Periodic Report Summary 2 - RESOLUTIOMIR (Mechanisms of Inflammation Resolution: Role of miRNAs) Context and Objectives In the present research project, we are investigating how does acute inflammation resolve. This is a key question in medicine since unresolved inflammation underlies the pathogenesis of several widely recurrent human diseases such as asthma, arthritis, cardiovascular diseases, and cancer. Since these and many other inflammation-related conditions, significantly affect the quality of life of millions of individuals worldwide and represent a costly burden for public health, treating inflammation is of wide interest. The elucidation of the basic mechanisms that orchestrate the resolution of inflammation represents a critical step to generate a new class of anti-inflammatory drugs, which, by stimulating endogenous circuits of inflammation resolution, may be devoid of serious side effects. In this respect, this proposal stands at the intersection of multiple disciplines, such as general pathology, molecular biology, pharmacology, haematology, and immunology. The specific aims that are being addressed in the present research are to determine whether resolution of acute inflammation is regulated at the bone marrow (BM) level by select microRNAs (miRNA), whose expression can be modulated by resolvin (Rv) D1 and lipoxin (LX) A4. Inflammation is a host defence mechanism to tissue damage or microbial infection. Characteristic of this rapid response is leukocyte recruitment at the site of injury to remove the pathogenic noxa. In order for this process to be beneficial to the host, a resolution program must take place and homeostasis be re-established. Several adaptive modifications in leukocyte type and phenotype occur within the inflammatory exudates during the initiation and resolution of inflammation, going from PMN, which are the first leukocyte population that infiltrate the inflamed tissue, towards monocyte/macrophages, the master regulators of tissue resolution. These changes in leukocyte subtypes during inflammatory responses reflect those in the bone BM compartment and likely depend on the re-orientation of the HSC differentiation and proliferation programs. Hematopoietic stem cells (HSC) constitute the common precursor of blood leukocytes. They mainly reside in a non proliferative quiescent state within the BM niche and in response to specific stimuli (e.g. peripheral inflammation) give rise to blood leukocytes. An emerging line of investigation indicates that many HSC processes are finely tuned by miRNAs, small (22–24 nt) noncoding RNA sequences that act primarily as translational repressors of gene transcripts to regulate physiological and pathological hematopoiesis as well as the immune response. Whether specific miRNAs are temporally regulated in HSC and control immune responses during inflammation and resolution is of extreme interest since this knowledge will help to identify pro-resolution miRNA signatures and target genes that can be exploited for the development of innovative anti-inflammatory approaches. Resolvin D1 (7S, 8R, 17S-trihydroxy-4Z, 9E, 11E, 13Z, 15E, 19Z-docosahexaenoic acid) and lipoxin A4 (5S, 6R, 15S-trihydroxy-7E, 9E, 11Z, 13E-eicosateraenoic acid) are endogenous autacoids biosynthesized respectively from docosahexanoic and arachidonic acid via lipoxygenases and subsequent reactions during inflammation and resolution. They carry specific actions to stop excessive neutrophil infiltration, stimulate the non phlogistic phagocytosis of microbes and apoptotic cells by macrophages, and regulate cellular pathways that control host inflammatory response and actively expedite the return to homeostasis. Cell specific actions of LXA4 and RvD1 encompass regulation of specific miRNAs (e.g. miR-146b, miR-208a, miR-219, let-7c) and target genes (e.g. proteins involved in NF-kB activation, chemokines such as IL-8, 5-lipoxygenase, and members of the TGF-β1 signalling pathway). Accruing evidence from pre-clinical studies and clinical trials signifies now that RvD1 and LXA4 can be exploited for the pharmacological treatment of inflammation-related diseases. Therefore, elucidating pharmacological properties of LXA4 and RvD1 and determining if their mechanisms of action involve regulation of specific miRNAs in HSC that control immune responses is of paramount interest as this knowledge can provide novel pathways that can be harnessed to treat inflammation-related diseases in humans by promoting resolution. Acute inflammation triggered at mucosal tissues is a paramount defence mechanism to infections that should ideally culminate with resolution, an active termination program whose histological signs are stop of leukocyte infiltration and their active removal by monocyte/macrophages (MΦ) via phagocytosis (also termed efferocytosis), along with elimination of pathogenic noxae and restor/repair of tissue integrity. On the contrary, failure to resolve inflammation leads to collateral tissue damage and loss of function. Pseudomonas aeruginosa is a leading cause of nosocomial infections associated with a high mortality rate and is the main etiologic agent of chronic respiratory infections in patients with cystic fibrosis, bronchiectasis, and chronic bronchitis. P. aeruginosa exploits adaptation mechanisms to lower