University of Groningen Regulatory properties of lactic acid bacteria for improving immune homeostasis Ren, Shengcheng

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Regulatory properties of lactic acid bacteria for improving immune homeostasis

Ren, Shengcheng

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Chapter 7

General discussion and future perspective

Chengcheng Ren

1,2

1

Immunoendocrinology, Division of Medical Biology, Department of

Pathology and Medical Biology, University of Groningen and University

Medical Center Groningen, Hanzeplein 1, 9700 RB Groningen, The

Netherlands.

2

School of Food Science and Technology, Jiangnan University, 1800 Lihu

Road, Wuxi 214122, China

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Lactic acid bacteria (LAB) are a group of lactic acid-producing Gram-positive bacteria and are important food-industrial microorganisms owing to their long-history of application in manufacturing fermented foods [1]. A wide range of functional effects have been associated with consumption of LABs [2] but varied effects of specific LAB strains under different physiological conditions were often observed. The effects of LABs were also found to be highly species- and strain-dependent. Therefore, mechanistic studies for elaborating the mechanisms underlying the effects of individual strains are warranted. Considering currently the limited amount of LAB strains with well-defined functional properties, there are great needs for standard high-throughput evaluation systems, which will ensure more convenient and accurate selections of optimal strain candidates for achieving certain health benefits.

A range of LAB strains from different species were investigated in this thesis to unravel the potential species- and strain-dependency of the effects of LABs. To gain more molecular insights in the species- and strain-dependent effects of LABs, we studied the interactions between different LAB strains and Toll-like receptors (TLRs), which are well-characterized pattern recognition receptors (PRRs) primarily responsible for the immune signalling of LABs [3]. In addition, we developed technology platforms to investigate the immune-regulating and gut barrier-protective effects of single LAB strains.

1. TLR-signalling capabilities of LABs shape their species- and strain-dependent immunomodulatory properties

In chapter 2, we found that the stimulating effects of different LAB strains in immune cells were species- and strain-specific. By employing a stepwise strategy, we assessed the TLR signalling effects of various LAB strains and demonstrated that TLR-signalling bacteria signaled mainly via TLR2. The TLR2-stimulating effects of LABs were again shown to be species- and strain-dependent. This suggests that direct interactions of LABs with TLRs are fundamental mechanisms that dictate the immunomodulatory activities of LABs. In addition, the TLR2-activating effects of the tested strains were contingent on stimulation dose, which indicates that administration dose of LABs is a key parameter that influences the effects of LABs. Moreover, only activating but not inhibiting effects of LAB strains on TLR signalling pathways were observed.

Intriguingly, we observed that the identified TLR2-signalling LAB strains specifically signaled via TLR2/TLR6 based on results from antibody blocking assay. It should be noted that among these TLR2/TLR6 signalling strains,

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Lactobacillus (L.) fermentum CCFM787 not only signaled through TLR2/TLR6 but

also activated TLR2/TLR1. Furthermore, TLR2/TLR6 was shown as the dominant pathway involved in the signalling of L. fermentum CCFM787 when compared with TLR2/TLR1. It is known that TLR2/TLR6 signalling contributes to the induction of anti-inflammatory immune responses whereas TLR2/TLR1 pathway elicits inflammatory responses [4,5]. Hence, L. fermentum CCFM787 possesses both pro- and anti-inflammatory potentials and is not suitable to be applied in inflammatory situations. Other selected strains that signal solely through TLR2/TLR6 may be promising candidates for controlling inflammatory disorders. In the follow-up research, their inflammation-attenuating effects should be first tested in animals such as in intestinal inflammation mouse models.

2. LAB-secreted bioactive factors exhibited immune active properties different from bacterial strains

Apart from LAB strains, their secreted bioactive factors have also been reported to exert health-promoting functions [6,7]. To deliver better understanding of the involved mechanisms underlying the effects of LAB-secreted components, culture supernatants of different LAB strains were examined in chapter 3 for their immune regulating potentials using a similar technology platform as in

chapter 2. Our results unravel species- and strain-specific secretion of soluble

components that can directly interact with TLRs and regulate immune signals. Notably, secreted products of several LAB strains such as L. reuteri CCFM14 and

L. fermentum CCFM620 were demonstrated to be immune- and TLR-activating.

In contrast, in chapter 2 these two strains were shown to be incapable to elicit immune stimulation. This inconsistency between the effects of bacterial strains and their released molecules reveals that secreted components may further enhance the beneficial functions of LAB strains. These findings provide novel insights in the molecular mechanisms involved in the effects of LABs, and will be instrumental in developing novel functional food products enriched with LAB-secreted products of bioactive functions.

