A Study of the Influence of Macrophages Activation on its Capacity to Bind

Bacterial Antigensusing Atomic Force Microscopy

Marta Targosz1,

Pawel Czuba1, Magdalena Strus3, Janusz Marcinkiewicz2, and Marek Szymonski1

1Institute of Physics, Jagiellonian University, 2Chair of Immunology, Jagiellonian University Medical College, 3Chair of Microbiology, Jagiellonian University Medical College,

„Probiotic bacteria enhancing the immune response. Studies have suggested that consumption of yogurt or milk that contains specific strains of Lactobacillus or supplements with Lactobacillus or Bifidobacterium may improve the natural immuneresponse. Further research is needed to confirm these early findingsand to best understand how the improved immune function may or may not help inwarding off infections.” (University of Maryland Medical Center http://www.umm.edu)

Our results can increase understanding of the role of probiotic bacteria in organisms and their positive influence on the protective function of immune cells,like macrophages.

Motivation for studies of the influence of probiotic bacteria

like Lactobacillus on the function of macrophages to bind bacterial antigens:

... are located in the outer part of bacterial walls

... have polymer structure

... cause inflammation

... trigger macrophage phagocytosis

Bacterial antigens

bacterial antigen

(endotoxin)

Bacterial antigens in our experiments

Lipopolysaccharides (LPS):

pathogenic bacteria

elicit inflammation

extracted from Escherichia coli

Exopolysaccharides (EPS)

probiotic bacteria

do not elicit inflammation

extracted from Lactobacillus

Macrophages

... play an important role in immunological system

... recognise pathogenic factors and kill them (in

phagocytosis process)

... have receptors on the membrane that recognize and

bind bacterial antigens.

Macrophage activation by bacterial antigen

After bindingbacterial antigens

Before bindingbacterial antigens

The activation of the macrophage is a consequence of binding bacterial antigen by receptors

5 m

Experimental set-up tip of the AFM modification and sample preparation

Force spectroscopy

force-distance curve

polymer extension

Specific interaction

Non-specific interaction

Specific interaction

Example of a force-distance curve

By analyzing the force-distance curves it is possible to directly measure the force needed to destroy a ligand-receptor bond (rupture force)

x 500

Probability of adhesion events

PA = Nwith adhesive jump / Ntotal

N – number of force-distance curves

Non-specific interaction

Results obtained for the EPS-activated macrophages

1. Modified system I: EPS-activated macrophages - LPS on the tip

2. Modified system II:Lactobacillus-activated macrophages – LPS on the tip

3. Reference systems: non-activated macrophages - LPS on the tip

PA = 0.58 / PA = 0.4 PA = 0.58 / PA = 0.46

EPS-activated macrophages – LPS on the tip

Lactobacillus-activated macrophages – LPS on the tip

EPS/Lactobacillus activated macrophages maintain or even increase their ability to bind LPS.

Results obtained for the LPS-activated macrophages

1. Modified system I: LPS-activated macrophages - EPS on the tip

2. Modified system II: E coli-activated macrophages - EPS on the tip

3. Reference systems: non-activated macrophages - EPS on the tip

LPS-activated macrophages – EPS on the tip

E. coli-activated macrophages – EPS on the tip

The activation of macrophages by both LPS molecules and bacteria E.coli decreases their ability to bind EPS

PA = 0.7 / PA = 0.22 PA = 0.7 / PA = 0.14

Conclusions

By using atomic force spectroscopy we were able to directly determine the changes in interactions between bacterial antigens and receptors after activation of macrophages.

LPS / Escherichia coli activated macrophages decrease their ability to bind EPS.

EPS / Lactobacillus activated macrophages maintain or even increase their ability to bind LPS. This may suggest that in vivo probiotic bacteriawill enhance the defence potential of local macrophages against pathogens expressing LPS.

These results are promising for improving the understanding of the role of probiotic bacteria in organisms and their interaction with immune cells, like macrophages (main motivation of this work)

Collaborations

Department of Immunology, Jagiellonian University Medical College

Department of Microbiology, Jagiellonian University Medical College

Institute of Immunology and Experimental Therapy, Polish Academy of Sciences,

This work was supported by the Grants: 1 P03B 098 28 and 3 T11E 033 26, from the Committee for Scientific Research (MNII) of Poland.

Atomic Force Microscopy (AFM)

liquid cell with sample

cantilever

piezo-scanner

detector

laser

•AFM provides three dimensional

images of biological specimens surfaces in ambient liquid and in gas environments

•AFM doesn’t require destructive methods of sample preparation,

such as coating or freezing

•AFM can probe elastic or adhesion

properties on surfaces by measurement of a so called

“force-distance curves”

Experimental set-up

PA = 0.58 PA = 0.12

Reference systems: non-activated macrophages - LPS on the tip

Receptor TLR4 is responsible for the bio-recognition of the LPS by macrophages.

PA = 0.7 PA = 0.37

Reference system: non-activated macrophages - EPS on the tip

The lack of significant changes in the average rupture force obtainedfor both systems suggest that TLR4 receptor doesn’t (or very weakly)contributed to the EPS binding.