TY - JOUR
AU - Schubert-Unkmeir, Alexandra
AB - Abstract The interaction of Neisseria meningitidis with both peripheral and brain endothelial cells is a critical event in the development of invasive meningococcal disease. In this study, we used in vitro models based on human brain microvascular endothelial cells (HBMEC), and peripheral endothelial EA.hy926 cells, to investigate their roles in the inflammatory response towards meningococcal infection. Both cell lines were infected with two pathogenic N. meningitidis isolates and secretion of the cytokine interleukin-6 (IL-6), the CXC chemokine IL-8 and the monocyte chemoattractant protein-1 (MCP-1) were estimated by ELISA. Neisseria meningitidis was able to stimulate the production of IL-6 and IL-8 by HBMEC and EA.hy926 cells in a time- and concentration-dependent manner. Interestingly, HBMEC released significant higher amounts of IL-6 and IL-8. Moreover, we observed that heat-killed bacteria stimulated high levels of IL-8. In addition, capsule expression had an inhibitory effect on IL-8 release. We extended our study and included serogroup C strains belonging to sequence type 11 clonal complex (cc) from a recent outbreak in France, as well as isolates belonging to the hypervirulent clonal complexes cc8, cc18, cc32 and cc269 and analyzed their ability to induce the secretion of IL-8 from both cell lines. Although individual variations were observed among different isolates, no clear correlations were observed between strain origin, clinical presentation and IL-8 levels. Neisseria meningitidis, brain and peripheral endothelial cells, IL-8, IL-6, MCP-1, hypervirulent clonal complexes INTRODUCTION Neisseria meningitidis (meningococcus) is a member of the bacterial family Neisseriaceae and appears microscopically as a Gram-negative diplococcus. Meningococci infect exclusively humans and are the causative agent of epidemic meningitis and rapidly progressing septic shock worldwide (Rosenstein et al.2001). While about 8%–25% of the population harbor meningococci as commensals in the nasopharynx, only few develop invasive meningococcal disease (IMD). During infection, N. meningitidis is able to cross the mucosal barrier and spread through the bloodstream, leading to meningococcemia and consecutive sepsis and/or meningitis. The interaction between bacteria and microvasculature endothelial cells is a prerequisite for the development of IMD. To mediate association with endothelial cells, meningococci express a variety of adhesins and invasins, including type IV pili (TfP), the outer membrane proteins, Opa and Opc (Virji, Makepeace et al.1993; Virji, Saunders et al.1993), and a number of newly identified minor adhesion or adhesion-like proteins, such as the adhesin complex protein, Neisserial adhesin A, Neisseria hia homolog A protein or the autotransporter meningococcal serine protease A (reviewed in Hung and Christodoulides 2013). At the cerebrovascular endothelium TfP bind to CD147 and activate the β2-adrenergic receptor that serves primarily as a signaling receptor (Coureuil et al.2010; Bernard et al.2014). Within the bloodstream, meningococci produce a strong inflammatory response and activate the complement and the coagulation cascades. A key inducer of cellular inflammatory responses, the lipooligosaccharide (LOS), is pivotal in causing secretion of various cytokines and chemokines within the vasculature leading to endothelial damage and capillary leakage. Importantly, a relationship between circulating levels of LOS and mortality rates in meningococcal disease has been demonstrated (Waage et al.1989; van Deuren et al.1995). However, non-LOS components of N. meningitidis have also been shown to significantly contribute to cytokine production (Sprong et al.2001; Christodoulides et al.2002; Møller et al.2005; Hellerud et al.2015). Both cytokines and chemokines are small molecules secreted by a variety of cell types. Chemokines (chemoattractant cytokines) resemble a subclass of cytokines and control the migration of monocytes, neutrophils and lymphocytes as well as chemotaxis induction. Cytokines can act as proinflammatory stimulus during an immune response at the site of infection; they also have homeostatic properties and are involved in the control of cells (Deshmane et al.2009; Palomino and Marti 2015). The chemokine interleukin-8 (IL-8) has been described as a stimulus for the degranulation of neutrophil granulocytes; hence, the synonym ‘neutrophil activating protein-1 (NAP-1)’ has been elected. Among many other functions, IL-8 is involved in rearrangement of the cytoskeleton, changes in intracellular calcium levels, integrin-receptor activation and respiratory burst (Paccaud, Schifferli and Baggiolini 1990; Baggiolini, Dewald and Moser 1994; Palomino and Marti 2015). Elevated levels of IL-8 in patients’ samples have been found in chronic inflammatory diseases as well as in systemic infections. A key function for inducing migration and infiltration of monocytes and macrophages is fulfilled by the chemokine monocyte chemoattractant protein-1 (MCP-1). Monocytes migrate from the blood into tissue, where they further differentiate into tissue macrophages and serve as mediators for immune surveillance and inflammatory response (Deshmane et al.2009; Ziegler-Heitbrock et al.2010). The cytokine IL-6 is commonly known to be involved in inflammation processes and is used as a marker protein in human serum samples to determine inflammatory reactions in patients. Recent studies indicate that IL-6 is furthermore involved in regenerative processes as well as in the regulation of metabolism, in the maintenance of bone homeostasis and in many neural functions (Scheller et al.2011). IL-6 antagonists have been developed as therapeutic drugs for systemic inflammatory diseases as