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Increased Cytokine mRNA Stability by Toll-Like Receptor (TLR) Ligands
CRITICAL CARE AND TRAUMA ligands produced during sepsis on inflammatory cytokine mRNA stability.
Extracellular Adenosine Triphosphate Protects Against Sepsis by Enhancing the Intracellular Killing of Bacteria Zoltan H Nemeth, MD, PhD, Balazs Csoka, PhD, Zoltan Spolarics, MD, PhD, Louis T DiFazio, MD, Rolando H Rolandelli, MD, FACS, Gyorgy Hasko, MD, PhD Morristown Medical Center, Morristown, NJ, Rutgers; New Jersey Medical School, Newark, NJ
METHODS: Murine peritoneal macrophages were stimulated with LPS, LTA or mtDNA, and levels of TNF-a, IL-6, and IL-1b mRNA were measured via RT- PCR. Actinomycin D treatment was used to calculate mRNA stability. The macrophage cell lines RAW and J774.2 were also tested. The RNA binding protein, HuR, was immunoprecipitated from cell extracts and binding to cytokine mRNA was tested. Knockdown with siRNA targeting HuR was performed followed by mRNA stability.
INTRODUCTION: Adenosine triphosphate (ATP) is a biologically active signaling molecule that is released into the extracellular space during inflammation, shock, and sepsis. ATP regulates a wide variety of immunological processes by binding to its receptors. P2X7 is the most highly expressed ATP receptor on cells of hematopoietic origin, and has emerged as an important regulator of immunity and inflammation.
RESULTS: All 3 TLR ligands induced cytokine mRNAs, and increased mRNA stability in primary macrophages. The effect was less pronounced in cell lines, especially for mtDNA. HuR binding to mRNA correlated with increased mRNA stability, and knockdown decreased the stabilizing effect of LTA and LPS on IL-6 and IL-1b, but not TNF-a. HuR phosphorylation was increased as was cytosolic HuR accumulation, which was inhibited with pretreatment with rottlerin.
METHODS: In this study, P2X7(-/-) mice, the pharmacological agonists ATP-magnesium salt (Mg-ATP, 100 mg/kg, EC50 z 1.32 mM) and ben-zoylbenzoyl-ATP (Bz-ATP, 10 mg/kg, EC50 z 285 mM), and antagonist oxidized ATP (oxi-ATP, 40 mg/kg, IC50 z 100 mM) were used to show that P2X7R activation is crucial for the control of mortality, bacterial dissemination, and inflammation in cecal ligation and puncture-induced polymicrobial sepsis in mice.
CONCLUSIONS: We demonstrate the novel finding that the TLR ligands LTA and mtDNA regulate different cytokine mRNA expression at the post-transcriptional level through HuR activity. Indicators of Radiographic Progression after Positive Initial Head CT in Trauma: A Multi-Institutional Retrospective Review Abid D Khan, MD, Anna J Elseth, Jaqueline A Brosius, MD, Thomas J Schroeppel, MD, FACS, Richard P Gonzalez, MD, FACS University of Colorado Health-Memorial Hospital, Colorado Springs, CO; Loyola University Medical Center, Maywood, IL
RESULTS: Our results with P2X7(-/-) bone marrow chimeric mice, adoptive transfer of peritoneal macrophages, and myeloidspecific P2X7(-/-) mice indicate that P2X7R signaling on macrophages is essential for the protective effect of P2X7Rs. P2X7R signaling protects through enhancing bacterial killing by macrophages, which is independent of the inflammasome. By using the connexin (Cx) channel inhibitor Gap27 (0.1 mg/kg, IC50 z 0.25 mM) and pannexin channel inhibitor probenecid (10 mg/ kg, IC50 z 11.7 mM), we showed that ATP release through Cx is important for inhibiting inflammation and bacterial growth.
INTRODUCTION: Brain injury guidelines (BIG) have suggested that repeat head CT (RHCT) is not required for all patients with positive findings on initial head CT after trauma. Patients categorized as BIG 1 and 2 would not have a RHCT, according to the guidelines. Our objective is to determine which factors are associated with an increased risk of radiographic progression after a positive initial CT and which patients may benefit from RHCT.
