Biocompatible Chitosan-Based Nanoparticles for the Delivery of Lactobacillus johnsonii Lipoteichoic Acid: Enhancing Stability and Biofunctional Properties.

Lipoteichoic acid (LTA) represents immunostimulatory molecules expressed by Gram-positive bacteria. They activate the innate immune system via Toll-like receptors. We have investigated the role of serum proteins in activation of human macrophages by LTA from Staphylococcus aureus and found it to be strongly attenuated by serum. In contrast, the same cells showed a sensitive response to LTA and a significantly enhanced production of tumor necrosis factor α under serum-free conditions. We show that LTA interacts with the serum protein lipopolysaccharide-binding protein (LBP) and inhibits the integration of LBP into phospholipid membranes, indicating the formation of complexes of LTA and soluble LBP. The addition of recombinant human LBP to serum-free medium inhibited the production of tumor necrosis factor α and interleukins 6 and 8 after stimulation of human macrophages with LTA in a dose-dependent manner. Using anti-LBP antibodies, this inhibitory effect could be attributed to soluble LBP, whereas LBP in its recently described transmembrane configuration did not modulate cell activation. Also, using primary alveolar macrophages from rats, we show a sensitive cytokine response to LTA under serum-free culture conditions that was strongly attenuated in the presence of serum. In summary, our data suggest that innate immune recognition of LTA is organ-specific with negative regulation by LBP in serum-containing compartments and sensitive recognition in serum-free compartments like the lung. Lipoteichoic acid (LTA) represents immunostimulatory molecules expressed by Gram-positive bacteria. They activate the innate immune system via Toll-like receptors. We have investigated the role of serum proteins in activation of human macrophages by LTA from Staphylococcus aureus and found it to be strongly attenuated by serum. In contrast, the same cells showed a sensitive response to LTA and a significantly enhanced production of tumor necrosis factor α under serum-free conditions. We show that LTA interacts with the serum protein lipopolysaccharide-binding protein (LBP) and inhibits the integration of LBP into phospholipid membranes, indicating the formation of complexes of LTA and soluble LBP. The addition of recombinant human LBP to serum-free medium inhibited the production of tumor necrosis factor α and interleukins 6 and 8 after stimulation of human macrophages with LTA in a dose-dependent manner. Using anti-LBP antibodies, this inhibitory effect could be attributed to soluble LBP, whereas LBP in its recently described transmembrane configuration did not modulate cell activation. Also, using primary alveolar macrophages from rats, we show a sensitive cytokine response to LTA under serum-free culture conditions that was strongly attenuated in the presence of serum. In summary, our data suggest that innate immune recognition of LTA is organ-specific with negative regulation by LBP in serum-containing compartments and sensitive recognition in serum-free compartments like the lung. The key to a successful pathogen defense is the recognition of pathogen-associated molecular structures by receptors of the innate immune system leading to a proinflammatory response. Systemic production of proinflammatory mediators, however, can also lead to sepsis, a complex clinical syndrome caused by an overshooting host response (1Cohen J. Nature. 2002; 420: 885-891Crossref PubMed Scopus (2157) Google Scholar). According to epidemiological studies, infections by Gram-positive bacteria are responsible for about half of the cases of systemic infections in the United States and Europe, and Staphylococcus aureus is the most frequently isolated Gram-positive pathogen in invasive infections and trauma patients (2Stillwell M. Caplan E.S. Infect. Dis. Clin. North Am. 1989; 3: 155-183Abstract Full Text PDF PubMed Google Scholar, 3Villavicencio R.T. Wall Jr., M.J. Am. J. Surg. 1996; 172: 291-296Abstract Full Text PDF PubMed Scopus (9) Google Scholar). For Gram-negative bacteria, the pathogenesis of septic shock has been attributed to the activation of the innate immune system by lipopolysaccharide (LPS) 2The abbreviations used are: LPS, lipopolysaccharide; FRET, fluorescence resonance energy transfer; IL, interleukin; LBP, lipopolysaccharide-binding protein; LTA, lipoteichoic acid; PI-PLC, phosphatidylinositol-specific phospholipase C; PS, phosphatidylserine; TLR, toll-like receptor; TNF, tumor necrosis factor; sLBP, soluble LBP; mLBP, transmembrane configuration of LBP; NBD-PE, N-(7-nitro-2,1,3-benzoxadiazol-4-yl)-PE; Rh-PE, N-(rhodamine B sulfonyl)-PE; PE, phosphatidylethanolamine. 2The abbreviations used are: LPS, lipopolysaccharide; FRET, fluorescence resonance energy transfer; IL, interleukin; LBP, lipopolysaccharide-binding protein; LTA, lipoteichoic acid; PI-PLC, phosphatidylinositol-specific phospholipase C; PS, phosphatidylserine; TLR, toll-like receptor; TNF, tumor necrosis factor; sLBP, soluble LBP; mLBP, transmembrane configuration of LBP; NBD-PE, N-(7-nitro-2,1,3-benzoxadiazol-4-yl)-PE; Rh-PE, N-(rhodamine B sulfonyl)-PE; PE, phosphatidylethanolamine. from the outer leaflet of the outer membrane of the organisms (4Beutler B. Rietschel E.T. Nat. Rev. Immunol. 2003; 3: 169-176Crossref PubMed Scopus (1046) Google Scholar). Cell activation by LPS is mediated by a complex receptor cluster consisting of Toll-like receptor (TLR) 4 and MD-2 (5Poltorak A. He X. Smirnova I. Liu M.-Y. Van Huffel C. Du X. Birdwell D. Alejos E. Silva M. Galanos C. Freudenberg M. Ricciardi-Castagnoli P. Layton B. Beutler B. Science. 1998; 282: 2085-2088Crossref PubMed Scopus (6421) Google Scholar, 6Takeuchi O. Hoshino K. Kawai T. Sanjo H. Takada H. Ogawa T. Takeda K. Akira S. Immunity. 1999; 11: 443-451Abstract Full Text Full Text PDF PubMed Scopus (2780) Google Scholar, 7Shimazu R. Akashi S. Ogata H. Nagai Y. Fukudome K. Miyake K. Kimoto M. J. Exp. Med. 1999; 189: 1777-1782Crossref PubMed Scopus (1746) Google Scholar, 8Schromm A.B. Lien E. Henneke P. Chow J.C. Yoshimura A. Heine H. Latz E. Monks B.G. Schwartz D.A. Miyake K. Golenbock D.T. J. Exp. Med. 2001; 194: 79-88Crossref PubMed Scopus (240) Google Scholar) and a number of further proteins such as CD11/CD18, CD55, CD81, GDF5, and CXCR4 (9Triantafilou K. Triantafilou M. Dedrick R.L. Nat. Immunol. 2001; 2: 338-345Crossref PubMed Scopus (355) Google Scholar, 10Heine H. El Samalouti V.T. Notzel C. Pfeiffer A. Lentschat A. Kusumoto S. Schmitz G. Hamann L. Ulmer A.J. Eur. J. Immunol. 2003; 33: 1399-1408Crossref PubMed Scopus (41) Google Scholar, 11Triantafilou M. Brandenburg K. Kusumoto S. Fukase K. Mackie A. Seydel U. Triantafilou K. Biochem. J. 2004; 381: 527-536Crossref PubMed Scopus (131) Google Scholar) and the ion channel MaxiK (12Seydel U. Scheel O. Mueller M. Brandenburg K. Blunck R. J. Endotoxin. Res. 2001; 7: 243-247Crossref PubMed Scopus (36) Google Scholar, 13Blunck R. Scheel O. Mueller M. Brandenburg K. Seitzer U. Seydel U. J. Immunol. 2001; 166: 1009-1015Crossref PubMed Scopus (125) Google Scholar). Because of its amphiphilic structure, LPS forms supramolecular aggregates in an aqueous environment. The supramolecular structure of these aggregates critically determines the biological activity of LPS (14Schromm A.B. Brandenburg K. Loppnow H. Moran A.P. Koch M.H. Rietschel E.T. Seydel U. Eur. J. Biochem. 2000; 267: 2008-2013Crossref PubMed Scopus (263) Google Scholar, 15Seydel U. Hawkins L. Schromm A.B. Heine H. Scheel O. Koch M.H. Brandenburg K. Eur. J. Immunol. 2003; 33: 1586-1592Crossref PubMed Scopus (78) Google Scholar). Transport of LPS aggregates to signaling proteins on the surface of mononuclear phagocytes represents the first step in cell activation. Multiple transport pathways for LPS exist to target host cells, including the transport of LPS in the serum by the soluble acute phase serum protein LPS-binding protein (LBP) (16Schumann R.R. Leong S.R. Flaggs G.W. Gray P.W. Wright S.D. Mathison J.C. Tobias P.S. Ulevitch R.J. Science. 1990; 249: 1429-1431Crossref PubMed Scopus (1378) Google Scholar, 17Schromm A.B. Brandenburg K. Rietschel E.T. Flad H.D. Carroll S.F. Seydel U. FEBS Lett. 1996; 399: 267-271Crossref PubMed Scopus (111) Google Scholar) or the soluble CD14 (sCD14) receptor (18Blondin C. Le Dur A. Cholley B. Caroff M. Haeffner-Cavaillon N. Eur. J. Immunol. 1997; 27: 3303-3309Crossref PubMed Scopus (30) Google Scholar) to the membrane-bound CD14 (mCD14) (19Wright S.D. Ramos R.A. Tobias P.S. Ulevitch R.J. Mathison J.C. Science. 