Adriana C. Soares

Increased Mortality and Inflammation in Tumor Necrosis Factor-Stimulated Gene-14 Transgenic Mice after Ischemia and Reperfusion Injury

TSG-14/PTX3 is a gene inducible by tumor necrosis factor (TNF)-alpha, interleukin-1 beta, and lipopolysaccharide in fibroblasts, macrophages, and endothelial cells. It encodes a 42-kd secreted glycoprotein that belongs to the pentraxin family of acute-phase proteins. Recently, we demonstrated that TSG-14 transgenic mice (TSG-14tg) overexpressing the murine TSG-14 gene under control of its own promoter are more resistant to lipopolysaccharide-induced shock and to polymicrobial sepsis caused by cecal ligation and puncture. Here we show that after ischemia and reperfusion (I/R) injury, TSG-14tg mice have an impaired survival rate, which appeared secondary to a markedly increased inflammatory response, as assessed by the local (duodenum and ileum) and remote (lung) enhancement in vascular permeability, hemorrhage, and neutrophil accumulation. Moreover, tissue concentrations of TNF-alpha, interleukin-1 beta, KC, and MCP-1 were higher in TSG-14tg as compared to wild-type mice after I/R injury. Of note, elevated TNF-alpha concentrations in serum were only observed in TSG-14tg mice and blockage of TNF-alpha action prevented lethality of TSG-14tg mice. These results demonstrate that transgenic expression of TSG-14 induces an enhanced local and systemic injury and TNF-alpha-dependent lethality after I/R. Taken together, our data point to a critical role of TSG-14 in controlling acute inflammatory response in part via the modulation of TNF-alpha expression.

The Essential Role of the Intestinal Microbiota in Facilitating Acute Inflammatory Responses

The restoration of blood flow, i.e., reperfusion, is the treatment of choice to save viable tissue following acute ischemia of a vascular territory. Nevertheless, reperfusion can be accompanied by significant inflammatory events that limit the beneficial effects of blood flow restoration. To evaluate the potential role of the intestinal microbiota in facilitating the development of tissue injury and systemic inflammation, germ-free and conventional mice were compared in their ability to respond to ischemia and reperfusion injury. In conventional mice, there was marked local (intestine) and remote (lung) edema formation, neutrophil influx, hemorrhage, and production of TNF-α, KC, MIP-2, and MCP-1. Moreover, there was an increase in the concentration of serum TNF-α and 100% lethality. In germ-free mice, there was no local, remote, or systemic inflammatory response or lethality after intestinal ischemia and reperfusion and, in contrast to conventional mice, germ-free animals produced greater amounts of IL-10. Similar results were obtained after administration of LPS, i.e., little production of TNF-α or lethality and production of IL-10 after LPS in germ-free mice. Blockade of IL-10 with Abs induced marked inflammation and lethality in germ-free mice after ischemia and reperfusion or LPS administration, demonstrating that the ability of these mice to produce IL-10 was largely responsible for their “no inflammation” phenotype. This was consistent with the prevention of reperfusion-associated injury by the exogenous administration of IL-10 to conventional mice. Thus, the lack of intestinal microbiota is accompanied by a state of active IL-10-mediated inflammatory hyporesponsiveness.

Activation of ATP-sensitive K(+) channels: mechanism of peripheral antinociceptive action of the nitric oxide donor, sodium nitroprusside.

Using the rat paw pressure test, in which sensitivity is increased by intraplantar injection of prostaglandin E(2) (PGE(2)), we conducted a study using several K(+) channel blockers. The objective was to determine what types of K(+) channels could be involved in the peripheral antinociceptive action of the nitric oxide donor sodium nitroprusside (SNP). SNP elicited a dose-dependent (250 and 500 microgram/paw) peripheral antinociceptive effect, which was considered local, since only higher doses produced an effect in the contralateral paw. The effect of SNP (500 microgram/paw) was dose-dependently antagonized by intraplantar administration of the sulfonylureas tolbutamide (20, 40 and 160 microgram) and glibenclamide (40, 80 and 160 microgram), selective blockers of ATP-sensitive K(+) channels. Charybdotoxin (2 microgram/paw), a selective blocker of high conductance Ca(2+)-activated K(+) channels, and apamin (10 microgram/paw), a selective blocker of low conductance Ca(2+)-activated K(+) channels, did not modify the peripheral antinociception induced by SNP. Tetraethylammonium (2 mg/paw), 4-aminopyridine (200 microgram/paw) and cesium (800 microgram/paw) also had no effect. Based on this experimental evidence, we conclude that the activation of ATP-sensitive K(+) channels could be the mechanism by which nitric oxide, donated by SNP, induces peripheral antinociception, and that Ca(2+)-activated K(+) channels and voltage-dependent K(+) channels appear not to be involved in the process.

