Cooperation of TLR2 with MyD88, PI3K, and Rac1 in lipoteichoic acid-induced cPLA2/COX-2-dependent airway inflammatory responses

Abstract

Lipoteichoic acid (LTA) plays a role in the pathogenesis of severe inflammatory responses induced by Gram-positive bacterial infection. Cytosolic phospholipase A(2) (cPLA(2)), cyclooxygenase-2 (COX-2), prostaglandin E(2) (PGE(2)), and interleukin (IL)-6 have been demonstrated to engage in airway inflammation. In this study, LTA-induced cPLA(2) and COX-2 expression and PGE(2) or IL-6 synthesis were attenuated by transfection with siRNAs of TLR2, MyD88, Akt, p42, p38, JNK2, and p65 or pretreatment with the inhibitors of PI3K (LY294002), p38 (SB202190), MEK1/2 (U0126), JNK1/2 (SP600125), and NF-kappaB (helenalin) in human tracheal smooth muscle cells (HTSMCs). LTA also induced cPLA(2) and COX-2 expression and leukocyte count in bronchoalveolar lavage fluid in mice. LTA-regulated PGE(2) or IL-6 production was inhibited by pretreatment with the inhibitors of cPLA(2) (AACOCF(3)) and COX-2 (NS-398) or transfection with cPLA(2) siRNA or COX-2 siRNA, respectively. LTA-stimulated NF-kappaB translocation or cPLA(2) phosphorylation was attenuated by pretreatment with LY294002, SB202190, U0126, or SP600125. Furthermore, LTA could stimulate TLR2, MyD88, PI3K, and Rac1 complex formation. We also demonstrated that Staphylococcus aureus could trigger these responses through a similar signaling cascade in HTSMCs. It was found that PGE(2) could directly stimulate IL-6 production in HTSMCs or leukocyte count in bronchoalveolar lavage fluid in mice. These results demonstrate that LTA-induced MAPKs activation is mediated through the TLR2/MyD88/PI3K/Rac1/Akt pathway, which in turn initiates the activation of NF-kappaB, and ultimately induces cPLA(2)/COX-2-dependent PGE(2) and IL-6 generation.

Figure 1

LTA and S. aureus induce PGE₂ and IL-6 generation in a cPLA₂/COX-2–dependent manner. A: HTSMCs were incubated with 50 μg/ml LTA or 10⁷ CFU/ml live or heat-killed S. aureus for the indicated time intervals. The expression of cPLA₂ and COX-2 were determined by Western blot. B: HTSMCs were pretreated with the inhibitor of cPLA₂ (AACOCF₃) or COX-2 (NS-398) for 1 hour, and then incubated with 50 μg/ml LTA or 10⁷ CFU/ml heat-killed S. aureus for 24 hours. The generation of PGE₂ was determined. C: HTSMCs were stimulated with 50 μg/ml LTA for the indicated time intervals. The cell lysates were subjected to Western blot using an anti–phospho-cPLA₂ or anti-GAPDH Ab. HTSMCs were transfected with scrambled siRNA, cPLA₂ siRNA, or COX-2 siRNA, and then challenged with LTA (D) or heat-killed S. aureus (E) for 24 hours. The generation of IL-6 was determined. Data are expressed as mean ± SEM of at least three independent experiments. *P < 0.05; **P < 0.01 as compared with the basal level (A). Significant differences between the compared groups are indicated: *P < 0.05; **P < 0.01 (B, D, E).

