Aim1: Understand the functional heterogeneity of CNS barrier macrophages upon challenge
1A/ Single cell heterogeneity of barrier macrophages upon CNS challenge
Meningeal macrophages are highly responsive to CNS pathogens. I hypothesize that MF1 and MF2 barrier macrophages are not equally potent to protect the CNS, and differ in their ability to detect pathogens, to switch on their activation program and to recruit immune cells from the periphery.
To test this hypothesis, I compared MF1 and MF2 ability to regulate an immune response in vivo, using intracranial injection of LPS. Our data indicate that the MF2 population is more responsive and up-regulates neutrophil chemoattractants more efficiently. To have a broader view of macrophage function, I performed single cell RNA seq on MF1 and MF2 population after LPS or mock- injection and analysed the changes in myeloid populations and gene expression.
1B/ Functional specialization of barrier macrophages upon CNS challenge
I also performed intravital imaging of meningeal macrophages in LPS and mock-injected reporter mice (MHC-II-GFP mice injected with red dextran). I have found that 10kDa red fluorescent dextran injected in the vasculature can specifically and efficiently target meningeal macrophages, based on the affinity for the mannose receptor CD206. Thus, MF1 will be RedDextran+ MHC-II-GFP+ and MF2 will be RedDextran+ MHC-II-GFP-. Of note, even though LPS can affect MHC-II expression in activated macrophages in a dose and time-dependent manner, we found that MHCII-GFP positive and negative populations were stable over the course of our 6 hours challenge. I started to assess MF1 and MF2 dynamic behavior following LPS challenge using Imaris Imaging software, but we had issues wih MHC-II GFP mice and had to pause this part of the research.
1C/ Mechanism allowing specialization of barrier macrophages upon CNS challenge
To understand the mechanism allowing higher MF2 responsiveness, I used the single cell analysis performed in 1A/ to identify candidate signalling pathways specifically up-regulated in MF2 compared to MF1 after LPS challenge. I then used transgenic mice to validate those results, such as STAT1 signaling.
Aim2: Reverse the age-driven impairment of barrier macrophages
2A/Effect of aging on barrier macrophages
While looking at the evolution of the myeloid landscape upon natural aging, I noticed a progressive loss of the MF2 macrophage subpopulation, together with an enrichment of the MF1 population.
I identified the contribution of the bone-marrow to the MF1 population over time, using tamoxifen-inducible CX3CR1-CreER mouse lines. Those transgenic mice are commonly used to distinguish resident macrophages and peripheral blood myeloid cells . Preliminary data using lineage tracing experiments in mice pulsed with tamoxifen at 1 MO indicate that peripheral cells engraft the tissue continually over time and convert into age-associated MF1 macrophages (not shown).
2B/Rejuvenation of barrier macrophages
To know if failure to recruit neutrophils upon aging is linked with the shift from the initial MF2 population to the age-associated MF1 population, I focused on the mechanism leading to this MF2 -> MF1 transition in order to block it. Our data using LysM-Cre DTRfl/fl mice (in which all macrophages, monocytes and neutrophils express DTR) indicate that diphteria toxin (DT) can efficiently deplete meningeal macrophages in addition to the transient loss (for 2-3 days) of monocytes and neutrophils. This is followed by progressive and preferential repopulation of the meninges by the potent MF2 population within two weeks. This phenocopies the myeloid landscape of young mice, and I refer to those mice as ‘rejuvenated’ mice.
2C/Blocking the aging of barrier macrophages
I expect that macrophage turnover and aging is regulated by local signals from other meningeal populations. To understand the signals driving macrophage aging, I performed a microarray of MF1 versus MF2 population in adult mice (d50) in the steady state. Upstream regulator analysis revealed that even in the steady state, inflammatory cytokines (e.g. IFNγ) are major upstream regulators of MF1 transcriptome (not shown). Our data confirmed that transgenic aged mice lacking those cytokines have a decrease proportion of MF1, and that the myeloid landscape in their meninges looks ‘younger’ (not shown).
2D/Effect of reversing macrophage aging on CNS barriers
I injected DT in aged control mice and LysM DTRfl/fl mice to ‘rejuvenate’ them, and challenge them with LPS. 24 hours later, I assessed antimicrobial functions and leucocyte chemoattraction as previously described. Unexpectedly, mice in which MF2 population is restored are more sensitive to LCMV.
