TXNIP regulates pulmonary inflammation induced by Asian sand dust

Abstract

Asian sand dust (ASD), a seasonal dust storm originating from the deserts of China and Mongolia, affects Korea and Japan during the spring, carrying soil particles and a variety of biochemical components. Exposure to ASD has been associated with the onset and exacerbation of respiratory disorders, although the underlying mechanisms remain unclear. This study investigates ASD-induced pulmonary toxicity and its mechanistic pathways, focusing on the role of thioredoxin-interacting protein (TXNIP). Using TXNIP knock-out (KO) mice and adeno-associated virus (AAV)-mediated TXNIP overexpression transgenic mice, we explored how TXNIP modulates ASD-induced pulmonary inflammation. Mice were exposed to ASD via intranasal administration on days 1, 3, and 5 to induce inflammation. ASD exposure led to significant pulmonary inflammation, evidenced by increased inflammatory cell counts and elevated cytokine levels in bronchoalveolar lavage fluid, as well as heightened protein expression of the TXNIP/NOD-like receptor pyrin domain-containing 3 (NLRP3) inflammasome. TXNIP KO mice exhibited attenuated airway inflammation and downregulation of the NLRP3 inflammasome compared to wild-type controls, while AAV-mediated TXNIP overexpression mice showed exacerbated inflammatory responses, including elevated NLRP3 inflammasome expression, compared to AAV-GFP controls. These findings suggest that TXNIP is a key regulator of ASD-induced pulmonary inflammation.

Keywords: Asian sand dust; Inflammasome; NLRP3; Pulmonary inflammation; TXNIP.

Graphical abstract

Fig. 1

Physicochemical characteristics of ASD. (A) Transmission electron microscopy and scanning electron microscopy images of ASD. (B) Hydrodynamic size of ASD was measured by dynamic light scattering method. (C) Zeta potential of ASD. (D) Represented images and (E) calculated composition of chemical elements of ASD which were measured by energy-dispersive X-ray spectroscopy.

Fig. 2

Effects of ASD exposure on inflammatory cell counts and cytokines in BALF. (A–D) Differential and total inflammatory cell counts. (E–G) IL-6, IL-1β, and TNF-α levels. NCPBS intranasal instillation; ASD10, 20, and 40 = 10, 20, and 40 mg/kg of ASD intranasal instillation, respectively. Data are presented as means ± SD (n = 5). ∗p < 0.05 and ∗∗p < 0.01.

Fig. 3

Effects of ASD exposure on inflammatory cell infiltration and expression of TXNIP, NLRP3 and IL-1β in the lungs. (A) Representative images of H&E staining for assessing airway inflammation and IHC staining for evaluating protein expression. (B) Inflammation index. (C–E) Quantification of TXNIP, NLRP3, IL-1β expression. NCPBS intranasal instillation; ASD10, 20, and 40 = 10, 20, and 40 mg/kg of ASD intranasal instillation, respectively. Scale bar = 100 μm. Data are presented as means ± SD (n = 5). ∗p < 0.05 and ∗∗p < 0.01.

Fig. 4

Effects of ASD exposure on TXNIP/NLRP3 inflammasome pathway. (A) Representative western blots. (B–G) Quantification of TXNIP, NLRP3, pro-caspase-1, caspase-1 (p20), pro-IL-1β, and IL-1β (p17) expression. NCPBS intranasal instillation; ASD10, 20, and 40 = 10, 20, and 40 mg/kg of ASD intranasal instillation, respectively. Data are presented as means ± SD (n = 5). ∗p < 0.05 and ∗∗p < 0.01.

Fig. 5

Effects of ASD exposure on inflammatory cell counts and cytokines in BALF of TXNIP KO mice. (A–D) Differential and total inflammatory cell counts. (E–G) IL-6, IL-1β, and TNF-α levels. WT NCPBS intranasal instillation in wild type (WT) mice; WT ASD = ASD intranasal instillation in WT mice; KO NCPBS intranasal instillation in TXNIP knockout (KO) mice; KO ASD = ASD intranasal instillation in TXNIP (KO) mice. Data are presented as means ± SD (n = 5). ∗p < 0.05 and ∗∗p < 0.01.

