Repairing damaged lungs using regenerative therapy

There is an urgent need for better treatment of lung diseases that are a major cause of morbidity and mortality worldwide. This urgency is illustrated by the current COVID-19 health crisis. Moderate-to-extensive lung injury characterizes several lung diseases, and not only therapies that reduce such lung injury are needed but also those that regenerate lung tissue and repair existing lung injury. At present, such therapies are not available, but as a result of a rapid increase in our understanding of lung development and repair, lung regenerative therapies are on the horizon. Here, we discuss existing targets for treatment, as well as novel strategies for development of pharmacological and cell therapy-based regenerative treatment for a variety of lung diseases and clinical studies. We discuss how both patient-relevant in vitro disease models using innovative culture techniques and other advanced new technologies aid in the development of pulmonary regenerative medicine

Cigarette smoke restricts the ability of mesenchymal cells to support lung epithelial organoid formation

Adequate lung epithelial repair relies on supportive interactions within the epithelial niche, including interactions with WNT-responsive fibroblasts. In fibroblasts from patients with chronic obstructive pulmonary disease (COPD) or upon in vitro cigarette smoke exposure, Wnt/β-catenin signalling is distorted, which may affect interactions between epithelial cells and fibroblasts resulting in inadequate lung repair. We hypothesized that cigarette smoke (CS), the main risk factor for COPD, interferes with Wnt/β-catenin signalling in fibroblasts through induction of cellular stress responses, including oxidative- and endoplasmic reticulum (ER) stress, and thereby alters epithelial repair support potential. Therefore, we assessed the effect of CS-exposure and the ER stress inducer Thapsigargin (Tg) on Wnt/β-catenin signalling activation in MRC-5 fibroblasts, and on their ability to support lung epithelial organoid formation. Exposure of MRC-5 cells for 15 min with 5 AU/mL CS extract (CSE), and subsequent 6 h incubation induced oxidative stress (HMOX1). Whereas stimulation with 100 nM Tg increased markers of both the integrated stress response (ISR - GADD34/PPP1R15A, CHOP) and the unfolded protein response (UPR - XBP1spl, GADD34/PPP1R15A, CHOP and HSPA5/BIP), CSE only induced GADD34/PPP1R15A expression. Strikingly, although treatment of MRC-5 cells with the Wnt activator CHIR99021 upregulated the Wnt/β-catenin target gene AXIN2, this response was diminished upon CSE or Tg pre-exposure, which was confirmed using a Wnt-reporter. Furthermore, pre-exposure of MRC-5 cells to CSE or Tg, restricted their ability to support organoid formation upon co-culture with murine pulmonary EpCam+ cells in Matrigel at day 14. This restriction was alleviated by pre-treatment with CHIR99021. We conclude that exposure of MRC-5 cells to CSE increases oxidative stress, GADD34/PPP1R15A expression and impairs their ability to support organoid formation. This inhibitory effect may be restored by activating the Wnt/β-catenin signalling pathway

Modulation of airway epithelial innate immunity and wound repair by M(GM-CSF) and M(M-CSF) macrophages

Airway epithelial cells and macrophages participate in inflammatory responses to external noxious stimuli, which can cause epithelial injury. Upon injury, epithelial cells and macrophages act in concert to ensure rapid restoration of epithelial integrity. The nature of the interactions between these cell types during epithelial repair is incompletely understood. We used an in vitro human coculture model of primary bronchial epithelial cells cultured at the air-liquid interface (ALI-PBEC) and polarized primary monocyte-derived macrophages. Using this coculture, we studied the contribution of macrophages to epithelial innate immunity, wound healing capacity, and epithelial exposure to whole cigarette smoke (WCS). Coculture of ALI-PBEC with lipopolysaccharide (LPS)-activated M(GM-CSF) macrophages increased the expression ofDEFB4A,CXCL8, andIL6at 24 h in the ALI-PBEC, whereas LPS-activated M(M-CSF) macrophages only increased epithelialIL6expression. Furthermore, wound repair was accelerated by coculture with both activated M(GM-CSF) and M(M-CSF) macrophages, also following WCS exposure. Coculture of ALI-PBEC and M(GM-CSF) macrophages resulted in increasedCAMPexpression in M(GM-CSF) macrophages, which was absent in M(M-CSF) macrophages.CAMPencodes LL-37, an antimicrobial peptide with immune-modulating and repair-enhancing activities. In conclusion, dynamic crosstalk between ALI-PBEC and macrophages enhances epithelial innate immunity and wound repair, even upon concomitant cigarette smoke exposure.Pathogenesis and treatment of chronic pulmonary disease

