Alterations of mitochondrial bioenergetics, dynamics, and morphology support the theory of oxidative damage involvement in autism spectrum disorder.

Autism spectrum disorder (ASD) has been hypothesized to be a result of the interplay between genetic predisposition and increased vulnerability to early environmental insults. Mitochondrial dysfunctions appear also involved in ASD pathophysiology, but the mechanisms by which such alterations develop are not completely understood. Here, we analyzed ASD primary fibroblasts by measuring mitochondrial bioenergetics, ultrastructural and dynamic parameters to investigate the hypothesis that defects in these pathways could be interconnected phenomena responsible or consequence for the redox imbalance observed in ASD. High levels of 4-hydroxynonenal protein adducts together with increased NADPH (nicotinamide adenine dinucleotide phosphateoxidase) activity and mitochondrial superoxide production coupled with a compromised antioxidant response guided by a defective Nuclear Factor Erythroid 2-Related Factor 2 pathway confirmed an unbalanced redox homeostasis in ASD. Moreover, ASD fibroblasts showed overactive mitochondrial bioenergetics associated with atypical morphology and altered expression of mitochondrial electron transport chain complexes and dynamics-regulating factors. We suggest that many of the changes observed in mitochondria could represent compensatory mechanisms by which ASD cells try to adapt to altered energy demand, possibly resulting from a chronic oxinflammatory status.

Cutaneous antimicrobial peptides: New "actors" in pollution related inflammatory conditions.

Ozone (O3) exposure has been reported to contribute to various cutaneous inflammatory conditions, such as eczema, psoriasis, rush etc. via a redox-inflammatory pathway. O3 is too reactive to penetrate cutaneous tissue; it interacts with lipids present in the outermost layer of skin, resulting in formation of oxidized molecules and hydrogen peroxide (H2O2). Interestingly, several inflammatory skin pathologies demonstrate altered levels of antimicrobial peptides (AMPs). These small, cationic peptides are found in various cells, including keratinocytes, eccrine gland cells, and seboctyes. Classically, AMPs function as antimicrobial agents. Recent studies indicate that AMPs also play roles in inflammation, angiogenesis, and wound healing. Since altered levels of AMPs have been detected in pollution-associated skin pathologies, we hypothesized that exposure to O3 could affect the levels of AMPs in the skin. We examined levels of AMPs using qRT-PCR, Western blotting, and immunofluorescence in vitro (human keratinocytes), ex vivo (human skin explants), and in vivo (human volunteer subjects exposed to O3) and observed increased levels of all the measured AMPs upon O3 exposure. In addition, in vitro studies have confirmed the redox regulation of AMPs in keratinocytes. This novel finding suggests that targeting AMPs could be a possible defensive strategy to combat pollution-associated skin conditions.

Evaluating the effect of ozone in UV induced skin damage.

Air pollution represents one of the main risks for both environment and human health. The rapid urbanization has been leading to a continuous release of harmful manmade substances into the atmosphere which are associated to the exacerbation of several pathologies. The skin is the main barrier of our body against the external environment and it is the main target for the outdoor stressors. Among the pollutants, Ozone (O3) is one of the most toxic, able to initiate oxidative reactions and activate inflammatory response, leading to the onset of several skin conditions. Moreover, skin is daily subjected to the activity of Ultraviolet Radiation which are well known to induce harmful cutaneous effects including skin aging and sunburn. Even though both UV and O3 are able to affect the skin homeostasis, very few studies have investigated their possible additive effect. Therefore, in this study we evaluated the effect of the combined exposure of O3 and UV in inducing skin damage, by exposing human skin explants to UV alone or in combination with O3 for 4-days. Markers related to inflammation, redox homeostasis and tissue structure were analyzed. Our results demonstrated that O3 is able to amplify the UV induced skin oxinflammation markers.

Additive effect of combined pollutants to UV induced skin OxInflammation damage. Evaluating the protective topical application of a cosmeceutical mixture formulation.

