Katrin Reppe

The NLRP3 Inflammasome Is Differentially Activated by Pneumolysin Variants and Contributes to Host Defense in Pneumococcal Pneumonia

Streptococcus pneumoniae is a leading cause of pneumonia, meningitis, and sepsis. Pneumococci can be divided into >90 serotypes that show differences in the pathogenicity and invasiveness. We tested the hypotheses that the innate immune inflammasome pathway is involved in fighting pneumococcal pneumonia and that some invasive pneumococcal types are not recognized by this pathway. We show that human and murine mononuclear cells responded to S. pneumoniae expressing hemolytic pneumolysin by producing IL-1β. This IL-1β production depended on the NOD-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome. Some serotype 1, serotype 8, and serotype 7F bacteria, which have previously been associated with increased invasiveness and with production of toxins with reduced hemolytic activity, or bacterial mutants lacking pneumolysin did not stimulate notable IL-1β production. We further found that NLRP3 was beneficial for mice during pneumonia caused by pneumococci expressing hemolytic pneumolysin and was involved in cytokine production and maintenance of the pulmonary microvascular barrier. Overall, the inflammasome pathway is protective in pneumonia caused by pneumococci expressing hemolytic toxin but is not activated by clinically important pneumococcal sequence types causing invasive disease. The study indicates that a virulence factor polymorphism may substantially affect the recognition of bacteria by the innate immune system.

Immunostimulation with Macrophage-Activating Lipopeptide-2 Increased Survival in Murine Pneumonia

Community-acquired pneumonia (CAP) is associated with high morbidity and mortality, and Streptococcus pneumoniae is the most prevalent causal pathogen identified in CAP. Impaired pulmonary host defense increases susceptibility to pneumococcal pneumonia. S. pneumoniae may up-regulate Toll-like receptor (TLR)-2 expression and activate TLR-2, contributing to pneumococcus-induced immune responses. In the current study, the course of severe murine pneumococcal pneumonia after pulmonary TLR-2-mediated immunostimulation with synthetic macrophage-activating lipopeptide-2 (MALP-2) was examined. Intratracheal MALP-2 application evoked enhanced proinflammatory cytokine and chemokine release, resulting in recruitment of polymorphonuclear neutrophils (PMN), macrophages, and lymphocytes into the alveolar space in WT, but not in TLR-2-deficient mice. In murine lungs as well as in human alveolar epithelial cells (A549), MALP-2 increased TLR-2 expression at both mRNA and protein level. Blood leukocyte numbers and populations remained unchanged. MALP-2 application 24 hours before intranasal pneumococcal infection resulted in increased levels of CCL5 associated with augmented leukocyte recruitment, and decreased levels of anti-inflammatory IL-10 in bronchoalveolar lavage fluid. Clinically, MALP-2-treated as compared with untreated mice showed increased survival, reduced hypothermia, and increased body weight. MALP-2 also reduced bacteremia and improved bacterial clearance in lung parenchyma, as examined by immunohistochemistry. In conclusion, pulmonary immunostimulation with MALP-2 before infection with S. pneumoniae improved local host defense and increased survival in murine pneumococcal pneumonia.

RNAi-mediated suppression of constitutive pulmonary gene expression by small interfering RNA in mice

The ability of synthetic small interfering RNA (siRNA) to silence gene expression makes it a useful tool in biomedical research. However, effective and non-toxic functional siRNA delivery to mouse lungs in vivo is still a key challenge, and regulation of constitutively expressed genes is poorly characterized. Following in vitro validation of siRNA molecules, naked, stabilized siRNA (AtuRNAi) was applied intranasally (i.n.) by droplets or intratracheally (i.t.) by MicroSprayer in female C57BL/6 mice. Distribution of Cy3-tagged siRNAs was examined. Pulmonary expression of ubiquitously (lamin B1) or cell-specific (E-cadherin, VE-cadherin), constitutive genes was analysed by TaqMan-realtime-PCR. Further, formulated lipoplex-siRNA, which has enhanced transfection efficiency, was applied i.t. or intravenously (i.v.). Single i.t. as compared to i.n. application of unformulated siRNA resulted in higher delivery efficiency and homogenous pulmonary distribution. After inhalation of target-specific siRNA, reduction of epithelial E-cadherin by 21%, but no significant reduction of endothelial VE-cadherin or ubiquitously expressed lamin B1 was observed. Pharmacokinetic analysis revealed rapid transfer of intact siRNA molecules into the vascular system and accumulation in the kidneys, calling lung specificity into question. I.t. application of lipoplex-siRNA evoked inflammation. In contrast, i.v. application of lipoplex-siRNA specifically reduced expression of VE-cadherin mRNA by about 50% in lungs without evoking lung cellular influx. In conclusion, sufficient pulmonary distribution of aerosolized siRNA was attained in mice by MicroSprayer, however development of appropriate siRNA carriers is highly desirable to improve lung-specific functional inhalative siRNA delivery. Pulmonary knockdown of constitutive endothelial targets by 50% was achieved by i.v. application of lipoplex-siRNA.

