Susceptibility to the development of experimental periodontal disease in the offspring of mice with a history of prenatal and perinatal probiotic supplementation

Author(s)

Pedro Henrique Felix Silva

• Document type: Doctoral Thesis
• Press: Ribeirão Preto.
• Institution: Universidade de São Paulo (USP). Faculdade de Odontologia de Ribeirão Preto (PCARP/BC)
• Defense date: 2024-01-15

Examining board members

Michel Reis Messora; Juliano Milanezi de Almeida; Joni Augusto Cirelli; Sergio Luiz de Souza Salvatore

• Advisor: Michel Reis Messora

Abstract

The main objective of this study was to analyze whether periodontitis, during pregnancy, associated or not with prenatal and perinatal supplementation with probiotic (PROB), affects the susceptibility of offspring to alveolar bone loss when challenged or not with Porphyromonas gingivalis w83 in life. adult. We used 64 pregnant mice divided into 4 groups (n=16): CM (animals without PD and not treated with probiotics); DPM (animals with PD and not treated with probiotics); CMP (animals without PD and treated with probiotics) and DPMP (animals with PD and treated with probiotics). The offspring conceived by each of these groups were divided into 2 subgroups: P1 (without PD induction; n=24) and P2 (with PD induction; n=24). After mating and confirmation of pregnancy, animals in the DPM and DPMP groups received gavages with 5x109 colony forming units (CFU)/mL of Porphyromonas gingivalis w83 for 37 days. In the offspring animals (P2 subgroups), PD was induced following the same protocol adopted for the DPM and DPMP groups, with gavages carried out for 21 days. Animals in the CMP and DPMP groups received 1 x 109 CFU/mL of Bifidobacterium animalis subsp. lactis HN019 administered in water for 56 days, starting 14 days before mating. On the 40th experimental day (19th day of pregnancy), 8 animals from each of the maternal groups CM, CMP, DPM and DPMP were euthanized to collect amniotic fluid and placental tissue. The remaining animals in the CM, CMP, DPM and DPMP groups were euthanized on the 63rd day. day of the experiment. Animals from subgroups P1 and P2 were euthanized 63 days after birth. The following were evaluated: 1) immunoinflammatory profile (IL-6, TNF-&alpha;, INF-&gamma;, IL-10, IL1&beta;, IL-8) present in periodontal tissues and amniotic fluid using immunoenzymatic assays (Luminex); 2) intestinal and oral microbial composition through metagenomic sequencing of the 16S gene; 3) immunofluorescent expression of DNA methyltransferase 3b (DNMT3b), acetylation of histones 3 and 4 (AcH3 and AcH4), e-cadherin, junctional adhesion molecule (JAM) and Toll-like receptors (TLR-2 and TLR-4) in intestinal, placental and periodontal tissues; 4) severity of inflammation present in the interproximal region of the 2nd. upper molar through histomorphometric analysis; 5) alveolar bone loss in the 2nd region. upper molar using x-ray microcomputed tomography (Micro-CT). The data obtained were subjected to statistical analysis (p<0.05). In amniotic fluid, on the 19th day of pregnancy, the DPM group demonstrated lower levels of IL-4 and IL-10, as well as higher levels of IL-1&beta;, IL-17, IL-8, INF-&gamma; and TNF-&alpha; when compared to the CM and CMP groups (p<0.05). The DPMP group had lower levels of IL-1&beta; when compared to the DPM group (p<0.05). In placental tissue, the DPM group showed lower expression of DNMT3b, e-cadherin and JAM, as well as higher expression of H3Ac, H4Ac TLR-2 and TLR-4 when compared to the CM, CMP and DPMP groups (p<0.05) . The DPMP group did not show significant differences in these markers when compared to the CM group (p>0.05). A lower expression of TLR-2 and TLR-4 was observed in the DPMP group when compared to the DPM group (p<0.05). At 63 experimental days, considering the maternal groups, it was observed that the DPMP group showed less alveolar and conjunctival bone loss when compared to the DPM group (p<0.05), as well as higher expressions of e-cadherin and JAM and lower levels of IL-1&beta;, IL-17, TNF-&alpha;, DNMT3b and H3Ac in periodontal tissues (p<0.05). In intestinal tissues, the DPMP group, when compared to the DPM group, showed greater villus height, lower expression of H3Ac and H4Ac and higher expressions of e-cadherin and JAM (p<0.05). In the P1 offspring groups, no significant differences were observed in bone volume and conjunctival attachment level between all experimental groups. However, the DPM-P1 group presented, in periodontal tissues, higher expression of DNMT3b, H3Ac and TLR-2, as well as lower expression of JAM when compared to the other P1 groups (p<0.05). In intestinal tissues, the DPM-P1 group showed higher expression of DNMT3b, H3Ac and TLR-2, as well as lower expression of JAM when compared to the CM-P1 and CMP-P1 groups (p<0.05). In the P2 offspring groups, the DPMP-P2 group presented greater bone volume and less loss of conjunctival attachment when compared to the DPM-P2 group (p<0.05). The DPMP-P2 group showed lower expression of DNMT3b and H3Ac in periodontal and intestinal tissues, as well as greater height of intestinal villi and greater expression of JAM in the small intestine when compared to the DPM-P2 group (p<0.05). The main results obtained in the analysis of the oral and intestinal microbiome were: i) Maternal groups - differences (p<0.05) were observed in the comparisons CM vs DPM (beta diversity - intestinal), CM vs CMP (beta diversity - oral and intestinal ) and DPM vs DPMP (beta - intestinal diversity); ii) P1 offspring groups - differences were observed in the comparisons DPM-P1 vs CM-P1 (beta - intestinal diversity), CM-P1 vs CMP-P1 (alpha and beta - intestinal diversity) and DPM-P1 vs DPMP-P1 ( alpha and beta diversity - intestinal); iii) P2 offspring groups - differences were observed in the comparisons CM-P2 and DPM-P2 (beta diversity - oral and intestinal), CM-P2 vs CMP-P2 (beta diversity - oral and intestinal) and DPM-P2 vs DPMP- P2 (beta diversity - intestinal). It can be concluded that i) the challenge with P. gingivalis w83 during pregnancy promoted microbiological, immunological, tissue barrier and epigenetic changes in the maternal and offspring intestinal and periodontal environments; ii) offspring from mothers challenged with Porphyromonas gingivalis w83 during pregnancy were more susceptible to loss of periodontal tissues when challenged in adulthood with Porphyromonas gingivalis w83; iii) prenatal and perinatal probiotic supplementation modulated microbiological, immunological, tissue barrier and epigenetic patterns in maternal and offspring periodontal and intestinal tissues, as well as reduced offspring susceptibility to periodontal destruction when challenged with Porphyromonas gingivalis w83 in adulthood. (AU)

• FAPESP's process: 19/12269-0 - Susceptibility to the development of experimental periodontal disease in the offspring of mice with a history of prenatal and perinatal probiotic supplementation
• Grantee: Pedro Henrique Felix Silva
• Support Opportunities: Scholarships in Brazil - Doctorate