rheumatoid arthritis, whereby this therapy is accompanied with elevated risks for infections. Endothelial cells have long been viewed as a passive lining of blood vessels endowed essentially with negative properties such as that of being non-reactive to blood components. It is now evident that upon exposure to different environmental stimuli vascular cells undergo profound changes in gene expression and function that allow these cells to participate actively in inflammatory reactions, immunity and thrombosis. Since both the interaction with brain microvascular and the interaction with peripheral macrovascular endothelial cells are pivotal for the development of IMD, we set out to compare the profiles of cytokine and chemokine secretion from both microvascular and macrovascular endothelial cells. A previous study from our group based on microarray technology revealed the early expression of several cytokine genes, including those encoding IL-6, IL-8 and MCP-1, in human brain microvascular endothelial cells (HBMEC) following infection with N. meningitidis (Schubert-Unkmeir et al.2007). However, the differences in gene expression may not reflect the biological significance and secretion of cytokines was only investigated for IL-6 in this previous study. The aim of this study was to compare the amount of proinflammatory cytokine and chemokine protein secretion from brain microvascular and peripheral endothelial cells after meningococcal challenge and to determine the effect of particular meningococcal components (capsule) on the production of IL-8 release. We extended our study and included a panel of well characterized clinical meningococcal isolates, including serogroup C isolates from a recent outbreak in France belonging to sequence type 11 (ST-11) clonal complex (cc), as well as meningococcal isolates belonging to the hypervirulent clonal complexes cc8, cc18, cc32 and cc269, and analyzed a possible correlation between the strain origin, clinical presentation and IL-8 levels released from endothelial cells. MATERIALS AND METHODS Bacterial strains and growth conditions Neisseria meningitidis strain MC58 is a serogroup (Sg) B strain of the sequence type (ST)-74 (ST-32 clonal complex [cc]), which was isolated in 1985 in the UK and was kindly provided by E. R. Moxon (McGuinness et al.1991). Neisseria meningitidis serogroup C strain 8013/clone12 is a SgC strain and belongs to ST-18cc (ST-177) (Nassif et al.1993).The construction of the capsule-deficient mutant of strain MC58 (MC58 siaD) was described earlier (Unkmeir et al.2002). Neisseria meningitidis SgC strains LNP24198 (C:P1.7–1,1:F3–6:cc11), LNP26643 (C:P1.7–1,1:F3–6:cc11) and LNP27256 (C:P1.5–1,10–8:F3–6:cc11) and N. meningitidis SgB strains LNP13763 (B:P1.7,16:F3–3:cc32), LNP21362 (B:P1.7,16:F3–3:cc32), LNP26900 (B:P1.7–2,16–13:F3–3:cc32), LNP26087 (B:P1.7–2,16:F3–3:cc32) and LNP26639 (B:P1.19–1,15–11:F5–36:cc269) were isolated during several recent outbreaks in France (Deghmane et al.2010). Clinical N. meningitidis isolates used in this study are summarized in Table 1. Table 1. List of N. meningitidis strains used in this study. ID  Serogroup  Sequence type (ST)  Clonal complex (cc)  Genotype typing group:P1.VR1,VR2, FetA:cc  Year isolated  Geography  Isolated (site)  Clinical presentation  Reference  DE7901  B  18  cc18  B:P1.12–1,2–2, F4–2:cc18  2001  Germany  n.s.  IMD  Brehony, Jolley and Maiden (2007)  LNP13763  B  32  cc32  B:P1.7,16, F3–3:cc32  1995  France  Blood  Meningococcemia  This work  LNP21362  B  32  cc32  B:P1.7,16, F3–3:cc32  2006  France  CSF  Meningococcemia+meningitis  Caron et al. (2011)  LNP26087  B  32  cc32  B:P1.7–2,16, F3–3:cc32  2011  France  Blood  Meningococcemia+meningitis  This work  LNP26639  B  269  cc269  B:P1.19–1,15–11: F5–36:cc269  2012  France  CSF  Meningitis  This work  LNP26900  B  32  cc32  B:P1.7–2,16–13, F3–3:cc32  2012  France  Blood  Meningococcemia  This work  MC58  B  74  cc32  B:P1.7,16–2, F1–5:cc32  1985  UK  n.s.  IMD  McGuinness et al. (1991)  8013/clone12  C  177  cc18  C:P1.21,26–2, F1–5:cc18  1989  France  n.s.  IMD  Rusniok et al. (2009)  DE6904  C  8  cc8  C:P1.5,2, F5–8:cc8  2002  Germany  n.s.  IMD  Brehony, Jolley and Maiden (2007)  DE7017  C  11  cc11  C:P1.22,14, F3–3:cc11  2000  Germany  n.s.  IMD  Brehony, Jolley and Maiden (2007)  FAM18  C  11  cc11  C:P1.5,22, F1–30:cc11  1980  USA  n.s.  IMD  Bentley et al. (2007)  LNP24198  C  11  cc11  C:P1.7–1:1:F3–6:cc11  2007  France  Blood  Septic shock  Deghmane et al. (2010)  LNP26643  C  11  cc11  C:P1.7–1:1:F3–6:cc11  2012  France  CSF  Meningitis  This work  LNP27256  C  11  cc11  C:P1.5–1,10–8:F3–6:cc11  2013  France  Skin biopsy  Septic shock  Taha et al. (2016)  WUE2121  C  11  cc11  C:P1.5,2, F3–9:cc11  1997  Germany  n.s.  IMD  Vogel et al. (1998)  ID  Serogroup  Sequence type (ST)  Clonal complex (cc)  Genotype typing group:P1.VR1,VR2, FetA:cc  Year isolated  Geography  Isolated (site)  Clinical presentation  Reference  DE7901  B  18  cc18  B:P1.12–1,2–2, F4–2:cc18  2001  Germany  n.s.  IMD  Brehony, Jolley and Maiden (2007)  LNP13763  B  32  cc32  B:P1.7,16, F3–3:cc32  1995  France  Blood  Meningococcemia  This work  LNP21362  B  32  cc32  B:P1.7,16, F3–3:cc32  2006  France  CSF  Meningococcemia+meningitis  Caron et al. (2011)  LNP26087  B  32  cc32  B:P1.7–2,16, F3–3:cc32  2011  France  Blood  Meningococcemia+meningitis  This work  LNP26639  B  269  cc269  B:P1.19–1,15–11: F5–36:cc269  2012  France  CSF  Meningitis  This work  LNP26900  B  32  cc32  B:P1.7–2,16–13, F3–3:cc32  2012  France  Blood  Meningococcemia  This work  MC58  B  74  cc32  B:P1.7,16–2, F1–5:cc32  1985  UK  n.s.  IMD  McGuinness et al. (1991)  8013/clone12  C  177  cc18  C:P1.21,26–2, F1–5:cc18  1989  France  n.s.  IMD  Rusniok et al. (2009)  DE6904  C  8  cc8  C:P1.5,2, F5–8:cc8  2002  Germany  n.s.  