Acute exposure to e-cigarette vapor (EV) is cytotoxic to airway cells in vitro. Acute exposure to EV decreases macrophage and neutrophil antimicrobial function. Inhalation of EV alters immunomodulating cytokines in the airways of mice. Inhalation of EV leads to increased markers of inflammation in BAL and serum. Staphylococcus aureus become more virulent when exposed to EV.

Rapid and efficient removal of apoptotic cells by phagocytes plays a key role during development, tissue homeostasis, and in controlling immune responses1–5. An important feature of efficient clearance is the capacity of a single phagocyte to ingest multiple apoptotic cells successively, and to process the increased load of corpse-derived cellular material6–9. However, factors that influence sustained phagocytic capacity or how they in turn influence continued clearance by phagocytes are not known. Here we identify that the ability of a phagocyte to control its mitochondrial membrane potential is a critical factor in the capacity of a phagocyte to engulf apoptotic cells. Changing the phagocyte mitochondrial membrane potential (genetically or pharmacologically) significantly affected phagocytosis, with lower potential enhancing engulfment and higher membrane potential inhibiting uptake. We then identified that Ucp2, a mitochondrial membrane protein that acts to lower the mitochondrial membrane potential10–12, is upregulated in phagocytes engulfing apoptotic cells (but not synthetic targets, bacteria, or yeast). Loss of Ucp2 limited the capacity of phagocytes to continually ingest apoptotic cells, while overexpression of Ucp2 increased the capacity for engulfment and the ability to engulf multiple apoptotic cells. Mutational and pharmacological inhibition of Ucp2 uncoupling activity reversed the positive effect of Ucp2 on engulfment capacity, suggesting a direct role for Ucp2-mediated mitochondrial function in phagocytosis. Macrophages from Ucp2-deficient mice13, 14 were impaired in their capacity to engulf apoptotic cells in vitro, and Ucp2-deficient mice displayed profound in vivo defects in clearing dying cells in the thymus and the testes. Collectively, these data suggest that phagocytes alter the mitochondrial membrane potential during engulfment to regulate uptake of sequential apoptotic cells, and that Ucp2 is a key molecular determinant of this step in vivo. Since Ucp2 function has also been linked to metabolic diseases and atherosclerosis14–16, these data identifying a new role for Ucp2 in regulating apoptotic cell clearance may provide additional insights toward understanding the complex etiology and pathogenesis of these diseases.

Oxidatively induced DNA lesions have been implicated in the etiology of many diseases (including cancer) and in aging. Repair of oxidatively damaged bases in all organisms occurs primarily via the DNA base excision repair (BER) pathway, initiated with their excision by DNA glycosylases. Only two mammalian DNA glycosylases, OGG1 and NTH1 of E. coli Nth family, were previously characterized, which excise majority of the oxidatively damaged base lesions. We recently discovered and characterized two human orthologs of E. coli Nei, the prototype of the second family of oxidized base-specific glycosylases and named them NEIL (Nei-like)-1 and 2. NEILs are distinct from NTH1 and OGG1 in structural features and reaction mechanism but act on many of the same substrates. Nth-type DNA glycosylases after base excision, cleave the DNA strand at the resulting AP-site to produce a 3′-αβ unsaturated aldehyde whereas Nei-type enzymes produce 3′-phosphate terminus. E. coli APEs efficiently remove both types of termini in addition to cleaving AP sites to generate 3′-OH, the primer terminus for subsequent DNA repair synthesis. In contrast, the mammalian APE, APE1, which has an essential role in NTH1/OGG1-initiated BER, has negligible 3′-phosphatase activity and is dispensable for NEIL-initiated BER. Polynucleotide kinase (PNK), present in mammalian cells but not in E. coli, removes the 3′ phosphate, and is involved in NEILinitiated BER. NEILs show a unique preference for excising lesions from a DNA bubble, while most DNA glycosylases, including OGG1 and NTH1, are active only with duplex DNA. The dichotomy in the preference of NEILs and NTH1/OGG1 for bubble versus duplex DNA substrates suggests that NEILs function preferentially in repair of base lesions during replication and/or transcription and hence play a unique role in maintaining the functional integrity of mammalian genomes.

