Poly(I:C)-Encapsulating Nanoparticles Enhance Innate Immune

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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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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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ACS Paragon Plus Environment


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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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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

256

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

260

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

274

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)

280

Poly(I:C)-encapsulating

nanoparticles

(pIC-NPs)

were

prepared

by

polyelectrolyte

281

complexation, exploiting the ability of the anionic poly(I:C) to physically cross-link the cationic

282

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

284

(+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

286

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

288

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)-

290

encapsulating chitosan nanoparticles.28 Transmission electron microscopy analysis revealed that

291

pIC-NPs consist of a rather loose irregularly shaped core and fiber-like polymer-structures

292

protruding from the core (Figure 1B). A well-known, major disadvantage of poly(I:C) is its

293

vulnerability to rapid degeneration by ribonucleases, which are ubiquitously present in vivo.29 To

294

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

296

analyzed by measuring the increase in UV-light absorption at 260 nm (hyperchromic effect).30

297

Whereas soluble poly(I:C) showed a rapid increase in absorbance within 2-3 min, indicating

298

degradation, absorbance of pIC-NPs did not increase but remained stable over the course of 60

299

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

303

soluble red-fluorescent poly(I:C) (both 2 µg/ml) by BMDM at different time points. Uptake was

304

measured as percentage of cells positive for red fluorescence. Data are presented as the mean ±

305

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

307

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.

310 311 312

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

314

pathway or receptor-mediated endocytosis rather than by passive uptake via the fluid phase.31 To

315

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

317

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)-

322

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).

327



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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

332

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

340

with boxes in (D) are shown at high magnification (Di and Dii). Thick arrows indicate electron-

341

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

343

(C, D); 500nm (E).

344 345 346

Intracellular BCG-bacteria and pIC-NPs are located to different compartments of the

347

endocytic pathway in BMDM

348

Aiming to study the interaction of BCG and pIC-NPs on the immunological response, we first

349

investigated if and how pIC-NPs might interact with BCG bacteria intracellularly with respect to

350

subsequent intracellular trafficking. By using dual-labelled pIC-NPs (red and magenta) and

351

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

354

terms of magnification and resolution, which makes it challenging to analyze subcellular

355

localization precisely. To circumvent these limitations, we exploited the electrostatic interaction

356

between cationic chitosan and negatively charged, 15 nm-sized gold particles to develop pIC-NPs

357

encapsulating gold particles (Au-pIC-NPs), which could be reliably identified within

358

macrophages by transmission electron microscopy (TEM) (Figure 3B and 3C, inlet). In addition,



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359

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,

362

Au-pIC-NPs were predominantly located in 5 nm gold-labelled late endosomes or lysosomes,

363

whereas 24 h after uptake, they were found to be exclusively in these compartments (Figure 3D,

364

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

371

for 24 h. As controls, BMDM were left untreated, infected with BCG only or treated with 20

372

µg/ml poly(I:C) in soluble form or pIC-NPs for 24 h. Production of NO was measured by

373

determining the nitrite levels in the culture supernatants using the Griess assay (B) NO

374

production in BMDM infected with BCG and subsequently incubated for 24 h with 2 µg/ml pIC-

375

NPs or Chi/TPP-NPs, which were produced by replacing poly(I:C) with sodium triphosphate

376

(TPP). The concentration of Chi/TPP-NPs was matched to the chitosan content of the pIC-NPs

377

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.