Delivery of the endolysin Cpl-1 by inhalation rescues mice with fatal pneumococcal pneumonia

OBJECTIVES Pneumonia is associated with a high morbidity and mortality worldwide. Streptococcus pneumoniae remains the most common cause of pneumonia, and pneumococcal antibiotic resistance is increasing. The purified bacteriophage endolysin Cpl-1 rapidly and specifically kills pneumococci. We tested the hypothesis that a single dose of recombinant aerosolized Cpl-1 would rescue mice with severe pneumococcal pneumonia. METHODS Female C57Bl/6 mice (aged 8-12 weeks) were transnasally infected with pneumococci. When severe pneumonia was established 24 h after infection, mice were treated with 25 μL of aerosolized Cpl-1. Survival was monitored for 10 days and the pulmonary and systemic bacterial burdens were assessed. Furthermore, cytokines were quantified in bronchoalveolar lavage fluid, and lung morphology was analysed histologically. RESULTS The endolysin efficiently reduced pulmonary bacterial counts and averted bacteraemia. Although concentrations of inflammatory cytokines were increased shortly after Cpl-1 inhalation, mice recovered rapidly, as shown by increasing body weight, and inflammatory infiltrates resolved in the lungs, leading to a reduction in mortality of 80%. CONCLUSIONS Administration of Cpl-1 by inhalation may offer a new therapeutic perspective for the treatment of pneumococcal lung infection.

The C-Type Lectin Receptor Mincle Binds to Streptococcus pneumoniae but Plays a Limited Role in the Anti-Pneumococcal Innate Immune Response

The innate immune system employs C-type lectin receptors (CLRs) to recognize carbohydrate structures on pathogens and self-antigens. The Macrophage-inducible C-type lectin (Mincle) is a FcRγ-coupled CLR that was shown to bind to mycobacterial cord factor as well as certain fungal species. However, since CLR functions during bacterial infections have not yet been investigated thoroughly, we aimed to examine their function in Streptococcus pneumonia infection. Binding studies using a library of recombinantly expressed CLR-Fc fusion proteins indicated a specific, Ca2+-dependent, and serotype-specific binding of Mincle to S. pneumonia. Subsequent experiments with different Mincle-expressing cells as well as Mincle-deficient mice, however, revealed a limited role of this receptor in bacterial phagocytosis, neutrophil-mediated killing, cytokine production, and antibacterial immune response during pneumonia. Collectively, our results indicate that Mincle is able to recognize S. pneumonia but is not required for the anti-pneumococcal innate immune response.

mCLCA3 Modulates IL-17 and CXCL-1 Induction and Leukocyte Recruitment in Murine Staphylococcus aureus Pneumonia

