Chronic Active Multiple Sclerosis Research Articles

B cell depletion therapy does not resolve chronic active multiple sclerosis lesions.

Chronic active lesions (CAL) in multiple sclerosis (MS) have been observed even in patients taking high-efficacy disease-modifying therapy, including B-cell depletion. Given that CAL are a major determinant of clinical progression, including progression independent of relapse activity (PIRA), understanding the predicted activity and real-world effects of targeting specific lymphocyte populations is critical for designing next-generation treatments to mitigate chronic inflammation in MS. We analyzed published lymphocyte single-cell transcriptomes from MS lesions and bioinformatically predicted the effects of depleting lymphocyte subpopulations (including CD20 B-cells) from CAL via gene-regulatory-network machine-learning analysis. Motivated by the results, we performed invivo MRI assessment of PRL changes in 72 adults with MS, 46 treated with anti-CD20 antibodies and 26 untreated, over ∼2 years. Although only 4.3% of lymphocytes in CAL were CD20 B-cells, their depletion is predicted to affect microglial genes involved in iron/heme metabolism, hypoxia, and antigen presentation. Invivo, tracking 202 PRL (150 treated) and 175 non-PRL (124 treated), none of the treated paramagnetic rims disappeared at follow-up, nor was there a treatment effect on PRL for lesion volume, magnetic susceptibility, or T1 time. PIRA occurred in 20% of treated patients, more frequently in those with ≥4 PRL (p=0.027). Despite predicted effects on microglia-mediated inflammatory networks in CAL and iron metabolism, anti-CD20 therapies do not fully resolve PRL after 2-year MRI follow up. Limited tissue turnover of B-cells, inefficient passage of anti-CD20 antibodies across the blood-brain-barrier, and a paucity of B-cells in CAL could explain our findings. Intramural Research Program of NINDS, NIH; NINDS grants R01NS082347 and R01NS082347; Dr. Miriam and Sheldon G. Adelson Medical Research Foundation; Cariplo Foundation (grant #1677), FRRB Early Career Award (grant #1750327); Fund for Scientific Research (FNRS).

African Americans with multiple sclerosis (MS) are more likely to have paramagnetic rim lesions (PRLs) compared to Caucasians (P11-3.017)

<h3>Objective:</h3> To determine the prevalence of paramagnetic rim lesions (PRLs) in African American patients with multiple sclerosis (AAwMS) compared to Caucasian (CAwMS). <h3>Background:</h3> PRLs are a subset of chronic active MS lesions with a rim of iron-laden innate immune cells and their presence has been associated with higher disability. AAwMS have been clinically observed to have higher disability and more severe symptoms as compared to other racial groups. <h3>Design/Methods:</h3> In this retrospective study of 139 AAwMS and 132 CAwMS quantitative susceptibility mapping (QSM), an MRI approach sensitive to iron, was used to identify PRLs. A linear model assessed the relationship between EDSS and race, adjusting for total lesion number (TLN), sex, age and disease duration (DD). The association of PRL per patient and race was modeled, adjusting for clinical variables and total lesions, using a zero-inflated Poisson regression. <h3>Results:</h3> The cohorts were well matched on all clinical variables and TLN. In a linear model, AAwMS had a higher EDSS (p-value &lt;0.001) with, on average, a 1.16-point higher EDSS (95% CI: [0.73, 1.60] than CAwMS. AAwMS had a mean of 1.3 (2.72) PRL whereas CAwMS had 0.82 (1.69), p=0.085. In the zero-inflated Poisson model, the log odds ratio of having no PRL was 0.53 (95% CI: [0.29, 0.97], p-value = 0.039) for AAwMS, indicating more patients have at least one PRL. In the Poisson model, only TLN (p = 0.021) and gender (p = 0.046) were found to be significant, which indicates that AAwMS were more likely to have one PRL, however among all patients with PRL, the total number of PRL did not depend on race. <h3>Conclusions:</h3> This is the first study to explore racial differences in the occurrence of chronic active lesions and provides preliminary evidence that PRL could relate to racial specific differences in disease pathology, an area which has been significantly understudied. <b>Disclosure:</b> Dr. Michaelson has nothing to disclose. Ms. Marcille has nothing to disclose. Dr. Sweeney has nothing to disclose. Dr. Kaunzner has received personal compensation in the range of $500-$4,999 for serving on a Scientific Advisory or Data Safety Monitoring board for Novartis. Dr. Kaunzner has received personal compensation in the range of $5,000-$9,999 for serving on a Speakers Bureau for Medlearning. Jai Perumal has nothing to disclose. Dr. Nealon has nothing to disclose. Thanh Nguyen has nothing to disclose. Dr. Gauthier has received personal compensation in the range of $500-$4,999 for serving as a Consultant for Gnenetech. The institution of Dr. Gauthier has received research support from Genentech.