IMD  Brehony, Jolley and Maiden (2007)  DE7017  C  11  cc11  C:P1.22,14, F3–3:cc11  2000  Germany  n.s.  IMD  Brehony, Jolley and Maiden (2007)  FAM18  C  11  cc11  C:P1.5,22, F1–30:cc11  1980  USA  n.s.  IMD  Bentley et al. (2007)  LNP24198  C  11  cc11  C:P1.7–1:1:F3–6:cc11  2007  France  Blood  Septic shock  Deghmane et al. (2010)  LNP26643  C  11  cc11  C:P1.7–1:1:F3–6:cc11  2012  France  CSF  Meningitis  This work  LNP27256  C  11  cc11  C:P1.5–1,10–8:F3–6:cc11  2013  France  Skin biopsy  Septic shock  Taha et al. (2016)  WUE2121  C  11  cc11  C:P1.5,2, F3–9:cc11  1997  Germany  n.s.  IMD  Vogel et al. (1998)  n.s.: not specified. IMD: invasive meningococcal disease. CSF: cerebrospinal fluid. View Large Table 1. List of N. meningitidis strains used in this study. ID  Serogroup  Sequence type (ST)  Clonal complex (cc)  Genotype typing group:P1.VR1,VR2, FetA:cc  Year isolated  Geography  Isolated (site)  Clinical presentation  Reference  DE7901  B  18  cc18  B:P1.12–1,2–2, F4–2:cc18  2001  Germany  n.s.  IMD  Brehony, Jolley and Maiden (2007)  LNP13763  B  32  cc32  B:P1.7,16, F3–3:cc32  1995  France  Blood  Meningococcemia  This work  LNP21362  B  32  cc32  B:P1.7,16, F3–3:cc32  2006  France  CSF  Meningococcemia+meningitis  Caron et al. (2011)  LNP26087  B  32  cc32  B:P1.7–2,16, F3–3:cc32  2011  France  Blood  Meningococcemia+meningitis  This work  LNP26639  B  269  cc269  B:P1.19–1,15–11: F5–36:cc269  2012  France  CSF  Meningitis  This work  LNP26900  B  32  cc32  B:P1.7–2,16–13, F3–3:cc32  2012  France  Blood  Meningococcemia  This work  MC58  B  74  cc32  B:P1.7,16–2, F1–5:cc32  1985  UK  n.s.  IMD  McGuinness et al. (1991)  8013/clone12  C  177  cc18  C:P1.21,26–2, F1–5:cc18  1989  France  n.s.  IMD  Rusniok et al. (2009)  DE6904  C  8  cc8  C:P1.5,2, F5–8:cc8  2002  Germany  n.s.  IMD  Brehony, Jolley and Maiden (2007)  DE7017  C  11  cc11  C:P1.22,14, F3–3:cc11  2000  Germany  n.s.  IMD  Brehony, Jolley and Maiden (2007)  FAM18  C  11  cc11  C:P1.5,22, F1–30:cc11  1980  USA  n.s.  IMD  Bentley et al. (2007)  LNP24198  C  11  cc11  C:P1.7–1:1:F3–6:cc11  2007  France  Blood  Septic shock  Deghmane et al. (2010)  LNP26643  C  11  cc11  C:P1.7–1:1:F3–6:cc11  2012  France  CSF  Meningitis  This work  LNP27256  C  11  cc11  C:P1.5–1,10–8:F3–6:cc11  2013  France  Skin biopsy  Septic shock  Taha et al. (2016)  WUE2121  C  11  cc11  C:P1.5,2, F3–9:cc11  1997  Germany  n.s.  IMD  Vogel et al. (1998)  ID  Serogroup  Sequence type (ST)  Clonal complex (cc)  Genotype typing group:P1.VR1,VR2, FetA:cc  Year isolated  Geography  Isolated (site)  Clinical presentation  Reference  DE7901  B  18  cc18  B:P1.12–1,2–2, F4–2:cc18  2001  Germany  n.s.  IMD  Brehony, Jolley and Maiden (2007)  LNP13763  B  32  cc32  B:P1.7,16, F3–3:cc32  1995  France  Blood  Meningococcemia  This work  LNP21362  B  32  cc32  B:P1.7,16, F3–3:cc32  2006  France  CSF  Meningococcemia+meningitis  Caron et al. (2011)  LNP26087  B  32  cc32  B:P1.7–2,16, F3–3:cc32  2011  France  Blood  Meningococcemia+meningitis  This work  LNP26639  B  269  cc269  B:P1.19–1,15–11: F5–36:cc269  2012  France  CSF  Meningitis  This work  LNP26900  B  32  cc32  B:P1.7–2,16–13, F3–3:cc32  2012  France  Blood  Meningococcemia  This work  MC58  B  74  cc32  B:P1.7,16–2, F1–5:cc32  1985  UK  n.s.  IMD  McGuinness et al. (1991)  8013/clone12  C  177  cc18  C:P1.21,26–2, F1–5:cc18  1989  France  n.s.  IMD  Rusniok et al. (2009)  DE6904  C  8  cc8  C:P1.5,2, F5–8:cc8  2002  Germany  n.s.  IMD  Brehony, Jolley and Maiden (2007)  DE7017  C  11  cc11  C:P1.22,14, F3–3:cc11  2000  Germany  n.s.  IMD  Brehony, Jolley and Maiden (2007)  FAM18  C  11  cc11  C:P1.5,22, F1–30:cc11  1980  USA  n.s.  IMD  Bentley et al. (2007)  LNP24198  C  11  cc11  C:P1.7–1:1:F3–6:cc11  2007  France  Blood  Septic shock  Deghmane et al. (2010)  LNP26643  C  11  cc11  C:P1.7–1:1:F3–6:cc11  2012  France  CSF  Meningitis  This work  LNP27256  C  11  cc11  C:P1.5–1,10–8:F3–6:cc11  2013  France  Skin biopsy  Septic shock  Taha et al. (2016)  WUE2121  C  11  cc11  C:P1.5,2, F3–9:cc11  1997  Germany  n.s.  IMD  Vogel et al. (1998)  n.s.: not specified. IMD: invasive meningococcal disease. CSF: cerebrospinal fluid. View Large Meningococci were cultured on Columbia Agar with 5% sheep blood (COS; bioMérieux, Lyon, France) and incubated at 37°C with 5% CO2 overnight. Liquid culturing was performed in proteose-peptone medium (PPM) plus 1% Kellogg's supplement I and II. The medium was inoculated with N. meningitidis strains from overnight cultures and was subsequently shaken at 37°C for 90 min until an OD of 1.0 (±0.1) was reached. For experiments with heat-killed meningococci, bacteria were then incubated at 65°C for 2 h. Killing was verified by streaking on Columbia Agar with 5% sheep blood (COS; bioMérieux, Lyon, France) and incubating at 37°C with 5% CO2 overnight. Cell lines and cell culturing HBMEC were kindly provided by K.S. Kim. HBMEC were isolated from brain biopsies of children suffering from epilepsy and immortalized by transfection with simian 40 large T antigen (Stins, Gilles and Kim 1997). HBMEC were cultured in culture flasks or 24-well plates coated with 0.2% gelatin. Culture medium contained RPMI 1640 medium (gibco life technologies, Karlsruhe, Germany) supplemented with 10% fetal calf serum (FCS, Gibco Life Technologies, Karlsruhe, Germany), 10% NuSerum® IV (Corning, NY, USA), 1% sodium pyruvate (1 mM), 1% L-Glutamine (2 mM), 1% non-essential amino acids (all purchased from GE Healthcare, Little Chalfont, UK), 5 U mL−1 heparin (BIOCHROM, Berlin, Germany) and 30 μg mL−1 endothelial cell growth supplement (ECGS, CellSystems, Troisdorf, Germany). EA.hy926 cells were purchased from ATCC (ATCC-CRL-2922, Manassas, VA, USA). EA.hy926 cells are hybridoma cells