1990; 249: 1431-1433Crossref PubMed Scopus (3398) Google Scholar). However, only recently it has been shown that LBP also assumes a transmembrane configuration (mLBP) and in this configuration incorporates LPS aggregates into the cell membrane (20Gutsmann T. Haberer N. Carroll S.F. Seydel U. Wiese A. Biol. Chem. 2001; 382: 425-434Crossref PubMed Scopus (38) Google Scholar, 21Gutsmann T. Mueller M. Carroll S.F. MacKenzie R.C. Wiese A. Seydel U. Infect. Immun. 2001; 69: 6942-6950Crossref PubMed Scopus (166) Google Scholar, 22Mueller M. Lindner B. Kusumoto S. Fukase K. Schromm A.B. Seydel U. J. Biol. Chem. 2004; 279: 26307-26313Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar). Other reports show complex vectorial transport chains for LPS monomers (23Gioannini T.L. Teghanemt A. Zhang D. Coussens N.P. Dockstader W. Ramaswamy S. Weiss J.P. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 4186-4191Crossref PubMed Scopus (296) Google Scholar). In contrast to the well investigated receptor system for LPS from Gram-negative bacteria, little is known about the molecular mechanisms involved in the recognition of Gram-positive pathogens. Several studies suggest that also molecules of the cell wall mediate inflammatory responses of the host, namely peptidoglycan of the murein layer and lipoteichoic acid (LTA). LTA is an amphiphilic molecule anchored to the outer surface of the cytoplasmic membrane by a glycerolipid. Peptidoglycan and LTA are released from the cell wall during growth and especially under antibiotic treatment, and both molecules have been shown to express immune stimulatory activity (24Gupta D. Kirkland T.N. Viriyakosol S. Dziarski R. J. Biol. Chem. 1996; 271: 23310-23316Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar, 25Morath S. Geyer A. Hartung T. J. Exp. Med. 2001; 193: 393-397Crossref PubMed Scopus (373) Google Scholar). After a long controversy about the immune stimulatory capacity and purity of commercial LTA preparations, establishment of a new purification protocol for LTA based on butanol extraction revealed that pure, LPS-free preparations of LTA from S. aureus exhibit immune stimulatory activity in human whole blood in high concentrations (25Morath S. Geyer A. Hartung T. J. Exp. Med. 2001; 193: 393-397Crossref PubMed Scopus (373) Google Scholar). LTA derived by this purification protocol was shown to be composed of a diacylglycerol lipid anchor covalently linked to a gentiobiose and a polymeric backbone that in S. aureus is composed of a central 1–3-linked glycerophosphate chain substituted with d-alanine, α-d-N-acetylglucosamine, and to a lesser extent unsubstituted 1–3-linked glycerophosphate residues. The immune stimulatory capacity of LTA could be confirmed by chemical synthesis of the lipid anchor containing a backbone of six glycerophosphate units carrying d-alanine and N-acetylglucosamine as substituents (26Morath S. Stadelmaier A. Geyer A. Schmidt R.R. Hartung T. J. Exp. Med. 2002; 195: 1635-1640Crossref PubMed Scopus (208) Google Scholar). However, the structural basis of cell activation by molecules derived from the Gram-positive cell wall is not conclusively defined. Although the molecular structures leading to activation of NOD proteins by peptidoglycan have recently been identified (27Girardin S.E. Boneca I.G. Carneiro L.A. Antignac A. Jehanno M. Viala J. Tedin K. Taha M.K. Labigne A. Zahringer U. Coyle A.J. DiStefano P.S. Bertin J. Sansonetti P.J. Philpott D.J. Science. 2003; 300: 1584-1587Crossref PubMed Scopus (1248) Google Scholar, 28Girardin S.E. Boneca I.G. Viala J. Chamaillard M. Labigne A. Thomas G. Philpott D.J. Sansonetti P.J. J. Biol. Chem. 2003; 278: 8869-8872Abstract Full Text Full Text PDF PubMed Scopus (1906) Google Scholar), the molecular basis of its TLR2 activity is not fully understood (29Travassos L.H. Girardin S.E. Philpott D.J. Blanot D. Nahori M.A. Werts C. Boneca I.G. EMBO Rep. 2004; 5: 1000-1006Crossref PubMed Scopus (392) Google Scholar, 30Dziarski R. Gupta D. Infect. Immun. 2005; 73: 5212-5216Crossref PubMed Scopus (194) Google Scholar). Also, recent data indicate that alanine substitution of LTA might not be required for its biological activity (31Henneke P. Morath S. Uematsu S. Weichert S. Pfitzenmaier M. Takeuchi O. Muller A. Poyart C. Akira S. Berner R. Teti G. Geyer A. Hartung T. Trieu-Cuot P. Kasper D.L. Golenbock D.T. J. Immunol. 2005; 174: 6449-6455Crossref PubMed Scopus (101) Google Scholar). The mechanisms of cell activation by LTA today are not understood. However, it is now generally accepted that LTA activates immune responses via a TLR2/TLR6 heterodimer (31Henneke P. Morath S. Uematsu S. Weichert S. Pfitzenmaier M. Takeuchi O. Muller A. Poyart C. Akira S. Berner R. Teti G. Geyer A. Hartung T. Trieu-Cuot P. Kasper D.L. Golenbock D.T. J. Immunol. 2005; 174: 6449-6455Crossref PubMed Scopus (101) Google Scholar, 32Schroder N.W. Morath S. Alexander C. Hamann L. Hartung T. Zahringer U. Gobel U.B. Weber J.R. Schumann R.R. J. Biol. Chem. 2003; 278: 15587-15594Abstract Full Text Full Text PDF PubMed Scopus (498) Google Scholar). There are, nevertheless, still conflicting reports on the requirement of CD14 (33Rietschel E.T. Schletter J. Weidemann B. El Samalouti V. Mattern T. Zahringer U. Seydel U. Brade H. Flad H.D. Kusumoto S. Gupta D. Dziarski R. Ulmer A.J. Microb. Drug Resist. 1998; 4: 37-44Crossref PubMed Scopus (77) Google Scholar, 34Hoebe K. Georgel P. Rutschmann S. Du X. Mudd S. Crozat K. Sovath S. Shamel L. Hartung T. Zahringer U. Beutler B. Nature. 2005; 433: 523-527Crossref PubMed Scopus (704) Google Scholar) and LBP in LTA-mediated cellular responses, ranging from enhancing to no effects of LBP on cell activation by LTA (32Schroder N.W. Morath S. Alexander C. Hamann L. Hartung T. Zahringer U. Gobel U.B. Weber J.R. Schumann R.R. J. Biol. Chem. 2003; 278: 15587-15594Abstract Full Text Full Text PDF PubMed Scopus (498) Google Scholar, 35Lehner M.D. Morath S. Michelsen K.S. Schumann R.R. Hartung T. J. Immunol. 2001; 166: 5161-5167Crossref PubMed Scopus (247) Google Scholar, 36Hermann C. Spreitzer I. Schroder N.W. Morath S. Lehner M.D. Fischer W. Schutt C. Schumann R.R. Hartung T. Eur. J. Immunol. 2002; 32: 541-551Crossref PubMed Scopus (109) Google Scholar). In the current study, we investigated the role of serum and LBP in cell activation by LTA. Our data show that LTA interacts with LBP and that cell activation is strongly attenuated by this interaction. We present evidence for a differential regulation of LTA recognition by macrophages dependent on the absence and presence of LBP. Reagents—Deep rough mutant LPS (Re LPS) was extracted from Salmonella enterica sv. Minnesota strain R595 according to the phenol/chloroform/petrol ether procedure (37Galanos C. Luderitz O. Rietschel E.T. Westphal O. Brade H. Brade L. Freudenberg M. Schade U. Imoto M. Yoshimura H. Kusumoto S. Shiba T. Eur. J. Biochem. 1985; 148: 1-5Crossref PubMed Scopus (401) Google Scholar). The LPS preparation was lyophilized and used in the natural salt form. The chemical purity of the LPS preparation was confirmed by mass spectrometry. Highly purified lipoteichoic acid was isolated from S. aureus as described previously (25Morath S. Geyer A. Hartung T. J. Exp. Med. 2001; 193: 393-397Crossref PubMed Scopus (373) Google Scholar). LPS and LTA were suspended in phosphate-buffered saline (Biochrom, Berlin, Germany) by thorough vortexing. The suspensions were temperature cycled at least twice between 4 and 56 °C, with each cycle followed by intense vortexing for a few min, and then stored at 4 °C for at least 12 h prior to measurement. Suspensions were aliquoted and stored at –20 °C. Phosphatidylserine (PS) from bovine brain was purchased from Avanti-Polar Lipids (Alabaster, AL) and used without further purification. The fluorescent dyes N-(7-nitro-2,1,3-benzoxadiazol-4-yl)-PE (NBD-PE) and N-(rhodamine B sulfonyl)-PE (Rh-PE) were purchased from Molecular Probes (Eugene, OR). Recombinant human LBP (456-amino acid holoprotein rLBP50) in 10 mm HEPES, pH 7.5, was a kind gift of XOMA LLC (Berkeley, CA). The monoclonal mouse anti-mouse LBP antibody biG33 cross-reacting with human LBP and the monoclonal anti-human CD14 antibody biG 14 were obtained from Biometec (Greifswald, Germany). Isotype-matched IgG1 control antibody was obtained from BD Biosciences (Heidelberg, Germany). PI-PLC was from Sigma. Preparation of Macrophages and Incubation Conditions—Monocytes were isolated from human peripheral blood of healthy donors