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PRELP enhances host innate immunity against respiratory tract pathogen Moraxella catarrhalis
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Abstracts / Immunobiology 221 (2016) 1131–1225
53 PRELP enhances host innate immunity against respiratory tract pathogen Moraxella catarrhalis Guanghui Liu 1,∗ , David Ermert 1 , Martin E. Johansson 2 , Kristian Riesbeck 3 , Anna M. Blom 1 1 Division of Medical Protein Chemistry, Department of Translational Medicine, Lund University, SE-205 02 Malmö, Sweden 2 Pathology, Department of Translational Medicine, Lund University, SE-205 02 Malmö, Sweden 3 Clinical Microbiology, Department of Translational Medicine, Lund University, SE-205 02 Malmö, Sweden
Respiratory tract infections are the leading cause of deaths globally. Increasing antibiotic resistance and low coverage of available vaccines require alternative therapeutics to treat respiratory tract infections. Here we report that proline/arginine-rich end leucine-rich repeat protein (PRELP) is a novel component of innate immune response. We show using immunochemistry that PRELP is present in alveolar fluid, resident macrophages/monocytes, myofibroblasts, and adventitia of blood vessels of lung tissue. PRELP specifically binds respiratory tract pathogens Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis, but not other tested bacterial pathogens. PRELP binds the majority of clinical isolates of M. catarrhalis (n = 49), and interacts directly with the ubiquitous surface protein A2/A2H of M. catarrhalis. PRELP increases the formation of membrane attack complex on M. catarrhalis, thereby enhancing the killing of M. catarrhalis by human serum. Moreover, PRELP competitively inhibits binding of complement inhibitor C4b-binding protein to M. catarrhalis. Furthermore, PRELP reduces Moraxella adhesion to and invasion of human lung epithelial cells. Taken together, PRELP enhances complement attack against M. catarrhalis and prevents bacterial colonization of lung epithelial cells. Thus, PRELP could have therapeutic potential to modulate the innate immune responses toward respiratory tract pathogens, particularly M. catarrhalis. http://dx.doi.org/10.1016/j.imbio.2016.06.068 54 Production of C3 and its secretion into the mucus by intestinal epithelial cells correlates with disease activity in experimental chronic colitis Kerstin Skibbe ∗ , Karen Ebbert, Sophie Preisker, Annika Sünderhauf, Petra Langenstrassen, Christian Sina, Stefanie Derer Molecular Gastroenterology, Department of Medicine 1, University of Lübeck, Germany Introduction: The complement system plays a critical role in the efficient detection and clearance of bacteria. However, uncontrolled and sustained complement activation exacerbates inflammatory processes, resulting in tissue damage as seen in inflammatory bowel diseases (IBD), such as Crohn’s disease (CD) and ulcerative colitis (UC). Therefore, the aim of the present study was to unravel whether bacteria binding to intestinal epithelial cells (IECs) impacts the expression of complement components by IEC under chronic inflammatory conditions. Methods: Chronic DSS colitis was induced in C57BL/6JRj mice through administration of 4% w/v DSS in 3 cycles of 5 days inter-
mitted by 5 days of water. Disease severity was determined by the disease activity index (DAI). IEC mRNA expression of complement components C1q, C2-C9, C3aR, C5aR1 and C5aR2 was measured by real-time PCR (RT-PCR) and immunohistochemistry (IHC) was performed to determine the localization of C3 in colonic tissues as well as the mucus. Additionally, inflamed and non-inflamed colonic biopsy samples collected from IBD patients as well as from healthy individuals were analyzed for C3 mRNA expression levels. Results: Our data demonstrate that primary murine IECs and intestinal epithelial cell lines exclusively express C3 and, to a lower extent, C3aR. However, no IEC mRNA expression of C1q, C2-C9, C5aR1 and C5aR2 was detected. Furthermore, LPS stimulation upregulated IEC expression and secretion of C3. In chronic DSS-induced colitis models, compared to untreated controls, C3 was significantly upregulated in ileal IECs as determined by both IHC and RT-PCR. Notably, no differences in C5, C3aR, C5aR1 or C5aR2 mRNA expression were observed. IHC analyses further revealed elevated C3 levels in the lamina propria, IECs and the