Nrf2 deficiency in dendritic cells enhances the adjuvant effect of ambient ultrafine particles on allergic sensitization

Abstract

Particulate matter (PM) is an important risk factor for asthma. Generation of oxidative stress by PM is a major mechanism of its health effects. Transcription factor nuclear factor (erythroid-derived 2)-like 2 (Nrf2) mediates antioxidant and phase II enzymes and is essential in protecting against oxidative stress and lung inflammation. We have previously shown that ambient ultrafine particles (UFP) could exert a potent adjuvant effect on allergic sensitization to ovalbumin (OVA) in mice. We hypothesized that Nrf2 deficiency in dendritic cells (DC) could enhance the adjuvant potential of UFP on allergic sensitization. We show that the adjuvant effect of intranasally instilled UFP is significantly enhanced in Nrf2 knockout (Nrf2(-/-)) mice compared with their wild-type (Nrf2(+/+)) counterparts. Under resting conditions, Nrf2(-/-) DC displayed an intrinsic predilection to a T helper 2-favoring cytokine profile characterized by a low level of IL-12p70 and a high level of IL-6 as compared to Nrf2(+/+) DC. Adoptive transfer of OVA/UFP-treated Nrf2(-/-) DC provoked a more severe allergic inflammation in the lung than Nrf2(+/+) DC in the same treatment group. We conclude that Nrf2 deficiency in DC may promote a constitutive immune-polarizing cytokine milieu, which we propose may have contributed to the augmented adjuvant effect of UFP on allergic sensitization.

Fig. 1

Nrf2 genotyping by PCR. Deletion of Nrf2 in BALB/c mice was confirmed by genotyping. PCR fragments of 262 and 214 bp represent Nrf2^+/+ and Nrf2^−/− alleles, respectively.

Fig. 2

The adjuvant effect of intranasally administered ambient UFP on OVA sensitization was further enhanced in Nrf2^−/− mice. a Increased BAL cell count including total cell number, eosinophils and lymphocytes; * p < 0.02 compared with respective saline control, ^a p < 0.05 compared with respective OVA. b Elevated OVA-IgG1 level in the blood; * p < 0.01 compared with respective saline control, ^a p < 0.01 compared with respective OVA. c Increased OVA-IgE production; * p < 0.01 compared with respective saline control, ^a p < 0.05 compared with respective OVA. d Increased IL-13 level in the BAL fluid; * p < 0.05 compared with all other groups.

Fig. 3

Histological analysis showing that Nrf2^−/− mice developed a more severe inflammation in the lung when exposed to UFP during OVA sensitization and smaller airways were more involved (indicated by arrows).

Fig. 4

UFP increased functional molecule expression on Nrf2^+/+ and Nrf2^−/− DC surface in a similar fashion. Surface molecule expression was analyzed by flow cytometry as described in Materials and Methods. a MHC II. b CD80. c CD86. * p < 0.01 compared with respective control.

Fig. 5

The effect of UFP on cytokine production by Nrf2^+/+ and Nrf2^−/− DC after 16-hour exposure. Cytokine levels in the DC culture media were measured by ELISA as described in Materials and Methods. a IL-12p70; * p < 0.05 compared with respective control. b IL-6; * p < 0.01 compared with respective control. c IL-1β; * p < 0.001 compared with respective control.

Fig. 6

Recipient mice developed a stronger allergic airway inflammation when sensitized by OVA/UFP-exposed Nrf2^−/− DC via adoptive transfer. Adoptive transfer and secondary OVA aerosol challenge were performed as described in Materials and Methods. a BAL cellular differentiation showing that sensitization by OVA/UFP-treated Nrf2^−/− DC led to eosinophilic inflammation in the lungs; * p < 0.01 compared with respective saline control, ^a p < 0.01 compared with respective OVA group. b Increased OVA-IgG1 in the blood of recipient mice sensitized by OVA/UFP-exposed DC; * p < 0.02 compared with all other groups. c Elevated IL-13 level in the BAL fluid; * p < 0.01 compared with respective saline control, ^a p < 0.05 compared with respective OVA group.

Fig. 7

a-g Lung histology showing a severe inflammation in the mice sensitized by OVA/UFP-pulsed Nrf2^−/− DC. Note the multiple inflammatory foci along the conducting airway (g).