by the Hypaque-Ficoll gradient method and cultivated at 37 °C and 6% CO2 in Teflon bags in RPMI 1640 medium (endotoxin ≤0.01 EU/ml; Biochrom) containing 100 units/ml penicillin, 100 μg/ml streptomycin, 2 mm l-glutamine, and 4% heat-inactivated human serum type AB from healthy donors. The cells were cultured in the presence of 2 ng/ml macrophage colony-stimulating factor for 7 days to differentiate monocytes to macrophages. To determine cytokine induction after cell stimulation, the cells were seeded at 200-μl aliquots of a suspension of 1 × 106 cells/ml in 96-well tissue culture dishes (Nunc, Wiesbaden, Germany) in RPMI 1640 medium containing 100 units/ml penicillin, 100 μg/ml streptomycin, 2 mm l-glutamine, with or without 4% human serum. Cell-free supernatants were collected 4 h after stimulation for TNFα determination or 24 h after stimulation for IL-6 and IL-8 determination, respectively, and stored at –20 °C until determination of cytokine content. The data shown are the means and standard deviations (±S.D.) of triplicate samples of one experiment and representative of at least three independent experiments. Alveolar macrophages of the rat were isolated by lung lavage of male Sprague-Dawley rats (Charles River, Sulzfeld, Germany) as described (38Wu Y. Adam S. Hamann L. Heine H. Ulmer A.J. Buwitt-Beckmann U. Stamme C. Am. J. Respir. Cell Mol. Biol. 2004; 31: 587-594Crossref PubMed Scopus (34) Google Scholar). Cell viability was checked by erythrosin B exclusion and routinely averaged 94–98%. The cells were washed once in RPMI and resuspended in RPMI containing 100 units/ml penicillin, 100 μg/ml streptomycin, 2 mm l-glutamine, with or without 10% heat-inactivated fetal calf serum (Linaris, Bettingen, Germany). To determine cytokine induction by LTA, the cells were seeded at 0.5 × 106 cells/well in 96-well tissue culture dishes and stimulated with LTA in the absence or presence of serum. To cleave cell-bound CD14 from the cell surface, the cells were treated with 0.5 unit/ml PI-PLC for 60 min at 37 °C prior to stimulation. Cell-free supernatants were harvested after 4 h of stimulation for the determination of TNFα. Cytokine Determination—Human TNFα and human IL-6 were determined in pooled cell-free supernatants of stimulated cells by sandwich enzyme-linked immunosorbent assay using monoclonal mouse antibody against human IL-6 and POD-conjugated rabbit anti-human IL-6 antibody and monoclonal mouse antibody against human TNFα and POD-conjugated rabbit anti-human TNFα antibody, respectively (Intex, Muttant, Switzerland) as stated in detail elsewhere (39Mueller M. Brandenburg K. Dedrick R. Schromm A.B. Seydel U. J. Immunol. 2005; 174: 1091-1096Crossref PubMed Scopus (62) Google Scholar). Rat TNFα and human IL-8 were determined in pooled cell-free supernatants of stimulated cells by sandwich enzyme-linked immunosorbent assay using Cytosets from BIOSOURCE (Solingen, Germany) exactly according to the manufacturer’s protocol. The data shown are the means ± S.D. of triplicate samples of one representative experiment. Fluorescence Resonance Energy Transfer Spectroscopy—The fluorescence resonance energy transfer was used as a assay A.B. Brandenburg K. Rietschel E.T. Flad H.D. Carroll S.F. Seydel U. FEBS Lett. 1996; 399: 267-271Crossref PubMed Scopus (111) Google Scholar, D. PubMed Scopus Google Scholar) to on the of LBP and LTA into from the phospholipid For the were with and in of The was under a of and the were resuspended in phosphate-buffered and with a for 1 min of the preparation was at least twice between 4 and 56 °C, with each cycle followed by intense vortexing for a few min, and then stored at 4 °C for at least 12 h prior to measurement. preparation of of the at 37 °C was at of and the of the of the and were on the fluorescence LBP and LTA aggregates were to after and 100 Because is used as a of molecules such as LBP or LTA an of the between and and to a energy an of the and a of the For a of the of the of the and the are against in the as the The data shown are representative for three independent experiments. Resonance surface resonance M. Nature. PubMed Scopus Google Scholar) was used as a assay to of LBP and LPS with from was with a 10 suspension of to an lipid for with LBP and LTA. LBP and LTA were at concentrations of 10 and 100 The was phosphate-buffered saline at pH and the were at 37 °C at a of 10 in a The data are as response units in on for one representative experiment of a of Cell of Macrophages by LTA in the of the immune stimulatory activity of LPS-free LTA preparations using human macrophages that were in from peripheral blood mononuclear of macrophages with LTA in the presence of 4% human serum to a dose-dependent production of the proinflammatory cytokine TNFα these μg/ml LTA were required to TNFα with the of TNFα by the same cells after stimulation with 1 ng/ml LPS under serum-free conditions ± 24 the of TNFα by these high concentrations of LTA were about three ± by 10 μg/ml However, stimulation of the cells with LTA under serum-free culture conditions to a of the cytokine these high concentrations of LTA of TNFα ± by 1 μg/ml as by 1 In contrast to serum-containing TNFα production was by ng/ml LTA under serum-free conditions and with concentrations of LTA, of cell activation at ng/ml LTA. could be for the IL-6 and both of showed a dose-dependent in the presence of serum after 24 h of stimulation suggest that a in serum strongly cell activation by of by LTA in human macrophages is sensitive under serum-free conditions. blood macrophages were stimulated in the absence or presence of 4% AB serum with the concentrations of LTA. Cell-free supernatants were harvested after 24 h for the determination of IL-6 and and IL-8 and Cytokine and concentrations are the means ± S.D. of The data shown are representative of three independent experiments. not LTA with LBP and the of LBP into is an serum protein involved in the innate immune recognition of a of pathogen-associated has been shown to with LPS and cell activation at concentrations of However, at high concentrations of LBP as in acute phase inhibitory effects of LBP on cellular responses to LPS have been T. Mueller M. Carroll S.F. MacKenzie R.C. Wiese A. Seydel U. Infect. Immun. 2001; 69: 6942-6950Crossref PubMed Scopus (166) Google Scholar, J. K. Schumann R.R. 2001; PubMed Scopus Google Scholar, L. Alexander C. Stamme C. U. Schumann R.R. Infect. Immun. 2005; 73: PubMed Scopus Google Scholar). Because LBP has been found to also with N.W. B. N. Michelsen K.S. U. Gobel U.B. Schumann R.R. J. Immunol. 2000; PubMed Scopus Google Scholar, J.R. D. Alexander C. Schroder N.W. A. C. D. Schumann R.R. Immunity. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar, N.W. Heine H. Alexander C. M. J. Hamann L. Gobel U.B. Schumann R.R. J. Immunol. 2004; PubMed Scopus Google Scholar), we that LBP be the in serum cellular response to LTA. We used a assay based on fluorescence resonance energy transfer to on the of LTA with LBP. We have shown in reports that LBP is not only a soluble protein into (20Gutsmann T. Haberer N. Carroll S.F. Seydel U. Wiese A. Biol. Chem. 2001; 382: 425-434Crossref PubMed Scopus (38) Google Scholar, 21Gutsmann T. Mueller M. Carroll S.F. MacKenzie R.C. Wiese A. Seydel U. Infect. Immun. 2001; 69: 6942-6950Crossref PubMed Scopus (166) Google Scholar). of LBP with phospholipid is enhanced for In the presence of LPS, LBP a transport of LPS into the phospholipid of A.B. Brandenburg K. Rietschel E.T. Flad H.D. Carroll S.F. Seydel U. FEBS Lett. 1996; 399: 267-271Crossref PubMed Scopus (111) Google Scholar). We used this assay to the of LTA on the of LBP with recombinant human LBP is to composed of the phospholipid it into the as can be from the in the of and LTA is to the in the absence of LBP, no in the could be indicating that LTA not into the phospholipid membrane of LBP to the of and LTA did not lead to in the LTA with in the or of further the of LTA and LBP with phospholipid membranes, we resonance method on the of in a We the surface of the and LTA and LBP. can be from the addition of LTA did not lead to an of response units indicating that LTA did not to the phospholipid LBP was of LBP to the phospholipid membrane in an in response Because the were under LTA was not present at the of addition of LBP, the absence of inhibitory effects of LTA on the of LBP with the In to the in the the addition of LTA to membrane-bound LBP 6 and did not lead to an of response that LTA not to phospholipid in the absence or presence of LBP. In is no for of LTA to LBP. LBP

