Miriam H.P. van Lieshout

R-roscovitine reduces lung inflammation induced by lipoteichoic acid and Streptococcus pneumoniae.

Bacterial pneumonia remains associated with high morbidity and mortality. The gram-positive pathogen Streptococcus pneumoniae is the most common cause of community-acquired pneumonia. Lipoteichoic acid (LTA) is an important proinflammatory component of the gram-positive bacterial cell wall. R-roscovitine, a purine analog, is a potent cyclin-dependent kinase (CDK)-1, -2, -5 and -7 inhibitor that has the ability to inhibit the cell cycle and to induce polymorphonuclear cell (PMN) apoptosis. We sought to investigate the effect of R-roscovitine on LTA-induced activation of cell lines with relevance for lung inflammation in vitro and on lung inflammation elicited by either LTA or viable S. pneumoniaein vivo. In vitro R-roscovitine enhanced apoptosis in PMNs and reduced tumor necrosis factor (TNF)-α and keratinocyte chemoattractant (KC) production in MH-S (alveolar macrophage) and MLE-12/MLE-15 (respiratory epithelial) cell lines. In vivo R-roscovitine treatment reduced PMN numbers in bronchoalveolar lavage fluid during LTA-induced lung inflammation; this effect was reversed by inhibiting apoptosis. Postponed treatment with R-roscovitine (24 and 72 h) diminished PMN numbers in lung tissue during gram-positive pneumonia; this step was associated with a transient increase in pulmonary bacterial loads. R-roscovitine inhibits proinflammatory responses induced by the gram-positive stimuli LTA and S. pneumoniae. R-roscovitine reduces PMN numbers in lungs upon LTA administration by enhancing apoptosis. The reduction in PMN numbers caused by R-roscovitine during S. pneumoniae pneumonia may hamper antibacterial defense.

NLRP3 and ASC Differentially Affect the Lung Transcriptome during Pneumococcal Pneumonia

Streptococcus pneumoniae is the most frequently isolated causative pathogen of community-acquired pneumonia, a leading cause of mortality worldwide. Inflammasomes are multiprotein complexes that play crucial roles in the regulation of inflammation. Nod-like receptor family, pyrin domain containing (NLRP) 3 is a sensor that functions in a single inflammasome, whereas adaptor apoptosis-associated speck-like protein containing a caspase activation and recruitment domain (ASC) is a common adaptor of several inflammasomes. We investigated the role of NLRP3 and ASC during S. pneumoniae pneumonia by comparing bacterial growth and spreading, and host innate immune responses in wild-type mice and mice deficient for either NLRP3 (Nlrp3(-/-)) or ASC (Asc(-/-)). Asc(-/-) mice had increased bacterial dissemination and lethality compared with Nlrp3(-/-) mice, although the cytokine response was impaired in both mouse strains. By detailed analysis of the early inflammatory response in the lung by whole-genome transcriptional profiling, we identified several mediators that were differentially expressed between Nlrp3(-/-) and Asc(-/-) mice. Of these, IL-17, granulocyte/macrophage colony-stimulating factor, and integrin-αM were significantly attenuated in Asc(-/-) relative to Nlrp3(-/-) mice, as well as a number of genes involved in the adaptive immune response. These differences may explain the increased susceptibility of Asc(-/ -) mice during S. pneumoniae infection, and suggest that either ASC-dependent NLRP3-independent inflammasomes or inflammasome-independent ASC functions may be involved.

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-β (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 pneumonia.

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

Klebsiella pneumoniae 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 Klebsiella. Here we investigated the role of MyD88 in myeloid and endothelial cells during Klebsiella pneumosepsis. Mice deficient for MyD88 in myeloid (LysM-Myd88−/−) and myeloid plus endothelial (Tie2-Myd88−/−) cells showed enhanced lethality and bacterial growth. Tie2-Myd88−/− 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-Myd88−/− 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.

