Probiotic modulation of immune responses in an in vitro
mucosal co-culture model
By
Neama Yasser Habil
A thesis submitted to the University of Plymouth
In partial fulfilment for the degree of
Doctor of Philosophy
School of Biomedical and Biological Sciences
Faculty of Science and Technology
This project funded by Ministry of Higher Education
Baghdad, Iraq
February 2013
Abstract
Probiotics confer health benefits through many mechanisms including modulation of the
gut immune system. Gut mucosal macrophages play a pivotal role in driving mucosal
immune responses. The local environment and macrophage subset determine immune
response: tolerance, associated with an M2-like, regulatory macrophage phenotype and
inflammatory activation with an M1-like phenotype. The aims of this study were firstly to
investigate the immunomodulatory effects of a panel of heat-killed (HK) probiotic
bacteria and their secreted proteins (SP) of Bifidobacterium breve (BB), Lactobacillus
rhamnosus GG (LR), L. salivarius (LS), L. plantarum (LP), L. ferrmentum (LF), and L.
casei strain Shirota (LcS) on cytokine production and TLR expression in monocultures
of monocytes, macrophage subsets, and intestinal epithelial cells. Normally, mucosal gut
macrophages resemble the M2 subset and fail to express CD14, a co-receptor for LPS
signalling. Therefore, probiotic modulation of LPS-induced NF-kB activity and cytokine
expression was investigated using a THP-1 monocyte-derived reporter cell line, model of
CD14hi/lo M1 and M2 macrophages. Secondly, a transwell co-culture system was
developed to investigate probiotic modulation of macrophage-influenced epithelial
barrier function. Parameters investigated included cytokine, TLR and hBD-2 expression,
TEER and IHC staining of the tight junction protein, ZO-1. Probiotics selectively
modulated monocyte and macrophage subset cytokine expression. Probiotics (HK and
SP) suppress CD14lo, augment CD14hi M1, and differentially regulated TNF-α
production in M2s. M2 macrophage IL-6 production was suppressed by both HK and
SPs, and differentially regulated in CD14lo and CD14hi M1s. NF-κB activation failed to
parallel probiotic regulation of TNF-α and IL-6. Probiotics (HK-LF and HK-LcS)
selectively modulated both endogenous and exogenous TNF-α and IL-10, as well as their
induction of epithelial cell expression of TLR and hBD-2. Epithelial expression of TEER,
ZO-1 and the endogenous TLR signal regulator, Tollip, were suppressed upon co-culture
with pro-inflammatory M1 macrophages paralleled by a suppression of IL-10 and upregulation of TNF-α and IL-8. In the presence of LPS, HK-LF enhanced TEER, ZO-1
and partially rescued Tollip expression, whereas HK-LcS had no effect on TEER and
ZO-1 and displayed a weaker rescue effect on Tollip compared with LF. In the
M2/epithelial cell co-culture, both probiotics enhanced TEER and ZO-1 in the presence
of LPS, whilst displaying a differential modulation of Tollip, dependant on the format of
probiotic (HK or SP). In conclusion, probiotic strains can differentially exert immune
activatory or suppressive functions and immunomodulation is determined by strain,
inflammatory environment, and mucosal macrophage effector phenotype. Future
probiotic development must consider prophylactic use in healthy individuals or
therapeutic treatment of defined pathological conditions, strain-specific effects, gut
mucosal integrity, and immune phenotype of mucosal macrophages.