Regina de Beer

The gut microbiota plays a protective role in the host defence against pneumococcal pneumonia

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. Design 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. Results 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. Conclusions 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.

Inflammation patterns induced by different Burkholderia species in mice

Burkholderia pseudomallei, which causes melioidosis, a severe, mainly pulmonary disease endemic in South‐East Asia, is considered to be the most pathogenic of the Burkholderia genus. B. thailandensis, however, is considered avirulent. We determined differences in patterns of inflammation of B. pseudomallei 1026b (clinical virulent isolate), B. pseudomallei AJ1D8 (an in vitro invasion‐deficient mutant generated from strain 1026b by Tn5‐OT182 mutagenesis) and B. thailandensis by intranasally inoculating C57BL/6 mice with each strain. Mice infected with B. thailandensis showed a markedly decreased bacterial outgrowth from lungs, spleen and blood 24 h after inoculation, compared with infection with B. pseudomallei and the invasion mutant AJ1D8. Forty‐eight hours after inoculation, B. thailandensis was no longer detectable. This was consistent with elevated pulmonary cytokine and chemokine concentrations after infection with B. pseudomallei 1026b and AJ1D8, and the absence of these mediators 48 h, but not 24 h, after inoculation with B. thailandensis. Histological examination, however, did show marked pulmonary inflammation in the mice infected with B. thailandensis, corresponding with substantial granulocyte influx and raised myeloperoxidase levels. Survival experiments showed that infection with 1 × 103 cfu B. thailandensis was not lethal, whereas inoculation with 1 × 106 cfu B. thailandensis was equally lethal as 1 × 103 cfu B. pseudomallei 1026b or AJ1D8. These data show that B. pseudomallei AJ1D8 is just as lethal as wild‐type B. pseudomallei in an in vivo mouse model, and B. thailandensis is perhaps more virulent than is often recognized.

Receptor for Advanced Glycation End Products Facilitates Host Defense during Escherichia coli–Induced Abdominal Sepsis in Mice

BACKGROUND The receptor for advanced glycation end products (RAGE) mediates a variety of inflammatory responses. METHODS To determine the role of RAGE in the innate immune response to abdominal sepsis caused by Escherichia coli, RAGE-deficient (RAGE(-/-)) and normal wild-type mice were intraperitoneally injected with E. coli. In a separate experiment, wild-type mice received either anti-RAGE immunoglobulin (Ig) G or control IgG. RESULTS E. coli sepsis resulted in an up-regulation of RAGE in the liver but not in the lungs. RAGE-deficient mice demonstrated an enhanced bacterial outgrowth in their peritoneal cavity and increased dissemination of the infection, accompanied by increased hepatocellular injury and exaggerated systemic cytokine release and coagulation activation, 20 h after intraperitoneal administration of E. coli. Wild-type mice treated with anti-RAGE IgG also displayed a diminished defense against the growth and/or dissemination of E. coli. RAGE was important for the initiation of the host response, as reflected by a reduced immune and procoagulant response early after intraperitoneal injection of E. coli lipopolysaccharide. CONCLUSION These data are the first to suggest that intact RAGE signaling contributes to an effective antibacterial defense during E. coli sepsis, thereby limiting the accompanying inflammatory and procoagulant response.

Lipopolysaccharide Inhibits Th2 Lung Inflammation Induced by House Dust Mite Allergens in Mice

The complex biology of asthma compels the use of more relevant human allergens, such as house dust mite (HDM), to improve the translation of animal models into human asthma. LPS exposure is associated with aggravations of asthma, but the mechanisms remain unclear. Here, we studied the effects of increasing LPS doses on HDM-evoked allergic lung inflammation. To this end, mice were intranasally sensitized and challenged with HDM with or without increasing doses of LPS (0.001-10 μg). LPS dose-dependently inhibited HDM-induced eosinophil recruitment into the lungs and mucus production in the airways. LPS attenuated the production of Th2 cytokines (IL-4, IL-5, IL-10, and IL-13) in HDM-challenged lungs, while enhancing the HDM-induced release of IL-17, IL-33, IFN-γ, and TNF-α. The shift toward a Th1 inflammatory response was further illustrated by predominant neutrophilic lung inflammation after LPS administration at higher doses. LPS did not influence HDM-induced plasma IgE concentrations. Although LPS did not significantly affect the activation of coagulation or complement in HDM-challenged lungs, it reduced HDM-initiated endothelial cell activation. This study is the first to provide insights into the effects of LPS in an allergic lung inflammation model making use of a clinically relevant allergen without a systemic adjuvant, revealing that LPS dose-dependently inhibits HDM-induced pulmonary Th2 responses.

