Muribaculum intestinale negatively impacts glioma growth in mice through the toll-like receptor 2

Abstract

Glioblastoma (GBM) is the most common and malignant brain tumor in adult humans. Recent studies have demonstrated a link between the composition of the gut microbiota and glioma progression. Here, we describe that the growth of glioma in mice is inversely correlated with the relative abundance of the anaerobic bacterium Muribaculum intestinale in the feces. We found that M. intestinale administration: 1) induced an inflammatory environment in the gut; 2) reduced glioma growth; 3) increased the pro-inflammatory profile of tumor-associated microglial cells and the frequency of CD8+ T cells; and 4) increased the peripheral TNF-α levels. The effects induced by M. intestinale administration were significantly attenuated upon toll-like receptor 2 (TLR2) silencing using TLR2-targeting siRNA. As a pattern-recognition receptor, TLR2 detects microbial-associated molecular patterns and orchestrates host immune responses to infection. Collectively, these data demonstrate that M. intestinale induces a pro-inflammatory response in glioma bearing mice, inhibiting tumor growth via TLR2-dependent signaling.

Keywords: Glioma; Muribaculum intestinale; gut microbiota; immune cell activation.

Figure 1.

Fecal microbiota in mice with glioma. A. Experimental design. B Alfa-diversity: biodiversity (inverse Simpson) and richness (number of bacterial species) among healthy (Healthy, gray, n = 22 biologically independent samples), sham-operated (sham, green, n = 7 biologically independent samples) and glioma mice (glioma, blue, n = 15 biologically independent samples). C. Beta-diversity following the Bray-Curtis dissimilarity distance algorithm, among healthy, sham and glioma mice. D. Experimental design. E Spearman correlation coefficient analysis between the relative abundance of all bacterial species found in glioma-bearing mice and tumor volumes (23 xy pairs) F. Spearman correlation coefficient graph between tumor volume and Acetatifactor muris abundance in glioma mice (ρ = 0.619, p = 0.0016, n = 23 xy pairs). G. Analysis of the relative abundance of fecal Acetatifactor muris among healthy (gray, n = 30, 0.156 ± 0.057, *p = 0.012 vs glioma), sham-operated (green, n = 7, 0.207 ± 0.070, *p = 0.017 vs glioma by Brown-Forsythe ANOVA test) and glioma mice (blue, n = 23 1.450 ± 0.470). H. Spearman correlation coefficient graph between tumor volume and Muribaculum intestinale abundance in glioma mice (ρ = -0.613, p = 0.0019, n = 23 xy pairs). I. Analysis of the relative abundance of fecal Muribaculum intestinale among healthy (gray, 26.75 ± 3.73, n = 30), sham-operated (green, n = 7, 29.40 ± 2.38) and glioma (blue, n = 23, 31.75 ± 3.17 ns by Brown-Forsythe and Welch ANOVA test).

Figure 2.

Bacterial administration affects tumor growth. Experimental setup for A. muris (A) or M. intestinale (B) administration. The black arrows depict the oral gavage of fresh broth (vehicle) or bacterium. C. Tumor volumes in vehicle (blue, n = 11, 100.0 ± 6.4%) and A. muris treated mice (orange, n = 11, 168.9 ± 28.8%, * p = 0.032 by unpaired Student’s t-test). Right, representative images of brain coronal slices with glioma. D. Tumor volumes in vehicle (blue, n = 9, 100.0 ± 15.4%) and M. intestinale treated mice (red, n = 10, 61.5 ± 6.5%, * p = 0.014 by unpaired Student’s t-test). Right, representative images of brain coronal slices with glioma stained with Haematoxylin & Eosin.

Figure 3.

