Akkermansia modulates microglia and astrocytes in a strain specific manner

doi:10.21203/rs.3.rs-4714226/v1

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Akkermansia modulates microglia and astrocytes in a strain specific manner

https://doi.org/10.21203/rs.3.rs-4714226/v1

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Microglia and astrocytes are the primary glial cells in the central nervous system (CNS) and their function is shaped by multiple factors. Regulation of CNS glia by the microbiota have been reported, although the role of specific bacteria has not been identified. We colonized germ-free mice with the type strain Akkermansia muciniphila (Am^T) and a novel strain of A. muciniphila (BWH-H3) isolated from a subject with multiple sclerosis and compared to mice colonized with Bacteroides cellulosilyticus (BWH-E5) isolated from a healthy control subject. We then investigated the effect of these bacteria on microglia and astrocyte gene expression by RNA sequencing. We found altered gene expression profiles in brain microglia, with Akkermansia downregulating genes related to antigen presentation and cell migration. Furthermore, we observed strain specific effects, with Akkermansia H3 upregulating histone and protein binding associated genes and downregulating channel and ion transport genes. Astrocyte pathways that were altered by Akkermansia H3 mono-colonization included upregulation of proliferation pathways and downregulation in cytoskeletal associated genes. Furthermore, animals colonized with type strain Akkermansia and strain H3 had effects on the immune system including elevated splenic γδ-T cells and increased IFNg production in CD4 + T cells. We also measured intestinal short chain fatty acids and found that both A. muciniphila strains produced proprionate while B. cellulosilyticus produced acetate, proprionate, and isovalerate. Taken together, our study shows that specific members of the intestinal microbiota influence both microglial and astroyctes which may be mediated by changes in short chain fatty acids and peripheral immune signaling.

microglia

astrocyte

germ-free

mono-colonization

akkermansia

RNAseq

Microbes and their metabolic products are key regulators of the gut-brain axis. How gut resident bacterial species influence central nervous system glia including microglia and astrocytes is not well understood. Eluciating this relationship has important implications for developing microbiome based therapies to modulate nervous system disease. Germ free (GF) mice are a valuable tool for investigating microbes and their effect on both central and peripheral immunity. GF mice do not harbor a native microbiota and thus can be mono-colonized with individual bacterial strains [1, 2]. Mono-association studies have shown a protective capacity of microbes in inflammatory bowel disease models [3, 4] and immune effects including increased Foxp3 + Treg production in mice mono-colonized with B. fragilis [2].

In multiple sclerosis (MS), one of the most consistent findings is an elevation in Akkermansia [1, 5–10], which we have previously associated with improved clinical outcomes [6]. These findings raise the question of whether Akkermansia has a beneficial or detrimental effect on the disease. In one study, investigators found that incubating Akkermansia with PBMCs increased IFNg production by CD4 + T cells in vitro. However, mono-colonization with Akkermansia did not increase inflammatory cytokines in vivo [1]. In the autoimmune encephalomyelitis (EAE) model we have found that administering Akkermansia to SPF mice ameliorated disease [6, 11]. There are 4 clades of Akkermansia which have differential metabolic and immunologic effects [12]. We identified novel MS-derived Akkermansia strains and found that A.m. BWH-H3 had the greatest effect in EAE and reduced IL-17 producing γδ-T cell [13, 14]. Although it has been demonstrated that Akkermansia strains can affect immunity and CNS disease, their effect on microglia and astrocytes, the major glial cells in the CNS, is unknown.

The development of microglial and their homeostatic function is dependent on the gut microbiota [15]. Microglial in GF mice have an immature phenotype and are inefficient in clearing pathogens, which can be restored by reconstitution of the gut microbiota or treatment with several microbial metabolites including short chain fatty acids (SCFAs) [15]. In Alzheimer’s and Parkinson’s models, GF mice have showed decreased disease pathology and immature microglial. The addition of SCFAs increased microglial activation and disease pathology to the level of SPF models [16]. These data demonstrate a critical role of gut microbiota and their metabolites in maintaining CNS immune homeostasis in both health and disease.

