Simon D. Pouwels

DAMPs activating innate and adaptive immune responses in COPD.

Chronic obstructive pulmonary disease (COPD), a progressive lung disease characterized by sustained neutrophilic airway inflammation, is caused by chronic exposure to noxious stimuli, e.g., cigarette smoke. This chronic exposure can induce immunogenic cell death of structural airway cells, inducing the release of damage-associated molecular patterns (DAMPs). Levels of several DAMPs, including S100 proteins, defensins, and high-mobility group box-1 (HMGB1), are increased in extracellular lung fluids of COPD patients. As DAMPs can attract and activate immune cells upon binding to pattern recognition receptors, we propose that their release may contribute to neutrophilic airway inflammation. In this review, we discuss the novel role of DAMPs in COPD pathogenesis. Relevant DAMPs are categorized based on their subcellular origin, i.e. cytoplasm, endoplasmic reticulum, nucleus, and mitochondria. Furthermore, their potential role in the pathophysiology of COPD will be discussed.

Cigarette Smoke-Induced Damage-Associated Molecular Pattern Release from Necrotic Neutrophils Triggers Proinflammatory Mediator Release

Cigarette smoking, the major causative factor for the development of chronic obstructive pulmonary disease, is associated with neutrophilic airway inflammation. Cigarette smoke (CS) exposure can induce a switch from apoptotic to necrotic cell death in airway epithelium. Therefore, we hypothesized that CS promotes neutrophil necrosis with subsequent release of damage-associated molecular patterns (DAMPs), including high mobility group box 1 (HMGB1), alarming the innate immune system. We studied the effect of smoking two cigarettes on sputum neutrophils in healthy individuals and of 5-day CS or air exposure on neutrophil counts, myeloperoxidase, and HMGB1 levels in bronchoalveolar lavage fluid of BALB/c mice. In human peripheral blood neutrophils, mitochondrial membrane potential, apoptosis/necrosis markers, caspase activity, and DAMP release were studied after CS exposure. Finally, we assessed the effect of neutrophil-derived supernatants on the release of chemoattractant CXCL8 in normal human bronchial epithelial cells. Cigarette smoking caused a significant decrease in sputum neutrophil numbers after 3 hours. In mice, neutrophil counts were significantly increased 16 hours after repeated CS exposure but reduced 2 hours after an additional exposure. In vitro, CS induced necrotic neutrophil cell death, as indicated by mitochondrial dysfunction, inhibition of apoptosis, and DAMP release. Supernatants from CS-treated neutrophils significantly increased the release of CXCL8 in normal human bronchial epithelial cells. Together, these observations show, for the first time, that CS exposure induces neutrophil necrosis, leading to DAMP release, which may amplify CS-induced airway inflammation by promoting airway epithelial proinflammatory responses.

Cigarette smoke-induced necroptosis and DAMP release trigger neutrophilic airway inflammation in mice.

Recent data indicate a role for airway epithelial necroptosis, a regulated form of necrosis, and the associated release of damage-associated molecular patterns (DAMPs) in the development of chronic obstructive pulmonary disease (COPD). DAMPs can activate pattern recognition receptors (PRRs), triggering innate immune responses. We hypothesized that cigarette smoke (CS)-induced epithelial necroptosis and DAMP release initiate airway inflammation in COPD. Human bronchial epithelial BEAS-2B cells were exposed to cigarette smoke extract (CSE), and necrotic cell death (membrane integrity by propidium iodide staining) and DAMP release (i.e., double-stranded DNA, high-mobility group box 1, heat shock protein 70, mitochondrial DNA, ATP) were analyzed. Subsequently, BEAS-2B cells were exposed to DAMP-containing supernatant of CS-induced necrotic cells, and the release of proinflammatory mediators [C-X-C motif ligand 8 (CXCL-8), IL-6] was evaluated. Furthermore, mice were exposed to CS in the presence and absence of the necroptosis inhibitor necrostatin-1, and levels of DAMPs and inflammatory cell numbers were determined in bronchoalveolar lavage fluid. CSE induced a significant increase in the percentage of necrotic cells and DAMP release in BEAS-2B cells. Stimulation of BEAS-2B cells with supernatant of CS-induced necrotic cells induced a significant increase in the release of CXCL8 and IL-6, in a myeloid differentiation primary response gene 88-dependent fashion. In mice, exposure of CS increased the levels of DAMPs and numbers of neutrophils in bronchoalveolar lavage fluid, which was statistically reduced upon treatment with necrostatin-1. Together, we showed that CS exposure induces necrosis of bronchial epithelial cells and subsequent DAMP release in vitro, inducing the production of proinflammatory cytokines. In vivo, CS exposure induces neutrophilic airway inflammation that is sensitive to necroptosis inhibition.

