Cross-talk between B cells, microglia and macrophages, and implications to central nervous system compartmentalized inflammation and progressive multiple sclerosis

Summary

Background

B cells can be enriched within meningeal immune-cell aggregates of multiple sclerosis (MS) patients, adjacent to subpial cortical demyelinating lesions now recognized as important contributors to progressive disease. This subpial demyelination is notable for a ‘surface-in’ gradient of neuronal loss and microglial activation, potentially reflecting the effects of soluble factors secreted into the CSF. We previously demonstrated that MS B-cell secreted products are toxic to oligodendrocytes and neurons. The potential for B-cell–myeloid cell interactions to propagate progressive MS is of considerable interest.

Methods

Secreted products of MS-implicated pro-inflammatory effector B cells or IL-10-expressing B cells with regulatory potential were applied to human brain-derived microglia or monocyte-derived macrophages, with subsequent assessment of myeloid phenotype and function through measurement of their expression of pro-inflammatory, anti-inflammatory and homeostatic/quiescent molecules, and phagocytosis (using flow cytometry, ELISA and fluorescently-labeled myelin). Effects of secreted products of differentially activated microglia on B-cell survival and activation were further studied.

Findings

Secreted products of MS-implicated pro-inflammatory B cells (but not IL-10 expressing B cells) substantially induce pro-inflammatory cytokine (IL-12, IL-6, TNFα) expression by both human microglia and macrophage (in a GM-CSF dependent manner), while down-regulating their expression of IL-10 and of quiescence-associated molecules, and suppressing their myelin phagocytosis. In contrast, secreted products of IL-10 expressing B cells upregulate both human microglia and macrophage expression of quiescence-associated molecules and enhance their myelin phagocytosis. Secreted factors from pro-inflammatory microglia enhance B-cell activation.

Interpretation

Potential cross-talk between disease-relevant human B-cell subsets and both resident CNS microglia and infiltrating macrophages may propagate CNS-compartmentalized inflammation and injury associated with MS disease progression. These interaction represents an attractive therapeutic target for agents such as Bruton's tyrosine kinase inhibitors (BTKi) that modulate responses of both B cells and myeloid cells.

Funding

Stated in Acknowledgments section of manuscript.

Research in context.

Evidence before this study

While pro-inflammatory memory B cells are now considered important contributors to peripheral cellular interactions involved in relapses of multiple sclerosis (MS), it is also well-established that B cells are fostered inside the MS CNS, with the potential that they contribute to CNS-compartmentalized inflammation and progressive disease. Within the CNS, B cells can be found enriched in meningeal immune-cell infiltrates that are associated with subjacent areas of subpial cortical demyelination, recognized in turn as important contributors to progressive disease. Hallmarks of this subpial cortical injury include a graded pattern of neuronal loss as well as microglial activation (both of which are greatest in the superficial cortical layer adjacent to the pia/meninges, and become less pronounced as one moves into deeper layers of the cortex), establishing a ‘surface-in’ pattern of pathology that could be consistent with one or more soluble toxic factor(s) that diffuse from the CSF (potentially secreted by the immune cells in the meninges). Supporting this possibility is prior work demonstrating that MS B-cell secreted products can be toxic to rat oligodendrocytes and both rat and human neurons. In addition, activated microglia/macrophages are a hallmark of the chronic-active (also known as active/inactive) perivascular lesions, which are also considered to be involved in disease progression. Given the importance of activated microglia and macrophages to CNS compartmentalized injury and progressive disease, as well as prior work indicating that B cells of MS patients may interact with myeloid cells in the periphery (as part of relapsing disease biology), we examined how secreted products of MS-implicated pro-inflammatory B cells (as well as IL-10 expressing B cells that may be induced with emerging therapies), may potentially impact disease-relevant responses of both human CNS-resident microglia and monocyte-derived macrophages.

Added value of this study

We demonstrate that products secreted by pro-inflammatory MS-implicated B cells can induce pro-inflammatory activation of both human brain-derived microglia and peripheral monocyte-derived macrophages. The pro-inflammatory myeloid cell activation mediated by these B cells (which is partly GM-CSF dependent) is associated with inhibition of homeostatic/quiescence molecules and induction of costimulatory molecules and inflammatory cytokines by the myeloid cells, which would be expected to propagate CNS-compartmentalized inflammation and injury. Secreted products of the MS-implicated B cells also decrease myeloid-cell myelin phagocytosis, which is important for clearance of myelin debris that can otherwise hinder repair. In turn, secreted factors from pro-inflammatory human microglia and macrophage promote B-cell activation. In contrast to the effects of pro-inflammatory B cells on the myeloid cells, soluble products of IL-10 expressing B cells induce myeloid cell molecules associated with quiescence and downregulate their pro-inflammatory responses.

Implications of all the available evidence

The prior work showing that soluble factors of MS-implicated B cells can be toxic to oligodendrocytes and neurons, together with our current demonstration that they can mediate pro-inflammatory activation of human CNS resident microglia, indicate that B-cell secreted products of MS patients may contribute to all the hallmark features of the subpial cortical injury in MS (ie. demyelination and the ‘surface-in’ neuronal loss and microglial activation). Our findings also raise the possibility that secreted products of MS patient B cell might activate infiltrating macrophage in the context of acute perivascular lesions, as well as resident microglia involved in the chronic-active lesions implicated in progressive MS pathology. Our further demonstration that activated pro-inflammatory microglia and macrophages induce activation of the B cells, points to bi-directional interactions that we propose exist between infiltrating B cells and CNS resident and infiltrating myeloid cells, which together would be expected to propagate CNS-compartmentalized inflammation and injury. The capacity we show of IL-10 expressing B cells to promote quiescence and mediate anti-inflammatory responses of microglia and macrophages highlights the opportunity to therapeutically mitigate CNS-compartmentalized inflammation by either: (i) limiting interactions between pro-inflammatory B cells and myeloid cells; or (ii) modulating B cells of patients from a pro-inflammatory to anti-inflammatory response profile that, in turn, may mitigate pro-inflammatory myeloid cells involved in progressive disease. Of particular interest in this regard are CNS-penetrating Bruton's tyrosine kinase inhibitors (BTKi) that modulate responses of both B cells and myeloid cells.

