Titanium Dioxide Nanoparticles Exacerbate Allergic Airway Inflammation via TXNIP Upregulation in a Mouse Model of Asthma

Titanium dioxide nanoparticles (TiO2NPs) are widely used in industrial and medicinal fields and in various consumer products, and their increasing use has led to an increase in the number of toxicity studies; however, studies investigating the underlying toxicity mechanism have been rare. In this study, we evaluated potential toxic effects of TiO2NPs exposure on lungs as well as the development of asthma through the ovalbumin (OVA)-induced mouse model of asthma. Furthermore, we also investigated the associated toxic mechanism. TiO2NPs caused pulmonary toxicity by exacerbating the inflammatory response, indicated by an increase in the number and level of inflammatory cells and mediators, respectively. OVA-induced asthma exposed mice to TiO2NPs led to significant increases in inflammatory mediators, cytokines, and airway hyperresponsiveness compared with those in non-exposed asthmatic mice. This was also accompanied by increased inflammatory cell infiltration and mucus production in the lung tissues. Additionally, TiO2NPs decreased the expression of B-cell lymphoma 2 (Bcl2) and the expressions of thioredoxin-interacting protein (TXNIP), phospho-apoptosis signal-regulating kinase 1, Bcl2-associated X, and cleaved-caspase 3 were escalated in the lungs of asthmatic mice compared with those in non-exposed asthmatic mice. These responses were consistent with in vitro results obtained using human airway epithelial cells. TiO2NPs treated cells exhibited an increase in the mRNA and protein expression of interleukin (IL)-1β, IL-6, and tumor necrosis factor-α with an elevation of TXNIP signaling compared to non-treated cells. Moreover, pathophysiological changes induced by TiO2NP treatment were significantly decreased by TXNIP knockdown in airway epithelial cells. Overall, TiO2NP exposure induced toxicological changes in the respiratory tract and exacerbated the development of asthma via activation of the TXNIP-apoptosis pathway. These results provide insights into the underlying mechanism of TiO2NP-mediated respiratory toxicity.


Introduction
Air pollutants such as yellow and fine dust, have become a critical social issue and are life-threatening to patients with respiratory diseases. Several studies [1][2][3][4] have particularly reported that titanium dioxide nanoparticles (TiO 2 NPs), components of Asian dust and air pollutants, exacerbate respiratory distress [1][2][3][4]. Moreover, TiO 2 NPs induce an intensive inflammatory response by triggering inflammatory cell migration and pro-inflammatory 2 of 16 cytokine secretion, consequently contributing to the development and exacerbation of respiratory diseases [5,6]. However, mechanisms associated with the toxic effects of titanium on the respiratory system and its diseases have not been clearly identified.
Of the known respiratory diseases, asthma is an inflammatory disease of the respiratory airways that affects approximately 300 million people globally [7]. It is characterized by excessive inflammation of bronchi and obstruction of the airflow due to increased immune responses, resulting in varying respiratory symptoms, mainly difficulty in breathing, wheezing, coughing, and tightness in the chest [3,8,9]. Based on its main characteristics, asthma has been divided into molecular pathways/clinical presentations or atopic/nonatopic asthma and more recently, into eosinophilic or non-eosinophilic phenotypes based on biological mechanisms [10]. Proinflammatory cytokines are key players in the development and progression of asthma, and they induce elevated immune responses, resulting in characteristic asthmatic responses such as eosinophilia, airway hyperresponsiveness (AHR), airway remodeling, and mucus production [11]. Previous studies have shown that fine dust and air pollutants aggravate asthma; however, the underlying toxicity mechanism is not well established.
Thioredoxin-interacting protein (TXNIP), a critical regulator of pathological responses, is induced by various of stressors including inflammation, metabolic dysfunction, apoptosis, and lung dysfunction [12][13][14]. TXNIP has been expressed in the lungs of experimental animals exposed to lipopolysaccharide and ovalbumin (OVA), which induce increased inflammatory responses via the activation of inflammasomes [15][16][17]. In contrast, TXNIP is involved in the apoptotic response of lung tissues. Elevation of TXNIP expression by various stimuli results in the activation of apoptotic signaling molecules such as mitochondrial apoptosis signal-regulating kinase 1 (ASK1), B-cell lymphoma 2 associated X (Bax), p38 mitogen-activated protein kinase, and cleaved-caspase 3 (Cas3), which eventually trigger the apoptosis of lung tissues [18,19]. The association between TiO 2 NPs and apoptosis under asthmatic conditions has not been clearly established. Therefore, an in-depth study is needed to understand the apoptosis mechanisms triggered via TXNIP in OVA-induced mice and how TiO 2 NPs pathologically exacerbate the development of asthma.
In this study, we investigated the exacerbation of asthma in response to TiO 2 NP exposure in OVA-induced asthmatic mice and explored the underlying mechanisms involving TXNIP and apoptosis.

