CD11b^high B Cells Increase After Stroke and Regulate Microglia: CD11b^high B Cells Increase After Stroke and Regulate MG

Abstract

Recent studies have highlighted the deleterious contributions of B cells to post-stroke recovery and cognitive decline. Different B cell subsets have been proposed based on expression levels of transcription factors (e.g. T-bet) as well as specific surface proteins. CD11b (α-chain of integrin) is expressed by several immune cell types and is involved in regulation of cell motility, phagocytosis, and other essential functions of host immunity. Although B cells express CD11b, the CD11b^high subset of B cells has not been well characterized, especially in immune dysregulation seen with aging and after stroke. Here, we investigate the role of CD11b^high B cells in immune responses after stroke in young and aged mice. We evaluated the ability of CD11b^high B cells to influence pro- and anti-inflammatory phenotypes of young and aged microglia (MG). We hypothesized that CD11b^high B cells accumulate in the brain and contribute to neuroinflammation in aging and after stroke. We found that CD11b^high B cells are a heterogeneous subpopulation of B cells, predominantly present in naïve aged mice. Their frequency increases in the brain after stroke in young and aged mice. Importantly, CD11b^high B cells regulate MG phenotype and increase MG phagocytosis in both ex vivo and in vivo settings, likely by production of regulatory cytokines (e.g., TNF-α). As both APCs and adaptive immune cells with long-term memory function, B cells are uniquely positioned to regulate acute and chronic phases of the post-stroke immune response, and their influence is subset-specific.

Introduction

Age-related changes in the immune system are a determining factor in clinical outcomes of neurological diseases including stroke (1, 2). Work from our lab and others has shown that immune cell populations from aged animals can be significantly different in relative frequency and tissue distribution compared to those from young animals, even in the absence of tissue injury (2–4). After ischemic stroke, aged mice have a smaller infarction size than young mice, however aged mice demonstrate worse neurological outcomes (5–8). Peripheral immune cells are critical to stroke recovery, and aged peripheral immune cells contribute to greater neurological deficits and dystrophic changes in microglia (9, 10). Microglia mount a pro-inflammatory response to infarction, perform phagocytosis, and present antigens for the gradual recruitment of adaptive immune cells (11). After injury, the dural meninges and eventually the brain parenchyma are infiltrated by peripheral leukocytes, further amplifying the immune response (12). Damage to the blood brain barrier (BBB) results in loss of integrity, allowing for a greater influx of peripheral leukocytes into the brain parenchyma (13, 14). Importantly, both brain-resident microglia (MG) and long-lived peripheral B cells undergo significant age-related changes (15–18).

Aged MG contribute to detrimental immune responses, chronic inflammation, and poorer outcomes after brain injury (2, 19). Aged MG have a transcriptomic profile indicative of a chronic pro-inflammatory state, characterized by increased pro-inflammatory cytokine production (e.g., TNF-α, IL-1β, and IL-6), genes associated with host defense, and cell adhesion (20–23). MG are key producers of and are regulated by TNF-α after injury (24). TNF-α participates in injury-mediated MG and astrocyte activation and MG phagocytosis (24–27). TNF-α can be secreted from variety of cell types including long-lived adaptive immune cells such as B cells (28).

B cells have multiple subtypes and diverse roles after stroke (29). For example, IL-10 secretion by regulatory B cells reduces infarct size and inflammation in mice (29, 30), and B cell production of IgG facilitates opsonization of myelin debris for clearance by microvascular endothelial cells and macrophage recruitment (31). Conversely, detrimental responses also arise from B cells. After stroke, brain antigens are exposed to the periphery, and antigen presenting cells from the brain migrate to the cervical lymph nodes, leading to the amplification of harmful auto-reactive lymphocytes and chronic inflammation (32).

Most myeloid cells, as well as B cells, express CD11b for regulation of cell motility and phagocytosis (33, 34). A distinct subset of B cells have been found to accumulate in aged mice and have been studied in the context of autoimmune diseases (35–37). However, less is known regarding the role of aged B cell subsets after stroke (38, 39). Different aged B cells subsets have been proposed based on expression levels of transcription factors (e.g. T-bet) as well as surface proteins including CD11b (40–43). CD11b^high B cells are often excluded in common approaches in flow cytometric brain immunophenotyping (44–48). Our data shows that relative frequency of CD11b^high B cells significantly increases with aging and after stroke in both young and aged mice. Sorting peripheral B cells into two subsets of CD11b^low and CD11b^high revealed significant differences in their surface phenotype, inflammatory cytokine production, and phagocytic activity (Fig. 1). We hypothesized that CD11b^high B cells increase in the brain and contribute to neuroinflammation in aging and after stroke by regulating MG activation and phagocytosis. We demonstrated that CD11b^high B cells can produce higher levels of TNF-α compared to their CD11b^low counterparts, which can activate MG phagocytosis. Taken together, our results emphasize the nuances of immunophenotyping of brain CD11b^high and CD11b^low B cell subsets in age-related neuroinflammation.

FIGURE 1. CD11b^high B cells increase with aging and have a distinct surface phenotype, increased TNF-α production, and increased phagocytosis.