Introduction

Multiple sclerosis (MS) is a potentially debilitating chronic inflammatory condition affecting the central nervous system (CNS). Early disease is characterized by relapses and remissions, with most patients eventually experiencing progressive deterioration. While MS was traditionally viewed as a T-cell mediated disease, the demonstration that anti-CD20 therapies (which selectively deplete circulating B cells) are highly effective at limiting new MS relapses,1, 2, 3, 4, 5, 6, 7, 8, 9 catalyzed studies that have implicated important antibody-independent peripheral contributions of memory B-cells to relapse biology. In particular, frequencies of circulating memory B cells that express high levels of costimulatory molecules and secrete exaggerated levels of pro-inflammatory cytokines (including GM-CSF, TNFα, LT and IL-6) are increased in untreated patients with MS. These pro-inflammatory B cells are thought to contribute as efficient antigen presentating cells (APCs), and/or through bystander effects of pro-inflammatory cytokine secretion, to aberrant peripheral activation of both T cells and myeloid cells that consequently traffic to the CNS and participate in relapsing disease biology.^3,10, 11, 12, 13, 14, 15, 16 At the same time, IL-10 expressing B cells (that have been implicated in downregulation of CNS inflammation in animal models in vivo, and can down-regulate both human T-cell and myeloid-cell responses in vitro), are diminished in MS patients.^10,11,15

In contrast to the contributions of peripheral immune cell interactions to relapsing MS disease activity, the biology underlying (non-relapsing) disease progression in MS is thought to involve inflammatory and degenerative mechanisms compartmentalized within the CNS. As part of the CNS-compartmentalized inflammation, B cells are known to be chronically fostered in the MS CNS, including within B-cell rich leptomeningeal and ependymal immune-cell collections17, 18, 19, 20, 21, 22, 23; reviewed in.^24,25 Pathological studies highlight the potential association between such B-cell rich leptomeningeal inflammation and subpial cortical, as well as spinal cord pathology,17, 18, 19^,21,26,27 and between ependymal inflammation and thalamic MS pathology,²³ which are now all thought to be important contributors to progressive disease.^23,26, 27, 28 The subpial cortical and thalamic injuries are both characterized by graded neuronal loss and microglial activation, which are most pronounced in the superficial layers of the cortex (immediately underlying the pia/meninges and CSF) and in the CSF-facing aspect of the thalamus (adjacent to ependymal structures), with a lesser degree of pathology in the deeper layers.^19,23,27 Of considerable interest is the possibility that immune cells, and particularly B cells, contribute to the sub-pial cortical and thalamic pathologic changes through release of soluble factors.

In this context, we have previously shown that secreted factors of MS-derived B cells can be cytotoxic to both oligodendrocytes and neurons.^29,30 We have also shown that activated human astrocytes can promote a B-cell fostering environment through secreted factors,³¹ thus emphasizing the potential role for bi-directional glial cell:B cell interactions in promoting CNS-compartmentalized inflammation. Herein, we consider whether activated CNS-myeloid cells can promote B cell activation and whether, in turn, MS-implicated B cells have the potential to impact myeloid cells, including both infiltrating monocyte-derived macrophages and resident microglia. Our overarching hypothesis is that such bi-directional cross-talk between disease-implicated B cells and microglia/macrophages mediated by the release of soluble factors, contributes to the propagation of CNS compartmentalized inflammatory injury and disease progression in MS.

Methods

Participants

Healthy control (HC) donors were recruited at the Montréal Neurological Institute, or at the University of Pennsylvania (Supplementary Table S1). Well characterized cohorts of self-reported male and female patients with MS were recruited at the MS clinic of the Montreal Neurological Institute (MNI), and the MS division, Department of Neurology, at the University of Pennsylvania (Supplementary Table S2). At the time of phlebotomy, all patients were untreated, had no exposure to immune-modulating treatments for at least six months, and no steroid exposure for at least 30 days prior the blood draws.

Human B cell isolation and culture

Peripheral blood mononuclear cells (PBMCs) were isolated by density centrifugation using Ficoll (GE Healthcare) using our well-established standardized protocols,¹⁵ and B cells were purified from PBMCs by positive selection using CD19 beads (Miltenyi Biotec) according to the manufacturer's protocol. B cell purity was regularly verified using flow cytometry, with typical purities of >98%. B cells were freshly plated in U-bottom 96 well-plates at a density of 2 × 10⁵ cells/well, in a total volume of 200 μL of serum free x-vivo culture media (Gibeco, Life Technologies). B-cell survival and activation profiles were assessed by flow cytometry following two days in culture, after exposure to conditioned media derived from microglia. We considered live B cells to be Annexin V^-/7AAD^- CD20⁺. To generate pro-inflammatory B cells, we activated B cells with a combination of CD40L (1 mg/mL, Enzo Life Sciences), IgM BCR cross-linking antibody (10 mg/mL, Jackson ImmunoResearch) and IL-4 (20 ng/mL, R&D System); and to generate IL-10 expressing B cells, we activated B cells with CpG DNA (1 mM; ODN2006, InvivoGen), as previously described.^11,13,15,32 All B cells were washed after 12 h of culture and serum free media was replaced. After a total of three days of culture, the non-activated B cell supernatant (referred to as ‘B sup’); the pro-inflammatory B cell supernatants (referred to as ‘B40 × 4 sup’); and the IL-10 expressing B cell supernatant (referred to as ‘BCpG sup’) were collected, and subsequently added at a 1:1 ratio to non-polarized microglia and macrophage cultures, as described below.

Human brain-derived microglia isolation and culture

Human adult microglia were isolated from temporal lobe brain tissue obtained from patients undergoing brain surgery for intractable epilepsy, following the Canadian Institutes of Health Research (CIHR) guidelines, and using our previously described protocol.³³ All tissue used was outside of the suspected focal epileptiform site. Briefly, surgically resected brain tissue is rapidly processed (within 1 h), blood clots are removed, and CNS tissue is rinsed with PBS and digested with 0.5% trypsin and 5 mL DNase for 30 min in a 37 °C shaking water bath. To block trypsinization, 10 mL FCS is then added and mashed brain tissue is subjected to Percoll gradient and several washes to remove myelin. The CNS cell suspension is then washed in MEM media 5% FCS, 1% penicillin/streptomycin and glutamine and kept in culture in T25 flasks. Culturing for 24 h allows microglia to adhere to the flask and the floating cells (including potentially contaminating oligodendrocytes and progenitor cells) are removed. Microglia purity is routinely assessed using flow cytometry after 3–5 days in culture (representative preparation shown in Supplementary Figure S1). For each experimental condition, 150,000–200,000 isolated human microglia were used. Under basal media conditions, the isolated microglia were cultured with Minimum Essential Media (MEM) supplemented with 5% FBS, and 1% penicillin/streptomycin and glutamine and cultured in either 12-well plates (1 mL/well) or 96 well-plates (200 μL/well). Where noted, microglia were pre-activated as previously published,³³ using either pro-inflammatory activation conditions with GM-CSF (5 ng/mL, Peprotech), IFNg (20 ng/mL, Gibco Lifetechnologies) and lipopolysaccharide LPS (100 ng/mL, Sigma); or alternate activation conditions with macrophage colony-stimulating factor M-CSF (25 ng/mL Peprotech), IL-4 (20 ng/mL, Gibco Lifetechnologies) and IL-13 (20 ng/mL, Gibco Lifetechnologies). The microglia were then washed three times with PBS, and replenished with fresh x-vivo serum-free media for another 24–30 h before collecting the microglia-conditioned media. We subsequently added 25% of either pro-inflammatory microglia-conditioned media (designated GM/I/L) or alternately activated microglia-conditioned media (designated M/4/13) to B-cell cultures, to assess the effects of differentially activated microglia-secreted products on the B cells. To assess the effects of B cell-secreted products on the differentially-treated microglia, the microglia were first either polarized or kept under basal culture media conditions as described above, then exposed to B-cell supernatants for two days, and characterized as described below.