379

Untreated cells, BMDM treated with 20 µg/ml soluble poly(I:C) or pIC-NPs alone, or cells only

380

infected with BCG were used as controls. Data are presented as means ± SD of three (B, C) or

381

four (A) independent experiments; nd: not detected; ns: not significant; * p < 0.05; ** p < 0.01;

382

*** p < 0.001.

383 384 385

BCG and pIC-NPs interact synergistically to stimulate pro-inflammatory activation of

386

BMDM

387

To test if pIC-NPs, or soluble poly(I:C) were able to enhance BCG-induced pro-inflammatory

388

activation of primary mouse macrophages, we measured the accumulation of nitrite, a stable

389

metabolite of nitric oxide (NO), in BMDM culture supernatants. Production of NO is a strong and

390

reliable marker for macrophage pro-inflammatory activation and usually correlates with

391

production of pro-inflammatory cytokines.32 BMDM were first infected with BCG and

392

immediately after treated with pIC-NP or soluble poly(I:C) for 24 h, when culture supernatants

393

were harvested and analyzed. There was a striking synergistic interaction between BCG and pIC-

394

NPs in inducing NO production (Figure 4A). But also simultaneous co-stimulation of BMDM



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Page 22 of 46

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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

397

Figure S1A). Neither BCG nor pIC-NPs alone, even at the highest concentration, were able to

398

trigger significant NO production. Soluble poly(I:C) used at the same concentrations as pIC-NPs

399

likewise failed to induce any significant NO production in BCG-infected macrophages. Chitosan

400

itself had no effect on NO production, as neither chitosan alone nor in combination with BCG

401

induced NO production (data not shown). In agreement with this, poly(I:C) proved to be the

402

essential component of pIC-NPs as chitosan nanoparticles produced using the crosslinker sodium

403

triphosphate (TPP) instead of poly(I:C) were unable to induce NO production in WT

404

macrophages in conjunction with BCG (Figure 4B).

405

In a similar manner, pIC-NPs synergistically enhanced the production of the pro-inflammatory

406

cytokines TNF-α, IL-12(p40/p70), IL-6, IL-1β and GM-CSF in BCG-infected macrophages

407

(Figure 4C). Although soluble poly(I:C) also was able to increase the levels of some cytokines

408

(IL-6 and GM-CSF), it did so to a much lesser degree than pIC-NPs. The only exception was IL-

409

12(p40/p70), whose levels in BCG-infected macrophages were elevated by both soluble poly(I:C)

410

and pIC-NPs to a similar extent. Neither soluble poly(I:C) nor pIC-NPs alone induced any

411

significant cytokine production. The production of the anti-inflammatory cytokine IL-10 was

412

likewise enhanced by pIC-NPs in combination with BCG, although at a low level. Like the

413

induction of NO, already a short period of co-stimulation with pIC-NPs was sufficient to

414

significantly enhance cytokine production in BCG-infected macrophages (Supplementary Figure

415

S1B). Compared with short co-treatment for 3 h, stimulation with pIC-NPs for 24 h after BCG

416

infection further increased levels of TNF-α, IL-6 and IL-1β substantially, whereas the levels of

417

IL-12(p40/p70), GM-CSF and IL-10 remained unchanged (Supplementary Figure S1C). In


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418

conclusion, the synergistic interaction between BCG and pIC-NPs, seen for NO production, was

419

also reflected in the enhanced production of pro-inflammatory cytokines.

420

421 422

Figure 5. Role of TLR- and non-TLR signaling pathways in synergistic induction of NO

423

production and enhancement of cytokine secretion in BMDM by pIC-NPs and BCG. (A) NO

424

production in BCG-infected WT and the indicated KO-BMDM macrophages in response to

425

stimulation with 2 µg/ml pIC-NPs for 24 h. (B) Secretion of pro-inflammatory cytokines in BCG-

426

infected WT- and the KO-BMDM stimulated with 2 µg/ml pIC-NPs for 24 h. Different letters

427

indicate statistically significant differences between groups (p ≤ 0.05). (C) NO production in WT-

428

BMDM after co-stimulation with 2 µg/ml pIC-NPs and different concentrations of lipomannan

429

for 24 h compared to co-stimulation with 2 µg/ml soluble poly(I:C) or to lipomannan treatment

430

alone. Data represent means ± SD of three (C) or four (A, B) independent experiments; ** p

Poly(I:C)-Encapsulating Nanoparticles Enhance Innate Immune

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