In order to investigate the strain-specific role of Akkermansia on the CNS we mono-colonized germ free mice with type strain Akkermansia muciniphila (Am^T) and a strain of Akkermansia isolated from an MS subject at Brigham and Women’s Hospital, Akkermansia strain H3 (Am-H3) [6], and analyzed their effects on microglia and astrocytes as measured by transcriptomics.

Animals:

All animal procedures were approved by the Brigham and Women’s Hospital IACUC. Germ-free mice on the C57BL/6 background (Jackson Laboratory, Bar Harbor, ME) were bred at the Massachusetts Host-Microbiome Center’s gnotobiotic core and were maintained under a 12 h light-dark cycle and received water and chow ad libitum.

Mono-colonization:

At 8-weeks of age, mice received a single oral gavage with 200 µl of one of four treatments: vehicle (brain heart infusion broth; Veh), Akkermansia muciniphila type strain (Am^T; ATCC BAA-835), Akkermansia muciniphila strain BWH-H3 (Am-H3), or Bacteroides cellulosyliticus strain BWH-E5 (Bc; Fig. 1A). All bacterial cultures were diluted to 1x10⁸ CFU/mL in brain heart infusion broth. Mice were maintained germ-free for 2-weeks with one cage change 7 days into the experiment. Confirmation of mono-association was performed at the end of experiment via microbiological culture of cecal samples at the Host-Microbiome Center. Animals that were not mono-colonized as defined by any non-species specific growth patterns were omitted from the study.

Tissue processing:

After sacrifice via CO₂ inhalation, mice received thoracotomy and subsequent transcardial perfusion with ice cold HBSS. Brains were removed and the whole left hemisphere was placed into 7mL glass tissue homogonizer with cold HBSS. Tissue was homogenized prior to centrifugation in 4° C at 1200 g for 7 min and subsequent aspiration of supernatant. Pellets were resuspended in 5mL of 37% Percoll before centrifugation at 4° C at 800 xg for 10 min. Percoll was washed with 5 mL FACS buffer (v:v in HBSS: 2% FBS, 2.5% HEPES, 0.4% EDTA) before centrifugation in 4° C at 1200 g for 7 min and supernatant was aspirated until ~ 50 µl remained in the tube. Cells were stained for FACS using anti-CD45 FITC (1:400; BD, Franklin Lakes, NJ, USA), anti-CD11b PeCy7 (1:400; Biolegend), anti-ACSA-2 PE (1:200; Miltenyi Biotec, Bergisch Gladbach, Germany). Lineage negative markers were used including PerCP conjugated antibodies at 1:800 dilution to anti-CD3, anti-CD19, anti-Nk1.1, anti-B220, and anti-Ly6G (Biolegend). Cells were incubated with antibodies for 25 min at RT in the dark before being washed with 5 mL FACS buffer and subsequently centrifuged in 4° C at 1200 xg for 7 min. Supernatants were aspirated and pellets resuspended in 100 µl of FACS buffer. Cells were transferred to flow polystyrene tubes and washed with 50 µl FACS buffer. Finally, cells were transferred into clean Eppendorf tubes containing 5 µl of 7-AAD. Microglia and astrocytes were sorted on a BD FACSAria II cell sorter. After sorting, cells were spun at 3500 xg for 10 min at 4° C before being processed for RNAseq.

SCFA quantification:

Cecal contents were removed and placed into a sterile Eppendorf tube prior to analysis of SCFAs via gas chromatography-mass spectrometry at the Massachusetts Host-Microbiome Center. Samples were analyzed for the following short-chain acids: acetate, propionate, isobutyrate, butyrate, isovalerate, valerate, isocaproate, caproate, and heptanoate. Detectable levels were observed in a majority of samples for 4 SCFAs: acetate, propionate, isobutyrate, and isovalerate which were quantified and analyzed via one-way ANOVA across mono-colonization groups.

