An anti-inflammatory phenotype with pronounced immunosuppression develops during sepsis, during which time neutrophils and monocytes/macrophages limit their Toll-like receptor 4 responses to bacterial lipopolysaccharide (LPS/endotoxin). We previously reported that during this endotoxin-tolerant state, distinct signaling pathways differentially repress transcription and translation of proinflammatory cytokines such as TNFα and IL-6. Sustained endotoxin tolerance contributes to sepsis mortality. While transcription repression requires chromatin modifications, a translational repressor complex of Argonaute 2 (Ago2) and RNA-binding motif protein 4 (RBM4), which bind the 3′-UTR of TNFα and IL-6 mRNA, limits protein synthesis. Here, we show that Dcp1 supports the assembly of the Ago2 and RBM4 repressor complex into cytoplasmic processing bodies (p-bodies) in endotoxin-tolerant THP-1 human monocytes following stimulation with LPS, resulting in translational repression and limiting protein synthesis. Importantly, this translocation process is reversed by Dcp1 knockdown, which restores TNFα and IL-6 protein levels. We also find this translational repression mechanism in primary macrophages of septic mice. Because p-body formation is a critical step in mRNA translation repression, we conclude that Dcp1 is a major component of the translational repression machinery of endotoxin tolerance and may contribute to sepsis outcome.

Toll-like receptors (TLRs) are critical components of the innate immune response of fish. In a phylogenetic analysis, TLR27 from three fish species, which belongs to TLR family 1, clustered with TLR14/18 and TLR25 on the evolutionary tree. The ectodomain of TLR27 is predicted to include 19 leucine-rich repeat (LRR) modules. Structural modeling showed that the TLR27 ectodomain can be divided into three distinctive sections. The lack of conserved asparagines on the concave surface of the central subdomain causes a structural transition in the middle of the ectodomain, forming a distinct hydrophobic pocket at the border between the central subdomain and the C-terminal subdomain. We infer that, like other functionally characterized TLRs in family 1, the hydrophobic pocket located between LRR11 and LRR12 participates in ligand recognition by TLR27. An evolutionary analysis showed that the dN/dS value at the TLR27 locus was very low. Approximately one quarter of the total number of TLR27 sites are under significant negatively selection pressure, whereas only two sites are under positive selection. Consequently, TLR27 is highly evolutionarily conserved and probably plays an extremely important role in the innate immune systems of fishes.

OBJECTIVE: To determine the role of Toll-like receptor 3 in cardiac dysfunction during polymicrobial sepsis. DESIGN: Controlled animal study. SETTING: University research laboratory. SUBJECTS: Male C57BL/6, wild-type, Toll-like receptor 3. INTERVENTION: Myocardial dysfunction is a major consequence of septic shock and contributes to the high mortality of sepsis. Toll-like receptors (TLRs) play a critical role in the pathophysiology of sepsis/septic shock. TLR3 is located in intracellular endosomes, and recognizes double-stranded RNA. This study examined the role of TLR3 in cardiac dysfunction following cecal ligation and puncture (CLP)-induced sepsis. TLR3 knockout (TLR3, n = 12) and age-matched wild-type (n = 12) mice were subjected to CLP. Cardiac function was measured by echocardiography before and 6 hrs after CLP. MEASUREMENTS AND MAIN RESULTS: CLP resulted in significant cardiac dysfunction as evidenced by decreased ejection fraction by 25.7% and fractional shortening by 29.8%, respectively. However, TLR3 mice showed a maintenance of cardiac function at pre-CLP levels. Wild-type mice showed 50% mortality at 58 hrs and 100% mortality at 154 hrs after CLP. In striking contrast, 70% of TLR3 mice survived indefinitely, that is, >200 hrs. TLR3 deficiency significantly decreased CLP-induced cardiac-myocyte apoptosis and attenuated CLP-induced Fas and Fas ligand expression in the myocardium. CLP-activation of TLR4-mediated nuclear factor-κB and Toll/IL-1 receptor-domain-containing adapter-inducing interferon-β-dependant interferon signaling pathways was prevented by TLR3 deficiency. In addition, CLP-increased vascular cell adhesion molecule-1 and intercellular adhesion molecule-1 expression, and neutrophil and macrophage sequestration in the myocardium were also attenuated in septic TLR3 mice. More significantly, adoptive transfer of wild-type bone-marrow stromal cells to TLR3 mice abolished the cardioprotective effect in sepsis. CONCLUSIONS: These data indicate that TLR3 plays a deleterious role in mediating cardiac dysfunction in sepsis. Thus, modulation of the TLR3 activity may be useful in preventing cardiac dysfunction in sepsis.

