Effectiveness of Different Antimicrobial Strategies for Staphylococcal Prosthetic Joint Infection: Results From a Large Prospective Registry-Based Cohort Study.

Background: Treatment of staphylococcal prosthetic joint infection (PJI) usually consists of surgical debridement and prolonged rifampicin combination therapy. Tailored antimicrobial treatment alternatives are needed due to frequent side effects and drug-drug interactions with rifampicin combination therapy. We aimed to assess the effectiveness of several alternative antibiotic strategies in patients with staphylococcal PJI. Methods: In this prospective, multicenter registry-based study, all consecutive patients with a staphylococcal PJI, treated with debridement, antibiotics and implant retention (DAIR) or 1-stage revision surgery between January 1, 2015 and November 3, 2020, were included. Patients were treated with a long-term rifampicin combination strategy (in 2 centers) or a short-term rifampicin combination strategy (in 3 centers). Antimicrobial treatment strategies in these centers were defined before the start of the registry. Patients were stratified in different groups, depending on the used antimicrobial strategy. Cox proportional hazards models were used to compare outcome between the groups. Results: Two hundred patients were included and stratified in 1 long-term rifampicin group (traditional rifampicin combination therapy) or 1 of 3 short-term rifampicin groups (clindamycin or flucloxacillin or vancomycin monotherapy, including rifampicin for only 5 postoperative days). Adjusted hazard ratios (aHRs) for failure in patients treated with short-term rifampicin and either flucloxacillin or clindamycin were almost equal to patients treated with long-term rifampicin combination therapy (aHR = 1.21; 95% confidence interval, .34-4.40). Conclusions: A short-term rifampicin strategy with either clindamycin or flucloxacillin and only 5 days of rifampicin was found to be as effective as traditional long-term rifampicin combination therapy. A randomized controlled trial is needed to further address efficacy and safety of alternative treatment strategies for staphylococcal PJI.

Genomic Investigation of Two Acinetobacter baumannii Outbreaks in a Veterinary Intensive Care Unit in The Netherlands.

Acinetobacter baumannii is a nosocomial pathogen that frequently causes healthcare-acquired infections. The global spread of multidrug-resistant (MDR) strains with its ability to survive in the environment for extended periods imposes a pressing public health threat. Two MDR A. baumannii outbreaks occurred in 2012 and 2014 in a companion animal intensive care unit (caICU) in the Netherlands. Whole-genome sequencing (WGS) was performed on dog clinical isolates (n = 6), environmental isolates (n = 5), and human reference strains (n = 3) to investigate if the isolates of the two outbreaks were related. All clinical isolates shared identical resistance phenotypes displaying multidrug resistance. Multi-locus Sequence Typing (MLST) revealed that all clinical isolates belonged to sequence type ST2. The core genome MLST (cgMLST) results confirmed that the isolates of the two outbreaks were not related. Comparative genome analysis showed that the outbreak isolates contained different gene contents, including mobile genetic elements associated with antimicrobial resistance genes (ARGs). The time-measured phylogenetic reconstruction revealed that the outbreak isolates diverged approximately 30 years before 2014. Our study shows the importance of WGS analyses combined with molecular clock investigations to reduce transmission of MDR A. baumannii infections in companion animal clinics.

Early differentiation between uncomplicated and complicated Staphylococcus aureus bacteraemia: Potential value and limitations of a clinical risk score.

OBJECTIVE: A cornerstone in the management of Staphylococcus aureus bacteraemia (SAB) is the differentiation between a complicated and an uncomplicated SAB course. The ability to early and accurately identify patients with - and without - complicated bacteraemia may optimise the utility of diagnostics and prevent unnecessary prolonged antibiotic therapy. METHODS: Development and validation of a prediction score in SAB using demographic, clinical, and laboratory data from two independent Dutch cohorts; estimating the risk of complicated disease at the time of the first positive blood culture. Models were developed using logistic regression and evaluated by c-statistics, ie area under the ROC-curve, and negative predictive values (NPV). RESULTS: The development- and validation cohorts included 150 and 183 patients, respectively. The most optimal prediction model included: mean arterial pressure, signs of metastatic infection on physical examination, leucocyte count, urea level and time to positivity of blood cultures (c-statistic 0.82, 95% CI 0.74-0.89). In the validation cohort, the c-statistic of the prediction score was 0,77 (95% CI 0.69-0.84). The NPV for complicated disease for patients with a score of ≤2 was 0.83 (95% CI 0.68-0.92), with a negative likelihood ratio of 0.14 (95% CI 0.06-0.31). CONCLUSION: The early SAB risk score helps to identify patients with high probability of uncomplicated SAB. However, the risk score's lacked absolute discriminative power to guide decisions on the management of all patients with SAB on its own. The heterogenicity of the disease and inconsistency in definitions of complicated SAB are important challenges in the development of clinical rules to guide the management of SAB.

