Ny Svensk forskningsartikel (med ackuratess)

En av våra supergrävare mejlar in en ytterst intressant forskningsrapport.Det handlar om en studie 4 Svenska forskare med bas från Lunds Universitet genomfört som tar upp ett område där huvudämnet (Mast Cells) spelar en större roll än tidigare befarat.Mastceller är en sorts vita blodceller som ingår i människas immunförsvar.

När en infektion drabbar människa händer en hel kedja av aktiviteter i kroppen.Immunförsvaret "drar igång" snabbt och försöker neutralisera det ämne (beskrivet som parasit) som letat sig in i kroppen.

 

Mastceller är bland de första som aktiveras och frisätter ämnen som är toxiska för parasiten.

Sen tar andra delar av immunförsvaret vid och förhoppningsvis gör sitt jobb.Men Mastceller har även identifierats som potentiellt farliga för vissa grupper av människor.Det handlar då om människor som har kroniska luftvägsbesvär och är mycket känsliga för astma och allergier.För den gruppen kan aktiverade Mastceller i värsta fall leda till Anaphylaxis dvs allergisk chock.Det kan resultera i dödsfall.Anaphylaxis beskrivs lättast som livshotande andnöd och cirkulationskollaps.Vanliga orsakssamband är getingstick,ormbett och vissa födoämnen individ är känslig för som ex jordnötter.Nu visar våra 4 braighta Svenska forskare att Mastceller kan vara ett hot mot dessa individer hyperkänsliga för starka/kraftiga ämnen.En kunskap världens alla allergi/astmadrabbade kommer ha nytta av där läkemedelssektorn då kan erbjuda de identifierade ett motmedel de har lättillgängligt att snabbt sätta in om tecken kommer på att individ är på väg att glida in i det stadiet.Och här pratar vi snabbt,en anafylaktisk chock kan på minuter leda till dödsfall.Ni har säkert hört talas om eller läst om människor som avlidit pga att ha ätit nån maträtt med spår av jordnötter.Den dödskampen att sakta kvävas till döds samtidigt som kroppens organ ger vika..*huga*

Nåväl,vi lämnar sådana hemskheter och går vidare till forskarnas studier istället.

Mastceller är för merparten av människor enbart av godo.De gör sitt jobb vid en infektion och hjälper även till vid lätt allergisk reaktion och häver denna.Man kan se det som samma händelse = ett främmande ämne tar sig in i kropp och Mastceller aktiveras för att neutralisera, eller förintgöra ämnet om det är av allvarligare karaktär.

Men för människor med kroniska luftvägsbesvär kan Mastceller utgöra ett hot visar denna studie.

Forskare och läkare har kunskap att Mastceller kan ha en negativ påverkan för de med svår astma och allergi.

De som ofta benämns ha kroniska luftvägsbesvär.Man har till dags dato inte riktigt kunnat lista ut varför deras eget immunsystem utsätter dem för fara.

Men nu har våra Svenska forskare genom sina studier bidragit med ny kunskap som kommer gagna andra forskare med deras egna forskningsprojekt inom området.I slutändan kommer naturligtvis de med svår astma och allergi dra nytta av denna kunskap.

Innan jag går in på själva studien vill jag nämna att den kommer ingå i en specialutgåva benämnd :

Special Issue "Mast Cells: From Host Defense to Pathology"

Den Barcelonabaserade forskaren Professor Margarita Martin står som redaktör för denna utgåva.

Redan vid inbjudan till världens alla ämnesspecifika forskare att komma in med sina studier nämner hon behovet av att få in nya kunskaper om Mastceller kontra Anaphylaxis.Just det ger våra Lundaforskare sina kollegor med sin forskningsrapport. Saxat från inbjudan:

However, mast cells are best known and widely recognized for their role in pathology as they are the main effector cell in allergies and anaphylaxis, a severe allergic reaction that may cause death. In the last few decades, the incidence of allergies and anaphylaxis has risen at an alarming rate and continues to rise. Therefore, a better understanding of the molecular basis for mast cell function will have repercussions that range from host defense to pathology.