Monocyte-derived macrophage isolation and culture

Monocytes were purified from PBMCs using positive selection with CD14 beads (Miltenyi Biotec) according to the manufacturer's protocol, and their purity was routinely confirmed by flow cytometry (consistently >98%). Freshly purified monocytes were then suspended at a density of 5 × 10⁵ cells/mL and plated in either flat bottom 12-well plates (1 mL/well) or 96 well-plates (200 μL/well). Cells were differentiated into macrophage for a period of five days in culture, using Roswell Park Memorial Institute (RPMI) media containing 10% fetal bovine serum (FBS), 1% penicillin/streptomycin and glutamine. Where noted, monocyte-derived macrophage were either left unpolarized in the presence of M-CSF or polarized in vitro as described for the microglia above. To characterize the effects of B-cell secreted products on macrophages, the differentially polarized or unpolarized macrophages were washed and exposed to the distinct B-cell supernatants, and characterized as described below.

Flow cytometry characterization of B cells, microglia and macrophages

Following two days in culture, B cells exposed to microglia conditioned media were collected and washed with PBS containing 5% FBS, and incubated with surface antibody staining: CD20 (anti-CD20, 2H7, BD Bioscience), CD69 (anti-CD69, L78 BD Bioscience), CD86 (anti-CD86, FUN-1, BD Bioscience) and CD95 (anti-CD95, DX2, BD Bioscience) for 20 min at 4 °C. B cells were then washed twice and stained with Annexin V (BD Bioscience) and 7AAD (BD Bioscience) for 10 min at room temperature using Annexin V buffer (BD Bioscience). To phenotype human microglia and macrophages under different differentiation conditions or in response to supernatants derived from functionally distinct B cell subtypes, we used Annexin V (BD Bioscience), 7AAD (BD Bioscience), CD11 (3.9, BD Bioscience), CD80 (2D10, Biolegend), CD115 (9-4D2-1E4), CD172 (15-1729-42), CD200R (OX-108, Biolegend), HLA-DR (G46-6, BD Bioscience), CD274 (MIH1, BD Bioscience), MerTK (125518; R&D System), mouse anti-human TREM-2 monoclonal antibody (10B11, generated in house by Dr. Laura Piccio; original clone generated in the laboratory of Dr. Marco Colonna), SIRP1⍺ (SE5A5, Biolegend) (Supplementary Table S3). In short, soluble TREM-2 was produced as a chimeric protein consisting of TREM-2 extracellular domain and human IgM constant regions (TREM-2-HuIgM). The latter, was amplified from the cloned full length cDNA by polymerase chain reaction (PCR) and used as FLAG peptide NH2-ter-minal fusion protein (Eastman Kodak Co.) to transiently transfect 293 HEK cells using (Bio-Rad Laboratories). For anti- TREM-2 huIgM production 6 week old BALB/c mice (Iffa-Credo) were immunized with purified TREM-2-HuIgM. Spleen cells were fused with the SP2/0 myeloma cells and using ELISA hybridoma supernatants were screened with TREM-2-HuIgM as capturing protein and human-absorbed horseradish peroxidase (HRP)-labeled goat anti-mouse IgG (BD PharMigen) as detecting Ab. ELISA-positive hybridoma supernatants were then tested by flow cytometry for staining 293 cells explressing FLAG-tagged TREM-2 mAb 29 EE (anti-TREM-2, IgG1), mAb 21c7 (control IgG, anti-TREM1). Briefly microglia were detached using dissociation buffer (Thermofisher), washed with PBS containing 5% FCS, and incubated with cell surface staining cocktail antibody, including CD11, CD80, CD115, CD200R, HLA-DR, CD274, CCR7, MerTK, TREM-2 and SIRP1⍺ for 20 min at 4 °C. Microglia were washed and stained with Annexin V and 7AAD for 10 min at room temperature using Annexin V buffer (BD Bioscience). All flow cytometry was performed by a single operator who followed the same standardized protocols for acquisition and analysis. All samples were acquired on LSR-Fortessa (BD Biosciences) and analyzed using FlowJo software.

The non-activated B cell supernatant (referred to as ‘B sup’); the pro-inflammatory B cell supernatants (referred to as ‘B40 × 4 sup’); and the IL-10 expressing B cell supernatant (referred to as ‘BCpG sup’)

Enzyme-linked immunosorbent assays

After exposure of myeloid cells to different B cell supernatants (either non-activated ‘B sup’, pro-inflammatory ‘B40 × 4 sup’ or IL-10 expressing ‘BCpG sup’), followed by the wash and further culture, the microglia and macrophage secreted products were then collected and used to quantify cytokines (IL-12, TNF⍺, IL-6 and IL-10) with OptEIA ELISA Kit (BD Bioscience) according to the manufacturer's protocol. Briefly, ELISA plates were pre-coated with capture antibody for at least 12 h. Non-specific binding sites were blocked for 1 h with blocking buffer (10% FCS and phosphate buffered saline (PBS)), and samples were added to the plate and incubated for 2 h at room temperatures. Detection antibody was added for 1 h at room temperature. The plates were developed by tetramethylbenzidine (BD Biosciences), the reaction was stopped by 0.005 M H₂SO₄ and read by a Bio-Rad microplate reader (Model 5550, Bio-Rad). Plates were washed with ELISA washing buffer (0.05% Tween 20 and PBS) between each step above.

Cytokine blocking assays

To functionally block GM-CSF within the B cell secreted products we added neutralizing antibody to GM-CSF (anti-GM-CSF, 3209, R&D System) or matching isotype control (monoclonal mouse IgG₁, R&D System) to the B cell supernatants at a concentration of 1 μg/mL, for 30 min at room temperature prior to adding the B cell supernatants to the monocyte-derived macrophages. After two days of culture, the macrophages were then activated using LPS (100 ng/mL, sigma) for the last day of culture, prior to measurement of myeloid cytokine production (IL-6, TNFα and IL-10) using ELISA, as described above.

Myelin purification

Human myelin was isolated from white matter obtained from post-mortem brain tissue, using sucrose gradient separation as previously described.³⁴ Briefly, white matter tissue was homogenized in 0.32 M sucrose, 1 mM MgCl, 10 mM Tris-HCL (pH = 7.5) and 0.1 mM PMSF. The homogenate was then layered with 1 M and 0.32 M sucrose containing 20 mM Tris- HCL (pH = 7.5) and 0.1 mM PMSF and centrifuged for 100,000 g over night at 4 °C. Crude myelin membrane and astrogliosome layers were carefully collected and diluted 1:1 in ice cold ultrapure H₂O (containing 20 mM Tris pH 7.5 + 0.1 mM PMSF). To concentrate myelin membranes, crude myelin membranes were pelleted at 100,000 g for 1 h and hyposomatically shocked by homogenization in 20 vol. of ice cold ultrapure H₂O (containing 20 mM Tris pH 7.5 + 0.1 mM PMSF). Shocked membranes were subjected to serial centrifugations (40,000 g for 20 min, 100,000 g for 3 h) and resuspended in 0.32 M sucrose at a final concentration of 1ug/mL for subsequent use in phagocytosis experiments. Myelin was confirmed to be endotoxin-free using the Limulus ambebocyte lysate test (Sigma Aldrich) and western-blot experiments confirmed the presence of myelin proteolipid protein (PLP), myelin basic protein (MBP) and myelin oligodendrocyte glycoprotein (MOG) within the isolated myelin preparation.

