Dendritic cell response after exposure to Salmonella enterica with different LPS structure.

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Final Thesis

Dendritic cell response after exposure to Salmonella

enterica with different LPS structure

Annika Engstrand

LiTH-IFM-A--Ex—09/2033-SE

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URL för elektronisk version 8 Datum Date xx/1/ 2009 Avdelning, Institution Division, Department Språk Language Rapporttyp Report category ISBN LiTH-IFM-A--Ex—09/2033-SE ________________________________________________ _____ ISRN LiU-Biol-Ex-634 ____________________________________ Licentiatavhandling x Examensarbete C-uppsats x D-uppsats Övrig rapport Svenska/Swedish x Engelska/English _ ________________ Titel Title

Dendritic cell response after exposure to Salmonella enterica with different LPS structure Författare

Author Annika Engstrand

Sammanfattning

Abstract

Lipopolysaccharide (LPS) is a structure of the gram-negative bacteria that protect from chemicals and works as a stabilization component for the membrane. Studies show that LPS also may have a function to avoid immune defense. In this project we investigate two Salmonella enterica variants with different LPS conformation. The wild-type Salmonella got an originally LPS structure and the mutant form had a defect one. The bacteria were transfected with a green fluorescent protein (GFP) to allow measuring of phagocytosis. Monocytes were isolated from human blood and were incubated for several days with cytokines to give dendritic cells. The cells were exposed to each type of Salmonella and incubated for different times. After labeling with phalloidin and studies with fluorescent microscopy, phagocytosis and F-actin were measured. The results show that it is a difference in phagocytosis and F-actin depending on LPS conformation. That means that LPS may have a decisive role for the pathogenicity of Salmonella.

Nyckelord

Keyword

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1 Abstract

Lipopolysaccharide (LPS) is a structure of the gram-negative bacteria that protect from chemicals and works as a stabilization component for the

membrane. Studies show that LPS also may have a function to avoid immune defense. In this project we investigate two Salmonella enterica variants with different LPS conformation. The wild-type Salmonella got an originally LPS structure and the mutant form had a defect one. The bacteria were transfected with a green fluorescent protein (GFP) to allow measuring of phagocytosis. Monocytes were isolated from human blood and were incubated for several days with cytokines to give dendritic cells. The cells were exposed to each type of Salmonella and incubated for different times. After labeling with phalloidin and studies with fluorescent microscopy, phagocytosis and F-actin were measured. The results show that it is a difference in phagocytosis and F-actin depending on LPS conformation. That means that LPS may have a decisive role for the

pathogenicity of Salmonella.

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2 Introduction

2.1 Dendritic cells

Dendritic cells (DCs) are antigen-presenting cells that have the ability to activate naïve T-cells [1, 2]. Before the cells can activate T-cells they must go through series of steps in a process called maturation. In the absence of signals the DCs are in a steady-state phase and under stimulation of certain cytokines and

microbial products they undergo phenotypic and functional changes which leads to maturation (fig. 1) [1,3]. Immature DCs have the ability to take up antigens by phagocytosis but cannot activate T-cells and start an immune response [2]. Immature DCs are found in both surface epithelia and other organs as heart and kidneys. Under infection they migrate to the lymph nodes and gradually change shape and undergo maturation. DCs have now a much higher ability to present antigens and attract naïve T-cells by certain chemokines [13].

Figure 1 Under influence of cytokines and microbial products DCs differentiate to

mature cells [20].

2.2 Salmonella enterica

Salmonella enterica is a gram-negative facultative intracellular bacterium that effect both animals and human. The bacteria cause a variety of diseases, from lethal diarrhea in calves to typhoid fever in humans [1]. Most isolates of

Salmonella are placed in the species enterica, which is further subdivided into serovars based on antigens on their surface; one of these serovars is

Typhimurium. Salmonella enterica serovar Typhimurium may multiply in the gastrointestinal tract of many animal species, especially in mice and rats, where it usually causes no disease, but in humans the bacteria causes gastroenteritis,

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which can result in many types of infections in the digestive tract and cause life-threatening diarrhea [4]. S. Typhimurium causes a systemic disease in mice that is similar to typhoid fever in humans, thus Typhimurium has been extensively used as a mouse model for Typhoid fever in humans [10, 11, 18].

Humans acquire Salmonella by the ingestion of contaminated foods, milk or water. The disease is treatable but in many affected developing countries there is poor health care and a scarcity of clean water, which complicates the situation [1]. After oral infection S. Typhimurium interacts with the intestinal mucosa, invades the mucosal tissue and triggers an inflammation response [10, 18, 19].

2.3 F-actin

Eukaryotic cells consist of three types of cytoskeleton filaments; actin filaments, microtubules and intermediate filaments. Actin consists of many globular G-actin monomers that build up a filamentous polymer that is called G-actin. F-actin is essential for all types of movement, cell division and to change the cell surface structure [12].

