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. 2014 Dec;6(4):219-26.
doi: 10.1038/ijos.2014.46. Epub 2014 Aug 8.

Oral microbiota and host innate immune response in bisphosphonate-related osteonecrosis of the jaw

Smruti Pushalkar  1 , Xin Li  1 , Zoya Kurago  2 , Lalitha V Ramanathapuram  2 , Satoko Matsumura  1 , Kenneth E Fleisher  3 , Robert Glickman  3 , Wenbo Yan  4 , Yihong Li  1 , Deepak Saxena  1
Affiliations

Oral microbiota and host innate immune response in bisphosphonate-related osteonecrosis of the jaw

Smruti Pushalkar et al. Int J Oral Sci. 2014 Dec.

Abstract

Bacterial biofilms have emerged as potential critical triggers in the pathogenesis of bisphosphonate (BP)-related osteonecrosis of the jaw (ONJ) or BRONJ. BRONJ lesions have shown to be heavily colonized by oral bacteria, most of these difficult to cultivate and presents many clinical challenges. The purpose of this study was to characterize the bacterial diversity in BRONJ lesions and to determine host immune response. We examined tissue specimens from three cohorts (n=30); patients with periodontal disease without a history of BP therapy (Control, n=10), patients with periodontal disease having history of BP therapy but without ONJ (BP, n=5) and patients with BRONJ (BRONJ, n=15). Denaturing gradient gel electrophoresis of polymerase chain reaction (PCR)-amplified 16S rRNA gene fragments revealed less bacterial diversity in BRONJ than BP and Control cohorts. Sequence analysis detected six phyla with predominant affiliation to Firmicutes in BRONJ (71.6%), BP (70.3%) and Control (59.1%). Significant differences (P<0.05) in genera were observed, between Control/BP, Control/BRONJ and BP/BRONJ cohorts. Enzyme-linked immunosorbent assay (ELISA) results indicated that the levels of myeloperoxidase were significantly lower, whereas interleukin-6 and tumor necrosis factor-alpha levels were moderately elevated in BRONJ patients as compared to Controls. PCR array showed significant changes in BRONJ patients with downregulation of host genes, such as nucleotide-binding oligomerization domain containing protein 2, and cathepsin G, the key modulators for antibacterial response and upregulation of secretory leukocyte protease inhibitor, proteinase 3 and conserved helix-loop-helix ubiquitous kinase. The results suggest that colonization of unique bacterial communities coupled with deficient innate immune response is likely to impact the pathogenesis of ONJ.

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Figures

Figure 1
Figure 1
Bacterial diversity in the tissues of Control, BP and BRONJ cohorts. (a) Band intensity profiles of DGGE gels. Marker I and II: DGGE reference markers corresponding to 16S rRNA gene fragments of the given bacterial species [Marker I: 1. F. nucleatum subsp. vincenti (ATCC 49256); 2. F. nucleatum subsp. nucleatum (ATCC 25586); 3. S. sanguinis (ATCC 10556); 4. S. oralis (ATCC 35037); 5. S. salivarus (ATCC 7073); 6. S. mutans (UA 159); 7. L. paracasei (ATCC 25598); 8. A. odontolyticus (ATCC 17929); 9. A naeslundii (ATCC 12104). Marker II: 1. F. nucleatum subsp. vincenti (ATCC 49256); 2. F. nucleatum subsp. nucleatum (ATCC 25586); 3. B. forsythus (ATCC 43037); 4. S. sanguinis (ATCC 10556); 5. S. oralis (ATCC 35037); 6. V. parvula (ATCC 17745); 7. P. intermedia (ATCC 25611); 8. A. actinomycemcomitans (ATCC 43717); 9. P. gingivalis (ATCC 33277); 10. A. odontolyticus (ATCC 17929); 11. A. naeslundii (ATCC 12104)]. (b) Cluster analysis by Dice coefficient of the bacterial fingerprints. BP, bisphosphonate; BRONJ, bisphosphonate-related osteonecrosis of the jaw; DGGE, denaturing gradient gel electrophoresis.
Figure 2
Figure 2
Taxonomic distribution of microbiota in the tissues of Control, BP and BRONJ cohorts. (a) Phylum level [significant differences were observed in relative distribution (%) of phyla between Control/BRONJ cohorts (P<0.05, Chi-square test)] (b) genus level [significant differences were observed in relative distribution (%) of genera between Control/BP, BP/BRONJ and Control/BRONJ cohorts (P<0.05, Chi-square test)] (c) frequency of gram-positive and gram-negative bacteria. BP, bisphosphonate; BRONJ, bisphosphonate-related osteonecrosis of the jaw.
Figure 3
Figure 3
Expression levels of MPO, IL-6 and TNF-alpha, in BRONJ and Control cohorts by ELISA. BRONJ, bisphosphonate-related osteonecrosis of the jaw; ELISA, enzyme-linked immunosorbent assay; IL, interleukin; MPO, myeloperoxidase; TNF, tumor necrosis factor.
Figure 4
Figure 4
Regulatory profiles of different genes in the tissues of BRONJ and Control patients generated by PCR array. BRONJ, bisphosphonate-related osteonecrosis of the jaw; CD, cluster of differentiation; CHUK, conserved helix-loop-helix ubiquitous kinase; CTSG, cathepsin G; IL, interleukin; MPO, myeloperoxidase; NOD, nucleotide-binding oligomerization domain; PCR, polymerase chain reaction; PRTN, proteinase; SLPI, secretory leukocytes protease inhibitor; TNF, tumor-necrosis factor.
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