Amorphous nanosilica particles block induction of oral tolerance in mice

Abstract

The mucosal immune system is exposed to non-self antigens in food and the gut microbiota. Therefore, the recognition of orally ingested non-self antigens is suppressed in healthy individuals to avoid excessive immune responses in a process called “oral tolerance”. The breakdown of oral tolerance has been cited as a possible cause of food allergy, and amorphous silica nanoparticles (nSP) have been implicated in this breakdown. As nSP are widely used in foodstuffs and other products, exposure to them is increasing; thus, investigations of any effects of nSP on oral tolerance are urgent. This study evaluated the effects of nSP30 (particle diameter = 39 nm) on immunological unresponsiveness induced in mice with oral ovalbumin (OVA). Specifically, production of OVA-specific antibodies, splenocyte proliferation in response to OVA, and effects on T-helper (T_H)-1, T_H2, and T_H17 responses (in terms of cytokine and IgG/IgE subclass expression) were evaluated. nSP30 increased the levels of OVA-specific IgG in OVA-tolerized mice and induced the proliferation of OVA-immunized splenocytes in response to OVA in a dose-related manner. nSP30 also increased the expression of OVA-specific IgG₁, IgE, and IgG_2a, indicating stimulation of the T_H1 and T_H2 responses. The expression of interferon (IFN)-γ (T_H1), interleukin (IL)-4 and IL-5 (T_H2), and IL-17 (T_H17) was also stimulated in a dose-related manner by nSP30 in splenocytes stimulated ex vivo with OVA. The induction of tolerance by OVA, the production of anti-OVA IgG antibodies, and proliferation of splenocytes in response to OVA was inhibited by nSP30 in conjunction with OVA and was dose-related. The nSP30 enhanced T_H1 and T_H2 responses that might prevent the induction of oral tolerance. Overall, this study showed that the abrogation of OVA-induced oral tolerance in mice by exposure to nSP30 was dose-related and that nSP30 stimulated T_H1, T_H2, and T_H17 responses.

Keywords:

• Amorphous silica
• nanomaterial
• oral tolerance

Introduction

The immune system distinguishes self from non-self and invasive pathogens (non-self antigens) (Rocha & von Boehmer 1991). However, in the mucosal immune system, immune responses to non-self antigens are suppressed to avoid excessive immune responses to harmless non-self antigens because the mucosal system is constantly exposed to non-pathogenic antigens, including dietary proteins and microbiota in the intestine. It has long been recognized that oral administration of an antigen induces immunological unresponsiveness to that antigen in healthy individuals, designated “oral tolerance” (Mowat 1987; Weiner et al. 1994). However, the exact mechanism underlying induction of oral tolerance is unknown and the dose of antigen consumed is considered to determine the type of tolerance induced. For example, low doses (≤1 mg) induce suppression mediated by the production of inhibitory cytokines, including transforming growth factor (TGF)-β and interleukin (IL)-10 secreted by T-regulatory (T_reg) cells (Khoury et al. 1992; Chen et al. 1994). In contrast, higher doses (>5 mg) induce the deletion or anergy of antigen-specific lymphocytes (Whitacre et al. 1991; Chen et al. 1995). The analysis of mice deficient in Peyer’s patches and mesenteric lymph nodes has demonstrated that these immune tissues are also involved in oral tolerance (Fujihashi et al. 2001).

Amorphous silica nanoparticles (nSP) are one of the most widely used nanomaterials and are commonly included in consumer products (Peters et al. 2012). nSP were introduced into food and consumer products in the 1990s, and are used as anticaking agents in food powders; in health-care products such as toothpastes, detergents, and cosmetics; as stabilizers and clarifying agents in liquors; and as antifoaming agents in the manufacture of decaffeinated coffee and tea (Contado 2015). A recent study demonstrated that orally administered amorphous nSP penetrate the intestinal barrier (Yoshida et al. 2014). Thus, because nSP are readily absorbed by mucosal tissues, oral tolerance might be modulated by the effect of nSP on the mucosal immune system in humans (Jani et al. 1990; Cross et al. 2007).

