Cellular and molecular mechanisms driving the expression of mobilizing cytokines
Sonata program. National Science Centre (2020-2023).
All blood cells are derived from hematopoietic stem cells (HSCs). HSCs differentiate to rapidly proliferating progenitor cells, that finally give rise to mature blood cells. The process of blood cells production (hematopoiesis) takes place in the bone marrow niches and is tightly regulated. In steady-state conditions only mature cells leave the bone marrow and supplement peripheral circulation. However, during hematopoietic stress, such as inflammation or intensified demand for the blood cells, hematopoiesis accelerates and more cells are released from bone marrow to peripheral blood in a process referred to as mobilization. The pool of mobilized cells includes cells on earlier differentiation stages and HSCs.
The mobilization allows for crucial adaptation of the organism to hematopoietic stress. Previous research resulted in the identification of several cytokines and growth factors that trigger mobilization, the mostly studied being G-CSF, SDF-1a, GM-CSF, SCF. This knowledge found significant clinical application and nowadays G-CSF is widely used to induce mobilization in patients with neutropenia or to harvest HSCs for subsequent transplantation.
Surprisingly, despite the successful clinical translation of the research on G-CSF and other mobilizing factors, the mechanisms that drive the first stages of mobilization were overlooked.
It still remains unknown i) which cell populations rapidly release G-CSF or other mobilizing cytokines upon stress signals in vivo and ii) which transcription factors regulate mobilization and drive their expression. Thus, we still do not understand the biological basics of the process that found crucial clinical application in modern hematology. Our project aims to address this gap in knowledge.
Our central hypothesis is that there are certain cell populations of monocytes, B cells and endothelial cells that transduce the hematopoietic stress signals to rapid production of mobilizing factors. We hypothesize that the rapid induction of mobilizing factors is mediated by inhibition of heme-sensing transcriptional repressors from Rev-erb and Bach families.
We have recently found that cobalt protophorphiryn IX (CoPP) strongly induces endogenous expression of G-CSF and other mobilizing cytokines in vivo. To our best knowledge, this is the only known compound that significantly induces endogenous G-CSF expression and mimics the hematopoietic stress signals, but does not induce systemic inflammation.
Here we use this advantage and propose a research model based on CoPP to investigate the cellular and molecular mechanisms that drive expression of G-CSF in vivo.
We propose to verify the central hypothesis of the project within three specific aims.
Aim 1 assumes the identification of the cell populations that produce G-CSF in response to CoPP. To check whether cells among monocytes, B-cells and endothelial cells induce expression of G-CSF, but also not to overlook other possible populations, we propose to generate the G-CSF-reporter mouse model.
Within Aim 2 we propose to verify our working hypothesis, that transcription factors from Rev-erb and Bach families drive the expression of G-CSF during mobilization in vivo. We plan to pharmacologically augment and inhibit the transcriptional repression mediated by Rev-erbs in vivo and check whether this will modulate CoPP-induced expression of G-CSF. We will also use the existing knock-out models of Bach 1 and 2 to verify their role in the hypothesized mechanisms.
In the Aim 3 we hypothesize that mobilization affects several interconnected pathways that are cell specific. Therefore, to explore what are the molecular mechanisms in distinct cell fractions we propose to perform single cell RNA sequencing. We will analyze which pathways are induced after CoPP in distinct cell types and therefore explore the investigated mechanism on systemic level with single-cell resolution.
As the expected outcome of the project, we hope to understand the initial stages of mobilization that were previously overlooked. This will provide the comprehensive picture of the mobilization process, including the mechanisms involved in hematopoietic response to stress signals. The results of the project may find the potential clinical application. Identification of mechanisms inducing the expression of endogenous mobilizing factors opens new possibilities to pharmacological induction of mobilization in patients, especially in those who do not respond to current protocols.