RNA interference-mediated silencing of Atp6i prevents both periapical bone erosion and inflammation in the mouse model of endodontic disease

Abstract

Dental caries is one of the most prevalent infectious diseases in the United States, affecting approximately 80% of children and the majority of adults. Dental caries may lead to endodontic disease, where the bacterial infection progresses to the root canal system of the tooth, leading to periapical inflammation, bone erosion, severe pain, and tooth loss. Periapical inflammation may also exacerbate inflammation in other parts of the body. Although conventional clinical therapies for this disease are successful in approximately 80% of cases, there is still an urgent need for increased efficacy of treatment. In this study, we applied a novel gene-therapeutic approach using recombinant adeno-associated virus (AAV)-mediated Atp6i RNA interference (RNAi) knockdown of Atp6i/TIRC7 gene expression to simultaneously target periapical bone resorption and periapical inflammation. We found that Atp6i inhibition impaired osteoclast function in vitro and in vivo and decreased the number of T cells in the periapical lesion. Notably, AAV-mediated Atp6i/TIRC7 knockdown gene therapy reduced bacterial infection-stimulated bone resorption by 80% in the mouse model of endodontic disease. Importantly, Atp6i(+/-) mice with haploinsufficiency of Atp6i exhibited protection similar to that in mice with bacterial infection-stimulated bone erosion and periapical inflammation, which confirms the potential therapeutic effect of AAV-small hairpin RNA (shRNA)-Atp6i/TIRC7. Our results demonstrate that AAV-mediated Atp6i/TIRC7 knockdown in periapical tissues can inhibit endodontic disease development, bone resorption, and inflammation, indicating for the first time that this potential gene therapy may significantly improve the health of those who suffer from endodontic disease.

Fig 1

AAV-sh-Atp6i simultaneously targets Atp6i and TIRC7 mRNA and efficiently knocks down the expression of Atp6i. (A) Diagram of loci illustrating Atp6i and TIRC7 zones of homology and shRNA specific for Atp6i/TIRC7 mRNA. (B) MBM stimulated with M-CSF/RANKL for 3 days and transduced with AAV-sh-Luc-YFP or AAV-sh-Atp6i. Fluorescence indicates effective transduction of preosteoclasts and osteoclasts (white arrows). (C) Western blot of Atp6i expression in MBM stimulated with M-CSF/RANKL for 3 days and transduced with AAV-sh-Luc-YFP or AAV-sh-Atp6i or left untreated (mock). Quantification of Western blot analysis demonstrates that AAV-sh-Atp6i-treated osteoclasts have significantly reduced expression of Atp6i. (D) Western blot showing that the Atp6i expression level was significantly decreased in Atp6i^+/− mice osteoclasts compared to Atp6i^+/+ mice. *, P < 0.01.

Fig 2

Depletion of Atp6i impaired extracellular acidification by osteoclasts and osteoclast-mediated bone resorption. (A and B) Acridine orange staining of osteoclasts, including cells without fusion (<3 nuclei). Both osteoclasts transduced with AAV-sh-Atp6i and osteoclasts from Atp6i^−/− mice show a lack of extracellular acidification (orange) compared to osteoclasts from the AAV-sh-Luc-YFP treatment group or Atp6i^+/+ mice. (C and D) Resorption lacunae were visualized by wheat germ agglutinin (WGA) (C) and scanning electron microscopy (SEM) (D). Depletion of Atp6i totally blocked osteoclast-mediated bone resorption. (E) Quantification of resorption pits on bone slices. ***, P < 0.005.

Fig 3

Infection induced periapical inflammation and bone resorption, which is reduced by AAV-sh-Atp6i treatment. (A) Fluorescent eGFP expression by AAV-infected cells in groups treated with PBS or AAV-sh-Atp6i. (B and C) Goldner's trichrome staining reveals that AAV-sh-Atp6i treatment (C), compared to AAV-sh-Luc-YFP treatment (B), protects infected mice from bone resorption in the periapical region. (D) Quantification of the ratio of bone volume (BV) to total volume (TV) of boxed areas in panels B and C. (E) TRAP staining and immunofluorescence staining for Atp6i (red) of sections from infected mice treated with AAV-sh-Luc-YFP or AAV-sh-Atp6i. (F) qPCR of Atp6i in uninfected mice (normal), infected mice treated with AAV-sh-Luc-YFP, and infected mice treated with AAV-sh-Atp6i. Hprt was used as an endogenous control. P, pulp. **, P < 0.01; ***, P < 0.005.

Fig 4

Atp6i depletion reduced infection-stimulated periapical bone resorption. (A) X-ray imaging of the crown and distal root of the mandibular first molar and patent apical foramen from WT BALB/cJ mice that did not receive polymicrobial infection or any form of treatment (normal), infected mice treated with AAV-sh-Luc-YFP (negative control), and infected mice treated with AAV-sh-Atp6i. (B) Micro-computed tomography (micro-CT) analysis of the mandibular first molar in WT BALB/cJ mice that did not receive polymicrobial infection or any form of treatment (normal), infected mice treated with AAV-sh-Luc-YFP, and infected mice treated with AAV-sh-Atp6i. Red arrows indicate severe periapical bone loss. (C) Quantification of the ratio of bone volume to total volume measured for periapical lesions in panel B. (D) Micro-CT analysis of the mandibular first molar in infected Atp6i^+/+ and Atp6i^+/− mice, and uninfected mice (normal). (E) Quantification of the bone volume/total volume ratio for periapical lesions in panel D. *, P < 0.05; **, P < 0.01. The top rows in panels B and D are typical 2D micro-computed tomography (micro-CT) images, while the bottom rows are 3D reconstitutions. Bar, 1 mm.

Fig 5

AAV-mediated Atp6i knockdown decreased the number of T cells in periapical lesions. (A to C) H&E staining of the periapical root sections from uninfected mice (normal) (A) or infected mice treated with AAV-sh-Luc-YFP (B) and AAV-sh-Atp6i (C). (D to R) Immunofluorescence staining of CD3-positive (red) T cells in periapical lesions in uninfected mice (normal) (G to I), infected mice treated with AAV-sh-Luc-YFP (J to L) or AAV-sh-Atp6i (M to O), or untreated infected mice (disease control) (P to R). Nuclei were labeled using the DNA stain DAPI (blue). (D to F) Negative control group (without primary antibody). AF, apical foramen; PDL, periodontal ligament.

Fig 6

AAV-sh-Atp6i reduced the expression of osteoclast marker genes and cytokines in periapical lesions. (A) qPCR of osteoclast marker genes (i.e., cathepsin K and Acp5), genes important for osteoclast differentiation (i.e., RANKL), a gene common to macrophages and osteoclasts (i.e., CD115), and cytokines (i.e., IL-6, IL-1α, IL-1β, and IL-17α) in the periapical lesion from uninfected mice (normal) or infected mice treated with AAV-sh-Luc-YFP or with AAV-sh-Atp6i. Hprt was used as an endogenous control. (B) IL-1α, IL-6, and IL-17α levels in the periapical lesion as detected by ELISA. NS, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.005.