We have also shown that nSP had enhanced adjuvant functions in mice treated with OVA plus nSP as assessed by more potent T_H1 and T_H2 immune responses compared with those treated with OVA alone (unpublished data). A previous study suggested that substances with adjuvant effects break down oral tolerance and induce allergic diseases (Ganeshan et al. 2009); however, to date there has been no report of the relationship between nanomaterials and food allergy. Therefore, we investigated the effects of nSP with a diameter of 30 nm (nSP30) on orally induced immunological unresponsiveness. To evaluate the effects of nSP30 on the suppression of responsiveness to oral ovalbumin (OVA), the production of OVA-specific anti-bodies and the proliferation of splenocytes in response to OVA were measured. The effects of nSP30 on T-helper (T_H)-1, T_H2, and T_H17 immune responses in mice were also investigated by measuring the OVA-specific immunoglobulin G (IgG) subclasses and cytokines expressed after exposure to nSP30 + OVA. Higher doses of OVA suppressed both T_H1 and T_H2 responses (Weiner 1997), whereas low doses predominantly suppressed the T_H2 response alone (Gonnella et al. 2003). Therefore, the induction of oral tolerance by the consumption of large amounts of antigen was judged a suitable strategy for the present study.

This study sought to provide further information about the effects of nSP on the immune system. Specifically, the present study investigated effects of nSP30 on orally induced immunologic unresponsiveness. The mean particle diameter of nSP30 is reported to be 39 nm based on a dynamic laser scattering analysis (Yamashita et al. 2011). Transmission electron microscopy demonstrated nSP30 are well-dispersed smooth-surfaced spheres. Therefore, because nSP30 remain stable, are well dispersed, and do not aggregate in phosphate-buffered saline (PBS), they were considered to be ideally suited to evaluate the biological effects of nSP in this study.

Materials and methods

Animals

BALB/c mice (male, 8-week-old) were purchased from Japan SLC, Inc. (Haruno Breeding branch, Shizuoka, Japan), and acclimated for 1 week before use. Throughout the study, the mice were maintained in polycarbonate cages in a pathogen-free environment maintained at 20–26 °C with 30–70% relative humidity and a 12-hr light-dark cycle. All mice had ad libitum access to standard rodent chow (CRF-1; Oriental Yeast Co., Ltd., Tokyo, Japan) and filtered UV-irradiated water. All experiments were performed according to the Ethical Guidelines established by Shionogi & Co., Ltd. and were approved by the Institutional Animal Care and Use Committee of Shionogi & Co., Ltd.

Induction of oral tolerance

OVA (Sigma, St. Louis, MO; 25 mg dissolved in 0.5 ml PBS, pH 7.2) was administered orally once daily by gastric intubation (gavage) for a period of 5 days before immunization with OVA (Day 0). As a non-specific foreign antigen control, 0.5 ml PBS alone or 0.5 ml PBS containing 25 mg hen egg lysozyme (HEL, Sigma) was administered instead of OVA for a period of 5 days before immunization. The gavage volume never exceeded 500 μl/dosing. Mice were weighed with an electronic balance (UX2200H, Shimadzu Corporation, Kyoto, Japan) on Days −6, −3, 0, 1, 4, 7, 14, and 21 of the study period.

Administration of antigen and nSP

As noted above, OVA was dissolved in PBS as a 50 mg/ml solution. The nSP30 were purchased from Micromod Partikeltechnologie GmbH (Rostock, Germany). Each nSP30 suspension was stored at room temperature, sonicated (400 W) for 5 min at 25 °C, and then vortexed for 1 min immediately before use. For the co-exposure experiments, the OVA solution was given orally immediately after oral administration of the nSP30 suspension (0.1, 1, or 10 mg/mouse) each day from Day −5 to Day −1. PBS alone was given as the control. Each treatment group consisted of five mice.

On Day 0, all mice were immunized in the base of the tail by subcutaneous injection of a solution containing 100 μg OVA dissolved in 50 μl PBS (pH 7.2) and emulsified with an equal volume of complete Freund’s adjuvant (CFA; Difco Laboratories, Detroit, MI). Three weeks later (i.e. Day 21), all mice were euthanized via isoflurane inhalation and blood and spleen specimens were collected for analysis. Blood was collected into SEPACLEAN A-5 tubes (Eiken Chemical Co., Ltd., Tokyo) and the tubes were centrifuged to separate sera, which were harvested and stored at −20 °C until analysis.

