Louise Hjerrild Zeuthen

Epithelial cells prime the immune response to an array of gut-derived commensals towards a tolerogenic phenotype through distinct actions of thymic stromal lymphopoietin and transforming growth factor-β

Humans and other mammals coexist with a diverse array of microbes colonizing the intestine, termed the microflora. The relationship is symbiotic, with the microbes benefiting from a stable environment and nutrient supply, and the host gaining competitive exclusion of pathogens and continuously maintenance of the gut immune homeostasis. Here we report novel crosstalk mechanisms between the human enterocyte cell line, Caco2, and underlying human monocyte‐derived DC in a transwell model where Gram‐positive (G+) commensals prevent Toll‐like receptor‐4 (TLR4)‐dependent Escherichia coli‐induced semimaturation in a TLR2‐dependent fashion. These findings add to our understanding of the hypo‐responsiveness of the gut epithelium towards the microflora. Gut DC posses a more tolerogenic phenotype than conventional DC. Here we show that Caco2 spent medium (SM) induces tolerogenic DC with lower expression of maturation markers, interleukin (IL)‐12p70, and tumour necrosis factor‐α when matured with G+ and Gram‐negative (G–) commensals, while IL‐10 production is enhanced in DC upon encountering G+ commensals and reduced upon encountering G– bacteria. The Caco2 SM‐induced tolerogenic phenotype is also seen in DC priming of naive T cells with elevated levels of transforming growth factor‐β (TGF‐β) and markedly reduced levels of bacteria‐induced interferon‐γ production. Caco2 cell production of IL‐8, thymic stromal lymphopoietin (TSLP) and TGF‐β increases upon microbial stimulation in a strain dependent manner. TSLP and TGF‐β co‐operate in inducing the tolerogenic DC phenotype but other mediators might be involved.

Lactic acid bacteria inducing a weak interleukin-12 and tumor necrosis factor alpha response in human dendritic cells inhibit strongly stimulating lactic acid bacteria but act synergistically with gram-negative bacteria.

ABSTRACT The development and maintenance of immune homeostasis indispensably depend on signals from the gut flora. Lactic acid bacteria (LAB), which are gram-positive (G+) organisms, are plausible significant players and have received much attention. Gram-negative (G−) commensals, such as members of the family Enterobacteriaceae, may, however, be immunomodulators that are as important as G+ organisms but tend to be overlooked. Dendritic cells (DCs) are crucial immune regulators, and therefore, the present study aimed at investigating differences among human gut flora-derived LAB and G− bacteria in their patterns of DC polarization. Human monocyte-derived DCs were exposed to UV-killed bacteria, and cytokine secretion and surface marker expression were analyzed. Profound differences in the DC polarization patterns were found among the strains. While strains of LAB varied greatly in their capacity to induce interleukin-12 (IL-12) and tumor necrosis factor alpha (TNF-α), G− strains were consistently weak IL-12 and TNF-α inducers. All strains induced significant amounts of IL-10, but G− bacteria were far more potent IL-10 inducers than LAB. Interestingly, we found that when weakly IL-12- and TNF-α-inducing LAB and strong IL-12- and TNF-α-inducing LAB were mixed, the weakly IL-12- and TNF-α-inducing LAB efficiently inhibited otherwise strong IL-12- and TNF-α-inducing LAB, yet when weakly IL-12- and TNF-α-inducing LAB were mixed with G− bacteria, they synergistically induced IL-12 and TNF-α. Furthermore, strong IL-12- and TNF-α-inducing LAB efficiently up-regulated surface markers (CD40, CD83, CD86, and HLA-DR), which were inhibited by weakly IL-12- and TNF-α-inducing LAB. All G− bacteria potently up-regulated surface markers; however, these markers were not inhibited by weakly IL-12- and TNF-α-inducing LAB. These much divergent DC stimulation patterns among intestinal bacteria, which encompass both antagonistic and synergistic relationships, support the growing evidence that the composition of the gut flora affects immune regulation and that compositional imbalances may be involved in disease etiology.

