Interleukin-1 and tumor necrosis factor receptor signaling is not required for bacteria-induced osteoclastogenesis and bone loss but is essential for protecting the host from a mixed anaerobic infection

Abstract

Bacterial infection causes significant morbidity, mediated in part by the up-regulation of inflammatory cytokines. Cytokine induction is thought to stimulate osteolysis in conditions such as periodontal disease and otitis media. To establish the relative importance of interleukin-1 (IL-1) and tumor necrosis factor (TNF) in mediating the response to a mixed anaerobic infection, we used an in vivo model in which the dental pulp was inoculated with six anaerobic pathogens, in mice with functional deletions of receptors to IL-1 (IL-1RI(-/-)), TNF (TNFRp55(-/-)-p75(-/-)), or both (TNFRp55(-/-)-IL-1RI(-/-)). Polymorphonuclear and mononuclear phagocyte recruitment occurred to the greatest extent in TNFRp55(-/-)-IL-1RI(-/-) mice, and to a lesser extent in IL-1RI(-/-) or TNFRp55(-/-)-p75(-/-) mice, and the least in wild-type mice, demonstrating that recruitment of these phagocytes is not dependent on IL-1 or TNF receptor signaling. A similar pattern was observed for bacterial penetration into host tissue. Because it had recently been reported that TNF played a critical role in mediating lipopolysaccharide-induced bone loss, we anticipated that mice with targeted deletions of TNFRp55(-/-) would have reduced osteoclastogenesis. Surprisingly, osteolytic lesion formation was greatest in animals lacking TNF and/or IL-1 receptors. These results indicate that IL-1 or TNF receptor signaling is not required for bacteria-induced osteoclastogenesis and bone loss, but does play a critical role in protecting the host against mixed anaerobic infections.

Figure 1.

Tissue necrosis is much more rapid in the absence of IL-1 and TNF activity. The dental pulp was surgically exposed and inoculated with 10 of five obligate anaerobes (Streptococcus intermedius, Peptostreptococcus micros, Porphyromonas gingivalis, Prevotella intermedius, and Fusobacterium nucleatum) and one facultative anaerobic bacteria (Streptococcus mutans), as described in Materials and Methods. Seven days later the animals were sacrificed and specimens were fixed in 4% paraformaldehyde, decalcified, prepared for cryostat sections, and stained with H&E. Intact tissues were still noted within the dental root in wild-type mice (A), whereas in the TNFRp55^−/−-IL-1RI^−/− mice (B), tissue necrosis had spread to the end of the root (original magnification ×200). The arrow indicates the junction between the middle and the apical third of the root.

Figure 2.

Quantitative analysis of tissue necrosis in mice lacking response to IL-1 and/or TNF. Surgical pulp exposure followed by inoculation with six oral pathogens was carried out as described in Figure 1 ▶ . H&E-stained cryostat sections were examined for the presence of tissue necrosis in the dental pulp. This tissue was divided into three equal parts: coronal third, middle third, and apical third, which follows the path of necrosis from the coronal third to the apical third of the dental root. Under microscopic examination (magnification, ×400), the following scale was used: 0, no necrosis; 1, partial necrosis of coronal third; 2, total necrosis of coronal third; 3, partial necrosis of middle third; 4, total necrosis of middle third; 5, partial necrosis of apical third; and 6, total necrosis of apical third. The highest score for the specimen represented the necrosis status of that tooth and was used in statistic analysis. Each value represents the mean ± SEM, n = 5 for each time point. Significant differences (P < 0.01) were noted between all receptor-deficient and wild-type mice at 7, 14, 21, and 38 days after bacterial challenge. There were no statistical differences between any of the groups at 3 days after pulpal insult. W, wild type; T, TNFRp55^−/−-p75^−/−; I, IL-1 RI^−/−; D, TNFRp55^−/−-IL-1RI^−/−.

Figure 3.

Bacterial penetration at tissue is greater in mice lacking IL-1 and/or TNF activity. The number of bacterial colonies that could be cultured from the apical portion of the distal root 8 days after exposure and inoculation with six oral pathogens was determined. Values represent the mean ± SEM, n = 10 for each group. All receptor-mutant mice showed significant differences (P < 0.01) when compared with wild-type mice. **Significantly (P < 0.01) larger values were observed in TNFRp55^−/−-IL-1RI^−/− mice compared to IL-1RI^−/− and TNFRp55^−/−-p75^−/− mice. *IL-1RI^−/− and TNFRp55^−/−-p75^−/− mice had values that were not significantly different from each other (P > 0.05) but were significantly higher than those of wild-type mice (P < 0.01).

Figure 4.

There is massive destruction of bone from osteolytic lesions in mice lacking IL-1 and/or TNF activity. The dental pulp was surgically exposed and inoculated with six different oral pathogens, as described in the legend to Figure 1 ▶ . Photomicrographs were taken of the osteolytic lesions from each group 38 days after inoculation. There was only slight bone loss in wild-type mice (A). In mice lacking either IL-1 (B) or TNF (C) activity, the amount of bone destruction was intermediate. Bone destruction was greatest in mice lacking both IL-1 and TNF activity (D). H&E-stained cryostat sections; original magnification, ×200.

Figure 5.

Osteolytic lesions form rapidly and to a greater extent in mice lacking IL-1 and/or TNF activity. The size of the osteolytic lesions around the distal root end of experimental molars was determined at the indicated time points after exposure of the dental pulp and inoculation by six oral pathogens. At all time points after day 7, the size of lesions in the mice lacking receptors for IL-1, TNF, or both was significantly larger (P < 0.01) than in wild-type mice. There were no significant differences in lesion size between mice lacking receptors for IL-1 or TNF at any time point. Values represent the mean ± SEM, n = 5 for each data point.

Figure 6.

Osteoclastogenesis occurs rapidly in mice lacking IL-1 and/or TNF activity. Osteoclastogenesis was measured as the number of osteoclasts per millimeter length of bone at the root end, after exposure of the dental pulp and inoculation by six oral pathogens. Osteoclasts were identified as TRAP-positive multinucleated cells lining bone at ×200 magnification. Values represent the mean ± SEM, n = 5, for each data point. In wild-type mice, osteoclastogenesis increased slowly and gradually. In mice lacking receptors for IL-1, TNF, or both, osteoclastogenesis increased dramatically by day 7. Thereafter, it decreased gradually. At day 38, osteoclastogenesis in wild-type mice was greater than that in mice lacking receptors for IL-1, TNF, or both (P < 0.05). At any time point, there was no significant difference in osteoclastogenesis between mice lacking receptors for IL-1 or TNF (P > 0.05).