Inhibition of inflammatory response in human keratinocytes by magnetic nanoparticles functionalized with PBP10 peptide derived from the PIP2-binding site of human plasma gelsolin

Background: Human plasma gelsolin (pGSN) is a multifunctional actin-binding protein involved in a variety of biological processes, including neutralization of pro-infammatory molecules such as lipopolysaccharide (LPS) and lipoteichoic acid (LTA) and modulation of host infammatory response. It was found that PBP10, a synthetic rhodamine B-conjugated peptide, based on the phosphoinositide-binding site of pGSN, exerts bactericidal activity against Grampositive and Gram-negative bacteria, interacts specifcally with LPS and LTA, and limits microbial-induced infammatory efects. The therapeutic efciency of PBP10 when immobilized on the surface of iron oxide-based magnetic nanoparticles was not evaluated, to date. Results: Using the human keratinocyte cell line HaCaT stimulated by bacterially-derived LPS and LTA as an in vitro model of bacterial infection, we examined the anti-infammatory efects of nanosystems consisting of iron oxidebased magnetic nanoparticles with aminosilane (MNP@NH2) or gold shells (MNP@Au) functionalized by a set of peptides, derived from the phosphatidylinositol 4,5-bisphosphate (PIP2)-binding site of the human plasma protein gelsolin, which also binds LPS and LTA. Our results indicate that these nanosystems can kill both Gram-positive and Gram-negative bacteria and limit the production of infammatory mediators, including nitric oxide (NO), reactive oxygen species (ROS), and interleukin-8 (IL-8) in the response to heat-killed microbes or extracted bacterial cell wall components. The nanoparticles possess the potential to improve therapeutic efcacy and are characterized by lower toxicity and improved hemocompatibility when compared to free peptides. Atomic force microscopy (AFM) showed that these PBP10-based nanosystems prevented changes in nanomechanical properties of cells that were otherwise stimulated by LPS. Conclusions: Neutralization of endotoxemia-mediated cellular efects by gelsolin-derived peptides and PBP10-containing nanosystems might be considered as potent therapeutic agents in the improved therapy of bacterial infections and microbial-induced infammation.This work was fnancially supported by the National Science Center, Poland under Grant: UMO-2015/17/B/NZ6/03473 (to RB) and Medical University of Bialystok (N/ST/ZB/18/002/1162 and N/ST/ZB/18/001/1162 (to RB) and N/ST/MN/18/002/1162 (to EP). Part of the study was conducted with the use of equipment purchased by the Medical University of Białystok as part of the RPOWP 2007-2013 funding, Priority I, Axis 1.1, contract No. UDARPPD.01.01.00-20-001/15-00 dated 26.06.2015. The physicochemical studies were performed in Centre of Synthesis and Analysis BioNanoTechno of the University of Bialystok (POPW.01.03.00-20-034/09-00 and POPW.01.03.00-20004/11 projects). EP acknowledges a doctoral scholarship from Polpharma Scientifc Foundation, Poland. PAJ and RB acknowledge support from NIH grant GM111942-01.Robert Bucki: [email protected] Piktel - Department of Microbiological and Nanobiomedical Engineering, Medical University of BialystokUrszula Wnorowska - Department of Microbiological and Nanobiomedical Engineering, Medical University of BialystokMateusz Cieśluk - Department of Microbiological and Nanobiomedical Engineering, Medical University of BialystokPiotr Deptula - Department of Microbiological and Nanobiomedical Engineering, Medical University of BialystokKatarzyna Pogoda - IInstitute of Nuclear Physics Polish Academy of SciencesIwona Misztalewska‑Turkowicz - Institute of Chemistry, University of BiałystokPaulina Paprocka - Department of Microbiology and Immunology, The Faculty of Medicine and Health Sciences of the Jan Kochanowski University in KielceKatarzyna Niemirowicz‑Laskowska - Department of Microbiological and Nanobiomedical Engineering, Medical University of BialystokAgnieszka Z. 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Recombinant Human Plasma Gelsolin Stimulates Phagocytosis while Diminishing Excessive Inflammatory Responses in Mice with Pseudomonas aeruginosa Sepsis

