Μηχανισμοί ανοσολογικής ρύθμισης του φλεγμονοσώματος σε ασθενείς με αλλεργικό άσθμα και σε πειραμαματικά μοντέλα

Εισαγωγή: Οι φλεγμονώδεις αποκρίσεις που προέρχονται απο την ενεργοποίηση του NLRP3 στα μονοκύτταρα του αίματος και στα μονοκύτταρα του πνεύμονα εμπλέκονται στην παθογένεια του άσθματος. Η αυτοφαγία είναι βασικός ρυθμιστής των φλεγμονωδών αποκρίσεων και ειδικότερα, αποτελεί έναν από τους πιο σημαντικούς μηχανισμούς ρύθμισης του φλεγμονοσώματος NLRP3. Ωστόσο, οι επιδράσεις της αυτοφαγίας και του κύριου ρυθμιστή της, του μεταγραφικού παράγοντα ΕΒ (TFEB), στα μονοκύτταρα ασθενών με Σοβαρό άσθμα (ΣΑ) παραμένει άγνωστη. Στη παρούσα εργασία, διερευνήσαμε εάν η ενεργοποίηση της αυτοφαγίας και του μεταγραφικού παράγοντα TFEB μπορούν να καταστέλλουν τις φλεγμονώδεις αποκρίσεις των μονοκυττάρων σε ασθενείς με άσθμα. Μέθοδοι: Απομονώσαμε CD14+ μονοκύτταρα από το περιφερικό αίμα ασθενών με άσθμα (ν=83) και υγιών δοτών (ν=46) και τα ενεργοποιήσαμε με λιποπολυσακχαρίτη και ΑΤΡ για να ενεργοποιήσουμε το φλεγμονόσωμα NLRP3 παρουσία ή όχι του επαγωγέα αυτοφαγίας, ραπαμυκίνη. Εξετάστηκαν ο σχηματισμός ASC speck, η ενεργοποίηση κασπάσης-1, τα επίπεδα IL-1β και IL-18, η μιτοχονδριακή λειτουργία, η απελευθέρωση ελευθέρων ριζών και η σηματοδότηση mTORC1. Η αυτοφαγία αξιολογήθηκε μετρώντας τον σχηματισμό LC3 puncta, με την αποικοδόμηση της πρωτεΐνης p62/SQSTM1 και την ενεργοποίηση του TFEB. Χρησιμοποιώντας ένα πειραματικό μοντέλο σοβαρού άσθματος, διερευνήσαμε τον ρόλο της σηματοδότησης του NLRP3 χρησιμοποιώντας Nlrp3−/− ποντίκια και/ή χορήγηση MCC950 και τον ρόλο της ενεργοποίησης TFEB χρησιμοποιώντας ποντίκια που υπερεκφράζουν TFEB στα κύτταρα μυελικής σειράς ή μέσω χορήγηση τρεαλόζης. Αποτελέσματα: Παρατηρήσαμε αυξημένη ενεργοποίηση του φλεγμονοσώματος NLRP3, με ταυτόχρονη μειωμένη αυτοφαγία στα μονοκύτταρα περιφερικού αίματος, αποτελέσματα τα αποία συσχετίστηκαν με παραμέτρους της σοβαρότητας του άσθματος. Οι ασθενείς με ΣΑ εμφάνισαν επίσης, μιτοχονδριακή δυσλειτουργία και συσσώρευση ελευθέρων ριζών. Η αυτοφαγία απέτυχε να αναστείλει τις φλεγμονώδεις αποκρίσεις των μονοκυττάρων που προέρχονται από το NLRP3, λόγω ελαττωματικής ενεργοποίησης του TFEB και της υπερβολικής σηματοδότησης mTORC1. Η καταστολή του NLRP3 φλεγμονοσώματος μείωσε την απελευθέρωση φλεγμονωδών κυρατοκινών και περιόρισε την φλεγμονή στους αεραγωγούς. Η ενεργοποίηση του TFEB αποκατέστησε την ελλατωματική αυτοφαγία, μείωσε την πνευμονική φλεγμονή και βελτίωσε τον φαινότυπο SA. Συμπεράσματα: Οι μελέτες μας αποκαλύπτουν οτι η ενεργοποίηση του μεταγραφικού παράγοντα TFEB στα μονοκύτταρα ασθενών με ΣΑ είναι σημαντική, θέτοντας τον ως ένα νεό θεραπευτικό στόχο, για τη διαχείριση της ΣΑ.Background Nucleotide oligomerization domain-like receptor 3 (NLRP3)-driven inflammatory responses by circulating and lung-resident monocytes are critical drivers of asthma pathogenesis. Autophagy restrains NLRP3-induced monocyte activation in asthma models. Yet, the effects of autophagy and its master regulator, transcription factor EB (TFEB), on monocyte responses in human asthma remain unexplored. Here, we investigated whether activation of autophagy and TFEB signaling suppress inflammatory monocyte responses in asthmatic individuals. Methods Peripheral blood CD14+ monocytes from asthmatic patients (n = 83) and healthy controls (n = 46) were stimulated with LPS/ATP to induce NLRP3 activation with or without the au-tophagy inducer, rapamycin. ASC specks, caspase-1 activation, IL-1β and IL-18 levels, mito-chondrial function, ROS release, and mTORC1 signaling were examined. Autophagy was eval-uated by LC3 puncta formation, p62/SQSTM1 degradation and TFEB activation. In a severe asthma (SA) model, we investigated the role of NLRP3 signaling using Nlrp3−/− mice and/or MCC950 administration, and the effects of TFEB activation using myeloid-specific TFEB-overexpressing mice or administration of the TFEB activator, trehalose. Results We observed increased NLRP3 inflammasome activation, concomitant with impaired autopha-gy in circulating monocytes that correlated with asthma severity. SA patients also exhibited mitochondrial dysfunction and ROS accumulation. Autophagy failed to inhibit NLRP3-driven monocyte responses, due to defective TFEB activation and excessive mTORC1 signaling. NLRP3 blockade restrained inflammatory cytokine release and linked airway disease. TFEB activation restored impaired autophagy, attenuated NLRP3-driven pulmonary inflammation, and ameliorated SA phenotype. Conclusions Our studies uncover a crucial role for TFEB-mediated reprogramming of monocyte inflamma-tory responses, raising the prospect that this pathway can be therapeutically harnessed for the management of SA

