Local and remote tissue injury upon intestinal ischemia and reperfusion depends on the TLR/MyD88 signaling pathway

Innate immune responses against microorganisms may be mediated by Toll-like receptors (TLRs). Intestinal ischemia-reperfusion (i-I/R) leads to the translocation of bacteria and/or bacterial products such as endotoxin, which activate TLRs leading to acute intestinal and lung injury and inflammation observed upon gut trauma. Here, we investigated the role of TLR activation by using mice deficient for the common TLR adaptor protein myeloid differentiation factor 88 (MyD88) on local and remote inflammation following intestinal ischemia. Balb/c and MyD88(-/-) mice were subjected to occlusion of the superior mesenteric artery (45 min) followed by intestinal reperfusion (4 h). Acute neutrophil recruitment into the intestinal wall and the lung was significantly diminished in MyD88(-/-) after i-I/R, which was confirmed microscopically. Diminished neutrophil recruitment was accompanied with reduced concentration of TNF-alpha and IL-1 beta level. Furthermore, diminished microvascular leak and bacteremia were associated with enhanced survival of MyD88(-/-) mice. However, neither TNF-alpha nor IL-1 beta neutralization prevented neutrophil recruitment into the lung but attenuated intestinal inflammation upon i-I/R. In conclusion, our data demonstrate that disruption of the TLR/MyD88 pathway in mice attenuates acute intestinal and lung injury, inflammation, and endothelial damage allowing enhanced survival.

Melatonin inhibits LPS-induced NO production in rat endothelial cells

Endothelial cells produce NO by activation of constitutive nitric oxide synthase (NOS) and transcription of inducible NOS (iNOS). We have previously shown that melatonin, in the nanomolar range, inhibits activation of constitutive NOS, and in the present paper, we evaluated whether it could interfere with the expression of iNOS, which is activated by lipopolysaccharide (LPS), a major component of gram-negative bacteria cell walls. Primary cultures of rat endothelial cells were loaded with fluorescent probe for NO detection. Nuclear factor kappa B (NF-kappa B) translocation in endothelial cells elicited by LPS was measured by electromobility shift assay, and the vasodilation of aortic rings was accessed by recording isometric contraction. Melatonin in a micromolar but not in a nanomolar range inhibits the NO production induced by LPS. This effect is not dependent on the activation of G protein-coupled melatonin receptors. The nuclear NF-kappa B translocation is a process necessary for iNOS transcription, and melatonin also inhibits its translocation. LPS induced vasodilation only in endothelium-intact aortic rings, and melatonin (10 mu m) inhibits the vasodilation. Here, we show that concentrations compatible with nocturnal melatonin surge (nm) did not interfere with the activity of iNOS. Considering that micromolar melatonin concentrations could be locally achieved through production by activated immune competent cells, extra-pineal melatonin could have a protective effect against tissue injury. We propose that melatonin blocked the LPS-induced vasodilation by inhibiting the NF-kappa B pathway. Finally, we propose that the effect of melatonin on vascular reactivity is one of the mechanisms that underlies the protective effect of this indolamine against LPS.

TUMOR NECROSIS FACTOR IS NOT ASSOCIATED WITH INTESTINAL ISCHEMIA/REPERFUSION-INDUCED LUNG INFLAMMATION

Intestinal ischemia-reperfusion (I/R) injury may cause acute systemic and lung inflammation. Here, we revisited the role of TNF-alpha in an intestinal I/R model in mice, showing that this cytokine is not required for the local and remote inflammatory response upon intestinal I/R injury using neutralizing TNF-alpha antibodies and TNF ligand-deficient mice. We demonstrate increased neutrophil recruitment in the lung as assessed by myeloperoxidase activity and augmented IL-6, granulocyte colony-stimulating factor, and KC levels, whereas TNF-alpha levels in serum were not increased and only minimally elevated in intestine and lung upon intestinal I/R injury. Importantly, TNF-alpha antibody neutralization neither diminished neutrophil recruitment nor any of the cytokines and chemokines evaluated. In addition, the inflammatory response was not abrogated in TNF and TNF receptors 1 and 2-deficient mice. However, in view of the damage on the intestinal barrier upon intestinal I/R with systemic bacterial translocation, we asked whether Toll-like receptor (TLR) activation is driving the inflammatory response. In fact, the inflammatory lung response is dramatically reduced in TLR2/4-deficient mice, confirming an important role of TLR receptor signaling causing the inflammatory lung response. In conclusion, endogenous TNF-alpha is not or minimally elevated and plays no role as a mediator for the inflammatory response upon ischemic tissue injury. By contrast, TLR2/4 signaling induces an orchestrated cytokine/chemokine response leading to local and remote pulmonary inflammation, and therefore disruption of TLR signaling may represent an alternative therapeutic target.

