Biology of patrolling monocytes in inflammation

The proposal addresses a novel and important question in physiology and disease: how do white blood cells help maintain a healthy blood vessel during responses to danger signal that may signify infection or inflammation. The endothelial cells are the first layer of cells that form the inside of blood vessels. They are submitted to various forms of stress, such as infection or damage in the underlying tissues. Endothelial cells causes blood vessel dysfunction and may result in clotting and increased inflammation, which in turn will compromise the delivery of oxygen and nutrients to tissues.

We have discovered that a subset of white blood cells called "Ly6C-low monocytes" (1) and their human homologues (2), continuously crawl on the endothelium of blood vessels (3). We also found that the nuclear receptor Nr4a1 is critical for the development of these Ly6Clow monocytes in mice (4). After some extensive work, we found that these monocytes survey endothelial cells, scavenge debris from the endothelial cells, and monitor their health. When the endothelial cells signal a 'danger' that could signify either cell death or viral infection, via a receptor that detects nucleic acids, the monocytes recruit and activate another type of white blood cells called neutrophils, which kills the endothelial cells. The monocytes then clean-up the endothelial cell debris. These findings are entirely novel and suggest important roles for monocytes in human diseases.

Therefore, we propose in this project to test this hypothesis, identify the underlying mechanisms, and investigate the role of Ly6C-low monocytes in kidney diseases or the growth of tumors, using mouse models and also investigating human monocytes.
The three aims of our projects are independent but complementary and should inform each other. We will investigate at the cellular level and at the molecular levels in vivo (aim 1 and 2), the mechanisms responsible for endothelial cell surveillance, orchestrated killing, and removal of endothelial cells by murine ly6C-low Nr4a1-dependent monocytes. Comparison will be made with human cells, to help identify conserved mechanisms.
The third aim of our study is to test the roles of ly6C-low Nr4a1-dependent monocytes in mouse models of kidney disease and tumor growth.
Although we currently hypothesize that Ly6Clow monocytes favour the renewal of the endothelium in a steady state, it is also possible that depending on the model, and on the magnitude and duration of the stimuli monocytes could also contribute to pathology via an increase in vascular or tissue damage.

The results from this programme of work should identify novel mechanisms of importance in the various diseases where the endothelium of blood vessels are affected, and thus original therapeutic approaches.


1. Geissmann F, Jung S, Littman DR. 2003. Blood monocytes consist of two principal subsets with distinct migratory properties. Immunity 19: 71-82
2. Cros J, Cagnard N, Woollard K, Patey N, Zhang SY, Senechal B, Puel A, Biswas SK, Moshous D, Picard C, Jais JP, D'Cruz D, Casanova JL, Trouillet C, Geissmann F. 2010. Human CD14dim monocytes patrol and sense nucleic acids and viruses via TLR7 and TLR8 receptors. Immunity 33: 375-86
3. Auffray C, Fogg D, Garfa M, Elain G, Join-Lambert O, Kayal S, Sarnacki S, Cumano A, Lauvau G, Geissmann F. 2007. Monitoring of blood vessels and tissues by a population of monocytes with patrolling behavior. Science 317: 666-70
4. Hanna RN, Carlin LM, Hubbeling HG, Nackiewicz D, Green AM, Punt JA, Geissmann F, Hedrick CC. 2011. The transcription factor NR4A1 (Nur77) controls bone marrow differentiation and the survival of Ly6C- monocytes. Nat Immunol 12: 778-85
5. Carlin LM, Stamatiades EG, Auffray C, Hanna RN, Glover L, Vizcay-Barrena G, Hedrick CC, Cook H.T, Diebold S & Geissmann F. 2013. Nr4a1-dependent Ly6Clow monocytes monitor endothelial cells and orchestrate their disposal. Cell 2013

