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Epithelial cells and macrophages represent two major cell populations in the lung. They reside in physical proximity and are influenced by inhaled substances, microbial- and host-derived factors, as well as by crosstalk between each other. Here, we report the first systematic study to compare the effects of apical and basolateral secretomes from primary human small airway epithelial cells (SAEC) on human macrophages. We exposed monocyte-derived macrophages (MDMs) to the secretome supernatants (SN) from the apical and basolateral chamber of SAEC culture in an air-liquid interface (ALI) setting and analyzed expression of macrophage surface markers. We found that the apical SN increased the expression of CD11c and CD16, whereas basolateral SN increased the expression of CD163 and CD300e, consistent with apical and basolateral epithelial secretions inducing an M1-biased and M2-biased macrophage polarization, respectively. Conversely, in the presence of Nontypeable Haemophilus influenzae (NTHi), apical SN from NTHi-exposed SAEC induced CD36, CD163 and CD300e and supressed CD11c expression suggesting a switch towards an M2-biased macrophage polarization. Analysis of SN from polarized epithelium revealed a number of factors with differential expression in the apical and basolateral secretome. Functional neutralization of IL6, IL8 or IL1α in the apical secretome led to a decrease in expression of 'M2-like' surface markers, supporting the concept of epithelial-derived secreted factors influencing macrophage phenotype. In conclusion, we show, for the first time to our knowledge, that SN from polarized epithelium, depending on the side of secretion, apical or basolateral, can elicit a differential influence on the macrophages polarization phenotype.

In many solid cancers, tumor-associated macrophages (TAM) represent the predominant myeloid cell population. Antigen (Ag) cross-presentation leading to tumor Ag-directed cytotoxic CD8+ T cell responses is crucial for anti-tumor immunity. However, the role of recruited monocyte-derived macrophages, including TAM, as potential cross-presenting cells is not well understood. Here, we show that primary human as well as mouse CD206+ macrophages are effective in functional cross-presentation of soluble self and non-self Ag, including tumor-associated Ag (TAA) as well as viral Ag. To confirm the presence of cross-presenting TAM in vivo, we performed phenotypic and functional analysis of TAM from B16-F10 and CT26 syngeneic tumor models and have identified CD11b+F4/80hiCD206+ TAM to effectively cross-present TAA. We show that CD11b+CD206+ TAM represent the dominant tumor-infiltrating myeloid cell population, expressing a unique cell surface repertoire, promoting Ag cross-presentation and Ag-specific CD8+ T cell activation comparable to cross-presenting CLEC9A+ dendritic cells (cDC1). The presence of cross-presenting CD206+ TAM is associated with reduced tumor burden in mouse syngeneic tumor models and with improved overall survival in cutaneous melanoma patients. Therefore, the demonstration of effective Ag cross-presentation capabilities of CD206+ TAM, including their clinical relevance, expands our understanding of TAM phenotypic diversity and functional versatility.

Unveiling the molecular mechanisms of tissue remodelling following injury is imperative to elucidate its regenerative capacity and aberrant repair in disease. Using different omics approaches, we identified enhancer of zester homolog 2 (EZH2) as a key regulator of fibrosis in injured lung epithelium. Epithelial injury drives an enrichment of nuclear transforming growth factor‐β‐activated kinase 1 (TAK1) that mediates EZH2 phosphorylation to facilitate its liberation from polycomb repressive complex 2 (PRC2). This process results in the establishment of a transcriptional complex of EZH2, RNA‐polymerase II (POL2) and nuclear actin, which orchestrates aberrant epithelial repair programmes. The liberation of EZH2 from PRC2 is accompanied by an EZH2‐EZH1 switch to preserve H3K27me3 deposition at non‐target genes. Loss of epithelial TAK1, EZH2 or blocking nuclear actin influx attenuates the fibrotic cascade and restores respiratory homeostasis. Accordingly, EZH2 inhibition significantly improves outcomes in a pulmonary fibrosis mouse model. Our results reveal an important non‐canonical function of EZH2, paving the way for new therapeutic interventions in fibrotic lung diseases., TAK1‐mediated phosphorylation of EZH2 regulates fibrotic epithelial remodelling after injury. EZH2 inhibition improves outcomes in a pulmonary fibrosis model, suggesting that targeting the TAK1‐EZH2 axis has therapeutic efficacy for idiopathic pulmonary fibrosis (IPF).

