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The Utilization of Oropharyngeal Intratracheal PAMP Administration and Bronchoalveolar Lavage to Evaluate the Host Immune Response in Mice
Published on: April 2, 2014
Effects of the TLR2 agonists MALP-2 and Pam3Cys in isolated mouse lungs
Martina Barrenschee1, Dennis Lex, Stefan Uhlig
1Institute of Pharmacology and Toxicology, University Hospital Aachen, RWTH Aachen University, Aachen, Germany.
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.
Insights
Toll-like receptor (TLR) activation in lungs by bacterial components triggers inflammatory responses. This study reveals that TLR2 and TLR4 agonists induce similar pro-inflammatory gene and mediator release, with some unique pathways identified for TLR2 activation.
Area of Science:
- Immunology
- Pulmonology
- Microbiology
Background:
- Gram-positive and Gram-negative bacteria are major causes of pneumonia and acute lung injury.
- The innate immune system recognizes these bacteria via toll-like receptors (TLRs), specifically TLR2 for Gram-positive and TLR4 for Gram-negative bacteria.
- While the lungs exhibit high TLR2 expression, the consequences of its activation remain poorly understood.
Purpose of the Study:
- To investigate the pro-inflammatory effects of TLR2/6 agonist MALP-2, TLR2/1 agonist Pam(3)Cys, and TLR4 agonist lipopolysaccharide (LPS) in isolated mouse lungs.
- To elucidate the downstream signaling pathways, including mitogen-activated protein kinase (MAPK) and AKT activation.
- To quantify the induction of key inflammatory genes and mediator release.
Main Methods:
- Isolated mouse lungs were perfused with MALP-2, Pam(3)Cys, or LPS.
- Mediator release was quantified using enzyme-linked immunosorbent assay (ELISA).
- MAPK and AKT activation were assessed by immunoblotting, and gene induction was measured by quantitative polymerase chain reaction (qPCR).
Main Results:
- All TLR agonists activated MAPK ERK1/2 and p38 pathways, but not JNK or AKT.
- TLR ligands upregulated key inflammation-related genes including Tnf, Il1β, Il6, Il10, Il12, Ifng, Cxcl2, and Ptgs2.
- MALP-2 demonstrated higher potency than Pam(3)Cys for Slpi, Cxcl10, and Parg induction, and uniquely induced Tnc (tenascin C). All agonists stimulated IL-6, TNF, CXCL2, and CXCL10 protein release.
Conclusions:
- TLR2 and TLR4 activation elicit comparable pulmonary responses in terms of MAPK activation, gene induction, and mediator release.
- Several genes, including Slpi, tenascin C, Parg, and Traf1, are newly identified as targets of TLR2 activation in the lungs.
- The distinct induction of tenascin C by MALP-2 suggests potential TLR2/6-specific signaling pathways in pulmonary inflammation.
