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The integrity of human intestinal epithelium is maintained by the intact TJ structures between the adjacent intestinal epithelial cells and essential for exclusion of the luminal pathogens and their toxins, as well as allergens [324]. Strains of some probiotic bacterial species, such as L. rhamnosus and L. plantarum, have been shown to have abilities of not

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only improving on the TJ integrity but also can compensate or even reverse the negative effects of the cytokine- or chemically-induced inflammation on the tight junction integrity [38-40, 260, 325]. However, apart from p40 and p75 characterised in L. rhamnosus GG

[38, 264], other effectors and the corresponding molecular mechanisms involved in the intestinal epithelial integrity are still unknown.

In this study, both GG and HN001 wild-type strains were shown in the TEER assays to be capable of positively impacting and also reversing the negative effects of TNFα on the TJ integrity. However, compared to the wild-type strain, GGΔspcB mutant interestingly demonstrated a much lower ability to reverse the TNFα–caused drop in TEER change across the polarised Caco-2 monolayers. This finding suggested that SpcB of GG might have a role in reversing the cytokine-induced destabilisation of the TJs in the Caco-2 monolayer.

The TNFα-mediated increase in Caco-2 cell layer’s permeability is proposed to result from impaired production and distribution of the TJ protein components (such as ZO1), in an NF-κB dependent manner [326]. The NF-κB p50/p65 has been shown to bind to the promoter of myosin light-chain kinase (MLCK)-encoding gene causing an increase in transcription, translation and enzymatic activity of MLCK, as well as the downstream signalling that results in the increased TJ permeability [327, 328]. Another report indicates that TNFα also induces phosphorylation of extracellular signal regulated kinases, ERK1 and ERK2 (MAPK group), in Caco-2. Apart from TNFα, the signalling pathways involved in maitenance of the TJ integrity between the intestinal cells also include a series of signalling molecules, including the Rho family GTPases, protein kinase C (PKC) and MAPK kinases [40, 325].

Lactobacillus strains, such as L. rhamnosus GG and L. plantarum ATCC8014 have been

shown to reverse one or more TNFα-dependent signalling pathways as described above to recover the intestinal epithelial integrity [258, 259, 325]. For example, L. rhamnosus

GG was shown to prevent the TNFα-induced nuclear translocation of NF-κB, blocking the downstream signalling pathways [259]. The recent studies on secreted proteins of L. rhamnosus GG, p40 and p75, have provided a putative molecular basis for probiotic

effects on the intestinal epithelial integrity. Both p40 and p75 have been shown to inhibit the TNFα-induced intestinal cell apoptosis and stimulate the intestinal cell growth,

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coupled to the PI3K/Akt-activation pathways [38]. Further studies also indicate that the previously observed protective effects of p40 on the intestinal cells are EGFR-dependent [90, 264].

However, not all signalling events mediated by L. rhamnosus GG are attributed to p40

and p75. For example, L. rhamnosus GG cell, but not p75 or p40, inhibit the TNFα-

stimulated p38/MAPK activation and induce ERK1/2 [38]. However, these effectors on

L. rhamnosus GG cell have not been identified so far. As the absence or presence of SpcB

in GG does not affect the TEER change in the absence of TNFα, it could specifically interfere with the TNFα-dependent signalling pathways. SpcB might be such a cell- surface-anchored immunomodulatory protein, which can reverse one or more TNFα- induced signalling cascades to protect the intestinal epithelial integrity. The SpcB interference could be direct or indirect, depending on whether the signalling cascades in Caco-2 are triggered by SpcB itself or a SpcB-dependent molecule.

In the strain HN001, no matter whether TNFα was present or absent, no difference in TEER change was observed between the ΔspcA or ΔspcB mutant and the wild-type, showing that the function of SpcB was strain-specific, and that in HN001 SpcB is redundant with respect to stabilising the TJs in the presence of TNFα. Different effects of SpcB from GG and HN001 on the stabilising the TJs could stem from potential differences in glycosylation. As the two glycosyltransferase genes are missing in the HN001 genome, SpcB in this strain could be nonglycosylated. Alternatively, SpcB is glycosylated in HN001 by other nondedicated glycosyltransferases, however compositionally or structurally the modification is different from the one in GG, resulting in different effects on cell signalling that controls the stability of the TJs.

Another research question relevant to L. rhamnosus-host interactions is whether SpcB

and/or SpcA promote adhesion of L. rhamnosus to Caco-2 cell layers. A few surface

proteins in GG have been demonstrated to mediate adhesion to Caco-2 cells, in particular SpaCBA pilus [41, 100, 102]. However, less information is available for HN001, a L. rhamnosus strain that does not have a pilus. In the in vitro adhesion assays, both GG and

HN001 strains adhere equally efficiently to the Caco-2 cells. Furthermore, no significant difference was observed between the wild-type strains and ΔspcA or ΔspcB mutant of GG and HN001, suggesting neither SpcA nor SpcB was a major adhesin in either of the two

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strains under the test conditions. As SpcB of GG has been shown to interfere with TNFα- induced destabilisation of the Caco-2 polarised monolayer integrity, this protein possibly has a much weaker interaction with the Caco-2 cells than that of the putative major adhesins of L. rhamnosus.

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Chapter VI: Conclusions and further