Phagocytosis assays

To assess the capacity of myeloid cells to ingest myelin, we first incubated the purified human myelin with a pH-sensitive dye (pH-Rodamine; Invitrogen) for 1 h in PBS (pH = 8). Fluorecently-labeled myelin was then added at a final concentration of 20 μg/mL to microglia or macrophage that were pre-exposed to distinct B-cell supernatants for two days. Following 1 h of incubation at 37 °C, epifluorescence microscopy (Leica Microsystems, Wetzlar, Germany) was used to visualize and quantify myelin phagocytosis by the microglia and macrophages, and flow cytometry (FACS Fortessa, BD Bioscience) was used to quantify internalized myelin. Macrophage myelin phagocytosis assays were performed to directly compare the impact of pro-inflammatory versus IL-10 expressing B cell supernatants derived from both RRMS patients and healthy controls (HC) on responses of the same HC donor macrophage.

Statistical analysis

All statistical analyses employed GraphPad Prism (version 7 and 9). Paired t tests were used for comparison across sex- and age-matched groups and either Friedman test with Dunn's multiple comparison test or one-way ANOVA were used for comparisons across groups or conditions, with two-way ANOVA used for comparisons between several groups across different conditions. The populations to which statistical models were applied included HC alone, MS alone and HC plus MS. The dependent variable in eah model was cell subset frequency or ingested myelin, while the fixed effects varied based on population of interest and study question. All statistical tests are indicated in the figure legends. p values of ≤0.05 were considered statistically significant. Sample size was determined based on the number of available human samples (primary microglia). No randomization or blinding were implemented. MS patients were only included if they were never exposed to immunomodulatory therapy or had no exposure for at least six months, and no steroid exposure for at least 30 days prior the blood draws. Additional exclusion criteria included MS patients with any history of malignancy, chronic infections or co-morbid autoimmune conditions, as ascertained by rigourous chart review in addition to self-report.

Ethics

All participants provided informed consent using protocols approved by the Montreal Neurological Institute, McGill University and the University of Pennsylvania institutional ethics review boards (IRB Protocol information: Neurological Disorders Tissue Bank, #816805).

Role of funders

Funding enabled the implementation of this study, however project concept, design, data collection, analysis and interpretation as well as manuscript writing were completed in an independent manner by the authors.

Results

Impact of human microglia-secreted factors on human B-cell survival and activation

Prior work assessing the potential role for glial cell:B cell interactions in promoting CNS-compartmentalized inflammation in MS demonstrated that secreted factors derived from activated human astrocytes can both enhance survival and pro-inflammatory activation of B cells. To extend this work, we first considered the effects that secreted factors of human CNS-resident myeloid cells (microglia) may have on B-cell survival and activation. Microglia are now recognized for their potential to exhibit diverse functional response profiles in both health and disease, that can range from pro-inflammatory to anti-inflammatory and potentially reparative activation states. To consider the possibility that differentially activated microglia may have different effects on B cells, we generated supernatants (conditioned media) from human microglia cultured under either basal conditions or pre-activated using GM-CSF/IFNg/LPS (‘GM/I/L’) as a proxy for microglia undergoing a form of pro-inflammatory activation, or using M-CSF/IL-4/IL-13 (‘M/4/13’) as a proxy for microglia undergoing alternate activation (with the activation conditions explicitly indicated in the figures as recommended by a recent microglia nomenclature white paper.³⁵ We then exposed human B cells in vitro to the different microglia supernatants and, using flow cytometry and Annexin V/7AAD staining, subsequently quantified B-cell survival for up to four days. Conditioned media of microglia cultured under basal conditions or under the distinct polarization conditions did not impact the survival of total (CD20⁺), memory (CD27⁺CD20⁺) or naïve (CD27⁻CD20⁺) human B cells, compared to the survival of corresponding B cells cultured with no exposure (Fig. 1 depicts B-cell survival assessed at 48 h following exposure to the different microglia secreted products; Supplementary Figure S2 depicts kinetic survival data of B cells over 96 h). We next assessed whether and how exposure of human B cells to the conditioned media of the differentially activated human microglia would influence B-cell activation. We observed that B cells exposed to ‘GM/I/L’- but not ‘M/3/4’-microglia supernatants substantially increased surface expression of the co-stimulatory molecule CD86 (Fig. 2a; p < 0.0001, n = 9) and the activation markers CD95 (Fig. 2b; p < 0.0001, n = 8) and CD69 (data not shown), while not impacting B-cell expression of HLA-DR (Fig. 2c; p > 0.99, n = 7).

Fig. 1.

No impact of human microglia-secreted factors on survival of human B cells and major B cell subsets. Human microglia were either left unpolarized under basal culture conditions (designated ‘Microglia Unp’), or polarized in vitro under pro-inflammatory activating conditions (using GM-CSF, IFNγ and LPS, designated ‘Microglia GM/I/L’); or under alternate-activation conditions (M-CSF, IL-4 and IL-13, designated ‘Microglia M/4/13’). Microglia were then washed and kept in culture for 48 h before collection of their conditioned media. B cells were cultured in serum-free media, either alone or with 25% microglia-conditioned media. Following 48 h of culture, B cell viability was quantified as the proportion of Annexin V^-/7AAD^- cells, gating on total CD20⁺ B cells or their memory (CD27+) and naïve (CD27−) subsets. (a) flow cytometry dot plots of representative experiment (b–d) summary survival data for (b) total B cells; and (c) memory or (d) naïve B cells subsets; n = 6–7 independent experiments. Data were analyzed with one-way ANOVA. See Supplementary Figure S2 for kinetic survival data of B cells over 96 h following exposure to the different microglia secreted products.

Fig. 2.

Soluble factors derived from human microglia undergoing prior pro-inflammatory activation induce B cell activation. B cells purified from peripheral blood mononuclear cells (PBMC) of healthy donors were cultured in serum-free media alone or in serum-free media with 25% conditioned media derived from microglia following either pro-inflammatory activation with GM-CSF, IFNg and LPS (designated ‘GM/I/L’) or alternate activation with M-CSF, IL-4 and IL-13 (designated ‘M/4/13’). The activation profile of live (Annexin V^- 7AAD^-) B cells was assessed after 48 h using flow cytometry. Conditioned media of microglia previously undergoing pro-inflammatory activation increases B cell surface expression of (a) the CD86 co-stimulatory-molecule; and (b) the activation marker CD95, (c) without an obvious change in their expression of HLA-DR. Levels of expression are shown as fold change in mean fluorescence intensity (MFI) as compared to B cells cultured in serum-free media alone; n = 7–9 independent experiments.

