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Article
Poly(I:C)-encapsulating nanoparticles enhance innate immune responses to the tuberculosis vaccine Bacille-Calmette-Guérin (BCG) via synergistic activation of innate immune receptors Martin T. Speth, Urska Repnik, Elisabeth Müller, Julia Spanier, Ulrich Kalinke, Alexandre Corthay, and Gareth Griffiths Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.7b00795 • Publication Date (Web): 03 Oct 2017 Downloaded from http://pubs.acs.org on October 5, 2017
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Molecular Pharmaceutics
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Poly(I:C)-encapsulating nanoparticles enhance
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innate immune responses to the tuberculosis vaccine
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Bacille-Calmette-Guérin (BCG) via synergistic
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activation of innate immune receptors
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Martin T. Speth1, Urska Repnik1, Elisabeth Müller1,2, Julia Spanier3, Ulrich Kalinke3, Alexandre
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Corthay2, Gareth Griffiths1*
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1
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Tumor Immunology lab, Department of Pathology, Rikshospitalet, Oslo University Hospital and University of Oslo, N-0424 Oslo, Norway,
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Department of Biosciences, University of Oslo, N-0371 Oslo, Norway,
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Institute for Experimental Infection Research, TWINCORE, Center for Experimental and
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Clinical Infection Research, a joint venture between the Helmholtz Centre for Infection Research,
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Braunschweig, and the Hannover Medical School, D-30625 Hannover, Germany,
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ABSTRACT
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The attenuated live vaccine strain Bacille Calmette-Guérin (BCG) is currently the only available
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vaccine against tuberculosis (TB), but is largely ineffective against adult pulmonary TB, the most
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common disease form. This is in part due to BCG’s ability to interfere with the host innate
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immune response, a feature that might be targeted to enhance the potency of this vaccine. Here,
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we investigated the ability of chitosan-based nanoparticles (pIC-NPs) containing polyinosinic-
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polycytidylic acid (poly(I:C)), an inducer of innate immunity via Toll-like receptor 3 (TLR3) to
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enhance the immunogenicity of BCG in mouse bone marrow-derived macrophages (BMDM) in
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vitro. Incorporation of poly(I:C) into NPs protected it against degradation by ribonucleases and
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increased its uptake by mouse BMDM. Whereas soluble poly(I:C) was ineffective, pIC-NPs
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strongly enhanced the pro-inflammatory immune response of BCG-infected macrophages in a
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synergistic fashion, as evident by increased production of cytokines and induction of nitric oxide
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synthesis. Using macrophages from mice deficient in key signaling molecules involved in the
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pathogen recognition response, we identified combined activation of MyD88- and TRIF-
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dependent TLR signaling pathways to be essential for the synergistic effect between BCG and
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NP. Moreover, synergy was strongly dependent on the order of the two stimuli, with TLR
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activation by BCG functioning as the priming event for the subsequent pIC-NP-stimulus, which
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acted through an auto-/paracrine type I interferon (IFN) feedback loop. Our results provide a
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foundation for a promising new approach to enhance BCG-vaccine immunogenicity by co-
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stimulation with NPs. They also contribute to a molecular understanding of the observed
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synergistic interaction between the pIC-NPs and BCG-vaccine.
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Molecular Pharmaceutics
KEYWORDS: adjuvant, Bacille Calmette-Guérin (BCG), chitosan, nanoparticles, poly(I:C), Toll-like receptor synergy, type I interferons (IFNs)
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INTRODUCTION
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Tuberculosis (TB), which is caused by the bacterial pathogen Mycobacterium tuberculosis
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(M.tb), is one of the most deadly infectious diseases globally. In pulmonary TB, which is the
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most common form of the disease, M.tb infects host macrophages and dendritic cells in the lung
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and resides intracellularly within maturation arrested, immature phagosomes. M.tb has developed
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a plethora of strategies to evade the host immune system including interference with dendritic
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cell (DC) maturation, antigen presentation and migration, resulting in a delayed and suboptimal
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adaptive immune response.1 Treatment of TB requires daily intake of multiple antibiotics over
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several months, which is expensive and challenged by patient non-compliance, M.tb drug
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tolerance and resistance. Therefore, prophylactic vaccination against TB remains a high priority
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in order to reduce the rate of infection.
