Trauma hemorrhagic shock-induced lung injury involves a gut-lymph-induced TLR4 pathway in mice.

BACKGROUND: Injurious non-microbial factors released from the stressed gut during shocked states contribute to the development of acute lung injury (ALI) and multiple organ dysfunction syndrome (MODS). Since Toll-like receptors (TLR) act as sensors of tissue injury as well as microbial invasion and TLR4 signaling occurs in both sepsis and noninfectious models of ischemia/reperfusion (I/R) injury, we hypothesized that factors in the intestinal mesenteric lymph after trauma hemorrhagic shock (T/HS) mediate gut-induced lung injury via TLR4 activation. METHODS/PRINCIPAL FINDINGS: The concept that factors in T/HS lymph exiting the gut recreates ALI is evidenced by our findings that the infusion of porcine lymph, collected from animals subjected to global T/HS injury, into naïve wildtype (WT) mice induced lung injury. Using C3H/HeJ mice that harbor a TLR4 mutation, we found that TLR4 activation was necessary for the development of T/HS porcine lymph-induced lung injury as determined by Evan's blue dye (EBD) lung permeability and myeloperoxidase (MPO) levels as well as the induction of the injurious pulmonary iNOS response. TRIF and Myd88 deficiency fully and partially attenuated T/HS lymph-induced increases in lung permeability respectively. Additional studies in TLR2 deficient mice showed that TLR2 activation was not involved in the pathology of T/HS lymph-induced lung injury. Lastly, the lymph samples were devoid of bacteria, endotoxin and bacterial DNA and passage of lymph through an endotoxin removal column did not abrogate the ability of T/HS lymph to cause lung injury in naïve mice. CONCLUSIONS/SIGNIFICANCE: Our findings suggest that non-microbial factors in the intestinal mesenteric lymph after T/HS are capable of recreating T/HS-induced lung injury via TLR4 activation.

HIF-1 mediates pathogenic inflammatory responses to intestinal ischemia-reperfusion injury.

Acute lung injury (ALI) and the development of the multiple organ dysfunction syndrome (MODS) are major causes of death in trauma patients. Gut inflammation and loss of gut barrier function as a consequence of splanchnic ischemia-reperfusion (I/R) have been implicated as the initial triggering events that contribute to the development of the systemic inflammatory response, ALI, and MODS. Since hypoxia-inducible factor (HIF-1) is a key regulator of the physiological and pathophysiological response to hypoxia, we asked whether HIF-1 plays a proximal role in the induction of gut injury and subsequent lung injury. Utilizing partially HIF-1α-deficient mice in a global trauma hemorrhagic shock (T/HS) model, we found that HIF-1 activation was necessary for the development of gut injury and that the prevention of gut injury was associated with an abrogation of lung injury. Specifically, in vivo studies demonstrated that partial HIF-1α deficiency ameliorated T/HS-induced increases in intestinal permeability, bacterial translocation, and caspase-3 activation. Lastly, partial HIF-1α deficiency reduced TNF-α, IL-1ß, cyclooxygenase-2, and inducible nitric oxide synthase levels in the ileal mucosa after T/HS whereas IL-1ß mRNA levels were reduced in the lung after T/HS. This study indicates that prolonged intestinal HIF-1 activation is a proximal regulator of I/R-induced gut mucosal injury and gut-induced lung injury. Consequently, these results provide unique information on the initiating events in trauma-hemorrhagic shock-induced ALI and MODS as well as potential therapeutic insights.

Evaluation of effect of specimen-handling parameters for plasma preparation tubes on viral load measurements obtained by using the Abbott RealTime HIV-1 load assay.

HIV-1 viral load testing is essential to the management of HIV-1-infected patients, and proper specimen handling ensures accurate viral load (VL) results. This study was performed to (i) evaluate the effect of freezing plasma in situ in BD Vacutainer plasma preparation tubes (PPT) on the accuracy of HIV-1 viral load results using the Abbott RealTime HIV-1 assay and (ii) evaluate the effect of whole-blood storage in the PPT for 6 h at room temperature prior to centrifugation (PPT6H) rather than 2 h as specified in the PPT product insert. Of the 64 HIV-positive subjects evaluated, 29 had average viral load counts of >40 copies/ml in at least one of the tubes tested and 35 subjects had a result of either "undetected target" or "below the limit of quantification" (LOQ) for all or some of the tubes regardless of handling condition. For the 29 subjects with VLs that were >LOQ, the mean biases between plasma from Vacutainer K(2)EDTA tubes and plasma frozen in situ in PPT and between K(2)EDTA tube plasma and plasma from PPT6H (log(10) copies/ml) were 0.005 and -0.001, respectively, and r(2) was >0.92 for all correlations. We conclude that VLs determined from plasma frozen in situ in PPT are equivalent to VLs in K(2)EDTA tube plasma and can be used for accurate quantification of HIV-1 RNA in the Abbott RealTime HIV-1 assay. Furthermore specimens collected in PPT can be stored for 6 h at room temperature with no effect on viral load results as measured by the Abbott RealTime HIV-1 assay.