RNAseq:

Microglia and astrocytes were processed on the same plate at the Broad Institute using their mRNA SMART-Seq2 Cell Population v1 sequencing pipeline. Transcriptome alignments were conducted using the Salmon tool with its standard parameters [17]. The R software (version 4.2.1) and DESeq2 package (version 1.36.0) [18] were employed to analyze differential gene expression. Genes that showed significant differential expression (with a p-value < 0.05, adjusted p-value < 0.05, and an absolute value of the log₂ fold change greater than 0.25) were chosen for pathway analysis. For this analysis, the gp.enrichr function, which pulled from GO Biological Process 2023 from GSEApy (version 1.1.1) was utilized, and the principal top pathways were identified for subsequent plotting [19–21]. The genes for the heatmap were filtered and selected based on a p-value < 0.05, a baseMean greater than 50, and a log₂ fold change exceeding 1.5.

Flow Cytometry:

Spleens were homogeized and passed through a 70 µm mesh filter on a 96-well culture plate containing complete media composed of: v:v; Iscove’s modified dulbecco’s medium (Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS; Thermo Fisher Scientific, Waltham, MA, USA), 1% penicillin/streptomycin (Thermo Fisher Scientific, Waltham, MA, USA), 1% Non-essential amino acid solution (Millipore-sigma, St. Louis, MO, USA), and 0.1% 2-Mercaptoethanol (Gibco, Grand Island, NY, USA). Splenocytes were centrifuged at 2000 rcf for 3 min. Supernatant was aspirated and cells were incubated for 2 min at RT in 1 mL ACK lysing buffer (Gibco, Grand Island, NY, USA). Cell pellet then received 10 mL of FACS buffer before being centrifuged at 2000 rcf for 3 minutes. Supernant was then aspirated prior to resuspension in 1 mL of complete media. Cells were filtered a second time before being plated at 50 µL per well and subsequently centrifuged at 2000 rcf for 3 min and decanted to remove remaining FACS buffer. Cells were treated with Fc blocking antibody (BD) at 1:200 dilution in FACS buffer and incubated at 4C for 5 min. Next, 50 µL of zombie fixable viability stain (BioLegend, San Diego, CA, USA) was added prior to incubation at 4C for 20 min. Cells were then centrifuged and decanted prior to washing 1x in 150 µL FACS buffer before addition of 50 µL extracellular antibody solution or fluorescence minus one (FMO) controls. Antibodies for extracellular staining were as follows: BUV395-anti-CD62L (1:800; BD), BUV496-anti-CD4 (1:800; BD), BV785-anti-CD8 (1:800; Biolegend), AF700-anti-CD44 (1:400; Biolegend), APC-Cy7-anti-CD3 (1:400; Biolegend), BV605-anti-PD-1 (1:400; Biolegend), PE-anti-LAP (1:400; Biolegend), PE-Dazzle-594-anti-CXCR3 (1:400; Biolegend), APC-anti-γδTCR (1:400; Carlsbad, CA, USA), BV650-anti-CXCR5 (1:200; Biolegend), Per-Cp-anti-CD107 (1:100; Biolegend). Cells were incubated in antibody solution for 20 min at 4C before washing with 150 µL FACS buffer, centrifugation, and decanting. Intracellular staining was performed by resuspending splenocytes in 100 µL of fixation and permeabilization solution from a Foxp3 / transcription factor staining buffer set (eBioscience, San Diego, CA, USA) for 20 min at 4C. Cells were washed with 200 µL permeabilization buffer and centrifuged at 2000 rcf for 3 min. After decanting, 50 µL of intracellular antibody solution or FMO controls was added to appropriate wells. Splenocytes for cytokine analysis were stimulated at 37°C for 4 h with PMA/ionomycin and GolgiStop containing monensin (1:1000; BD) prior to being centrifuged at 1500 rcf for 5 min and decanted. Cells were washed with 200 µL FACS buffer before centrifugation at 2000 rcf for 3 min and subsequently decanted. Antibodies for cytokine staining were as follows: APC-Cy7-anti-CD3, BUV496-anti-CD4, BV785-anti-CD8, and BV421-anti-IFNg (1:400, Biolegend). Cells were incubated in antibody solution for 30 min at RT before being washed with 200 µL of permeabilization buffer, centrifuged, and decanted. Splenocytes were resuspended in 400 µL of FACS buffer and transferred to flow tubes. Cells were processed for flow cytometric acquisition on a Fortessa using DIVA software (BD Biosciences). Data was analyzed in FlowJo (v10.1, TreeStar Inc, Ashland, OR, USA).

