Fig 2: Effect of LPS and P. gingivalis on TLR4 (CD284) and TLR2 (CD282) expression in monocytes of healthy, SMC, MCI and AD individuals. Monocytes were cultured in the absence ( - ) or presence ( + ) of LPS (100ng/ml) or Pg supernatant (20µg) for 1 hr at 37°C, 5% CO2 air mixture, stained for TLR4 (CD284) TLR2 (CD282) and analyzed by flow cytometry as in M&M. a TLR4 ± LPS. b TLR4 ± Pg. c TLR2 ± LPS. d TLR2 ± Pg. Each dot corresponds to one individual. Statistical analyses were performed by Student t-testing to assess differences within each patient group. Data are a combination of 18 independent experiments and are shown as mean of MFI ± SD. The asterisks correspond to *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001

Fig 3: Heme decreases bacterial clearance in the lung and decreases TLR2 expression in PMN in vivo.(A and B) Fold change of TLR2 MFI in PMN in the blood and the BM, 4 hours after heme challenge alone (50 mg/kg, i.p.) in vivo (n = 6/group). (C) Fold change of S. aureus CFU in BALF 24 hours after infection with and without heme challenge. Mice were challenged with heme 4 hours before the infection (50 mg/kg, i.p.; n = 10–12/group). (D) Fold change of TLR2 MFI in PMN in BAL 24 hours after infection with and without heme challenge (n = 4/group). (E) A representative flow cytometry analysis image showing the MFI of TLR2 in PMN in the BAL. Blue represents PMN in BAL from control mice, and red represents PMN from mice with heme challenge. Gray represents unstained cells. (F and G) Fold change of TLR2 MFI in PMN in the blood and the BM 24 hours after infection, with and without heme challenge (n = 4/group). (H–J) Levels of KC, IP-10, and MIP-1a in the BALF at the baseline (n = 3), 24 hours after lung infection with and without heme challenge (n = 4/group). (K) Fold change of reactive oxygen species (ROS) release in PMN treated with naive or trauma serum in response to S. aureus exposure in vitro (3 independent experiments, each with n = 3–4/group). ROS release is measured by the AUC. All data are presented as mean ± SEM. Statistical analyses for H, I, and J were performed by 1-way ANOVA with post hoc Tukey’s test. All other P values were calculated by unpaired 2-tailed Student’s t test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig 4: Trauma injury downregulates CD16, TLR2, and TLR4 expression in circulating PMN in patients.(A–D) MFI of CD16, TLR2, TLR4, and CD66b in circulating PMN collected from control patients (n = 6) and trauma patients on day 0 or 1 (n = 9) measured by flow cytometry. A representative flow cytometry analysis image is shown on the right (except for CD66b). Gray represents unstained cells, blue the control, and red the trauma. All data are presented as mean ± SEM. Statistical analysis was performed by using unpaired 2-tailed Student’s t test. *P < 0.05, **P < 0.01.