Lactobacilli and bifidobacteria induce differential interferon-β profiles in dendritic cells
Highlights
► Lactobacilli induce high amounts of IFN-β (differential strain specific expression profiles) in dendritic cells. ► The JNK pathway (MAP kinases) is a prerequisite for the induction of IFN-β by lactobacilli. ► Lactobacilli induce high levels of IL-12 and TNF-α. ► Bifidobacteria and certain lactobacilli strains are low IL-12 and TNF-α inducers. ► Low IL-12 inducers are able to completely abrogate the IFN-β production induced by lactobacilli.
Introduction
Lactobacilli and bifidobacteria are generally referred to as probiotics due to their health promoting properties, encompassing a variety of effects such as exclusion and inhibition of pathogens in the gut, increase of the integrity of the gut epithelium barrier, and modulation of the host immune system both locally and systemically [1]. Regarding the immunomodulating properties, the clinical applications of lactobacilli and bifidobacteria comprise prevention and treatment of allergic diseases in particular atopic dermatitis in children and inflammatory bowel diseases as well as prevention of virus infection and use as an adjuvant in vaccination [2]. Despite great improvements in the understanding of how lactobacilli and bifidobacteria interact with cells of the immune system it is still unknown how they exert their immunomodulatory effects. To date, no general picture of the immunemodulatory properties of these bacteria on a genus, specie or strain level is available.
Lactobacilli and bifidobacteria are recognized by dendritic cells (DC) which are pivotal in maintaining immunological homeostasis in the gut (reviewed by Coombes and Powrie [3]). DC are scattered throughout the gut mucosa, where they are in close proximity to the microbiota. Subepithelial DC can sample luminal bacteria antigens by passing their dendrites between epithelial tight junctions into the gut lumen [4], [5]. Furthermore, DC interact directly with bacteria that have gained access via M cells [6]; they use pattern recognition receptors (PRRs), such as Toll-like receptors (TLRs), to sense microbial motifs and thereby recognize the different members of the microbiota. In earlier work, we have shown that lactobacilli and bifidobacteria induce distinct strain specific DC maturation patterns [7], [8] and distinct effects on NK cells [9] and T cells [10]. We and others have shown that TLR2 plays a key role upon stimulation of DC and macrophages with lactobacilli [11], [12], [13].
Type I interferons (IFNs) are secreted cytokines that orchestrate diverse immune responses to infection [14]. Although discovered by their ability to protect cells from viral infections, recent reports have described bacterial induction of IFN-β (reviewed by Monroe et al. [15] and Trinchieri [16]). We have previously shown that Lactobacillus acidophilus strains induce high amounts of IFN-β in DC, leading to a strong Th1 response including the expression of hundreds of interferon-stimulated genes (ISGs) and other genes involved in the innate host response against [12]. Furthermore, human mucosal transcriptome analysis revealed that IFN regulatory factors and IFN-induced or -regulated genes are strongly up-regulated after consumption of L. acidophilus, Lactobacillus casei and Lactobacillus rhamnosus [17].
Hence, it is indicated that the IFN-β inducing capacity of L. acidophilus may explain the virus preventive effect reported for the probiotic bacterium L. acidophilus NCFM [18]. We also demonstrated that this induction of IFN-β is dependent on the MAP kinase c-Jun N-terminal kinase (JNK) [19] and a prerequisite for a strong expression of IL-12 [12]. Furthermore, Bifidobacterium bifidum Z9, not inducing IFN-β, inhibited the L. acidophilus triggered IFN-β expression [19]. It is suggested that the Jun dimerization protein 2 (JPD2), via blocking of Ifn-β transcription, plays a central role in this regulatory mechanism [19].
The main aim of the present study was to screen a large number of lactobacilli and bifidobacteria to determine differences and similarities between the specific strains and species. We investigated the ability of lactobacilli and bifidobacteria to induce a strong Th1 polarized DC phenotype characterized by a high IL-12 production and to which extent this IL-12 induction is dependent on IFN-β. Moreover, we wanted to characterize the IL-12 inhibitory effect of lactobacilli and bifidobacteria and to determine whether these strains use a general mechanism for the inhibition. We therefore tested a panel of 27 lactobacilli strains (8 species) and 16 bifidobacteria strains (5 species) for their effects on DC maturation and polarization. Our results reveal that strains from four of the five lactobacilli species induce IL-12, and that Lactobacillus reuteri strains inhibit IL-12. All bifidobacteria strains were clearly IL-12 inhibitory. We demonstrate that IFN-β induced by lactobacilli is a key factor for a strong Th1 polarized phenotype of DCs including a high IL-12 production, and that the MAP kinase JNK is mandatory for the induction of IFN-β. Addition of inhibitory strains (bifidobacteria and L. reuteri) to L. acidophilus NCFM stimulated DC almost completely blocked the induction of IFN-β. Our results indicate that the transcription factor JDP-2 plays a decisive role in this inhibitory mechanism.
Section snippets
Bacterial strains, growth conditions and preparation of lactobacilli and bifidobacteria
The gastrointestinal microbiota-derived bacteria used are listed in Table 1. The lactobacilli and bifidobacteria strains were inoculated from frozen glycerol stocks in 50 ml de Man Rogosa Sharp (MRS) broth (Merck, Darmstadt, Germany) and sub-cultured twice in MRS under anaerobic conditions at 37 °C for 16 h. The cultures were harvested at the stationary growth phase by centrifugation (4.200g, 5 min), washed twice (4.200g, 5 min) in sterile PBS (Lonza, Basel, Switzerland), re-suspended in 1/10 the
Theory
The comparison of a large panel of lactobacilli and bifidobacteria will serve as a reference for key studies of the immune modulating effects of these strains, especially when they are used in clinical trials and different strains are combined.
Distinct cytokine profiles upon stimulation of dendritic cells with lactobacilli and bifidobacteria
The immunomodulatory effects of a large panel of lactobacilli and bifidobacteria were determined in murine bone-marrow derived DC (Fig. 1). In preliminary experiments, we observed a dose–response curve with a rise in cytokine production with increasing bacterial concentration up to a maximum level from where the cytokine production dropped again (unpublished data). The dose–response curve differs not only between bacterial strains but also between the different cytokines. Therefore, stimulation
Discussion
It is well-established that a high IL-12 production of DC matured by microbial stimuli gives rise to Th1 polarization and thus a strong stimulation of the adaptive immune defense. In the present study, we screened a large number of lactobacilli and bifidobacteria genera for their immune stimulatory properties. Our data shows that strains of the lactobacillus genus are heterogeneous in their immune stimulatory capacity when added in vitro to DC, whereas strains of the bifidobacterium genus are
Role of funding
This study was supported by the Danish Dairy Board and The Danish Food Industry Agency (The FØTEK 3 and FØSU programme).
Acknowledgements
We thank Marianne K Pedersen and Anni Mehlsen for invaluable technical assistance.
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2021, Molecular ImmunologyCitation Excerpt :Both signaling pathways leading to IL-12 induction may also involve stimulation and signaling through MAP Kinases (Arthur and Ley, 2013). As an example, IFN-β and IL-12 induction by the Gram-positive L. acidophilus was fully dependent on c-Jun N-terminal kinase (JNK) phosphorylation (Weiss et al., 2011, 2010a). In contrast to JNK, phosphorylation of the p38 MAP Kinase leads to the induction of Dual Specificity Phosphatase (DUSP)-1, which dephosphorylates and hereby inactivates JNK and p38 (Seternes et al., 2019).
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Shared first author.
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Present address: Immunotechnology, Biopharmaceuticals Research Unit, Novo Nordisk A/S, Måløv, Denmark.