(A) Representative gating strategy from Cells/Singlets/Live/CD45⁺/CD19⁺ B cells in naïve Yg and Ag mice. (B) The ratio of CD11b^high to CD11b^low B cells isolated from spleen and brain in Yg (n=4) and Ag (n=5) naïve animals (spleen p=0.0089, brain p=0.0466, unpaired T-test). Absolute counts listed in Table 1. Results reproduced in two independent experiments. (C) The first column represents tSNE plot of CD45⁺/CD19⁺ cells demonstrating the CD11b^high (red) and CD11b^low (blue) subpopulations of B cells in Yg, Ag, and combined populations as well as the increase in CD11b^high B cells with aging. The second column demonstrates a heatmap of the co-expression of B-cell associated surface markers (CD11b, CD80, CD138, and CD73) in Ag B cells. (D) Surface phenotype in MFI of CD45⁺/CD19⁺/CD11b^high compared to CD45⁺/CD19⁺/CD11b^low B cells isolated from splenocytes of naïve Ag (n=6) and Yg (n=4) mice. Data reproduced in 2 independent experiments. The top two rows show a significant increase in CD268 in Ag (p=0.0011) and Yg (p<0.0001) cells, no significant change in CD27 in Ag (p=0.3944) with an increase in Yg (p=0.0002) cells, a decrease in IgD (Ag p <0.0001 and Yg p= 0.0008) cells, and no significant changes in IgM in Ag (p=0.2075) and Yg (p=0.00529) cells. The bottom two rows show a significant increase in CD80 in Ag (p=0.0208) and Yg (p=0.0064) cells, and an increase in CD138 in Yg (p= 0.0006) with no change in Ag (p = 0.1786) cells, and an increase in CD73 in Ag (p=0.0002) and Yg (p<0.0001) cells. (E) Phagocytosis of 1μm FITC fluorescent beads by sorted Cells/Singlets/Live/CD45⁺/CD19⁺ CD11b^high and CD11b^low B-cells from splenocytes isolated from Ag naïve animals (n=8). Representative images of 1μm FITC fluorescent beads with CD11b^low (left) CD11b^high (right) B cells stained with DAPI. (F) FACs detection of TNFα production in CD11b^high and CD11b^low B-cells from sorted CD45⁺/CD19⁺/CD11b^high, sorted CD45⁺/CD19⁺/CD11b^low, sorted CD45⁺/CD19⁺, and splenocytes isolated from Ag naïve animals (n=5). (G) FACs analysis of phagocytosis of fluorescent beads by splenocytes, sorted Cells/Singlets/Live/CD45⁺/CD19⁺ B cells, and sorted Cells/Singlets/Live/CD45⁺/CD19⁺ CD11b^high and sorted Cells/Singlets/Live/CD45⁺/CD19⁺ CD11b^low B-cells from splenocytes isolated from Ag naïve animals (n=5) B, C, D, F, and G results reproduced in two independent experiments. Unpaired T-test. Mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, N.S. not significant.

Materials and Methods

Mice:

Young (2–4 months) and aged (18–22 months) C57BL/6J male wild-type (WT) mice from the National Institute on Aging (NIA) were used in this study. PepBoy mice (from Jackson Laboratory) are a congenic strain which carry the differential Ptprc pan leukocyte marker commonly known as CD45.1. C57BL/6 inbred mice express the Ptprc (CD45.2) allele. CD45 is a common b antigen expressed in all leukocytes. It has two different alleles, CD45.1 and CD45. 2, which are functionally identical. This allows for the differentiation of transferred B cells from (CD45.2) mice (donor) to recipient PepBoy (CD45.1) mice, due to the different CD45 alleles (CD45.1 vs CD45.2). All animals were group housed in Tecniplast individually ventilated cage racks, fed a commercially available irradiated, balanced mouse diet (no. 5058, LabDiet, St Louis, MO) and provided corncob bedding. Rooms were maintained at 21–24°C and under a 12:12 h light:dark cycle. All animals were maintained specific pathogen free (see Supplemental Table 1 for list of monitored pathogens). Animal procedures were performed at an Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) accredited facility and were approved by the Animal Welfare Committee at the University of Texas Health Science Center at Houston, TX, USA.

Middle cerebral artery occlusion (MCAO):

Transient focal ischemia was induced under isoflurane anesthesia in young or aged mice for 60 minutes by occlusion of the right middle cerebral artery (49). Body temperature was maintained at 37.0 ± 1.0°C throughout the surgery by an automated temperature control feedback system (TC1000, mouse, CWE Inc., USA). A midline ventral neck incision was made, and unilateral MCAO was performed by inserting a Doccol monofilament (Doccol Corp, Redlands, CA, USA) into the right internal carotid. One hour after ischemia, animals were anesthetized again, and reperfusion was established by withdrawal of the monofilament. Animals were then placed in a recovery cage and were euthanized 72 hours after reperfusion. Sham controls were subjected to same procedure except the suture was not introduced into the middle cerebral artery. Animals were randomly assigned into the stroke and sham surgery groups and single housed in their recovery cages for the first two hours after surgery. Sham and stroke mice were then housed together in their home cages to minimize detrimental effects of social isolation (50). All mice were selected for sham or stroke surgery in a randomized manner and all analyses were performed blinded to surgical conditions. Animals that died due to hemorrhagic transformation or NDS > 3 included 13 aged (out of 35) and 6 young (out of 23). Representative Kaplan-Meier curve included in Fig. 2D.

FIGURE 2. CD11b^high B cells are increased to both young and aged brains as early as post-MCAO day 7.

(A) Schematic of the MCAO model used for inducing neurological injury in mice. (B) tSNE plots of live CD45^high cells (non-MG immune cells) from sham and stroked homogenized brain from Yg and Ag mice, demonstrating CD19⁺ B cells (pink), CD11b^high B cells (red), CD11b^low B cells (blue), CD3⁺ T cells (orange), Ly6C⁺ monocytes (green), and ungated populations (gray), indicating the increase in CD11b^high B cells after stroke when compared to sham in both Yg and Ag MCAO mice. (C) Quantification of CD11b^high and CD11b^low B cells isolated from brain, comparing the absolute counts and percentage of CD19⁺ cells from sham controls to the ipsilateral hemisphere in stroked animals. Conducted in both Yg (n=5–15) and Ag mice (n=13–21). Left the ipsilateral stroked hemisphere in Ag animals demonstrate a significant increase in CD11b^high B cells (p=0.0019) with Yg cells trending upwards in number (p=0.0573). Right the ipsilateral stroked hemisphere in Ag and Yg animals demonstrate a significant increase in the percentage of CD11b^high B cells within the total CD45⁺/CD19⁺ B cell population (p<0.0001, p=0.0027 respectively). (D) Representative Kaplan-Meier Curve for MCAO mortality in aged and young mice. Results compiled from 6 independent experiments. Outlier test ROUT Q=1% was performed on Yg and Ag groups, resulting in the removal of 1 Yg sham and 2 Yg stroke samples. Outliers where removed before analysis. Unpaired analysis T-test, mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, N.S. not significant.

