Bifidobacterium bifidum Actively Changes the Gene Expression Profile Induced by Lactobacillus acidophilus in Murine Dendritic Cells

Dendritic cells (DC) play a pivotal regulatory role in activation of both the innate as well as the adaptive immune system by responding to environmental microorganisms. We have previously shown that Lactobacillus acidophilus induces a strong production of the pro-inflammatory and Th1 polarizing cytokine IL-12 in DC, whereas bifidobacteria do not induce IL-12 but inhibit the IL-12 production induced by lactobacilli. In the present study, genome-wide microarrays were used to investigate the gene expression pattern of murine DC stimulated with Lactobacillus acidophilus NCFM and Bifidobacterium bifidum Z9. L. acidophilus NCFM strongly induced expression of interferon (IFN)-β, other virus defence genes, and cytokine and chemokine genes related to the innate and the adaptive immune response. By contrast, B. bifidum Z9 up-regulated genes encoding cytokines and chemokines related to the innate immune response. Moreover, B. bifidum Z9 inhibited the expression of the Th1-promoting genes induced by L. acidophilus NCFM and had an additive effect on genes of the innate immune response and Th2 skewing genes. The gene encoding Jun dimerization protein 2 (JDP2), a transcription factor regulating the activation of JNK, was one of the few genes only induced by B. bifidum Z9. Neutralization of IFN-β abrogated L. acidophilus NCFM-induced expression of Th1-skewing genes, and blocking of the JNK pathway completely inhibited the expression of IFN-β. Our results indicate that B. bifidum Z9 actively inhibits the expression of genes related to the adaptive immune system in murine dendritic cells and that JPD2 via blocking of IFN-β plays a central role in this regulatory mechanism

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

Additive Effects of <i>Bifidobacterium bifidum</i> Z9 on the Chemokine Expression in <i>Lactobacillus acidophilus</i> NCFM stimulated Dendritic Cells.

<p>Data indicates gene expression fold changes of dendritic cells stimulated with <i>L. acidophilus</i> NCFM, <i>B. bifidum</i> Z9 and both strains in combination in comparison with un-stimulated dendritic cells measured on Affymetrix microarrays. Foldchanges are log₂ relative to control.</p

<i>Jdp2</i> and MAPK kinases control immune responses in dendritic cells.

<p>Expression of <i>Jdp2</i> and inhibition of MAPK kinases in dendritic cells upon stimulation of <i>L. acidophilus</i> NCFM. <b>A</b>. <i>B. bifidum</i> Z9 exerts an additive effect on the expression of <i>Jdp2</i> in DC stimulated with <i>L. acidophilus</i> NCFM at timepoint 10 h measured by RT-PCR (<i>L. acidophilus</i> NCFM+<i>B. bifidum</i> Z9 versus <i>L. acidophilus</i> NCFM, <i>P</i><0.01). <b>B</b>: Protein expression of IFN-β, Cxcl10 and IL-12 in murine <i>L. acidophilus</i> NCFM stimulated dendritic cells measured by ELISA in the presence of the MAPK inhibitors p38, JNK1/2 and MEK1/2 (<i>L. acidophilus</i> NCFM versus inhibitors, <i>P</i><0.01).</p

Primers and Probes used for Real-Time PCR Analysis.

<p>Primers and Probes used for Real-Time PCR Analysis.</p

Microarray analysis of dendritic cells stimulated with <i>Lactobacillus acidophilus</i> NCFM and <i>Bifidobacterium bifidum</i> Z9.

<p>Up- and downregulation of genes upon incubation of dendritic cells with <i>L. acidophilus</i> NCFM and <i>B. bifidum</i> Z9, respectively, and both strains in combination. A. Venn diagram depicting the distinct up- and down regulation and overlap of genes. Only genes with an absolute fold change greater than 2 were included. B. Parametric Gene Set Enrichment of Gene Ontology (GO) Biological Process terms of the top 30 most significant GO terms comparing <i>L. acidophilus</i> NCFM vs. control (no stimulation). Blue indicates repression of the genes, white indicates no change and red indicates induction of the genes in the GO term. Only the GO term “response to virus” was significantly different (*) when comparing <i>L. acidophilus NCFM</i> and <i>B. bifidum Z9</i> responses.</p

<i>Lactobacillus acidophilus</i> NCFM induces IFN-β in dendritic cells.

<p>The IFN-β encoding gene is strongly up-regulated in <i>L .acidophilus NCFM</i> stimulated dendritic cells and responsible for the expression of the chemokines Cxcl10 and Ccl12. <b>A</b>. Expression of the IFN-β encoding gene over time in dendritic cells upon stimulation with <i>L. acidophilus</i> NCFM and <i>B. bifidum</i> Z9. <b>B</b>. Inhibition of IFN-β activity induced by <i>L. acidophilus</i> NCFM by addition of polyclonal IFN-β antibodies measured via the gene expression of the chemokine <i>Cxcl10</i> after 10 h and <b>C</b>. via the gene expression of the chemokine <i>Ccl12</i> after 10 h and <b>D</b>. via protein production of Cxcl10 in the supernatant after 24 h of stimulation by ELISA (<i>P</i><0.01).</p

Induction of cytokines in <i>Lactobacillus acidophilus</i> NCFM and <i>Bifidobacterium bifidum</i> Z9 stimulated dendritic cells.

<p>Protein expression of cytokines was measured by ELISA in supernatants from dendritic cells after 20 h of stimulation with <i>L. acidophilus</i> NCFM, <i>B. bifidum</i> Z9, or with both strains in combination. Supernatant from unstimulated DC were used as controls (<i>L. acidophilus</i> NCFM+<i>B. bifidum</i> Z9 versus <i>L. acidophilus</i> NCFM <i>P</i><0.01). Results are representative of 2 experiments.</p

Induction of chemokines in <i>Lactobacillus acidophilus</i> NCFM and <i>Bifidobacterium bifidum</i> Z9 stimulated dendritic cells.

<p>Gene expression analysis of the chemokines <i>Cxcl10</i>, <i>Ccl12</i>, <i>Cxcl1</i> and <i>Cxcl2</i> in dendritic cells upon stimulation with <i>L. acidophilus</i> NCFM, <i>B. bifidum</i> Z9 or the two bacteria in combination and unstimulated dendritic cells (control) was performed by RT-PCR after 10 h of stimulation (<i>L. acidophilus</i> NCFM+<i>B. bifidum</i> Z9 versus <i>L. acidophilus</i> NCFM, <i>P</i><0.01).</p