TLR4 inhibition impairs bacterial clearance in a therapeutic setting in murine abdominal sepsis

To investigate the therapeutic effect of E5564 (a clinically used TLR4 inhibitor) in murine abdominal sepsis elicited by intraperitoneal infection with a highly virulent Escherichia coli in the context of concurrent antibiotic therapy. Mice were infected with different doses (~2 × 10(4)-2 × 10(6) CFU) of E. coli O18:K1 and treated after 8 h with ceftriaxone 20 mg/kg i.p. combined with either E5564 10 mg/kg i.v. or vehicle. For survival studies this treatment was repeated every 12 h. Bacterial loads and inflammatory parameters were determined after 20 h in peritoneal lavage fluid, blood, liver and lung tissue. Plasma creatinin, AST, ALT and LDH were determined to assess organ injury. E5564 impaired bacterial clearance under the antibiotic regime after infection with a low dose E. coli (1.7 × 10(4) CFU) while renal function was slightly preserved. No differences were observed in bacterial load and organ damage after infection with a tenfold higher (1.7 × 10(5) E. coli) bacterial dose. While treatment with E5564 slightly attenuated inflammatory markers provoked by the sublethal doses of 104-105 E. coli under the antibiotic regime, it did not affect lethality evoked by infection with 1.7 × 106 E. coli. The impact of TLR4 inhibition during abdominal sepsis by virulent E. coli bacteria is only beneficial at low infection grade at cost of bactericidal activit

Modular Transcriptional Networks of the Host Pulmonary Response during Early and Late Pneumococcal Pneumonia

Streptococcus pneumoniae (Spneu) remains the most lethal bacterial pathogen and the dominant agent of community-acquired pneumonia. Treatment has perennially focused on the use of antibiotics, albeit scrutinized due to the occurrence of antibiotic-resistant Spneu strains. Immunomodulatory strategies have emerged as potential treatment options. Although promising, immunomodulation can lead to improper tissue functions either at steady state or upon infectious challenge. This argues for the availability of tools to enable a detailed assessment of whole pulmonary functions during the course of infection, not only those functions biased to the defense response. Thus, through the use of an unbiased tissue microarray and bioinformatics approach, we aimed to construct a comprehensive map of whole-lung transcriptional activity and cellular pathways during the course of pneumococcal pneumonia. We performed genome-wide transcriptional analysis of whole lungs before and 6 and 48 h after Spneu infection in mice. The 4,000 most variable transcripts across all samples were used to assemble a gene coexpression network comprising 13 intercorrelating modules (clusters of genes). Fifty-four percent of this whole-lung transcriptional network was altered 6 and 48 h after Spneu infection. Canonical signaling pathway analysis uncovered known pathways imparting protection, including IL17A/IL17F signaling and previously undetected mechanisms that included lipid metabolism. Through in silico prediction of cell types, pathways were observed to enrich for distinct cell types such as a novel stromal cell lipid metabolism pathway. These cellular mechanisms were furthermore anchored at functional hub genes of cellular fate, differentiation, growth and transcription. Collectively, we provide a benchmark unsupervised map of whole-lung transcriptional relationships and cellular activity during early and late pneumococcal pneumoni

TIR-Domain-Containing Adaptor-Inducing Interferon-β (TRIF) Mediates Antibacterial Defense during Gram-Negative Pneumonia by Inducing Interferon-x03B3

Klebsiella pneumoniae is an important cause of Gram-negative pneumonia and sepsis. Mice deficient for TIR-domain-containing adaptor-inducing interferon-β (TRIF) demonstrate enhanced bacterial growth and dissemination during Klebsiella pneumonia. We show here that the impaired antibacterial defense of TRIF mutant mice is associated with absent interferon (IFN)-x03B3; production in the lungs. IFN-x03B3; production by splenocytes in response to K. pneumoniae in vitro was critically dependent on Toll-like receptor 4 (TLR4), the common TLR adaptor myeloid differentiation primary response gene (MyD88) and TRIF. Reconstitution of TRIF mutant mice with recombinant IFN-x03B3; via the airways reduced bacterial loads in lungs and distant body sites to levels measured in wild-type mice, and partially restored pulmonary cytokine levels. The IFN-x03B3;-induced, improved, enhanced antibacterial response in TRIF mutant mice occurred at the expense of increased hepatocellular injury. These data indicate that TRIF mediates antibacterial defense during Gram-negative pneumonia, at least in part, by inducing IFN-x03B3; at the primary site of infectio

