Automated Filtering of Intrinsic Movement Artifacts during Two-Photon Intravital Microscopy

<div><p><i>In vivo</i> imaging using two-photon microscopy is an essential tool to explore the dynamic of physiological events deep within biological tissues for short or extended periods of time. The new capabilities offered by this technology (e.g. high tissue penetrance, low toxicity) have opened a whole new era of investigations in modern biomedical research. However, the potential of using this promising technique in tissues of living animals is greatly limited by the intrinsic irregular movements that are caused by cardiac and respiratory cycles and muscular and vascular tone. Here, we show real-time imaging of the brain, spinal cord, sciatic nerve and myenteric plexus of living mice using a new automated program, named Intravital_Microscopy_Toolbox, that removes frames corrupted with motion artifacts from time-lapse videos. Our approach involves generating a dissimilarity score against precalculated reference frames in a specific reference channel, thus allowing the gating of distorted, out-of-focus or translated frames. Since the algorithm detects the uneven peaks of image distortion caused by irregular animal movements, the macro allows a fast and efficient filtering of the image sequence. In addition, extra features have been implemented in the macro, such as XY registration, channel subtraction, extended field of view with maximum intensity projection, noise reduction with average intensity projections, and automated timestamp and scale bar overlay. Thus, the Intravital_Microscopy_Toolbox macro for ImageJ provides convenient tools for biologists who are performing <i>in vivo</i> two-photon imaging in tissues prone to motion artifacts.</p></div

Calculation of dissimilarity scores using the automatic reference frame generation (RFG) option, and subsequent removal of artifactual frames from the video sequence.

<p>(<b>a</b>) Graph plotting of the total number of frames in a 1000-frame video against the dissimilarity score, identified as ‘square difference’ score on the y-axis. (<b>b</b>) Relative frequency distribution of the number of frames per dissimilarity score. (<b>c</b>) Graph showing the frame-by-frame analysis of a 1000-frame video sequence. Artifactual frames were eliminated (identified by red crosses) using a cutoff percentile of 60% (defined by the horizontal blue line). Major artifacts (shown as black dots) are typically artifacts resulting in a full field distortion of the image, while minor artifacts (shown as green dots) are small glitches in frames that do not impair the image interpretation. (<b>d</b>) Images corresponding to local maxima (upper panels) and minima (lower panels) in the first 200 frames of the video sequence. Note that frames corresponding to a local maximum are associated with heavy artifacts, while frames corresponding to a local minimum are typically very similar to the reference frame. All images are derived from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053942#pone.0053942.s009" target="_blank">Video S3</a>.</p

The Inflammasome Pyrin Contributes to Pertussis Toxin-Induced IL-1β Synthesis, Neutrophil Intravascular Crawling and Autoimmune Encephalomyelitis

<div><p>Microbial agents can aggravate inflammatory diseases, such as multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE). An example is pertussis toxin (PTX), a bacterial virulence factor commonly used as an adjuvant to promote EAE, but whose mechanism of action is unclear. We have reported that PTX triggers an IL-6-mediated signaling cascade that increases the number of leukocytes that patrol the vasculature by crawling on its luminal surface. In the present study, we examined this response in mice lacking either TLR4 or inflammasome components and using enzymatically active and inactive forms of PTX. Our results indicate that PTX, through its ADP-ribosyltransferase activity, induces two series of events upstream of IL-6: 1) the activation of TLR4 signaling in myeloid cells, leading to pro-IL-1β synthesis; and 2) the formation of a pyrin-dependent inflammasome that cleaves pro-IL-1β into its active form. In turn, IL-1β stimulates nearby stromal cells to secrete IL-6, which is known to induce vascular changes required for leukocyte adhesion. Without pyrin, PTX does not induce neutrophil adhesion to cerebral capillaries and is less effective at inducing EAE in transgenic mice with encephalitogenic T lymphocytes. This study identifies the first microbial molecule that activates pyrin, a mechanism by which infections may influence MS and a potential therapeutic target for immune disorders.</p></div

PTX increases pyrin expression in myeloid cells via a TLR4-dependent pathway.

