Danielle G. Souza

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.

Commensal microbiota is fundamental for the development of inflammatory pain

The ability of an individual to sense pain is fundamental for its capacity to adapt to its environment and to avoid damage. The sensation of pain can be enhanced by acute or chronic inflammation. In the present study, we have investigated whether inflammatory pain, as measured by hypernociceptive responses, was modified in the absence of the microbiota. To this end, we evaluated mechanical nociceptive responses induced by a range of inflammatory stimuli in germ-free and conventional mice. Our experiments show that inflammatory hypernociception induced by carrageenan, lipopolysaccharide, TNF-α, IL-1β, and the chemokine CXCL1 was reduced in germ-free mice. In contrast, hypernociception induced by prostaglandins and dopamine was similar in germ-free or conventional mice. Reduction of hypernociception induced by carrageenan was associated with reduced tissue inflammation and could be reversed by reposition of the microbiota or systemic administration of lipopolysaccharide. Significantly, decreased hypernociception in germ-free mice was accompanied by enhanced IL-10 expression upon stimulation and could be reversed by treatment with an anti-IL-10 antibody. Therefore, these results show that contact with commensal microbiota is necessary for mice to develop inflammatory hypernociception. These findings implicate an important role of the interaction between the commensal microbiota and the host in favoring adaptation to environmental stresses, including those that cause pain.

Effects of inhibition of PDE4 and TNF‐α on local and remote injuries following ischaemia and reperfusion injury

The effects of phosphodiesterase (PDE)4 and TNF‐α inhibition were assessed on the local and remote injuries following intestinal ischaemia and reperfusion (I/R) injury in rats. The PDE4 inhibitor rolipram dose‐dependently (1 – 10 mg kg−1) suppressed the local (intestine) and remote (lung) increases in vascular permeability and neutrophil recruitment following mild I/R injury. SB207499 (ariflo), a structurally‐distinct PDE4 inhibitor, also suppressed the injuries following mild I/R injury. In a severe model of I/R injury, treatment with rolipram (10 mg kg−1) partially reversed the local and remote increases in vascular permeability, neutrophil recruitment, intestinal haemorrhage and intestinal LTB4 concentrations. The anti‐TNF‐α anti‐serum was more effective than rolipram at inhibiting local and remote injuries and prevented the lethality associated with severe I/R. Rolipram and anti‐TNF‐α prevented the increase in the concentrations of TNF‐α in the lung and intestine, but rolipram only partially inhibited the elevation of this cytokine in serum. Rolipram had little effect on the increases of IL‐1ß concentrations in lung and serum, whereas treatment with anti‐TNF‐α markedly increased the concentration of this cytokine. Concentrations of IL‐10 rose significantly in the lung and serum and these increases were blocked by rolipram or anti‐TNF‐α. The capacity of PDE4 inhibitors to block the recruitment of neutrophils into tissues, the production of LTB4 and of the pro‐inflammatory cytokines TNF‐α, IL‐1ß and IL‐6 appear to underlie their anti‐inflammatory effects in our model of I/R injury. Overall, PDE4 inhibition was less effective than inhibition of TNF‐α for protection against I/R injury.

The Required Role of Endogenously Produced Lipoxin A4 and Annexin-1 for the Production of IL-10 and Inflammatory Hyporesponsiveness in Mice

The appropriate development of an inflammatory response is central for the ability of a host to deal with any infectious insult. However, excessive, misplaced, or uncontrolled inflammation may lead to acute or chronic diseases. The microbiota plays an important role in the control of inflammatory responsiveness. In this study, we investigated the role of lipoxin A4 and annexin-1 for the IL-10-dependent inflammatory hyporesponsiveness observed in germfree mice. Administration of a 15-epi-lipoxin A4 analog or an annexin-1-derived peptide to conventional mice prevented tissue injury, TNF-α production, and lethality after intestinal ischemia/reperfusion. This was associated with enhanced IL-10 production. Lipoxin A4 and annexin-1 failed to prevent reperfusion injury in IL-10-deficient mice. In germfree mice, there was enhanced expression of both lipoxin A4 and annexin-1. Blockade of lipoxin A4 synthesis with a 5-lipoxygenase inhibitor or Abs against annexin-1 partially prevented IL-10 production and this was accompanied by partial reversion of inflammatory hyporesponsiveness in germfree mice. Administration of BOC-1, an antagonist of ALX receptors (at which both lipoxin A4 and annexin-1 act), or simultaneous administration of 5-lipoxygenase inhibitor and anti-annexin-1 Abs, was associated with tissue injury, TNF-α production, and lethality similar to that found in conventional mice. Thus, our data demonstrate that inflammatory responsiveness is tightly controlled by the presence of the microbiota and that the innate capacity of germfree mice to produce IL-10 is secondary to their endogenous greater ability to produce lipoxin A4 and annexin-1.

