Netherlands

Emphysema in patients with obstructive pulmonary disease (COPD) is characterized by progressive inflammation and causes irreversible alveolar tissue destruction. The mechanism leading to alveolar damage is not yet clear but to date consensus remains that neutrophils, monocytes and macrophages are key inflammatory cells mediating tissue destruction, and regulation involves various types of lymphocytes. Therefore, we performed a first mass cytometry (cytometry by time-of-flight, CyTOF) single cell analysis to compare immune cells derived from non-emphysematous (n=5) and severe emphysematous (n=15) lung tissue and paired blood samples. We applied a lymphoid and a myeloid antibody panel with 76 markers to obtain an overview of the immune cell profile in the lung and blood. Data were analyzed with Hierarchical Stochastic Neighbor Embedding (HSNE) and dual t-SNE algorithm to identify tissue-specific cell clusters. CyTOF analysis revealed distinct immune cell populations in human lung tissue (Fig. 1), which clustered differently from blood-derived cells. Moreover, CyTOF analysis revealed distinct features of several immune cell populations in emphysematous tissue, and we are now exploring emphysematous lung-specific cell clusters. To determine disease associated cellular interactions in emphysema, we are now applying imaging CyTOF, allowing simultaneous detection of up to 40 markers in a single lung tissue section.

Dutch Lung Congress

High-dimensional mass cytometry analysis of immune cells in emphysematous lung tissue

immune cells

mass cytometry

emphysema

[![](https://assets.underline.io/uploads/markdown_image/1/image/261902fdf187a88bfa74da226ff77186.png)](https://drive.google.com/file/d/1gUViQJJHCSgM38OhwpDoTdN89evCsZ-7/view?usp=sharing)
[![](https://assets.underline.io/uploads/markdown_image/1/image/1605292c232e6f185c7f87ee38e8502a.png)](https://drive.google.com/file/d/1kPkznVX_6cx-n846z_pUtyVDZql9KImc/view?usp=sharing)


### **Welcome Message**

Welcome to the inaugural **Dutch Lung Congress.**  We have all missed our opportunities to network and interact with our colleagues in the last twelve months or more.  While our desire was to meet face to face once again, when it became evident that this would not be possible until much later in 2021 (or even into 2022) the hard working committee organizing the Dutch Lung Congress found, what we hope will be, an excellent alternative to facilitate our annual meeting.

The Dutch Lung Congress aims to bring together everyone interested in research related to pulmonary diseases in the Netherlands.  We hope this event will provide opportunities for multidisciplinary interactions to enhance our approaches for addressing these challenging diseases.  Each session will include input from patients, clinical and basic and/or translational scientists discussing the latest information around the central themes chosen by the organizing committee.  One advantage of holding our congress as a virtual event this year is that we will have the opportunity to hear from world leading experts from all over the globe.  We hope you enjoy the programme the organizing committee have put together and will engage in dynamic discussions with all the speakers.

One highlight of our congress will be the Young Investigator Session.  Remember to join us for this exciting session when we will hear from the presenters who submitted the top scoring abstracts.  This promises to be a session full of exciting science that will display the strengths that constitute the future talent in our field.

When we are not listening to these great presentations we can all meet in Gathertown.  Within this interactive platform you will be able to meet colleagues and friends for networking. Enjoy a private chat over a virtual coffee, visit the booths of our sponsors and other parties, chat with the Young Investigator Board members or take some timeout in the relaxation spaces.  While in Gathertown take the opportunity to visit all the diverse posters on display. Chat live with the presenters or leave them a question on the discussion board for them to engage with you.  Make sure you check for any messages each time you enter Gathertown to make the most of your networking opportunities.

We look forward to seeing you during the DLC and the great scientific (and other) discussions we are anticipating during our 2021 meeting.