Dimethyl Fumarate Reduces Inflammation in Chronic Active Multiple Sclerosis Lesions.

Background and ObjectivesTo determine the effects of dimethyl fumarate (DMF) and glatiramer acetate on iron content in chronic active lesions in patients with multiple sclerosis (MS) and in human microglia in vitro.MethodsThis was a retrospective observational study of 34 patients with relapsing-remitting MS and clinically isolated syndrome treated with DMF or glatiramer acetate. Patients had lesions with hyperintense rims on quantitative susceptibility mapping, were treated with DMF or glatiramer acetate (GA), and had a minimum of 2 on-treatment scans. Changes in susceptibility in rim lesions were compared among treatment groups in a linear mixed effects model. In a separate in vitro study, induced pluripotent stem cell–derived human microglia were treated with DMF or GA, and treatment-induced changes in iron content and activation state of microglia were compared.ResultsRim lesions in patients treated with DMF had on average a 2.77-unit reduction in susceptibility per year over rim lesions in patients treated with GA (bootstrapped 95% CI −5.87 to −0.01), holding all other variables constant. Moreover, DMF but not GA reduced inflammatory activation and concomitantly iron content in human microglia in vitro.DiscussionTogether, our data indicate that DMF-induced reduction of susceptibility in MS lesions is associated with a decreased activation state in microglial cells. We have demonstrated that a specific disease modifying therapy, DMF, decreases glial activity in chronic active lesions. Susceptibility changes in rim lesions provide an in vivo biomarker for the effect of DMF on microglial activity.Classification of EvidenceThis study provided Class III evidence that DMF is superior to GA in the presence of iron as a marker of inflammation as measured by MRI quantitative susceptibility mapping.

QSMRim-Net: Imbalance-aware learning for identification of chronic active multiple sclerosis lesions on quantitative susceptibility maps

Background and PurposeChronic active multiple sclerosis (MS) lesions are characterized by a paramagnetic rim at the edge of the lesion and are associated with increased disability in patients. Quantitative susceptibility mapping (QSM) is an MRI technique that is sensitive to chronic active lesions, termed rim + lesions on the QSM. We present QSMRim-Net, a data imbalance-aware deep neural network that fuses lesion-level radiomic and convolutional image features for automated identification of rim + lesions on QSM. MethodsQSM and T2-weighted-Fluid-Attenuated Inversion Recovery (T2-FLAIR) MRI of the brain were collected at 3 T for 172 MS patients. Rim + lesions were manually annotated by two human experts, followed by consensus from a third expert, for a total of 177 rim + and 3986 rim negative (rim−) lesions. Our automated rim + detection algorithm, QSMRim-Net, consists of a two-branch feature extraction network and a synthetic minority oversampling network to classify rim + lesions. The first network branch is for image feature extraction from the QSM and T2-FLAIR, and the second network branch is a fully connected network for QSM lesion-level radiomic feature extraction. The oversampling network is designed to increase classification performance with imbalanced data. ResultsOn a lesion-level, in a five-fold cross validation framework, the proposed QSMRim-Net detected rim + lesions with a partial area under the receiver operating characteristic curve (pROC AUC) of 0.760, where clinically relevant false positive rates of less than 0.1 were considered. The method attained an area under the precision recall curve (PR AUC) of 0.704. QSMRim-Net out-performed other state-of-the-art methods applied to the QSM on both pROC AUC and PR AUC. On a subject-level, comparing the predicted rim + lesion count and the human expert annotated count, QSMRim-Net achieved the lowest mean square error of 0.98 and the highest correlation of 0.89 (95% CI: 0.86, 0.92). ConclusionThis study develops a novel automated deep neural network for rim + MS lesion identification using T2-FLAIR and QSM images.

