Modulation of Alzheimer’s disease brain pathology in mice by gut bacterial depletion: the role of IL-17a

Figures & data

Figure 1. Antibiotic treatment successfully depletes the bacteria in the gut of APP-transgenic mice.

Three-month-old APP-transgenic female mice were treated with and without antibiotics in drinking water for 2 months. Intestinal content (total 100 mg) for the isolation of bacterial DNA was harvested from the appendix and neighboring colon. Bacterial DNA was first measured for the amount with real-time PCR (a; t test; n = 20 and 6 for mice receiving normal water and antibiotic supplement), and then sequenced for the V3-V4 region of 16S rRNA-encoding gene (n = 5 and 4 for control and antibiotic-treated mice, respectively). Using Sobs, Shannon, Ace, Chao and Simpson’s indices, α-diversity analysis shows that treatment with an antibiotic cocktail significantly reduces bacterial richness and diversity within each mouse (b–f; t test). Principal coordinate analysis (PCoA) was used for β-diversity analysis of bacterial composition at the genus level in APP-transgenic mice with and without antibiotic treatment (g; Each symbol represents the gut bacteria of an individual mouse). As expected, the structure of gut bacterial community of antibiotics-treated APP-transgenic mice differed significantly from those of APP-transgenic littermates with normal drinking water (g; ANOSIM analysis between +antibiotics and control mice). Bar plots depict abundance (% of total) of the indicated genera. Wilcoxon rank-sum tests show that antibiotic treatment increases the relative abundance of bacteria in the genera Escherichia-Shigella and Parasutterella (h). ***: p < 0.001 and ****: p < 0.0001.

Figure 2. Depletion of gut bacteria reduces Il-17a-expressing CD4-positive lymphocytes.

Three-month-old APP-transgenic female mice, expressing or not Il-17a-eGFP reporter, were treated with and without antibiotics in drinking water for 2 months. CD4-positive splenocytes were selected and detected for transcription of T lymphocyte marker genes. The transcription of Il-17a gene was significantly down-regulated by antibiotic treatment, while the transcription of Ifn-γ and Il-4 genes was up-regulated in CD4+ splenocytes compared to APP-transgenic control mice with normal drinking water (a–c; t test, n = 6 - 7 per group). The transcription of Il-10 gene in spleen cells was not changed by antibiotic treatment (d; t test, n = 6 - 7 per group). In addition, single cell suspensions were prepared from both lamina propria and Peyer’s patches of 5-month-old Il-17a-eGFP-expressing APP-transgenic mice and analyzed by flow cytometry after fluorescent staining of CD4 (e and g). Intestinal cells prepared from APP-transgenic mice without expressing Il-17a-eGFP reporter were stained with APC-conjugated rat IgG2b as an isotype control (Isotype w/o GFP). The expression of IL-17a-associated eGFP was decreased in CD4+ lymphocytes in the intestine of APP-transgenic mice by antibiotic treatments (f and h; t test, n = 7 - 10 per group). *p < 0.05; **p < 0.01.

Figure 3. Depletion of gut bacteria reduces bacterial DNA in the brains of both Il-17a-deficient and wild-type APP-transgenic mice.

Three-month-old Il-17a-deficient (ko) and wild-type (wt) APP-transgenic female mice received drinking water with and without supplement of antibiotics for 2 months. The hippocampus and cortex were collected and homogenized in Trizol for DNA isolation. The amount of bacterial DNA was evaluated by real-time PCR using mouse Gapdh gene as an internal control. Depletion of gut bacteria significantly decreased bacterial DNA in both Il-17a-deficient and wild-type APP-transgenic mice; interestingly, deficiency of Il-17a also reduced bacterial DNA in the brain compared with Il-17a wild-type AD mice (a and b; Mann-Whitney-U-Test, n = 8 - 9 per group for Il-17a-wildtype mice and 6 - 8 per group for Il-17a-deficient mice). *, p< 0.05.

Figure 4. Depletion of gut bacteria reduces inflammatory activation in the brain of Il-17a-wildtype, but not Il17a-deficient APP-transgenic mice.