Measurement of OVA-specific antibodies

The assay to measure OVA-specific antibodies was performed as described previously (Matsumura et al. 2010). IgG, IgG₁, IgG_2a, and IgE antibodies specific for OVA were each measured by ELISA. To measure the levels of IgG, IgG₁, and IgG_2a antibodies, 100 μl/well of OVA (100 μg/ml) was incubated overnight at 4 °C in 96-well flat-bottomed microtiter plates, and the wells were then washed three times with PBS. The wells were blocked with 200 μl PBS containing 1% (w/v) casein (Sigma) by incubating at 37 °C for 1 hr. After the wells were washed, each received either 100 μl of a 1:10,000 (for IgG or IgG₁ measurements) or 1:50 (for IgG_2a) dilution of each serum sample and the plates were incubated at 37 °C for 1 hr. Thereafter, the wells were gently washed, and 100 μl/well of solution containing alkaline phosphatase (AP)-conjugated goat anti-mouse IgG (1:1000 dilution; Sigma) or AP-conjugated rat anti-mouse IgG₁ (or IgG_2a) (1:1000 dilution; BD Pharmingen, San Diego, CA) was added, and the plates were incubated at 37 °C for 1 hr. The wells were then gently washed and 100 μl of a 3 mM p-nitrophenyl phosphate solution (BioRad, Hercules, CA) was added to each well and the plates were incubated in the dark (at room temperature) until a bright yellow reaction product was observed. The absorbance in each well was measured at 405 nm with a VersamMax microplate reader (MDS Inc., Sunnyvale, CA). All results were expressed as the mean absorbance at OD₄₀₅ (A₄₀₅) ± SEM.

To measure IgE, 100 μl of 10 μg/ml rat anti-mouse IgE antibody (Monosan, Uden, the Netherlands) was loaded into each well of a 96-well flat-bottomed microtiter plate and incubated at 37 °C for 1 hr. After the wells were washed three times with SuperBlock Blocking Buffer (Pierce Biotechnology, Inc., Rockford, IL), 100 μl of each serum sample (diluted 1:5) was added to the wells, and the plate was incubated for 1 hr at 37 °C. The wells were then washed with PBS containing 0.05% (v/v) Tween 20 before each received 100 μl of a biotinylated-OVA (1 μg/ml) solution. After incubation at 37 °C for 1 hr, the wells were washed, and 100 μl of a 1 μg/ml solution of streptavidin conjugated with horseradish peroxidase (HRP; Vector Laboratories, Inc., Burlingame, CA) was added to each well. The plate was incubated at 37 °C for 1 hr, the wells were washed, 100 μl of TMB (3,3′,5,5′-tetramethylbenzidine) peroxide substrate solution (Pierce) was added to each well and the plate was incubated for 30 min at room temperature. The reaction in each well was stopped by the addition of 100 μl of 2 M sulfuric acid and absorbance values were then measured at 450 nm in the microplate reader. All results were expressed as mean absorbance at OD₄₅₀ (A₄₅₀) ± SEM.

Proliferation assay

Spleens removed on Day 21 were processed to yield cell suspensions as previously described (Yamaki et al. 2003). Erythrocytes in the suspensions were lysed with a Tris-NH₄Cl solution (0.75% [w/v] NH₄Cl and 0.2% Tris, pH 7.2, room temperature, 5 min) and then all non-erythrocytes were collected by centrifugation and re-suspended in complete medium (RPMI 1640 containing 1 mM l-glutamine, 40 μM 2-mercaptoethanol (all Sigma), 100 U penicillin/ml, 100 μg streptomycin/ml (both from Life Technologies Co., Carlsbad, CA), and 10% [v/v] heat-inactivated fetal bovine serum (FBS; Thermo Fisher Scientific Inc., Waltham, MA)). After the cells were counted and viability assessed (using trypan blue), 5 × 10⁵ cells in 0.1 ml complete medium were placed into each well of a 96-well plate. The cells were then either treated with a bolus of OVA (500 μg OVA/ml final concentration in each well), or medium alone in place of the OVA bolus (unstimulated cells). After 48 hr in culture at 37 °C and 5% CO₂ atmosphere, each well was pulsed with 0.5 μCi of [³H]-thymidine (GE Healthcare UK Ltd., Buckinghamshire, UK) and the cells were cultured for 6 h. Cells in each well were harvested onto fiberglass filters using a multi-harvester (Perkin Elmer, Waltham, MA) and total [³H]-radioactivity was determined by liquid scintillation counting. All results were expressed as the mean total counts per minute (CPM) of triplicate cultures of cells pooled from five mice.