In this study the potential of liposomes as a vitamin E (VE) and β-carotene (βC) delivery system was examined. The co-encapsulated liposomes of βC and VE (L-VE-βC) were prepared and characterized. Their antioxidant properties were evaluated by free radical scavenging activities of 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS), hydroxyl radical and lipid peroxidation assay. The in vitro sustained release behaviour was then investigated and discussed. VE and βC were co-encapsulated in liposomes with high encapsulation efficiency, up to 92.49% and 86.16% for βC and VE, respectively. The antioxidant activities of L-VE-βC samples were significantly higher than that of single loaded liposome. Among different ratios of VE/βC, L-VE-βC at 5:3 exhibited the highest radical scavenging rates, with 66.80%, 56.58% and 34.39% for DPPH, ABTS and OH radical, respectively. L-VE-βC samples also had a good ability to inhibit lipid peroxidation, especially the sample with ratios of VE/βC at 5:3 and 3:1. In simulated gastrointestinal release, L-VE-βC exhibited an excellent sustained release behaviour in SGF with the accumulated rate at about 20%, while the release rate in SIF increased to over 80%, where they should be absorbed. The release kinetics analysis indicated that βC was released in the Higuchi model in stomach, and the Korsmeyr-Peppas model in intestine. Compared to single loaded liposomes, the combined-loaded liposomes exhibited higher antioxidant activity and bioavailability, suggesting the potential applications in functional foods. © 2022 Society of Chemical Industry.

