Blocking proinflammatory cytokine release modulates peripheral blood mononuclear cell response to Porphyromonas gingivalis

Abstract

Background: Chronic periodontitis (CP) is an inflammatory disease in which cytokines play a major role in the progression of disease. Anti-inflammatory cytokines (interleukin 4 [IL-4] and IL-10) were reported to be absent or reduced in diseased periodontal tissues, suggesting an imbalance between the proinflammatory and anti-inflammatory mediators. This study tests the hypothesis that there is cellular crosstalk mediated by proinflammatory and anti-inflammatory cytokines and that blocking proinflammatory cytokine (tumor necrosis factor-α [TNF-α] and IL-1) production will enhance anti-inflammatory cytokine (IL-4 and IL-10) production from peripheral blood mononuclear cells (PBMCs) in response to Porphyromonas gingivalis.

Methods: PBMCs were isolated from individuals diagnosed with CP or healthy individuals and cultured for 24 hours. Concanavalin A (ConA) was used as an activator of lymphocyte function. Live and heat-killed P. gingivalis or lipopolysaccharide from P. gingivalis were used as the bacterial stimulants. TNF-α and IL-1 production was neutralized by specific antibodies against TNF-α and IL-1α or IL-β. Culture supernatants were evaluated by enzyme-linked immunosorbent assay for TNF-α, IL-1β, IL-4, and IL-10 production.

Results: Live P. gingivalis did not result in any significant IL-10 or IL-4 release, whereas heat-killed P. gingivalis led to a significant increase in IL-10 levels compared with unstimulated or live P. gingivalis-stimulated cells from both healthy individuals or those with CP. Overall, PBMCs from patients with CP produced significantly lower IL-10 in response to ConA and P. gingivalis, suggesting chronic suppression of the anti-inflammatory cytokine production. Blocking the proinflammatory cytokine response did not result in any substantial change in IL-10 or IL-4 response to live P. gingivalis. Blocking the proinflammatory cytokine response restored IL-10 production by cells from CP in response to P. gingivalis lipopolysaccharide.

Conclusions: These findings suggest that PBMCs from patients with CP have suppressed anti-inflammatory cytokine production that can, in part, be restored by neutralizing proinflammatory cytokines. Monocytes are an important source of IL-10 production, and monocyte-derived IL-10 might play a regulatory role in the pathogenesis of CP.

Figure 1. IL-10 and IL-4 release by PBMCs from healthy donors

PBMC were stimulated in the presence and absence of a combination of neutralizing antibodies to the pro-inflammatory cytokines TNF-α, IL-1α, and IL-1β [0.25µg/ml (anti-TNF-α), 0.31µg/ml (anti-IL-1α), and 0.031µg/ml (anti-IL-1β)]. Each experiment was repeated in duplicate; results represent mean and standard deviations. Panels A and B: A specific T cell-activator (ConA; 500µg/ml) significantly increased cytokine release compared to unstimulated (resting) cells (*p<0.05). Blocking pro-inflammatory cytokine release did not significantly change the ConA-induced IL-10 and IL-4 release by healthy cells (p>0.05). Panels C and D: Live P. gingivalis did not significantly induce IL-10 or IL-4 production by the PBMC (p>0.05); heat-killed P. gingivalis led to a significant increase in IL-10 levels compared to unstimulated (*p<0.05) or live P. gingivalis-stimulated cells (^#p<0.05). Blocking pro-inflammatory cytokine response did not result in any substantial change in IL-10 or IL-4 response (p>0.05). Panels E and F: P. gingivalis LPS (100ng/ml) induced a significant increase in IL-10 (*p<0.05) with no impact on IL-4 levels (p>0.05). E. coli LPS (100ng/ml) induced significant IL-10 production (*p<0.05) with no substantial change in IL-4 levels (p>0.05).

Figure 2. IL-10 and IL-4 release by PBMC from patients with chronic periodontitis

PBMC were stimulated in the presence and absence of a combination of neutralizing antibodies to the pro-inflammatory cytokines TNF-α, IL-1α, and IL-1β [0.25µg/ml (anti-TNF-α), 0.31µg/ml (anti-IL-1α), and 0.031µg/ml (anti-IL-1β)]. Each experiment was repeated in duplicate; results represent mean and standard deviations. Panels A and B: Con A (500µg/ml) stimulation did not produce significant IL-10 in PBMC from chronic periodontitis patients compared to unstimulated cells (p>0.05). The difference was significant compared to cells from healthy donors (^¥p<0.05). IL-4 production was significantly increased compared to unstimulated cells (*p<0.05), while significantly less than healthy cells (^¥p<0.05). Panels C and D: Live P. gingivalis did not significantly induce IL-10 or IL-4 production (p>0.05); heat-killed P. gingivalis led to a significant increase in IL-10 levels compared to unstimulated (*p<0.05) or live P. gingivalis-stimulated cells (^#p<0.05). Blocking the pro-inflammatory cytokine response did not result in any substantial change in IL-10 or IL-4 response (p>0.05). IL-10 production by PBMC from periodontitis patients in response to heat-killed P. gingivalis was significantly less than the cells from healthy donors (^¥p<0.05). Panels E and F: P. gingivalis LPS (100ng/ml) did not induce a significant increase in IL-10 or in IL-4 levels (p>0.05) compared to unstimulated cells. When IL-1 and TNF-α were blocked, P. gingivalis LPS-induced IL-10 production was significantly higher than unstimulated cells (*p<0.05) with no change in Il-4 release. P. gingivalis LPS-induced IL-10 production by the PBMC from periodontitis patients was significantly less than the healthy cells with or without neutralization of pro-inflammatory cytokines (^¥p<0.05). E. coli (100ng/ml) LPS stimulated IL-10 by the PBMC significantly compared to resting (*p<0.05) and P. gingivalis-LPS-stimulated (^#p<0.05) cells. When the TNF-α and IL-1 responses were blocked, E. coli LPS-induced IL-10 production was significantly reduced (^†p<0.05), significantly lower than the P. gingivalis LPS (^#p<0.05) or healthy cells (^¥p<0.05).

Figure 3. IL-10 production from monocytes in response to P. gingivalis

Each experiment was repeated in duplicate; results represent mean and standard deviations. Panels A and B: Live P. gingivalis did not generate any significant IL-10 production by the monocytes; heat-killed P. gingivalis led to a statistically significant increase in IL-10 production compared to resting (*p<0.05) or live bacteria-stimulated (^#p<0.05) cells. Blocking the pro-inflammatory cytokine activity did not result any significant change compared to cells that were not exposed to neutralization, while statistically significant compared to resting (*p<0.05) or live- bacteria-stimulated (^#p<0.05) cells with neutralization. Panels C and D: LPS (100ng/ml) from both E. coli and P. gingivalis caused significant IL-10 production from monocytes compared to resting cells (*p<0.05). Blocking pro-inflammatory cytokines did not lead to any significant change in IL-10 production in response to E. coli LPS (p>0.05), while there was a significant increase in IL-10 response to LPS from P. gingivalis after 48 hours of neutralization (^†p<0.05). At both 24 and 48 hours, P. gingivalis LPS-stimulated IL-10 production by the monocytes was significantly higher than the E. coli LPS-treated cells under neutralization (^#p<0.05).