Differential activation of immune cells by commensal versus pathogen-derived bacterial DNA


Thesis Type: Postgraduate

Institution Of The Thesis: Middle East Technical University, Faculty of Arts and Sciences, Department of Biology, Turkey

Approval Date: 2015

Thesis Language: English

Student: Sinem Günalp

Consultant: MAYDA GÜRSEL

Abstract:

Immunological mechanisms making contribution to discriminating signals obtained from commensal versus pathogenic bacteria is an active area of research and recent evidence proposes that commensals and pathogens might express discrete variants of pathogen associated molecular patterns (PAMP). We hypothesized that as a major member of PAMP, bacterial DNA (bacDNA) originating from commensals versus pathogens might possess distinct immunostimulatory activities, enabling their dis- crimination by the immune system. To test this hypothesis, DNAs derived from two bona fide commensal bacteria, Lactobacillus fermentum, Lactobacillus salivarus, one commensal strain of Enterococcus faecium, one virulent clinical isolate of Enterococcus faecium and 2 strict pathogens; Listeria monocytogenes, Streptococcus pyogenes were used for human peripheral blood mononuclear cells, human neutrophils, murine splenocytes, murine fibroblasts, THP-1 monocytes, human (h)TLR9 expressing HEK cells, STING expressing and STING knockout interferon reporter cell lines were stimulated with various doses of human commensal or pathogen-derived purified DNAs as such or following their complexation with the transfection reagent Lipofectamine 2000. Results showed that commensal but not pathogen derived bacDNA induced IP-10 and IFN secretion from hPBMC. However, there was no significant difference in STING expressing B16 IFN-reporter cell line and murine fibroblast L929 cells. Almost 30-60% of the observed response was STING dependent. In contrast, DNAs derived from pathogens triggered significantly higher levels of TNF-α, IL-6 and/or IL-1 production from hPBMCs, human neutrophils and murine splenocytes. Consistent with this, NF-κB activation by hTLR9 expressing HEK cells was significantly higher upon stimulation with DNAs derived from pathogens. In conclusion, our findings suggest that pathogen and commensal bacterial DNAs are recognized by distinct sets of receptors and induce differential activation of immune cells. Collectively, commensal versus pathogen derived DNA’s route of entry into cells, their recognition by selective receptors /sensors and also their resistance to plasma nucleases might be important factors that contribute to such distinct responses.