The proinflammatory effects of particulate pollutants, including diesel exhaust particles (DEP), are related to their content of redox cycling chemicals and their ability to generate oxidative stress in the respiratory tract. An antioxidant defense pathway, which involves phase II enzyme expression, protects against the pro-oxidative and proinflammatory effects of DEP. The expression of enzymes, including heme oxygenase-1 (HO-1) and GST, is dependent on the activity of a genetic antioxidant response element in their promoters. In this study we investigated the mechanism by which redox cycling organic chemicals, prepared from DEP, induce phase II enzyme expression as a protective response. We demonstrate that aromatic and polar DEP fractions, which are enriched in polycyclic aromatic hydrocarbons and quinones, respectively, induce the expression of HO-1, GST, and other phase II enzymes in macrophages and epithelial cells. We show that HO-1 expression is mediated through accumulation of the bZIP transcription factor, Nrf2, in the nucleus, and that Nrf2 gene targeting significantly weakens this response. Nrf2 accumulation and subsequent activation of the antioxidant response element is regulated by the proteasomal degradation of Nrf2. This pathway is sensitive to pro-oxidative and electrophilic DEP chemicals and is also activated by ambient ultrafine particles. We propose that Nrf2-mediated phase II enzyme expression protects against the proinflammatory effects of particulate pollutants in the setting of allergic inflammation and asthma.

Lipid rafts are detergent-insoluble membrane domains that play a key role in signal transduction by the T-cell antigen receptor. Proteome analysis revealed the presence of amidosulfobetaine-soluble signal transducing, integral membrane, cytoskeletal, heat shock, and GTP-binding proteins in rafts prepared from Jurkat cells. Several of these proteins were recruited to rafts by CD3/CD28 costimulation. Of particular interest is the inducible association of activated IkappaB kinase complexes with raft vesicles that could be captured with anti-flotillin-1 antibodies. Following amidosulfobetaine solubilization, flotillin-beta and IKKbeta underwent reciprocal co-immunoprecipitation. Treatment of Jurkat cells with methyl-beta-cyclodextrin disrupted the assembly and activation of this raft complex and also interfered in CD3/ CD28-induced activation of a NF-kappaB response element in the IL-2 promoter.

Proteomic profiling of accessible bodily fluids, such as plasma, has the potential to accelerate biomarker/biosignature development for human diseases. The HUPO Plasma Proteome Project pilot phase examined human plasma with distinct proteomic approaches across multiple laboratories worldwide. Through this effort, we confidently identified 3020 proteins, each requiring a minimum of two high-scoring MS/MS spectra. A critical step subsequent to protein identification is functional annotation, in particular with regard to organ systems and disease. Performing exhaustive literature searches, we have manually annotated a subset of these 3020 proteins that have cardiovascular-related functions on the basis of an existing body of published information. These cardiovascular-related proteins can be organized into eight groups: markers of inflammation and/or cardiovascular disease, vascular and coagulation, signaling, growth and differentiation, cytoskeletal, transcription factors, channels/receptors and heart failure and remodeling. In addition, analysis of the peptide per protein ratio for MS/MS identification reveals group-specific trends. These findings serve as a resource to interrogate the functions of plasma proteins, and moreover, the list of cardiovascular-related proteins in plasma constitutes a baseline proteomic blueprint for the future development of biosignatures for diseases such as myocardial ischemia and atherosclerosis.

Currently no treatments exist for preterm infants with diffuse white matter injury (DWMI) caused by hypoxia. Due to the improved care of preterm neonates and increased recognition by advanced imaging techniques, the prevalence of DWMI is increasing. A better understanding of the pathophysiology of DWMI is therefore of critical importance. The integrated stress response (ISR), a conserved eukaryotic response to myriad stressors including hypoxia, may play a role in hypoxia-induced DWMI and may represent a novel target for much needed therapies. In this study, we use in vitro and in vivo hypoxic models of DWMI to investigate whether the ISR is involved in DWMI. We demonstrate that hypoxia activates the ISR in primary mouse oligodendrocyte precursor cells (OPCs) in vitro and that genetically inhibiting the ISR in differentiating OPCs increases their susceptibility to in vitro hypoxia. We also show that a well established in vivo mild chronic hypoxia (MCH) mouse model and a new severe acute hypoxia (SAH) mouse model of DWMI activates the initial step of the ISR. Nonetheless, genetic inhibition of the ISR has no detectable effect on either MCH-or SAH-induced DWMI. In addition, we demonstrate that genetic enhancement of the ISR does not ameliorate MCH-or SAH-induced DWMI. These studies suggest that, while the ISR protects OPCs from hypoxia in vitro, it does not appear to play a major role in either MCH-or SAH-induced DWMI and is therefore not a likely target for therapies aimed at improving neurological outcome in preterm neonates with hypoxia-induced DWMI.

Although the adverse effects of neonatal hypoxia associated with premature birth on the central nervous system are well known, the contribution of hypoxic damage to the peripheral nervous system (PNS) has not been addressed. We demonstrate that neonatal hypoxia results in hypomyelination and delayed axonal sorting in mice leading to electrophysiological and motor deficits that persist into adulthood. These findings support a potential role for PNS hypoxic damage in the motor impairment that results from premature birth and suggest that therapies designed to protect the PNS may provide clinical benefit.

