[Show abstract][Hide abstract] ABSTRACT: The Gram-negative bacterium Burkholderia pseudomallei causes melioidosis and is a CDC category B bioterrorism agent. Toll-like receptor (TLR)-2 impairs host defense during pulmonary B.pseudomallei infection while TLR4 only has limited impact. We investigated the role of TLRs in B.pseudomallei-lipopolysaccharide (LPS) induced inflammation. Purified B.pseudomallei-LPS activated only TLR2-transfected-HEK-cells during short stimulation but both HEK-TLR2 and HEK-TLR4-cells after 24 h. In human blood, an additive effect of TLR2 on TLR4-mediated signalling induced by B.pseudomallei-LPS was observed. In contrast, murine peritoneal macrophages recognized B.pseudomallei-LPS solely through TLR4. Intranasal inoculation of B.pseudomallei-LPS showed that both TLR4-knockout(-/-) and TLR2x4-/-, but not TLR2-/- mice, displayed diminished cytokine responses and neutrophil influx compared to wild-type controls. These data suggest that B.pseudomallei-LPS signalling occurs solely through murine TLR4, while in human models TLR2 plays an additional role, highlighting important differences between specificity of human and murine models that may have important consequences for B.pseudomallei-LPS sensing by TLRs and subsequent susceptibility to melioidosis.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: Objective:
Pneumonia accounts for more deaths than any other infectious disease worldwide. The intestinal microbiota supports local mucosal immunity and is increasingly recognised as an important modulator of the systemic immune system. The precise role of the gut microbiota in bacterial pneumonia, however, is unknown. Here, we investigate the function of the gut microbiota in the host defence against Streptococcus pneumoniae infections.
We depleted the gut microbiota in C57BL/6 mice and subsequently infected them intranasally with S. pneumoniae. We then performed survival and faecal microbiota transplantation (FMT) experiments and measured parameters of inflammation and alveolar macrophage whole-genome responses.
We found that the gut microbiota protects the host during pneumococcal pneumonia, as reflected by increased bacterial dissemination, inflammation, organ damage and mortality in microbiota-depleted mice compared with controls. FMT in gut microbiota-depleted mice led to a normalisation of pulmonary bacterial counts and tumour necrosis factor-α and interleukin-10 levels 6 h after pneumococcal infection. Whole-genome mapping of alveolar macrophages showed upregulation of metabolic pathways in the absence of a healthy gut microbiota. This upregulation correlated with an altered cellular responsiveness, reflected by a reduced responsiveness to lipopolysaccharide and lipoteichoic acid. Compared with controls, alveolar macrophages derived from gut microbiota-depleted mice showed a diminished capacity to phagocytose S. pneumoniae.
This study identifies the intestinal microbiota as a protective mediator during pneumococcal pneumonia. The gut microbiota enhances primary alveolar macrophage function. Novel therapeutic strategies could exploit the gut-lung axis in bacterial infections.
[Show abstract][Hide abstract] ABSTRACT: Asthma patients show evidence of a procoagulant state in their airways, accompanied by an impaired function of the anticoagulant protein C system. We aimed to study the effect of recombinant human activated protein C (rhAPC) in allergic asthma patients.We conducted a randomised, double-blind, placebo-controlled, proof-of-concept study in house dust mite (HDM) allergic asthma patients. Patients were randomised to receive intravenous rhAPC (24 microg.kg-1.h-1; n=12) or placebo (n=12) for 11 h. 4 h after the start of infusion, a first bronchoscopy was performed to challenge one lung segment with saline (control) and a contralateral segment with a combination of HDM extract and lipopolysaccharide (HDM+LPS), thereby mimicking environmental house dust exposure. A second bronchoscopy was conducted 8 h after intrabronchial challenge to obtain bronchoalveolar lavage fluid (BALF).rhAPC did not influence HDM+LPS induced procoagulant changes in the lung. In contrast, rhAPC reduced BALF leukocyte counts by 43% relative to placebo, caused by an inhibitory effect on neutrophil influx (64% reduction), while leaving eosinophil influx unaltered. rhAPC also reduced neutrophil degranulation products in the airways.Intravenous rhAPC attenuates HDM+LPS-induced neutrophil migration and protein release in allergic asthma patients by an effect that does not rely on coagulation inhibition.