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The live attenuated vaccine strain Mycobacterium bovis Bacille Calmette-Guérin (BCG)
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confers some protection against more severe forms of tuberculosis (TB) during childhood, but is
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largely ineffective against pulmonary TB in adults.2 Despite its limited and variable efficacy in
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adults, the BCG-vaccine remains to date the only one in use against TB. The BCG-strain is
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considered to be one of the safest vaccines currently in use, but has retained some undesirable
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properties from its virulent ancestor, such as interference with professional antigen presenting
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cell (APC) activation and antigen presentation and DC migration.3-4 These are believed to
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contribute to the suboptimal induction of T cell-mediated immunity by the BCG-vaccine.5-6 The
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cell-mediated arm of the immune system, including type I helper CD4+ (TH1) and in particular
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cytotoxic CD8+ T lymphocytes (CTL) is widely accepted to be very important for conveying
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protection against intracellular pathogens in general, and M.tb in particular.7
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Thus, a major challenge for either improving the BCG-vaccine or developing new TB vaccines
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is to evoke immune responses that are able to generate a strong, broad and long-lived T cell-
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mediated immunity. For this purpose, several new immune modulating or stimulating agents are
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currently under investigation as adjuvants for vaccines against TB as well as other diseases.8-9
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The most potent and promising candidates are agonists of Toll-like receptors (TLRs), which are
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involved in pathogen recognition by APCs, especially macrophages and dendritic cells. TLRs
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recognize a set of pathogen associated molecular patterns (PAMPs) and trigger an immune
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response through two main intracellular signaling pathways, either involving the adaptor
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molecule MyD88 (all TLRs except TLR3), the adaptor molecule TRIF (TLR3), or both (TLR4).
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Activation of the MyD88-dependent pathway drives production of pro-inflammatory cytokines,
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whereas the TRIF-dependent pathway induces the transcription of antiviral proteins and type I
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interferons (IFN).
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The TLR3 agonist polyriboinosinic acid-polyribocytidylic acid (poly(I:C)) is a synthetic double-
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stranded RNA derivate, which induces a robust and strong TH1- and CTL polarized immune
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response in vivo.10-11 As a consequence, poly(I:C) is considered to be a promising adjuvant
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candidate for new vaccines against intracellular pathogens such as viruses and invasive bacteria.12
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However, soluble poly(I:C) is prone to rapid degradation by ribonucleases, which are
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ubiquitously present in serum and tissues in vivo and higher dosage can cause detrimental side-
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effects, including fever and anemia.13
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Nanotechnology has further propelled the development of vaccines by providing delivery
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platforms for vaccines with multiple advantageous properties. Nano-and micro-particles can
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protect both immune-stimulatory adjuvants and vaccine antigens against rapid degradation,
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enhance their cellular uptake and increase their specificity for innate immune cells. Most
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importantly, these delivery formulations often function as a localized depot for adjuvants and
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antigens, enabling sustained immune stimulation.14 Nanoparticle- and liposome-based
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formulations in particular have been considered promising with several candidates currently in
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advanced clinical trials.15-16
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In recent years, special focus has been put on combining multiple immune-stimulatory
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adjuvants with delivery platforms to multi-adjuvanted vaccine formulations.17 This strategy holds
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great potential not only for enhancing the strength, but also for optimizing the quality of the
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immune responses induced by new vaccine formulations. Different combinations of TLR
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agonists were shown to act synergistically, whereas others have modulating or even antagonistic
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effects on immune activation. Especially strong synergistic immune cell activation can be
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achieved by combining MyD88-dependent and TRIF-dependent TLR-agonists.18 However, due
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to their potency, especially when combined with nanotechnology delivery systems, it is crucial to
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understand the mechanisms underlying such adjuvant interactions in order to take advantage of
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these for multi-adjuvanted vaccines.
