Lipopolysaccharide clearance, bacterial clearance, and systemic inflammatory responses are regulated by cell type-specific functions of TLR4 during sepsis

Abstract

The morbidity associated with bacterial sepsis is the result of host immune responses to pathogens, which are dependent on pathogen recognition by pattern recognition receptors, such as TLR4. TLR4 is expressed on a range of cell types, yet the mechanisms by which cell-specific functions of TLR4 lead to an integrated sepsis response are poorly understood. To address this, we generated mice in which TLR4 was specifically deleted from myeloid cells (LysMTLR4KO) or hepatocytes (HCTLR4KO) and then determined survival, bacterial counts, host inflammatory responses, and organ injury in a model of cecal ligation and puncture (CLP), with or without antibiotics. LysM-TLR4 was required for phagocytosis and efficient bacterial clearance in the absence of antibiotics. Survival, the magnitude of the systemic and local inflammatory responses, and liver damage were associated with bacterial levels. HCTLR4 was required for efficient LPS clearance from the circulation, and deletion of HCTLR4 was associated with enhanced macrophage phagocytosis, lower bacterial levels, and improved survival in CLP without antibiotics. Antibiotic administration during CLP revealed an important role for hepatocyte LPS clearance in limiting sepsis-induced inflammation and organ injury. Our work defines cell type-selective roles for TLR4 in coordinating complex immune responses to bacterial sepsis and suggests that future strategies for modulating microbial molecule recognition should account for varying roles of pattern recognition receptors in multiple cell populations.

FIGURE 1.

LysMTLR4 is required for efficient bacterial clearance. (A) Seven day survival after CLP. Bacterial counts in blood (B), liver (C), and peritoneal lavage fluid (D) 18 h after CLP. Plasma (E) endotoxin and (F) ALT level at 6 and 18 h after CLP. Data represent mean ± SD (n = 8–11 mice/group [time points]; n = 12–20 mice/group [survival]). *p < 0.05.

FIGURE 2.

Neutrophil influx and circulating IL-6 levels are independent of TLR. MPO activity in peritoneal lavage fluid (A), lung (B), and liver (C) at baseline (0), 6, and 18 h after CLP. (D) Plasma IL-6 at 6 and 18 h after CLP. Data represent mean ± SD (n = 8–11 mice/group). *p < 0.05.

FIGURE 3.

HCTLR4 plays an important role in endotoxin clearance in mice after CLP. Bacterial counts in liver (A) and peritoneal lavage fluid (B) of mice after CLP + Imipenem (25 mg/kg, twice a day s.c.). Plasma levels of endotoxin (C), IL-6 (D), and ALT (E) at 18 h after CLP + Imipenem. Data represent mean ± SD (n = 6–8 mice/group). *p < 0.05 between indicated groups.

FIGURE 4.

Hepatocyte LPS uptake is TLR4 dependent in vitro. Hepatocytes were isolated from WT and TLR4KO mice and treated with Alexa Fluor 488–labeled fluorescent LPS (1 mg/ml). LPS uptake was monitored by live cell microscopy. (A) Photomicrographs at 0 and 90 min after LPS treatment. Arrows indicate internalized LPS. (B) Quantification of fluorescent cytoplasmic particle number represented as fold change of cytoplasmic particle number at 90 min compared with time 0. (C) Uptake of LPS and transfer into lysosomes. LPS vesicle = green arrow; lysosome = red arrow, LysoTracker Red; colocalization = orange arrow. Original magnification ×40. Data represent mean ± SD. Images are representative of at least three separate experiments. Experiments were performed in at least duplicate.

FIGURE 5.

LPS clearance is dependent on HCTLR4 during endotoxemia in mice, and endotoxin regulates the systemic inflammatory response. (A) Liver immunofluorescence in mice 6 h after i.v. LPS (5 mg/kg) LPS. Original magnification ×20. Green = LPS; red = macrophage; blue = nucleus; white = actin. White arrow indicates LPS⁺ macrophages. Plasma levels of endotoxin (B) and IL-6 (C) at 6 and 24 h after LPS. Data represent mean ± SD (n = 6–8 mice/group). Images are representative of results from n = 6 mice/group. *p < 0.05.

FIGURE 6.

Low-dose LPS primes monocytes to improve bacterial clearance during CLP. (A) In vitro phagocytosis of WT peritoneal macrophages after 6 h of pretreatment with 1, 10, or 100 ng/ml LPS. (B) In vivo peritoneal monocyte phagocytosis of fluorescent-labeled heat-killed E. coli in WT, Flox, TLR4KO, HCTLR4KO, and LysMTLR4KO mice 6 h after i.p. LPS (5 mg/kg). (C) Plasma endotoxin (LPS) level at 6 h in WT mice given i.p. LPS (5 mg/kg) or saline (control). (D) Bacterial counts at 18 h in blood, liver, and peritoneal lavage fluid of WT mice given LPS (1 μg/kg, i.v.) of saline (control) 1 h after CLP. Plasma levels of endotoxin (E) and ALT (F) in mice given LPS or saline 1 h after CLP. Data represent mean ± SD (n = 4/group for phagocytosis assays; n = 7/group for CLP). *p < 0.05 versus control or as indicated.