Pathogenic Escherichia coli change the adhesion between neutrophils and endotheliocytes in the experimental bacteremia model

Abstract

Septicemia caused by gram-negative bacteria is characterized by high death rate due to the endotoxin release. Since the septicemia depends not only on biochemical aspects of interactions in the system bloodstream, the study of mechanical interactions is also important. Using a model of experimental septicemia caused by E. coli, a hyperproduction of integrins CD11a and CD11b by neutrophils was shown, but this did not lead to the establishment of strong adhesion contacts between endothelial cells and neutrophils. On the contrary, adhesion force and work, as assessed by FS spectroscopy, were statistically significantly reduced in the presence of bacteria. It has also been shown that exposure to the pathogenic strain E. coli 321 increases the stiffness of the membrane-cytoskeleton complex of endothelial cells and bacteria significantly change their morphology on long-term observation. At the same time, we observed the death of neutrophils by apoptosis. Thus, it was shown that besides lipopolysaccharide release there are other pathogenic factors of E. coli: decrease in the interaction between neutrophil and endothelial cell caused by an increase of the endothelial cell rigidity and apoptotic death of neutrophils probably as a result of adhesins and exotoxin effects. Obtained results should be taken in mind during the therapy of septicemia.

Keywords: Escherichia coli; FS spectroscopy; adhesion force; adhesion work; apoptosis; endothelial cells; neutrophils; stiffness of membrane-cytoskeleton complex.

Figure 1. Increased expression of integrins by neutrophils under the influence of E. coli 321.(A) CD11a; (B) CD11b; (C) CD18. Data represent the median of the mean fluorescence intensity (MFI) determined in six independent experiments, (* – p < 0.05, Mann-Whitney U test).

Figure 2. Expression dynamics of (A) ICAM 1 (intercellular adhesion molecule); (B) VCAM 1 (vascular cell adhesion molecule); (C) P- and E-selectin (ELAM); (D) endoglin. PBS – negative control (no exposure), TNF – positive control, E. coli 321 – pathogenic strain, E. coli HB101 – saprophyte strain. Data represent median of mean fluorescence intensity (MFI) ± SEM from five independent experiments (* – p < 0.05 Kruskal-Wallis H test).

Figure 3. Adhesion contacts between neutrophils and endotheliocytes of EA.hy926 cell line. Adhesion force (A) and adhesion work (B) after treatment with pathogenic strain E. coli 321; (C) and (D) adhesion force and work respectively after treatment with the non-pathogenic strain E. coli HB101. Data represent all means of the force (n=70) and work (n=20) of adhesion obtained for different donors in a series of 5 repetitions for each donor. (Paired Sample Wilcoxon Signed Ranks test (W), Pair-sample t-test (T)). * – p < 0.05.

Figure 4. Morphology and stiffness of endothelial cells assessed by scanning ion-conductance microscopy.(A) control; top line – morphology, bottom line – stiffness (dynamics taken at 7 min intervals); (B) after application of pathogenic strain E. coli 321; top line – morphology, bottom line – stiffness (dynamics taken at 20 min intervals); (C) – after application of non-pathogenic strain E. coli HB101; top line – morphology, bottom line – stiffness (dynamics taken at 20 min intervals).

Figure 5. Viability of endothelial cells (cell line Ea.hy926) before (A) and after (B) incubation with E. coli 321 during 120 min.

Figure 6. Decrease in neutrophil viability under the influence of pathogenic strain E. coli 321.(A) viability level; (B) necrosis level; (C) apoptosis level; (D) flow cytometry dot plots in control; (E) flow cytometry dot plots after treatment with pathogenic strain E. coli 321: the upper left quadrant indicates Annexin V negative, PI positive. * – p < 0.05, Mann-Whitney U test.

Figure 7. Apoptotic (A) and necrotic (B) death of neutrophil granulocytes in a model of septicopiemia as cells migrate along the E. coli 321 chemoattraction gradient to the lower chamber.