Generation of IL-23 Producing Dendritic Cells (DCs) by Airborne Fungi Regulates Fungal Pathogenicity via the Induction of TH-17 Responses

Interleukin-17 (IL-17) producing T helper cells (TH-17) comprise a newly recognized T cell subset with an emerging role in adaptive immunity to a variety of fungi. Whether different airborne fungi trigger a common signaling pathway for TH-17 induction, and whether this ability is related to the inherent pathogenic behavior of each fungus is currently unknown. Here we show that, as opposed to primary pathogenic fungi (Histoplasma capsulatum), opportunistic fungal pathogens (Aspergillus and Rhizopus) trigger a common innate sensing pathway in human dendritic cells (DCs) that results in robust production of IL-23 and drives TH-17 responses. This response requires activation of dectin-1 by the fungal cell wall polysaccharide b-glucan that is selectively exposed during the invasive growth of opportunistic fungi. Notably, unmasking of b-glucan in the cell wall of a mutant of Histoplasma not only abrogates the pathogenicity of this fungus, but also triggers the induction of IL-23 producing DCs. Thus, b-glucan exposure in the fungal cell wall is essential for the induction of IL-23/TH-17 axis and may represent a key factor that regulates protective immunity to opportunistic but not pathogenic fungi

Flotillin-dependent membrane microdomains are required for functional phagolysosomes against fungal infections

© 2020 The Author(s). This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/)Lipid rafts form signaling platforms on biological membranes with incompletely characterized role in immune response to infection. Here we report that lipid-raft microdomains are essential components of phagolysosomal membranes of macrophages and depend on flotillins. Genetic deletion of flotillins demonstrates that the assembly of both major defense complexes vATPase and NADPH oxidase requires membrane microdomains. Furthermore, we describe a virulence mechanism leading to dysregulation of membrane microdomains by melanized wild-type conidia of the important human-pathogenic fungus Aspergillus fumigatus resulting in reduced phagolysosomal acidification. We show that phagolysosomes with ingested melanized conidia contain a reduced amount of free Ca2+ ions and that inhibition of Ca2+-dependent calmodulin activity led to reduced lipid-raft formation. We identify a single-nucleotide polymorphism in the human FLOT1 gene resulting in heightened susceptibility for invasive aspergillosis in hematopoietic stem cell transplant recipients. Collectively, flotillin-dependent microdomains on the phagolysosomal membrane play an essential role in protective antifungal immunity.This work was supported by the International Leibniz Research School ( ILRS ) as part of the excellence graduate school Jena School for Microbial Communication (JSMC) funded by the Deutsche Forschungsgemeinschaft ( DFG ), the Leibniz Science Campus “InfectoOptics,” and the DFG -funded CRC 127 8 “PolyTarget” (project B02 to A.A.B. and Z01 to M.T.F.). C.C. and A.C were supported by the Northern Portugal Regional Operational Programme (NORTE 2020) under the Portugal 2020 Partnership Agreement, through the European Regional Development Fund ( FEDER ) ( NORTE-01-0145-FEDER-000013 ), and by the Fundação para a Ciência e Tecnologia ( FCT ) ( SFRH/BPD/96176/2013 to C.C. and IF/00735/2014 to A.C.).info:eu-repo/semantics/publishedVersio

Uncoupling of IL-6 signaling and LC3-associated phagocytosis drives immunoparalysis during sepsis

Contains fulltext : 238107.pdf (Publisher’s version ) (Closed access)Immune deactivation of phagocytes is a central event in the pathogenesis of sepsis. Herein, we identify a master regulatory role of IL-6 signaling on LC3-associated phagocytosis (LAP) and reveal that uncoupling of these two processes during sepsis induces immunoparalysis in monocytes/macrophages. In particular, we demonstrate that activation of LAP by the human fungal pathogen Aspergillus fumigatus depends on ERK1/2-mediated phosphorylation of p47phox subunit of NADPH oxidase. Physiologically, autocrine IL-6/JAK2/Ninein axis orchestrates microtubule organization and dynamics regulating ERK recruitment to the phagosome and LC3(+) phagosome (LAPosome) formation. In sepsis, loss of IL-6 signaling specifically abrogates microtubule-mediated trafficking of ERK, leading to defective activation of LAP and impaired killing of bacterial and fungal pathogens by monocytes/macrophages, which can be selectively restored by IL-6 supplementation. Our work uncovers a molecular pathway linking IL-6 signaling with LAP and provides insight into the mechanisms underlying immunoparalysis in sepsis

Calcium sequestration by fungal melanin inhibits calcium-calmodulin signalling to prevent LC3-associated phagocytosis