Since the skin is one of the targets of the harmful effects of environmental insults, several studies have investigated the effects of outdoor stressors on cutaneous tissue. Ozone (O3), particulate matter (PM), and ultraviolet radiation (UV) have all been shown to induce skin damage through disruption of tissue redox homeostasis, resulting in the so called "OxInflammation" condition. However, few studies have explored whether these stressors can act synergistically in cutaneous tissues. In the present work, we evaluated whether O3, PM, and UV, which are the most common environmental skin insults, act synergistically in inducing skin damage, and whether this effect could be prevented through topical application of a cosmeceutical formulation mixture (CF Mix) containing 15% vitamin C (l-ascorbic acid), 1% vitamin E (α-tocopherol), and 0.5% ferulic acid. Human skin explants obtained from three different subjects were sequentially exposed to 200 mJ UV light, 0.25 ppm O3 for 2 h, and 30 min of diesel engine exhaust (DEE), alone or in combination for 4 days (time point D1 and D4). We observed a clear additive effect of O3 and DEE in combination with UV in increasing levels of several oxidative (4HNE, HO-1) and inflammatory (COX2, NF-κB) markers and loss of barrier-associated proteins, such as filaggrin and involucrin. Furthermore, daily topical pre-treatment with the CF Mix prevented upregulation of the inflammatory and oxidative markers and the loss of both involucrin and filaggrin. In conclusion, this study is the first to investigate the combined effects of three of the most harmful outdoor stressors on human skin and suggests that daily topical application may prevent pollution-induced skin damage.

Altered inflammasome machinery as a key player in the perpetuation of Rett syndrome oxinflammation.

Rett syndrome (RTT) is a progressive neurodevelopmental disorder mainly caused by mutations in the X-linked MECP2 gene. RTT patients show multisystem disturbances associated with an oxinflammatory status. Inflammasomes are multi-protein complexes, responsible for host immune responses against pathogen infections and redox-related cellular stress. Assembly of NLRP3/ASC inflammasome triggers pro-caspase-1 activation, thus, resulting in IL-1ß and IL-18 maturation. However, an aberrant activation of inflammasome system has been implicated in several human diseases. Our aim was to investigate the possible role of inflammasome in the chronic subclinical inflammatory condition typical of RTT, by analyzing this complex in basal and lipopolysaccharide (LPS)+ATP-stimulated primary fibroblasts, as well as in serum from RTT patients and healthy volunteers. RTT cells showed increased levels of nuclear p65 and ASC proteins, pro-IL-1ß mRNA, and NLRP3/ASC interaction in basal condition, without any further response upon the LPS + ATP stimuli. Moreover, augmented levels of circulating ASC and IL-18 proteins were found in serum of RTT patients, which are likely able to amplify the inflammatory response. Taken together, our findings suggest that RTT patients exhibited a challenged inflammasome machinery at cellular and systemic level, which may contribute to the subclinical inflammatory state feedback observed in this pathology.

Redox regulation of cutaneous inflammasome by ozone exposure.

Several pollutants have been shown to affect skin physiology, among which ozone (O3) is one of the most toxic. Prolonged exposure to O3 leads to increased oxidative damage and cutaneous inflammation. The correlation between O3 exposure and inflammatory cutaneous conditions (atopic dermatitis, psoriasis, acne and eczema) has been already suggested, although the mechanism involved is still unclear. In the last few decades, a new multiprotein complex, the inflammasome, has been discovered and linked to tissue inflammation, including inflammatory skin conditions. The inflammasome activates inflammatory responses and contributes to the maturation of cytokines such as interleukin 1ß (IL-1ß) and interleukin 18. This complex is also responsive to reactive oxygen species (ROS), which plays a role in triggering the activation of the complex. On this basis it is possible hypothesize that the activation of the inflammasome could be the link between the inflammatory skin conditions associated to O3 exposure. In the present work, the ability of O3 to induce inflammasome activation was determined in different skin models, ranging from 2D (human keratinocytes) to 3D models in vitro and ex vivo. Results clearly showed that O3 exposure increased both transcript and protein levels of the main inflammasome complex, such as ASC and caspase-1. Furthermore, by using both immunofluorescence and an ASC oligomerization assay the formation of the complex was determined together with increased secreted levels of both IL-18 and IL-1ß. Of note is that H2O2 and to a less extent 4HNE (both considered the main mediators of O3 interaction with cellular membranes) were also able to activate skin inflammasome while the use of catalase prevents the activation. This study demonstrated that O3 can activate cutaneous inflammasome in a redox dependent manner suggesting a possible role of this new pathway in pollution induced inflammatory skin conditions.