Measurement of cytokines

Samples of RPMI 1640 (1 ml) containing 5 × 10⁶ splenocytes from OVA-tolerized mice treated with or without nSP30 were placed in 24-well tissue culture plates and then received medium alone or medium containing 500 μg OVA/ml. After 4 days of incubation at 37 °C, the supernatant in each well was harvested and stored at −20 °C until assay. Formation/secretion of interferon (IFN)-γ, interleukin (IL)-4, IL-5, and IL-17 by cells was quantified using commercial ELISA kits (Endogen, Inc., Woburn, MA for IFNγ, IL-4, IL-5; R&D Systems Inc. Minneapolis, MN, for IL-17), according to the manufacturer’s protocols. The level of sensitivity of the kits was 10 pg IFNγ/ml, 5 pg IL-4/ml, 5 pg IL-5/ml, and 5 pg IL-17/ml.

Statistical analysis

All data are reported as means ± SEM. A one-way analysis of variance (ANOVA) followed by a Dunnett’s parametric multiple t-test was used to compare the data obtained from the samples in each group with those from the PBS/PBS or OVA/PBS control groups. All analyses were done using Prism Software v.6.03 for Windows (GraphPad, La Jolla, CA). A p value <0.05 was accepted as significant.

Results

Effects of nSP30 on bodyweight

To investigate the effects of nSP30 on body weight, mice were weighed on Days −6, −3, 0, 1, 3, 7, 14, and 21 of the study period. The body weight changes in mice administered nSP30 and/or OVA are shown in Figure 1. No significant differences in bodyweight were observed in any group. The administration of nSP30 (10 mg) with OVA led to a transient and mild weight loss immediately after its administration (Days 0 and 1). However, this was not considered to have affected the study results because: (1) no significant differences in bodyweight were observed in any group; (2) weight loss was transient and very mild; and (3) no abnormal clinical signs were observed in the animals during the study.

Figure 1. Body weight changes after oral administration of antigen and/or nSP30. Mice were weighed on Days −3, 0, 1, 3, 7, 14, and 21 of the study period. Values are expressed as means (n = 5).

Effects of nSP30 on the oral OVA-induced suppression of the production of anti-OVA IgG antibodies and the proliferation of splenocytes in response to OVA

To induce oral tolerance, subsets of mice were given OVA by oral gavage once daily from Day −5 to Day −1 (five times). All mice were then immunized with OVA on Day 0. To examine the effects of nSP30 on the suppression of OVA-specific antibody production by oral OVA, six experimental groups were established and anti-OVA IgG antibodies were measured in sera collected on Day 21. As shown in Figure 2(A), anti-OVA IgG antibody production was significantly increased in mice orally administered 1 or 10 mg of nSP30 simultaneously with OVA, compared with that in mice that had been orally tolerized (p < 0.001 or p < 0.0001, respectively). Serum levels of anti-OVA IgG in mice administered OVA together with 1 or 10 mg of nSP30 were 2.1- and 3.7-fold higher, respectively, than those in mice given OVA alone from Day −5 to Day −1. Mice receiving the oral administration of OVA alone showed a significant suppression of anti-OVA IgG antibody production (p < 0.0001). In contrast, gavage with HEL rather than OVA (as a control) did not affect antibody production.

Figure 2. Effects of nSP30 on oral ovalbumin (OVA)-induced suppression of anti-OVA IgG antibody production and splenocyte proliferation in response to OVA. All mice were immunized with OVA on Day 0. To induce oral tolerance, mice received 25 mg OVA dissolved in PBS by gavage once daily from Day −5 to Day −1 (five times). As controls, PBS or 25 mg of hen egg lysozyme (HEL) was used instead of OVA. To examine the effects of nSP30 on oral tolerance, PBS or 0.1, 1.0, or 10 mg of nSP30 was administered orally just before mice were gavaged. (A) Effect of nSP30 on the oral OVA-induced suppression of the production of anti-OVA IgG antibodies. Serum samples were collected on Day 21 and assayed for anti-OVA IgG antibodies by ELISA. Values are expressed as means ± SEM of samples from 5 mice/regimen. (B) Effects of nSP30 on the oral OVA-induced suppression of splenocyte proliferation in response to OVA. On Day 21, spleens were removed and cells isolated, pooled, and incubated with 500 μg OVA/ml for 24 h to measures their proliferative capacity. Values shown are means ± SEM of triplicate cultures/cohort. CPM: counts per minute, ****p < 0.0001 vs. PBS/PBS, #p < 0.05, ###p < 0.001, and ####p < 0.0001 vs. OVA/PBS (Dunnett’s test).