Figure 2

LTA and S. aureus enhance leukocyte count in BAL in a cPLA₂/COX-2–dependent manner in mice. A: Mice were intratracheally injected with live or heat-killed S. aureus (10⁷ CFU/mouse) for 24 hours, and then the appearances of the lungs were observed. Mice were intratracheally injected with (B) LTA (4 mg/kg) for 24 hours or (C) live S. aureus (10⁷ CFU/mouse) for the indicated time intervals. BAL fluid was acquired and leukocyte count was determined by a hemocytometer. Mice were intratracheally injected with (D) LTA (4 mg/kg) or (E) live S. aureus (10⁷ CFU/mouse) for 24 hours. Airway and lung tissues were homogenized to extract proteins. The levels of cPLA₂ and COX-2 expression were determined by Western blot. Mice were i.p. given one dose of AACOCF₃ or NS-398 (2 mg/kg) for 1 hour before (F) LTA or (G) live S. aureus treatment, and sacrificed after 24 hours. BAL fluid was acquired and leukocyte count was determined by a hemocytometer. Data are expressed as mean ± SEM of at least three independent experiments. **P < 0.01 as compared with the sham group (B and C). Significant differences between the compared groups are indicated: *P < 0.05; **P < 0.01 (F and G).

Figure 3

LTA induces cPLA₂ and COX-2 expression through a MyD88-dependent TLR2 signaling pathway. A: HTSMCs were pretreated with an anti-TLR2 or anti-TLR4 Ab for 1 hour, and then incubated with 50 μg/ml LTA for 24 hours. The expression of cPLA₂ and COX-2 were determined by Western blot. B: HTSMCs were transfected with scrambled siRNA or TLR2 siRNA, and then challenged with LTA for 24 hours. The protein levels of TLR2, cPLA₂, and COX-2 were determined by Western blot. C: HTSMCs were transfected with scrambled siRNA or MyD88 siRNA, and then challenged with LTA for 24 hours. The protein levels of MyD88, cPLA₂, and COX-2 were determined by Western blot. D: HTSMCs were co-transfected with TLR2 siRNA, MyD88 siRNA, or scrambled siRNA and cPLA₂-luc or COX-2-luc reporter gene, and then treated with 50 μg/ml LTA for 4 hours. The cPLA₂ and COX-2 promoter activities were determined in the cell lysates. E and F: HTSMCs were transfected with scrambled siRNA, TLR2 siRNA, or MyD88 siRNA, and then challenged with LTA for 24 hours. The media were collected and analyzed for PGE₂ and IL-6 production. Data are expressed as mean ± SEM of at least three independent experiments. *P < 0.05; **P < 0.01 as compared with those of the scrambled siRNA-transfected cells exposed to LTA.

Figure 4

LTA-induced cPLA₂ and COX-2 expression are mediated via PI3K/Akt. A: HTSMCs were pretreated with LY294002 for 1 hour, and then incubated with LTA for 24 hours. The levels of cPLA₂ and COX-2 expression were determined by Western blot. B: HTSMCs were pretreated with LY294002 (30 μmol/L) for 1 hour, and then stimulated with 50 μg/ml LTA for the indicated time intervals. The cell lysates were subjected to Western blot using an anti–phospho-Akt Ab at Ser⁴⁷³ HTSMCs were transfected with scrambled siRNA or Akt siRNA, and then challenged with LTA for 24 hours (C) or 4 hours (D). C: The levels of Akt, cPLA₂, and COX-2 expression were determined by Western blot. D: The RNA samples were analyzed by RT-PCR for the levels of cPLA₂ and COX-2 mRNA expression. E: HTSMCs were co-transfected with Akt siRNA or scrambled siRNA and cPLA₂-luc or COX-2–luc reporter gene, and then treated with 50 μg/ml LTA for 4 hours. The cPLA₂ and COX-2 promoter activities were determined in the cell lysates. F and G: HTSMCs were transfected with scrambled siRNA or Akt siRNA, and then challenged with LTA for 24 hours. The media were collected and analyzed for PGE₂ and IL-6 production. Data are expressed as mean ± SEM of at least three independent experiments. *P < 0.05; **P < 0.01 as compared with those of the scrambled siRNA-transfected cells exposed to LTA.