Lactobacillus acidophilus induces virus immune defence genes in murine dendritic cells by a Toll‐like receptor‐2‐dependent mechanism

Lactobacilli are probiotics that, among other health‐promoting effects, have been ascribed immunostimulating and virus‐preventive properties. Certain Lactobacillus spp. have been shown to possess strong interleukin‐12 (IL‐12) ‐inducing properties. As IL‐12 production depends on the up‐regulation of type I interferons (IFNs), we hypothesized that the strong IL‐12‐inducing capacity of Lactobacillus acidophilus NCFM in murine bone‐marrow‐derived dendritic cells (DCs) is caused by an up‐regulation of IFN‐β, which subsequently induces IL‐12 and the double‐stranded RNA binding Toll‐like receptor‐3 (TLR‐3). The expression of the genes encoding IFN‐β, TLR‐3, IL‐12 and IL‐10 in DCs upon stimulation with L. acidophilus NCFM was determined. Lactobacillus acidophilus NCFM induced a much stronger expression of Ifn‐β, Il‐12 and Il‐10 compared with the synthetic double‐stranded RNA ligand Poly I:C, whereas the levels of expressed Tlr‐3 were similar. Whole genome microarray gene expression analysis revealed that other genes related to viral defence were significantly up‐regulated and among the strongest induced genes in DCs stimulated with L. acidophilus NCFM. The ability to induce IFN‐β was also detected in another L. acidophilus strain (X37), but was not a property of other probiotic strains tested, i.e. Bifidobacterium bifidum Z9 and Escherichia coli Nissle 1917. The IFN‐β expression was markedly reduced in TLR‐2−/− DCs, dependent on endocytosis, and the major cause of the induction of Il‐12 and Tlr‐3 in DCs stimulated with L. acidophilus NCFM. Collectively, our results reveal that certain lactobacilli trigger the expression of viral defence genes in DCs in a TLR‐2 manner dependent on IFN‐β.

Pseudomonas aeruginosa quorum‐sensing signal molecules interfere with dendritic cell‐induced T‐cell proliferation

Pseudomonas aeruginosa releases a wide array of toxins and tissue-degrading enzymes. Production of these malicious virulence factors is controlled by interbacterial communication in a process known as quorum sensing. An increasing body of evidence reveals that the bacterial signal molecule N-(3-oxododecanoyl)-L-homoserine lactone (OdDHL) exhibits both quorum-sensing signalling and immune-modulating properties. Recently, yet another quorum-sensing signal molecule, the Pseudomonas quinolone signal (PQS), has been shown to affect cytokine release by mitogen-stimulated human T cells. In the present article we demonstrate that both OdDHL and PQS decrease the production of interleukin-12 (IL-12) by Escherichia coli lipopolysaccharide-stimulated bone marrow-derived dendritic cells (BM-DCs) without altering their IL-10 release. Moreover, BM-DCs exposed to PQS and OdDHL during antigen stimulation exhibit a decreased ability to induce T-cell proliferation in vitro. Collectively, this suggests that OdDHL and PQS change the maturation pattern of stimulated DCs away from a proinflammatory T-helper type I directing response, thereby decreasing the antibacterial activity of the adaptive immune defence. OdDHL and PQS thus seem to possess dual activities in the infection process: as inducers of virulence factors as well as immune-modulators facilitating the infective properties of this pathogen.

Lactobacilli and bifidobacteria induce differential interferon-β profiles in dendritic cells.