Autophagy: A Friend or Foe in Allergic Asthma?

Autophagy is a major self-degradative process through which cytoplasmic material, including damaged organelles and proteins, are delivered and degraded in the lysosome. Autophagy represents a dynamic recycling system that produces new building blocks and energy, essential for cellular renovation, physiology, and homeostasis. Principal autophagy triggers include starvation, pathogens, and stress. Autophagy plays also a pivotal role in immune response regulation, including immune cell differentiation, antigen presentation and the generation of T effector responses, the development of protective immunity against pathogens, and the coordination of immunometabolic signals. A plethora of studies propose that both impaired and overactive autophagic processes contribute to the pathogenesis of human disorders, including infections, cancer, atherosclerosis, autoimmune and neurodegenerative diseases. Autophagy has been also implicated in the development and progression of allergen-driven airway inflammation and remodeling. Here, we provide an overview of recent studies pertinent to the biology of autophagy and molecular pathways controlling its activation, we discuss autophagy-mediated beneficial and detrimental effects in animal models of allergic diseases and illuminate new advances on the role of autophagy in the pathogenesis of human asthma. We conclude contemplating the potential of targeting autophagy as a novel therapeutic approach for the management of allergic responses and linked asthmatic disease

Severe Asthmatic Responses: The Impact of TSLP

Asthma is a chronic inflammatory disease that affects the lower respiratory system and includes several categories of patients with varying features or phenotypes. Patients with severe asthma (SA) represent a group of asthmatics that are poorly responsive to medium-to-high doses of inhaled corticosteroids and additional controllers, thus leading in some cases to life-threatening disease exacerbations. To elaborate on SA heterogeneity, the concept of asthma endotypes has been developed, with the latter being characterized as T2-high or low, depending on the type of inflammation implicated in disease pathogenesis. As SA patients exhibit curtailed responses to standard-of-care treatment, biologic therapies are prescribed as adjunctive treatments. To date, several biologics that target specific downstream effector molecules involved in disease pathophysiology have displayed superior efficacy only in patients with T2-high, eosinophilic inflammation, suggesting that upstream mediators of the inflammatory cascade could constitute an attractive therapeutic approach for difficult-to-treat asthma. One such appealing therapeutic target is thymic stromal lymphopoietin (TSLP), an epithelial-derived cytokine with critical functions in allergic diseases, including asthma. Numerous studies in both humans and mice have provided major insights pertinent to the role of TSLP in the initiation and propagation of asthmatic responses. Undoubtedly, the magnitude of TSLP in asthma pathogenesis is highlighted by the fact that the FDA recently approved tezepelumab (Tezspire), a human monoclonal antibody that targets TSLP, for SA treatment. Nevertheless, further research focusing on the biology and mode of function of TSLP in SA will considerably advance disease management

Targeting NLRP3 Inflammasome Activation in Severe Asthma

Severe asthma (SA) is a chronic lung disease characterized by recurring symptoms of reversible airflow obstruction, airway hyper-responsiveness (AHR), and inflammation that is resistant to currently employed treatments. The nucleotide-binding oligomerization domain-like Receptor Family Pyrin Domain Containing 3 (NLRP3) inflammasome is an intracellular sensor that detects microbial motifs and endogenous danger signals and represents a key component of innate immune responses in the airways. Assembly of the NLRP3 inflammasome leads to caspase 1-dependent release of the pro-inflammatory cytokines IL-1β and IL-18 as well as pyroptosis. Accumulating evidence proposes that NLRP3 activation is critically involved in asthma pathogenesis. In fact, although NLRP3 facilitates the clearance of pathogens in the airways, persistent NLRP3 activation by inhaled irritants and/or innocuous environmental allergens can lead to overt pulmonary inflammation and exacerbation of asthma manifestations. Notably, administration of NLRP3 inhibitors in asthma models restrains AHR and pulmonary inflammation. Here, we provide an overview of the pathophysiology of SA, present molecular mechanisms underlying aberrant inflammatory responses in the airways, summarize recent studies pertinent to the biology and functions of NLRP3, and discuss the role of NLRP3 in the pathogenesis of asthma. Finally, we contemplate the potential of targeting NLRP3 as a novel therapeutic approach for the management of SA