Nele S. Pauwels

Role of IL-1α and the Nlrp3/caspase-1/IL-1β axis in cigarette smoke-induced pulmonary inflammation and COPD

Cigarette smoke (CS), the primary risk factor of chronic obstructive pulmonary disease (COPD), leads to pulmonary inflammation through interleukin-1 receptor (IL-1R)I signalling, as determined using COPD mouse models. It is unclear whether interleukin (IL)-1&agr; or IL-1&bgr;, activated by the Nlrp3/caspase-1 axis, is the predominant ligand for IL-1RI in CS-induced responses. We exposed wild-type mice (treated with anti-IL-1&agr; or anti-IL-1&bgr; antibodies), and IL-1RI knockout (KO), Nlrp3 KO and caspase-1 KO mice to CS for 3 days or 4 weeks and evaluated pulmonary inflammation. Additionally, we measured the levels of IL-1&agr; and IL-1&bgr; mRNA (in total lung tissue by RT-PCR) and protein (in induced sputum by ELISA) of never-smokers, smokers without COPD and patients with COPD. In CS-exposed mice, pulmonary inflammation was dependent on IL-1RI and could be significantly attenuated by neutralising IL-1&agr; or IL-1&bgr;. Interestingly, CS-induced inflammation occurred independently of IL-1&bgr; activation by the Nlrp3/caspase-1 axis. In human subjects, IL-1&agr; and IL-1&bgr; were significantly increased in total lung tissue and induced sputum of patients with COPD, respectively, compared with never-smokers. These results suggest that not only IL-1&bgr; but also IL-1&agr; should be considered as an important mediator in CS-induced inflammation and COPD.

NLRP3/Caspase-1–Independent IL-1β Production Mediates Diesel Exhaust Particle-Induced Pulmonary Inflammation

Inhalation of diesel exhaust particles (DEP) induces an inflammatory reaction in the lung; however, the mechanisms are largely unclear. IL-1β/IL-1RI signaling is crucial in several lung inflammatory responses. Typically, caspase-1 is activated within the NLRP3 inflammasome that recognizes several damage-associated molecular patterns, which results in cleavage of pro–IL-1β into mature IL-1β. In this study, we hypothesized that the NLRP3/caspase-1/IL-1β pathway is critical in DEP-induced lung inflammation. Upon DEP exposure, IL-1RI knockout mice had reduced pulmonary inflammation compared with wild-type mice. Similarly, treatment with rIL-1R antagonist (anakinra) and IL-1β neutralization impaired the DEP-induced lung inflammatory response. Upon DEP exposure, NLRP3 and caspase-1 knockout mice, however, showed similar IL-1β levels and comparable pulmonary inflammation compared with wild-type mice. In conclusion, these data show that the DEP-induced pulmonary inflammation acts through the IL-1β/IL-1RI axis. In addition, DEP initiates inflammation independent of the classical NLRP3/caspase-1 pathway, suggesting that other proteases might be involved.

Leptin Modulates Innate and Adaptive Immune Cell Recruitment after Cigarette Smoke Exposure in Mice

