R. Savanh Chanthaphavong

Current theories on the pathophysiology of multiple organ failure after trauma

Despite the enormous efforts to elucidate the mechanisms of the development of multiple organ failure (MOF) following trauma, MOF following trauma is still a leading cause of late post-injury death and morbidity. Now, it has been proven that excessive systemic inflammation following trauma participates in the development of MOF. Fundamentally, the inflammatory response is a host-defence response; however, on occasion, this response turns around to cause deterioration to host depending on exo- and endogenic factors. Through this review we aim to describe the pathophysiological approach for MOF after trauma studied so far and also introduce the prospects of this issue for the future.

Distribution and neurochemical identification of pancreatic afferents in the mouse

Dysfunction of primary afferents innervating the pancreas has been shown to contribute to the development of painful symptoms during acute and chronic pancreatitis. To investigate the distribution and neurochemical phenotype of pancreatic afferents, Alexa Fluor‐conjugated cholera toxin B (CTB) was injected into the pancreatic head (CTB‐488) and tail (CTB‐555) of adult male mice to label neurons retrogradely in both the dorsal root ganglia (DRG) and nodose ganglia (NG). The NG and DRG (T5–T13) were processed for fluorescent immunohistochemistry and visualized by using confocal microscopy. Spinal pancreatic afferents were observed from T5 to T13, with the greatest contribution coming from T9–T12. The pancreatic afferents were equally distributed between right and left spinal ganglia; however, the innervation from the left NG was significantly greater than from the right. For both spinal and vagal afferents there was significantly greater innervation of the pancreatic head relative to the tail. The total number of retrogradely labeled afferents in the nodose was very similar to the total number of DRG afferents. The neurochemical phenotype of DRG neurons was dominated by transient receptor potential vanilloid 1 (TRPV1)‐positive neurons (75%), GDNF family receptor alpha‐3 (GFRα3)‐positive neurons (67%), and calcitonin gene‐related peptide (CGRP)‐positive neurons(65%) neurons. In the NG, TRPV1‐, GFRα3‐, and CGRP‐positive neurons constituted only 35%, 1%, and 15% of labeled afferents, respectively. The disparity in peptide and receptor expression between pancreatic afferents in the NG and DRG suggests that even though they contribute a similar number of primary afferents to the pancreas, these two populations may differ in regard to their nociceptive properties and growth factor dependency. J. Comp. Neurol. 509:42–52, 2008.

Proinflammatory Role of Leukocyte-Derived Egr-1 in the Development of Murine Postoperative Ileus

BACKGROUND & AIMS Early growth response gene-1 (Egr-1) is an important inflammatory transcription factor. We hypothesize that leukocyte-derived Egr-1 plays a key inflammatory role in causing postoperative ileus. METHODS Wild-type, Egr-1 knockout, and chimera mice (constructed by irradiation followed by injection with Egr-1(+/+) or Egr-1(-/-) bone marrow) were subjected to surgical manipulation of the gastrointestinal tract to induce ileus. Reverse-transcription polymerase chain reaction, Western blot, and immunohistochemistry quantified and localized Egr-1. Lumenal transit of nonabsorbable fluorescein isothiocyanate-labeled dextran and in vitro organ bath techniques measured functional gastrointestinal motility. Inflammatory mediator expressions were measured by Griess reaction, enzyme-linked immunosorbent assay, and multiplex Luminex assay. RESULTS Intestinal manipulation rapidly and significantly induced Egr-1 messenger RNA and protein within the inflamed muscularis externa. Egr-1 was colocalized early to smooth muscle and enteric neurons and later in extravasated monocytes after surgery when postoperative ileus was functionally prominent. The functional severity of postoperative ileus was significantly ameliorated in mice deficient in Egr-1(-/-) and chimera wild-type mice transplanted with Egr-1(-/-) bone marrow, whereas knockout mice with Egr-1(+/+) bone marrow again displayed significant ileus. Motility was mechanistically associated in Egr-1(-/-) gene deficiency with a down-regulation in the release of nitric oxide, prostanoids, monocyte chemoattractant protein-1, macrophage inflammatory protein-1alpha, interleukin-6, interleukin-1, and granulocyte colony-stimulating factor, as well as a decrease in the recruitment of leukocytes into the manipulated muscle wall of the intestine compared with wild-type mice. CONCLUSIONS Leukocyte-derived Egr-1 plays an early critical inflammatory role in the initiation of the postoperative inflammatory response, which leads to a prolonged decreased in gastrointestinal motility after intestinal surgery.