Flow cytometry:

A previously published brain single cell suspension protocol was used (2, 51). In brief, mice were euthanized by intraperitoneal avertin injection. Blood was drawn by cardiac puncture with heparinized needles. Red blood cell lysis was achieved by two consecutive 10-min incubations with tris–ammonium chloride (Stem Cell Technologies). Mice were transcardially perfused with 20 ml cold, sterile PBS prior to aseptic removal of brain tissues. Brain tissue was placed in complete Roswell Park Memorial Institute 1640 (Lonza) medium then mechanically and enzymatically digested in Collagenase/Dispase (1 mg/mL; Roche Diagnostics) and DNase (10 mg/mL; Roche Diagnostics) for 45 minutes at 37 °C with gentle shaking (80 RPM). The cell suspension was filtered through a 70μm cell strainer. Leukocytes were harvested from the interphase of a 70%–30% Percoll gradients for the brain tissue after a 20-minute centrifugation at room temperature with no brakes. Cells were washed and blocked with Fc receptor block (BioLegend, Lot: B298973) before staining with the following pre-conjugated fluorophores: CD45.2-eF450 (eBioscience, Cat#: 48-0451-82), CD45.1- FITC (eBioscience, Cat#: 11045385), CD11b-APC (BioLegend, Cat#: 101212), CD19-FITC (BioLegend, Cat# 101506), Ly6C-PerCP-Cy5.5 (BioLegend, Cat#: 128011), Tmem119-PE-Cy7 (eBioscience, Cat#: 25-6119-82), P2RY12-PE (BioLegend, Cat#: 848003), MHCII-APC-Fire750 (BioLegend, Cat#: 107652), IgM-PE-Cy7 (BioLegend, Cat#: 406514), IgD-PE (BioLegend Cat#: 405706), CD268-FITC (ThermoFischerScientific Cat#: 11-5943-82), CD27-PerCP-Cy5.5 (BioLegend, Cat#: 124214), CD80-FITC, CD138-APC (BioLegend, Cat#: 142506), CD73-PE-cy7 (BioLegend, Cat#: 127224), T-bet-PE (BioLegend, Cat#: 644810), TNF-alpha-APC (BioLegend Cat#: 506308), and Zombie Aqua (BioLegend, Cat#: 423102). Cell stimulation for cytokine staining was done using Cell Activation Cocktail (with Brefeldin A) (BioLegend, Cat#:423304). Cell isolation, Percoll gradient, and immunostaining steps were carried out at once for both controls and injury models to minimize experimental variability, i.e. all sham and stroke samples were processed together, and all naïve young and aged samples were processed together. Data were acquired on Cytoflex-S (Beckman Coulter) or BD FACSMelody (BD) and analyzed using FlowJo (Treestar Inc.). No less than 300,000 events were recorded for each sample. Tissue-matched and injury-matched fluorescence-minus-one and unstained controls were used to aid in positive gating strategy (Fig S1A). t-distributed stochastic neighbor embedding (tSNE) plots were generated in FlowJo using DownSample plug-in (3,000 cells per sample for each study group) followed by tSNE algorithm on all compensated parameters (except viability) at 1,000 iterations, perplexity of 30, learning rate of 5040, and Barnes-Hut gradient algorithm.

Cell sorting:

Single cell suspension and surface staining were performed as described above. After viability and single cell selections, MG gated as Live Tmem119⁺ (verified to be CD45^intCD11b⁺) were sorted under an aseptic hood from the single cell suspension prepared from naïve aged male brains (full brains, n=8) stroked aged male brains (full brains, n=4) naïve young male brains (full brains, n=4) using BD FACSMelody.

After viability and single cell selections, B cells, gated as Live CD45⁺CD19⁺ were sorted under the hood from the single cell suspension prepared from naïve or stroked aged male spleens (spleen, n=14) using BD FACSMelody. Fluorescence-minus-one and unstained controls were used to aid in positive gating strategy to delineate CD11b^high from CD11b^low and CD11b^neg B cells. Sorting was performed from tube directly into 96-well plate used for co-incubation experiments to preserve cell counts, and into FACs tubes for adoptive transfer experiments. Cell counts were conducted in ratios similar to what was analyzed from our post-stroke data (i.e. 1:1000 MG to CD11b^high B cells and 1:100 MG to CD11b^low B cells), to maintain physiologic relevance of our ex vivo experimental conditions. Each sorted sample was then divided (by volume) into control or co-incubated cohorts with each B cell subset for 4 hours under sterile cell-culture environment. Cells were then washed with PBS, stained for surface markers and viability after the 4-hour co-incubation and then analyzed by flow cytometry.

Adoptive B cell Transfer:

Donor mice were 18–20 months old so that they would a have sufficient number of cells to collect for transfer. Naïve or stroked mice were euthanized and immune cells collected post mortem. Using BD Melody cell sorting system, a pure cell population of approximately 50,000 CD11b expressing B lymphocytes were collected for adoptive transfer into a single recipient mouse. 1 donor mouse for every 1 recipient mouse was required in order to have an adequate number of cells. The number of cells (50,000) was determined by the physiologic amount we have detected in aged and stroked mice. Cells were resuspended in 37°C PBS, and retro-orbitally injected into anesthetized recipient using a BD Insulin Syringe with the BD Ultra-Fine™ needle 12.7mm x 30G 3/10 mL/cc.