On behalf of the DLC organizing Committee
<br>**Janette Burgess
<br>Chair, DLC Organizing Committee**
![](https://assets.underline.io/uploads/markdown_image/1/image/f461fb275d9667fb2a76a30813388e90.png)



### Special Session: Publieksmiddag
![](https://assets.underline.io/uploads/markdown_image/1/image/77e768dd500e6205175e5cafba59abdc.png)






![](https://assets.underline.io/uploads/markdown_image/1/image/f461fb275d9667fb2a76a30813388e90.png)
**More about the committee team:**:
[Barbro N. Melgert, PhD] (https://drive.google.com/file/d/1q_IIH3JvTivumCUzzsZGX7BgD41HxQmk/view?usp=sharing)
// [Bas Holverda, PhD] (https://drive.google.com/file/d/1QKXHmTTQjgs4OZVuM8doXU055S-fu_uU/view?usp=sharing)
// [Frits Franssen, PhD] (https://drive.google.com/file/d/1d7HRUJaqGuRFzkPN3SVgkbBfqFkDkWq2/view?usp=sharing)
// [Janette Burgess, PhD] (https://drive.google.com/file/d/1VHFlrEN6uCQGeeW3Lj4I8aTw7HsB0GFr/view?usp=sharing)
// [Michel van den Heuvel, PhD] (https://drive.google.com/file/d/1JeOEP8MHxiK_4lONr8UM2HaxdxpkAmA2/view?usp=sharing)
// [Patrick Bhairosing] (https://drive.google.com/file/d/1xI8ORq-g1bbVcrlRDviZ9NhAwanc4dFl/view?usp=sharing)
// [Gimano Amatngalim, PhD] (https://drive.google.com/file/d/1fyVDDKARLNjRk_JZk8_GyhGMjmp6DWHp/view?usp=sharing)
// [Peter Kunst, PhD] (https://drive.google.com/file/d/1QhtB4nQezpAjbfUAcBnINc-Bm3Shy2Lf/view?usp=sharing)
// [Dr. Michel van den Heuvel, Prof., MD, PhD] (https://drive.google.com/file/d/1JBdNEBXZCEikEZhJ6MoD5DhQfXMz3bP6/view?usp=sharing)
![](https://assets.underline.io/uploads/markdown_image/1/image/f461fb275d9667fb2a76a30813388e90.png)

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poster

Background: 

The Asthma Optimiser is a web-based application to support primary care health care professionals during their asthma consultations by structured guidance through step-by-step questioning and capture of patients’ answers. Using a built-in asthma management decision tree, the tool suggests how they may optimize patients’ asthma management or facilitate referral to asthma specialists. This is in accordance with GINA and NHG guidelines. Data can be stored in the EMR. The CAPTURE study aims to evaluate the feasibility and utility of the Asthma Optimiser in primary care. 

Examination of the feasibility:

Qualitative semi-structured interviews with 20 primary care nurses will be held to examine barriers and facilitators of using the Asthma Optimiser (also known as Refer ID). Purposive sampling will be adopted to attain a sample of primary care nurses who differ in work experience, age, and who work in different GP practices. Interviews will be held using a ‘topic guide’. Data will be collected in an iterative manner (i.e., by updating the topic guide after each interview). Experiences will be examined with thematic analysis and axial coding. This will result in an exhaustive taxonomy of ‘tips and tricks’. 

Examination of the utility: 

A prospective observational study of 354 adult primary care patients with asthma will be conducted. All patients receive standard medical care with additional blood eosinophilic measurement using point-of-care testing and complete the Work Productivity and Activity Impairment (WPAI) questionnaire. Primary outcome is the distribution of asthma patients over the management suggestions of the decision-tree. We will study the association of this distribution with blood eosinophil counts, self-reported WPAI, as well as other pre-existing patient characteristics. Patients referred to secondary care will be followed-up for an additional 26 weeks, analyzing these difficult-to-treat asthma cases.


