Background Activins are members of the TGFβ-superfamily implicated in the pathogenesis of several immuno-inflammatory disorders. Based on our previous studies demonstrating that over-expression of Activin-A in murine lung causes pathology sharing key features of COVID-19, we hypothesized that Activins and their natural inhibitor Follistatin might be particularly relevant to COVID-19 pathophysiology. Methods Activin-A, Activin-B and Follistatin levels were retrospectively analyzed in 574 serum samples from 263 COVID-19 patients hospitalized in three independent centers, and compared with common demographic, clinical and laboratory parameters. Optimal-scaling with ridge-regression was used to screen variables and establish a prediction model. Result The Activin/Follistatin-axis was significantly deregulated during the course of COVID-19, correlated with severity and independently associated with mortality. FACT-CLINYCoD, a novel disease scoring system, adding one point for each of Follistatin>6235pg/ml, Activin-A>591pg/ml, Activin-B>249pg/ml, CRP>10.3mg/dL, LDH>427U/L, Intensive Care Unit (ICU) admission, Neutrophil/Lymphocyte-Ratio>5.6, Age>61, Comorbidities>1 and D-dimers>1097ng/ml, efficiently predicted fatal outcome in an initial cohort (AUC: 0.951; 95%CI: 0.919-0.983, p<10 -6). Two independent cohorts that were used for validation indicated similar AUC values. Conclusions This study unravels strong link between Activin/Follistatin-axis and COVID-19 mortality and introduces FACT-CLINYCoD, a novel pathophysiology-based tool that allows dynamic prediction of disease outcome, supporting clinical decision making.

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Background: Hypoxia is a prominent feature of the BM microenvironment, influencing both normal and malignant hematopoiesis. HIF-1α, which is a key regulator of hypoxia responses by mediating the transition to glycolytic metabolism, serves as a cell cycle checkpoint of HSC quiescence and function. It has been proposed that differential HIF-1α protein expression between hypoxic endosteal and less hypoxic vascular niche finely regulates normal hematopoiesis by promoting both quiescence and survival of HSCs, as well as proliferation and differentiation of HPCs. DNA damage response 1 gene (REDD1) is a direct transcriptional target of HIF-1α linking hypoxia to energy regulation and autophagy. Recent evidence suggests that metabolism and autophagy are developmentally programmed and essential for effective hematopoiesis. Aims: To study the implication of HIF-1α/REDD1/autophagy/metabolism axis in differentiation/maturation of hematopoietic BM cells of MDS patients. Methods: BM aspiration and biopsy samples were collected from 15 untreated MDS patients from all subtypes except MDS-RARS and 7 age-matched controls with non-malignant hematologic disorder. Demographic, clinical, laboratory and karyotypic parameters were recorded. BM biopsies were immunohistochemicallly stained by fluorescent-labeled 2-nitroimidazole to assess hypoxic areas in BM. CD34 and myeloid lineage cells were isolated using magnetic beads and ficoll double-layer protocol, respectively. BM cell populations were determined by FACS analysis using standard gating strategies. HIF-1α and REDD1 gene and protein expression was evaluated by qRT-PCR and FACS analysis, respectively. Autophagy was determined by immunofluorescence for LAMP-1/LC3B and immunoblotting for LC3B/p62 (SQSTM1), whereas mitophagy by immunofluorescence for LC3B/TOMM20. Mitochondrial membrane potential (ΔΨ) and mitochondrial mass were analyzed by FACS analysis using mitotrackers. Metabolomic analysis of myeloid lineage cells was performed by liquid chromatography mass spectrometry (LC-MSn). Raw data files were processed using several chemo-informatics tools. Results: We found a preferential strong accumulation of 2-nitroimidazole in intrasinusoidal regions of MDS BM, indicating that hypoxia is a fundamental feature of BM in MDS. We demonstrated a statistically significant REDD1 gene overexpression and an increased intracellular protein co-expression of HIF-1α and REDD1 protein levels in both CD34 and myeloid cells from MDS compared to controls, as determined by RT-qPCR and FACS analysis, respectively. Higher REDD1 protein expression was shown in patients with high grade dysplasia as assessed by the Ogata classification system. Moreover, both CD34 and myeloid cells from MDS demonstrated increased LC3B puncta compared to controls with concurrent staining for CD34 and MPO. The quantitative evaluation of LC3B by Western blot revealed high level of expression of LC3B-II in the MDS myeloid cells compared to controls indicating increased autophagic activity. The observed p62/SQSTM1 degradation along with the colocalization pattern of LC3B/LAMP-1 suggest increased autophagic flux. Metabolomic analysis of MDS myeloid lineage cells compared to controls revealed excessive glycolysis, defective oxidative phosphorylation and increased reductive carboxylation glutaminolysis associated with elevated level of intracellular 2-hydroxyglutarate, all indicative of HIF-1α driven metabolism. The co-localization between TOMM20 marker and autophagosomes in MDS myeloid cells was compatible with increased mitophagy whereas, MDS myeloid cells, were characterized by a reduction of mitochondrial mass and membrane potential in comparison to controls, as determined by FACS analysis. Conclusion: Our results provide evidence for the first time of the hypoxia-driven HIF-1α/REDD1/autophagy axis in the pathophysiology of MDS. Our study suggests that this deregulated pathway is responsible for the production of 2-hydroxyglutarate, an oncometabolite, which is implicated in dysregulated epigenetic homeostasis. All the above may lead to the dysregulated metabolism and differentiation potential of the myeloid cells, thus unraveling a new pathogenetic mechanism for the MDS development. Disclosures No relevant conflicts of interest to declare.

Rationale: Activins are inflammatory and tissue-repair-related members of the TGFβ-superfamily that have been implicated in the pathogenesis of several immuno-inflammatory disorders including sepsis/acute respiratory distress syndrome (ARDS). We hypothesized that they might be of particular relevance to COVID-19 pathophysiology. Objectives: To assess the involvement of the Activin-Follistatin-axis in COVID-19 pathophysiology. Methods: Levels of Activins -A, -B and their physiological inhibitor Follistatin, were retrospectively analyzed in 314 serum samples from 117 COVID-19 patients derived from two independent centers and compared with common demographic, clinical and laboratory parameters. Optimal-scaling with ridge-regression was used to screen variables and establish a prediction model. Main Results: The Activin/Follistatin-axis was significantly deregulated during the course of COVID-19 and was independently associated with severity and in-hospital mortality. FACT-CLINYCoD, a novel disease scoring system, adding one point for each of Follistatin >6235 pg/ml, Activin-A >591 pg/ml, Activin-B >249 pg/ml, CRP >10.3 mg/dL, LDH >427 U/L, Intensive Care Unit (ICU) admission, Neutrophil/Lymphocyte-Ratio >5.6, Years of Age >61, Comorbidities >1 and D-dimers >1097 ng/ml, efficiently predicted and monitored fatal outcome independently of multiplicity and timing of sampling (AUC: 0.951±0.032, p<10-6). Validation in 35 samples derived from a third hospital indicated comparable AUC (0.958±0.086, p=0.032). Conclusion: This study unravels the link between Activin/Folistatin-axis and COVID-19 mortality and introduces FACT-CLINYCoD, a novel pathophysiology-based tool that copes with the dynamic and heterogeneous nature of COCVID-19, predicts disease outcome and supports clinical decision making. Prospective large-scale validation of this calculator, as well as investigation of the mechanisms linking Activin/Folistatin-axis to COVID-19 pathogenesis is warranted.

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