Porphyromonas Gingivalis and E-coli induce different cytokine production patterns in pregnant women

Abstract

Objective: Pregnant individuals of many species, including humans, are more sensitive to various bacteria or their products as compared with non-pregnant individuals. Pregnant individuals also respond differently to different bacteria or their products. Therefore, in the present study, we evaluated whether the increased sensitivity of pregnant women to bacterial products and their heterogeneous response to different bacteria was associated with differences in whole blood cytokine production upon stimulation with bacteria or their products.

Methods: Blood samples were taken from healthy pregnant and age-matched non-pregnant women and ex vivo stimulated with bacteria or LPS from Porphyromonas Gingivalis (Pg) or E-coli for 24 hrs. TNFα, IL-1ß, IL-6, IL-12 and IL-10 were measured using a multiplex Luminex system.

Results: We observed a generally lower cytokine production after stimulation with Pg bacteria or it's LPS as compared with E-coli bacteria. However, there was also an effect of pregnancy upon cytokine production: in pregnant women the production of IL-6 upon Pg stimulation was decreased as compared with non-pregnant women. After stimulation with E-coli, the production of IL-12 and TNFα was decreased in pregnant women as compared with non-pregnant women.

Conclusion: Our results showed that cytokine production upon bacterial stimulation of whole blood differed between pregnant and non-pregnant women, showing that the increased sensitivity of pregnant women may be due to differences in cytokine production. Moreover, pregnancy also affected whole blood cytokine production upon Pg or E-coli stimulation differently. Thus, the different responses of pregnant women to different bacteria or their products may result from variations in cytokine production.

Figure 1. Gating strategy for leukocyte subpopulations and TLR expression.

Leukocytes were selected in the forward-sidescatter plot (fig. 1A) and copied to a sidescatter-CD14 plot. Monocytes (CD14 positive cells), granulocytes (CD14 negative cells with high SSC) and lymphocytes (CD14 negative cells with low SSC) were gated (fig. 1B). CD14 positive cells were copied to a TLR2/TLR4 plot. Using the isotype control sample, gates were set in the TLR2/TLR4 plot so that at least 99% of the isotype controls were negative for TLR2/TLR4 expression (fig. 1C). This gate was then used to identify the percentages of TLR4/TLR2 double positive, TLR2 single and TLR4 single positive monocytes as well as their mean fluorescence intensity (MFI), in the antibody incubated samples (fig. 1D).

Figure 2. Cytokine concentrations following stimulation with bacteria.

Concentrations (ng/ml) of IL-1b, IL-6, IL-12, TFNα and IL-10 in plasma of pregnant and non-pregnant women following stimulation of whole blood with E-coli (black dots) or P. Gingivalis (PG) (open dots) bacteria (5*10⁷ bacteria/ml) for 24 hr. *significantly different from E-coli (two-way ANOVA followed by Bonferroni post-tests, p<0.05)). a: significantly different from pregnant women after the same stimulation (two-way ANOVA followed by Bonferroni post-tests, p<0.05).

Figure 3. Cytokine concentrations following stimulation with LPS.

Concentrations (ng/ml) of IL-1b, IL-6, IL-12, TFNα and IL-10 in plasma of pregnant and non-pregnant women following stimulation of whole blood with E-coli (black dots) or P. Gingivalis (PG) (open dots) LPS (2 µg/ml) for 24 hr. *significantly different from E-coli (two-way ANOVA followed by Bonferroni post-tests, p<0.05). a: significantly different from pregnant women after the same stimulation (two-way ANOVA followed by Bonferroni post-tests, p<0.05).

Figure 4. Pro-inflammatory/anti-inflammatory cytokine ratio following stimulation with bacteria.

Ratio of Il-12/IL-10, TNFα/IL-10 and IL-6/IL-10 cytokine production in plasma of pregnant and non-pregnant women following stimulation of whole blood with E-coli (black dots) or P. Gingivalis (PG) (open dots) bacteria (5*10⁷ bacteria/ml) for 24 hr. *significantly different from E-coli (two-way ANOVA followed by Bonferroni post-tests, p<0.05). a: significantly different from pregnant women after the same stimulation (two-way ANOVA followed by Bonferroni post-tests, p<0.05).

Figure 5. Pro-inflammatory/anti-inflammatory cytokine ratio following stimulation with LPS.

Ratio of IL-12/IL-10, TNFα/IL-10 and IL-6/IL-10 cytokine production in plasma of pregnant and non-pregnant women following stimulation of whole blood with E-coli (black dots) or P. Gingivalis (PG) (open dots) LPS (2 µg/ml) for 24 hr. *significantly different from E-coli (two-way ANOVA followed by Bonferroni post-tests, p<0.05). a: significantly different from pregnant women after the same stimulation (two-way ANOVA followed by Bonferroni post-tests, p<0.05).

Figure 6. Expression of TLR2 and TLR4 in pregnant and non-pregnant women.

Expression of TLR2 and TLR4 on monocytes of pregnant (open squares) and non-pregnant women (black squares). A: Percentage of TLR2 positive monocytes (left graph) and mean fluorescent intensity of TLR2 staining of monocytes (right graph). B: Percentage of TLR4 positive monocytes (left graph) and mean fluorescent intensity of TLR4 staining of monocytes (right graph). *: significantly increased vs pregnant women (student’s T test, p<0.05).