A role for myeloid cell interplay in meningeal inflammation during central nervous system autoimmunity

Neuroinflammation is a complex process that contributes to the development of several neurodegenerative conditions. The pathogenesis of inflammatory central nervous system (CNS) diseases involves the recruitment of peripheral immune cells and the production of inflammatory mediators such as cytokines, chemokines, and reactive oxygen species. In addition, meningeal inflammation can also favor CNS dysfunction and has been recently described as a key pathological feature of several neurodegenerative pathologies, such as multiple sclerosis (MS). A growing body of evidence suggests a pivotal contribution of neutrophils to MS development. The link between neutrophil infiltration of the leptomeninges and MS pathogenesis has been shown by pioneer studies using either samples from MS patients or experimental autoimmune encephalomyelitis (EAE), the animal model of MS. Particularly, neutrophil extravasation in the sub-arachnoid space (SAS) correlates with a more severe disease outcome. The accumulation of neutrophils into the CNS and meninges is associated with the activation of CNS-resident sentinel cells (e.g., CNS resident borderassociated macrophages and parenchymal microglia), mediated by tissue danger signals. Previous studies suggested that both border-associated macrophages and microglia may recall peripheral neutrophils and regulate their pro-inflammatory functions. On the other hand, CNS-invading neutrophils could influence the function of resident phagocytes inducing the release of inflammatory mediators further boosting the pathological processes sustained by local immune cells. This project aimed at characterizing the role of the crosstalk between neutrophils infiltrating the spinal cord (SC) SAS during early EAE and leptomeningeal macrophages in the disease pathogenesis, taking advantage of our cutting-edge live imaging platform. Firstly, neutrophil accumulation studied in EAE mice by flow cytometry highlighted a peak of infiltration at EAE onset, both in the SC leptomeninges and parenchyma. Interestingly, immunofluorescence staining on whole mount preparations of SC leptomeninges revealed that infiltrating neutrophils remained mainly compartmentalized close to meningeal vessels. To better understand the phenotype of infiltrating neutrophils and their role during EAE-associated neuroinflammation, we performed single-cell RNAsequencing (scRNA-seq) experiments at disease onset and during the chronic phase. Our data showed that SC-infiltrating neutrophils at EAE onset were enriched in cells with a phenotype characterized by migration and cell-cell interactions compared to those from the chronic phase of the disease. This data was supported by in vivo two-photon laser scanning microscopy (TPLSM) functional studies during early EAE, confirming the highly migrating neutrophil phenotype, with a predominant fraction of infiltrating neutrophils moving close to leptomeningeal blood vessels and performing swarming, suggesting their potential engagement in cell-cell contacts. We next performed TPLSM experiments and demonstrated the capacity of neutrophils to interact with leptomeningeal macrophages in CX3CR1-GFP mice with EAE. In addition, we performed whole mount preparations of SC leptomeninges, in which we further confirmed that infiltrating neutrophils compartmentalized near blood vessels are engaged in close contacts with leptomeningeal macrophages. Moreover, our scRNA-seq results identified leukocyte function associated antigen-1 (LFA-1) integrin as a potential molecular mediator of neutrophil compartmentalization and myeloid cellular interplay within the SC SAS. Indeed, TPLSM experiments showed that in vivo LFA-1 blockade led to a progressive decompartmentalization of leptomeningeal neutrophils, together with a time-dependent decrease in the number and duration of neutrophil-macrophage contacts. In addition, by using wide-field microscopy and a customized live imaging system of in vitro co-cultures, we confirmed that neutrophil contacts with leptomeningeal macrophages are controlled by the LFA-1 integrin. Finally, we checked the therapeutic relevance of blocking the LFA-1-dependent interactions between neutrophil and leptomeningeal macrophages by performing intrathecal treatment with an anti-LFA-1 antibody in EAE mice. Our local therapeutic approach at disease onset determined a significant amelioration of EAE clinical course and a reduction of the classical neuropathological hallmarks, such as areas of inflammation, demyelination, and activation of meningeal and parenchymal resident macrophages. Collectively, our data indicate that myeloid cell interactions at CNS borders may contribute to inflammation amplification and disease development suggesting that interfering with meningeal neutrophil-macrophage crosstalk may have therapeutic importance for CNS neuroinflammatory and autoimmune diseases.