Plasma gelsolin (pGSN) is a highly conserved abundant circulating protein, characterized by diverse immunomodulatory activities including macrophage activation and the ability to neutralize pro-inflammatory molecules produced by the host and pathogen. Using a murine model of Gram-negative sepsis initiated by the peritoneal instillation of Pseudomonas aeruginosa Xen 5, we observed a decrease in the tissue uptake of IRDye®800CW 2-deoxyglucose, an indicator of inflammation, and a decrease in bacterial growth from ascitic fluid in mice treated with intravenous recombinant human plasma gelsolin (pGSN) compared to the control vehicle. Pretreatment of the murine macrophage line RAW264.7 with pGSN, followed by addition of Pseudomonas aeruginosa Xen 5, resulted in a dose-dependent increase in the proportion of macrophages with internalized bacteria. This increased uptake was less pronounced when cells were pretreated with pGSN and then centrifuged to remove unbound pGSN before addition of bacteria to macrophages. These observations suggest that recombinant plasma gelsolin can modulate the inflammatory response while at the same time augmenting host antibacterial activity.This work was supported by the National Science Center, Poland under Grant: UMO-2015/17/B/NZ6/03473 (to RB), National Institutes of Health: GM111942 (to PAJ) and Medical University of Bialystok: SUB/1/DN/19/001/1162 (to RB), N/ST/MN/18/001/1162 (to MC). Part of the study was conducted with the use of equipment purchased by the Medical University of Białystok as part of the RPOWP 2007-2013 funding, Priority I, Axis 1.1, contract No. UDA- RPPD.01.01.00-20-001/15-00 dated 26.06.2015. This work was supported by the program of the Minister of Science and Higher Education under the name “Regional Initiative of Excellence in 2019–2022”, project number: 024/RID/2018/19, financing amount: 11.999.000,00 PLN.Ewelina Piktel: [email protected] Wnorowska: [email protected] Cieśluk: [email protected] Deptuła: [email protected] V. Prasad: [email protected] Król: [email protected] Durnaś: [email protected] Namiot: [email protected] H. Markiewicz: [email protected] Niemirowicz-Laskowska: [email protected] Z. Wilczewska: [email protected] A. Janmey: [email protected] Reszeć: [email protected] Bucki: [email protected] Piktel - Department of Medical Microbiology and Nanobiomedical Engineering, Medical University of BialystokUrszula Wnorowska - Department of Medical Microbiology and Nanobiomedical Engineering, Medical University of BialystokMateusz Cieśluk - Department of Medical Microbiology and Nanobiomedical Engineering, Medical University of BialystokPiotr Deptuła - Department of Medical Microbiology and Nanobiomedical Engineering, Medical University of BialystokSuhanya V. Prasad - Department of Medical Microbiology and Nanobiomedical Engineering, Medical University of BialystokGrzegorz Król - Department of Microbiology and Immunology, the Faculty of Medicine and Health Sciences of the Jan Kochanowski University in KielceBonita Durnaś - Department of Microbiology and Immunology, the Faculty of Medicine and Health Sciences of the Jan Kochanowski University in KielceAndrzej Namiot - Department of Anatomy, Medical University of BialystokKarolina H. Markiewicz - Institute of Chemistry, University of BiałystokKatarzyna Niemirowicz-Laskowska - Department of Medical Microbiology and Nanobiomedical Engineering, Medical University of BialystokAgnieszka Z. Wilczewska - Institute of Chemistry, University of BiałystokPaul A. Janmey - Institute for Medicine and Engineering, University of PennsylvaniaJoanna Reszeć - Department of Pathology, Medical University of BialystokRobert Bucki - Department of Medical Microbiology and Nanobiomedical Engineering, Medical University of Bialystok; Department of Microbiology and Immunology, the Faculty of Medicine and Health Sciences of the Jan Kochanowski University in KielceLee, P.S.; Patel, S.R.; Christiani, D.C.; Bajwa, E.; Stossel, T.P.; Waxman, A.B. Plasma gelsolin depletion and circulating actin in sepsis: A pilot study. PLoS ONE 2008, 3, e3712.Li-ChunHsieh, K.; Schob, S.; Zeller, M.W.; Pulli, B.; Ali, M.; Wang, C.; Chiou, T.T.; Tsang, Y.M.; Lee, P.S.; Stossel, T.P.; et al. Gelsolin decreases actin toxicity and inflammation in murine multiple sclerosis. J. Neuroimmunol. 2015, 287, 36–42.Bucki, R.; Kulakowska, A.; Byfield, F.J.; Zendzian-Piotrowska, M.; Baranowski, M.; Marzec, M.; Winer, J.P.; Ciccarelli, N.J.; Górski, J.; Drozdowski, W.; et al. 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Unique Role of Vimentin Networks in Compression Stiffening of Cells and Protection of Nuclei from Compressive Stress