Differential Roles of MyD88 and TRIF in Hematopoietic and Resident Cells During Murine Gram-Negative Pneumonia

Background. Pneumonia is frequently caused by gram-negative pathogens, among which Klebsiella pneumoniae prominently features. Recognition of pathogen-associated molecular patterns by Toll-like receptors (TLRs) is important for an appropriate immune response during infection. TLR signaling can proceed via 2 distinct routes that are dependent on the adaptor proteins Myeloid differentiation primary response gene (88) (MyD88) and TIR-domain-containing adaptor-inducing interferon-beta (TRIF). The aim of the study was to determine the relative contribution of MyD88 and TRIF signaling in resident and hematopoietic cells to host defense during pneumonia. Methods. Bone marrow chimeras of MyD88 deficient/wild type and TRIF mutant/wild type mice were created and infected with K. pneumoniae via the airways. Results. MyD88 in both resident and hematopoietic cells contributed to survival and antibacterial defense in late-stage infection, whereas only TRIF in hematopoietic cells was protective. On the other hand, resident MyD88 and hematopoietic TRIF contributed to distant cellular injury. Resident MyD88 was pivotal for early chemokine release and neutrophil recruitment in the bronchoalveolar space. Conclusions. MyD88- and TRIF-dependent signaling has a differential contribution to host defense in different cell types that changes from early-to late-stage gram-negative pneumoni

Hematopoietic but Not Endothelial Cell MyD88 Contributes to Host Defense during Gram-negative Pneumonia Derived Sepsis

<div><p><i>Klebsiella pneumoniae</i> is an important cause of sepsis. The common Toll-like receptor adapter myeloid differentiation primary response gene (MyD)88 is crucial for host defense against <i>Klebsiella</i>. Here we investigated the role of MyD88 in myeloid and endothelial cells during <i>Klebsiella</i> pneumosepsis. Mice deficient for MyD88 in myeloid (LysM-<i>Myd88^−/−</i>) and myeloid plus endothelial (Tie2-<i>Myd88^−/−</i>) cells showed enhanced lethality and bacterial growth. Tie2-<i>Myd88^−/−</i> mice reconstituted with control bone marrow, representing mice with a selective MyD88 deficiency in endothelial cells, showed an unremarkable antibacterial defense. Myeloid or endothelial cell MyD88 deficiency did not impact on lung pathology or distant organ injury during late stage sepsis, while LysM-<i>Myd88^−/−</i> mice demonstrated a strongly attenuated inflammatory response in the airways early after infection. These data suggest that myeloid but not endothelial MyD88 is important for host defense during gram-negative pneumonia derived sepsis.</p></div

Lung epithelial MyD88 drives early pulmonary clearance of Pseudomonas aeruginosa by a flagellin dependent mechanism

Pseudomonas aeruginosa is a flagellated pathogen frequently causing pneumonia in hospitalized patients and sufferers of chronic lung disease. Here we investigated the role of the common Toll-like receptor (TLR) adaptor myeloid differentiation factor (MyD)88 in myeloid vs. lung epithelial cells in clearance of P. aeruginosa from the airways. Mice deficient for MyD88 in lung epithelial cells (Sftpccre-MyD88-lox mice) or myeloid cells (LysMcre-MyD88-lox mice) and bone marrow chimeric mice deficient for TLR5 (the receptor recognizing Pseudomonas flagellin) in either parenchymal or hematopoietic cells were infected with P. aeruginosa via the airways. Sftpccre-MyD88-lox mice demonstrated a reduced influx of neutrophils into the bronchoalveolar space and an impaired early antibacterial defense after infection with P. aeruginosa, whereas the response of LysMcre-MyD88-lox mice did not differ from control mice. The immune-enhancing role of epithelial MyD88 was dependent on recognition of pathogen-derived flagellin by epithelial TLR5, as demonstrated by an unaltered clearance of mutant P. aeruginosa lacking flagellin from the lungs of Sftpccre-MyD88-lox mice and an impaired bacterial clearance in bone marrow chimeric mice lacking TLR5 in parenchymal cells. These data indicate that early clearance of P. aeruginosa from the airways is dependent on flagellin-TLR5-MyD88-dependent signaling in respiratory epithelial cell