Figure 5

LTA- and S. aureus–induced cPLA₂ and COX-2 expression are mediated through the formation of a TLR2/MyD88/PI3K/Rac1 complex. HTSMCs were stimulated with 50 μg/ml LTA or 10⁷ CFU/ml heat-killed S. aureus for the indicated time intervals. The cell lysates were subjected to immunoprecipitation with anti-TLR2 (A) or anti-MyD88 Ab (B), and then the immunoprecipitates were analyzed by SDS-PAGE and immunoblotted using an anti-TLR2, anti-MyD88, anti-p85α, anti-p110, or anti-Rac1 Ab.

Figure 6

Involvement of TLR2-dependent MAPKs phosphorylation in LTA-mediated cPLA₂ and COX-2 expression in HTSMCs. Cells were transfected with the siRNA of scrambled, p42, p38α, p38β, p38δ, p38γ, or JNK2, and then challenged with LTA for 24 hours. A: The levels of p42, p38α, p38β, p38δ, p38γ, JNK2, cPLA₂, and COX-2 expression were determined by Western blot. B and C: The media were collected and analyzed for PGE₂ and IL-6 release, respectively. D: HTSMCs were pretreated without or with 10 μmol/L U0126, 10 μmol/L SB202190, or 10 μmol/L SP600125 for 1 hour, and then stimulated with 50 μg/ml LTA for the indicated time intervals. The cell lysates were subjected to Western blot using an anti–phospho-p42/p44 MAPK, anti-phospho-p38, anti-phospho-JNK1/2, or anti-GAPDH Ab. E: HTSMCs were transfected with the siRNA of scrambled, TLR2, MyD88, or Akt, and then incubated with 50 μg/ml LTA for 5 minutes. The cell lysates were subjected to Western blot using an anti–phospho-p42/p44 MAPK, anti–phospho-p38, anti–phospho-JNK1/2, or anti-GAPDH Ab. F: HTSMCs were pretreated with 30 μmol/L LY294002, 10 μmol/L U0126, 10 μmol/L SB202190, or 10 μmol/L SP600125 for 1 hour, and then stimulated with LTA for 10 minutes. The cell lysates were subjected to Western blot using an anti–phospho-cPLA₂ or anti–β-actin Ab. Data are expressed as mean ± SEM of at least three independent experiments. **P < 0.01 as compared with those of the scrambled siRNA-transfected cells. *P < 0.05 as compared with those of the scrambled siRNA-transfected cells exposed to LTA.

Figure 7

LTA induces cPLA₂ and COX-2 expression via NF-κB. A–C: HTSMCs were transfected with scrambled siRNA or p65 siRNA, and then challenged with LTA for 24 hours. A: The levels of p65, cPLA₂, and COX-2 expression were determined by Western blot. B and C: The media were collected and analyzed for PGE₂ and IL-6 production. D: HTSMCs were transiently transfected with NF-κB-luc reporter gene, and then challenged with LTA for the indicated time intervals. The NF-κB promoter activity was determined in the cell lysates. E: HTSMCs were stimulated with LTA for the indicated time intervals. The cell lysates were subjected to Western blot using an anti–phospho-p65 (Ser⁵³⁶) Ab. The cytosolic and nuclear extracts were prepared and subjected to Western blot using an anti-p65 Ab. Lamin A and GAPDH were used as a marker protein for nuclear and cytosolic fractions, respectively. F: HTSMCs were transiently transfected with NF-κB-luc reporter gene, pretreated with an anti-TLR2 Ab (5 μg/ml), LY294002 (30 μmol/L), U0126 (10 μmol/L), SB202190 (10 μmol/L), SP600125 (10 μmol/L), or helenalin (1 μmol/L) for 1 hour, and then incubated with LTA for 1 hour. The NF-κB promoter activity was determined in the cell lysates. G: HTSMCs were pretreated with an anti-TLR2 Ab (5 μg/ml), LY294002 (30 μmol/L), U0126 (10 μmol/L), SB202190 (10 μmol/L), SP600125 (10 μmol/L), or helenalin (1 μmol/L) for 1 hour, and then stimulated with 50 μg/ml of LTA for 1 hour. HTSMCs were fixed, and then labeled with anti-p65 Ab and followed with an FITC-conjugated secondary antibody. Individual cells were imaged. The red arrow indicates p65 expression. Data are expressed as mean ± SEM of at least three independent experiments. Significant differences between the compared groups are indicated: *P < 0.05; **P < 0.01 (B, C, F). *P < 0.05 as compared with the basal level (D).