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We have recently shown that complementing the BCG-vaccine with poly(I:C) using
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nanotechnology methods can efficiently enhance pro-inflammatory activation in mouse primary
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macrophages.19 Nanocoating of poly(I:C) onto the surface of live BCG bacteria using layer-by-
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layer (LbL) self-assembly both enhanced the production of pro-inflammatory cytokines and the
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expression of molecules involved in macrophage – T-cell interactions including CD80, CD86 and
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CD40. Strikingly, poly(I:C) nanocoated onto BCG, but not poly(I:C) co-delivered in its soluble
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form along with BCG induced significant production of nitric oxide (NO) and bactericidal
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activity in macrophages. These results are in accordance with earlier reports by others showing
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strongly enhanced immunostimulatory potency of nanoparticle (NP) surface-bound poly(I:C)
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compared to poly(I:C) in its soluble form.20-21 Together, poly(I:C)-nanocoated BCG were able to
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induce a distinct pro-inflammatory macrophage phenotype including IL-12 production, which is
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commonly associated with promotion of a strongly TH1-skewed adaptive immune response,
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involving secretion of IFN-γ.22-23
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In this study, we developed and characterized poly(I:C)-encapsulating nanoparticles (pIC-NPs)
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using the simple method of polyelectrolyte complexation between the anionic poly(I:C) and
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cationic chitosan, a non-toxic, biocompatible and biodegradable natural polymer.24 We tested
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these pIC-NPs for their ability to induce and amplify a pro-inflammatory phenotype in mouse
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bone marrow-derived macrophages (BMDM), in conjunction with (separately administered)
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BCG bacteria. Based on experiments using macrophages derived from transgenic mice deficient
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in adaptor molecules of central pathogen recognition pathways, we also provide a model for the
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molecular mechanisms involved, including those responsible for the underlying synergistic
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interaction between BCG and poly(I:C)-NPs. Our results provide a promising foundation for a
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new approach that combines the BCG-vaccine with NP-based delivery of the adjuvant poly(I:C)
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that has the potential to improve the potency of the BCG-vaccine.
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EXPERIMENTAL SECTION
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Preparation and characterization of pIC-NPs
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To prepare poly(I:C) encapsulating chitosan-nanoparticles (pIC-NPs), endotoxin-free low
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molecular chitosan (KiOmedine-CSU®, MW 60,000–120,000; Sigma-Aldrich) was dissolved in
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1 % acetic acid, diluted with dH2O to a concentration of 2 mg/ml and adjusted to pH 6. An equal
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volume of 1 mg/ml low molecular weight poly(I:C) (Invivogen) in 0.9 % NaCl was then added
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dropwise to the chitosan solution under stirring, and the solution was left stirring for 20 min. The
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pIC-NPs were then collected on a glycerol bed by centrifugation at 10,000 g for 20 min, re-
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suspended in 0.9 % NaCl and dissociated by water bath sonication for 10 min before use. All
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reagents used for pIC-NP production were certified endotoxin-free by the manufacturers, and all
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steps of the procedure were done in a laminar flow hood to ensure aseptic conditions. Red
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fluorescent pIC-NPs and soluble red-fluorescent poly(I:C) were produced by supplementing the
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poly(I:C)-solution with rhodamine-labeled poly(I:C) (Invivogen) in a ratio of 50:1. Dual-labelled
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fluorescent pIC-NPs were prepared by using red-fluorescent poly(I:C) in combination with
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chitosan conjugated with near-infrared fluorochrome IRDye® 680RD (LI-COR Biosciences).
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NPs encapsulating 15 nm gold particles in their matrix (Au-pIC-NP) were produced as follows:
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15 nm colloidal gold particles were prepared as described previously.25 Unconjugated 15 nm
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colloidal gold particles were diluted in 2 mg/ml chitosan and concentrated 10 times by
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centrifugation at 17,000 g for 30 min. Au-pIC-NPs were then prepared using the chitosan-gold
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particle solution as described above. Chitosan nanoparticles without poly(I:C) were produced by
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replacing poly(I:C) with 1 mg/ml sodium tripolyphosphate (TPP; Sigma-Aldrich).
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Hydrodynamic size and the ζ-potential, representing the surface charge of the particles, were
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measured on a Nano ZS Zeta-sizer instrument (Malvern Instruments). Encapsulation efficiency
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and the amount of poly(I:C) encapsulated in pIC-NPs was determined indirectly by measuring
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unbound poly(I:C) after NP collection based on the absorbance of soluble poly(I:C) at 260 nm
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using a Nanodrop (Thermo Fisher Scientific). Likewise, the amount of chitosan in pIC-NPs was
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calculated indirectly from the amount of unbound chitosan in the supernatant. Free chitosan was
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measured using a fluorescamine assay.26 Throughout the article, the indicated concentrations of
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pIC-NPs refer to the concentration of poly(I:C) in form of pIC-NPs.
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The morphology of pIC-NPs was analyzed by transmission electron microscopy (TEM). The
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pIC-NPs were adsorbed to a formvar-coated TEM grid and briefly stained with 3% uranyl acetate
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and imaged with a CM100 transmission electron microscope (FEI). Images were recorded with a
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Quemesa camera using iTEM software (Olympus Soft Imaging Solutions).
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For the nuclease protection assay, 10 U RNase (RNaseOne™; Promega) was added to a
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solution of soluble poly(I:C) or pIC-NPs, both at a concentration of 20 µg/ml poly(I:C), and the
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absorbance at 260 nm was measured at different time points using a spectrophotometer
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(Eppendorf).
