Aluminum hydroxide

Mice were randomly divided into eight groups of 6 animals in each group (Table 1). In the first group of mice, allergic airway inflammation (Asthma) caused by OVA was induced. For this, mice were sensitized on day 0 with an intraperitoneal injection (ip) of 20 μg OVA (Merck, Germany) and 1 mg aluminum hydroxide (Merck, Germany) in 0.2 mL of sterile saline (PBS) on days 0, 14, and 21, and then, 80 μg of OVA in 50 μL of sterile PBS was injected intranasal on days 27, 28, and 35. On day 37, bronchoalveolar lavage, blood, and lungs were collected.
In groups 2, 3, 6, and 7, animals were injected intraperitoneally 5 days before sensitization with 5 μg/animal of GMDP (JSC Peptek, Russia) in PBS or 1 μg/animal of LPS (Ultra-pure, Invivogen) in PBS. In groups 2 and 3, OVAlbumin was then administered similarly to group 1 (Asthma). In groups 4 and 5, GMDP and LPS, during sensitization animals were intraperitoneally injected at 5 μg/animal GMDP in PBS or 1 μg/animal LPS in PBS together with 20 μg OVA and 1 mg aluminum hydroxide in 0.2 mL of sterile saline solution. The control group of mice was injected intraperitoneally and intranasal with sterile PBS according to the protocol.

Outer membrane vesicles were provided by Janssen Vaccines and Prevention B.V. Briefly, OMVs were produced from N. meningitidis H44/76 (B:P1.7,16;F3-3) WT or derivatives. nOMVs were produced from a H44/76 ΔRΔL (15 (link), 16 (link), 27 (link)), resulting in a pentaacylated meningococcal LPS from the lpxL1 mutant N. meningitidis strain, lacking the secondary C12:0 acyl chain at the 2′-position of the lipid A. Detergent-extracted OMVs were produced from the WT strain or the ΔRΔL mutant (28 ). Adsorption to aluminum hydroxide (Sigma-Aldrich) was performed, as described elsewhere (29 (link)). Before use, OMVs were diluted in 10 mM Tris pH 7.4/3% (w/v) sucrose to 50 μg total protein/ml. OMV size was determined by dynamic light scattering (DLS) using the Zetasizer nanoseries (Malvern Nano-ZS, l 1/4 532 nm, Westborough, MA, USA).

Tetraethyl orthosilicate (TEOS) was purchased from Merck (Darmstadt, Germany). Ethanol with a purity of 99% was purchased from Chemsolute (Th. Geyer, Lohmar, Germany) and used without further purification. Hydrochloride acid (2 M) was purchased from Bernd Kraft (Duisburg, Germany) and ammonium hydroxide solution (1 M) was purchased from Alfa Aesar (Thermo Fisher Schientific, Kandel, Germany). The different ceramic opacifiers used were: aluminum oxide (Al₂O₃) from Alfa Aesar (Thermo Fisher Schientific, Kandel, Germany); aluminum hydroxide from Sigma–Aldrich (Steinheim, Germany); boehmite B30, 200 SM, and ASPEXT all from Nabaltec (Schwandorf, Germany). The glass fiber mat “Insulfrax S Matte” was purchased from Insulcon GmbH (Neuss, Germany), and the Insulfrax S (Insulcon GmbH, Neuss, Germany) is a binderless needled blanket produced by mechanical needling of spun fibers.
The differences among the boehmite particles were mainly based on the particle sizes and the specific surface areas (see Table 1). Thus, the influence of these boehmite particles was examined more closely.

Mice in the three diet groups were randomly divided into three additional groups (n=8 per group): controls, OVA and OVA + ozone. The mice in the OVA and OVA + ozone groups were sensitized intraperitoneally (IP) with 20 µg OVA (Grade V, Sigma-Aldrich) diluted in 0.2 mL of Dulbecco’s phosphate buffered saline (PBS) and 2 mg aluminum hydroxide (Sigma-Aldrich) in a total volume of 20 mL on days 0, 7, and 14, and challenged via aerosol nebulization with 1% OVA for 30 min each day from day 21 to day 25. Mice in the control group were treated with PBS at both timepoints in the same manner as previously described (33 (link)). After the OVA challenge, the same number of mice in the OVA + ozone group were exposed to 2.5 ppm ozone for 2 h daily from day 21 to day 25 as previously described (34 (link), 35 (link)). The control mice were exposed to room air during this period.

The animal study protocol (VD2153.2) was approved by the Service Vétérinaire du Canton de Vaud, Switzerland (Figure 1A). The C57BL/6J CRL mice strain was chosen because it has been successfully used in OVA-induced food allergy and oral tolerance models [32 (link),33 (link),34 (link),35 (link),36 (link)]. Briefly, 5-week-old C57BL/6J CRL female mice (Charles River Laboratories, Lyon, France) were sensitized using two subcutaneous injections of 100 µg (Sigma) and 1 mg aluminum hydroxide (Sigma) in 50 µL of phosphate buffer saline (PBS; Sigma) on two separate sites (neck and back) at days 13 and 26. Oral challenge was performed using a gavage with 50 mg OVA in 250 µL of PBS at day 33. At day 35, mice were anaesthetized using isoflurane and euthanized after collecting blood from the abdominal aorta. Axillary/brachial lymph nodes were harvested for further analyses. Mice had free access to food pellets (Kliba 3437, Kliba Nafag, Kaiseraugst, Switzerland) containing wheat, barley, soy, corn, and amino acids as a source of protein. No egg protein was present in the diet.