Neuroinflammation is a complex inflammatory process occurring within the central nervous system (CNS). It is mediated by the production of cytokines, chemokines, reactive oxygen species and secondary messengers produced and secreted by CNS-resident cells and recruited immune cells. Recent studies highlighted the key role of neutrophils as crucial players in chronic inflammatory diseases including CNS disorders such as Alzheimer’s disease (AD) and multiple sclerosis (MS). Previous studies on experimental autoimmune encephalomyelitis (EAE), the animal model of MS, have shown that neutrophils in the CNS are localized in close proximity to damaged tissues, such as demyelinated areas and leaked vessels, suggesting their potential detrimental role in the pathogenesis and progression of MS/EAE. The extravasation of neutrophils into the CNS from the peripheral circulation is suggested to be induced by CNS-resident sentinel cells (e.g., astrocytes, resident macrophages and microglia), which are activated by tissue danger signals release. Some studies suggested that both astrocytes and microglia might regulate neutrophil functions. Inversely, CNS-invading neutrophils could release factors that affect reactivity of glial cells. The aim of this project is to shed light on the cellular interplay exerted by infiltrating neutrophils in the spinal cord (SC) during EAE, taking advantage in particular of our cutting-edge live imaging techniques. At first, flow cytometry studies carried on EAE mice showed neutrophil infiltration during all the clinical phases of disease but especially at the peak, during which a massive neutrophil infiltration within the parenchyma was observed. Immunofluorescence staining on SC sections revealed also numerous extravasated neutrophils establishing physical contacts with astrocytes and microglia/macrophages, suggesting that neutrophil-glial cell crosstalk may represent a previously unknown key axis during EAE. To corroborate this hypothesis, we developed a protocol of target validation on a dynamic in vitro neutrophil-glial cell co-culture system. We observed that neutrophils display different motility parameters when co-cultured with astrocytes or microglia/macrophages, and that Leukocyte function associated antigen-1 (LFA-1) blockade affected exclusively interactions with myeloid cells when isolated at the peak of disease. These data were also confirmed using neutrophils isolated from LFA-1 knock-out mice. Interestingly, we obtained similar results in vivo performing two-photon laser microscopy in the subarachnoid space (SAS) of lumbar SC at the peak of disease. Weakly motile extravasated neutrophils were found, moving preferentially located near to the vessels. Interestingly, the blockade of LFA-1 led to a progressive reduction in the portion of neutrophils that were moving slowly near the blood vessels, resulting in an increased portion of cells that, once left vessel compartmentalization, drastically enhanced velocity and directness moving deep in spinal SAS space. In CX3CR1-eGFP EAE mice, the local administration of anti-LFA-1 antibody clearly inhibited perivascular macrophage-neutrophil interactions, suggesting that LFA-1 integrin is a key integrin controlling neutrophil dynamics at the interface with SC parenchyma during acute EAE. To check a therapeutic relevance of our in vitro and in vivo findings, we performed intrathecal injection of anti-LFA-1 antibody at disease onset and 4 days later in EAE-induced mice. We observed that anti-LFA-1 treatment determined a significant inhibition of EAE clinical symptoms, together with amelioration in neuropathological hallmarks, in terms of infiltrated leukocytes, demyelination, microgliosis and astrogliosis. Collectively, the molecular mechanisms controlling neutrophil crosstalk with glial cells could represent an innovative field of study, thus contributing to develop new therapeutic strategies for the treatment of MS.

The integrin LFA-1 as a potential target for neutrophil interactions during neuroinflammation

Nicola Lopez
Writing – Original Draft Preparation
2022-01-01

Abstract

Neuroinflammation is a complex inflammatory process occurring within the central nervous system (CNS). It is mediated by the production of cytokines, chemokines, reactive oxygen species and secondary messengers produced and secreted by CNS-resident cells and recruited immune cells. Recent studies highlighted the key role of neutrophils as crucial players in chronic inflammatory diseases including CNS disorders such as Alzheimer’s disease (AD) and multiple sclerosis (MS). Previous studies on experimental autoimmune encephalomyelitis (EAE), the animal model of MS, have shown that neutrophils in the CNS are localized in close proximity to damaged tissues, such as demyelinated areas and leaked vessels, suggesting their potential detrimental role in the pathogenesis and progression of MS/EAE. The extravasation of neutrophils into the CNS from the peripheral circulation is suggested to be induced by CNS-resident sentinel cells (e.g., astrocytes, resident macrophages and microglia), which are activated by tissue danger signals release. Some studies suggested that both astrocytes and microglia might regulate neutrophil functions. Inversely, CNS-invading neutrophils could release factors that affect reactivity of glial cells. The aim of this project is to shed light on the cellular interplay exerted by infiltrating neutrophils in the spinal cord (SC) during EAE, taking advantage in particular of our cutting-edge live imaging techniques. At first, flow cytometry studies carried on EAE mice showed neutrophil infiltration during all the clinical phases of disease but especially at the peak, during which a massive neutrophil infiltration within the parenchyma was observed. Immunofluorescence staining on SC sections revealed also numerous extravasated neutrophils establishing physical contacts with astrocytes and microglia/macrophages, suggesting that neutrophil-glial cell crosstalk may represent a previously unknown key axis during EAE. To corroborate this hypothesis, we developed a protocol of target validation on a dynamic in vitro neutrophil-glial cell co-culture system. We observed that neutrophils display different motility parameters when co-cultured with astrocytes or microglia/macrophages, and that Leukocyte function associated antigen-1 (LFA-1) blockade affected exclusively interactions with myeloid cells when isolated at the peak of disease. These data were also confirmed using neutrophils isolated from LFA-1 knock-out mice. Interestingly, we obtained similar results in vivo performing two-photon laser microscopy in the subarachnoid space (SAS) of lumbar SC at the peak of disease. Weakly motile extravasated neutrophils were found, moving preferentially located near to the vessels. Interestingly, the blockade of LFA-1 led to a progressive reduction in the portion of neutrophils that were moving slowly near the blood vessels, resulting in an increased portion of cells that, once left vessel compartmentalization, drastically enhanced velocity and directness moving deep in spinal SAS space. In CX3CR1-eGFP EAE mice, the local administration of anti-LFA-1 antibody clearly inhibited perivascular macrophage-neutrophil interactions, suggesting that LFA-1 integrin is a key integrin controlling neutrophil dynamics at the interface with SC parenchyma during acute EAE. To check a therapeutic relevance of our in vitro and in vivo findings, we performed intrathecal injection of anti-LFA-1 antibody at disease onset and 4 days later in EAE-induced mice. We observed that anti-LFA-1 treatment determined a significant inhibition of EAE clinical symptoms, together with amelioration in neuropathological hallmarks, in terms of infiltrated leukocytes, demyelination, microgliosis and astrogliosis. Collectively, the molecular mechanisms controlling neutrophil crosstalk with glial cells could represent an innovative field of study, thus contributing to develop new therapeutic strategies for the treatment of MS.
Neuroinflammation, EAE, neutrophils, glial cells, neuroimmune interactions
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11562/1066265
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