Ex vivo co-incubation experiments:

Collection and isolation of brain monocytes was performed by optimized enzymatic digested followed by Percoll gradient protocol (2, 51). Brain tissue was harvested after cardiac perfusion with PBS to eliminate blood from the brain tissue. There is a possibility that PBS-perfused samples contain some blood-sourced immune cells prior to ex vivo co-incubation. Thus, we performed digestion and Percoll-gradient based separation of MG at which point, each individual single cell suspension of CNS mononuclear cells was divided (by equal volumes) into a two or three co-incubation conditions with different B cell subsets (Fig. 1). Co-incubated cells were kept in a sterile cell-culture environment at 37°C for 4 hours and 24 hours.

Phagocytosis Assay:

0.5μm Fluoresbrite® 641-conjugated carboxylate microspheres (1 μl stock solution/200 μl in 96-well plate) (52) were added to sorted MG or to CNS mononuclear cells enriched in MG (from brain that is post-digestion and post-Percoll gradient protocol) and incubated for 4- or 24-hours. Cell mixtures were incubated with beads for 30 minutes at 37°C. After, cells were washed twice with PBS, and then stained for flow cytometry following the previous protocol.

Microscopy:

Images were obtained on a Keyence BZ-X810 all in one fluorescence microscope at 40x and 60x magnification. Images were processed for brightness and contrast correction, cropping, and addition of scale bars with Keyence BZ-X800 Analyzer 1.1.1.8. software.

Statistical analysis:

Statistical analysis for flow cytometry data was performed using unpaired t test, One-way ANOVA, and paired one-way ANOVA with post hoc analysis with all related p values adjusted by Dunnett’s or Tukey’s methods for multiple comparisons (specified in figure legends). Statistical significance was considered at p < 0.05 and *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001 convention was used in the presented figures. All statistical analyses were performed with GraphPad Prism 7.

Results

CD11b^high B cells increase with aging and have a distinct surface phenotype, increased TNF-α production, and increased phagocytosis.

First, we provided a representative gating strategy from Cells/Singlets/Live/CD45⁺/CD19⁺ B cells in naïve aged and young mice to demonstrate the significant increase in relative frequency of CD11b^high B cells in aged mice (Fig. 1A and Fig. S1A). The increased ratio of CD11b^high to CD11b^low B cells with aging was independent of tissue origin (spleen or brain) (Fig. 1B and Table 1). We then performed multidimensional flow cytometric phenotyping of CD11b^high B cells. We asked if CD11b^high B cells were predominated by mature B cells, memory B cells, or class switched B cells using expression of CD73, CD138, and CD80, respectively. Results are depicted in a tSNE map superimposed with heat maps for each surface marker (Fig. 1C). Individual gating revealed an increase in CD80 and CD73 median fluorescent intensities (MFIs) in CD45⁺CD19⁺CD11b^high compared to CD45⁺CD19⁺CD11b^low splenic B cells of young and aged naïve mice, with plasma cell marker CD138 MFI also significantly increased in CD45⁺CD19⁺CD11b^high of young naïve mice (Fig. 1D). A significant increase in CD27 (young p=0.0002) supports a memory B cell phenotype within the CD11b^high B cell population. To understand the functional role of CD11b^high B cells, we examined surface phenotype markers CD268, IgD, and IgM expression (Fig. 1D) on CD45⁺CD19⁺CD11b^high compared to CD45⁺CD19⁺CD11b^low B cells isolated from splenocytes of young (n=4) and aged (n=6) naïve mice. CD45⁺CD19⁺CD11b^high B cells showed a significant increase in CD268 (aged p=0.0011 and young p< 0.0001), a significant decrease in IgD (aged p<0.0001 and young p=0.0008), and no significant changes in IgM in young or aged B cells, compared to aged-matched CD45⁺CD19⁺CD11b^low (Fig. 1D). The relative frequency of these populations varied (Fig. S2A–B). Together, these observations indicated that the CD11b^high B cells are a mixed population of B cell subsets. We also examined CD11b^high B cells from the spleen, blood, and skull bone marrow of young and aged stroked mice, which showed a distinct surface phenotype from CD11b^low B cells (Fig. S2C–E). These observations suggest that CD11b^high B cells increase in the periphery and in the brain with aging, in the absence of any brain injury. Further, CD11b^high B cells are a highly activated, heterogeneous subset of B cells with distinct surface phenotype from their CD11b^low B cell counterparts.

Table 1:

Absolute counts of naïve brain and spleen B cell populations for Fig. 1B

Absolute Count of Naïve Brain and Spleen B cell Populations
Tissue	Naïve Group	CD11bhigh B Cells (Average ± SD)	CD11blow B Cells (Average ± SD)	Sample size
Spleen	Aged	28485.6 ± 5895.95	102279.80 ± 20288.60	5
	Young	23032.25 ± 3647.26	95702.75 ± 11175.64	4
Brain	Aged	96.40 ± 64.92	595.40 ± 441.76	5
	Young	4.75 ± 5.19	166.75 ± 62.78	4

To verify that B cells can increase their expression of CD11b under pro-inflammatory conditions, we incubated sorted CD45⁺CD19⁺ B cells with LPS for 48 hours. Indeed, our data showed that CD11b expression is increased on sorted B cells after a 48-hour LPS stimulation when compared to B cells not treated with LPS isolated from the same animal (Fig. S1B). We also examined T-bet expression levels of CD45⁺CD19⁺CD11b^high B cells and found that their relative frequency of T-bet+ cells are significantly higher than that of their CD45⁺CD19⁺CD11b^low B cell counterparts (Fig. S1C), which is consistent with their previously proposed age-associated B cell phenotype (43).

