The results of the present study indicate that airway exposure to engineered SNP during sensitization of mice to OVA enhances pathologic aspects of allergic airway disease upon secondary OVA challenge. This effect was more profound with increasing SNP doses and was characterized by enhancement of OVA-induced eosinophilic and lymphocytic inflammation, Th2 and Th17 cytokine expression, elevation of serum OVA-specific IgE and IgG1 levels, as well as an enhanced manifestation of mucous cell metaplasia in pulmonary airways. The fact that SNP exposure during allergen sensitization enhanced the pulmonary allergic response to the secondary OVA challenge indicates that SNP exhibits adjuvant-like characteristics in the development of allergic airway disease. Adjuvants are defined as compounds that are not immunogenic themselves, but increase and/or modulate the intrinsic immunogenicity of an antigen. Adjuvants are used in vaccines to induce potent and persistent immune responses, with the additional benefits that less antigen and/or fewer injections are needed . In this study, however, SNP promoted the immunologic response towards the allergen (OVA) and thereby potentiated the adverse allergic responses in the pulmonary airways.
Our results suggest that workplace exposures to engineered NP could have similar adverse health consequences as those reported for UFP in outdoor air pollution associated with high traffic roadways. UFP collected from the Los Angeles Air Basin have been shown to act as adjuvants to enhance the development and severity of allergic airway disease, using a similar OVA-induced murine model of asthma . In addition, epidemiologic studies have reported an increased incidence of asthma in children living in close proximity to highway traffic [26, 27], where ambient UFP concentrations are high . By comparison, relatively few studies have investigated the exacerbation of allergic airway disease by other types of NP exposures. Exacerbation in allergic airway disease, with increased Th2 responses, have been reported in OVA-sensitized and -challenged mice when co-exposed with 50 μg multi-walled carbon nanotubes (MWCNT)  or different sized carbon black NP . Similar findings were reported by Hussain et al. in mice sensitized with toluene diisocyanate (TDI) via skin and exposed to TiO2 NP and gold NP via instillation (0.8 mg/kg body weight) . Unlike our study design, mice in these studies were co-exposed to NPs during both the sensitization and challenge phases of the allergen administration. In our study we found that inhaled SNP during sensitization phase alone can act as adjuvants to markedly increase the magnitude of the host’s secondary immune response upon subsequent allergen challenge. A similar effect was also found previously for nano-sized crystalline silica particles . However, a recent study by Ban and colleagues  addressed the same question in a similar OVA mouse model with iron oxide NP, and found that iron NP exposure during sensitization with OVA results in attenuation of OVA mediated allergic airway disease. This suggests that the adjuvant effect of SNP in the development of allergic airway disease, we observed in our study, is likely to be particle specific. Further research, however, is needed to understand the immune modulatory effects of different NP and the impact of NP material, size and surface coating.
In our study, SNP exposure alone, without the antigen, caused a dose-dependent pro-inflammatory response in non-allergic animals as indicated by a modest increase in BALF neutrophils and elevations of neutrophil-related chemokines and innate immune response genes, namely Kc, Mip-1α, Mip-2, Itln1 and Irg1. These effects were evident at SNP exposure doses of 100 and 400 μg and demonstrate that these engineered SNP at high dose cause a minimal, yet sustained, innate immune responses up to 16 days post-instillation. By comparison, many rodent SNP toxicity studies describe airway neutrophilic inflammation that is accompanied by overt toxicity and tissue injury. For example, persistent pulmonary toxicity [13, 34], including neutrophilic inflammation, apoptosis, tissue injury [13, 35], the induction of pro-inflammatory BALF cytokines such as IL1β, IL6 and TNFα  as well as cardiovascular effects  have been reported. These studies used smaller sized SNP (14 nm) at similar or higher exposure doses (100 μg/mouse ; 3 mg/mouse ). Compared to these in vivo studies, we used larger SNP (90 nm), which appear to be less toxic than smaller SNP reported by others . The SNP used in ours study were further modified with a PEG shell which prevents them from agglomeration and gives them the ability to penetrate rapidly through airway mucus barriers . PEG-coatings have been reported to decrease systemic NP interactions and overall toxicity [38–40], which might explain the less severe acute inflammatory response we observed compared to those reported by others.
Adjuvant effects described in the current study had a mixed Th2/Th17 cytokine response, similar to findings by Li et al. who used ambient UFP in a similar OVA model . Mechanisms by which SNP may induce an adjuvant Th2/Th17 cytokine response can only be speculated, since this study was not designed to investigate the underlying mechanisms of adjuvancy in detail. SNP are known to induce oxidative stress , which plays an important role in the pathogenesis of asthma [7, 41]. This connection has been shown by Li et al. for ambient UFP where co-administration with the anti-oxidant N-acetylcysteine in OVA challenged mice diminished the adjuvant allergic airway response of UFP . In another study, oxidative stress, caused by diesel enriched PM, was also shown to skew the immune response from a Th1 to a Th2 cytokine profile . It has been suggested that oxidative stress-induced activation of transcription factor NF-E2-related factor 2 (NRF2), during the sensitization phase with an allergen, can down regulate the production of Th1 cytokines IL12 and IFNγ  and thereby leads to a Th1/Th2 imbalance. Furthermore results from in vitro studies on amorphous, colloidal SNP in a size range of 14 to 80 nm show a size- and dose-dependent cytotoxicity of SNP with induction of oxidative stress and/or glutathione (GSH) depletion [44, 45]. At the time point after challenge, however, we did not detect any signs of oxidative stress. Further investigations are therefore needed to assess oxidative stress and Th1/Th2 cytokine balance during the sensitization phase of our protocol in order to address these potential mechanisms of SNP-associated adjuvancy.