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Bacterial cultures and preparation of BCG for infection
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Bacille Calmette-Guérin (Pasteur strain) expressing GFP (BCG-GFP; generous gift of Michael
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Niederweis) were grown in Middlebrook's 7H9 broth medium (BD Biosciences, Heidelberg,
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Germany) supplemented with 10% OADC, 0.02% glycerol, 0.05% Tween 80 and 50 µg/ml
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Hygromycin B at 37 °C without agitation. To prepare BCG-GFP cultures for infection of
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BMDM, bacteria in the exponential growth phase with an optical density (OD600) of 0.6–0.9
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were harvested by centrifugation at 3,000 g for 10 min, washed twice in 0.9% NaCl solution and
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resuspended in RPMI-medium supplemented with 10% FCS. The bacterial suspension was then
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sonicated for 10 min in a bath sonicator and passed 10 times through a 23-gauge needle to break
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up clumps and to ensure a homogenous suspension of single bacteria.
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Isolation and treatment of Bone Marrow-Derived Macrophages (BMDM)
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Wild-type C57BL/6 (WT) mice were purchased from Taconic Biosciences and transgenic mice
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deficient in MyD88 (MyD88-/-), TRIF (TRIF-/-), CARDIF (CARDIF-/-) or the IFNAR 1 chain
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(IFNAR-/-) were reared at the central mouse facility of the Helmholtz Centre for Infection
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Research, Braunschweig, and at TWINCORE, Centre for Experimental and Clinical Infection
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Research, Hannover, Germany. Mice deficient in ASC (ASC-/-) came from the Oslo University
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Hospital, Rikshospitalet.27 Bone Marrow-Derived Macrophages were prepared as described
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previously.19 Briefly, bone marrow was flushed from the tibias and femurs of 8-12 week old mice
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and red blood cells were removed by incubation in RBC lysis buffer. Bone marrow cells were
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then incubated in RPMI-medium supplemented with 10% FCS, 30% L929 conditioned medium
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and 50 µM β-mercaptoethanol in non-tissue culture treated dishes at 37 °C for 7 days with
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medium changes after 3 and 6 days. Differentiated BMDM were then detached by incubating the
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cell monolayer in Ca2+/Mg2+-free PBS at 4 °C and harvested by centrifugation. Throughout all
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experiments, cells were then maintained in RPMI medium supplemented with 10% FCS, 10%
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L929-conditioned medium and 50 µM β-mercaptoethanol.
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For infection with live BCG, WT- and KO-BMDM cultures were incubated with BCG-GFP at
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a MOI of 10 for 3 h and then washed twice with warm Ca2+/Mg2+-free PBS in order to remove
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any extracellular bacteria. Uninfected or BCG-infected BMDM were then incubated with
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medium only, soluble poly(I:C), pIC-NPs or Chi/TPP-NPs at the indicated concentrations for 24
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h. For short term stimulation, BMDM were infected with BCG for 3 h in the presence of soluble
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poly(I:C) or pIC-NPs at the indicated concentrations, washed and cultured for further 24 h in cell
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culture medium.
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For selective stimulation of TLR2, BMDM were treated with lipomannan or the synthetic
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triacylated lipopeptide Pam3CSK4 (both Invivogen) at the indicated concentrations and for the
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indicated time periods.
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For stimulation experiments with recombinant IFN-β (rIFN-β), WT-BMDM were treated with
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different concentrations (given in international units (IU) per ml) of mouse rIFN-β (R&D
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Systems) for 24 h either before or after infection with BCG. Cell culture supernatants were then
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collected for analysis 24 h after BCG infection. For rIFN-β rescue experiments, BCG-infected
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WT- and KO-BMDM were treated with 2 µg/ml pIC-NPs alone or in presence of 3000 IU/ml
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rIFN-β for 24 h.
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Flow Cytometry (FACS) Assay
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To analyze the uptake of poly(I:C) by BMDM, cells were incubated with 10 µg/ml red-
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fluorescent pIC-NPs or soluble poly(I:C), which corresponds to a concentration of 0.2 µg/ml pure
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rhodamine-labeled poly(I:C), for the indicated time periods. BMDM cultures were then washed 3
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times with warm PBS, collected by incubation in Ca2+/Mg2+ free PBS at 4°C, fixed with 2%
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paraformaldehyde for 15 min and analyzed using a BD FACsCalibur flow cytometer (BD
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Biosciences).
