Abstract
In sepsis, toll-like receptor (TLR)-4 modulates the migration of neutrophils to infectious foci, favoring bacteremia and mortality. In experimental sepsis, organ dysfunction and cytokines released by activated macrophages can be reduced by gastrin-releasing peptide (GRP) receptor (GRPR) antagonist RC-3095. Here we report a link between GRPR and TLR-4 in experimental models and in sepsis patients. RAW 264.7 culture cells were exposed to lipopolysaccharide (LPS) or tumor necrosis factor (TNF)-α and RC-3095 (10 ng/mL). Male Wistar rats were subjected to cecal ligation and puncture (CLP), and RC-3095 was administered (3 mg/kg, subcutaneously); after 6 h, we removed the blood, bronchoalveolar lavage, peritoneal lavage and lung. Human patients with a clinical diagnosis of sepsis received a continuous infusion with RC-3095 (3 mg/kg, intravenous) over a period of 12 h, and plasma was collected before and after RC-3095 administration and, in a different set of patients with systemic inflammatory response syndrome (SIRS) or sepsis, GRP plasma levels were determined. RC-3095 inhibited TLR-4, extracellular-signal-related kinase (ERK)-1/2, Jun NH(2)-terminal kinase (JNK) and Akt and decreased activation of activator protein 1 (AP-1), nuclear factor (NF)-κB and interleukin (IL)-6 in macrophages stimulated by LPS. It also decreased IL-6 release from macrophages stimulated by TNF-α. RC-3095 treatment in CLP rats decreased lung TLR-4, reduced the migration of cells to the lung and reduced systemic cytokines and bacterial dissemination. Patients with sepsis and systemic inflammatory response syndrome have elevated plasma levels of GRP, which associates with clinical outcome in the sepsis patients. These findings highlight the role of GRPR signaling in sepsis outcome and the beneficial action of GRPR antagonists in controlling the inflammatory response in sepsis through a mechanism involving at least inhibition of TLR-4 signaling.
Highlights
Sepsis remains an important problem with high rates of morbidity and mortality, despite modern advances in critical care management
This binding activity was suppressed by exposure to RC-3095, suggesting that suppression of nuclear factor (NF)-κB and nuclear translocation of activator protein 1 (AP-1) by RC-3095 was associated with decreased gene expression of toll-like receptor (TLR)-4 and MAP kinase activation (Figure 1C, F = 54 [group versus time], F = 442 [group versus result], F = 135 [time versus result], p < 0.001 to all interactions; Figure 1D, F = 7.6 [group versus time], F = 31 [group versus result], F = 17 [time versus result], p < 0.001 to all interactions; and Figure 1E, F = 0.04, p = 1.0 [group versus time], F = 277 [group versus result], p < 0.001, F = 0.04 [time versus result], p = 1.0)
Our analysis shows direct interaction of RC3095 only with gastrin-releasing peptide receptor (GRPR) and gastrin-releasing peptide (GRP), and LPS is connected with the network at first level by interaction with TLR4 and the lymphocyte antigen 96 (LY96)
Summary
Sepsis remains an important problem with high rates of morbidity and mortality, despite modern advances in critical care management. Molecules involved in the TLR-4–activated pathway include the adaptor molecule, myeloid differentiation primary response protein 88 (MyD88), interleukin (IL)-1 receptor–associated kinases and TNF receptor–associated factor 6 [9] This pathway results in activation of several mitogen-activated protein kinases (MAPKs), as well as activation of the transcription factors such as nuclear factor (NF)-κB and activator protein 1 (AP-1), which contribute to the development of septic shock and multiple organ failure with transcriptional regulation of inflammatory genes [10]. In this context, TLR-4–defective mice presented neutrophil migration to the peritoneal cavity during sepsis induced by lethal cecal ligation and puncture (CLP) and, as a consequence, are more resistant to sepsis than controls [11]. Given its central role in the pathogenesis of sepsis, TLR-4 is a target for the development of novel therapies against sepsis
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