Biology subjects

van der Does, A. M.

Publications and source records attributed to van der Does, A. M..

7 recordsLinked to original sources

bioRxiv2026

Ageing of the Upper Airway Epithelial Niche Limits Tissue-Resident T Cell Immunity

Respiratory tract infections are a major cause of morbidity and mortality among older adults worldwide. Yet, how ageing shapes protective immunity within the upper respiratory tract (URT) remains poorly understood. Here, we profiled the nasal immune landscape of young adults and older adults with and without frailty, using high-dimensional cytometry, proteomics, single-cell transcriptomics and T cell receptor (TCR) sequencing. Ageing was associated with a selective reduction of tissue-resident memory T (Trm) cells, independent of effector T cell numbers, inflammageing levels or frailty and was accompanied by a decline in T cell receptor repertoire stability. Trm cells from older adults also showed decreased steady-state IFN{gamma} expression, accompanied by reduced antiviral transcriptional programs in the mucosa. Mechanistically, in vitro PBMC-epithelium co-culture models revealed that older adults have a reduced capacity for Trm differentiation, which was associated with a diminished epithelial TGF-{beta} secretion, driven by reduced expression in ciliated epithelial cells. Together, these findings reveal that URT immunity deteriorates with age due to disrupted epithelial-immune crosstalk. This identifies the epithelial niche as a key regulator of mucosal immune ageing and suggests that enhancing TGF{beta} signaling may improve the effectiveness of mucosal airway vaccination strategies in older populations.

immunology↗

bioRxiv2025

Modelling cigarette smoke-induced lung vascular dysfunction using an alveolus-on-chip

The alveolus is central to gas exchange in the lung, and alveolar damage is a characteristics of a variety of lung diseases. Understanding alveolar microvascular dynamics and epithelial-endothelial interactions is essential for accurately modeling alveolar physiology and its dysfunction in lung diseases such as Chronic Obstructive Pulmonary Disease (COPD), pulmonary fibrosis and acute respiratory distress syndrome (ARDS). In this study, we present an open-top, membrane-free alveolus-on-chip platform incorporating self-assembled, perfusable 3D vascular networks by primary human lung endothelial cells and pericytes, co-cultured with alveolar epithelial type 2 (AEC2) cells. These vascular networks were developed within 6 days under continuous flow and remained stable for at least 12 days. The inclusion of pericytes supported capillary-like vessel formation and increased gene expression of EDNRB1, a gene enriched in alveolar microvascular endothelial cells. Furthermore, CD31 gene expression was higher in 3D endothelial networks compared to 2D endothelial monolayers, suggesting increased cell-cell adhesion. Monocytes could be successfully perfused through the networks, expanding the platforms potential for studying immune interactions in lung disorders. Culturing AEC2 monolayers directly on the vascularized hydrogel enabled physiologically relevant cell-cell interactions without artificial membranes, while maintaining air-liquid interface conditions. Importantly, exposure of the AEC2 layer to whole cigarette smoke (WCS) led to complete disintegration of the underlying vascular network, an effect not observed in the absence of AEC2. This chip model provides a human-relevant system for investigating vascular-epithelial crosstalk in the alveolus, smoke-induced lung injury, and immune recruitment, offering a valuable platform for future disease modeling and drug testing applications.

bioengineering↗

bioRxiv2025

LL-37 and citrullinated-LL-37 enhance disparate oxylipins: LL-37-mediated chemokine response is dependent on COX-2 and the P2X7 receptor in human bronchial epithelial cells