Single immunoglobulin interleukin-1 receptor-related molecule impairs host defense during pneumonia and sepsis caused by Streptococcus pneumoniae

Streptococcus pneumoniae is a common cause of pneumonia and sepsis. Toll-like receptors (TLRs) play a pivotal role in the host defense against infection. In this study, we sought to determine the role of single immunoglobulin interleukin-1 receptor-related molecule (SIGIRR a.k.a. TIR8), a negative regulator of TLR signaling, in pneumococcal pneumonia and sepsis. Wild-type and SIGIRR-deficient (sigirr-/-) mice were infected intranasally (to induce pneumonia) or intravenously (to induce primary sepsis) with S. pneumoniae and euthanized after 6, 24, or 48 h for analyses. Additionally, survival studies were performed. sigirr-/- mice showed delayed mortality during lethal pneumococcal pneumonia. Accordingly, sigirr-/- mice displayed lower bacterial loads in lungs and less dissemination of the infection 24 h after the induction of pneumonia. SIGIRR deficiency was associated with increased interstitial and perivascular inflammation in lung tissue early after infection, with no impact on neutrophil recruitment or cytokine production. sigirr-/- mice also demonstrated reduced bacterial burdens at multiple body sites during S. pneumoniae sepsis. sigirr-/- alveolar macrophages and neutrophils exhibited an increased capacity to phagocytose viable pneumococci. These results suggest that SIGIRR impairs the antibacterial host defense during pneumonia and sepsis caused by S. pneumonia

Genetic and functional characterization of primary cells from LysM-<i>Myd88^−/−</i> and Tie2-<i>Myd88^−/−</i> mice.

<p>The residual amount of the MyD88^fl/fl allel in blood and primary cells LysM-<i>Myd88^−/−</i> and Tek-<i>Myd88^−/−</i> mice was quantified via qRT-PCR relative to the unaffected <i>Socs-3</i> gene. The amount of remaining “floxed” MyD88 region in LysM-MyD88^−/− and Tek-MyD88^−/− mice was calculated using the 2^-deltaCt (ΔΔCt) method using the amount of genomic DNA from <i>Myd88^fl/fl</i> mice for the no-deletion control. The deletion efficiency was calculated as (1 - residual Myd88^fl) ×100% <b>(A)</b>. Whole blood <b>(B)</b>, alveolar and peritoneal macrophages <b>(C,D)</b> and splenocytes <b>(E)</b> derived from control, LysM-<i>Myd88^−/−</i> and Tie2-<i>Myd88^−/−</i> mice (n = 3 per group) were in vitro stimulated with LPS derived from <i>Klebsiella pneumoniae</i> (1 µg/ml) or heat killed <i>K. pneumoniae</i> in two concentrations (2×10⁵ CFU/ml or 2×10⁷/ml) for 20 hours. Data are expressed as mean (SE). * <i>p</i><0.05, ** <i>p</i><0.01, *** <i>p</i><0.001.</p

Inflammatory response in <i>LysM-MyD88^−/−</i> and <i>Tie2-MyD88^−/−</i> during <i>K. pneumonia</i> pulmonary tract infection.

<p>Control, LysM-MyD88^−/− and Tie2-MyD88^−/− mice were inoculated with ∼6×10³ CFU <i>K. pneumoniae</i> and sacrificed 24 hours later. Homogenates were prepared from right lungs. Cytokine and chemokine levels are presented in pg/ml lung homogenate or plasma. Data are mean (SE) of 5–8 mice per group.</p><p>*<i> p</i><0.05,</p><p>** <i>p</i><0.01 vs control mice.</p><p>Bd = below detection.</p><p>Inflammatory response in <i>LysM-MyD88^−/−</i> and <i>Tie2-MyD88^−/−</i> during <i>K. pneumonia</i> pulmonary tract infection.</p