CONCLUSIONS: Our findings that P2X7 receptor inactivation leads to higher mortality and cell death in sepsis suggest that perhaps stimulation of this signaling pathway could convey an endogenous protective immune mechanism in the treatment of sepsis.
METHODS: A multi-institutional retrospective review of all traumatic head injury patients with positive CT findings was conducted. Patients were classified as BIG 1, 2, or 3. Data were then analyzed with Chi-square, Fisher’s exact test, Wilcoxon test, or univariable logistic regression, where appropriate.
Increased Cytokine mRNA Stability by Toll-Like Receptor (TLR) Ligands Bedabrata Sarkar, MD, PhD, FACS, Jun-Yuan Huang, PhD, Alex Rothkrug, Michael Ghio, Tejal S Brahmbhatt, MD, George Kasotakis, MD, MPH, Chaitan K Narsule, MD, FACS Boston University School of Medicine, Boston, MA
RESULTS: Radiographic progression occurred in 11.0% of BIG 1 and 2 patients (n¼218). BIG 1 and 2 patients with intraparenchymal hemorrhage (IPH) (OR¼3.20 [95% CI 1.29-7.93]) or with multiple CT findings (OR¼2.74 [95% CI 1.12-6.74]) had the highest risk of progression. One BIG 2 patient had a clinically significant decompensation and required neurosurgical intervention. Overall, 19.3% of BIG 1, 2, and 3 patients had radiographic progression (n¼652). Again, those with IPH (OR¼1.70 [95% CI 1.03-2.79]) and multiple findings (OR¼2.50 [95% CI 1.303.90] were most likely to progress. Eight of the 126 patients that had radiographic progression required neurosurgical intervention.
INTRODUCTION: Lipoteichoic acid (LTA) exclusive to Grampositive bacteria is functionally equivalent to lipopolysaccharide (LPS), of Gram-negative bacteria. Distinct patterns of cytokine expression have been identified after binding to their respective receptors, TLR2 and TLR4. In septic patients, mitochondrial DNA (mtDNA) correlates with mortality, and binds TLR9. Inflammatory cytokines induced by TLR signaling are upregulated at the level of mRNA stability, through the actions of RNA binding proteins such as HuR. Our focus was to determine the effects of TLR
e15
http://dx.doi.org/10.1016/j.jamcollsurg.2017.07.562 ISSN 1072-7515/17
Extracellular Adenosine Triphosphate Protects Against Sepsis by Enhancing the Intracellular Killing of Bacteria Zoltan H Nemeth, MD, PhD, Balazs Csoka, PhD, Zoltan Spolarics, MD, PhD, Louis T DiFazio, MD, Rolando H Rolandelli, MD, FACS, Gyorgy Hasko, MD, PhD Morristown Medical Center, Morristown, NJ, Rutgers; New Jersey Medical School, Newark, NJ
METHODS: Murine peritoneal macrophages were stimulated with LPS, LTA or mtDNA, and levels of TNF-a, IL-6, and IL-1b mRNA were measured via RT- PCR. Actinomycin D treatment was used to calculate mRNA stability. The macrophage cell lines RAW and J774.2 were also tested. The RNA binding protein, HuR, was immunoprecipitated from cell extracts and binding to cytokine mRNA was tested. Knockdown with siRNA targeting HuR was performed followed by mRNA stability.
INTRODUCTION: Adenosine triphosphate (ATP) is a biologically active signaling molecule that is released into the extracellular space during inflammation, shock, and sepsis. ATP regulates a wide variety of immunological processes by binding to its receptors. P2X7 is the most highly expressed ATP receptor on cells of hematopoietic origin, and has emerged as an important regulator of immunity and inflammation.
RESULTS: All 3 TLR ligands induced cytokine mRNAs, and increased mRNA stability in primary macrophages. The effect was less pronounced in cell lines, especially for mtDNA. HuR binding to mRNA correlated with increased mRNA stability, and knockdown decreased the stabilizing effect of LTA and LPS on IL-6 and IL-1b, but not TNF-a. HuR phosphorylation was increased as was cytosolic HuR accumulation, which was inhibited with pretreatment with rottlerin.