Physicochemical Characterization of TiO 2 NPs
The morphology, primary, and hydrodynamic sizes of TiO 2 NPs are shown in Figure 1. The primary and hydrodynamic sizes in phosphate-buffered saline (PBS) were 48.89 ± 15.49 and 238.94 ± 57.94 nm, respectively. The specific surface area of TiO 2 NPs was 40.45 and 39.38 m 2 /g as determined using the Brunauer-Emmett-Teller (BET) and single point methods, respectively (Table 1). The zeta potential of the TiO 2 NPs was −31.01 mV (Figure 1c). Purity of TiO 2 NPs was measured as 21.35% of Ti and 78.65% of O using energy-dispersive X-ray spectroscopy (Figure 1d). TiO 2 NP suspensions did not show detectable endotoxin levels (data not shown). TiO 2 NP concentrations in the lung tissues were determined using inductively coupled plasma mass spectrometry (ICP-MS) (Table 1); the amount of TiO 2 NPs in the TiO 2 NPs-treated groups was markedly increased in a dose-dependent manner compared with that in the vehicle control (VC) group.

Effects of TiO 2 NPs on AHR and Inflammatory Cell Counts
In the pulmonary toxicity study, exposure to TiO 2 NPs significantly increased inflammatory cell counts in the bronchoalveolar lavage fluid (BALF) of mice compared with the BALF of VC mice. In particular, a marked increase in the number of neutrophils and macrophages was observed ( Figure S1). BALF of the OVA group exhibited a significant increase in inflammatory cell counts, especially that of eosinophils, compared to that of BALF of the VC group ( Figure 2a). OVA-induced mice exposed to TiO 2 NPs exhibited elevated counts of eosinophils, macrophages, and neutrophils compared with the OVA group, and this increase occurred in a dose-dependent manner. As shown in Figure 2b, the mean Penh value was augmented in the OVA group compared with the VC group. Furthermore, Penh values exhibited dose-dependent increases in the OVA + TiO 2 NP mice compared with the OVA mice ( Figure 2b).

Effects of TiO 2 NPs on Cytokine Production and OVA-Specific IgE Levels in Serum
To determine whether TiO 2 NPs affect the production of inflammatory cytokines in addition to increasing the number of inflammatory cells, cytokine levels in BALF samples were measured. The pulmonary toxicity study revealed that levels of proinflammatory cytokines, namely tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-1β, were significantly augmented in TiO 2 NP-treated groups compared with those in the VC group ( Figure S2). Levels of TNF-α, IL-6, and IL-1β in BALF were also significantly greater in the OVA group compared with those in the VC group. Exposure to TiO 2 NPs increased levels of TNF-α, IL-6, and IL-1β compared with those in the OVA group; this increase was dose-dependent (Figure 3a-c). Similar to levels of TNF-α, IL-6, and IL-1β, levels of IL-5 and IL-13 in BALF were also higher in the OVA group. Moreover, levels of IL-5 and IL-13 were increased dose-dependently (Figure 3d,e) and the OVA-specific IgE level in the serum was elevated in the TiO 2 NP-treated groups compared to that in the OVA group ( Figure 3f).