Leptin, a pleiotropic type I cytokine, was recently demonstrated to be expressed by resident lung cells in chronic obstructive pulmonary disease patients and asymptomatic smokers. To elucidate the functional role of leptin in the onset of chronic obstructive pulmonary disease, we tested leptin-deficient ob/ob mice (C57BL/6), leptin receptor-deficient db/db mice (C57BKS), and littermates in a model of cigarette smoke (CS)-induced pulmonary inflammation. Wild-type (WT) C57BL/6 mice were exposed for 4 or 24 wk to control air or CS. Pulmonary leptin expression was analyzed by immunohistochemistry and real-time PCR. Pulmonary inflammation upon 4 wk CS exposure was evaluated in bronchoalveolar lavage fluid (BALF) and lung tissue of WT, ob/ob, and db/db mice. Immunohistochemical analysis revealed leptin expression in bronchial epithelial cells, pneumocytes, alveolar macrophages, and bronchial/vascular smooth muscle cells. The 4 and 24 wk CS exposure increased leptin expression in bronchial epithelial cells and pneumocytes versus air-exposed WT mice (p < 0.05). The 4 wk CS exposure resulted in increased accumulation of neutrophils, dendritic cells, macrophages, and lymphocytes in BALF and lung tissue of WT, ob/ob, and db/db mice. CS-exposed ob/ob and db/db mice showed in general higher numbers of neutrophils and lower numbers of CD4+, CD8+, and dendritic cells versus CS-exposed WT mice. Consistently, CXCL1 levels were enhanced in BALF of CS-exposed ob/ob and db/db mice versus WT mice (p < 0.05). Exogenous leptin administration completely restored the skewed inflammatory profile in ob/ob mice. These data reveal an important role of leptin in modulating innate and adaptive immunity after CS inhalation in mice.

Enhanced deposition of low-molecular-weight hyaluronan in lungs of cigarette smoke-exposed mice

Chronic obstructive pulmonary disease (COPD) is characterized by infiltration of inflammatory cells, destruction of lung parenchyma, and airway wall remodeling. Hyaluronan (HA) is a component of the extracellular matrix, and low-molecular-weight (LMW) HA fragments have proinflammatory capacities. We evaluated the presence of HA in alveolar and airway walls of C57BL/6 mice that were exposed to air or cigarette smoke (CS) for 4 weeks (subacute) or 24 weeks (chronic). We measured deposition of the extracellular matrix proteins collagen and fibronectin in airway walls and determined the molecular weight of HA purified from lung tissue. In addition, we studied the expression of HA-modulating genes by RT-PCR. HA staining in alveolar walls was significantly enhanced upon chronic CS exposure, whereas HA levels in the airway walls were already significantly higher upon subacute CS exposure and remained elevated upon chronic CS exposure. This differed from the deposition of collagen and fibronectin, which are only elevated at the chronic time point. In lungs of CS-exposed mice, the molecular weight of HA clearly shifted toward more LMW HA fragments. CS exposure significantly increased the mRNA expression of the HA synthase gene Has3 in total lung tissue, whereas the expression of Has1 was decreased. These in vivo studies in an experimental model of COPD show that CS exposure leads to enhanced deposition of (mostly LMW) HA in alveolar and bronchial walls by altering the expression of HA-modulating enzymes. This may contribute to airway wall remodeling and pulmonary inflammation in COPD.

Different regulation of cigarette smoke induced inflammation in upper versus lower airways

BackgroundCigarette smoke (CS) is known to initiate a cascade of mediator release and accumulation of immune and inflammatory cells in the lower airways. We investigated and compared the effects of CS on upper and lower airways, in a mouse model of subacute and chronic CS exposure.MethodsC57BL/6 mice were whole-body exposed to mainstream CS or air, for 2, 4 and 24 weeks. Bronchoalveolar lavage fluid (BAL) was obtained and tissue cryosections from nasal turbinates were stained for neutrophils and T cells. Furthermore, we evaluated GCP-2, KC, MCP-1, MIP-3α, RORc, IL-17, FoxP3, and TGF-β1 in nasal turbinates and lungs by RT-PCR.ResultsIn both upper and lower airways, subacute CS-exposure induced the expression of GCP-2, MCP-1, MIP-3α and resulted in a neutrophilic influx. However, after chronic CS-exposure, there was a significant downregulation of inflammation in the upper airways, while on the contrary, lower airway inflammation remained present. Whereas nasal FoxP3 mRNA levels already increased after 2 weeks, lung FoxP3 mRNA increased only after 4 weeks, suggesting that mechanisms to suppress inflammation occur earlier and are more efficient in nose than in lungs.ConclusionsAltogether, these data demonstrate that CS induced inflammation may be differently regulated in the upper versus lower airways in mice. Furthermore, these data may help to identify new therapeutic targets in this disease model.

