High-parameter cytometry unmasks microglial cell spatio-temporal response kinetics in severe neuroinflammatory disease

Abstract

Background Differentiating infiltrating myeloid cells from resident microglia in neuroinflammatory disease is challenging, because bone marrow-derived inflammatory monocytes infiltrating the inflamed brain adopt a ‘microglia-like’ phenotype. This precludes the accurate identification of either cell type without genetic manipulation, which is important to understand their temporal contribution to disease and inform effective intervention in its pathogenesis. During West Nile virus (WNV) encephalitis, widespread neuronal infection drives substantial CNS infiltration of inflammatory monocytes, causing severe immunopathology and/or death, but the role of microglia in this remains unclear. Methods Using high-parameter cytometry and dimensionality-reduction, we devised a simple, novel gating strategy to identify microglia and infiltrating myeloid cells during WNV-infection. Validating our strategy, we (1) blocked the entry of infiltrating myeloid populations from peripheral blood using monoclonal blocking antibodies, (2) adoptively transferred BM-derived monocytes and tracked their phenotypic changes after infiltration and (3) labelled peripheral leukocytes that infiltrate into the brain with an intravenous dye. We demonstrated that myeloid immigrants populated only the identified macrophage gates, while PLX5622 depletion reduced all 4 subsets defined by the microglial gates. Results Using this gating approach, we identified four consistent microglia subsets in the homeostatic and WNV-infected brain. These were P2RY12hi CD86−, P2RY12hi CD86+ and P2RY12lo CD86− P2RY12lo CD86+. During infection, 2 further populations were identified as 'inflammatory' and 'microglia-like' macrophages, recruited from the bone marrow. Detailed kinetic analysis showed significant increases in the proportions of both P2RY12lo microglia subsets in all anatomical areas, largely at the expense of the P2RY12hi CD86− subset, with the latter undergoing compensatory proliferation, suggesting replenishment of, and differentiation from this subset in response to infection. Microglia altered their morphology early in infection, with all cells adopting temporal and regional disease-specific phenotypes. Late in disease, microglia produced IL-12, downregulated CX3CR1, F4/80 and TMEM119 and underwent apoptosis. Infiltrating macrophages expressed both TMEM119 and P2RY12 de novo, with the microglia-like subset notably exhibiting the highest proportional myeloid population death. Conclusions Our approach enables detailed kinetic analysis of resident vs infiltrating myeloid cells in a wide range of neuroinflammatory models without non-physiological manipulation. This will more clearly inform potential therapeutic approaches that specifically modulate these cells.

Impact of Dietary Fiber on West Nile Virus Infection

“…For immunohistochemistry, colon tissue was fixed overnight in 2% PFA and subsequently placed in a series of solutions of progressively increasing sucrose concentration (10%, 20% and 30% sucrose in PBS), before being embedded in optimum cutting temperature compound (O.C.T. ; Tissue-Tek) and frozen in hexane pre-chilled in liquid nitrogen, as previously described ( 26 , 31 ). Tissue blocks were sectioned (8–9 µm), fixed in methanol, rinsed in tris-buffered saline with 0.05% Tween 20 (TBST) and blocked with 10% FCS before being stained with primary fluorophore-conjugated antibodies targeting WNV non-structural protein 1 (NS-1) and FOX-3 in neuronal nuclei (NeuN).…”

Early microglial response, myelin deterioration and lethality in mice deficient for very long chain ceramide synthesis in oligodendrocytes

“…Brains were processed into single cell suspensions for spectral cytometry, as previously described (Spiteri et al, 2022; Spiteri et al, 2021). Briefly, brain cells were isolated using a 30%/80% Percoll gradient after homogenizing tissue on a gentleMACS dissociator (Miltenyi Biotec).…”

Microglia and monocytes in inflammatory CNS disease: integrating phenotype and function

“…However, during inflammation in response to CNS perturbation, microglia become reactive or activated , a state in which they upregulate CD45, partially or totally retract their cytoplasmic extensions and increase their somatic volume to adopt a more amoeboid morphology [ 266 , 303 ]. Microglia are also joined by a substantial infiltrate of BM-derived monocytes [ 298 ], both of which similarly express typical myeloid markers [ 19 , 204 , 298 , 299 , 332 ] (such as CD68, Fig. 1 ).…”

“…Such infiltrating monocyte-derived cells (MC), like monocyte-derived macrophages (MDM) and dendritic cells (moDC), can adopt a microglia-like phenotype upon entry into the inflamed brain. While these cells do not necessarily upregulate genes expressed by homeostatic microglia [ 25 , 66 ], the phenotypic similarities between populations of resident and infiltrating myeloid cells may approximate one another in neuroinflammation, hampering accurate identification of their respective functions in disease [ 298 , 299 ].…”

“…↓↓ CX3CR1, F4/80, TMEM119 and CD68; ↓ P2RY12 (WNV infection at dpi 7, spectral and conventional flow cytometry [ 298 ])…”