Effects of TiO 2 NPs on Airway Inflammation and Mucous Secretion in OVA-Induced Mice
Mice exposed to TiO 2 NPs showed an accumulation of inflammatory cells around the alveoli and bronchi as well as increased mucus production ( Figure S3). OVA-induced mice showed a significantly higher degree of airway inflammation than VC mice. When the OVA-induced mice were treated with TiO 2 NPs, airway inflammation worsened in a dose-dependent manner compared to that in OVA-induced mice (Figure 4a). Changes in the mucus production index exhibited a pattern similar to changes in airway inflammation wherein mucus production in the TiO 2 NP-treated groups was markedly escalated in a dosedependent manner compared with that in the OVA group ( Figure 4b). Moreover, thickening of the basement membrane and airway smooth muscle, vasodilatation, increased number of blood vessels, and goblet cell metaplasia were observed in OVA-induced mice. These pathological changes were more severe in TiO 2 NP-treated groups (Figure 4a 10, and 20 mg/kg of TiO2NPs intranasal instillation, respectively. Data are represented as means ± SD, n = 6. ## p < 0.01, significantly different from the VC group; * p < 0.05, ** p < 0.01, significantly different from the OVA group.

Effects of TiO2NPs on Cytokine Production and OVA-Specific IgE Levels in Serum
To determine whether TiO2NPs affect the production of inflammatory cytokines in addition to increasing the number of inflammatory cells, cytokine levels in BALF samples were measured. The pulmonary toxicity study revealed that levels of proinflammatory cytokines, namely tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-1β, were significantly augmented in TiO2NP-treated groups compared with those in the VC group ( Figure S2). Levels of TNF-α, IL-6, and IL-1β in BALF were also significantly greater in the OVA group compared with those in the VC group. Exposure to TiO2NPs increased levels of TNF-α, IL-6, and IL-1β compared with those in the OVA group; this increase was dosedependent (Figure 3a-c). Similar to levels of TNF-α, IL-6, and IL-1β, levels of IL-5 and IL-13 in BALF were also higher in the OVA group. Moreover, levels of IL-5 and IL-13 were  OVA, OVA challenge + PBS intranasal instillation; TiO2NPs 5, 10, and 20, OVA challenge +5, 10, and 20 mg/kg of TiO2NPs intranasal instillation, respectively. Data are represented as means ± SD, n = 6. # p < 0.05, ## p < 0.01, significantly different from the VC group; * p < 0.05, ** p < 0.01, significantly different from the OVA group.

Effects of TiO2NPs on Airway Inflammation and Mucous Secretion in OVA-Induced Mice
Mice exposed to TiO2NPs showed an accumulation of inflammatory cells around the alveoli and bronchi as well as increased mucus production ( Figure S3). OVA-induced mice showed a significantly higher degree of airway inflammation than VC mice. When the OVA-induced mice were treated with TiO2NPs, airway inflammation worsened in a dose-dependent manner compared to that in OVA-induced mice ( Figure 4a). Changes in the mucus production index exhibited a pattern similar to changes in airway inflammation wherein mucus production in the TiO2NP-treated groups was markedly escalated in a dose-dependent manner compared with that in the OVA group ( Figure 4b). Moreover, thickening of the basement membrane and airway smooth muscle, vasodilatation, increased number of blood vessels, and goblet cell metaplasia were observed in OVA-induced mice. These pathological changes were more severe in TiO2NP-treated groups (   Black arrows indicate inflammatory cell infiltration. Black arrowheads indicate mucus within lung epithelial goblet cells. Data are represented as means ± SD, n = 6. # p < 0.05, ## p < 0.01, significantly different from the VC group; * p < 0.05, ** p < 0.01, significantly different from the OVA group. Bar = 50 μm.