Limited role for ASC and NLRP3 during in vivo Salmonella Typhimurium infection

BackgroundThe inflammasome is an intracellular protein complex triggered by exposure to intracellular pathogens, its components or other endogenous proteins. It leads to the activation of and subsequent release of proinflammatory cytokines such as IL-1β and IL-18. S. Typhimurium is a Gram-negative intracellular bacterium, which is known to trigger inflammasome assembly via recognition by the cytosolic receptors, NLRP3 and NLRC4 (which act via the adaptor protein, ASC) to induce cell death and cytokine release. We sought to characterize the role of ASC and NLRP3 in two different murine models (typhoid and colitis) of systemic Salmonella infection.ResultsRelease of the inflammasome cytokine IL-18 was hampered in Asc−/− but not Nlrp3−/− mice (background C57BL/6) during S. Typhimurium infection. Unexpectedly, neither ASC nor NLRP3 played a significant role in host defense against S. Typhimurium infection, as reflected by equal bacterial counts in WT, Asc−/− and Nlrp3−/− mice at all time points, in both the typhoid and colitis models. Proinflammatory cytokine levels (TNF-α, IL-6) and the extent of hepatic and splenic pathology did not differ between groups in the typhoid model. In the colitis model small differences were seen with regard to splenic and hepatic inflammation, although this was IL-18 independent.ConclusionsIL-18 release was reduced in Asc−/− but not Nlrp3−/− mice during S. Typhimurium infection. Despite this reduction, bacterial counts, cytokine levels and histological inflammation did not differ between wild-type and knockout mice in either model. Our results reveal a limited role for ASC and NLRP3 during in vivo S. Typhimurium infection despite its role in cytokine maturation.

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. pneumoniae.

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 cells.

Endoplasmic reticulum chaperone gp96 in macrophages is essential for protective immunity during Gram-negative pneumonia.

Klebsiella pneumoniae is among the most common Gram‐negative bacteria that cause pneumonia. Gp96 is an endoplasmic reticulum chaperone that is essential for the trafficking and function of Toll‐like receptors (TLRs) and integrins. To determine the role of gp96 in myeloid cells in host defence during Klebsiella pneumonia, mice homozygous for the conditional Hsp90b1 allele encoding gp96 were crossed with mice expressing Cre‐recombinase under control of the LysM promoter to generate LysMcre‐Hsp90b1‐flox mice. LysMcre‐Hsp90b1‐flox mice showed absence of gp96 protein in macrophages and partial depletion in monocytes and granulocytes. This was accompanied by almost complete absence of TLR2 and TLR4 on macrophages. Likewise, integrin subunits CD11b and CD18 were not detectable on macrophages, while being only slightly reduced on monocytes and granulocytes. Gp96‐deficient macrophages did not release pro‐inflammatory cytokines in response to Klebsiella and displayed reduced phagocytic capacity independent of CD18. LysMcre‐Hsp90b1‐flox mice were highly vulnerable to lower airway infection induced by K. pneumoniae, as reflected by enhanced bacterial growth and a higher mortality rate. The early inflammatory response in Hsp90b1‐flox mice was characterized by strongly impaired recruitment of granulocytes into the lungs, accompanied by attenuated production of pro‐inflammatory cytokines, while the inflammatory response during late‐stage pneumonia was not dependent on the presence of gp96. Blocking CD18 did not reproduce the impaired host defence of LysMcre‐Hsp90b1‐flox mice during Klebsiella pneumonia. These data indicate that macrophage gp96 is essential for protective immunity during Gram‐negative pneumonia by regulating TLR expression. Copyright

Role of Nucleotide-Binding Oligomerization Domain-Containing (NOD) 2 in Host Defense during Pneumococcal Pneumonia

Streptococcus (S.) pneumoniae is the most common causative pathogen in community-acquired pneumonia. Nucleotide-binding oligomerization domain-containing (NOD) 2 is a pattern recognition receptor located in the cytosol of myeloid cells that is able to detect peptidoglycan fragments of S. pneumoniae. We here aimed to investigate the role of NOD2 in the host response during pneumococcal pneumonia. Phagocytosis of S. pneumoniae was studied in NOD2 deficient (Nod2 -/-) and wild-type (Wt) alveolar macrophages and neutrophils in vitro. In subsequent in vivo experiments Nod2 -/- and Wt mice were inoculated with serotype 2 S. pneumoniae (D39), an isogenic capsule locus deletion mutant (D39Δcps) or serotype 3 S. pneumoniae (6303) via the airways, and bacterial growth and dissemination and the lung inflammatory response were evaluated. Nod2 -/- alveolar macrophages and blood neutrophils displayed a reduced capacity to internalize pneumococci in vitro. During pneumonia caused by S. pneumoniae D39 Nod2 -/- mice were indistinguishable from Wt mice with regard to bacterial loads in lungs and distant organs, lung pathology and neutrophil recruitment. While Nod2 -/- and Wt mice also had similar bacterial loads after infection with the more virulent S. pneumoniae 6303 strain, Nod2 -/- mice displayed a reduced bacterial clearance of the normally avirulent unencapsulated D39Δcps strain. These results suggest that NOD2 does not contribute to host defense during pneumococcal pneumonia and that the pneumococcal capsule impairs recognition of S. pneumoniae by NOD2.

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 pneumonia.