The influence of cell-free Lactobacillus rhamnosus GG conditioned medium on macrophage functional activity

  • Yashaswini Seenappanahalli Nanjundaiah

    Student thesis: Doctoral Thesis


    Macrophages as professional phagocytes have been considered as prominent participants in the response to acute infection and play a significant role in preventing infecting bacteria multiplying and damaging the host environment. Macrophages have complex mechanisms equipped with specialised receptors to recognise their targets. Upon internalisation of receptor-bacterial complex, macrophages initiate killing of the ingested microbes partly through the generation of free radicals species. However, the excessive production of reactive oxygen species (ROS) and nitric oxide (NO) during phagocytic activity may also contribute to the inflammatory process which although is beneficial in killing bacteria may result in deleterious consequences, especially following an interaction of NO with superoxide radicals to form reactive nitrogen species such as peroxynitrite.Probiotics are beneficial bacteria which have been shown to activate host innate immune response by influencing phagocytic activity of macrophages. This immunomodulatory effect has also been shown due to release of a number of soluble proteins by probiotic bacteria in the culture medium. However, the mechanism of regulation is not clear. In this study, we have therefore evaluated the influence of cell free Lactobacillus rhamnosus GG conditioned medium (LGG-CM) on macrophage phagocytosis and also on ROS and NO production by the J774 macrophage cell line, and further evaluated the acute effect of LGG-CM on NO and ROS production during the process of macrophage phagocytosis of E.coli in real time.Murine macrophage J774 cells were used in the study as these cells are readily activated by bacterial lipopolysaccharide (LPS) and produce NO. Gentamicin protection assay was employed in order to investigate the effect of LGG-CM on the ingestion and digestion phases of macrophages phagocytosis of E. coli HfrC. The cells were exposed to different dilutions of LGG-CM in the absence and presence of bacterial LPS, E. coli and the inhibitors or scavengers for ROS and NO. The J774 macrophages were loaded with free radicals sensitive fluorescent dyes, namely, H2DCFDA for monitoring ROS or with DAFFM-DA for NO detection. Acute free radical production was measured using a fluorescence microplate reader and changes were analysed by cumulative sum (CuSum) calculations. The fluorescence measurements were taken every two minutes for the first 60 minutes to monitor free radicals production during the ingestion period and from 60 minutes to 280 minutes to monitor free radicals production during the bacterial digestion period. The fluorescence was measured at 485 nm excitation and 528 nm emissions.The LGG-CM did not have any toxic effect on E. coli or macrophage viability, as determined by antibacterial studies and cell proliferation assays. The LGG-CM treated macrophages ingested significantly less bacteria both at 30 minutes and 60 minutes of incubation (p< 0.05). However, only higher concentrations of LGG-CM (75 % LGG-CM and 100 % LGG-CM) significantly increased the bacterial digestion rate of E. coli by the macrophages (p< 0.01). The LPS on its own did not have any effect on macrophage phagocytosis. The LGG-CM mediated enhanced bacterial digestion was inhibited by ROS and NO inhibitors and ROS scavengers. The LGG-CM at higher concentration induced increase expression of NADPH oxidase subunits (p47 phox and gp91 phox) at 6 hours (p< 0.01).A low concentration of LGG-CM (10 % LGG-CM) or LPS did not cause any significant change in the basal levels of ROS or NO production. In contrast, a high concentration ofiiLGG-CM significantly enhanced ROS generation but also significantly reduced the NO level in both the ingestion and digestion phases of phagocytosis.These findings are novel and suggest for the first time that probiotics may release factors in culture medium which enhances macrophage digestion capability. The LGG-CM also has the capability to increase ROS production and may additionall
    Date of Award16 Mar 2016
    Original languageEnglish
    Awarding Institution
    • Teesside University
    SupervisorMosharraf Sarker (Supervisor), David Wright (Supervisor) & Zulfiqur Ali (Supervisor)

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