Mono-colonization with Akkermansia modulates microglia gene expression. We previously found a differential effect of Akkermansia strains and B. cellulosyliticus on CNS inflammation in the experimental autoimmune encephalomyelitis (EAE) model [6]. In order to identify strain-specific effects on glia, we mono-colonized mice with these strains and investigated transcriptional profiles of microglia and astrocytes. Principal component analysis (PCA) demonstrated differential clustering based on the mono-colonization group vs. vehicle controls (Fig. 1). Microglia gene expression demonstrated a downregulation of Ndufaf7 and Mmgt2 in Am^T treated mice vs. vehicle GF animals and an upregulation of Dgkq and Nhsl2 (Fig. 2A-B). Gene set enrichment pathway analysis on DEGs from Veh vs Am^T treated mice identified downregulated pathways including lymphocyte mediated immunity, endothelial development and stem cell differentiation as being significantly altered (Fig. 2C). No upregulated pathways were observed. When we compared mice mono-colonized with Veh vs. a gram negative control bacteria, Bc, 1 gene was significantly altered, Dennd1a and in microglia from mice mono-colonized with Am-H3 vs. Bc, 5 genes were downregulated and 2 upregulated (Fig. 2D-E). Pathway analysis of Am-H3 vs Bc identified the enrichment of pathways related to γδ-TCR T cell activation and integrin mediated signaling. We also observed downregulation of pathways involved in cell proliferation including ras and TOR signaling in Am-H3 vs Bc groups (Fig. 2F). Furthermore, Am-H3 mono-colonized mice had 7 downregulated and 6 upregulated genes vs. Veh (Fig. 2G-H). Pathway analysis of Veh vs Am-H3 identified upregulated pathways largely associated with cell differentiation, and downregulated pathways involved in membrane and extracellular matrix organization (Fig. 2I). When we compared Am^T treated mice vs. Am-H3, we found 1 significantly altered gene, Tbc1d24.

Akkermansia mono-colonization modulates astrocyte gene expression. We then investigated the impact of mono-colonization with MS associated microbes on astrocyte gene expression and found altered gene expressions profiles in both Am^T and Am-H3 treated mice vs. Veh controls. Tmpo and Ppp1r7 were upregulated and Sec24a, Gpt, and B3galt5 were downregulated in Am^T vs. Veh treated mice (Fig. 3A-C). Pathway analysis of Veh vs Am^T treated animals identified vesicle packaging and endocytosis pathways upregulated and protein kinase and phosphorylation activity downregulated (Fig. 3B). When comparing Am-H3 astrocytes vs. Veh mono-colonized, we found 8 genes downregulated and 10 upregulated (Fig. 3D-F) including Top2a and Ermn (Fig. 3F). We identified pathways involved in cell projections and proliferation upregulated and vesicular transport and catabolic process genes downregulated (Fig. 3E). Analysis of astrocytes isolated from mice mono-colonized with Am-H3 vs. Bc, identified 5 downregulated and 24 upregulated genes (Fig. 3G-I). Pathway analysis of Am-H3 v Bc showed several upregulated pathways involved in cytoskeletal and dendritic morphology alterations (Fig. 3H).