A lymphocyte-microglia-astrocyte axis in chronic active multiple sclerosis.

Multiple sclerosis (MS) lesions that do not resolve in the months after they form harbour ongoing demyelination and axon degeneration, and are identifiable in vivo by their paramagnetic rims on MRI scans1-3. Here, to define mechanisms underlying this disabling, progressive neurodegenerative state4-6 and foster development of new therapeutic agents, we used MRI-informed single-nucleus RNA sequencing to profile the edge of demyelinated white matter lesions at various stages of inflammation. We uncovered notable glial and immune cell diversity, especially at the chronically inflamed lesion edge. We define 'microglia inflamed in MS' (MIMS) and 'astrocytes inflamed in MS', glial phenotypes that demonstrate neurodegenerative programming. The MIMS transcriptional profile overlaps with that of microglia in other neurodegenerative diseases, suggesting that primary and secondary neurodegeneration share common mechanisms and could benefit from similar therapeutic approaches. We identify complement component 1q (C1q) as a critical mediator of MIMS activation, validated immunohistochemically in MS tissue, genetically by microglia-specific C1q ablation in mice with experimental autoimmune encephalomyelitis, and therapeutically by treating chronic experimental autoimmune encephalomyelitis with C1q blockade. C1q inhibition is a potential therapeutic avenue to address chronic white matter inflammation, which could be monitored by longitudinal assessment of its dynamic biomarker, paramagnetic rim lesions, using advanced MRI methods.

Characterization of chronic active multiple sclerosis lesions with sodium (23 Na) magnetic resonance imaging-preliminary observations.

There has been an increasing interest in chronic active multiple sclerosis (MS) lesions as a new magnetic resonance imaging (MRI) marker of disease progression. Chronic active lesions are characterized by progressive tissue matrix damage, axonal loss and chronic inflammation. Sodium (23 Na) MRI provides a biochemical marker of cell integrity and tissue viability in a quantitative manner. The aim of this study was to investigate with 23 Na MRI tissue abnormalities in chronic active lesions as indicators of tissue destruction. To identify chronic active lesions, two 3D magnetization-prepared rapid acquisition gradient-echo datasets obtained 12months apart were processed using the voxel-guided morphometry algorithm. Cross-sectional 23 Na MRI was performed during the 12-month follow-up period. Total sodium concentration was calculated in chronic active lesions compared to shrinking, chronic stable and acute contrast-enhancing lesions. Overall, 70 MS lesions (21 chronic active, 10 shrinking, 29 chronic stable lesions, 10 acute contrast-enhancing lesions) in 12 patients were included. Total sodium concentration in chronic active lesions (49.57±8.47mM) was significantly higher than in shrinking (42.16±3.9mM; p=0.03) and chronic stable lesions (39.92±4.82mM; p<0.001). Chronic active lesions showed similar sodium values compared to acute contrast-enhancing lesions (48.06±6.65mM; p=0.97). No differences between shrinking and chronic stable lesions were observed (p=0.89). High sodium values in chronic active MS lesions may be an indicator of ongoing inflammation and tissue damage.

Illumination of Molecular Pathways in Multiple Sclerosis Lesions and the Immune Mechanism of Matrine Treatment in EAE, a Mouse Model of MS.

The etiology of multiple sclerosis (MS) is not clear, and the treatment of MS presents a great challenge. This study aimed to investigate the pathogenesis and potential therapeutic targets of MS and to define target genes of matrine, a quinolizidine alkaloid component derived from the root of Sophorae flavescens that effectively suppressed experimental autoimmune encephalomyelitis (EAE), an animal model of MS. To this end, the GSE108000 gene data set in the Gene Expression Omnibus Database, which included 7 chronic active MS lesions and 10 control samples of white matter, was analyzed for differentially expressed genes (DEGs). X cell was used to analyze the microenvironmental differences in brain tissue samples of MS patients, including 64 types of immune cells and stromal cells. The biological functions and enriched signaling pathways of DEGs were analyzed by multiple approaches, including GO, KEGG, GSEA, and GSVA. The results by X cell showed significantly increased numbers of immune cell populations in the MS lesions, with decreased erythrocytes, megakaryocytes, adipocytes, keratinocytes, endothelial cells, Th1 cells and Tregs. In GSE108000, there were 637 DEGs, including 428 up-regulated and 209 down-regulated genes. Potential target genes of matrine were then predicted by the network pharmacology method of Traditional Chinese medicine, and 12 key genes were obtained by cross analysis of the target genes of matrine and DEGs in MS lesions. Finally, we confirmed by RT-PCR the predicted expression of these genes in brain tissues of matrine-treated EAE mice. Among these genes, 2 were significantly downregulated and 6 upregulated by matrine treatment, and the significance of this gene regulation was further investigated. In conclusion, our study defined several possible matrine target genes, which can be further elucidated as mechanism(s) of matrine action, and novel targets in the treatment of MS.

