All animal experiments were conducted in accordance with the European guidelines for animal care. To facilitate mechanistic investigation of particle biodistribution, mice of the B57/B6 genetic background, that are used to generate genetically-manipulated models, were preferred to more toxic-sensitive mouse strains. Male eight- to ten-week-old C57BL/6, mdx (with leaky blood brain barrier (BBB)), CX3CR1
GFP/+ (with GFP reporter gene insertion allowing visualization of microglia), and CCL2
−/− mice were used (Jackson, West Grove, PA, USA). Mice were protected from Al-containing materials, fed with manufactured animal food and water ad libitum, and exposed to 12:12 light/dark cycles. Experiments using fluorescent particles were extremely labor intensive and expensive to perform. All of them were done in triplicate. Homogeneity of results made it unnecessary to use more than three mice per point.
The dose of alum-containing vaccine administered to mice was calibrated to mimic the mean number of doses received by MMF patients. One dose of commercially available anti-hepatitis B vaccine contains 0.5 mg Al according to the product data sheet. Based on an average of human body weight of 60 kg (most patients being women), the amount received for each immunization is 8.33 μg/kg. The allometric conversion from human to mouse (FDA Guidance 5541) gives a final amount of approximately 100 μg/kg. A dose of 36 μL vaccine, which corresponds to 18 μg Al, was injected to mimic the cumulative effect induced by 5.2 human doses to 35 g mice (the mean weight at the d180 midtime of brain analysis). This dose represents an equivalent 6.8 human doses in the youngest animal (27 g body weight, 11 weeks of age at sacrifice) and 4.3 in the oldest one (42 g at 62 weeks).
Furnace atomic absorption spectrometry
Al concentrations were determined in whole tibialis anterior (TA) muscles and brains dried at 37°C and digested with concentrated HNO3 (14 mol/L). Digests were allowed to cool before dilution to 10% HNO3 with ultra-pure water. The total aluminum in each digest was measured by transversely heated graphite atomizer graphite furnace atomic absorption spectrometry (TH GFAAS) and results were expressed as Al mg/g tissue dry weight.
As in normal conditions Al may be detected with marked interindividual variations in tissues, de novo incorporation of aluminum in too low doses does not cause easily detectable changes when global conventional approaches are used
. Here we used particle induced X-ray emission (PIXE), a procedure analyzing radiation emitted from the interaction of a proton beam with the matter
, to detect areas enclosing small Al spots. Sections (20 μm-thick) carefully protected from environmental Al were mounted on fresh formvar films, kept in the cryostat for 6 hours and stored under Al-free silica gel. Mineral and metal ions were detected using the nuclear microprobe of the Centre d’Etudes Nucléaires de Bordeaux-Gradignan. A 1 MeV proton beam focused down to a 2 μm spot was randomly scanned over multiple 500 × 500 μm fields of tissue sections. In the case of an Al signal, a re-test of 100 × 100 μm areas of interest was performed. PIXE and Rutherford backscattering spectrometry analyses were employed simultaneously and quantitative results were computed, as previously described
. Al spots were considered eligible on three criteria: a size of more than 3 pixels (that is, above the background noise), a depot not colocalized with Si, and a depot surrounded by a rounded halo of decreased intensity (both characteristics limiting confusion with contamination by external dust overcoming the protection procedures).
Synthesis of Al-Rho particles
Gadolinium oxide nanohybrids with Al(OH)3 coating were obtained in three steps: (i) gadolinium oxide nanoparticles were first synthesized; (ii) polysiloxane shell growth was then induced by hydrolysis-condensation of convenient silane precursors in the presence of the nanoparticles; and (iii) the nanohybrids were coated by the addition of aluminum nitrate and soda in stoichiometric conditions.
