1. A Study of the Influence of Macrophages Activation on its Capacity to Bind Bacterial Antigens using Atomic Force Microscopy Marta Targosz 1, Pawel Czuba 1, Magdalena Strus 3, Janusz Marcinkiewicz 2, and Marek Szymonski 1 1 Institute of Physics, Jagiellonian University, 2 Chair of Immunology, Jagiellonian University Medical College, 3 Chair of Microbiology, Jagiellonian University Medical College,

2. „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 immune response. Further research is needed to confirm these early findings and 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:

3. ... are located in the outer part of bacterial walls... have polymer structure... cause inflammation... trigger macrophage phagocytosis Bacterial antigens bacterial antigen (endotoxin )

4. 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

5. 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.

6. Macrophage activation by bacterial antigen After binding bacterial antigens Before binding bacterial antigens The activation of the macrophage is a consequence of binding bacterial antigen by receptors 5  m

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

8. Force spectroscopy force-distance curve

9. 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 P A = N with adhesive jump / N total N – number of force-distance curves Non-specific interaction

10. 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

11. P A = 0.58 / P A = 0.4P A = 0.58 / P A = 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.

12. 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

13. 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 P A = 0.7 / P A = 0.22 P A = 0.7 / P A = 0.14

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 bacteria will 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)

15. 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.

16. 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”

17. Experimental set-up

18. P A = 0.58P A = 0.12 Reference systems: non-activated macrophages - LPS on the tip Receptor TLR4 is responsible for the bio-recognition of the LPS by macrophages.

19. P A = 0.7P A = 0.37 Reference system: non-activated macrophages - EPS on the tip The lack of significant changes in the average rupture force obtained for both systems suggest that TLR4 receptor doesn’t (or very weakly) contributed to the EPS binding.