Peptidoglycan Recognition Protein 4 Limits Bacterial Clearance and Inflammation in Lungs by Control of the Gut Microbiota.

Streptococcus pneumoniae is the most frequent cause of community-acquired pneumonia. Endogenous host defense molecules such as peptidoglycan recognition protein 4 (PGLYRP4) might influence the course of this disease. To the best of our knowledge, there are no reports on the relevance of PGLYRP4 in pneumonia. Therefore, wild type (WT) and PGLYRP4-deficient (PGLYRP4KO) mice were analyzed in an in vivo and in vitro experimental setting to examine the influence of PGLYRP4 on the course of pneumococcal pneumonia. Furthermore, caecal 16S rRNA microbiome analysis was performed, and microbiota were transferred to germfree WT mice to assess the influence of microbiotal communities on the bacterial burden. Mice lacking PGLYRP4 displayed an enhanced bacterial clearance in the lungs, and fewer mice developed bacteremia. In addition, an increased recruitment of immune cells to the site of infection, and an enhanced bacterial killing by stronger activation of phagocytes could be shown. This may depend partly on the detected higher expression of complement factors, interferon-associated genes, and the higher pro-inflammatory cytokine response in isolated primary PGLYRP4KO vs. WT cells. This phenotype is underlined by changes in the complexity and composition of the caecal microbiota of PGLYRP4KO compared to WT mice. Strikingly, we provided evidence, by cohousing and stable transfer of the respective WT or PGLYRP4KO mice microbiota into germfree WT mice, that the changes of the microbiota are responsible for the improved clearance of S. pneumoniae lung infection. In conclusion, the deficiency of PGLYRP4, a known antibacterial protein, leads to changes in the gut microbiota. Thus, alterations in the microbiota can change the susceptibility to S. pneumoniae lung infection independently of the host genotype.

Peptidoglycan Recognition Protein 2 Regulates Neutrophil Recruitment Into the Lungs After Streptococcus pneumoniae Infection.

Peptidoglycan (PGN) recognition proteins (PGLYRPs) are a highly conserved group of host defense proteins in insects and mammals that sense bacterial cell wall PGN and act bactericidally or cleave PGN by amidase function. Streptococcus (S.) pneumoniae is one of the top five killers worldwide and causes, e.g., pneumonia, endocarditis, meningitis and sepsis. S. pneumoniae accounts for approximately 1.5-2 million deaths every year. The risk of antibiotic resistance and a general poor prognosis in young children and elderly people have led to the need for new treatment approaches. To the best of our knowledge, there is no report on the relevance of PGLYRP2 in lung infections. Therefore, we infected mice deficient for PGLYRP2 transnasally with S. pneumoniae and examined the innate immune response in comparison to WT animals. As expected, PGLYRP2-KO animals had to be sacrificed earlier than their WT counterparts, and this was due to higher bacteremia. The higher bacterial load in the PGLYRP2-KO mice was accomplished with lower amounts of proinflammatory cytokines in the lungs. This led to an abolished recruitment of neutrophils into the lungs, the spread of bacteria and the subsequent aggravated course of the disease and early mortality of the PGLYRP2-KO mice. These data suggest a substantial role of PGLYRP2 in the early defense against S. pneumoniae infection, and PGLYRP2 might also affect other infections in the lungs.

Peptidoglycan Recognition Protein 3 Does Not Alter the Outcome of Pneumococcal Pneumonia in Mice.

Pneumococci frequently cause community-acquired pneumonia, a disease with high mortality rates, particularly in young children and in the elderly. Endogenous antimicrobial peptides and proteins such as PGLYRP3 may contribute to the progression and outcome of this disease. Since increasing antibiotic resistant strains occur all over the world, these endogenous antimicrobial molecules are interesting new targets for future therapies. In this study, the expression pattern of PGLYRP3 was analyzed in alveolar epithelial cells, alveolar macrophages and neutrophils. Additionally, the function of PGLYRP3 during Streptococcus pneumoniae-induced pneumonia was investigated in a murine pneumococcal pneumonia model using PGLYRP3KO mice. PGLYRP3 is expressed in all selected cell types but pneumococcus-dependent induction of PGLYRP3 was observed only in neutrophils and alveolar macrophages. Interestingly, there were no significant differences in the bacterial loads within the lungs, the blood or the spleens, in the cytokine response, the composition of immune cells and the histopathology between wild type and PGLYRP3KO mice. Finally, we could neither observe significant differences in the clinical symptoms nor in the overall survival. Collectively, PGLYRP3 seems to be dispensable for the antibacterial defense during pneumococcal pneumonia.

