Alternative Pathways of IL-1 Activation, and Its Role in Health and Disease.

Cytokines activate or inhibit immune cell behavior and are thus integral to all immune responses. IL-1α and IL-1β are powerful apical cytokines that instigate multiple downstream processes to affect both innate and adaptive immunity. Multiple studies show that IL-1β is typically activated in macrophages after inflammasome sensing of infection or danger, leading to caspase-1 processing of IL-1β and its release. However, many alternative mechanisms activate IL-1α and IL-1β in atypical cell types, and IL-1 function is also important for homeostatic processes that maintain a physiological state. This review focuses on the less studied, yet arguably more interesting biology of IL-1. We detail the production by, and effects of IL-1 on specific innate and adaptive immune cells, report how IL-1 is required for barrier function at multiple sites, and discuss how perturbation of IL-1 pathways can drive disease. Thus, although IL-1 is primarily studied for driving inflammation after release from macrophages, it is clear that it has a multifaceted role that extends far beyond this, with various unconventional effects of IL-1 vital for health. However, much is still unknown, and a detailed understanding of cell-type and context-dependent actions of IL-1 is required to truly understand this enigmatic cytokine, and safely deploy therapeutics for the betterment of human health

Dexamethasone induces ω3-derived immunoresolvents driving resolution of allergic airway inflammation.

To the Editor: Resolution of inflammation has long been considered to be a passive process that ensues in an inflammatory response following the dilution of proinflammatory cues. However, this paradigm is shifting as it is becoming increasingly evident that resolution is an active reaction that uses a complex molecular machinery that orderly terminates inflammation. Among the multiple mechanisms involved, specialized proresolving lipid mediators (SPMs) such as resolvins, protectins, and lipoxins have taken center stage.1 These are derived from ω3 and ω6 polyunsaturated fatty acids through complex intracellular and transcellular biosynthetic pathways, and promote resolution of inflammation by their manifold and concerted functions inhibiting leukocyte trafficking, dampening proinflammatory signaling, inducing apoptosis, and promoting anti-inflammatory M2-like macrophage phenotypes, efferocytosis, and tissue restitution.1This work was supported by core funding from the Hellenic Ministry of Education, Research and Religious Affairs and the Hellenic Ministry of Health, and by the research grants PREDICTA (contract no. 260895) from the European Commission, MIDAS (contract no. MIS 377047), and RESOLVE-ASTHMA (contract no. 2601) from the Hellenic Ministry of Education, Research and Religious Affairs

Progression of mouse skin carcinogenesis is associated with increased ERα levels and is repressed by a dominant negative form of ERα.

Estrogen receptors (ER), namely ERα and ERβ, are hormone-activated transcription factors with an important role in carcinogenesis. In the present study, we aimed at elucidating the implication of ERα in skin cancer, using chemically-induced mouse skin tumours, as well as cell lines representing distinct stages of mouse skin oncogenesis. First, using immunohistochemical staining we showed that ERα is markedly increased in aggressive mouse skin tumours in vivo as compared to the papilloma tumours, whereas ERβ levels are low and become even lower in the aggressive spindle tumours of carcinogen-treated mice. Then, using the multistage mouse skin carcinogenesis model, we showed that ERα gradually increases during promotion and progression stages of mouse skin carcinogenesis, peaking at the most aggressive stage, whereas ERβ levels only slightly change throughout skin carcinogenesis. Stable transfection of the aggressive, spindle CarB cells with a dominant negative form of ERα (dnERα) resulted in reduced ERα levels and reduced binding to estrogen responsive elements (ERE)-containing sequences. We characterized two highly conserved EREs on the mouse ERα promoter through which dnERα decreased endogenous ERα levels. The dnERα-transfected CarB cells presented altered protein levels of cytoskeletal and cell adhesion molecules, slower growth rate and impaired anchorage-independent growth in vitro, whereas they gave smaller tumours with extended latency period of tumour onset in vivo. Our findings suggest an implication of ERα in the aggressiveness of spindle mouse skin cancer cells, possibly through regulation of genes affecting cell shape and adhesion, and they also provide hints for the effective targeting of spindle cancer cells by dnERα