Is blood of uncomplicated hemochromatosis patients safe and effective for blood transfusion? A systematic review

Hemochromatosis is a disorder of the iron metabolism, characterized by high body iron content, necessitating frequent phlebotomies to remove excess iron. In some countries, this blood is discarded and not used for blood transfusion because of the non-voluntary character of this donation, and because a potential risk of microbial contamination of the donor blood is assumed. A systematic review was performed in order to collect and critically examine solid evidence with regard to the effectiveness and safety of blood for transfusion when derived from hemochromatosis patients who do not suffer from complications or organ damage. Using three databases (The Cochrane Library, MEDLINE, and Embase) we searched for studies from date of inception until January 2012. Out of 3470 articles, 80 references that were relevant to our question were selected, including many opinion pieces, comments, letters, and narrative reviews. Based on our selection criteria, we finally retained only six observational studies, so evidence on this subject is scarce and furthermore, the strength of the available evidence is low to very low, due to poor study designs. We found no evidence that red blood cell concentrates from hemochromatosis patients without complications of iron overload do not comply with the physiological quality requirements for transfusion, nor that their blood would present a greater risk to recipient safety than blood from non-hemochromatosis donors. However, in vitro findings from two studies suggest that iron-overloaded patients would be more susceptible to bacterial growth, but future in vivo studies are warranted to confirm this. Based on this, we call for harmonization of the blood donor selection policy among countries allowing hemochromatosis patients who do not suffer from complications of iron overload to donate blood, once iron levels are normalized.

Part 9: First Aid 2015 International Consensus on First Aid Science With Treatment Recommendations

### Definition of First Aid The International Liaison Committee on Resuscitation (ILCOR) First Aid Task Force first met in June 2013. Comprising nominated members from around the globe appointed by each ILCOR member organization, the task force members first agreed to the goals of first aid and produced a definition of first aid as it might apply to the international setting. Task force members considered an agreed-upon definition essential for the subsequent development of research questions, evidence evaluation, and treatment recommendations. First aid is defined as the helping behaviors and initial care provided for an acute illness or injury. First aid can be initiated by anyone in any situation. A first aid provider is defined as someone trained in first aid who should The goals of first aid are to preserve life, alleviate suffering, prevent further illness or injury, and promote recovery. This definition of first aid addresses the need to recognize injury and illness, the requirement to develop a specific skill base, and the need for first aid providers to simultaneously provide immediate care and activate emergency medical services (EMS) or other medical care as required. First aid assessments and interventions should be medically sound and based on evidence-based medicine or, in the absence of such evidence, on expert medical consensus. The scope of first aid is not purely scientific, as both training and regulatory requirements will influence it. Because the scope of first aid varies among countries, states, and provinces, the treatment recommendations contained herein may need to be refined according to circumstances, need, and regulatory constraints. One difference between this 2015 definition and that used for the 2010 process is that the …

Cigarette smoke induces PTX3 expression in pulmonary veins of mice in an IL-1 dependent manner.