Quantitative MRI texture analysis in chronic active multiple sclerosis lesions

ObjectiveRecently, there has been an increasing interest in “chronic enlarging” or “chronic active” multiple sclerosis (MS) lesions that are associated with clinical disability. However, investigation of dynamic lesion volume changes requires longitudinal MRI data from two or more time points. The aim of this study was to investigate the application of texture analysis (TA) on baseline T1-weighted 3D magnetization-prepared rapid acquisition gradient-echo (MPRAGE) images to differentiate chronic active from chronic stable MS lesions. Material and methodsTo identify chronic active lesions as compared to non-enhancing stable lesions, two MPRAGE datasets acquired on a 3 T MRI at baseline and after 12 months follow-up were applied to the Voxel-Guided Morphometry (VGM) algorithm. TA was performed on the baseline MPRAGE images, 36 texture features were extracted for each lesion. ResultsOverall, 374 chronic MS lesions (155 chronic active and 219 chronic stable lesions) from 60 MS patients were included in the final analysis. Multiple texture features including “DISCRETIZED_HISTO_Energy”, “GLCM_Energy”, “GLCM_Contrast” and “GLCM_Dissimilarity” were significantly higher in chronic active as compared to chronic stable lesions. Partial least squares regression yielded an area under the curve of 0.7 to differentiate both lesion types. ConclusionOur results suggest that multiple texture features extracted from MPRAGE images indicate higher intralesional heterogeneity, however they demonstrate only a fair accuracy to differentiate chronic active from chronic stable MS lesions.

Estimation of Multiple Sclerosis lesion age on magnetic resonance imaging

We introduce the first-ever statistical framework for estimating the age of Multiple Sclerosis (MS) lesions from magnetic resonance imaging (MRI). Estimating lesion age is an important step when studying the longitudinal behavior of MS lesions and can be used in applications such as studying the temporal dynamics of chronic active MS lesions. Our lesion age estimation models use first order radiomic features over a lesion derived from conventional T1 (T1w) and T2 weighted (T2w) and fluid attenuated inversion recovery (FLAIR), T1w with gadolinium contrast (T1w+c), and Quantitative Susceptibility Mapping (QSM) MRI sequences as well as demographic information. For this analysis, we have a total of 32 patients with 53 new lesions observed at 244 time points. A one or two step random forest model for lesion age is fit on a training set using a lesion volume cutoff of 15 mm3 or 50 mm3. We explore the performance of nine different modeling scenarios that included various combinations of the MRI sequences and demographic information and a one or two step random forest models, as well as simpler models that only uses the mean radiomic feature from each MRI sequence. The best performing model on a validation set is a model that uses a two-step random forest model on the radiomic features from all of the MRI sequences with demographic information using a lesion volume cutoff of 50 mm3. This model has a mean absolute error of 7.23 months (95% CI: [6.98, 13.43]) and a median absolute error of 5.98 months (95% CI: [5.26, 13.25]) in the validation set. For this model, the predicted age and actual age have a statistically significant association (p-value <0.001) in the validation set.

Continuous Immune-Modulatory Effects of Human Olig2+ Precursor Cells Attenuating a Chronic-Active Model of Multiple Sclerosis.