Pleurodynia caused by an echovirus 1 brought back from the tropics.

A 31 year-old woman presented with acute pain on the left side of the thorax and abdomen, radiating to the back together with fever, after she had returned from traveling in Southeast Asia. Except for pleural friction rub auscultated on the left hemithorax, no physical abnormalities were detected. We diagnosed a classical course of Bornholm disease, caused by an echovirus type 1. While described as a classical pathogen causing Bornholm disease, this genotype has not been reported frequently in Surveillance data in the Western World.

The Enterococcus faecium enterococcal biofilm regulator, EbrB, regulates the esp operon and is implicated in biofilm formation and intestinal colonization.

Nowadays, Enterococcus faecium is one of the leading nosocomial pathogens worldwide. Strains causing clinical infections or hospital outbreaks are enriched in the enterococcal surface protein (Esp) encoding ICEEfm1 mobile genetic element. Previous studies showed that Esp is involved in biofilm formation, endocarditis and urinary tract infections. In this study, we characterized the role of the putative AraC type of regulator (locus tag EfmE1162_2351), which we renamed ebrB and which is, based on the currently available whole genome sequences, always located upstream of the esp gene, and studied its role in Esp surface exposure during growth. A markerless deletion mutant of ebrB resulted in reduced esp expression and complete abolishment of Esp surface exposure, while Esp cell-surface exposure was restored when this mutant was complemented with an intact copy of ebrB. This demonstrates a role for EbrB in esp expression. However, during growth, ebrB expression levels did not change over time, while an increase in esp expression at both RNA and protein level was observed during mid-log and late-log phase. These results indicate the existence of a secondary regulation system for esp, which might be an unknown quorum sensing system as the enhanced esp expression seems to be cell density dependent. Furthermore, we determined that esp is part of an operon of at least 3 genes putatively involved in biofilm formation. A semi-static biofilm model revealed reduced biofilm formation for the EbrB deficient mutant, while dynamics of biofilm formation using a flow cell system revealed delayed biofilm formation in the ebrB mutant. In a mouse intestinal colonization model the ebrB mutant was less able to colonize the gut compared to wild-type strain, especially in the small intestine. These data indicate that EbrB positively regulates the esp operon and is implicated in biofilm formation and intestinal colonization.

Identification of a genetic determinant in clinical Enterococcus faecium strains that contributes to intestinal colonization during antibiotic treatment.

Intestinal colonization by antibiotic-resistant Enterococcus faecium is the first step in a process that can lead to infections in hospitalized patients. By comparative genome analysis and subsequent polymerase chain reaction screening, we identified a locus that encodes a putative phosphotransferase system (PTS). The PTS locus was widespread in isolates from hospital outbreaks of infection (84.2%) and nonoutbreak clinical infections (66.0%) but absent from human commensal isolates. Deletion of pstD, which is predicted to encode the enzyme IID subunit of this PTS, significantly impaired the ability of E. faecium to colonize the murine intestinal tract during antibiotic treatment. This is the first description of a determinant that contributes to intestinal colonization in clinical E. faecium strains.

Loss of suppression of tumorigenicity 2 (ST2) gene reverses sepsis-induced inhibition of lung host defense in mice.

RATIONALE: After surviving the initial hyperinflammatory phase, patients with sepsis display features consistent with immunosuppression, which renders the host susceptible to nosocomial infections, in particular bacterial pneumonia. Suppression of tumorigenicity 2 (ST2) is a negative regulator of Toll-like receptor signaling implicated in endotoxin tolerance. OBJECTIVES: The present study sought to determine the role of ST2 in modulating host defense in the lung during sepsis, using a murine model of cecal ligation and puncture (CLP)-induced sepsis followed by a secondary infection with Pseudomonas aeruginosa via the airways. METHODS: CLP or sham surgery was performed on BALB/c wild-type (WT) and ST2 knockout (KO) mice, and 24 hours later animals were challenged with 10(8) live P. aeruginosa. MEASUREMENTS AND MAIN RESULTS: CLP mice demonstrated impaired clearance of Pseudomonas from their lungs and reduced pulmonary levels of tumor necrosis factor-α and IL-6 compared with sham mice. After CLP, ST2KO mice with secondary pneumonia displayed a strongly improved survival and a better bacterial clearance compared with WT mice, which was accompanied by enhanced lung inflammation. CLP did not influence the responsiveness of alveolar macrophages toward P. aeruginosa ex vivo irrespective of the st2 genotype. In contrast, CLP resulted in a reduced capacity of WT CD4(+) and CD8(+) T cells to produce IFN-γ and tumor necrosis factor-α, an immune suppressive effect that was not seen in ST2KO mice. CONCLUSIONS: These findings indicate that gene products of ST2 contribute to the immune-compromised state during sepsis and the ensuing disturbed homeostasis of lung host defense.