Full-text · Article · Sep 2015 · European Respiratory Journal
[Show abstract][Hide abstract] ABSTRACT: 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 an enhanced bacterial growth and a higher mortality rate. The early inflammatory response in Hsp90b1-flox mice was characterized by a strongly impaired recruitment of granulocytes into the lungs, accompanied by an attenuated production of proinflammatory 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.
No preview · Article · Sep 2015 · The Journal of Pathology
[Show abstract][Hide abstract] ABSTRACT: Triggering Receptor Expressed on Myeloid cells (TREM)-1 and -2 can affect Toll-like receptor mediated activation of immune cells. Klebsiella (K.) pneumoniae is a common cause of pneumonia-derived sepsis. Here we studied the role of TREM-1/3 and TREM-2 in the host response during Klebsiella pneumonia. Macrophages lacking either TREM-1/3 or TREM-2 were tested for their responsiveness towards K. pneumoniae as well as their capacity to internalize this pathogen in vitro. TREM-1/3 and TREM-2 deficient mice were infected with K. pneumoniae via the airways and their responses were compared with those in wild type mice. TREM-1/3 deficient macrophages produced lower cytokine levels upon exposure to K. pneumoniae, while on the opposite TREM-2 deficient macrophages released higher cytokine concentrations. TREM-2 deficient, but not TREM-1/3 deficient, macrophages showed a reduced capacity to phagocytose K. pneumoniae. TREM-1/3 deficient mice showed an impaired host defense during Klebsiella pneumonia, as reflected by worsened survival and increased bacterial growth and dissemination. In contrast, TREM-2 deficiency did not impact on disease outcome. Although TREM-1/3 and TREM-2 influence macrophage responsiveness to K. pneumoniae in vitro, only TREM-1/3 contribute to the host response during Klebsiella pneumonia in vivo, serving a protective role.
No preview · Article · Apr 2015 · American Journal of Respiratory Cell and Molecular Biology
[Show abstract][Hide abstract] ABSTRACT: Typhoid fever, caused by the Gram-negative bacterium Salmonella enterica serovar Typhi, is a major cause of community-acquired bacteremia and death worldwide. S100A8 (MRP8) and S100A9 (MRP14) form bioactive antimicrobial heterodimers (calprotectin) that can activate Toll-like receptor 4, promoting lethal, endotoxin-induced shock and multi-organ failure. We aimed to characterize the expression and function of S100A8/A9 in patients with typhoid fever and in a murine invasive Salmonella model.
S100A8/A9 protein levels were determined in acute phase plasma or feces from 28 Bangladeshi patients, and convalescent phase plasma from 60 Indonesian patients with blood culture or PCR-confirmed typhoid fever, and compared to 98 healthy control subjects. To functionally characterize the role of S100A8/A9, we challenged wildtype (WT) and S100A9-/- mice with S. Typhimurium and determined bacterial loads and inflammation 2- and 5- days post infection. We further assessed the antimicrobial function of recombinant S100A8/A9 on S. Typhimurium and S. Typhi replication in vitro. Typhoid fever patients demonstrated a marked increase of S100A8/A9 in acute phase plasma and feces and this increases correlated with duration of fever prior to admission. S100A8/A9 directly inhibited the growth of S. Typhimurium and S. Typhi in vitro in a dose and time dependent fashion. WT mice inoculated with S. Typhimurium showed increased levels of S100A8/A9 in both the liver and the systemic compartment but S100A9-/- mice were indistinguishable from WT mice with respect to bacterial growth, survival, and inflammatory responses, as determined by cytokine release, histopathology and organ injury.
S100A8/A9 is markedly elevated in human typhoid, correlates with duration of fever prior to admission and directly inhibits the growth of S. Typhimurium and S. Typhi in vitro. Despite elevated levels in the murine invasive Salmonella model, S100A8/A9 does not contribute to an effective host response against S. Typhimurium in mice.
[Show abstract][Hide abstract] ABSTRACT: Tuberculosis (TB) is an important cause of morbidity and mortality worldwide. Toll-like-receptors (TLRs) are important for the recognition of the causative agent Mycobacterium tuberculosis. Negative regulation of TLRs is necessary to control deleterious inflammatory damage, but could provide a means of immune evasion by M. tuberculosis as well.