Am ^T and Am-H3 modulate T cells. Because the microbiome may also influence glia by modulating peripheral immunity, we investigated with effect of mono-colonization of Am^T and Am-H3 on splenic T cells. Total CD3 + T cells were increased in Am^T (p < 0.01) and Am-H3 (p < 0.05) mono-colonized compared to Veh treated animals (Fig. 4A). Effector CD4 + T cells (p < 0.01) and CD44(Hi) CD62L(lo) activated T cells (p < 0.05) were increased in Am^T compared to Veh (Fig. 4B-C). We also found increased IFNg production by CD4 + T cells in mice colonized with Am-H3 and Bc. We observed an increase in γδ-TCR T cells in Am^T (p < 0.05) and Am-H3 (p < 0.001) vs. vehicle, whereas Am-H3 was also increased compared to Bc (p < 0.05; Fig. 4D,E). However, total CD4 + and CD8 + frequency were not altered (Figure S1A & C-D).

Differential production of SCFAs in monocolonized mice and gut mucosal integrity. Studies have shown that SCFAs modulate microglia [16]. We found that specific SCFAs were elevated in the cecum from mono-colonized animals vs. Veh treated GF mice. Bc mono-colonized animals had increased acetate (p < 0.0001) propionate (p < 0.0001) and isovalerate (p < 0.0001) compared to Veh, Am^T, and Am-H3 mono-colonized animals (Fig. 5A-C). Both Am^T and Am-H3 produced propionate as the primary SCFA (p < 0.001; Fig. 5B). Since Akkermansia and Bc are known to degrade mucus [22, 23], we investigated the gut mucus barrier and epithelial integrity by measuring goblet cells labelled for UEA-1 + and analyzed cell quantity and size. Colonic goblet cells were labeled with UEA-1 lectin and we found similar quantities and sizes in Veh, Bc, Am^T, and Am-H3 monocolonized mice. Furthermore, we found cecal weights to be similar between mono-colonization groups (Figure S2).

The gut microbiota influences peripheral and central immune function in health and disease [24–26]. It is not known whether strain-specific effects of single bacteria or their metabolites can augment protective pathways involved in CNS homeostasis, or conversely promote pathogenic processes in the the CNS. To address these questions, we colonized GF mice with MS associated microbes [6] and investigated their effects on gene expression in brain microglia and astrocytes and linked these effects to changes in peripheral immunity and intestinal microbial metabolites.

Multiple studies have reported microbiota alternations in neurologic diseases [1, 5–9, 27–31]. Akkermansia has been shown to ameliorate disease in animal models of MS, AD, ALS, and epilepsy [6, 32–35], whereas disease related pathways are worsened when Akkermansia is cultured with PBMCs from MS subjects [1] and when cultured with an enteroendocrine cell line [36]. There are 4 clades of Akkermansia with distinct metabolic and immunologic properties [12]. We found that Akkermansia BWH-H3 had a greater protective effect in EAE than the 3 other Akkermansia strains. In addition, strain specific asssociations of Akkermansia with MS and MS clinical measures have been described [5, 6].

To investigate the properties of these Akkermansia strains, we mono-colonized mice with either Akkermansia muciniphila type strain, or our recently isolated Akkermansia BWH-H3 strain [6]. We observed strain specific effects on microglial gene expression after mono-colonization. Minimal effects were observed with the Am^T strain compared to vehicle treated animals. We found that Am-H3 had a greater effect on microglia gene expression including those involved in cell adhesion (e.g., Nid2), none of which were altered by Am^T mono-colonization. Srsf1, a nuclear export adapter whose depletion prevents neurodegeneration in the drosophila model of C9ORF72 related diseases [37, 38] was downregulated in Am-H3 treated animals. Eif2s3y, a Y-chromosomal gene that causes autism-like behaviors in male mice [39] was also downregulated in Am-H3 mono-colonized microglia. Bc, a Gram-negative anaerobic microbe that we used as a non-Akkermansia control elevated Eif2s3y compared to Am-H3, supporting Am-H3’s beneficial influence on brain microglia. Taken together, these data provide evidence of strain specific effects on microglial gene expression.

