Combining in vivo spectral biphoton imaging, multiparametric cytometry and mouse genetics to characterize neuroinflammation dynamics and function in EAE models. – NeuroInflamDyn

1) The aim is to obtain precise timelines of microglial cell (expressing CD11c-EYFP) activation and myeloid cell (expressing LysM-EGFP) recruitment into the parenchyma, their relative spatial distribution, changes in morphology as they evolve towards a macrophagic activity inside the spinal cord tissue as well as the consequence of their interactions with neurons in terms of axon (Thy-CFP) degeneration.
2) The aim is to further characterize the immune cascade and axon losses concomitant with the amplification of motor deficits and with the disease remission phase.
We perform multiparametric flow cytometry on dissociated spinal cord and blood samples collected from individual animals at remarkable stages of EAE progression defined in aim 1. To identify the cell subpopulations that control disease progression, we dynamically correlate the relative frequencies and phenotypes of those cells with the occurrence of the imaging biomarkers found in 1.
3) The aim is to undertake a functional characterization of the DC/monocytes subtypes7 involved during the whole cascade of neuroinflammation spanning from the initiation of neuronal damage to the remission phase. By using bone marrow-reconstituted mice, we will modulate only bone marrow derived leukocytes without affecting resident microglia. We will take advantage of innovative mouse models in which multi-task recombinant cassettes allow either cell tagging with a fluorescent protein or conditional/constitutive ablation of a given cell type to characterize the selective contribution of each subtype on the disease progression.
4) The aim is to provide the demonstration that the methodology set up in 1 & 2 objectives is adequate to perform preclinical research and refined drug testing. As a proof of concept we will search whether VEGF interferes with EAE immunity and modifies the EAE spinal cord immune microenvironment.

In the human disorder multiple sclerosis (MS) and in the model experimental autoimmune encephalomyelitis (EAE), macrophages predominate in demyelinated areas and their numbers correlate to tissue damage. They arise from resident microglia and infiltrating, peripherally derived monocytes. We tracked the dynamics of the innate response in relation with axon damage and clinical signs. We induced EAE in transgenic LysM-EGFP//CD11c-EYFP//Thy1CFP reporter mice and used a combination of high content flow cytometry, immunofluorescence and dynamic spectral two-photon imaging to characterize the distribution, morphology and activity of microglia and blood-derived infiltrating myeloid cells in live mice. We identified a stepwise innate immune program with circulating neutrophils and P1 monocytes invading the spinal cord through the meninges. Plaques development followed a gradient from the external part of the white matter towards deeper area, contrary to microglial activation. Maturation of P1 monocytes towards P3 monocyte-derived dendritic cells occurred in plaques. The occurrence of this major and transient cell subset characterized a stabilization of the axon degradation and clinical signs and the predominance of microglia to process axon debris in plaques.

The appreciation of the distinct dynamics and functions of innate cell subsets is a crucial issue in CNS inflammatory diseases. We precisely discriminate components of the innate immune response in individual LysM-EGFP//CD11c-EYFP//Thy1-CFP EAE-induced mice, over time. These data were combined with slices imaging data and in vivo non invasive recurrent observations of the immune, neuronal and vascular compartments of the spinal cord. This multiparametric data set revealed patterns of spatial and temporal distributions of different cell subsets of the innate immune system. For example, neutrophils and circulating monocytes invade the external part of the spinal cord likely through the meninges, then distribute all over the white matter whereas using the vascular scaffold where part of them differenciate into dendritic cells (moDCs) within plaques. This prominent accumulation correlates with axon degeneration in the superficial dorsal columns. By contrast, microglial cells became activated all over the spinal cord before accumulating to plaques at the time of the maximal accumulation of moDcs. Strickingly, these events are coincident with a stabilization of the clinical signs.
Therefore the monitoring of the distinct dynamics and functions of these innate subpopulations might help designing rational strategies to manipulate them for therapeutic purposes. This represents our future prospects.

Collaborative: Alexandre Jaouen, Céline Caravagna, Sophie Desplat-Jégo, Keith Fenrich, Hervé Luche, Genevieve Rougon, Marie Malissen & Franck Debarbieux. Dynamics and distribution of microglia and myeloid cells infiltrating the central nervous system during MOG-induced EAE (submitted)
Rougon G., Brasselet S., Debarbieux F. (in press) Advances in intravital optical imaging of the central nervous system in rodents. Brain Plasticity ISSN:2213-6304
Chrobok NL, Jaouen A, Fenrich KK, Bol JG, Wilhelmus MM, Drukarch B, Debarbieux F, van Dam AM (2016) Monocyte behaviour and tissue transglutaminase expression during experimental autoimmune encephalomyelitis in transgenic CX3CR1(gfp/gfp) mice. Amino Acids. 2016 Nov 9. [Epub ahead of print] ISSN: 0939-4451 ;
Caravagna C., Jaouen A., Debarbieux F., Rougon G.(2016) Innovative mouse models for Imaging neuroinflammation Current Protocols in Mouse Biology 6(2):131-47. doi: 10.1002. ISBN :978-0-470-94239-0

This application which gathers neurobiologists and immunologists has for objective to analyze the interdependence of inflammation, neurodegeneration and disease progression in multiple sclerosis. As a model, experimental autoimmune encephalomyelitis, which recapitulates the pathological hallmarks of MS, is induced in Thy1-CFP//LysM-EGFP//CD11c-EYFP triple mutant reporter mice. We will combine our recent methodological developments in (1) advanced spinal cord in vivo 2 photon spectral imaging, (2) high content flow cytometry analysis and (3) mouse genetics to (1) characterize imaging markers of disease progression, (2) obtain a global picture of the immune cells in spinal tissue over time as well as (3) a functional characterization of the dendritic cells/monocytes subtypes involved, (4) explore the effect of VEGF on the immune signature to provide the proof that the methodology is suited to perform refined drug testing.