Effects of the TLR2 agonists MALP-2 and Pam3Cys in isolated mouse lungs

Abstract

Background: Gram-positive and Gram-negative bacteria are main causes of pneumonia or acute lung injury. They are recognized by the innate immune system via toll-like receptor-2 (TLR2) or TLR4, respectively. Among all organs, the lungs have the highest expression of TLR2 receptors, but little is known about the pulmonary consequences of their activation. Here we studied the effects of the TLR2/6 agonist MALP-2, the TLR2/1 agonist Pam(3)Cys and the TLR4 agonist lipopolysaccharide (LPS) on pro-inflammatory responses in isolated lungs.

Methodology/principal findings: Isolated perfused mouse lungs were perfused for 60 min or 180 min with MALP-2 (25 ng/mL), Pam(3)Cys (160 ng/mL) or LPS (1 µg/mL). We studied mediator release by enzyme linked immunosorbent assay (ELISA), the activation of mitogen activated protein kinase (MAPK) and AKT/protein kinase B by immunoblotting, and gene induction by quantitative polymerase chain reaction. All agonists activated the MAPK ERK1/2 and p38, but neither JNK or AKT kinase. The TLR ligands upregulated the inflammation related genes Tnf, Il1β, Il6, Il10, Il12, Ifng, Cxcl2 (MIP-2α) and Ptgs2. MALP-2 was more potent than Pam(3)Cys in inducing Slpi, Cxcl10 (IP10) and Parg. Remarkable was the strong induction of Tnc by MALP2, which was not seen with Pam(3)Cys or LPS. The growth factor related genes Areg and Hbegf were not affected. In addition, all three TLR agonists stimulated the release of IL-6, TNF, CXCL2 and CXCL10 protein from the lungs.

Conclusions/significance: TLR2 and TLR4 activation leads to similar reactions in the lungs regarding MAPK activation, gene induction and mediator release. Several genes studied here have not yet been appreciated as targets of TLR2-activation in the lungs before, i.e., Slpi, tenascin C, Parg and Traf1. In addition, the MALP-2 dependent induction of Tnc may indicate the existence of TLR2/6-specific pathways.

Figure 1. Lung functions.

(A) Tidal volume and (B) pulmonary resistance in isolated perfused mouse lungs exposed to Pam₃Cys (160 ng/mL, black squares), MALP-2 (25 ng/mL, grey squares) or LPS (1 µg/mL, black circles) from 60 min to the end of the experiment; control lungs are shown as white circles. Data are expressed as mean ± SEM, n = 3–5.

Figure 2. Mitogen activated protein kinase and Akt kinase activation.

After 60 min of perfusion under baseline conditions, isolated mouse lungs were perfused for another 60 or 180 min with Pam₃Cys (160 ng/mL, n = 5), MALP-2 (25 ng/mL, n = 5), LPS (1 µg/mL, n = 3) or under control conditions (n = 5). The basal and phosphorylated forms of several kinases were analyzed by immunoblotting after 60 min or 180 min: (A) Erk1/2, (B) p38, (C) Jnk, and (D) Akt kinase. Data were calculated as the ratio of the phosphorylated protein to the total amount of the protein and then referenced to the control on the same gel. Data are shown as mean ± SEM. The micrographs show one representative immunoblot from 3 (LPS) or 5 (C; Pam₃Cys, MALP-2) independent experiments.

Figure 3. Cluster analysis of the gene expression data.

After 60 min of perfusion under baseline conditions, isolated mouse lungs were perfused for another 180 min with Pam₃Cys (160 ng/mL, n = 5), MALP-2 (25 ng/mL, n = 5), LPS (1 µg/mL, n = 3) or under control conditions (n = 5). The different colors (red, brown, green, blue) identify genes that clustered together.

Figure 4. Expression of genes in cluster 1.

After 60 min of perfusion under baseline conditions, isolated mouse lungs were perfused for another 180 min with Pam₃Cys (160 ng/mL, n = 5), MALP-2 (25 ng/mL, n = 5), LPS (1 µg/mL, n = 3) or under control conditions (n = 4). Genes: amphiregulin (Areg), angiopoietin-like 2 (Angptl2), heparin-binding epithelial growth fator (HBegf), poly(ADP-ribose)glycohydrolase (Parg), TNF receptor-associated factor 1 (Traf1), and secretory leukocyte peptidase inhibitor (Slpi). Data were normalized to the experimental control and are shown as mean ± SEM. *, p<0.05 vs control; **, p<0.01 vs control; ***, p<0.001 vs control.

Figure 5. Expression of genes in cluster 2.

After 60 min of perfusion under baseline conditions, isolated mouse lungs were perfused for another 180 min with Pam₃Cys (160 ng/mL, n = 5), MALP-2 (25 ng/mL, n = 5), LPS (1 µg/mL, n = 3) or under control conditions (n = 5). Genes: interleukin 4 (Il4), interleukin 12p35 (Il12a), tenascin C (Tnc), interleukin 10 (Il10), cyclooxygenase 2 (Ptgs2), and IP10 (Cxcl10). Data were normalized to the experimental control and are shown as mean ± SEM. *, p<0.05 vs control; **, p<0.01 vs control; ***, p<0.001 vs control.

Figure 6. Expression of genes in cluster 3 and 4.

After 60 min of perfusion under baseline conditions, isolated mouse lungs were perfused for another 180 min with Pam₃Cys (160 ng/mL, n = 5), MALP-2 (25 ng/mL, n = 5), LPS (1 µg/mL, n = 3) or under control conditions (n = 5). Genes: interleukin 1β (Il1b), macrophage inflammatory protein 2α (Cxcl2), interleukin 6 (Il6), tumor necrosis factor (Tnf), interferon γ (Ifng), and IL12p40 (IL12b). Data were normalized to the experimental control and are shown as mean ± SEM. *, p<0.05 vs control; **, p<0.01 vs control; ***, p<0.001 vs control.

Figure 7. Mediator release into the perfusate.

After 60 min of perfusion under baseline conditions, isolated mouse lungs were perfused for another 180 min with Pam₃Cys (black squares, 160 ng/mL, n = 5), MALP-2 (grey squares, 25 ng/mL, n = 5), LPS (black circles, 1 µg/mL, n = 3) or under control conditions (white circles, n = 4). Perfusate samples were taken every 30 min and analyzed by ELISA for TNF (A), IL-6 (B), MIP-2α (C) and IP-10 (D). Data are expressed as mean ± SEM. *, p<0.05 vs. control.

Figure 8. Reference genes for RT-qPCR experiments.

After 60 min of perfusion under baseline conditions, isolated mouse lungs were perfused for another 180 min with Pam₃Cys (160 ng/mL, n = 5), MALP-2 (25 ng/mL, n = 5), LPS (1 µg/mL, n = 3) or under control conditions (n = 4). Shown are four potential callibrator genes: β2-microglobulin (b2m), ribosomal protein L 32 (rpl32), hypoxhantine phosphoribosyl transferase I (hprt1) and tyrosine kinase 2 (tyk2). Data were normalized to the experimental control and presented as mean ± SEM.