A Role for cGMP in Inducible Nitric-oxide Synthase (iNOS)-induced Tumor Necrosis Factor (TNF) α-converting Enzyme (TACE/ADAM17) Activation, Translocation, and TNF Receptor 1 (TNFR1) Shedding in Hepatocytes

Background: iNOS/NO blocks TNFα-induced apoptosis by cGMP-dependent and cGMP-independent mechanisms in hepatocytes. Result: TLR4-dependent iNOS expression in hepatocytes leads to NO/cGMP/PKG-dependent TACE/ADAM17 and iRhom2 phosphorylation and interaction, TACE/ADAM17 activation/surface translocation, and TNFR1 shedding. Conclusion: iNOS expression leads to rapid TNFR1 shedding through NO/cGMP/PKG-dependent translocation and activation of TACE/ADAM17. Significance: This novel mechanism for iNOS/NO/cGMP-induced TACE activation and translocation may limit TNFα-mediated signaling. We and others have previously shown that the inducible nitric-oxide synthase (iNOS) and nitric oxide (NO) are hepatoprotective in a number of circumstances, including endotoxemia. In vitro, hepatocytes are protected from tumor necrosis factor (TNF) α-induced apoptosis via cGMP-dependent and cGMP-independent mechanisms. We have shown that the cGMP-dependent protective mechanisms involve the inhibition of death-inducing signaling complex formation. We show here that LPS-induced iNOS expression leads to rapid TNF receptor shedding from the surface of hepatocytes via NO/cGMP/protein kinase G-dependent activation and surface translocation of TNFα-converting enzyme (TACE/ADAM17). The activation of TACE is associated with the up-regulation of iRhom2 as well as the interaction and phosphorylation of TACE and iRhom2, which are also NO/cGMP/protein kinase G-dependent. These findings suggest that one mechanism of iNOS/NO-mediated protection of hepatocytes involves the rapid shedding of TNF receptor 1 to limit TNFα signaling.

Nonhemopoietic Cell TLR4 Signaling Is Critical in Causing Early Lipopolysaccharide-Induced Ileus

Endotoxin-mediated ileus is poorly understood. Our objective was to mechanistically investigate the role of cell-specific TLR4 expression/signaling in causing gastrointestinal dysmotility. TLR4 chimeras and CSF-1-dependent macrophage-deficient mice were subjected to i.p. ultrapure (UP)-LPS (5 mg/kg). At 6 h, gastric emptying and gastrointestinal transit assessed in vivo motility, and jejunal circular muscle contractility was measured in vitro. Muscularis infiltration of neutrophils and monocytes were counted, and intestinal muscularis inflammatory mediators were quantified by quantitative PCR. Demonstrating TLR4 dependency, UP-LPS-induced gastric stasis and ileus of TLR4WT mice were absent in mutant TLR4LPS-d mice. Unexpectedly, engraftment of TLR4-mutant bone marrow into TLR4-competent mice (bmTLR4LPS-d/TLR4WT) exhibited a significant transit delay to UP-LPS similar to bmTLR4WT/TLR4WT mice. CSF-1−/− mice were not protected from ileus. Contrary, UP-LPS-treated bmTLR4WT/TLR4LPS-d and bmTLR4LPS-d/TLR4LPS-d mice had normal transit. No leukocytic infiltration was detected at 6 h. Spontaneous jejunal contractions were markedly suppressed in UP-LPS-treated TLR4-competent mice, but bethanechol-stimulated contractions were not altered by UP-LPS in any group. UP-LPS-induced inflammatory mRNAs in a TLR4-dependent manner, but TLR4 mRNA itself was not significantly altered. In chimera mice, UP-LPS induction of IL-1β and IL-10 were hemopoietic dependent, and GM-CSF was nonhemopoietic dependent, whereas IL-6 and inducible NO synthase were derived from both cell types. Hemopoietic and nonhemopoietic cells contribute to TLR4-sensitive muscularis inflammatory signaling, but nonhemopoietic TLR4 signaling plays an exclusive primary role in causing functional UP-LPS-induced gastric stasis and ileus. Direct LPS suppression of spontaneous contractility participates in mediating early TLR4-transduced dysmotility.