BackgroundDuring airway inflammation, chemokines, oxylipins (bioactive lipids) and cationic host defence peptides (CHDP) are enhanced in the lungs. However, the interplay of these molecules in the process of airway inflammation is not fully resolved. The human cathelicidin CHDP, LL-37, can enhance the expression of chemokines which is turn facilitates influx of leukocytes into the lungs. Moreover, LL-37 can get citrullinated during inflammation and the effect of this post-translational modification on LL-37-mediated immunomodulatory functions remains unclear. Therefore, in this study we aimed to define the impact of LL-37 and citrullinated-LL-37 (citLL-37) on oxylipins and its association with downstream chemokine production in human bronchial epithelial cells (HBEC), and its functional impact on leukocyte influx. MethodsWe used a lipidomics approach to identify oxylipins that are enhanced in response to LL-37 and citLL-37 in HBEC. We further examined the role of selected oxylipins in LL-37- and citLL-37-mediated chemokine production by ELISA, and related leukocyte migration using a transwell migration assay. ResultsWe showed that LL-37, but not citLL-37, enhances oxylipins that are known to promote inflammation such as prostaglandins regulated by the cyclooxygenase (COX pathway). Although both LL-37 and citLL-37 upregulated COX-2, LL-37-mediated increase in COX-2 expression was significantly higher than that mediated by citLL-37. We showed that upregulation of COX-2 expression was dependent on the P2X7 purinergic receptor. Our mechanistic studies revealed that LL-37-mediated increase in chemokines, GRO, IL-8 and MIP-3, was dependent on the COX-2 pathway. Our results also indicated that COX-2-induced PGE2 may act in an autocrine manner signaling through its EP receptors to facilitate LL-37-induced chemokine production. We functionally confirmed that factors secreted from HBEC in response to LL-37, but not citLL-37, promotes neutrophil migration which is COX-2 dependent. ConclusionThe results of this study indicate that pro-inflammatory responses mediated by LL-37 is alleviated by citrullination of the peptide. These findings suggest that citrullination of LL-37 may be a post-translational regulatory mechanism to control inflammation. Overall, this study underscores the role of LL-37 in influencing the enhancement of bioactive lipids and metabolic pathways such as COX-2 and its link to the peptide-mediated immunomodulatory functions in the lungs.

immunology↗

bioRxiv2024

Cigarette smoke induces PPP1R15A via oxidative stress to modulate inflammatory cytokine production by bronchial epithelial cells

Cigarette smoke is the principal cause of chronic obstructive pulmonary disease (COPD) in western societies. It induces pulmonary inflammation and activates multiple stress signaling pathways in bronchial epithelial cells, including the integrated stress response (ISR). The ISR is triggered by phosphorylation of the translation initiation factor eIF2 in response to a variety of insults and is functionally antagonised by the eIF2 phosphatase subunit PPP1R15A. Deletion of PPP1R15A exaggerates the ISR and in many models protects against a cellular stress. However, loss of PPP1R15A was recently shown to exaggerate pulmonary fibrosis in bleomycin-challenged mice. We therefore wished to understand the role of PPP1R15A in the pulmonary response to cigarette smoke, which can cause both COPD and pulmonary fibrosis. Whole cigarette smoke rapidly activated the ISR in mucociliary differentiated human primary bronchial epithelial cells with those from COPD patients exhibiting higher levels of ISR. Acute exposure to cigarette smoke condensate induced a pro-inflammatory cytokine response accompanied by PPP1R15A expression. In this model, PPP1R15A induction was independent of eIF2 phosphorylation but was suppressed by antioxidants suggesting a response to oxidative stress. In vivo studies in mice revealed that PPP1R15A deficiency enhanced the secretion of proinflammatory cytokines in mice, suggesting a regulatory role for PPP1R15A in modulating the inflammatory response to smoke. Taken together, these data suggest that PPP1R15A is induced by cigarette smoke by an ISR-independent oxidative stress mechanism and ameliorates the pro-inflammatory cytokine response to cigarette smoke.

cell biology↗

bioRxiv2024

LL-37 modulates IL-17A/F-mediated airway inflammation by selectively suppressing Lipocalin-2.

BackgroundLevels of the human cationic host defence peptide (CHDP) LL-37 is enhanced in the lungs during neutrophilic airway inflammation. LL-37 drives Th17 differentiation, and Th17 cells produce IL-17A and IL-17F which forms the biologically active heterodimer IL-17A/F. While IL-17 is a critical mediator of neutrophilic airway inflammation, LL-37 exhibits contradictory functions; LL-37 can both promote and mitigate neutrophil recruitment depending on the inflammatory milieu. The impact of LL-37 on IL-17-induced responses in the context of airway inflammation remains largely unknown. Therefore, we examined signaling intermediates and downstream responses mediated by the interplay of IL-17A/F and LL-37, in human bronchial epithelial cells (HBEC). As LL-37 can get citrullinated during airway inflammation, we also examined LL-37-mediated downstream response compared to that with citrullinated LL-37 (citLL- 37), in HBEC. ResultsUsing an aptamer-based proteomics approach, we identified proteins that are altered in response to IL-17A/F in HBEC. Proteins enhanced in response to IL-17A/F were primarily neutrophil chemoattractants, including chemokines and proteins associated with neutrophil migration such as lipocalin-2 (LCN-2) and Elafin. We showed that selective depletion of LCN-2 mitigated neutrophil migration, thus functionally demonstrating LCN-2 as a critical neutrophil chemoattractant. We further demonstrated that LL-37 and citLL-37 selectively suppresses IL- 17A/F-induced LCN-2 production, in bronchial epithelial cells. Mechanistic studies revealed that LL-37 and citLL-37 suppressed IL-17A/F-mediated C/EBP{beta}, a transcription factor required for LCN-2 production. In contrast, LL-37 and citLL-37 enhanced the ribonuclease Regnase-1, which is a negative regulator of IL-17 and LCN-2. In an animal model of neutrophilic airway inflammation with elevated IL-17A/F in the lungs, we demonstrated that CRAMP (mouse ortholog of LL-37) negatively correlates with LCN-2. ConclusionsOverall, our findings showed that LL-37 and citLL-37 can selectively suppress the abundance of IL-17A/F-mediated LCN-2, a protein that is critical for neutrophil migration, in bronchial epithelial cells. These results suggest that LL-37, and its modified citrullinated form, has the potential to negatively regulate IL-17-mediated neutrophil migration to control airway inflammation. To our knowledge, this is the first study to report that the immunomodulatory function of LL-37 engages an RNA binding protein, Regnase-1, indicating post-transcriptional regulation of airway inflammation by the peptide.