BackgroundChronic obstructive pulmonary disease (COPD) is associated with abnormal inflammatory responses and structural alterations of the airways, lung parenchyma and pulmonary vasculature. Since Pentraxin-3 (PTX3) is a tuner of inflammatory responses and is produced by endothelial and inflammatory cells upon stimuli such as interleukin-1β (IL-1β), we hypothesized that PTX3 is involved in COPD pathogenesis.Methods and ResultsWe evaluated whether cigarette smoke (CS) triggers pulmonary and systemic PTX3 expression in vivo in a murine model of COPD. Using immunohistochemical (IHC) staining, we observed PTX3 expression in endothelial cells of lung venules and veins but not in lung arteries, airways and parenchyma. Moreover, ELISA on lung homogenates and semi-quantitative scoring of IHC-stained sections revealed a significant upregulation of PTX3 upon subacute and chronic CS exposure. Interestingly, PTX3 expression was not enhanced upon subacute CS exposure in IL-1RI KO mice, suggesting that the IL-1 pathway is implicated in CS-induced expression of vascular PTX3. Serum PTX3 levels increased rapidly but transiently after acute CS exposure.To elucidate the functional role of PTX3 in CS-induced responses, we examined pulmonary inflammation, protease/antiprotease balance, emphysema and body weight changes in WT and Ptx3 KO mice. CS-induced pulmonary inflammation, peribronchial lymphoid aggregates, increase in MMP-12/TIMP-1 mRNA ratio, emphysema and failure to gain weight were not significantly different in Ptx3 KO mice compared to WT mice. In addition, Ptx3 deficiency did not affect the CS-induced alterations in the pulmonary (mRNA and protein) expression of VEGF-A and FGF-2, which are crucial regulators of angiogenesis.ConclusionsCS increases pulmonary PTX3 expression in an IL-1 dependent manner. However, our results suggest that either PTX3 is not critical in CS-induced pulmonary inflammation, emphysema and body weight changes, or that its role can be fulfilled by other mediators with overlapping activities.

Part 9: First aid

### Definition of First Aid The International Liaison Committee on Resuscitation (ILCOR) First Aid Task Force first met in June 2013. Comprising nominated members from around the globe appointed by each ILCOR member organization, the task force members first agreed to the goals of first aid and produced a definition of first aid as it might apply to the international setting. Task force members considered an agreed-upon definition essential for the subsequent development of research questions, evidence evaluation, and treatment recommendations. First aid is defined as the helping behaviors and initial care provided for an acute illness or injury. First aid can be initiated by anyone in any situation. A first aid provider is defined as someone trained in first aid who should The goals of first aid are to preserve life, alleviate suffering, prevent further illness or injury, and promote recovery. This definition of first aid addresses the need to recognize injury and illness, the requirement to develop a specific skill base, and the need for first aid providers to simultaneously provide immediate care and activate emergency medical services (EMS) or other medical care as required. First aid assessments and interventions should be medically sound and based on evidence-based medicine or, in the absence of such evidence, on expert medical consensus. The scope of first aid is not purely scientific, as both training and regulatory requirements will influence it. Because the scope of first aid varies among countries, states, and provinces, the treatment recommendations contained herein may need to be refined according to circumstances, need, and regulatory constraints. One difference between this 2015 definition and that used for the 2010 process is that the …

COPD is associated with reduced pulmonary interstitial expression of pentraxin-3.

Pentraxin (PTX)3 is involved in antimicrobial defence, apoptotic cell clearance and extracellular matrix stability. As these processes are altered in chronic obstructive pulmonary disease (COPD), we aimed to investigate PTX3 expression in patients with this disease. PTX3 expression was quantified by immunohistochemical staining of lung tissue from never-smokers, smokers without COPD, and in patients with COPD of Global Initiative for Chronic Obstructive Lung Disease (GOLD) stage I, II and III–IV. mRNA expression was examined in total lung tissue by quantitative RT-PCR. PTX3 concentration was measured in induced sputum and plasma by ELISA. PTX3 is mainly localised in the interstitium of the small airways and alveolar walls. There were no significant differences in pulmonary, sputum and plasma PTX3 expression between study groups. However, PTX3 expression in small airways correlated significantly with forced expiratory volume in 1 s (r=0.35, p=0.004). In the alveolar walls, PTX3 expression correlated significantly with carbon monoxide transfer coefficient (r=0.28, p=0.04). In sputum, PTX3 levels were highly correlated with the number of neutrophils. Finally, systemic levels of PTX3 tended to be lower in severe COPD compared with mild COPD. In COPD, airflow limitation and reduced transfer coefficient for carbon monoxide are associated with lower pulmonary interstitial expression of PTX3.