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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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CLP surgery induced local and systemic expression of TLR3. (A) Representative immunochemistry analysis of TLR3 expression in peritoneal cells from sham and CLP 6 h after surgery. TLR3 expression is observed in brown. Bars, 20 μm. (B) Immunochemistry analysis of TLR3 expression in thioglycollate-elicited macrophages of tlr3−/− mice. Bar, 20 μm. (C and D) Histograms exhibit TLR3 expression in permeabilized peritoneal (C) and splenic (D) CD11b+ cells. (E and F) Nonpermeabilized peritoneal (E) and splenic (F) CD11b+ cells (gray line, isotype control antibody; shaded, sham mice; black line, CLP mice). Data are means ± SEM from three independent experiments. (G) Immunoreactive CCL3 and MIP-2 supernatant levels from thioglycollate-elicited WT and tlr3−/− peritoneal neutrophils exposed to poly(I:C). *, P < 0.05 compared with sham mice; §, P < 0.05 compared with WT mice.
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fig1: CLP surgery induced local and systemic expression of TLR3. (A) Representative immunochemistry analysis of TLR3 expression in peritoneal cells from sham and CLP 6 h after surgery. TLR3 expression is observed in brown. Bars, 20 μm. (B) Immunochemistry analysis of TLR3 expression in thioglycollate-elicited macrophages of tlr3−/− mice. Bar, 20 μm. (C and D) Histograms exhibit TLR3 expression in permeabilized peritoneal (C) and splenic (D) CD11b+ cells. (E and F) Nonpermeabilized peritoneal (E) and splenic (F) CD11b+ cells (gray line, isotype control antibody; shaded, sham mice; black line, CLP mice). Data are means ± SEM from three independent experiments. (G) Immunoreactive CCL3 and MIP-2 supernatant levels from thioglycollate-elicited WT and tlr3−/− peritoneal neutrophils exposed to poly(I:C). *, P < 0.05 compared with sham mice; §, P < 0.05 compared with WT mice.

Mentions: PAMPs activate TLR-induced signaling pathways, leading to the induction of innate immune responses. A mixed pathological picture is associated with CLP, which includes the elicitation of various leukocyte populations, vascular permeability changes, and significant necrosis of the gut tissue (14). This latter pathology may hold an important key in understanding endogenous signals that amplify the innate response, as growing evidence has suggested that host-derived RNA (9, 13) from necrotic cells serve as major stimuli for TLR3 activation. To assess the expression pattern of TLR3 during the development of CLP sepsis, we initially localized the expression of TLR3. Although peritoneal macrophages and neutrophils recovered from the sham WT mice did not express TLR3, the expression of TLR3, via immunohistochemistry, was detected in these cell populations from mice undergoing experimental sepsis induced by CLP (Fig. 1 A). As a control, no expression of this receptor was detected in thioglycollate-elicited peritoneal macrophages from tlr3−/− mice (Fig. 1 B), confirming the specificity of this antibody. At 24 h, CLP mice showed increased intracellular expression of TLR3 in peritoneal and splenic CD11b+ cells (Fig. 1, C and D, respectively) when compared with sham mice. Surprisingly, CLP also induced the extracellular expression of TLR3 in CD11b+ cells isolated from peritoneal lavage (Fig. 1 E). No statistical differences were observed at the extracellular expression of this molecule in spleen cells from CLP and sham surgery mice (Fig. 1 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)

CLP surgery induced local and systemic expression of TLR3. (A) Representative immunochemistry analysis of TLR3 expression in peritoneal cells from sham and CLP 6 h after surgery. TLR3 expression is observed in brown. Bars, 20 μm. (B) Immunochemistry analysis of TLR3 expression in thioglycollate-elicited macrophages of tlr3−/− mice. Bar, 20 μm. (C and D) Histograms exhibit TLR3 expression in permeabilized peritoneal (C) and splenic (D) CD11b+ cells. (E and F) Nonpermeabilized peritoneal (E) and splenic (F) CD11b+ cells (gray line, isotype control antibody; shaded, sham mice; black line, CLP mice). Data are means ± SEM from three independent experiments. (G) Immunoreactive CCL3 and MIP-2 supernatant levels from thioglycollate-elicited WT and tlr3−/− peritoneal neutrophils exposed to poly(I:C). *, P < 0.05 compared with sham mice; §, P < 0.05 compared with WT mice.
© Copyright Policy
Related In: Results  -  Collection

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

fig1: CLP surgery induced local and systemic expression of TLR3. (A) Representative immunochemistry analysis of TLR3 expression in peritoneal cells from sham and CLP 6 h after surgery. TLR3 expression is observed in brown. Bars, 20 μm. (B) Immunochemistry analysis of TLR3 expression in thioglycollate-elicited macrophages of tlr3−/− mice. Bar, 20 μm. (C and D) Histograms exhibit TLR3 expression in permeabilized peritoneal (C) and splenic (D) CD11b+ cells. (E and F) Nonpermeabilized peritoneal (E) and splenic (F) CD11b+ cells (gray line, isotype control antibody; shaded, sham mice; black line, CLP mice). Data are means ± SEM from three independent experiments. (G) Immunoreactive CCL3 and MIP-2 supernatant levels from thioglycollate-elicited WT and tlr3−/− peritoneal neutrophils exposed to poly(I:C). *, P < 0.05 compared with sham mice; §, P < 0.05 compared with WT mice.
Mentions: PAMPs activate TLR-induced signaling pathways, leading to the induction of innate immune responses. A mixed pathological picture is associated with CLP, which includes the elicitation of various leukocyte populations, vascular permeability changes, and significant necrosis of the gut tissue (14). This latter pathology may hold an important key in understanding endogenous signals that amplify the innate response, as growing evidence has suggested that host-derived RNA (9, 13) from necrotic cells serve as major stimuli for TLR3 activation. To assess the expression pattern of TLR3 during the development of CLP sepsis, we initially localized the expression of TLR3. Although peritoneal macrophages and neutrophils recovered from the sham WT mice did not express TLR3, the expression of TLR3, via immunohistochemistry, was detected in these cell populations from mice undergoing experimental sepsis induced by CLP (Fig. 1 A). As a control, no expression of this receptor was detected in thioglycollate-elicited peritoneal macrophages from tlr3−/− mice (Fig. 1 B), confirming the specificity of this antibody. At 24 h, CLP mice showed increased intracellular expression of TLR3 in peritoneal and splenic CD11b+ cells (Fig. 1, C and D, respectively) when compared with sham mice. Surprisingly, CLP also induced the extracellular expression of TLR3 in CD11b+ cells isolated from peritoneal lavage (Fig. 1 E). No statistical differences were observed at the extracellular expression of this molecule in spleen cells from CLP and sham surgery mice (Fig. 1 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