Prof. Dr. Margarita Martín
Guest Editor

I nedanstående forskningsrapport kommer vi se hur stor användning Lundaforskarna haft av PHI`s eminenta HoloMonitorsystem. (fetningar och understrykningar är mina egna)

Mast Cell Proteases Tryptase and Chymase Induce Migratory and Morphological Alterations in Bronchial Epithelial Cells

Published: 16 May 2021 

Frida Berlin

,

Sofia Mogren

,

Julia Tutzauer

and

Cecilia K. Andersson

^*

Abstract

Chronic respiratory diseases are often characterized by impaired epithelial function and remodeling. Mast cells (MCs) are known to home into the epithelium in respiratory diseases, but the MC-epithelial interactions remain less understood. 

Therefore, this study aimed to investigate the effect of MC proteases on bronchial epithelial morphology and function. Bronchial epithelial cells were stimulated with MC tryptase and/or chymase. Morphology and epithelial function were performed using cell tracking analysis and holographic live-cell imaging. Samples were also analyzed for motility-associated gene expression. Immunocytochemistry was performed to compare cytoskeletal arrangement. Stimulated cells showed strong alterations on gene, protein and functional levels in several parameters important for maintaining epithelial function. The most significant increases were found in cell motility, cellular speed and cell elongation compared to non-stimulated cells. Also, cell morphology was significantly altered in chymase treated compared to non-stimulated cells. In the current study, we show that MC proteases can induce cell migration and morphological and proliferative alterations in epithelial cells. 

Thus, our data imply that MC release of proteases may play a critical role in airway epithelial remodeling and disruption of epithelial function.

 

Keywords: mast cell; tryptase; chymase; bronchial epithelial cells; morphology; migration; proliferation; holomonitor

1. Introduction

Mast cells (MCs) have long been recognized as key cells in different pathological conditions and are mainly acknowledged for their detrimental roles in allergies and asthma. 

Both IgE-mediated and non-IgE-mediated activation of MCs can trigger the release of various immunologically active substances. 

Firstly, MC activation causes the release of preformed mediators that are stored within the mast cell secretory granules (containing, e.g., histamine, tumor necrosis factor and proteases chymase, tryptase and carboxypeptidase A3), followed by the release of lipid mediators (leukotrienes and prostaglandins) and lastly, de novo production of, e.g., chemokines, cytokines and growth factors. 

Evidence for their role in pathology is the increased presence and activation in or near structures involved in pathophysiology of the lung, such as smooth muscle, glands and epithelium.

Based on their granule content of serine proteases tryptase and chymase, MCs are divided into two major subpopulations in humans: MC_T (tryptase⁺) and MC_TC (tryptase⁺, chymase⁺). 

In the healthy human lung, the MC_T is the dominating subtype and is readily found in structures such as bronchial mucosa and alveolar parenchyma. 

In various pathological conditions of the lung, such as severe asthma, chronic obstructive pulmonary disease (COPD), cystic fibrosis (CF) and idiopathic pulmonary fibrosis (IPF) the number of MC_TC increase and also home into structures that normally have very few MCs, such as the bronchial epithelium. 

The role of intraepithelial MCs and the phenotypic change from MC_T to MC_TC in airway pathology is, to this point, unknown.

 

Another main component in the development of chronic respiratory diseases is an abnormal epithelial airway barrier, which produces excessive amounts of pro-inflammatory mediators in response to pathogens and noxious stimuli, resulting in a cycle of early and permanent lung damage, and ultimately chronic lung disease. 

It is established that the airway epithelium undergoes dramatic remodeling and loss of function in various respiratory diseases. The remodeling might initially have a protective function but may lead to loss of function if persisting into a chronic disease. Changes of the airway epithelium in airway disease include transformation towards a more proliferative, less differentiated cell type with loss of cell-cell contact.

The in vivo function of MC proteases on various types of epithelium is still under investigation and both detrimental and protective roles have been found. Tryptase has, in vitro, been shown to stimulate epithelial cell proliferation, chemotaxis and differentiation. 

