TLR2 deficiency promotes IgE and inhibits IgG1 class-switching following ovalbumin sensitization

Abstract

Background: To explore the roles of Toll-like receptor (TLR)2 in Th2 cytokine production and immunoglobulin (Ig) class switching following ovalbumin (OVA) sensitization.

Methods: TLR2^-/- and wild-type C57BL/6 mice were sensitized by intraperitoneal injection with OVA. Lung pathology was assessed by hematoxylin and eosin staining. Abundance of interleukin (IL)4, IL5, IL13, and IL21 transcripts in the lungs was quantified by RT-PCR. OVA-specific IgG1, IgG2a, IgG2b, IgE and IgM were quantified by enzyme-linked immunosorbent assay. Phosphorylated signal transducer and activator of transcription (STAT)3 in lung tissue was detected by immunohistochemistry staining and nuclear factor (NF) κB activation was measured by immunofluorescence staining. STAT3 activation was inhibited using cryptotanshinone (CPT) treatment. Germline transcripts (Iμ-Cμ, Iγ-Cγ, Iα-Cα or Iε-Cε), post-recombination transcripts (Iμ-Cγ, Iμ-Cα or Iμ- Cε) and mature transcripts (V_HDJ_H-Cγ, V_HDJ_H-Cα or V_HDJ_H-Cε) were analyzed from splenic B cells of OVA-sensitized wild-type mice (with or without CPT treatment) and TLR2^-/- mice (with or without IL21 treatment).

Results: The lungs of TLR2^-/- mice showed a lesser degree of inflammation than wild-type mice after OVA sensitization. Following OVA sensitization, levels of IL4, IL13, and IL21, but not IL5, were significantly lower in TLR2^-/- compared with wild-type mice. Moreover, OVA-specific IgG1 and IgE titers were markedly lower and higher, respectively, in TLR2^-/- mice. TLR2 deficiency inhibited STAT3 activation but not NF-κB p65 activation. CPT treatment reduced IgG1 titers via inhibition of Stat3 phosphorylation. Both TLR2 knockout and CPT treatment reduced the frequencies of Iγ1-Cγ1, Iγ3-Cγ3 and Iα-Cα transcripts, but IL21 treatment compensated for the effects of TLR2 deficiency.

Conclusion: These results suggest a role of TLR2 in restricting OVA-sensitized lung inflammation via promotion of IgG1 and inhibition of IgE class switching regulated by IL21 and STAT3.

Keywords: IL21; IgE; IgG1; Ovalbumin; STAT3; Sensitization; TLR2.

Fig. 1

The lungs of TLR2^−/− mice showed less inflammation following OVA sensitization compared with wild-type mice. A Lungs were fixed, embedded in paraffin and cut into 3-μm thick sections. Lung sections were stained with hematoxylin and eosin (HE) and photographed using an Olympus microscope. Magnification: 200-fold; B Cytokines were measured in Lung tissue or B-cell by RT-PCR. Data are shown individually and as the mean ± s.e.m. IL4, IL13 and IL21 expression decreased more significantly following OVA sensitization in TLR2^−/− mice compared with wild-type mice as shown. Data for IL4, IL5, IL13 and IL21 were normalized to wild-type mice treated with normal saline (NS). *: p < 0.05, n = 6 mice per group

Fig. 2

Serum titers of OVA-specific IgG₁(1), IgA (2), IgG_2a (3), IgM (4), IgG_2b (5) and IgE (6) in wild-type and TLR2^−/− mice were detected by ELISA. Data are shown individually and as the mean ± s.e.m. OVA-specific IgG1 and IgE titers were markedly lower and higher, respectively, in TLR2^−/− mice. *: p < 0.05, n = 6 mice per group

Fig. 3

Immunofluorescence staining of NF-κB p65 (PE, red) showed that p65 was not detectable in nuclei (DAPI, blue). Bar = 30 μm

Fig. 4

Immunohistochemistry staining of p-STAT3 showed lower numbers of positive epithelial cells in the lungs of OVA-sensitized TLR2^−/− mice compared with those of OVA-sensitized wild-type mice. Magnification: 200-fold. *: p < 0.05, n = 6 mice per group

Fig. 5

CPT administered at doses of 200 mg/kg/day on days 0 and 7 30 min prior to OVA administration decreased levels of p-STAT3 in lung epithelial cells of wild-type mice (A, immunohistochemistry), decreased serum titers of OVA-specific IgG₁ (B, ELISA) and alleviated lung inflammation (C, hematoxylin and eosin staining). A and C magnification: 200-fold. *: p < 0.05, n = 6 mice per group

Fig. 6

RT-PCR showed that addition of IL21 addition Iα-Cα, Iγ1-Cγ1, and Iγ3-Cγ3 fgermline transcription in CPT-treated wild-type or TLR2^−/− mice, n = 6 mice per group, *, p < 0.05