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To study the ability of BCG, lipomannan and pIC-NP to enhance the expression of TLR2 and
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TLR3, BMDM cultures were treated with BCG or lipomannan for 3 h, washed and incubated for
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additional 24 h in normal medium or they were incubated with 2 µg/ml pIC-NPs for 24 h. Cells
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were then collected, washed with PBS and treated with a Fc receptor blocking solution (Macs).
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To analyze TLR2 expression, cells were stained with a PE-conjugated antibody against TLR2
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(clone CB225, Biolegend) or a matching isotype control using standard protocols for surface
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staining. In contrast, TLR3 is exclusively expressed intracellularly. To analyze expression of
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TLR3, which is exclusively located intracellularly, BMDM were fixed with 2%
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paraformaldehyde after incubation with FC receptor blocking solution, permeabilized with 0.5%
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Tween20 in PBS/0.5% BSA/2 mM EDTA and stained with a PE-conjugated antibody against
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TLR3 (clone 11F8, Biolegend) or a matching isotype control. After staining, cells were fixed
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with 2% paraformaldehyde for 15 min and analyzed using a BD FACsCalibur flow cytometer.
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Confocal microscopy
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For Confocal microscopy analysis, BMDM were seeded on poly-L-lysine coated glass
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coverslips. To study pIC-NP uptake, cells were incubated with 10 µg/ml red fluorescent pIC-NPs
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or soluble poly(I:C) for different time periods. For co-localization studies, BMDM cultures were
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treated with 20 µg/ml dual-labeled pIC-NPs alone or together with BCG-GFP for 3 h, washed 3
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times with warm Ca2+/Mg2+ free PBS and fixed immediately or after additional 24 h culture
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with 2% paraformaldehyde for 15 min. Cells were finally imaged with an Olympus FluoView
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1000 inverted IX81 confocal laser scanning microscope.
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Transmission electron microscopy (TEM) of thin eponsections
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To label lysosomes, BMDM were pulsed with 5nm colloidal gold for 2 h, washed and
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incubated in fresh medium for additional 2 h. Cells were then incubated with BCG and 10 µg/ml
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pIC-NPs encapsulating 15 nm gold (Au-pIC-NPs) for 3 h, washed 3 times with warm
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Ca2+/Mg2+ free PBS and incubated in normal medium for additional 3 or 24 h. Cells were then
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fixed with 1% glutaraldehyde in 0.2 M HEPES buffer, pH 7.4, overnight. Cell pellets were
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embedded in 1% low melting point agarose and post-fixed with 2% osmium tetroxide for 2 h,
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followed by staining with 2% uranyl acetate for 2 h, dehydration with a graded ethanol series
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(70%, 80%, 90%, 96%, 100%), progressive infiltration with epoxy resin and finally heat
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polymerization at 60 °C overnight. Thin sections (70 nm) were cut using an ultramicrotome
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Ultracut UCT (Leica Microsystems) and a diamond knife (Diatome) and then contrasted with
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0.2% lead citrate for 15 s. Samples were analyzed with JEM-1400 transmission electron
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microscope (JEOL). Images were recorded with TemCam-F216 camera and EM-MENU software
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(TVIPS).
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Nitric oxide and cytokine detection
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Cell culture medium were collected from WT or KO-BMDM cultures after the indicated
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treatments, centrifuged at 400 g for 10 min and the cell-free supernatants were used immediately
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(NO measurement) or frozen at −80 °C until use (cytokine detection). NO production in
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macrophage cultures was determined by measuring the accumulation of the stable NO metabolite
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nitrite in the supernatants using the colorimetric Griess assay (Thermo Fisher Scientific)
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according to the producer's protocol. Absorption at 570 nm was measured with a Wallac Victor2
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plate reader (Perkin Elmer) and the concentration of nitrite was calculated from a nitrite standard
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curve. For determination of cytokine production, cell culture supernatants were thawed and
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multiplex bead array assays were used to measure the levels of IL-1β, IL-6, IL-10, IL-
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12(p40/p70), GM-CSF, TNF-α (Thermo Fisher Scientific) and the type I IFNs, IFN-α and IFN-β
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(eBioscience) according to the producer's instructions. Sample were then were analyzed using a
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Luminex® xMAP platform (BD Biosciences).
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Statistics
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Multiple groups were compared by using one-way or two-way ANOVA where appropriate and
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differences between groups were analyzed post hoc by Tuckey's pairwise comparisons. Statistical
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tests were performed using Minitab 17 (Minitab).