Considering the complexity in the composition and biochemical properties of different types of soluble factors in LAB-secreted products, it is of great significance to characterize the TLR2 ligands present in the secreted products that are responsible for priming immune responses. This will further decipher the underlying molecular mechanisms, and will facilitate the production and potentiate the feasibility of secreted products-enriched dietary or pharmaceutical products. To achieve this goal, sophisticated strategic

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approaches for defining responsible molecules need to be developed in the future study.

TLR signallings are the focus of the current thesis as TLRs are believed as one crucial host receptor involved in microbial recognition [3]. Our results showed that bioactive molecules released by all tested LAB strains activated exclusively TLR2 signalling. It is worth noting that secreted products of two strains, i.e. L. reuteri CCFM14 and L. fermentum CCFM787, were shown to signal through other PRRs aside from TLRs. Previous studies have shown that the final physiological efficacy of some LAB strains involves the collaborative actions of various PRR signallings such as TLR and nucleotide-binding oligomerization domain (NOD)-like receptor (NLR) families [8,9]. Therefore, other PRR families such as C-type lectin receptors (CLRs) and NLRs that are known to participate in the signalling responses of LABs [8-10] should be tested in future studies. This will further enrich the current knowledge regarding LAB-host crosstalk and provide more molecular insights in the mechanisms of the effects of LABs.

3. TLR2-signalling LAB strains and gut epithelial barrier protection

Enhancement of intestinal epithelial barrier function is suggested as an essential mechanism by which LABs support gut health [3]. TLR2 has been widely recognized as a pivotal player in gut barrier regulation [11]. Therefore, in

chapter 4 the identified TLR2-signalling LAB strains were evaluated for their

potentials in protecting barrier function of T84 human intestinal epithelial cells. In order to explore the mechanisms involved in the barrier-protective effects of LABs, we applied two disruptors that cause barrier damage via acting on varied pathways, i.e. mitogen-activated protein kinase (MAPK) or protein kinase C (PKC) pathway. It was shown that TLR2-activating LAB strains specifically prevented disruption of barrier integrity induced by PKC-dependent disruptor but not MAPK-dependent stressor. This illustrates that these TLR2-activating strains exerted barrier protection by acting on PKC pathway.

PKC is a classic pathway that is involved in barrier function modulation [12]. PKC family is comprised of various PKC isoforms, and differential effects of different PKC isoforms on epithelial barrier integrity have been reported [12]. In follow-up studies it would be tempting to identify the specific PKC isoform that was targeted by LAB strains and to investigate how TLR2 is involved in LAB-conferred barrier protection. These knowledge will deliver a deeper

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179 understanding of the molecular mechanisms involved in barrier-regulating effects of LABs.

4. Species- and strain-specific properties of LABs on mucus function-related genes in goblet cells are contingent on the physiological situations

LABs are also known to modulate mucus barrier thereby improving gut health [13], but the mechanisms involved are still not completely clear as yet. Moreover, species-dependent effects of LABs on mucus production have been found in previous studies [13]. In order to gain more insights in the species- and strain-dependency of mucus-modulating properties of LABs and the underlying mechanisms, in chapter 5 we assessed the effects of LAB strains from different species on mucus function-related genes in goblet cells. Our results suggest that the expression of goblet cell genes were profoundly affected by various LAB strains in a species- and strain-dependent fashion. Besides, the effects were confirmed to depend on duration of LAB stimulation, which needs to be considered in LAB application for modulating mucus function.

Furthermore, our results suggest that living bacteria are required for modifying goblet cell genes, revealing that for some LAB strains it is necessary to monitor their in vivo viabilities after consumption for achieving desirable nutritional or therapeutic benefits. We also found that for some strains their released bioactive factors partially contributed to their effects on goblet cell genes. This further corroborates the notion illustrated in chapter 3 that LAB-secreted bioactive molecules can strengthen the effects of bacterial strains. These findings also indicate that effector molecules of LABs that mediate the functional effects of LABs can be components present on bacterial cells or secreted bioactive molecules.