The objectives of the proposed integrated programme of work are:
Aim 1 will investigate the mechanisms responsible for endothelial cell killing by Ly6Clow monocytes. We will analyse in vivo using intravital microscopy and electron microscopy the role of membrane dependent necroptosis, reactive oxygen species, NETs formation, IFNAR, and platelets, in Ly6Clow monocyte retention inside capillaries and TLR7 dependent necrosis of endothelial cells.
Aim 2 will test the hypothesis that NR4A1 controls the differentiation and homeostasis of murine and human Ly6Clow/CD14 dim monocytes. We will examine the role of NR4A1 in a conditional hematopoietic Nr4a1 deficient model, and perform RNA-seq and ChIP-seq analysis to analyse and compare the target genes of Nr4a1 in mice and human monocytes.
Aim 3 will test the hypothesis that Ly6Clow monocytes control endothelial damage in glomerulonephritis due to immune complex deposition and in a TLR7/8 sensitive tumor model. We will examine the role of Ly6Clow monocytes and TLR signalling in glomerulonephritis following injection of an antibody against the glomerular basement membrane (GBM) which generates a glomerular inflammatory response via immune complex formation in the glomerulus and in the vascularisation of B16F10 tumors.

Aim 1 and 2 will be started in parallel at the start of the project. They address at the cellular and molecular levels the mechanisms that control the differentiation and functions of ly6C-low monocytes and their human CD14dim counterparts. The experimental systems, technological approaches and mouse lines we will be using are in place in the laboratory, and we have assembled a team of outstanding collaborators with whom we have established collaborations.
Aim 3 will be started in year 3, to best incorporate results from the previous aims. This aim will investigate the roles of ly6C-low monocytes in pathological conditions such as glomerulonephritis and tumor growth, and the mechanisms involved

This project aims to characterise directly in situ and in vivo the molecular mechanisms and effector functions of a subset of monocytes that patrol the endothelium of blood vessels. The project will use a combination of genetically modified fluorescent protein reporter mice, gene expression studies, and cutting-edge intravital microscopy techniques to image mechanisms directly in situ in a living host in 'normal' steady state and inflammatory conditions.

Who will benefit from the proposed research?
In the short term, the main beneficiaries of this project internationally will be scientists in the fields of i) immunology, ii) cell motility and iii) biological imaging and locally iv) the immunology and medical communities at KCL v) existing students in the departments at KCL plus those joining the school of medicine as part of doctoral training programs.
In the longer term, the general public and patients with inflammatory disease should benefit from discoveries of the cellular and molecular mechanisms of inflammation, in terms of new drugs and diagnostic/prognostic tools.

How they will benefit
Communications and Engagement
As the project uses an innovative approach to attempt to describe a potentially novel form of leukocyte interaction with the endothelium it is envisioned that the work will result in the sharing of knowledge via a series of high impact publications, members of the team presenting data at international conferences and additional efforts detailed below to ensure open access to data

Data sharing
In addition to publications and conferences, imaging data formats will be kept consistent with the Open Microscopy Environment (OME; www.openmicroscopy.org) to make sharing of data and accession in future imaging databases simple. Images and data will also be deposited in the macrophage community website, www.macrophages.com an online resource for those interested in macrophages and their role as major effector cells in innate and adaptive immunity, of which Prof. Geissmann is a member of the steering committee.
Gene expression arrays, RNAseq and chromatin immunoprecipitation deep-sequencing projects will generate extensive amount of data. We will conform to instruction from the MRC for the proper management and Sharing of data. As per our current practice, raw expression data in a MIAME-compliant format, short sequence reads aligned to the latest human genome assembly, and relevant (anonymised) informations will be made publicly available using existing resources including www.ebi.ac.uk/arrayexpress for gene expression data, and the UCSC Genome Browser web interface to be easily accessible to the wider scientific community. Data will be uploaded at the time they will be generated, but access will be password protected until publication, at which time they will become freely accessible. Data sets will be also preserved in a centralised and secure back-up storage organised for this purpose by the KCL IT department. Hard drives are stored in a dedicated room with its own temperature control in our centre and data also backed up on tapes at another location in KCL.

Engaging with the general public:
Our work in this area will have three specific goals:
1) To promote a better understanding of science, health and research-related issues within the general population with specific reference to inflammation
2) To demonstrate that science and research is an exciting discipline.
3) To provide opportunities for younger people to learn about science as a future career.
- Engage students from local schools in research
- Host Open Days for the public to visit our Centre to show how basic science opens up new opportunities for diagnosis and treatment for people with arthritis.
-Provide opportunities for the post-doctoral fellows to learn about and actively participate in public engagement.