The crystal structure of titanium dioxide nanoparticles influences immune activity in vitro and in vivo

Abstract

Background: The use of engineered nanoparticles (NP) is widespread and still increasing. There is a great need to assess their safety. Newly engineered NP enter the market in a large variety; therefore safety evaluation should preferably be in a high-throughput fashion. In vitro screening is suitable for this purpose. TiO ₂ NP exist in a large variety (crystal structure, coating and size), but information on their relative toxicities is scarce. TiO ₂ NP may be inhaled by workers in e.g. paint production and application. In mice, inhalation of TiO ₂ NP increases allergic reactions. Dendritic cells (DC) form an important part of the lung immune system, and are essential in adjuvant activity. The present study aimed to establish the effect of a variety of TiO ₂ NP on DC maturation in vitro. Two NP of different crystal structure but similar in size, uncoated and from the same supplier, were evaluated for their adjuvant activity in vivo.

Methods: Immature DC were differentiated in vitro from human peripheral blood monocytes. Exposure effects of a series of fourteen TiO ₂ NP on cell viability, CD83 and CD86 expression, and IL-12p40 and TNF-α production were measured. BALB/c mice were intranasally sensitized with ovalbumin (OVA) alone, OVA plus anatase TiO ₂ NP, OVA plus rutile TiO ₂ NP, and OVA plus Carbon Black (CB; positive control). The mice were intranasally challenged with OVA. OVA-specific IgE and IgG1 in serum, cellular inflammation in bronchoalveolar lavage fluid (BALF) and IL-4 and IL-5 production in draining bronchial lymph nodes were evaluated.

Results: All NP dispersions contained NP aggregates. The anatase NP and anatase/rutile mixture NP induced a higher CD83 and CD86 expression and a higher IL-12p40 production in vitro than the rutile NP (including coated rutile NP and a rutile NP of a 10-fold larger primary diameter). OVA-specific serum IgE and IgG1 were increased by anatase NP, rutile NP, and CB, in the order rutile<anatase<CB. The three particles similarly increased IL-4 and IL-5 production by bronchial LN cells and eosinophils and lymphocytes in the BALF. Neutrophils were induced by rutile NP and CB but not by anatase NP.

Conclusions: Our data show that measuring CD83 and CD86 expression and IL-12p40 and TNF-α production in DC in vitro may provide an efficient way to screen NP for potential adjuvant activity; future studies should establish whether this also holds for other NP. Based on antigen-specific IgE and IgG1, anatase NP have higher adjuvant activity than rutile NP, confirming our in vitro data. Other parameters of the allergic response showed a similar response for the two NP crystal structures. From the viewpoint of safe(r) by design products, rutile NP may be preferred over anatase NP, especially when inhalation exposure can be expected during production or application of the product.

Keywords: Adjuvant; Anatase; Dendritic cell; Inhalation; LgE; LgG1; Maturation; Ovalbumin; Rutile; Titanium dioxide.

Fig. 1

a. OVA-specific IgE in serum. b. OVA-specific IgG1 in serum. Mice were sensitized with OVA alone, OVA + rutile TiO₂ NP, OVA + anatase TiO₂ NP, or OVA + Carbon Black (CB), and challenged with OVA. N = 6, mean ± SEM is shown. (*), (**), and (***) P < 0.05, P < 0.01, and P < 0.001vs. OVA alone; (+), (++), and (+++) P < 0.05, P < 0.01, and P < 0.001 vs. OVA + rutile NP; (###) P < 0.001 vs. OVA + anatase NP

Fig. 2

a. IL-4 production by LN cells. b. IL-5 production by LN cells. Mice were sensitized with OVA alone, OVA + rutile NP, OVA + anatase NP, or OVA + CB, and challenged with OVA. LN cell preparations were made and incubated with Con A for 24 h. N = 6, mean ± SEM is shown. (*) P < 0.05 vs. OVA alone

Fig. 3

a. Percentage of eosinophils in the BALF after OVA challenge. b. Number of eosinophils in the BALF after OVA challenge. c. Percentage of eosinophils in the BALF after OVA sensitization and challenge. d. Number of eosinophils in the BALF after OVA sensitization and challenge. A, B. Mice were sensitized with PBS, rutile NP, anatase NP, or CB, and challenged with OVA. C, D. Mice were sensitized with OVA alone, OVA + rutile NP, OVA + anatase NP, or OVA + CB, and challenged with OVA. N = 6, mean ± SEM is shown. In A and B, (*) P < 0.05 vs. PBS alone; (+) P < 0.05 vs. rutile NP; (#) P < 0.05 vs. anatase NP. In C and D, (*) P < 0.05 vs. OVA alone

Fig. 4

a. Percentage of lymphocytes in the BALF after OVA challenge. b. Number of lymphocytes in the BALF after OVA challenge. c. Percentage of lymphocytes in the balf after ova sensitization and challenge. d. Number of lymphocytes in the BALF after OVA sensitization and challenge. See legend to Fig. 3. In A and B, (*) and (**) P < 0.05 and P < 0.01 vs. PBS alone; (+) P < 0.05 vs. rutile NP. In C and D, (*), (**) and (**) P < 0.05, P < 0.01 and P < 0.01 vs. OVA alone; (+) P < 0.05 vs. OVA + rutile NP; (##) P < 0.05 vs. OVA + anatase NP

Fig. 5

a. Percentage of neutrophils in the BALF after OVA challenge. b. Number of neutrophils in the BALF after OVA challenge. c. Percentage of neutrophils in the BALF after OVA sensitization and challenge. d. Number of neutrophils in the BALF after OVA sensitization and challenge. See legend to Fig. 3. In A and B, (*) and (***) P < 0.05 and P < 0.001 vs. PBS alone; (++) P < 0.01 vs. rutile NP; (#) and (###) P < 0.05 and P < 0.001 vs. anatase NP. In C and D, (**) P < 0.01 vs. OVA alone; (++) P < 0.01 vs. OVA + rutile NP; (#) P < 0.05 vs. OVA + anatase NP

Fig. 6

a. Percentage of macrophages in the BALF after OVA challenge. b. Number of macrophages in the BALF after OVA challenge. c. Percentage of macrophages in the BALF after OVA sensitization and challenge. d. Number of macrophages in the BALF after OVA sensitization and challenge. See legend to Fig. 3. In A and B, (*) P < 0.05 vs. PBS alone; (+) and (++) P < 0.05 and P < 0.01 vs. rutile NP. In C and D, (***) P < 0.001 vs. OVA alone; (+) P < 0.05 vs. OVA + rutile NP; (##) P < 0.01 vs. OVA + anatase NP