Three-month-old APP-transgenic female mice with (ko) and without (wt) knockout of Il-17a gene, and 3 or 22-month-old C57BL/6J female mice were treated with and without antibiotics in drinking water for 2 months. Thereafter, brain tissues were sectioned and microglia were counted with stereological method, Optical Fractionator, after immunohistochemical staining of Iba1 (in brown color) (a–e), or homogenized for RNA isolation (f, h, and j–m) and measurement of inflammatory gene transcripts with real-time PCR, as well as for ELISA assays of Tnf-α and Il-1β concentrations (g and i). Depletion of gut bacteria significantly decreased Iba1-positive microglia in both the hippocampus and cortex of Il-17a-wildtype, but not in Il17a-deficient APP-transgenic mice (b, c and e; t test, n = 5 - 9 per group). Similarly, depletion of gut bacteria significantly reduced the transcripts of Il-1β and Ccl-2, and increased Il-10 transcript in the brain of Il-17a-wildtype, but not in Il17a-deficient APP-transgenic mice (h, j, and k; t test, n = 6 - 17 per group). Transcriptional regulation in AD mice deleted of gut bacteria was confirmed by the reduction in protein levels of Il-1β but not Tnf-α in brain homogenate compared to APP mice receiving normal water (g and i; t test, n = 4 - 9 per group). However, depletion of gut bacteria significantly reduced the transcript of Chi3l3 in the brain of Il17a-deficient APP-transgenic mice (l; t test, n = 6 - 7 per group). Moreover, depletion of gut bacteria did not change the transcription of various inflammatory genes (Tnf-α, Il-1β, Ccl-2, Il-10, Chi3l3, and Mrc1) in in the brain of 5-month-old C57BL/6J female mice (n; t test, n = 3 - 9 per group), however, significantly increased Tnf-α transcription and decreased Chi3l3 transcription in 24-month-old C57BL/6J female mice (O; t test, n = 5 - 6 per group). *: p < 0.05; **: p < 0.01.

Figure 5. Depletion of gut bacteria inhibits inflammatory activation in microglia in the brain of Il-17a-wildtype, but not Il17a-deficient APP-transgenic mice.

Three-month-old APP-transgenic female mice with (ko) and without (wt) knockout of Il-17a gene were treated with and without antibiotics in drinking water for 2 months. CD11b-positive brain cells were selected and quantified for the transcription of DAM marker genes. Depletion of gut significantly decreased the transcription of Il-1β, and Ccl-2 genes, and tended to down-regulate the transcription of Tnf-α, Il-10, and Itgax genes in the brain of Il-17a-wildtype, but not in Il17a-deficient APP-transgenic mice (a–d, and k; t test, n = 4 – 7 per group). Depletion of gut bacteria significantly reduced transcription of Apoe gene, but not Trem2, Cx3cr1, P2ry12, Clec7a, and Lpl genes in both Il-17a-wildtype and deficient APP-transgenic mice (e–j; t test, n = 4 - 8 per group). In the analysis of microglial morphology after immunofluorescent staining of Iba1 (l), depletion of gut bacteria increased the number of branches and endpoints of the processes of microglia in Il-17a-wildtype, but not in Il17a-deficient APP-transgenic mice (m – o; t test, n = 5-7 per group). The Iba1-positive cells marked with “*” without showing clear processes were excluded for analysis of microglial morphology (l). *: p < 0.05; **: p < 0.01; #: 0.05 < p < 0.10.

Figure 6. Depletion of gut bacteria reduces Aβ in the brain of Il-17a-wildtype, but not Il17a-deficient APP-transgenic mice.

Three-month-old APP-transgenic female mice with (ko) and without (wt) knockout of Il-17a gene were treated with and without antibiotics in drinking water for 2 months. Brain tissues were sectioned and stained with immunofluoresce-conjugated Aβ antibodies (a and d) and methoxy-XO4, a fluorescent dye for Aβ aggregates (g and j). Depletion of gut bacteria decreased Aβ deposits in both the hippocampus and cortex after staining of Aβ either with antibody or dye (b, c, h and i; t test, n = 6 - 13 per group). However, in the Il17a-deficient AD mice, depletion of gut bacteria did not change the cerebral Aβ loads (e, f, k and l; t test, n = 4-7 per group). Brain tissues were also serially homogenized and extracted in TBS, TBS plus 1% Triton-100 (TBS-T) and guanidine-HCl, and then measured for Aβ40 and Aβ42 with ELISA (m–r). Depletion of gut bacteria decreased the concentrations of both Aβ40 and Aβ42 in TBS- and TBS-T-soluble brain fractions, but not in guanidine-HCl-soluble fraction of Il-17a-wildtype APP-transgenic mice (m–r; t test, n = 7-9 per group). Depletion of gut bacteria did not change the concentrations of Aβ40 and Aβ42 in various fractions of brain homogenates of Il-17a-deficient AD mice (n–r; t test, n = 4 - 5 per group), except that it significantly increased the concentration of Aβ42 in TBS-soluble fraction (m). *: p < 0.05; **: p < 0.01; ***: p < 0.001; #: 0.05 < p < 0.10.

