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Abstract

Hematopoietic stem cells (HSCs) are critical components of the hematopoietic system and are responsible for renewing all blood cell lineages throughout life. These cells are quiescent and reside in niches in the bone marrow (BM). Over the past decade, our group and others have discovered that inflammatory stress impacts quiescent HSCs in vivo, leading to their activation. However, the dynamics, heterogeneity, and mechanisms underlying stress-induced activation of HSCs remain unclear. In this thesis, I unraveled the mechanisms regulating HSCs proliferation and recovery in response to acute treatment with the proinflammatory cytokine interferon alpha(IFNα) by initially determining three-time points representing the sensing, proliferation, and recovery phases of HSCs' proliferative response to acute IFNα treatment. Using time series bulk RNA sequencing (RNAseq), I identified distinct molecular patterns and changes in the activation and repression of various biological categories in HSCs. Surprisingly, even after returning to a quiescent state 72 hours (h) post-treatment, HSCs remained metabolically active and underwent a significant metabolic shift towards oxidative phosphorylation (OXPHOS). In addition, the tricarboxylic acid cycle (TCA), pentose phosphate pathway (PPP), fatty acid, and purine metabolism were reduced, and HSCs showed decreased myeloid priming and bias. Thus far, little is known about the dynamics and heterogeneity of these stress responses in the whole hematopoietic stem and progenitor (HSPC) cells. Inflammation-induced marker changes in the HSPCs compartment make it challenging to investigate the heterogeneity in the inflammatory response in HSPCs. Thus, I employed a single-cell (Sc) time series RNAseq experiment to study the heterogeneous and dynamic impacts of IFNα on HSPCs. The results showed heterogeneity in the response of HSPCs to IFNα, with HSCs being the strongest responders based on their gene expression changes. In collaboration with Brigitte Bouman and Dr. Laleh Haghverdi at the MDC in Berlin, we developed and used a response-pseudotime inference approach to analyze the scRNAseq data and identified global and cell type-specific inflammation signatures, revealing unique molecular patterns of gene expression and biological processes in response to IFNα. Interestingly, we were able to associate reduced myeloid differentiation programs in HSPCs with a reduced abundance of myeloid progenitors and differentiated cells following IFNα treatment. Taken together, the single-cell time series analyses have allowed us to unbiasedly study the heterogeneous and dynamic impact of IFNα on the HSPCs. In addition to investigating the dynamics and heterogeneity of the response of HSCs to IFNα, I compared the immediate transcriptional response of HSCs to various other proinflammatory cytokines. This analysis showed that IFNs, TNFα, ILs, and mimetics of viral and bacterial infections induced unique gene alterations in HSCs, underscoring the diversity of cytokine responses in these cells. Finally, I investigated how the baseline levels of these proinflammatory cytokines regulate hematopoiesis. Analysis of the hematopoietic system in Ifnar-/-Ifngr-/- (2KO) and Ifnar-/-Ifngr-/-Tnfrsf1dKOIl1r-/- (5KO) mice under homeostatic conditions revealed a decrease in HSCs and LSKs compared with wild-type (WT) mice. Furthermore, HSCs from these cytokine receptors knockout (KO) mice showed impaired colony-forming capacity and early competitive advantage. Interestingly, 5KO mice also showed a delayed recovery of HSCs cycling following 5-FU treatment. In addition, bulk RNA sequencing of 5KO HSCs revealed altered cell cycle pathways. Overall, these results underscore the essential role of proinflammatory cytokines in regulating HSC function during homeostasis. In conclusion, this thesis comprehensively explains the transcriptional changes within the HSPCs population in response to proinflammatory cytokines, focusing on IFNα.