Neuroglial precursor cells (NPC) possess immune-modulatory properties by which they prevent immune-mediated injury in experimental autoimmune encephalomyelitis (EAE). It is unclear whether cell transplantation in a clinical-relevant setup induces ongoing therapeutic effects in a chronic-active model of progressive multiple sclerosis (MS). We examined whether human embryonic stem cell (hESC)-derived NPCs inhibit progressive EAE in Biozzi AB/H mice, manifesting with chronic-active neuroinflammation and demyelinated plaques. hESC-derived NPCs were propagated for 6-8 weeks as spheres enriched for Olig2+ cells to switch from neuronal to glial commitment and to enrich for oligodendrocyte progenitor cells. NPC were transplanted intracerebroventricularly at 30 days post-EAE induction, after the acute relapse. We evaluated effects of cell transplantation on clinical parameters, neuroinflammation, myelination, and axonal loss. Transplanted animals exhibited a significantly milder disease, reduced neuroinflammation, reduced demyelination, and reduced axonal loss as compared to control EAE mice. Toluidine-blue semi-thin staining showed a bystander neuroprotective effect of human precursor cells preventing the loss of myelinated fibers in superficial layer of the cervical dorsal funiculus. Human Olig2+ cells were detected along spinal cord meninges after 65 days of follow-up. In co-cultures in vitro, Olig2+ human precursors inhibited Concanavalin A-induced murine T cell activation and proliferation. To conclude, glial-committed human NPC induce ongoing immune-regulatory and neuroprotective effects, following transplantation into mice with a clinical-relevant model of chronic-active MS and during established disease, entering the chronic phase. These properties highlight the therapeutic potential of human NPC transplantation in chronic MS and their delivery via the cerebrospinal fluid.

Astrocytes in the Pathogenesis of Multiple Sclerosis: An In Situ MicroRNA Study

Astrocytes are increasingly recognized as active contributors to the disease process in multiple sclerosis (MS), rather than being merely reactive. We investigated the expression of a selected microRNA (miRNA) panel that could contribute both to the injury and to the recovery phases of the disease. Individual astrocytes were laser microdissected from brain sections. We then compared the miRNAs' expressions in MS and control brain samples at different lesional stages in white versus grey matter regions. In active MS lesions, we found upregulation of ischemia-related miRNAs in white but not grey matter, often with reversion to the normal state in inactive lesions. In contrast to our previous findings on MS macrophages, expression of 2 classical inflammatory-related miRNAs, miRNA-155 and miRNA-146a, was reduced in astrocytes from active and chronic active MS lesions in white and grey matter, suggesting a lesser direct pathogenetic role for these miRNAs in astrocytes. miRNAs within the categories regulating aquaporin4 (-100, -145, -320) and glutamate transport/apoptosis/neuroprotection (-124a, -181a, and -29a) showed some contrasting responses. The regional and lesion-stage differences of expression of these miRNAs indicate the remarkable ability of astrocytes to show a wide range of selective responses in the face of differing insults and phases of resolution.

Association of Chronic Active Multiple Sclerosis Lesions With Disability In Vivo

In multiple sclerosis (MS), chronic active lesions, which previously could only be detected at autopsy, can now be identified on susceptibility-based magnetic resonance imaging (MRI) in vivo as non-gadolinium-enhancing lesions with paramagnetic rims. Pathologically, they feature smoldering inflammatory demyelination at the edge, remyelination failure, and axonal degeneration. To our knowledge, the prospect of long-term in vivo monitoring makes it possible for the first time to determine their contribution to disability and value as a treatment target. To assess whether rim lesions are associated with patient disability and long-term lesion outcomes. We performed 3 studies at the National Institutes of Health Clinical Center: (1) a prospective clinical/radiological cohort of 209 patients with MS (diagnosis according to the 2010 McDonald revised MS criteria, age ≥18 years, with 7-T or 3-T susceptibility-based brain MRI results) who were enrolled from January 2012 to March 2018 (of 209, 17 patients [8%] were excluded because of uninterpretable MRI scans); (2) a radiological/pathological analysis of expanding lesions featuring rims; and (3) a retrospective longitudinal radiological study assessing long-term lesion evolution in 23 patients with MS with yearly MRI scans for 10 years or more (earliest scan, 1992). (1) Identification of chronic rim lesions on 7-T or 3-T susceptibility-based brain MRI in 192 patients with MS and the association of rim counts with clinical disability (primary analysis) and brain volume changes (exploratory analysis). (2) Pathological characterization of 10 expanding lesions from an adult with progressive MS who came to autopsy after 7 years of receiving serial in vivo MRI scans. (3) Evaluation of annual lesion volume change (primary analysis) and T1 times (exploratory analysis) in 27 rim lesions vs 27 rimless lesions. Of 209 participants, 104 (50%) were women and 32 (15%) were African American. One hundred seventeen patients (56%) had at least 1 rim lesion regardless of prior or ongoing treatment. Further, 84 patients (40%) had no rims (mean [SD] age, 47 [14] years), 66 (32%) had 1 to 3 rims (mean [SD] age, 47 [11] years), and 42 (20%) had 4 rims or more (mean [SD] age, 44 [11] years). Individuals with 4 rim lesions or more reached motor and cognitive disability at an earlier age. Normalized volumes of brain, white matter, and basal ganglia were lower in those with rim lesions. Whereas rimless lesions shrank over time (-3.6%/year), rim lesions were stable in size or expanded (2.2%/year; P < .001). Rim lesions had longer T1 times, suggesting more tissue destruction, than rimless lesions. On histopathological analysis, all 10 rim lesions that expanded in vivo had chronic active inflammation. Chronic active lesions are common, are associated with more aggressive disease, exert ongoing tissue damage, and occur even in individuals treated with effective disease-modifying therapies. These results prompt the planning of MRI-based clinical trials aimed at treating perilesional chronic inflammation in MS.

