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Antonia Becker,1 Karoline Röhrich,1 Amanda Leske,1 Ulrike Heinicke,1 Tilo Knape,2 Aimo Kannt,2,3 Verena Trümper,4 Kai Sohn,5 Annett Wilken-Schmitz,1 Holger Neb,1 Elisabeth H Adam,1 Volker Laux,2 Michael J Parnham,2 Valerie Onasch,4 Andreas Weigert,4 Kai Zacharowski,1,2 Andreas von Knethen1 1Goethe University Frankfurt, Department of Anaesthesiology, Intensive Care Medicine, and Pain Therapy, University Hospital Frankfurt, Frankfurt, 60590, Germany; 2Fraunhofer Institute for Translational Medicine and Pharmacology ITMP, Frankfurt, 60596, Germany; 3Institute of Clinical Pharmacology, Goethe University, Frankfurt, 60590, Germany; 4Institute of Biochemistry I, Faculty of Medicine, Goethe-University Frankfurt, Frankfurt, 60590, Germany; 5Innovation Field in-vitro Diagnostics, Fraunhofer Institute for Interfacial Engineering and Biotechnology IGB, Stuttgart, 70569, GermanyCorrespondence: Andreas von Knethen, Email andreas.vonknethen@ukffm.deBackground: COVID-19 is a serious viral infection, which is often associated with a lethal outcome. Therefore, understanding mechanisms, which affect the immune response during SARS-CoV2 infection, are important.Methods: To address this, we determined the number of T cells in peripheral blood derived from intensive care COVID-19 patients. Based on our previous studies, evaluating PPARγ-dependent T cell apoptosis in sepsis patients, we monitored PPARγ expression. We performed a next generation sequencing approach to identify putative PPARγ-target genes in Jurkat T cells and used a PPARγ transactivation assay in HEK293T cells. Finally, we translated these data to primary T cells derived from healthy donors.Results: A significantly reduced count of total CD3+ T lymphocytes and the CD4+ and CD8+ subpopulations was observed. Also, the numbers of anti-inflammatory, resolutive Th 2 cells and FoxP3-positive regulatory T cells (Treg) were decreased. We observed an augmented PPARγ expression in CD4+ T cells of intensive care COVID-19 patients. Adapted from a next generation sequencing approach in Jurkat T cells, we found the chemoattractant receptor‐homologous molecule expressed on T helper type 2 cells (CRTH2) as one gene regulated by PPARγ in T cells. This Th 2 marker is a receptor for prostaglandin D and its metabolic degradation product 15-deoxy-∆12,14-prostaglandin J2 (15d-PGJ2), an established endogenous PPARγ agonist. In line, we observed an increased PPARγ transactivation in response to 15d-PGJ2 treatment in HEK293T cells overexpressing CRTH2. Translating these data to primary T cells, we found that Th 2 differentiation was associated with an increased expression of CRTH2. Interestingly, these CRTH2+ T cells were prone to apoptosis.Conclusion: These mechanistic data suggest an involvement of PPARγ in Th 2 differentiation and T cell depletion in COVID-19 patients.Keywords: Treg, PPARγ, Th 2, COVID-19, NGS, IL-4, CRTH2, FoxP3

Coronavirus disease 2019 (COVID-19) is caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) and can affect multiple organs, among which is the circulatory system. Inflammation and mortality risk markers were previously detected in COVID-19 plasma and red blood cells (RBCs) metabolic and proteomic profiles. Additionally, biophysical properties, such as deformability, were found to be changed during the infection. Based on such data, we aim to better characterize RBC functions in COVID-19. We evaluate the flow properties of RBCs in severe COVID-19 patients admitted to the intensive care unit by using microfluidic techniques and automated methods, including artificial neural networks, for an unbiased RBC analysis. We find strong flow and RBC shape impairment in COVID-19 samples and demonstrate that such changes are reversible upon suspension of COVID-19 RBCs in healthy plasma. Vice versa, healthy RBCs resemble COVID-19 RBCs when suspended in COVID-19 plasma. Proteomics and metabolomics analyses allow us to detect the effect of plasma exchanges on both plasma and RBCs and demonstrate a new role of RBCs in maintaining plasma equilibria at the expense of their flow properties. Our findings provide a framework for further investigations of clinical relevance for therapies against COVID-19 and possibly other infectious diseases.

Good science in translational research requires good animal welfare according to the principles of 3Rs. In many countries, determining animal welfare is a mandatory legal requirement, implying a categorization of animal suffering, traditionally dominated by subjective scorings. However, how such methods can be objectified and refined to compare impairments between animals, subgroups, and animal models remained unclear. Therefore, we developed the RELative Severity Assessment (RELSA) procedure to establish an evidence-based method based on quantitative outcome measures such as body weight, burrowing behavior, heart rate, heart rate variability, temperature, and activity to obtain a relative metric for severity comparisons. The RELSA procedure provided the necessary framework to get severity gradings in TM-implanted mice, yielding four distinct RELSA thresholds L1 surgery > restraint stress > colitis. The bootstrapped 95% confidence intervals reliably show that RELSA estimates are conditionally invariant against missing information but precise in ranking the quantitative severity information to the moderate context of the transmitter-implantation model. In conclusion, we propose the RELSA as a validated tool for an objective, computational approach to comparative and quantitative severity assessment and grading. The RELSA procedure will fundamentally improve animal welfare, data quality, and reproducibility. It is also the first step toward translational risk assessment in biomedical research.

The bioactive lipid sphingosine-1-phosphate (S1P), along with its receptors, modulates lymphocyte trafficking and immune responses to regulate skin inflammation. Macrophages are important in the pathogenesis of psoriasiform skin inflammation and express various S1P receptors. How they respond to S1P in skin inflammation remains unknown. We show that myeloid specific S1P receptor 1 (S1PR1) deletion enhances early inflammation in a mouse model of imiquimod-induced psoriasis, without altering the immune cell infiltrate. Mechanistically, myeloid S1PR1 deletion altered the formation of IL-1β, VEGF-A, and VEGF-C, and their receptors’ expression in psoriatic skin, which subsequently lead to reciprocal regulation of neoangiogenesis and neolymphangiogenesis. Experimental findings were corroborated in human clinical datasets and in knockout macrophages in vitro. Increased blood vessel but reduced lymph vessel density may explain the exacerbated inflammatory phenotype in conditional knockout mice. These findings assign a novel role to macrophage S1PR1 and provide a rationale for therapeutically targeting local S1P during skin inflammation.