Characterization of neutrophil interactions with meningeal macrophages during central nervous system autoimmunity

Neuroinflammation represents a crucial aspect of multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE). In this context, an intricate network of cellular interactions, involving both central nervous system (CNS)-invading leukocytes and -resident cells, may contribute to disease pathogenesis and clinical disease manifestation. In particular, neuroinflammatory processes compartmentalized in the meninges are gaining recognition as important drivers of autoimmune brain pathology. Indeed, subpial demyelinating lesions, an exclusive hallmark of MS, are generally associated with overlying foci of meningeal inflammation. In line with this, recent studies found that the presence of neutrophils in the leptomeninges of MS patients correlated with more severe subpial demyelination. However, how neutrophils contribute to MS and EAE pathogenesis is not clearly understood, and the MAIN AIM of this thesis was to study neutrophil communication with other immune cells in the context of meningeal inflammation. Starting from the analysis of single cell RNA sequencing (scRNA-seq) data, we unveiled the transcriptional plasticity of neutrophils at the onset and chronic phase of EAE. Indeed, they partitioned into three clusters along a maturation trajectory, each one characterized by a distinct functional profile. Overall, neutrophils at EAE onset turned out to be more activated, motile, and prone to interact with other cells compared to the chronic stage. In a bid to validate these transcriptomic observations and gain pathophysiological insight into the role of neutrophils during meningeal inflammation, we evaluated their motility behavior in the spinal cord (SC) subarachnoid space at EAE onset using two-photon laser scanning microscopy (TPLSM). Approximately two-thirds of extravasated neutrophils were retained in proximity to meningeal blood vessels, where they performed swarming movements suggestive of interactions with local cells. Given their abundance in the perivascular area, we hypothesized that meningeal macrophages could represent candidate cellular interactors with extravasated neutrophils. Indeed, TPLSM and immunofluorescence experiments confirmed that neutrophils and macrophages establish contacts in the SC leptomeninges in the acute EAE phase, suggesting that this myeloid interplay could contribute to amplify the inflammatory process during CNS autoimmunity. In line with this hypothesis, neutrophil systemic depletion starting from the EAE pre-clinical phase was associated with a weaker activation state of leptomeningeal macrophages. Therefore, we examined scRNA-seq data to identify candidate molecular mediators of this detrimental interaction. After finding that neutrophil clusters expressed specific patterns of chemotactic receptors and adhesion molecules, potentially underlying different migratory and interactive behaviors, we decided to test the role of LFA-1 integrin in the neutrophil-macrophage axis. Interfering with LFA-1 function both in vivo and in vitro resulted in a general increase of neutrophil motility parameters and a significant reduction of contacts with macrophages, corroborating the involvement of LFA-1 in mediating this myeloid crosstalk. Finally, post-mortem brain sections of MS patients were stained to verify that such interactions could occur during human pathology. Our preliminary results showed co-localization of neutrophils and MHCII+ cells, likely macrophages, in leptomeningeal areas overlying subpial demyelinating lesions. Moreover, neutrophils were often found in close vicinity to MHCII/CD68+ cells in the leptomeninges, hinting at possible interactions between these cells also in MS. Overall, our EAE and MS data show an interplay between invading neutrophils and leptomeningeal macrophages and suggest that interfering with this myeloid cell-cell communication may reduce neuroinflammation and neurodegeneration and have a therapeutic effect in CNS autoimmune diseases.