Background: Environmental factors must play a significant role in the emergence of clonal hematopoiesis since only a fraction of individuals harboring clonal hematopoiesis of indeterminate potential (CHIP) mutations develop hematologic malignancy. Mouse models of chronic inflammation have demonstrated clonal expansion of Tet2 and Dnmt3a knockout hematopoietic cells while Ppm1d mutated clones exhibit clonal dominance in response to cytotoxic DNA damaging chemotherapy stress. Epidemiologic studies have associated smoking behavior with clonal hematopoiesis but the leukemogenic effects of cigarette smoke on hematopoietic stem cells (HSCs) are poorly defined. In addition, the exploding use of electronic (e)-cigarettes has led to significant concern on their detrimental health effects plus studies of E-cigarettes on the hematopoietic system are non-existent. Here, we investigated the role of cigarette smoke and E-cigarette aerosols in promoting clonal expansion of common CHIP mutations. We hypothesize that one or more specific somatic CHIP mutation displays a fitness advantage in the presence of cigarette smoke and/or e-cigarette aerosols. Methods: Competitive bone marrow transplant assays were used to determine the development of clonal expansion in response to cigarette smoke and E-cigarette aerosols using Tet2 knockout (Tet2 -/-), Dnmt3a R878H and Jak2 V617F genetically modified mice. Lethally irradiated recipient mice in the CD45.1/2 background were transplanted with whole bone marrow cells from wild type (WT) (CD45.1) and mutant (CD45.2) mice. Ratio of cells transplanted were 1:10 for Tet2 -/- and WT; 1:5 for Dnmt3a R878H and WT and 1:1 for Jak2 V617F and WT. Transplanted mice were exposed to cigarette smoke or E-cigarette aerosols using a nose-only inhalation exposures system for 2 hours/day, 4 days/week for 2 or 3 months. Control mice were exposed to room air using the nose-only inhalation approach. Results: After 2 months of exposure, we observed that Tet2 -/- cells had significantly expanded in the smoke group (paired t-test, p<0.05) while there was no significant difference in the air group (Fig. 1A). Furthermore, this increase in Tet2 -/- cells was more pronounced in the myeloid cell subset (Fig. 1B). While the knock-in mouse model of Jak2 V617F does not display a competitive advantage in a lethally irradiated bone marrow transplant setting, we observed persisting levels of Jak2 V617F mutant cells following smoke exposure but significantly reduced levels in the air group, illustrating that the mutant cells prevail in a smoke environment (Fig. 1C). During sacrifice of the Jak2 V617F transplanted and exposed mice, long-term HSCs in the bone marrow exhibited a trend towards increased bromodeoxyuridine (BrdU) incorporation and increased DNA damage as determined by H2AX staining (Fig 1D). Meanwhile, E-cigarette aerosol exposure of mice transplanted with Dnmt3a R878H cells, displayed increased levels of circulating mutant cells compared to the air group (Fig. 1E) (repeated measures, 2-way ANOVA). Conclusion: In vivo exposure of mouse models of CHIP to cigarette smoke and E-cigarette aerosols promotes mutant cell expansion over time. Our data indicate that more than one mutation is selected by environmental factors in the form of tobacco products. This data is important to guide us in preventive medicine and early detection of clonal hematopoiesis. Future research is aimed at deciphering the molecular responses of WT cells to cigarette smoke and E-cigarette aerosols and strategies to preserve WT stem cell fitness in the context of smoking and E-cigarette usage. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

IntroductionSomatic mutations in myeloid growth factor pathway genes, such as JAK2, and genes involved in epigenetic regulation, such as TET2, in hematopoietic stem cells (HSCs) leads to clonal hematopoiesis of indeterminate potential (CHIP) which presents a risk factor for hematologic malignancy and cardiovascular disease. Smoking behavior has been repeatedly associated with the occurrence of CHIP but whether smoking is an environmental inflammatory stressor in promoting clonal expansion has not been investigated.MethodsWe performed in vivo smoke exposures in both wildtype (WT) mice and transplanted mice carrying Jak2V617F mutant and Tet2 knockout (Tet-/-) cells to determine the impact of cigarette smoke (CS) in the HSC compartment as well as favoring mutant cell expansion.ResultsWT mice exposed to smoke displayed increased oxidative stress in long-term HSCs and suppression of the hematopoietic stem and progenitor compartment but smoke exposure did not translate to impaired hematopoietic reconstitution in primary bone marrow transplants. Gene expression analysis of hematopoietic cells in the bone marrow identified an imbalance between Th17 and Treg immune cells suggesting a local inflammatory environment. We also observed enhanced survival of Jak2V617F cells exposed to CS in vivo and cigarette smoke extract (CSE) in vitro. WT bone marrow hematopoietic cells from WT/Jak2V617F chimeric mice exposed to CS demonstrated an increase in neutrophil abundance and distinct overexpression of bone marrow stromal antigen 2 (Bst2) and retinoic acid early transcript 1 (Raet1) targets. Bst2 and Raet1 are indicative of increased interferon signaling and cellular stress including oxidative stress and DNA damage, respectively. In chimeric mice containing both WT and Tet2-/- cells, we observed an increased percentage of circulating mutant cells in peripheral blood post-cigarette smoke exposure when compared to pre-exposure levels while this difference was absent in air-exposed controls.ConclusionAltogether, these findings demonstrate that CS results in an inflamed bone marrow environment that provides a selection pressure for existing CHIP mutations such as Jak2V617F and Tet2 loss-of-function.