Effects of enamel matrix derivative and transforming growth factor-ß1 on connective tissue growth factor in human periodontal ligament fibroblasts.

BACKGROUND: Enamel matrix derivative (EMD) is suggested to stimulate transforming growth factor-ß (TGF-ß) production. Connective tissue growth factor (CTGF) is a downstream mediator of TGF-ß. This study explores the effects of EMD and TGF-ß1 on CTGF in periodontal ligament (PDL) fibroblasts and their interactions in PDL proliferation and development. METHODS: Human PDL cells were stimulated with EMD. To explore the effects of EMD and TGF-ß1 on CTGF expression, cells were treated with and without TGF-ß inhibitor, and CTGF protein levels were assayed by Western blot analysis. To study the role of CTGF in PDL development, cells were treated with CTGF inhibitor. DNA synthesis was analyzed by bromodeoxyuridine enzyme-linked immunosorbent assay. Reverse-transcription polymerase chain reaction was performed to examine messenger RNA expression of PDL osteoblastic differentiation markers: type I collagen, alkaline phosphatase, and osteocalcin. RESULTS: EMD induced a concentration-dependent increase of CTGF protein expression in PDL cells. EMD- and TGF-ß1-stimulated CTGF expression was significantly reduced in the presence of TGF-ß inhibitor. CTGF inhibition downregulated both EMD- and TGF-ß1-induced DNA synthesis. The effect of CTGF and EMD on osteoblastic mRNA expression in PDL cells is not obvious. CONCLUSIONS: EMD stimulates CTGF expression in human PDL cells, a process modulated by the TGF-ß pathway. CTGF can affect EMD- and TGF-ß1-induced PDL cell proliferation, but its effects on PDL with regard to osteoblastic differentiation remain inconclusive. The results provide novel insights into EMD-CTGF interaction in PDL cells.

Streptococcus pneumoniae-induced regulation of cyclooxygenase-2 in human lung tissue.

The majority of cases of community-acquired pneumonia are caused by Streptococcus pneumoniae and most studies on pneumococcal host interaction are based on cell culture or animal experiments. Thus, little is known about infections in human lung tissue. Cyclooxygenase-2 and its metabolites play an important regulatory role in lung inflammation. Therefore, we established a pneumococcal infection model on human lung tissue demonstrating mitogen-activated protein kinase (MAPK)-dependent induction of cyclooxygenase-2 and its related metabolites. In addition to alveolar macrophages and the vascular endothelium, cyclooxygenase-2 was upregulated in alveolar type II but not type I epithelial cells, which was confirmed in lungs of patients suffering from acute pneumonia. Moreover, we demonstrated the expression profile of all four E prostanoid receptors at the mRNA level and showed functionality of the E prostanoid(4) receptor by cyclic adenosine monophosphate production. Additionally, in comparison to previous studies, cyclooxygenase-2/prostaglandin E(2) related pro- and anti-inflammatory mediator regulation was partly confirmed in human lung tissue after pneumococcal infection. Overall, cell type-specific and MAPK-dependent cyclooxygenase-2 expression and prostaglandin E(2) formation in human lung tissue may play an important role in the early phase of pneumococcal infections.

Legionella pneumophila induces human beta defensin-3 in pulmonary cells.

