Resident Cellular Components of the Human Lung Current Knowledge and Goals for Research on Cell Phenotyping and Function

The purpose of the workshop was to identify still obscure or novel cellular components of the lung, to determine cell function in lung development and in health that impacts on disease, and to decide promising avenues for future research to extract and phenotype these cells. Since robust technologies are now available to identify, sort, purify, culture, and phenotype cells, progress is now within sight to unravel the origins and functional capabilities of lung cells in developmental stages and in disease. The Workshop's agenda was to first discuss the lung's embryologic development, including progenitor and stem cells, and then assess the functional and structural cells in three main compartments of the lung: (1) airway cells in bronchial and bronchiolar epithelium and bronchial glands (basal, secretory, ciliated, Clara, and neuroendocrine cells); (2) alveolar unit cells (Type 1 cells, Type 2 cells, and fibroblasts in the interstitium); and (3) pulmonary vascular cells (endothelial cells from different vascular structures, smooth muscle cells, and adventitial fibroblasts). The main recommendations were to: (1) characterize with better cell markers, both surface and nonsurface, the various cells within the lung, including progenitor cells and stem cells; (2) obtain more knowledge about gene expression in specific cell types in health and disease, which will provide insights into biological and pathologic processes; (3) develop more methodologies for cell culture, isolation, sorting, co-culture, and immortalization; and (4) promote tissue banks to facilitate the procurement of tissue from normal and from diseased lung for analysis at all levels

Spdef null mice lack conjunctival goblet cells and provide a model of dry eye

Goblet cell numbers decrease within the conjunctival epithelium in drying and cicatrizing ocular surface diseases. Factors regulating goblet cell differentiation in conjunctival epithelium are unknown. Recent data indicate that the transcription factor SAM-pointed domain epithelial-specific transcription factor (Spdef) is essential for goblet cell differentiation in tracheobronchial and gastrointestinal epithelium of mice. Using Spdef(-/-) mice, we determined that Spdef is required for conjunctival goblet cell differentiation and that Spdef(-/-) mice, which lack conjunctival goblet cells, have significantly increased corneal surface fluorescein staining and tear volume, a phenotype consistent with dry eye. Microarray analysis of conjunctival epithelium in Spdef(-/-) mice revealed down-regulation of goblet cell-specific genes (Muc5ac, Tff1, Gcnt3). Up-regulated genes included epithelial cell differentiation/keratinization genes (Sprr2h, Tgm1) and proinflammatory genes (Il1-alpha, Il-1beta, Tnf-alpha), all of which are up-regulated in dry eye. Interestingly, four Wnt pathway genes were down-regulated. SPDEF expression was significantly decreased in the conjunctival epithelium of Sjogren syndrome patients with dry eye and decreased goblet cell mucin expression. These data demonstrate that Spdef is required for conjunctival goblet cell differentiation and down-regulation of SPDEF may play a role in human dry eye with goblet cell loss. Spdef(-/-) mice have an ocular surface phenotype similar to that in moderate dry eye, providing a new, more convenient model for the disease

Corticosteroid-resistant neutrophilic airway inflammation and hyperresponsiveness caused by il-13