Figure 7. Depletion of gut bacteria reduces β-secretase activity in the brain of Il-17a-wildtype, but not Il17a-deficient APP-transgenic mice.

Three-month-old Il-17a-deficient (ko) and wild-type (wt) APP-transgenic female mice were treated with and without antibiotics in drinking water for 2 months. The brain tissue was stained with Cy3-conjugated CD68 antibody (a). Antibiotic treatment significantly reduces the density of CD68-immunofluorescence in both cortex and hippocampus of APP-transgenic mice compared to AD mice drinking normal water (b and c; t test, n = 6 per group). APP-transgenic mice were also injected (i.p.) with methoxy-XO4 for the detection of Aβ-associated fluorescence in CD11b-positive brain cells by flow cytometry (d). Depletion of gut bacteria significantly decreased both the percentage of fluorescent cells among CD11b-positive brain cells, and the mean fluorescence intensity (mFI) of CD11b-positive cell population (e and f; t test, n = 4 - 5 per group). Brain tissues were further collected from AD mice for quantification of Neprilysin and Ide gene transcripts (g–j), and for β- and γ-secretase assays (k – n). Depletion of gut bacteria did not change the transcription of Neprilysin and Ide genes (g–j; t test, n = 6-8 per group); however, significantly inhibited the activity of β-secretase in the brain of Il-17a-wildtype but not Il17a-deficient APP-transgenic mice (k and m; two-way ANOVA, n = 6-7 per group for Il-17a-wildtype mice, and n = 4-5 per group for Il-17a knockout mice). Depletion of gut bacteria did not change γ-secretase activity in AD mice (l and n; two-way ANOVA, n = 6-7 per group for Il-17a-wildtype mice, and n = 4-5 per group for Il-17a knockout mice). *: p < 0.05; **: p < 0.01.

Figure 8. Depletion of gut bacteria increases Abcb1 and Lrp1 expression in the blood-brain-barrier of Il-17a-wildtype, but not Il17a-deficient APP-transgenic mice.

Three-month-old Il-17a-deficient (ko) and wild-type (wt) APP-transgenic female mice were treated with and without antibiotics in drinking water for 2 months. Brain homogenates were quantified for protein levels of Abcb1 and Lrp1 with Western blot (a and d). Depletion of gut bacteria significantly increased expression of Abcb1 and Lrp1 in brains of Il-17a-wildtype (b and c), but not in Il-17a-deficient (e and f) APP-transgenic mice (t test, n = 13, and 11 - 12 per group for Il-17a wt and ko mice, respectively). Cerebral vessels were also isolated from Il17a-wildtype AD mice and detected for Abcb1 and Lrp1 at the BBB (g). Depletion of gut bacteria significantly increased the expression of Abcb1 and Lrp1 in blood vessels of APP-transgenic mice (h and I; t test, n = 11-12 per group); however, it did not change the protein level of claudin-5 (j; t test, n = 4-5 per group). In further experiments, Abcb1 and Lrp1 were detected with quantitative Western blot in the brain homogenates from 5-month-old non-APP-transgenic female mice with different expression of Il-17a (k; wild-type [wt], heterozygote [het] and knockout [ko]), and in the isolated blood vessels from 5-month-old Il17a-wt and ko APP-transgenic female mice (n). Deficiency of Il-17a significantly increased Abcb1 but not Lrp1 in the non-APP-transgenic mouse brain in a gene dose-dependent manner (l and m; one-way ANOVA followed by Bonferroni post-hoc test, n = 4-9 per group), and increased protein levels of both Abcb1 and Lrp1 in the blood vessels of APP-transgenic mice (o and p; t test, n = 15 - 19 per group). *: p < 0.05; **: p < 0.01; ****: p < 0.0001.

Figure 9. Depletion of gut bacteria increases Arc transcription in the brain of Il-17a-wildtype, but not Il17a-deficient APP-transgenic mice.

Three-month-old Il-17a-deficient (ko) and wild-type (wt) APP-transgenic female mice were treated with and without antibiotics in drinking water for 2 months. Thereafter, brain tissues were homogenized and measured for transcripts of Arc, Grin1 and Bdnf genes. Antibiotic treatment significantly increased the transcription of Arc gene in Il-17a-wildtype, but not in Il17a-deficient APP-transgenic mice (a; t test, n = 6 and 7 - 8 per group for Il-17a wt and ko mice, respectively). Antibiotic treatment did not change the transcription of Grin1 and Bdnf genes (b and c; t test, n = 6 and 7 - 8 per group for Il-17a wt and ko mice, respectively). *: p < 0.05.

Data availability statement

Our study has not generated new datasets. All data generated or analyzed during this study are included in this published article and its supplementary information files.