The health promoting effects of probiotics are well-documented; however, current knowledge on immunostimulatory effects is based on data from a single strain or a limited selection of strains or species. Here, we compared the capacity of 27 lactobacilli and 16 bifidobacteria strains to stimulate bone marrow-derived dendritic cells (DC). Most lactobacilli strains, including Lactobacillus acidophilus, Lactobacillus gasseri, Lactobacillus casei and Lactobacillus plantarum, induced strong IL-12 and TNF-α production and up-regulation of maturation markers. In contrast, all bifidobacteria and certain lactobacilli strains were low IL-12 and TNF-α inducers. IL-10 and IL-6 levels showed less variation and no correlation with IL-12 and TNF-α. DC matured by strong IL-12-inducing strains also produced high levels of interferon (IFN)-β. When combining two strains, low IL-12 inducers inhibited this IFN-β production as well as IL-12 and Th1-skewing chemokines. The IFN-β induction was mediated through c-Jun N-terminal kinase (JNK) irrespective of the stimulating strain. The inhibitory bacteria induced higher levels of the transcription factor c-Jun dimerization protein (JDP)-2, thereby counteracting the effect of JNK. Our data demonstrate that lactobacilli can be divided into two groups of bacteria featuring contrasting effects, while all bifidobacteria exhibit uniform effects. This underlines the importance of selecting the proper strain(s) for probiotic purposes.

Lactobacillus acidophilus induces a slow but more sustained chemokine and cytokine response in naïve foetal enterocytes compared to commensal Escherichia coli

BackgroundThe first exposure to microorganisms at mucosal surfaces is critical for immune maturation and gut health. Facultative anaerobic bacteria are the first to colonise the infant gut, and the impact of these bacteria on intestinal epithelial cells (IEC) may be determinant for how the immune system subsequently tolerates gut bacteria.ResultsTo mirror the influence of the very first bacterial stimuli on infant IEC, we isolated IEC from mouse foetuses at gestational day 19 and from germfree neonates. IEC were stimulated with gut-derived bacteria, Gram-negative Escherichia coli Nissle and Gram-positive Lactobacillus acidophilus NCFM, and expression of genes important for immune regulation was measured together with cytokine production. E. coli Nissle and L. acidophilus NCFM strongly induced chemokines and cytokines, but with different kinetics, and only E. coli Nissle induced down-regulation of Toll-like receptor 4 and up-regulation of Toll-like receptor 2. The sensitivity to stimulation was similar before and after birth in germ-free IEC, although Toll-like receptor 2 expression was higher before birth than immediately after.ConclusionsIn conclusion, IEC isolated before gut colonisation occurs at birth, are highly responsive to stimulation with gut commensals, with L. acidophilus NCFM inducing a slower, but more sustained response than E. coli Nissle. E. coli may induce intestinal tolerance through very rapid up-regulation of chemokine and cytokine genes and down-regulation of Toll-like receptor 4, while regulating also responsiveness to Gram-positive bacteria.

Establishment of tolerance to commensal bacteria requires a complex microbiota and is accompanied by decreased intestinal chemokine expression

Intricate regulation of tolerance to the intestinal commensal microbiota acquired at birth is critical. We hypothesized that epithelial cell tolerance toward early gram-positive and gram-negative colonizing bacteria is established immediately after birth, as has previously been shown for endotoxin. Gene expression in the intestine of mouse pups born to dams that were either colonized with a conventional microbiota or monocolonized (Lactobacillus acidophilus or Eschericia coli) or germ free was examined on day 1 and day 6 after birth. Intestinal epithelial cells from all groups of pups were stimulated ex vivo with L. acidophilus and E. coli to assess tolerance establishment. Intestine from pups exposed to a conventional microbiota displayed lower expression of Ccl2, Ccl3, Cxcl1, Cxcl2, and Tslp than germ-free mice, whereas genes encoding proteins in Toll-like receptor signaling pathways and cytokines were upregulated. When comparing pups on day 1 and day 6 after birth, a specific change in gene expression pattern was evident in all groups of mice. Tolerance to ex vivo stimulation with E. coli was only established in conventional animals. Colonization of the intestine was reflected in the spleen displaying downregulation of Cxcl2 compared with germ-free animals on day 1 after birth. Colonization reduced the expression of genes involved in antigen presentation in the intestine-draining mesenteric lymph nodes, but not in the popliteal lymph nodes, as evidenced by gene expression on day 23 after birth. We propose that microbial detection systems in the intestine are upregulated by colonization with a diverse microbiota, whereas expression of proinflammatory chemokines is reduced to avoid excess recruitment of immune cells to the maturing intestine.