Bacterial recognition by host cells is essential for initiation of infection and the host response. Bacteria interact with host cells via multiple pattern recognition receptors that recognize microbial products or pathogen-associated molecular patterns. In response to this interaction, host cell signaling cascades are activated that lead to inflammatory responses and/or phagocytic clearance of attached bacteria. Brain angiogenesis inhibitor 1 (BAI1) is a receptor that recognizes apoptotic cells through its conserved type I thrombospondin repeats and triggers their engulfment through an ELMO1/Dock/Rac1 signaling module. Because thrombospondin repeats in other proteins have been shown to bind bacterial surface components, we hypothesized that BAI1 may also mediate the recognition and clearance of pathogenic bacteria. We found that preincubation of bacteria with recombinant soluble BAI1 ectodomain or knockdown of endogenous BAI1 in primary macrophages significantly reduced binding and internalization of the Gram-negative pathogen Salmonella typhimurium. Conversely, overexpression of BAI1 enhanced attachment and engulfment of Salmonella in macrophages and in heterologous nonphagocytic cells. Bacterial uptake is triggered by the BAI1-mediated activation of Rac through an ELMO/Dock-dependent mechanism, and inhibition of the BAI1/ELMO1 interaction prevents both Rac activation and bacterial uptake. Moreover, inhibition of ELMO1 or Rac function significantly impairs the proinflammatory response to infection. Finally, we show that BAI1 interacts with a variety of Gramnegative, but not Gram-positive, bacteria through recognition of their surface lipopolysaccharide. Together these findings identify BAI1 as a pattern recognition receptor that mediates nonopsonic phagocytosis of Gram-negative bacteria by macrophages and directly affects the host response to infection.innate immunity | lipopolysaccharide-binding | TNF-α

Preferential repair of bulky DNA adducts from the transcribed genes via nucleotide excision repair is well characterized in mammalian cells. However, definitive evidence is lacking for similar repair of oxidized bases, the major endogenous DNA lesions. Here we show that the oxidized base-specific human DNA glycosylase NEIL2 associates with RNA polymerase II and the transcriptional regulator heterogeneous nuclear ribonucleoprotein-U (hnRNP-U), both in vitro and in cells. NEIL2 immunocomplexes from cell extracts preferentially repaired the mutagenic cytosine oxidation product 5-hydroxyuracil in the transcribed strand. In a reconstituted system, we also observed NEIL2-initiated transcription-dependent base excision repair of 5-hydroxyuracil in the transcribed strand, with hnRNP-U playing a critical role. Chromatin immunoprecipitation/reimmunoprecipitation studies showed association of NEIL2, RNA polymerase II, and hnRNP-U on transcribed but not on transcriptionally silent genes. Furthermore, NEIL2-depleted cells accumulated more DNA damage in active than in silent genes. These results strongly support the preferential role of NEIL2 in repairing oxidized bases in the transcribed genes of mammalian cells.

Helicobacter pylori infection is associated with gastritis, ulcers, and gastric cancer. The infection becomes chronic as the host response is unable to clear it. Gastric epithelial cells (GEC) play an important role during the host response, and their expression of class II MHC and costimulatory molecules such as CD80 and CD86 suggests their role in local Ag presentation. Although T cells are recruited to the infected gastric mucosa, they have been reported to be hyporesponsive. In this study, we detected the expression of B7-H1 (programmed death-1 ligand 1), a member of B7 family of proteins associated with T cell inhibition on GEC. Quantitative real-time RT-PCR revealed that B7-H1 expression increased significantly on GEC after H. pylori infection. Western blot analysis showed that B7-H1 expression was induced by various H. pylori strains and was independent of H. pylori virulence factors such as Cag, VacA, and Urease. The functional role of B7-H1 in the cross talk between GEC and T cells was assessed by coculturing GEC or H. pylori-infected GEC with CD4+ T cells isolated from peripheral blood. Using blocking Abs to B7-H1 revealed that B7-H1 was involved in the suppression of T cell proliferation and IL-2 synthesis, and thus suggested a role for B7-H1 on the epithelium as a contributor in the chronicity of H. pylori infection.