mucus. In addition, colonic C3 mRNA expression levels positively correlated with disease activity index (DAI) of DSS-treated mice. Of significant interest, inflamed colonic biopsy samples from CD, but not UC, patients compared to non-inflamed tissue or healthy control samples displayed exacerbated C3 mRNA expression. Conclusion: These findings demonstrate high intestinal C3 levels to occur during severe chronic colitis and hence support the idea that IEC derived C3 plays a role in the pathogenesis of chronic colitis. Furthermore, determination of intestinal C3 levels may be helpful in discriminating CD from UC patients. http://dx.doi.org/10.1016/j.imbio.2016.06.069 55 A highly specific ELISA based assay for screening of complement capacity using EDTA plasma related to different pathways of the complement system Jytte Bryde Clausen ∗ , Jesper Andresen, Katrine Pilely, Peter Garred Laboratory of Molecular Medicine, Department of Clinical Immunology Sect. 7631, Rigshospitalet, University Hospital of Copenhagen, Denmark The complement system is an important part of the innate immune system. Deficiencies in many of the complement proteins and their regulatory molecules can cause severe and recurrent infections, systemic diseases, such as systemic lupus erythematosus and renal diseases. Therefore, it is highly important to screen for complement deficiencies in the different complement pathways; the classical pathway (CP), the lectin pathway (LP), and the alternative pathway (AP). The purpose of this study was to develop specific and standardized ELISA based complement assays to assess the functional capacity of the CP, LP, and the AP at the level of C4, C3 and the terminal complement complex. Traditionally, serum is used in these types of assays. However, it would be desirable also to use EDTA plasma in kinetic complement analyses. We were able to develop ELISA based assays to assess the functional capacity of the CP, the MBL or ficolin mediated LP, and the AP in which EDTA plasma could successfully be used instead of serum. The assays are highly specific using coatings for each pathway and specific buffer systems to eliminate contributions from the other pathways. The assays can be used as a diagnostic tool to assess suspected immunodeficiencies related to the comple-
Abstracts / Immunobiology 221 (2016) 1131–1225
53 PRELP enhances host innate immunity against respiratory tract pathogen Moraxella catarrhalis Guanghui Liu 1,∗ , David Ermert 1 , Martin E. Johansson 2 , Kristian Riesbeck 3 , Anna M. Blom 1 1 Division of Medical Protein Chemistry, Department of Translational Medicine, Lund University, SE-205 02 Malmö, Sweden 2 Pathology, Department of Translational Medicine, Lund University, SE-205 02 Malmö, Sweden 3 Clinical Microbiology, Department of Translational Medicine, Lund University, SE-205 02 Malmö, Sweden
Respiratory tract infections are the leading cause of deaths globally. Increasing antibiotic resistance and low coverage of available vaccines require alternative therapeutics to treat respiratory tract infections. Here we report that proline/arginine-rich end leucine-rich repeat protein (PRELP) is a novel component of innate immune response. We show using immunochemistry that PRELP is present in alveolar fluid, resident macrophages/monocytes, myofibroblasts, and adventitia of blood vessels of lung tissue. PRELP specifically binds respiratory tract pathogens Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis, but not other tested bacterial pathogens. PRELP binds the majority of clinical isolates of M. catarrhalis (n = 49), and interacts directly with the ubiquitous surface protein A2/A2H of M. catarrhalis. PRELP increases the formation of membrane attack complex on M. catarrhalis, thereby enhancing the killing of M. catarrhalis by human serum. Moreover, PRELP competitively inhibits binding of complement inhibitor C4b-binding protein to M. catarrhalis. Furthermore, PRELP reduces Moraxella adhesion to and invasion of human lung epithelial cells. Taken together, PRELP enhances complement attack against M. catarrhalis and prevents bacterial colonization of lung epithelial cells. Thus, PRELP could have therapeutic potential to modulate the innate immune responses toward respiratory tract pathogens, particularly M. catarrhalis. http://dx.doi.org/10.1016/j.imbio.2016.06.068 54 Production of C3 and its secretion into the mucus