Toll-Like Receptor 2 Modulates Pulmonary Inflammation and TNF-α Release Mediated by Mycoplasma pneumoniae

Abstract

Objectives: To investigate the roles that Toll-like receptors (TLRs) play in lung inflammation mediated by Mycoplasma pneumoniae (MP).

Methods: The changes in TLRs and tumor necrosis factor alpha (TNF-α) in peripheral blood of children with M. pneumoniae pneumonia (MPP) were monitored, and the interactions of signaling molecules regulating TNF-α release in A549 cells and neutrophils after M. pneumoniae stimulation were investigated. In TLR2 knockout (TLR2-/-) mice, the levels of TNF-α in bronchial alveolar lavage fluid (BALF) and peripheral blood after mycoplasma infection and the pathological changes in the lung tissue of mice were detected.

Results: TNF-α levels in peripheral blood of children with MPP were higher than those in non-infected children, and children with refractory MPP had the highest levels of TNF-α and TLR2. TNF-α secretion and TLR2, myeloid differentiation primary response 88 (MyD88) and phospho-p65(p-p65) levels were increased in stimulated cells. TNF-α secretion was suppressed upon siRNA-mediated TLR2 silencing. Pharmacological inhibition of nuclear factor-kappa B (NF-κB) and MyD88 effectively reduced TNF-α expression. Compared with wild-type mice, the TNF-α in serum and BALF decreased, and lung pro-inflammatory response was partially suppressed in TLR2-/- mice.

Conclusion: We concluded that TLR2 regulates M. pneumoniae-mediated lung inflammation and TNF-α release through the TLR2-MyD88-NF-κB signaling pathway.

Keywords: A549 cells; MyD88; Mycoplasma pneumoniae; NF-κB; TLR2; TNF-α; neutrophils.

Figure 1

Serum TNF-α levels in the three study groups. (A) The levels of serum TNF-α was determined by ELISA. (B–E) TLR1, TLR2, TLR4, and TLR6 mRNA relative expression level in children’s blood neutrophils were determined by RT-qPCR and were normalized by human GAPDH gene. *P < 0.05 is statistically significant. MPP, Mycoplasma pneumoniae pneumonia; RMPP, refractory M. pneumoniae pneumonia.

Figure 2

TNF-α secretion by (A) neutrophils and (B) A549 cells upon M. pneumoniae stimulation when compared with the NC group as assessed by ELISA. *P < 0.05 is statistically significant. NC, normal control; MP, Mycoplasma pneumoniae.

Figure 3

TNF-α secretion measured by ELISA in control or MP-infected A549 cells with or without knockdown of TLR2 by siRNA. **P < 0.01, *P < 0.05. P < 0.05 is statistically significant. siNC = A549 cells were transfected with negative control small interfering RNA (siRNA); siTLR-2 = A549 cells were transfected with siRNA targeting TLR2. MP, Mycoplasma pneumoniae.

Figure 4

Expression changes of MyD88 and p-p65 in A549 cells stimulated with M. pneumoniae. (A, B) A representative Western blot image. (C, D) Densitometry analysis of band intensity was performed. Quantitative densitometry of western blots was performed using Image J analysis. The expression was normalized to GADPH, GADPH was used as a loading control. Experiments were performed in biological triplicates. *P < 0.05 vs normal control group. P < 0.05 is statistically significant. NC, normal control; MP, Mycoplasma pneumoniae; PDTC, Pyrrolidinedithiocarbamic acid (a NF-κB inhibitor).

Figure 5

TNF-α secretion measured by ELISA in control or MP-infected A549 cells incubated with (A) MyD88 or (B) NF-κB inhibitors. *P < 0.05 is statistically significant. NC, normal control; MP, Mycoplasma pneumoniae; PDTC, Pyrrolidinedithiocarbamic acid, a NF-κB inhibitor; NBP2-29328, a MyD88 Inhibitor Peptide.

Figure 6

Numbers of (A) BALF total leukocytes, (B) neutrophils, and TNF-α concentration in (C) BALF and (D) serum in wild-type and TLR2 knockout mice. *P < 0.05 is statistically significant. WT, wild-type mice; TLR2 KO, TLR2 knockout mice; NS, normal saline; MP, Mycoplasma pneumoniae; BALF, Bronchoalveolar Fluid.

Figure 7

Hematoxylin and eosin staining of lung sections of wild-type and TLR2 knockout mice. Scale bar, 50μm. WT, wild-type mice; TLR2 KO, TLR2 knockout mice; NS, normal saline; MP, Mycoplasma pneumoniae.