Secreted factors derived from pro-inflammatory B cells induce a pro-inflammatory profile of microglia and macrophages

Both resident microglia and infiltrating monocyte-derived macrophages are present within the chronically inflamed CNS of patients with MS. We therefore evaluated the consequences of exposing such myeloid cells to supernatants derived from functionally distinct B cells. We generated supernatants from B cells that were either non-activated or pre-activated as previously described to generate pro-inflammatory B cells or IL-10 expressing B cells.^{10,11,13,15,32} In keeping with the prior reports, B cells activated under pro-inflammatory conditions secreted considerably higher levels of GM-CSF and IL-6, while B cells activated under IL-10 promoting conditions induced higher levels of IL-10 (Supplementary Figure S3). We then transiently cultured purified human microglia or monocyte derived-macrophages with the various B cell supernatants. After two days, the myeloid cells were washed thoroughly with fresh media added, and then activated with LPS for another day in culture to assess their cytokine expression profiles. Compared to microglia pre-exposure to non-activated B cell supernatants (designated as ‘B sup’) or IL-10 expressing B cell supernatants (designated based on the B cell activation conditions as ‘BCpG sup’), pre-exposure of microglia to pro-inflammatory B cell supernatants (designated as ‘B40 × 4 sup’) significantly increased the capacity of microglia to secrete the pro-inflammatory cytokines IL-12 (Fig. 3a; p = 0.03, n = 5), IL-6 (Fig. 3b; p = 0.01, n = 5) and TNF⍺ (Fig. 3c; p = 0.001, n = 5), while the pro-inflammatory B cell supernatants significantly down-regulated the microglia IL-10 production (Fig. 3d; p = 0.002, n = 6). Similarly, secreted products of pro-inflammatory B cells significantly enhanced the capacity of monocyte-derived macrophages to secrete IL-12 (Fig. 3e; p = 0.02, n = 6), IL-6 (Fig. 3f; p = 0.0005, n = 4) and TNF⍺ (Fig. 3g; p = 0.002, n = 3) while down-regulating their IL-10 production (Fig. 3h; p = 0.002, n = 5). To address the molecular mechanism underlying these effects of B-cell supernatants on myeloid cell responses, and given the limiting number of available human-derived microglia, we carried out a series of experiments focusing on monocyte-derived macrophages, using neutralizing antibodies to selected B cell cytokines. We found that the capacity of secreted products from pro-inflammatory B cells to induce macrophage pro-inflammatory cytokine responses was GM-CSF-dependent, as the selective antibody neutralization of GM-CSF within pro-inflammatory B cell supernatants (compared to control antibody) abrogated the enhanced myeloid cell production of IL-12 (Fig. 3e; p = 0.006, n = 3), IL-6 (Fig. 3f; p = 0.01, n = 5) and TNF⍺ (Fig. 3g; p = 0.001, n = 3). GM-CSF blockade did not affect the ability of the pro-inflammatory B cell supernatants to downregulate IL-10 production by myeloid cells (data not shown). Similar experiments neutralizing IL-10 within the supernatants of B cells activated under the IL-10 inducing conditions revealed that the IL-10 in these B cell supernatants was partially responsible for inhibiting myeloid secretion of TNF⍺, but was not responsible for inhibiting myeloid secretion of IL-12 and IL-6 (data not shown). The differential modulation of myeloid cytokine responses by the different B cell supernatants occurred without influencing the myeloid cell survival (Supplementary Figure S4).

Fig. 3.

Soluble factors derived from pro-inflammatory B cells induce a pro-inflammatory cytokine response profile of human microglia and macrophage. Human microglia (a–d) were purified from CNS tissue obtained during epilepsy surgery and macrophages (e–h) were generated by M-CSF treatment of CD14⁺ peripheral monocytes freshly isolated from healthy controls. Microglia and Macrophage were then exposed in a 1:1 ratio to B-cell supernatants derived from either non-activated B cells (designated ‘B sup’); pro-inflammatory B cells activated with a combination of CD40L, IgM BCR cross-linking antibody and IL-4 (designated ‘B40 × 4 sup’); or IL-10 expressing B cells activated with CpG DNA (designated ‘BCpG sup’), for 24 h, then washed and activated with LPS for an additional 24 h in fresh media. Myeloid-cell supernatants were then collected and IL-12, IL-6, TNFα and IL-10 secretion was measured using ELISA. (a-c) Supernatants derived from pro-inflammatory B cells increase IL-12, IL-6 and TNFα production by microglia; while (d) down-regulating their IL-10 production. (e) The pro-inflammatory B cell-secreted products similarly enhance macrophage production of IL-12; (f) IL-6 and (g) TNFα, while (h) decreasing their IL-10 production. Each column represents mean data from n = 6 (microglia) and n = 6–7 (macrophage) independent experiments; data were analyzed with one-way ANOVA.

Secreted products of pro-inflammatory B cells or IL-10 expressing B cells differentially modulate the balance between quiescent and activation markers of human microglia and macrophages

The contribution of myeloid cells (both CNS-resident microglia and infiltrating monocyte-derived macrophages) to CNS compartmentalized inflammation in MS is thought to be influenced in part by the balance between the levels of their surface expression of activation-associated molecules (eg. CD80; HLA-DR) and quiescence-associated molecules (eg. TREM-2, MerTK, M-CSFR, SIRP1⍺, CD200R). To evaluate the potential for functionally distinct B cell subsets to impact the balance between myeloid cell activation-associated and quiescence-associated molecules, we first wished to establish that the expression of these molecules by myeloid cells could be modulated in vitro. To this end, we demonstrated that a traditional pro-inflammatory activation paradigm (‘GM/I/L’) induced the expected myeloid surface expression of CD80 in both microglia (Supplementary Figure S5a and b; p = 0.02, n = 6) and monocyte derived-macrophages (Supplementary Figure S5h and i; p < 0.0001, n = 12), and also induced HLA-DR expression by macrophage (Supplementary Figure 5j; p < 0.001, n = 8) with a tendency to do so also for microglia. The same pro-inflammatory activation also tended to decrease surface expression of the quiescence molecules TREM-2 and MerTK by microglia (Supplementary Figure S5e and f) and also decreased expression of several quiescence molecules by macrophage (Supplementary Figure S5l–o). In contrast, a classic alternative activation paradigm (‘M/4/13’) enhanced expression of the quiescence molecules TREM-2 and M-CSFR by microglia (Supplementary Figure S5e and g), and of the quiescence molecules TREM-2, SIRP1⍺ and CD200R by macrophage (Supplementary Figure S5l, p and q). With this established readout system that could capture reciprocal modulation of myeloid activation and quiescence markers, we next assessed how conditioned media derived from pro-inflammatory B cells or IL-10 expressing B cells might impact the balance between these markers, on either macrophages or microglia. As shown in Fig. 4, pro-inflammatory B cell derived conditioned media (‘B40 × 4 sup’) increased expression of the activation marker CD80 on both microglia (Fig. 4a and b; p = 0.01, n = 4) and macrophages (Fig. 4g and h; p = 0.003, n = 12) without significantly changing their expression of HLA-DR (Fig. 4c and i). The pro-inflammatory B cell secreted products also tended to decrease expression of the quiescence molecule TREM-2 by microglia (Fig. 4d and e) and decreased expression of MerTK by macrophages (Fig. 4l). In contrast, IL-10 expressing B-cell conditioned media (‘BCpG sup’) substantially up-regulated TREM-2 surface expression by both microglia (Fig. 4d and e; p = 0.04, n = 5) and macrophages (Fig. 4j and k; p = 0.00008, n = 11) relative to the effects of pro-inflammatory B-cell derived conditioned media. Overall, these findings indicate that secreted products of functionally distinct B-cell subsets can differentially modulate the balance between quiescence and activation markers of microglia and macrophages. Specifically, pro-inflammatory B cells (as implicated in MS) could induce, through their secreted products, an activated, pro-inflammatory state of both CNS-resident and potentially infiltrating myeloid cells, while B cells modulated towards an anti-inflammatory and potentially regulatory response profile, did not activate the myeloid cells and could even serve to upregulate myeloid cell expression of molecules associated with a more quiescent or homeostatic state.