Effects of TiO2NPs on TXNIP and Apoptotic Protein Expression
Immunohistochemistry (IHC) was used to estimate expression levels of TXNIP and cleaved-Cas3 in lung tissues in response to OVA and TiO2NP treatment. Lungs of normal mice exposed to TiO2NPs showed a dose-dependent increase in the expression of TXNIP and cleaved-Cas3 compared with those of VC mice. Similarly, exposure to TiO2NPs led to a surge in the expressions of TXNIP, phospho-ASK1 (p-ASK1), Bax, and cleaved-Cas3 and a Black arrows indicate inflammatory cell infiltration. Black arrowheads indicate mucus within lung epithelial goblet cells. Data are represented as means ± SD, n = 6. # p < 0.05, ## p < 0.01, significantly different from the VC group; * p < 0.05, ** p < 0.01, significantly different from the OVA group. Bar = 50 µm.

Effects of TiO 2 NPs on TXNIP and Apoptotic Protein Expression
Immunohistochemistry (IHC) was used to estimate expression levels of TXNIP and cleaved-Cas3 in lung tissues in response to OVA and TiO 2 NP treatment. Lungs of normal mice exposed to TiO 2 NPs showed a dose-dependent increase in the expression of TXNIP and cleaved-Cas3 compared with those of VC mice. Similarly, exposure to TiO 2 NPs led to a surge in the expressions of TXNIP, phospho-ASK1 (p-ASK1), Bax, and cleaved-Cas3 and a decline in the expression of B-cell lymphoma 2 (Bcl2) compared with that in VC mice ( Figure S4). TXNIP expression in the OVA group was greater than that in the VC group and increased in a dose-dependent manner in TiO 2 NP-treated groups than in the OVA group (Figure 5a). Similarly, cleaved-Cas3 expression in the OVA group increased compared to that in the VC group and in the TiO 2 NP-treated groups compared with that in the OVA group (Figure 5b). The effects of TiO 2 NPs on TXNIP activation were determined using immunoblotting. As shown in Figure 6a,b, TXNIP expression in the lungs was increased in the OVA group compared with the VC group as were the expressions of p-ASK1, Bax, and cleaved-Cas3. However, Bcl2 expression decreased in the OVA group compared with the VC group. Compared with the OVA group, exposure to TiO 2 NPs induced a marked increase in the expression of TXNIP, p-ASK1, Bax, and cleaved-Cas3 in a dose-dependent manner while the Bcl2 level in TiO 2 NP-treated groups was lower than that in the OVA group.    ). VC, PBS intranasal instillation; OVA, OVA challenge + PBS intranasal instillation; TiO2NPs 5, 10, and 20, OVA challenge +5, 10, and 20 mg/kg of TiO2NPs intranasal instillation, respectively. Data are represented as means ± SD, n = 6. ## p < 0.01, significantly different from the VC group; ** p < 0.01, significantly different from the OVA group. Bar = 50 μm.

Effects of TiO 2 NPs on the Production of Proinflammatory Mediators in NCI-H292 Cells
For the in vitro experiments, TiO 2 NP concentrations for treatment groups were decided based on results of the cell viability assay (Figure 7a). Treatment of the TiO 2 NPs significantly elevated the levels of IL-1β, IL-6, and TNF-α in NCI-H292 cells in a dosedependent manner compared to the untreated cells (Figure 7b-d).

Effects of TiO2NPs on the Production of Proinflammatory Mediators in NCI-H292 Cells
For the in vitro experiments, TiO2NP concentrations for treatment groups were decided based on results of the cell viability assay (Figure 7a). Treatment of the TiO2NPs significantly elevated the levels of IL-1β, IL-6, and TNF-α in NCI-H292 cells in a dosedependent manner compared to the untreated cells (Figure 7b-d).   3.125, 6.25, 12.5, and 25 μg/mL of TiO2NP treatment; respectively. Data are represented as means ± SD, n = 3. ** p < 0.01, significantly different from the control group.
As shown in Figure 8a-d, real-time reverse-transcription polymerase chain reaction (qRT-PCR) results revealed that expression levels of IL-1β, IL-6, IL-8, and TNF-α were sig nificantly increased in a dose-dependent manner in NCI-H292 cells treated with TiO2NPs compared with those in the control group.