derived from human umbilical vein endothelial cells fused with the permanent human cell line A549 (Edgell, McDonald and Graham 1983; Edgell et al.1990). EA.hy926 cells were cultured in culture flasks or 24-well plates in Dulbecco's Modified Eagle Medium (DMEM high glucose GlutaMAX® supplement pyruvate, Gibco Life Technologies) supplemented with 10% FCS. Growth conditions for both cell lines contained humid atmosphere with 5% CO2 at 37°C. Infection of cells Cells were grown to confluence on a 24-well plate, with 4 × 105 cells per well. Prior to infection, cells were washed with RPMI or DMEM, respectively. RPMI or DMEM plus 10% heat-inactivated (30 min at 56°C) human serum (TCS Biosciences, Botolph Claydon, UK) was added as infection medium, and cells were infected with different concentrations of bacteria ranging from 4 × 106 to 8 × 107 CFU per monolayer, corresponding to a multiplicity of infection (MOI) of 10, 100 and 200, respectively. Cells treated with infection medium and sterile PPM+ medium served as mock control. Adherence and invasion Adherence of meningococci to endothelial cells was determined by colony-forming unit (CFU) counting with ProtoCOL colony counter (Synbiosis, Cambridge, UK). Two, 4 and 6 h post-infection(p.i.), cell culture supernatant was analyzed for unbound bacteria by streaking on COS agar plates which were then incubated at 37°C with 5% CO2 overnight. Cells were washed with RPMI or DMEM without supplements. One panel of cells was incubated with 1% saponin diluted in cell culture medium for 15 min. Cells with adherent bacteria were then scratched off the well bottom and streaked on COS agar plates to determine adherence. The second panel was incubated for 2 h with 200 μg mL−1 gentamicin (BIOCHROM, Berlin, Germany) solved in RPMI or DMEM to kill any remaining extracellular bacteria. Cells were washed again and treated with saponin as described above. Cells containing invaded bacteria were collected and streaked on COS agar plates to determine the number of invasive meningococci. Subsequently, efficiency of antibiotic treatment was verified by incubating the gentamicin solution overnight on COS agar. Results of adherence and invasion CFU counts were only further evaluated if the gentamicin control had remained sterile. Cytokine ELISA BD OptEIA® Human IL-8 ELISA Kit II (BD, San Jose, CA, USA) was used according to the manufacturer's instructions to determine IL-8 concentration in supernatants of infected cells. IL-6 and MCP-1 were detected by BD OptEIA Set Human IL-6 or BD OptEIA Set Human MCP-1, respectively (both BD Biosciences, San Diego, USA). Cell culture supernatants were diluted with sample diluent (PBS plus 10% FCS) 1:10 both for EA.hy926 and HBMEC supernatants for IL-6-, IL-8- and MCP-1-ELISA. Supernatants of cells infected with MC58 siaD for 6 h were diluted 1:40. Statistical analysis Statistical differences between groups were calculated using the Student's unpaired t-test (two-tailed) using Excel (Microsoft Office). P-values ≤ 0.05 were considered significant, whereas P-values ≤ 0.01 were considered highly significant. RESULTS Determination of cytokine and chemokine secretion by brain microvascular and peripheral endothelial cells challenged with Neisseria meningitidis In order to compare the properties of brain microvascular endothelial cells with peripheral endothelial cells in terms of the inflammatory response during meningococcal infection, two cell lines were implemented: HBMEC and the peripheral endothelial cell line EA.hy926. The EA.hy926 cell line is a hybrid cell, derived by the fusion of HUVEC with the continuous human lung carcinoma cell line A549 and is one of the best characterized macrovascular endothelial cell lines (Bouïs et al.2001). Both cell lines were infected with the serogroup B disease strain MC58, a corresponding isogenic unencapsulated mutant strain (MC58 siaD) and the serogroup C disease isolate 8013/clone12, and release of cytokine and chemokine proteins was estimated by commercially available enzyme-linked immunosorbent assays (ELISA). Neisseria meningitidis MC58 was chosen as a prototype strain belonging to ST-32 clonal complex (cc), while N. meningitidis isolate 8013/clone 12 (also known as clone 12 or 2C4.3) was included as a prototype ST-18 cc disease strain. HBMEC and EA.hy926 cell monolayers were infected with various concentrations of bacteria (ranging from 4 × 106 to 8 × 107 colony-forming units (CFU)/monolayer, respectively), and a dose-dependent release of cytokine and chemokine secretion was measured over a time period from 2 h p.i. up to 6 h p.i. (Figs 1 and 2). Challenge with increased concentrations of N. meningitidis induced a time and dose-dependent secretion of IL-6 and IL-8, compared to the results obtained with uninfected control cells. In particular, challenge of the HBMEC cell line with N. meningitidis MC58 induced high levels of IL-8, ranging from 153 pg mL−1 at 2 h p.i. to 1700 pg mL­1 at 6 h when cells were infected with 4 × 106 CFU/monolayer and 292 pg mL−1 at 2 h p.i. to 2962 pg mL−1 at 6 h when cells were infected with a high infection dose of 8 × 107 CFU/monolayer (Fig. 1A). Smaller amounts of IL-6 release in response to MC58 infection were observed, ranging from 0.21 pg mL−1 at 2 h p.i. to 2.47 pg mL−1 at 6 h when cells were infected with 4 × 106 CFU/monolayer and 1.96 pg mL−1 at 2 h p.i. to 5.24 pg mL−1 at 6 h p.i. when cells were infected with 8 × 107 CFU/monolayer (Fig. 1B). In contrast, MCP-1 was not detectable in significant amounts in supernatants derived from MC58-infected HBMEC compared to supernatants from the uninfected control cells (Fig. 1C). Infection with N. meningitidis strain 8013/clone12 resulted in comparable release of IL-6, IL-8 and MCP-1. Figure 1. View largeDownload slide Chemokine and cytokine