Oral delivery of pVAX-OMP and pVAX-hly DNA vaccine using chitosan-tripolyphosphate (Cs-TPP) nanoparticles in Rohu, (Labeo rohita) for protection against Aeromonas hydrophila infection

Bacterial infections have been shown to be involved in several inflammatory diseases such as brain inflammation. A major factor for these findings is due to the secretion of pro-inflammatory mediators by host cells triggered by the components released from the bacteria. Among these components, lipoteichoic acid (LTA), a component of Gram-positive bacterial cell wall, has been found to be elevated in cerebrospinal fluid of patients suffering from meningitis. Moreover, increased plasma levels of matrix metalloproteinases (MMPs), in particular MMP-9, have been observed in patients with brain inflammatory diseases and may contribute to disease pathology. However, the molecular mechanisms underlying LTA-induced MMP-9 expression in rat brain astrocytes (RBA-1 cells) remain poorly defined. Here, the data with zymographic, Western blotting, RT-PCR, and immunofluorescent staining analyses showed that LTA induced MMP-9 expression and activation via a TLR2-activated c-Src-dependent transactivation of PDGFR pathway. Transactivation of PDGFR led to activation of PI3K/Akt and p42/p44 MAPK and then activated the IKK/NF-kappaB cascade. The activated-NF-kappaB translocated into nucleus which bound to kappaB-binding site of MMP-9 promoter, and thereby turned on transcription of MMP-9. Eventually, upregulation of MMP-9 by LTA enhanced cell migration of astrocytes. Taken together, these results suggested that in RBA-1 cells, activation of NF-kappaB by a c-Src-dependent PI3K/Akt-p42/p44 MAPK activation mediated through transactivation of PDGFR is essential for MMP-9 gene upregulation induced by LTA. Understanding the regulation of MMP-9 expression and functional changes by LTA/TLR system on astrocytes may provide potential therapeutic targets of Gram-positive bacterial infection in brain disorders.

