2 nya forskningsrapporter från Lund

Svenska forskare briljerar ! Bloggen presenterar här 2 nya forskningsrapporter utförda vid Lunds Universitet företrädesvis.Först har vi Marcus Järås research group som levererar en studie om,som jag förstår det, blodcancer. Leukemi som är en riktigt riktig eländig cancerform. Tvi vale.

IL4 promotes phagocytosis of murine leukemia cells counteracted by CD47 upregulation

Abstract

Cytokines are key regulators of tumor immune surveillance by controlling immune cell activity. 

Here, we investigated whether interleukin 4 (IL4) has antileukemic activity via immune-mediated mechanisms in an in vivo murine model of acute myeloid leukemia driven by the MLL-AF9 fusion gene. Although IL4 strongly inhibited leukemia development in immunocompetent mice, the effect was diminished in immune-deficient recipient mice, demonstrating that the antileukemic effect of IL4 in vivo is dependent on the host immune system. 

Using flow cytometric analysis and immunohistochemistry, we revealed that the antileukemic effect of IL4 coincided with an expansion of F4/80+ macrophages in the bone marrow and spleen. 

To elucidate whether this macrophage expansion was responsible of the antileukemic effect, we depleted macrophages in vivo with clodronate liposomes. Macrophage depletion eliminated the antileukemic effect of IL4, showing that macrophages mediated the IL4-induced killing of leukemia cells. 

In addition, IL4 enhanced murine macrophage-mediated phagocytosis of leukemia cells in vitro. 

Global transcriptomic analysis of macrophages revealed an enrichment of signatures associated with alternatively activated macrophages and increased phagocytosis upon IL4 stimulation. Notably, IL4 concurrently induced Stat6-dependent upregulation of CD47 on leukemia cells, which suppressed macrophage activity. 

Consistent with this finding, combining CD47 blockade with IL4 stimulation enhanced macrophage-mediated phagocytosis of leukemia cells. 

Thus, IL4 has two counteracting roles in regulating phagocytosis in mice; enhancing macrophage-mediated killing of leukemia cells, but also inducing CD47 expression that protects target cells from excessive phagocytosis. 

Taken together, our data suggests that combined strategies that activate macrophages and block CD47 have therapeutic potential in AML.

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Phase holographic imaging 

For morphologic analysis, a total of 5 000 macrophages were seeded per well in a 24-well plate and placed in a Holomonitor® M4 (Phase Holographic Imaging AB, Lund, Sweden). 

The microscope was located in an incubator at 37°C and 5% CO2. 

Cells were allowed to attach for one hour, and then images were acquired and analyzed with the software Hstudio™ (Phase Holographic Imaging AB). 

Individual cells were measured for volume and irregularity, a parameter based on the roundness of the cell.

 

Denna studie publicerades 6 Maj och finns inte med på PHI`s lista över publikationer.

 

Nästa forskningsrapport publicerades för 5 dagar sen, 24 Juli.

Lundaforskare i samarbete med Bispebjergs och Frederiksbergs sjukhus i Danmark.

Studien tar upp ett helt annat ämne än den uschliga typen cancer.Nämligen allergi och astma. 

 

Mast cell tryptase enhances wound healing by promoting migration in human bronchial epithelial cells

ABSTRACT

Epithelial damage and increase of intraepithelial mast cells (MC) are characteristics of asthma.
The role of MC mediator tryptase and the protease-activated receptor-2 (PAR2) on epithelial wound healing is not fully investigated.
Stimulation of bronchial epithelial cells (BECs) with tryptase promoted gap closure, migration and cellular speed compared to controls.
Stimulated BECs had higher expression of migration marker CD151 compared to controls.
Proliferation marker KI67 was upregulated in tryptase-stimulated BECs compared to controls. Treatment with PAR2 antagonist I-191 reduced gap closure, migration and cell speed compared to BECs stimulated with tryptase.
We found that tryptase enhances epithelial wound healing by increased migration and proliferation, which is in part regulated via PAR2.
Our data suggest that tryptase might be beneficial in tissue repair under baseline conditions.
However, in a pathological context such as asthma with increased numbers of activated MCs, it might lead to epithelial remodeling and loss of function.

I inledningstexten vid Introduction ska ni läsa det här stycket med välbehag.

The aim of this study is to investigate the normal physiological role and mechanism of MC tryptase on epithelial cell migration and proliferation in a wound gap model. 