Genetic Deletion of Toll-Like Receptor 4 on Platelets Attenuates Experimental Pulmonary Hypertension

Rationale: Recent studies demonstrate a role for toll-like receptor 4 (TLR4) in the pathogenesis of pulmonary hypertension (PH); however, the cell types involved in mediating the effects of TLR4 remain unknown. Objectives: The objective of this study was to determine the contribution of TLR4 expressed on nonparenchymal cells to the pathogenesis of PH. Methods and Results: TLR4 bone marrow chimeric mice revealed an equal contribution of TLR4 on nonparenchymal and parenchymal cells in the pathogenesis of PH as determined by measuring right ventricular (RV) systolic pressure and RV hypertrophy. However, the deletion of TLR4 from myeloid lineage cells had no effect on the development of PH because we found no difference in RV systolic pressure or RV hypertrophy in wild-type versus LysM-TLR4−/− mice. To explore the potential role of platelet TLR4 in the pathogenesis of PH, platelet-specific TLR4−/− mice were generated (PF4-TLR4−/− mice). TLR4−/− platelets from either global TLR4−/− or PF4-TLR4−/− mice were functional but failed to respond to lipopolysaccharide, demonstrating a lack of TLR4. PF4-TLR4−/− mice demonstrated significant protection from hypoxia-induced PH, including attenuated increases in RV systolic pressure and RV hypertrophy, decreased platelet activation, and less pulmonary vascular remodeling. The deletion of TLR4 from platelets attenuated serotonin release after chronic hypoxia, and lipopolysaccharide-stimulated platelets released serotonin and promoted pulmonary artery smooth muscle cell proliferation in a serotonin-dependent manner. Conclusions: Our data demonstrate that TLR4 on platelets contributes to the pathogenesis of PH and further highlights the role of platelets in PH.

Differential Molecular and Cellular Immune Mechanisms of Postoperative and Lps-Induced Ileus in Mice and Rats

Ileus is caused by the initiation of a complex cascade of molecular and cellular inflammatory responses within the intestinal muscularis, which might be species specific. Our objective was to investigate a possible immunological divergence in the mechanisms of postoperative- and endotoxin-induced ileus in C57BL/6 mice and Sprague-Dawley rats. Gastrointestinal transit (GIT) was measured at 24 h after the injurious stimulus. MPO-staining and F4/80 immunohistochemistry were used to quantify polymorphonuclear and monocyte infiltration of jejunal muscularis whole-mounts, and intestinal muscularis MCP-1, ICAM-1 and iNOS gene expression was assessed by RT-PCR. Intestinal muscularis subjected to in vivo surgical manipulation (SM) or LPS treatment was cultured for 24 h, and the liberation of nitric oxide and chemokines/cytokines into the culture medium was analyzed by Griess reaction and Luminex multiplex assay. Intestinal SM and lipopolysaccharide (LPS) (15 mg/kg) caused a significant delay in gastrointestinal transit, which was more severe in mice compared to rats in both injury models. Both SM- and LPS-triggered neutrophil and monocytic extravasation into the rat jejunal muscularis exceeded the cellular infiltration seen in mice. These results correlated with significantly greater increases in rat muscularis MCP-1 (syn. CCL2), ICAM-1 and iNOS message with more subsequent NO production after SM or LPS compared to mouse. The cultured muscularis obtained from SM mice released significantly more inflammatory proteins such as TNF-α, IL-1-α, IL-4 and GM-CSF compared to the manipulated rat muscularis. In contrast, LPS initiated the secretion of significantly more IL-1β by the inflamed rat muscularis compared to the mouse, but GM-CSF (syn. CSF2) liberation from mouse muscularis was markedly higher compared to LPS-treated rat muscularis. The data indicate that mechanistically the development of ileus in rat is mediated predominately through a leukocytic pathway consisting of chemotaxis, cellular extravasation and NO liberation. Whereas, the more intense mouse ileus evolves via a potent but injury-specific local cytokine response.