Modulation of Airway Epithelial Innate Immunity and Wound Repair by M(GM-CSF) and M(M-CSF) Macrophages

Airway epithelial cells and macrophages participate in inflammatory responses to external noxious stimuli, which can cause epithelial injury. Upon injury, epithelial cells and macrophages act in concert to ensure rapid restoration of epithelial integrity. The nature of the interactions between these cell types during epithelial repair is incompletely understood. We used an in vitro human coculture model of primary bronchial epithelial cells cultured at the air-liquid interface (ALI-PBEC) and polarized primary monocyte-derived macrophages. Using this coculture, we studied the contribution of macrophages to epithelial innate immunity, wound healing capacity, and epithelial exposure to whole cigarette smok

Cigarette smoke restricts the ability of mesenchymal cells to support lung epithelial organoid formation

Peer reviewed: TrueAcknowledgements: The authors thank Sophie Bos, Xinhui Wu and Chiara Ciminieri (Dept. of Molecular Pharmacology, Groningen Research Institute of Pharmacy, University of Groningen, Groningen, the Netherlands) for their technical support during the experiments.Adequate lung epithelial repair relies on supportive interactions within the epithelial niche, including interactions with WNT-responsive fibroblasts. In fibroblasts from patients with chronic obstructive pulmonary disease (COPD) or upon in vitro cigarette smoke exposure, Wnt/β-catenin signalling is distorted, which may affect interactions between epithelial cells and fibroblasts resulting in inadequate lung repair. We hypothesized that cigarette smoke (CS), the main risk factor for COPD, interferes with Wnt/β-catenin signalling in fibroblasts through induction of cellular stress responses, including oxidative- and endoplasmic reticulum (ER) stress, and thereby alters epithelial repair support potential. Therefore, we assessed the effect of CS-exposure and the ER stress inducer Thapsigargin (Tg) on Wnt/β-catenin signalling activation in MRC-5 fibroblasts, and on their ability to support lung epithelial organoid formation. Exposure of MRC-5 cells for 15 min with 5 AU/mL CS extract (CSE), and subsequent 6 h incubation induced oxidative stress (HMOX1). Whereas stimulation with 100 nM Tg increased markers of both the integrated stress response (ISR - GADD34/PPP1R15A, CHOP) and the unfolded protein response (UPR - XBP1spl, GADD34/PPP1R15A, CHOP and HSPA5/BIP), CSE only induced GADD34/PPP1R15A expression. Strikingly, although treatment of MRC-5 cells with the Wnt activator CHIR99021 upregulated the Wnt/β-catenin target gene AXIN2, this response was diminished upon CSE or Tg pre-exposure, which was confirmed using a Wnt-reporter. Furthermore, pre-exposure of MRC-5 cells to CSE or Tg, restricted their ability to support organoid formation upon co-culture with murine pulmonary EpCam+ cells in Matrigel at day 14. This restriction was alleviated by pre-treatment with CHIR99021. We conclude that exposure of MRC-5 cells to CSE increases oxidative stress, GADD34/PPP1R15A expression and impairs their ability to support organoid formation. This inhibitory effect may be restored by activating the Wnt/β-catenin signalling pathway

Corrigendum: Cigarette smoke restricts the ability of mesenchymal cells to support lung epithelial organoid formation

Peer reviewed: Tru

Acute and chronic effects of treatment with mesenchymal stromal cells on LPS-induced pulmonary inflammation, emphysema and atherosclerosis development.

