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-alpha 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.

Figure 1

Application of mAb T2.5 for specific detection of TLR2. (A–D) Results of flow cytometry of HEK293 cells stably overexpressing Flag-tagged mTLR2 (A) or human TLR2 (B), as well as primary TLR2^–/– (C) and wild-type murine macrophages (D), by staining with mAb T2.5 (bold line). Negative controls represent cells incubated with a mouse IgG-specific secondary antibody only (filled areas). For positive controls, Flag-specific (A and B) and mTLR2-specific (C and D) polyclonal antisera were used (thin line). (E) For immunoprecipitation with T2.5, lysates of HEK293 cells overexpressing murine or human TLR2, as well as of murine RAW264.7 macrophages, were applied as indicated. TLR2 precipitates were visualized by application of Flag-specific (HEK293) or mTLR2-specific (RAW264.7) polyclonal antisera. Flag-specific beads (αFlag) and protein G beads in the absence of antibodies (pG), as well as vector-transfected HEK293 cells, were used as controls. The size of TLR2 was 97 kDa.

Figure 2

Subcellular localization of TLR2 in vitro. Monoclonal antibody T2.5 was used for cytochemical detection of overexpressed mTLR2 and human TLR2 (hTLR2) (A), as well as endogenous murine (TLR2^+/+, wild-type) or human TLR2 in primary macrophages (B). Vector-transfected HEK293 cells as well as TLR2^–/– primary macrophages were analyzed as controls. Concanavalin A (ConA) was used for staining of cellular membranes. The bar in the lower right corner of each field represents a distance of 20 ∝m (A) or 10 ∝m (B) on the slides analyzed.

Figure 3

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).

Figure 4

Molecular analysis of the effects of mAb T2.5 on TLR2ECD-P₃CSK₄ interaction. (A and B) Binding of recombinant TLR2ECD-Fc fusion protein (T2EC, positive controls) to immobilized P₃CSK₄ upon preincubation with T2.5 (T2EC + T2.5) at different molar excesses (A, ∞1, ∞3.3, ∞10) or with an isotype-matched control mAb (T2EC + con) at tenfold molar excess only (B, ∞10). Binding was continuously monitored in an SPR biosensor device, and amounts of antibodies used to gain high molecular excess over T2EC (coincubation) were applied alone as negative controls (A, T2.5; B, Con). Response units at 300 seconds are a measure for P₃CSK₄-binding capacities of T2EC and T2EC plus mAb. (C) For analysis of approximate localization of T2.5 epitope within the TLR2ECD, a mutant human TLR2 construct lacking the N-terminal third of the LRR-rich ECD (hTLR2-mutH) was used for NF-κB–dependent luciferase assay upon transient transfection, preincubation with mAb (T2.5, conT2), and P₃CSK₄ challenge (black bars). Absence of mAb treatment (No mAb) and/or of P₃CSK₄ challenge (white bars), and empty vector (Vector), represent respective controls.

Figure 5

TLR2 expression ex vivo immediately after primary cell isolation. Flow cytometry of splenocytes and peritoneal washout cells from wild-type (TLR2^+/+) and TLR2^–/– mice ex vivo immediately after isolation (n = 5, cells pooled for each sample). (A) CD11b⁺ splenocytes from mice challenged with LPS for 24 hours were analyzed for surface and intracellular TLR2 expression by staining with T2.5 (bold line, TLR2^+/+; filled area, TLR2^–/–). (B and C) For analysis of TLR2 regulation upon infection, mice were either left uninfected (–) or infected with B. subtilis and sacrificed after 24 hours (+). Upon staining of CD11b, cells were stained with T2.5 (TLR2) either without permeabilization (B) or after permeabilization (C). Numbers in quadrants represent the percentage of single- or double-stained cells with respect to the total number of viable cells analyzed.

Figure 6

Inhibitory effect of mAb T2.5 on host activation by microbial challenge in vivo. Mice were pretreated i.p. with 1 mg mAb T2.5 (black bars) or left untreated (white bars). Mice were challenged i.p. with P₃CSK₄ and D-galactosamine after 1 hour and sacrificed 2 or 4 hours later (n = 4 for each group at each time point). Serum concentrations of TNF-α (A), GROα/KC (human IL-8 homolog) (B), IL-6 (C), and IL-12p40 (D) were analyzed by ELISA. *P < 0.05, **P < 0.005, ***P < 0.001, Student’s t test for unconnected samples.

Figure 7

Effects of mAb T2.5 administration on viability after TLR2-specific systemic challenge. (A) IFN-γ– and D-galactosamine–sensitized mice received no mAb, 1 mg of mAb T2.5, or 1 mg of conT2 i.p. 30 minutes prior to microbial challenge with bacterial lipopeptide analogue P₃CSK₄ (open circles, no mAb, n = 4; open triangles, mAb conT2, n = 3; filled squares, mAb T2.5, n = 4). (B–D) Mice challenged with a high dose of h.i. B. subtilis were left untreated, treated 1 hour later with the indicated dosages of mAb T2.5 (B; filled diamonds, 1 mg, n = 3; open squares, 0.5 mg, n = 3; open triangles, 0.25 mg, n = 4; ∞’s, 0.13 mg, n = 4; open circles, no mAb T2.5, n = 4), or treated with 1 mg of mAb’s at the different time points indicated below (C and D). (C) TLR2-specific mAb was administered before (–) or after (+) bacterial challenge (filled inverted triangles, no mAb, n = 8; open circles, mAb conT2, –1 hour, n = 3; filled diamonds, mAb T2.5, –1 hour, n = 4; open squares, mAb T2.5, +1 hour, n = 3; ∞’s, mAb T2.5, +2 hours, n = 3; open diamonds, mAb T2.5, +3 hours, n = 4; open triangles, mAb T2.5, +4 hours, n = 3). (D) TLR2-specific mAb T2.5 was administered before (–) bacterial challenge (open triangles, no mAb; filled squares, mAb T2.5, –3 hours; open diamonds, mAb T2.5, –4 hours; open circles, mAb T2.5, –5 hours; filled inverted triangles, mAb T2.5, –6 hours; n = 3 for all groups).