The suppression of ex vivo splenocyte proliferation in response to OVA was also increased significantly in the nSP30 (1 and 10 mg) + OVA groups compared with that in the OVA-alone group (p < 0.05 and p < 0.0001, respectively) (Figure 2B). Proliferation in response to OVA in the group treated with nSP30 (0.1 mg) + OVA was similar to that in the OVA-alone group. The rates of cell proliferation in response to OVA (500 μg/ml) in mice treated with 1 or 10 mg of nSP30 + OVA were 214% and 697%, respectively, relative to the control.

Effects of nSP30 on the oral OVA-induced suppression of the production of anti-OVA IgG₁, IgE, and IgG_2a antibodies

To assess the effects of nSP30 on T_H1 and T_H2 immune responses, serum anti-OVA IgG subclasses and IgE were measured (Figure 3). IgG subclasses and IgE reflect the T_H1 and T_H2 immune responses because IFNγ secreted by T_H1 cells promotes IgG_2a production by B cells, whereas IL-4 secreted by T_H2 cells promotes IgG₁ and IgE production. Anti-OVA IgG₁, IgE, and IgG_2a production was significantly enhanced in the nSP30 (10 mg) + OVA group compared with that in the OVA-alone group (p < 0.0001, p < 0.001, and p < 0.01, respectively); production levels in the nSP30 (0.1 mg) + OVA group were similar to those in the OVA-alone group. Only anti-OVA IgG₁ production was also significantly enhanced in the nSP30 (1 mg) + OVA group (p < 0.001).

Figure 3. Effects of nSP30 on suppression of anti-ovalbumin (anti-OVA) IgG_2a, IgG₁, and IgE antibody production. All mice were immunized with OVA on Day 0. To induce oral tolerance, the mice received 25 mg of OVA dissolved in PBS by gavage once daily from Day −5 to Day −1 (five times). As controls, PBS or 25 mg of hen egg lysozyme (HEL) was used for gavage instead of OVA. To examine the effects of nSP30 on oral tolerance, PBS or 0.1, 1.0, or 10 mg of nSP30 was administered orally immediately before the mice were gavaged. Serum samples were collected on Day 21 and assayed for anti-OVA (A) IgG_2a, (B) IgG₁, and (C) IgE antibodies by ELISA. Values are expressed as means ± SEM of samples from 5 mice/regimen. **p < 0.01, ***p < 0.001, ****p < 0.0001 vs. PBS/PBS, ##p < 0.01, ###p < 0.001, ####p < 0.0001 vs. OVA/PBS (Dunnett’s test).

Effects of nSP30 on the oral OVA-induced suppression of cytokine secretion

To assess the types of immune responses affected in terms of cytokine levels, the levels of IFN-γ (T_H1 cytokine), IL-4 and IL-5 (T_H2 cytokines), and IL-17 (T_H17 cytokine) secreted by splenocytes from OVA-immunized mice were measured after stimulation with OVA in vitro (Figure 4). The OVA-suppressed secretion of IFNγ, IL-4, IL-5, and IL-17 was reversed (p < 0.001 or p < 0.0001) in splenocytes from mice that received nSP30 (1 or 10 mg) + OVA. These results were consistent with the changes noted for Ig isotype levels, and suggested that nSP30 blocked oral tolerance by promoting T_H1, T_H2, and T_H17 immune responses.

Figure 4. Effects of nSP30 on suppression of IFNγ, IL-4, IL-5, and IL-17 production. All mice were immunized with ovalbumin (OVA) on Day 0. To induce oral tolerance, the mice received 25 mg OVA dissolved in PBS by gavage once daily from Day −5 to Day −1 (five times). As controls, PBS or 25 mg of hen egg lysozyme (HEL) were used for gavage instead of OVA. To examine the effects of nSP30 on oral tolerance, PBS or 0.1, 1, or 10 mg of nSP30 was administered orally immediately before the mice were gavaged. On Day 21, the mouse spleens were removed and the splenocytes were incubated with (solid columns) or without (open columns) 500 μg/ml OVA, and (A) IFNγ, (B) IL-4, (C) IL-5, and (D) IL-17 were measured in the culture supernatants. Values are expressed as means ± SEM concentration (of each cytokine) of triplicate samples from the culture supernatants of cells pooled from five mice. ****p < 0.0001 vs. PBS/PBS, ###p < 0.001 and ####p < 0.0001 vs. OVA/PBS (Dunnett’s test).