Contribution of macrophage migration inhibitory factor to the pathogenesis of dengue virus infection

Dengue fever is an emerging viral disease transmitted by arthropods to humans in tropical countries. Dengue hemorrhagic fever (DHF) is escalating in frequency and mortality rates. Here we studied the involvement of macrophage migration inhibitory factor (MIF) in dengue virus (DENV) infection and its pathogenesis. Patients with DHF had elevated plasma concentrations of MIF. Both leukocytes from these patients and macrophages from healthy donors infected in vitro with DENV showed a substantial amount of MIF within lipid droplets. The secretion of MIF by macrophages and hepatocytes required a productive infection and occurred without an increase in gene transcription or cell death, thus indicating active secretion from preformed stocks. In vivo infection of wild‐type and miFdeficient (Mif−/−) mice demonstrated a role of MIF in dengue pathogenesis. Clinical disease was less severe in Mif−/− mice, and they exhibited a significant delay in lethality, lower viremia, and lower viral load in the spleen than wild‐type mice. This reduction in all parameters of severity on DENV infection in Mif−/− mice correlated with reduced proinflam‐matory cytokine concentrations. These results demon‐strated the contribution of MIF to the pathogenesis of dengue and pointed to a possible beneficial role of neutralizing MIF as an adjunctive therapeutic approach to treat the severe forms of the disease.—Assuncäo‐Miranda, I., Amaral, F. A., Bozza, F. A., Fagundes, C. T., Sousa, L. P., Souza, D. G., Pacheco, P., Barbosa‐Lima, G., Gomes, R. N., Bozza, P. T., Da Poian, A. T., Teixeira, M. M., Bozza, M. T. Contribution of macro‐phage migration inhibitory factor to the pathogenesis of dengue virus infection. FASEB J. 24, 218–228 (2010). www.fasebj.org

Repertaxin, a novel inhibitor of rat CXCR2 function, inhibits inflammatory responses that follow intestinal ischaemia and reperfusion injury

Neutrophils are thought to play a major role in the mediation of reperfusion injury. CXC chemokines are known inducers of neutrophil recruitment. Here, we assessed the effects of Repertaxin, a novel low molecular weight inhibitor of human CXCL8 receptor activation, on the local, remote and systemic injuries following intestinal ischaemia and reperfusion (I/R) in the rat. Pre‐incubation of rat neutrophils with Repertaxin (10−11–10−6M) inhibited the chemotaxis of neutrophils induced by human CXCL8 or rat CINC‐1, but not that induced by fMLP, PAF or LTB4, in a concentration‐dependent manner. Repertaxin also prevented CXCL8‐induced calcium influx but not CXCL8 binding to purified rat neutrophils. In a model of mild I/R injury (30 min of ischaemia and 30 min of reperfusion), Repertaxin dose‐dependently (3–30 mg kg−1) inhibited the increase in vascular permeability and neutrophil influx. Maximal inhibition occurred at 30 mg kg−1. Following severe I/R injury (120 min of ischaemia and 120 min of reperfusion), Repertaxin (30 mg kg−1) markedly prevented neutrophil influx, the increase in vascular permeability both in the intestine and the lungs. Moreover, there was prevention of haemorrhage in the intestine of reperfused animals. Repertaxin effectively suppressed the increase in tissue (intestine and lungs) and serum concentrations of TNF‐α and the reperfusion‐associated lethality. For comparison, we also evaluated the effects of an anti‐CINC‐1 antibody in the model of severe I/R injury. Overall, the antibody effectively prevented tissue injury, systemic inflammation and lethality. However, the effects of the antibody were in general of lower magnitude than those of Repertaxin. In conclusion, CINC‐1 and possibly other CXC chemokines, acting on CXCR2, have an important role during I/R injury. Thus, drugs, such as Repertaxin, developed to block the function of the CXCR2 receptor may be effective at preventing reperfusion injury in relevant clinical situations.