secretion by HBMEC. Chemokine and cytokine protein secretion by human brain microvascular endothelial cells (HBMEC) infected with 4 × 106, 4 × 107 and 8 × 107 CFU of N. meningitidis strains MC58, MC58 siaD and 8013/clone12. The data are mean chemokine/cytokine levels, and the error bars indicate standard errors of the mean of three independent experiments performed in duplicate. *P ≤ 0.05, **P ≤ 0.01. Figure 1. View largeDownload slide Chemokine and cytokine secretion by HBMEC. Chemokine and cytokine protein secretion by human brain microvascular endothelial cells (HBMEC) infected with 4 × 106, 4 × 107 and 8 × 107 CFU of N. meningitidis strains MC58, MC58 siaD and 8013/clone12. The data are mean chemokine/cytokine levels, and the error bars indicate standard errors of the mean of three independent experiments performed in duplicate. *P ≤ 0.05, **P ≤ 0.01. Figure 2. View largeDownload slide Chemokine and cytokine secretion by EA.hy926. Chemokine and cytokine protein secretion by EA.hy926 cells infected with 4 × 106, 4 × 107 and 8 × 107 CFU of N. meningitidis strains MC58, MC58 siaD and 8013/clone12. The data are mean chemokine/cytokine levels, and the error bars indicate standard errors of the mean of three independent experiments performed in duplicate. * P ≤ 0.05, ** P ≤ 0.01. Figure 2. View largeDownload slide Chemokine and cytokine secretion by EA.hy926. Chemokine and cytokine protein secretion by EA.hy926 cells infected with 4 × 106, 4 × 107 and 8 × 107 CFU of N. meningitidis strains MC58, MC58 siaD and 8013/clone12. The data are mean chemokine/cytokine levels, and the error bars indicate standard errors of the mean of three independent experiments performed in duplicate. * P ≤ 0.05, ** P ≤ 0.01. To compare the properties of brain microvascular endothelial cells with peripheral endothelial cells next the inflammatory response of EA.hy926 cells, a peripheral macrovascular endothelial cell line after meningococcal challenge was analyzed. Therefore, EA.hy926 cells were infected with N. meningitidis strains MC58 and 8013/clone12, and release of cytokine and chemokine proteins was estimated according to the time points used for the HBMEC cell line. As observed with HBMEC, challenge with increasing concentrations of N. meningitidis induced a time- and dose-dependent secretion of IL-6 and IL-8, and in addition MCP-1 (Fig. 2A–C). Significant differences compared to the uninfected control cells were again observed at 6 h p.i. when cells were infected with a high infection dose of 8 × 107 CFU/monolayer (Fig. 2). Compared to the HBMEC cell line, the amount of detectable IL-6 in EA.hy926 supernatants with a maximum of 1.36 pg mL−1 was negligible, although the time- and dose-dependent increase was statistically significant (Fig. 2B). In contrast to HBMEC, meningococcal challenge of EA.hy926 with MC58 triggered a significant release of MCP-1 at 4 and 6 h p.i. ranging from 2.73 pg mL−1 at 4 h p.i. to 4.00 pg mL−1 at 6 h using an infection dose of 4 × 106 CFU/monolayer and 5.59 pg mL−1 at 4 h p.i. to 13.69 pg mL−1 at 6 h using an infection dose of 8 × 107 CFU/monolayer (Fig. 2C). In parallel, the contribution of capsule expression in modulating the induction of cytokine and chemokine secretion was determined by infecting both cell lines with the corresponding isogenic unencapsulated mutant strain MC58 siaD. Interestingly, challenge with N. meningitidis MC58 siaD induced significant higher levels of IL-6 and IL-8 secretion at 4 and 6 h p.i. from both HBMEC and EA.hy926 cells compared to the encapsulated parental strain suggesting an inhibitory effect of capsule expression in terms of cytokine release (Figs 1 and 2). Adherence and invasion of different Nm strains to/into endothelial cells To examine whether cytokine and chemokine release could be correlated to the adhesive and/or invasive properties of meningococci, gentamicin protection assays were performed as described before (Unkmeir et al.2002). Both cell lines were infected with three different doses of bacteria as described above and adhesion and invasion were estimated after 2 h, 4 h and 6 h p.i. Meningococci adhered very effectively to both the HBMEC and the EA.hy926 cell line reaching a maximum of about 5 × 107 adherent bacteria per cell monolayer for strain MC58 at 6 h p.i. (Fig. 3). In addition to adherence, the meningococcal wild-type strain MC58 elicited a time-dependent increase of bacterial uptake ranging from 5.3 × 101 at 2 h p.i. up to 1.2 × 103 at 6 h p.i. for HBMEC and from 2.1 × 102 at 2 h p.i. up to 5.2 × 103 at 6 h p.i. for EA.hy926 cells. The unencapsulated mutant strain MC58 siaD was significantly more invasive than the wild-type strain (P < 0.01), according to previously published data (Unkmeir et al.2002; Fowler et al.2006). Whereas for HBMEC no significant differences between strain MC58 and strain 8013/12 were observed in terms of invasiveness, strain MC58 was significantly more invasive in EA.hy926 compared to strain 8013/12. Taken together, our findings suggested that the potency of the inflammatory response might be either controlled by capsule expression or might be due to higher invasive properties of unencapsulated N. meningitidis. Figure 3. View largeDownload slide Adherence to and invasion into HBMEC and EA.hy926 cells. Adherence to and invasion into HBMEC (A) and EA.hy926 cells (B) were assessed over a 6 h infection time using 4 × 106, 4 × 107 and 8 × 107 CFU of N. meningitidis strains MC58 (black bars), MC58 siaD (gray bars) and 8013/clone12 (white bars) by gentamicin protection assays at the time interval indicated. The data show mean values ± SD of three independent experiments conducted in duplicate. Figure 3. View largeDownload slide Adherence to and invasion into HBMEC and EA.hy926 cells. Adherence to and invasion into HBMEC (A) and EA.hy926 cells (B) were assessed over a 6 h infection time using 4 × 106, 4 × 107 and 8 × 107 CFU of N. meningitidis strains MC58 (black bars), MC58 siaD (gray bars) and 8013/clone12 (white bars) by gentamicin protection assays at the time interval indicated. The data show mean values ± SD of three independent experiments conducted in duplicate. IL-8 release in response to Neisseria meningitidis isolates belonging to hypervirulent clonal complexes cc32, cc18, cc8, cc11 and cc269 Recently, the emergence of new virulent N. meningitidis serogroup C ST-11 cc isolates in France has been reported (Deghmane et al.2010). These isolates displayed a new phenotype (C:2a:P1.7,1), caused infections that occurred as clusters and were associated with high virulence in mice (Deghmane et al.2010). We next determined the levels of human IL-8 release in vitro by endothelial cell lines in response to those isolates and extended the analyses towards further isolates belonging to the hyperinvasive clonal complexes cc8, cc18, cc32 and cc269 (Table 1). Both HBMEC and EA.hy926 were infected with bacteria for 6 h using an infection dose of 8 × 107 CFU/monolayer. However, although individual variations were observed among different N. meningitidis isolates, no correlations were observed between the strain origin, clinical presentation and IL-8 levels released from endothelial cell lines (Fig. 4): strains LNP26639 and LNP26643 (both found to cause meningitis) induced significantly elevated levels of IL-8 secreted from both cell lines; strains LNP13763, LNP21362, LNP26087, LNP24198 and LNP27256 were associated with septicemia or septicemia and meningitis and also triggered high IL-8 release from HBMEC, slightly less high as the levels in response to the meningitis causing isolates LNP26639 and LNP26643. This trend of meningitis-associated isolates to induce higher levels of IL-8 seemed clearer if only the three cc11 isolates were considered (Fig. 4). Compared to HBMEC, strains LNP13763, LNP21362, LNP26087, LNP24198 and LNP27256 triggered low-intermediate IL-8 levels from EA.hy926 cells (P < 0.01). Figure 4. View largeDownload slide Differential IL-8 release of HBMEC and EA.hy926 cells. Differential IL-8 release of HBMEC (A) and EA.hy926 cells (B) infected with 8 × 107 CFU of different N. meningitidis strains belonging to the hypervirulent clonal complexes cc32, cc18, cc8, cc11 and cc269. Shown are mean chemokine levels, and the error bars indicate standard errors of the mean of three independent experiments performed in duplicate. Figure 4. View largeDownload slide Differential IL-8 release of HBMEC and EA.hy926 cells. Differential IL-8 release of HBMEC (A) and EA.hy926 cells (B) infected with 8 × 107 CFU of different N. meningitidis strains belonging to the hypervirulent clonal complexes cc32, cc18, cc8, cc11 and cc269. Shown are mean chemokine levels, and the error bars indicate standard errors of the mean of three independent experiments performed in duplicate. Adhesive and invasive properties of Neisseria meningitidis belonging to different hyperinvasive clonal complexes As described above, we examined whether chemokine release in response to Sg C ST-11 cc isolates and isolates of cc8, cc18, cc32 and cc269 could be correlated to adhesive and/or invasive properties of the tested isolates and performed gentamicin protection assays. Both cell lines were infected using a concentration of 8 × 107 CFU/monolayer for 6 h as described above, and adhesion and invasion were estimated after 2, 4 and 6 h p.i. All N. meningitidis isolates adhered effectively to both HBMEC and EA.hy926 cells ranging between about 8 × 106 and 9 × 108 adherent bacteria per cell monolayer (Fig. 5, only data for 6 h p.i. are shown). In line with the data found for chemokine release, individual variations were observed among different N. meningitidis isolates. However, no correlations between the strain origin, clinical picture and adhesive or invasive properties were detected (Fig. 5). Figure 5. View largeDownload slide Adherence to and invasion into HBMEC and EA.hy926 cells. Adherence to and invasion into HBMEC (A) and EA.hy926 cells (B) were determined after 6 h of infection using 8 × 107 CFU of N. meningitidis strains of the hypervirulent clonal complexes cc32, cc18, cc8, cc11 and cc269. The data show mean values ± SD of three independent experiments conducted in duplicate. Figure 5. View largeDownload slide Adherence to and invasion into HBMEC and EA.hy926 cells. Adherence to and invasion into HBMEC (A) and EA.hy926 cells (B) were determined after 6 h of infection using 8 × 107 CFU of N. meningitidis strains of the hypervirulent clonal complexes cc32, cc18, cc8, cc11 and cc269. The data show mean values ± SD of three independent experiments conducted in duplicate. Effect of heat-killed meningococci on IL-8 release from both HBMEC and EA.hy926 We next examined whether killed or not killed N. meningitidis can induce the release of IL-8 from both HBMEC and EA.hy926 cells. Both cell lines were exposed to live and heat-killed (65°C for 2 h) meningococcal isolates MC58 and the unencapsulated mutant strain MC58 siaD using three different doses (4 × 106, 4 × 107 and 8 × 107 CFU/monolayer), and IL-8 release was measured after 6 h p.i., as described above. A dose-dependent response relationship of heat-killed N. meningitidis for IL-8 production by HBMEC and EA.hy926 cells after 6 h p.i. was observed (Fig. 6A and B). IL-8 release from HBMEC did not significantly differ between cells infected with viable MC58 and cells infected with heat-killed MC58, suggesting that mainly heat-stable molecules, including LOS, heat-stable proteins or peptidoglycan, contribute to IL-8 release. Of interest, heat-killed meningococci were able to induce substantial higher amounts of