The uptake, transport, and presentation of Ags by lung dendritic cells (DCs) are central to the initiation of CD8 T cell responses against respiratory viruses. Although several studies have demonstrated a critical role of CD11blow/negCD103+ DCs for the initiation of cytotoxic T cell responses against the influenza virus, the underlying mechanisms for its potent ability to prime CD8 T cells remain poorly understood. Using a novel approach of fluorescent lipophilic dye-labeled influenza virus, we demonstrate that CD11blow/negCD103+ DCs are the dominant lung DC population transporting influenza virus to the posterior mediastinal lymph node as early as 20 h postinfection. By contrast, CD11bhighCD103neg DCs, although more efficient for taking up the virus within the lung, migrate poorly to the lymph node and remain in the lung to produce proinflammatory cytokines instead. CD11blow/negCD103+ DCs efficiently load viral peptide onto MHC class I complexes and therefore uniquely possess the capacity to potently induce proliferation of naive CD8 T cells. In addition, the peptide transporters TAP1 and TAP2 are constitutively expressed at higher levels in CD11blow/negCD103+ DCs, providing, to our knowledge, the first evidence of a distinct regulation of the Ag-processing pathway in these cells. Collectively, these results show that CD11blow/negCD103+ DCs are functionally specialized for the transport of Ag from the lung to the lymph node and also for efficient processing and presentation of viral Ags to CD8 T cells.

Foxp3(+) CD4(+) regulatory T cells (Tregs) represent a highly suppressive T cell subset with well-characterized immunosuppressive effects during immune homeostasis and chronic infections, although the role of these cells in acute viral infections is poorly understood. The present study sought to examine the induction of Foxp3(+) CD4(+) Tregs in a nonlethal murine model of pulmonary viral infection by the use of the prototypical respiratory virus influenza A. We establish that influenza A virus infection results in a robust Foxp3(+) CD4(+) T cell response and that regulatory T cell induction at the site of inflammation precedes the effector T cell response. Induced Foxp3(+) CD4(+) T cells are highly suppressive ex vivo, demonstrating that influenza virus-induced Foxp3(+) CD4(+) T cells are phenotypically regulatory. Influenza A virus-induced regulatory T cells proliferate vigorously in response to influenza virus antigen, are disseminated throughout the site of infection and primary and secondary lymphoid organs, and retain Foxp3 expression in vitro, suggesting that acute viral infection is capable of inducing a foreign-antigen-specific Treg response. The ability of influenza virus-induced regulatory T cells to suppress antigen-specific CD4(+) and CD8(+) T cell proliferation and cytokine production correlates closely to their ability to respond to influenza virus antigens, suggesting that virus-induced Tregs are capable of attenuating effector responses in an antigen-dependent manner. Collectively, these data demonstrate that primary acute viral infection is capable of inducing a robust, antigen-responsive, and suppressive regulatory T cell response.

Background: Polycyclic aromatic hydrocarbons (PAHs) are of environmental and public health concerns and contribute to adverse skin attributes such as premature skin aging and pigmentary disorder. However, little information is available on the potential roles of chronic urban PAH pollutant exposure on the cutaneous microbiota. Given the roles of the skin microbiota have on healthy and undesirable skin phenotypes and the relationships between PAHs and skin properties, we hypothesize that exposure of PAHs may be associated with changes in the cutaneous microbiota. In this study, the skin microbiota of over two hundred Chinese individuals from two cities in China with varying exposure levels of PAHs were characterized by bacterial and fungal amplicon and shotgun metagenomics sequencing. Results: Skin site and city were strong parameters in changing microbial communities and their assembly processes. Reductions of bacterial-fungal microbial network structural integrity and stability were associated with skin conditions (acne and dandruff). Multivariate analysis revealed associations between abundances of Propionibacterium and Malassezia with host properties and pollutant exposure levels. Shannon diversity increase was correlated to exposure levels of PAHs in a dose-dependent manner. Shotgun metagenomics analysis of samples (n = 32) from individuals of the lowest and highest exposure levels of PAHs further highlighted associations between the PAHs quantified and decrease in abundances of skin commensals and increase in oral bacteria. Functional analysis identified associations between levels of PAHs and abundance of microbial genes of metabolic and other pathways with potential importance in host-microbe interactions as well as degradation of aromatic compounds. Conclusions: The results in this study demonstrated the changes in composition and functional capacities of the cutaneous microbiota associated with chronic exposure levels of PAHs. Findings from this study will aid the development of strategies to harness the microbiota in protecting the skin against pollutants.