Essential role of platelet-activating factor receptor in the pathogenesis of Dengue virus infection

Severe dengue infection in humans causes a disease characterized by thrombocytopenia, increased levels of cytokines, increased vascular permeability, hemorrhage, and shock. Treatment is supportive. Activation of platelet-activating factor (PAF) receptor (PAFR) on endothelial cells and leukocytes induces increase in vascular permeability, hypotension, and production of cytokines. We hypothesized that activation of PAFR could account for the major systemic manifestations of dengue infection. Inoculation of adult mice with an adapted strain of Dengue virus caused a systemic disease, with several features of the infection in humans. In PAFR−/− mice, there was decreased thrombocytopenia, hemoconcentration, decreased systemic levels of cytokines, and delay of lethality, when compared with WT infected mice. Treatment with UK-74,505, an orally active PAFR antagonist, prevented the above-mentioned manifestations, as well as hypotension and increased vascular permeability, and decreased lethality, even when started 5 days after virus inoculation. Similar results were obtained with a distinct PAFR antagonist, PCA-4246. Despite decreased disease manifestation, viral loads were similar (PAFR−/−) or lower (PAFR antagonist) than in WT mice. Thus, activation of PAFR plays a major role in the pathogenesis of experimental dengue infection, and its blockade prevents more severe disease manifestation after infection with no increase in systemic viral titers, suggesting that there is no interference in the ability of the murine host to deal with the infection. PAFR antagonists are disease-modifying agents in experimental dengue infection.

Effects of a BLT receptor antagonist on local and remote reperfusion injuries after transient ischemia of the superior mesenteric artery in rats.

Reperfusion of ischemic vascular beds may lead to recruitment and activation of leukocytes, release of mediators of the inflammatory process and further injury to the affected vascular bed and to remote sites. Neutrophils appear to play a major role in the pathophysiology of reperfusion injury. Amongst inflammatory mediators shown to activate neutrophils and induce their recruitment in vivo, much interest has been placed on the role of leukotriene (LT)B(4). Here, we have assessed the effects of the BLT receptor antagonist (+)-1-(3S, 4R)-[3-(4-phenyl-benzyl)-4-hydroxy-chroman-7-yl]-cyclopentane carboxylic acid (CP 105,696) in a model of neutrophil-dependent ischemia and reperfusion injury in the rat. The superior mesenteric artery was isolated and ischemia was induced by its total occlusion for 30 min. After 30 min of reperfusion, injury was assessed by evaluating the extravasation of Evans blue, an index of vascular permeability, and the levels of myeloperoxidase, an index of neutrophil accumulation, in the intestine, mesentery and lung. The neutrophil-dependence of the local (intestine and mesentery) and remote (lung) injury was confirmed by using fucoidin, a selectin blocker, and WT-3, an anti-CD18 monoclonal antibody. Post-ischemic treatment with CP 105,696 dose-dependently inhibited vascular permeability and neutrophil accumulation in the intestine and mesentery. CP 105,696 also blocked the vascular permeability changes, but not neutrophil accumulation, in the lungs after reperfusion injury. Virtually identical results were obtained with another BLT receptor antagonist, 1-(5-ethyl-2-hydroxy-4-(6-methyl-6-(1H-tetrazol-5-yl)-heptoxy++ +)-phenyl )ethanone (LY255283). Our results suggest that post-ischemic treatment with BLT receptor antagonists may inhibit local and remote ischemia and reperfusion injury by blocking both the accumulation and/or activation of neutrophils.

Effects of the PAF receptor antagonist UK74505 on local and remote reperfusion injuries following ischaemia of the superior mesenteric artery in the rat

The effects of the long lasting and potent PAF receptor antagonist UK74505 were assessed on the local and remote injuries following ischaemia and reperfusion (I/R) of the superior mesenteric artery (SMA) in rats. In a severe model of ischaemia (120 min) and reperfusion (120) injury, in addition to the local and remote increases in vascular permeability and neutrophil accumulation, there was significant tissue haemorrhage, blood neutropenia, systemic hypotension and elevated local and systemic TNF‐α levels. Post‐ischaemic treatment with the selectin blocker fucoidin (10 mg kg−1) prevented neutrophil accumulation in tissue and, in consequence, all the local and systemic injuries following severe I/R. Treatment with an optimal dose of UK74505 (1 mg kg−1) also reversed local and remote neutrophil accumulation, increases in vascular permeability and intestinal haemorrhage. UK74505 partially inhibited blood neutropenia and reperfusion‐induced hypotension. Interestingly, both fucoidin and UK74505 prevented the local, but not systemic, increases of TNF‐α levels following severe I/R injury, demonstrating an important role of migrating cells for the local production of TNF‐α. However, the results do not support a role for PAF as an intermediate molecule in the production of systemic TNF‐α. The beneficial effects of UK74505 and other PAF receptor antagonists in models of I/R injury in animals and the safety of UK74505 use in man warrant further investigations of the use of this drug as preventive measure for I/R injury in humans.