Netherlands

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. 


Dutch Lung Congress

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

il-8

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### **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**
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### Special Session: Publieksmiddag
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**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)
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poster

Introduction Small fiber neuropathy (SFN) may presents as complication in amongst others sarcoidosis.1 SFN can potentially result into a large range of symptoms with a high impact on quality of life.2 Although treatment of the underlying disease of SFN is advised for the past decades, little research has been performed to investigate SFN improvement as consequence of sarcoidosis treatment. This retrospective study investigates treatment effects of Infliximab on SFN symptoms.
Methods The Small Fiber Neuropathy Screening List (SFNSL) was used to identify changes in SFN symptoms. Maximal standardized uptake value (SUVmax) from Fluordeoxyglucose Positron Emission Tomography (FDG-PET) was used as inflammatory activity measure. To investigate the effects of Infliximab on inflammatory activity, a subdivision was made defined as improved, stable and worsened SFNSL-score.
Results 45 sarcoidosis patients were eligible for analysis. SFNSL-score showed a mean decrease of -3 points (p = 0.207). SUVmax did improve with a mean of 3.44 (p=<0.001). When looking at the subdivision based on the change in SFNSL, only in the group with worsening of SFN-related symptoms, no clinical relevant improvement of sarcoidosis inflammatory activity was induced.
Discussion In patients with active inflammatory sarcoidosis defined by FDG-PET, Infliximab treatment showed an improvement of the SFNSL-score in 47% of patients. In this subgroup we found a significant decrease of sarcoidosis inflammation measured by FDG-PET. In patients without improvement of SFNSL, the SUVmax did not improve clinically relevant. These data could open a discussion about whether persistent inflammation in sarcoidosis can be detrimental to symptoms of SFN.


Effects of Infliximab on small fiber neuropathy symptoms in sarcoidosis patients


Allergen-specific subcutaneous immunotherapy (SCIT) is a treatment for allergic airway disease that depends on regulatory T cell (Tregs) induction by dendritic cells (DCs) to achieve long-term tolerance. Linkage of specific sialic acid residues to antigens (sialylation) enhances their binding to immune inhibitory Siglec receptors on DCs, instructing a tolerogenic phenotype. Here, we test whether use of sialylated rPhleum pratense 5a (Sia-rPhl p5a), or Sia-Phl p5a- and Phl p1-derived peptides, can enhance efficacy of SCIT in a grass pollen (GP)-driven mouse model of allergic asthma.
CFSE labeled CD4+ T-cell activation by (un)modified peptide-loaded DCs was measured in vitro to select responsive peptides. Next, BALB/cByJ mice were GP sensitized, treated with injections of saline, (un)sialylated rPhl p5a, Phl p1 or Phl p5a-peptides, or GP extract, followed by GP-challenges. We measured immunoglobulins, ear swelling responses (ESR), inflammatory cells and cytokines in broncho-alveolar lavage (BAL) and lung tissue and airway hyperresponsiveness (AHR).
T-cells isolated from GP-sensitized mice showed increased proliferation and higher production of TGF-β1 in response to sia-peptide-loaded DCs in vitro, as compared to their unsialylated controls. In vivo, SCIT using GP and rPhl p5a effectively suppressed AHR and airway inflammation. Sia-rPhl p5a SCIT significantly enhanced suppression of ESR and AHR as well as IL-5 and IL-13 levels and eosinophilia in BALF compared to unmodified rPhl p5a. In addition, sialylation of Phl p5a-peptides, but not of Phl p1a-peptides, enhanced suppression of ESR and AHR.
Sialylation of rPhl p5a (full protein or peptides) leads to superior suppression of allergic inflammation, while Phl p1-peptides were ineffective, also after sialylation. Use of the recombinant full protein induces neutralizing antibodies, which was not enhanced by sialylation. SCIT with Sia-rPhl p5a and or peptides is safe in our experimental mouse model. Glycan-allergen conjugates have the potential to be developed as novel improved SCIT treatment.


