Fine particulate matter induces airway inflammation by disturbing the balance between Th1/Th2 and regulation of GATA3 and Runx3 expression in BALB/c mice

Abstract

The present study aimed to examine the effects of 2.5 µm particulate matter (PM2.5) on airway inflammation and to investigate the possible underlying mechanism. Specifically, the focus was on the imbalance of T helper (Th)1/Th2 cells and the dysregulated expression of transcription factors, including trans‑acting T cell‑specific transcription factor 3 (GATA3), runt‑related transcription factor 3 (Runx3) and T‑box transcription factor TBX21 (T‑bet). In this study, ambient PM2.5 was collected and analyzed, male BALB/c mice were sensitized and treated with PBS, ovalbumin (OVA), PM2.5 or OVA + PM2.5. The effects of PM2.5 alone or PM2.5 + OVA on immunopathological changes, the expression of transcription factors GATA3, Runx3 and T‑bet, and the imbalance of Th1/Th2 were investigated. It was found that PM2.5 + OVA co‑exposure significantly enhanced inflammatory cell infiltration, increased higher tracheal secretions in lung tissue and upregulated respiratory resistance response to acetylcholine compared with PM2.5 or OVA single exposure and control groups. In addition, higher protein and mRNA expression levels of Th2 inflammatory mediators interleukin (IL)‑4, IL‑5 and IL‑13 in bronchoalveolar lavage fluid were observed in PM2.5 + OVA treated mice, whereas the expression levels of GATA3 and STAT6 were exhibited in mice exposed to OVA + PM2.5 compared with the OVA and PM2.5 groups. By contrast, PM2.5 exposure decreased the protein and mRNA expression levels of Th1 cytokine interferon‑γ and transcription factors Runx3 and T‑bet, especially among asthmatic mice, different from OVA group, PM2.5 exposure only failed to influence the expression of T‑bet. To conclude, PM2.5 exposure evoked the allergic airway inflammation response, especially in the asthmatic mouse model and led to Th1/Th2 imbalance. These effects worked mainly by upregulating GATA3 and downregulating Runx3. These data suggested that Runx3 may play an important role in PM2.5‑aggravated asthma in BALB/c mice.

Keywords: 2.5 µm particulate matter; runt‑related transcription factor 3; trans‑acting T cell‑specific transcription factor GATA‑3; T helper 1/2; asthma.

Figure 1.

Overview of the experimental design. On days 0, 7 and 14 mice in the OVA and OVA + PM2.5 groups were i.p. injected with 100 µg OVA and alum adjuvant, whereas mice in the control and PM2.5 groups were administered an equivalent volume of PBS (i.p.). A total of 100 µg PM2.5 particles in 50 µl physiological saline was administered intranasally to mice in the PM2.5 and OVA + PM2.5 groups from days 16–20, an equivalent volume of PBS was administered intranasally to mice in the control and OVA group. Mice, except for those in the control group, were challenged with aerosolized 1% OVA (total volume, 100 ml) for 30 min from days 21–25. OVA, ovalbumin; PM2.5, 2.5 µm particulate matter; i.p., intraperitoneally; PBS, phosphate-buffer saline.

Figure 2.

PM2.5 exposure exacerbates airway hyper-responsiveness, especially in asthmatic mice. Data are expressed as the mean ± SD; n=12; *P<0.05 vs. Control; ^∆P<0.05 vs. PM2.5; ^#P<0.05 vs. OVA. OVA, ovalbumin; PM2.5, 2.5 µm particulate matter; RL, resistance of the lung.

Figure 3.

Expression of serum OVA-specific IgE and inflammatory cytokines in BALF. (A) Expression of OVA-specific IgE in serum. Expression of (B) IL-4, (C) IL-5, (D) IL-13 and (E) IFN-γ in BALF. Data are expressed as the mean ± SD; n=12; *P<0.05 vs. Control; ^∆P<0.05 vs. PM2.5; ^#P<0.05 vs. OVA. OVA, ovalbumin; BALF, bronchoalveolar lavage fluid; PM2.5, 2.5 µm particulate matter; IFN-γ, interferon; IL-, interleukin.

Figure 4.

Total number of inflammatory cells in the BALF from the right lungs of mice. Wright-Giemsa staining method was performed to show the (A) total number of inflammatory cells, and the differential expression of types of inflammatory cells, including (B) macrophages, (C) neutrophils, (D) eosinophils and (E) lymphocytes, in the BALF from the right lungs of mice. Data are expressed as the mean ± SD; n=12; *P<0.05 vs. Control; ^∆P<0.05 vs. PM2.5; ^#P<0.05 vs. OVA. BALF, bronchoalveolar lavage fluid; OVA, ovalbumin; PM2.5, 2.5 µm particulate matter.

Figure 5.

PM2.5 exposure aggravates airway inflammation and mucus secretion, especially in asthmatic mice. (A) Hematoxylin and eosin and PAS staining images (magnification, ×200). Inflammation indicated by red arrows. Quantitative analysis of mean (B) linear intercept, (C) matrix membrane layer, (D) smooth muscle layer and (E) PAS⁺ cells per bronchus using Axiovision Viewer Image software. Data are expressed as the mean ± SD; n=6; *P<0.05 vs. Control; ^∆P<0.05 vs. PM2.5; ^#P<0.05 vs. OVA. OVA, ovalbumin; PM2.5, 2.5 µm particulate matter; PAS, Periodic Acid Schiff.

Figure 6.

Protein expression of GATA3, STAT6, T-bet and Runx3 in the lung tissues of mice. (A) Western blotting images and (B) semi-quantification of the protein expression of GATA3, STAT6, T-bet and Runx3. Data are expressed as the mean ± SD; n=6; *P<0.05 vs. Control; ^∆P<0.05 vs. PM2.5; ^#P<0.05 vs. OVA. OVA, ovalbumin; PM2.5, 2.5 µm particulate matter; GATA3, trans-acting T cell-specific transcription factor GATA-3; T-bet, T-box transcription factor TBX21; Runx3, runt-related transcription factor 3.

Figure 7.

mRNA expression levels of IL-4, IL-5, IL-13, IFN-γ, GATA3, T-bet and Runx3 in the lung tissues of mice. Data are expressed as the mean ± SD; n=6; *P<0.05 vs. Control; ^∆P<0.05 vs. PM2.5; ^#P<0.05 vs. OVA. OVA, ovalbumin; PM2.5, 2.5 µm particulate matter; GATA3, trans-acting T cell-specific transcription factor GATA-3; T-bet, T-box transcription factor TBX21; Runx3, runt-related transcription factor 3; IFN-γ, interferon; IL-, interleukin.

Figure 8.

Percentage of Th1 and Th2 cells was measured using flow cytometry and the ratio of Th1 and Th2 cells was assessed. (A) Flow cytometry plots and quantification of (B) the percentage of Th1 cells, (C) the percentage of Th2 cells and (D) the Th1/Th2 ratio. Data are expressed as the mean ± SD; n=6; *P<0.05 vs. Control; ^∆P<0.05 vs. PM2.5; ^#P<0.05 vs. OVA. OVA, ovalbumin; PM2.5, 2.5 µm particulate matter; Th, T helper cells.