COPD is a pulmonary disorder often accompanied by cardiovascular disease (CVD), and current treatment of this comorbidity is suboptimal. Systemic inflammation in COPD triggered by smoke and microbial exposure is suggested to link COPD and CVD. Mesenchymal stromal cells (MSC) possess anti-inflammatory capacities and MSC treatment is considered an attractive treatment option for various chronic inflammatory diseases. Therefore, we investigated the immunomodulatory properties of MSC in an acute and chronic model of lipopolysaccharide (LPS)-induced inflammation, emphysema and atherosclerosis development in APOE*3-Leiden (E3L) mice.Hyperlipidemic E3L mice were intranasally instilled with 10 μg LPS or vehicle twice in an acute 4-day study, or twice weekly during 20 weeks Western-type diet feeding in a chronic study. Mice received 0.5x106 MSC or vehicle intravenously twice after the first LPS instillation (acute study) or in week 14, 16, 18 and 20 (chronic study). Inflammatory parameters were measured in bronchoalveolar lavage (BAL) and lung tissue. Emphysema, pulmonary inflammation and atherosclerosis were assessed in the chronic study.In the acute study, intranasal LPS administration induced a marked systemic IL-6 response on day 3, which was inhibited after MSC treatment. Furthermore, MSC treatment reduced LPS-induced total cell count in BAL due to reduced neutrophil numbers. In the chronic study, LPS increased emphysema but did not aggravate atherosclerosis. Emphysema and atherosclerosis development were unaffected after MSC treatment.These data show that MSC inhibit LPS-induced pulmonary and systemic inflammation in the acute study, whereas MSC treatment had no effect on inflammation, emphysema and atherosclerosis development in the chronic study

The effect of PPE-induced emphysema and chronic LPS-induced pulmonary inflammation on atherosclerosis development in APOE*3-LEIDEN mice.

BackgroundChronic obstructive pulmonary disease (COPD) is characterized by pulmonary inflammation, airways obstruction and emphysema, and is a risk factor for cardiovascular disease (CVD). However, the contribution of these individual COPD components to this increased risk is unknown. Therefore, the aim of this study was to determine the contribution of emphysema in the presence or absence of pulmonary inflammation to the increased risk of CVD, using a mouse model for atherosclerosis. Because smoke is a known risk factor for both COPD and CVD, emphysema was induced by intratracheal instillation of porcine pancreatic elastase (PPE).MethodsHyperlipidemic APOE*3-Leiden mice were intratracheally instilled with vehicle, 15 or 30 µg PPE and after 4 weeks, mice received a Western-type diet (WTD). To study the effect of emphysema combined with pulmonary inflammation on atherosclerosis, mice received 30 µg PPE and during WTD feeding, mice were intranasally instilled with vehicle or low-dose lipopolysaccharide (LPS; 1 µg/mouse, twice weekly). After 20 weeks WTD, mice were sacrificed and emphysema, pulmonary inflammation and atherosclerosis were analysed.ResultsIntratracheal PPE administration resulted in a dose-dependent increase in emphysema, whereas atherosclerotic lesion area was not affected by PPE treatment. Additional low-dose intranasal LPS administration induced a low-grade systemic IL-6 response, as compared to vehicle. Combining intratracheal PPE with intranasal LPS instillation significantly increased the number of pulmonary macrophages and neutrophils. Plasma lipids during the study were not different. LPS instillation caused a limited, but significant increase in the atherosclerotic lesion area. This increase was not further enhanced by PPE.ConclusionThis study shows for the first time that PPE-induced emphysema both in the presence and absence of pulmonary inflammation does not affect atherosclerotic lesion development