Increased serum levels of LL37, HMGB1 and S100A9 during exacerbation in COPD patients

Chronic obstructive pulmonary disease (COPD) is a severe and progressive lung disease characterised by destruction of lung parenchyma and chronic airway inflammation [1]. A major cause of COPD is chronic exposure to noxious gases and particles, including cigarette smoke. During exacerbation, COPD patients experience a worsening of symptoms that coincides with increased inflammation and accelerated decline in lung function, resulting in a decrease in quality of life and increased healthcare costs. Approximately half of the COPD exacerbations causing hospitalisation are associated with respiratory viral and/or bacterial infections [2]. However, the underlying mechanisms causing COPD exacerbations are unknown [3]. Molecules derived from viruses and bacteria during airway infection can trigger the activation of pattern recognition receptors (PRRs) on lung structural and innate immune cells, and may thus contribute to the aggravation of inflammation during COPD exacerbations [1]. Interestingly, damage-associated molecular patterns (DAMPs) released from damaged or necrotic cells are also known to activate PRRs, including Toll-like receptors (TLRs) and the receptor for advanced glycation end-products (RAGE) [4]. A role for DAMPs has been proposed in the pathogenesis of COPD, as various DAMPs have been found to be increased in lung fluids and serum of COPD patients [5, 6]. Furthermore, Ager, the gene encoding RAGE, has been identified by genome-wide association studies as a susceptibility gene for COPD [7, 8]. Moreover, the serum levels of soluble RAGE (sRAGE), a decoy receptor for RAGE, were shown to be significantly lower in COPD patients, while the RAGE ligand EN-RAGE (also known as S100 calcium-binding protein (S100)A12) was significantly higher in COPD patients compared with smoking and nonsmoking controls [9]. It is currently unknown, however, whether DAMPs play a role in COPD exacerbations. Here, we hypothesised that the release of DAMPs is increased during exacerbations of COPD. Serum levels of the RAGE-activating DAMPs LL37, HMGB1 and S100A9 are increased during exacerbation in COPD patients http://ow.ly/Idh3r

Pim1 kinase protects airway epithelial cells from cigarette smoke-induced damage and airway inflammation

Exposure to cigarette smoke (CS) is the main risk factor for developing chronic obstructive pulmonary disease and can induce airway epithelial cell damage, innate immune responses, and airway inflammation. We hypothesized that cell survival factors might decrease the sensitivity of airway epithelial cells to CS-induced damage, thereby protecting the airways against inflammation upon CS exposure. Here, we tested whether Pim survival kinases could protect from CS-induced inflammation. We determined expression of Pim kinases in lung tissue, airway inflammation, and levels of keratinocyte-derived cytokine (KC) and several damage-associated molecular patterns in bronchoalveolar lavage in mice exposed to CS or air. Human bronchial epithelial BEAS-2B cells were treated with CS extract (CSE) in the presence or absence of Pim1 inhibitor and assessed for loss of mitochondrial membrane potential, induction of cell death, and release of heat shock protein 70 (HSP70). We observed increased expression of Pim1, but not of Pim2 and Pim3, in lung tissue after exposure to CS. Pim1-deficient mice displayed a strongly enhanced neutrophilic airway inflammation upon CS exposure compared with wild-type controls. Inhibition of Pim1 activity in BEAS-2B cells increased the loss of mitochondrial membrane potential and reduced cell viability upon CSE treatment, whereas release of HSP70 was enhanced. Interestingly, we observed release of S100A8 but not of double-strand DNA or HSP70 in Pim1-deficient mice compared with wild-type controls upon CS exposure. In conclusion, we show that expression of Pim1 protects against CS-induced cell death in vitro and neutrophilic airway inflammation in vivo. Our data suggest that the underlying mechanism involves CS-induced release of S100A8 and KC.