Idiopathic pulmonary fibrosis (IPF) is a chronic and progressive fibrosing interstitial lung disease with poor prognosis. The current standard-of-care for IPF is the kinase inhibitor nintedanib, which has a distinct inhibitory profile targeting a range of tyrosine kinases. The mechanism of action of nintedanib has been well characterized for myofibroblast activity, yet remains less clear for the immune populations distributed throughout the lung. Given the proposed role of heterogeneous pulmonary macrophage populations in mediating both protective and pathogenic roles in lung fibrosis, we sought to identify repair-associated macrophage populations in mice influenced by nintedanib after 7 and 14 days post-bleomycin challenge using single cell RNA sequencing. Bleomycin exposure triggered expansion of inflammatory MHCIIhigh macrophage populations, which was partially reversed after nintedanib treatment. Concurrently, nintedanib promoted the expansion of MHCIIlow macrophages, which were linked to attenuation of lung fibrosis. Concomitantly, nintedanib promoted an increased expression of canonical macrophage repair markers in MHCIIlow macrophages. Finally, exposure of inflammatory macrophages to nintedanib in vitro resulted in attenuated expression of MHCII transcripts. In conclusion, a component of nintedanib’s protective mode of action in lung fibrosis relies on expanding distinct MHCIIlow macrophage populations and redirecting them toward a reparative phenotype. This study provides a rationale for further refinement of therapeutic kinase inhibition in fibrotic diseases.

The introduction of N6-methyladenosine (m6 A) into siRNA targeting Factor VII impacts its potency in cells and has a significant influence on the selectivity of siRNA, including reduced off-targeting. These effects are dependent on the position of m6 A in the siRNA duplex, with some of the sequences identified as more potent and/or selective than their non-methylated counterpart. These findings broaden the repertoire of available chemical modifications for siRNA therapeutics and imply potential regulatory role of N6-methyladenosine in the RNAi pathways.

To restore organ homeostasis, a myriad of cell types need to activate rapid and transient programs to adjust cell fate decisions and elicit a collective behaviour. Characterisation of such programs are imperative to elucidate an organ’s regenerative capacity and its aberrant repair in disease. By modelling epithelial-mesenchymal crosstalk, we provide direct evidence for transforming growth factor β1 (TGFβ1)-damaged epithelium initiating a bi-directional fibrotic cascade with the mesenchyme. Strikingly, TGFβ1-damaged epithelia facilitates the release of Enhancer of Zester Homolog 2 (EZH2) from Polycomb Repressive Complex 2 (PRC2) to establish a novel fibrotic transcriptional complex of EZH2, RNA-polymerase II (POL2) and nuclear actin. Perturbing this complex by disrupting epithelial EZH2 or actomyosin remodelling abrogates the fibrotic crosstalk. The liberation of EZH2 from PRC2 is accompanied by an EZH2-EZH1 switch to preserve global H3K27me3 occupancy. Our results reveal an important non-canonical function of EZH2, paving the way for therapeutic interventions in fibrotic disease.

BackgroundElevated counts of alveolar macrophages and attenuated phagocytic capacity are associated with chronic obstructive pulmonary disease (COPD). Factors governing macrophage phagocytosis are poorly understood. In this study we aimed to compare the influence of airway epithelial cell secretions from individuals with COPD and without COPD (non-COPD) on macrophage phagocytic activity, and the role of antimicrobial peptides (AMPs).MethodsSupernatants from non-COPD and COPD small airway epithelial cell (SAEC) cultures exposed to non-typeable Haemophilus influenzae (NTHi) were applied to human monocyte-derived macrophages (MDMs) to assess their influence on phagocytosis. SAECs were analysed for changes in AMP expression by quantitative reverse transcription PCR, and the influence of select AMPs on macrophage phenotype and function was assessed by flow cytometry and metabolic activity assay.ResultsSecretions from the apical and basolateral surface of NTHi-exposed SAECs from non-COPD donors elicited superior phagocytic capacity in MDMs. Moreover, NTHi exposure led to a rapid increase in the expression of a range of AMPs by non-COPD SAECs, but this response was delayed in COPD SAECs. We demonstrate that treatment with AMPs β-defensin 2 and S100 calcium binding protein A8/S100 calcium binding protein A9 (S100A8/A9) improved the phagocytic capacity of MDMs. In-depth analysis of the influence of S100A8/A9 on MDMs revealed a role for this AMP in macrophage phenotype and function. Furthermore, we show that the expression of S100A8 and S100A9 is directly regulated by WNT/β-catenin signalling, a known deregulated pathway in COPD.ConclusionIn conclusion, for the first time, we demonstrate that airway epithelium from patients with COPD has a reduced capacity to support the phagocytic function of macrophages in response to acute NTHi exposure, and we identify the WNT/β-catenin signalling-modulated and epithelium-derived S100A8/A9 as a potent regulator of macrophage phenotype and function.

