Fig 1.
Composition and structure of the stool microbial community from control and levofloxacin-treated mice (n = 6 mice per group) at the a. Phylum and b. Class level; c. Richness (Chao-1) and alpha diversity (Shannon and Simpson) indices, Statistical test Mann-Whitney (p<0.05); d. Beta diversity at the OTU level is represented spatially by a principal coordinate diagram (PCoA) using Bray Curtis dissimilarity distances, Statistical test PERMANOVA and ANOSIM (p<0.05).
Fig 2.
a. Venn diagram indicating the significantly different taxa in the stool microbiota of control and levofloxacin-treated mice (p<0.05) (n = 6 mice per group). Mann-Whitney/Kruskal-Wallis (M-W/K-W), Zero-inflated Gaussian fit (ZiG fit), and EdgeR analysis methods. b. Each box indicates the taxon’s abundance represented with a logarithmic transformation (Log-transformed Count). Full name obtained from Silva database †: Lachnospiraceae_NK4A136_group, *: Prevotellaceae_UCG_001, §: Clostridia_vadinBB60_group, ¥: Rikenellaceae_RC9_gut_group. The last available classification level was used when no genus-level identification was obtained. The letter before the name indicates the taxonomic level reached in identification (i.e., c: class, o: order, f: family, g: genus). c. Linear discriminant analysis effect size (LEfSe) of the metabolic pathways obtained in PICRUSt between control and levofloxacin-treated mice (LDA> 2; p<0.05). The annotation of the metabolic pathways was performed in MetaCyC, available at https://biocyc.org.
Fig 3.
Effects of levofloxacin on the intestines of DBA/1 mice.
A. Histological findings in the ileocolic junction by H&E staining (n = 6 mice per group). Inflammatory cell infiltration was scored according to severity and extent. Epithelial changes were scored by quantifying hyperplasia, goblet cell loss, crypt abscesses, erosion, and ulceration. The mucosal architecture was evaluated according to granulation tissue, irregular crypts, crypt loss, and villous blunting. The student’s t-test was used to determine statistically significant differences when p<0.05. The images were acquired with 10X and 40X amplification using a digital camera (AmScope MU1803) and an optical microscope (AxioStar Plus, Carl Zeiss). II: Inflammatory infiltrate; HP: Hyperplasia; E: Epithelial erosion; U: Epithelial ulceration; GC: Goblet cell; GCL: Goblet cell loss; CA: Crypt abscesses; IC: Irregular crypts; CL: Crypt loss; VB: Villous blunting. B. Protein-protein interactions network of differentially expressed genes (DEG). The lists of the DEG (Z-score ≥ 1.5 SD) were analyzed on the STRING and Cytoscape platforms. The primary clusters of sub-networks were obtained using the Molecular Complex Detection (MCODE) complement (cutoff = 0.4). The network was obtained in STRING with the first three clusters for up- and down-expressed genes. The KEGG signaling pathways relevant to arthritis were selected (Table) and marked with different colors. Nodes with the most significant interaction in the pathways of interest are amplified. C. Expression of TNF-α, IL-23a, IL17-a, and JAK-3 in the ileocolic junction. Immunodetection was done using streptavidin-peroxidase conjugated and DAB as the chromogen. The images were acquired with 10X amplification using a digital camera (AmScope MU1803) coupled with an optical microscope (AxioStar Plus, Carl Zeiss); the scale bar corresponds to 100 μM. The quantification was carried out with the ImageJ program and the IHC toolbox in at least 20 microscopic field images from each study subject. The DAB color was extracted from each image, and the maximum and mean gray values were obtained. Each image’s optical density (OD) was obtained with log10 (maximum gray value / mean gray value). The OD means, and standard deviations were calculated and graphed by the study group. The student’s t-test was used to determine statistically significant differences when p<0.05. C: Control. L: Levofloxacin. FDR: False discovery rate.
Table 1.
Inflammatory signaling pathways differentially expressed by levofloxacin in the intestines, hind paws, and spines of DBA/1 mice with spontaneous arthritis.
Table 2.