2.4 Lipopolysaccharide

Gram-negative bacteria contain of an extra lipid bilayer, besides the phospholipid layer, Lipopolysaccharide (LPS). It is composed of three components (lipid A, basal core, O-polysaccharide) and is embedded in the outer layer of the outer membrane of the bacteria (fig 2). Endotoxins consist of Lipid A, the inner component of LPS, of the gram-negative bacteria and have an ability to start a variety of inflammatory responses in humans, which can be lethal [14, 15].

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2.5 Aim

The primary aim of this project was to study the interaction between dendritic cells and Salmonella enterica serovar Typhimurium. Our expectations were to find out whether there was a difference in phagocytosis and quantity of F-actin between Salmonella wild-type and mutant bacteria. This research can lead to further understanding of the importance of LPS in pathogenicity and the discovery of new target sites for anti-infective measures.

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3 Material and methods

3.1 Culture medium and cytokines

The monocytes was cultured in Iscove`s modified Dulbecco`s medium (IMDM) supplemented with 4 mM L-glutamin, 10 % heat-inactivated fetal calf serum (FCS), 100 U ml-1 penicillin and 100 µg mL-1 streptomycin (Gibco

BRL/Invitrogen). Interleukin-4 (IL-4) and recombinant human granulocyte macrophage colony-stimulating factor (GM-CSF) were purchased from R&D Systems.

The Salmonella culture medium consisted of 3 mL Luria Bertani (LB) broth, from a stock solution, supplemented with 4 µL ampicillin.

3.2 Cell isolation

The monocytes were isolated from human whole blood according to Bøyum [5]. After sedimentation on a gradient with 2, 5 % dextran and separated with

Lymphoprep (Axis-Shield PoC AS), centrifugation in a swing –out rotor for 30 min at 400 g at 4 °C and a brief hypotonic lyses, the cells were harvested and washed repeatedly in ice-cold Krebs Ringer Glucose (KRG; 120 mM NaCl, 4.9 mM KCl, 1.2 mM MgSO4, 8.3 mM KH2PO4 and 10 mM glucose) to remove

density gradient residue and platelets. After the final wash, the monocytes were isolated by negative selection using a cocktail of biotin-conjugated antibodies to CD3, CD7, CD19, CD45RA, CD56 and IgE respectively and MACS CD14 micro beads coupled to antibiotin monoclonal antibodies (Miltenyi Biotec). The resulting monocyte-enriched fractions were put in cryogenic vials in a Cryo freezing container(Nalgene) over night and for further storage they were placed in a cryonic container (-199 °C).

3.3 Dendritic cells

Monocytes were seeded on sterile glass cover slips in cell culture plates (Nunc) at 2× cells per well. To count the cells a 10-chamber counting grid (Fast-Read 102 TM_{; ISL) was used. The plates was incubated in 37 °C in 5% CO}

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2 h then were non adherent cells washed away with room tempered KRG with 1mM CaCl2. DCs were generated by culturing the cells in 1mL IMDM

containing 1000 U mL-1 GM-CSF and 500 U mL-1 IL-4. The cytokines were added to the cultures at day 0 and 3 to generate immature DCs at day 5 [6, 7].

3.4 Bacteria

The strains used, S. typhimurium wild-type 395 MS (smooth) and a mutant 395 MR10 (rough), differ with respect to structure and physicochemical surface

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characteristics; the mutant has a different Lipopolysaccharide chain compare to the wild-type Salmonella (fig.3) [8].The bacteria were cultured and transfected with a green fluorescent protein (pEGFP). After culturing, Salmonella were stored in -70 °C until they were used. The day before an experiment the bacteria were thawed and cultured in sterile test-tubes in 3 mL LB broth supplemented with 4µL ampicillin. The bacteria were grown at 37 °C for 16 h on an orbital shaker.

Figure 3 The differences in structure between Salmonella MS and MR10 [22].

3.5 Phagocytosis and labeling of F-actin

The bacteria were suspended in IMDM at room temperature and added to the DCs at a ratio of 5:1, except from the control group there no bacteria was added. The cells were incubated with the bacteria for 10 min at 37 °C, 5 % CO2 and

were washed three times with IMDM. Incubation was continued at 37 °C for 10 or 60 min respectively for each Salmonella strain. 2 % paraformaldehyde

(Sigma – Aldrich) in KRG was used for fixation and the plates were incubated in room temperature for 15 min and washed three times in PBS (7, 6). The cells were incubated for 30 min with agitation at room temperature with Alexa568 Fluor-phalloidin (Invitrogen-Molecular Probes) to stain F-actin. Alexa568 Fluor-Phalloidin from a stock solution (200 U mL-1 in methanol kept at -20 °C) was diluted 1:40 in PBS (pH 7, 6) supplemented with 100 µg mL-1

lysophosphatidylcholine (Sigma-Aldrich) as a membrane permeabilizing agent. The glass cover ships were fixated on microscope slides and were left over night in room temperature and were stored in 4 °C until they were used.

3.6 Microscopy

Phagocytosis was imaging with a confocal laser scanning microscope (Sarastro 2000; Molecular Dynamics) and a 60× oil immersion objective. The 514 nm line of the Argon laser was used for simultaneous excitation of GFP and Alexa568 Fluor. Dichronic mirrors with cut-off wavelengths of 535 and 595 nm were used for the excited and emitted light, respectively. A 545DF30 nm band-pass filter

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was employed for the green signal and a 600 nm long-pass emission filter for the red signal.