The epithelial polarity regulator LGALS9/galectin-9 induces fatal frustrated autophagy in KRAS mutant colon carcinoma that depends on elevated basal autophagic flux

Oncogenic mutation of KRAS (Kirsten rat sarcoma viral oncogene homolog) in colorectal cancer (CRC) confers resistance to both chemotherapy and EGFR (epidermal growth factor receptor)-targeted therapy. We uncovered that KRAS mutant (KRASmut) CRC is uniquely sensitive to treatment with recombinant LGALS9/Galectin-9 (rLGALS9), a recently established regulator of epithelial polarity. Upon treatment of CRC cells, rLGALS9 rapidly internalizes via early- and late-endosomes and accumulates in the lysosomal compartment. Treatment with rLGALS9 is accompanied by induction of frustrated autophagy in KRASmut CRC, but not in CRC with BRAF (B-Raf proto-oncogene, serine/threonine kinase) mutations (BRAFmut). In KRASmut CRC, rLGALS9 acts as a lysosomal inhibitor that inhibits autophagosome-lysosome fusion, leading to autophagosome accumulation, excessive lysosomal swelling and cell death. This antitumor activity of rLGALS9 directly correlates with elevated basal autophagic flux in KRASmut cancer cells. Thus, rLGALS9 has potent antitumor activity toward refractory KRASmut CRC cells that may be exploitable for therapeutic use.

Crohn's Disease Patients Have More IgG-Binding Fecal Bacteria than Controls

ABSTRACT In Crohns disease (CD), chronic gut inflammation leads to loss of mucosal barrier integrity. Subsequent leakage of IgG to the gut could produce an increase of IgG coating of intestinal bacteria. We investigated if there is more IgG coating in patients than in volunteers and whether this is dependent on the host IgG response or on the gut bacteria. Fecal and serum samples were obtained from 23 CD patients and 11 healthy volunteers. Both the in vivo IgG-coated fecal bacteria and in vitro IgG coating after serum addition were measured by flow cytometry and related to disease activity. The bacterial composition in feces was determined using fluorescence in situ hybridization. The IgG-binding capacities of Escherichia coli strains isolated from feces of patients and volunteers were assessed. The results showed that the in vivo IgG-coated fraction of fecal bacteria of patients was slightly larger than that of volunteers but significantly larger after incubation with either autologous or heterologous serum. This was dependent on the bacteria and independent of disease activity or the serum used. The presence of more Enterobacteriaceae and fewer faecalibacteria in patient feces was confirmed. E. coli isolates from patients bound more IgG than isolates from volunteers (P < 0.05) after the addition of autologous serum. Together, these results indicate that CD patients have more IgG-binding gut bacteria than healthy volunteers. We showed that the level of IgG coating depends on the bacteria and not on the serum used. Furthermore, CD patients have a strong specific immune response to their own E. coli bacteria.