Effects of TiO 2 NPs on TXNIP and Apoptosis Protein Expression in NCI-H292 Cells
Immunoblotting revealed a dose-dependent rise in TXNIP expression and in levels of p-ASK1, Bax, and cleaved-Cas3 in TiO 2 NP-treated cells compared to those in untreated cells (Figure 9a,b). To determine the role of TXNIP in mediating effects of TiO 2 NPs, we transfected NCI-H292 cells with TXNIP-specific small interfering RNA (siRNA). The control siRNA did not have any effect on the increased expression of p-ASK1, Bax, and cleaved-Cas3 and Bcl2 expression decreased with TiO 2 NP treatment; however, treatment with TXNIP-specific siRNA diminished the expression of Bax and cleaved-Cas3 and increased the expression of Bcl2 in TiO 2 NP-treated cells, restoring them to levels similar to those in the control group (Figure 10a,b).

Effects of TiO2NPs on TXNIP and Apoptosis Protein Expression in NCI-H292 Cells
Immunoblotting revealed a dose-dependent rise in TXNIP expression and in levels of p-ASK1, Bax, and cleaved-Cas3 in TiO2NP-treated cells compared to those in untreated cells (Figure 9a,b). To determine the role of TXNIP in mediating effects of TiO2NPs, we transfected NCI-H292 cells with TXNIP-specific small interfering RNA (siRNA). The control siRNA did not have any effect on the increased expression of p-ASK1, Bax, and cleaved-Cas3 and Bcl2 expression decreased with TiO2NP treatment; however, treatment with TXNIP-specific siRNA diminished the expression of Bax and cleaved-Cas3 and increased the expression of Bcl2 in TiO2NP-treated cells, restoring them to levels similar to those in the control group (Figure 10a,b).   Relative densitometric values of protein expression. Control, PBS treatment; 3.125, 6.25, 12.5, and 25 μg/mL of TiO2NPs treatment; respectively. Data are represented as means ± SD, n = 3. ** p < 0.01, significantly different from the control group.

Discussion
There has been an increase in the number of patients with underlying respiratory diseases, a vulnerable subpopulation who warrant consideration when evaluating the potential respiratory toxicity of various substances [20]. The aim of this study was to examine