Chronic obstructive pulmonary disease (COPD), in particular emphysema, is characterized by loss of parenchymal alveolar tissue and impaired tissue repair. Wingless and INT-1 (WNT)/β-catenin signaling is reduced in COPD; however, the mechanisms thereof, specifically the role of the frizzled (FZD) family of WNT receptors, remain unexplored.To identify and functionally characterize specific FZD receptors that control downstream WNT signaling in impaired lung repair in COPD.FZD expression was analyzed in lung homogenates and alveolar epithelial type II (ATII) cells of never-smokers, smokers, patients with COPD, and two experimental COPD models by quantitative reverse transcriptase-polymerase chain reaction, immunoblotting, and immunofluorescence. The functional effects of cigarette smoke on FZD4, WNT/β-catenin signaling, and elastogenic components were investigated in primary ATII cells in vitro and in three-dimensional lung tissue cultures ex vivo. Gain- and loss-of-function approaches were applied to determine the effects of FZD4 signaling on alveolar epithelial cell wound healing and repair, as well as on expression of elastogenic components.FZD4 expression was reduced in human and experimental COPD lung tissues as well as in primary human ATII cells from patients with COPD. Cigarette smoke exposure down-regulated FZD4 expression in vitro and in vivo, along with reduced WNT/β-catenin activity. Inhibition of FZD4 decreased WNT/β-catenin-driven epithelial cell proliferation and wound closure, and it interfered with ATII-to-ATI cell transdifferentiation and organoid formation, which were augmented by FZD4 overexpression. Moreover, FZD4 restoration by overexpression or pharmacological induction led to induction of WNT/β-catenin signaling and expression of elastogenic components in three-dimensional lung tissue cultures ex vivo.Reduced FZD4 expression in COPD contributes to impaired alveolar repair capacity.

Baarsma et al. report increased expression and posttranslational modification of the noncanonical ligand WNT-5A in COPD. Fibroblast-derived WNT-5A inhibits canonical WNT–β-catenin–driven alveolar epithelial cell–mediated wound healing and transdifferentiation, and thus contributes to impaired lung regeneration and COPD pathogenesis., Chronic obstructive pulmonary disease (COPD) is a leading cause of death worldwide. One main pathological feature of COPD is the loss of functional alveolar tissue without adequate repair (emphysema), yet the underlying mechanisms are poorly defined. Reduced WNT–β-catenin signaling is linked to impaired lung repair in COPD; however, the factors responsible for attenuating this pathway remain to be elucidated. Here, we identify a canonical to noncanonical WNT signaling shift contributing to COPD pathogenesis. We demonstrate enhanced expression of noncanonical WNT-5A in two experimental models of COPD and increased posttranslationally modified WNT-5A in human COPD tissue specimens. WNT-5A was increased in primary lung fibroblasts from COPD patients and induced by COPD-related stimuli, such as TGF-β, cigarette smoke (CS), and cellular senescence. Functionally, mature WNT-5A attenuated canonical WNT-driven alveolar epithelial cell wound healing and transdifferentiation in vitro. Lung-specific WNT-5A overexpression exacerbated airspace enlargement in elastase-induced emphysema in vivo. Accordingly, inhibition of WNT-5A in vivo attenuated lung tissue destruction, improved lung function, and restored expression of β-catenin–driven target genes and alveolar epithelial cell markers in the elastase, as well as in CS-induced models of COPD. We thus identify a novel essential mechanism involved in impaired mesenchymal–epithelial cross talk in COPD pathogenesis, which is amenable to therapy.

Chronic obstructive pulmonary disease (COPD) is characterized by emphysema, chronic inflammation and increased bacterial colonization of airways associated with exacerbations. While the number of macrophages is elevated in COPD, their phagocytic capacity is attenuated. Antimicrobial peptides (AMP) are naturally occurring proteins with anti-pathogen and proposed immunomodulatory functions. We hypothesize that the airway epithelium-derived AMPs regulate the function of macrophages in COPD. We stimulated non-COPD and COPD small airway epithelial cells (SAEC) with heat-inactivated non-typeable Haemophilus influenzae (Nthi) in air-liquid-interface cultures. We subjected the secretome from apical and basolateral compartment to mass spectrometry and identified a number of secreted AMPs, differentiating between the site of secretion, but not with the Nthi challenge or disease status. We found, however, that Nthi regulated the mRNA expression of AMPs. We observed upregulation of S100A8 and S100A9 after 3h stimulation in non-COPD- to a higher extent than in COPD-derived SAEC but the difference was leveled out after 24h stimulation. This suggests the delayed response in COPD epithelium. Additionally, we used the supernatants collected from SAECs to stimulate primary human monocyte-derived macrophages (MDM). Supernatants from non-COPD cultures had higher impact on phagocytosis in comparison to COPD after 3h Nthi stimulation, but again after 24h this effect was diminished. We next stimulated MDM with S100A8/A9 and observed significant improvement in phagocytosis. Thus, lung epithelium-derived S100A8/A9 could improve phagocytosis in macrophages and this pathway could be explored for therapeutic utility in COPD.