Slowly expanding/evolving lesions as a magnetic resonance imaging marker of chronic active multiple sclerosis lesions

Background: Chronic lesion activity driven by smoldering inflammation is a pathological hallmark of progressive forms of multiple sclerosis (MS). Objective: To develop a method for automatic detection of slowly expanding/evolving lesions (SELs) on conventional brain magnetic resonance imaging (MRI) and characterize such SELs in primary progressive MS (PPMS) and relapsing MS (RMS) populations. Methods: We defined SELs as contiguous regions of existing T2 lesions showing local expansion assessed by the Jacobian determinant of the deformation between reference and follow-up scans. SEL candidates were assigned a heuristic score based on concentricity and constancy of change in T2- and T1-weighted MRIs. SELs were examined in 1334 RMS patients and 555 PPMS patients. Results: Compared with RMS patients, PPMS patients had higher numbers of SELs (p = 0.002) and higher T2 volumes of SELs (p < 0.001). SELs were devoid of gadolinium enhancement. Compared with areas of T2 lesions not classified as SEL, SELs had significantly lower T1 intensity at baseline and larger decrease in T1 intensity over time. Conclusion: We suggest that SELs reflect chronic tissue loss in the absence of ongoing acute inflammation. SELs may represent a conventional brain MRI correlate of chronic active MS lesions and a candidate biomarker for smoldering inflammation in MS.

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.

Comparison of two different methods of image analysis for the assessment of microglial activation in patients with multiple sclerosis using (R)-[N-methyl-carbon-11]PK11195

Chronic active multiple sclerosis (MS) lesions have a rim of activated microglia/macrophages (m/M) leading to ongoing tissue damage, and thus represent a potential treatment target. Activation of this innate immune response in MS has been visualized and quantified using PET imaging with [11C]-(R)-PK11195 (PK). Accurate identification of m/M activation in chronic MS lesions requires the sensitivity to detect lower levels of activity within a small tissue volume. We assessed the ability of kinetic modeling of PK PET data to detect m/M activity in different central nervous system (CNS) tissue regions of varying sizes and in chronic MS lesions. Ten patients with MS underwent a single brain MRI and two PK PET scans 2 hours apart. Volume of interest (VOI) masks were generated for the white matter (WM), cortical gray matter (CGM), and thalamus (TH). The distribution volume (VT) was calculated with the Logan graphical method (LGM-VT) utilizing an image-derived input function (IDIF). The binding potential (BPND) was calculated with the reference Logan graphical method (RLGM) utilizing a supervised clustering algorithm (SuperPK) to determine the non-specific binding region. Masks of varying volume were created in the CNS to assess the impact of region size on the various metrics among high and low uptake regions. Chronic MS lesions were also evaluated and individual lesion masks were generated. The highest PK uptake occurred the TH and lowest within the WM, as demonstrated by the mean time activity curves. In the TH, both reference and IDIF based methods resulted in estimates that did not significantly depend on VOI size. However, in the WM, the test-retest reliability of BPND was significantly lower in the smallest VOI, compared to the estimates of LGM-VT. These observations were consistent for all chronic MS lesions examined. In this study, we demonstrate that BPND and LGM-VT are both reliable for quantifying m/M activation in regions of high uptake, however with blood input function LGM-VT is preferred to assess longitudinal m/M activation in regions of relatively low uptake, such as chronic MS lesions.