Rationale: severe corticosteroid refractory asthma is a significant unmet medical need. It accounts for 10% of the asthma population and 50% of the health economic burden. Recent understanding of asthma heterogeneity has evolved beyond clinical characteristics, allowing definition of distinct disease phenotypes such as those defined by levels of Type 2 inflammation (Type-2 high ‘eosinophilic’ disease and Type-2 low ‘neutrophilic’ disease). However, a recent study using dupilumab (an antibody that blocks the common IL-4 and IL-13 receptor chain, IL-4Rα) as an add-on therapy in adults with uncontrolled persistent asthma showed efficacy irrespective of baseline eosinophil count (Wenzel et al, Lancet 2016). The aim of this work was to use IL-13 transgenic mice to test the hypothesis that a subset of IL-13 mediated airway responses are corticosteroid-unresponsive and contribute to ongoing airways symptoms.Methods: IL-13 expression in the lungs was induced using Doxycycline (DOX) in Ccsp-rtTA/Otet-Il-13 double-transgenic (Ccsp/Il-13) mice. Littermate control single transgenic mice also received DOX. Where indicated, mice received daily intra-peritoneal injections of 3 mg/kg Dexamethasone (Dex) for 3–7 days and control mice received saline. Methacholine challenge and lung function measurements were performed and lungs harvested for mRNA analysis and immunohistochemistry (IHC). BALF was obtained for ELISA and differential cell counts.Results: compared to controls, Ccsp/Il-13 mice showed significantly increased airway hyperresponsiveness (AHR) to methacholine and IHC revealed increased bronchial smooth muscle and goblet cell metaplasia. The BALF of these mice contained mixed eosinophilic and neutrophilic inflammation, but neutrophils predominated. Characteristic Th2-responsive genes (Il-13, Eotaxin, Muc5ac, Periostin and SerpinB2) as well as genes more characteristic of Th17 responses (Cxcl1/Kc, Cxcl2 and Csf3) were significantly elevated. Treatment with Dex did not abrogate AHR, even though eosinophilia and the ‘Th2’ gene signature were significantly reduced. However, neutrophils and the ‘Th17’ signature remained elevated.Conclusion: although IL-13 promotes eosinophilic airways disease, it can also drive corticosteroid refractory inflammation characterised by persistent neutrophilia, Th17 cytokines and maintenance of AHR. These findings may help explain the beneficial effect of dupilumab in uncontrolled asthma. The Ccsp/Il-13 mouse may be a useful model for dissecting the molecular pathways and mechanisms associated with predominant neutrophilic, corticosteroid refractory disease

Soluble adam33 augments the pulmonary immune response promoting allergic airway sensitivity

Rational: a disintegrin and metalloproteinase 33 (ADAM33) was discovered in 2002 as an asthma susceptibility gene. Genetic associations have been made between ADAM33 polymorphisms and asthma disease severity, bronchial hyperresponsivenss (BHR) and rate of lung function decline in both adults and children. A soluble, catalytically active form of the protein (sADAM33) has been identified in the bronchoalveolar lavage fluid of patients, levels of which correlate with disease severity (Lee JY et al, AJRCCM 2006). To study the role sADAM33, a lung specific, doxycycline (Dox) inducible, transgenic mouse, expressing the human pro and metalloproteinase domains of the full-length protein was generated (Ccsp-rtTA/Otet-ADAM33-Pro-MP). Induction of sADAM33, followed by house dust mite (HDM) sensitisation and challenge, resulted in increased BHR and airway inflammation (Davies ER et al, JCI-Insight 2016). The mechanisms by which ADAM33 promotes this susceptibility are unclear. The aim of this work is to identify pathways that are augmented by the induction of sADAM33, which promote increased sensitivity to allergen.Methods: RNA samples from whole lung of adult mice, where sADAM33 had been induced for 4 or 8 weeks, were analysed by next generation RNA sequencing. Identified genes were confirmed across experimental time points (72 hour, 7 day, 4 and 8 weeks on Dox) in wider sample cohorts of Ccsp-rtTA/Otet-ADAM33-Pro-MP and control mice through RTqPCR.Results: the predominant signal from the RNAseq output was for modulation of immune response genes at 4 weeks of sADAM33 expression (GO:0006955 Immune response: 31 genes, 58.49% coverage, FDR p value=7.09 E-22). Genes associated with an immune activation signature (Ccl5, Irgm1, Gm12250, Gzmb, Ncr1) were validated at least one time point. By 8 weeks of sADAM33 expression genes associated with negative regulation of the response were also validated (Ido1, Cd274).Conclusion: induction of sADAM33 in murine lungs, without allergic sensitisation, augmented underlying immune processes in this transgenic mouse model, which may contribute to increased susceptibility to allergic airway inflammation and BHR when challenged with allergen. Further work is required to delineate how sADAM33 affects immune cell populations and their behaviour in the lung

Inhibitory effects of surfactant protein A on surfactant phospholipid hydrolysis by secreted phospholipases A2