Differential regulation of HIF-1alpha isoforms in murine macrophages by TLR4 and adenosine A(2A) receptor agonists.

Adenosine A(2A)R and TLR agonists synergize to induce an "angiogenic switch" in macrophages, down-regulating TNF-alpha and up-regulating VEGF expression. This switch involves transcriptional regulation of VEGF by HIF-1, transcriptional induction of HIF-1alpha by LPS (TLR4 agonist), and A(2A)R-dependent post-transcriptional regulation of HIF-1alpha stability. Murine HIF-1alpha is expressed as two mRNA isoforms: HIF-1alphaI.1 and -I.2, which contain alternative first exons and promoters. HIF-1alphaI.2 is expressed ubiquitously, and HIF-1alphaI.1 is tissue-specific. We investigated the regulation of these isoforms in macrophages by TLR4 and A(2A)R agonists. HIF-1alphaI.1 is induced strongly compared with HIF-1alphaI.2 upon costimulation with LPS and A(2A)R agonists (NECA or CGS21680). In unstimulated cells, the I.1 isoform constituted approximately 4% of HIF-1alpha transcripts; in LPS and NECA- or CGS21680-treated macrophages, this level was approximately 15%, indicating a substantial contribution of HIF-1alphaI.1 to total HIF-1alpha expression. The promoters of both isoforms were induced by LPS but not enhanced further by NECA, suggesting A(2A)R-mediated post-transcriptional regulation. LPS/NECA-induced expression of HIF-1alphaI.1 was down-regulated by Bay 11-7085 (NF-kappaB inhibitor) and ZM241385 (A(2A)R antagonist). Although VEGF and IL-10 expression by HIF-1alphaI.1-/- macrophages was equivalent to that of wild-type macrophages, TNF-alpha, MIP-1alpha, IL-6, IL-12p40, and IL-1beta expression was significantly greater, suggesting a role for HIF-1alphaI.1 in modulating expression of these cytokines. A(2A)R expression in unstimulated macrophages was low but was induced rapidly by LPS in a NF-kappaB-dependent manner. LPS-induced expression of A(2A)Rs and HIF-1alpha and A(2A)R-dependent HIF-1alpha mRNA and protein stabilization provide mechanisms for the synergistic effects of LPS and A(2A)R agonists on macrophage VEGF expression.

Synergistic up-regulation of vascular endothelial growth factor (VEGF) expression in macrophages by adenosine A2A receptor agonists and endotoxin involves transcriptional regulation via the hypoxia response element in the VEGF promoter.

Macrophages are an important source of vascular endothelial growth factor (VEGF). Adenosine A2A receptor (A2AR) agonists with Toll-like receptor (TLR) 2, 4, 7, and 9 agonists synergistically induce macrophage VEGF expression. We show here using VEGF promoter-luciferase reporter constructs that the TLR4 agonist Escherichia coli lipopolysaccharide (LPS) and the A2AR agonists NECA and CGS21680 synergistically augment VEGF transcription in macrophages and that the HRE in the VEGF promoter is essential for this transcription. We examined whether LPS and/or NECA induce HIF-1alpha expression. HIF-1alpha mRNA levels were increased in LPS-treated macrophages in an NF-kappaB-dependent manner; NECA strongly increased these levels in an A2AR-dependent manner. LPS induced luciferase expression from a HIF-1alpha promoter-luciferase construct in an A2AR-independent manner. Further stimulation with NECA did not increase HIF-1alpha promoter activity, indicating that the A2AR-dependent increase in HIF-1alpha mRNA is post-transcriptional. LPS/NECA treatment also increased HIF-1alpha protein and DNA binding levels. Deletion of putative NF-kappaB-binding sites from the VEGF promoter did not affect LPS/NECA-induced VEGF promoter activity, suggesting that NF-kappaB is not directly involved in VEGF transcription. Taken together, these data indicate that LPS/NECA-induced VEGF expression involves transcriptional regulation of the VEGF promoter by HIF-1alpha through the HRE. HIF-1alpha is transcriptionally induced by LPS and post-transcriptionally up-regulated in an A2AR-dependent manner.