LC3-associated phagocytosis (LAP) is a non-canonical autophagy pathway regulated by Rubicon, with an emerging role in immune homeostasis and antifungal host defence. Aspergillus cell wall melanin protects conidia (spores) from killing by phagocytes and promotes pathogenicity through blocking nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-dependent activation of LAP. However, the signalling regulating LAP upstream of Rubicon and the mechanism of melanin-induced inhibition of this pathway remain incompletely understood. Herein, we identify a Ca2+ signalling pathway that depends on intracellular Ca2+ sources from endoplasmic reticulum, endoplasmic reticulum-phagosome communication, Ca2+ release from phagosome lumen and calmodulin (CaM) recruitment, as a master regulator of Rubicon, the phagocyte NADPH oxidase NOX2 and other molecular components of LAP. Furthermore, we provide genetic evidence for the physiological importance of Ca2+-CaM signalling in aspergillosis. Finally, we demonstrate that Ca2+ sequestration by Aspergillus melanin inside the phagosome abrogates activation of Ca2+-CaM signalling to inhibit LAP. These findings reveal the important role of Ca2+-CaM signalling in antifungal immunity and identify an immunological function of Ca2+ binding by melanin pigments with broad physiological implications beyond fungal disease pathogenesis.Onassis Foundation under the ‘Special Grant and Support Program for Scholars’ Association Members’ (Grant no. R ZM 003-1/2016-2017); G.C. was supported by grants from the Greek State Scholarship Foundation (I.K.Y.), the Hellenic General Secretariat for Research and Technology-Excellence program (ARISTEIA) and a Research Grant from Institut Mérieux; J.P.L. was supported by European Community’s Seventh Framework Programme (FP7/2007-2013) under grant agreement 260338 ALLFUN and ANR-10-BLAN-1309 HYDROPHOBIN, and the Association Vaincre La Mucoviscidose (RF20140501052/1/1/141); H.F. and N.M.N. were supported by the project FROnTHERA (NORTE-01-0145-FEDER-000023), supported by Northern Portugal Regional Operational Programme (NORTE 2020), under the Portugal 2020 Partnership Agreement, through the European Regional Development Fund (ERDF), and by Fundação para a Ciência e Tecnologia (FCT) project SPARTAN (PTDC/CTM-BIO/4388/2014), funded through the PIDDAC Program. A.C. and C.C. were supported by NORTE 2020, under the Portugal 2020 Partnership Agreement, through the ERDF (NORTE-01-0145-FEDER-000013), and by FCT (IF/00735/2014 and SFRH/BPD/96176/2013). G.S.D. and J.L.F. were supported by NIH grant AI-106269. K.J.K-C is supported by the Division of Intramural Research (DIR), NIAID, NIHinfo:eu-repo/semantics/publishedVersio

Self-RNA–antimicrobial peptide complexes activate human dendritic cells through TLR7 and TLR8

Dendritic cell (DC) responses to extracellular self-DNA and self-RNA are prevented by the endosomal seclusion of nucleic acid–recognizing Toll-like receptors (TLRs). In psoriasis, however, plasmacytoid DCs (pDCs) sense self-DNA that is transported to endosomal TLR9 upon forming a complex with the antimicrobial peptide LL37. Whether LL37 also interacts with extracellular self-RNA and how this may contribute to DC activation in psoriasis is not known. Here, we report that LL37 can bind self-RNA released by dying cells, protect it from extracellular degradation, and transport it into endosomal compartments of DCs. In pDC, self-RNA–LL37 complexes activate TLR7 and, like self-DNA–LL37 complexes, trigger the secretion of IFN-α without inducing maturation or the production of IL-6 and TNF-α. In contrast to self-DNA–LL37 complexes, self-RNA–LL37 complexes also trigger the activation of classical myeloid DCs (mDCs). This occurs through TLR8 and leads to the production of TNF-α and IL-6, and the differentiation of mDCs into mature DCs. We also found that self-RNA–LL37 complexes are present in psoriatic skin lesions and are associated with mature mDCs in vivo. Our results demonstrate that the cationic antimicrobial peptide LL37 converts self-RNA into a trigger of TLR7 and TLR8 in human DCs, and provide new insights into the mechanism that drives the auto-inflammatory responses in psoriasis

Phagosomal removal of fungal melanin reprograms macrophage metabolism to promote antifungal immunity

Acknowledgements This work was supported by the Northern Portugal Regional Operational Programme (NORTE 2020), under the Portugal 2020 Partnership Agreement, through the European Regional Development Fund (FEDER) (NORTE-01- 0145-FEDER-000013), the Fundação para a Ciência e Tecnologia (FCT) (SFRH/BD/136814/2018 to S.M.G., SFRH/BD/141127/2018 to C.D.O., PD/BD/137680/2018 to D.A., IF/00474/2014 to N.S.O., IF/01390/2014 to E.T., IF/00959/2014 to S.C., IF/00021/2014 to R.S., PTDC/SAU-SER/29635/2017 and CEECIND/04601/2017 to C.C., and CEECIND/03628/2017 to A.C.), the Institut Mérieux (Mérieux Research Grant 2017 to C.C.), and the European Society of Clinical Microbiology and Infectious Diseases (ESCMID Research Grant 2017 to A.C.). M.G.N. was supported by a Spinoza grant of the Netherlands Organization for Scientific Research. A.A.B. was supported by the Deutsche Forschungsgemeinschaft Collaborative Research Center/Transregio TR124 FungiNet (project A1). G.D.B. was funded by the Wellcome Trust (102705), the MRC Centre for Medical Mycology and the University of Aberdeen (MR/N006364/1).Peer reviewedPublisher PD

