The effects of neutropenia and G-CSF on the hematopoietic stem cell

In chapter one an introduction to this thesis is given. Hematopoiesis is introduced,
as a process of blood formation from hematopoietic stem cell (HSC) to effector cell;
a differentiation process which takes place in the bone marrow. The indispensable
contribution of the HSC’s microenvironment (HSC niche) within this process is discussed.
Neutrophils, one of the effector cells coming forth from myeloid differentiation, is presented
as a possible contributor to the HSC niche. In the chapters that follow we aim to interrogate
the neutrophil as an HSC regulating niche cell by studying a neutropenia mouse model,
G-CSF contribution and zooming in on a neutropenic patient.
In chapter two we exploit our conditional knockout mouse model of neutropenia, where
HSCs remain genetically intact, to thoroughly test the neutrophil as an HSC regulating niche
cell. We observed that HSCs were kept quiescent in the absence of neutrophils, in itself
an intriguing counterintuitive finding. Upon transcriptional analysis of HSCs residing in a
neutropenic environment it was clear that HSCs were myeloid biased, yet not excessively
cycling to fill the void in the periphery. Interestingly HSCs did not show an age-related
immunophenotype as their control counterparts did. This spiked our interest, and we thus
compared transcriptomes of our model to readily available aged murine transcriptomes.
Leading us to the finding that neutrophils play a role in steady-state interferon tonus,
through natural killer cells, speculating on the consequential role of neutrophils in aging.
Chapter three focuses on the role of chronic exposure of G-CSF, one of the main
hematopoietic cytokines responsible for neutrophil production, in steady state
hematopoiesis and neutropenia. We demonstrate that long-term exposure to G-CSF at doses
relevant to the treatment of neutropenia does not attenuate the long-term competitive
repopulation ability of HSCs in a mouse model of neutropenia. HSCs in neutropenic mice
display transcriptional wiring consistent with endogenously elevated levels of G-CSF, but
administration of exogenous G-CSF does not induce exit of quiescence, DNA damage, or loss
of serial repopulation ability of HSCs.
In chapter four we describe a congenital neutropenic patient with a homozygous
mutation in the hinge motif of the G-CSF receptor (CSF3R). By analysis of the bone marrow
we conclude that the di-proline hinge motif in the extracellular cytokine receptor homology
domain of CSF3R is critical for adequate neutrophil production, but dispensable for in vivo
terminal neutrophil maturation. Our in vitro experiments suggest that crippling mutations
of the hinge motif cause hypo-responsiveness to G-CSF treatment, marking them as a
separate clinical entity.
Finally, in chapter five, divers aspects of the thesis are summarized, discussed and put
into future perspective.