The complement system facilitates clearance of Enterococcus faecium during murine peritonitis.

BACKGROUND: Infections with multidrug-resistant enterococci are a growing problem worldwide. Little is known about the host defense against enterococcal diseases. In vitro studies have demonstrated an important role played by complement proteins in neutrophil-mediated phagocytosis. In this study, we investigated the importance of complement in an in vivo model of Enterococcus faecium peritonitis. METHODS: Peripheral neutrophils and peritoneal macrophages were incubated with E. faecium that had been preincubated with decomplemented or normal plasma, and phagocytosis and killing were examined. E. faecium peritonitis was induced in C57BL/6 mice rendered complement deficient by intraperitoneal injection with cobra venom factor (CVF) and in complement 3 (C3) knockout mice. The course of the infection was compared with that in saline control and wild-type mice, respectively, at several time points up to 48 h after infection. RESULTS: Opsonization by complement enhanced phagocytosis by neutrophils and macrophages. CVF-treated and C3 knockout mice were severely hampered in clearing E. faecium from all organs and tissues under study (peritoneal fluid, blood, lungs, and liver). Higher peritoneal cytokine and chemokine levels were measured in decomplemented mice, whereas no differences in systemic or peritoneal cell kinetics were detected. CONCLUSION: Complement deficiency severely hampers the clearance of E. faecium peritonitis and subsequent systemic infection.

Intestinal colonization with Enterococcus faecium does not influence pulmonary defense against Pseudomonas aeruginosa in mice.

BACKGROUND: Enterococci, and especially multiresistant Enterococcus faecium, are increasingly found colonizing hospitalized patients. This increased prevalence of colonization is not only associated with an increased prevalence of infections caused by enterococci, but also by infections with other nosocomial pathogens. In this study we investigated the causality of this observed relationship, by determining the influence of intestinal colonization with E. faecium on pulmonary defense against Pseudomonas aeruginosa. METHODOLOGY/PRINCIPAL FINDINGS: Three groups of mice were tested; 2 groups of mice were pre-treated with vancomycin, of which one group was subsequently treated by oral gavage of vancomycin-resistant E. faecium (VRE). The third group did not receive any pre-treatment. P. aeruginosa pneumonia was induced in all mice. Vancomycin treatment resulted in intestinal gram-negative bacterial overgrowth and VRE treatment resulted in colonization throughout the intestines. All 3 groups of mice were able to clear P. aeruginosa from the lungs and circulation, with comparable lung cytokine responses and lung damage. Mice treated with vancomycin without VRE colonization displayed modestly increased plasma levels of TNF-alpha and IL-10. CONCLUSION: Overgrowth of E. faecium and/or gram-negative bacteria does not impact importantly on pulmonary defense against P. aeruginosa pneumonia.

Enterococcal surface protein transiently aggravates Enterococcus faecium-induced urinary tract infection in mice.

The role that the enterococcal surface protein Esp plays in the capacity of Enterococcus faecium to adhere to uroepithelial cells and the role that it plays in urinary tract infection and peritonitis was investigated in vitro and in vivo, respectively, using Esp-expressing E. faecium (E1162) and its isogenic Esp-deficient mutant (E1162 Delta esp). Esp expression enhanced in vitro binding to bladder and kidney epithelial cells. In mice, higher numbers of E1162 were cultured from kidneys and bladders after the induction of urinary tract infection, compared with E1162 Delta esp numbers. This was accompanied by a higher frequency of bacteremia, higher cytokine levels in kidney tissue, and renal insufficiency. Esp had no effect on the course of E. faecium peritonitis.

Intestinal Enterococcus faecium colonization improves host defense during polymicrobial peritonitis.