the inflammatory reaction and escape recognition and killing by the innate and adaptive immune system. Of interest, both US and European health surveillance agencies classify clinical strains of P. aeruginosa as “serious threat”, emphasizing the urgent need for new strategies to combat its infections, limit subsequent exacerbated inflammation without immune suppression, and stimulate microbial clearance by the immune system. Since RvD1 regulated miRNAs involved in leukocyte differentiation (miR-223), recruitment, and activation (miR-146b), we therefore tested anti-microbial, anti-inflammatory, and pro-resolutive actions of RvD1 in acute and chronic lung infection triggered by a multi-drug resistant clinical strain of P. aeruginosa that causes pulmonary dysfunction and death of patients.Tasks Performed and Main Results Achieved Task Q1. To determine whether hematopoiesis regulated during inflammation and resolutionTask Q2. To establish which miRNAs are regulated in HSC during inflammation and resolutionTask Q3. To investigate whether LXA4 and RvD1 regulate miRNAs in HSC during inflammation and resolutionTask Q4. To demonstrate if LXA4 and RvD1 regulate miRNAs in HSC cells in a GPCR-dependent manner To address these tasks we have employed zymosan-induced murine acute peritonitis as model of self-limited acute inflammatory reaction as well as acute and chronic lung infection with a clinical strain (RP73) of P. aeruginosa to determine RvD1 actions in long-term inflammation. To monitor the temporal progression of inflammation, peritoneal and bronchoalveolar lavage fluid infiltrating leukocytes were collected, counted, and characterized at several time intervals to exstablis resolution indices. To determine which miRNAs were differentially expressed in HSC, c-kit (CD117)+ Sca-1+ Lin- (KSL) HSC were isolated from bone marrow aspirates obtained from mice at 6 h peritonitis or control mice using fluorescence-activated cell sorting and miRNA fractions were purified for real time PCR analysis to evaluate expression levels of ~ 300 of the best characterized mouse miRNAs. In order to determine whether oral RvD1 regulates the miRNA signature of HSC during acute inflammation, KLS HSC from mice receiving injection of zymosan and oral administration of RvD1 or vehicle were sorted and miRNA fractions were isolated to determine expression levels of ~ 50 miRNAs using a real time PCR array. HSC will be transfected to over-express RvD1 receptors ALX/FPR2 and GRP32 and treated in vitro with RvD1 to establish whether RvD1 regulate miRNA in HSCA via receptor interactions.Main results from tasks performed in the reporting period we:1. Established the kinetics of PMN infiltration and numbers in peripheral blood and bone marrow, demonstrating that hematopoiesis is regulated in response to an acute inflammatory challenge;2. Determined which miRNAs are differentially expressed in HSC during acute inflammation compared to naïve cells and if RvD1 regulates specific miRNAs in HSC during acute inflammation; 3. Demonstrated for the first time that RvD1 is biologically active upon oral delivery in regulating expression of select miRNAs (miR-223, miR-146b, and miR-125b) in HSC involved in leukocyte recruitment and activation;4. Proved that oral treatment with RvD1 significantly reduced death, airway bacterial, and neutrophil infiltration in P. aeruginosa chronically infected mice. RvD1 also enhanced efferocytosis by macrophages and protected from lung damage, mucous metaplasia, and bronchiolar hyperplasia.Collectively, our result provide novel insights into endogenous and RvD1-driven pro-resolution mechanisms that can be further explored and harnessed for treating human inflammation-related diseases.Prospects of Research Career and Re-Integration of the FellowDuring the second two years of the Career Integration Grant, Dr. Recchiuti has consolidated scientific qualities acquired in his doctoral and post-doctoral tenure in the field of cell biology, pharmacology, immunology, and molecular biology and transferred knowledge and expertise to the host institution. He has also improved his independent thinking and leadership attitudes, learned how to manage a multipronged research project with complete autonomy, administer grant funds, and coordinate work activities of collaborators. Dr. Recchiuti obtained intramural and extramural funding as Prinicpal Investigator and consolidated his own research group.These qualities allowed Dr. Recchiuti to successfully achieve the main goals of the project and to submit, in less than two years, two manuscripts as corresponding author. Dr. Recchiuti obtained a mid-term position of four years at the “G. d’Annunzio” University of Chieti as “Marie Curie Researcher” for the duration of the Career Integration Grant and was assigned teaching activities to students of the School of Medicine of the “G. d’Annunzio” University. At the end of the Career Integration Grant (2014) Dr. Recchiuti is under evaluation for recruitment as Associate Professor according to the current national legislation. Therefore, the Career Integration Grant has fulfilled the objective to contribute to the re-integration of Dr. Recchiuti and his career development as independent scientist after a period of mobility. 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