Endogenous MCP-1 promotes lung inflammation induced by LPS and LTA.

Monocyte chemoattractant protein 1 (MCP-1) plays an important role in leukocyte recruitment to sites of infection and inflammation. In addition, MCP-1 may attenuate inflammation by virtue of its capacity to inhibit the production of proinflammatory cytokines. We here investigated the role of MCP-1 in lung inflammation induced by lipopolysaccharide (LPS) or lipoteichoic acid (LTA), constituents of the gram-negative and gram-positive bacterial cell wall, respectively. Healthy humans demonstrated elevated MCP-1 concentrations in their bronchoalveolar lavage fluid (BALF) 6h after inhalation of LPS. Similarly, intranasal administration of LPS or LTA to mice resulted in a rise in BALF MCP-1 levels. Murine alveolar macrophage-like cells released significant amounts of MCP-1 upon stimulation with LPS or LTA in vitro. Compared to Wt mice, MCP-1(-/-) mice demonstrated lower TNF-α levels and a diminished neutrophil influx into their bronchoalveolar space after either LPS or LTA instillation. After intrapulmonary delivery of LPS MCP-1(-/-) mice had decreased interleukin-6 and KC concentrations and less severe lung inflammation upon histopathological examination. Remarkably, MCP-1 deficiency was associated with an early enhancement of interleukin-10 release in BALF after both LPS and LTA instillation. These data suggest that MCP-1 is a proinflammatory mediator during pulmonary inflammation induced by either LPS or LTA.

The urokinase receptor (uPAR) facilitates clearance of Borrelia burgdorferi

The causative agent of Lyme borreliosis, the spirochete Borrelia burgdorferi, has been shown to induce expression of the urokinase receptor (uPAR); however, the role of uPAR in the immune response against Borrelia has never been investigated. uPAR not only acts as a proteinase receptor, but can also, dependently or independently of ligation to uPA, directly affect leukocyte function. We here demonstrate that uPAR is upregulated on murine and human leukocytes upon exposure to B. burgdorferi both in vitro as well as in vivo. Notably, B. burgdorferi-inoculated C57BL/6 uPAR knock-out mice harbored significantly higher Borrelia numbers compared to WT controls. This was associated with impaired phagocytotic capacity of B. burgdorferi by uPAR knock-out leukocytes in vitro. B. burgdorferi numbers in vivo, and phagocytotic capacity in vitro, were unaltered in uPA, tPA (low fibrinolytic activity) and PAI-1 (high fibrinolytic activity) knock-out mice compared to WT controls. Strikingly, in uPAR knock-out mice partially backcrossed to a B. burgdorferi susceptible C3H/HeN background, higher B. burgdorferi numbers were associated with more severe carditis and increased local TLR2 and IL-1β mRNA expression. In conclusion, in B. burgdorferi infection, uPAR is required for phagocytosis and adequate eradication of the spirochete from the heart by a mechanism that is independent of binding of uPAR to uPA or its role in the fibrinolytic system.

Platelet-Activating Factor Receptor Contributes to Host Defense against Pseudomonas aeruginosa Pneumonia but Is Not Essential for the Accompanying Inflammatory and Procoagulant Response