Metabolic signaling pathways differentially expressed by levofloxacin in the intestines, hind paws, and spines of DBA/1 mice with spontaneous arthritis.
Fig 4.
Effects of levofloxacin on the hind paws of DBA/1 mice.
A. Histological findings in the hind paws by H&E staining. Inflammatory infiltrate (II), synovial membrane (SM) hyperplasia (SH), cartilage (C) damage (CD), enthesitis (E), cartilage neoformation (CN), bone (B) neoformation (BN), and ankylosis was evaluated using the semiquantitative scale: 0, absent; 1, mild; 2, moderate; or 3, severe. The mean score and standard deviation were calculated and graphed for each group (n = 6 mice per group). The student’s t-test was used to determine statistically significant differences when p<0.05. The images were acquired with 4X amplification using a digital camera (AmScope MU1803) and an optical microscope (AxioStar Plus, Carl Zeiss). B. Protein-protein interactions network of DEG. The lists of the differentially expressed genes (Z-score ≥ 1.5 SD) were analyzed on the STRING and Cytoscape platforms. The primary clusters of sub-networks were obtained using the Molecular Complex Detection (MCODE) complement (cutoff = 0.4). The network was obtained in STRING with the first three clusters of up- and down-expressed genes. The KEGG signaling pathways relevant to arthritis were selected (Table) and marked with different colors. Nodes with the most significant interaction in the pathways of interest are amplified. C. Expression of TNF-α, IL-23a, IL17-a, and JAK-3 in synovial membranes, enthesis, and neoformation areas of hind paws. Immunodetection was done using streptavidin-peroxidase conjugated and DAB as the chromogen. The images were acquired with 10X and 40X amplification using a digital camera (AmScope MU1803) and an optical microscope (AxioStar Plus, Carl Zeiss). The quantification was carried out with the ImageJ program and the IHC toolbox in at least 20 microscopic field images from each study subject. The DAB color was extracted from each image, and the maximum and mean gray values were obtained. Each image’s optical density (OD) was obtained with log10 (maximum gray value / mean gray value). The OD means, and standard deviations were calculated and graphed by the study group. The student’s t-test was used to determine statistically significant differences when p<0.05. C: Control. L: Levofloxacin. FDR: False discovery rate.
Fig 5.
Effects of levofloxacin on the spines of DBA/1 mice.
A. Protein-protein interactions network of differentially expressed genes. The lists of the differentially expressed genes (Z-score ≥ 1.5 SD) were analyzed on the STRING and Cytoscape platforms. The primary clusters of sub-networks were obtained using the Molecular Complex Detection (MCODE) complement (cutoff = 0.4). The network was obtained in STRING with the first three clusters of up- and down-expressed genes. The KEGG signaling pathways relevant to arthritis were selected (Table) and marked with different colors. Nodes with the most significant interaction in the pathways of interest are amplified. B. Expression of TNF-α, IL-23a, IL17-a, and JAK-3 in the spine. Immunodetection was done using streptavidin-peroxidase conjugated and DAB as the chromogen. The images were acquired with 10X and 40X amplification using a digital camera (AmScope MU1803) and an optical microscope (AxioStar Plus, Carl Zeiss). The quantification was carried out with the ImageJ program and the IHC toolbox in at least 20 microscopic field images from each study subject. The DAB color was extracted from each image, and the maximum and mean gray values were obtained. Each image’s optical density (OD) was obtained with log10 (maximum gray value / mean gray value). The OD means, and standard deviations were calculated and graphed by the study group. The student’s t-test was used to determine statistically significant differences when p<0.05. C: Control. L: Levofloxacin. FDR: False discovery rate. C. Effect of levofloxacin on serum cytokines levels in DBA/1 mice. TNF-α, IFNγ, IL-6, IL-10, and monocyte chemoattractant protein-1 (MCP-1) concentrations (pg/ml) were determined by flow cytometry in the serum of control and levofloxacin-treated mice. Bars show each cytokine’s means and standard deviations in the study groups. The student’s t-test was used to determine statistically significant differences when p<0.05.
Table 3.
Inflammatory cytokines differentially expressed by levofloxacin in the intestine, hind paws, and spine of DBA/1 mice.