Fig. 4.

Differential modulation of activation and quiescence marker expression by human microglia and macrophage, following exposure to secreted products of pro-inflammatory versus IL-10 expressing B cells. Human microglia (a–f) and monocyte-derived macrophage (g–l) were either cultured under basal media conditions (‘Media’), or exposed at a 1:1 ratio to either pro-inflammatory B cell supernatants (‘B40 × 4 sup’) or IL-10 expressing B cell supernatants (‘BCpG sup’) for 48 h, and myeloid cell surface marker expression were then assessed by flow cytometry. Representative flow cytometry histogram plots of expression of the activation marker and co-stimulatory molecule CD80 by (a) microglia and (g) macrophage. (a, b) Pro-inflammatroy B cell supernatants substantially induce CD80 expression by both microglia (a,b) and (g, h) macrophage while downregulating the expression of (e) quiescence molecules TREM-2 by microglia and (i) MerTK by macrophage. In contrast, IL-10 expressing B cell supernatants significantly induce TREM-2 expression by both (e) microglia and (k) macrophage. Summary data depict average results from n = 4 (microglia) and n = 8–12 (macrophage) independent experiments; data were analyzed with the Friedman and Dunn's multiple comparison tests.

Secreted factors derived from pro-inflammatory and IL-10 expressing B cells differentially modulate myelin phagocytosis by microglia and macrophages

To assess the impact of myeloid cell exposure to B cell secreted products on the potential capacity of CNS resident microglia and infiltrating macrophages to phagocytose human myelin, purified human microglia and macrophages were pre-exposed to conditioned media derived from pro-inflammatory and IL-10 expressing B cells, and flow cytometry was subsequently used to quantify fluorescently-tagged myelin ingestion by the myeloid cells (Fig. 5). We observed that the pro-inflammatory (‘B40 × 4 sup’) and IL-10 expressing (‘BCpG sup’) B cell supernatants differentially impacted microglia phagocytosis of human-derived myelin. While exposure to pro-inflammatory B cell supernatants tended to decrease myelin phagocytosis by both microglia (Fig. 5h) and macrophages (Fig. 5i), exposure to supernatants of IL-10 expressing B cells substantially increased phagocytic functions of both microglia (p = 0.01, n = 4) and macrophages (p = 0.009, n = 6) compared to the effects of the pro-inflammatory B cell supernatants.

Fig. 5.

Secreted factors derived from pro-inflammatory versus IL-10 expressing B cells differentially modulate myelin phagocytosis by microglia and macrophage. Purified human microglia (a–h) or macrophages (i), were either cultured under basal media conditions (‘Media’), or exposed to pro-inflammatory B cell supernatants (‘B40 × 4 sup’) or IL-10 expressing B cell supernatants (‘BCpG sup’) for 48 h with human pH-rhodamine stained myelin added to the last hour of culture (a–c). Representative immunofluorescence images (20×) of microglia phagocytosis of myelin depicting the impact of exposure to (b) pro-inflamatory and (c) IL-10 expressing B-cell supernatants, compared to microglia phagocytosis under (a) basal media conditions. (d, e, f) Representative flow cytometry dot plots and gating strategy used to quantify myelin-ingesting microglia based on mean fluorescence intensity (MFI) of pH-rhodo staining (d–f) and (g) MFI histogram plots of a representative experiment. While pro-inflammatory B cell supernatants tend to decrease myelin phagocytosis, supernatants of IL-10 expressing B cells substantially increase myelin phagocytosis by both (h) microglia and (i) macrophage. Data depict the average of n = 4 (microglia) and n = 6 (macrophage) independent experiments; analyzed with the Friedman and Dunn's multiple comparison test.

Soluble products of pro-inflammatory B cells derived from untreated patients with MS induce heightened pro-inflammatory myeloid cell responses

Since patients with MS have been shown to harbor B cells with an increased propensity for proinflammatory responses following activation,^11,13,15,32 we wished to examine how secreted products of MS B cells may impact myeloid cells. Using the same experimental approach described above, we purified circulating B cells from untreated patients with MS and age- and sex-balanced healthy controls (Supplementary Tables S1 and Table S2), then differentially activated their B cells as described above, and collected their supernatants. The supernatants were applied in parallel to macrophages derived from monocytes that were isolated from the same healthy control individuals. We observed that, compared to secreted products of pro-inflammatory B cells generated from healthy controls (‘HC B40 × 4 sup’), secreted products of pro-inflammatory B cells generated from patients with MS (‘MS B40 × 4 sup’) induced significantly higher secretion of the macrophage pro-inflammatory cytokines IL-12p40 (Fig. 6a: p = 0.01, n = 7) and TNFα (Fig. 6c: p = 0.009, n = 7). Secreted products of IL-10 expressing B cells generated from both patients with MS (‘MS B4CpG sup’) and healthy controls (‘HC B4CpG sup’) induced lower levels of pro-inflammatory myeloid cytokines (Fig. 6a–c) while inducing similarly increased production of myeloid IL-10 (Fig. 6d; p = 0.027 and p = 0.0066, n = 7). Similar effects were also noted when assessing the impact of supernatants of pro-inflammatory and IL-10 expressing B cells generated from MS patients and controls on expression of the myeloid cell activation marker CD80, which was increased by exposure to the pro-inflammatory B cell supernatants but not the IL-10 expressing B cell supernatants (Fig. 6e; n = 7), and the quiescence marker TREM-2 which was increased by exposure to the IL-10 expressing B cell supernatants but not the pro-inflammatory B cell supernatants (Fig. 6f; n = 7). There were no diffrences between the effects of MS or healthy control derived B-cell secreted products on myeloid cell expression of HLA-DR or MerTk (data not shown). Comparison of the effects of secreted products of MS and healthy control derived B cells also revealed similar effects on phagocytosis of monocyte-derived macrophages, with products of IL-10 expressing B cells from both MS and controls inducing greater phagocytosis than their pro-inflammatory B cell products (data not shown). We could further recapitulate the reciprocal modulation of human microglial cell phagocytosis by secreted products of untreated MS patient pro-inflammatory versus IL-10 expressing B cells (Supplementary Figure S6).

Fig. 6.

