A low dose of Mycoplasma pneumoniae infection enhances an established allergic inflammation in mice: the role of the prostaglandin E2 pathway

Abstract

Background: Over 40% of chronic stable asthma patients have evidence of respiratory Mycoplasma pneumoniae (Mp) infection as detected by PCR, but not by serology and culture, suggesting that a low-level Mp is involved in chronic asthma. However, the role of such a low-level Mp infection in the regulation of allergic inflammation remains unknown.

Objective: To determine the impact of a low-level Mp infection in mice with established airway allergic inflammation on allergic responses such as eosinophilia and chemokine eotaxin-2, and the underlying mechanisms [i.e. the prostaglandin E(2) (PGE(2)) pathway] since PGE(2) inhalation before an allergen challenge suppressed the eosinophil infiltration in human airways.

Methods: BALB/c mouse models of ovalbumin (OVA)-induced allergic asthma with an ensuing low- or high-dose Mp were used to assess IL-4 expression, bronchoalveolar lavage (BAL) eosinophil, eotaxin-2 and PGE(2) levels, and lung mRNA levels of microsomal prostaglandin E synthase-1 (mPGES-1). Primary alveolar macrophages (pAMs) from naïve BALB/c mice were cultured to determine whether Mp-induced PGE(2) or exogenous PGE(2) down-regulates IL-4/IL-13-induced eotaxin-2.

Results: Low-dose Mp in allergic mice significantly enhanced IL-4 and eotaxin-2, and moderately promoted lung eosinophilia, whereas high-dose Mp significantly reduced lung eosinophilia and tended to decrease IL-4 and eotaxin-2. Moreover, in both OVA-naïve and allergic mice, lung mPGES-1 mRNA and BAL PGE(2) levels were elevated in mice infected with high-dose, but not low-dose Mp. In pAMs, IL-4/IL-13 significantly increased eotaxin-2, which was reduced by Mp infection accompanied by dose-dependent PGE(2) induction. Exogenous PGE(2) inhibited IL-4/IL-13-induced eotaxin-2 in a dose-dependent manner.

Conclusions: This study highlights a novel concept on how different bacterial loads in the lung modify the established allergic airway inflammation and thus interact with an allergen to further induce Th2 responses. That is, unlike high-level Mp, low-level Mp fails to effectively induce PGE(2) to down-regulate allergic responses (e.g. eotaxin-2), thus maintaining or even worsening allergic inflammation in asthmatic airways.

Fig. 1

Low-dose Mp infection in allergic mice enhances IL-4 expression. Intranasal inoculation of low-dose (10³ CFU/mouse), high-dose (10⁷ CFU/mouse) Mp or saline was performed in OVA-allergic mouse models (n=6/group) as described in the Methods. Lungs were collected on day 7 post-Mp or –saline. (A) IL-4 mRNA relative levels in the lung tissue. (B) IL-4 mRNA relative levels in isolated lung CD4+ T cells. Gene expression was evaluated by quantitative real-time RT-PCR. Data are expressed as medians (25–75% range). (C) Upper panel: representative dot-plot of gating and data analysis of the CD4+ T cells expressing IL-4 protein in whole lung cells were quantified using intracellular cytokine staining and flow cytometry (a: gating for viable lymphocytes; b: gating for CD3+CD4+ T cells; c: % of CD3+CD4+ T cells expressing IL-4 protein). Lower panel: The percentages of CD3+CD4+ T cells expressing IL-4 protein. Data are expressed as means ± SEM. CFU, colony forming unit; Mp, Mycoplasma pneumoniae; Sal, saline.

Fig. 2

Low-dose Mp infection in allergic mice enhances eotaxin-2 production. Intranasal inoculation of low-dose (10³ CFU/mouse), high-dose (10⁷ CFU/mouse) Mp or saline was performed in OVA-allergic mouse models (n=6/group) as described in the Methods. BAL fluid was collected on day 7 post-Mp or -saline for eotaxin-2 measurement by using ELISA. Data are expressed as means ± SEM. BAL, bronchoalveolar lavage; CFU, colony forming unit; Mp, Mycoplasma pneumoniae; Sal, saline.

Fig. 3

High-dose Mp infection in allergic mice induces lung mPGES-1 mRNA expression. Intranasal inoculation of low-dose (10³ CFU/mouse), high-dose (10⁷ CFU/mouse) Mp or saline was performed in OVA-allergic mouse models (n=6/group) as described in the Methods. Lungs were collected on day 7 post-Mp or –saline for mPGES-1 mRNA quantification by quantitative real-time RT-PCR. Data are expressed as medians (25–75% range). CFU, colony forming unit; Mp, Mycoplasma pneumoniae; mPGES-1, microsomal prostaglandin E synthase-1; Sal, saline.

Fig. 4

Mp infection inhibits eotaxin-2, but increases PGE₂ production in IL-4/IL-13-treated alveolar macrophages. BAL cells from naïve BALB/c mice were seeded in triplicate (5 × 10⁵ cells/well) per condition into 48-well culture plates, and incubated for 2 h at 37°C, 5% CO₂. Thereafter, adherent cells (alveolar macrophages) were washed in phosphate-buffered saline (PBS) and treated with recombinant mouse IL-4 and IL-13 (10 ng/ml each) or BSA (20 ng/ml) 2 h prior to PBS or Mp infection (0.01, 0.1, 1 and 10 CFU/cell), and further incubated for 24 h. Cell culture supernatants were collected for eotaxin-2 (A) or PGE₂ (B) ELISA. Data are from five independent experiments and expressed as means ± SEM. BSA, bovine serum albumin; CFU, colony forming unit; Mp, Mycoplasma pneumoniae; PGE₂, prostaglandin E₂.

Fig. 5

Exogenous PGE₂ directly inhibits IL-4/IL-13-induced eotaxin-2 production in alveolar macrophages. BAL cells from naïve BALB/c mice were seeded in triplicate (5 × 10⁵ cells/well) per condition into 48-well culture plates, and incubated for 2 h at 37°C, 5% CO₂. Thereafter, adherent cells were washed in phosphate-buffered saline (PBS) and treated with recombinant mouse IL-4 and IL-13 (10 ng/ml each) or BSA (20 ng/ml) 2 h prior to dimethyl sulfoxide (DMSO, control) or PGE₂ (1- 1000nM) in DMSO, and further incubated for 24 h. Cell culture supernatants were collected for eotaxin-2 ELISA. Data are from three independent experiments and expressed as means ± SEM. BSA, bovine serum albumin; PGE₂, prostaglandin E₂.