Glycan modification of rPhl p5a leads to superior suppression of allergic inflammation in a SCIT mouse model of allergic asthma

Spirometry is essential in the diagnosis of airway disease and can be useful in monitoring patients. Despite the essential role, spirometry remains largely underused in primary care. Currently, due to COVID-19, the use of office spirometry is contraindicated in many countries. Furthermore, post COVID-19 spirometry in general practices can be optimalized. Spirometric devices are costly and personnel requires special training. Referral for spirometry increases the cost for patients and lowers the feasibility. Spirometry takes up significant amounts of time that could be used for extra information gathering via more extensive conversation and questionnaires. Spirometry in general practices can be a problem for airway disease that is presenting in specific situations, such as exercise-induced asthma. In recent years, handheld spirometry linked to phones/apps has been developed for study purposes and remote monitoring. 

In this implementation study, we will evaluate whether a spirometry test performed at home with NuvoAir would be a feasible and good alternative to spirometry performed at the general practice office.


Home spirometry for the diagnosis and monitoring of asthma and COPD in primary care: an implementation study

Aim
We aimed to identify facilitators and barriers of adherence to multimorbid exacerbation action plans in patients with COPD and comorbidities.

Methods
Individual semi-structured interviews on patients’ experiences with use of multimorbid symptom diaries and exacerbation action plans in a self-management trial (the COPE-III study) were conducted with patients with moderate to severe COPD and ≥1 comorbidity. Interview data were thematically analysed using the Capability, Opportunity and Motivation-B model to understand behaviour regarding patient adherence to multimorbid exacerbation action plans. 

Results
Ten patients (5 Australian, 5 Dutch; 6 men, age 59-83 years) were interviewed (Table 1). Patients indicated several distinctive perspectives on their own role and healthcare providers’ roles towards disease management (Table 2). Facilitators of adherence included the availability of social support, taking responsibility for disease management, and improved disease comprehension. Barriers to adherence included aversion against daily symptom monitoring and medication use, complexity of symptom diaries and action plans, and difficulties to recognise increased symptoms and to differentiate between COPD symptoms and comorbid symptoms.

Conclusion
To optimise adherence to multimorbid exacerbation action plans, self-management training should be tailored to patients’ level of understanding, directed towards comprehension of COPD, comorbidities and medication, symptom recognition and differentiation between COPD and comorbid symptoms, and focus on individual patients’ roles towards disease management to enhance patients’ responsibility. Our results also highlight the importance of implementation of effective social support strategies in COPD self-management interventions to further improve adherence.


Facilitators and barriers of adherence to exacerbation action plans in patients with COPD and comorbidities: a qualitative study

Background: IL11 was recently reported to induce the formation of myofibroblasts and their secretion of collagen, and its subcutaneous administration drives the development of pulmonary fibrosis in mice. While the role of IL11 on myofibroblast differentiation is described, less is known as to how IL11 affects epithelial-mesenchymal communication associated with epithelial repair. Furthermore, it is not yet well-established which cell types are the main producers and responders to IL11 in vivo.

Here we aim to investigate the role of IL11 in alveolar regeneration.

Methods: Epithelial progenitor cell behavior was studied using an organoid model, comprised of a co-culture of primary mouse CD31-CD45-Epcam+ cells with either CCL206 fibroblasts or primary human fibroblasts. After 14 days of growth, the number and size of the organoids were determined. Additionally, immunofluorescent staining for Surfactant protein C (SPC) and acetylated tubulin (ACT) was performed to define alveolar vs airway organoids. Immunohistochemical staining of IL11 and IL11 receptor was performed on human lung tissue to observe producer and responder cells of IL11 signaling.

Results: The presence of rhIL11 significantly decreased the number of both mouse and human organoids formed in a concentration-dependent manner. Additionally, the fraction of SPC expressing organoids was concentration-dependently reduced by rhIL11 whereas the fraction of SPC-ACT- organoids was increased. Organoid size was not affected by rhIL11. Finally, immunohistochemistry revealed that IL11R is found in the smooth muscle, airway epithelium, endothelium and macrophages, whereas IL11 was primarily observed in the airway epithelium, alveolar type 2 cells and macrophages.

