Adenosine Diphosphate Improves Wound Healing in Diabetic Mice Through P2Y12 Receptor Activation.

Chronic wounds are a public health problem worldwide, especially those related to diabetes. Besides being an enormous burden to patients, it challenges wound care professionals and causes a great financial cost to health system. Considering the absence of effective treatments for chronic wounds, our aim was to better understand the pathophysiology of tissue repair in diabetes in order to find alternative strategies to accelerate wound healing. Nucleotides have been described as extracellular signaling molecules in different inflammatory processes, including tissue repair. Adenosine-5'-diphosphate (ADP) plays important roles in vascular and cellular response and is immediately released after tissue injury, mainly from platelets. However, despite the well described effect on platelet aggregation during inflammation and injury, little is known about the role of ADP on the multiple steps of tissue repair, particularly in skin wounds. Therefore, we used the full-thickness excisional wound model to evaluate the effect of local ADP application in wounds of diabetic mice. ADP accelerated cutaneous wound healing, improved new tissue formation, and increased both collagen deposition and transforming growth factor-ß (TGF-ß) production in the wound. These effects were mediated by P2Y12 receptor activation since they were inhibited by Clopidogrel (Clop) treatment, a P2Y12 receptor antagonist. Furthermore, P2Y1 receptor antagonist also blocked ADP-induced wound closure until day 7, suggesting its involvement early in repair process. Interestingly, ADP treatment increased the expression of P2Y12 and P2Y1 receptors in the wound. In parallel, ADP reduced reactive oxygen species (ROS) formation and tumor necrosis factor-α (TNF-α) levels, while increased IL-13 levels in the skin. Also, ADP increased the counts of neutrophils, eosinophils, mast cells, and gamma delta (γδ) T cells (Vγ4+ and Vγ5+ cells subtypes of γδ+ T cells), although reduced regulatory T (Tregs) cells in the lesion. In accordance, ADP increased fibroblast proliferation and migration, myofibroblast differentiation, and keratinocyte proliferation. In conclusion, we provide strong evidence that ADP acts as a pro-resolution mediator in diabetes-associated skin wounds and is a promising intervention target for this worldwide problem.

Neutrophil and Eosinophil DNA Extracellular Trap Formation: Lessons From Pathogenic Fungi.

Fungal infections represent a worldwide health problem. Fungal pathogens are responsible for a variety of conditions, including superficial diseases, allergic pathologies and potentially lethal invasive infections. Neutrophils and eosinophils have been implicated as effector cells in several pathologies. Neutrophils are major effector cells involved in the control of fungal infections and exhibit a plethora of antifungal mechanisms, such as phagocytosis, reactive oxygen species production, degranulation, extracellular vesicle formation, and DNA extracellular trap (ET) release. Eosinophils are polymorphonuclear cells classically implicated as effector cells in the pathogenesis of allergic diseases and helminthic infections, although their roles as immunomodulatory players in both innate and adaptive immunity are currently recognized. Eosinophils are also endowed with antifungal activities and are abundantly found in allergic conditions associated with fungal colonization and sensitization. Neutrophils and eosinophils have been demonstrated to release their nuclear and mitochondrial DNA in response to many pathogens and pro-inflammatory stimuli. ETs have been implicated in the killing and control of many pathogens, as well as in promoting inflammation and tissue damage. The formation of ETs by neutrophils and eosinophils has been described in response to pathogenic fungi. Here, we provide an overview of the mechanisms involved in the release of neutrophil and eosinophil ETs in response to fungal pathogens. General implications for understanding the formation of ETs and the roles of ETs in fungal infections are discussed.

Detection of Eosinophil Extracellular DNA Traps.

The process of extracellular DNA trap release by leukocytes, including eosinophils, has been considered as an important cell-mediated immune response to different inflammatory stimuli helping to understand the physiopathology of many diseases. Here we describe in detail two useful and simple protocols for a semiquantitative and a qualitative analysis of extracellular DNA traps released by human eosinophils, based on fluorimetry and fluorescence microscopy, respectively. These methods can also be applied to detect the DNA trap release by other leukocytes such as neutrophils and even other cell types.

Pharmacological modulation of reactive oxygen species (ROS) improves the airway hyperresponsiveness by shifting the Th1 response in allergic inflammation induced by ovalbumin.

Asthma is an allergic inflammation driven by the Th2 immune response with release of cytokines such as IL-4 and IL-13, which contribute to the airflow limitations and airway hyperresponsiveness (AHR). The involvement of oxidative stress in this process is well-established, but the specific role of the superoxide anion and nitric oxide in asthma are poorly understood. Thus, the aim of this study was to investigate the mechanisms underlying the superoxide anion/nitric oxide production and detoxification in a murine asthma model. BALB/c male mice were sensitised and challenged with ovalbumin (OVA). Pretreatments with either apocynin (14 mg/kg) or allopurinol (25 mg/kg) (superoxide anion synthesis inhibitors), aminoguanidine (50 mg/kg) (nitric oxide synthesis inhibitor) or diethyldithiocarbamate (100 mg/kg) (superoxide dismutase inhibitor) were performed 1 h before the challenge. Our data showed that apocynin and allopurinol ameliorated AHR and reduced eosinophil peroxidase, as well as IL-4 and IL-13 levels. Apocynin also abrogated leukocyte peribronchiolar infiltrate and increased IL-1ß secretion. Aminoguanidine preserved lung function and shifted the Th2 to the Th1 response with a reduction of IL-4 and IL-13 and increase in IL-1ß production. Diethyldithiocarbamate prevented neither allergen-induced AHR nor eosinophil peroxidase (EPO) generation. All treatments protected against oxidative damage observed by a reduction in TBARS levels. Taken together, these results suggest that AHR in an asthma model can be avoided by the down-regulation of superoxide anion and nitric oxide synthesis in a mechanism that is independent of a redox response. This down-regulation is also associated with a transition in the typical immunological Th2 response toward the Th1 profile.

Antianaphylactic properties of 7-epiclusianone, a tetraprenylated benzophenone isolated from Garcinia brasiliensis.

Prior studies have emphasized the anti-inflammatory and antioxidant properties of polyisoprenylated benzophenone derivatives, but their putative effect on allergic conditions has not yet been addressed. In the current study, the naturally occurring 7-epiclusianone, isolated from Garcinia brasiliensis, was investigated to check its effectiveness on allergen-evoked intestinal spasm. The standard antiallergic azelastine was used for comparison. We found that 7-epiclusianone and azelastine inhibited antigen-induced contractions of guinea pig ileum with similar IC (50) values (2.3 +/- 1.1 microM and 3.3 +/- 1.2 microM, respectively). A similar blockade of anaphylactic histamine release from the ileum was also noted. In contrast, azelastine was more potent than 7-epiclusianone to prevent spasms induced by histamine (IC (50) = 6.3 +/- 0.2 nM and 3.7 +/- 0.1 microM, respectively). These findings reveal that 7-epiclusianone is clearly active against the anaphylactic response and should be considered as a molecular template in drug discovery for allergic syndromes.