Myocardial dysfunction is a major consequence of septic shock and contributes to the high mortality of sepsis. In the present study, we examined the effect of Toll-like receptor 2 (TLR2) ligands, peptidoglycan (PGN), and Pam3CSK4 (Pam3) on cardiac function in cecal ligation and puncture (CLP)-induced sepsis in mice. We also investigated whether the phosphoinositide 3-kinase (PI3K)/Akt signaling pathway is involved in the effect of TLR2 ligands on cardiac function in CLP mice. PGN was administered to C57B6/L mice 1 h before the induction of CLP. Sham surgically operated mice served as a control. Cardiac function indexes (rate of change in left ventricular pressure, stroke work, cardiac output, and ejection fraction) were examined by a microconductance pressure catheter. Cardiac function was significantly decreased 6 h after CLP-induced sepsis compared with shamoperated control. In contrast, PGN administration attenuated CLPinduced cardiac dysfunction. Importantly, the therapeutic treatment with Pam3 1 h after CLP also significantly attenuated cardiac dysfunction in CLP mice. However, the beneficial effect of TLR2 ligands on cardiac dysfunction in CLP-mice was abolished in TLR2-deficient mice. PGN administration significantly increased the levels of phospho-Akt and phospho-GSK-3β in the myocardium compared with the levels in untreated CLP mice. PI3K inhibition abolished the PGNinduced attenuation of cardiac dysfunction in CLP mice. In conclusion, these data demonstrate that the administration of TLR2 ligands, PGN, or Pam3 attenuates cardiac dysfunction in septic mice via a TLR2/PI3K-dependent mechanism. More significantly, Pam3 therapeutic treatment will have a potential clinical relevance.

We have previously reported that morphine induces apoptosis. However, the underlying molecular mechanisms remain to be elucidated. Toll-like receptor 2 (TLR2), a key immune receptor in the TLR family, modulates cell survival and cell death in various systems. Evidence indicates that β-arrestin 2 acts as a negative regulator of innate immune activation by TLRs. Here, we investigated the roles of TLR2, the downstreaming mediator MyD88, and β-arrestin 2 in morphine-induced apoptosis. We showed that overexpression of TLR2 in HEK293 cells caused a significant increase in apoptosis after morphine treatment. Inhibition of MyD88 by transfecting dominant negative MyD88 or overexpression of β-arrestin 2 by transfecting β-arrestin 2 full length plasmid in TLR2 overexpressing HEK293 cells attenuated morphine-induced apoptosis. Our study thus demonstrates that TLR2 signaling mediates the morphine-induced apoptosis, and β-arrestin 2 is a negative regulator in morphine-induced, TLR2-mediated apoptosis.

The brain's resistance to ischemic injury can be transiently augmented by prior exposure to a sub-lethal stress stimulus, i.e. preconditioning. It has been reported that Toll-like receptors (TLRs) are involved in the preconditioning-induced protective effect against ischemic brain injury. In this study, we investigated the effect of preconditioning with a TLR2 specific ligand, Pam3CSK4, on focal cerebral ischemia/reperfusion (I/R) injury in mice. Pam3CSK4 was administered systemically 24 h before the mice were subjected to focal cerebral ischemia (1 h) followed by reperfusion. Cerebral infarct size was determined, blood brain barrier (BBB) permeability was evaluated, and expression of tight-junction proteins were examined after focal cerebral I/R. Results showed that pre-treatment with Pam3CSK significantly reduced brain infarct size (1.9 ± 0.5% vs 9.4 ± 2.2%) compared with the untreated I/R group. Pam3CSK4 pre-treatment also significantly reduced acute mortality (4.3% vs 24.2%), preserved neurological function (8.22 ± 0.64 vs 3.91 ± 0.57), and attenuated brain edema (84.61 ± 0.08% vs 85.29 ± 0.09%) after cerebral I/R. In addition, Pam3CSK4 pre-treatment preserved BBB function as evidenced by decreased leakage of serum albumin (0.528 ± 0.026 vs 0.771 ± 0.059) and Evans Blue (9.23 ± 0.72 μg/mg vs 12.56 ± 0.65 μg/mg) into brain tissue. Pam3CSK4 pre-treatment also attenuated the loss of the tight junction protein occludin in response to brain I/R injury. These results suggest that TLR2 is a new target of ischemic preconditioning in the brain and preconditioning with a TLR2 specific ligand will protect the brain from I/R injury.

