Our objective was to evaluate the effects of non-biodegradable/biopersistant solid TiO2 NPs on the classical hypersensitivity reaction to a well-known potent dermal sensitizer (DNCB). To the best of our knowledge, there is no information available on the effect of manufactured NPs on the dermal sensitization potential of chemicals, in general, and of DNCB in particular. Recently, sensitizing potentials of biodegradable particles (ethosomes) was described, but such information about environmentally relevant solid NPs is lacking [19–21]. Here we demonstrate that TiO2 NPs (0.04 mg/ml) act as an immune-stimulator on the dermal sensitization capacity of DNCB. The stimulation of the dermal sensitization was coupled with a significant change in cytokine release which corresponds with the induction of a Th2 response. The experiments confirmed our hypothesis that pre-treatment with TiO2 NPs modulate sensitization to DNCB.
There is conflicting evidence about the skin penetration of nanomaterials [22, 23]. However, it has been suggested that NPs could pass through the stratum corneum of the skin using intercellular channels or hair follicles and penetrate into deeper skin layers [24, 25]. A thorough search of the available literature indicates that stratum corneum is an effective barrier against the uptake of TiO2 NPs in healthy skin. However, various research publications anticipate the possibility of penetration of TiO2 NPs to the deeper layers of skin/viable skin in case of local damage (sun burns etc) or when lipid vesicles formulations are involved [19, 20, 26]. For example, a sub-erythemal dose ultraviolet radiation exposure (UVB 270 mJ/cm2) in mice has been shown to allow the penetration of 45 nm quantum dot NPs to deep dermis . In another study, it has been observed that 7 nm TiO2 NP exposure with UV irradiation can lead to increased skin barrier dysfunction and possible aggravation of contact dermatitis due to increased invasion of Staphylococcus aureus
. Interestingly, people tend to apply more sun screens containing NPs in case they have damaged skin (sunburns, burns) when the barrier function of skin is already impaired. It has also been reported that in a skin barrier dysfunction conditions (mite allergen exposure) intradermal exposure to rutile TiO2 NPs can lead to aggravation of the atopic skin lesions . Particles which penetrate normal or damaged skin are taken up by antigen presenting cells (APC), such as Langerhans cells or dendritic cells, and subsequently removed via the lymphatic system [9, 30]. In view of these considerations, along with the possibility of exposure in individuals with pathological lesions of the skin, subcutaneous route becomes a relevant route to study NP induced effects on dermal sensitization. TiO2 NP concentrations used in this study are based on literature reports by us and others describing these as non-cytotoxic concentrations in vitro and in vivo
[31, 32]. Moreover, we did not observe any type of local injury around the site of injection of NPs.
Our results indicate that TiO2 NPs do not show dermal sensitization potential after a single subcutaneous injection, which is in accordance with findings in literature . However, subcutaneous presence of TiO2 NPs significantly increases the skin sensitization potential of DNCB, which is not the case with subcutaneous presence of pigment TiO2 particles before dermal sensitization. It is possible that increased sensitization after NP exposures is due to binding of the antigen with the NPs, leading to formation of depot of antigen which is better recognized by skin APCs. It has been proposed that ultrafine TiO2 particles (below 30 nm) bind more ovalbumin per mass unit than fine particles and this binding may lead to a depot of antigen leading to increased antigenicity . Moreover, it is a well accepted fact that professional APCs are more readily stimulated by the particulate antigens, thus adsorption of antigen per se can increase the antigenicity . Other possible explanation might be physical interactions between the NPs and APCs. Impurities cannot be a confounding factor in our experiments as there were no detectable amounts present in TiO2 NPs utilized in this study.
The results of the present study demonstrate that pre-exposure to TiO2 NPs does not interfere with the immune system if followed by a sham dermal treatment (TiO2/Veh). However, when pre-exposure to TiO2 NPs is followed by DNCB sensitization, a Th2 favoured immune response in regional lymph node cells develops, with increased IL-4 and decreased IL-10 levels, while DNCB itself is a known potent Th1 responder . The apparent IFN-γ secretion confirms the DNCB-induced Th1 response, even with prior injection of TiO2 NPs. Nevertheless, TiO2 NPs injection followed by DNCB sensitization results in significantly increased levels of IL-4, demonstrating Th2 stimulation. In addition, we found a decrease in IL-10 secretion. IL-10 is a cytokine released by several cell types, such as monocytes, activated T cells, Th2 cells, mast cells and regulatory T cells. IL-10 is capable of inhibiting pro-inflammatory responses and is suggested to play a major role in maintenance of self-tolerance . We think that sub-cutaneous exposure to TiO2 in DNCB sensitized mice, decreased IL-10, and thereby allowing the development of a Th2 response, independent of the presence of the Th1 response (levels of IFN-γ are maintained).
Allergic sensitization reactions are the first step against the “foreign” materials, and are either Th1 or Th2 polarized. It has been shown that particles themselves can act as modulating agents in skewing the Th responses. Impact of particles on the skewing of Th response is largely dependent on the chemical nature and characteristics of the materials. Larsen et al, found that TiO2 NPs promote allergic sensitization to ovalbumin (IgE and IgG1 levels) and thus primes a Th2 dominated immune response . Diesel exhaust can promote both Th1 and Th2 responses [37, 38]. Carbon nanotubes either amplify Th1 (MWCNT) or Th2 (SWCNT) or both (MWCNT) responses [39, 40]. These studies are done in the models of respiratory allergy using ovalbumin as sensitizing agent. However, respiratory allergic responses have already been shown to be more prone to Th2 skewing while skin sensitization responses are mostly Th1 dependent [41, 42]. It is interesting to note that although we observe a skewing of immune response towards Th2 we still observe a shift in the potency of DNCB, with almost a 3-fold fold decrease in EC3.