Figure 8

LTA and S. aureus induce leukocytes in BAL and cPLA₂ and COX-2 expression in mice via PI3K/Akt, MAPKs, and NF-κB. Mice were given one dose of LY294002, U0126, SB202190, SP600125, or helenalin (2 mg/kg) for 1 hour before LTA (A) or live S. aureus treatment (B), and sacrificed after 24 hours. BAL fluid was acquired and leukocyte count was determined by a hemocytometer. Mice were i.p. given one dose of LY294002, U0126, SB202190, SP600125, or helenalin (2 mg/kg) for 1 hour before LTA (C) or heat-killed S. aureus treatment (D), and sacrificed after 24 hours. Airway tissues were homogenized to extract protein. The levels of cPLA₂ and COX-2 expression were determined by Western blot. E: HTSMCs were transfected with siRNAs of TLR2, MyD88, Akt, p38α, p42, JNK2, or p65, and then incubated with heat-killed S. aureus for 24 hours. The levels of cPLA₂ and COX-2 expression were determined by Western blot. Data are expressed as mean ± SEM of three independent experiments. **P < 0.01 as compared with the sham group. *P < 0.05 as compared with the mice exposed to LTA (A) or live S. aureus (B) alone.

Figure 9

Arachidonic acid induces cPLA₂ phosphorylation and cPLA₂ and COX-2 expression in HTSMCs. Cells were incubated with 30 μmol/L arachidonic acid (AA) for the indicated times. A: The cell lysates were subjected to Western blot using an anti–phospho-cPLA₂ or anti–β-actin Ab. B: The levels of cPLA₂ and COX-2 expression were determined by Western blot analysis. Data are expressed as mean ± SEM of three independent experiments. *P < 0.05; **P < 0.01 as compared with the basal level.

Figure 10

PGE₂ induces IL-6 production in HTSMCs and leukocytes in BAL in mice. HTSMCs were pretreated with 10 μmol/L AH 6809 (an EP1 and EP2 receptor antagonist), 10 μmol/L SC-19220 (an EP1 receptor antagonist), or 10 μmol/L GW627368X (an EP4 receptor antagonist) for 1 hour, and then incubated with LTA (A) or heat-killed S. aureus (B) for 24 hours. The media were collected and analyzed for IL-6 production. C: HTSMCs were incubated with 30 μmol/L PGE₂ for 16 hours and 24 hours, and then IL-6 generation was measured. D: Mice were intratracheally injected with PGE₂ (2 mg/kg) for 24 hours, and then BAL fluid was acquired and leukocyte count was determined by a hemocytometer. Data are expressed as mean ± SEM of three independent experiments. **P < 0.01 as compared with the basal level (A–C) or the sham group (D). *P < 0.05 as compared with the cells exposed to LTA (A) or heat-killed S. aureus (B) alone.

Figure 11

Schematic diagram illustrating the proposed signaling pathway involved in LTA-induced cPLA₂/COX-2–dependent airway inflammation. LTA activates the TLR2/MyD88/PI3K/Rac1/Akt pathway to enhance ERK1/2, p38 MAPK, and JNK1/2 phosphorylation, which in turn initiates the activation of NF-κB and ultimately induces cPLA₂/COX-2–dependent PGE₂ and IL-6 generation in HTSMCs.