Skin TLR7 triggering promotes accumulation of respiratory dendritic cells and natural killer cells.

The TLR7 agonist imiquimod has been used successfully as adjuvant for skin treatment of virus-associated warts and basal cell carcinoma. The effects of skin TLR7 triggering on respiratory leukocyte populations are unknown. In a placebo-controlled experimental animal study we have used multicolour flow cytometry to systematically analyze the modulation of respiratory leukocyte subsets after skin administration of imiquimod. Compared to placebo, skin administration of imiquimod significantly increased respiratory dendritic cells (DC) and natural killer cells, whereas total respiratory leukocyte, alveolar macrophages, classical CD4+ T helper and CD8+ T killer cell numbers were not or only moderately affected. DC subpopulation analyses revealed that elevation of respiratory DC was caused by an increase of respiratory monocytic DC and CD11b(hi) DC subsets. Lymphocyte subpopulation analyses indicated a marked elevation of respiratory natural killer cells and a significant reduction of B lymphocytes. Analysis of cytokine responses of respiratory leukocytes after stimulation with Klebsiella pneumonia indicated reduced IFN-γ and TNF-α expression and increased IL-10 and IL-12p70 production after 7 day low dose skin TLR7 triggering. Additionally, respiratory NK cytotoxic activity was increased after 7d skin TLR7 triggering. In contrast, lung histology and bronchoalveolar cell counts were not affected suggesting that skin TLR7 stimulation modulated respiratory leukocyte composition without inducing overt pulmonary inflammation. These data suggest the possibility to modulate respiratory leukocyte composition and respiratory cytokine responses against pathogens like Klebsiella pneumonia through skin administration of a clinically approved TLR7 ligand. Skin administration of synthetic TLR7 ligands may represent a novel, noninvasive means to modulate respiratory immunity

Hypoxia drives glucose transporter 3 expression through HIF-mediated induction of the long non-coding RNA NICI

Hypoxia inducible transcription factors (HIFs) directly dictate the expression of multiple RNA species including novel and as yet uncharacterized long non-coding transcripts with unknown function. We used pan-genomic HIF-binding and transcriptomic data to identify a novel long non-coding RNA NICI (Non-coding Intergenic Co-Induced transcript) on chromosome 12p13.31 which is regulated by hypoxia via HIF-1 promoter-binding in multiple cell types. CRISPR/Cas9-mediated deletion of the hypoxia-response element revealed co-regulation of NICI and the neighboring protein-coding gene, solute carrier family 2 member 3 (SLC2A3) which encodes the high-affinity glucose transporter 3 (GLUT3). Knock-down or knock-out of NICI attenuated hypoxic induction of SLC2A3 indicating a direct regulatory role of NICI in SLC2A3 expression, which was further evidenced by CRISPR/Cas9-VPR mediated activation of NICI expression. We also demonstrate that regulation of SLC2A3 is mediated through transcriptional activation rather than post-transcriptional mechanisms since knock-out of NICI leads to reduced recruitment of RNA polymerase 2 to the SLC2A3 promoter. Consistent with this we observe NICI-dependent regulation of glucose consumption and cell proliferation. Furthermore, NICI expression is regulated by the VHL tumour suppressor and is highly expressed in clear cell renal cancer, where SLC2A3 expression is associated with patient prognosis, implying an important role for the HIF/NICI/SLC2A3 axis in this malignancy

Southern Ocean phytoplankton physiology in a changing climate

The Southern Ocean (SO) is a major sink for anthropogenic atmospheric carbon dioxide(CO2), potentially harbouring even greater potential for additional sequestration of CO2 through enhanced phytoplankton productivity. In the SO, primary productivity is primarily driven by bottom up processes (physical and chemical conditions) which are spatially and temporally heterogeneous. Due to a paucity of trace metals (such as iron) and high variability in light, much of the SO is characterised by an ecological paradox of high macronutrient concentrations yet uncharacteristically low chlorophyll concentrations. It is expected that with increased anthropogenic CO2 emissions and the coincident warming, the major physical and chemical process that govern the SO will alter, influencing the biological capacity and functioning of the ecosystem. This review focuses on the SO primary producers and the bottom up processes that underpin their health and productivity. It looks at the major physicochemical drivers of change in the SO, and based on current physiological knowledge, explores how these changes will likely manifest in phytoplankton, specifically, what are the physiological changes and floristic shifts that are likely to ensue and how this may translate into changes in the carbon sink capacity, net primary productivity and functionality of the SO