<p><b><i>a</i></b>, Quantification of pyrin mRNA by qRT-PCR in peritoneal leukocytes from TLR4-knockout and wild-type mice harvested 6 h after intraperitoneal injection of PTX (20 µg/kg) or PBS. *Significantly different from all the other groups according to Wilcoxon tests (<i>P</i>≤0.014). Sample size: 8 (PTX groups) or 4 (PBS groups). <b><i>b</i></b>, Bivariate analysis showing a positive correlation between the amounts of pyrin and IL-1β mRNAs (Spearman's test, <i>P</i><0.0001, R = 0.98). <b><i>c</i></b>, Quantification of pyrin mRNA by qRT-PCR in peritoneal leukocytes harvested from mice 6 h after injection of PTX or PBS, and sorted using the CD11b, F4/80 and Ly6G markers. *Significantly different from the corresponding PBS group according to the Wilcoxon test (<i>P</i>≤0.0081). Sample size: 6–7 per group. <b><i>d</i></b>, Pyrin detection by Western blotting in peritoneal cells from mice killed 6 h after injection of PTX or PBS. β-actin was used as loading control.</p

Both IL-6 and IL-1β are produced in tissues exposed to PTX, but only IL-6 reaches increased levels in the circulation.

<p><b><i>a</i></b>, Quantification of the mRNAs encoding IL-6 and IL-1β by qRT-PCR in different tissues from mice killed 3 or 6 h after intraperitoneal injection of PTX (20 µg/kg) or PBS. *Significantly different from the corresponding PBS group according to the Wilcoxon test (<i>P</i><0.05). Sample size: 4–10 (PBS groups) or 7–10 (PTX groups). <b><i>b</i></b>, Quantification of IL-6 and IL-1β by ELISA in peritoneal fluid and plasma samples. *Significantly different from the corresponding PBS group according to the Wilcoxon test (<i>P</i>≤0.0003). Sample size: 3–9 (PBS groups) or 4–10 (PTX groups).</p

Pyrin is not required for the development of EAE induced by adoptive transfer of encephalitogenic T cells.

<p>Monitoring of EAE in C57BL/6 mice expressing (black circles) or lacking pyrin (white squares) after adoptive transfer of lymphocytes isolated from MOG-immunized wild-type mice and reactivated <i>in vitro</i>. All mice were included in the analyses, except for the clinical scoring (bottom graph), which included only mice that had developed clinical signs of EAE at the end of the study (i.e., after 21 days). No significant intergenotype difference was detected according to Wilcoxon tests (<i>P</i>≥0.2). Additional statistics are provided in the table. Sample size: 8 (Pyrin^+/+) or 7 (Pyrin^−/−).</p

PTX induces the IL-1β−IL-6 cascade through the TLR4 pathway and pyrin inflammasome.

<p><b><i>a</i></b>, Quantification of IL-6 by ELISA in plasma from mice of different genotypes killed 6 h after intraperitoneal injection of PTX (20 µg/kg) or PBS. *Significantly different from the corresponding PBS group according to Wilcoxon tests (<i>P</i>≤0.0066). Sample size: 4–17 (PBS groups) or 7–15 (PTX groups). <b><i>b</i></b>, Quantification of IL-1β by ELISA in peritoneal fluid from mice treated or not with PTX. *Significantly different from the corresponding PBS group according to Wilcoxon tests (<i>P</i>≤0.017). <b><i>c</i></b>, Quantification of IL-1β mRNA by qRT-PCR in peritoneal leukocytes from mice treated or not with PTX. *Significantly different from the corresponding PBS group according to Wilcoxon tests (<i>P</i>≤0.022).</p

PTX requires pyrin to induce neutrophil intravascular crawling and to maximally promote EAE.

<p><b><i>a</i></b>, Micrographs showing rod-shaped leukocytes stained for Ly6G or CD3 by immunohistochemistry in brain sections from a PTX-treated mouse (C57BL/6 background). Scale bar: 5 µm. <b><i>b</i></b>, Stereological counts of rod-shaped Ly6G⁺ and CD3⁺ leukocytes in the cerebral cortex of C57BL/6 mice deficient (white bars) or not (black bars) in pyrin and killed 24 h after treatment with PTX (2 injections of 20 µg/kg given 2 days apart) or PBS. *Significantly different from the corresponding pyrin-deficient group according to the Wilcoxon test (<i>P</i> = 0.0050). Sample size: 6–7 (PBS groups) or 7–10 (PTX groups). <b><i>c</i></b>, Monitoring of EAE in PTX-treated 2D2 mice expressing (black circles) or lacking pyrin (white squares). All mice were included in the analyses, except for the clinical scoring (right graph), which included only mice that had developed clinical signs of EAE at the end of the study (i.e., after 21 days). *Time at which a significant intergenotype difference was found according to Wilcoxon tests (<i>P</i>≤0.044). The Kaplan-Meier curves (EAE incidence) were significantly different according to the Wilcoxon test (<i>P</i> = 0.013). Additional statistics are provided in the table. Sample size: 12 (2D2 Pyrin^+/+) or 19 (2D2 Pyrin^−/−). <b><i>d</i></b>, Micrographs of spinal cord sections showing comparable infiltration of CD3⁺ T cells in 2D2 mice expressing or not pyrin with an EAE score of 3, but few cells in a mouse that did not develop EAE. These mice were killed 21 days after the first PTX injection. Scale bar: 100 µm.</p

Radiosensitive peritoneal macrophages and neutrophils respond to PTX by producing IL-1β, which stimulates IL-6 production by radioresistant stromal cells.

