Inhibition of the sphingosine-1-phosphate pathway promotes the resolution of neutrophilic inflammation.

Sphingosine-1-phosphate (S1P) is an important sphingolipid derived from plasma membrane and has a known role in productive phase of inflammation, but its role in neutrophil survival and resolution phase of inflammation is unknown. Here, we investigated the effects of inhibition of S1P receptors and the blockade of S1P synthesis in BALB/c mice and human neutrophils. S1P and S1PR1-3 receptors expression were increased in cells from the pleural cavity stimulated with LPS. Using different antagonists of S1PRs and inhibitors of different steps of the metabolic pathway of S1P production, we show that S1P and its receptors are involved in regulating neutrophil survival and resolution of inflammation in the pleural cavity. Given the role of the S1P-S1PR axis in resolution of inflammation, we sought to identify whether blockade at different levels of the sphingosine-1-phosphate synthesis pathway could affect neutrophil survival in vitro. Inhibitors of the S1P pathway were also able to induce human neutrophil apoptosis. In addition, blockade of S1P synthesis or its receptor facilitated the efferocytosis of apoptotic neutrophil. Taken together, our data demonstrate a fundamental role for S1P in regulating the outcome of inflammatory responses, and position S1P-S1PR axis as a potential target for treatment of neutrophilic inflammation.

ROCK Inhibition Drives Resolution of Acute Inflammation by Enhancing Neutrophil Apoptosis.

Uncontrolled inflammation leads to tissue damage and it is central for the development of chronic inflammatory diseases and autoimmunity. An acute inflammatory response is finely regulated by the action of anti-inflammatory and pro-resolutive mediators, culminating in the resolution of inflammation and restoration of homeostasis. There are few studies investigating intracellular signaling pathways associated with the resolution of inflammation. Here, we investigate the role of Rho-associated kinase (ROCK), a serine/threonine kinase, in a model of self-resolving neutrophilic inflammatory. We show that ROCK activity, evaluated by P-MYPT-1 kinetics, was higher during the peak of lipopolysaccharide-induced neutrophil influx in the pleural cavity of mice. ROCK inhibition by treatment with Y-27632 decreased the accumulation of neutrophils in the pleural cavity and was associated with an increase in apoptotic events and efferocytosis, as evaluated by an in vivo assay. In a model of gout, treatment with Y-27632 reduced neutrophil accumulation, IL-1ß levels and hypernociception in the joint. These were associated with reduced MYPT and IκBα phosphorylation levels and increased apoptosis. Finally, inhibition of ROCK activity also induced apoptosis in human neutrophils and destabilized cytoskeleton, extending the observed effects to human cells. Taken together, these data show that inhibition of the ROCK pathway might represent a potential therapeutic target for neutrophilic inflammatory diseases.

Phosphoinositide-3 kinase gamma regulates caspase-1 activation and leukocyte recruitment in acute murine gout.

This study investigates the participation of PI3Kγ in the development of joint inflammation and dysfunction in an experimental model of acute gout in mice. Acute gout was induced by injection of monosodium urate (MSU) crystals into the tibiofemoral joint of mice. The involvement of PI3Kγ was evaluated using a selective inhibitor and mice deficient for PI3Kγ (PI3Kγ-/- ) or with loss of kinase activity. Neutrophils recovered from the inflamed joint were quantified and stained for phosphorylated Akt (pAkt) and production of reactive oxygen species (ROS). The adherence of leukocytes to the joint microvasculature was assessed by intravital microscopy and cleaved caspase-1 by Western blot. Injection of MSU crystals induced massive accumulation of neutrophils expressing phosphorylated Akt. In the absence of PI3Kγ, there was reduction of pAkt expression, chemokine production, and neutrophil recruitment. Genetic or pharmacological inhibition of PI3Kγ reduced the adherence of leukocytes to the joint microvasculature, even in joints with established inflammation. Neutrophils from PI3Kγ-/- mice produced less ROS than wild-type neutrophils. There was decreased joint damage and dysfunction in the absence of PI3Kγ. In addition, in the absence of PI3Kγ activity, there was reduction of cleaved caspase-1 and IL-1ß production in synovial tissue after injection of MSU crystals and leukotriene B4 . Our studies suggest that PI3Kγ is crucial for MSU crystal-induced acute joint inflammation. It is necessary for regulating caspase-1 activation and for mediating neutrophil migration and activation. Drugs that impair PI3Kγ function may be useful to control acute gout inflammation.

