Imbalances in the Oral Microbiota associated with Oral Mucosal Diseases

Transcription

1 2nd Microbiome R&D and Business Collaboration Congress: Asia Imbalances in the Oral Microbiota associated with Oral Mucosal Diseases Youngnim Choi, D.D.S., Ph.D.

2 C Huttenhower et al. Nature 486, (212) doi:1.138/nature11234 Characteristics of Oral microbiome 5 ~ 7 species

3 Plaque biofilm Actinomyces species V. parvula A. odontolyticus S. mutans C. gracillis S. mitis C. rectus S. constellatus S. oralis S. sanguinis F. periodonticum Streptococcus P. intermedia sp. E. nodatum P. nigrescens C. showae S. gordonii P. micros S. intermedius F. nuc. vincentii C. gingivalis F. nuc. nucleatum C. sputigena F. nuc. Polymorphum C. ochracea C. concisus A. actino. a E. corrodens A. actino. b S. noxia P. gingivalis T. forsythia T. denticola Early colonizer Bridging colonizer Late colonizer Adapted from Periodontology 2, 28: by Socransky and Haffajee

4 Oral lichen planus (OLP) A chronic mucocutaneous disease of unknown etiology White striation, erosive/ulcerative lesions Histopathologic features Liquefaction of basal cell layer of epithelia Band-like lymphocytic infiltration (mostly T cells) Lesions diminish but not eliminated T-cell mediated pathogenesis

5 The presence of bacteria in gingival tissue H&E Eubacterial probe Negative probe Healthy site Periodontitis Lesion J Periodontol 85: e312-e322, 214

6 Bacterial invasion (NBT-BCIP stain intensity / ROI) Bacterial invasion (NBT-BCIP stain intensity / ROI) Increased bacterial invasion into OLP tissues Con OLP Con (n = 1) OLP (n = 36) H&E Eubacterial probe Negative probe 1% 72.5% OR 23.7, P = Epithelia 1 1 Lamina propria Control r =.149 p =.371 OLP r =.563 p < Control OLP.1 Control OLP

7 Bacteria detected within T cells

8 Bacteria detected within T cells

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10 Correlations between the amount of bacteria and CD4 + /CD8 + T cells in the lamina propria CD8+ T cells (DAB stain intensity / ROI) Macrophage (DAB stain intensity / ROI) CD4+ T cells (DAB stain intensity / ROI) CD4 Con OLP 1 1 r =.46 p < CD Control OLP r =.379 p < Mac Control OLP r = p = Control OLP

11 Shanon index Chao1 index PCo2( 19.8%of total variation) Altered mucosal microbiota in OLP lesions a 6 P=.293 b 4 2 HC OLP 6 P= HC OLP HC OLP (n = 18) (n = 13) PCo1( 29.9%of total variation)

12 Relative abundance (%) Relative abundance (%) a OR.428 p=.35or.428 p=.35 b c d P4975_s HC 4 species/phylotypes HQ75798_s Streptococcus vestibularis HC OLP Streptococcaceae_uc_s OLP c OLP Fusobacterium nucleatum 1 Neisseria oralis Porphyromonas gingivalis Phylum Firmicutes Proteobacteria Actinobacteria Bacteroidetes Fusobacteria Spirochaetes TM7 Tenericutes ETC 38 species/phylotypes Streptococcus gordonii Leptotrichia wadei Genus Streptococcus Rothia Lautropia Prevotella Leptotrichia Capnocytophaga Porphyromonas Aggregatibacter Acinetobacter TM7_g Treponema Others ETC HC OLP Capnocytophaga gingivalis OLP OR 3E+9 p=.47 Streptococcus sanguinis OR.428 p=.35

13 Normalized TER (%) Normalized O.D. (%) Effect on oral epithelial barrier function TER Viability Vehicle S.s S.g C.g h 6h 12h 24h Vehicle S.s S.g C.g

14 Bacterial invasion into epithelial cells S. sanguinis S. gordonii C. gingivalis M1 M1 1% 24% 28% Mol Cells 3: , 21

15 Bacterial invasion into T cells 1 h infection CD4 CD8 CD14 S. sanguinis S. sanguinis S. sanguinis S. gordonii S. gordonii S. gordonii C. gingivalis C. gingivalis C. gingivalis

16 Bacterial invasion into T cells 1 h infection CD4 CD8 CD14 S. sanguinis S. sanguinis S. sanguinis S. gordonii S. gordonii S. gordonii C. gingivalis C. gingivalis C. gingivalis

