Chronic low dose chlorine exposure aggravates allergic inflammation and airway hyperresponsiveness and activates inflammasome pathway

Abstract

Background: Epidemiologic clinical studies suggested that chronic exposure to chlorine products is associated with development of asthma and aggravation of asthmatic symptoms. However, its underlying mechanism was not clearly understood. Studies were undertaken to define the effects and mechanisms of chronic low-dose chlorine exposure in the pathogenesis of airway inflammation and airway hyperresponsiveness (AHR).

Methods: Six week-old female BALB/c mice were sensitized and challenged with OVA in the presence and absence of chronic low dose chlorine exposure of naturally vaporized gas of 5% sodium hypochlorite solution. Airway inflammation and AHR were evaluated by bronchoalveolar lavage (BAL) cell recovery and non-invasive phlethysmography, respectively. Real-time qPCR, Western blot assay, and ELISA were used to evaluate the mRNA and protein expressions of cytokines and other inflammatory mediators. Human A549 and murine epithelial (A549 and MLE12) and macrophage (AMJ2-C11) cells were used to define the responses to low dose chlorine exposure in vitro.

Results: Chronic low dose chlorine exposure significantly augmented airway inflammation and AHR in OVA-sensitized and challenged mice. The expression of Th2 cytokines IL-4 and IL-5 and proinflammatory cytokine IL-1β and IL-33 were significantly increased in OVA/Cl group compared with OVA group. The chlorine exposure also activates the major molecules associated with inflammasome pathway in the macrophages with increased expression of epithelial alarmins IL-33 and TSLP in vitro.

Conclusion: Chronic low dose exposure of chlorine aggravates allergic Th2 inflammation and AHR potentially through activation of inflammasome danger signaling pathways.

Figure 1. Mouse chlorine exposure system.

A, a container filled with NaOCl solution was placed in the cage and mice were exposed to naturally vaporizing chlorine gas 8 hours a day, 5 times a week, for 4 weeks. B, a container with holes on the top surface to avoid direct skin contact with the NaOCl solution.

Figure 2. Effect of low dose chronic chlorine exposure on allergen-induced airway hyperresponsiveness and inflammation 6 weeks old BALB/c mice were sensitized and challenged with OVA allergen with or without low dose chlorine exposure.

A, Airway hyperresponsiveness was determined by whole body phlethysmography *p<0.05 vs. other groups. B, Lung inflammatory response were measured by BAL cell recovery. Macro, macrophages; Neutro, neutrophils; Eosino, eosinophils; Lympho, lymphocytes. *p<0.05 vs. other groups. C, H&E staining (x200). D, Quantification of eosinophil in the lung tissue. E, PAS staining (x200), F: Semi-quantified mucus index score in the PAS staining. Values in panels A, B, D and F are mean ± SEM of evaluations in a minimum of 5 mice. Panel C and E is a representative of a minimum of four similar experiments.

Figure 3. Effect of low dose chronic chlorine exposure on Th2 cytokine and IL-17 expression in OVA sensitized and challenged mice.

A-C, the mRNA expression of IL-4 (A), IL-5(B) and IL-13(C) was evaluated by RT-qPCR. D. The level of IL-17 in BAL was measured by ELISA. Values in these panels are mean ± SEM of evaluations in a minimum of 5 mice. *p<0.05 vs. OVA only group.

Figure 4. Effect of low dose chronic chlorine exposure on the molecules associated with inflammasome activation.

A, IL-1β levels in the BAL from OVA-sensitized and challenged mice with or without chlorine exposure were measured by ELISA. *p<0.05 vs. OVA only group. B, The mRNA expressions of IL-1β were measured by RT-qPCR after 48 and 96 hrs stimulation with 0.001% NaOCl. *p<0.05 vs. control. C, IL-1β levels were measured using ELISA after low dose NaOCl treatments in AMJ2-C11 mouse macrophage cells. *p<0.05 vs. control. D, Western blot evaluation on the molecules associated with inflammasome activation in the macrophages with and without exposure of NaOCl (IL-1β and caspase-1 in the cell supernatant, and IL-18 in the cell lysates). Values in panels in A, B and C are mean ± SEM. Panel D is a representative of a minimum of three similar experiments.

Figure 5. Effect of low dose chronic chlorine exposure on the expression of epithelial alarmines.

A, The levels of IL-33 expression in the lungs from the mice sensitized and challenged by OVA with or without chlorine exposure were evaluated by RT-qPCR. *p<0.05 vs. groups without chlorine exposure. B, The mRNA expressions of IL-33 in murine MLE12 epithelial cells were measured by qRT-PCR after 48 and 96 hrs stimulation of 0.001% NaOCl. *p<0.05 vs. control. C, TSLP protein expression was evaluated by Western blot analysis. Values in panels A and B are mean ± SEM of evaluations in a minimum of 5 mice. Panel C is a representative of a minimum of three similar experiments.