Analysis of signal transduction pathways in macrophages using expression vectors with CMV promoters: a cautionary tale.

The cytomegalovirus (CMV) major immediate-early promoter is a strong promoter used for both in vitro and in vivo expression of proteins in signal transduction and gene therapy studies. CMV activity is induced by external stimuli such as endotoxin from Gram-negative bacteria (LPS), TNF-alpha and phorbol esters. This inducibility poses problems when this promoter is used to drive the expression of either wild type or dominant negative mutated proteins as tools in signal transduction studies. This report draws attention to the problem associated with this widely used approach. The role of NF-kappaB and Hypoxia Inducible Factor-1alpha (HIF-1alpha) in the transcriptional regulation of Vascular Endothelial Growth Factor (VEGF) in macrophages was investigated using CMV-promoter-driven expression of either wild type or dominant negative proteins involved in these pathways. Difficulties encountered while interpreting the data due to the inducibility of the CMV promoter by LPS are highlighted in this report and provide a cautionary note for the evaluation of data acquired using this approach.

An angiogenic switch in macrophages involving synergy between Toll-like receptors 2, 4, 7, and 9 and adenosine A(2A) receptors.

Adenosine A(2A) receptor (A(2A)R) agonists synergize with Escherichia coli (E. coli) LPS [toll-like receptor (TLR)4 agonist] to up-regulate vascular endothelial growth factor (VEGF) expression in murine macrophages. Here, we demonstrate that TLR2, TLR7, and TLR9, but not TLR3 and TLR5 agonists, also synergize with A(2A)R agonists and adenosine to up-regulate VEGF, while simultaneously strongly down-regulating TNFalpha expression. In the absence of adenosine or A(2A)R agonists, Porphyromonas gingivalis (P. gingivalis) LPS and PAM(3)CAG (TLR2 agonists), resiquimod (R848) (TLR7 agonist), and non-methylated CpG DNA (TLR9 agonist) strongly up-regulate TNFalpha expression, with no effect on VEGF. In the presence of adenosine or A(2A)R agonists, but not A(1)R agonists, TLR2, 4, 7, and 9 agonists strongly up-regulate VEGF expression, while simultaneously down-regulating TNFalpha. C57BL/10ScN (TLR4 deletion mutant) macrophages produce TNFalpha in response to TLR2, 3, 7, and 9 agonists, but not the TLR4 agonist E. coli LPS. With adenosine or A(2A)R agonists, TLR2, 7, and 9, but not TLR4 agonists, also synergistically up-regulate VEGF, while down-regulating TNFalpha expression. Polyinosinic-polycytidilic acid (poly(I:C)) (TLR3 agonist) stimulates TNFalpha expression in macrophages from both C57BL/10ScSn and C57BL/10ScN mice, but has little effect on VEGF expression in the presence of adenosine or A(2A)R agonists. R-flagellins from Serratia marcescens (S. marcescens) and Salmonella muenchen (S. muenchen) do not stimulate TNFalpha expression in either C57BL/10ScSn or C57BL10/ScN mice, and have no effect on VEGF production in the presence of adenosine or A(2A)R agonists. While adenosine and A(2A)R agonists strongly down-regulate TNFalpha protein expression induced by TLR2, 3, 4, 7, and 9 agonists, TNFalpha mRNA and NF-kappaB activation are not reduced. We propose a novel signaling pathway in murine macrophages involving synergy between TLRs 2, 4, 7, and 9 and A(2A)Rs, that up-regulates VEGF and down-regulates TNFalpha expression, thus acting as an angiogenic switch. This angiogenic switch may play an important role in ischemia when TLR agonists are present, providing an interface between innate immunity and wound healing.

Regulation of vascular endothelial growth factor gene expression in murine macrophages by nitric oxide and hypoxia.