BACKGROUND: Vancomycin-resistant (VR) Enterococcus faecium is increasingly found to colonize and infect hospitalized patients. Enterococci are frequently isolated from polymicrobial infections originating from the intestines. The impact of VR E. faecium on these infections and vice versa is not clear. METHODS: Mice were intestinally colonized with VR E. faecium during oral vancomycin treatment; control mice received oral vancomycin only. Fourteen days later, cecal ligation and puncture (CLP) was performed in all mice to induce polymicrobial peritonitis in the presence or absence of VR E. faecium colonization. RESULTS: VR E. faecium colonization per se was not associated with systemic dissemination of VR E. faecium. CLP resulted in systemic VR E. faecium infection in all VR E. faecium-colonized mice, with high VR E. faecium loads in peritoneal lavage fluid, blood, liver, and lungs. Forty-eight hours after CLP, mice infected with VR E. faecium had significantly lower bacterial loads in all organs tested than mice not infected with VR E. faecium. Additionally, lower inflammatory parameters were measured in VR E. faecium-infected mice. CLP induced transient liver and kidney damage, with a faster recovery in VR E. faecium-colonized mice. CONCLUSIONS: VR E. faecium infection, originating from a natural source (the intestinal tract), does not worsen the outcome of CLP-induced polymicrobial peritonitis and sepsis but rather facilitates bacterial clearance and attenuates host inflammatory responses.

Cecal ligation and puncture induced sepsis impairs host defense against Enterococcus faecium peritonitis.

PURPOSE: Multiresistant and vancomycin resistant Enterococcus faecium (VRE) can cause serious infections in hospitalized patients with various co-morbid diseases. We investigated the course of VRE peritonitis after cecal ligation and puncture (CLP)-induced sepsis and compared this to sham operated mice. METHODS: Mice were subjected to CLP or sham surgery. Forty-eight hours thereafter four groups were created by subjecting mice to peritoneal injection of either VRE or saline. RESULTS: Mice infected with VRE after CLP were severely impaired in eliminating VRE from the peritoneal cavity and distant body sites. These mice failed to mount an early inflammatory response at the primary site of VRE infection. VRE superinfection did not influence CLP-induced organ damage or polymicrobial bacterial loads. CONCLUSIONS: Sublethal polymicrobial sepsis greatly facilitates infection and dissemination of VRE. VRE does not influence the course of CLP-induced sepsis.

Peritoneal macrophages are important for the early containment of Enterococcus faecium peritonitis in mice.

The increasing incidence of infections with multi-drug resistant Enterococcus faecium necessitates studies to increase knowledge on the pathogenesis of these infections. In this study, the contribution of peritoneal macrophages during E. faecium peritonitis was investigated. In an ex vivo setting, peritoneal macrophages harvested from C57BL/6 mice were responsive to, and able to phagocytose and kill, E. faecium. In vivo, peritoneal macrophages were depleted by intraperitoneal injection of clodronate-encapsulated liposomes, prior to inducing E. faecium peritonitis. Depletion of resident peritoneal macrophages caused a clear delay in peritoneal clearance of E. faecium with increased systemic dissemination. Mice depleted of peritoneal macrophages were able to recruit macrophages and neutrophils to the peritoneal cavity after infection, comparable to control mice. Furthermore, increased levels of peritoneal cytokines and chemokines were found in mice depleted of peritoneal macrophages. This study indicates that peritoneal macrophages are important in the early containment of E. faecium peritonitis and for the regulation of the inflammatory response.

Enterococcal surface protein Esp is not essential for cell adhesion and intestinal colonization of Enterococcus faecium in mice.

BACKGROUND: Enterococcus faecium has globally emerged as a cause of hospital-acquired infections with high colonization rates in hospitalized patients. The enterococcal surface protein Esp, identified as a potential virulence factor, is specifically linked to nosocomial clonal lineages that are genetically distinct from indigenous E. faecium strains. To investigate whether Esp facilitates bacterial adherence and intestinal colonization of E. faecium, we used human colorectal adenocarcinoma cells (Caco-2 cells) and an experimental colonization model in mice. RESULTS: No differences in adherence to Caco-2 cells were found between an Esp expressing strain of E. faecium (E1162) and its isogenic Esp-deficient mutant (E1162Deltaesp). Mice, kept under ceftriaxone treatment, were inoculated orally with either E1162, E1162Deltaesp or both strains simultaneously. Both E1162 and E1162Deltaesp were able to colonize the murine intestines with high and comparable numbers. No differences were found in the contents of cecum and colon. Both E1162 and E1162Deltaesp were able to translocate to the mesenteric lymph nodes. CONCLUSION: These results suggest that Esp is not essential for Caco-2 cell adherence and intestinal colonization or translocation of E. faecium in mice.

