Different airborne particulates trigger distinct immune pathways leading to peanut allergy in a mouse model

Abstract

Background: Environmental exposure to peanut through non-oral routes is a risk factor for peanut allergy. Early-life exposure to air pollutants, including particulate matter (PM), is associated with sensitization to foods through unknown mechanisms. We investigated whether PM promotes sensitization to environmental peanut and the development of peanut allergy in a mouse model.

Methods: C57BL/6J mice were co-exposed to peanut and either urban particulate matter (UPM) or diesel exhaust particles (DEP) via the airways and assessed for peanut sensitization and development of anaphylaxis following peanut challenge. Peanut-specific CD4⁺ T helper (Th) cell responses were characterized by flow cytometry and Th cytokine production. Mice lacking select innate immune signaling genes were used to study mechanisms of PM-induced peanut allergy.

Results: Airway co-exposure to peanut and either UPM- or DEP-induced systemic sensitization to peanut and anaphylaxis following peanut challenge. Exposure to UPM or DEP triggered activation and migration of lung dendritic cells to draining lymph nodes and induction of peanut-specific CD4⁺ Th cells. UPM- and DEP-induced distinct Th responses, but both stimulated expansion of T follicular helper (Tfh) cells essential for peanut allergy development. MyD88 signaling was critical for UPM- and DEP-induced peanut allergy, whereas TLR4 signaling was dispensable. DEP-induced peanut allergy and Tfh-cell differentiation depended on IL-1 but not IL-33 signaling, whereas neither cytokine alone was necessary for UPM-mediated sensitization.

Conclusion: Environmental co-exposure to peanut and PM induces peanut-specific Tfh cells and peanut allergy in mice.

Keywords: T follicular helper cells; air pollution; exposome; particulate matter; peanut allergy.

FIGURE 1

Airway co-exposure to peanut and particulate matter induces peanut allergy in mice. (A) Experimental model for airway co-exposure to peanut (PN) and either UPM or DEP. (B) Serum peanut-specific IgE and IgG1 were measured on Day 14. Bars represent means ± SEM (n = 10–13 mice per group). Shown is the combined data from three experiments. *p < .05, ***p < .001 (compared to the PN group), one-way ANOVA. (C) Core body temperatures after i.p. challenge to peanut on Day 17. Symbols represent means ± SEM (n = 9–10 mice per group). Shown is the combined data from three experiments. *p < .05, ***p < .001 (compared to the PN group), two-way ANOVA.

FIGURE 2

Inhaled PM induces activation and migration of peanut-laden cDCs to lung-draining lymph nodes. (A–C). Mice were co-exposed once to fluorescently labeled peanut (PN-AF647) and either UPM or DEP. After 24 h, mediastinal lymph nodes (mLN) were collected for analysis. (A) Gating strategy for identification of peanut-laden (PN-AF647⁺) migratory cDCs in mLN by flow cytometry. The representative cytograms show peanut uptake by cDC1 and cDC2 from mice co-exposed to UPM and PN-AF647 (red histograms). Mice treated with UPM and unlabeled PN were included for gating controls (gray histograms). (B) Numbers and percentages of migratory PN-AF647⁺ cDC1s and cDC2s in mLN. (C) Median fluorescence intensity (MFI) of CD40 and CD86 display by PN-AF647⁺ cDC1s and cDC2s in mLN. Bars represent means ± SEM (n = 6–10 per group). Shown is the combined data from two experiments. *p < .05, **p < .01, ***p < .001 (compared to the PN group), two-way ANOVA.

FIGURE 3

UPM and DEP induce distinct T helper cell responses to peanut. (A–C). Mice were co-exposed to peanut (PN) and either UPM or DEP, as in Figure 1A. On Day 14, mLN were collected for analysis. (A) Representative cytograms (gated on TCRβ⁺CD4⁺ cells) showing the frequency of peanut-specific (CD154⁺CD44^hi) T cells following stimulation with peanut protein or media for 6 h. Numbers indicate the percentage of gated cells. (B) Percentages of peanut-specific CD4⁺ T cells following stimulation with media or peanut. **p < .01, paired t-test. (C) Th cytokine levels in mLN cell culture supernatants following stimulation with peanut protein for 4 days. Bars represent means ± SEM (n = 8 = 9 mice per group). Shown is the combined data from two experiments. *p < .05, **p < .01, ***p < .001 (compared to the PN group), one-way ANOVA.