Gadolinium chloride hexahydrate ((GdCl3, 6H2O]) 99.99%), sodium hydroxide (NaOH, 99.99%), tetraethyl orthosilicate (Si(OC2H5)4, TEOS, 98%), (3-aminopropyl) triethoxysilane (H2N(CH2)3-Si(OC2H5)3, APTES, 99%), triethylamine (TEA, 99.5%), rhodamine B isothiocyanate (RBITC), aluminium nitrate nonahydrate (Al(NO3)3.9H2O, ACS reagent ≥ 98%) and dimethyl sulfoxide (DMSO, 99.5%) were purchased from Sigma-Aldrich (St Louis, MO, USA). Diethylene glycol (DEG, 99%) was purchased from SDS Carlo Erba, Val de Reuil (France).
Preparation of gadolinium oxide core
A first solution was prepared by dissolving GdCl3, 6H2O (0.56 g) in 50 mL DEG at room temperature. A second solution was prepared by adding a NaOH solution (0.49 mL, 10 M) in 50 mL DEG. The second solution was progressively added to the first one, at room temperature, for 15 hours. A transparent colloid of gadolinium oxide nanoparticles in DEG was obtained.
Encapsulation of Gd2O3 cores by polysiloxane shell
A total of 105 μL of APTES and 67 μL of TEOS was added to 100 mL of the gadolinium oxide nanoparticle solution under stirring at 40°C. A total of 5 μL of APTES was previously coupled to 1 mg RBITC in DMSO (1 mL) used as solvent and then added to the colloidal solution. After 1 hour, 1,913 μL of a DEG solution (0.1 M of TEA, 10 M of water) was added. The whole coating procedure was repeated three more times (with no more addition of RBITC), every 24 hours. The final mixture was stirred for 48 hours at 40°C. The obtained solution could be stored at room temperature for weeks without alteration.
Coating of fluorescent nanohybrids with a Al(OH)3 shell
A total of 2.5 mL of the colloidal solution was diluted by 2 to obtain a 5 mL solution in DEG. A total of 75 mg of aluminum nitrate nonahydrate was dissolved in 10 mL of water before addition to the colloidal solution. The resulting mixture was stirred for 5 minutes and 4 mL of a soda solution (0.2 M) was added before stirring for 1 hour.
Purification of Al-Rho was performed by tangential filtration through Vivaspin filtration membranes (MWCO = 10 kDa) purchased from Sartorius Stedim Biotech (Aubagne, France). The colloidal solution was introduced into 20 mL Vivaspin tubes and centrifuged at 4,100 rpm. This step was repeated several times, by filling the tubes with water and centrifuging again, until the desired purification rate was reached (≥100). The purified colloidal solution was freeze dried for storage in five pillboxes, using a Christ Alpha 1–2 lyophilisator. The compound contained 4 μg Al per μL of Al-Rho suspension. Control transmission electron microscopy showed non-fibrous particles about 10 nm in size, typical of aluminum hydroxyde (traditional precipated alum). Similarly to vaccine alum, they formed agglomerates of submicronic/micronic size. The immunological properties of such traditional alum-protein precipitates are quite similar to those of the reference adjuvant approved by the FDA (Al oxyhydroxyde: Alhydrogel®, Invivogen, Toulouse France) and differ from other formulations not licensed for human use (18).
Peripheral injections of fluorescent nanomaterials
Two types of fluorescent nanomaterials were used: exploratory polychromatic fluorescent latex beads (FLBs) (500 nm fluorospheres, Polysciences, Warrington, PA, USA) and confirmatory Al-Rho nanohybrids constructed with a rhodamine containing core and an Al(OH)3 shell. FLBs were used first because they offer several characteristics that facilitate their detection in tissues, including strong fluorescence, spheric appearance and homogeneous size. This allowed us to get a clear picture of what was happening in terms of biodistribution of these avidly phagocytosed particles. Al Rho particles were less fluorescent and more heterogeneous in shape and size than FLBs but better represented alum adjuvant surrogates. Almost all biodistribution experiments performed with FLBs in wild type mice were also done with Al-Rho. In contrast, FLBs and Al Rho were differentially used in mutated/genetically-modified mice: FLBs were preferred to study particle biodistribution in mdx mice with BBB alterations and when the GFP marker was used (that is, CX3CR1
GFP/+ mice with fluorescent microglia, GFP + BMT studies); Al-Rho particles were preferred in gain/loss of CCL2/MCP-1 function studies designed on the basis of preliminary results on the CCL2 status of alum-intolerant humans.
