Social stress enhances IL-1beta and TNF-alpha production by Porphyromonas gingivalis lipopolysaccharide-stimulated CD11b+ cells

Abstract

Psychological stress is associated with an increased expression of markers of peripheral inflammation, and there is a growing literature describing a link between periodontal pathogens and systemic inflammation. The hypothesis of the present work is that exposing mice to the social stressor, called social disruption (SDR), would enhance the inflammatory response to lipopolysaccharide (LPS) derived from the oral pathogen, Porphyromonas gingivalis. Mice were exposed to SDR for 2h per day on 6 consecutive days. On the morning following the last cycle of SDR, mice were tested for anxiety-like behavior in the open field test and novel object test. The mice were sacrificed the following day and their spleens harvested. Spleen cells were stimulated with LPS derived from P. gingivalis in the absence or presence of increasing doses of corticosterone. Social disruption resulted in anxiety-like behavior, and the production of IL-1beta and TNF-alpha was significantly higher in spleen cells from mice exposed to SDR in comparison to levels from non-stressed control mice. In addition, the viability of spleen cells from mice exposed to SDR was significantly greater than the viability of cells from non-stressed control mice, even in the presence of high doses of corticosterone. The use of cultures enriched for CD11b+ cells indicated that the stressor was affecting the activity of splenic myeloid cells. This study demonstrates that social stress enhances the inflammatory response to an oral pathogen and could provide a critical clue in the reported associations between stress, inflammation, and oral pathogens.

Figure 1

The social disruption stressor significantly increased anxiety-like behavior. Behavior was assessed in the open field, and mice exposed to SDR were found to spend less time in the center of the open field (Fig. 1A). This was not dependent upon a reduction in the overall locomotion of the mice, since mice exposed to SDR traveled as far as the non-stressed HCC mice while in the open field (Fig. 1B). Mice exposed to SDR also spent a significantly reduced amount of time on the same side of the open field as a novel object (Fig. 1C), especially during the first three minutes of testing. n = 9 mice per group. * p < .05 vs. non-stressed HCC mice.

Figure 2

Exposure to social disruption significantly affects the cellular composition of the spleen. Spleens were removed from mice the morning following the last cycle of SDR, weighed (Fig. 2A), and macerated to create a single cell suspension. Flow cytometry was used to quantify the number of splenic monocytes/macrophages (Fig. 2B) and granulocytes (Fig. 2C). n = 4 − 5 per group. * p < .05 vs. non-stressed HCC mice.

Figure 3

Cell viability was significantly enhanced after exposure to social disruption. 2.5×10⁵ spleen cells were cultured with 10 μg/ml of P. gingivalis LPS with increasing doses of corticosterone for 48 h. Cell viability was assessed using a colorometric assay, with data expressed as the optical density. n = 5 per group. * p < .05 vs. non-stressed HCC mice at the same corticosterone concentration.

Figure 4

Cytokine production was significantly enhanced by exposure to social disruption. 5×10⁵ spleen cells were culture with 10 μg/ml of P. gingivalis LPS with increasing doses of corticosterone for 18 h. Cytokine levels in the supernatants were determine via ELISA. For both IL-1β and TNF-α, cytokine levels were higher in cultures containing splenocytes from stressed mice at all dose of corticosterone (p < .05). n = 5 per group.

Figure 5

Stress-induced increases in spleen cell viability were dependent upon CD11b+ myeloid cells. Splenic CD11b+ myeloid cells were enriched from spleen cell cultures and stimulated with 1 μg/ml of P. gingivalis LPS and increasing doses of corticosterone for 48 h prior to assessing cell viability. Data are the optical density of the assay. * p < .05 vs non-stressed HCC mice. n = 5 per group.

Figure 6

Stress-induced increases in IL-1β and TNF-α were partly due to CD11b+ myeloid cells. Splenic CD11b+ myeloid cells were enriched from spleen cell cultures and stimulated with 1 μg/ml of P. gingivalis LPS for 18 h. Cytokine levels in the supernatants were assessed via ELISA and are expressed as pg/ml. n = 5 per group. * p < .05 vs non-stressed HCC mice.