Quantitative changes in cytochrome P450 (CYP) proteins involved in drug metabolism as a consequence of drug treatment are important parameters in predicting the fates and pharmacological consequences of xenobiotics and drugs. In this study we undertook comparative P450 proteomics using liver from control and 1,4-bis-2-(3,5-dichloropyridyloxybenzene) (TCPOBOP)-dosed mice. The method involved separation of microsomal proteins by SDS-PAGE, trypsin digestion, and postdigest 18 O/ 16 O labeling followed by nano-LC-MS/MS for peptide identification and LC-MS for relative quantification. Seventeen P450 proteins were identified from mouse liver of which 16 yielded data sufficient for relative quantification. All the P450s detected were unambiguously identified except the highly homologous CYP2A4/2A5. With the exception of CYP2A12, -2D10, and -2F2, the levels of all the P450s quantified were affected by treatment with TCPOBOP (3 mg/kg). CYP1A2, -2A4/5, -2B10, -2B20, -2C29, -2C37, -2C38, -3A11, and -39A1 were up-regulated, and CYP2C40, -2E1, -3A41, and -27A1 down-regulated. The response of CYP2B20 to stimulation has not been distinguished previously from that of CYP2B10 because of the poor discrimination between these two proteins (they share 87% sequence identity). Differential response to chemical stimulation by closely related members of the CYP2C subfamily was also observed. Molecular & Cellular Proteomics 6:953-962, 2007.The cytochromes P450 (P450s or CYPs 1 ) are a superfamily of mixed function oxidases, members of which are present in virtually all living organisms. P450s are characterized into families and subfamilies by their sequence similarities. Currently there are over 360 families and more than 3100 sequenced and named enzymes; these numbers are continuing to increase. In human there are 57 putatively functional P450 genes, whereas in mouse there are 102 such genes.2 P450s are thought to have evolved, in part, as a protective adaptive response against the toxic effects of environmental chemicals (1). They are the most important drug-metabolizing enzymes in mammals and in humans are responsible for the phase I metabolism of 70 -80% of all clinically used drugs (2). In addition to their detoxification role, P450s can also be responsible for the conversion of chemical toxins and procarcinogens to their toxic or carcinogenic forms (3). The ability of P450s to activate chemical toxins has been exploited in cancer chemotherapy where several anticancer drugs, including cyclophosphamide, ifosfamide, dacarbazine, and AQ4N are known to be metabolically activated by P450 isoforms to their respective cytotoxic species (4). A method that would enable not only the non-selective identification of the P450 enzymes present in diseased tissue but also the quantification of expression levels relative to that of surrounding normal tissue would provide invaluable information regarding the metabolic fate of the selected drugs and hence the outcome of therapy.Traditional approaches to the analysis of P450 enzymes rely on immunoblot...

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Allergic contact dermatitis is a primarily T-cell-mediated inflammatory skin disease induced by exposure to small molecular-weight haptens, which covalently bind to proteins. The abundance of cutaneous T cells that recognize CD1a antigen-presenting molecules raises the possibility that MHC-independent antigen presentation may be relevant in some hapten-driven immune responses. Here we examine the ability of contact sensitizers to influence CD1-restricted immunity. Exposure of human antigen-presenting cells such as monocyte-derived dendritic cells and THP-1 cells to the prototypical contact sensitizer dinitrochlorobenzene potentiated the response of CD1a- and CD1d-autoreactive T cells, which released a vast array of cytokines in a CD1- and TCR-dependent manner. The potentiating effects of dinitrochlorobenzene depended upon newly synthesized CD1 molecules and the presence of endogenous stimulatory lipids. Further examination of a broad panel of contact sensitizers revealed 1,4-benzoquinone, resorcinol, isoeugenol and cinnamaldehyde to activate the same type of CD1-restricted responses. These findings provide a basis for the antigen-specific activation of skin-associated CD1-restricted T cells by small molecules and may have implications for contact sensitizer-induced inflammatory skin diseases.

Foxp3+ CD4+ regulatory T cells represent a T cell subset with well-characterized immunosuppressive effects during immune homeostasis and chronic infections, and there is emerging evidence to suggest these cells temper pulmonary inflammation in response to acute viral infection. Recent studies have demonstrated treatment with PC61 CD25-depleting antibody potentiates inflammation in a murine model of RSV infection, while paradoxically delaying recruitment of CD8+ T cells to the site of inflammation. The present study therefore sought to examine the role of these cells in a murine model of acute influenza A virus infection through the administration of PC61 CD25-depleting antibody. PC61 antibody is able to partially deplete CD25+Foxp3+ regulatory T cells to a comparable degree as seen within previous work examining RSV, however this does not alter influenza A-virus induced mortality, weight loss, viral clearance and cellularity within the lung. Collectively, these data demonstrate that partial depletion of CD4+CD25+ regulatory T cells with PC61 antibody does not alter the course of influenza A virus infection.

Airway epithelium functions not only as a physical barrier, but also a regulator of lung inflammation. IFN-γ plays a critical role in airway inflammation associated with respiratory viral infection. We investigated differential protein profiling in IFN-γ-stimulated normal human bronchial epithelial cells (HBEC) using a 2-dimensional gel electrophoresis followed by MALDI-TOF-MS/MS. IFN-γ markedly stimulated apolipoprotein L2 (ApoL2) protein expression in normal HBEC. ApoL2 mRNA expression was also elevated in normal human lung fibroblasts and smooth muscle cells stimulated with IFN-γ, in lung tissues from an IFN-γ-predominant influenza A virus-infected mouse lung injury model, and in cancer lung tissues from human patients. Normal HBEC showed strong resistance to IFN-γ-induced cytotoxicity. ApoL2 knockdown by siRNA promoted IFN-γ-induced cytotoxicity as revealed by a significant drop in cell viability using MTT and CyQUANT NF cell proliferation assays, and a marked increase in hypodiploid sub-G1 cell population in cell cycle analysis. Furthermore, depletion of ApoL2 facilitated IFN-γ-induced membrane damage and chromatin condensation as observed in Hoechst and propidium iodide-double staining and in transmission electron microscopy, and DNA fragmentation using a DNA laddering assay, in a caspase-dependent manner. Our results reveal a novel function for ApoL2 in conferring anti-apoptotic ability of human bronchial epithelium to the cytotoxic effects of IFN-γ, in maintaining airway epithelial layer integrity.