Angiotensin-(1-7) Promotes Resolution of Eosinophilic Inflammation in an Experimental Model of Asthma.

Defective apoptosis of eosinophils, the main leukocyte in the pathogenesis of asthma, and delay in its removal lead to lung damage and loss of pulmonary function due to failure in the resolution of inflammation. Here, we investigated the ability of angiotensin-(1-7) [Ang-(1-7)], a pivotal peptide of the renin-angiotensin system, to promote resolution of an allergic lung inflammatory response. Balb/c mice were sensitized and challenged with ovalbumin and treated with Ang-(1-7) at the peak of the inflammatory process. Bronchoalveolar lavage (BAL) fluid and lungs were collected 24 h after treatment. Different lung lobes were processed for histology to evaluate inflammatory cell infiltration, airway and pulmonary remodeling, total collagen staining, and measurements of (i) collagen I and III mRNA expression by qRT-PCR; (ii) ERK1/2, IκB-α, and GATA3 protein levels by Western blotting; and (iii) eosinophilic peroxidase activity. Total number of inflammatory cells, proportion of apoptotic eosinophils and immunofluorescence for caspase 3 and NF-κB in leukocytes were evaluated in the BAL. Mas receptor immunostaining was evaluated in mouse and human eosinophils. Engulfment of human polimorphonuclear cells by macrophages, efferocytosis, was evaluated in vivo. Ang-(1-7) reduced eosinophils in the lung and in the BAL, increased the number of apoptotic eosinophils, shown by histology criteria and by increase in caspase 3 immunostaining. Furthermore, Ang-(1-7) decreased NF-kB immunostaining in eosinophils, reduced GATA3, ERK1/2, and IκB-α expression in the lung and decreased pulmonary remodeling and collagen deposition. Importantly, Ang-(1-7) increased efferocytosis. Our results demonstrate, for the first time, Ang-(1-7) activates events that are crucial for resolution of the inflammatory process of asthma and promotion of the return of lung homeostasis, indicating Ang-(1-7) as novel endogenous inflammation-resolving mediator.

Nanocomposite treatment reduces disease and lethality in a murine model of acute graft-versus-host disease and preserves anti-tumor effects.

Graft versus host disease (GVHD) is an immunological disorder triggered by bone marrow transplantation that affects several organs, including the gastrointestinal tract and liver. Fullerenes and their soluble forms, fullerols, are nanocomposites with a closed symmetrical structure with anti-inflammatory and anti-oxidant properties. The present study evaluated the effects of treatment with the fullerol (C60(OH)18-20) in the development and pathogenesis of GVHD in a murine model. Mice with experimental GVHD that were treated with the fullerol showed reduced clinical signs of disease and mortality compared with untreated mice. Treatment with the fullerol decreased the hepatic damage associated with reduced hepatic levels of reactive oxygen species, pro-inflammatory cytokines and chemokines (IFN-γ TNF-α, CCL2, CCL3 and CCL5) and reduced leukocyte accumulation. The amelioration of GVHD after treatment with the fullerol was also associated with reduced intestinal lesions and consequent bacterial translocation to the blood, liver and peritoneal cavity. Moreover, the fullerol treatment alleviated the GVHD while preserving effects of the graft against a leukemia cell line (GFP+P815). In summary, the fullerol was effective in reducing the GVHD inflammatory response in mice and may suggest novel ways to treat this disease.