Exposure to Air Pollution Disrupts Circadian Rhythm through Alterations in Chromatin Dynamics.

Particulate matter ≤2.5µm (PM2.5) air pollution is a leading environmental risk factor contributing disproportionately to the global burden of non-communicable disease. We compared impact of chronic exposure to PM2.5 alone, or with light at night exposure (LL) on metabolism. PM2.5 induced peripheral insulin resistance, circadian rhythm (CR) dysfunction, and metabolic and brown adipose tissue (BAT) dysfunction, akin to LL (with no additive interaction between PM2.5 and LL). Transcriptomic analysis of liver and BAT revealed widespread but unique alterations in CR genes, with evidence for differentially accessible promoters and enhancers of CR genes in response to PM2.5 by ATAC-seq. The histone deacetylases 2, 3, and 4 were downregulated with PM2.5 exposure, with increased promoter occupancy by the histone acetyltransferase p300 as evidenced by ChIP-seq. These findings suggest a previously unrecognized role of PM2.5 in promoting CR disruption and metabolic dysfunction through epigenetic regulation of circadian targets.

Differential contribution of bone marrow-derived infiltrating monocytes and resident macrophages to persistent lung inflammation in chronic air pollution exposure.

Chronic exposure to particulate matter < 2.5µ (PM2.5) has been linked to cardiopulmonary disease. Tissue-resident (TR) alveolar macrophages (AΦ) are long-lived, self-renew and critical to the health impact of inhalational insults. There is an inadequate understanding of the impact of PM2.5 exposure on the nature/time course of transcriptional responses, self-renewal of AΦ, and the contribution from bone marrow (BM) to this population. Accordingly, we exposed chimeric (CD45.2/CD45.1) mice to concentrated PM2.5 or filtered air (FA) to evaluate the impact on these end-points. PM2.5 exposure for 4-weeks induced an influx of BM-derived monocytes into the lungs with no contribution to the overall TR-AΦ pool. Chronic (32-weeks) PM2.5 exposure on the other hand while associated with increased recruitment of BM-derived monocytes and their incorporation into the AΦ population, resulted in enhanced apoptosis and decreased proliferation of TR-AΦ. RNA-seq analysis of isolated TR-AΦ and BM-AΦ from 4- and 32-weeks exposed mice revealed a unique time-dependent pattern of differentially expressed genes. PM2.5 exposure resulted in altered histological changes in the lungs, a reduced alveolar fraction which corresponded to protracted lung inflammation. Our findings suggest a time-dependent entrainment of BM-derived monocytes into the AΦ population of PM2.5 exposed mice, that together with enhanced apoptosis of TR-AΦ and reorganization of transcriptional responses, could collectively contribute to the perpetuation of chronic inflammation.

Metabolic effects of air pollution exposure and reversibility.

Air pollution involving particulate matter smaller than 2.5 µm in size (PM2.5) is the world's leading environmental risk factor contributing to mortality through cardiometabolic pathways. In this study, we modeled early life exposure using chow-fed C57BL/6J male mice that were exposed to real-world inhaled, concentrated PM2.5 (~10 times ambient levels/~60-120 µg/m3) or filtered air over a 14-week period. We investigated the effects of PM2.5 on phenotype, the transcriptome, and chromatin accessibility and compared these with the effects of a prototypical high-fat diet (HFD) as well as cessation of exposure on phenotype reversibility. Exposure to PM2.5 impaired glucose and insulin tolerance and reduced energy expenditure and 18FDG-PET uptake in brown adipose tissue. Multiple differentially expressed gene clusters in pathways involving metabolism and circadian rhythm were noted in insulin-responsive tissues. Although the magnitude of transcriptional change detected with PM2.5 exposure was lower than that observed with a HFD, the degree of alteration in chromatin accessibility after PM2.5 exposure was significant. The novel chromatin remodeler SMARCA5 (SWI/SNF complex) was regulated in response to PM2.5 exposure, the cessation of which was associated with a reversal of insulin resistance and restoration of chromatin accessibility and nucleosome positioning near transcription start sites, as well as a reversal of exposure-induced changes in the transcriptome, including SMARCA5. These changes indicate pliable epigenetic control mechanisms following cessation of exposure.

