DEPs Induce Local Ige Class Switching Independent of Their Ability to Stimulate iBALT de Novo Formation

Abstract

Background: Diesel exhaust particles (DEPs) are leading to a general increase in atopic diseases worldwide. However, it is still unknown whether DEPs induce systemic B-cell IgE class switching in secondary lymphoid organs or locally in the lungs in inducible bronchus-associated lymphoid tissue (iBALT). The aim of this work was to identify the exact site of DEP-mediated B-cell IgE class switching and pro-allergic antibody production.

Methods: We immunized BALB/c mice with different OVA doses (0.3 and 30 µg) intranasally in the presence and absence of two types of DEPs, SRM1650B and SRM2786. We used low (30 µg) and high (150 µg) DEP doses.

Results: Only a high DEP dose induced IgE production, regardless of the particle type. Local IgE class switching was stimulated upon treatment with both types of particles with both low and high OVA doses. Despite the similar ability of the two standard DEPs to stimulate IgE production, their ability to induce iBALT formation and growth was markedly different upon co-administration with low OVA doses.

Conclusions: DEP-induced local IgE class switching takes place in preexisting iBALTs independent of de novo iBALT formation, at least in the case of SRM1650B co-administered with low OVA doses.

Keywords: antibody production; antigen doses; diesel particulate matter; local Ig class switch; lungs; tertiary lymphoid structures.

Figure 1

Study design. Black arrows indicate i.n. immunization of mice with OVA alone at a 0.3 µg or 30 µg dose or in combination with DEPs at a low (30 µg) or high (150 µg) dose. Animals immunized with saline alone without OVA were used as a control group. Green arrows indicate a high-dose (250 µg) OVA i.n. challenge. The time points when the mice were killed and when the samples were collected are indicated by red arrows.

Figure 2

Antibody production in response to low OVA doses, DEPs, or their combination. BALB/c mice were immunized 2–3 times a week for 8 weeks (a total of 18 immunizations) via the i.n. route with 0.3 µg of OVA alone or with different types of DEP at 30 or 150 µg. The titers of OVA-specific IgE (A), IgG1 (C), IgG2a (D), and total IgE (B) were measured. The statistically significant differences of the groups immunized with OVA with particles vs. the OVA control are shown with blue bars, and the statistically significant differences of the groups immunized with particles alone vs. the saline control are shown by black bars. (*—p < 0.05; **—p < 0.01). The experiment was performed three times. The graphs show the data from three representative experiments (total number of mice included in analysis from all experiments n = 6–7 per group). The data indicate biological replicates obtained from individual mice.

Figure 3

Cell response to DEPs. BALB/c mice were immunized with a low dose of OVA, 0.3 µg (A–C), or high dose, 30 µg (D–F), alone or with 150 µg of DEP1. After this, the mice were challenged three times a week for two weeks with 250 µg of OVA and sacrificed. BALs were taken, and the eosinophils (Eo), neutrophils (Neu), and alveolar macrophages (Mϕ) were quantified with flow cytometry. The experiment was performed three times. Representative data are from one out of three independent experiments. Significant differences vs. PBS are shown with black bars; those vs. 0.3 µg of OVA with blue ones; and those vs. 30 µg of OVA with red bars (*—p < 0.05; **—p < 0.01). The graphs show the data from three representative experiments (total number of mice included in analysis from all experiments n = 5 per group). The data indicate biological replicates obtained from individual mice.