Fig. 6

Effects of ASD exposure on inflammatory cell infiltration and expression of NLRP3 inflammasome in the lungs of TXNIP KO mice. (A) Representative images of H&E staining for assessing airway inflammation and IHC staining for evaluating protein expression. (B) Inflammation index. (C–E) Quantification of NLRP3, Caspase-1 and IL-1β expression. WT NCPBS intranasal instillation in wild type (WT) mice; WT ASD = ASD intranasal instillation in WT mice; KO NCPBS intranasal instillation in TXNIP knockout (KO) mice; KO ASD = ASD intranasal instillation in TXNIP (KO) mice. Scale bar = 100 μm. Data are presented as means ± SD (n = 5). ∗p < 0.05 and ∗∗p < 0.01.

Fig. 7

Effects of ASD exposure on NLRP3 inflammasome in TXNIP KO mice. (A) Representative western blots. (B–F) Quantification of NLRP3, pro-caspase-1, caspase-1 (p20), pro-IL-1β, and IL-1β (p17) expression. WT NCPBS intranasal instillation in wild type (WT) mice; WT ASD = ASD intranasal instillation in WT mice; KO NCPBS intranasal instillation in TXNIP knockout (KO) mice; KO ASD = ASD intranasal instillation in TXNIP (KO) mice. Data are presented as means ± SD (n = 5). ∗p < 0.05 and ∗∗p < 0.01.

Fig. 8

Effects of ASD treatment on inflammatory cytokine and protein expression of TXNIP and NLRP3 in NCI–H292 cells. (A) Cell viability. (B) IL-6 level. (C) Immunofluorescence for TXNIP and NLRP3 in siRNA mediated TXNIP knockdown cells. Scale bar = 20 μm. ASD were treated with 6.25, 12.5, 25, 50 μg/mL, respectively. Scrambled siRNA and TXNIP siRNA were treated with 20 nM ∗p < 0.05.

Fig. 9

Effects of ASD exposure on inflammatory cell counts and cytokines in BALF of TXNIP overexpression mice. (A–D) Differential and total inflammatory cell counts. (E–G) IL-6, IL-1β, and TNF-α levels. NCPBS intranasal instillation; Uninfected ASD = ASD intranasal instillation; AAV-GFP ASD = AAV-GFP intratracheal injection + ASD intranasal instillation. AAV-TXNIP ASD = AAV-TXNIP intratracheal injection + ASD intranasal instillation. Data are presented as means ± SD (n = 5). ∗p < 0.05 and ∗∗p < 0.01.

Fig. 10

Effects of ASD exposure on inflammatory cell infiltration and mucus production in the lungs of TXNIP overexpression mice. (A) Representative images of H&E staining for assessing airway inflammation and PAS staining for measuring mucus production. (B) Inflammation index. (C) Mucus production. NCPBS intranasal instillation; Uninfected ASD = ASD intranasal instillation; AAV-GFP ASD = AAV-GFP intratracheal injection + ASD intranasal instillation. AAV-TXNIP ASD = AAV-TXNIP intratracheal injection + ASD intranasal instillation. Scale bar = 100 μm. Data are presented as means ± SD (n = 5). ∗p < 0.05 and ∗∗p < 0.01.

Fig. 11

Effects of ASD exposure on expression of TXNIP/NLRP3 inflammasome in lungs of TXNIP overexpression mice. (A) Immunofluorescence for TXNIP. Scale bar = 50 μm. (B) Representative images of IHC staining for evaluating protein expression. Scale bar = 100 μm. (C–E) Quantification of NLRP3, Caspase-1 and IL-1β expression. NCPBS intranasal instillation; Uninfected ASD = ASD intranasal instillation; AAV-GFP ASD = AAV-GFP intratracheal injection + ASD intranasal instillation. AAV-TXNIP ASD = AAV-TXNIP intratracheal injection + ASD intranasal instillation. Data are presented as means ± SD (n = 5). ∗p < 0.05 and ∗∗p < 0.01.

Fig. 12

Effects of ASD exposure on expression of TXNIP/NLRP3 inflammasome in TXNIP overexpression mice. (A) Representative western blots. (B–G) Quantification of TXNIP, NLRP3, pro-caspase-1, caspase-1 (p20), pro-IL-1β, and IL-1β (p17) expression. NCPBS intranasal instillation; Uninfected ASD = ASD intranasal instillation; AAV-GFP ASD = AAV-GFP intratracheal injection + ASD intranasal instillation. AAV-TXNIP ASD = AAV-TXNIP intratracheal injection + ASD intranasal instillation. Data are presented as means ± SD (n = 5). ∗p < 0.05 and ∗∗p < 0.01.