The role traditionally assigned to astrocytes in the pathogenesis of multiple sclerosis (MS) lesions has been the formation of the glial scar once inflammation has subsided. Astrocytes are now recognized to be early and highly active players during lesion formation and key for providing peripheral immune cells access to the central nervous system. Here, we review the role of astrocytes in the formation and evolution of MS lesions, including the recently described functional polarization of astrocytes, discuss prototypical pathways for astrocyte activation, and summarize mechanisms by which MS treatments affect astrocyte function.

Chronic active multiple sclerosis lesions, characterized by a hyperintense rim of iron-enriched, activated microglia and macrophages, have been linked to greater tissue damage. Post-mortem studies have determined that chronic active lesions are primarily related to the later stages of multiple sclerosis; however, the occurrence of these lesions, and their relationship to earlier disease stages may be greatly underestimated. Detection of chronic active lesions across the patient spectrum of multiple sclerosis requires a validated imaging tool to accurately identify lesions with persistent inflammation. Quantitative susceptibility mapping provides efficient in vivo quantification of susceptibility changes related to iron deposition and the potential to identify lesions harbouring iron-laden inflammatory cells. The PET tracer 11 C-PK11195 targets the translocator protein expressed by activated microglia and infiltrating macrophages. Accordingly, this study aimed to validate that lesions with a hyperintense rim on quantitative susceptibility mapping from both relapsing and progressive patients demonstrate a higher level of innate immune activation as measured on 11 C-PK11195 PET. Thirty patients were enrolled in this study, 24 patients had relapsing remitting multiple sclerosis, six had progressive multiple sclerosis, and all patients had concomitant MRI with a gradient echo sequence and PET with 11 C-PK11195. A total of 406 chronic lesions were detected, and 43 chronic lesions with a hyperintense rim on quantitative susceptibility mapping were identified as rim + lesions. Susceptibility (relative to CSF) was higher in rim + (2.42 AE 17.45 ppb) compared to rimÀ lesions (À14.6 AE 19.3 ppb, P 5 0.0001). Among rim + lesions, susceptibility within the rim (20.04 AE 14.28 ppb) was significantly higher compared to the core (À5.49 AE 14.44 ppb, P 5 0.0001), consistent with the presence of iron. In a mixed-effects model, 11 C-PK11195 uptake, representing activated microglia/macrophages, was higher in rim + lesions compared to rimÀ lesions (P = 0.015). Validating our in vivo imaging results, multiple sclerosis brain slabs were imaged with quantitative susceptibility mapping and processed for immunohistochemistry. These results showed a positive translocator protein signal throughout the expansive hyperintense border of rim + lesions, which co-localized with iron containing CD68 + microglia and macrophages. In conclusion, this study provides evidence that suggests that a hyperintense rim on quantitative susceptibility measure within a chronic lesion is a correlate for persistent inflammatory activity and that these lesions can be identified in the relapsing patients. Utilizing quantitative susceptibility measure to differentiate chronic multiple sclerosis lesion subtypes, especially chronic active lesions, would provide a method to assess the impact of these lesions on disease progression.

Multiple sclerosis (MS) is characterized by demyelinated and inflammatory lesions in the brain and spinal cord that are highly variable in terms of cellular content. Here, we used imaging mass cytometry (IMC) to enable the simultaneous imaging of 15+ proteins within staged MS lesions. To test the potential for IMC to discriminate between different types of lesions, we selected a case with severe rebound MS disease activity after natalizumab cessation. With post-acquisition analysis pipelines we were able to: (1) Discriminate demyelinating macrophages from the resident microglial pool; (2) Determine which types of lymphocytes reside closest to blood vessels; (3) Identify multiple subsets of T and B cells, and (4) Ascertain dynamics of T cell phenotypes vis-à-vis lesion type and location. We propose that IMC will enable a comprehensive analysis of single-cell phenotypes, their functional states and cell-cell interactions in relation to lesion morphometry and demyelinating activity in MS patients.

Background Inflammation in chronic active lesions occurs behind a closed blood–brain barrier and cannot be detected with MRI. Activated microglia are highly enriched for iron and can be visualized with quantitative susceptibility mapping (QSM), an MRI technique used to delineate iron. Objective To characterize the histopathological correlates of different QSM hyperintensity patterns in MS lesions. Methods MS brain slabs were imaged with MRI and QSM, and processed for histology. Immunolabeled cells were quantified in the lesion rim, center, and adjacent normal‐appearing white matter (NAWM). Iron+ myeloid cell densities at the rims were correlated with susceptibilities. Human‐induced pluripotent stem cell (iPSC)‐derived microglia were used to determine the effect of iron on the production of reactive oxygen species (ROS) and pro‐inflammatory cytokines. Results QSM hyperintensity at the lesion perimeter correlated with activated iron+ myeloid cells in the rim and NAWM. Lesions with high punctate or homogenous QSM signal contained no or minimally activated iron− myeloid cells. In vitro, iron accumulation was highest in M1‐polarized human iPSC‐derived microglia, but it did not enhance ROS or cytokine production. Conclusion A high QSM signal outlining the lesion rim but not punctate signal in the center is a biomarker for chronic inflammation in white matter lesions.

