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Triggering receptor expressed on myeloid cells (TREM)-1 and -2 can affect Toll-like receptor-mediated activation of immune cells. Klebsiella 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 toward K. pneumoniae and for 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, whereas 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 affect 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.

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–/– neutrophils 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 h of infection, Trem-1/3–/– mice 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–/– alveolar macrophages produced fewer 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 a key player in protective innate immunity during pneumococcal pneumonia, most likely by enhancing the early immune response of alveolar macrophages. Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

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.

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.

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(-/-) neutrophils 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 h of infection, Trem-1/3(-/-) mice 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(-/-) alveolar macrophages produced fewer 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 a key player in protective innate immunity during pneumococcal pneumonia, most likely by enhancing the early immune response of alveolar macrophages.

BACKGROUND Streptococcus pneumoniae is the most frequently isolated pathogen responsible for community-acquired pneumonia. Osteopontin is involved in inflammation during both innate and adaptive immunity. METHODS To determine the role of osteopontin in the host response during pneumococcal pneumonia, osteopontin knockout (KO) and normal wild-type (WT) mice were intranasally infected with viable S. pneumoniae. RESULTS Pneumonia was associated with a rapid increase in pulmonary osteopontin concentrations in WT mice from 6 h onward. Osteopontin KO mice showed a prolonged survival relative to WT mice, which was accompanied by diminished pulmonary bacterial growth and reduced dissemination to distant body sites. In addition, at 48 h after infection pulmonary inflammation was decreased in osteopontin KO mice as reflected by lower inflammation scores and reduced chemokine concentrations. In contrast to pneumococcal pneumonia, osteopontin deficiency did not influence bacterial growth in primary pneumococcal sepsis induced by direct intravenous infection, suggesting that the detrimental effect of osteopontin on antibacterial defense during pneumonia primarily is exerted in the pulmonary compartment. Moreover, recombinant osteopontin stabilized S. pneumoniae viability in vitro. CONCLUSIONS These results suggest that the pneumococcus misuses osteopontin in the airways for optimal growth and to cause invasive disease after entering the lower airways.

Sir William Osler, probably the most influential physician in the English-speaking world at the turn of the century more than a hundred years ago, wrote the following about sepsis in his famous text book, The Evolution of Modern Medicine (1904): “Except on few occasions, the patient appears to die from the body’s response to infection rather than from it”. The assumption that sepsis is the consequence of an overwhelming inflammatory reaction of the patient to microorganisms was widely accepted for many years. Current knowledge indicates that this paradigm is oversimplified and only partially true. The original theory that sepsis mortality is caused by an overstimulated immune system was based on studies in animals that were infused with large doses of bacteria or bacterial products, in particular lipopolysaccharide (LPS), the toxic component of the Gram-negative bacterial cell wall. Such infusions result in a brisk systemic release of an array of pro-inflammatory mediators of which many have been found to be directly responsible for the death of the host. In a hallmark manuscript published in 1985, Beutler and colleagues reported that elimination of the early activity of the pro-inflammatory cytokine, tumor necrosis factor (TNF)-α, after intravenous injection of LPS prevented death in mice [1]. Two years later, these results were confirmed by Tracey and colleagues, who showed that a monoclonal anti-TNF-α antibody protected baboons against lethal Gram-negative sepsis [2]. Since then, anti-TNF-α therapies have been found to be protective in a number of sepsis models in which bacteria or bacterial products were administered systemically as a bolus or a brief infusion [3].