IL-8 from EA.hy926 than viable bacteria. As determined above heat-killed capsule-deficient MC58 siaD meningococci induced a higher amount of chemokine release compared to the heat-killed encapsulated wild-type strain. Figure 6. View largeDownload slide Dose-dependent release of IL-8 by HBMEC and EA.hy926 cells. Dose-dependent release of IL-8 by HBMEC (A) and EA.hy926 cells (B) incubated with viable and heat-killed N. meningitidis for 6 h. The data are mean values and the error bars indicate standard errors of the mean of three independent experiments performed in duplicate. Figure 6. View largeDownload slide Dose-dependent release of IL-8 by HBMEC and EA.hy926 cells. Dose-dependent release of IL-8 by HBMEC (A) and EA.hy926 cells (B) incubated with viable and heat-killed N. meningitidis for 6 h. The data are mean values and the error bars indicate standard errors of the mean of three independent experiments performed in duplicate. DISCUSSION Endothelial cells have long been viewed as a passive lining of blood vessels and non-reactive to blood components. During the last years, it became evident that upon exposure to different environmental stimuli vascular cells undergo profound changes in gene expression and function that allow these cells to participate actively in inflammatory reactions, immunity and thrombosis. During meningococcal infection, both the interaction with brain microvascular and the interaction with peripheral macrovascular endothelial cells are pivotal for the development of IMD. In this study, we therefore investigated and compared the inflammatory response of HBMEC, a microvascular brain endothelial cell line, with EA.hy926, a peripheral macrovascular endothelial cell line, in response to N. meningitidis infection. We demonstrate that N. meningitidis was able to stimulate the production of IL-6 and IL-8 from HBMEC and from EA.hy926 cells in a time- and concentration-dependent manner. Of note, our findings showed that the brain microvascular endothelial cell line released significant higher amounts of IL-6 and IL-8 compared to the peripheral endothelial cell line following infection with N. meningitidis. Moreover, we observed that heat-killed bacteria stimulated high levels of IL-8 from EA.hy926 cells. In addition, capsule expression had an inhibitory effect on IL-8 release. The production of key inflammatory mediators is closely associated with organ failure and outcome in patients suffering from IMD (Waage et al.1989; Halstensen et al.1993; van Deuren et al.1995; Møller et al.2005). The major proinflammatory cytokines which are upregulated during IMD are TNF-α, IL-1β, IL-6, IL-8, MCP-1, MCP-1α and G-CSF. These different cytokines are produced at multiple sites in the body and released to the circulation. The liver, spleen, circulating leukocytes and endothelial cells all have the capacity to produce inflammatory mediators, and several studies have been conducted to demonstrate the ability of N. meningitidis to induce secretion of proinflammatory cytokines and chemokines from different cell types, including monocytes, dendritic cells, endothelial cells and meningothelial meningioma cells (Taha 2000; Dixon et al.2001; Wells et al.2001; Christodoulides et al.2002). Herein we compared the inflammatory response from a brain endothelial cell line with a peripheral endothelial cell line after meningococcal infection. Interestingly, our data showed that the brain microvascular endothelial cell line released significant higher amounts of IL-6 and IL-8 compared to the peripheral endothelial cell line following infection with N. meningitidis, whereas MCP-1 was only significantly released from infected EA.hy926 cells. A possible explanation for the higher cytokine release from HBMEC is that the induction of cytokines is the result of bacteria-cell adhesion. When comparing the adhesive and invasive properties of N. meningitidis to HBMEC and EA.hy926 cells we found a slight higher number of bacteria adhering to HBMEC. Moreover, the number of invasive bacteria was slightly higher for HBMEC compared to EA.hy926 cells. It has already been shown for bacteria that adhesion to endothelial cells is a prerequisite for cytokine production. For example, pilus-mediated adhesion of N. gonorrhoeae has been shown to upregulate the expression and secretion of IL-8, GM-CSF and TNF-α (Naumann et al.1997; Christodoulides et al.2000). In line with our data, it has been shown that only adherent meningococci induce the expression of TNF-α by endothelial cells (Taha 2000); however, TNF­α is only released in the presence of monocytes (Taha 2000). Another explanation for the differential activation of cytokines might be that receptors such as the Toll-like receptor 4, which has been shown to confer responsiveness to Gram-negative lipopolysaccharides (LPS), are differentially expressed on HBMEC and EA.hy926 cells. LPS has long been recognized as a key factor in inducing a strong immune response in mammalian cells. However, several reports have shown that in vitro cytokine release is not only caused by meningococcal LPS, and non-LPS molecules significantly contribute to the inflammatory reaction of the host (Sprong et al.2001; Christodoulides et al.2002; Zarantonelli et al.2006). Herein we observed that heat-killed bacteria stimulated as high levels of IL-8 release by HBMEC as did viable bacteria, suggesting that mainly heat-stable molecules, including LPS, heat-stable proteins and peptidoglycan, contributed to IL-8 release from both macrovascular and microvascular endothelial cell lines. Interestingly, heat-killed bacteria induced higher IL-8 protein levels released by EA.hy926 cells compared to the levels induced by live bacteria. Accordingly, heat-killed Streptococcus suis has been reported to induce extremely high and