We detected differences in phagocytic activity and cytokine production of CD11b^high B cells when compared to CD11b^low B cells. A phagocytosis assay was performed using 0.5μm fluorescent beads incubated for 30 minutes with sorted CD45⁺CD19⁺CD11b^high and CD11b^low B subsets from naïve aged spleens, and then analyzed by flow cytometry. Representative images of fluorescent beads with CD11b^low (left) compared to CD11b^high (right) B cells stained with DAPI were generated to visualize the uptake of beads by the B cells (Fig. 1E). Flow cytometric analysis of B cell phagocytic activity revealed a significant increase in the uptake of fluorescent beads in CD11b^high B cells compared to CD11b^low B cells (Fig. 1G). Previously described activated B cells in mice showed increased TNF-α production (53). Next, to determine if CD11b^high B cells produce cytokines capable of mediating pro-inflammatory activity in MG, TNF-α production was quantified in CD11b^high and CD11b^low B cells sorted as CD45⁺CD19⁺CD11b^high cells, sorted CD45⁺CD19⁺CD11b^low cells, sorted CD45⁺CD19⁺ cells, and un-sorted splenocytes isolated from naïve aged animals. This analysis revealed CD11b^high B cells have higher capacity for TNF-α production when compared to CD11b^low B cells (Fig. 1F). Importantly, CD11b^high B cells did not depend on presence of other immune cells for this effect (Fig. 1F). These findings indicate activated state of these CD11b^high B cells in both antigen uptake and cytokine production.

CD11b^high B cells are increased in both young and aged brains as early as post-MCAO day 7.

To test whether CD11b^high B cells increase in the brain after stroke, both young and aged mice underwent 60-min middle cerebral artery occlusion (MCAO) (Fig. 2A). At post-MCAO day 7, brain CD11b^high and CD11b^low B cells were compared in contralateral and ipsilateral hemispheres in stroke and sham animals. After stroke, both young and aged samples show an increase the density of immune populations, notably in CD11b^high B cells (Fig. 2B, red island). The ipsilateral stroke hemisphere in aged animals demonstrated a significant increase in absolute CD11b^high B cell counts (p = 0.0019) with young cells trending upwards in number (p = 0.0573). The ipsilateral stroke hemisphere in aged animals demonstrated that stroke resulted in a significant increase in the percentage of CD11b^high B cells within the total CD45⁺/CD19⁺ B cell population (aged p < 0.0001 and young p = 0.0027) (Fig. 2C). Absolute counts of B cell populations are provided in Table 2, and a representative Kaplan-Meier curve of MCAO mortality in young and aged mice is included in Fig. 2D.

Table 2:

Absolute cell counts from right brain hemisphere in Figure 2.

Absolute Cell Counts from Right Brain Hemisphere
	Group	CD45+/CD19+ B Cell (Average ± SEM)	CD11bhigh B Cell (Average ± SEM)	CD11blow B Cell (Average ± SEM)
Aged	MCAO	1111 ± 201.3	557.6 ± 113.2	367.7 ± 101.2
	Sham	830.3 ± 251.1	63.31 ± 21.85	735.9 ± 235.9
Young	MCAO	1321 ± 449.2	635 ± 176.6	595.2 ± 383.3
	sham	63.29 ± 22.03	3 ± 0.55	54 ± 21.59

Differential MG surface phenotype after stroke and after incubation with sorted CD11b^high or CD11b^low B cells.

Next, CD45, CD11b, MHC-II, P2RY12, and Tmem119 were analyzed to assess MG activation from ipsilateral hemispheres in young (Fig. 3A) and aged (Fig. 3B) mice compared to either contralateral hemisphere or sham brain samples. The gating strategy for MG is provided (Fig. S1D). Consistent with previous studies (2, 3, 54, 55), MFIs of CD45 and MHC-II were significantly increased while P2RY12 and Tmem119 were significantly decreased in young post-stroke MG (Fig. 3A). The surface profile of MG from aged mice after MCAO also revealed a significant increase in CD45 in the ipsilateral hemisphere as well as similar trends in MHC-II, P2RY12, and Tmem119 expressions after MCAO (Fig. 3B). These results confirmed activation of MG after stroke in our model, which was also associated with significantly increased presence of CD11b^high B cells in the brain.

FIGURE 3. Differential MG surface phenotype after stroke and after incubation with sorted CD11b^high or CD11b^low B cells.

(A) Surface profile of MG from Yg mice after MCAO comparing contralateral hemisphere and ipsilateral hemispheres. CD45 MFI increased in MG in the ipsilateral hemisphere (p<0.0001), CD11b MFI in MG had no significant change in the ipsilateral hemisphere, MHCII MFI significantly increased in MG in the ipsilateral hemisphere (P=0.0048), P2RY12 MFI decreased in MG in the ipsilateral hemisphere (p=0.0026), Tmem119 MFI decreased in MG in the ipsilateral hemisphere (p=0.0098). (B) Surface profile of MG from Ag mice after MCAO comparing contralateral hemisphere and ipsilateral hemispheres. CD45 MFI increased in MG in the ipsilateral hemisphere (p=0.0119), CD11b, MHCII, Tmem119, and P2RY12 MFI in MG had no significant change in the ipsilateral hemisphere. Ag MG-enriched CNS mononuclear cells respond differently after incubation with CD11b^high or CD11b^low B cells sorted from Ag splenocytes after 4-hour and 24-hour incubations. (C) Analysis of MG derived from Ag mice and co-incubated with B cells sorted from the same respective animal. Post-percoll brain homogenate was divided across three incubation conditions for paired comparison. MG after a 4-hour co-incubation with CD11b^low B cells, CD11b^high B cells, or alone resulted in no significant changes in CD45 or CD11b MFI, and Tmem119 MFI significantly increasing (p=0.0323) in CD11b^high B cells compared to MG alone. (D) Using the same design as C with a longer, 24-hour co-incubation with CD11b^low B cells, CD11b^high B cells, or alone resulted in CD45 MFI significantly increasing (p=0.0463), no significant changes in CD11b MFI or Tmem119 MFI in CD11b^high B cells compared to MG alone. A and B were reproduced in two independent experiments underwent unpaired one-way ANOVA. C and D were performed once and underwent paired one-way ANOVA with Dunnett’s multiple comparisons test. mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, N.S. not significant.