The Coagulation and Immune Systems are Directly Linked through the Activation of Interleukin-1α by Thrombin

Ancient organisms have a combined coagulation and immune system, and although links between inflammation and haemostasis exist in mammals, they are indirect and slower to act. Here we investigated direct links between mammalian immune and coagulation systems by examining cytokine proproteins for potential thrombin protease consensus sites. We found that interleukin (IL)-1α is directly activated by thrombin. Thrombin cleaved pro-IL-1α at a site perfectly conserved across disparate species, indicating functional importance. Surface pro-IL-1α on macrophages and activated platelets was cleaved and activated by thrombin, while tissue factor, a potent thrombin activator, colocalised with pro-IL-1α in the epidermis. Mice bearing a mutation in the IL-1α thrombin cleavage site (R114Q) exhibited defects in efficient wound healing and rapid thrombopoiesis after acute platelet loss. Thrombin-cleaved IL-1α was detected in humans during sepsis, pointing to the relevance of this pathway for normal physiology and the pathogenesis of inflammatory and thrombotic diseases.This work was funded by British Heart Foundation Grants FS/09/005/26845, FS/13/3/30038, FS/18/19/33371 and RG/16/8/32388 to MC, RG/13/14/30314 to MB, the BHF Cambridge Centre for Research Excellence RE/13/6/30180, the Oxbridge BHF Regenerative Medicine Centre RM/13/3/30159, and the Cambridge NIHR Biomedical Research Centre

Toll-like receptor 7 stimulates production of specialized pro-resolving lipid mediators and promotes resolution of airway inflammation.

Although specialized pro-resolving mediators (SPMs) biosynthesized from polyunsaturated fatty acids are critical for the resolution of acute inflammation, the molecules and pathways that induce their production remain elusive. Here, we show that TLR7, a receptor recognizing viral ssRNA and damaged self-RNA, mobilizes the docosahexaenoic acid (DHA)-derived biosynthetic pathways that lead to the generation of D-series SPMs. In mouse macrophages and human monocytes, TLR7 activation triggered production of DHA-derived monohydroxy metabolome markers and generation of protectin D1 (PD1) and resolvin D1 (RvD1). In mouse allergic airway inflammation, TLR7 activation enhanced production of DHA-derived SPMs including PD1 and accelerated the catabasis of Th2-mediated inflammation. D-series SPMs were critical for TLR7-mediated resolution of airway inflammation as this effect was lost in Alox15(-/-) mice, while resolution was enhanced after local administration of PD1 or RvD1. Together, our findings reveal a new previously unsuspected role of TLR7 in the generation of D-series SPMs and the resolution of allergic airway inflammation. They also identify TLR stimulation as a new approach to drive SPMs and resolution of inflammatory diseases

Temporal inhibition of autophagy reveals segmental reversal of ageing with increased cancer risk

Abstract: Autophagy is an important cellular degradation pathway with a central role in metabolism as well as basic quality control, two processes inextricably linked to ageing. A decrease in autophagy is associated with increasing age, yet it is unknown if this is causal in the ageing process, and whether autophagy restoration can counteract these ageing effects. Here we demonstrate that systemic autophagy inhibition induces the premature acquisition of age-associated phenotypes and pathologies in mammals. Remarkably, autophagy restoration provides a near complete recovery of morbidity and a significant extension of lifespan; however, at the molecular level this rescue appears incomplete. Importantly autophagy-restored mice still succumb earlier due to an increase in spontaneous tumour formation. Thus, our data suggest that chronic autophagy inhibition confers an irreversible increase in cancer risk and uncovers a biphasic role of autophagy in cancer development being both tumour suppressive and oncogenic, sequentially

The role of lipid mediators in the resolution of experimental allergic airway inflammation