A specific DAMP profile identifies susceptibility to smoke-induced airway inflammation

To the Editor: Chronic obstructive pulmonary disease (COPD) is a major cause of morbidity and mortality, with a worldwide prevalence of 9–10% [1]. COPD is associated with chronic, neutrophilic inflammation in the lungs, causing destruction of lung parenchyma (emphysema) and/or remodelling of the airways with mucus hypersecretion (bronchitis) [2]. Chronic exposure to noxious particles and gases, such as cigarette smoke, is the major risk factor for COPD, while susceptibility to the disease has a strong genetic component [2]. While the activity of the innate immune system increases with disease progression during early stages of COPD, the precise nature of the factors that trigger innate immune responses in COPD is currently unknown. Cell damage and death upon exposure to cigarette smoke in COPD may induce the release of damage associated molecular patterns (DAMPs) [3]. Elevated levels of several prototypic DAMPs, including high-mobility group box (HMGB)1, heat shock proteins (HSPs) and S100A8, have been observed in bronchoalveolar lavage (BAL) fluid, serum and epithelial lining fluid of COPD patients [4–6]. DAMPs activate cells of the innate immune system upon binding to pattern recognition receptors, such as toll-like receptors (TLRs) and receptor for advanced glycation end-products (RAGE). Importantly, the AGER gene, which encodes the RAGE receptor, is a genome-wide association study susceptibility gene for COPD [7]. To date, studies investigating the role of DAMPs in COPD have focused on the analysis of a single DAMP. However, while each individual DAMP triggers …

Genetic variation associates with susceptibility for cigarette smoke-induced neutrophilia in mice

Neutrophilic airway inflammation is one of the major hallmarks of chronic obstructive pulmonary disease and is also seen in steroid resistant asthma. Neutrophilic airway inflammation can be induced by different stimuli including cigarette smoke (CS). Short-term exposure to CS induces neutrophilic airway inflammation in both mice and humans. Since not all individuals develop extensive neutrophilic airway inflammation upon smoking, we hypothesized that this CS-induced innate inflammation has a genetic component. This hypothesis was addressed by exposing 30 different inbred mouse strains to CS or control air for 5 consecutive days, followed by analysis of neutrophilic lung inflammation. By genomewide haplotype association mapping, we identified four susceptibility genes with a significant association to lung tissue levels of the neutrophil marker myeloperoxidase under basal conditions and an additional five genes specifically associated with CS-induced tissue MPO levels. Analysis of the expression levels of the susceptibility genes by quantitative RT-PCR revealed that three of the four genes associated with CS-induced tissue MPO levels had CS-induced changes in gene expression levels that correlate with CS-induced airway inflammation. Most notably, CS exposure induces an increased expression of the coiled-coil domain containing gene, Ccdc93, in mouse strains susceptible for CS-induced airway inflammation whereas Ccdc93 expression was decreased upon CS exposure in nonsusceptible mouse strains. In conclusion, this study shows that CS-induced neutrophilic airway inflammation has a genetic component and that several genes contribute to the susceptibility for this response.

Susceptibility for cigarette smoke-induced DAMP release and DAMP-induced inflammation in COPD

Cigarette smoke (CS) exposure is a major risk factor for chronic obstructive pulmonary disease (COPD). We investigated whether CS-induced damage-associated molecular pattern (DAMP) release or DAMP-mediated inflammation contributes to susceptibility for COPD. Samples, including bronchial brushings, were collected from young and old individuals, susceptible and nonsusceptible for the development of COPD, before and after smoking, and used for gene profiling and airway epithelial cell (AEC) culture. AECs were exposed to CS extract (CSE) or specific DAMPs. BALB/cByJ and DBA/2J mice were intranasally exposed to LL-37 and mitochondrial (mt)DAMPs. Functional gene-set enrichment analysis showed that CS significantly increases the airway epithelial gene expression of DAMPs and DAMP receptors in COPD patients. In cultured AECs, we observed that CSE induces necrosis and DAMP release, with specifically higher galectin-3 release from COPD-derived compared with control-derived cells. Galectin-3, LL-37, and mtDAMPs increased CXCL8 secretion in AECs. LL-37 and mtDAMPs induced neutrophilic airway inflammation, exclusively in mice susceptible for CS-induced airway inflammation. Collectively, we show that in airway epithelium from COPD patients, the CS-induced expression of DAMPs and DAMP receptors in vivo and the release of galectin-3 in vitro is exaggerated. Furthermore, our studies indicate that a predisposition to release DAMPs and subsequent induction of inflammation may contribute to the development of COPD.