BackgroundLiver kinase B1 (Lkb1, gene name Stk11) functions as a tumor suppressor in cancer. Myeloid cell Lkb1 potentiates lung inflammation induced by the Gram-negative bacterial cell wall component lipopolysaccharide and in host defense during Gram-negative pneumonia. Here, we sought to investigate the role of myeloid Lkb1 in lung inflammation elicited by the Gram-positive bacterial cell wall component lipoteichoic acid (LTA) and during pneumonia caused by the Gram-positive respiratory pathogen Streptococcus pneumoniae (Spneu).MethodsAlveolar and bone marrow derived macrophages (AMs, BMDMs) harvested from myeloid-specific Lkb1 deficient (Stk11-ΔM) and littermate control mice were stimulated with LTA or Spneu in vitro. Stk11-ΔM and control mice were challenged via the airways with LTA or infected with Spneu in vivo.ResultsLkb1 deficient AMs and BMDMs produced less tumor necrosis factor (TNF)α upon activation by LTA or Spneu. During LTA-induced lung inflammation, Stk11-ΔM mice had reduced numbers of AMs in the lungs, as well as diminished cytokine release and neutrophil recruitment into the airways. During pneumonia induced by either encapsulated or non-encapsulated Spneu, Stk11-ΔM and control mice had comparable bacterial loads and inflammatory responses in the lung, with the exception of lower TNFα levels in Stk11-ΔM mice after infection with the non-encapsulated strain.ConclusionMyeloid Lkb1 contributes to LTA-induced lung inflammation, but is not important for host defense during pneumococcal pneumonia.