In this work, we investigate whether stiffening in compression is a feature of single cells and whether the intracellular polymer networks that comprise the cytoskeleton (all of which stiffen with increasing shear strain) stiffen or soften when subjected to compressive strains. We find that individual cells, such as fibroblasts, stiffen at physiologically relevant compressive strains, but genetic ablation of vimentin diminishes this effect. Further, we show that unlike networks of purified F-actin or microtubules, which soften in compression, vimentin intermediate filament networks stiffen in both compression and extension, and we present a theoretical model to explain this response based on the flexibility of vimentin filaments and their surface charge, which resists volume changes of the network under compression. These results provide a new framework by which to understand the mechanical responses of cells and point to a central role of intermediate filaments in response to compression

Neutrophil extracellular traps as the main source of eDNA

Neutrophil extracellular traps (NETs) are web-like structures consisting of decondensed DNA together with accompanying proteins, including histones and antimicrobial peptides released from activated neutrophils as part of the first-line defence against pathogens. Despite the protective role of neutrophils, a number of studies indicate that overproduction of NETs followed by accumulation of extracellular DNA (eDNA) and other negatively-charged polyelectrolytes (PE) such as F-actin, contribute to the pathogenesis of some diseases. Neutrophil extracellular traps are also recognised as the structural and functional support of microbial biofilms and should thus be considered as therapeutic targets. Importantly, the chemical nature of PE permits aggregate formation induced by a number of polycations occurring naturally in the human body, including cationic antimicrobial peptides. This review summarises recent reports focused on the clinical significance of NET-derived eDNA and PE and discusses the potential therapeutic strategies to limit the negative consequences of eDNA accumulation

Physics Comes to the Aid of Medicine—Clinically-Relevant Microorganisms through the Eyes of Atomic Force Microscope

Despite the hope that was raised with the implementation of antibiotics to the treatment of infections in medical practice, the initial enthusiasm has substantially faded due to increasing drug resistance in pathogenic microorganisms. Therefore, there is a need for novel analytical and diagnostic methods in order to extend our knowledge regarding the mode of action of the conventional and novel antimicrobial agents from a perspective of single microbial cells as well as their communities growing in infected sites, i.e., biofilms. In recent years, atomic force microscopy (AFM) has been mostly used to study different aspects of the pathophysiology of noninfectious conditions with attempts to characterize morphological and rheological properties of tissues, individual mammalian cells as well as their organelles and extracellular matrix, and cells’ mechanical changes upon exposure to different stimuli. At the same time, an ever-growing number of studies have demonstrated AFM as a valuable approach in studying microorganisms in regard to changes in their morphology and nanomechanical properties, e.g., stiffness in response to antimicrobial treatment or interaction with a substrate as well as the mechanisms behind their virulence. This review summarizes recent developments and the authors’ point of view on AFM-based evaluation of microorganisms’ response to applied antimicrobial treatment within a group of selected bacteria, fungi, and viruses. The AFM potential in development of modern diagnostic and therapeutic methods for combating of infections caused by drug-resistant bacterial strains is also discussed

Use of magnetic nanoparticles as a drug delivery system to improve chlorohexidine antimicrobial activity