The human hCLCA1 and its murine ortholog mCLCA3 (calcium-activated chloride channel regulators) are exclusively expressed in mucus cells and linked to inflammatory airway diseases with increased mucus production, such as asthma, cystic fibrosis and chronic obstructive pulmonary disease. Both proteins have a known impact on the mucus cell metaplasia trait in these diseases. However, growing evidence points towards an additional role in innate immune responses. In the current study, we analyzed Staphylococcus aureus pneumonia, an established model to study pulmonary innate immunity, in mCLCA3-deficient and wild-type mice, focusing on the cellular and cytokine-driven innate inflammatory response. We compared clinical signs, bacterial clearance, leukocyte immigration and cytokine responses in the bronchoalveolar compartment, as well as pulmonary vascular permeability, histopathology, mucus cell number and mRNA expression levels of selected genes (mClca1 to 7, Muc5ac, Muc5b, Muc2, Cxcl-1, Cxcl-2, Il-17). Deficiency of mCLCA3 resulted in decreased neutrophilic infiltration into the bronchoalveolar space during bacterial infection. Only the cytokines IL-17 and the murine CXCL-8 homolog CXCL-1 were decreased on mRNA and protein levels during bacterial infection in mCLCA3-deficient mice compared to wild-type controls. However, no differences in clinical outcome, histopathology or mucus cell metaplasia were observed. We did not find evidence for regulation of any other CLCA homolog that would putatively compensate for the lack of mCLCA3. In conclusion, mCLCA3 appears to modulate leukocyte response via IL-17 and murine CXCL-8 homologs in acute Staphylococcus aureus pneumonia which is well in line with the proposed function of hCLCA1 as a signaling molecule acting on alveolar macrophages.

A semisynthetic Streptococcus pneumoniae serotype 8 glycoconjugate vaccine

Automated glycan assembly enabled antibody reverse engineering to develop a semisynthetic carbohydrate–based vaccine against the highly virulent Streptococcus pneumoniae serotype 8. Pruning out nonprotective glycotopes Pediatric vaccines targeting bacterial capsular polysaccharides are more effective for certain types of bugs than others, and the manufacturing process as well as immunodominance of different glycan epitopes (glycotopes) can lead to a mixed immune response that does not protect against disease. To directly identify glycotopes that induce a protective response, Schumann et al. combined antibody reverse engineering with automated glycan assembly using Streptococcus pneumoniae serotype 8 as a proof of concept. Promising glycotopes conjugated to a carrier protein induced protective antibodies in mice and were also immunogenic in rabbits. When combined with a commercially available pneumococcal vaccine, these glycoconjugates were able to boost the opsonophagocytic bacterial killing ability of sera from immunized rabbits. This approach leveraging semisynthetic glycoconjugates could lead to the design of more effective bacterial vaccines. Glycoconjugate vaccines based on capsular polysaccharides (CPSs) of pathogenic bacteria such as Streptococcus pneumoniae successfully protect from disease but suffer from incomplete coverage, are troublesome to manufacture from isolated CPSs, and lack efficacy against certain serotypes. Defined, synthetic oligosaccharides are an attractive alternative to isolated CPSs but require the identification of immunogenic and protective oligosaccharide antigens. We describe a medicinal chemistry strategy based on a combination of automated glycan assembly (AGA), glycan microarray–based monoclonal antibody (mAb) reverse engineering, and immunological evaluation in vivo to uncover a protective glycan epitope (glycotope) for S. pneumoniae serotype 8 (ST8). All four tetrasaccharide frameshifts of ST8 CPS were prepared by AGA and used in glycan microarray experiments to identify the glycotopes recognized by antibodies against ST8. One tetrasaccharide frameshift that was preferentially recognized by a protective, CPS-directed mAb was conjugated to the carrier protein CRM197. Immunization of mice with this semisynthetic glycoconjugate followed by generation and characterization of a protective mAb identified protective and nonprotective glycotopes. Immunization of rabbits with semisynthetic ST8 glycoconjugates containing protective glycotopes induced an antibacterial immune response. Coformulation of ST8 glycoconjugates with the marketed 13-valent glycoconjugate vaccine Prevnar 13 yielded a potent 14-valent S. pneumoniae vaccine. Our strategy presents a facile approach to develop efficient semisynthetic glycoconjugate vaccines.

Pulmonary immunostimulation with MALP-2 in influenza virus-infected mice increases survival after pneumococcal superinfection.

