Ex Vivo Immunomodulatory Effects of Lactobacillus-, Lacticaseibacillus-, and Bifidobacterium-Containing Synbiotics on Human Peripheral Blood Mononuclear Cells and Monocyte-Derived Dendritic Cells in the Context of Grass Pollen Allergy.

Sensing of the intestinal microbiota by the host immune system is important to induce protective immune responses. Hence, modification of the gut microbiota might be able to prevent or treat allergies, mediated by proinflammatory Th2 immune responses. The aim was to investigate the ex vivo immunomodulatory effects of the synbiotics Pollagen® and Kallergen®, containing the probiotic bacterial strains Lactobacillus, Lacticaseibacillus and Bifidobacterium, in the context of grass pollen allergy. Peripheral blood mononuclear cells (PBMCs) from grass pollen-allergic patients and healthy controls were stimulated with grass pollen extract (GPE) and synbiotics and Gata3 expression and cytokine secretion analyzed. Monocyte-derived dendritic cells (MoDCs) cells were matured in the presence of GPE and synbiotics, co-cultured with autologous naïve T cells and maturation markers and cytokine secretion analyzed. GPE stimulation of PBMCs from grass pollen-allergic patients resulted in a significant higher production of the Th2 cytokines IL-4, IL-5, IL-9 and IL-13 compared to healthy controls. Gata3+CD4+ T cell induction was independent of the allergic status. The synbiotics promoted IL-10 and IFN-γ secretion and downregulated the GPE-induced Th2-like phenotype. Co-culturing naïve T cells with MoDCs, matured in the presence of GPE and synbiotics, shifted the GPE-induced Th2 cytokine release towards Th1-Th17-promoting conditions in allergic subjects. The investigated synbiotics are effective in downregulating the GPE-induced Th2 immune response in PBMCs from grass pollen-allergic patients as well as in autologous MoDC-T cell stimulation assays. In addition to increased IL-10 release, the data indicates a shift from a Th2- to a more Th1- and Th17-like phenotype.

Hydroalcoholic extract from Origanum vulgare induces a combined anti-mycobacterial and anti-inflammatory response in innate immune cells.

In nature, many plants or their extracted compounds have been found to possess anti-inflammatory features and therapeutic properties against infectious as well as non-infectious diseases, including cancer. In this study, we analysed the immunomodulatory effects on innate immune cells of hydroalcoholic extract from Origanum vulgare L. ssp. hirtum (HyE-Ov), a plant traditionally known for its anti-oxidative properties. The effects of HyE-Ov were tested on human monocyte derived dendritic cells (DC), type-1 (M1) and type-2 macrophages (M2) infected with M. bovis Bacille Calmette-Guérin (BCG), used as a model of persistent intracellular bacterium. DC, M1 and M2 treated with HyE-Ov significantly enhanced their mycobactericidal activity, which was associated with phagosomal acidification in M1 and M2 and increase of phagosomal, but not mitochondrial ROS production in M1, M2, and DC. Treatment of BCG-infected DC with HyE-Ov significantly reduced TNF-α and IL-12 production and increased TGF-ß synthesis. Finally, experiments were repeated using eight different HPLC fractions of HyE-Ov. Results showed that the capability to activate anti-microbial and anti-inflammatory response is shared by different fractions, suggesting that diverse bioactive molecules are present within the hydroalcoholic extract. Altogether, these results show that HyE-Ov promotes anti-mycobacterial innate immunity and limits inflammatory response in vitro and suggest that this plant extract may be exploitable as phytocomplex or nutraceutical for novel host-directed therapeutic approaches.

Activités immunomodulatrice, anti-inflammatoire, antiprurigineuse et tolérogénique induites par I-modulia®, un extrait issu de culture d'Aquaphilus dolomiae, dans les modèles pharmacologiques de dermatite atopique: Immunomodulatory, anti-inflammatory, anti-pruritus and tolerogenic activities induced by I-modulia®, an Aquaphilus dolomiae culture extract, in atopic dermatitis pharmacology models.

Atopic dermatitis (AD) is an inflammatory and pruritic dermatosis of multifactorial origin. Topical steroids are the first line treatment for severe AD however alternatives treatment are increasingly needed. A biological concentrate was elaborated from culture of an Avène aquatic microflora isolate namely Aquaphilus dolomiae. Numerous extracts were evaluated in relevant AD in vitro models with human keratinocytes. Among these extracts, a particular one I-modulia® was found to be remarkable in terms of pharmacological activities: innate immunity modulating by agonizing Toll like receptor (TLR)2, TLR4 and TLR5, induction of anti-microbial peptides, inhibition of cytokines characteristics of T helper (Th)1, Th2 and Th17 responses, inhibition of Protease-activated-receptor (PAR) 2 and Thymic-stromal-lymphopoeitin (TSLP) both being known to be upregulated in pruritus. Additionally, when human dendritic cells (DC) were stimulated in vitro by Staphylococcus aureus secretomes from AD children lesions, I-modulia® was capable to induce IL-10 secretion to activate regular T lymphocytes and rendered DC tolerogenic. I-modulia®, extract of biotech origin incorporated in emollient, displays anti-inflammatory, anti-pruritus activities, restores homeostasis immune and ameliorates AD in young infant.

