Physiological and motility parameters measured during intravital imaging of influenza infected mouse tracheas by multiphoton microscopy.

<p>Mice that received adoptively transferred GFP+ OT-I spleen cells and subsequently infected with X31-OVA-I virus as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005881#ppat.1005881.g003" target="_blank">Fig 3</a> were prepared and imaged on sequential days after infection by intravital multiphoton microscopy (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005881#sec002" target="_blank">Methods</a>). <b>(A)</b> Rectal body temperatures of infected mice measured after sedation just prior to imaging. Each point is the average of 3–5 mice ± standard deviation. <b>(B)</b> Pulse-oximetry measurements of heart rate (HR) and percent blood oxygenation with and without respiratory support. Green and blue symbols represent blood O₂ and HR, respectively, in a mouse on respiratory support. Black and red symbols represent O₂ and HR, respectively from a mouse not receiving respiratory support. <b>(C-F)</b> Cell motility parameters calculated using Volocity software (Perkin-Elmer). Each symbol represents a single cell, and the plots contain 3–5 replicate experiments on individual mice. Horizontal gray bars denote the mean. C = velocity; D = Meandering index (also called confinement ratio); E = Displacement (distance form origin) for an entire cell track; F = average turning angle for each imaging step in a track. <b>(G-H)</b> Spider plots showing individual cell tracks plotted from a common origin using data from days 7 and 9 after infection.</p

Intravital imaging site of the mouse trachea.

<p>B6 mice were infected as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005881#ppat.1005881.g001" target="_blank">Fig 1</a> or T cell receptor transgenic OT-I x GFP spleen cells (10⁵) were adoptively transferred into recipient B6 mice one day prior to infection with 3x10³ EID₅₀ recombinant influenza A/HK/X31-OVA expressing the ovalbumin peptide siinfekl recognized by the OT-I T cells. <b>(A)</b> Exposed trachea just prior to imaging reveals its translucent appearance. <b>(B)</b> Whole mount imaging of excised trachea viewed from the inside. Tissue was stained with antibodies to CD8 (red) and Col IV (green). <b>(C-E)</b> Intact trachea from mice that received adoptive transfer of OT-I GFP+ cells, intravenous dextran (red) to label the blood vessels followed by imaging by multiphoton microscopy. Images C and D show 0 and 90 degree points of view through the trachea, with the SHG signal towards the outer wall of the trachea appearing in the distal layer of C and bottom of D.</p

Cellular composition of influenza infected trachea, lung, and BAL.

<p>Mice were infected with influenza (as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005881#ppat.1005881.g001" target="_blank">Fig 1</a>) and tissues harvested on different days after infection. Lung tissue was taken after broncho-alveolar lavage and perfusion of the blood vessels. Experiments consisted of pooled groups of 3 mice per time point, and 3–4 experiments per time point. Lung and trachea were enzyme digested and gently homogenized to make a single cell suspension and counted. Cells were stained with a panel of antibodies against T- and B-cell markers and MHC-Class I tetramers corresponding to immunodominant CD8+ T cell epitopes and analyzed by flow cytometry (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005881#sec002" target="_blank">Methods</a>). Trachea = A-E, BAL = F-J, Lung = K-O. Each point represents the average of 3–4 experiments ± standard error of the mean.</p

Influenza infection of the mouse trachea.

<p>C57BL/6 mice at 8 weeks of age were infected with 10⁵ EID₅₀ of H3N2 influenza A/HK/X31 by an intranasal route. On days 2 and 8 the trachea was excised, snap frozen, and later sectioned and stained with antibodies to influenza nucleoprotein (NP, blue), collagen IV (Col IV, green), and CD8 (red); A = day 2, B = day 8. Separate cohorts of mice (n = 3–4) were similarly infected and virus titers in the trachea and lung were determined after homogenization of the tissues followed by dilution and inoculation of embryonated hen eggs (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005881#sec002" target="_blank">Methods</a>). Titers of influenza virus in lung and trachea expressed as EID₅₀ per ml of tissue homogenate (C) or per gram of tissue weighed prior to homogenization in 1 ml serum-free media (D). Closed circles = lung, open circles = trachea.</p

Virus clearance in mice infected with X31-OVA and the effect of OVA-peptide-MHC blocking antibodies on cell velocities.