Despite the fact that gram-positive infections constitute around 50% of all cases leading to septic shock, little is yet known about the mechanisms involved. This study was carried out to find out more about the effects of cell wall components peptidoglycan (PepG) and lipoteichoic acid (LTA) of the gram-positive bacterium Streptococcus pyogenes in the pig. Specific pathogen-free pigs (20 kg bodyweight) were pretreated with metyrapone (a cortisol-synthesis inhibitor) and then were given 2-h infusions of 160 microg/kg of PepG (n = 5), 160 microg/kg LTA (n=5), or a combination of both (LTA + PepG, 160 microg/kg each, n = 5). Four hours after start of the infusions, the PepG, LTA, and LTA + PepG groups showed decreases in mean arterial pressure (change of -11%, -25%, and -47% from baseline, respectively), dynamic lung compliance (-18%, -24%, and -38%), arterial oxygen tension (-10%, -16%, and -37%), changes in blood leukocyte numbers (+11%, -27%, and -67%), and increases in pulmonary vascular resistance index (+7%, +106%, and +307% from baseline) and metabolic acidosis (base excess values decreased with 1.8, 2.3 and 8.1 units). The differences between the PepG and LTA + PepG groups were statistically significant (P < 0.05, Kruskal-Wallis tests), but not between LTA and LTA + PepG groups. However, no changes in systemic nitric oxide (NO) production could be detected, which is much in contrast to studies on lower order animals. Moreover, comparison of the results obtained using this model with those obtained in a model of endotoxin-induced septic shock showed distinct difference in the mechanisms by which gram-positive and gram-negative bacterial components exert their actions. For example, a marked fall in systemic blood pressure and dynamic lung compliance is seen in both models, but in the present gram-positive sepsis model, much less interleukin-8 and tumor necrosis factor-alpha are produced. In conclusion, this study showed that PepG and LTA act synergistically to cause respiratory failure and septic shock in the pig. The infusion of the combination of PepG and LTA in the pig could serve as a new, well-controlled model for studies of gram-positive sepsis.

Intrapulmonary administration of a p38 mitogen activated protein kinase inhibitor partially prevents pulmonary inflammation

Histamine is a major inflammatory molecule released from the mast cell, and is known to activate endothelial cells. However, its ability to modulate endothelial responses to bacterial products has not been evaluated. In this study we determined the ability of histamine to modulate inflammatory responses of endothelial cells to Gram-negative and Gram-positive bacterial cell wall components and assessed the role of Toll-like receptors (TLR) 2 and 4 in the co-operation between histamine and bacterial pathogens. Human umbilical vein endothelial cells (HUVEC) were incubated with lipopolysaccharide (LPS), lipoteichoic acid (LTA), or peptidoglycan (PGN) in the presence or absence of histamine, and the expression and release of interleukin-6 (IL-6), and NF-kappaB translocation were determined. The effect of histamine on the expression of mRNA and proteins for TLR2 and TLR4 was also evaluated. Incubation of HUVEC with LPS, LTA and PGN resulted in marked enhancement of IL-6 mRNA expression and IL-6 secretion. Histamine alone markedly enhanced IL-6 mRNA expression in HUVEC, but it did not stimulate proportional IL-6 release. When HUVEC were incubated with LPS, LTA, or PGN in the presence of histamine marked amplification of both IL-6 production and mRNA expression was noted. HUVEC constitutively expressed TLR2 and TLR4 mRNA and proteins, and these were further enhanced by histamine. The expression of mRNAs encoding MD-2 and MyD88, the accessory molecules associated with TLR signalling, were unchanged by histamine treatment. These results demonstrate that histamine up-regulates the expression of TLR2 and TLR4 and amplifies endothelial cell inflammatory responses to Gram-negative and Gram-positive bacterial components.

The induction of tumor necrosis factor alpha (TNF-alpha) by polytetrafluoroethylene (PTFE) particles (5-50 microns) and by bacterial lipopolysaccharide (LPS) and lipoteichoic acid (LTA) was examined in RAW cell cultures. Twenty-four-hour culture supernatants from the treated and control cells were assayed for TNF-alpha using a mouse L929 cell cytotoxicity assay. Untreated RAW cells produced low levels of endogenous TNF-alpha in the culture supernatants. Addition of 0.5 ng to 1 microgram/ mL LPS or 1 ng to 1 microgram/ml LTA increased the TNF-alpha production by 7-3570-fold and 2-815-fold, respectively. Addition of 1-5 mg PTFE increased the TNF-alpha production by 6-17-fold over the untreated control cell levels. The cells exposed to PTFE and 0.5 ng/mL LPS or 5 ng/mL LTA produced TNF-alpha levels that were significantly higher than those produced by any inducer alone. Thus, both LTA, a Gram-positive bacterial cell wall component and LPS, a Gram-negative bacterial cell wall component, can induce TNF-alpha production, which is further enhanced by PTFE particles in RAW cells.

Liposomes play a significant role in encapsulation of various bioactive compounds (BACs), including functional food ingredients to improve the stability of core. This technology can be used for promoting an effective application in functional food and nutraceuticals. Incorporation of traditional and emerging methods for the developments of liposome for loading BACs resulted in viable and stable liposome formulations for industrial applications. Thus, the advance technologies such as supercritical fluidic methods, microfluidization, ultrasonication with traditional methods are revisited. Liposomes loaded with plant and animal BACs have been introduced for functional food and nutraceutical applications. In general, application of liposome systems improves stability, delivery, and bioavailability of BACs in functional food systems and nutraceuticals. This review covers the current techniques and methodologies developed and practiced in liposomal preparation and application in functional foods.

