Organoids are self‐organizing 3D structures grown from stem cells that recapitulate essential aspects of organ structure and function. Here, we describe a method to establish long‐term‐expanding human airway organoids from broncho‐alveolar resections or lavage material. The pseudostratified airway organoids consist of basal cells, functional multi‐ciliated cells, mucus‐producing secretory cells, and CC10‐secreting club cells. Airway organoids derived from cystic fibrosis (CF) patients allow assessment of CFTR function in an organoid swelling assay. Organoids established from lung cancer resections and metastasis biopsies retain tumor histopathology as well as cancer gene mutations and are amenable to drug screening. Respiratory syncytial virus (RSV) infection recapitulates central disease features, dramatically increases organoid cell motility via the non‐structural viral NS2 protein, and preferentially recruits neutrophils upon co‐culturing. We conclude that human airway organoids represent versatile models for the in vitro study of hereditary, malignant, and infectious pulmonary disease.

Excessive release of neutrophil extracellular traps (NETs) is associated with disease severity and contributes to tissue injury, followed by severe organ damage. Pharmacological or genetic inhibition of NET release reduces pathology in multiple inflammatory disease models, indicating that NETs are potential therapeutic targets. Here, we demonstrate using a preclinical basket approach that our therapeutic anti-citrullinated protein antibody (tACPA) has broad therapeutic potential. Treatment with tACPA prevents disease symptoms in various mouse models with plausible NET-mediated pathology, including inflammatory arthritis (IA), pulmonary fibrosis, inflammatory bowel disease and sepsis. We show that citrulline residues in the N-termini of histones 2A and 4 are specific targets for therapeutic intervention, whereas antibodies against other N-terminal post-translational histone modifications have no therapeutic effects. Because citrullinated histones are generated during NET release, we investigated the ability of tACPA to inhibit NET formation. tACPA suppressed NET release from human neutrophils triggered with physiologically relevant human diseaserelated stimuli. Moreover, tACPA diminished NET release and potentially initiated NET uptake by macrophages in vivo, which was associated with reduced tissue damage in the joints of a chronic arthritis mouse model of IA. To our knowledge, we are the first to describe an antibody with NET-inhibiting properties and thereby propose tACPA as a drug candidate for NET-mediated inflammatory diseases, as it eliminates the noxious triggers that lead to continued inflammation and tissue damage in a multidimensional manner.

A wide variety of microbial and inflammatory factors induce DNA release from neutrophils as neutrophil extracellular traps (NETs). Consensus on the kinetics and mechanism of NET release has been hindered by the lack of distinctive methods to specifically quantify NET release in time. Here, we validate and refine a semi-automatic live imaging approach for quantification of NET release. Importantly, our approach is able to correct for neutrophil input and distinguishes NET release from neutrophil death by other means, aspects that are lacking in many NET quantification methods. Real time visualization shows that opsonized S. aureus rapidly induces cell death by toxins, while actual NET formation occurs after 90 minutes, similar to the kinetics of NET release by immune complexes and PMA. Inhibition of SYK, PI3K and mTORC2 attenuates NET release upon challenge with physiological stimuli but not with PMA. In contrast, neutrophils from chronic granulomatous disease patients show decreased NET release only in response to PMA. With this refined method, we conclude that NET release in primary human neutrophils is dependent on the SYK-PI3K-mTORC2 pathway and that PMA stimulation should be regarded as mechanistically distinct from NET formation induced by natural triggers.

Increased NET release is associated with autoimmunity and inflammation in SLE and APS. Inhibition of NET release thus could be of potential benefit in a subset of patients with SLE and APS.