Mechanisms of injury and repair in alveolar epithelial cells (AECs) are critically involved in the progression of various lung diseases including idiopathic pulmonary fibrosis (IPF). Homeobox only protein x (HOPX) contributes to the formation of distal lung during development. In adult lung, alveolar epithelial type (AT) I cells express HOPX and lineage-labeled Hopx+ cells give rise to both ATI and ATII cells after pneumonectomy. However, the cell function of HOPX-expressing cells in adult fibrotic lung diseases has not been investigated. In this study, we have established a flow cytometry-based method to evaluate HOPX-expressing cells in the lung. HOPX expression in cultured ATII cells increased over culture time, which was accompanied by a decrease of proSP-C, an ATII marker. Moreover, HOPX expression was increased in AECs from bleomycin-instilled mouse lungs in vivo. Small interfering RNA-based knockdown of Hopx resulted in suppressing ATII-ATI trans-differentiation and activating cellular proliferation in vitro. In IPF lungs, HOPX expression was decreased in whole lungs and significantly correlated to a decline in lung function and progression of IPF. In conclusion, HOPX is upregulated during early alveolar injury and repair process in the lung. Decreased HOPX expression might contribute to failed regenerative processes in end-stage IPF lungs.

The WNT signalling cascades have emerged as critical regulators of a wide variety of biological aspects involved in lung development as well as in physiological and pathophysiological processes in the adult lung. WNTs (secreted glycoproteins) interact with various transmembrane receptors and co-receptors to activate signalling pathways that regulate transcriptional as well as non-transcriptional responses within cells. In physiological conditions, the majority of WNT receptors and co-receptors can be detected in the adult lung. However, dysregulation of WNT signalling pathways contributes to the development and progression of chronic lung pathologies, including idiopathic pulmonary fibrosis (IPF), chronic obstructive pulmonary disease (COPD), asthma and lung cancer. The interaction between a WNT and the (co-)receptor(s) present at the cell surface is the initial step in transducing an extracellular signal into an intracellular response. This proximal event in WNT signal transduction with (cell-specific) ligand-receptor interactions is of great interest as a potential target for pharmacological intervention. In this review we highlight the diverse expression of various WNT receptors and co-receptors in the aforementioned chronic lung diseases and discuss the currently available biologicals and pharmacological tools to modify proximal WNT signalling.

Idiopathic pulmonary fibrosis (IPF) is a devastating lung disease with poor prognosis and limited therapeutic options. The incidence of IPF increases with age, and ageing-related mechanisms such as cellular senescence have been proposed as pathogenic drivers. The lung alveolar epithelium represents a major site of tissue injury in IPF and senescence of this cell population is probably detrimental to lung repair. However, the potential pathomechanisms of alveolar epithelial cell senescence and the impact of senolytic drugs on senescent lung cells and fibrosis remain unknown. Here we demonstrate that lung epithelial cells exhibit increased P16 and P21 expression as well as senescence-associated β-galactosidase activity in experimental and human lung fibrosis tissue and primary cells. Primary fibrotic mouse alveolar epithelial type (AT)II cells secreted increased amounts of senescence-associated secretory phenotype (SASP) factors in vitro, as analysed using quantitative PCR, mass spectrometry and ELISA. Importantly, pharmacological clearance of senescent cells by induction of apoptosis in fibrotic ATII cells or ex vivo three-dimensional lung tissue cultures reduced SASP factors and extracellular matrix markers, while increasing alveolar epithelial markers. These data indicate that alveolar epithelial cell senescence contributes to lung fibrosis development and that senolytic drugs may be a viable therapeutic option for IPF., Alveolar epithelial cell senescence occurs in IPF and senolytic treatment attenuates experimental lung fibrosis http://ow.ly/nFlz30bsmNm

Vol. 214, No. 1, January 2017. Pages [143–163][1]. The authors regret that in their original paper, the left two micrographs in Fig. 7 C appeared as duplicates. The corrected figure appears below. The online HTML and PDF versions of this article have been corrected. The error only remains in the