Discussion
There has been an increase in the number of patients with underlying respiratory diseases, a vulnerable subpopulation who warrant consideration when evaluating the potential respiratory toxicity of various substances [20]. The aim of this study was to examine the effect of TiO 2 NPs on asthma exacerbation and to elucidate its underlying mechanism. We showed that exposure to TiO 2 NPs aggravated asthma, increased TXNIP expression, and activated apoptosis in lungs of OVA-induced mice. Furthermore, TiO 2 NP treatment of NCI-H292 cells led to an upregulation of apoptotic machinery via upregulation in TXNIP.
In this study, exposure to TiO 2 NPs increased the inflammatory response in the respiratory tract and aggravated major asthma symptoms, namely, airway inflammation, airway remodeling, mucus overproduction, and AHR, in mice with OVA-induced asthma. Eosinophilic inflammatory response, which is characteristic of asthma, is known to be induced by IL-4, IL-5, and IL-13 produced by CD4 + T helper type 2 (Th2) cells [21]. Furthermore, proinflammatory cytokines such as TNF-α, IL-6, and IL-1β, function as growth factors for B cells and play an important role in the differentiation of CD4 + Th2 cells [22]. These cytokines have been reported to increase the secretion of mucus by stimulating goblet cells of bronchi, resulting in AHR [23,24]. Asthmatic mice exposed to TiO 2 NPs exhibited a characteristic increase in cytokines, further heightening the aforementioned major asthma symptoms. Moreover, in this study, IgE level was elevated upon exposure to TiO 2 NPs in asthmatic mice, and such an increase has been reported to be associated with the overproduction of Th2 type cytokines [25]. Overproduction of IgE and Th2 cytokines is observed in asthmatic patients with the eosinophilic asthma phenotype as well as in asthmatic animals [15,25]. We observed that cytokine production was significantly increased in NCI-H292 cells treated with TiO 2 NPs, which was similar to our in vivo observations. These results were consistent with histological evidence. Exposure to TiO 2 NPs induced inflammatory cell infiltration into lung tissue and mucus production from goblet cells in a dose-dependent manner and aggravated airway inflammatory responses and mucus production in OVA-induced asthmatic animals. Thus, we demonstrated that TiO 2 NPs caused respiratory toxicity and exacerbated asthma, which is consistent with the findings of previous reports [1, 6,26].
TXNIP can directly bind to thioredoxin (TRX) and inhibit its function, leading to the activation of an apoptotic signaling pathway. Under normal conditions, TRX inhibits the activation of ASK1 via formation of a complex. However, activated TXNIP induces the dissociation of this TRX-ASK1 complex, resulting in the activation of ASK1 and, consequently, apoptosis [27,28]. The association between nanoparticles and TXNIP has been reported in several studies [15,16]. For example, exposure to silica dioxide nanoparticles has been shown to worsen asthma and increase pulmonary toxicity via upregulation of TXNIP. In this study, exposure to TiO 2 NPs increased TXNIP expression and activated TXNIP downstream signaling in lungs of normal and asthmatic mice. Furthermore, we found that the TiO 2 NP-activated apoptosis was suppressed by downregulation of the TXNIP gene in human airway epithelial cells. In contrast, upregulation of TXNIP promotes apoptosis by increasing the Bax/Bcl2 ratio and cleaved-caspase 3 expression [19]. Overall, exposure of TiO 2 NPs to mice increased the expression of TXNIP in lungs, demonstrating that TXNIP might be involved in the molecular pathogenesis of asthma. This suggests that TXNIP may be responsible for the aggravating effect of TiO 2 NP-induced apoptosis in asthmatic lungs.
In conclusion, exposure to TiO 2 NPs revealed a marked increase in AHR, inflammatory cytokines and responses, and mucus overproduction, which are typical characteristics of asthma, and these characteristic were correlated with those in the apoptotic signaling pathway ( Figure 11). Moreover, we elucidated the aggravating effect of TiO 2 NP inhalation in respiratory tracts with asthma, along with the molecular pathogenesis through the regulation of TXNIP. Thus, our present study provides useful information on new target signaling not yet observed in TiO 2 NP-induced asthmatic lung tissues.  Figure 11. Proposed signaling pathway for TiO2NP-induced apoptosis in the lung of asthmatic mic TXNIP levels are increased by exposure to TiO2NP phosphorylates ASK1, thereby increasing apop tosis. TXNIP plays a key mediator role in the exacerbation of asthma by TiO2NPs.

Characterization of TiO2NPs
TiO2NPs were purchased from Sigma-Aldrich (particle size < 25 nm, 637254, S Louis, MO, USA). We quantified the morphology and size of TiO2NPs using transmissio electron microscopy (JEM-1210, JEOL, Tokyo, Japan) and scanning electron microscop (Zeiss EVO-MA10; Carl Zeiss Meditec AG, Jena, Germany) at accelerating voltages of 20 kV and 15 kV, respectively. The specific surface area of TiO2NPs was measured by nitro gen absorption methods based on the multipoint BET method (ASAP2020; Micromeritic Norcross, GA, USA). The hydrodynamic size and zeta potential of TiO2NPs were dete mined by ELS-8000 (Otsuka Electronic, Tokyo, Japan). The purity of TiO2NPs used in th Figure 11. Proposed signaling pathway for TiO 2 NP-induced apoptosis in the lung of asthmatic mice. TXNIP levels are increased by exposure to TiO 2 NP phosphorylates ASK1, thereby increasing apoptosis. TXNIP plays a key mediator role in the exacerbation of asthma by TiO 2 NPs.

