoking is the main risk factor in this disease development. S100A8 is a member of the S100 protein family, with an oxidative stress-related and antiinflammatory role. The mechanisms of human alveolar type II (ATII) cell injury contributing to emphysema pathophysiology are not completely understood. We wanted to determine whether S100A8 can protect ATII cells against injury induced by cigarette smoke and this disease development. We used freshly isolated ATII cells from nonsmoking and smoking organ donors, as well as patients with emphysema to determine S100A8 function. S100A8 protein and mRNA levels were low in individuals with this disease and correlated with its severity as determined by using lung tissue from areas with mild and severe emphysema obtained from the same patient. Its expression negatively correlated with high oxidative stress as observed by 4-hydroxynonenal levels. We also detected decreased serine phosphorylation within S100A8 by PKAα in this disease. This correlated with increased S100A8 ubiquitination by SYVN1. Moreover, we cultured ATII cells isolated from nonsmokers followed by treatment with cigarette smoke extract. We found that this exposure upregulated S100A8 expression. We also confirmed the cytoprotective role of S100A8 against cell injury using gain- and loss-of-function approaches in vitro. S100A8 knockdown sensitized cells to apoptosis induced by cigarette smoke. In contrast, S100A8 overexpression rescued cell injury. Our results suggest that S100A8 protects ATII cells against injury and cigarette smoke-induced emphysema. Targeting S100A8 may provide a potential therapeutic strategy for this disease.

Protein Tyrosine Phosphatase-N13 Promotes Myofibroblast Resistance to Apoptosis in Idiopathic Pulmonary Fibrosis.

RATIONALE: Idiopathic pulmonary fibrosis (IPF) is a progressive, fibrotic interstitial lung disease characterized by (myo)fibroblast accumulation and collagen deposition. Resistance to Fas-induced apoptosis is thought to facilitate (myo)fibroblast persistence in fibrotic lung tissues by poorly understood mechanisms. OBJECTIVES: To test the hypothesis that PTPN13 (protein tyrosine phosphatase-N13) is expressed by IPF lung (myo)fibroblasts, promotes their resistance to Fas-induced apoptosis, and contributes to the development of pulmonary fibrosis. METHODS: PTPN13 was localized in lung tissues from patients with IPF and control subjects by immunohistochemical staining. Inhibition of PTPN13 function in primary IPF and normal lung (myo)fibroblasts was accomplished by: 1) downregulation with TNF-α (tumor necrosis factor-α)/IFN-γ, 2) siRNA knockdown, or 3) a cell-permeable Fas/PTPN13 interaction inhibitory peptide. The role of PTPN13 in the development of pulmonary fibrosis was assessed in mice with genetic deficiency of PTP-BL, the murine ortholog of PTPN13. MEASUREMENTS AND MAIN RESULTS: PTPN13 was constitutively expressed by (myo)fibroblasts in the fibroblastic foci of patients with IPF. Human lung (myo)fibroblasts, which are resistant to Fas-induced apoptosis, basally expressed PTPN13 in vitro. TNF-α/IFN-γ or siRNA-mediated PTPN13 downregulation and peptide-mediated inhibition of the Fas/PTPN13 interaction in human lung (myo)fibroblasts promoted Fas-induced apoptosis. Bleomycin-challenged PTP-BL-/- mice, while developing inflammatory lung injury, exhibited reduced pulmonary fibrosis compared with wild-type mice. CONCLUSIONS: These findings suggest that PTPN13 mediates the resistance of human lung (myo)fibroblasts to Fas-induced apoptosis and promotes pulmonary fibrosis in mice. Our results suggest that strategies aimed at interfering with PTPN13 expression or function may represent a novel strategy to reduce fibrosis in IPF.

Epithelial Heparan Sulfate Contributes to Alveolar Barrier Function and Is Shed during Lung Injury.

The lung epithelial glycocalyx is a carbohydrate-enriched layer lining the pulmonary epithelial surface. Although epithelial glycocalyx visualization has been reported, its composition and function remain unknown. Using immunofluorescence and mass spectrometry, we identified heparan sulfate (HS) and chondroitin sulfate within the lung epithelial glycocalyx. In vivo selective enzymatic degradation of epithelial HS, but not chondroitin sulfate, increased lung permeability. Using mass spectrometry and gel electrophoresis approaches to determine the fate of epithelial HS during lung injury, we detected shedding of 20 saccharide-long or greater HS into BAL fluid in intratracheal LPS-treated mice. Furthermore, airspace HS in clinical samples from patients with acute respiratory distress syndrome correlated with indices of alveolar permeability, reflecting the clinical relevance of these findings. The length of HS shed during intratracheal LPS-induced injury (≥20 saccharides) suggests cleavage of the proteoglycan anchoring HS to the epithelial surface, rather than cleavage of HS itself. We used pharmacologic and transgenic animal approaches to determine that matrix metalloproteinases partially mediate HS shedding during intratracheal LPS-induced lung injury. Although there was a trend toward decreased alveolar permeability after treatment with the matrix metalloproteinase inhibitor, doxycycline, this did not reach statistical significance. These studies suggest that epithelial HS contributes to the lung epithelial barrier and its degradation is sufficient to increase lung permeability. The partial reduction of HS shedding achieved with doxycycline is not sufficient to rescue epithelial barrier function during intratracheal LPS-induced lung injury; however, whether complete attenuation of HS shedding is sufficient to rescue epithelial barrier function remains unknown.