Oxidative stress pathways of air pollution mediated toxicity: Recent insights.

Ambient air pollution is a leading environmental cause of morbidity and mortality globally with most of the outcomes of cardiovascular origin. While numerous mechanisms are proposed to explain the link between air pollutants and cardiovascular events, the evidence supports a role for oxidative stress as a critical intermediary pathway in the transduction of systemic responses in the cardiovascular system. Indeed, alterations in vascular function are a critical step in the development of cardiometabolic disorders such as hypertension, diabetes, and atherosclerosis. This review will provide an overview of the impact of particulate and gaseous pollutants on oxidative stress from human and animal studies published in the last five years. We discuss current gaps in knowledge and evidence to date implicating the role of oxidative stress with an emphasis on inhalational exposures. We conclude with the identification of gaps, and an exhortation for further studies to elucidate the impact of oxidative stress in air pollution mediated effects.

Vitamin D improves sunburns by increasing autophagy in M2 macrophages.

Cutaneous inflammation from UV radiation exposure causes epidermal damage, cellular infiltration, and secretion of pro-inflammatory mediators that exacerbate tissue destruction. Recovery is mediated chiefly by anti-inflammatory M2 macrophages that suppress inflammation and augment epidermal regeneration. Vitamin D enables anti-inflammation to promote tissue repair in response to injury. Since vitamin D enhances cellular macroautophagy/autophagy, we investigated the role of autophagy in vitamin D protection of UV-mediated sunburn and inflammation. Using a UV-mediated acute skin injury mouse model, we demonstrate that a single dose of vitamin D resolves injury with sustained inhibition of inflammatory cytokines associated with enhanced autophagy in myeloid anti-inflammatory M2 macs. Increased MAP1LC3B/LC3 expression corroborated with complete autolysosome formation detected by electron microscopy and correlated with degradation of SQSTM1/p62 in the skin following vitamin D treatment. Specifically, pharmacological inhibition of autophagy increased UV-induced apoptosis, suppressed M2 macs recruitment, and prevented vitamin D downregulation of Tnf and Mmp9 in the skin. Furthermore, selective deletion of autophagy in myeloid cells of atg7 cKO mice abrogated vitamin D-mediated protection and recapitulated UV-induced inflammation. Mechanistically, vitamin D signaling activated M2-autophagy regulators Klf4, Pparg, and Arg1. Lastly, analysis of UV-exposed human skin biopsies detected a similar increase in macrophage autophagy following vitamin D intervention, identifying an essential role for autophagy in vitamin D-mediated protection of skin from UV damage. Abbreviations: ARG1: arginase 1; ATG7 cKO: autophagy related 7 conditional knockout; HPF: high powered field; KLF4: Kruppel like factor 4; MAP1LC3B/LC3: microtubule-associated protein 1 light chain 3 beta; macs: macrophage; 3-MA: 3-methyladenine; MMP9: matrix metallopeptidase 9; NOS2: nitric oxide synthase 2, inducible; PPARG: peroxisome proliferator activated receptor gamma; SQSTM1/p62: sequestosome 1; TNF: tumor necrosis factor; UV: ultraviolet; VD: vitamin D, 25-hydroxy vitamin D3; 1,25-VD: 1, 25-dihydroxy vitamin D3.

Defining the timing of 25(OH)D rescue following nitrogen mustard exposure.