To study this, we used a novel digital holographic cytometry (DHC) called Holomonitor M4 (Phase Holographic Imaging, Lund, Sweden). 

The advantage of this compared to traditional gap closure analyses is that it is a high throughput system, uses standard culture conditions with an automated image capture, segmentation and tracking of cells and combines functional aspects of cellular behavior with morphological properties on cell level. 

We hypothesize that tryptase will enhance gap closure in bronchial epithelial cells since earlier studies have established tryptase as a potent mitogenic for structural cells such as fibroblasts in the respiratory system.

Material and methods

Cell culture

Bronchial epithelial cell line BEAS-2B was purchased from ATCC (Walkersville, MD, USA).
Cells were cultured in RPMI-medium 1640 (Gibco, 61,870–010) with addition of 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin Life Technologies (Stockholm, Sweden).
For harvest of supernatant, RNA and protein cells were cultured in six well nunc multidish (Nunc Technologies, Carlsbad USA).
The cells were cultured at 37°C in 5% CO2 and were grown until confluency reached approximately 80–90%, automatically determined in the live cell imaging system Holomonitor Phase Holographic Imaging (Lund, Sweden) before start of the experiment.
Thereafter, cells were treated with 0.5 μg/mL (equivalent to 31 mU/mL) human lung tryptase (Merck Millipore, Darmstadt, Germany). During stimulation of the cells starvation medium was used (RPMI medium with 1% FBS and 1% penicillin-streptomycin). Supernatants were collected after 1 hr, 6 hr and 24 hr post stimulation.

Live cell imaging: gap closure and migration assay

For live cell imaging analysis for epithelial gap closure, migration and morphology, BEAS-2B were cultured in Sarstedt TC 6-well plate (Nümbrecht Germany).
Scratch was created with HoloLid 71,120 (Lund, Sweden) culture-insert 2 well (Ibidi, Martinsried, Germany) where 70 μL cell suspension (5*105 cells/mL) was applied into each well and cultured for 24 hours to reach confluency, thereafter the inserts were removed.

Cells were treated with tryptase 0.5 μg/mL for 24 hours and monitored in live cell imaging system Holomonitor Phase Holographic Imaging (Lund, Sweden).
For assay with PAR2 inhibition BEAS-2B were pre-treated for 30 minutes in 37°C in 5% CO2 with antagonist I-191 HY117793 (Sollentuna, Sweden).

After incubation with I-191 BEAS-2B were washed in PBS and thereafter 0.5 μg/mL tryptase or starvation RPMI-medium only was added to the wells.
Functional studies of epithelial gap closure, migration and morphology were performed using HoloMonitor M4 live cell imaging system Phase Holographic Imaging (Lund, Sweden).

In the present study following parameters was investigated in order to evaluate the wound gap closure: gap width (micrometers), cell covered area (%, statistically quantified as fold change), motility (micrometers), motility speed (micrometer/hour) and morphology (boxed length).
A capture pattern for each well was selected, in this project three randomly chosen positions in each well were selected.

Time-lapse was selected to 24 hours monitoring with images taken every 7 minutes of the three selected positions in each well.
The monitoring of one six-well plate resulted in approximately 3700 images.
Quantitative phase imaging of wound healing followed by automatic cell tracking to determine migration was performed using automated acquisition and image segmentation of multiple time-lapse image sequences in multiple wells in HStudio. 

In conclusion, this study showed that tryptase has a potent mitogenic effect on both migration and proliferation in normal bronchial epithelial cells BEAS-2B as well as increased epithelial production of several growth factors. We can also report that the pro-migratory, but not proliferative, effect of tryptase is PAR2 dependent. This suggest a pivotal role of MC tryptase in healthy tissues in terms of rapid gap closure in the damaged epithelial barrier. However, in a pathological context tryptase could be harmful, having a catalytic role in chronic inflammation and remodeling as well as enhancing tumor progression via pro-migratory effects. Our results highlight that further research is warranted given the possibility to target tryptase and PAR2 for clinical therapeutic use.

 

Den som kan översätta sista stycket till begriplig svenska får en guldstjärna.

Min tolkning är att Lundaforskarna tillsammans med läkare från de danska sjukhusen kommit nånting på spåren till fromma för allergi och astmadrabbade.Och det med användande av PHI`s eminenta instrument. 

- I give you the HoloMonitor. 

 

 

                                            Mvh the99

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