Shaping the neutrophil: morphological, genomic, and functional maturation

Neutrophils are the most numerous white blood cell (millions per millilitre of blood) and are crucial for defending us against pathogens. At the same time the neutrophil is the most short-lived white blood cell (hours to days). Hence, billions of neutrophils need to be produced by the bone marrow every day to keep the numbers in the blood stable. When these numbers drop due to disease and/or drug treatments, the patient is exposed to an acute threat of severe infections. Additionally, changes in the neutrophil production process, leading to the release of immature, dysfunctional, or hyperactive neutrophils, have been implicated in many common chronic inflammatory diseases and cancer. . Despite the importance of healthy neutrophil production, also called granulopoiesis, the molecular mechanisms driving this process are poorly understood.

During granulopoiesis, progenitor cells go through changes and specializations to become mature, fully functional neutrophils. One of the most notable and extensive changes happens to the cell's nucleus. Over the course of multiple days, a simple, round nucleus changes shape to become a complex, segmented nucleus consisting of multiple lobes. The shape of the nucleus has been used for decades to signal how far the neutrophil maturation in the bone marrow has progressed, yet no-one understands why or how this unique shape is formed. This is partly because it has been difficult to directly link the nuclear shape to the more detailed descriptions of neutrophil maturation stage provided by other technologies.

Our research group has developed a novel method to define nuclear shape in much more detail and in live cells, aided by state-of-the-art 3D microscopy and Artificial Intelligence. Here, we propose to elucidate the molecular programming of neutrophil nuclear shape for the first time. We suspect the gradual changes in nuclear shape are correlated to the gradual acquisition of key neutrophil functionalities during granulopoiesis, as has been suggested before by us and others. Hence an improved understanding of the molecular pathways that initiate and drive these spectacular changes will improve our understanding of the crucial process of granulopoiesis and how we might manipulate or stimulate it.

The nucleus' main function is to safely and tightly package the DNA, while also allowing the activation of specific genes encoded in the DNA. The activation of a unique set of genes determines cellular functionality and behaviour. To better understand the molecular mechanisms behind the changes seen during granulopoiesis, we propose to study the nucleus at different levels of magnitude. We will analyse interactions between DNA regions both far apart and DNA regions closer together. This will flag the genes that likely have a role in driving granulopoiesis. We expect for the first time to link the nuclear shape, DNA interactions, and genetic programming during neutrophil maturation, to obtain a comprehensive picture of the drivers of granulopoiesis. We will use bioinformatics solutions to tie these different novel datasets together for improved interpretation.

Next, the most promising drivers of granulopoiesis will be removed from neutrophils by gene editing techniques. This will allow us to directly assess the nuclear shape, functionality, and behaviour of the edited cells, validating the role of the putative drivers in changing nuclear shape and/or neutrophil function during granulopoiesis.

The proposed work will greatly improve our understanding of the molecular programming of neutrophil production, a fundamental process critical to maintaining our health. This will ultimately bridge the existent sporadic mouse and human datasets and will open up possibilities for more selective interference with neutrophil functions.

During healthy neutrophil development its nucleus goes through dramatic morphological changes, from a simple round to a multi-segmented, lobulated nucleus. Additionally, neutrophils acquire different functions at different stages of development. We have identified and validated several transcription factors (TFs) that play a role in neutrophil morphological development and/or functional responses.

Here we will use a combination of cutting-edge microscopy and genomic approaches to explore the hypothesis that the changes in nuclear morphology and the acquisition of effector functions during neutrophil maturation are transcriptionally driven. To test this, detailed tracking of 3D nuclear morphology in live cells will be paired with scRNAseq of heterogeneous populations of neutrophils. We will also elucidate how chromatin spatial rearrangements help with obtaining a multi-segmented nuclear shape, by capturing changes in long- and short-range chromatin interactions over the time course of in vitro neutrophil maturation.

We pose that distinct TF-target modules control specific neutrophil features. Computational approaches, including multivariate time series analysis, will determine relationships between time, morphological features (nuclear size, shape, segmentation, etc), and genomic factors (gene expression, accessibility, chromatin topology) in the process of neutrophil maturation. Moreover, the multi-omics analyses will construct a TF-Target interaction network and predict key TFs likely to be involved. These predictions will be validated by assessing how genetic deletion of the TFs affects morphological or functional maturation of neutrophils.

This project will provide new answers to fundamental questions of neutrophil biology, generate new standards for high resolution morphological, genomic, and functional phenotype assignment across the neutrophil maturation trajectory, and identify key regulatory modules controlling neutrophil morphology and functions.