OBJECTIVE: Mass exposure to alkylating agents such as nitrogen mustard (NM), whether accidental or intentional as during warfare, are known to cause systemic toxicity and severe blistering from cutaneous exposure. Thus, establishing the timing and appropriate dose of any potential drug designed to reverse or impede these toxicities is critical for wound repair and survival. Our previous data demonstrates that a single intraperitoneal injection of low-dose 25-hydroxyvitamin D3 (25(OH)D) given as early as 1 h following NM exposure is sufficient to rescue mice from pancytopenia and death. However, the duration of time following exposure where intervention is still effective as a countermeasure is unknown. In this study, we sought to assess the maximal time permissible following NM exposure where 25(OH)D still affords protection against NM-induced cutaneous injury. Additionally, we determined if a higher dose of 25(OH)D would be more efficacious at time interval where low dose 25(OH)D is no longer effective. METHODS: Low (5 ng) and high (50 ng) doses of 25(OH)D were administered intraperitoneally to mice following exposure to topical NM to assess wound resolution and survival. Mice were imaged and weighed daily to measure wound healing and to monitor systemic toxicity. RESULTS: We demonstrated that 5 ng 25(OH)D administered as early as 1 h and as late as 24 h post-NM exposure is able to achieve 100% recovery in mice. In contrast, intervention at and beyond 48 h of NM exposure failed to achieve full recovery and resulted in ≥60% death between days 6 and 12, demonstrating the critical nature of timely intervention with 25(OH)D at each respective dose. In order to circumvent the observed failure at >48 h exposure, we provided two consecutive doses of 5 ng or 50 ng of 25(OH)D at 48 h and 72 h post-NM exposure. Repeat dosing with 25(OH)D at 48 h and beyond led to marked improvement of lesion size with 75% recovery from mortality. CONCLUSIONS: The opportunity to use 25(OH)D as a medical countermeasure for NM-induced toxicity has a finite of window for intervention. However, modifications such as repeat dosing can be an effective strategy to extend the intervention potential of 25(OH)D.

Oral Vitamin D Rapidly Attenuates Inflammation from Sunburn: An Interventional Study.

The diverse immunomodulatory effects of vitamin D are increasingly being recognized. However, the ability of oral vitamin D to modulate acute inflammation in vivo has not been established in humans. In a double-blinded, placebo-controlled interventional trial, 20 healthy adults were randomized to receive either placebo or a high dose of vitamin D3 (cholecalciferol) one hour after experimental sunburn induced by an erythemogenic dose of UVR. Compared with placebo, participants receiving vitamin D3 (200,000 international units) demonstrated reduced expression of proinflammatory mediators tumor necrosis factor-α (P = 0.04) and inducible nitric oxide synthase (P = 0.02) in skin biopsy specimens 48 hours after experimental sunburn. A blinded, unsupervised hierarchical clustering of participants based on global gene expression profiles revealed that participants with significantly higher serum vitamin D3 levels after treatment (P = 0.007) demonstrated increased skin expression of the anti-inflammatory mediator arginase-1 (P = 0.005), and a sustained reduction in skin redness (P = 0.02), correlating with significant expression of genes related to skin barrier repair. In contrast, participants with lower serum vitamin D3 levels had significant expression of proinflammatory genes. Together the data may have broad implications for the immunotherapeutic properties of vitamin D in skin homeostasis, and implicate arginase-1 upregulation as a previously unreported mechanism by which vitamin D exerts anti-inflammatory effects in humans.

Early indicators of survival following exposure to mustard gas: Protective role of 25(OH)D.

The use of sulfur mustard (SM) as a chemical weapon for warfare has once again assumed center stage, endangering civilian and the military safety. SM causes rapid local skin vesication and late-onset systemic toxicity. Most studies on SM rely on obtaining tissue and blood for characterizing burn pathogenesis and assessment of systemic pathology, respectively. However the present study focuses on developing a non-invasive method to predict mortality from high dose skin SM exposure. We demonstrate that exposure to SM leads to a dose dependent increase in wound area size on the dorsal surface of mice that is accompanied by a progressive loss in body weight loss, blood cytopenia, bone marrow destruction, and death. Thus our model utilizes local skin destruction and systemic outcome measures as variables to predict mortality in a novel skin-based model of tissue injury. Based on our recent work using vitamin D (25(OH)D) as an intervention to treat toxicity from SM-related compounds, we explored the use of 25(OH)D in mitigating the toxic effects of SM. Here we show that 25(OH)D offers protection against SM and is the first known demonstration of an intervention that prevents SM-induced mortality. Furthermore, 25(OH)D represents a safe, novel, and readily translatable potential countermeasure following mass toxic exposure.