The purpose of this doctoral thesis is the study of the role of LM of inflammation and resolution in experimental models of allergic airway inflammation (AAI). Towards this end, state-of-the-art Liquid Chromatography – Tandem Mass Spectrometry based methodologies were developed for the determination of LM of inflammation and resolution in different biological matrices (mouse lung tissue, mouse/human serum and culture medium), thus facilitating the multi-faceted study of AAI.More specifically, the LC-MS methodologies were applied for the determination of the lipid mediator profile in the lungs of mice with AAI-induced by ovalbumin (OVA). Lipidomic analysis was performed both at the induction phase as well as during resolution which ensues. The lipidomic profiling was also performed in corresponding serum obtained by these mice in order for us to determine the circulating levels of LM and correlation between tissue and serum. These experiments demonstrated the tissue-specific activation of specialized pro-resolving pathways and provided insight toward to possible use of these mediators as biomarkers indicating the point where resolution mechanisms have been initiated and how does this relateto the outcome of disease.Within the same context, we explored possible agents/drugs which are either already being used in the clinical setting as anti-inflammatory therapeutics or they have demonstrated their anti-inflammatory potential in experimental models of AAI. Dexamethasone, as a representative drug of the corticosteroids family, and Resiquimod (R-848), a TLR7 agonist were examined in vivo for their capacity to dampen the inflammatory process as a function of their potential effects on regulating the endogenous resolution machinery. After the in vivo therapeutic administration of these agents, we monitored the fluctuations of the lipidomic signature at the tissue level and concluded that these agents accelerated resolution of AAI by directly regulating the pro-resolving LM pathways. Our results demonstrated that conventional asthma therapies influence the endogenous resolution molecular pathways and as a consequence significantly affect the final outcome of the disease.Σκοπός της παρούσας διατριβής είναι η μελέτη των ΛΜ της φλεγμονής και της λύσης σε πειραματικά μοντέλα αλλεργικού άσθματος. Για την επίτευξη των στόχων προχωρήσαμε σε ανάπτυξη καινοτόμων μεθοδολογιών με βάση την Υγρή Χρωματογραφία σε σύζευξη με Φασματομετρία Μάζας για τον προσδιορισμό ΛΜ της φλεγμονής και της λύσης σε διαφορετικά βιολογικά υγρά (ορός) και ιστούς (πνεύμονας).Πιο συγκεκριμένα, πραγματοποιήθηκε ανάλυση σε επίπεδο παραγωγής ΛΜ (λιπιδομική ανάλυση) στους πνεύμονες μυών με αλλεργική φλεγμονή στους αεραγωγούς επαγόμενη από οβαλβουμίνη, τόσο στη φάση της επαγωγής της φλεγμονής όσο και στη φάση της επακόλουθης λύσης της. Παράλληλα πραγματοποιήθηκε η ίδια ανάλυση και στον ορό των μυών με σκοπό να καθοριστεί η συγκέντρωση τους και στη συστηματική κυκλοφορία. Τα δεδομένα αυτά μας έδωσαν πληροφορίες για τη σχετική συγκέντρωση των ΛΜ στον ιστό προς τον ορό και συνέβαλαν στην αξιολόγηση για το κατά πόσο κάποια απ’ αυτά τα μόρια μπορούν να λειτουργήσουν ως βιοδείκτες της πορείας της νόσου.Στο ίδιο πλαίσιο, της μελέτης της ενδεχόμενης χρήσης τους ως βιοδείκτες,πραγματοποιήθηκε χορήγηση κάποιων κλασικών αντι-φλεγμονωδών φαρμάκων (πιο συγκεκριμένα, δεξαμεθαζόνη) τα οποία χρησιμοποιούνται ευρέως στη διαχείριση των συμπτωμάτων του άσθματος. Eπιπλέον, δοκιμάστηκαν παράγοντες οι οποίοι έχουν αποδείξει στην ισχυρή αντι-φλεγμονώδη δράση τους σε μοντέλα αλλεργικού άσθματος όπως είναι ο αγωνιστής του TLR7, Resiquimod (R-848). Μετά τη χορήγηση των παραγόντων αυτών καθορίστηκε το λιπιδικό προφίλ το οποίο φάνηκε να τροποποιείται παρουσία του φαρμάκου, επιταχύνοντας σε πιο πρώιμο χρονικό σημείο τη παραγωγή αντι-φλεγμονωδών ΛΜ. Τα αποτελέσματά μας έδειξαν ότι οι συμβατικές θεραπείες για το άσθμα επιδρούν στους ενδογενείς μηχανισμούς της λύσης και κατ’ επέκταση έχουν μεγάλη συμμετοχή στη τελική έκβαση της νόσου