NITRIC OXIDE MEDIATES LUNG VASCULAR PERMEABILITY AND LYMPH-BORNE IL-6 AFTER AN INTESTINAL ISCHEMIC INSULT

Acute lung injury following intestinal I/R depends on neutrophil-endothelial cell interactions and on cytokines drained from the gut through the lymph. Among the mediators generated during I/R, increased serum levels of IL-6 and NO are also found and might be involved in acute lung injury. Once intestinal ischemia itself may be a factor of tissue injury, in this study, we investigated the presence of IL-6 in lymph after intestinal ischemia and its effects on human umbilical vein endothelial cells (HUVECs) detachment. The involvement of NO on the increase of lung and intestinal microvascular permeability and the lymph effects on HUVEC detachment were also studied. Upon anesthesia, male Wistar rats were subjected to occlusion of the superior mesenteric artery during 45 min, followed by 2-h intestinal reperfusion. Rats were treated with the nonselective NO synthase (NOS) inhibitor L-NAME (N(omega)-nitro-L-arginine methyl ester) or with the selective inhibitor of iNOS aminoguanidine 1 h before superior mesenteric artery occlusion. Whereas treatment with L-NAME during ischemia increased both IL-6 levels in lymph and lung microvascular permeability, aminoguanidine restored the augmented intestinal plasma extravasation due to ischemia and did not induce IL-6 in lymph. On the other hand, IL-6 and lymph of intestinal I/R detached the HUVECs, whereas lymph of ischemic rats upon L-NAME treatment when incubated with anti-IL-6 prevented HUVEC detachment. It is shown that the intestinal ischemia itself is sufficient to increase intestinal microvascular permeability with involvement of iNOS activation. Intestinal ischemia and absence of constitutive NOS activity leading to additional intestinal stress both cause release of IL-6 and increase of lung microvascular permeability. Because anti-IL-6 prevented the endothelial cell injury caused by lymph at the ischemia period, the lymph-borne IL-6 might be involved with endothelial cell activation. At the reperfusion period, this cytokine does not seem to be modulated by NO.

Histopathology and laser autofluorescence of ischemic kidneys of rats

The purpose of this research was to evaluate the severity of renal ischemia/reperfusion injury as determined by histology and by laser-induced fluorescence (LIF) with excitation wavelengths of 442 nm and 532 nm. Wistar rats (four groups of six animals) were subjected to left renal warm ischemia for 20, 40, 60 and 80 min followed by 10 min of reperfusion. Autofluorescence was determined before ischemia (control) and then every 5-10 min thereafter. Tissue samples for histology were harvested from the right kidney (control) and from the left kidney after reperfusion. LIF and ischemia time showed a significant correlation (p < 0.0001 and r (2)=0.47, and p=0.006 and r (2)=0.25, respectively, for the excitation wavelengths of 442 nm and 532 nm). Histological scores showed a good correlation with ischemia time (p < 0.0001). The correlations between optical spectroscopy values and histological damage were: LIF at 442 nm p < 0.0001, LIF at 532 nm p=0.001; IFF (peak of back scattered light/LIF) at 442 nm p > 0.05, and IFF at 532 nm p > 0.05. After reperfusion LIF tended to return to preischemic basal levels which occurred in the presence of histological damage. This suggests that factors other than morphological alterations may have a more relevant effect on changes observed in LIF. In conclusion, renal ischemia/reperfusion changed tissue fluorescence induced by laser. The excitation light of 442 nm showed a better correlation with the ischemia time and with the severity of tissue injury.

Inhibition of myeloperoxidase-mediated protein nitration by tempol: Kinetics, mechanism, and implications