<p>As described in Methods, OT-1-GFP CD8+ T cell receptor transgenic T cells were adoptively transferred into mice prior to infection with X31-OVA virus expressing the siinfekl OVA peptide. <b>(A)</b> Lungs and trachea were excised and titered by indirect immunofluorescence assay <b>(B-D)</b> The day 7 velocities of OT-I-GFP T cells are plotted over time during a 25-minute long imaging session. Control mice <b>(B)</b> were untreated. Experimental mice were treated with monoclonal antibody 25-D1.16 against Kb-siinfekl peptide-MHC complex, and imaged 2h later at 37°C <b>(C)</b> or at 35°C <b>(D)</b> with the warming objective turned off. The red lines indicate the average velocities at each increment over time, while the black horizontal line in <b>(C)</b> and <b>(D)</b> indicates the overall average of the day 7 control mice.</p

Meandering index versus velocity plots on different days after infection.

<p>The values for individual cells of meandering index and velocity are graphed to depict changing cell population behavior over the course of infection. Each symbol is data from a single cell. Histograms along the top and right axes indicate the cell densities. Crosshairs defining the quadrants are based on the densities in (D). A = day 6; B = day 7, C = day 8; D = day 9; E = day 10, and F = day 14.</p

Frequency distributions of the arrest coefficients for each day after infection.

<p>For each day after infection measured, the arrest coefficients (defined as the fraction of time that a cell has a speed less than 2μm per minute) were calculated for individual cell tracks and the values of the population were binned into 0.1 increments. A value of 1 indicates full arrest. Y-axis indicates the number of coefficient values in each bin. A = day 6; B = day 7, C = day 8; D = day 9; E = day 10, and F = day 14.</p

Additional file 3: Table S3. of Stress granules and RNA processing bodies are novel autoantibody targets in systemic sclerosis

Systemic sclerosis (SSc)-specific enrichment of processes and components. Proteins differentially detected in SSc were analyzed using gProfiler. Statistically significant processes and components are shown. A) Peptides detected at any level across all four groups. B) Peptides identified in all SSc groups, but absent in controls. C) Analysis of 137 proteins differentially detected in SSc. BP biological process, CC cellular component, MF molecular function, ke KEGG pathway, re REACTOME pathway. (ODS 70 kb

Akt signaling contributes to autocrine TNFα production in multiple cell types.

<p>FADD deficient Jurkat cells were treated with TNFα followed by measurement of (A) human TNFα mRNA levels by qRT-PCR and normalized using human 18S RNA or (B) western blot at 9 hr. RAW 264.7 or J774A.1 cells were treated with zVAD.fmk (100 uM or 50 uM respectively) followed by (C,E) measurement of TNFα mRNA levels by qRT-PCR or (D,F) western blot at 9 hr. (G) Akt null mouse lung fibroblasts expressing Myr-Akt or K179M were treated with zVAD.fmk and TNFα followed by measurement of TNFα mRNA levels by qRT-PCR at 9 hr. (H) Mouse lung fibroblasts expressing only endogenous Akt1 or Akt2 were treated with zVAD.fmk and TNFα followed by measurement of TNFα mRNA levels by qRT-PCR at 9 hr.</p

mTORC1 contributes to the regulation of necroptosis.

<p>(A) L929 cells were treated with zVAD.fmk or TNFα for 9 hr and harvested for western blot. (B) Cell under serum free condition were treated with bFGF or bFGF/zVAD.fmk for the indicated amounts of time, followed by western blotting using the indicated antibodies. (C) Necroptosis was induced by zVAD.fmk or TNFα in L929 cell in the presence of inhibitors of Akt(Akt inh. VIII) and mTOR (rapamycin, Torin-1 and PI-103). (D) L929 cells with mTOR siRNA knockdown were harvested for western blot or treated with zVAD.fmk or TNFα for 24 hrs. Cell viability was determined 24 hr after activation of necroptosis. In all graphs, average±SD was plotted.</p