<p><b><i>a</i></b>, Quantification of IL-1β and IL-6 by ELISA in peritoneal fluid or plasma collected from bone marrow chimeric mice 6 h after intraperitoneal injection of PTX (20 µg/kg) or PBS. *Significantly different from the controls (i.e., wild-type mice lethally irradiated and transplanted with bone marrow cells obtained from wild-type mice; WT→WT) according to <i>post hoc</i> Wilcoxon tests (Kruskal-Wallis test, <i>P</i>≤0.004). Sample size: 4–10 per group. <b><i>b</i></b>, Quantification of IL-1β by ELISA in peritoneal fluid from CD11b-TK^mt-30 mice treated twice daily for 6 days with GCV or saline, and killed 6 h after injection of PTX or PBS. *Significantly different from saline-treated mice injected with PTX according to the Wilcoxon test (<i>P</i> = 0.0017). Sample size: 4–7 per group. <b><i>c</i></b>, Quantification of IL-1β mRNA by qRT-PCR in peritoneal leukocytes harvested from mice 6 h after injection of PTX or PBS, and sorted using the CD11b, F4/80 and Ly6G markers. *Significantly different from the corresponding PBS group according to the Wilcoxon test (<i>P</i>≤0.05). Sample size: 6–7 per group. <b><i>d</i></b>, Comparison of DsRed fluorescence in macrophages (CD11b⁺F4/80⁺), neutrophils (CD11b⁺Ly6G⁺) and non-myeloid leukocytes (CD11b⁻) harvested from the peritoneum of pIL1-DsRed transgenic mice treated or not with PTX. Cells were analyzed by flow cytometry and gated on CD45. *Significantly different from the corresponding PBS group according to the Wilcoxon test (<i>P</i>≤0.008). Sample size: 5 (PBS groups) or 6 (PTX groups). <b><i>e</i></b>, Micrographs showing <i>in situ</i> hybridization signals for IL-6 mRNA (clusters of black grains, arrows) in the abdominal wall of a mouse exposed for 3 h to PTX. The sections were counterstained for the mesenchymal/fibroblast marker S100A4 by immunohistochemistry (brown). Abbreviation: M, muscle bundle. Scale bars: 50 µm (main images) or 10 µm (insert). <b><i>f</i></b>, Quantification of IL-6 expression by qRT-PCR or ELISA in primary cultures of S100A4⁺ abdominal stromal cells exposed for 3 h to PTX (100 ng/ml), IL-1β (10 ng/ml) or PBS. *Significantly different from the PBS group according to <i>post hoc</i> Wilcoxon tests (Kruskal-Wallis test, <i>P</i> = 0.0009). Sample size: 5–7 per group.</p

The ability of PTX to induce the IL-1β−IL-6 cascade depends on the integrity of its multimeric structure and enzymatic activity.

<p><b><i>a</i></b>, Quantification of IL-1β mRNA in peritoneal leukocytes by qRT-PCR, and of IL-1β and IL-6 protein in peritoneal fluid by ELISA. The samples were from mice killed 6 h after intraperitoneal injection PBS or equimolar doses of PTX (20 µg/kg), PTX-A (5.3 µg/kg) or PTX-B (14.5 µg/kg). *Significantly different from all the other groups according to <i>post hoc</i> Wilcoxon tests (Kruskal-Wallis test, <i>P</i>≤0.001). Sample size: 16 (PBS group) or 8–11 (toxin groups). <b><i>b</i></b>, Same analyzes as in <i>a</i>, except that mice received PTX^mut (20 µg/kg). *Significantly different from all the other groups according to <i>post hoc</i> Wilcoxon tests (Kruskal-Wallis test, <i>P</i>≤0.0012). Sample size: 10–12 per group. <b><i>c</i></b>, Western blots revealing the presence of mature IL-1β, cleaved Casp1, TLR4 and Myd88 in peritoneal fluid or peritoneal cell lysates from mice killed 6 h after injection of PBS, wild-type PTX or PTX^mut. Arrowheads indicate the cleaved forms. β-actin was used as loading control. <b><i>d</i></b>, Quantification of the Western blots by optical densitometry. *Significantly different from all the other groups according to <i>post hoc</i> Wilcoxon tests (Kruskal-Wallis test, <i>P</i>≤0.02). Sample size: 8 per group.</p