Dibutyryl-cyclic GMP induces peripheral antinociception via activation of ATP-sensitive K+ channels in the rat PGE2-induced hyperalgesic paw

Using the rat paw pressure test, in which increased sensitivity is induced by intraplantar injection of prostaglandin E2, we studied the action of several K+ channel blockers in order to determine what types of K+ channels could be involved in the peripheral antinociception induced by dibutyrylguanosine 3 : 5′‐cyclic monophosphate (DbcGMP), a membrane permeable analogue of cyclic GMP. DbcGMP elicited a dose‐dependent (50, 75, 100 and 200 μg paw−1) peripheral antinociceptive effect. The effect of the 100 μg dose of DbcGMP was considered to be local since only a higher dose (300 μg paw−1) produced antinociception in the contralateral paw. The antinociceptive effect of DbcGMP (100 μg paw−1) was dose‐dependently antagonized by intraplantar administration of the sulphonylureas tolbutamide (20, 40 and 160 μg) and glibenclamide (40, 80 and 160 μg), selective blockers of ATP‐sensitive K+ channels. Charybdotoxin (2 μg paw−1), a selective blocker of high conductance Ca2+‐activated K+ channels, and apamin (10 μg paw−1), a selective blocker of low conductance Ca2+‐activated K+ channels, did not modify the peripheral antinociception induced by DbcGMP. Tetraethylammonium (2 mg paw−1), 4‐aminopyridine (200 μg paw−1) and cesium (800 paw−1), non‐selective voltage‐gated potassium channel blockers, also had no effect. Based on this experimental evidence, we conclude that the activation of ATP‐sensitive K+ channels could be the mechanism by which DbcGMP induces peripheral antinociception, and that Ca2+‐activated K+ channels and voltage‐dependent K+ channels appear not to be involved in the process.

Transient TLR Activation Restores Inflammatory Response and Ability To Control Pulmonary Bacterial Infection in Germfree Mice

Mammals are colonized by an astronomical number of commensal microorganisms on their environmental exposed surfaces. These symbiotic species build up a complex community that aids their hosts in several physiological activities. We have shown that lack of intestinal microbiota is accompanied by a state of active IL-10–mediated inflammatory hyporesponsiveness. The present study investigated whether the germfree state and its hyporesponsive phenotype alter host resistance to an infectious bacterial insult. Experiments performed in germfree mice infected with Klebsiella pneumoniae showed that these animals are drastically susceptible to bacterial infection in an IL-10–dependent manner. In germfree mice, IL-10 restrains proinflammatory mediator production and neutrophil recruitment and favors pathogen growth and dissemination. Germfree mice were resistant to LPS treatment. However, priming of these animals with several TLR agonists recovered their inflammatory responsiveness to sterile injury. LPS pretreatment also rendered germfree mice resistant to pulmonary K. pneumoniae infection, abrogated IL-10 production, and restored TNF-α and CXCL1 production and neutrophil mobilization into lungs of infected germfree mice. This effective inflammatory response mounted by LPS-treated germfree mice resulted in bacterial clearance and enhanced survival upon infection. Therefore, host colonization by indigenous microbiota alters the way the host reacts to environmental infectious stimuli, probably through activation of TLR-dependent pathways. Symbiotic gut colonization enables proper inflammatory response to harmful insults to the host, and increases resilience of the entire mammal-microbiota consortium to environmental pressures.