To obtain insight in the extent of expression of inhibitory regulators of immunity in patients with active TB, peripheral-blood-mononuclear-cells (PBMCs) and plasma were obtained from 54 TB patients and 29 healthy blood donors from Chittagong, Bangladesh. Bilateral alveolar macrophages were obtained from an infected versus a contralateral normal lung segment of 9 patients. Statistical analyses were performed using Mann–Whitney U and Wilcoxon matched pairs testing. Correlations were calculated using the Spearman rho test.
PBMCs harvested from TB patients demonstrated increased mRNA expression of IL-1-receptor-associated-kinase-M, suppressor-of-cytokine-signalling-3 and Toll-interacting-protein. Flow cytometry revealed enhanced expression of IL-1-receptor-like-1 (ST2) on lymphocytes. Plasma soluble ST2 was elevated in patients with TB and correlated with established TB biomarkers, most strongly with soluble interleukin-2 receptor subunit α and interleukin-8. Alveolar macrophage mRNA expression of negative TLR regulators did not differ between the infected and contralateral lung side.
These results show enhanced expression of distinct negative regulators of innate immunity in PBMCs of patients with TB and identify plasma soluble ST2 as a potential novel biomarker for TB disease activity.
Full-text · Article · Feb 2015 · BMC Infectious Diseases
[Show abstract][Hide abstract] ABSTRACT: Toll-like receptors (TLR) and the downstream adaptor protein MyD88 are considered crucial for protective immunity during bacterial infections. Streptococcus (S.) pneumoniae is a human respiratory pathogen and a large majority of clinical pneumococcal isolates expresses an external polysaccharide capsule. We here sought to determine the role of pneumococcal capsule in MyD88-mediated antibacterial defense during S. pneumonia pneumonia. Wild type (WT) and Myd88-/- mice were inoculated intranasally with serotype 2 S. pneumoniae D39 or with an isogenic capsule locus deletion mutant (D39∆cps), and analysed for bacterial outgrowth and inflammatory responses in the lung. As compared to WT mice, Myd88-/- mice infected with D39 demonstrated a modestly impaired bacterial clearance accompanied by decreased inflammatory responses in the lung. Strikingly, while WT mice rapidly cleared D39∆cps, Myd88-/- mice showed 105-fold higher bacterial burdens in their lungs and dissemination to blood 24 hours after infection. These data suggest that the pneumococcal capsule impairs recognition of TLR ligands expressed by S. pneumoniae and thereby partially impedes MyD88-mediated antibacterial defense.
[Show abstract][Hide abstract] ABSTRACT: Objectives: Streptococcus pneumoniae is the most common causative organism in community-acquired pneumonia responsible for millions of deaths every year. DNAX-activating protein of 12 kDa is an adaptor molecule for different myeloid expressed receptors involved in innate immunity. Design: Animal study. Setting: University research laboratory. Subjects: DNAX-activating protein of 12 kDa–deficient (dap12–/–) and wild-type mice. Interventions: Mice were intranasally infected with S. pneumoniae. In addition, ex vivo responsiveness of alveolar macrophages was examined. Measurements and Main Results: dap12–/– alveolar macrophages released more tumor necrosis factor-α upon stimulation with S. pneumoniae and displayed increased phagocytosis of this pathogen compared with wild-type cells. After infection with S. pneumoniae via the airways, dap12–/– mice demonstrated reduced bacterial outgrowth in the lungs together with delayed dissemination to distant body sites relative to wild-type mice. This favorable response in dap12–/– mice was accompanied by reduced lung inflammation and an improved survival. Conclusions: These data suggest that DNAX-activating protein of 12 kDa impairs host defense during pneumococcal pneumonia at the primary site of infection at least in part by inhibiting phagocytosis by alveolar macrophages.
No preview · Article · Dec 2014 · Critical Care Medicine
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: Background Streptococcus pneumoniae is the most commonly identified pathogen in community-acquired pneumonia (CAP). Myeloid-related protein (MRP) 8/14 is a major component of neutrophils that is released upon infection or injury. MRP8/14 is essential for protective immunity during infection by a variety of micro-organisms through its capacity to chelate manganese and zinc. Here, we aimed to determine the role of MRP8/14 in pneumococcal pneumonia.