immunology↗

bioRxiv2021

Impact of human airway epithelial cellular composition on SARS-CoV-2 infection biology

Infection biology and pathogenesis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the coronavirus disease 2019 (COVID-19), are incompletely understood. Here, we assessed the impact of airway epithelial cellular composition on infection in air-liquid interface (ALI) cultures of differentiated primary human tracheal (PTEC) and bronchial epithelial cells (PBEC). We first compared SARS-CoV-2 infection kinetics, related antiviral and inflammatory responses, and viral entry factors in PTEC and PBEC. Next, the contribution of differentiation time was investigated by differentiating ALI-PTEC/PBEC for 3-5 weeks and comparing dynamics of viral replication/spread, cellular composition and epithelial responses. We observed a gradual increase in viral load with prolonged culture duration. Ciliated and goblet cells were predominantly infected in both PTEC and PBEC. Immunofluorescence analysis and RT-qPCR showed that compared to other cell types mainly ciliated and goblet cell numbers were affected by increased culture duration. An increased proportion of these two target cell types was associated with increased viral load. Furthermore, modulation of cellular composition using IL-13 and the Notch signaling inhibitor DAPT, underlined the importance of both ciliated and goblet cells for infection. DAPT treatment resulted in a lower viral load and a relative increase in ciliated cells at the expense of goblet cells, compared to IL-13 treated cultures in which both cell types were present and viral load was higher. In conclusion, our results identify cellular composition as a contributing factor to airway epithelial susceptibility to SARS-CoV-2. IMPORTANCEIn this study, we determined an effect of culture duration and airway cellular composition of ALI-PBEC and ALI-PTEC cultures on SARS-CoV-2 infection. We found that SARS-CoV-2 infection was increased with prolonged cell culture time and the total percentage and proportion of ciliated and goblet cells played an important role in infection level, suggesting that airway epithelial differentiation/maturation levels may in part determine susceptibility of SARS-CoV-2 infection. The development of effective therapies either targeting virus replication or pathogenesis against SARS-CoV-2 requires robust cell culture-based infection models to test small molecules and biologicals. Therefore, it is important to identify factors that are essential for reliably modeling SARS-CoV-2-airway epithelial cell interactions. This study sheds light on virus-airway epithelial cell interactions and adds to the complexity of SARS-CoV-2 cell tropism in the airways. In addition, the effect of IL-13 on viral infection hints at a causal connection between SARS-CoV-2 infection and (allergic) asthma.

cell biology↗

bioRxiv2021

Breathing on Chip: Biomechanical forces change airway epithelial cell biology ina human Airway Lung-Chip

Human lung function is intricately linked to blood flow and breathing cycles, but it remains unknown how these dynamic cues shape human airway epithelial biology. Here we report a state-of-the-art protocol for studying effects of physiological airflow and stretch on differentiation, cellular composition, and mucociliary clearance of human primary airway epithelial cells cultured on a perfused airway chip. Perfused epithelial tissue cultures developed a large airway-like cellular composition with accelerated maturation and polarization of mucociliary clearance when compared to traditional (static) culture methods. Additional application of airflow and stretch to the airway chip resulted in a cellular composition more comparable to the small(er) airways, reduced baseline secretion of interleukin-8 and other inflammatory proteins, and reduced gene expression of matrix metalloproteinase (MMP) 9, fibronectin, and other extracellular matrix factors. These results indicate that breathing-like mechanical stimuli are important modulators of airway epithelial cell differentiation and homeostasis and that their fine-tuned application could generate models of specific epithelial regions, pathologies, and mucociliary (dys)function.

cell biology↗