Role of PAF receptors during intestinal ischemia and reperfusion injury. A comparative study between PAF receptor‐deficient mice and PAF receptor antagonist treatment

The reperfusion of ischemic tissues may be associated with local and systemic inflammation that prevents the full benefit of blood flow restoration. The present study aimed to confirm a role for platelet‐activating factor receptor(s) (PAFR) during ischemia and reperfusion injury by using genetically modified mice deficient in the PAFR (PAFR−/− mice) and to evaluate comparatively the effectiveness of pharmacological treatment using the PAFR antagonist UK‐74,505 (modipafant). The reperfusion of the ischemic superior mesenteric artery (SMA) induced marked local (intestine) and remote (lungs) tissue injury, as assessed by the increase in vascular permeability, neutrophil influx and intestinal hemorrhage and in the production of TNF‐α. There was also a systemic inflammatory response, as shown by the increase in serum TNF‐α concentrations and marked reperfusion‐associated lethality. After reperfusion of the ischemic SMA, PAFR−/− mice had little tissue or systemic inflammation and lethality was delayed, but not prevented, in these mice. Interestingly, the reperfusion‐associated increases in tissue concentrations of IL‐10 were significantly greater in PAFR−/− than wild‐type mice. Pretreatment with PAFR antagonist UK‐74,505 (1 mg kg−1) markedly prevented tissue injury, as assessed by the increase in vascular permeability, neutrophil accumulation, hemorrhage and TNF‐α concentrations in the intestine and lungs. In contrast, UK‐74,505 failed to affect reperfusion‐associated lethality and increases in serum TNF‐α when used at 1 mg kg−1. Reperfusion‐associated lethality and increase in serum TNF‐α were only affected when a supra‐maximal dose of the antagonist was used (10 mg kg−1). At this dose, UK‐74,505 also induced a marked enhancement of reperfusion‐associated increases in tissue concentrations of IL‐10. However, at the same dose, UK‐74,505 failed to prevent reperfusion‐associated lethality in PAFR−/− mice any further. The present studies using genetically modified animals and a receptor antagonist firmly establish a role of PAFR activation for the local, remote and systemic inflammatory injury and lethality which follows reperfusion of the ischemic SMA in mice. Moreover, it is suggested that high doses of PAFR antagonists need to be used if the real efficacy of these compounds is to be tested clinically.

Role of the platelet‐activating factor (PAF) receptor during pulmonary infection with gram negative bacteria

The lipid mediator PAF plays an important role in the phagocytosis of particles, including bacteria, and consequent production of pro‐inflammatory cytokines, such as TNF‐α and IL‐8. Using a PAF receptor antagonist (UK‐74,505) and PAF receptor knock‐out mice, we have investigated the relevance of PAF for the inflammatory changes and lethality after pulmonary infection with the gram‐negative bacteria Klebsiella pneumoniae in mice. At an inoculum of 3×106 bacteria, there was marked pulmonary (bronchoalveolar lavage and lung) neutrophilia that started early (2.5 h after infection) and peaked at 48 h. All animals were dead by day 4 of infection. The chemokine KC and the pro‐inflammatory cytokine TNF‐α increased rapidly and persisted for 48 h in the lungs. Pretreatment with UK‐74,505 (30 mg kg−1 per day, p.o.) had no significant effects on the number of infiltrating neutrophils in BAL fluid or lung tissue, as assessed by histology and measuring myeloperoxidase, or on the concentrations of KC. In contrast, concentrations of TNF‐α and the number of bacteria inside neutrophils were significantly diminished. In order to support a role for the PAF during K. pneumoniae infection, experiments were also carried out in PAFR‐deficient mice. In the latter animals, lethality occurred earlier than in wild‐type controls. This was associated with greater number of bacteria in lung tissue and diminished percentage of neutrophils containing bacteria in their cytoplasm. Our results suggest that PAF, acting on its receptor, plays a protective role during infection with K. pneumoniae in mice.

The role of salivary nitrophorins in the ingestion of blood by the triatomine bug Rhodnius prolixus (Reduviidae: Triatominae)

To assist haematophagy, Rhodnius prolixus produces several bioactive molecules in its saliva which it injects into the host skin. The most abundant of these molecules are the nitrophorins (NPs). In this work, we reduced the expression of NP1-4 in the saliva of R. prolixus by RNAi and evaluated the subsequent feeding performance of the bugs using the cibarial pump electromyogram either on the dorsal skin or on the tail vein of the mice. NPs salivary mRNA was reduced by >99% in comparison to controls. Saliva from knockdown nymphs also presented 82% less haemproteins while the total protein was not reduced. Knockdown nymphs feeding on the skin had lower ingestion rates mainly due to the longer cumulative probing time and lower cibarial pump frequency. Another difference was that knockdown insects bit approximately 5 times more. No differences were observed between groups fed on the tail vein. When the feeding sites were compared, nymphs fed on the tail vein had higher effective ingestion rates. These findings endorse the importance of the NPs for the ability of bugs to complete the meal in a short total contact time with a low number of bites, decreasing the perception of the insect by the host.