Characterization of TiO 2 NPs
TiO 2 NPs were purchased from Sigma-Aldrich (particle size <25 nm, 637254, St. Louis, MO, USA). We quantified the morphology and size of TiO 2 NPs using transmission electron microscopy (JEM-1210, JEOL, Tokyo, Japan) and scanning electron microscopy (Zeiss EVO-MA10; Carl Zeiss Meditec AG, Jena, Germany) at accelerating voltages of 200 kV and 15 kV, respectively. The specific surface area of TiO 2 NPs was measured by nitrogen absorption methods based on the multipoint BET method (ASAP2020; Micromeritics, Norcross, GA, USA). The hydrodynamic size and zeta potential of TiO 2 NPs were determined by ELS-8000 (Otsuka Electronic, Tokyo, Japan). The purity of TiO 2 NPs used in the experiment was determined by energy-dispersive X-ray spectroscopy (Rayny EDX-700, Shimadzu, Kyoto, Japan). The endotoxin levels in thee TiO 2 NP suspension were determined using a Pierce LAL Chromogenic Endotoxin Quantitation Kit (Thermo Fisher Scientific, Waltham, MA, USA). After completion of treatment procedures, lung tissue was harvested, weighed, and digested overnight with concentrated nitric acid, and the resultant samples were analyzed for elemental TiO 2 NPs using ICP-MS (Perkin Elmer, Waltham, MA, USA).

Experimental Procedure for Allergic Asthma Induction
Specific pathogen-free female BALB/c mice (six weeks old) were purchased from Samtako Co. (Osan, Korea) quarantined and acclimatized for seven days. Animals were confined at 22 ± 2 • C in a room with a relative humidity of 50 ± 5%, artificial lighting from 08:00-20:00, and 13-18 air changes per hour. Animals were provided ad libitum access to a standard laboratory diet (Samtako Co., Osan, Korea) and water. All experimental procedures were carried out in accordance with the National Institute Health Guidelines for the Care and Use of Laboratory Animals. The Institutional Animal Care and Use Committee of Chonnam National University approved experimental protocols involving animals (CNU IACUC-YB-2020-19).
To investigate the effect of TiO 2 NPs on the development of asthma, 30 animals were randomly assigned to five experimental groups (each group, n = 6); VC group, OVA group, and three OVA+ TiO 2 NPs (5, 10, and 20 mg/kg) groups. On days 1 and 15, mice were sensitized with an intraperitoneal injection of 20 µg of OVA (Sigma-Aldrich, St. Louis, MO, USA) emulsified with 2 mg of aluminum hydroxide (Thermo Scientific, Waltham, MA, USA) in 200 µL of PBS (pH 7.4). On days 22, 24, and 26, mice received a 1 h airway challenge with 1% (w/v) OVA solution aerosolized using an ultrasonic nebulizer (NE-U12; Omron Corp., Tokyo, Japan). On days 21, 23, and 25, mice of the TiO 2 NP treatment groups (5, 10, and 20 mg/kg doses in 50 µL of PBS, respectively) received TiO 2 NPs via intranasal instillation under light anesthesia using Zoletil 50 ® (Virbac Laboratories). The VC and OVA group animals received 50 µL of PBS via intranasal instillation. TiO 2 NPs were prepared in PBS and sonicated in an ultrasonicator for 3 min before intranasal instillation.

Measurement of Airway AHR
Penh values were indirectly assessed 24 h after the final intranasal instillation via single-chamber whole body plethysmography (Allmedicus, Seoul, Korea). Mice were anesthetized for a brief period with an intraperitoneal injection of a mixture of Zoletil and Xylazine (40 mg/kg and 10 mg/kg, respectively), subsequently placed in a chamber, and nebulized with aerosolized PBS or methacholine in increasing concentrations (10, 20, and 40 mg/mL).

