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TLR3 is an endogenous sensor of tissue necrosis during acute inflammatory events.

Cavassani KA, Ishii M, Wen H, Schaller MA, Lincoln PM, Lukacs NW, Hogaboam CM, Kunkel SL - J. Exp. Med. (2008)

Bottom Line: We observed the involvement of TLR3 activation during experimental polymicrobial septic peritonitis and ischemic gut injury in the absence of an exogenous viral stimulus.Importantly, an immunoneutralizing antibody directed against TLR3 attenuated the generation of inflammatory chemokines evoked by byproducts from necrotic neutrophils cultured with wild-type macrophages.In vivo, anti-TLR3 antibody attenuated the tissue injury associated with gut ischemia and significantly decreased sepsis-induced mortality.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, University of Michigan, Ann Arbor, MI 48109, USA. kcavassa@med.umich.edu

ABSTRACT
Ligands from dying cells are a source of Toll-like receptor (TLR) activating agents. Although TLR3 is known to respond to RNA from necrotic cells, the relative importance of this response in vivo during acute inflammatory processes has not been fully explored. We observed the involvement of TLR3 activation during experimental polymicrobial septic peritonitis and ischemic gut injury in the absence of an exogenous viral stimulus. In TLR3-deficient mice, increased chemokine/cytokine levels and neutrophil recruitment characterized the initial inflammatory responses in both injury models. However, the levels of inflammatory chemokines and tumor necrosis factor alpha quickly returned to baseline in tlr3(-/-) mice, and these mice were protected from the lethal effects of sustained inflammation. Macrophages from tlr3(-/-) mice responded normally to other TLR ligands but did not respond to RNA from necrotic neutrophils. Importantly, an immunoneutralizing antibody directed against TLR3 attenuated the generation of inflammatory chemokines evoked by byproducts from necrotic neutrophils cultured with wild-type macrophages. In vivo, anti-TLR3 antibody attenuated the tissue injury associated with gut ischemia and significantly decreased sepsis-induced mortality. Collectively, these data show that TLR3 is a regulator of the amplification of immune response and serves an endogenous sensor of necrosis, independent of viral activation.

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TLR3 was required for chemokine generation by WT peritoneal macrophages after co-culture with necrotic neutrophils. (A) The specificity of polyclonal rabbit anti-TLR3 was determined by Western blot analysis of TLR3 expression in the following samples: lane 1, WT macrophages; lane 2, tlr7−/− macrophages; lane 3, tlr3−/− macrophages; and lane 4, recombinant TLR3. The black line indicates that intervening lanes have been spliced out. (B-E) Chemokine concentrations in the supernatants of WT peritoneal macrophages co-cultured with necrotic neutrophils with IgG or anti-TLR3 polyclonal antibody were quantified using Bio-Plex. The data are means ± SEM of triplicate wells and are representative of three independent experiments. *, P < 0.05; **, P < 0.01; and ***, P < 0.001 compared with IgG group.
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fig7: TLR3 was required for chemokine generation by WT peritoneal macrophages after co-culture with necrotic neutrophils. (A) The specificity of polyclonal rabbit anti-TLR3 was determined by Western blot analysis of TLR3 expression in the following samples: lane 1, WT macrophages; lane 2, tlr7−/− macrophages; lane 3, tlr3−/− macrophages; and lane 4, recombinant TLR3. The black line indicates that intervening lanes have been spliced out. (B-E) Chemokine concentrations in the supernatants of WT peritoneal macrophages co-cultured with necrotic neutrophils with IgG or anti-TLR3 polyclonal antibody were quantified using Bio-Plex. The data are means ± SEM of triplicate wells and are representative of three independent experiments. *, P < 0.05; **, P < 0.01; and ***, P < 0.001 compared with IgG group.

Mentions: Using Western blot analysis, we examined the specificity of our polyclonal antibody directed against mouse TLR3. As shown in Fig. 7 A, this antibody detected recombinant TLR3 protein (a band at ∼140 kD), as well as in poly(I:C)-stimulated macrophages from WT (lane 1) and tlr7−/− (lane 2) mice, but not in tlr3−/− (lane 3) macrophages (Fig. 7 A). As would be expected given the polyclonal nature of this antibody, other bands were detected in the macrophage samples from the WT and tlr7−/− mice, but it appeared that these additional bands (∼60–80 kD) were specific for TLR3 because they were not detected by this antibody in tlr3−/− macrophages. Next, a functional analysis of TLR3 signaling was performed in purified WT peritoneal macrophage cultures exposed to necrotic neutrophils or stimulated by poly(I:C) (Fig. 7 B and Fig. S4 [available at http://www.jem.org/cgi/content/full/jem.20081370/DC1], respectively). After exposure to poly(I:C) alone or poly(I:C) and control IgG for 24 h, CCL2, CCL3, CCL5, MIP-2, KC, and TNF-α were prominently expressed in supernatants from these cultures. In contrast, the presence of anti-TLR3 antibody significantly reduced the chemokine generation by poly(I:C)-activated peritoneal macrophages (Fig. S4 A). However, the anti-TLR3 antibody did not have any effect on CCL2, CCL3, CCL5, and TNF-α production induced by LPS, CpG-DNA, and PamCys3 (Fig. S4 B).