Pseudomonas aeruginosa is a major cause of nosocomial pneumonia, which is associated with high morbidity and mortality. Because of its ubiquitous nature and its ability to develop resistance to antibiotics, it is a problematic pathogen from a treatment perspective. Platelet-activating factor receptor (PAFR) is involved in phagocytosis of several pathogens. To determine the role of PAFR in the innate immune response to P. aeruginosa pneumonia, pafr gene-deficient (PAFR−/−) mice and normal wild-type (Wt) mice were intranasally inoculated with P. aeruginosa. PAFR deficiency impaired host defense as reflected by increased bacterial outgrowth and dissemination in mice with a targeted deletion of the PAFR gene. PAFR−/− neutrophils showed a diminished phagocytosing capacity of P. aeruginosa in vitro. Relative to Wt mice, PAFR−/− mice demonstrated increased lung inflammation and injury as reflected by histopathology, relative lung weights and total protein concentrations in bronchoalveolar lavage fluid, which was accompanied by higher levels of proinflammatory cytokines in lung homogenates and plasma. In addition, PAFR deficiency was associated with exaggerated local and systemic activation of coagulation as determined by fibrin staining of lung tissue and pulmonary and plasma concentrations of thrombin-antithrombin complexes and D-dimer. These data suggest that PAFR is an essential component of an effective host response to P. aeruginosa pneumonia, at least partly via its contribution to the phagocytic properties of professional granulocytes. Additionally, our results indicate that PAFR signaling is not essential for the induction of a local and systemic inflammatory and procoagulant response to Pseudomonas pneumonia.

The Polysaccharide Capsule of Streptococcus pneumonia Partially Impedes MyD88-Mediated Immunity during Pneumonia in Mice

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.

Urokinase Plasminogen Activator Receptor-Deficient Mice Demonstrate Reduced Hyperoxia-Induced Lung Injury

Patients with respiratory failure often require supplemental oxygen therapy and mechanical ventilation. Although both supportive measures are necessary to guarantee adequate oxygen uptake, they can also cause or worsen lung inflammation and injury. Hyperoxia-induced lung injury is characterized by neutrophil infiltration into the lungs. The urokinase plasminogen activator receptor (uPAR) has been deemed important for leukocyte trafficking. To determine the expression and function of neutrophil uPAR during hyperoxia-induced lung injury, uPAR expression was determined on pulmonary neutrophils of mice exposed to hyperoxia. Hyperoxia exposure (O2>80%) for 4 days elicited a pulmonary inflammatory response as reflected by a profound rise in the number of neutrophils that were recovered from bronchoalveolar lavage fluid and lung cell suspensions, as well as increased bronchoalveolar keratinocyte-derived chemokine, interleukin-6, total protein, and alkaline phosphatase levels. In addition, hyperoxia induced the migration of uPAR-positive granulocytes into lungs from wild-type mice compared with healthy control mice (exposed to room air). uPAR deficiency was associated with diminished neutrophil influx into both lung tissues and bronchoalveolar spaces, which was accompanied by a strong reduction in lung injury. Furthermore, in uPAR(-/-) mice, activation of coagulation was diminished. These data suggest that uPAR plays a detrimental role in hyperoxia-induced lung injury and that uPAR deficiency is associated with diminished neutrophil influx into both lung tissues and bronchoalveolar spaces, accompanied by decreased pulmonary injury.

Mast Cells Impair Host Defense During Murine Streptococcus pneumoniae Pneumonia

BACKGROUND Streptococcus pneumoniae is the most common causative pathogen in community-acquired pneumonia. Mast cells (MCs) are located mainly at the host-environment interface where they function as sentinels. OBJECTIVE Our goal was to study the role of MCs during pneumonia caused by S. pneumoniae. METHODS Lung tissue of patients who had died from pneumococcal pneumonia or a nonpulmonary cause was stained for MCs and tryptase. Wild-type (WT) and MC-deficient (Kit(W-sh/W-sh)) mice were observed or sacrificed after induction of pneumonia by intranasal inoculation of S. pneumoniae. In separate experiments, WT mice were treated with doxantrazole or cromoglycate, which are MC stabilizing agents. RESULTS The constitutive presence of tryptase-positive MCs was reduced in affected lungs from pneumonia patients. Kit(W-sh/W-sh) mice showed a prolonged survival during the first few days after median lethal dose (LD)100 and LD50 infection, while overall mortality did not differ from that in WT mice. Relative to WT mice, Kit(W-sh/W-sh) mice showed reduced bacterial counts with less bacterial dissemination to distant organs and less inflammation. Neither doxantrazole nor cromoglycate influenced antibacterial defense or inflammatory responses after airway infection with S. pneumoniae. CONCLUSIONS MCs exhibit an unfavorable role in host defense during pneumococcal pneumonia by a mechanism independent of degranulation.