Collection of Bronchoalveolar Lavage Fluid (BALF) and Cell Counting
Mice were sacrificed at 24 h after measurement of AHR via an intraperitoneal injection of Zoletil 50 ® (Virbac Laboratories), and a tracheostomy was performed. To obtain BALF, ice-cold PBS (0.7 mL) was infused into their lungs twice and subsequently withdrawn each time using a tracheal cannula (a total volume of 1.4 mL). BALF samples were centrifuged, and its supernatant was collected for biochemical analysis. Collected cells were resuspended in ice-cold PBS (0.5 mL), and 200 µL of the resuspended solution was centrifuged (200× g, 4 • C, 10 min) onto slides using a cytospin (Hanil Science Industrial Co., Ltd., Seoul, Korea). Slides were dried, and the cells were fixed and stained. Differential cell counts were performed using the Diff-Quik ® staining reagent (Sysmex Corporation, Kobe, Japan) according to the manufacturer's instructions.

Cytokines Assay
Levels of several cytokines in BALF, namely TNF-α, IL-6, IL-1β, IL-5, and IL-13, were measured using commercial enzyme-linked immunosorbent assay (ELISA) kits (BD Biosciences, San Jose, CA, USA) according to the manufacturer's protocol. The serum level of OVA-specific IgE was measured using an ELISA kit (BioLegend, San Diego, CA, USA). Absorbance was measured at 450 nm using an ELISA reader (Bio-Rad Laboratories, Hercules, CA, USA).

Histopathology and IHC
After BALF samples were collected, the lung tissue was fixed with 4% (v/v) paraformaldehyde for 48 h. Tissues were paraffin-embedded, sectioned at a thickness of 4 µm and stained using hematoxylin and eosin (Sigma-Aldrich, St. Louis, MO, USA) or periodic acid-Schiff solution (IMEB Inc., San Marcos, CA, USA) to evaluate airway inflammation and mucus production, respectively. Furthermore, sectioned tissues were processed for IHC, as previously described [29]. The primary antibodies used for the detection of protein expression were anti-TXNIP (NBP1-54578; 1:200 dilution; Novus Biologicals, Littleton, CO, USA) and anti-cleaved-Cas3 (#9661; 1:200 dilution; Cell signaling, Danvers, MA, USA). Each slide was examined manually by investigators blind to the treatment groups using a light microscope (Leica, Wetzlar, Germany) with 10× and 20× objective lenses and a 100× oil immersion lens. Ten randomly selected nonoverlapping areas per slide were captured with a digital camera (IMTcamCCD5; IMT Inc., Daejeon, Korea), and quantitative analyses of airway inflammation, mucus production, and protein expression were performed using an image analyzer (IMT i-Solution software, Vancouver, BC, Canada).

Cell Culture
The human airway epithelial cell line NCI-H292 was obtained from the American Type Culture Collection (Manassas, VA, USA). Cells were grown in RPMI 1640 medium (WELGENE, Gyeongsan, Korea) with 10% fetal bovine serum, streptomycin (100 µg/mL), and penicillin (100 U/mL) and incubated in a humidified chamber maintained at 37 • C with 5% CO 2 . Cells were serum-starved for 1 h before use.

Cell Viability Assay
Cell viability was performed using the EZ-Cytox cell viability assay kit (DAELIL lab, Seoul, Korea), and concentration of TiO 2 NPs was chosen with reference to a previous study [31]. NCI-H292 cells were seeded in 96 well-plates (4 × 10 4 cells/well). After 24 h, fresh medium and various concentrations of TiO 2 NPs (1.56, 3.13, 6.25, 12.5, 25 µg/mL) were added. The culture plate was incubated for another 24 h. Eventually, viable cells were determined by adding 10 µL of the kit solution to each well before incubating for