Fig 2: 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 3: Liver crush injury downregulates TLR2 expression in PMN in vivo.(A and B) Fold change of TLR2 MFI in PMN 24 hours after S. aureus lung infection in the blood (n = 10–16/group) and the BM (n = 7–11/group). (C and D) Fold change of TLR2 MFI in PMN 4 hours after liver crush injury in the blood (n = 6–7/group) and the BM (n = 8–11/group). (E) Fold change of TLR2 MFI in PMN in BAL, 24 hours after infection with and without liver crush injury (n = 14–15/group). (F) A representative flow cytometry analysis image showing the MFI of TLR2 in PMN in BAL. Blue represents the PMN in the BAL from the “Infection” group, and red represents the PMN from the “Trauma + Infection” group. (G and H) Fold change of TLR2 MFI in PMN in the blood (n = 14–16/group) and the BM (n = 11/group), 24 hours after infection, with and without liver crush injury. (I) TLR2 mRNA expression levels in lung tissues, 24 hours after S. aureus infection with and without liver crush injury (normalized to HPRT1 mRNA expression; n = 3–4/group). All data are presented as mean ± SEM. Statistical analysis was performed 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.