International audienceHydrolysis of surfactant phospholipids by secreted phospholipases A(2) (sPLA(2)) contributes to surfactant dysfunction in acute respiratory distress syndrome. The present study demonstrates that sPLA(2)-IIA, sPLA(2)-V, and sPLA(2)-X efficiently hydrolyze surfactant phospholipids in vitro. In contrast, sPLA(2)-IIC, -IID, -IIE, and -IIF have no effect. Since purified surfactant protein A (SP-A) has been shown to inhibit sPLA(2)-IIA activity, we investigated the in vitro effect of SP-A on the other active sPLA(2) and the consequences of sPLA(2)-IIA inhibition by SP-A on surfactant phospholipid hydrolysis. SP-A inhibits sPLA(2)-X activity, but fails to interfere with that of sPLA(2)-V. Moreover, in vitro inhibition of sPLA(2)-IIA-induces surfactant phospholipid hydrolysis correlates with the concentration of SP-A in surfactant. Intratracheal administration of sPLA(2)-IIA to mice causes hydrolysis of surfactant phosphatidylglycerol. Interestingly, such hydrolysis is significantly higher for SP-A gene-targeted mice, showing the in vivo inhibitory effect of SP-A on sPLA(2)-IIA activity. Administration of sPLA(2)-IIA also induces respiratory distress, which is more pronounced in SP-A gene-targeted mice than in wild-type mice. We conclude that SP-A inhibits sPLA(2) activity, which may play a protective role by maintaining surfactant integrity during lung injury

β-catenin is required for specification of proximal/distal cell fate during lung morphogenesis

The lungs are divided, both structurally and functionally, into two distinct components, the proximal airways, which conduct air, and the peripheral airways, which mediate gas exchange. The mechanisms that control the specification of these two structures during lung development are currently unknown. Here we show that {beta}-catenin signaling is required for the formation of the distal, but not the proximal, airways. When the gene for {beta}-catenin was conditionally excised in epithelial cells of the developing mouse lung prior to embryonic day 14.5, the proximal lung tubules grew and differentiated appropriately. The mice, however, died at birth because of respiratory failure. Analysis of the lungs by in situ hybridization and immunohistochemistry, using molecular markers of the epithelial and mesenchymal components of both proximal and peripheral airways, showed that the lungs were composed primarily of proximal airways. These observations establish, for the first time, both the sites and timing of specification of the proximal and peripheral airways in the developing lung, and that {beta}-catenin is one of the essential components of this specification

SAM-pointed domain ETS factor mediates epithelial cell-intrinsic innate immune signaling during airway mucous metaplasia

Airway mucus plays a critical role in clearing inhaled toxins, particles, and pathogens. Diverse toxic, inflammatory, and infectious insults induce airway mucus secretion and goblet cell metaplasia to preserve airway sterility and homeostasis. However, goblet cell metaplasia, mucus hypersecretion, and airway obstruction are integral features of inflammatory lung diseases, including asthma, chronic obstructive lung disease, and cystic fibrosis, which cause an immense burden of morbidity and mortality. These chronic lung diseases are united by susceptibility to microbial colonization and recurrent airway infections. Whether these twinned phenomena (mucous metaplasia, compromised host defenses) are causally related has been unclear. Here, we demonstrate that SAM pointed domain ETS factor (SPDEF) was induced by rhinoviral infection of primary human airway cells and that cytoplasmic activities of SPDEF, a transcriptional regulator of airway goblet cell metaplasia, inhibited Toll-like receptor (TLR) activation of epithelial cells. SPDEF bound to and inhibited activities of TLR signaling adapters, MyD88 and TRIF, inhibiting MyD88-induced cytokine production and TRIF-induced interferon ? production. Conditional expression of SPDEF in airway epithelial cells in vivo inhibited LPS-induced neutrophilic infiltration and bacterial clearance. SPDEF-mediated inhibition of both TLR and type I interferon signaling likely protects the lung against inflammatory damage when inciting stimuli are not eradicated. Present findings provide, at least in part, a molecular explanation for increased susceptibility to infection in lung diseases associated with mucous metaplasia and a mechanism by which patients with florid mucous metaplasia may tolerate microbial burdens that are usually associated with fulminant inflammatory disease in normal hosts