Identification of Chronic Active Multiple Sclerosis Lesions on 3T MRI.

MR imaging-pathologic studies have reported that paramagnetic rims on 7T susceptibility-based MR imaging identify, in vivo, the subset of MS lesions with compartmentalized inflammation at the lesion edge and associated remyelination failure. Here, we assessed the reliability of detecting these rims on high-resolution 3T phase images. High-resolution T2* and phase MR imaging was collected in 20 patients with MS at 3T (3D segmented EPI, 0.65 mm3) and 7T (2D gradient-echo, 0.2 × 0.2 × 1 mm) MR imaging. In each case, 5 discrete chronic (nonenhancing) MS lesions were selected on T2 FLAIR images for rim evaluation. Five raters experienced in MS imaging contributed to the rim assessment, of whom 3 worked independently on 3T data, and 2, on 7T data. Consensus agreement was reached for both 3T and 7T rim evaluations. Discrepancies between 3T and 7T were discussed, and consensus was reached. Phase rims were seen in 34 lesions at 7T and in 36 lesions at 3T by consensus. Inter- and intrarater reliability were "substantial/good" both at 3T and 7T analysis (Cohen κ, >0.71). Based on consensus agreement, the reliability of rim visualization at 3T versus 7T was 0.78 (κ) with a pair-wise agreement of 90%. More lesions were judged to be false-positive or false-negative at 3T than at 7T. Nearly all 7T paramagnetic rims can also be seen at 3T. Imaging at 3T opens the possibility of implementing paramagnetic rims as an outcome measure in multicenter, MR imaging-based clinical trials aimed at treating perilesional persistent inflammation and its potential effects on remyelination.

MMP7 cleaves remyelination-impairing fibronectin aggregates and its expression is reduced in chronic multiple sclerosis lesions.

Upon demyelination, transient expression of fibronectin precedes successful remyelination. However, in chronic demyelination observed in multiple sclerosis (MS), aggregates of fibronectin persist and contribute to remyelination failure. Accordingly, removing fibronectin (aggregates) would constitute an effective strategy for promoting remyelination. Matrix metalloproteinases (MMPs) are enzymes known to remodel extracellular matrix components, including fibronectin. Here, we examined the ability of MMPs to degrade fibronectin aggregates. Our findings reveal that MMP7 cleaved fibronectin aggregates resulting into a prominent 13 kDa EIIIA (16 kDa EDA)‐containing fragment. MMP7 was upregulated during lysolecithin‐induced demyelination, indicating its potential for endogenous fibronectin clearance. In contrast, the expression of proMMP7 was substantially decreased in chronic active and inactive MS lesions compared with control white matter and remyelinated MS lesions. Microglia and macrophages were major cellular sources of proMMP7 and IL‐4‐activated, but not IFNγ+LPS‐activated, microglia and macrophages secreted significant levels of proMMP7. Also, conditioned medium of IL‐4‐activated macrophages most efficiently cleaved fibronectin aggregates upon MMP‐activating conditions. Yet, coatings of MMP7‐cleaved fibronectin aggregate fragments inhibited oligodendrocyte maturation, indicating that further degradation and/or clearance by phagocytosis is essential. These findings suggest that MMP7 cleaves fibronectin aggregates, while reduced (pro)MMP7 levels in MS lesions contribute to their persistent presence. Therefore, upregulating MMP7 levels may be key to remove remyelination‐impairing fibronectin aggregates in MS lesions.

Myeloid cell plasticity in the evolution of central nervous system autoimmunity.