Conclusions: IL11 may impair epithelial progenitor function by suppressing their activation and by suppressing the formation of mature alveolar cell types. IL11R localization suggests mesenchymal activation by IL11 may contribute to the observed effects on progenitor function, possibly through mesenchyme-epithelial crosstalk.

IL11 negatively impacts adult lung alveolar organoid formation

Nasal high-flow therapy (NHFT) appears to be a suitable treatment in COVID-19 patients, but healthcare professionals are reluctant to use the therapy on the ward. This observational study included 86 COVID-19 patients in whom NHFT was initiated on the ward. As a control group, 43 patients were retrospectively included. In patients with a restricted medical policy, 51% of the patients in the NHFT group recovered compared to 20% in the control group. In patients with a full medical policy, ICU admission was 71% in the NHFT group compared to 46% in the control group. Overall, NHFT did not lead to increased or delayed intubations. Furthermore, the overall mortality rate and the mortality rate after intubation is similar in both groups (control 15%, NHFT 17%). The study shows that NHFT on the ward is feasible and effective in both patients with a full medical policy and in patients with a restricted policy.

The effectiveness of nasal high-flow therapy on the COVID-19 ward

Particulate matter is a widespread pollutants, consisting of a complex mixture of organic and inorganic substances. Epidemiological and experimental exposure studies have clearly associated chronic exposure of airborne particulate matter (PM) with increased respiratory diseases. Although several in vitro studies have been performed to elucidate toxicological effect of particulate matter on airway epithelial cells, the majority of studies do not represent the correlation and applicability to realistic in vivo systems. In the current study, we developed and implemented an in vitro model to assess the chronic PM exposure with lower concentrations in order to provide more realistic exposure condition and illuminate the implications of chronic PM exposure. This model was further used to study how chronic PM exposure might predispose to the development of allergy or allergic reactions. Human bronchial epithelial cells 16HBE cells were exposed to increasing concentrations of diesel exhaust particles (12.5-800 µg/ml) for 96h followed by 24h exposure to house dust mite. Afterwards, effect on cell cytotoxicity and barrier integrity were investigated. When 16HBE cells were exposed to 12.5 µg/ml DEP, we determined that chronically exposed cells showed an augmented stress and modified functionality in comparison to acute exposed cells. The current study shows that chronic exposure to low concentrations of PM (12.5 ug/ml) leads to loss of airway barrier integrity compared to 24h exposure. Moreover, in this study we showed that pre-exposure to DEP (12.5 and 25µg/ml) aggravated the HDM-induced loss of TEER and an increased flux of LY to the basolateral compartment compared to HDM or DEP alone. Pre-exposure to DEP additionally increased increased HDM-induced IL-6 and CCL-22. In summary, in the current study we provided for the first an in vitro chronic model which might be helpful for further understanding of the interplay between allergens and chronic PM exposure.

Repeated exposure of bronchial epithelial cells to particular matter increases allergen-induced cytokine release and permeability 

Human lung function is intricately linked to the mechanics of breathing, but how breathing mechanics influence lung epithelial cell functioning is incompletely understood. While respiration-related strains and fluid flows have been suggested to promote alveolar epithelial cell function, the study of such fundamental mechanisms in the airway epithelium has been hindered by the lack of suitable in vitro airway models.

Here, we developed a well-differentiated primary cell culture model of the human airway epithelium on a commercial organs-on-chips platform that enables the application of breathing-associated airflow and cyclic strain, and features optional endothelial cell co-culture to allow for crosstalk with the vascular compartment.

Using this model, we evaluated the impact of airflow and physiological levels of cyclic strain on airway epithelial cell differentiation and function. Our findings suggest that breathing-associated mechanical stimulation changes epithelial cell type composition, reduces secretion of IL-8, and downregulates gene expression of matrix metalloproteinase 9, fibronectin, and other extracellular matrix (remodeling) factors.