Implementation of the Asthma Optimiser e-health tool in primary care: a mixed methods approach

Multiple studies have indicated that growing up on a traditional farm is associated with protection against virus-induced wheezing, allergies and asthma, suggesting protective effects on the airway epithelial barrier. Environmental triggers such as allergens, pollutants and pathogens can damage the airway epithelial barrier and contribute to both the development and the progression of asthma. This is mediated in part by airway epithelial alarmins such as IL-25, IL-33 and TSLP, that activate an array of innate immune cells. The aim of the present study is to investigate if (pre-)treatment using farm dust extract (FD) inhibits expression of IL-25, IL-33 and TSLP in primary bronchial epithelial cells (PBEC). Both submerged (S) (n=5 donors) and differentiated (ALI-)PBEC (n=3 donor mixes; 5 donors/mix) were pre-treated with FD for 24 h. Next, cells were stimulated with TNF-α/IFN-γ and poly(I:C)/IL-4 (in S-PBEC only) for 6 h to induce alarmin expression in presence or absence of FD. FD reduced TNF-α/IFN-γ-mediated gene expression of IL33 and IL25 and Poly(I:C)/IL-4-mediated gene expression of TSLP after 6 h in S-PBEC, whereas in ALI-PBEC there was a trend towards a decrease in TNF-α/IFN-γ-mediated gene expression of IL33 and no effect on IL25. The results indicate that FD inhibits inflammation-mediated induction of expression of alarmins in PBEC. However, mechanisms underlying the inhibitory effects of FD on alarmin expression require further investigation. 
This study was supported a grant from the Lung Foundation Netherlands: AWWA grant # 12.0.17.001


Gene expression of epithelial alarmins is decreased by farm dust exposure in primary bronchial epithelial cells

Background:
With the continued advancement of CFTR modulator therapies there is likely to be a burgeoning population of adult cystic fibrosis (CF) patients unable to expectorate sputum. Consequently, the detection and surveillance of pulmonary colonisation, previously reliant on sputum culture, needs re-examining. We hypothesised that cough swabs analysed with culture-independent analysis of the 16S gene could serve as a surrogate for colonisation of the lower airways. 
Methods:
Cough swabs and sputum samples were prospectively collected from consecutive adults and children with CF across two sites at regular outpatient appointments. Conventional culture analysis and next generation sequencing were used to compare paired same day samples. 
Results:
Twenty-two adults and 8 paediatric patients provided 75 paired cough swabs and sputum samples. Alpha diversity measures showed increased bacterial richness in sputum, while evenness and Simpson’s index were higher in cough swabs. Within each sampling technique, microbial composition showed greater similarity when considering intra-patient variation. Poor concordance was observed between culture independent cough swabs and culture dependent/independent sputum analysis for specific pathogens, with cough swabs unable to accurately identify commonly associated CF pathogens (AUROCC range: 0.51 to 0.64). 

Conclusion:
Culture independent analysis of cough swabs provides an inaccurate diagnosis of lower respiratory tract colonization and should not be used as a diagnostic test in patients with CF.


Comparison of microbial composition of cough swabs and sputum for pathogen detection in patients with cystic fibrosis

Rationale: In chronic obstructive pulmonary disease (COPD), the airways are characterized by goblet cell hyperplasia, decreased ciliated cell numbers and an altered turnover of the extracellular matrix (ECM). Current 2D (co-)culture systems lack the spatial in vivo environment that is formed by ECM. In this study we aimed to develop a lung ECM hydrogel-based co-culture model to investigate bidirectional interactions between fibroblasts and epithelial cells in relation to ECM changes. 

Methods: Porcine lungs were decellularized, lyophilized and ground to a powder. This powder was porcine pepsin-solubilized for 48h, pH neutralized, buffered with PBS and incubated at 37°C for 30 min to generate ECM hydrogels. Human primary lung fibroblasts (50,000/ml) from non-COPD donors were mixed with hydrogels prior to gelation in transwells. The next day, the hydrogels were coated with collagen type IV or left uncoated. After 24h, 100,000 primary bronchial epithelial cells (PBEC) from non-COPD donors were seeded on top of fibroblast seeded and unseeded gels. The cells were cultured submerged for 4 days and subsequently at air liquid interface for 14 days. The hydrogels were imaged using inverted light microscopy and by histology on paraffin-embedded sections

Results: PBECs attached and formed a mono layer on the collagen-IV coated and uncoated hydrogels. All gels contracted after 48 hours. The absence of fibroblasts appeared to reduce the degree of contraction. After 4 days of submerged culture the epithelial layer formed a stacked multicellular confluent layer on the hydrogels. After 14 days of air exposure the multicellular epithelial cells on the hydrogels further expanded and the fibroblasts remodeled their environment, as shown by a change in structure and staining intensity around the fibroblasts in the ECM hydrogel seen in the H&E staining.

