Periodic Reporting for period 1 - MacMeninges (Control of Central Nervous Sytem inflammation by meningeal macrophages, and its impairment upon aging)
Reporting period: 2019-05-01 to 2021-04-30
Recent data indicate that the immune system in the central nervous system (CNS) is linked with inflammatory and neurodegenerative diseases . Due to the presence of the cranial bone, which limits tissue expansion, brain edema and CNS inflammation have to be controlled . Similar to other barrier surfaces, the surface of the CNS is connected to the periphery by layers of highly vascularized membranes, the meninges, populated by a myriad of resident immune sentinels (such as macrophages) that block threatening pathogens . Due to their strategic location at the interface between the periphery and the brain, the meninges function as the first line of protection of the CNS and represent a major site of immune cells recruitment to block further neuroinvasion . A breach in this protective system can allow the spread of neuroinvasive pathogens (e.g. HIV, Zika) with consequent CNS damage4. However, as meninges get easily inflamed and support robust inflammatory reactions, their overactivation can drive neuroinflammation in different contexts such as autoimmune diseases, migraine, stroke or microbial infections4.
Waisman A, Liblau RS, Becher B. Lancet Neurol. 2015;14(9):945-955.
Manglani M, McGavern DB. Curr Opin Virol. 2017;28:116-126.
Mowat AM, Scott CL, Bain CC, 2017 Nat Med. 2017 Nov 7;23(11):1258-1270.
Rua R, McGavern DB. Trends Mol Med. 2018.
Waisman A, Liblau RS, Becher B. Lancet Neurol. 2015;14(9):945-955.
Manglani M, McGavern DB. Curr Opin Virol. 2017;28:116-126.
Mowat AM, Scott CL, Bain CC, 2017 Nat Med. 2017 Nov 7;23(11):1258-1270.
Rua R, McGavern DB. Trends Mol Med. 2018.
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.
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.
This project is novel as it explores the functional defects occurring at the brain borders, that could create a breach and contribute to cognitive disorders in the aging population.
This project is thus expected to lead to a breakthrough in the context of age-driven neurocognitive disorders and to pioneer a new field of neuro-immunology. Advancements should lead to the development of new therapeutic strategies focusing on brain surface and resident sentinels to restore CNS immunity in the aging population.
This project is thus expected to lead to a breakthrough in the context of age-driven neurocognitive disorders and to pioneer a new field of neuro-immunology. Advancements should lead to the development of new therapeutic strategies focusing on brain surface and resident sentinels to restore CNS immunity in the aging population.