Chymase, conversely, has in some studies been shown to downregulate skin epithelial cell proliferation. Mechanistically, a number of studies have shown that tryptase can cleave intestinal and corneal epithelial tight junction proteins and degrade hemidesmosomes. 

In addition, several studies have shown that tryptase can induce the release of various pro-inflammatory mediators, such as prostaglandin E2 and IL-8 from epithelial cells, and chymase can additionally stimulate mucin expression in airway epithelial cells. 

Overall, these findings suggest that MC proteases also might have profound effects on bronchial epithelial cells. Less is however known regarding the effects of these proteases on bronchial epithelial cell function and morphology, or if these changes are beneficial or detrimental.

 

Therefore, this study aimed to investigate the effect of MC proteases on bronchial epithelial morphology and function using a novel holographic live cell imaging technique in combination with motility-associated gene arrays and studies of cytoskeletal arrangement. 

In the current study, we show that MC proteases induce cell migration and morphological and proliferative alterations in epithelial cells. 

Thus, our data implies that MC release of proteases may play a critical role in airway epithelial remodeling and disruption of epithelial function.

 

Ur studien klipper jag in HoloMonitorrelevant info.

 

2. Results

2.1. Mast Cell Proteases Alter Cell Growth and Division Rate in Bronchial Epithelial Cells

To study the effect of MC proteases tryptase and chymase on epithelial cells, an overall investigation on cell proliferation and cytotoxicity in response to tryptase, chymase or tryptase and chymase in combination was performed. The percentage of cells relative to starting point (t₀) over time and at 36 h  were obtained using the holographic live cell imaging system. At 36 h, chymase stimulation decreased cell growth (122.5% ± 66.8, p = 0.008) and a similar trend was seen for the combination of tryptase and chymase (211.0% ± 65.6, p = 0.2). Tryptase induced no significant growth change at 36 h relative to t₀ (311.6% ± 101.1) in comparison to non-stimulated cells (279.3% ± 84.2, p = 0.6). To ensure that the lowered cell growth in chymase treated cells were not due to cell cytotoxicity, a lactate dehydrogenase (LDH) assay was performed, which indicated no cytotoxic effect at the chosen protease concentrations (0.5 µg/mL). We also examined images obtained in the Holomonitor to validate the health of the cells and we did not observe any signs of cell death, detachment or floating cells in the wells.

 

 

Figure 1. Tryptase induce mitogenic properties whereas chymase attenuate the proliferation rate in bronchial epithelial cells. (A) Percentage cell growth over 36 h for non-stimulated cells (NS) or cells treated with tryptase (T), chymase (C) or tryptase and chymase (TC) over time and the percentage of cell increase at 36 h relative to the starting point (B) was established using the holographic live cell imaging. LDH enzymatic activity was measured in a dose-response setup (C) to detect potential cytotoxicity. (D) Percentage of dividing cells at a 12 h interval. (E) Representation of MTT signal at 24 h, 48 h and 72 h after treatment. (F) Quantification of Ki67 expression (pixels⁺/cells^total) in BEAS-2B using fluorescence immunocytochemistry and ImageJ. (G) Representative micrographs of immunofluorescence stain for KI67 (FITC, green) and nuclei (DAPI, blue) in BEAS-2b stimulated with tryptase, chymase and in combination compared to non-stimulated cells. Scale bar in G: 50 µm. White arrowheads show Ki67 positive cells. Data represent mean ± SEM. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001 using Mann–Whitney test. LDH, KI67, MTT: results are based on three independent experiments. Holomonitor: results are based on five focus points per experiment and stimulation, in three independent repeats.  

