Quantitative susceptibility mapping identifies inflammation in a subset of chronic multiple sclerosis lesions.

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 11C-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 11C-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 11C-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 ± 17.45 ppb) compared to rim- lesions (-14.6 ± 19.3 ppb, P < 0.0001). Among rim+ lesions, susceptibility within the rim (20.04 ± 14.28 ppb) was significantly higher compared to the core (-5.49 ± 14.44 ppb, P < 0.0001), consistent with the presence of iron. In a mixed-effects model, 11C-PK11195 uptake, representing activated microglia/macrophages, was higher in rim+ lesions compared to rim- 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.

CNS cell-derived exosome signatures as blood-based biomarkers of neurodegenerative diseases.

Molecular biomarkers require the reproducible capture of disease-associated changes and are ideally sensitive, specific and accessible with minimal invasiveness to patients. Exosomes are a subtype of extracellular vesicles that have gained attention as potential biomarkers. They are released by all cell types and carry molecular cargo that reflects the functional state of the cells of origin. These characteristics make them an attractive means of measuring disease-related processes within the central nervous system (CNS), as they cross the blood-brain barrier (BBB) and can be captured in peripheral blood. In this review, we discuss recent progress made toward identifying blood-based protein and RNA biomarkers of several neurodegenerative diseases from circulating, CNS cell-derived exosomes. Given the lack of standardized methodology for exosome isolation and characterization, we discuss the challenges of capturing and quantifying the molecular content of exosome populations from blood for translation to clinical use.

Faecal incontinence with concurrent disorders of gut-brain interaction: A worse outcome.

BACKGROUND: Faecal incontinence is a common debilitating condition associated with poor quality of life that generates substantial economic strain on healthcare systems. OBJECTIVES: We aimed to evaluate, in a tertiary referral population presenting with faecal incontinence, the impact of suffering additional disorders of gut-brain interaction (DGBI) on symptom severity, anxiety, depression and quality of life. METHODS: Design: Retrospective cohort study. SETTING: Tertiary referral Neurogastroenterology centre. PATIENTS: All patients presenting with faecal incontinence from 2007 to 2020 were included. MAIN OUTCOME MEASURES: The results from structured medical and surgical questionnaires including Rome III Integrative Questionnaire, Faecal Incontinence Severity Index, Hospital Anxiety and Depression Scale, SF-36, and anorectal physiology were analysed using Stata version 17. Patients were categorised into 3 groups: 0-1 additional DGBI, 2 DGBIs, and 3+ DGBI. Statistical significance was defined as p < 0.05 (two-tailed). KEY RESULTS: Faecal incontinence patients (n = 249; mean age 63.4 ± 12.6 years; 93.6% female, 48.1% urge subtype) met diagnostic criteria for mean 2.2 additional DGBI each, mostly affecting bowel (n = 231, 42.4%) and anorectal (n = 150, 27.5%) regions. A greater number of DGBIs was associated with higher faecal incontinence symptom severity (p < 0.001), higher anxiety (p = 0.002) and depression (p = 0.003), and worse quality of life in areas of mental health (p = 0.037) and social effect (p < 0.001). Patients with a greater number of concurrent DGBI demonstrated a greater family history of gastrointestinal problems (p = 0.004). There were no associations found between a greater amount of DGBIs and anorectal physiology. CONCLUSIONS AND INFERENCES: A greater number of additional DGBIs in faecal incontinence patients was associated with worse faecal incontinence symptoms, higher anxiety and depression scores, and worse quality of life but was unrelated to physiology. This highlights the need to proactively search for comorbid DGBI in patients presenting with faecal incontinence.

QSM is an imaging biomarker for chronic glial activation in multiple sclerosis lesions.

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.

Differential expression of the T-cell inhibitor TIGIT in glioblastoma and MS.

OBJECTIVE: To 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. METHODS: We 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. RESULTS: We 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. CONCLUSIONS: The 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.

Multiplexed imaging of immune cells in staged multiple sclerosis lesions by mass cytometry.

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.

The landscape of myeloid and astrocyte phenotypes in acute multiple sclerosis lesions.

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.

The Role of Astrocytes in Multiple Sclerosis.

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.

Enhanced astrocyte responses are driven by a genetic risk allele associated with multiple sclerosis.

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.