DC with labeled F-actin was visualized with a Zeiss Axioskop and a 63× oil immersion objective. The cells were visualized with Easy Image Analysis 2000 (Version 2.7.2.2, Tekno Optik AB) and the images were captured with a CCD camera with ZVS-47E amplifier. The free data program Image J was used to measure the quantity of F-actin in the cells.

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4 Results

4.1 F-actin

After every experimental day pictures were taken of on a hundred cells with labeled actin. The pictures were further analyzed and a drawing board was used to mark the area of the cells to be able to measure the total F-actin in every photograph of the DCs. The results of different experiment show a varying pattern but the total results of all five experiments demonstrate an increased F- actin activity in all cells compared to the control group. Salmonella MR10 seems to affect the DCs to produce F-actin in a higher amount than Salmonella MS (fig. 4). Group Significance Control vs. MS(10 ) *** Control vs. MS(60) *** Control vs. MR10(10) *** Control vs. MR10(60) *** MS10 vs. MR10(10) - MS60 vs. MR10(60) ***

Figure 4 F-actin in DCs after interaction with Salmonella MS or MR10 for 10/60 min.

The graph shows the mean values of the fluorescence of all estimated cells. Each group contains data from a hundred dendritic cells from five separate experiments; errors bars indicate SD; ***P< 0.001. The numbers in parenthesis indicate incubation time.

4.2 Phagocytosis

Microscope slides with cells and bacteria were studied with a fluorescence microscope. Phagocytosed Salmonella were counted for a hundred DCs and grouped in three categories; 0, 1-10 or >10 bacteria in a single cell. The results showed that DCs have a much higher ability to phagocytose Salmonella MR10 then MS. There is also a difference between the incubation time 10 and 60 min. Each DC tend to phagocytose more Salmonella MR10 after a prolonged

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Figure 5 Phagocytosed Salmonella MS or MR10 for 10/60 min. The different colors

of the staples indicate the number of phagocytosed bacteria for each cell. Each group contains data from 520-547 randomly selected cells. The numbers in parenthesis indicate incubation time.

4.3 Statistics

A student`s T-test (two-tailed) were made from the results of F-actin. Error bars indicate SEM (standard error of the mean). The calculated values of differences between control and experimental group (t-value) were compared with fixed values from a table (P-value). A P-value of <0.001 was considered significant.

Table 1 Statistical P and t values for measured F-actin. The numbers in the

parenthesis indicate incubation time.

Group t Pvalue (∞) Control vs. MS(10) Control vs. MS(60) Control vs. MR10(10) Control vs. MR10(60) 1_{MS10 vs. MR10(10)} MS60 vs. MR10(60) 1_{There is no statistic significance.}

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5 Discussion

It has been described in earlier studies that Salmonella mutants with deficiencies in polysaccharide side-chains have a higher tendency to be phagocytosed

[16,17]. According to other research the results shows that immature DCs work as phagocytosing cells and mature DCs have a roll in the immune system [2,19]. In this study we only used immature DCs to see the phagocytosing effect. The reason why MR10 seems to be more attractive to DCs may depend on that the structure make it easier to be phagocytosed. This type of bacteria lack parts of the outer layer, which seems to enable adhesion for the DCs. It is also possible that Salmonella MS is more toxic for the cells than MR10 are. Under the experiment the cells seem to be negative affected of the wild-type, especially after a longer incubation time. The shape of the cells became abnormal and certain cells underwent apoptosis (not shown). For that reason the capacity for the cells to phagocytose perhaps became smaller.

The relationship between phagocytosis and F-actin production is viewable in the results. The relation likely depends on the cells need to change shape during phagocytosis and therefor have to produce F-actin. When the phagocytosis rates become higher, the quantity of F-actin also increases. The cells also use

dendrites to catch bacteria, which is an actin-dependent process.

The uptake of Salmonella enterica serovar Typhimurium by DCs is very small described and further research requires for the understanding of the first

interaction. What we can see according to the results is that LPS plays an

important role in the interaction and uptake of phagocyte cells but it is necessary to continue and develop additional experiments to understand the reason. That F-actin should increase was expected but we did not know if there would be a difference between the different Salmonella strains. The difference could have a meaning but it is uncertain without more repetitions of the experiment. The results of measured F-actin were different from the five experiments and it is possible that external factors have a role in the final results. There were many different elements before it was possible to measure quantity of F-actin and it could influence the cells and their ability to produce actin.

There is many ways to develop the experiment further. One idea is to use mature DCs to see if the phagocytosing efficacy is different. Another way is to vary the incubation time to see if my assumptions of that Salmonella MS is more toxic to DCs is true. A third alternative is to use other mutant Salmonella

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6 Acknowledgements

I would like to thank Katarina Tejle for help with laboratory experiments and Karl-Eric Magnusson for answering my questions. Most of all I would thank Björn for all support and useful discussions.

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Dendritic cell response after exposure to Salmonella enterica with different LPS structure.