Myeloid cells, including macrophages and dendritic cells, are a prominent component of central nervous system (CNS) infiltrates during multiple sclerosis (MS) and the animal model experimental autoimmune encephalomyelitis (EAE). Although myeloid cells are generally thought to be proinflammatory, alternatively polarized subsets can serve noninflammatory and/or reparative functions. Here we investigate the heterogeneity and biological properties of myeloid cells during central nervous system autoimmunity. Myeloid cell phenotypes in chronic active MS lesions were analyzed by immunohistochemistry. In addition, immune cells were isolated from the CNS during exacerbations and remissions of EAE and characterized by flow cytometric, genetic, and functional assays. Myeloid cells expressing inducible nitric oxide synthase (iNOS), indicative of a proinflammatory phenotype, were detected in the actively demyelinating rim of chronic active MS lesions, whereas macrophages expressing mannose receptor (CD206), a marker of alternatively polarized human myeloid cells, were enriched in the quiescent lesion core. During EAE, CNS-infiltrating myeloid cells, as well as microglia, shifted from expression of proinflammatory markers to expression of noninflammatory markers immediately prior to clinical remissions. Murine CNS myeloid cells expressing the alternative lineage marker arginase-1 (Arg1) were partially derived from iNOS+ precursors and were deficient in activating encephalitogenic T cells compared with their Arg1- counterparts. These observations demonstrate the heterogeneity of CNS myeloid cells, their evolution during the course of autoimmune demyelinating disease, and their plasticity on the single cell level. Future therapeutic strategies for disease modification in individuals with MS may be focused on accelerating the transition of CNS myeloid cells from a proinflammatory to a noninflammatory phenotype. Ann Neurol 2018;83:131-141.

Gene Expression Profiling of Multiple Sclerosis Pathology Identifies Early Patterns of Demyelination Surrounding Chronic Active Lesions.

In multiple sclerosis (MS), activated microglia and infiltrating macrophages phagocytose myelin focally in (chronic) active lesions. These demyelinating sites expand in time, but at some point turn inactive into a sclerotic scar. To identify molecular mechanisms underlying lesion activity and halt, we analyzed genome-wide gene expression in rim and peri-lesional regions of chronic active and inactive MS lesions, as well as in control tissue. Gene clustering revealed patterns of gene expression specifically associated with MS and with the presumed, subsequent stages of lesion development. Next to genes involved in immune functions, we found regulation of novel genes in and around the rim of chronic active lesions, such as NPY, KANK4, NCAN, TKTL1, and ANO4. Of note, the presence of many foamy macrophages in active rims was accompanied by a congruent upregulation of genes related to lipid binding, such as MSR1, CD68, CXCL16, and OLR1, and lipid uptake, such as CHIT1, GPNMB, and CCL18. Except CCL18, these genes were already upregulated in regions around active MS lesions, showing that such lesions are indeed expanding. In vitro downregulation of the scavenger receptors MSR1 and CXCL16 reduced myelin uptake. In conclusion, this study provides the gene expression profile of different aspects of MS pathology and indicates that early demyelination, mediated by scavenger receptors, is already present in regions around active MS lesions. Genes involved in early demyelination events in regions surrounding chronic active MS lesions might be promising therapeutic targets to stop lesion expansion.

Staining of HLA-DR, Iba1 and CD68 in human microglia reveals partially overlapping expression depending on cellular morphology and pathology

HLA-DR, Iba1 and CD68 are widely used microglia markers in human tissue. However, due to differences in gene regulation, they may identify different activation stages of microglia. Here, we directly compared the expression of HLA-DR, Iba1 and CD68 in microglia with different phenotypes, ranging from ramified to amoeboid, to foamy phagocytizing macrophages, in adjacent sections immunocytochemically double stained for two of the markers. Material was used from patients diagnosed with multiple sclerosis (MS) and Alzheimer's disease (AD) patients and control subjects because together they contain all the microglia activation stages in an acute and a chronic inflammatory setting. We found a similar, yet not identical, overall expression pattern. All three markers were expressed by ramified/amoeboid microglia around chronic active MS lesions, but overlap between HLA-DR and Iba1 was limited. Foamy macrophages in the demyelinating rims of active MS lesions of MS expressed more HLA-DR and CD68 than Iba1. All markers were expressed by small microglia accumulations (nodules) in MS NAWM. Dense core AD plaques in the hippocampus were mostly associated with microglia expressing HLA-DR. Diffuse AD plaques were not specifically associated with microglia at all. These results indicate that microglia markers have different potential for neuropathological analysis, with HLA-DR and CD68 reflecting immune activation and response to tissue damage, and Iba1 providing a marker more suited for structural studies in the absence of pathology.