Suppression of Hyperactive Immune Responses Protects against Nitrogen Mustard Injury.

DNA alkylating agents like nitrogen mustard (NM) are easily absorbed through the skin and exposure to such agents manifest not only in direct cellular death but also in triggering inflammation. We show that toxicity resulting from topical mustard exposure is mediated in part by initiating exaggerated host innate immune responses. Using an experimental model of skin exposure to NM we observe activation of inflammatory dermal macrophages that exacerbate local tissue damage in an inducible nitric oxide synthase (iNOS)-dependent manner. Subsequently these activated dermal macrophages reappear in the bone marrow to aid in disruption of hematopoiesis and contribute ultimately to mortality in an experimental mouse model of topical NM exposure. Intervention with a single dose of 25-hydroxyvitamin D3 (25(OH)D) is capable of suppressing macrophage-mediated iNOS production resulting in mitigation of local skin destruction, enhanced tissue repair, protection from marrow depletion, and rescue from severe precipitous wasting. These protective effects are recapitulated experimentally using pharmacological inhibitors of iNOS or by compounds that locally deplete skin macrophages. Taken together, these data highlight a critical unappreciated role of the host innate immune system in exacerbating injury following exposure to NM and support the translation of 25(OH)D in the therapeutic use against these chemical agents.

Hyper-inflammation and skin destruction mediated by rosiglitazone activation of macrophages in IL-6 deficiency.

Injury initiates recruitment of macrophages to support tissue repair; however, excessive macrophage activity may exacerbate tissue damage causing further destruction and subsequent delay in wound repair. Here we show that the peroxisome proliferation-activated receptor-γ agonist, rosiglitazone (Rosi), a medication recently reintroduced as a drug to treat diabetes and with known anti-inflammatory properties, paradoxically generates pro-inflammatory macrophages. This is observed in both IL-6-deficient mice and control wild-type mice experimentally induced to produce high titers of auto-antibodies against IL-6, mimicking IL-6 deficiency in human diseases. IL-6 deficiency when combined with Rosi-mediated upregulation of suppressor of cytokine signaling 3 leads to an altered ratio of nuclear signal transducer and activator of transcription 3/NF-κB that allows hyper-induction of inducible nitric oxide synthase (iNOS). Macrophages activated in this manner cause de novo tissue destruction, recapitulating human chronic wounds, and can be reversed in vivo by recombinant IL-6, blocking macrophage infiltration, or neutralizing iNOS. This study provides insight into an unanticipated paradoxical role of Rosi in mediating hyper-inflammatory macrophage activation significant for diseases associated with IL-6 deficiency.

The virtues of oxygenation: low tissue oxygen adversely affects the killing of Leishmania.

Hypoxia contributes to the persistence of infections through altered immune responses. Studies examining skin O2 changes at the site of a lesion are limited. The prevailing methods require the use of electrochemical O2 sensors or radiolabeled electrodes that utilize O2 and may interfere with the precision at low O2 levels. In this issue, Mahnke et al. (2014) demonstrate, using a novel fluorescence-based imaging technology, that low oxygen tension (pO2) impairs NO-mediated anti-leishmanial immunity, leading to increased parasite burden. Replenishing tissue oxygen profoundly enhanced NO-mediated leishmanial killing, underscoring the need to accurately assess oxygenation in infected tissues as a novel strategy to challenge intracellular infection. The technology presented here may have clinical-translational potential in noninvasively assessing disease burden and response to treatment.

Junior doctors' understanding of alcohol units remains poor.

A survey of 586 trainee doctors in Yorkshire was undertaken to ascertain current junior doctors' knowledge and understanding of alcohol units. Approximately 18% of trainees had no knowledge of alcohol units despite the fact that 82% believed they had a good knowledge. Once again, those who did not drink alcohol knew less about alcohol units than those who did. Little progress seems to have been made on this important subject since our previous survey 7 years ago. Further steps must be taken to ensure that junior doctors are taught about alcohol units during the course of their training so that they are able to counsel patients appropriately.