Soluble products of pro-inflammatory B cells derived from untreated patients with MS induce heightened pro-inflammatory myeloid cell responses. B cells were purified from healthy controls (HC) or from age- and sex-balanced untreated patients with MS (Supplementary Tables S1 and S2) and activated as described above. B cell supernatants were then applied in parallel to monocyte-derived macrophage isolated from the same healthy control individuals. Supernatants of pro-inflamatory B cells generated from MS patients (deignated ‘MS B40 × 4 sup’) induced (a) higher secretion of IL-12p40, (b) IL-6 and (c) TNFα by myeloid compared to levels induced by pro-inflamatory B cells generated from healthy controls (deignated ‘HC B40 × 4 sup’). Supernatants B cells generated under IL-10-promoting activation conditions from MS patients (‘MS BCpG sup’) and healthy controls (‘HC BCpG sup’) both induced (d) increased secretion of myeloid cell IL-10. The supernatants of pro-inflammatiory and IL-10 expressing B cells generated from MS patients had similar reciprocal effects on myeloid cell expression of (e) the activation marker CD80 and (f) quiescence marker TREM-2 as did the supernatants of pro-inflammatiory and IL-10 expressing B cells generated from healthy controls. Data shown from n = 7 independent experiments each using different sources of MS and control B cells and control macrophage; data were analyzed with one-way ANOVA.

Discussion

In addition to the peripheral contribution of abnormal pro-inflammatory memory B cells to relapse biology in patients with MS, B cells are chronically fostered in the CNS of patients, including within B-cell rich leptomeningeal immune-cell collections17, 18, 19 and ependymal infiltrates^18,20,22,23; reviewed in.^24,25 These leptomeningeal and ependymal infiltrates represent key features of CNS-compartmentalized inflammation, and are thought to contribute to the graded ‘surface in’ injuries described in the subpial cortex, and in the CSF aspect of the thalamus, respectively—which, in turn, have been associated with more rapid and/or severe clinical disease progression.^21,23 The intriguing prospect that B cells fostered within meningeal and ependymal immune cell infiltrates contribute to progressive pathology is predicated on such effects being mediated by secreted factors. This prospect is supported by a study that assessed the relationship between subpial cortical injury and soluble factors in the CSF of the same patients, and found that the extent of subpial cortical injury best correlated with CSF factors associated with B cell biology.³⁶ Our prior observations that secreted factors (potentially exosomes) released from B cells of untreated MS patients can induce cell death of both oligodendrocytes and neurons via apoptosis (independent of B-cell derived antibodies),^29,30,37 and that secreted factors of activated human astrocytes can support both the survival and activation of B cells which in turn can function as efficient APC to T cells,³¹ provide further indirect support for potential contributions of B cells fostered within the MS CNS to key pathologic features involved in propagating subpial cortical and thalamic inflammatory injury.

Our current study extends prior work, and considers the potential that bi-directional interactions between B cells and CNS resident (or infiltrating) myeloid cells that are mediated through secretion of soluble factors may further contribute to propagation of chronic compartmentalized inflammation and progressive CNS injury in MS. We examined how, on one hand, soluble products released by differentially activated myeloid cells can impact B-cell survival and their responses, and how soluble products released by functionally distinct B cells including both MS-implicated pro-inflammatory B cells and IL-10 expressing B cells that may have regulatory properties, may impact both resident CNS microglia, as well as infiltrating monocyte-derived macrophages.

Supporting the potential for such bi-directional cross-talk, we observed that secreted products of pro-inflammatory but not control or alternatively activated microglia, can activate B cells. In turn, functionally distinct B cells can differentially impact the state of activation and disease-relevant functional responses of both human CNS resident microglia and monocyte-derived macrophages. In particular, we observed that soluble products released by MS-implicated pro-inflammatory B cells limited subsequent myeloid cell secretion of the anti-infammatory cytokine IL-10, while enhancing pro-inflammatory responses of both microglia and macrophages, including the induction of myeloid cell expression of the T-cell costimulatory molecule CD80 and secretion of the pro-inflammatory cytokines IL-12, IL-6 and TNF⍺; the latter of which are reportedly increased in CSF and within meningeal tissue of patients presenting with more aggressive forms of progressive MS.³⁶ This pro-inflammatory modulation of myeloid cells was partly driven by GM-CSF secreted by the MS implicated B cells, indicating one or more additional molecule/s in the B-cell secreted products influence/s the myeloid cell pro-inflammatory response. Production of IL-12 and IL-6 by antigen presenting cells including myeloid cells is well known to respectively contribute to Th1 and Th17 T cell differentiation,³⁸ providing a potential mechanism by which the B cells could indirectly contribute to pro-inflammatory T cell responses. Thus, pro-inflammatory B cells fostered in the inflamed MS CNS compartment may contribute to propagating pro-inflammatory T-cell responses both directly as shown previously,³¹ and indirectly through modulation of myeloid cells, as shown here.

In contrast to the impact of pro-inflammatory B-cell supernatants, we found that products secreted by IL-10 expressing B cells do not induce pro-inflammatory myeloid responses, but rather can induce microglia and macrophage expression of TREM-2, a molecule implicated in myeloid cell quiescence and phagocytosis rather than pro-inflammatory activation. TREM-2 is highly expressed by foamy myeloid cells in actively demyelinating MS lesions.³⁹ It is known that murine microglia phagocytic function can be enhanced through TREM-2 and in response to IL-10,⁴⁰ and upregulated expression of TREM-2 by microglia has been shown to promote myelin debris clearance and remyelination in a model of CNS inflammation.⁴¹ In keeping with this, we noted substantially increased myelin phagocytosis by both microglia and macrophages following exposure to the IL-10 expressing B cell-secreted products. On the other hand, exposure of myeloid cells to pro-inflammatory B cell-secreted products selectively diminished myelin uptake and MerTK expression by macrophages, though the same was not observed for microglia, raising the possibility that in certain contexts resident CNS microlgia and infiltrating macrophage contribute differently to myelin clearance.

Our suggestion that bi-directional interactions between CNS-infiltrating immune cells and resident CNS cells may be involved in progressive disease in MS is in line with recent work highlighting potential cross-talk between CNS infiltrating lymphocytes and CNS glial cells (particularly microglia and astrocytes) in the context of chronic/active (also known as mixed active/inactive) white matter lesions,⁴² that have also been implicated in progressive MS.43, 44, 45, 46 A study reporting that meningeal lymphocyte accumulation correlates with chronic white matter lesion activity in progressive MS²² raises the intriguing possibility that some common mechanism may contribute to driving progressive disease at both sites of ‘surface-in’ pathology (such as the subpial cortex and thalamus), as well as the chronic active parenchymal lesions. Future work may elucidate in situ gradients of particular molecules involved in driving tissue injury.

Overall, our findings that human microglia can influence the activation profile of B cells and that, in turn, pro-inflammatory and anti-inflammatory activated B cells can differently influence responses of microglia and macrophages, provide insights into bi-directional immune cell:glial interactions that may be involved in CNS-compartmentalized inflammation and injury underlying MS disease progression. The prospect that B cell-myeloid cell interactions may be therapeutically targeted in MS is of particular interest in the context of CNS-penetrating Bruton's tyrosine kinase inhibitors (BTKi), known to modulate responses of both B cells and myeloid cells, as well as in the context of emerging cell-based therapies including IL-10 secreting regulatory B cells that are being developed for autoimmune diseases.

