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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 peritoneal macrophages after co-culture with necrotic but not apoptotic cells. (A) The necrotic cells were identified by analyzing the side scatter (SSC) and PI+ staining. (B–F) Chemokine concentrations in the supernatants of WT or tlr3−/− peritoneal macrophages co-cultured with necrotic neutrophils, apoptotic splenocytes, poly(I:C), CpG-ODN, Pam3Cys, or LPS and quantified using ELISA and/or Bio-Plex. The data are means ± SEM from three to four combined experiments. *, P < 0.05; **, P < 0.01; and ***, P < 0.001 compared with tlr3−/− mice. (G and H) KC (G) and MIP-2 (H) protein levels in 96-well tissue culture plates containing WT or tlr3−/− macrophages and one of untreated, RNase-, or Benzonase-treated necrotic PMNs. **, P < 0.01 when necrotic PMNs were compared with medium; #, P < 0.05 when enzyme-treated necrotic PMNs were compared with nontreated necrotic PMNs.
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fig6: TLR3 was required for chemokine generation by peritoneal macrophages after co-culture with necrotic but not apoptotic cells. (A) The necrotic cells were identified by analyzing the side scatter (SSC) and PI+ staining. (B–F) Chemokine concentrations in the supernatants of WT or tlr3−/− peritoneal macrophages co-cultured with necrotic neutrophils, apoptotic splenocytes, poly(I:C), CpG-ODN, Pam3Cys, or LPS and quantified using ELISA and/or Bio-Plex. The data are means ± SEM from three to four combined experiments. *, P < 0.05; **, P < 0.01; and ***, P < 0.001 compared with tlr3−/− mice. (G and H) KC (G) and MIP-2 (H) protein levels in 96-well tissue culture plates containing WT or tlr3−/− macrophages and one of untreated, RNase-, or Benzonase-treated necrotic PMNs. **, P < 0.01 when necrotic PMNs were compared with medium; #, P < 0.05 when enzyme-treated necrotic PMNs were compared with nontreated necrotic PMNs.

Mentions: We addressed the possibility that the lack of amplification of the inflammatory response in tlr3−/− mice after injury was caused by their inability to respond to byproducts from necrotic cells. Accordingly, we examined the role of TLR3 in the generation of inflammatory chemokines by isolated peritoneal WT and tlr3−/− macrophages after their co-culture with necrotic polymorphonuclear neutrophils (PMNs) or apoptotic splenocytes. In cultures of WT macrophages, the addition of necrotic neutrophils (97% of cells were propidium iodide+ (PI+), as shown in Fig. 6 A) for 24 h significantly increased the levels of CCL5 (Fig. 6 B), MIP-2 (Fig. 6 C), CCL3 (Fig. 6 D), KC (Fig. 6 E), and CXCL10 (Fig. 6 F) above control (medium alone) levels. In marked contrast, the addition of necrotic neutrophils to cultures of tlr3−/− macrophages did not induce the expression of these chemokines (Fig. 6, B–F). However, chemokine responses evoked by the addition of apoptotic spleen cells (depicted) or neutrophils (not depicted) did not appear to be TLR3 dependent (Fig. 6, B–F).


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 peritoneal macrophages after co-culture with necrotic but not apoptotic cells. (A) The necrotic cells were identified by analyzing the side scatter (SSC) and PI+ staining. (B–F) Chemokine concentrations in the supernatants of WT or tlr3−/− peritoneal macrophages co-cultured with necrotic neutrophils, apoptotic splenocytes, poly(I:C), CpG-ODN, Pam3Cys, or LPS and quantified using ELISA and/or Bio-Plex. The data are means ± SEM from three to four combined experiments. *, P < 0.05; **, P < 0.01; and ***, P < 0.001 compared with tlr3−/− mice. (G and H) KC (G) and MIP-2 (H) protein levels in 96-well tissue culture plates containing WT or tlr3−/− macrophages and one of untreated, RNase-, or Benzonase-treated necrotic PMNs. **, P < 0.01 when necrotic PMNs were compared with medium; #, P < 0.05 when enzyme-treated necrotic PMNs were compared with nontreated necrotic PMNs.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2571935&req=5

fig6: TLR3 was required for chemokine generation by peritoneal macrophages after co-culture with necrotic but not apoptotic cells. (A) The necrotic cells were identified by analyzing the side scatter (SSC) and PI+ staining. (B–F) Chemokine concentrations in the supernatants of WT or tlr3−/− peritoneal macrophages co-cultured with necrotic neutrophils, apoptotic splenocytes, poly(I:C), CpG-ODN, Pam3Cys, or LPS and quantified using ELISA and/or Bio-Plex. The data are means ± SEM from three to four combined experiments. *, P < 0.05; **, P < 0.01; and ***, P < 0.001 compared with tlr3−/− mice. (G and H) KC (G) and MIP-2 (H) protein levels in 96-well tissue culture plates containing WT or tlr3−/− macrophages and one of untreated, RNase-, or Benzonase-treated necrotic PMNs. **, P < 0.01 when necrotic PMNs were compared with medium; #, P < 0.05 when enzyme-treated necrotic PMNs were compared with nontreated necrotic PMNs.
Mentions: We addressed the possibility that the lack of amplification of the inflammatory response in tlr3−/− mice after injury was caused by their inability to respond to byproducts from necrotic cells. Accordingly, we examined the role of TLR3 in the generation of inflammatory chemokines by isolated peritoneal WT and tlr3−/− macrophages after their co-culture with necrotic polymorphonuclear neutrophils (PMNs) or apoptotic splenocytes. In cultures of WT macrophages, the addition of necrotic neutrophils (97% of cells were propidium iodide+ (PI+), as shown in Fig. 6 A) for 24 h significantly increased the levels of CCL5 (Fig. 6 B), MIP-2 (Fig. 6 C), CCL3 (Fig. 6 D), KC (Fig. 6 E), and CXCL10 (Fig. 6 F) above control (medium alone) levels. In marked contrast, the addition of necrotic neutrophils to cultures of tlr3−/− macrophages did not induce the expression of these chemokines (Fig. 6, B–F). However, chemokine responses evoked by the addition of apoptotic spleen cells (depicted) or neutrophils (not depicted) did not appear to be TLR3 dependent (Fig. 6, B–F).

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