METHODS: In this study, P2X7(-/-) mice, the pharmacological agonists ATP-magnesium salt (Mg-ATP, 100 mg/kg, EC50 z 1.32 mM) and ben-zoylbenzoyl-ATP (Bz-ATP, 10 mg/kg, EC50 z 285 mM), and antagonist oxidized ATP (oxi-ATP, 40 mg/kg, IC50 z 100 mM) were used to show that P2X7R activation is crucial for the control of mortality, bacterial dissemination, and inflammation in cecal ligation and puncture-induced polymicrobial sepsis in mice.
CONCLUSIONS: We demonstrate the novel finding that the TLR ligands LTA and mtDNA regulate different cytokine mRNA expression at the post-transcriptional level through HuR activity. Indicators of Radiographic Progression after Positive Initial Head CT in Trauma: A Multi-Institutional Retrospective Review Abid D Khan, MD, Anna J Elseth, Jaqueline A Brosius, MD, Thomas J Schroeppel, MD, FACS, Richard P Gonzalez, MD, FACS University of Colorado Health-Memorial Hospital, Colorado Springs, CO; Loyola University Medical Center, Maywood, IL
RESULTS: Our results with P2X7(-/-) bone marrow chimeric mice, adoptive transfer of peritoneal macrophages, and myeloidspecific P2X7(-/-) mice indicate that P2X7R signaling on macrophages is essential for the protective effect of P2X7Rs. P2X7R signaling protects through enhancing bacterial killing by macrophages, which is independent of the inflammasome. By using the connexin (Cx) channel inhibitor Gap27 (0.1 mg/kg, IC50 z 0.25 mM) and pannexin channel inhibitor probenecid (10 mg/ kg, IC50 z 11.7 mM), we showed that ATP release through Cx is important for inhibiting inflammation and bacterial growth.
INTRODUCTION: Brain injury guidelines (BIG) have suggested that repeat head CT (RHCT) is not required for all patients with positive findings on initial head CT after trauma. Patients categorized as BIG 1 and 2 would not have a RHCT, according to the guidelines. Our objective is to determine which factors are associated with an increased risk of radiographic progression after a positive initial CT and which patients may benefit from RHCT.
CONCLUSIONS: Our findings that P2X7 receptor inactivation leads to higher mortality and cell death in sepsis suggest that perhaps stimulation of this signaling pathway could convey an endogenous protective immune mechanism in the treatment of sepsis.
METHODS: A multi-institutional retrospective review of all traumatic head injury patients with positive CT findings was conducted. Patients were classified as BIG 1, 2, or 3. Data were then analyzed with Chi-square, Fisher’s exact test, Wilcoxon test, or univariable logistic regression, where appropriate.
Increased Cytokine mRNA Stability by Toll-Like Receptor (TLR) Ligands Bedabrata Sarkar, MD, PhD, FACS, Jun-Yuan Huang, PhD, Alex Rothkrug, Michael Ghio, Tejal S Brahmbhatt, MD, George Kasotakis, MD, MPH, Chaitan K Narsule, MD, FACS Boston University School of Medicine, Boston, MA
RESULTS: Radiographic progression occurred in 11.0% of BIG 1 and 2 patients (n¼218). BIG 1 and 2 patients with intraparenchymal hemorrhage (IPH) (OR¼3.20 [95% CI 1.29-7.93]) or with multiple CT findings (OR¼2.74 [95% CI 1.12-6.74]) had the highest risk of progression. One BIG 2 patient had a clinically significant decompensation and required neurosurgical intervention. Overall, 19.3% of BIG 1, 2, and 3 patients had radiographic progression (n¼652). Again, those with IPH (OR¼1.70 [95% CI 1.03-2.79]) and multiple findings (OR¼2.50 [95% CI 1.303.90] were most likely to progress. Eight of the 126 patients that had radiographic progression required neurosurgical intervention.
INTRODUCTION: Lipoteichoic acid (LTA) exclusive to Grampositive bacteria is functionally equivalent to lipopolysaccharide (LPS), of Gram-negative bacteria. Distinct patterns of cytokine expression have been identified after binding to their respective receptors, TLR2 and TLR4. In septic patients, mitochondrial DNA (mtDNA) correlates with mortality, and binds TLR9. Inflammatory cytokines induced by TLR signaling are upregulated at the level of mRNA stability, through the actions of RNA binding proteins such as HuR. Our focus was to determine the effects of TLR
e15
http://dx.doi.org/10.1016/j.jamcollsurg.2017.07.562 ISSN 1072-7515/17