Bacterial Lipoprotein Induces Resistance to Gram-Negative Sepsis in TLR4-Deficient Mice via Enhanced Bacterial Clearance

Abstract

TLRs are highly conserved pathogen recognition receptors. As a result, TLR4-deficient C3H/HeJ mice are highly susceptible to Gram-negative sepsis. We have previously demonstrated that tolerance induced by bacterial lipoprotein (BLP) protects wild-type mice against polymicrobial sepsis-induced lethality. In this study, we assessed whether pretreatment of C3H/HeJ mice with BLP could induce resistance to a subsequent Gram-negative Salmonella typhimurium infection. Pretreatment with BLP resulted in a significant survival benefit in TLR4-deficient C3H/HeJ mice (p < 0.0002 vs control C3H/HeJ) after challenge with live S. typhimurium (0.25 × 106 CFU/mouse). This survival benefit was associated with enhanced bacterial clearance from the circulation and in the visceral organs (p < 0.05 vs control C3H/HeJ). Furthermore, pretreatment with BLP resulted in significant increases in complement receptor type 3 (CR3) and FcγIII/IIR expression on polymorphonuclear neutrophils (PMNs) and macrophages (p < 0.05 vs control C3H/HeJ). There was impaired bacterial recognition and phagocytosis in TLR4-deficient mice compared with wild-type mice. However, a significant augmented uptake, ingestion, and intracellular killing of S. typhimurium by PMNs and peritoneal macrophages was evident in BLP-pretreated C3H/HeJ mice (p < 0.05 vs control C3H/HeJ). An up-regulation of inducible NO synthase and increased production of intracellular NO were observed in peritoneal macrophages from BLP-pretreated C3H/HeJ mice (p < 0.05 vs control C3H/HeJ). Depletion of PMNs did not diminish the beneficial effects of BLP with regard to both animal survival and bacterial clearance. These results indicate that BLP, a TLR2 ligand, protects highly susceptible TLR4-deficient mice from Gram-negative sepsis via enhanced bacterial clearance.

ST2 Negatively Regulates TLR2 Signaling, but Is Not Required for Bacterial Lipoprotein-Induced Tolerance

“…Our in vivo work demonstrated that pretreatment with a nonlethal dose of BLP protects mice against not only a subsequent lethal BLP challenge, but also a subsequent lethal LPS challenge through a cross-tolerance to LPS (8). Unlike LPS tolerance, BLP tolerance-afforded protection against microbial sepsis was observed in both wild-type (WT) (8) and TLR4-deficient (9) mice. This is closely associated with BLP-induced reprogramming in phagocytes characterized by hypo-responsiveness in producing proinflammatory cytokines and simultaneously an enhanced antimicrobial activity (7)(8)(9).…”

“…with 10 mg/kg BLP to induce BLP tolerance (BLP-tolerized), as described previously (8,9). Nontolerized mice received an equal volume (200 ml) of i.p.…”

“…Peritoneal macrophages were collected from WT and ST2-deficient mice by peritoneal lavage and incubated with DMEM containing 10% heat-inactivated FCS in 24-well plates (Falcon, Lincoln Park, NJ) for 90 min to remove nonadherent cells, as described previously (9). Bone marrow-derived macrophages (BMDM) were isolated from the femurs of WT and ST2-deficient mice and cultured in DMEM containing 20% heat-inactivated FCS, penicillin (100 U/ml), streptomycin sulfate (100 mg/ml), and supplemented with 10 ng/ml recombinant mouse macrophage-CSF (R&D Systems, Minneapolis, MN) for 7 d at 37˚C in a humidified 5% CO 2 atmosphere, as described previously (20).…”

“…Unlike LPS tolerance, BLP tolerance-afforded protection against microbial sepsis was observed in both wild-type (WT) (8) and TLR4-deficient (9) mice. This is closely associated with BLP-induced reprogramming in phagocytes characterized by hypo-responsiveness in producing proinflammatory cytokines and simultaneously an enhanced antimicrobial activity (7)(8)(9). Our in vitro work found that BLP-induced tolerance is associated with suppressed TLR2 expression and an inhibition in both MAP kinase phosphorylation and NFkB activation (10).…”

Injury Enhances Resistance to Escherichia coli Infection by Boosting Innate Immune System Function

“…The adherence and phagocytic uptake of FITC-labeled, heat-killed E. coli (100°C for 1 h) were determined by flow cytometry as previously described by O'Brien et al (20). In brief, heat-killed E. coli were thawed and incubated in 0.1 M carbonate buffer (pH 9.5) in the presence 0.1% FITC (Sigma-Aldrich) for 60 min at 37°C.…”