M. intestinale administration alters gut environment. A. Analysis of M. intestinale enrichment in feces of mice at the end of treatment (blue, vehicle = 0.62 ± 0.08% n = 7; red, M. intestinale = 1.05 ± 0.16% n = 8, * p = 0.050 by unpaired Student’s t-test). B. Volcano Plot illustrating the differential gene expression in distal colon tissue for M. intestinale (n = 3 independent biological samples) vs vehicle-treated glioma mice (n = 3 independent biological samples). C. Gene ontology enrichment analysis showing the upregulated and downregulated genes and relative biological processes in distal colon tissues of M. intestinale (n = 3 independent biological samples) vs vehicle-treated glioma mice (n = 3 independent biological samples). D. Representative images of H&E-staining of colon tissue showing changes in inflammatory cell infiltrates. Colonic samples from M.intestinale mice (upper, blue squared) displayed higher amount of inflammatory infiltrate in lamina propria (yellow arrow) compared to vehicle mice (lower, red squared). Original magnification 10x. Right: quantification of inflamed colon fraction (blue, vehicle 11.3 ± 2.6%, n = 3 mice per group/44 total slice; red, M. intestinale 22.6 ± 2.3%, n = 3 mice/48 total slice, *p = 0.032 by unpaired Student’s t-test). E. Experimental setup for M. intestinale and siRNA loaded dendrimers administration. The black arrows depict the weekly oral gavage of fresh broth (vehicle) or bacterium. Dark yellow triangles depict the intraperitoneal injection (every four days) of scramble-siRNA (scramble) or TLR2-siRNA loaded dendrimers. F. Tlr2 transcript level evaluated as fold increase by real time qPCR over vehicle (blue, 0.99 ± 0.09, n = 10), M.intestinale + scramble-siRNA (red, 1.48 ± 0.17, n = 10, *p = 0.048 vs Vehicle) and M.intestinale + TLR2-siRNA (red stripes, 0.84 ± 0.10, n = 11, *p = 0. 0014 vs M.intestinale + scramble-siRNA by Brown-Forsythe and Welch ANOVA) at the end of experiments. G. Quantification by ELIZA of TNF-α level (pg/ml) in the serum of vehicle (blue, n = 4, 22.3 ± 3.6, **p = 0.0065 vs siRNA-scramble), M. intestinale/scramble siRNA (red, n = 4, 46.6 ± 3.2, **p = 0.0012 vs siRNA-TLR2 by Brown-Forsythe and Welch ANOVA tests) and M. intestinale/siRNA TLR2 (red stripes, n = 4, 10.0 ± 1.2) treated mice. H. Quantification of immune cell subset frequencies in colon lamina propria of vehicle (blue, n = 8-9), M. intestinale/scramble siRNA (red, n = 10-11) and M. intestinale/siRNA TLR2 (red stripes, n = 10-11) treated mice. Statistics by Brown-Forsythe and Welch ANOVA tests.

Figure 4.

M. intestinale affects tumor growth through TLR2. A. Experimental setup for M. intestinale and siRNA loaded dendrimers administration. The black arrows depict the weekly oral gavage of fresh broth (vehicle) or bacterium. Dark yellow triangles depict the intraperitoneal injection (every four days) of scramble-siRNA (scramble) or TLR2-siRNA loaded dendrimers. B. Tumor volume evaluation in vehicle (blue, 17.0 ± 2.2mm³, n = 6), M. intestinale/scramble-siRNA (red, 7.2 ± 1.2mm³, n = 7, **p = 0.005 vs vehicle) and M. intestinale/ TLR2- siRNA (red stripes, 17.1 ± 2.9 mm³, n = 6, *p = 0.01 vs scramble-siRNA) treated mice. Right, Representative images of brain-glioma coronal slices with glioma. C. Protein expression level (MFI, mean fluorescence intensity) as evaluated by flow cytometric analysis of Inos and Arg1 in microglia isolated from vehicle (blue, iNOS = 4869 ± 159, n = 10, *p = 0.020 vs M. intestinale/scramble-siRNA; Arg1 = 1506 ± 82, n = 10, **p = 0.0054 vs M. intestinale/scramble-siRNA), M. intestinale/scramble-siRNA (red, Inos = 5834 ± 327, n = 10; Arg1 = 1152 ± 75, n = 10) and M. intestinale/ TLR2- siRNA (red stripes, Inos = 4275 ± 228, n = 5, **p = 0.0018 vs M. intestinale/scramble-siRNA; Arg1 = 1174 ± 97, n = 5, *p = 0.027 vs Vehicle) treated glioma mice. Frequencies of monocytes/macrophages (D) and CD8 positive cells (E) isolated from vehicle (blue, monocytes/macrophages = 12.72 ± 1.86%, n = 9, *p = 0.047 vs M. intestinale/scramble-siRNA; CD8 = 3.79 ± 0.48%, n = 6, *p = 0.030 vs M. intestinale/scramble-siRNA), M. intestinale/scramble-siRNA (red, monocytes/macrophages = 7.30 ± 1.70%, n = 9; CD8 = 6.16 ± 0.77%, n = 6) and M. intestinale/ TLR2- siRNA (red stripes, monocytes/macrophages = 14.88 ± 2.18%, n = 4, *p = 0.029 vs vs M. intestinale/scramble-siRNA; CD8 = 4.20 ± 1.08%, n = 4) treated glioma mice. F. Frequency of CD107 positive cells among CD8 positive cells isolated from vehicle (blue, 2.38 ± 0.93%, n = 6, **p = 0.003 vs M. intestinale/scramble-siRNA), M. intestinale/scramble-siRNA (red, 9.22 ± 1.45%, n = 6) and M. intestinale/ TLR2- siRNA (red stripes, 4.43 ± 1.11, n = 4, *p = 0.031 vs M. intestinale/scramble-siRNA) treated glioma mice. Statistics by Brown-Forsythe and Welch ANOVA tests.