These results indicate that breathing-associated forces might be important modulators of airway epithelial cell composition and function and that their fine-tuned application could generate models of specific epithelial phenotypes and pathologies. 


Breathing on Chip: studying the effect of biomechanical forces on airway epithelial cell biology using a modular Airway Lung-Chip

Background: To complement telephone or video consultations during the ongoing COVID-19 pandemic we provided our children with cystic fibrosis (CF) a smartphone connected home spirometer (Spirobank® Smart (Medical International Research, Roma, Italy)). During lockdown, or when children cannot attend the hospital, they perform a pulmonary function test (PFT) guided by the lung function technician via a video connection, before the doctor’s e-consult. Via the smartphone app (version: 1.1) a pdf of the results is sent to the hospital. The children brought the Spirobank with them at their first live hospital visit to get extra training and instructions.
Aim: To test the reliability of spirometry results with the home spirometer Spirobank compared to conventional PFT in the hospital.
Methods: Children with CF (n=16) with median age 11.5 yrs. (IQR: 5) and a 50/50 male/ female ratio were included. At their live visit children performed conventional PFT (MasterScreen™ PFT (Vyaire medical, Würzburg, Germany)) and subsequently Spirobank spirometry guided by the lung function technician, with the same instruction as during video guided consultations. Wilcoxon signed-rank test was used to test the differences between percent predicted (pp)FVC, ppFEV1 and ppMFEF25-75 of both measurements.
Results: Median (IQR) of ΔppFVC, ΔppFEV1, and ΔppMFEF25-75 were 2% (6.25), 2.5% (4.25) and 4% (4.75) respectively. ppFEV1 of Spirobank was significantly lower compared to PFT (p = 0.049), whereas ppFVC (p = 0.73) and ppMFEF25-75 (p= 0.10) were not.
Conclusions: We showed a statistically significant lower ppFEV1 measured by the home spirometer Spirobank compared to conventional PFT, however with a median difference of 2% this was in most cases not a clinically significant difference. We conclude that the Spirobank is a useful home spirometer with reliable results that are slightly lower compared to conventional PFT, when it is performed under (video)guidance of a lung function technician.

Funded by: Sophia foundation & Yaren Foundation

Reliability of a home spirometer for monitoring children with cystic fibrosis during the COVID-19 pandemic.

Introduction: Adult-onset asthma (AOA) is usually more severe compared to childhood onset asthma (CoA). Given the increasing evidence that AoA is associated with obesity, we investigated the relationship of other related metabolic comorbid conditions with AoA compared to CoA. 
Study design and methods: This cross-sectional study compared the metabolic syndrome and lipid derived inflammatory markers in patients with AoA, CoA and age- and sex-matched control subjects without asthma. Participants were asthma patients visiting the outpatient clinic of two teaching hospitals in Rotterdam, The Netherlands. All participants underwent lung function tests, blood tests and physical activity tracking. AoA was defined as asthma age of onset after the age of 18. Metabolic syndrome was defined according to the international joint interim statement criteria.
Results: Eighty-one participants were included (27 AoA, 25 CoA, 29 controls). AoA was associated with the metabolic syndrome (Odds Ratio=4.04 95% CI (1.04-15.68) p=0.028, Nagelkerke R2=0.268), adjusted for age, sex, body mass index and smoking habits. AoA patients had higher median serum IL-6 and leptin-adiponectin (LA) ratio compared to controls (IL-6 (pg/mL): 3.10 [1.11-4.30] vs. 1.13 [0.72-1.58], p=0.002 and LA ratio (pg/mL): 6.21 [2.45-14.11] vs. 2.24 [0.67-4.71], p=0.0390). This was not observed in CoA and controls. 
Conclusion: AoA was associated with the metabolic syndrome and its related pro-inflammatory endocrine and cytokine status. This may suggest lipid inflammatory markers play a role in the pathophysiology of AoA.

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

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Effects of Infliximab on small fiber neuropathy symptoms in sarcoidosis patients

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