To understand the role of CD11b^high B cells in the context of neuroinflammation, we assessed the ex vivo ability of CD11b^high B cells to induce changes to MG surface phenotype. Aged MG-enriched CNS mononuclear cell suspensions were divided across three co-incubation conditions with: sorted CD11b^high B cells, sorted CD11b^low B cells, and a control group. Sorted cells were derived from the spleen of the same animal. We hypothesized that CD11b^high B cells are capable of inducing an activated state in MG. 4hrs of co-incubation with CD11b^high B cells compared to the control resulted in significantly increased Tmem119 expression levels, while CD11b^low B cells induced no changes (Fig. 3C). After a 24-hour co-incubation, there was a significant increase in CD45 expression in MG co-incubated with sorted CD11b^high B cells compared to control, and the addition of CD11b^low B cells had no significant differences from the control group (Fig. 3D). Our data suggests CD11b^high B cells can regulate MG activation. The increase of CD45 and decrease of Tmem119 indicate an activation state(3, 56–58).

To directly compare the capacity of CD11b^high and CD11b^low B cells to differentially influence MG surface phenotype responses, young and aged MG-enriched CNS mononuclear cells and sorted MG underwent 4hr co-incubation using a broader panel of MG surface markers (CD45, CD11b, Tmem119, P2RY12, and MHC-II) (Fig. S3A–D). Young MG after 4hr co-incubation with CD11b^high B cells compared to CD11b^low B cells resulted in relatively decreased CD45, CD11b, Tmem119, and P2RY12 expression levels (Fig. S3A top). Analysis of aged MG co-incubated with B cells sorted from the same animals resulted in relatively decreased CD45 and Tmem119 expression (Fig. S3A bottom). Our data suggests CD11b^high B cells can regulate MG activation unlike CD11b^low B cells. Reduction of CD45 and CD11b indicate reduced activation state while decrease in Tmem119 and P2RY12 suggest an increased activation state of MG. Furthermore, aged MG appeared more resistant to changes in their surface phenotype compared to young MG.

We then evaluated surface expression of pure MG (sorted as Live Tmem119+) from both young and aged mice after a 4-hour co-incubation with sorted CD11b^high or CD11b^low B cells from aged splenocytes. Young MG after co-incubation with CD11b^high B cells compared to CD11b^low B cells resulted in a relative decrease in CD45 and CD11b, no significant changes in Tmem119 and P2RY12 expression levels, and MHC-II had a relative decrease (Fig. S3Btop). Aged MG showed no significant changes for CD45, CD11b, Tmem119, P2RY12, and MHC-II after co-incubation with CD11b^high B cells compared to CD11b^low B cells (Fig. S3Bbottom). These observations indicate that CD11b^high B cells can, independently of other immune cells, influence young MG but not aged MG. MG have been shown to alter their surface expression in ex vivo conditions (59). Therefore, our next step in understanding the effects of CD11b^high B cells on MG was to use an in vivo model of adoptive transfer.

Differential MG surface phenotype after adoptive transfer of CD11b^high or CD11b^low B cells into young naïve host.

Adoptive transfer can determine if increasing the number of CD11b^high B cells alters MG activation. We used PepBoy mice with a distinguishable CD45 haplotype (i.e. CD45.1) to carry out our adoptive B cell transfer experiments. First, spleen, blood, brain and skull bone marrow underwent analysis to detect the presence of CD45.2⁺ cells in recipient mice 24hrs after retroorbital injection. This validated that the cells from the donor mice were successfully transferred to the recipient in all tissues, and it indicated that CD11b^high B cells distribute throughout the host in the absence of injury (Table 3). Next, sorted CD11b^high and CD11b^low B cells from the spleens of aged wildtype mice were transferred into young PepBoy mice (Fig. 4A). The brains of the recipient underwent analysis 24hrs after injection. CD11b increased in MG in CD11b^high B cell recipients compared to CD11b^low B cell recipients (Fig. S3D). CD11b increased in MG in CD11b^high B cell recipients compared to CD11b^low B cell recipients. CD45, CD11b, and MHCII MFI had no significant difference in MG between vehicle control and CD11b^high or CD11b^low B cell recipients. Tmem119 and P2RY12 MFI increased in MG in CD11b^high and CD11b^low B cell recipients compared to vehicle control (Fig 4B). Our data suggests while there were responses in MG due to the presence of additional B cells, there was no clear pattern of MG activation based on surface phenotyping alone. Next, we investigated the phagocytotic activity in MG as a response to ex vivo and in vivo stimulation with CD11b^high or CD11b^low B cells.

Table 3:

Absolute counts of total live and donor cells (CD45.2) in recipient mouse (CD45.1) 24hrs after adoptive transfer by retroorbital injection.