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RESULTS
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Figure 1. Characterization of poly(I:C) encapsulating chitosan nanoparticles (pIC-NPs). (A)
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Schematic illustration of the preparation process of pIC-NPs. (B) Transmission electron
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microscopy (TEM) of pIC-NPs; scale bar 200 nm. (C) Nuclease protection assay with soluble
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poly(I:C) and pIC-NPs. Degradation of poly(I:C) was measured as increase in absorbance at 260
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nm (hyperchromatic effect). Data are presented as the mean ± SD of 3 independent experiments.
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Preparation and characterization of poly(I:C)-encapsulating chitosan nanoparticles (pIC-
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NPs)
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Poly(I:C)-encapsulating
nanoparticles
(pIC-NPs)
were
prepared
by
polyelectrolyte
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complexation, exploiting the ability of the anionic poly(I:C) to physically cross-link the cationic
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polyelectrolyte chitosan via electrostatic interactions (Figure 1A). Nanoparticles (NPs) formed
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spontaneously after mixing the polyelectrolytes in aqueous solution and were positively charged
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(+37.7 ± 1.8 mV) with a hydrodynamic size of 384 ± 46 nm and a polydispersity index (PDI) of
285
0.145 ± 0.06. Poly(I:C) was efficiently incorporated into the pIC-NPs with an encapsulation
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efficacy of 88.7 ± 2.6 %. By measuring the amount of both the unbound poly(I:C) and chitosan
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remaining in the supernatant after collection of pIC-NPs, the loading percentage of poly(I:C) in
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pIC-NPs was determined. Poly(I:C) loading percentage was 58.7 ± 3.1 % based on weight. These
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results are in accordance with other studies showing very similar characteristics for poly(I:C)-
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encapsulating chitosan nanoparticles.28 Transmission electron microscopy analysis revealed that
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pIC-NPs consist of a rather loose irregularly shaped core and fiber-like polymer-structures
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protruding from the core (Figure 1B). A well-known, major disadvantage of poly(I:C) is its
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vulnerability to rapid degeneration by ribonucleases, which are ubiquitously present in vivo.29 To
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test if pIC-NP are able to protect poly(I:C) against ribonuclease degradation, both soluble
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poly(I:C) and pIC-NPs were exposed to recombinant ribonuclease. Degradation of poly(I:C) was
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analyzed by measuring the increase in UV-light absorption at 260 nm (hyperchromic effect).30
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Whereas soluble poly(I:C) showed a rapid increase in absorbance within 2-3 min, indicating
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degradation, absorbance of pIC-NPs did not increase but remained stable over the course of 60
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min, indicating full protection of poly(I:C) against nuclease degradation (Figure 1C).
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Figure 2. Uptake of soluble poly(I:C) or pIC-NPs and intracellular stability of pIC-NPs in
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BMDM. (A) Flow cytometry analysis of the uptake of red-fluorescent pIC-NPs in comparison to
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soluble red-fluorescent poly(I:C) (both 2 µg/ml) by BMDM at different time points. Uptake was
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measured as percentage of cells positive for red fluorescence. Data are presented as the mean ±
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SD of 3 independent experiments; *** p < 0.001. (B) Confocal microscopy images of the uptake
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of red-fluorescent pIC-NPs compared to soluble red-fluorescent poly(I:C) by BMDM after
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different time periods; scale bars: 20 µm. (C) Confocal microscopy images of dual-labeled pIC-
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NPs with red-fluorescent poly(I:C) and IRDye680™-conjugated chitosan. (D) Co-localization of
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poly(I:C) and chitosan directly or 24 h after intracellular uptake by BMDM; scale bars: 5 µm.
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Uptake of poly(I:C) by mouse BMDM is enhanced by pIC-NPs
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NPs are in general preferentially and effectively taken up by APCs usually via the phagocytic
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pathway or receptor-mediated endocytosis rather than by passive uptake via the fluid phase.31 To
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investigate if pIC-NPs enhance uptake of poly(I:C) in primary macrophages, we followed the
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uptake of fluorescently labelled poly(I:C) in soluble form or in pIC-NPs over time by flow
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cytometry and confocal microscopy. Incorporation of poly(I:C) into NPs greatly increased uptake
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of poly(I:C) by macrophages compared to soluble poly(I:C) and resulted in different intracellular
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distribution patterns (Figure 2A and B). After a 12 h and 24 h incubation, pIC-NPs were
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distinctly concentrated in numerous bright fluorescent foci, whereas foci representing
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accumulated soluble poly(I:C) were considerably fainter. To study the stability of the poly(I:C)-
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chitosan interaction in the pIC-NPs within macrophages, we combined rhodamine-labelled
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poly(I:C) and chitosan conjugated with the near-infrared fluorochrome IRDye® to create dual-
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labelled pIC-NPs. Using laser confocal microscopy, dual-labelled pIC-NPs revealed essentially
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complete co-localization of poly(I:C) and chitosan in the NPs, which persisted for at least 24 h
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after uptake by macrophages (Figure 2C and D).