Nanotechnology offers new tools for developing therapies to prevent and treat oral infections, particularly biofilm-dependent disorders, such as dental plaques and endodontic and periodontal diseases. Chlorhexidine (CHX) is a well-characterized antiseptic agent used in dentistry with broad spectrum activity. However, its application is limited due to inactivation in body fluid and cytotoxicity toward human cells, particularly at high concentrations. To overcome these limitations, we synthesized nanosystems composed of aminosilane-coated magnetic nanoparticles functionalized with chlorhexidine (MNP@CHX). In the presence of human saliva, MNPs@ CHX displayed significantly greater bactericidal and fungicidal activity against planktonic and biofilm-forming microorganisms than free CHX. In addition, CHX attached to MNPs has an increased ability to restrict the growth of mixed-species biofilms compared to free CHX. The observed depolarization of mitochondria in fungal cells treated with MNP@CHX suggests that induction of oxidative stress and oxidation of fungal structures may be a part of the mechanism responsible for pathogen killing. Nanoparticles functionalized by CHX did not affect host cell proliferation or their ability to release the proinflammatory cytokine, IL-8. The use of MNPs as a carrier of CHX has great potential for the development of antiseptic nanosystems.This work was supported by the National Science Center, Poland, under grant UMO-2014/15/D/NZ6/02665 (to KN). In 2016, KN was awarded a fellowship from the Foundation for Polish Science. This study was conducted with the use of equipment purchased by the Medical University of Białystok as part of the RPOWP 2007–2013 funding, Priority I, Axis 1.1, contract number UDA-RPPD.01.01.00-20-001/15-00, dated 26.06.2015.Grażyna Tokajuk - Department of Microbiological and Nanobiomedical Engineering, Medical University of Białystok; Department of Intergrated Dentistry, Medical University of BiałystokKatarzyna Niemirowicz - Department of Microbiological and Nanobiomedical Engineering, Medical University of BiałystokPiotr Deptuła - Department of Microbiological and Nanobiomedical Engineering, Medical University of Białystok; Department of Materials and Biomedical Engineering, Białystok University of TechnologyEwelina Piktel - Department of Microbiological and Nanobiomedical Engineering, Medical University of BiałystokMateusz Cieśluk - Department of Microbiological and Nanobiomedical Engineering, Medical University of BiałystokAgnieszka Z. Wilczewska - Institute of Chemistry, University of Białystok, Białystok, PolandJan R. Dąbrowski - Department of Materials and Biomedical Engineering, Białystok University of TechnologyRobert Bucki - Department of Microbiological and Nanobiomedical Engineering, Medical University of BiałystokKarpiński TM, Szkaradkiewicz AK. Chlorhexidine – pharmacobiological activity and application. Eur Rev Med Pharmacol Sci. 2015; 19(7):1321–1326.Wood A, Payne D. The action of three antiseptics/disinfectants against enveloped and non-enveloped viruses. J Hosp Infect. 1998;38(4): 283–295.Fathilah AR, Himratul-Aznita WH, Fatheen AR, Suriani KR. The antifungal properties of chlorhexidine digluconate and cetylpyrinidinium chloride on oral Candida. J Dent. 2012;40(7):609–615.Kuyyakanond T, Quesnel LB. The mechanism of action of chlorhexidine. FEMS Microbiol Lett. 1992;100(1–3):211–215.Zorko M, Jerala R. 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Bacteria Residing at Root Canals Can Induce Cell Proliferation and Alter the Mechanical Properties of Gingival and Cancer Cells

Understanding the importance of oral microbiota in human health and disease also leads to an expansion of the knowledge on functional, metabolic, and molecular alterations directly contributing to oral and systemic pathologies. To date, a compelling number of studies have documented the crucial role of some oral cavity-occurring microbes in the initiation and progression of cancers. Although this effect was noted primarily for Fusobacterium spp., the potential impact of other oral microbes is also worthy of investigation. In this study, we aimed to assess the effect of Enterococcus faecalis, Actinomyces odontolyticus, and Propionibacterium acnes on the proliferation capability and mechanical features of gingival cells and cell lines derived from lung, breast, and ovarian cancers. For this purpose, we incubated selected cell lines with heat-inactivated bacteria and supernatants collected from biofilms, cultured in both anaerobic and aerobic conditions, in the presence of surgically removed teeth and human saliva. The effect of oral bacteria on cell population growth is variable, with the highest growth-promoting abilities observed for E. faecalis in relation to human primary gingival fibroblasts (HGF) and lung cancer A549 cells, and P. acnes in relation to breast cancer MCF-7 and ovarian cancer SKOV-3 cells. Notably, this effect seems to depend on a delicate balance between the pro-stimulatory and toxic effects of bacterial-derived products. Regardless of the diverse effect of bacterial products on cellular proliferation capability, we observed significant alterations in stiffness of gingival and lung cancer cells stimulated with E. faecalis bacteria and corresponding biofilm supernatants, suggesting a novel molecular mechanism involved in the pathogenesis of diseases in oral cavities and tooth tissues. Accordingly, it is proposed that analysis of cancerogenic features of oral cavity bacteria should be multivariable and should include investigation of potential alterations in cell mechanical properties. These findings corroborate the important role of oral hygiene and root canal treatment to assure the healthy stage of oral microbiota

Biofilm Growth Causes Damage to Silicone Voice Prostheses in Patients after Surgical Treatment of Locally Advanced Laryngeal Cancer