Hemodialysis patients with central venous catheters (CVCs) have chronic systemic inflammation, the source of which may be related to intraluminal bacterial biofilm. There is currently no non-invasive method to adequately evaluate intraluminal biofilm. Lipoteichoic acid (LTA) is a Gram-positive bacterial cell wall component that is spontaneously shed. The purpose of this study was to determine whether LTA could be quantified in biological samples and to evaluate potential relationships to markers of inflammation. Heparin-locked catheter aspirate was drawn from both the arterial and venous ports of each CVC prior to dialysis initiation. Venous blood from the dialysis circuit was collected 30 min after dialysis initiation. LTA was quantified in aspirate and plasma. Key markers of inflammation (interleukin-6, and hepcidin) and endothelial dysfunction (soluble vascular endothelial cadherin) were also determined in plasma samples. Catheter aspirate and systemic blood samples were obtained from 40 hemodialysis patients. The median (range) duration of catheter use was 130 (20–1635) days. Unexpectedly, median (range) plasma LTA concentrations (ng/mL) were significantly higher than catheter aspirate LTA concentrations [3.93 (0.25–15) vs. 2.38 (0.1–8.1), respectively, p = 0.01] in the majority (70%) of patients. Area under the receiver operator characteristic (ROC) curve showed good potential prognostic value of catheter aspirate LTA predicting systemic LTA concentrations with an area under the curve of 0.815 (95% CI, 0.68–0.95). A significant correlation was found between LTA and serum ferritin (r = 0.32, p = 0.04), however, there were no significant correlations between LTA and the other inflammation biomarkers assessed. LTA is quantifiable in aspirate and plasma of hemodialysis patients with CVCs and warrants further investigation to determine potential clinical application to intraluminal biofilm evaluation.

Bioaccessibility and antioxidant activity of curcumin after encapsulated by nano and Pickering emulsion based on chitosan-tripolyphosphate nanoparticles

Background The aim was to investigate the influence of Gram-positive cell wall components on monocyte surface inflammatory receptors compared with lipopolysaccharide (LPS) in human whole blood. Methods Human whole blood from six healthy donors was incubated with peptidoglycan (PepG) 10 μg ml−1 from Staphylococcus aureus and Bacillus subtilis, lipoteichoic acid (LTA) 100 μg ml−1 from S. aureus and LPS 10 ng ml−1 from Escherichia coli in microcentrifuge tubes at 37°C with slow rotation for 4 h. Subsequent to incubation with fluorescein isothiocyanate (FITC)- and phycoerythrine (PE)-labelled monoclonal antibodies, surface molecule (CD14, CD54, CD58, CD64, CD80 and human leucocyte antigen (HLA) DR) expression was analysed by flow cytometry performed on the FACScan using the CellQuest software. Results All surface molecules except CD80 were expressed constitutively on monocytes in whole blood. Stimulation with either type of PepG, LTA and LPS did not induce any changes in expression of CD58 or CD64, nor did they induce expression of CD80. However, significant upregulation of CD54 and HLA-DR was observed in all donors. Contrary to LPS, PepG and LTA also upregulated the expression of monocyte surface CD14. Conclusion The Gram-positive cell wall products PepG and LTA strongly upregulated the expression of intercellular adhesion molecule 1 (CD54) and HLA-DR on monocytes in human whole blood, as was seen in LPS-stimulated whole blood. In contrast to Gram-negative endotoxin, the Gram-positive cell wall products also upregulated the expression of the LPS receptor CD14. This suggests possible differences in intracellular signalling between Gram-positive cell wall products and Gram-negative LPS in monocytes.

Highly purified lipoteichoic acid from gram-positive bacteria induces in vitro blood–brain barrier disruption through glia activation: Role of pro-inflammatory cytokines and nitric oxide

Bacterial pneumonia remains associated with high morbidity and mortality. The gram-positive pathogen Streptococcus pneumoniae is the most common cause of community-acquired pneumonia. Lipoteichoic acid (LTA) is an important proinflammatory component of the gram-positive bacterial cell wall. R-roscovitine, a purine analog, is a potent cyclin-dependent kinase (CDK)-1, -2, -5 and -7 inhibitor that has the ability to inhibit the cell cycle and to induce polymorphonuclear cell (PMN) apoptosis. We sought to investigate the effect of R-roscovitine on LTA-induced activation of cell lines with relevance for lung inflammation in vitro and on lung inflammation elicited by either LTA or viable S. pneumoniae in vivo. In vitro R-roscovitine enhanced apoptosis in PMNs and reduced tumor necrosis factor (TNF)-α and keratinocyte chemoattractant (KC) production in MH-S (alveolar macrophage) and MLE-12/MLE-15 (respiratory epithelial) cell lines. In vivo R-roscovitine treatment reduced PMN numbers in bronchoalveolar lavage fluid during LTA-induced lung inflammation; this effect was reversed by inhibiting apoptosis. Postponed treatment with R-roscovitine (24 and 72 h) diminished PMN numbers in lung tissue during gram-positive pneumonia; this step was associated with a transient increase in pulmonary bacterial loads. R-roscovitine inhibits proinflammatory responses induced by the gram-positive stimuli LTA and S. pneumoniae. R-roscovitine reduces PMN numbers in lungs upon LTA administration by enhancing apoptosis. The reduction in PMN numbers caused by R-roscovitine during S. pneumoniae pneumonia may hamper antibacterial defense.