Pore-formation by adenylate cyclase toxoid activates dendritic cells to prime CD8+ and CD4+ T cells.

The adenylate cyclase toxin-hemolysin (CyaA) of Bordetella pertussis is a bi-functional leukotoxin. It penetrates myeloid phagocytes expressing the complement receptor 3 and delivers into their cytosol its N-terminal adenylate cyclase enzyme domain (~400 residues). In parallel, ~1300 residue-long RTX hemolysin moiety of CyaA forms cation-selective pores and permeabilizes target cell membrane for efflux of cytosolic potassium ions. The non-enzymatic CyaA-AC(-) toxoid, has repeatedly been successfully exploited as an antigen delivery tool for stimulation of adaptive T-cell immune responses. We show that the pore-forming activity confers on the CyaA-AC(-) toxoid a capacity to trigger Toll-like receptor and inflammasome signaling-independent maturation of CD11b-expressing dendritic cells (DC). The DC maturation-inducing potency of mutant toxoid variants in vitro reflected their specifically enhanced or reduced pore-forming activity and K(+) efflux. The toxoid-induced in vitro phenotypic maturation of DC involved the activity of mitogen activated protein kinases p38 and JNK and comprised increased expression of maturation markers, interleukin 6, chemokines KC and LIX and granulocyte-colony-stimulating factor secretion, prostaglandin E2 production and enhancement of chemotactic migration of DC. Moreover, i.v. injected toxoids induced maturation of splenic DC in function of their cell-permeabilizing capacity. Similarly, the capacity of DC to stimulate CD8(+) and CD4(+) T-cell responses in vitro and in vivo was dependent on the pore-forming activity of CyaA-AC(-). This reveals a novel self-adjuvanting capacity of the CyaA-AC(-) toxoid that is currently under clinical evaluation as a tool for delivery of immunotherapeutic anti-cancer CD8(+) T-cell vaccines into DC.

Cecum lymph node dendritic cells harbor slow-growing bacteria phenotypically tolerant to antibiotic treatment.

In vivo, antibiotics are often much less efficient than ex vivo and relapses can occur. The reasons for poor in vivo activity are still not completely understood. We have studied the fluoroquinolone antibiotic ciprofloxacin in an animal model for complicated Salmonellosis. High-dose ciprofloxacin treatment efficiently reduced pathogen loads in feces and most organs. However, the cecum draining lymph node (cLN), the gut tissue, and the spleen retained surviving bacteria. In cLN, approximately 10%-20% of the bacteria remained viable. These phenotypically tolerant bacteria lodged mostly within CD103⁺CX3CR1⁻CD11c⁺ dendritic cells, remained genetically susceptible to ciprofloxacin, were sufficient to reinitiate infection after the end of the therapy, and displayed an extremely slow growth rate, as shown by mathematical analysis of infections with mixed inocula and segregative plasmid experiments. The slow growth was sufficient to explain recalcitrance to antibiotics treatment. Therefore, slow-growing antibiotic-tolerant bacteria lodged within dendritic cells can explain poor in vivo antibiotic activity and relapse. Administration of LPS or CpG, known elicitors of innate immune defense, reduced the loads of tolerant bacteria. Thus, manipulating innate immunity may augment the in vivo activity of antibiotics.

Regulation of macrophage and dendritic cell function by pathogens and through immunomodulation in the avian mucosa.

Macrophages (MPh) and dendritic cells (DC) are members of the mononuclear phagocyte system. In chickens, markers to distinguish MPh from DC are lacking, but whether MPh and DC can be distinguished in humans and mice is under debate, despite the availability of numerous markers. Mucosal MPh and DC are strategically located to ingest foreign antigens, suggesting they can rapidly respond to invading pathogens. This review addresses our current understanding of DC and MPh function, the receptors expressed by MPh and DC involved in pathogen recognition, and the responses of DC and MPh against respiratory and intestinal pathogens in the chicken. Furthermore, potential opportunities are described to modulate MPh and DC responses to enhance disease resistance, highlighting modulation through nutraceuticals and vaccination.

Microbes and microbial effector molecules in treatment of inflammatory disorders.

The healthy gut tolerates very large numbers of diverse bacterial species belonging mainly to the Bacteroidetes and Firmicutes phyla. These bacteria normally coexist peacefully with the gut and help maintain immune homeostasis and tolerance. The mechanisms promoting tolerance affect various cell populations, including the epithelial cells lining the gut, resident dendritic cells (DCs), and gut-homing T cells. Gut bacteria also influence multiple signaling pathways from Toll-like receptors to nuclear factor κB and regulate the functionality of DCs and T cells. Several bacterial species have been identified that promote T-cell differentiation, in particular T-helper 17 and T-regulatory cells. Insight into the molecular mechanisms by which bacteria mediate these effects will be very important in identifying new ways of treating intestinal and extra-intestinal immune-mediated diseases. These diseases are increasing dramatically in the human population and require new treatments. It may be possible in the future to identify specific bacterial species or strains that can correct for T-cell imbalances in the gut and promote immune homeostasis, both locally and systemically. In addition, new information describing microbial genomes affords the opportunity to mine for functional genes that may lead to new generation drugs relevant to a range of inflammatory disease conditions.

