Contribution of cytokines to the etiology and progression of Primary Myelofibrosis

Primary Myelofibrosis (PMF) is the end-stage of Philadelphia-negative myeloproliferative neoplasms (MPN) and is characterized by fibrosis and hematopoietic failure in bone marrow, with a consequential migration of the malignant hematopoietic stem cells (HSC) in the spleen where they induce ineffective haematopoiesis. To date, available therapies for PMF are still palliative and do not halt the progression of this neoplasm. During my PhD years, our laboratory investigated the factors promoting the onset and progression of PMF. In our PMF mice model, Gata1low mouse, we studied the role of the interaction of HSC niche with megakaryocytes and HSC localization in the bone marrow during their division and cycle. We observed the inflammation and the main protagonists (LNC-2, CXCL1, and TGF-β) of this process and how their level changes before and after the onset of the disease. We investigated the different megakaryocyte populations in the fibrotic environment in different organs (lung and bone marrow) to define the megakaryocytes implicated in this process. In human samples, we described different ultrastructural abnormalities of megakaryocytes from the bone marrow and the spleen, identifying a possible different metabolism in those two populations. In conclusion, we highlighted the intricated crosstalk between the megakaryocytes, the niche and HSC in PMF. We identified megakaryocytes-dependent cytokines altering the homeostasis of the niche and HSC. Those cytokines could be used as alternative therapeutic targets. Furthermore, we observed different megakaryocytic populations in different organs, providing new prospective on the role of megakaryocytes in different microenvironments.

Circulating Fibrocytes, their role in renal fibrosis and molecular pathways involved. Possible biomarkers of fibrogenesis in chronic kidney disease

Circulating Fibrocytes (CFs) are bone marrow-derived mesenchymal progenitor cells that express a similar pattern of surface markers related to leukocytes, hematopoietic progenitor cells and fibroblasts. CFs precursor display an ability to differentiate into fibroblasts and Myofibroblasts, as well as adipocytes. Fibrocytes have been shown to contribute to tissue fibrosis in the end-stage renal disease (ESRD), as well as in other fibrotic diseases, leading to fibrogenic process in other organs including lung, cardiac, gut and liver. This evidence has been confirmed by several experimental proofs in mice models of kidney injury. In the present study, we developed a protocol for the study of CFs, by using peripheral blood monocytes cells (PBMCs) samples collected from healthy human volunteers. Thanks to a flow cytometry method, in vitro culture assays and the gene expression assays, we are able to study and characterize this CFs population. Moreover, results confirmed that these approaches are reliable and reproducible for the investigation of the circulating fibrocytes population in whole blood samples. Our final aim is to confirm the presence of a correlation between the renal fibrosis progression, and the different circulating fibrocyte levels in Chronic Kidney Disease (CKD) patients. Thanks to a protocol study presented and accepted by the Ethic Committee we are continuing the study of CFs induction in a cohort of sixty patients affected by CKD, divided in three distinct groups for different glomerular filtration rate (GFR) levels, plus a control group of thirty healthy subjects. Ongoing experiments will determine whether circulating fibrocytes represent novel biomarkers for the study of CKD progression, in the early and late phases of this disease.

Design and synthesis of E3 ligase RNF5 inhibitors as innovative strategy to trigger mutant CFTR rescue in Cystic Fibrosis

In Cystic Fibrosis (CF) the deletion of phenylalanine 508 (F508del) in the CFTR anion channel is associated to misfolding and defective gating of the mutant protein. Among the known proteins involved in CFTR processing, one of the most promising drug target is the ubiquitin ligase RNF5, which normally promotes F508del-CFTR degradation. In this context, a small molecule RNF5 inhibitor is expected to chemically mimic a condition of RNF5 silencing, thus preventing mutant CFTR degradation and causing its stabilization and plasma membrane trafficking. Hence, by exploiting a virtual screening (VS) campaign, the hit compound inh-2 was discovered as the first-in-class inhibitor of RNF5. Evaluation of inh-2 efficacy on CFTR rescue showed that it efficiently decreases ubiquitination of mutant CFTR and increases chloride current in human primary bronchial epithelia. Based on the promising biological results obtained with inh-2, this thesis reports the structure-based design of potential RNF5 inhibitors having improved potency and efficacy. The optimization of general synthetic strategies gave access to a library of analogues of the 1,2,4-thiadiazol-5-ylidene inh-2 for SAR investigation. The new analogues were tested for their corrector activity in CFBE41o- cells by using the microfluorimetric HS-YFP assay as a primary screen. Then, the effect of putative RNF5 inhibitors on proliferation, apoptosis and the formation of autophagic vacuoles was evaluated. Some of the new analogs significantly increased the basal level of autophagy, reproducing RNF5 silencing effect in cell. Among them, one compound also displayed a greater rescue of the F508del-CFTR trafficking defect than inh-2. Our preliminary results suggest that the 1,2,4-thiadiazolylidene could be a suitable scaffold for the discovery of potential RNF5 inhibitors able to rescue mutant CFTRs. Biological tests are still ongoing to acquire in-depth knowledge about the mechanism of action and therapeutic relevance of this unprecedented pharmacological strategy.

Longitudinal Micro CT-driven biomarkers as a tool to evaluate lung fibrosis progression and antifibrotic efficacy in a refined Bleomycin mouse model

Idiopathic pulmonary fibrosis (IPF) is a chronic progressive disease with no curative pharmacological treatment. Animal models play an essential role in revealing molecular mechanisms involved in the pathogenesis of the disease. Bleomycin (BLM)-induced lung fibrosis is the most widely used and characterized model for anti-fibrotic drugs screening. However, several issues have been reported, such as the identification of an optimal BLM dose and administration scheme as well as gender-specificity. Moreover, the balance between disease resolution, an appropriate time window for therapeutic intervention and animal welfare remains critical aspects yet to be fully elucidated. In this thesis, Micro CT imaging has been used as a tool to identify the ideal BLM dose regimen to induce sustained lung fibrosis in mice as well as to assess the anti-fibrotic effect of Nintedanib (NINT) treatment upon this BLM administration regimen. In order to select the optimal BLM dose scheme, C57bl/6 male mice were treated with BLM via oropharyngeal aspiration (OA), following either double or triple BLM administration. The triple BLM administration resulted in the most promising scheme, able to balance disease resolution, appropriate time-window for therapeutic intervention and animal welfare. The fibrosis progression was longitudinally assessed by micro-CT every 7 days for 5 weeks after BLM administration and 5 animals were sacrificed at each timepoint for the BALF and histological evaluation. The antifibrotic effect of NINT was assessed following different treatment regimens in this model. Herein, we have developed an optimized mouse model of pulmonary fibrosis, enabling three weeks of the therapeutic window to screen putative anti-fibrotic drugs. micro-CT scanning, allowed us to monitor the progression of lung fibrosis and the therapeutical response longitudinally in the same subject, drastically reducing the number of animals involved in the experiment.