by intestinal epithelial cells correlates with disease activity in experimental chronic colitis Kerstin Skibbe ∗ , Karen Ebbert, Sophie Preisker, Annika Sünderhauf, Petra Langenstrassen, Christian Sina, Stefanie Derer Molecular Gastroenterology, Department of Medicine 1, University of Lübeck, Germany Introduction: The complement system plays a critical role in the efficient detection and clearance of bacteria. However, uncontrolled and sustained complement activation exacerbates inflammatory processes, resulting in tissue damage as seen in inflammatory bowel diseases (IBD), such as Crohn’s disease (CD) and ulcerative colitis (UC). Therefore, the aim of the present study was to unravel whether bacteria binding to intestinal epithelial cells (IECs) impacts the expression of complement components by IEC under chronic inflammatory conditions. Methods: Chronic DSS colitis was induced in C57BL/6JRj mice through administration of 4% w/v DSS in 3 cycles of 5 days inter-
mitted by 5 days of water. Disease severity was determined by the disease activity index (DAI). IEC mRNA expression of complement components C1q, C2-C9, C3aR, C5aR1 and C5aR2 was measured by real-time PCR (RT-PCR) and immunohistochemistry (IHC) was performed to determine the localization of C3 in colonic tissues as well as the mucus. Additionally, inflamed and non-inflamed colonic biopsy samples collected from IBD patients as well as from healthy individuals were analyzed for C3 mRNA expression levels. Results: Our data demonstrate that primary murine IECs and intestinal epithelial cell lines exclusively express C3 and, to a lower extent, C3aR. However, no IEC mRNA expression of C1q, C2-C9, C5aR1 and C5aR2 was detected. Furthermore, LPS stimulation upregulated IEC expression and secretion of C3. In chronic DSS-induced colitis models, compared to untreated controls, C3 was significantly upregulated in ileal IECs as determined by both IHC and RT-PCR. Notably, no differences in C5, C3aR, C5aR1 or C5aR2 mRNA expression were observed. IHC analyses further revealed elevated C3 levels in the lamina propria, IECs and the mucus. In addition, colonic C3 mRNA expression levels positively correlated with disease activity index (DAI) of DSS-treated mice. Of significant interest, inflamed colonic biopsy samples from CD, but not UC, patients compared to non-inflamed tissue or healthy control samples displayed exacerbated C3 mRNA expression. Conclusion: These findings demonstrate high intestinal C3 levels to occur during severe chronic colitis and hence support the idea that IEC derived C3 plays a role in the pathogenesis of chronic colitis. Furthermore, determination of intestinal C3 levels may be helpful in discriminating CD from UC patients. http://dx.doi.org/10.1016/j.imbio.2016.06.069 55 A highly specific ELISA based assay for screening of complement capacity using EDTA plasma related to different pathways of the complement system Jytte Bryde Clausen ∗ , Jesper Andresen, Katrine Pilely, Peter Garred Laboratory of Molecular Medicine, Department of Clinical Immunology Sect. 7631, Rigshospitalet, University Hospital of Copenhagen, Denmark The complement system is an important part of the innate immune system. Deficiencies in many of the complement proteins and their regulatory molecules can cause severe and recurrent infections, systemic diseases, such as systemic lupus erythematosus and renal diseases. Therefore, it is highly important to screen for complement deficiencies in the different complement pathways; the classical pathway (CP), the lectin pathway (LP), and the alternative pathway (AP). The purpose of this study was to develop specific and standardized ELISA based complement assays to assess the functional capacity of the CP, LP, and the AP at the level of C4, C3 and the terminal complement complex. Traditionally, serum is used in these types of assays. However, it would be desirable also to use EDTA plasma in kinetic complement analyses. We were able to develop ELISA based assays to assess the functional capacity of the CP, the MBL or ficolin mediated LP, and the AP in which EDTA plasma could successfully be used instead of serum. The assays are highly specific using coatings for each pathway and specific buffer systems to eliminate contributions from the other pathways. The assays can be used as a diagnostic tool to assess suspected immunodeficiencies related to the comple-