Additional file 1: of Mucosal and salivary microbiota associated with recurrent aphthous stomatitis

The comparison of the oral mucosa and saliva microbiota between the control subjects of current study and HMP subjects at the phylum level. (PDF 428 kb

Relative amounts of F. nucleatum and T. denticola in saliva.

<p>The relative proportion of <i>F. nucleatum</i> or <i>T. denticola</i> among the total bacteria in saliva was determined by real-time PCR of 16S rRNA. Bars indicate median values.</p

Antibody and T Cell Responses to <em>Fusobacterium nucleatum</em> and <em>Treponema denticola</em> in Health and Chronic Periodontitis

<div><p>The characteristics of the T cell response to the members of oral flora are poorly understood. We characterized the antibody and T cell responses to FadA and Td92, adhesins from <em>Fusobacterium nucleatum</em>, an oral commensal, and <em>Treponema denticola</em>, a periodontal pathogen, respectively. Peripheral blood and saliva were obtained from healthy individuals and patients with untreated chronic periodontitis (CP, n = 11 paris) and after successful treatment of the disease (n = 9). The levels of antigen-specific antibody were measured by ELISA. In plasma, IgG1 was the most abundant isotype of Ab for both Ags, followed by IgA and then IgG4. The levels of FadA-specific salivary IgA (sIgA) were higher than Td92-specific sIgA and the FadA-specific IgA levels observed in plasma. However, the periodontal health status of the individuals did not affect the levels of FadA- or Td92-specific antibody. Even healthy individuals contained FadA- and Td92-specific CD4⁺ T cells, as determined by the detection of intracytoplasmic CD154 after short-term <em>in vitro</em> stimulation of peripheral blood mononuclear cells (PBMCs) with the antigens. Patients with CP tended to possess increased numbers of FadA- and Td92-specific CD4⁺ T cells but reduced numbers of Td92-specific Foxp3⁺CD4⁺ Tregs than the healthy subjects. Both FadA and Td92 induced the production of IFNγ and IL-10 but inhibited the secretion of IL-4 by PBMCs. In conclusion, <em>F. nucleatum</em> induced Th3 (sIgA)- and Th1 (IFNγ and IgG1)-dominant immune responses, whereas <em>T. denticola</em> induced a Th1 (IFNγ and IgG1)-dominant response. This IFNγ-dominant cytokine response was impaired in CP patients, and the Td92-induced IFNγ levels were negatively associated with periodontal destruction in patients. These findings may provide new insights into the homeostatic interaction between the immune system and oral bacteria and the pathogenesis of periodontitis.</p> </div

FadA-, Td92-, and TT-specific Abs in saliva and plasma.

<p>The levels of FadA-, Td92-, and TT-specific-IgA in saliva (a) and the Ag-specific-IgA (b), -IgG1 (c), and -IgG4 (d) in plasma were determined by ELISA. Bars indicate median values. CP (B): patients with chronic periodontitis before treatment. CP (A): patients with chronic periodontitis after treatment.</p

Correlations of the examined immunologic parameters between the Ags.

†<p>Spearman’s rho calculated using all values from healthy individuals and patients with CP before and after therapy.</p

Cytokine production by PBMCs in response to FadA, Td92, or TT PBMCs from healthy subjects (n = 9), patients with CP before (B) treatment (n = 9), and patients with CP after (A) treatment (n = 7) were stimulated with medium alone (No Ag), 30 µg/ml FadA, Td92, or TT for 48 hours in the presence of purified anti-CD28 mAb and anti-CD49d mAb.

<p>(a–d) The amount of IFNγ, IL-4, IL-10, and IL-17 secreted into the supernatant was measured by ELISA. (e) The ratio of IFNγ/IL-4 was calculated and plotted. For the calculation of this ratio, half of the lowest detectable concentration (0.1 pg/ml) was arbitrarily assigned to IL-4 samples that had a zero value. Bars indicate median values. *, <i>P</i><0.05; **, <i>P</i><0.01 compared to No Ag.</p

Characteristics and clinical parameters of subjects.

†<p>Mann-Whitney U test between healthy and patients with CP before treatment.</p>‡<p>Mann-Whitney U test between healthy and patients with CP after treatment.</p>§<p>Mean ± standard deviation.</p

Bacteria composition analysis in human brain tissues at the phylum, genus, and species levels.

(A) Compositions of phyla from brain samples are presented. (B) Heatmap of top 20 genera. The numbers indicate relative abundance. (C) Relative abundance of the genus across distinct brain areas was examined and the significantly enriched genera, KE159538_g and Proteus, are presented. (D) Relative abundance of the genus between N and AD was examined and the significantly enriched in AD, Cloacibacterium, is presented. (E, F) Rela G tive abundance of the species was analyzed between N and AD. The relative abundance of (E) Cloacibacterium normanense and (F) Porphyromonas gingivalis across the samples are presented. The significance among the HT, F, and HC and between N and AD was examined by the Kruskal–Wallis test and Mann–Whitney U test, respectively.</p

Classification of species sources.

Alzheimer’s disease (AD) is a neurodegenerative disease accompanied by neuroimmune inflammation in the frontal cortex and hippocampus. Recently, the presence of bacteria in AD-affected brains has been documented, prompting speculation about their potential role in AD-associated neuroinflammation. However, the characterization of bacteriota in human brains affected by AD remains inconclusive. This study aimed to investigate potential associations between specific bacteria and AD pathology by examining brain tissues from AD-associated neurodegenerative regions (frontal cortex and hippocampus) and the non-AD-associated hypothalamus. Employing 16S rRNA gene sequencing, 30 postmortem brain tissue samples from four individuals with normal brain histology (N) and four AD patients were analyzed, along with three blank controls. A remarkably low biomass characterized the brain bacteriota, with their overall structures delineated primarily by brain regions rather than the presence of AD. While most analyzed parameters exhibited no significant distinction in the brain bacteriota between the N and AD groups, the unique detection of Cloacibacterium normanense in the AD-associated neurodegenerative regions stood out. Additionally, infection-associated bacteria, as opposed to periodontal pathogens, were notably enriched in AD brains. This study’s findings provide valuable insights into potential link between bacterial infection and neuroinflammation in AD.</div

Potential contaminants ratio from total reads in human brain tissues.

Total reads, non-specific amplicon/total reads, and contaminants/total reads in different brain areas are depicted. The significance among the HT, F, and HC was examined by the Kruskal–Wallis test. (TIF)</p