Discussion

The present study showed that suppression of the production of anti-OVA IgG antibodies and proliferation of splenocytes in response to OVA by consumption of OVA was blocked by the administration of nSP30 in conjunction with OVA and was dose-related. These findings suggest that nSP30 effectively abrogates oral tolerance. This study also demonstrated that treatment with nSP30 reversed the suppression of anti-OVA IgG₁, IgG_2a, and IgE antibody production that occurred when mice were fed OVA. These findings in nSP30-treated mice suggested that nSP30 induced both T_H1 and T_H2 immune responses because IgG₁ and IgE production are dependent on T_H2 cells and IgG_2a on T_H1 cells. These conclusions were also supported by the findings that the secretion of a T_H1 cytokine (IFNγ and T_H2 cytokines (IL-4 and IL-5) was augmented by nSP30. Our studies have already shown that nSP30 exerts an adjuvant effect on T_H1 and T_H2 immune responses (unpublished data). The present data suggest that the ability of nSP30 to enhance T_H1 and T_H2 immune responses might contribute, at least in part, to their blockade of the induction of oral tolerance. Thus, nSP30 may induce both T_H1 and T_H2 immune responses which subsequently induce cell-mediated and humoral immunity, respectively, by inducing cross-presentation, which might explain the low specificity of nSP for T_H-related immune responses (Hirai et al. 2012).

In addition, nSP30 blocked the suppression of T_H17 IL-17 production induced by the consumption of OVA in this study. T_H17 cells present in the small intestinal lamina propria are induced by enteric bacteria such as segmented filamentous bacteria. T_H17 cells differentiate from naïve CD4⁺ T-cells when stimulated by IL-6 and TGFβ (Infante-Duarte et al. 2000; Bettelli et al. 2006). T_H17 cells secrete IL-17 and IL-22, which have important roles in the elimination of fungi and extracellular bacteria from epithelial cells via neutrophil migration and antibacterial peptides (Honda & Littman 2012). Other studies have previously noted that the administration of nSP induced the expression of inflammatory cytokines, including IL-6 and tumor necrosis factor-α (Wottrich et al. 2004; Nishimori et al. 2009). Thus, the induction of IL-6 production by nSP might be involved in the differentiation of T_H17 cells. These data suggest that the ability of nSP30 to enhance both T_H1 and T_H2 immune responses as well as T_H17 immune responses might contribute, at least in part, to the suppression of oral tolerance induction. It is also worth noting that nanoparticles are reactive and can avidly bind proteins. Thus, another possible mechanism for the effect observed in this study is the altered transport or cellular processing of OVA antigens upon co-exposure to nanoparticles and OVA.

Oral tolerance is modulated by environmental factors and biologically active substances. For example, diesel exhaust particles, which have adjuvant effects on immune responses (Yoshino & Sagai 1999), blocked the induction of oral tolerance and excessive immune responses to dietary proteins and microbiota in the intestine (Yoshino et al. 1998). Citrus pectin derived from food also blocked the suppression of antigen-specific IgG1 and IgE antibody production (Khramova et al. 2009). The breakdown of oral tolerance might cause allergic diseases, such as food allergy and asthma, and autoimmune diseases such as inflammatory bowel disease (Sato et al. 2014). Therefore, allergen-specific immunotherapies, developed based on the rationale of oral tolerance are expected to become a new innovative approach to treat these diseases.nSP are widely used in the production of food products (e.g. as anti-caking agents). Although the predominant particle size of nSP included in foods is ≈30 nm and the estimated daily intake (EDI) of nSP via food is currently 20–50 mg by a 70-kg person, smaller particle sizes and greater exposure to nSP by various routes are predicted to increase in the future (Dekkers et al. 2011). In the present study, 10 mg nSP30/mouse/day significantly blocked oral tolerance induced by consumption of OVA; this dose corresponded to 30 g nSP30/day for a 70-kg reference adult.

Individuals are rarely exposed to 30 g silica/d in practice, because 30 g nSP30/d corresponds to at least 600 times the EDI in food. However, the safety of nSP must be maintained because nSP have rapidly become widespread in daily life, and men and women of all ages (including pregnant women) cannot avoid exposure to them. Therefore, the absorption, distribution, metabolism, and excretion information for nSP, including their biokinetics, accumulation, and excretion routes, should be continually analyzed so that a more appropriate “no observed adverse effect level” can be determined and an “acceptable” daily intake criteria for nSP can be set.

Conclusions

This study showed that the oral administration of nSP30 blocked the suppression of anti-OVA IgG antibody production and the proliferation of splenocytes in response to orally administered OVA and was dose-related. Therefore, there is a possibility that the effect of nSP30 on the immunological response may induce food allergy.

Disclosure statement

The authors declare no conflicts of interest. The authors alone are responsible for the content of this manuscript.