Conclusions: The combination of a lung ECM hydrogel with a basement membrane like coating made it possible to co-culture primary lung fibroblasts and PBECs in 3D at ALI, generating a morphology resembling the in vivo airway wall. 


Airway epithelial cells, fibroblasts and the extracellular matrix: a ménage à trois resembling the airway wall in vivo


Introduction: 
Nowadays, identifying disease progression in an early stage has direct treatment implications in patients with fibrotic interstitial lung diseases (F-ILDs). eHealth could be a solution for close monitoring and detecting disease progression, at low burden for patients and clinicians. The main aims of this study are to assess long-term FVC change in patients with F-ILD measured with home spirometry and to evaluate the feasibility of a multinational patient-led registry in F-ILD. In addition, we aim to better validate patient-reported outcomes (PROMs) in patients with F-ILD.
Methods: 
The I-FILE is a prospective multinational observational study, conducted in seven European countries. We aim to include 700 treatment naive patients with newly diagnosed F-ILD. Patients perform daily home spirometry in the first three months followed by once weekly measurements for a period of 2 years. In addition, PROMs will be completed online every 6 months, including the King’s brief Interstitial Lung Disease Health Status, EQ5D-5L, Visual Analogue Scale on cough, dyspnea, fatigue and general complaints, Leicester Cough Questionnaire, Fatigue Assessment Scale , Work Productivity and Activity Impairment Questionnaire, Global Rating of Change Scale and Living with Pulmonary Fibrosis questionnaire. Results are encrypted and directly sent to a secured server through the online I-FILE application.
Outcomes: 
The primary endpoint is change in FVC after 12 months assessed by home monitoring. Measurements will be compared to in-hospital FVC obtained during regular follow up visits. Other endpoints are adherence to daily and weekly home spirometry and validity, consistency and responsiveness of PROMs in patients with F-ILD. 
Conclusions: 
The I-FILE study will be the ultimate collaboration between patients and doctors to provide much needed insights in disease behavior and progression in a large and diverse patient group with F-ILD. Results can be used to optimize monitoring of dieasese course to enable tailored treatment for patients with F-ILD

Study design of an international patient-led registry in fibrotic interstitial lung diseases using eHealth technology: I-FILE

Objective: To determine the cause of work-related asthma.
Method: A city transit bus driver (57 yrs.) of a public transport company with non-allergic, non-eosinophilic asthma developed progressive work-related symptoms of productive cough, wheezing and dyspnea when driving in buses with a crude polyurethane filter (Bus A). Symptoms did not worsen when driving on the only bus with a different type of climate control, including a filter for particles > 0,1 μm (Bus B).
Lung function was monitored during and after passenger transport services with Bus A, on regular routes in a city (Breda) and suburban areas. Subsequently, air quality was measured in 2 buses of type A and in Bus B, on the same day and under standardized conditions during rides without passengers, but following regular routes.
Results: Increased symptoms without significant changes in FEV1 were observed during 3 successive regular rides with Bus A. However, two days afterwards, FEV1 dropped from 2,7 l to 1,9 l (30%). Real time measurements showed constantly lower levels of soot in Bus B. Mean soot concentrations in Buses A were 2,4 and 2,1 μg/m3 and in Bus B 1,2 μg/m3 (Fig. 1). Levels of PM 2,5 did not differ between buses A and B (28-32 μg/m3). In all buses, levels of ultrafine particles depended on outdoor air pollution.
Conclusion: Soot concentrations in Bus A were comparable with levels present in busy streets and urban centres and may be responsible for work-related asthma. 
(Guarnieri M, Balmes JR. Lancet 2014; 383:1581-92).