Figure 4

Expression of transcripts corresponding to B-cell Ig class switching and antibody production in lung tissue and regional lymph nodes. BALB/c mice were immunized 2–3 times a week for eight weeks (a total of 18 immunizations) via the i.n. route without OVA (Saline), with a low (0.3 µg) OVA dose or high (30 µg) OVA dose, without particles (control), or with either DEP1 or DEP2 in high (150 µg) dose. After immunization, the mice were sacrificed. The expression of transcripts corresponding to IgE (germline ε) (A,E) and IgG1 (germline γ1) (B,F) class switching, as well as transcripts corresponding to mature IgE (postswitch ε) (C,G)- and IgG1 (postswitch γ1) (D,H)-producing cells were measured in lung tissue (A–D) and regional lymph nodes (E–H). Black bars indicate significant (*—p < 0.05; **—p < 0.01) differences between mice immunized without OVA with different DEPs and the respective control without DEPs; green bars indicate significant differences between mice immunized with low OVA doses with or without DEPs and the respective control groups; red bars indicate significant differences between mice immunized with high OVA doses with or without DEPs and the respective control groups. The experiment was performed three times. The graphs show the data from three representative experiments (total number of mice included in analysis from all experiments n = 6 per group). The data indicate biological replicates obtained from individual mice.

Figure 5

Histological analysis of lung tissue after prolonged OVA and DEP administration. BALB/c mice were immunized 2–3 times a week for eight weeks (a total of 18 immunizations) via the i.n. route with the indicated doses of OVA and different doses of DEP1 or DEP2. Following immunization, the mice were sacrificed and the lungs were isolated and stained for histological analysis (H&E). Relative areas of iBALT on histological sections of mice immunized with PBS and low OVA doses with or without DEPs in either 30 or 150 µg dose (A) or with high OVA doses with or without the same doses of DEPs (B), and representative histological images (100×) (samples from mice immunized with or without DEPs in 150 µg dose) (C) are shown. Red arrows correspond to DEPs; blue arrows show iBALTs. Black bars show significant differences between the groups immunized with particles alone and PBS control (*—p < 0.05; **—p < 0.01). Blue and red bars show differences between the mice immunized with low (blue) or high (red) OVA doses together with DEPs and mice immunized with OVA alone. The experiment was performed twice. The graphs show the data from three representative experiments (total number of mice included in analysis from all experiments n = 6–7 per group). The data indicate biological replicates obtained from individual mice.

Figure 6

Production of iBALT-inducing chemokines and expression of cytokines in the lung tissue of immunized mice. BALB/c mice were immunized as described in Figure 4 with or without high (150 µg) DEPs doses. Control mice were immunized in the absence of DEPs. The production of CXCL13 (A), the expression of the iBALT-promoting cytokines Tnfa (B) and Ifna1 (C), type-2 immune responses promoting the cytokines Il4 (D) and Il13 (E), and type-1 immune responses promoting cytokine Ifng (F) were measured in the lung tissue. Black bars indicate significant (*—p < 0.05; **—p < 0.01) differences between the mice immunized without OVA with different DEPs and the respective control without DEPs; green bars indicate significant differences between the mice immunized with a low OVA dose with or without DEPs and the respective control groups; red bars indicate significant differences between mice immunized with a high OVA dose with or without DEPs and respective control groups. The experiment was performed three times. The graphs show the data from three representative experiments (total number of mice included in analysis from all experiments n = 6 per group). The data indicate biological replicates obtained from individual mice.

Figure 7

Expression of B-cell-related genes in lung tissue. BALB/c mice were immunized as described in Figure 4 with or without high (150 µg) DEPs doses. Control mice were immunized in the absence of DEPs. Expression of markers for relative B-cell numbers (Cd19; (A)), germinal center formation (Bcl6; (B)), and B-cell extrafollicular activation (Ebi2; (C)) is shown. Black bars indicate significant (*—p < 0.05; **—p < 0.01) differences between the mice immunized without OVA with different DEPs and the respective control without DEPs; green bars indicate significant differences between the mice immunized with low OVA doses with or without DEPs and the respective control groups; red bars indicate significant differences between the mice immunized with high OVA doses with or without DEPs and the respective control groups. The experiment was performed three times. The graphs show the data from three representative experiments (total number of mice included in analysis from all experiments n = 6 per group). The data indicate biological replicates obtained from individual mice.