Stimulation of allergen-loaded macrophages by TLR9-ligand potentiates IL-10-mediated suppression of allergic airway inflammation in mice

Abstract

Background: Previously, we demonstrated that OVA-loaded macrophages (OVA-Mphi) partially suppress OVA-induced airway manifestations of asthma in BALB/c mice. In vitro studies showed that OVA-Mphi start to produce IL-10 upon interaction with allergen-specific T cells, which might mediate their immunosuppressive effects. Herein, we examined whether IL-10 is essential for the immunosuppressive effects of OVA-Mphi in vivo, and whether ex vivo stimulation of the IL-10 production by OVA-Mphi could enhance these effects.

Methods: Peritoneal Mphi were loaded with OVA and stimulated with LPS or immunostimulatory sequence oligodeoxynucleotide (ISS-ODN) in vitro. The increase of IL-10 production was examined and, subsequently, ex vivo stimulated OVA-Mphi were used to treat (i.v.) OVA-sensitized mice. To further explore whether Mphi-derived IL-10 mediates the immunosuppressive effects, Mphi isolated from IL-10-/- mice were used for treatment.

Results: We found that stimulation with LPS or ISS-ODN highly increased the IL-10 production by OVA-Mphi (2.5-fold and 4.5-fold increase, respectively). ISS-ODN stimulation of OVA-Mphi significantly potentiated the suppressive effects on allergic airway inflammation. Compared to sham-treatment, ISS-ODN-stimulated OVA-Mphi suppressed the airway eosinophilia by 85% (vs. 30% by unstimulated OVA-Mphi), IL-5 levels in bronchoalveolar lavage fluid by 80% (vs. 50%) and serum OVA-specific IgE levels by 60% (vs. 30%). Importantly, IL-10-/-Mphi that were loaded with OVA and stimulated with ISS-ODN ex vivo, failed to suppress OVA-induced airway inflammation.

Conclusions: These results demonstrate that Mphi-derived IL-10 mediates anti-inflammatory responses in a mouse model of allergic asthma, which both can be potentiated by stimulation with ISS-ODN.

Figure 1

LPS and immunostimulatory sequence oligodeoxynucleotide (ISS-ODN) enhance the IL-10 production by OVA-Mφ. 1 × 10⁵ Mφ/well were loaded with 2 mg/ml OVA and stimulated with either 10 μg/ml LPS or 3 μg/ml ISS-ODN for 20 h. As a control, Mφ were stimulated with mutated-ODN (M-ODN, 3 μg/ml). One of four representative experiments is shown.

Figure 2

OVA-specific IgE levels in serum are significantly suppressed upon treatment with ISS-ODN-stimulated and OVA-loaded Mφ. OVA-sensitized BALB/c mice were treated (i.v.) with saline (sham), OVA-Mφ, ISS-ODN-stimulated OVA-Mφ (ISS-ODN/OVA-Mφ), or LPS-stimulated OVA-Mφ (LPS/OVA-Mφ). Subsequently, these mice were challenged by OVA inhalation. Serum OVA-specific IgE levels were measured prior to and after challenge. Values are expressed as the mean ± SEM (n = 6 to 8). *P < .05 compared with sham-treated and OVA-challenged mice. ^†P < .05 compared with mice treated with OVA-Mφ and that were OVA-challenged.

Figure 3

ISS-ODN-stimulated and OVA-loaded Mφ (ISS-ODN/OVA-Mφ) significantly suppress airway eosinophilia and IL-5 levels in the bronchoalveolar lavage fluid. The number of eosinophils (eo), neutrophils (neutro) and mononuclear cells (MNC) in the BALF (A), and IL-5 levels in the BALF (B) after OVA inhalation challenge. Values are expressed as the mean ± SEM (n = 6 to 8). *P < .01 and ^†P < .05 compared with sham-treated mice. ^‡P < .01 and ^§P < .05 compared with mice treated with OVA-Mφ.

Figure 4

The suppression of OVA-specific IgE in serum by ISS-ODN-stimulated and OVA-loaded Mφ is partially mediated by IL-10. Sensitized mice were treated (i.v.) with saline (sham), ISS-ODN-stimulated OVA-Mφ (ISS-ODN/OVA-Mφ), ISS-ODN-stimulated IL-10^-/- Mφ (ISS-ODN/IL10^-/-Mφ), or ISS-ODN-stimulated IL-10^-/- OVA-Mφ (ISS-ODN/IL10^-/-OVA-Mφ). Serum OVA-specific IgE levels were measured prior to and after OVA challenge. Values are expressed as the mean ± SEM (n = 6 to 8 per group). *P < .05 compared with sham-treated and OVA-challenged mice. ^†P < .05 compared with mice treated with ISS-ODN/IL10^-/-OVA-Mφ and that were OVA-challenged.

Figure 5

IL-10 is crucial in the suppression of airway eosinophilia and IL-5 levels in the bronchoalveolar lavage fluid by ISS-ODN-stimulated and OVA-loaded Mφ (ISS-ODN/OVA-Mφ). (A) Number of eosinophils (eo), neutrophils (neutro) and mononuclear cells (MNC) in the BALF after OVA challenge. (B) Levels of IL-5 in the BALF after OVA challenge. Values are expressed as the mean ± SEM (n = 6 to 8 per group). *P < .05 compared with sham-treated mice. ^†P < .05 compared with mice treated with ISS-ODN/IL10^-/-OVA-Mφ.