Dissertation committee:
Μπούμπας Δημήτριος, Καθηγητής, Ιατρική Σχολή, ΕΚΠΑ
Θυφρονίτης Γεώργιος, Καθηγητής, Τμήμα Βιολογικών Εφαρμογών κ Τεχνολογιών, Πανεπιστήμιο Ιωαννίνων
Καψογεώργου Ευσταθία, Αναπληρώτρια Καθηγήτρια, Ιατρική Σχολή, ΕΚΠΑ
Ρουμπελάκη Μαρία, Επίκουρη Καθηγήτρια, Ιατρική Σχολή, ΕΚΠΑ
Εμμανουηλίδου Ευαγγελία, Επίκουρη Καθηγήτρια, Σχολή Θετικών Επιστημών, ΕΚΠΑ
Ξάνθου-Τσιγκόγλου Γεωργία, Β' Ερευνήτρια, ΙΙΒΕΑΑ
Βεκρέλλης Κωνσταντίνος, Β' Ερευνητής, ΙΙΒΕΑΑ
Summary:
Introduction and Purpose: T helper type cells 17 (Th17) exhibit functional heterogeneity fostering both pathogenic and nonpathogenic, tissue-protective functions. In multiple sclerosis (MS), pathogenic Th17 cells are critical drivers of autoimmune central nervous system (CNS) inflammation and demyelination. Still, the factors that control Th17 cell pathogenicity remain incompletely defined. Activin-A is a pleiotropic cytokine that is involved in a variety of biological processes and exerts both pro- and anti- inflammatory functions. Our previous studies revealed that activin-A restrains Th2 allergic responses in asthmatic individuals and protects against experimental asthma. Still, the role of activin-A in autoimmune responses that are mediated with auto-reactive Τh1 and Th17 cells remains to be elucidated. Our hypothesis is that activin-A plays a key role in Th17 pathogenicity and linked CNS autoimmune inflammation. Our aims are to investigate a) the role of activin-A in the regulation of autoimmune CNS inflammation in vivo and b) to explore the molecular mechanisms that are involved in the suppressive effects of activin-A on the pathogenicity of Th17 cells.
Materials and Methods: The in vivo role of activin-A in MS was explored following therapeutic administration in a C57BL/6 mouse model of MS, experimental autoimmune encephalomyelitis (EAE). The effects of activin-A on the regulation of Th17 pathogenicity in vivo were examined through adoptive transfer Τh17 cells in to immunosuppressed Rag-/- mice following EAE induction. Functional experiments were also performed in Nt5e and Phd2 deficient mice. Th17 cells were generated in vitro under highly-pathogenic conditions (IL-6, IL-1β, IL-23) and the effects of activin-A on their transcriptional and phenotypic profile were examined by flow cytometry, ELISA, qPCR, RNA-seq, immunofluorescence, chromatin immunoprecipitation (ChIP) assays and confocal microscopy analyses. Bioinformatics, proteomics and metabolomics analyses deciphered the molecular mechanisms underlying the effects of activin-A on Th17 pathogenicity. The levels of activin-A were measured in the serum and cerebrospinal fluid of individuals with RRMS, (n=23), healthy donors (n=10) and patients with other non-autoimmune diseases (n=20). Finally, the role of activin-A on human Th17 differentiation and effector CD4+T cells responses of patients with MS was examined.
Results: Our findings demonstrated that in vivo therapeutic administration of activin-A in EAE mice, ameliorated disease severity and alleviated CNS immunopathology and demyelination, associated with decreased activation of pathogenic Th17 cells. During Th17 cell differentiation, activin-A stimulation repressed genes linked to Th17 pathogenicity, including Ifng, Csf2, Il1b, Tbx21 and Batf, concomitant with an upregulation of genes associated with non-pathogenic Th17 cells, such as, Il10, Maf and Ahr. The aforementioned effects were mediated by activin-A’s canonical signaling pathway, involving activin-like kinase-4 (ALK) receptor and the transcriptional factors phosphoSmad2/3. Extended analysis of the whole genome (RNAseq), combined with in vivo studies, revealed that activation of the ATP-depleting CD39 and CD73 ectonucleotidases is essential for activin-A–induced suppression of the pathogenic signature and the encephalitogenic functions of Th17 cells. Mechanistical studies, found that transcription factor aryl hydrocarbon receptor (Ahr), along with STAT3 and c-Maf, are recruited to promoter elements on Entpd1 and Nt5e (encoding CD39 and CD73 ectonucleotidases, respectively) and on anti-inflammatory cytokine IL-10 and control their expression in Th17 cells in response to activin-A. Moreover, we showed that activin-A negatively regulates the expression and stability of the metabolic sensor, hypoxia-inducible factor-1α, and key inflammatory proteins linked to pathogenic Th17 cell states. Finally, we demonstrated that activin-A is induced in the CNS of individuals with MS and its presence restrains human Th17 cell-mediated responses.
Conclusions: Our studies uncovered activin-A as a critical controller of Th17 cell pathogenicity that can be targeted for the suppression of autoimmune CNS inflammation.
Keywords:
Cytokines, Th17 cell differentiation, Ectonucleotidases, Activin-A
