Additional file 1 of IL-1β and TNF-α play an important role in modulating the risk of periodontitis and Alzheimer’s disease
Creators: name: Wang, Rachel Pei-Hsuan
name: Huang, Jianpan
name: Chan, Kannie Wai Yan
name: Leung, Wai Keung
name: Goto, Tetsuya
name: Ho, Yuen-Shan
name: Chang, Raymond Chuen-Chung
Publisher: name: figshare
Year: 2024
Subjects: subject: Medicine
subject: Microbiology
subject: FOS: Biological sciences
subject: Neuroscience
subject: Immunology
subject: FOS: Clinical medicine
subject: Biological Sciences not elsewhere classified
subject: Mental Health
subject: Infectious Diseases
subject: FOS: Health sciences

description: Additional file 1: Table S1. List of primer sequences used in this study. Fig. S1. Assessment of bone mineral density from the right mandibular jaws of C57BL/6J and 3×Tg-AD mice at 3 months, 8 months, and 15-16 months of age. (a) A representative two-dimensional sagittal image of a right mandibular jaw depicting the region of interest (highlighted in yellow) for the quantitative measurement of bone mineral density. (b) Quantitative analysis of bone mineral density. Data are presented as mean ± SEM (C57BL/6J mice: n = 5/age group, 3×Tg-AD mice: n = 9-11/age group). Statistical analysis was done using one-way ANOVA with Tukey’s post-hoc test. ***p &lt; 0.001. Fig. S2. Periodontitis induced by the injection of heat-killed Porphyromonas gingivalis resulted in elevated CRP levels in the plasma. (a) Plasma CRP concentration in nTg mice from each group. (b) Plasma CRP concentration in 3×Tg-AD mice from each group. Data are presented as mean ± SEM (n = 5-7). Statistical analysis was done using unpaired t test. *p &lt; 0.05. Fig. S3. Periodontitis induced by the injection of heat-killed Porphyromonas gingivalis resulted in increased phosphorylated tau proteins immunoreactivity in the brains of non-transgenic (nTg) mice. (a) Representative images of immunofluorescence staining for phosphorylated tau Ser396 (green) and DAPI (blue) in the cortex, sub-regions of the hippocampus, and thalamus of nTg mice in each group. (b) Representative images of immunofluorescence staining for phosphorylated tau Ser404 (green) and DAPI (blue) in the cortex, sub-regions of the hippocampus, and thalamus of nTg mice in each group. (c, d) Quantification of (c) phosphorylated tau Ser396 and (d) phosphorylated tau Ser404 immunofluorescence intensity in the cortex, sub-regions of the hippocampus, and thalamus of nTg mice in each group. Data are presented as mean ± SEM (n = 6). Statistical analysis was done using unpaired t test. *p &lt; 0.05, **p &lt; 0.01. Fig. S4. Non-transgenic (nTg) mice body weight expressed as a percent change from baseline during the experimental period of heat-killed Porphyromonas gingivalis injections. Data are presented as mean ± SEM (n = 7-8). Statistical analysis was done using two-way ANOVA with Bonferroni’s post-hoc test. Fig. S5. Periodontitis induced by the injection of heat-killed Pg resulted in impairment of long-term memory in non-transgenic (nTg) mice. (a, b) Open field test for the assessment of general exploration and anxiety behavior. (a) Total distance traveled and (b) the percentage of time spent in center were examined. (c, d) Spontaneous Y maze test for the assessment of short-term spatial working memory. (c) Percentage spontaneous alternation and (d) the number of total arm entries were examined. (e) Puzzle box test for the assessment of problem-solving abilities and executive functions. Data are presented as mean ± SEM (n = 12-14). Statistical analysis was done using unpaired t test. **p &lt; 0.01. Fig. S6. Non-transgenic (nTg) mice body weight expressed as a percent change from baseline during transgenic (nTg) mice body weight expressed as a percent change from baseline during transgenic (nTg) mice body weight expressed as a percent change from baseline during the experimental period of mixed cytokines injections. Fig. S7. Injections of mixed cytokines resulted in hyperactivity and impairment of short-term memory in non-transgenic (nTg) mice. (a, b) Open field test for the assessment of general exploration and anxiety behavior. (a) Total distance traveled and (b) the percentage of time spent in center were examined. (c, d) Spontaneous Y maze test for the