Bacterial-Specific Induction of Inflammatory Cytokines Significantly Decreases upon Dual Species Infections of Implant Materials with Periodontal Pathogens in a Mouse Model

Abstract

Cytokine profiles are often perturbed after infections of medical implants. With a non-invasive in vivo imaging system, we report in a mouse model that interferon expression after infection of subcutaneous implants with Streptococcus oralis, Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, and Treponema denticola (alone or as a combination) was species-specific, persisted longer in the presence of implants, and notably decreased upon dual species infections. This type I interferon expression disappeared within two weeks; however, histology of implant-tissue interface indicated high recruitment of immune cells even after three weeks. This was suggestive that biomaterial-associated infections could have prolonged effects, including the systemic stimulation of inflammatory cytokines. The present study investigated the systemic impact of this chronic peri-implant inflammation on the systemic expression of inflammatory cytokines (23) using a multiplex assay. Initially, the cytokine measurement in murine fibroblasts exposed to periodontal pathogens remained limited to the expression of five cytokines, namely, IL-6, G-CSF, CXCL-1/KC, MCP-1 (MCAF), and IL-12 (p40). The systemic determination of cytokines in mice increased to 19 cytokines (IL-1α, IL-2, IL-3, IL-5, IL-6, IL-9, IL-12 (p40), IL-12 (p70), IL-13, IL-17A, CCL-11/Eotaxin, G-CSF, IFN-γ, CXCL1/KC, MCP-1 (MCAF), MIP-1α/CCL3, MIP-1β/CCL4, CCL5/RANTES, and TNF-α). Systemic induction of cytokines was species-specific in the mouse model. The cytokine induction from infected implants differed significantly from sole tissue infections and sterile implants. Notably, systemic cytokine induction decreased after infections with dual species compared to single species infections. These findings describe the systemic effect of chronic peri-implant inflammation on the systemic induction of inflammatory cytokines, and this effect was strongly correlated to the type and composition of initial infection. Systemic modulations in cytokine expression upon dual species infections exhibit an exciting pattern that might explain the complications associated with biomaterial-related infection in patients. Moreover, these findings validate the requirement of multispecies infections for pre-clinical studies involving animal models.

Keywords: biomaterial-associated infections; cytokine; fibroblasts; mouse model; periodontal pathogens.

Figure 1

Morphology of titanium cylinders designed for implantation into mice (a). Implants allowed accumulation of various host cells visible after 21 days of implantation ((b), white asterisks). Mouse showing the impression of subcutaneously implanted titanium cylinders ((c), white arrows indicate implanted titanium).

Figure 2

Morphology and the expression of G-CSF, CXCL-1, and MCP1 in fibroblasts. Microscopic documentation of unstimulated fibroblasts (a) as well as those stimulated with S. oralis surrounding murine fibroblasts (b), A. actinomycetemcomitans (c), P. gingivalis (d), and T. denticola (e), or a mixture of all four bacterial species (f). White arrows target remaining fibroblasts (b,f). The box and whisker graphs indicate the expression of G-CSF (g), CXCL-1 (h), and MCP-1 (i) in uninfected fibroblasts (CT) or after co-cultivation with S. oralis (So), A. actinomycetemcomitans (Aa), P. gingivalis (Pg), and T. denticola (Td), or a mixture of all species (So-Aa-Pg-Td). Significant changes according to the uninfected control were adjusted by Bonferroni correction for multiple tests. Symbols ***, **, and * indicate p < 0.001, p < 0.01, and p < 0.05, respectively.

Figure 3

Modulations in the systemic expression of interleukins in mice. The box and whiskers graphs indicate serum interleukin expression for IL-9 (a), IL-12p40 (b), IL-12p70 (c), IL-13 (d), and IL-17 (e) in animals with sterile implants (empty bars), infected implants (filled colored bars), and infected tissues (empty bars with colored borders). Data were analyzed animal-wise (biological replicates (n = 3), technical replicates (3 each)). Significant values have been adjusted by the Bonferroni correction for multiple tests. Symbols ***, **, *, and # indicate p < 0.001, p < 0.01, p < 0.05, and significant p-value prior to Bonferroni correction, respectively, between sterile and infected implants (black symbols). Significant differences between infections in the presence and absence of implants are indicated by colored symbols in each case. Abbreviations: So (S. oralis), Aa (A. actinomycetemcomitans), Pg (P. gingivalis), Td (T. denticola).

Figure 4

Systemic expression of chemokines in mice. Black bars represent sterile implants, filled bars indicate infected implants, and empty colored bars indicate infected tissues for CXCL1/KC (a), CCL4/MIP1β (b), CCL5/RANTES (c), and CCL11 (d). Data were analyzed animal-wise (biological replicates (n = 3), technical replicates (3 each)). Significant values were adjusted by the Bonferroni correction for multiple tests. Symbols **, *, and # indicate p < 0.01, p < 0.05, and significant p-value prior to Bonferroni correction, respectively. Black symbols represent significant differences between sterile and infected implants. Significant differences between infected implants and infected sham-operated tissues are indicated by colored symbols. Abbreviations: So (S. oralis), Aa (A. actinomycetemcomitans), Pg (P. gingivalis), Td (T. denticola).

Figure 5

Systemic levels of inflammatory cytokines. Graphs show serum levels of IFN-γ (a), G-CSF (b), TNF-α (c), and CCL2/MCP-1 (d) 21 days after implantations and infections. Empty bars represent sterile implants, colored bars indicate infected implants, and empty bars with colored borders indicate infected tissues. Data from three independent experiments are shown. Data were analyzed animal-wise (biological replicates (n = 3), technical replicates (3 each)). Significant values were adjusted by the Bonferroni correction for multiple tests. Symbols ***, **, *, and # indicate p < 0.001, p < 0.01, p < 0.05, and significant p-value prior to Bonferroni correction, respectively. Black symbols represent significant differences between sterile and infected implants. Colored symbols indicate significant differences between infected implants and infected tissues in the absence of implants. Abbreviation: So (S. oralis), Aa (A. actinomycetemcomitans), Pg (P. gingivalis), Td (T. denticola).