Impaired host defense to Klebsiella pneumoniae infection in mice treated with the PDE4 inhibitor rolipram

The increase in levels of cAMP in leukocytes by selective inhibitors of PDE4 may result in reduction of inflammation, and may be useful in the treatment of pulmonary inflammatory disorders in humans. Here, we have assessed whether oral treatment with the prototype PDE4 inhibitor, rolipram, interfered with the antibacterial host response following pulmonary infection of mice with Klebsiella pneumoniae. K. pneumoniae infection induced a marked increase in the recruitment of neutrophils to the lungs and the production of proinflammatory cytokines and chemokines, including tumor necrosis factor‐α (TNF‐α) and keratinocyte‐derived chemokine (KC), in bronchoalveolar (BAL) fluid and lung tissue. There were also detectable amounts of interleukin‐10 (IL‐10) and significant lethality. Treatment with rolipram (3–30 mg kg−1) was associated with earlier lethality and significant inhibition of the TNF‐α production. This was associated with enhanced production of IL‐10 in lung tissue of rolipram‐treated animals. Rolipram treatment did not affect KC expression and the recruitment of neutrophils in the lung tissue. Over 70% of neutrophils that migrated into the BAL fluid following K. pneumoniae infection ingested bacteria. Treatment with rolipram inhibited the percentage of neutrophils undergoing phagocytosis of K. pneumoniae in a dose‐dependent manner. Maximal inhibition (62%) occurred at doses equal to or greater than 10 mg kg−1. Thus, treatment of mice with the PDE4 inhibitor rolipram is accompanied by earlier lethality, enhanced bacterial load and decreased capacity of the responding host to produce TNF‐α and of neutrophils to phagocytose bacteria. It will be important to investigate whether the shown ability of PDE4 inhibitors to inhibit neutrophil phagocytosis and control experimental bacterial infection will translate into an inhibition of the ability of neutrophils to deal with infectious microorganisms in the clinical setting.

Influence of the intestinal anticoagulant in the feeding performance of triatomine bugs (Hemiptera; Reduviidae)

Triatomines are haematophagous insects in all post-embryonic life stages. They are vectors of Trypanosoma cruzi, the causative agent of Chagas disease. Their vectorial ability is influenced by their feeding performance, which varies greatly amongst species. Recent work showed that inhibition of the coagulation process in the anterior midgut (crop) environment considerably influences the blood meal size. In this work, we performed a comparative study of the level of anticoagulant activity in the saliva and crop contents of three triatomine species -Triatoma infestans, Triatoma brasiliensis and Rhodnius prolixus - and correlated this with their feeding performance on live hosts. Moreover, the feeding parameters on a large diameter vessel influenced by the crop anticoagulants were evaluated in detail. The anticoagulant activity was significantly higher in the crop contents than in salivary glands, varying from 1.6-fold higher for R. prolixus to 70-fold higher for T. brasiliensis. Amongst the species, T. brasiliensis had the lowest crop anticoagulant activity, the lowest concentration of thrombin inhibitor, and took the longest to feed. Triatoma brasiliensis nymphs that had their intestinal anticoagulant (brasiliensin) knocked down by RNA interference had the lowest capacity to maintain cibarial pump frequency at higher levels throughout the feeding process and consequently a lower ingestion rate (mg/min), even when fed under favourable conditions (large diameter vessel). However, the feeding difficulty for brasiliensin knockdown T. brasiliensis nymphs was reversed by treating the host mice with heparin (a potent systemic anticoagulant) before blood feeding. The results indicate that crop anticoagulant activity influences modulation of the blood-pumping frequency to the intestine and significantly affects the feeding efficiency of triatomine spp. on live hosts.