17 Invaded cells (%) CFU (X 1 5 cell) CFU (X 1 3 cell) Bacterial invasion into T cells 1 h infection 1 h infection + 1 h or 24 h with gentamicin d 5 e h 24 h CD4 CD8 CD14 Ss Sg Cg CD4 CD8 CD14 Ss Sg Cg CD4 CD8 CD14 Ss Sg Cg

18 (Pg/ml) (Pg/ml) (ng/ml) (ng/ml) Chemokine induction CXCL8 (IL-8) HOK CD4 CD8 CD CXCL1 (IP-1) HOK CD4 CD8 CD14 Vehicle Ss Sg Cg CCL5 (RANTES) CCL3 (MIP-1α) HOK CD4 CD8 CD14 HOK CD4 CD8 CD14 Vehicle Ss Sg Cg

19 Proposed pathogenesis model for OLP Epithelial barrier dysfunction Dysbiosis of mucosal microbiota Liquefaction of basal cell layer Bacterial invasion of epithelia and lamina propria Recruit CD4 and CD8 T cells Survival of bacteria within host cells (?) Continuous bacterial invasion (?) Chronic inflammation Under revision in Scientific Reports

20 Recurrent aphthous stomatitis (RAS) One of the most common oral mucosal disorders Recurrent occurrence of single or multiple ulcers Extremely painful and heal more slowly than traumatic ulcers No cure to stop recurrence Suspected contributing factors Genetic predisposition Immunologic disturbance Viral and bacterial infection Vitamin deficiency stress

21 Shannon diversity index Chao1 PCo2 (14.5% of total variation) Mucosal and salivary microbiota in RAS A Saliva Mucosa B labial mucosa buccal mucosa tongue tip saliva Control RAS Control RAS Control RAS (n = 7) (n = 8) Control RAS (n = 18) (n = 18) PCo1 (37.5% of total variation) Control saliva RAS saliva Control mucosa RAS mucosa

22 Shannon diversity index Chao1 PCo2 (14.5% of total variation) Mucosal and salivary microbiota in RAS A Saliva Mucosa B labial mucosa buccal mucosa tongue tip saliva Control RAS Control RAS Control RAS (n = 7) (n = 8) Control RAS (n = 18) (n = 18) PCo1 (37.5% of total variation) Control saliva RAS saliva Control mucosa RAS mucosa

23 Shannon diversity index Chao1 PCo2 (14.5% of total variation) Mucosal and salivary microbiota in RAS A Saliva Mucosa B labial mucosa buccal mucosa tongue tip saliva Control RAS Control RAS Control RAS (n = 7) (n = 8) Control RAS (n = 18) (n = 18) PCo1 (37.5% of total variation) Control saliva RAS saliva Control mucosa RAS mucosa

24 Taxanomic composition

25 Proliferation (% to control) Viability (%) Species associated with RAS in the members of healthy core microbiota - 15 species/phylotypes (mucosa) - 1 species/phylotypes (saliva) in rare species - 7 species/phylotypes (mucosa) - 11 species/phylotypes (saliva) MOI1 MOI5 MOI1 Con Aj Ss Pg Control A. johnsonii S. salivarius P. gingivalis S. salivarius OR.734 per 1% increase, CI 95% , P=.2 A. johnsonii OR 211 per 1% increase, CI 95% E4, P= Conditionally accepted to BMC microbiology Con Aj Ss Pg Control A. johnsonii S. salivarius P. gingivalis

26 OD value OD value OD value OD value OD value OD value Potential new therapeutics for RAS Accinetobacter johnsonii Antibiotic (mg/ml) Streptococcus salivarius (1) Antibiotic Gentamicin (mg/ml) μg / ml ) Streptococcus salivarius (2) Antibiotic Gentamicin ( μg (mg/ml) / ml ) Streptcoccus gordonii Antibiotic (mg/ml) Neisseria oralis o Gentamicin ( μg / ml ) Antibiotic (mg/ml) Rothia mucilaginosa Antibiotic Gentamicin ((mg/ml) μg / ml )

27 Acknowledgements Funding NRF Korea R1A1A K2A2A4619 Ministry of Health & Welfare, Korea - HI13C16 Choi s Lab Yun Sik Choi Jehan Alam Yun-Ji Kim Keum Jin Baek Areum Lee Dong IL Kwon Collaborators Prof. Hee Kyung Park, Dept. of Oral Medicine, SNUDH Prof. Hei Jung Yoon, Dept. of Oral Pathology, SNUDH

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31 Correlations between the amount of bacteria and CD3 + T cells in the lamina propria

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