FLB suspension diluted at 1:1 in PBS contained 1.8 × 1011 particles per mL. A total volume of 40 μL (20 μL in each TA muscle) was injected, corresponding to a total amount of 7.2 × 109 particles. The same volume of Al-Rho suspension was injected in TA muscles. PBS-injected mice were used as controls. Tissues, including popliteal and inguinal DLNs, spleen, brain and blood, were collected at various time points post injection. Three mice were included per group at each time point for both injected materials and their controls. Other administration routes were compared to the standard i.m. injection, including s.c. injection of 20 μL FLBs in each hindlimb, and i.v. injection of 40 μL FLBs in the tail vein.
Stereotactic cerebral injections
Mice were anaesthetized with ketamine and xylazine. Al-Rho suspension (0.5 μL) was stereotactically injected in the striatum using a 1 μL Hamilton syringe. Biodistribution of i.c. injected Al-Rho to cervical DLNs, assessed by serial sectioning of the whole cervical region, and spleen, was compared to biodistribution to the popliteal DLN and spleen of the same amount of Al-Rho injected in the TA muscle.
Pharmacological and physical migration blockade
The prostaglandin analog BW245C, an agonist of the PGD2 receptor, was used to inhibit APC migration as previously reported
. Since BW245C is active for two days after injection, BW245C (100 nM, Cat.no.12050, Cayman Chemical, Ann Arbor, MI, USA) was injected twice in the TA muscle: it was first co-injected with FLBs at d0 and a second time alone at d2, and DLNs were removed for examination at d4. Untreated FLB-injected mice were used as controls. In another set of experiments DLNs were surgically ablated and mice immediately injected with FLBs in the TA muscle.
Loss and gain of CCL2 function experiments
Exploratory analyses performed in MMF patients with ASIA [see Additional file
1: supplementary information section] yielded a CCL2 signal in the form of: (1) a selective increase of CCL2 in the serum of MMF patients compared to healthy controls; and (2) a given haplotype in the CCL2 gene tending to be more frequent in MMF patients than in the general population. These results led us to use mouse models to explore the role of CCL2 in the biodisposition of particulate materials. Loss of CCL2 function studies were done using CCL2
−/− mice injected i.m. with 40 μL Al-Rho. Gain of CCL2 function experiments consisted first of i.m. co-injection of 10 μL murine rCCL2 (100 μg/ml; R&D, Minneapolis, MN, USA) with 40 μL Al-Rho. DLNs were removed at d4, spleen, brain and blood at d21. In other experiments murine rCCL2 was infused into the brain through a catheter stereotactically inserted into the striatum at d7 post-Al-Rho, fed by a subcutaneously implanted osmotic micropump fixed into the neck (0.25 μL/hour Alzet brain infusion kit, Charles River, L’Arbresle, France). rCCL2 was infused for 14 days (diffusion rate 180 pg/day), with or without rCCL2 i.m. injection concurrent with Al-Rho injection. At d21 post Al-Rho injection, animals were sacrificed, and blood and tissues were collected. For controls, osmotic pumps filled with PBS were used.
Tissue preparation and particle counting
Mice under terminal anesthesia were transcardially perfused with PBS followed by ice-cold 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer. Tissues and organs were removed, post-fixed in PFA for 4 hours at 4°C, immersed overnight at 4°C in a 30% sucrose solution, and quickly frozen. Whole brains were serially cut into coronal cryosections of 40 μm, spleen and muscle into 20 μm, and DLNs into 10 μm, and stored at −20°C until particle counting or treatment. Brain sections were successively deposited on 10 different Superfrost® slides in order to obtain 10 identical series, thus allowing determination of total particle content by multiplying by 10 the number of particles found in one series. A similar approach was used for DLNs and spleen. Blood was collected by heart puncture and 100 μL were smeared for particle counting.