sustained levels of IL-8 secretion by human THP-1 monocytes and in a whole-blood culture system (Segura, Vadeboncoeur and Gottschalk 2002; Segura et al.2006). A further major virulence factor of N. meningitidis is the capsule. To explore the role of capsule in cytokine release, we included a non-encapsulated isogenic mutant strain of MC58 in our study. The absence of the polysaccharide capsule resulted in a significant increase of both IL-6 and IL-8 from HBMEC and EA.hy926 cells, as well as MCP-1 from EA.hy926 cells. The impact of capsule expression has been studied in other human pathogens, such as S. bovis, S. pneumoniae, S. suis and Escherichia coli K1 (Tuomanen et al.1985; Ellmerich et al.2000; Vadeboncoeur et al.2003; Metkar et al.2007). For example, for S. suis it has been shown that purified cell wall material is a more potent inducer of cytokine release than purified capsule, suggesting that the presence of a capsule might therefore indirectly downregulate cytokine production by masking the stimulatory effect of surface antigens directly responsible for host cytokine response (Segura et al.2006). On the other hand, the unencapsulated mutant strain MC58 siaD was significantly more invasive than the wild-type strain, suggesting that the potency of the inflammatory response might also be due to the invasive properties resulting in induction of intracellular signal pathways leading to transcription of genes encoding for inflammatory mediators. In addition to comparing brain endothelial cells with peripheral endothelial cells, we also analyzed the effect of bacterial concentrations on cytokine production. Cytokine release varied directly with bacterial concentration and only a high concentration of bacteria was able to induce cytokine production. Therefore, cytokines appear to be gradually upregulated as the proliferation of meningococci proceeds. Interestingly, this finding correlates with the clinical presentation when patients with fulminant septicemia with massive microbial proliferation in the circulation are characterized by extremely high levels of inflammatory mediators in plasma. Recently, the emergence of new virulent N. meningitidis serogroup C ST-11 cc isolates in France has been reported (Deghmane et al.2010). These isolates displayed a new phenotype (C:2a:P1.7,1), caused infections that occurred as clusters and were associated with high virulence in mice (Deghmane et al.2010). We extended our study and included three isolates of this cluster as well as meningococcal isolates belonging to the hypervirulent clonal complexes cc8, cc18, cc32 and cc269, and analyzed their ability to induce the secretion of IL-8 from both cell lines as well as their adhesive and invasive properties. Although individual variations were observed among different N. meningitidis isolates, no clear correlations could be attributed to the origin of the strains, clinical presentation and IL-8 levels released from either brain endothelial or peripheral endothelial cells. The IL-8 levels released from HBMEC in response to strains LNP26639, 8013/clone12 and LNP26643, which are associated with the clinical picture of meningitis, were slightly higher than those in response to the other strains, however, statistically not significant (P > 0.05). Strains from the clonal complexes cc32, cc11 and cc269 were sorted by clinical manifestations, but no direct correlation between clonal complex, clinical presentation and IL-8 release from peripheral endothelial cells could be observed (Fig. 7). This difference may need to be reanalyzed with higher number of isolates that can then be stratified according to their clonal complexes. In addition, there were no clear correlations between meningitis, septic shock or meningococcemia causing isolates in their adhesion or invasion levels to brain or peripheral endothelial cells. In particular, isolate LNP24198 (C:2a:P1.7,1) failed to induce high levels of IL-8 from both HBMEC and EA.hy926 cells, although this isolate provoked significant high levels of the chemokine keratinocyte-derived cytokine (KC, the functional murine homolog of human IL-8) in a transgenic mouse model (Deghmane et al.2010). A possible explanation for this discrepancy might be due to the fact that for this study endothelial cell lines were used as an in vitro model, and primary human cells freshly isolated might show different results when infected with meningococci. Furthermore, circulating leukocytes or monocytes also significantly contribute to cytokine secretion in response to meningococcal infection in vivo (Hackett, Thomson and Hart 2001). Figure 7. View largeDownload slide Correlation between clonal complex (cc), clinical manifestation and IL-8 release of EA.hy926 cells. Outbreak strains from France belonging to cc11, cc32 or cc269 were classified upon their clinical manifestation correlated with IL-8 release of infected EA.hy926 cells. Figure 7. View largeDownload slide Correlation between clonal complex (cc), clinical manifestation and IL-8 release of EA.hy926 cells. Outbreak strains from France belonging to cc11, cc32 or cc269 were classified upon their clinical manifestation correlated with IL-8 release of infected EA.hy926 cells. Taken together, our data show that both microvascular and macrovascular endothelial cells are active participants in the inflammatory response. 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TI - Comparison of the inflammatory response of brain microvascular and peripheral endothelial cells following infection with Neisseria meningitidis
JF - Pathogens and Disease
DO - 10.1093/femspd/ftx038
DA - 2017-07-01
UR - https://www.deepdyve.com/lp/oxford-university-press/comparison-of-the-inflammatory-response-of-brain-microvascular-and-7di1rzer04
SP - ftx038
VL - 75
IS - 5
DP - DeepDyve
ER -