Voice prosthesis implantation with the creation of a tracheoesophageal fistula is the gold standard procedure for voice rehabilitation in patients after a total laryngectomy. All patients implanted with a voice prosthesis (VP) have biofilms of fungi and bacteria grow on their surface. Biofilm colonization is one of the main reasons for VP degradation that can lead to VP dysfunction, which increases the high risk of pneumonia. In a 20-month evaluation period, 129 cases of prostheses after replacement procedures were investigated. Microbiological examination of the biofilms revealed that there were four of the most common fungi species (Candida spp.) and a large variety of bacterial species present. We studied the relationship between the time of proper function of Provox VP, the microorganism composition of the biofilm present on it, and the degradation level of the silicone material. Evaluation of the surface of the removed VP using an atomic force microscope (AFM) has demonstrated that biofilm growth might drastically change the silicone’s mechanical properties. Changes in silicone stiffness and thermal properties might contribute to the failure of VP function. Our data can serve in future studies for the development of methods to prevent or inhibit biofilm formation on the VP surface that would translate to an increase in their durability and safety

Inhibition of inflammatory response in human keratinocytes by magnetic nanoparticles functionalized with PBP10 peptide derived from the PIP2-binding site of human plasma gelsolin

Abstract Background Human plasma gelsolin (pGSN) is a multifunctional actin-binding protein involved in a variety of biological processes, including neutralization of pro-inflammatory molecules such as lipopolysaccharide (LPS) and lipoteichoic acid (LTA) and modulation of host inflammatory response. It was found that PBP10, a synthetic rhodamine B-conjugated peptide, based on the phosphoinositide-binding site of pGSN, exerts bactericidal activity against Gram-positive and Gram-negative bacteria, interacts specifically with LPS and LTA, and limits microbial-induced inflammatory effects. The therapeutic efficiency of PBP10 when immobilized on the surface of iron oxide-based magnetic nanoparticles was not evaluated, to date. Results Using the human keratinocyte cell line HaCaT stimulated by bacterially-derived LPS and LTA as an in vitro model of bacterial infection, we examined the anti-inflammatory effects of nanosystems consisting of iron oxide-based magnetic nanoparticles with aminosilane (MNP@NH2) or gold shells (MNP@Au) functionalized by a set of peptides, derived from the phosphatidylinositol 4,5-bisphosphate (PIP2)-binding site of the human plasma protein gelsolin, which also binds LPS and LTA. Our results indicate that these nanosystems can kill both Gram-positive and Gram-negative bacteria and limit the production of inflammatory mediators, including nitric oxide (NO), reactive oxygen species (ROS), and interleukin-8 (IL-8) in the response to heat-killed microbes or extracted bacterial cell wall components. The nanoparticles possess the potential to improve therapeutic efficacy and are characterized by lower toxicity and improved hemocompatibility when compared to free peptides. Atomic force microscopy (AFM) showed that these PBP10-based nanosystems prevented changes in nanomechanical properties of cells that were otherwise stimulated by LPS. Conclusions Neutralization of endotoxemia-mediated cellular effects by gelsolin-derived peptides and PBP10-containing nanosystems might be considered as potent therapeutic agents in the improved therapy of bacterial infections and microbial-induced inflammation

Assessment of Ceragenins in Prevention of Damage to Voice Prostheses Caused by Candida Biofilm Formation

This study aimed to investigate the potential application of ceragenins (CSAs) as new candidacidal agents to prevent biofilm formation on voice prostheses (VPs). The deterioration of the silicone material of VPs is caused by biofilm growth on the device which leads to frequent replacement procedures and sometimes serious complications. A significant proportion of these failures is caused by Candida species. We found that CSAs have significant candidacidal activities in vitro (MIC; MFC; MBIC), and they effectively eradicate species of yeast responsible for VP failure. Additionally, in our in vitro experimental setting, when different Candida species were subjected to CSA-13 and CSA-131 during 25 passages, no tested Candida strain showed the significant development of resistance. Using liquid chromatography–mass spectrometry (LC-MS), we found that VP immersion in an ethanol solution containing CSA-131 results in silicon impregnation with CSA-131 molecules, and in vitro testing revealed that fungal biofilm formation on such VP surfaces was inhibited by embedded ceragenins. Future in vivo studies will validate the use of ceragenin-coated VP for improvement in the life quality and safety of patients after a total laryngectomy