Epigenetic annotation studies of genetic risk variants for multiple sclerosis (MS) implicate dysfunctional lymphocytes in MS susceptibility; however, the role of central nervous system (CNS) cells remains unclear. We investigated the effect of the risk variant, rs7665090G, located near NFKB1, on astrocytes. We demonstrated that chromatin is accessible at the risk locus, a prerequisite for its impact on astroglial function. The risk variant was associated with increased NF-κB signaling and target gene expression, driving lymphocyte recruitment, in cultured human astrocytes and astrocytes within MS lesions, and with increased lesional lymphocytic infiltrates and lesion sizes. Thus, our study establishes a link between genetic risk for MS (rs7665090G) and dysfunctional astrocyte responses associated with increased CNS access for peripheral immune cells. MS may therefore result from variant-driven dysregulation of the peripheral immune system and of the CNS, where perturbed CNS cell function aids in establishing local autoimmune inflammation.

Activated myeloid cells and astrocytes are the predominant cell types in active multiple sclerosis (MS) lesions. Both cell types can adopt diverse functional states that play critical roles in lesion formation and resolution. In order to identify phenotypic subsets of myeloid cells and astrocytes, we profiled two active MS lesions with thirteen glial activation markers using imaging mass cytometry (IMC), a method for multiplexed labeling of histological sections. In the acutely demyelinating lesion, we found multiple distinct myeloid and astrocyte phenotypes that populated separate lesion zones. In the post-demyelinating lesion, phenotypes were less distinct and more uniformly distributed. In both lesions cell-to-cell interactions were not random, but occurred between specific glial subpopulations and lymphocytes. Finally, we demonstrated that myeloid, but not astrocyte phenotypes were activated along a lesion rim-to-center gradient, and that marker expression in glial cells at the lesion rim was driven more by cell-extrinsic factors than in cells at the center. This proof-of-concept study demonstrates that highly multiplexed tissue imaging, combined with the appropriate computational tools, is a powerful approach to study heterogeneity, spatial distribution and cellular interactions in the context of MS lesions. Identifying glial phenotypes and their interactions at different lesion stages may provide novel therapeutic targets for inhibiting acute demyelination and low-grade, chronic inflammation. Electronic supplementary material The online version of this article (10.1186/s40478-019-0779-2) contains supplementary material, which is available to authorized users.

ObjectiveTo identify coinhibitory immune pathways important in the brain, we hypothesized that comparison of T cells in lesions from patients with MS with tumor-infiltrating T cells (TILs) from patients with glioblastoma multiforme may reveal novel targets for immunotherapy.MethodsWe collected fresh surgical resections and matched blood from patients with glioblastoma, blood and unmatched postmortem CNS tissue from patients with MS, and blood from healthy donors. The expression of TIGIT, CD226, and their shared ligand CD155 as well as PD-1 and PDL1 was assessed by both immunohistochemistry and flow cytometry.ResultsWe found that TIGIT was highly expressed on glioblastoma-infiltrating T cells, but was near-absent from MS lesions. Conversely, lymphocytic expression of PD-1/PD-L1 was comparable between the 2 diseases. Moreover, TIGIT was significantly upregulated in circulating lymphocytes of patients with glioblastoma compared with healthy controls, suggesting recirculation of TILs. Expression of CD226 was also increased in glioblastoma, but this costimulatory receptor was expressed alongside TIGIT in the majority of tumor-infiltrating T cells, suggesting functional counteraction.ConclusionsThe opposite patterns of TIGIT expression in the CNS between MS and glioblastoma reflects the divergent features of the immune response in these 2 CNS diseases. These data raise the possibility that anti-TIGIT therapy may be beneficial for patients with glioblastoma.

23Activated myeloid cells and astrocytes are the predominant cell types in acute multiple sclerosis (MS) lesions. The 24 polarization states of these cells play a critical role in lesion formation and resolution. To identify myeloid and 25 astrocyte subsets present during lesion development, we profiled the cellular composition of an acutely demyelinating 26 and an acute post-demyelinating MS lesion with thirteen immune markers, using imaging mass cytometry (IMC). In 27 the early demyelinating lesion, distinct myeloid and astrocyte phenotypes populated separate lesion zones and engaged 28 in specific interactions. In the late active lesion, phenotypes were less diverse and more uniformly distributed 29 throughout the lesion. Our study demonstrates that multiplexed tissue imaging, combined with new computational 30 tools, is a powerful approach to study cellular heterogeneity and spatial organization in MS lesions. Identifying glial 31 phenotypes and their key interactions at different lesion stages may elucidate novel therapeutic targets for inhibiting 32 acute demyelination and low-grade neuro-inflammation.