FIGURE 4

UPM and DEP stimulate the expansion of T follicular helper (Tfh) cells, which are essential for PM-induced peanut allergy. (A) Tfh cell numbers (left) and percentages (right) in mLN from mice co-exposed to peanut (PN) and either UPM or DEP on Day 14 of sensitization. Bars represent means ± SEM (n = 13–14 mice per group). Shown is the combined data from three experiments. *p < .05, **p < .01 (compared to the PN group), one-way ANOVA. (B) Representative cytograms (gated on CD4⁺CD44^hiPD-1^hiCXCR5^hi cells) showing the frequency of IL-4⁺IL-13⁺ Tfh (Tfh13) and IL-4⁺IL-13⁻ Tfh (Tfh2) cells in mLN on Day 14. (C) Percentages of Tfh13 and Tfh2 cells on Day 14. Bars represent means ± SEM (n = 5 mice per group). *p < .05, **p < .01, ***p < .001 (compared to PN group), one-way ANOVA. (D and E) Cd4^WTBcl6^fl/fl or Cd4^CreBcl6^fl/fl mice were co-exposed to PN and either UPM or DEP, as in Figure 1A. (D) Serum peanut-specific IgE and IgG1 were measured on Day 14. Bars represent means ± SEM (n = 6–9 mice per group), **p < .01, ***p < .001, unpaired t-test. (E) Core body temperatures after i.p. challenge to peanut on Day 17. Symbols represent means ± SEM (n = 6–9 mice per group). **p < .01, ***p < .001, two-way ANOVA.

FIGURE 5

PM-mediated sensitization to peanut is dependent upon MyD88 signaling. (A and B). WT, Myd88^−/−, or Tlr4^−/− mice were co-exposed to peanut (PN) and either UPM or DEP, as in Figure 1A. (A) Serum peanut-specific IgE and IgG1 were measured on Day 14. Bars represent means ± SEM (n = 7–13 mice per group). *p < .05, **p < .01, ***p < .001 (compared to WT), one-way ANOVA. (B) Core body temperatures after i.p. challenge to peanut on Day 17. Symbols represent means ± SEM (n = 7–13 mice per group). Shown is the combined data from three experiments. *p < .05, **p < .01, ***p < .001 (compared to WT), two-way ANOVA.

FIGURE 6

IL-1 signaling is required for DEP-mediated airway sensitization to peanut and induction of peanut-specific Tfh cells. (A–E). WT, Il1r1^−/−, or Il33^−/− mice were co-exposed to peanut (PN) and either UPM or DEP, as in Figure 1A. (A) Serum peanut-specific IgE and IgG1 were measured on Day 14. Bars represent means ± SEM (n = 6–13 mice per group). **p < .01, ***p < .001, one-way ANOVA. (B) Core body temperatures after i.p. challenge to peanut on Day 17. Symbols represent means ± SEM (n = 6–13 mice per group). Shown is the combined data from five experiments. *p < .05, **p < .01, ***p < .001, two-way ANOVA. (C) Percentages of total Tfh cells in mLN on Day 14. (D) Representative cytograms (gated on Tfh cells) showing the frequency of peanut-specific (CD154⁺CD44^hi) Tfh cells following stimulation with peanut protein or media for 6 h. Numbers indicate the percentage of gated cells. (E) Percentages of peanut-specific Tfh cells in mLN on Day 14. Shown is the combined data from two experiments. (F–G). WT, Il1r1^−/− or Il33^−/− mice were co-exposed to PN-AF647 and DEP. After 24 h, mLN were collected for analysis. (F) Number of PN-AF647⁺ cDC1s and cDC2s in mLN. (G) Median fluorescence intensity (MFI) of CD86 display by PN-AF647⁺ cDC1s and cDC2s in mLN. Bars represent means ± SEM (n = 6–13 per group). Shown is the combined data from three experiments. *p < .05, **p < .01, ***p < .001, one-way ANOVA. ns, not significant.