Recently IL17 has been associated with more severe forms of asthma, especially those cases complicated by persistent airway neutrophils . Th17 responses in allergic airway disease are promoted by IL6, tumor growth factor β (TGFβ), IL23, SAA3 as well as IL1β [47, 48]. In our study we detected significant increases in allergic SNP/OVA-mice of IL17A, IL6 and IL1β (cytokine and gene expression) as well as Saa3 (gene expression). SAA3 has been shown to activate the NLRP3 inflammasome and promote an allergic Th17 response in mice in combination with other mediators . The NLRP3 inflammasome is a protein complex required for splicing pro-IL1β into its active form , and was recently found to have an immuno-stimulatory function for aluminum adjuvants in vaccination . Though we did not measure inflammasome activation in the present study, SNP treatment caused a dose- dependent increase in BALF IL1β, and a recent study has shown that SNP can activate the inflammasome . It is therefore possible that the SNP-mediated adjuvant effects in allergic models are related to inflammasome activation and/or oxidative stress. However, further studies are needed to elucidate their roles in the adjuvant effects of SNP in more detail.
Besides Th2 and Th17 cytokine responses, we also detected an increase in TNFα and Th1 cytokine IFNγ in the SNP/OVA-mice which was not detectable in SNP- or OVA-mice. Therefore it may be possible that a Th1 component is present as well in the adjuvant response of SNP/OVA-mice. It has been shown previously, that Th1 cells do not attenuate Th2 cell–induced airway hyperreactivity in OVA-immunized BALB/c mice, but rather cause severe airway inflammation . We therefore suggest that the increase in BALF cytokines TNFα and IFNγ are augmenting rather than attenuating the allergic response in our model.
Besides stimulation of Th2 response, NP have also been shown to influence maturation, antigen presentation and co-stimulation of DC , and the analysis of TBLN cell populations in our study confirmed these findings. In response to DNA and RNA viruses, pDC secrete large amounts of IFN-α and IFN-β that play important roles in activating other cells in the immune system. For example, IFN-α and IFN-β produced by pDC have been shown to increase CD69 expression and IFN-γ production from CD4+ T cells [54, 55], and also activate CD8+ T cells upon influenza challenge . Up-regulation of CD69 on pDC in response to influenza infection has been found to cause down-regulation of sphingosine-1-phosphate (S1P) on pDC, resulting in elongated transit time of pDC and their accumulation in LN . An OVA-induced CD69 up-regulation on pDC might therefore lead to retention of pDC in TBLN where they could interact with lymphocytes and stimulate an immune response. The CD69 expression on pDC was further increased by co-exposure with SNP. Although AM have been suggested to prevent development of airway hyperresponsiveness upon OVA challenge , AM are also known to produce proinflammatory cytokines that enhance Th2 cytokine production by pulmonary CD4+ T lymphocytes . In our study, the activation of AM was further increased in the presence of SNP and an increase in BALF Th2 cytokines was measured. In addition, OVA-induced maturation of CD11c+ cells, including APC, such as DC and macrophages, was also exaggerated by SNP. Koike et al. further confirmed that effects on APC parallel those on allergic pathology in vivo in their overall trend . These mechanisms might partly explain the SNP-mediated immune enhancement; however, further research is still required to understand interaction of NPs with the immune system, which could include additional surface markers to further refine effects of NPs on various myeloid subpopulations.
In our treatment protocol, mice were IN instilled with a wide range of SNP doses (0, 10, 100 or 400 μg SNP per instillation). The applied doses of SNP used in our study are comparable to those used in similar studies performing intratracheal (IT) or IN administration [13, 29, 30, 32, 33]. IN has been shown to be an easily applicable and efficient method to administer particle suspensions in murine models . Nevertheless, particle inhalation represents a more realistic model for NP workplace exposure. Intrapulmonary NP delivery and distribution might differ between inhalation and instillation exposure which may result in different dose responses. The potential workplace exposure levels for SNP, however, are currently not known and it is therefore difficult to estimate a realistic dosing regimen. It has been reported that airborne, crystalline silica particles may reach as high as 0.28 mg/m3 in some workplace conditions such as cement mason/concrete finisher .
In summary, the results of our study indicate that engineered SNP can act as adjuvants to enhance the development of allergic airway disease in mice. This finding further suggests that individuals exposed to SNP might be more prone to develop allergic airway diseases, establishing a new aspect of NP toxicity that has particular relevance to occupational NP exposure. More research, however, is needed to clarify the potential risks of NP exposure in the development of allergic airway diseases in humans. Nevertheless, the murine allergic OVA model we used in our study, which involved IN instillation of allergen and SNPs during sensitization followed by IN challenge with allergen only, may be used to test the adjuvant potential of other NPs in allergic airway disease.