The common IL1A single nucleotide polymorphism rs17561 is a hypomorphic mutation that significantly reduces interleukin-1α release from human blood cells.

Funder: Cambridge NIHR Biomedical Research CentreInterleukin-1 alpha (IL-1α) is a powerful cytokine that drives inflammation and modulates adaptive immunity. Due to these powerful effects, IL-1α is controlled at multiple levels from transcription to cleavage and release from the cell. Genome-wide association studies can identify loci that drive important diseases, although often the functional effect of the variant on phenotype remains unknown or small, with most risk variants in non-coding regions. We find that the common variant rs17561 changes a conserved amino acid in the central region of IL-1α linking the pro piece to the cytokine domain. Using a recall-by-genotype study and whole blood stimulation, we find that minor allele homozygotes release ~50% less IL-1α than the major allele, with IL-1β release equivalent. IL-1α transcript level was identical between groups, implying a post-transcriptional effect, whilst cleavage of recombinant pro-IL-1α by multiple proteases was also equivalent for both forms. Importantly, transfected macrophages also release less minor allele IL-1α upon inflammasome activation, revealing that reduced secretion is directly caused by the missense amino acid substitution and more minor allele IL-1α was retained within the cell. Thus, rs17561 represents a very common hypomorphic mutation in IL-1α. We believe this novel data will be important for determining the potential contribution of IL-1α to disease and/or physiological processes, for example, by Mendelian randomisation, and may aid patient stratification when considering anti-IL-1 therapies.This work was funded by BHF Grants FS/13/3/30038, FS/18/19/33371 and RG/16/8/32388 to MCHC, the BHF Cambridge Centre for Research Excellence RE/13/6/30180, and the Cambridge NIHR Biomedical Research Centr

The common IL1A SNP rs17561 is a hypomorphic mutation that significantly reduces IL-1α release from human blood cells.

Interleukin-1 alpha (IL-1α) is a powerful cytokine that drives inflammation and modulates adaptive immunity. Due to these powerful effects IL-1α is controlled at multiple levels from transcription to cleavage and release from the cell. Genome-wide association studies can identify loci that drive important diseases, although often the functional effect of the variant on phenotype remains unknown or small, with most risk variants in non-coding regions. We find that the common variant rs17561 changes a conserved amino acid in the central region of IL-1α linking the pro piece to the cytokine domain. Using a recall-by-genotype study and whole blood stimulation, we find that minor allele homozygotes release ~50% less IL-1α than the major allele, with IL-1β release equivalent. IL-1α transcript level was identical between groups, implying a post-transcriptional effect, whilst cleavage of recombinant pro-IL-1α by multiple proteases was also equivalent for both forms. Importantly, transfected macrophages also release less minor allele IL-1α upon inflammasome activation, revealing that reduced secretion is directly caused by the missense amino acid substitution, and more minor allele IL-1α was retained within the cell. Thus, rs17561 represents a very common hypomorphic mutation in IL-1α. We believe this novel data will be important for determining the potential contribution of IL-1α to disease and/or physiological processes, e.g. by Mendelian randomisation, and may aid patient stratification when considering anti-IL-1 therapies.This work was funded by BHF Grants FS/13/3/30038, FS/18/19/33371 and RG/16/8/32388 to MCHC, the BHF Cambridge Centre for Research Excellence RE/13/6/30180, and the Cambridge NIHR Biomedical Research Centr