Despite the therapeutic potential of tempol (4-hydroxy-2,2,6,6-tetra-methyl-1-piperidinyloxy) and related nitroxides as antioxidants, their effects on peroxidase-mediated protein tyrosine nitration remain unexplored. This posttranslational protein modification is a biomarker of nitric oxide-derived oxidants, and, relevantly, it parallels tissue injury in animal models of inflammation and is attenuated by tempol treatment. Here, we examine tempol effects on ribonuclease (RNase) nitration mediated by myeloperoxidase (MPO), a mammalian enzyme that plays a central role in various inflammatory processes.. Some experiments were also performed with horseradish peroxidase (HRP). We show that tempol efficiently inhibits peroxidase-mediated RNase nitration. For instance, 10 mu M tempol was able to inhibit by 90% the yield of 290 mu M 3-nitrotyrosine produced from 370 mu M RNase. The effect of tempol was not completely catalytic because part of it was consumed by recombination with RNase-tyrosyl radicals. The second-order rate constant of the reaction of tempol with MPO compound I and 11 were determined by stopped-flow kinetics as 3.3 x 10(6) and 2.6 x 10(4) M-1 s(-1), respectively (pH 7.4, 25 degrees C); the corresponding HRP constants were orders of magnitude smaller. Time-dependent hydrogen peroxide and nitrite consumption and oxygen production in the incubations were quantified experimentally and modeled by kinetic simulations. The results indicate that tempol inhibits peroxidase-mediated RNase nitration mainly because of its reaction with nitrogen dioxide to produce the oxammonium cation, which, in turn, recycles back to tempol by reacting with hydrogen peroxide and superoxide radical to produce oxygen and regenerate nitrite. The implications for nitroxide antioxidant mechanisms are discussed.

Oncostatin M is a novel glucocorticoid-dependent neuroinflammatory factor that enhances oligodendrocyte precursor cell activity in demyelinated sites

The innate immune reaction to tissue injury is a natural process, which can have detrimental effects in the absence of negative feedbacks by glucocorticoids (GCs). Although acute lipopolysaccharide (LPS) challenge is relatively harmless to the brain parenchyma of adult animals, the endotoxin is highly neurotoxic in animals that are treated with the GC receptor antagonist RU486. This study investigated the role of cytokines of the gp130-related family in these effects, because they are essential components of the inflammatory process that provide survival signals to neurons. Intracerebral LPS injection stimulated expression of several members of this family of cytokines, but oncostatin M (Osm) was the unique ligand to be completely inhibited by the RU486 treatment. OSM receptor (Osmr) is expressed mainly in astrocytes and endothelial cells following LPS administration and GCs are directly responsible for its transcriptional activation in the presence of the endotoxin. In a mouse model of demyelination, exogenous OSM significantly modulated the expression of genes involved in the mobilization of oligodendrocyte precursor cells (OPCs), differentiation of oligodendrocyte, and production of myelin. In conclusion, the activation of OSM signaling is a mechanism activated by TLR4 in the presence of negative feedback by GCs on the innate immune system of the brain. OSM absence is associated with detrimental effects of LPS, whereas exogenous OSM favors repair response to demyelinated regions. (C) 2010 Elsevier Inc. All rights reserved.

A Role for galectin-3 in renal tissue damage triggered by ischemia and reperfusion injury

Ischemic-reperfusion injury (IRI) triggers an inflammatory response involving neutrophils/macrophages, lymphocytes and endothelial cells. Galectin-3 is a multi-functional lectin with a broad range of action such as promotion of neutrophil adhesion, induction of oxidative stress, mastocyte migration and degranulation, and production of pro-inflammatory cytokines. The aim of this study was evaluate the role of galectin-3 in the inflammation triggered by IRI. Galectin-3 knockout (KO) and wild type (wt) mice were subjected to 45 min of renal pedicle occlusion. Blood and kidney samples were collected at 6, 24, 48 and 120 h. Blood urea was analyzed enzymatically, while MCP-1, IL-6 and IL-1 beta were studied by real-time PCR. Reactive oxygen species (ROS) was investigated by flow cytometry. Morphometric analyses were performed at 6, 24, 48 and 120 h after reperfusion. Urea peaked at 24 h, being significantly lower in knockout animals (wt = 264.4 +/- 85.21 mg/dl vs. gal-3 KO = 123.74 +/- 29.64 mg/dl, P = 0.001). Galectin-3 knockout animals presented less acute tubular necrosis and a more prominent tubular regeneration when compared with controls concurrently with lower expression of MCP-1, IL-6, IL-1 beta, less macrophage infiltration and lower ROS production at early time points. Galectin-3 seems to play a role in renal IRI involving the secretion of macrophage-related chemokine, pro-inflammatory cytokines and ROS production.