Work-related meets environmental asthma in a bus driver

Background
Approximately 8% of all births occur between 30-36 weeks of pregnancy. Respiratory tract infections (RTI) and wheezing illnesses disproportionally affect such preterms in early life. Next to the relatively underdeveloped lungs at birth, an unfavourable microbiome associated with an aberrant immune maturation might underlie the susceptibility to RTI. Early- and severe RTI increase the likelihood of subsequent poor respiratory health. Oral bacterial lysates have shown to be effective in the prevention of RTI in children through the interaction with the gut immune system and stimulation of non-specific immune responses. Our systematic review showed reduction of pre-school wheezing episodes and asthma exacerbations after bacterial lysate use (mean difference of -1.40 episodes (95% CI -2.01--0.80 ;figure 1). Also, primary prevention of RTI and wheezing episodes in very young infants is described and currently being studied (Orbex study, USA). However, studies so far excluded preterms despite their extra-ordinary disease burden.

Aim To reduce RTI and wheezing in moderate-late preterms in the first year of life by bacterial lysate administration and to determine the correlation of biological markers with respiratory symptoms, immune protection and treatment effect. 

Methods
This randomised placebo-controlled trial will include 500 otherwise healthy preterm infants (30+0-35+6 weeks). OM-85 or placebo powder will be given from 6-10 weeks after birth until 12 months of age. Clinical data will be continuously collected by e-Health and study visits; with optional biosampling (blood; nasal fluid, brush and swab; faeces) and sleep impedance pneumography at baseline, 6 and 12 months. 

Outcome
Lower RTI and wheezing episodes in the first year of life. Immune maturation, as well as microbiome development in the respiratory tract and gut. Biomarkers for risk-group selection and/or treatment success. 

Impact
We envision finding an affordable therapy for a large patient group thereby improving life-long respiratory health and (family) quality of life. 


Protecting late-moderate preterm infants from respiratory tract infections and wheeze in their first year of life by using bacterial lysates: a study protocol


Chronic Obstructive Pulmonary Disease (COPD) patients frequently develop osteoporosis, severely limiting life quality and increasing hospitalisation. Accelerated bone degradation is traditionally explained by elevated Receptor Agonist of NFKB signalling Ligand (RANKL) levels in the bone, resulting in osteoclast activity, however, COPD patients with dominant emphysema also presented with elevated serological levels of RANKL (Bai et al, 2011). Recent data from Woldhuis et al shows that RANKL is produced by lung fibroblasts, and that in COPD, compared to a plethora of secreted cytokines, RANKL secretion has the highest fold increase. Also, local production of the inflammatory cytokine IL1β is known to be a potent inducer of RANKL in osteoblasts, which in the inflamed COPD lung could translate to increased RANKL production. However, it is currently unclear whether IL1β also induces local RANKL production in the lung. We hypothesize that IL1β induces RANKL expression in lung fibroblasts, resulting in higher production and secretion of RANKL. To test this, we first explored a single-cell sequencing dataset generated by Lee et al. comprising IL1β treated murine lung organoids. Re-analysis showed that stromal cells in this model strongly upregulated RANKL in response to IL1β. To further explore the effects of IL1b, primary fibroblasts from COPD lung were treated with il1b for 48 hours. Although free RANKL was not detected in culture supernatants in either condition, levels of the RANKL/osteoprotegerin (OPG) complex were increased under influence of IL1β, whereas OPG levels remained constant, indicating that RANKL production indeed increased. Combined, these results show that RANKL production is regulated by IL1β in lung fibroblasts. Also, these data mark the importance of measuring both free- and OPG-bound RANKL, as OPG might obscure any effects of RANKL. Building on this, future research should focus on exploring whether this regulatory effect is altered in COPD by checking both free- and OPG-bound RANKL in lung tissue and serum and examining the possible role of RANKL in lung tissue.