Vascular endothelial growth factor (VEGF) expression in murine peritoneal macrophages is strongly upregulated by hypoxia via transcriptional and posttranscriptional mechanisms. Interferon-gamma (IFN-gamma) with Escherichia coli lipopolysaccharide (LPS) also upregulates expression of VEGF, as well as of the inducible nitric oxide synthase (iNOS). Hypoxia (1% O(2)) upregulates VEGF expression in macrophages from both wild-type and iNOS knockout mice, indicating that hypoxic upregulation of VEGF is independent of iNOS. However, the iNOS inhibitor aminoguanidine (AG) decreases the VEGF expression induced by LPS/IFN-gamma, indicating an important role for NO. NO-dependent induction of VEGF is strongly dependent on cell density. LPS/IFN-gamma treatment induces minimal VEGF protein expression in macrophages cultured at low cell densities (<0.25 x 10(6) cells/cm(2)); at higher cell densities (>0.25 x 10(6) cells/cm(2)) that lead to conditions of pericellular hypoxia, however, induction of VEGF expression was strong. Transient transfection of RAW 264.7 cells with luciferase reporter constructs of the murine VEGF promoter indicates that both hypoxia and LPS/IFN-gamma independently induce VEGF promoter activity, irrespective of cell density. Although LPS/IFN-gamma treatment induces transcriptional activation of the VEGF promoter, significant levels of VEGF protein are only expressed by cells at high density under conditions of pericellular hypoxia. This suggests an important regulatory role for hypoxia at the posttranscriptional level. Deletion analysis of the VEGF promoter shows that the hypoxia response element region and its immediate flanking sequences are essential for both hypoxia and LPS/IFN-gamma-induced VEGF promoter activation.

Synergistic up-regulation of vascular endothelial growth factor expression in murine macrophages by adenosine A(2A) receptor agonists and endotoxin.

Under normoxic conditions, macrophages from C57BL mice produce low levels of vascular endothelial growth factor (VEGF). Hypoxia stimulates VEGF expression by approximately 500%; interferon-gamma (IFN-gamma) with endotoxin [lipopolysaccharide (LPS)] also stimulates VEGF expression by approximately 50 to 150% in an inducible nitric oxide synthase (iNOS)-dependent manner. Treatment of normoxic macrophages with 5'-N-ethyl-carboxamido-adenosine (NECA), a nonselective adenosine A(2) receptor agonist, or with 2-[p-(2-carboxyethyl)-phenylethyl amino]-5'-N-ethyl-carboxamido-adenosine (CGS21680), a specific adenosine A(2A) receptor agonist, modestly increases VEGF expression, whereas 2-chloro-N(6)-cyclopentyl adenosine (CCPA), an adenosine A(1) agonist, does not. Treatment with LPS (0 to 1000 ng/ml), or with IFN-gamma (0 to 300 U/ml), does not affect VEGF expression. In the presence of LPS (EC(50) < 10 ng/ml), but not of IFN-gamma, both NECA and CGS21680 synergistically up-regulate VEGF expression by as much as 10-fold. This VEGF is biologically active in vivo in the rat corneal bioassay of angiogenesis. Inhibitors of iNOS do not affect this synergistic induction of VEGF, and macrophages from iNOS-/- mice produce similar levels of VEGF as wild-type mice, indicating that NO does not play a role in this induction. Under hypoxic conditions, VEGF expression is slightly increased by adenosine receptor agonists but adenosine A(2) or A(1) receptor antagonists 3,7-dimethyl-1-propargyl xanthine (DMPX), ZM241385, and 8-cyclopentyl-1,3-dipropylxanthine (DCPCX) do not modulate VEGF expression. VEGF expression is also not reduced in hypoxic macrophages from A(3)-/- and A(2A)-/- mice. Thus, VEGF expression by hypoxic macrophages does not seem to depend on endogenously released or exogenous adenosine. VEGF expression is strongly up-regulated by LPS/NECA in macrophages from A(3)-/- but not A(2A)-/- mice, confirming the role of adenosine A(2A) receptors in this pathway. LPS with NECA strongly up-regulates VEGF expression by macrophages from C(3)H/HeN mice (with intact Tlr4 receptors), but not by macrophages from C(3)H/HeJ mice (with mutated, functionally inactive Tlr4 receptors), implicating signaling through the Tlr4 pathway in this synergistic up-regulation. Finally, Western blot analysis of adenosine A(2A) receptor expression indicated that the synergistic interaction of LPS with A(2A) receptor agonists does not involve up-regulation of A(2A) receptors by LPS. These results indicate that in murine macrophages there is a novel pathway regulating VEGF production, that involves the synergistic interaction of adenosine A(2A) receptor agonists through A(2A) receptors with LPS through the Tlr4 pathway, resulting in the strong up-regulation of VEGF expression by macrophages in a hypoxia- and NO-independent manner.