Genetic Deletion of Toll-Like Receptor 4 on Platelets Attenuates Experimental Pulmonary HypertensionNovelty and Significance

Rationale: Recent studies demonstrate a role for toll-like receptor 4 (TLR4) in the pathogenesis of pulmonary hypertension (PH); however, the cell types involved in mediating the effects of TLR4 remain unknown. Objectives: The objective of this study was to determine the contribution of TLR4 expressed on nonparenchymal cells to the pathogenesis of PH. Methods and Results: TLR4 bone marrow chimeric mice revealed an equal contribution of TLR4 on nonparenchymal and parenchymal cells in the pathogenesis of PH as determined by measuring right ventricular (RV) systolic pressure and RV hypertrophy. However, the deletion of TLR4 from myeloid lineage cells had no effect on the development of PH because we found no difference in RV systolic pressure or RV hypertrophy in wild-type versus LysM-TLR4−/− mice. To explore the potential role of platelet TLR4 in the pathogenesis of PH, platelet-specific TLR4−/− mice were generated (PF4-TLR4−/− mice). TLR4−/− platelets from either global TLR4−/− or PF4-TLR4−/− mice were functional but failed to respond to lipopolysaccharide, demonstrating a lack of TLR4. PF4-TLR4−/− mice demonstrated significant protection from hypoxia-induced PH, including attenuated increases in RV systolic pressure and RV hypertrophy, decreased platelet activation, and less pulmonary vascular remodeling. The deletion of TLR4 from platelets attenuated serotonin release after chronic hypoxia, and lipopolysaccharide-stimulated platelets released serotonin and promoted pulmonary artery smooth muscle cell proliferation in a serotonin-dependent manner. Conclusions: Our data demonstrate that TLR4 on platelets contributes to the pathogenesis of PH and further highlights the role of platelets in PH.

Inducible nitric oxide synthase contributes to immune dysfunction following trauma.

ABSTRACT Trauma results in a persistent depression in adaptive immunity, which contributes to patient morbidity and mortality. This state of immune paralysis following trauma is characterized by a change in cell-mediated immunity, specifically a depression in T-cell function and a shift toward TH2 T-cell phenotype. Upregulation of inducible nitric oxide synthase (iNOS) is well recognized after injury and contributes to the inflammatory response and organ damage early after trauma. However, it is unknown whether iNOS plays a role in adaptive immune dysfunction after trauma. This study utilized a murine model of severe peripheral tissue injury to show that iNOS is rapidly upregulated in macrophages and a (Gr-1hi–CD11bhi) myeloid-derived suppressor cell subpopulation in the spleen. Through the use of iNOS knockout mice, a specific iNOS inhibitor, and a nitric oxide (NO) scavenger, this study demonstrates that iNOS-derived NO is required for the depression in T-lymphocyte proliferation, interferon &ggr;, and interleukin 2 production within the spleen at 48 h after trauma. These findings support the hypothesis that iNOS regulates immune suppression following trauma and suggest that targeting the sustained production of NO by iNOS may attenuate posttraumatic immune depression.