Novel transcript profiling of diffuse alveolar damage induced by hyperoxia exposure in mice: Normalization by glyceraldehyde 3-phosphate dehydrogenase

Under mechanical ventilation with high-inspired oxygen concentration, diffuse alveolar damage (DAD) was found to take place in some patients. To clarify the molecular pathophysiology of this condition we investigated the time course of gene expression changes induced by hyperoxia exposure in mouse lung using real-time quantitative polymerase chain reaction (real-time qPCR). Our results normalized by glyceraldehyde 3-phosphate dehydrogenase showed that mRNA levels of cysteine rich protein 61 (CYR61) and connective tissue growth factor (CTGF) were significantly up-regulated, while those of surfactant-associated protein C (SFTPC), cytochrome P450, 2F2 (CYP2F2), Claudin 1, (CLDN1), membrane-associated zonula occludens protein-1 (ZO-1), lysozyme (LYZS), and P lysozyme structural (LZP-S) were significantly down-regulated. Increasing level of mRNAs, each encoding CYR61 and CTGF, suggests a serious risk of fibrosing alveolitis. Decrease in levels of mRNAs for SFTPC, CYP2F2, CLDN1, ZO-1, LYZS, and LZP-S suggests alveolar dysfunction and disruption of the immune system. Moreover we confirmed apoptotic conditions, such as significant up-regulations of mRNA levels in Myc and Galectin-3. Hyperoxic condition probably yielded reactive oxygen species (ROS), which resulted in a malignant cycle of ROS production by Myc overexpression

A Palindromic CpG-Containing Phosphodiester Oligodeoxynucleotide as a Mucosal Adjuvant Stimulates Plasmacytoid Dendritic Cell-Mediated T_H1 Immunity

<div><p>Background</p><p>CpG oligodeoxynucleotides (ODNs), resembling bacterial DNA, are currently tested in clinical trials as vaccine adjuvants. They have the nuclease-resistant phosphorothioate bond; the immune responses elicited differ according to the CpG ODN sequence and vaccination method. To develop a CpG ODN that can induce plasmacytoid dendritic cell (pDC)-mediated T_H1 immunity through the mucosa, we constructed phosphodiester G9.1 comprising one palindromic CpG motif with unique polyguanosine-runs that allows degradation similar to naturally occurring bacterial DNA.</p><p>Methods</p><p>T_H1 and T_H2 immunity activation was evaluated by cytokine production pattern and <i>T-bet/GATA-3</i> ratio in human peripheral blood mononuclear cells and mouse bone marrow cells. Adjuvanticity was evaluated in mice administered G9.1 with diphtheria toxoid (DT) through nasal vaccination.</p><p>Results</p><p>G9.1 exhibited stronger IFN-α-inducing activity than A-class CpG ODN2216 and increased <i>T-bet/GATA-3</i> ratio by enhancing <i>T-bet</i> expression. Nasally administered G9.1 plus DT induced DT-specific mucosal IgA and serum IgG, but not IgE, responses with antitoxin activity in C57BL/6 and BALB/c mice, possibly due to IFN/BAFF production. Induction of T_H1, but not T_H2, -type Abs depended completely on pDCs, the first <i>in vivo</i> demonstration by CpG ODNs.</p><p>Conclusions</p><p>G9.1 is a promising mucosal adjuvant for induction of pDC-mediated T_H1 immunity.</p></div

G9.1-induced T_H1-type Ab production was completely pDC-dependent.

<p>Anti-mPDCA-1-treated and isotype control-treated BALB/c mice (n = 4) were administered DT (2 Lf) plus G9.1 (20 µg) nasally twice at a 1-month interval. Serum titers of anti-DT IgG1 and IgG2a were measured as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088846#pone-0088846-g003" target="_blank">Fig. 3</a>. The percentage of pDCs in splenocytes decreased from 0.18 to 0.01 after anti-mPDCA-1 treatment (lower panel). The pDC-depleted mice exhibited no G9.1-induced IgG2a production (upper panel). *<i>p</i><0.05 and **<i>p</i><0.01 as determined by <i>t</i>-test. Three independent experiments yielded nearly identical results.</p

G9.1 stimulated T_H1-related cytokine production and transcription factor expression in human PBMCs.

<p><b><i>A</i></b>, PBMCs (1.5×10⁶/mL) were cultured for 16 h with various concentrations of CpG ODNs or their negative controls. The IFN-α concentration released in the culture supernatant was measured by ELISA. (five donors; each symbol reflects an individual donor; *<i>p</i><0.05 between G9.1 and ODN2216). <b><i>B</i></b>, PBMCs (1.5×10⁶/mL) were cultured for 2, 4, 6, 12, or 18 h with 1 µM G9.1 or ODN2216 in the presence/absence of anti-human IFN-α/β receptor-chain 2 Ab (4 µg/mL; PBL Interferon Source, NJ, USA) (upper panel, six donors); or treated with IFN-α/β receptor-chain 2 Ab (30 min), exposed to 1 µM G9.1 or ODN2216 (30 min), and re-cultured for 12 h after removing/not removing the CpG ODNs by extensive washing (lower panel, four donors). The addition of 4 µg/mL mouse IgG2a as isotype control did not alter G9.1-mediated IFN-α production. *<i>p</i><0.05 and ** <i>p</i><0.01 by a paired <i>t</i>-test. <b><i>C and D</i></b>, PBMCs (1.5×10⁶/mL) were cultured for 12–16 h (C) or overnight (D) in medium alone, 0.4 µM negG9.1 or 0.4 µM G9.1, cytokine concentrations in the culture supernatant measured (C), and RT-PCR performed for the cell pellet to estimate <i>T-bet</i> and <i>GATA-3</i> expression levels (D). **<i>p</i><0.01 by a paired <i>t</i>-test (six donors). <b><i>E</i></b>, PBMCs, CD304⁺ cell-depleted PBMCs, and pDCs untreated or pretreated with 1 µg/mL chloroquine or 2 µg/mL anti-CD303 Ab for 30 min were cultured for 12–16 h with 0.4 µM G9.1 and IFN-α concentrations in the supernatant compared with native G9.1-treated cultures. Data shown are three donors for PBMC, six donors for choloroquine-treated assay and seven donors for anti-CD303. **<i>p</i><0.01 by a paired <i>t</i>-test. In all the experiments, the IFN-α concentrations in the media from PBMCs or pDCs treated with negG9.1 or left untreated were negligible.</p

Nasally administered G9.1 promoted mucosal IgA response.

<p><b><i>A</i></b>, BALB/c and C57BL/6 mice were immunized intranasally with 2 Lf DT with or without 20 µg of G9.1 on days 0, 14, 21, and 28. Lavages from the lungs and nasal cavity and the supernatants of the feces mixture were prepared on day 35 and anti-DT IgA titers measured. **<i>p</i><0.01 by <i>t</i>-test (n = 5). Three independent experiments yielded nearly identical results. <b><i>B</i></b>, BM cells (5×10⁶/mL) prepared from C57BL/6 mice were cultured for 24 h in medium alone or with varying concentrations of G9.1 or negG9.1. The culture supernatants were evaluated for BAFF and the activated form of TGF-β1 by Quantikine immunoassay (ELISA Kit). Data are from one of two independent experiments yielding nearly identical results. Shown as the means from duplicate assays.</p