TLR3 is an endogenous sensor of tissue necrosis during acute inflammatory events.

Cavassani KA, Ishii M, Wen H, Schaller MA, Lincoln PM, Lukacs NW, Hogaboam CM, Kunkel SL - J. Exp. Med. (2008)

TLR3 was required for chemokine generation by WT peritoneal macrophages after co-culture with necrotic neutrophils. (A) The specificity of polyclonal rabbit anti-TLR3 was determined by Western blot analysis of TLR3 expression in the following samples: lane 1, WT macrophages; lane 2, tlr7−/− macrophages; lane 3, tlr3−/− macrophages; and lane 4, recombinant TLR3. The black line indicates that intervening lanes have been spliced out. (B-E) Chemokine concentrations in the supernatants of WT peritoneal macrophages co-cultured with necrotic neutrophils with IgG or anti-TLR3 polyclonal antibody were quantified using Bio-Plex. The data are means ± SEM of triplicate wells and are representative of three independent experiments. *, P < 0.05; **, P < 0.01; and ***, P < 0.001 compared with IgG group.
© Copyright Policy
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2571935&req=5

fig7: TLR3 was required for chemokine generation by WT peritoneal macrophages after co-culture with necrotic neutrophils. (A) The specificity of polyclonal rabbit anti-TLR3 was determined by Western blot analysis of TLR3 expression in the following samples: lane 1, WT macrophages; lane 2, tlr7−/− macrophages; lane 3, tlr3−/− macrophages; and lane 4, recombinant TLR3. The black line indicates that intervening lanes have been spliced out. (B-E) Chemokine concentrations in the supernatants of WT peritoneal macrophages co-cultured with necrotic neutrophils with IgG or anti-TLR3 polyclonal antibody were quantified using Bio-Plex. The data are means ± SEM of triplicate wells and are representative of three independent experiments. *, P < 0.05; **, P < 0.01; and ***, P < 0.001 compared with IgG group.
Mentions: Using Western blot analysis, we examined the specificity of our polyclonal antibody directed against mouse TLR3. As shown in Fig. 7 A, this antibody detected recombinant TLR3 protein (a band at ∼140 kD), as well as in poly(I:C)-stimulated macrophages from WT (lane 1) and tlr7−/− (lane 2) mice, but not in tlr3−/− (lane 3) macrophages (Fig. 7 A). As would be expected given the polyclonal nature of this antibody, other bands were detected in the macrophage samples from the WT and tlr7−/− mice, but it appeared that these additional bands (∼60–80 kD) were specific for TLR3 because they were not detected by this antibody in tlr3−/− macrophages. Next, a functional analysis of TLR3 signaling was performed in purified WT peritoneal macrophage cultures exposed to necrotic neutrophils or stimulated by poly(I:C) (Fig. 7 B and Fig. S4 [available at http://www.jem.org/cgi/content/full/jem.20081370/DC1], respectively). After exposure to poly(I:C) alone or poly(I:C) and control IgG for 24 h, CCL2, CCL3, CCL5, MIP-2, KC, and TNF-α were prominently expressed in supernatants from these cultures. In contrast, the presence of anti-TLR3 antibody significantly reduced the chemokine generation by poly(I:C)-activated peritoneal macrophages (Fig. S4 A). However, the anti-TLR3 antibody did not have any effect on CCL2, CCL3, CCL5, and TNF-α production induced by LPS, CpG-DNA, and PamCys3 (Fig. S4 B).

Bottom Line: We observed the involvement of TLR3 activation during experimental polymicrobial septic peritonitis and ischemic gut injury in the absence of an exogenous viral stimulus.Importantly, an immunoneutralizing antibody directed against TLR3 attenuated the generation of inflammatory chemokines evoked by byproducts from necrotic neutrophils cultured with wild-type macrophages.In vivo, anti-TLR3 antibody attenuated the tissue injury associated with gut ischemia and significantly decreased sepsis-induced mortality.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, University of Michigan, Ann Arbor, MI 48109, USA. kcavassa@med.umich.edu

ABSTRACT
Ligands from dying cells are a source of Toll-like receptor (TLR) activating agents. Although TLR3 is known to respond to RNA from necrotic cells, the relative importance of this response in vivo during acute inflammatory processes has not been fully explored. We observed the involvement of TLR3 activation during experimental polymicrobial septic peritonitis and ischemic gut injury in the absence of an exogenous viral stimulus. In TLR3-deficient mice, increased chemokine/cytokine levels and neutrophil recruitment characterized the initial inflammatory responses in both injury models. However, the levels of inflammatory chemokines and tumor necrosis factor alpha quickly returned to baseline in tlr3(-/-) mice, and these mice were protected from the lethal effects of sustained inflammation. Macrophages from tlr3(-/-) mice responded normally to other TLR ligands but did not respond to RNA from necrotic neutrophils. Importantly, an immunoneutralizing antibody directed against TLR3 attenuated the generation of inflammatory chemokines evoked by byproducts from necrotic neutrophils cultured with wild-type macrophages. In vivo, anti-TLR3 antibody attenuated the tissue injury associated with gut ischemia and significantly decreased sepsis-induced mortality. Collectively, these data show that TLR3 is a regulator of the amplification of immune response and serves an endogenous sensor of necrosis, independent of viral activation.

Show MeSH
Related in: MedlinePlus