The acute-phase response impairs host defence against Enterococcus faecium peritonitis.

Enterococcus faecium is an emerging pathogen that causes infections in hospitalized patients with various co-morbid diseases. These underlying diseases are often associated with an acute-phase response that renders patients vulnerable to nosocomial infections. To study the influence of the acute-phase response induced by sterile tissue injury on host defence against E. faecium, mice were injected subcutaneously with either turpentine or casein 1 day before intraperitoneal infection with E. faecium. Control mice were subcutaneously injected with saline or sodium bicarbonate, respectively. Turpentine and casein induced an acute-phase response as reflected by increases in the plasma concentrations of interleukin-6, serum amyloid P and C3. A pre-existent acute-phase response in mice was associated with a strongly reduced capacity to clear E. faecium, resulting in prolonged bacteraemia for several days. The inflammatory response to E. faecium was impaired in mice with an acute-phase response, as shown by reduced capacity to mount a neutrophilic leucocytosis in peripheral blood and by decreased local cytokine concentrations. These data indicate that the acute-phase response impairs host defence against E. faecium, suggesting that this condition may contribute to the increased vulnerability of critically ill patients to enterococcal infections.

Neutrophils are essential for rapid clearance of Enterococcus faecium in mice.

A progressive increase in infections with multiresistant Enterococcus faecium has been reported, especially in cancer patients and neutropenic patients. Despite its increasing importance as a nosocomial pathogen, knowledge of the pathogenesis of E. faecium infections is highly limited. In this study, we investigated the role of neutrophils during peritonitis with subsequent bacteremia caused by E. faecium. Therefore, we depleted neutrophils by intraperitoneal injections of monoclonal antibody RB6-8C5. Mice were followed for 5 days, and the enterococcal outgrowth and inflammatory response were compared between neutropenic mice and immunoglobulin G-injected control mice. Neutropenic mice demonstrated a severe delay in enterococcal clearance from all cultured organs (peritoneal fluid, blood, and lung and liver tissue). In particular, neutropenic mice remained bacteremic for up to 3 days, whereas all nonneutropenic mice had cleared the bacteria from circulation by 2 days. Furthermore, neutropenic mice displayed elevated peritoneal cytokine and chemokine levels 1 day after the infection and attracted fewer macrophages into the peritoneal cavity. In the circulation, a prolonged elevation of tumor necrosis factor alpha, interleukin-6, and the acute-phase proteins serum amyloid A and complement 3 were measured in neutropenic mice. In conclusion, attraction of neutrophils to the primary site of E. faecium infection is important for a rapid clearance of this bacterium, thereby attenuating a systemic inflammatory response.

CD14 impairs host defense against gram-negative sepsis caused by Burkholderia pseudomallei in mice.

BACKGROUND: CD14 is a pattern-recognition receptor that can facilitate the presentation of bacterial components to either Toll-like receptor 2 (TLR2) or TLR4. We have recently shown that during melioidosis, a severe infection caused by the gram-negative bacterium Burkholderia pseudomallei, TLR2 but not TLR4 impacts the immune response of the intact host in vivo. METHODS: The function of CD14 in melioidosis was analyzed by means of in vitro and in vivo approaches, using wild-type (WT) and CD14 knockout (KO) mice. RESULTS: CD14-deficient macrophages and whole blood leukocytes released less tumor necrosis factor (TNF)-alpha on stimulation with B. pseudomallei or B. pseudomallei lipopolysaccharide in vitro, compared with WT cells. Strikingly, CD14 KO mice intranasally inoculated with B. pseudomallei demonstrated reduced lethality and significantly decreased bacterial outgrowth, compared with WT mice. Administration of recombinant soluble CD14 to CD14 KO mice partially reversed their phenotype to that of WT mice. Lastly, CD14 deficiency did not alter the capacity of macrophages or neutrophils to phagocytose or kill B. pseudomallei. CONCLUSION: CD14 is crucially involved in the recognition of B. pseudomallei by innate immune cells but plays a remarkable detrimental role in the host response against B. pseudomallei. Inhibition of CD14 may be a novel treatment strategy in melioidosis.

Endogenous beta-adrenergic receptors inhibit lipopolysaccharide-induced pulmonary cytokine release and coagulation.