ABSTRACT Pulmonary infection with influenza virus is frequently complicated by bacterial superinfection, with Streptococcus pneumoniae being the most prevalent causal pathogen and hence often associated with high morbidity and mortality rates. Local immunosuppression due to pulmonary influenza virus infection has been identified as a major cause of the pathogenesis of secondary bacterial lung infection. Thus, specific local stimulation of the pulmonary innate immune system in subjects with influenza virus infection might improve the host defense against secondary bacterial pathogens. In the present study, we examined the effect of pulmonary immunostimulation with Toll-like receptor 2 (TLR-2)-stimulating macrophage-activating lipopeptide 2 (MALP-2) in influenza A virus (IAV)-infected mice on the course of subsequent pneumococcal superinfection. Female C57BL/6N mice infected with IAV were treated with MALP-2 on day 5 and challenged with S. pneumoniae on day 6. Intratracheal MALP-2 application increased proinflammatory cytokine and chemokine release and enhanced the recruitment of leukocytes, mainly neutrophils, into the alveolar space of IAV-infected mice, without detectable systemic side effects. Local pulmonary instillation of MALP-2 in IAV-infected mice 24 h before transnasal pneumococcal infection considerably reduced the bacterial number in the lung tissue without inducing exaggerated inflammation. The pulmonary viral load was not altered by MALP-2. Clinically, MALP-2 treatment of IAV-infected mice increased survival rates and reduced hypothermia and body weight loss after pneumococcal superinfection compared to those of untreated coinfected mice. In conclusion, local immunostimulation with MALP-2 in influenza virus-infected mice improved pulmonary bacterial elimination and increased survival after subsequent pneumococcal superinfection.

Murine CLCA5 is uniquely expressed in distinct niches of airway epithelial cells

The murine mCLCA5 protein is a member of the chloride channel regulators, calcium-activated (CLCA) family and is suspected to play a role in airway mucus cell differentiation. Although mCLCA5 mRNA was previously found in total lung extracts, the expressing cells and functions in the naive murine respiratory tract are unknown. Therefore, mCLCA5 protein expression was identified by immunohistochemistry and confocal laser scanning microscopy using entire lung sections of naive mice. Moreover, we determined mRNA levels of functionally related genes (mClca3, mClca5, Muc5ac and Muc5b) and quantified mCLCA5-, mCLCA3- and CC10-positive cells and periodic acid-Schiff-positive mucus cells in naive, PBS-treated or Staphylococcus aureus-infected mice. We also investigated mCLCA5 protein expression in Streptococcus pneumoniae and influenza virus lung infection models. Finally, we determined species-specific differences in the expression patterns of the murine mCLCA5 and its human and porcine orthologs, hCLCA2 and pCLCA2. The mCLCA5 protein is uniquely expressed in highly select bronchial epithelial cells and submucosal glands in naive mice, consistent with anatomical locations of progenitor cell niches. Under conditions of challenge (PBS, S. aureus, S. pneumoniae, influenza virus), mRNA and protein expression strongly declined with protein recovery only in models retaining intact epithelial cells. In contrast to mice, human and porcine bronchial epithelial cells do not express their respective mCLCA5 orthologs and submucosal glands had fewer expressing cells, indicative of fundamental differences in mice versus humans and pigs.

Antihistone Properties of C1 Esterase Inhibitor Protect against Lung Injury

&NA; Rationale: Acute respiratory distress syndrome is characterized by alveolar epithelial cell injury, edema formation, and intraalveolar contact phase activation. Objectives: To explore whether C1 esterase inhibitor (C1INH), an endogenous inhibitor of the contact phase, may protect from lung injury in vivo and to decipher the possible underlying mechanisms mediating protection. Methods: The ability of C1INH to control the inflammatory processes was studied in vitro and in vivo. Measurements and Main Results: Here, we demonstrate that application of C1INH alleviates bleomycin‐induced lung injury via direct interaction with extracellular histones. In vitro, C1INH was found to bind all histone types. Interaction with histones was independent of its protease inhibitory activity, as demonstrated by the use of reactive‐center‐cleaved C1INH, but dependent on its glycosylation status. C1INH sialylated‐N‐ and ‐O‐glycans were not only essential for its interaction with histones but also to protect against histone‐induced cell death. In vivo, histone‐C1INH complexes were detected in bronchoalveolar lavage fluid from patients with acute respiratory distress syndrome and multiple models of lung injury. Furthermore, reactive‐center‐cleaved C1INH attenuated pulmonary damage evoked by intravenous histone instillation. Conclusions: Collectively, C1INH administration provides a new therapeutic option for disorders associated with histone release.