Modulation of respiratory dendritic cells during Klebsiella pneumonia infection

Klebsiella pneumoniae is a leading cause of severe hospital-acquired respiratory tract infections and death but little is known regarding the modulation of respiratory dendritic cell (DC) subsets. Plasmacytoid DC (pDC) are specialized type 1 interferon producing cells and considered to be classical mediators of antiviral immunity. By using multiparameter flow cytometry analysis we have analysed the modulation of respiratory DC subsets after intratracheal Klebsiella pneumonia infection. Data indicate that pDCs and MoDC were markedly elevated in the post acute pneumonia phase when compared to mock-infected controls. Analysis of draining mediastinal lymph nodes revealed a rapid increase of activated CD103+ DC, CD11b+ DC and MoDC within 48 h post infection. Lung pDC identification during bacterial pneumonia was confirmed by extended phenotyping for 120G8, mPDCA-1 and Siglec-H expression and by demonstration of high Interferon-alpha producing capacity after cell sorting. Cytokine expression analysis of ex vivo-sorted respiratory DC subpopulations from infected animals revealed elevated Interferon-alpha in pDC, elevated IFN-gamma, IL-4 and IL-13 in CD103+ DC and IL-19 and IL-12p35 in CD11b+ DC subsets in comparison to CD11c+ MHC-class IIlow cells indicating distinct functional roles. Antigen-specific naive CD4+ T cell stimulatory capacity of purified respiratory DC subsets was analysed in a model system with purified ovalbumin T cell receptor transgenic naive CD4+ responder T cells and respiratory DC subsets, pulsed with ovalbumin and matured with Klebsiella pneumoniae lysate. CD103+ DC and CD11b+ DC subsets represented the most potent naive CD4+ T helper cell activators. These results provide novel insight into the activation of respiratory DC subsets during Klebsiella pneumonia infection. The detection of increased respiratory pDC numbers in bacterial pneumonia may indicate possible novel pDC functions with respect to lung repair and regeneration

Respiratory T and B lymphocyte numbers are suppressed after skin TLR7 stimulaton.

<p>Relative (A) and absolute (B) respiratory T and B lymphocyte subsets were quantitated by flow cytometry (see gating strategy <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043320#pone.0043320.s003" target="_blank">Fig. S3</a>) after skin imiquimod treatement. Mean ± SEM; n ≥4; *p<0.05; **p<0.01; ***p<0.001 versus placebo.</p

Topical TLR7 triggering affects skin dendritic cell CD86 and CD274 expression.

<p>Absolute skin DC subset numbers from 2cm² skin (A) and subset specific expression of CD86 (B) and CD274 (C) were determined by flow cytometry after 1d and 7d imiquimod exposure as described in the Materials and Methods. Among the CD11c⁺ MHC-class II⁺ CD45⁺ skin DC, Langerhans cells (LC) were identified based on CD207⁺, CD103^-, CD11b^int expression profile. Different dermal DC (DDC) subsets were discriminated based on heterogenous expression of CD207, CD103, CD11b. Mean ± SEM; n ≥2-3. *p<0.05; **p<0.01; ***p<0.001 versus placebo.</p

Skin TLR7 stimulation does not induce lung histopathology or alveolar leukocytosis.

<p>Lung sections (A) and BAL leukocyte numbers (B) of mice exposed to imiquimod or placebo. (A) shows histopathology of d7 animals. Histopathology of d1 animals exhibited similar results (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043320#pone.0043320.s005" target="_blank">Fig. S5</a>). BAL leukocyte numbers from d1 and d7 treated animals after imiquimod exposure (B). Mean ± SEM; n ≥3.</p

Skin application of imiquimod increases relative and absolute respiratory DC numbers.

<p>Skin of mice was treated with TLR7 ligand imiquimod or placebo in a daily fashion and relative and absolute respiratory leukocyte subsets were quantitated by flow cytometry at the indicated time points (see gating stategy in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043320#pone.0043320.s001" target="_blank">Fig. S1</a>). Total lung leukocytes (A), respiratory macrophages (B), respiratory granulocytes (C) and respiratory DC numbers after imiquimod exposure (D). Mean ± SEM; n ≥4; *p<0.05; **p<0.01; ***p<0.001 versus placebo.</p

Skin TLR7 triggering modulates respiratory cytokine production.

<p>Respiratory leukocytes were CD45⁺ microbead purified from 1d and 7d imiquimod or placebo-treated animals and IFN-γ (A), TNF-α (B), IL-10 (C), IL-2 (D), IL-12p70 (E) and IL-23 were quantitated after LPS, CpG or Klebsiella pneumonia lysate stimulation. IL-23 was below the detection limit (27 pg/ml) in all groups. In unstimulated cells, cytokines were not detectable (detection limit 27 pg/ml). Mean ± SEM; n ≥3; *p<0.05; **p<0.01; ***p<0.001 versus placebo.</p