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Figure 3. Intracellular location of pIC-NPs and BCG in BMDM subcellular compartments. (A)
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Confocal images of BMDM infected with green-fluorescent BCG (BCG-GFP) and treated with
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dual-labeled pIC-NPs for 3 h and cultured for additional 24 h. The outline of the cell nucleus is
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indicated with a stipulated line in the images; scale bar: 5 µm. (B) Schematic depiction of the
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preparation of 15 nm gold particle encapsulating pIC-NPs (Au-pIC-NPs). (C- E) Transmission
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electron micrographs of BMDM at different time points after treatment with BCG and Au-pIC-
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NPs for 3 h. (C) Uptake of Au-pIC-NPs by BMDM; 15 nm gold particles are indicated by
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arrowheads. Inlet in (C): example of an Au-pIC-NP; arrowheads indicate 15 nm gold particles
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within the nanoparticles matrix or closely associated with pIC-NPs; scale bar 200 nm.
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Intracellular location of BCG and Au-pIC-NPs (labeled in the figure as NP, arrowheads indicate
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15 nm gold) in BMDM 3 h (D) and 24 h (E) after treatment with Au-pIC-NPs. Areas indicated
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with boxes in (D) are shown at high magnification (Di and Dii). Thick arrows indicate electron-
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dense polyphosphate granules in the cytoplasm of BCG. Lysosomal compartments were labeled
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by pre-loading the cells with 5 nm gold particles (thin arrows) before treatment; scale bars: 1 µm
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(C, D); 500nm (E).
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Intracellular BCG-bacteria and pIC-NPs are located to different compartments of the
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endocytic pathway in BMDM
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Aiming to study the interaction of BCG and pIC-NPs on the immunological response, we first
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investigated if and how pIC-NPs might interact with BCG bacteria intracellularly with respect to
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subsequent intracellular trafficking. By using dual-labelled pIC-NPs (red and magenta) and
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fluorescent BCG-bacteria (green) we were able to visualize their intracellular distribution by
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confocal fluorescence microscopy; which revealed that both were located within the same cells,
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but without spatial overlap (Figure 3A). However, confocal fluorescence microscopy is limited in
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terms of magnification and resolution, which makes it challenging to analyze subcellular
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localization precisely. To circumvent these limitations, we exploited the electrostatic interaction
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between cationic chitosan and negatively charged, 15 nm-sized gold particles to develop pIC-NPs
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encapsulating gold particles (Au-pIC-NPs), which could be reliably identified within
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macrophages by transmission electron microscopy (TEM) (Figure 3B and 3C, inlet). In addition,
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to identify the endosomal compartments, in which Au-pIC-NPs and BCG accumulate, BMDM
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were pulse-chased with 5 nm sized gold-particles to label late endosomal and lysosomal
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compartments before they were incubated with Au-pIC-NPs and BCG. Shortly after their uptake,
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Au-pIC-NPs were predominantly located in 5 nm gold-labelled late endosomes or lysosomes,
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whereas 24 h after uptake, they were found to be exclusively in these compartments (Figure 3D,
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Di and Dii). In contrast, 24 h after their uptake the majority of BCG-bacteria were neither found
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in 5 nm gold containing compartments nor together with Au-pIC-NPs in the same compartment,
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suggesting that they resided in phagosomes that have failed to fuse with lysosomes (Figure 3E).
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Figure 4. Production of nitric oxide (NO) and cytokines in BMDM co-stimulated with BCG and
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pIC-NPs or soluble poly(I:C). (A) NO production by BMDM infected with BCG and
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subsequently incubated with different concentrations of poly(I:C) in soluble form or in pIC-NPs
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for 24 h. As controls, BMDM were left untreated, infected with BCG only or treated with 20
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µg/ml poly(I:C) in soluble form or pIC-NPs for 24 h. Production of NO was measured by
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determining the nitrite levels in the culture supernatants using the Griess assay (B) NO
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production in BMDM infected with BCG and subsequently incubated for 24 h with 2 µg/ml pIC-
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NPs or Chi/TPP-NPs, which were produced by replacing poly(I:C) with sodium triphosphate
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(TPP). The concentration of Chi/TPP-NPs was matched to the chitosan content of the pIC-NPs
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used. (C) Production of pro- and anti-inflammatory cytokines by BMDM infected with BCG and
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subsequently incubated with 20 µg/ml poly(I:C) in soluble form or in pIC-NPs for 24 h.
