Fig 1.
Angiostrongylus cantonensis infection induces brain inflammation.
(A) The body weight of mice. (B) Neurological score of mice. (C) Representative H&E-stained section of the mouse brain. C, cerebral cortex; CA, cornu ammonis; In, inflammatory infiltration. Scale bars correspond to 200 μm. (D) Histological score of the brain sections. (E-F) Serum (E) and brain (F) interleukin (IL)-1β levels. n = 6 mice in each group. Data in (A and B) are presented as mean ± S.E.M and (D-F) as mean ± S.D; area under curve (AUC) data are presented as mean ± S.D. A.U., arbitrary unit. Significance determined by unpaired T-test, with p < 0.05 considered significant.
Fig 2.
Angiostrongylus cantonensis infection induces changes in the gut microbiome.
(A-C) Alpha diversity of the microbiomes in uninfected and infected mice. (A) qPCR-based quantification of total 16S copy-number in 1 ng of stool DNA, as shown by Ct values per ng DNA. (B) Chao index, for estimation of the number of OTUs per sample. (C) Shannon index, for estimation of the microbial diversity in each sample. (D) Principal co-ordinates analysis (PCoA) based on Bray-Curtis distance on the mice’s microbiomes between uninfected and infected mice. The x and y axes represent the first, second, and third principal coordinates. The value in percentage in the axis label represents the contribution of the corresponding coordinate to the sample variance. (E-G) Taxa summary of bacterial phyla (E), class (F), and genera (G; showing only the top 32 most abundant classifications) from uninfected and infected mice. n = 6 mice in each group. Data in (A-C) are presented as mean ± S.D. A.U., arbitrary unit. Significance determined by unpaired T-test, with p < 0.05 considered significant.
Fig 3.
Angiostrongylus cantonensis infection alters intestinal Lactobacillus abundance.
(A) Relative abundance of Lactobacillus between uninfected and infected mice, obtained by 16S rDNA sequencing of stool samples. (B) Scatter plot of Pearson’s correlation coefficient (r) between Lactobacillus abundance and neurological or histological scores. (C-F) Relative abundance of Lactobacillus (C), Lactobacillus murinus (D), Lactobacillus johnsonii (E), and Lactobacillus reuteri (F) between uninfected and infected mice, obtained by qPCR of stool samples. n = 6 mice in each group. Data in (A, C-F) are presented as mean ± S.D. A.U., arbitrary unit. Significance determined by unpaired T-test (A, C-F) or Pearson correlation (B), with p < 0.05 considered significant.
Fig 4.
Inoculation of Lactobacillus johnsonii improves neuroangiostrongyliasis in mice.
(A) The body weight of mice. (B) Neurological score of mice. (C) Representative H&E-stained section of the mouse brain. C, cerebral cortex; CA, cornu ammonis; H, hemorrhage; In, inflammatory infiltration. Scale bars correspond to 200 μm. (D) Histological score of the brain sections. (E-F) Serum (E) and brain (F) interleukin (IL)-1β levels. (G) Kaplan-Meier survival curves of L. johnsonii-inoculated mice, with or without A. cantonensis infection. For (A-F), n = 4 uninfected mice (with or without L. johnsonii inoculation) and n = 6 infected mice (with or without L. johnsonii inoculation); for (G), n = 5 mice in each group except n = 3 uninfected mice with L. johnsonii inoculation. Data in (A and B) are presented as mean ± S.E.M and (D-F) as mean ± S.D; area under curve (AUC) data are presented as mean ± S.D. A.U., arbitrary unit. * p < 0.05; ** p < 0.01; *** p < 0.001; and **** p < 0.0001. Significance determined by one-way ANOVA (A-F) or Log-rank test (G), with p < 0.05 considered significant.
Fig 5.
Lactobacillus johnsonii inoculation alters immune pathways in mice with neuroangiostrongyliasis.
Transcriptomic analysis of the brain between A. cantonensis-infected mice with vehicle or Lactobacillus johnsonii inoculation. (A) Volcano plot of differentially expressed genes (DEGs) identified between vehicle-treated and L. johnsonii-treated, A. cantonensis-infected mice. Data are presented in log2 fold change of gene expression. Each dot represents a single gene. Grey dots represent non-significant genes, blue dots represent down-regulated DEGs, and red dots represent up-regulated DEGs. Significant DEGs are indicated as FC > 2 and FDR < 0.05. (B) Heatmap showing the 435 significant DEGs. Different color codes represent different clusters of genes with similar functions or participate in the same biological processes. Clustering was done with the Log10(FPKM + 1) values. (C) Gene ontology (GO) classifications based on significantly enriched GO terms. The x-axis shows the -log10(p-value) of each term. (D) GO classifications based on the number of DEGs in each GO category. The x-axis shows the number of DEGs. Only the top 30 most prominent GO categories are shown. Different color codes represent different GO categories. (E) KEGG enrichment histogram. The x-axis indicates the number of genes in each pathway term.
Fig 6.
Lactobacillus johnsonii inoculation alters immune response in mice with neuroangiostrongyliasis.
(A-E) Levels of IL-2 (A), IL-4 (B), IL-5 (C), IL-10 (D), and MCP-1 (E) in the mice brain. (F-J) Levels of IL-2 (E), IL-4 (F), IL-5 (G), IL-10 (H), and MCP-1 (I) in the mice serum. n = 4 uninfected mice (with or without L. johnsonii inoculation) and n = 6 infected mice (with or without L. johnsonii inoculation). Data are presented as mean ± S.D. * p < 0.05; ** p < 0.01; and *** p < 0.001. Significance as determined by one-way ANOVA, with p < 0.05 considered significant.
Fig 7.
Lactobacillus johnsonii inoculation did not affect metabolites in mice with Angiostrongylus cantonensis infection.
(A) Serum level of short-chain fatty acids. AA, acetic acid; PA, propionic acid; IsoBA, isobutyric acid; BA, butyric acid; 2M-BA, 2-methylbutyric acid; Iso-VA, isovaleric acid; VA, valeric acid. (B) Serum level of tryptophan metabolites. Trp, tryptophan; IAA, indole-3-acetic acid; IPA, indole-3-propionic acid; IBA, indole-3-butyric acid; ILA, indolelactic acid. (C) Serum level of hippuric acid. (D) Serum levels of cholic acid. Other bile acids, including ursodeoxycholic acid, chenodeoxycholic acid, deoxycholic acid, lithocholic acid, glycoursodeoxycholic acid, glycocholic acid, glycochenodeoxycholic acid, and glycodeoxycholic acid were not detected. n = 3 mice in each group. Values below the limit of detection are typed in as 0. Data are presented as mean ± S.E.M. * p < 0.05 and *** p < 0.001. Significance determined by one-way ANOVA, with p < 0.05 considered significant.