2.2. Bronchial Epithelial Cells Stimulated with MC Proteases Display Cell Elongation and Cytoskeletal Rearrangement

The Holomonitor system allowed us to capture 2D as well as 3D reconstructions of cells throughout the experiments. Hence, we compared the cell morphology over time, both within the treatment groups and between the groups. Cells in all treatment groups at 36 h indicated alterations in cell morphology, towards a more elongated shape when compared to non-stimulated cells. To further investigate if tryptase and chymase may induce elongation, cells were treated with tryptase and/or chymase for 24 h and analyzed using a scanning electron microscopy (SEM). High magnification images confirmed the observation from the Holomonitor and demonstrated an increased prevalence of strongly elongated cells in all three treated groups compared to non-stimulated cells. The non-stimulated cells displayed normal flat, shield-like shapes where cells in close proximity to each other formed a thin but dense monolayer. Cells stimulated with tryptase or chymase, alone or in combination, displayed an elongated, stretched shape in unorganized mono/multilayers. To examine if stimulation with MC proteases was associated with changes in the underlying actin cytoskeleton, we examined the F-actin organization using FITC-conjugated phalloidin and confocal microscopy. A rearrangement of the cytoskeleton was observed in all treated groups, which exhibited an elongated morphology with actin-rich filaments extending into the lamellipodia. Non-stimulated cells were less elongated with the actin arranged in multiple long, thin fibers, stretching across the whole cell and exhibited limited lamellipodial structures. The majority of cells stimulated with MC proteases contained numerous lamellipodia projections (T: 70%, C: 65%, TC: 70%), whereas only 3% of non-stimulated cells exhibited this phenotype.

Figure 2. Representative holographic images obtained with HolomonitorM4 at the starting point (A) and 36 h (B) of five focus points per experiment and stimulation, in three independent repeats. The top images in each panel represent high magnification images in 3D of the bottom 2D images. Scale bar (bottom panels): 100 µm. The colors representing the cell height, where yellow is the maximum cell height.

 

2.3. Tryptase and Chymase Induce Morphological Alterations in Bronchial Epithelial Cells

Using the Holomonitor M4 and its holographic live cell imaging technology, we obtained both 2D and 3D morphological data. When studying cell area over time, we found that the area of chymase treated cells were significantly decreased already after 6 h post-stimulation (260.3 µm² ± 132.7, p = 0.04) compared to non-stimulated cells (334.7 µm² ± 133.8) and remained significantly decreased throughout the whole experiment (36 h) (Figure 4A). In contrast, cell area in tryptase treated cells was significantly increased at 6 h (417.5 µm² ± 164.0, p = 0.01) compared to non-stimulated cells (Figure 4A). At 24 and 36 h, both chymase and tryptase-chymase treated cells were significantly smaller (24 h; C: 252.0 µm² ± 145.8, p ≤ 0.0001; TC: 324.2 µm² ± 173.3, p ≤ 0.0001 and 36 h; C: 231.4 µm² ± 135.4, p ≤ 0.0001; TC: 299.9 µm² ± 148.1, p ≤ 0.0001) in comparison to non-stimulated cells (24 h; 422.8 µm² ± 209.5, 36 h; 435.8 µm² ± 177.4) (Figure 4A). The total change in cell area over time (36 h vs. t₀) showed a reduction with chymase and a trend towards a reduction with a combination of tryptase-chymase treated cells (C: −24.6% ± 15.6, p = 0.03 and TC: −3.2% ± 28.9, p = 0.2) (Figure 4B) but no change between non-stimulated cells and tryptase treated cells (NS: 31.2% ± 31.8, T: 10.8% ± 30.4 p = 0.5) in relation to their individual starting point. Confluency represents the percentage of the total area of a frame that is covered by cells, and to study the impact of MC proteases, we analyzed the percentage of confluency at 36 h relative to starting time (Figure 4C). We found that chymase-treated cells had a significantly reduced change in confluency (66.8% ± 27.7, p = 0.008) at 36 h when compared to non-stimulated cells (NS: 342.4% ± 160.4). No difference was seen for tryptase or chymase and tryptase in combination compared to non-stimulated cells (T: 356.8% ± 149.6, p = 0.6; TC: 209.5% ± 100.9, p = 0.2). To further investigate 3D morphological alterations, the optical cell thickness at 36 h was analyzed (Figure 4D). We found a significant increased in all three treated groups (T: 2.0 µm ± 0.7, p = 0.0002; C: 3.0 µm ± 1.3, p ≤ 0.0001; TC: 3.1 µm ± 1.2, p ≤ 0.0001) when compared to non-stimulated cells (1.9 µm ± 0.9). When analyzing optical volume, we found no difference between non-stimulated (793.1 µm³ ± 371.3) and tryptase (819.6 µm³ ± 501.6, p = 0.34), but a significant decrease in chymase treated cells (670.3 µm³ ± 409.1, p = 0.001) as well as a significant increase in tryptase-chymase treated cells (885.4 µm³ ± 438.8, p = 0.009) (Figure 4E).