Microbial mono-colonization also influenced astrocyte gene expression in a strain specific manner. We observed an increase in Tmpo in mice mono-colonized with Am^T. Tmpo codes for thymopoietin which downregulates inflammatory responses in EAE [40]. Am-H3 altered astrocyte gene expression pathways involved in transport activity and cytoskeletal regulation. Downregulation of Ermn in Am-H3 in our mono-colonized animals is consistent with the altered astrocyte cytoskeletal rearrangement we observed in our pathway analysis. Ermn is downregulated in RRMS [41], and knocking-out Ermn leads to inflammation, microgliosis and increased astrocyte numbers [42]. This suggests that downregulation of Ermn in Am-H3 treated animals is associated with astrocyte restructuring. We also observed elevation of actin and cytoskeletal associated genes in Am-H3 vs. vehicle mono-colonized animals, including Fam107a, an actin associated gene that’s protein product functions to maintain astrocytes in a quisescent, nonproliferative state [43]. Together our findings identify the structural and functional regulation of astrocytes by Akkermansia strain Am-H3.

Alterations in immune function during mono-colonization are likely driven by a combination of factors. We observed strain specific alterations in peripheral immune populations from mono-colonized mice, with Am-H3 being associated with elevated splenic γδ-T cells. In MS, γδ-T cells have two distinct phenotypes, one characterized by IL-17 production and one driven by IFNγ producing cells [14]. We previously found that Am-H3 reduces IL-17 producing γδ-T cells in EAE [6]. We also found an elevation of IFNγ producing T cells in our Am-H3 and Bc mono-colonized animals but not type strain Am^T. Our data point towards Akkermansia mono-colonization, and specifically Am-H3, shifting peripheral T populations towards a more protective phenotype.

Gut epithelial and mucus barrier integrity are important components of intestinal homeostasis and SCFAs have an important physiologic role including modulating barrier function [44] and SCFAs have been shown to promote microglial homestasis and contribute to microglial maturation in germ free animals [15]. Bc produced multiple SCFAs [45] and both strains of Akkermansia we investigated produced propionate, which has been shown to be beneficial in MS [46]. It is well recognized that the microbiota is responsible for SCFA in the gut and as expected we did not detect SCFA in vehicle treated mice. Akkermansia is a mucin degrader [47] raising the question of whether monocoloinzation would impair gut barrier function. We found no differences in gut wall mucus in goblet cells in mono-colonized groups.

In conclusion, we found that Akkermansia mono-colonization modulates microglia and astrocyte gene expression, which may be mediated by changes in short chain fatty acids and peripheral immune signaling.

Conflict of interest:

None to declare.

Ethics Declaration

All animal procedures were approved by the Brigham and Women’s Hospital IACUC under protocol# 2016N000230.

Funding:

LAS – National Multiple Sclerosis Society, Grant #FG-2207-40162

Author Contribution

LAS conceived the study, performed the experiments, analyzed and interpreted the data and wrote the manuscript. TBL and FM, analyzed RNAseq data and edited the manuscript. DSL performed experiments and edited the manuscript. TM analyzed and interpreted immunology data and edited the manuscript. LMC and HLW conceived the study and oversaw the entirety of the project. All authors reviewed the manuscript.

Acknowledgement

The authors wish to express their gratitude to Dr. Lynn Bry and her exceptional team at the Massachusetts Host-Microbiome Center, including Vladimir Yeliseyev for overseeing and performing mono-colonization gavages, and Mary Delaney for processing cecal SCFAs.

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Akkermansia modulates microglia and astrocytes in a strain specific manner

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Introduction

Materials and Methods

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Mono-colonization:

Tissue processing:

SCFA quantification:

RNAseq:

Flow Cytometry:

Results

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