Astroglial TLR9 antagonism promotes chemotaxis and alternative activation of macrophages via modulation of astrocyte-derived signals: implications for spinal cord injury

Abstract

Background: The recruitment of immune system cells into the central nervous system (CNS) has a profound effect on the outcomes of injury and disease. Glia-derived chemoattractants, including chemokines, play a pivotal role in this process. In addition, cytokines and chemokines influence the phenotype of infiltrating immune cells. Depending on the stimuli present in the local milieu, infiltrating macrophages acquire the classically activated M1 or alternatively activated M2 phenotypes. The polarization of macrophages into detrimental M1 versus beneficial M2 phenotypes significantly influences CNS pathophysiology. Earlier studies indicated that a toll-like receptor 9 (TLR9) antagonist modulates astrocytederived cytokine and chemokine release. However, it is not known whether these molecular changes affect astrocyteinduced chemotaxis and polarization of macrophages. The present studies were undertaken to address these issues. Methods: The chemotaxis and polarization of mouse peritoneal macrophages by spinal cord astrocytes were evaluated in a Transwell co-culture system. Arrays and ELISA were utilized to quantify chemokines in the conditioned medium (CM) of pure astrocyte cultures. Immunostaining for M1-and M2-specific markers characterized the macrophage phenotype. The percentage of M2 macrophages at the glial scar was determined by stereological approaches in mice sustaining a mid-thoracic spinal cord contusion injury (SCI) and intrathecally treated with oligodeoxynucleotide 2088 (ODN 2088), the TLR9 antagonist. Statistical analyses used two-tailed independent-sample t-test and one-way analysis of variance (ANOVA) followed by Tukey's post hoc test. A p value < 0.05 was considered to be statistically significant. Results: ODN 2088-treated astrocytes significantly increased the chemotaxis of peritoneal macrophages via release of chemokine (C-C motif) ligand 1 (CCL1). Vehicle-treated astrocytes polarized macrophages into the M2 phenotype and ODN 2088-treated astrocytes promoted further M2 polarization. Reduced CCL2 and CCL9 release by astrocytes in response to ODN 2088 facilitated the acquisition of the M2 phenotype, suggesting that CCL2 and CCL9 are negative regulators of M2 polarization. The percentage of M2 macrophages at the glial scar was higher in mice sustaining a SCI and receiving ODN 2088 treatment as compared to vehicle-treated injured controls. Conclusions: TLR9 antagonism could create a favorable environment during SCI by supporting M2 macrophage polarization and chemotaxis via modulation of astrocyte-to-macrophage signals.

Dissecting the Dual Role of the Glial Scar and Scar-Forming Astrocytes in Spinal Cord Injury

“…This recruitment of macrophages lays a crucial foundation for recovery from SCI since these macrophages not only clear toxic debris from injured tissues through phagocytosis but also remove dying neutrophils through efferocytosis (Hawthorne and Popovich, 2011). Reactive astrocytes induce chemotaxis and polarization of macrophages to manipulate their fates (Li et al, 2020). In the subacute phase, macrophages amplify astrocytic activity and promote glial scar formation.…”

Role of astroglial toll-like receptors (TLRs) in central nervous system infections, injury and neurodegenerative diseases

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Dwellers and Trespassers: Mononuclear Phagocytes at the Borders of the Central Nervous System

“…Similar to what is observed in EAE models, sensing of CCL2 and CXCL12 and production of MMP9 are required for monocyte migration from the BBB to the parenchyma upon SCI ( 297 , 298 ). CCL2 and other chemokines released by glial cells such as astrocytes might also contribute to the acquisition of a functional phenotype by invading and local macrophages ( 299 ). Local TNF-α production increases MMP-9 expression highlighting a complex interplay of cytokines and proteases.…”

“…Indeed, one report described locally recruited MdMs as pro-inflammatory players surrounded by anti-inflammatory microglia distal to the injured area ( 306 ). Acquisition of an anti-inflammatory phenotype is also affected by the activation status of nearby astroglial cells ( 299 ). In another work, inhibition of monocyte infiltration (via splenectomy) accordingly resulted into an ameliorated clinical phenotype ( 307 ).…”