assessment of short-term spatial working memory. (c) Percentage spontaneous alternation and (d) the number of total arm entries were examined. (e) Puzzle box test for the assessment of problem-solving abilities and executive functions. Data are presented as mean ± SEM (n = 7). Statistical analysis was done using unpaired t test. *p &lt; 0.05, **p &lt; 0.01. Fig. S8. Total bacterial load in the gums of 3×Tg-AD mice measured by 16S rRNA real-time PCR at the end of the experimental period. Tissue DNA spiked with 100 ng of Pg DNA was included as positive controls. Data are presented as mean ± SEM (n = 12). Statistical analysis was done using one-way ANOVA with Tukey’s post-hoc test. *p &lt; 0.05. Fig. S9. Periodontitis induced by injection of heat-killed Pg as well as ligature placement resulted in increased 6E10 immunoreactivity in the brains of 3×Tg-AD mice. (a-b) Representative images of immunofluorescence staining for 6E10 (red) and DAPI (blue) in the cortex and sub-regions of hippocampus of 3×Tg-AD mice with and without (a) Pg injections and (b) ligature placement. (c-d) Quantification of 6E10 immunofluorescence intensity in the cortex and sub-regions of the hippocampus of 3×Tg-AD mice with and without (c) Pg injections and (d) ligature placement. Data are presented as mean ± SEM (n = 6-7). Statistical analysis was done using unpaired t test. *p &lt; 0.05, **p &lt; 0.01. Fig. S10. 3×Tg 3×Tg-AD mice body weight expressed as a percent change from baseline during the experimental period of heat-killed Pg injections. Fig. S11. Periodontitis induced by the injection of heat-killed Porphyromonas gingivalis resulted in impairment of both short- and long-term memory in 3×Tg-AD mice. (a, b) Open field test for the assessment of general exploration and anxiety behavior. (a) Total distance traveled and (b) the percentage of time spent in center were examined. Data are presented as mean ± SEM (n = 15-16). (c, d) Spontaneous Y maze test for the assessment of short-term spatial working memory. (c) Percentage spontaneous alternation and (d) the number of total arm entries were examined. Data are presented as mean ± SEM (n = 11-12). (e) Puzzle box test for the assessment of problem-solving abilities and executive functions. Data are presented as mean ± SEM (n = 12-13). Statistical analysis was done using unpaired t test. *p &lt; 0.05, **p &lt; 0.01. Fig. S12. 3×Tg-AD mice body weight expressed as a percent change from baseline during the experimental period of ligature placement. Data are presented as mean ± SEM (n = 12). Statistical analysis was done using two-way ANOVA with Bonferroni’s post-hoc test. **p &lt; 0.01. Fig. S13. Ligature-induced periodontitis resulted in impairment of both short- and long-term memory in 3×Tg-AD mice. (a, b) Open field test for the assessment of general exploration and anxiety behavior. (a) Total distance traveled and (b) the percentage of time spent in center were examined. Data are presented as mean ± SEM (n = 17-19). (c, d) Spontaneous Y maze test for the assessment of short-term spatial working memory. (c) Percentage spontaneous alternation and (d) the number of total arm entries were examined. Data are presented as mean ± SEM (n = 19). Data are presented as mean ± SEM (n = 18-19). Statistical analysis was done using unpaired t test. **p &lt; 0.01. Fig. S14. Injections of mixed cytokines resulted in decreased TH fluorescence intensity and increased Iba-1 intensity in the locus coeruleus of 3×Tg-AD mice. (a) Representative images of immunofluorescence staining for TH (green) and Iba-1 (red) in the locus coeruleus of 3×Tg-AD mice with and without mixed cytokines injection. (b) Representative images of immunofluorescence staining for TH (green) and GFAP (red) in the locus coeruleus of 3×Tg-AD mice with and without mixed cytokines injection. (c) Quantification of TH+ cells/field, TH, Iba-1, and GFAP immunofluorescence intensity in the locus coeruleus of 3×Tg-AD mice without and without mixed cytokines injection. Data are presented as mean ± SEM (n = 5). Statistical analysis was done using unpaired t test. *p &lt; 0.05, **p &lt; 0.01. Fig. S15. Total bacterial load in the whole brain homogenates of 3×TgT-AD mice at 7-day and 5-weeks postweeks post ligation measured by 16S rRNA real-time PCR.