Table 1.
Primer sequences.
Table 2.
Figure 1.
Elevated inflammatory profiles in the multiple organs of TAX1BP1-KO mice.
Mitral valve tissues from either 8 or 16(-wk) TAX1BP1-KO mice or their wild-type littermates were collected by Arcturus XT LCM system and total RNAs were prepared by RNeasy mini kit (Qiagen). Each cDNA pool was generated from the individual RNA sample and gene expression profiles were evaluated using Whole Mouse Genome Microarray Kit (Agilent). A) Principal component analysis (PCA) by conditions was performed on R statistical package (Version 2.15.1) and represented as a scatterplots of whole gene expression profiles of 8- or 16-wk TAX1BP1-KO mice (8wKO #1- #3 or 16wKO #1-#3, surrounded by red circles) and their WT littermates (8wWT #1- #3 or 16wWT #1-#3, blue circles). The PCA plot showed that samples clustered based on their genetic backgrounds. Data represent n = 12. Component % variance; PC1 = 34.95%, PC2 = 19.48%. B) Heat map representation of differentially expressed genes in the mitral valves from either 8- or 16-wk TAX1BP1-KO mice or their WT littermates. 588 genes were differentially expressed in TAX1BP1-KO vs. WT littermates (P<0.03). Each column represents the expression profile of either the TAX1BP1-KO mice or WT littermates. Red and green colors indicate high and low expression levels, respectively, relative to the mean (see color bar). C) Volcano plot analysis of microarray revealed that 588 probes were significantly expressed more than 2-fold vs control. Red and green areas indicate significant increasing and decreasing changes in gene expression (p<0.03).
Table 3.
Gene symbol, gene description, fold change and p-value for all genes up-regulated by >20-fold in TAX1BP1-KO mice.
Table 4.
Gene symbol, gene description, fold change and p-value for all genes down-regulated by >5 fold in TAX1BP1-KO mice.
Figure 2.
Validation of genes and proteins identified their expression alteration in the mitral valves of TAX1BP1-KO mice.
RT-PCR validation of genes identified their expression alteration in the mitral valves of TAX1BP1-KO mice, A) SAA3 B) EFCAB2 respectively. Gray bar: TAX1BP1-KO, black bar: WT. Mitral valve specimens were prepared from 16-wk TAX1BP1-KO mice or their WT littermates and stained by anti-Saa3 antibody (C and D) or anti-I-κBα antibody respectively (E and F).
Figure 3.
Inflammatory properties in the multiple organs of TAX1BP1-KO mice.
The morphologic and functional alterations of the environments of liver (A and B) and skin (C and D) were also examined with HE-staining. Red and white triangles indicate accumulated lymphocytes and Councilman bodies respectively.
Figure 4.
Massive infiltration of inflammatory cells causes severe tissue lesion in the mitral valves of TAX1BP1-KO mice.
Electron microscopy examinations on the mitral valves of 8-, 16- and 60-wk TAX1BP1-KO mice (A: 8wKO, and C: 60wKO) and their WT littermates (B: 8wWT and D: 60wWT). See Figure S1 for details. Each panel was duplicated with colorized areas in specific cell types and abbreviated descriptions (Fig. 4A' to 4D'). Abbreviations, CL: Collagen layer; EC: Endothelial cell; ED: Edema; FB: Fibroblast; FC: Fibrocyte; GD: Granule deposition; MΦ: Macrophage; NP: Neutrophil; PC: Plasma cell; TC: T cell.
Figure 5.
Enhanced expression of inflammatory genes after the LPS-stimulation to TAX1BP1-KO mice.
200 μg of Salmonella typhimurium lipopolysaccharide (LPS) in 100 μl sterile pyrogen-free saline were injected into a right footpads of TAX1BP1-KO or WT littermate mice. At the time 2, 6, 12, 24 and 48 hour post-injection (PT), each group of mouse were euthanized and tissues including serum, lymphocytes and eyes were collected. A) LPS-triggered induction of Tax1bp1 in eye tissue was monitored. Ten μg of cell lysates from WT BL6 mice at 2, 6, 12, 24 and 48 hour PT of LPS were probed with anti-Tax1bp1, -I-κBα and -Tubulin antibodies. B, C) Total RNAs of eye tissues from at 6, 12 and 48 hour PT of LPS to TAX1BP1-KO or WT littermates and their untreated controls were prepared and the expressions of IL-6 and CXCL1 were quantitated with RT-PCR. D, E) Sera from at 6, 12 and 48 hour PT of LPS to TAX1BP1-KO or WT littermates and their untreated controls were collected and the amount of Il-6 and Cxcl1 were quantitated with multiplex ELISA system (BioRad). Gray bar: TAX1BP1-KO, black bar: WT littermate.
Figure 6.
Amelioration of inflammatory valvulitis and conduction disturbance after the antibiotics treatment on TAX1BP1-KO mice.
TAX1BP1-KO or WT littermate mice (male) were first raised with the normal diets and water for 4 weeks, and then, antibiotic treatment group (C, D, G and H, n = 5/group) provided ampicillin (1 g/L; Wako), vancomycin Hydrochloride (500 mg/L; Wako), neomycin trisulfate salt hydrate (1 g/L; Sigma-Aldrich), and metronidazole (1 g/L; Wako) in drinking water for 12 weeks based on a protocal of the commensal depletion (Rakoff-Nahoum S., Cell 2004). The non-antibiotics controls (A, B, E and F, n = 5/group) were equally raised and maintained except for antibiotics treatment. Each group of mice were anesthetized and sacrificed at the end of 16 weeks experimental period and histochemical representatives of each group were displayed with HE-staining (A to D) or anti-Saa3 immuno-staining (IS, E to H). I). Heart rhythms of 16-week-old TAX1BP1-KO treated with antibiotics over 12 weeks (male, n = 5/group) were monitored with telemetric electrocardiogram (12-lead ECG). J) The average values of PQ-intervals were compared with those of untreated TAX1BP1-KO mice and their WT littermates.
Figure 7.
Reduction of the Saa3 and Cxcl1 expression in the sera of TAX1BP1-KO mice after the antibiotics treatment.
ELISA quantitation of Saa3 (A) or Cxcl13 (B) of the sera on four groups were performed. Gray bar: TAX1BP1-KO mice, black bar: WT littermates (n = 5/group).
Figure 8.
Splenic hypertrophy of TAX1BP1-KO mice and its cancellation by antibiotics treatment.
Examinations on the spleen volume (A) and the area of cecum (B) were performed. The average values of spleen volumes (C) and cecum areas (D) were displayed (n = 5/group).
Figure 9.
Cancelation of valvulitis in the MyD88/TAX1BP1 double-KO mice.
The HE-staining (A, B) and immunostaining of Saa3 (C, D) and I-κBα (E, F) were compared between TAX1BP1-KO and MyD88/TAX1BP1-KO mice. ELISA quantification of Saa3 (I) and Cxcl13 on the sera of both genetic background.