Beta2-adrenergic receptors are expressed on different cell types in the lung, including respiratory epithelial cells, smooth muscle cells, and macrophages. The aim of the current study was to determine the role of beta-adrenergic receptors in the regulation of lung inflammation induced by instillation via the airways of lipopolysaccharide (LPS) (a constituent of the gram-negative bacterial cell wall) or lipoteichoic acid (LTA) (a component of the gram-positive bacterial cell wall). Mice inhaled the beta-adrenergic antagonist propranolol or saline 30 minutes before and 3 hours after intranasal LPS or LTA administration. LPS and LTA induced a profound inflammatory response in the lungs as reflected by an influx of neutrophils and the release of proinflammatory cytokines and chemokines into bronchoalveolar lavage fluid (BALF). Propranolol inhalation resulted in enhanced LPS-induced lung inflammation, which was reflected by a stronger secretion of TNF-alpha, IL-6, and monocyte chemoattractant protein-1 into BALF and by enhanced coagulation activation (thrombin-antithrombin complexes). In LTA-induced lung inflammation, propranolol did not influence cytokine release but potentiated activation of coagulation. Propranolol did not alter neutrophil recruitment in either model. This study suggests that beta-adrenergic receptors, which are widely expressed in the lungs, serve as negative regulators of pulmonary cytokine release and coagulation induced by LPS and less so during LTA-induced pulmonary inflammation.

TLR2-dependent MyD88 signaling contributes to early host defense in murine Enterococcus faecium peritonitis.

The incidence of infections with Enterococcus faecium is increasing worldwide. TLRs have been implicated in the recognition of pathogens and the initiation of an adequate innate immune response. We here sought to determine the roles of MyD88, the common adaptor protein involved in TLR signaling, TLR2, TLR4, and CD14 in host defense against E. faecium peritonitis. MyD88 knockout (KO) mice demonstrated an impaired early response to E. faecium peritonitis, as reflected by higher bacterial loads in peritoneal fluid and liver accompanied by a markedly attenuated neutrophil influx into the abdominal cavity. In vitro, not only MyD88 KO macrophages but also TLR2 KO and CD14 KO macrophages displayed a reduced responsiveness to E. faecium. In accordance, transfection of TLR2 rendered human embryonic kidney 293 cells responsive to E. faecium, which was enhanced by cotransfection of CD14. TLR2 KO mice showed higher bacterial loads in peritoneal fluid after in vivo infection with E. faecium and a diminished influx of neutrophils, whereas CD14 KO mice had an unaltered host response. E. faecium phagocytosis and killing were not affected by MyD88, TLR2, or CD14 deficiency. TLR4 did not play a role in the immune response to E. faecium in vitro or in vivo. These data suggest that MyD88 contributes to the effective clearance of E. faecium during peritonitis at least in part via TLR2 and by facilitating neutrophil recruitment to the site of the infection.

Lipoteichoic acid-induced lung inflammation depends on TLR2 and the concerted action of TLR4 and the platelet-activating factor receptor.

Lipoteichoic acid (LTA) is a major outer cell wall component of Gram-positive bacteria that has been implicated as an important factor in the inflammatory response following bacterial infection. In vitro data indicate roles for TLR2, platelet-activating factor receptor (PAFR), CD14, and LPS-binding protein (LBP) in cellular responsiveness to LTA, whereas the mechanisms contributing to LTA effects in vivo have never been investigated. Using mice deficient for LBP, CD14, TLR2, TLR4, or PAFR, we now examined the role of these molecules in pulmonary inflammation induced by highly purified LTA in vivo. Although pulmonary LBP increased dose-dependently following administration of LTA, the inflammatory response was unaltered in LBP-/- mice. TLR2 proved to be indispensable for the initiation of an inflammatory response, as polymorphonuclear cell influx, TNF-alpha, keratinocyte-derived chemokine, and MIP-2 release were abolished in TLR2-/- mice. Minor effects such as moderately decreased TNF-alpha and MIP-2 levels were observed in the absence of CD14, indicating a role for CD14 as a coreceptor. Quite surprisingly, the absence of TLR4 greatly diminished pulmonary inflammation and the same phenotype was observed in PAFR-/- animals. In contrast to all other mice studied, only TLR4-/- and PAFR-/- mice displayed significantly elevated IL-10 pulmonary concentrations. These data suggest that TLR2 is the single most important receptor signaling the presence of LTA within the lungs in vivo, whereas TLR4 and PAFR may influence lung inflammation induced by LTA either by sensing LTA directly or through recognition and signaling of endogenous mediators induced by the interaction between LTA and TLR2.