Klebsiella (K.) pneumoniae is a common cause of pneumonia-derived sepsis. Myeloid related protein 8 (MRP8, S100A8) and MRP14 (S100A9) are the most abundant cytoplasmic proteins in neutrophils. They can form MRP8/14 heterodimers that are released upon cell stress stimuli. MRP8/14 reportedly exerts antimicrobial activity, but in acute fulminant sepsis models MRP8/14 has been found to contribute to organ damage and death. We here determined the role of MRP8/14 in K. pneumoniae sepsis originating from the lungs, using an established model characterized by gradual growth of bacteria with subsequent dissemination. Infection resulted in gradually increasing MRP8/14 levels in lungs and plasma. Mrp14 deficient (mrp14−/−) mice, unable to form MRP8/14 heterodimers, showed enhanced bacterial dissemination accompanied by increased organ damage and a reduced survival. Mrp14−/− macrophages were reduced in their capacity to phagocytose Klebsiella. In addition, recombinant MRP8/14 heterodimers, but not MRP8 or MRP14 alone, prevented growth of Klebsiella in vitro through chelation of divalent cations. Neutrophil extracellular traps (NETs) prepared from wildtype but not from mrp14−/− neutrophils inhibited Klebsiella growth; in accordance, the capacity of human NETs to kill Klebsiella was strongly impaired by an anti-MRP14 antibody or the addition of zinc. These results identify MRP8/14 as key player in protective innate immunity during Klebsiella pneumonia., Author Summary Neutrophils are phagocytes that are well known for their capacity to engulf and kill microbial pathogens. It has become increasingly clear that neutrophils also kill or inhibit growth extracellularly by releasing neutrophil extracellular traps (NETs), chromatin fibers decorated with neutrophil derived proteins. MRP8/14 has been identified as one of the major antimicrobial proteins herein. Previous investigations have shown that endogenously released MRP8/14 is also sensed by the host as a danger signal and able to potentiate the harmful systemic inflammatory response syndrome. Indeed, in the setting of fulminant systemic inflammation, such as induced by endotoxin or Escherichia coli administration, MRP8/14 contributed to organ injury and mortality. The clinical scenario of sepsis however, involves an initial infection at the primary site followed by bacterial spreading to other organs. In the present setting of pneumonia-derived sepsis using the common human respiratory and sepsis pathogen Klebsiella pneumoniae MRP8/14 clearly served a beneficial role in antimicrobial defense. We here provide a likely mechanism by showing that MRP8/14 plays a role in phagocytosis and that its presence is critical in both murine and human NETs to inhibit bacterial growth.

BACKGROUND: K. pneumoniae is a common cause of pneumonia-derived sepsis. MRP8 and MRP14 are the most abundant cytoplasmic proteins in neutrophils. They can form MRP8/14 heterodimers that are released during invasive infectious diseases. MRP8/14 reportedly exerts antimicrobial activity and enhances the pro-inflammatory response via Toll-like receptor (TLR) 4. Our objective was to determine the role of MRP8/14 in the host defense during murine Klebsiella pneumonia. METHODS: We intranasally challenged Wildtype (WT) and MRP14 knockout (KO) mice with K. pneumoniae. Mice were sacrificed 6, 24 and 48 hours post-infection for the determination of bacterial loads, MRP8/14 and cytokines in blood and organs or monitored in a survival study. Whole blood obtained from WT, MRP14 KO and TLR4 KO mice were stimulated with growth-arrested Klebsiella to determine the role of MRP8/14 on the cytokine response in vitro. Growth inhibitory effects of MRP8/14 were assessed by incubation of Klebsiella with recombinant MRP8/14 in the presence or absence of zinc. In addition we determined the contribution of MRP8/14 on antimicrobial activity in neutrophil extracellular traps (NETs) induced in WT and MRP14 KO neutrophils. RESULTS: Klebsiella infection resulted in local and systemic MRP8/14 release. MRP14 KO mice were more susceptible to K. pneumoniae, as reflected by a worsened survival and increased bacterial outgrowth in blood and organs. Cytokine measurements in WT and MRP14 KO displayed minor differences. Whole blood stimulation showed a minimal contribution of MRP8/14 on the TLR4 mediated Klebsiella cytokine response. Recombinant MRP8/14 strongly reduced Klebsiella growth, which was reversed by the addition of zinc. Accordingly, NETs induced in MRP14 KO neutrophils showed a reduced capacity to inhibit Klebsiella growth compared to WT NETs CONCLUSION: MRP8/14 contributes to a protective innate defense in Klebsiella pneumonia. Our data indicate that MRP8/14 exerts direct effects on Klebsiella trapped in NETs by chelation of zinc required for bacterial growth.