N-acetylcysteine protects against renal injury following bilateral ureteral obstruction

Background. Obstructive nephropathy decreases renal blood flow (RBF) and glomerular filtration rate (GFR), causing tubular abnormalities, such as urinary concentrating defect, as well as increasing oxidative stress. This study aimed to evaluate the effects of N-acetylcysteine (NAC) on renal function, as well as on the protein expression of aquaporin 2 (AQP2) and endothelial nitric oxide synthase (eNOS), after the relief of bilateral ureteral obstruction (BUO). Methods. Adult male Wistar rats were divided into four groups: sham (sham operated); sham operated + 440 mg/kg body weight (BW) of NAC daily in drinking water, started 2 days before and maintained until 48 h after the surgery; BUO (24-h BUO only); BUO + NAC-pre (24-h BUO plus 440 mg/kg BW of NAC daily in drinking water started 2 days before BUO); and BUO + NAC-post (24-h BUO plus 440 mg/kg BW of NAC daily in drinking water started on the day of BUO relief). Experiments were conducted 48 h after BUO relief. Results. Serum levels of thiobarbituric reactive substances, which are markers of lipid peroxidation, were significantly lower in NAC-treated rats than in the BUO group rats. The administration of NAC provided significant protection against post-BUO GFR drops and reductions in RBF. Renal cortices and BUO rats presented decreased eNOS protein expression of eNOS in the renal cortex of BUO group rats, whereas it was partially recovered in BUO + NAC-pre group rats. Urine osmolality was significantly lower in BUO rats than in sham group rats or NAC-treated rats, the last also presenting less interstitial fibrosis. Post-BUO downregulation of AQP2 protein expression was averted in the BUO + NAC-pre group rats. Conclusions. This study demonstrates that NAC administration ameliorates the renal function impairment observed 48 h after the relief of 24-h BUO. Oxidative stress is important for the suppression of GFR, RBF, tissue AQP2 and eNOS in the polyuric phase after the release of BUO.

Bone marrow mononuclear cells attenuate fibrosis development after severe acute kidney injury

One of the early phases that lead to fibrosis progression is inflammation. Once this stage is resolved, fibrosis might be prevented. Bone marrow mononuclear cells (BMMCs) are emerging as a new therapy for several pathologies, including autoimmune diseases, because they enact immunosuppression. In this study we aimed to evaluate the role of BMMC administration in a model of kidney fibrosis induced by an acute injury. C57Bl6 mice were subjected to unilateral severe ischemia by clamping the left renal pedicle for 1 h. BMMCs were isolated from femurs and tibia, and after 6 h of reperfusion, 1 x 10(6) cells were administrated intraperitoneally. At 24 h after surgery, treated animals showed a significant decrease in creatinine and urea levels when compared with untreated animals. Different administration routes were tested. Moreover, interferon (IFN) receptor knockout BMMCs were used, as this receptor is necessary for BMMC activation. Labeled BMMCs were found in ischemic kidney on FACS analysis. This improved outcome was associated with modulation of inflammation in the kidney and systemic modulation, as determined by cytokine expression profiling. Despite non-amelioration of functional parameters, kidney mRNA expression of interleukin (IL)-6 at 6 weeks was lower in BMMC-treated animals, as were levels of collagen 1, connective tissue growth factor (CTGF), transforming growth factor-beta (TGF-beta) and vimentin. Protective molecules, such as IL-10, heme oxygenase 1 (HO-1) and bone morphogenetic 7 (BMP-7), were increased in treated animals after 6 weeks. Moreover, Masson and Picrosirius red staining analyses showed less fibrotic areas in the kidneys of treated animals. Thus, early modulation of inflammation by BMMCs after an ischemic injury leads to reduced fibrosis through modulation of early inflammation.

Administration of Neural Precursor Cells Ameliorates Renal Ischemia-Reperfusion Injury

