Immunomodulatory effects of the microvesicles from bacterial cell wall of Pantoea agglomerans

Background: In the environment, Gram-negative bacteria are able to produce large amounts of spherical structures measuring 30-50 nm in diameter. These structures, referred to as "microvesicles" or "microglobules", emerge by fragmentation of the outer membrane of bacterial wall. Microvesicles are suspected of inducing inflammatory lung diseases (e.g. in workers exposed to organic dusts), but possibly they also stimulate anti-tumour defence mechanisms. The present study was aimed at assessing the immunomodulatory properties of microvesicles in vitro.

Methods: Microvesicles (MV) were made of the bacterial wall of Pantoea agglomerans (Erwinia herbicola). Peripheral blood mononuclear cells (PBMC) of five healthy volunteers were cultured for 6 h, 24 h, 3 days, and 5 days with a 5-fold dilution series of MV ranging from 0.48 - 1500 µg/ml. The following variables were observed: secretion of IFN-γ and TNF-α (ELISA and ELISPOT assays), intensity of cell divisions (LPT), expression of surface markers CD8, CD14, CD16, CD25, CD69, CD80, CD83, HLA-DR, the apoptosis marker Annexin V and PI permeability (FACS).

Results: After 24 hours, a clear dose-dependent response to MV was seen for IFN-γ production starting already with the lowest MV concentration of 0.48 µg/ml (p=0.042). As for TNF-α, a significant (p=0.05) increase in the production of this cytokine was seen after 3 days at MV concentrations of 300 µg/ml or higher. On the LPT, there was a clear and significant dose-response after 5 days (p=0.001). Regarding expression of cell surface markers, the only phenomenon seen in all donors was the decrease in the number of CD14(+)CD83(+) cells with increasing MV concentration (probably due to increased MoDC maturation)

Conclusion: We have shown that microvesicles are biologically active in PBMC cultures in a dose-dependent manner. Interestingly, IFN-γ production was increased already at lowest MV doses tested, whereas TNF-α became upregulated only by doses 600-times higher. This offers a possible explanation for the epidemiological and clinical observations mentioned in the introduction: Depending on the inhaled doses of MV, IFN-γ would be upregulated alone (inflammatory effects) or together with TNF-α (inflammatory and anti-tumour effects).

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Sensimun (formerly Celimun) biomedical research outsourcing in allergy and immunology

ELISpot - training and implementation

ELISpot - scanning and analysis of ELISpot plates

Sensimun (dawniej Celimun)- badania biomedyczne na zlecenie

Institute of Dermatology, Krakow, Poland

Instytut Dermatologii w Krakowie

Specjalista dermatolog i wenerolog, Kraków