Another important finding of this study is that the effects of LABs on mucus-associated genes are contingent on the physiological conditions. The tested genes were differentially regulated by LABs in the healthy states and during stimulation with TNF-α, IL-13 or the mucus disruptor tunicamycin. Thus, candidate strains for mucus reinforcement should be rationally selected based on the immune status of the applied populations. Here our approach of investigating LAB-elicited expression change of mucus barrier-associated genes in goblet cells renders a high-throughput profiling of various LAB strains in a cost- and time-effective way. This study offers a technology platform to preliminarily evaluate the mucus-regulatory potentials of LAB strains and will

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contribute to a more targeted in vivo studies based on indications from in vitro assay.

5. Fibroblasts influenced species- and strain-specific effects of LABs on goblet cell genes associated with mucus barrier

Subepithelial fibroblast is a type of stromal cells and can regulate epithelial functions through interacting with intestinal epithelium [14]. It still remains elusive how mucus function is impacted by fibroblasts and whether fibroblasts modify LAB-induced modulation on mucus. As a follow-up study of chapter 5, LAB strains of various species with confirmed effects on goblet cell-related genes were selected and the impact of fibroblasts on their effects were further investigated in chapter 6. It was found that fibroblasts differentially modified the expression of different related genes and their effects on mucus-related genes were dependent on treatment duration. Furthermore, LAB-conferred mucus-modulatory effects were altered by the presence of fibroblasts and the impacts of fibroblasts were bacterial species-dependent. These observations clearly prove the involvement of fibroblast-goblet cell interaction in mucus regulation.

Notably, differential impacts of fibroblasts on LAB-conferred regulatory effects on mucus genes were observed under healthy condition and in different disease situations. Moreover, fibroblasts tended to abrogate gene expression dysregulation associated with disease models. This further underscores the essential role of fibroblasts in preserving gut mucus barrier function, and indicates that the role of fibroblasts in mucus modulation should not be neglected when developing in vitro evaluation models to study the mucus-regulating potentials of LAB.

We sought to define fibroblast-derived factors that are responsible for the observed effects on mucus. This however was not well addressed in this study. In-depth investigations on identifying the responsible factors of fibroblasts will provide more molecular insights in fibroblast-goblet cell crosstalk and its effects on mucus regulation.

6. Conclusion and future perspective

In this thesis, in vitro technology platforms were established and were used to evaluate the immunomodulatory and gut barrier-regulating potentials of a range of LAB strains. These functional properties of LAB are strain-specific and contingent on the physiological situations applied. These findings suggest that it

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181 is imperative to screen proper strain candidates for boosting health status or coping with specific diseases. Strain selections need to be tailored for the targeted populations based on the physiological status of the host. Moreover, as illustrated in this thesis, timing, dose, and duration of bacterial supplementation as well as bacterial viability are crucial parameters profoundly affecting the effects of LAB and should be carefully considered and optimized in order to achieve the highest efficacy of specific LAB formulations.

The studies presented in this thesis also provide more insights in the mechanisms for the functional effects of LABs. We demonstrated that the direct interaction of LAB with PRRs specifically TLRs is the molecular basis for LAB-induced immune regulation. TLR2-signalling strains were found to support gut epithelial barrier function via specifically acting on PKC. Our results also showed that LABs might modulate gut mucus barrier via directly acting on goblet cells. Notably, novel proof of principle was established that fibroblasts play a profoundly important role in mucus barrier and their interactions with goblet cells markedly influence the mucus-regulating properties of LABs. These findings can assist in developing more effective in vitro evaluation systems for better identifications of optimal strain candidates.

It was also proved in this thesis that either bacterial cell components or secreted bioactive molecules can serve as effector molecules responsible for the observed effects of LAB. In-depth studies aiming to characterize these effector molecules of LAB are of great importance since they will lead to a deeper understanding of the variations in the efficacy of different LAB species and strains. These information contribute to the design of medical foods or dietary supplements comprising bacterial strains, bacterial cell components or excreted bioactive products to meet specialized medical or nutritional requirements.

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