Methods MRP8/14 was determined in bronchoalveolar lavage fluid (BALF) and serum of CAP patients, in lung tissue of patients who had succumbed to pneumococcal pneumonia, and in BALF of healthy subjects challenged with lipoteichoic acid (a component of the gram-positive bacterial cell wall) via the airways. Pneumonia was induced in MRP14 deficient and normal wildtype mice. The effect of MRP8/14 on S. pneumoniae growth was studied in vitro.
Results CAP patients displayed high MRP8/14 levels in BALF, lung tissue and serum. Healthy subjects challenged with lipoteichoic acid demonstrated elevated MRP8/14 in BALF. Likewise, mice with pneumococcal pneumonia had high MRP8/14 levels in lungs and the circulation. MRP14 deficiency, however, was associated with reduced bacterial growth and lethality, in the absence of notable effects on the inflammatory response. High zinc levels strongly inhibited growth of S. pneumoniae in vitro, which was partially reversed by MRP8/14.
Conclusions In sharp contrast to its previously reported host-protective role in several infections, the present results reveal that in a model of CAP, MRP8/14 is misused by S. pneumoniae, facilitating bacterial growth by attenuating zinc toxicity toward the pathogen.
[Show abstract][Hide abstract] ABSTRACT: Streptococcus (S.) pneumoniae is a common gram-positive pathogen in community-acquired pneumonia and sepsis. Triggering Receptor Expressed on Myeloid cells-1 (TREM-1) is a receptor on phagocytes known to amplify inflammatory responses. Previous studies showed that TREM-1 inhibition protects against lethality during experimental gram-negative sepsis. We here aimed to investigate the role of TREM-1 in an experimental model of pneumococcal pneumonia using TREM-1/3 deficient (Trem-1/3(-/-) ) and wild type (Wt) mice. Additionally ex vivo responsiveness of Trem-1/3(-/-n) eutrophils and macrophages was examined. S. pneumoniae infection resulted in a rapid recruitment of TREM-1 positive neutrophils into the bronchoalveolar space while high constitutive TREM-1 expression on alveolar macrophages remained unchanged. TREM-1/3 deficiency led to increased lethality accompanied by enhanced growth of S. pneumoniae at the primary site of infection and increased dissemination to distant organs. Within the first 3-6 hours of infection Trem-1/3(-/-m) ice demonstrated a strongly impaired innate immune response in the airways, as reflected by reduced local release of cytokines and chemokines and a delayed influx of neutrophils. Trem-1/3(-/-a) lveolar macrophages produced less cytokines upon exposure to S. pneumoniae in vitro and were less capable of phagocytosing this pathogen. TREM-1/3 deficiency did not influence neutrophil responsiveness to S. pneumoniae. These results identify TREM-1 as key player in protective innate immunity during pneumococcal pneumonia most likely by enhancing the early immune response of alveolar macrophages.
No preview · Article · Aug 2014 · The Journal of Pathology
[Show abstract][Hide abstract] ABSTRACT: Staphylococcus (S.) aureus has emerged as an important cause of necrotizing pneumonia. Lung injury during S. aureus pneumonia may be enhanced by local release of damage associated molecular patterns such as high-mobility group box 1 (HMGB1). In the current study we sought to determine the functional role of HMGB1 and its receptors, toll-like receptor 4 (TLR4) and the receptor for advanced glycation end products (RAGE), in the injurious host response to S. aureus pneumonia.
Pneumonia was induced in wild type (Wt), TLR4 deficient (tlr4-/-) and RAGE deficient (rage-/-) mice by intranasal inoculation of 1 x 107 colony-forming units (CFU) of a USA300 S. aureus. In a separate set of experiments, Wt mice were injected intraperitoneally with a monoclonal anti-HMGB1 antibody or an isotype matched control antibody immediately before and every 24 hours after intranasal infection of S. aureus. Mice were sacrificed at 6, 24, 48 or 72 hours after infection for harvesting of blood and organs.
S. aureus pneumonia was associated with HMGB1 release in the bronchoalveolar compartment peaking after 24 hours. Anti-HMGB1 attenuated lung pathology and protein leak and reduced interleukin-1beta release 6 hours after infection, but not at later time points. RAGE deficiency more modestly attenuated lung pathology without influencing protein leak, while TLR4 deficiency did not impact on lung injury.
These data suggest that HMGB1 and RAGE, but not TLR4, contribute to lung injury accompanying the early phase of S. aureus pneumonia.
Full-text · Article · Dec 2013 · Critical care (London, England)