Objective: We have previously demonstrated that nuclear factor kappa B (NFκB) activation is needed for the development of cardiac hypertrophy in vivo. NFκB is a downstream transcription factor in the Toll-like receptor (TLR)-mediated signaling pathway; therefore, we investigated a role of TLR4 in cardiac hypertrophy in vivo. Methods: TLR4-deficient mice (C.C3H-Tlr4 lps-d, n = 6), wild-type (WT) genetic background mice (BALB/c, n = 6), TLR4-deleted strain (C57BL/10ScCr, n = 8), and WT controls (C57BL/10ScSn, n = 8) were subjected to aortic banding for 2 weeks. Age-matched surgically operated mice served as controls. In a separate experiment, rapamycin (2 mg/kg, daily) was administered to TLR4-deficient mice and WT mice immediately following aortic banding. The ratio of heart weight/body weight (HW / BW) was calculated, and cardiac myocyte size was examined by FITC-labeled wheat germ agglutinin staining of membranes. NFκB binding activity and the levels of phospho-p70S6K in the myocardium were also examined. Results: Aortic banding significantly increased the ratio of HW / BW by 33.9% (0.601 ± 0.026 vs. 0.449 ± 0.004) and cell size by 68.4% in WT mice and by 10.00% (0.543 ± 0.011 vs. 0.495 ± 0.005) and by 11.8% in TLR4-deficient mice, respectively, compared with respective sham controls. NFκB binding activity and phospho-p70S6K levels were increased by 182.6% and 115.2% in aortic-banded WT mice and by 78.0% and 162.0% in aortic-banded TLR4-deficient mice compared with respective sham controls. In rapamycin-treated aortic-banded mice, the ratio of HW / BW was increased by 18.0% in WT mice and by 3.5% in TLR4-deficient mice compared with respective sham controls. Conclusion: Our results demonstrate that TLR4 is a novel receptor contributing to the development of cardiac hypertrophy in vivo and that both the TLR4-mediated pathway and PI3K/Akt/mTOR signaling are involved in the development of cardiac hypertrophy in vivo.

Gain-of-function polymorphisms in the gene encoding human interferon regulatory factor-5 (IRF5) are associated with an increase in risk for the development of the autoimmune disease Systemic Lupus Erythematosus (SLE). IRF5 is a transcription factor that participates in the activation of the immune system through its role in both innate and adaptive immune cells. To determine the role of IRF5 in lupus pathogenesis in vivo, we evaluated the effect of Irf5-deficiency in the MRL/lpr mouse lupus model. We find that Irf5-deficient (Irf5-/-) MRL/lpr mice develop much less severe disease than their Irf5-sufficient (Irf5+/+) littermates, demonstrating an important role for IRF5 in disease pathogenesis in vivo. Patients with SLE are at increased risk for the development of atherosclerosis due in large part to poorly-defined lupus-specific risk factors. One such lupus-specific risk factor is thought to be chronic inflammation associated with the autoimmune process. As IRF5 is involved in pro-inflammatory responses we hypothesized that Irf5-deficiency would ameliorate atherosclerosis development in the context of autoimmunity. We therefore examined the role of IRF5 in the gld.apoE-/- mouse model of lupus and lupus-associated atherosclerosis. Irf5-deficiency led to a decrease in splenomegaly, lymphadenopathy, anti-nuclear autoantibody production and the severity of kidney disease. Surprisingly, despite the reduction in systemic autoimmunity, Irf5-deficiency led to a marked increase in the severity of atherosclerosis and to metabolic dysregulation characterized by hyperlipidemia, increased adiposity and insulin-resistance. Bone marrow chimera studies revealed that the pathogenic role of IRF5 in lupus was solely due to its expression in hematopoietic cells. The atheroprotective effect of Irf5 and the suppression of adiposity were found to be due to Irf5 expression in both hematopoietic and non-hematopoietic cells, whereas protection from hyperlipidemia was solely due to the expression of Irf5 in non-hematopoietic cells. Together, our results reveal a role for IRF5 in metabolic homeostasis, as well as in protection against atherosclerosis even in the setting of reduced lupus severity.

京都大学 / 0048 / 新制・課程博士 / 博士(薬学) / 甲第18549号 / 薬博第811号 / 新制||薬||238(附属図書館) / 31449 / 京都大学大学院薬学研究科医療薬科学専攻 / (主査)教授 橋田 充, 教授 髙倉 喜信, 教授 佐治 英郎 / 学位規則第4条第1項該当 / Doctor of Pharmaceutical Sciences / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20267号 / 医博第4226号 / 新制||医||1021(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 妹尾 浩, 教授 坂井 義治, 教授 竹内 理 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DGAM