The mammalian AP-endonuclease APE1 is a ubiquitous and remarkably multifunctional protein. It plays a central role in the base excision repair (BER) pathway for repairing damaged bases and DNA single-strand breaks induced by reactive oxygen species and alkylating agents and also repairing AP sites that are generated spontaneously or after the excision of oxidized and alkylated bases by DNA glycosylases (12,40). APE1 was also shown to incise DNA 5Ј to oxidatively damaged bases including 5,6-dihydrothymidine and alpha-2Ј-deoxyadenosine in the nucleotide incision repair pathway (11,21). The deletion of the N-terminal 61 amino acid residues, which are dispensable for the AP-endonuclease activity, affected its incision activity as well (21, 28). Moreover, the ability of APE1 to incise DNA 5Ј to a bulky exocyclic adduct such as p-benozoquinone has also been reported (22). Besides its repair function, mammalian APE1 has two unique and apparently distinct transcriptional regulatory activities. It was independently identified as a reductive activator of c-Jun in vitro and named . Subsequently, several other transcription factors (including p53, NF-B, hypoxia-inducible factor 1-␣, PAX5, and PAX8) were also shown to be activated by APE1, presumably via the same redox process (13,31,55). A third and distinct function of APE1 as a trans-acting factor was discovered when APE1 was identified as being one of the regulatory proteins that binds to the negative Ca 2ϩ response elements (nCaRE-A and -B) in the Ca 2ϩ -dependent downregulation of the parathyroid hormone gene (48) and subsequently in the human renin gene (17). We later showed that human APE1 is acetylated at Lys6 and Lys7 by the histone acetyltransferase p300, both in vivo and in vitro, and that acetylation enhances APE1's binding to nCaRE-B, leading to the repression of the parathyroid hormone promoter (2). Our follow-up studies indicated that a small but significant fraction of APE1 in HeLa cells and mouse liver is present in the acetylated form (14,56). Recently, we have shown that the early growth response protein (Egr-1)-mediated activation of phosphoinositol phosphatase and tensin homologue (PTEN) is dependent on APE1 acetylation (14).Although APE1 heterozygous mice are viable and appear to be normal, APE1 nullizygous mice show early embryonic lethality (37,39,63). We recently showed that APE1 inactivation induced apoptosis in mouse embryo fibroblasts conditionally nullizygous for endogenous APE1, an effect that could be prevented by the ectopic expression of human APE1 (27). Using a complementation assay, we also showed that both the repair activity and acetylation-mediated transcriptional regulatory functions of APE1 are required to prevent the apoptosis of APE1-null mouse embryo fibroblasts. The unexpected essentiality of APE1's regulatory function suggests that APE1 is a coregulator of many critical genes.Resistance to many common anticancer drugs often occurs due to the enhanced expression of ATP-binding cassette (ABC) transporter proteins (6,20). Among the ...

Electronic (e)-cigarettes theoretically may be safer than conventional tobacco. However, our prior studies demonstrated direct adverse effects of e-cigarette vapor (EV) on airway cells, including decreased viability and function. We hypothesize that repetitive, chronic inhalation of EV will diminish airway barrier function, leading to inflammatory protein release into circulation, creating a systemic inflammatory state, ultimately leading to distant organ injury and dysfunction. C57BL/6 and CD-1 mice underwent nose only EV exposure daily for 3-6 mo, followed by cardiorenal physiological testing. Primary human bronchial epithelial cells were grown at an air-liquid interface and exposed to EV for 15 min daily for 3-5 days before functional testing. Daily inhalation of EV increased circulating proinflammatory and profibrotic proteins in both C57BL/6 and CD-1 mice: the greatest increases observed were in angiopoietin-1 (31-fold) and EGF (25-fold). Proinflammatory responses were recapitulated by daily EV exposures in vitro of human airway epithelium, with EV epithelium secreting higher IL-8 in response to infection (227 vs. 37 pg/ml, respectively; P < 0.05). Chronic EV inhalation in vivo reduced renal filtration by 20% ( P = 0.017). Fibrosis, assessed by Masson's trichrome and Picrosirius red staining, was increased in EV kidneys (1.86-fold, C57BL/6; 3.2-fold, CD-1; P < 0.05), heart (2.75-fold, C57BL/6 mice; P < 0.05), and liver (1.77-fold in CD-1; P < 0.0001). Gene expression changes demonstrated profibrotic pathway activation. EV inhalation altered cardiovascular function, with decreased heart rate ( P < 0.01), and elevated blood pressure ( P = 0.016). These data demonstrate that chronic inhalation of EV may lead to increased inflammation, organ damage, and cardiorenal and hepatic disease.