Adoptive Transfer: Absolute cell counts 24hrs after retroorbital injection
Sample:	Detectable CD45.2 cells	Number of Live cells
01-Spleen-C1.fcs	78	275637
01-Spleen-C2.fcs	31	285155
01-Spleen-C3.fcs	60	278033
01-Spleen-C4.fcs	65	270406
01-Blood-E1.fcs	661	23962
01-Blood-E2.fcs	208	11333
01-Blood-E3.fcs	105	4946
01-Blood-E4.fcs	152	17447
01-Skull-G1.fcs	347	27276
01-Skull-G2.fcs	288	48142
01-Skull-G3.fcs	318	62906
01-Skull-G4.fcs	628	89692
01-Brain-A1.fcs	234	10208
01-Brain-A2.fcs	105	19886
01-Brain-A3.fcs	178	15527
01-Brain-A4.fcs	252	10378
Tissue totals	Transferred immune cells	Number of live cells
Mouse 1	1320	337083
Mouse 2	632	364516
Mouse 3	661	361412
Mouse 4	1097	387923

FIGURE 4. Differential MG surface phenotype after adoptive transfer of CD11b^high or CD11b^low B cells into young naïve host.

(A) Schematic. Splenocytes from naïve and stroked Ag male C57BL/6 mice underwent cell sorting to isolate CD11b^high and CD11b^low B cells. Cells were then adoptively transferred to Yg Pepboy recipient mice via retroorbital injection to contribute to modulation of MG phenotype. (B) Surface profile of MG from Yg recipient mice after adoptive transfer from Ag stroke donors. CD45 and CD11b MFI had no significant difference in MG between vehicle control and CD11b^high or CD11b^low B cell recipients. Tmem119 MFI increased in MG in CD11b^high and CD11b^low B cell recipients compared to vehicle controls (CD11b^high p=0.0014 and CD11b^low p=0.0001). P2RY12 increased in MG in CD11b^high and CD11b^low B cell recipients compared to vehicle controls (CD11b^high p=0.0040 and CD11b^low p=0.0032). MHCII MFI had no significant difference in MG between CD11b^high and CD11b^low B cell recipients and vehicle control. Experiments were performed once. Unpaired one-way ANOVA with Tukey’s multiple comparisons test, mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, N.S. not significant.

CD11b^high B cells increase MG phagocytosis.

TNF-α has been shown to regulate MG phagocytosis (24, 26, 27, 60), thus we tested whether CD11b^high B cells increase MG phagocytosis, given their higher capacity for TNF-α production (Fig. 1F). Phagocytotic activity of MG-enriched CNS mononuclear cells after 4 (Fig. 5A) and 24hrs (Fig. 5B–C) of co-incubation with CD11b^high and CD11b^low B cell subsets was assessed. MG showed significantly increased phagocytosis after a 4hr ex vivo co-incubation with CD11b^high B cells compared to controls, while CD11b^low B cells had no significant effect on the median fluorescent intensity of bead⁺ MG (Fig. 5A–C). However, MG incubated with CD11b^low B cells had a greater percentage of bead⁺ MG (Fig. 5A–C). Taken together, MG incubated with CD11b^high had higher phagocytosis capacity, even though the overall number of phagocytosing MG were similar in both MG incubated with CD11b^low and MG incubated with CD11b^high (Fig. 5A). Young and Aged MG significantly increased their phagocytic activity after 24hrs co-incubation with CD11b^high B cells measured by relative frequency of bead positive MG (Fig. 5B–C).

FIGURE 5. CD11b^high B cells increase MG phagocytosis.

(A) Phagocytotic activity of 0.5μm fluorescent beads by Ag MG after 4-hour ex vivo co-incubation (n=5). Left measured bead MFI, with a significant increase (p=0.0089), in MG incubated with CD11b^high B cells compared to MG alone, right show an increased relative frequency of phagocytotic activity in MG incubated with CD11b^low B cells and compared to MG alone (p=0.0134). (B) Relative frequency of phagocytotic activity in Ag left and Yg right MG after 24-hours co-incubation with no significant differences in bead MFI, and a significant increased relative frequency of phagocytotic activity in MG incubated with CD11b^high B cells compared to MG alone (p=0.0065). (n=4). (C) Ag MG had no significant differences in bead MFI, and an increased relative frequency of phagocytotic activity in MG incubated with CD11b^low compared to MG alone (p=0.0504). Paired 1-way ANOVA with Dunnett’s multiple comparisons test. (D) Phagocytotic activity of Yg MG after adoptively transferred Ag CD11b^high and CD11b^low B cells 24hrs after transfer of B cell populations (n=3). Left measured bead MFI, with a significant increase in MG in mice who received CD11b^high B cells compared and CD11b^low B cells (p= 0.0116), right shows an increased relative frequency of phagocytotic activity in MG incubated with CD11b^high and CD11b^low B cells (p= 0.0140). Unpaired T-test. (E) Phagocytotic activity of Yg MG after adoptively transferred stroked Ag CD11b^high and CD11b^low B cells 24hrs after transfer of B cell populations (n=3–4). Left measured bead MFI, with a significant increase in MG incubated with CD11b^high B cells compared to MG alone (p=0.0417), right show an increased relative frequency of phagocytotic activity in MG incubated with CD11b^high and CD11b^low B cells compared to MG alone (p=0.0023 and 0.0154 respectively). A and C were independently reproduced in two experiments. B, D, and E were performed once. One-way ANOVA post hoc Tukey’s multiple comparison, Mean ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, N.S. not significant.

Finally, phagocytosis assays of MG after adoptive B cell transfer from aged (Fig. 5D) and aged stroke (Fig. 5E) donors demonstrated that phagocytosis capacity of MG is significantly increased in MG from mice that received CD11b^high B cells compared to MG from mice that received CD11b^low B cells and vehicle control groups. Both ex vivo and in vivo data in our study consistently show that CD11b^high B cells can independently increase MG phagocytosis.