In this study we evaluated whether administration of stem cells of neural origin (neural precursor cells, NPCs) could be protective against renal ischemia-reperfusion injury (IRI). We hypothesized that stem cell outcomes are not tissue-specific and that NPCs can improve tissue damage through paracrine mechanisms, especially due to immunomodulation. To this end, Wistar rats (200-250 g) were submitted to 1-hour ischemia and treated with NPCs (4 x 10(6) cells/animal) at 4 h of reperfusion. To serve as controls, ischemic animals were treated with cerebellum homogenate harvested from adult rat brain. All groups were sacrificed at 24 h of reperfusion. NPCs were isolated from rat fetus telencephalon and cultured until neurosphere formation (7 days). Before administration, NPCs were labeled with carboxyfluorescein diacetate succinimydylester (CFSE). Kidneys were harvested for analysis of cytokine profile and macrophage infiltration. At 24 h, NPC treatment resulted in a significant reduction in serum creatinine (IRI + NPC 1.21 + 0.18 vs. IRI 3.33 + 0.14 and IRI + cerebellum 2.95 + 0.78mg/dl, p < 0.05) and acute tubular necrosis (IRI + NPC 46.0 + 2.4% vs. IRI 79.7 + 14.2%, p < 0.05). NPC-CFSE and glial fibrillary acidic protein (GFAP)-positive cells (astrocyte marker) were found exclusively in renal parenchyma, which also presented GFAP and SOX-2 (an embryonic neural stem cell marker) mRNA expression. NPC treatment resulted in lower renal proinflammatory IL1-beta and TNF-alpha expression and higher anti-inflammatory IL-4 and IL-10 transcription. NPC-treated animals also had less macrophage infiltration and decreased serum proinflammatory cytokines (IL-1 beta, TNF-alpha and INF-gamma). Our data suggested that NPC therapy improved renal function by influencing immunological responses. Copyright (C) 2009 S. Karger AG, Basel

TLR2 and TLR4 expression after kidney ischemia and reperfusion injury in mice treated with FTY720

Ischemia and reperfusion injury (IR) is an antigen independent inflammatory process that causes tissue damage. After IR, kidneys up-regulate leukocyte adhesion molecules and toll-like receptors (TLRs). Moreover, injured kidneys can also secrete factors (i.e. heat shock protein) which bind to TLRs and trigger intracellular events culminating with the increase in the gene expression of inflammatory cytokines. FTY720 is an immunomodulatory compound and protects at least in part kidneys submitted to IR. The mechanisms associated with FTY720`s beneficial effects on kidneys after IR remain elusive. We investigated whether FTY720 administration in mice submitted to kidney IR is associated with modulation of TLR2 and TLR4 expression. C57BL/6 mice submitted to 30 min of renal pedicles clamp were evaluated for serum parameters (creatinine, urea and nitric oxide), kidney histology, spleen and kidney infiltrating cells expression of TLR2 and TLR4, resident kidney cells expression of TLR2 and TLR4 and IL-6 protein expression in kidney. FTY720-treated mice presented decrease in serum creatinine, urea and nitric oxide, diminished expression of TLR2 and TLR4 both in spleen and kidney infiltrating cells, and reduced kidney IL-6 protein expression in comparison with IR non-treated mice. However, acute tubular necrosis was present both in IR non-treated and IR + FTY720-treated groups. Also, FTY720 did not prevent TLR2 and TLR4 expression in kidney resident cells. In conclusion, FTY720 can promote kidney function recovery after IR by reducing the inflammatory process. Further studies are needed in order to establish whether TLR2 and TLR4 down regulation should be therapeutically addressed as protective targets of renal function and structure after IR. (C) 2011 Elsevier B.V. All rights reserved.

Bradykinin inducible receptor is essential to lipopolysaccharide-induced acute lung injury in mice

Lipopolysaccharides from gram-negative bacteria are amongst the most common causative agents of acute lung injury, which is characterized by an inflammatory response, with cellular infiltration and the release of mediators/cytokines. There is evidence that bradykinin plays a role in lung inflammation in asthma but in other types of lung inflammation its role is less clear. In the present study we evaluated the role of the bradykinin B(1) receptor in acute lung injury caused by lipopolysaccharide inhalation and the mechanisms behind bradykinin actions participating in the inflammatory response. We found that in C57BI/6 mice, the bradykinin B(1) receptor expression was up-regulated 24 h after lipopolysaccharide inhalation. At this time, the number of cells and protein concentration were significantly increased in the bronchoalveolar lavage fluid and the mice developed airway hyperreactivity to methacholine. In addition, there was an increased expression of tumor necrosis factor-alpha, interleukin-1 beta and interferon-gamma and chemokines (monocytes chemotactic protein-1 and KC) in the bronchoalveolar lavage fluid and in the lung tissue. We then treated the mice with a bradykinin B, receptor antagonist, R-954 (Ac-Orn-[Oic(2), alpha-MePhe(5), D-beta Nal(7), Ile(8)]desArg(9)-bradykinin), 30 min after lipopolysaccharide administration. We observed that this treatment prevented the airway hyperreactivity as well as the increased cellular infiltration and protein content in the bronchoalveolar lavage fluid. Moreover, R-954 inhibited the expression of cytokines/chemokines. These results implicate bradykinin, acting through B(1) receptor, in the development of acute lung injury caused by lipopolysaccharide inhalation. (C) 2010 Elsevier B.V. All rights reserved.