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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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Increased peritoneal neutrophil influx and a marked reduction in necrotic peritoneal cells during septic peritonitis in tlr3−/− mice. (A) WT and tlr3−/− peritoneal cells at 24 h after sham and CLP surgeries. (B) MPO levels in peritoneal lavage samples at 24 h after either sham or CLP surgery. Data are means ± SEM from two separate experiments. (C) Toluidine blue staining of peritoneal cells from WT and tlr3−/− mice at 24 h after CLP surgery. Bars, 50 μm. (D and E) Apoptotic and necrotic cells were identified by annexin V+/PI− and annexin V+/PI+ staining, respectively. The data represent means ± SEM (n = 5 mice per condition). *, P < 0.05; and **, P < 0.01 compared with C57BL/6 mice after CLP surgery.
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fig2: Increased peritoneal neutrophil influx and a marked reduction in necrotic peritoneal cells during septic peritonitis in tlr3−/− mice. (A) WT and tlr3−/− peritoneal cells at 24 h after sham and CLP surgeries. (B) MPO levels in peritoneal lavage samples at 24 h after either sham or CLP surgery. Data are means ± SEM from two separate experiments. (C) Toluidine blue staining of peritoneal cells from WT and tlr3−/− mice at 24 h after CLP surgery. Bars, 50 μm. (D and E) Apoptotic and necrotic cells were identified by annexin V+/PI− and annexin V+/PI+ staining, respectively. The data represent means ± SEM (n = 5 mice per condition). *, P < 0.05; and **, P < 0.01 compared with C57BL/6 mice after CLP surgery.

Mentions: The contribution of TLR3 to septic peritonitis in WT and tlr3−/− mice after sham or CLP surgery is summarized in Fig. 2. A significantly greater number of peritoneal cells were present in the tlr3−/− group compared with the WT group at 24 h after CLP surgery (Fig. 2 A). Significantly higher peritoneal levels of myeloperoxidase (MPO; a specific marker of neutrophil recruitment) were also present in tlr3−/− mice compared with WT mice (Fig. 2 B). Moreover, peritoneal washes from septic mice were characterized by considerable necrotic cellular debris and bacterial contamination, whereas similar washings from septic tlr3−/− mice contained greater numbers of neutrophils, little necrotic cellular debris, and fewer bacteria (Fig. 2 C). It is worth noting that although peritoneal neutrophil numbers were significantly greater in the tlr3−/− group compared with the WT group after 24 h following CLP surgery, neutrophil numbers returned to equivalent levels in the peritoneal cavity in the surviving WT and knockout groups at 72 h after CLP (unpublished data). Consistent with the decreased presence of bacteria contamination in peritoneal washes from the tlr3−/− group at 24 h after CLP surgery, we observed that bacteria were not detected in the peritoneal fluid and blood in any of the tlr3−/− mice, whereas WT mice exhibited 104–106 log bacteria/CFU/μl of peritoneal wash and 105 log bacteria/CFU/μl of blood.


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)

Increased peritoneal neutrophil influx and a marked reduction in necrotic peritoneal cells during septic peritonitis in tlr3−/− mice. (A) WT and tlr3−/− peritoneal cells at 24 h after sham and CLP surgeries. (B) MPO levels in peritoneal lavage samples at 24 h after either sham or CLP surgery. Data are means ± SEM from two separate experiments. (C) Toluidine blue staining of peritoneal cells from WT and tlr3−/− mice at 24 h after CLP surgery. Bars, 50 μm. (D and E) Apoptotic and necrotic cells were identified by annexin V+/PI− and annexin V+/PI+ staining, respectively. The data represent means ± SEM (n = 5 mice per condition). *, P < 0.05; and **, P < 0.01 compared with C57BL/6 mice after CLP surgery.
© Copyright Policy
Related In: Results  -  Collection

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

fig2: Increased peritoneal neutrophil influx and a marked reduction in necrotic peritoneal cells during septic peritonitis in tlr3−/− mice. (A) WT and tlr3−/− peritoneal cells at 24 h after sham and CLP surgeries. (B) MPO levels in peritoneal lavage samples at 24 h after either sham or CLP surgery. Data are means ± SEM from two separate experiments. (C) Toluidine blue staining of peritoneal cells from WT and tlr3−/− mice at 24 h after CLP surgery. Bars, 50 μm. (D and E) Apoptotic and necrotic cells were identified by annexin V+/PI− and annexin V+/PI+ staining, respectively. The data represent means ± SEM (n = 5 mice per condition). *, P < 0.05; and **, P < 0.01 compared with C57BL/6 mice after CLP surgery.
Mentions: The contribution of TLR3 to septic peritonitis in WT and tlr3−/− mice after sham or CLP surgery is summarized in Fig. 2. A significantly greater number of peritoneal cells were present in the tlr3−/− group compared with the WT group at 24 h after CLP surgery (Fig. 2 A). Significantly higher peritoneal levels of myeloperoxidase (MPO; a specific marker of neutrophil recruitment) were also present in tlr3−/− mice compared with WT mice (Fig. 2 B). Moreover, peritoneal washes from septic mice were characterized by considerable necrotic cellular debris and bacterial contamination, whereas similar washings from septic tlr3−/− mice contained greater numbers of neutrophils, little necrotic cellular debris, and fewer bacteria (Fig. 2 C). It is worth noting that although peritoneal neutrophil numbers were significantly greater in the tlr3−/− group compared with the WT group after 24 h following CLP surgery, neutrophil numbers returned to equivalent levels in the peritoneal cavity in the surviving WT and knockout groups at 72 h after CLP (unpublished data). Consistent with the decreased presence of bacteria contamination in peritoneal washes from the tlr3−/− group at 24 h after CLP surgery, we observed that bacteria were not detected in the peritoneal fluid and blood in any of the tlr3−/− mice, whereas WT mice exhibited 104–106 log bacteria/CFU/μl of peritoneal wash and 105 log bacteria/CFU/μl of blood.

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