 

Figure 4. Holographical analysis of morphological alterations in airway epithelial cells stimulated with MC proteases. (A) Demonstration of single cell area (µm²) over time where each dot represents one cell. (B) Cell area at 36 h, relative to starting point. (C) Confluency at 36 h relative to the starting point. (D) The optical thicknesses and (E) optical volume. (F) Representation of the measurements of cell box length and box breadth. (G) The ratio of box breadth and box length plotted at different time points over time. (H) The percentage of cells in each group at different time point that were over the cut-off threshold. The cut-off threshold represents the 10% percentile of the ratio of all NS cells at all time points. Data represent mean ± SEM. Statistical analysis was tested using Mann–Whitney test (* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001) and for (G) Chi-squared test * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001. Data are based on the same number of cells at each time point and stimulation throughout the different analyzed parameters. Results are based on five focus points per experiment and stimulation in independent repeats.

2.4. Tryptase and Chymase Induce Enhanced Cell Migration, Motility and Speed in Bronchial Epithelial Cells

Taken together, the migratory data obtained from the Holomonitor revealed that chymase induced the highest motility (total travel distance) and migration speed compared to non-stimulated cells at the later time interval. However, tryptase induced the furthest migration directed away from the starting position. The combined stimulation showed results similar to tryptase treated cells except for migration, which was comparable with chymase only treated cells.

 

Figure 5. Single-cell tracking showed enhanced cell migration, motility and speed in cells treated with mast cell proteases. (A) Representation of single-cell tracking XY-plots obtained from the HolomonitorM4 and compared migration at two different time intervals (0–12 h and 24–36 h) and between stimulation. Individual cells are seen in different colors. (B) Comparison of single-cell migration, (C) motility and (D) speed in tryptase (T), chymase (C) and in combination (TC) compared to controls (NS). Data represent mean ± SEM. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001 using Mann-Whitney test. Results based on cell tracking of at least five cells per monitored position and from three randomly chosen focus positions in each well, giving a total of >15 cells per time point and stimulation. 

 

Discussions

Previous studies have shown a proliferative effect of tryptase on airway epithelial cells. 

Chymase has instead been shown to inhibit epithelial cell growth in human skin epithelium. 

Therefore, we were particularly interested in the proliferative effect on bronchial epithelial cells of tryptase and chymase alone or when the two proteases were combined. 

Our proliferation data from HolomonitorM4 showed that tryptase alone induced an increase, although not significant, in the number of cells compared to non-stimulated cells during a 36 h experiment. Further, tryptase demonstrated a tendency towards an amplified proliferative result when analyzing division rate at 24–36 h, and a significant increase in proliferation marker KI67 as well as in metabolic activity (MTT), suggesting that tryptase can induce mitogenic properties in bronchial epithelial cells. However, a longer experiment might be required to verify a significantly increased effect at a cellular level. In accordance with previous studies in other epithelial cell types, our data showed a significant decrease in proliferation and cell division rate in chymase-treated cells. Using LDH cytotoxic assay and Holomonitor we found that the decreased cell growth was not due to cell death or cytotoxicity. 

These effects indicate that chymase might have a detrimental role in epithelial barrier function, leading to diminished healing capacity upon injury. 

Cells treated with MC proteases displayed a higher number of cells that had an elongated shape (ratio between cell length and breadth). This response was seen early (6 h) in chymase-treated cells, whereas an increase in both tryptase and chymase-treated groups was observed at 24 h post-stimulation. 

