Antagonistic antibody prevents toll-like receptor 2–driven lethal shock-like syndromes
Guangxun Meng, Mark Rutz, Matthias Schiemann, Jochen Metzger, Alina Grabiec, Ralf Schwandner, Peter B. Luppa, Frank Ebel, Dirk H. Busch, Stefan Bauer, Hermann Wagner, Carsten J. Kirschning
Guangxun Meng, Mark Rutz, Matthias Schiemann, Jochen Metzger, Alina Grabiec, Ralf Schwandner, Peter B. Luppa, Frank Ebel, Dirk H. Busch, Stefan Bauer, Hermann Wagner, Carsten J. Kirschning
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Article Infectious disease

Antagonistic antibody prevents toll-like receptor 2–driven lethal shock-like syndromes

Abstract

Hyperactivation of immune cells by bacterial products through toll-like receptors (TLRs) is thought of as a causative mechanism of septic shock pathology. Infections with Gram-negative or Gram-positive bacteria provide TLR2-specific agonists and are the major cause of severe sepsis. In order to intervene in TLR2-driven toxemia, we raised mAb’s against the extracellular domain of TLR2. Surface plasmon resonance analysis showed direct and specific interaction of TLR2 and immunostimulatory lipopeptide, which was blocked by T2.5 in a dose-dependent manner. Application of mAb T2.5 inhibited cell activation in experimental murine models of infection. T2.5 also antagonized TLR2-specific activation of primary human macrophages. TLR2 surface expression by murine macrophages was surprisingly weak, while both intra- and extracellular expression increased upon systemic microbial challenge. Systemic application of T2.5 upon lipopeptide challenge inhibited release of inflammatory mediators such as TNF-α and prevented lethal shock-like syndrome in mice. Twenty milligrams per kilogram of T2.5 was sufficient to protect mice, and administration of 40 mg/kg of T2.5 was protective even 3 hours after the start of otherwise lethal challenge with Bacillus subtilis. These results indicate that epitope-specific binding of exogenous ligands precedes specific TLR signaling and suggest therapeutic application of a neutralizing anti-TLR2 antibody in acute infection.

Authors

Guangxun Meng, Mark Rutz, Matthias Schiemann, Jochen Metzger, Alina Grabiec, Ralf Schwandner, Peter B. Luppa, Frank Ebel, Dirk H. Busch, Stefan Bauer, Hermann Wagner, Carsten J. Kirschning

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Inhibitory effect of mAb T2.5 on cell activation in vitro. (A–D) NF-κB–d...
Inhibitory effect of mAb T2.5 on cell activation in vitro. (A–D) NF-κB–dependent luciferase activities in HEK293 cells overexpressing either murine (A) or human TLR2 (B), as well as TNF-α concentrations in supernatants of RAW264.7 (C) or primary murine macrophages (D) challenged with inflammatory agonists. Rel. lucif. activity, relative luciferase activity; ND, not detectable. Cells were incubated with T2.5 or conT2 only (white bars), or additionally challenged with IL-1β (A and B, light gray bars), ultrapure LPS (C and D, medium gray bars), P3CSK4 (black bars), or h.i. B. subtilis (A–D, dark gray bars). (E) NF-κB/p65 nuclear translocation dependent on mAb, P3CSK4 challenge, or LPS challenge in human macrophages was analyzed by cytochemical staining. Unstim., unstimulated. Scale bar: 20 ∝m; magnification was equal for all recordings. (F and G) NF-κB–dependent EMSA was analyzed by application of nuclear extracts from RAW264.7 macrophages, and phosphorylation of MAPKs Erk1/2 (pErk1/2), p38 (pP38), and Akt (pAkt) was analyzed by application of total extracts from RAW264.7 macrophages. Cells were preincubated with the indicated amounts of mAb T2.5 or conT2 (∝g/ml) and challenged with P3CSK4 or LPS subsequently for 90 minutes (F; arrows indicate specific NF-κB–DNA complexes) or 30 minutes (G; phosphorylation-independent p38-specific immunoblot analysis as positive control). Untreated cells were analyzed as controls (Control).