IL1β induces RANKL secretion in human lung fibroblasts

DHA but not EPA reduces IL-8 (KC) cytokine release from lung alveolar epithelial cells
Dan Li, Danique Lobee, Melissa Van der Veen, Hoeke.A. Baarsma, Reinoud Gosens, Martina Schmidt 

Background:
The acute inflammatory response to injury or pathogens protects the host from systemic infection and initiates tissues repair. Recent research showed that resolution of acute inflammation is an active process which is initiated by the omega 3 fatty acids docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA). Here we aim to investigate in the role of DHA and EPA in models of inflammation in the lung epithelium. 
Method:
Human bronchial epithelial (BEAS-2B) cells and human alveolar epithelial (A549) cells were exposed to different concentrations and the indicated time periods (up to 24h) to DHA and EPA (up to 100 μM), followed by stimulation with lipopolysaccharide (LPS) (0.01-100 μg/ml). Cell viability was measured by MTT. The inflammatory markers interleukin-8 (IL-8, IL-6) were measured by ELISA. Expression of IkkB, IkB-a, p65 expression has been detected by western blot and immunofluorescence. To translate the findings from the 2D cell cultrues to the 3D cell cultures, a mouse organoid model comprised of a co-culture of primary mouse CD31-CD45-Epcam+ cells with CCL206 fibroblasts was used. 
Results:
DHA and EPA alone and in combination with LPS did not affect the cell viability in both BEAS-2B and A549 cells. In BEAS-2B cells, DHA and EPA did not reduce the LPS-stimulated IL-8 and IL-6 release. On the contrary, in A549 cells DHA (60μM) but not EPA significantly reduced the LPS-induced IL-8 release, leaving the IL-6 release unchanged. Total IKKB and p65 expression was not changed by DHA, EPA, measured alone or in combination with LPS. The p-IKKa/b, IkB-a expression and p-65 nuclear translocation are being researched now by Western blot and immunofluorescence. Importantly, we found in the organoid model that LPS induces mouse KC release, a process significantly reduced by DHA but not EPA. 
Conclusion: 
DHA but not EPA may reduce the inflammatory response in lung alveolar epithelial cells by suppressing IL-8 (KC) release. The mechanism involved still needs to be studied further. 


DHA but not EPA reduces IL-8 (KC) cytokine release from lung alveolar epithelial cells

Prednisone is first-line treatment for pulmonary sarcoidosis and leads to improvement of pulmonary function and symptoms on short-term. However, prednisone can also lead to side-effects, which may negatively affect quality of life. Hence, patients and doctors report an urgent need for better evidence based first line treatment options. Methotrexate is considered as second-line treatment and seems to have fewer side-effects as reported in a patient survey. In the PREDMETH study we investigate the effectiveness of methotrexate compared with prednisone as first line treatment for pulmonary sarcoidosis. Furthermore, we examine blood biomarkers to assess whether response to therapy can be predicted in individual patients.

Methods
The PREDMETH study is initiated by the Erasmus Medical Center and St. Antonius Hospital Nieuwegein and developed in collaboration with the sarcoidosis patient association. This non-inferiority study takes place in 18 hospitals across the Netherlands. Patients will be randomized to treatment with prednisone or methotrexate in a predefined schedule for 24 weeks. Data will be collected at baseline, 4 weeks, 16 weeks, 24 weeks, 1 year and 2 years. Additionally, patients perform weekly home spirometry with a handheld spirometer and report symptoms and side-effects through an online home monitoring program (developed in collaboration with patients with sarcoidosis) during the first six months of the study. The primary endpoint is change in forced vital capacity at six months. Secondary endpoints are adverse events, quality of life and blood biomarkers. The study is currently recruiting patients. 

Discussion
This unique national study is developed by doctors, researchers and patients to provide better evidence based treatment for pulmonary sarcoidosis. If our hypothesis is confirmed and methotrexate is as effective as prednisone with less side-effect this will initiate a change in practice. Furthermore, the translational research will provide more insight to tailored treatment and the pathophysiological mechanisms of sarcoidosis.

Design of a randomized controlled trial to evaluate effectiveness of methotrexate versus prednisone as first-line treatment for pulmonary sarcoidosis: the PREDMETH study

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