BACKGROUND: Legionella pneumophila is an important causative agent of severe pneumonia in humans. Human alveolar epithelium and macrophages are effective barriers for inhaled microorganisms and actively participate in the initiation of innate host defense. The beta defensin-3 (hBD-3), an antimicrobial peptide is an important component of the innate immune response of the human lung. Therefore we hypothesize that hBD-3 might be important for immune defense towards L. pneumophila. METHODS: We investigated the effects of L. pneumophila and different TLR agonists on pulmonary cells in regard to hBD-3 expression by ELISA. Furthermore, siRNA-mediated inhibition of TLRs as well as chemical inhibition of potential downstream signaling molecules was used for functional analysis. RESULTS: L. pneumophila induced release of hBD-3 in pulmonary epithelium and alveolar macrophages. A similar response was observed when epithelial cells were treated with different TLR agonists. Inhibition of TLR2, TLR5, and TLR9 expression led to a decreased hBD-3 expression. Furthermore expression of hBD-3 was mediated through a JNK dependent activation of AP-1 (c-Jun) but appeared to be independent of NF-kappaB. Additionally, we demonstrate that hBD-3 elicited a strong antimicrobial effect on L. pneumophila replication. CONCLUSIONS: Taken together, human pulmonary cells produce hBD-3 upon L. pneumophila infection via a TLR-JNK-AP-1-dependent pathway which may contribute to an efficient innate immune defense.

CEACAM1 inhibits Toll-like receptor 2-triggered antibacterial responses of human pulmonary epithelial cells.

Although Moraxella catarrhalis and Neisseria meningitidis are important human pathogens, they often colonize the human respiratory tract without causing overt clinical symptoms. Both pathogens express structurally unrelated proteins that share the ability to stimulate the adhesion molecule CEACAM1 expressed on human cells. Here we demonstrate that the interaction of CEACAM1 with ubiquitous surface protein A1 expressed on M. catarrhalis or with opacity-associated proteins on N. meningitidis resulted in reduced Toll-like receptor 2-initiated transcription factor NF-kappaB-dependent inflammatory responses of primary pulmonary epithelial cells. These inhibitory effects were mediated by tyrosine phosphorylation of the immunoreceptor tyrosine-based inhibitory motif of CEACAM1 and by recruitment of the phosphatase SHP-1, which negatively regulated Toll-like receptor 2-dependent activation of the phosphatidylinositol 3-OH kinase-Akt kinase pathway. Our results identify a CEACAM1-dependent immune-evasion strategy.

Enamel matrix derivative induces connective tissue growth factor expression in human osteoblastic cells.

BACKGROUND: Enamel matrix derivative (EMD) stimulates the production of transforming growth factor-beta (TGF-beta), which has been suggested to play a role in mediating the effects of EMD in periodontal tissue regeneration. Connective tissue growth factor (CTGF) is a mediator of TGF-beta and promotes cell development. The interaction between EMD and CTGF is unknown. This study explored the effects of EMD on CTGF expression in human osteoblastic cells and whether the interaction is modulated by the TGF-beta signaling pathway. Also, the roles of CTGF in cell proliferation, cell cycle progression, and mineralized nodule formation of EMD-induced osteoblastic cultures were examined. METHODS: Human osteoblastic cells (Saos-2) were treated with 25 to 100 microg/ml EMD with or without the addition of TGF-beta inhibitor. CTGF mRNA expression was detected by reverse transcription-polymerase chain reaction (RT-PCR), and CTGF protein levels were assayed by Western blot analysis. In addition, cell cycle progression and DNA synthesis were determined by flow cytometry and 5-bromo-2'-deoxyuridine (BrdU) incorporation following EMD treatment with or without CTGF antibody. Mineralization was examined by alizarin red staining and quantified by elution with cetylpyridinium chloride. RESULTS: Western blot and RT-PCR analysis demonstrated a dose-dependent increase of CTGF expression by EMD. EMD-induced CTGF expression was reduced significantly in the presence of TGF-beta inhibitor. Cell cycle and BrdU analysis revealed an increase in cell proliferation following EMD treatment, which was due to an increase in the percentage of cells in the G2/M phase of the cell cycle. No significant effect was found when anti-CTGF antibody was added. Conversely, mineralization was inhibited significantly in EMD-treated cultures in the presence of anti-CTGF antibody. CONCLUSIONS: EMD stimulates CTGF expression, and the interaction is modulated via TGF-beta in osteoblastic cells. Also, CTGF affects EMD-induced osteoblastic mineralization but not cell proliferation. To our knowledge, these results provide novel insight into EMD-CTGF interaction, two biomodifiers that have therapeutic relevance to tissue engineering and regeneration.