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Untreated cells, BMDM treated with 20 µg/ml soluble poly(I:C) or pIC-NPs alone, or cells only
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infected with BCG were used as controls. Data are presented as means ± SD of three (B, C) or
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four (A) independent experiments; nd: not detected; ns: not significant; * p < 0.05; ** p < 0.01;
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*** p < 0.001.
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BCG and pIC-NPs interact synergistically to stimulate pro-inflammatory activation of
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BMDM
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To test if pIC-NPs, or soluble poly(I:C) were able to enhance BCG-induced pro-inflammatory
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activation of primary mouse macrophages, we measured the accumulation of nitrite, a stable
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metabolite of nitric oxide (NO), in BMDM culture supernatants. Production of NO is a strong and
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reliable marker for macrophage pro-inflammatory activation and usually correlates with
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production of pro-inflammatory cytokines.32 BMDM were first infected with BCG and
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immediately after treated with pIC-NP or soluble poly(I:C) for 24 h, when culture supernatants
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were harvested and analyzed. There was a striking synergistic interaction between BCG and pIC-
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NPs in inducing NO production (Figure 4A). But also simultaneous co-stimulation of BMDM
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with pIC-NPs and BCG for only a short period (3 h), followed by wash-out and 24 h culture in
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the absence of pIC-NPs resulted in synergistic induction of significant NO levels (Supplementary
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Figure S1A). Neither BCG nor pIC-NPs alone, even at the highest concentration, were able to
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trigger significant NO production. Soluble poly(I:C) used at the same concentrations as pIC-NPs
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likewise failed to induce any significant NO production in BCG-infected macrophages. Chitosan
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itself had no effect on NO production, as neither chitosan alone nor in combination with BCG
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induced NO production (data not shown). In agreement with this, poly(I:C) proved to be the
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essential component of pIC-NPs as chitosan nanoparticles produced using the crosslinker sodium
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triphosphate (TPP) instead of poly(I:C) were unable to induce NO production in WT
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macrophages in conjunction with BCG (Figure 4B).
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In a similar manner, pIC-NPs synergistically enhanced the production of the pro-inflammatory
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cytokines TNF-α, IL-12(p40/p70), IL-6, IL-1β and GM-CSF in BCG-infected macrophages
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(Figure 4C). Although soluble poly(I:C) also was able to increase the levels of some cytokines
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(IL-6 and GM-CSF), it did so to a much lesser degree than pIC-NPs. The only exception was IL-
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12(p40/p70), whose levels in BCG-infected macrophages were elevated by both soluble poly(I:C)
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and pIC-NPs to a similar extent. Neither soluble poly(I:C) nor pIC-NPs alone induced any
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significant cytokine production. The production of the anti-inflammatory cytokine IL-10 was
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likewise enhanced by pIC-NPs in combination with BCG, although at a low level. Like the
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induction of NO, already a short period of co-stimulation with pIC-NPs was sufficient to
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significantly enhance cytokine production in BCG-infected macrophages (Supplementary Figure
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S1B). Compared with short co-treatment for 3 h, stimulation with pIC-NPs for 24 h after BCG
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infection further increased levels of TNF-α, IL-6 and IL-1β substantially, whereas the levels of
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IL-12(p40/p70), GM-CSF and IL-10 remained unchanged (Supplementary Figure S1C). In
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conclusion, the synergistic interaction between BCG and pIC-NPs, seen for NO production, was
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also reflected in the enhanced production of pro-inflammatory cytokines.
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Figure 5. Role of TLR- and non-TLR signaling pathways in synergistic induction of NO
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production and enhancement of cytokine secretion in BMDM by pIC-NPs and BCG. (A) NO
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production in BCG-infected WT and the indicated KO-BMDM macrophages in response to
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stimulation with 2 µg/ml pIC-NPs for 24 h. (B) Secretion of pro-inflammatory cytokines in BCG-
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infected WT- and the KO-BMDM stimulated with 2 µg/ml pIC-NPs for 24 h. Different letters
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indicate statistically significant differences between groups (p ≤ 0.05). (C) NO production in WT-
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BMDM after co-stimulation with 2 µg/ml pIC-NPs and different concentrations of lipomannan
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for 24 h compared to co-stimulation with 2 µg/ml soluble poly(I:C) or to lipomannan treatment
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alone. Data represent means ± SD of three (C) or four (A, B) independent experiments; ** p