Survival and prognostic factors after initiation of treatment in Waldenström's macroglobulinemia

Background: Waldenström's macroglobulinemia (WM) is an unusual lymphoplasmacytoid lymphoma characterized by the presence of a serum monoclonal immunoglobulin M. Although several studies have evaluated possible prognostic factors of this disease, few have focused on the survival and prognosis of symptomatic patients after initiation of treatment. Patients and methods: Our study included 122 previously untreated patients with a median age of 67 years who required systemic treatment. Multiple variables were analyzed for their prognostic value on survival after initiation of treatment using univariate and Cox regression multivariate analysis. Results: The median overall survival was 107 months. Pretreatment factors associated with shorter survival were age ≥ 65 years, splenomegaly, B-symptoms (weight loss, fever or night sweats), hemoglobin 50%. In the multivariate analysis, the two variables with independent prognostic value were age ≥ 65 years and hemoglobin < 10 g/dL. Furthermore, we were able to divide our patients into three risk groups based on the presence of two, one or none of these adverse prognostic factors. The median survival times in the high-, intermediate- and low-risk groups were 46 months, 107 months and 172 months, respectively (P < 0.0001). Discussion: Our findings suggest that advanced age and anemia appear to be the two dominant prognostic factors for survival after initiation of treatment in patients with WM. These two readily available parameters can stratify the patients into three distinct subgroups and may help the selection of appropriate treatment.Εισαγωγή: Η μακροσφαιριναιμία Waldenström (WM) αποτελεί ένα σπάνιο λεμφοπλασματοκυττοειδές λέμφωμα που χαρακτηρίζεται από την παρουσία μονοκλωνικής ανοσοσφαιρίνης Μ στον ορό. Αρκετές μελέτες στο παρελθόν έχουν εξετάσει πιθανούς προγνωστικούς παράγοντες για τη νόσο, ωστόσο ελάχιστες από αυτές αφορούσαν την επιβίωση και πρόγνωση συμπτωματικών ασθενών αμέσως μετά την έναρξη θεραπευτικής αγωγής. Ασθενείς και μεθοδολογία: Η μελέτη μας περιελάμβανε 122 συμπτωματικούς ασθενείς με διάμεση ηλικία 67 έτη, οι οποίοι είχαν ανάγκη έναρξης θεραπευτικής αγωγής κατά την αρχική διάγνωση. Ακολούθησε μονοπαραγοντική και πολυπαραγοντική ανάλυση με τη στατιστική μέθοδο Cox για τον καθορισμό παραγόντων με ανεξάρτητη προγνωστική αξία για την επιβίωση των ασθενών μετά την έναρξη θεραπείας. Αποτελέσματα: Η συνολική διάμεση επιβίωση ήταν 107 μήνες. Παράγοντες συσχετιζόμενοι με δυσμενή επιβίωση πριν από την έναρξη της θεραπείας ήταν ηλικία ≥ 65 έτη, σπληνομεγαλία, Β-συμπτώματα (απώλεια βάρους, πυρετός η νυκτερινοί ιδρώτες), αιμοσφαιρίνη < 10 g/dL, αιμοπετάλια < 100 x 10⁶/dL, αλβουμίνη < 3.5 g/dL, και μυελική διήθηση από λεμφοπλασματοκύτταρα ≥ 50%. Στην πολυπαραγοντική ανάλυση, δύο ήταν οι μεταβλητές με ανεξάρτητη αρνητική προγνωστική αξία, ηλικία ≥ 65 έτη και αιμοσφαιρίνη < 10 g/dL. Ακολούθως, καταφέραμε να διακρίνουμε τρεις κατηγορίες κινδύνου στον πληθυσμό των ασθενών της μελέτης, βασιζόμενοι στην παρουσία, δύο, ενός η κανενός από τους παραπάνω δυσμενείς προγνωστικούς παράγοντες. Η διάμεση επιβίωση στην υψηλού, ενδιάμεσου, και χαμηλού κινδύνου κατηγορία ασθενών ήταν 46 μήνες, 107 μήνες και 172 μήνες, αντίστοιχα (Ρ < 0.0001). Συμπεράσματα: Τα ευρήματα της μελέτης μας αναδεικνύουν την προχωρημένη ηλικία και την αιμοσφαιρίνη ως τους σημαντικότερους παράγοντες για την επιβίωση των ασθενών με WM πριν από την έναρξη θεραπείας . Αυτές οι δυο άμεσα διαθέσιμες παράμετροι μπορούν να διαχωρίσουν τους ασθενείς με WM σε τρεις ομάδες κινδύνου, βοηθώντας έτσι στην επιλογή κατάλληλης θεραπείας σε κάθε περίπτωση