We examined the effect of modulating phosphoinositide 3-kinase (PI3K) activity in a murine model of cecal ligation and puncture-induced polymicrobial sepsis. Inhibition of PI3K activity with wortmannin increased serum cytokine levels and decreased survival time in septic mice. We have reported that an immunomodulator, glucan phosphate, induces protection in murine polymicrobial sepsis. We observed that glucan stimulated tissue PI3K activity, which positively correlated with increased survival in septic mice. We investigated the effect of PI3K inhibition on survival in septic mice treated with glucan. Treatment of mice with the PI3K inhibitors, wortmannin and LY294002, completely eliminated the protective effect of glucan, indicating that protection against septic mortality was mediated through PI3K. Inhibition of PI3K resulted in increased serum levels of IL1-β, IL-2, IL-6, IL-10, IL-12, and TNF-α in septic mice. Apoptosis is thought to play a central role in the response to septic injury. We observed that inhibition of PI3K activity in septic mice resulted in increased splenocyte apoptosis and a change in the anatomic distribution of splenocyte apoptosis. We conclude that PI3K is a compensatory mechanism that suppresses proinflammatory and apoptotic processes in response to sepsis and/or inflammatory injury. Thus, PI3K may play a pivotal role in the maintenance of homeostasis and the integrity of the immune response during sepsis. We also observed that glucan phosphate decreased septic morbidity and mortality through a PI3K-dependent mechanism. This suggests that stimulation of the PI3K pathway may be an effective approach for preventing or treating sepsis and/or septic shock.
Toll-like receptors (TLRs) play a critical role in the induction of innate immune responses which have been implicated in neuronal death induced by global cerebral ischemia/reperfusion (GCI/R). The present study investigated the role and mechanisms-of-action of TLR4 signaling in ischemiainduced hippocampal neuronal death. Neuronal damage, activation of the TLR4 signaling pathway, expression of pro-inflammatory cytokines and activation of the PI3K/Akt signaling pathway in the hippocampal formation (HF) were assessed in wild type (WT) mice and TLR4 knockout mice (TLR4 -/-) mice after GCI/R. GCI/R increased expression of TLR4 protein in the hippocampal formation (HF) and other brain structures in WT mice. Phosphorylation of the inhibitor of kappa B (p-IκB) as well as activation of nuclear factor kappa B (NFκB) increased in the HF of WT mice. In contrast, there were lower levels of p-IκB and NFκB binding activity in TLR4 -/-mice subjected to GCI/R. Pro-inflammatory cytokine expression was also decreased, while phosphorylation of Akt and GSK3β were increased in the HF of TLR4 -/-mice after GCI/R. These changes correlated with decreased neuronal death/apoptosis in TLR4 -/-mice following GCI/R. These data suggest that activation of TLR4 signaling contributes to ischemia-induced hippocampal neuronal death. In addition, these data suggest that modulation of TLR4 signaling may attenuate ischemic injury in hippocampal neurons.
Recent studies have shown that Toll-like receptors (TLRs) are involved in cerebral ischemia/ reperfusion (I/R) injury. This study was to investigate the role of TLR2 and TLR4 in acute focal cerebral I/R injury. Cerebral infarct size, neurological function and mortality were evaluated. NFκB binding activity, phosphorylation of IκBα, Akt and ERK1/2 were examined in ischemic cerebral tissue by EMSA and Western blots. Compared to wild type (WT) mice, in TLR4 knockout (TLR4KO) mice, brain infarct size was decreased (2.6 ± 1.18% vs 11.6 ± 1.97% of whole cerebral volume, p<0.05) and neurological function was maintained (7.3 ± 0.79 vs 4.7 ± 0.68, p<0.05). However, compared to TLR4KO mice, TLR2 knockout (TLR2KO) mice showed higher mortality (38.2% vs 13.0%, p<0.05), decreased neurological function (2.9 ± 0.53 vs 7.3 ± 0.79, p<0.05) and increased brain infarct size (19.1 ± 1.33% vs 2.6 ± 1.18%, p<0.05). NFκB activation and IκBα phosphorylation were attenuated in TLR4KO mice (1.09 ± 0.02 and 1.2 ± 0.04) compared to TLR2KO mice (1.31 ± 0.02 and 2.2 ± 0.32) after cerebral ischemia. Compared to TLR4KO mice, in TLR2KO mice, the phosphorylation of Akt (0.2 ± 0.03 vs 0.9 ± 0.16, p<0.05) and ERK1/2 (0.8 ± 0.06 vs 1.3 ± 0.17) evoked by cerebral I/R was attenuated. The present study demonstrates that TLR2 and TLR4 play differential roles in acute cerebral I/R injury. Specifically, TLR4 contributes to cerebral I/R injury, while TLR2 appears to be neuroprotective by enhancing the activation of protective signaling in response to cerebral I/R.
Early increases in TLR2/4 gene and TLR4 protein expression correlated with mortality, whereas blunting TLR gene and protein expression correlated with improved long-term survival. This suggests that early up-regulation of tissue TLR2/4 may play a role in the proinflammatory response and pathophysiology of polymicrobial sepsis.
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