Since all cells, independent of stimulation, elongate over time due to biological cycles and naturally occurring migration when cells are not 100% confluent, the top 10 percentile based on non-stimulated cell elongation was used. Using this approach, more than 30% of all cells treated with chymase were considered elongated. To further evaluate the elongation, we manually assessed epithelial cell elongation using SEM and immunofluorescence microscopy. We found that the proportion of cells as well as the actual cell elongation ratio to be even more increased in all three treated groups when compared to the analyses obtained from the Holomonitor.

 

In the current study, we studied the effect of tryptase and chymase alone as well as the simultaneous effect of the two proteases on epithelial cell migration. In contrast to the few previous studies, we found a strong enhancement of cell migration, motility and speed when stimulating cells with either tryptase, chymase or the combination of the two proteases. By using the holographic live cell imaging system, we were able to carefully track individual cells at an interval of 15 min over 36 h (representing >5000 images in total). Hence, this method gave us a very precise illustration of cell movements, both visually through videos as well as in automated measurements of exact values which were used for analysis.

 

We have for the first time studied the role of MC tryptase and chymase on airway epithelial cell morphology and function by using the novel holographic live cell imaging system, Holomonitor M4. This method is label-free and is performed inside an incubator, allowing minimal interference with the cells. Some exclusive strengths of using this system are the ability to study 3D reconstructed cells over a certain time lap, obtain multiple morphological and quantitative cell parameters at each captured frame, and to get a great overall picture of cell behavior and health. Additionally, the cell tracking system enables a unique possibility to analyze the accurate cell migration, motility and speed over time.

 

4. Materials and Methods

4.1. Cell Culture

Human bronchial epithelial cells, BEAS-2B (ATCC, Walkersville, MD, USA) were maintained in RPMI-medium 1640 (Gibco, Paisley, UK, 61870-010) with a supplement of 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin (Life Technologies, Stockholm, Sweden). The cells were cultured in a humidified atmosphere at 37 °C in 5% CO₂ until approximately 80–90% confluency was reached. MC proteases were diluted in starvation medium containing RPMI medium with 1% FBS and 1% penicillin–streptomycin. A harvest of supernatant, RNA and protein cells were collected after 6 and 24 h post-stimulation. For Holomonitor experiments, cells were treated with tryptase and/or chymase diluted in starvation RPMI-medium and thereafter monitored for 36 h.

 

4.8. Live Cell Imaging: Migration, Morphology and Proliferation

Functional studies of epithelial migration, morphology and proliferation was per-formed with Holomonitor M4 live cell imaging system from Phase Holographic Imaging (Lund, Sweden). BEAS-2B were cultured in Sarstedt TC 6-well plate (Nümbrecht, Germany) and the imaging was performed inside an incubator at 37 °C in 5% CO₂. Prior to the start of monitoring, a capture pattern of 5 randomly chosen positions per well and time lapse for imaging (one image every 15 min over 36 h) for respective wells was selected. One 6-well plate resulted in approximately 5200 images. The quantification of the automatic time lapse and cell tracking to determine migration, morphology and proliferation in multiple independent repeats and was carried out using HStudio, a software system with a capacity of a wide range of applications designed for the analysis of holographic microscopy images of unstained adherent cells, using Holomonitor. For the automated cell recognition, the same settings and thresholds were used for treatment groups and non-stimulated cells.

 

 

Min kommentar

Med denna studie skapar forskarna Frida Berlin,Sofia Mogren,Julia Tutzauer och Cecilia K. Andersson sig ett namn inom forskarvärlden.Med ett fullt användande av HoloMonitorsystemets alla möjligheter har de gett alla svårt astmasjuka resp allergidito ett hopp om att resten av forskarvärlden ska ta över stafettpinnen och få fram effektiva läkemedel för att undvika Anaphylaxis.

Denna studie borde VD Egelberg rama in och hänga på sitt kontor.

                                            Mvh the99

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Ps.Gårdagens nyhet om säljet till Pandorum Technology,att användas inom regenerativ medicin ger många intressanta scenarion jag har för avsikt att kika närmare på. Grävspaden står redo. Stay tuned.