Our study is limited to in vitro experiments, and complementary pathological studies (for example demonstrating the presence of pro-inflammatory B cells in association with pro-inflammatory myeloid cell activation in the MS CNS) would re-enforce our findings. It is worth noting that not all interactions between infiltrating immune cells and glial cells in the MS CNS are necessarily involved in injury, as both immune and glial responses may also contribute to acquiescing pathologic inflammation and/or contributing to clearance of debris and even repair.47, 48, 49, 50, 51 Indeed, both resident microglia and infiltrating macrophages in MS have been found to acquire distinct activation phenotypes and functional properties that could be either pro-inflammatory or beneficial. The mechanisms contributing to differential myeloid cell activation (whether in the subpial cortex, the CSF-facing aspect of the thalamus, or in the context of chornic active parenchymal lesions), remain however largely unknown. Elucidating the specific mechanisms involved in bidirectioanl immune:glial interactions (including those involving B cells and myeloid cells) could point to innovative therapeutic targets aimed at either interrupting interactions that propagate injury, or promoting interactions that may generate anti-inflammatory/acquiescing or reparative responses of both infiltrating and resident-CNS cells. Ultimately, the importance of cascades of immune:glial interactions to progressive disease mechanisms will likely require successful biological-proof-of principle studies in patients, combining therapies that will prove effective at limiting disease progression, with biological measures that will help to ascertain how such effects are achieved.

Contributors

Concept and design H.T. and A.BO. Data acquisition and analysis H.T., R.L. and L.Z. Subject recruitment H.T., L.Z., D.J. and A.BO. C.M. trained H.T. to handle process microglia samples. F.C, L.P shared TREM-2 antibodies. L.H. shared the phagocytosis protocol. S.L., A.P., J.G., J.A.B., R.P.L., J.P.A. F.C.B., discussed findings. Initial preparation of manuscript and figures H.T. Manuscript Revisions H.T. and A.B.O. Final manuscript review & Editing: all authors. R.L. and A.BO. had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analyses. All authors read and approved the final version of the manuscript.

Data sharing statement

Anonymized data will be made available to all reasonable requests from qualified investigators.

Declaration of interests

H.T. and L.Z. received consulting fees from EMD Serono outside of the submitted work. D.J. has received consulting fees from Biogen, Genentech, Novartis, EMD Serono, Banner Life Sciences, Bristol Meyers Squibb, Horizon, Cycle and Sanofi Genzyme outside of the submitted work. A.BO. has received fees for advisory board participation and/or consulting from Abata, Accure, Atara Biotherapeutics, Biogen, BMS/Celgene/Receptos, GlaxoSmithKline, Gossamer, Immunic, Janssen/Actelion, Medimmune, Merck/EMD Serono, Novartis, Roche/Genentech, Sangamo, Sanofi-Genzyme, Viracta; and has received grant support to the University of Pennsylvania from Biogen Idec, Roche/Genentech, Merck/EMD Serono and Novartis. L.P. has recived grant support from Alector on projects not related to this work.

Appendix A. Supplementary data

Supplementary Figure S1

Assessment of Microglia purity. Microglia were purified using Percoll gradient and multiparametric flow cytometry assessed purity of microglia using CD11c

Supplementary Figure S2

Kinetics of B cell survival following exposure to conditioned media of differentially activated microglia. Purified B cells from healthy individuals were cultured for up to 4 days (96 hrs) with 25% supernatant of human microglia that were either previously left unpolarized under basal culture conditions (‘Unp Microglia sup’); or pre-exposed to either proinflammatory activating conditions (using GM-CSF, IFNγ and LPS; designated ‘GM/I/L’), or alternate activating conditions (using M-CSF, IL-4 and IL-13; designated ‘M/4/13’), as described in methods. B cell viability was subsequently assessed at different time points by gating on CD20⁺ Annexin V^- 7AAD^- viable B cells using flow cytometry. Graphics represent ranges of n = 2 independent experiments.

Supplementary Figure S3

Effects of different modes of B cell activation on their phenotype and cytokine secretion profiles. B cells were activated under either pro-inflammatory conditions (‘MS B40X4 sup’) or IL-10 inducing conditions (‘MS BCpG sup’), as described above. (a) Multiparametric flow cytometry demonstrated that B cells undergoing pro-inflammatory activation express considerably higher levels of the activation marker CD69, as well as the surface costimulatory molecules CD80 and CD86, while B cells activated under IL-10 inducing conditions tend to exhibit increased levels of CD24 and CD38. (b) Multiplex ELISA of the B cell supernatants confirmed that the pro-inflammatory B cells secreted considerably higher levels of GM-CSF and IL-6 while B cells activated under IL-10 inducing conditions secreted significantly higher level of IL-10.

Supplementary Figure S4

Different B cell supernatants do not impact microglia or macrophage survival. Human microglia were purified from samples obtained during epilepsy surgery and macrophage were generated by M-CSF treatment of freshly isolated CD14⁺ peripheral monocytes from healthy volunteers. Microglia and macrophage were then either left unpolarized under basal culture conditions (Unp), or differentially activated as described above, with or without exposure to pro-inflamatory B cell supernatants (‘+B40X4 sup’), or IL-10 expressing B cell supernatants (‘+BCpG sup’) for two days. (a) Microglia and (b) macrophage survival was assessed using 7AAD and ANNEXIN V staining. Data are shown for n = 4 (microglia) and n = 5 (macrophage) independent experiments. Data were analyzed with one-way ANOVA.

Supplementary Figure S5

Modulation of expression of activation and quiescence markers by human microglia and macrophage under pro-inflammatory and alternate activation conditions. Human microglia and monocyte-derived macrophage were polarized in vitro under either pro-inflammatory activating conditions using GM-CSF, IFNg and LPS (designated ‘GM/I/L’) or alternate activation conditions with M-CSF, IL-4 and IL-13 (designated ‘M/4/13’), as described above. Representative histogram plots are shown for the effects of the different polarization conditions on CD80 as an example of an activation marker and TREM-2 as an example of a quiescence marker, for (a,d) microglia and (h,k) macrophage, respectively. Summary data show the expected differential modulation of surface expression of activation (CD80, HLA-DR) and quiescence molecules (TREM-2, MerTK, M-CSFR, SIRP1a and CD200R) under the different activation conditions for (a-g) microglia and (h-q) macrophage. Summary data represent results of 3-6 independent experiments for both microglia) and macrophage. Data were analyzed with one-way ANOVA.

Supplementary Figure S6

Effects of secreted factors of MS-patient derived pro-inflammatory and IL-10 expressing B cells on human microglia phagocytosis. Supernatants were generated from MS patient-derived B cells that were activated under either pro-inflammatory conditions (‘MS B40X4 sup’) or IL-10 inducing conditions (‘MS BCpG sup’), as described above. The B cell supernatants were then applied to human CNS-derived microglia and the subsequent microglia phagocytosis of myelin was assessed using flow cytometry, quantifying the mean fluorescence intensity (MFI) of ingested pH-rhodo stained myelin. Representative data are shown for myelin phagocytosis by microglia that were cultured in either basal media alone (‘Media’) or following exposure to MS patient-derived B cell supernatants.