Immunohistochemistry and Morin staining
Immunostaining was done using commercial primary antibodies routinely used in the lab, raised against CD11b (1/200, AbD Serotec, Oxford, UK), F4/80 (1/50, AbCam, Cambridge, UK), GFAP (1/200, DakoCytomation, Trappes, France), vimentin (1/500 DakoCytomation), collagen IV (1/100 Millipore, Temecula, CA, USA), NG2 (1/200, Millipore, Molsheim, France), MAP2 (1/100, Sigma-Aldrich, Lyon, France), and IL1β (1/100, AbCam, Paris, France) or nonspecific mouse IgG (Jackson ImmunoResearch, Suffolk, UK). Then, biotinylated anti-rat and anti-rabbit antibodies (1/200, Vector Laboratories, Paris, France) were used accordingly and were revealed using Alexa fluor 488-conjugated streptavidin (1/200 Invitrogen, Cergy-Pontoise, France). Neuron labeling was done using NeuroTrace® blue fluorescent Nissl Stain according to the manufacturer” instructions (Invitrogen). Al was stained with Morin (M4008-2 G, Sigma-Aldrich) used as 0.2 g dissolved in a solution consisting of 0.5% acetic acid in 85% ethanol
. Formation of a fluorescent complex with Al was detected under a 420 nm excitation wavelength as an intense green fluorescence with a characteristic 520 nm emission. Notably, nanohybrids (Gd2O3) core encapsulated by polysiloxane shell were not positively stained by Morin. In contrast, when coated with Al(OH)3, these particles were strongly positive for Morin. Fluorescence microscopy and spectral analyses were done using Carl Zeiss light and confocal microscopes.
Cell isolation from blood and tissues and flow cytometry
For blood cell immunophenotyping, 100 μL blood was treated with ethylenediaminetetraacetic acid (EDTA) and stained with fluorescein isothiocyanate (FITC)-conjugated antibodies. Erythrocytes were lysed using hypotonic lysis solution, and then cells were washed with (D)MEM and sorted using a MoFlo cell sorter (Beckman Coulter, Villepinte, France). Cells were extracted from tissues of exsanguinated mice perfused with PBS. Tissues were removed and freshly dissociated in (D)MEM. DLNs and spleen were dissociated in (D)MEM containing 0.2% collagenase-B (Roche Diagnostics, Meylan, France) and 0.2% trypsine-EDTA at 37°C for 45 minutes twice. Brain tissue was dissociated in 1% Trypsin-HBSS (Thermo Scientific HyClone, South Logan, UK) containing 100 U/mL DNase (Roche Diagnostics). Cell suspensions were filtered and counted. CD45+ or CD11b+ cells were isolated using magnetic cell sorting (MACS, Miltenyi Biotec, Paris, France) and stained with one of the following antibodies and their isotypes: FITC-conjugated anti-CD11b, FITC–conjugated anti–Ly-6C (GR1), FITC–conjugated anti-CD11c (BD-Pharmingen Bioscience, San Diego, CA, USA). Cells were sorted using a cell sorter. Populations presenting >90% purity were used. Sorted cells were cytospinned and stained with Hoechst-33342 for nucleus. Particle loaded cells were counted under a fluorescence microscope.
Bone marrow transplantation experiments
GFP+ bone marrow (BM) cells were obtained by flushing the femurs of adult CAG-GFP mice and were injected retroorbitally (1 × 107 cells per mouse) to four-week-old C57BL/6 mice, as previously described
. Recipient mice were irradiated at 9.0 Gy on d1 before transplantation, and were treated with 10 mg/kg/day ciprofloxacin for 10 days. Blood chimerism of >90% was controlled at three to four weeks post-transplantation.
All experimental values are presented as means and standard deviation except when indicated. Statistical analyses used unpaired Student’s t-test (genotypes); P <0.05 was considered significant.