Discussion

The central nervous system (CNS) has long been considered “immune privileged,” a dogma that arose from studies by Medawar (61) and others, in which transplanting heterologous tissue into the CNS parenchyma failed to induce an effective immune response (62, 63). However, accumulating evidence of neuroimmune communication in the CNS in both homeostatic and pathological conditions has shifted this paradigm (64). The CNS contains microglia (MG) which play a central role in both the acute and chronic phases of neuroinflammation. Additionally, innate and adaptive immune cells in the CNS meninges take an active part in the brain immune surveillance (65, 66). Pioneering work by Kipnis (12, 14, 67) and others revealed an extensive network of meningeal lymphatics and their role in the neuroimmune interface, shedding light on the importance of central and peripheral immune interactions.

Age-associated B cells (ABCs) are a group of functionally distinct B cells that significantly increased in aged mice and in autoimmune diseases in humans (53). The surface phenotyping of these cells vary and they contribute up to 30% of the mature B cell pool of aged mice (40–42, 53, 68). Here, we demonstrate that the CD11b^high subset of B cells have a distinct surface phenotype in the CNS and periphery, and demonstrate increased TNF-α production and increased phagocytosis in ex vivo assays. The ABC phenotype has been associated with increased expression of T-bet (69, 70), a transcription factor essential for autoantibody production (71, 72). A significant increase in T-bet levels in the CD11b^high B cells was also seen in this study (Fig. S1C), supporting that these CD11b^high B cells could be a subset of ABCs. We then determined that CD11b^high B cells have a distinct surface phenotype and are a highly activated and heterogeneous as demonstrated by higher expression of CD138 (expressed by plasma cells (73)), CD80 and CD27 (expressed by memory B cells (74, 75)), CD73 (expressed by class-switched B cells (76)), and CD268 (aka BAFFR, expressed by B cells implicated in auto-reactivity (33, 77). This conclusion is further supported by the significant decrease in IgD with no significant change in IgM (Fig. 1D), a finding that is consistent with previous phenotyping of ABCs (37, 68). These findings combined along with accumulation of CD11b^high B cells in the brain with aging and after neurological injury suggest that these cells could be considered as a subset of ABCs.

The amount of CD11b^high B cells increase significantly by post-MCAO day 7 in both young and aged brain and this increase is associated with expected activation of MG after stroke when assessed by higher expression of CD45 and MHC-II while lower expression of Tmem119 and P2RY12 (3, 78). The function of Tmem119 is not yet understood, however its decreased expression has been associated with activation of MG (58, 79–81). The increased expression of CD45 and MHC-II is characteristic of increased MG activation and phagocytosis (82, 83). Next, we sought to induce a stroke-like MG activation state by co-incubating sorted MG from young and aged brains with either CD11b^high or CD11b^low sorted B cells. We found that the CD11b^high B cells can distinctly modify surface phenotype and increased MG phagocytosis when compared to their CD11b^low counterparts ex vivo. Furthermore, 24hrs after adoptively transferring CD11b^high B cells from both aged naïve and aged stroke WT mice into young PepBoy mice resulted in a significant increase in phagocytosis in MG compared to CD11b^low transferred B cells Given the increasing availability of FDA-approved B cell therapies for variety of inflammatory conditions, better insights into the interaction between B cells and innate CNS immune cells is of high clinical relevance (84).

The infiltration of peripheral lymphocytes after stroke is a key feature of the progression of neuroinflammation and B cell-derived cytokine production can influence outcomes after stroke (42, 85). Studies have shown that B cells contribute to post-stroke cognitive impairment (42, 85–87). Here, we demonstrated that CD11b^high B cells produce significantly higher amounts of TNF-α than CD11b^low B cells and this increased production of TNF-α is correlated with ex vivo increased MG phagocytic activity, which is consistent with prior reports (24, 26, 27, 60, 88). By affecting MG activation and phagocytosis and possessing the chronic memory function, CD11b^high B cells have the ability to regulate long-term neurological outcomes in aging and after cerebrovascular injury, as recently shown by a robust presence of ABCs cells in the meningeal layers (48). Therefore, we speculate that the increased activation of MG and their increased phagocytosis to be, at least in part, due to the presence of CD11b^high B cells and their high capacity to produce TNF-α. Future mechanistic studies using TNF-receptor knockouts in MG can shed more light on the regulatory influence of TNF-α produced by CD11b^high B cells on MG-mediated neuroinflammation.

Previous studies have shown that flow cytometric analysis exclude CD11b^high B cells (44–47) or “infiltrating monocytes” that include CD11b^high B cells (89). As we demonstrate here, this gating strategy would only exclude a negligible fraction of the B cell population in young mice (Fig. 1A), thus appropriate when using young uninjured animal models. However, in studies investigating B cells in brain injury or aging models, the exclusion of CD11b^high B cells will result in the exclusion of a significant subpopulation of B cells (Fig. 1A). Not only is this a large sum of cells to exclude, but also, we demonstrated here that CD11b^high B cells have increased cytokine production and phagocytic activity, and their exclusion may lead to skewed results in the assessment of B cell function in neuroinflammation. Therefore, careful validations should be performed when using flow cytometric identification of B cells in the context of neuroinflammation.

As both APCs and adaptive immune cells with memory function, B cells are uniquely positioned to regulate both acute and chronic phases of the post-stroke immune response, and their influence is subset-specific. Future studies are warranted to better understand the function of CD11b^high B cells in neuroinflammation.

Key Points:

1. CD11b^high B cells are a distinct subset of B cells that increase with aging.

2. CD11b^high B cells are increased in both young and aged brains after stroke.

3. CD11b^high B cells can regulate MG phenotypes and increase phagocytosis.

List of abbreviations:

MG

Microglia

CNS

central nervous system

CD

cluster of differentiation

Tmem119

trans-membrane protein 119

P2RY12

purinergic receptor P2Y12

MCAO

middle cerebral artery occlusion

LPS

lipopolysaccharide

tSNE

t-distributed stochastic neighbor embedding

AD

Alzheimer’s disease

EAE

experimental autoimmune encephalomyelitis