Bifidobacterium bifidum NCC 453 promotes tolerance induction in murine models of sublingual immunotherapy.

BACKGROUND: Enhancing clinical efficacy remains a major goal in allergen-specific immunotherapy. In this study, we tested three strains of bifidobacteria as candidate adjuvants for sublingual allergy vaccines. METHODS: Probiotic candidates were evaluated in human monocyte-derived dendritic cell (h-DC) maturation and CD4(+) T-cell polarization in vitro models and further tested in murine models of sublingual immunotherapy in BALB/c mice sensitized to either ovalbumin or birch pollen. RESULTS: Bifidobacterium adolescentis, B. bifidum and B. longum induced h-DC maturation and polarized naïve CD4(+) T cells toward interferon-γ and interleukin-10 production. B. bifidum increased CD25(high), Foxp3(+) cells within CD4(+) T lymphocytes and was the most potent inducer of interferon-γ in Th2-skewed peripheral blood mononuclear cells and h-DC T-cell cocultures. It also induced a significant decrease in airway hyperresponsiveness in BALB/c mice sensitized to ovalbumin. Sublingual administration of B. bifidum together with recombinant Bet v 1 enhanced tolerance induction in BALB/c mice sensitized to birch pollen, with a downregulation of both airway hyperresponsiveness, lung inflammation and Bet v 1-specific Th2 responses. CONCLUSIONS: Due to its capacity to reorient established Th2 responses toward Th1/regulatory T-cell profiles, B. bifidum represents a valid candidate adjuvant for specific immunotherapy of type I allergies.

Colostrum and amniotic fluid from different species exhibit similar immunomodulating effects in bacterium-stimulated dendritic cells.

The fetus and newborn are immunologically immature. Bioactive compounds in amniotic fluid (AF) and maternal milk therefore play a key role in the immunological development of the infant intestine. We hypothesized that colostrum and AF exert similar immunomodulatory effects on the developing immune system. Hence, bone marrow-derived murine dendritic cells (BMDCs) were co-incubated with Clostridium perfringens A or Escherichia coli Nissle 1917 and porcine, bovine, or human AF, colostrum/milk whey fractions. Interleukin (IL) 10, IL-12, IL-6, and tumor necrosis factor-? (TNF-?) production was measured. IL-12 production was reduced with all AFs and wheys, and IL-6 and TNF-? were also reduced by porcine AF. Porcine and bovine whey both reduced TNF-? production. Overall, the reductions were most pronounced with the porcine fluids. Only bovine fluids caused strong induction of IL-10. Overall, effects of AF and whey from same species were similar. Viability of stimulated BMDCs was not significantly affected by the fluids. Neutralization of IL-10, transforming growth factor ?, and epidermal growth factor (EGF) did not remove the IL-12-inhibiting effect, but EGF neutralization increased IL-10 production. Addition of EFG to DCs enhanced the bacterium-induced cytokine production contrary to the effect of AF and colostrums, ruling out EGF as the inhibitory component in the fluids.

MyD88-dependent SHIP1 regulates proinflammatory signaling pathways in dendritic cells after monophosphoryl lipid A stimulation of TLR4.

We previously showed that monophosphoryl lipid A (MLA) activates TLR4 in dendritic cells (DCs) in a Toll/IL-1R domain-containing adaptor inducing IFN-ß (TRIF)-biased manner: MLA produced from Salmonella minnesota Re595 induced signaling events and expression of gene products that were primarily TRIF dependent, whereas MyD88-dependent signaling was impaired. Moreover, when tested in TRIF-intact/MyD88-deficient DCs, synthetic MLA of the Escherichia coli chemotype (sMLA) showed the same activity as its diphosphoryl, inflammatory counterpart (synthetic diphosphoryl lipid A), indicating that TRIF-mediated signaling is fully induced by sMLA. Unexpectedly, we found that the transcript level of one proinflammatory cytokine was increased in sMLA-treated cells by MyD88 deficiency to the higher level induced by synthetic diphosphoryl lipid A, which suggested MyD88 may paradoxically help restrain proinflammatory signaling by TRIF-biased sMLA. In this article, we demonstrate that sMLA induces MyD88 recruitment to TLR4 and activates the anti-inflammatory lipid phosphatase SHIP1 in an MyD88-dependent manner. At the same time, MyD88-dependent signaling activity at the level of IL-1R-associated kinase 1 is markedly reduced. Increased SHIP1 activity is associated with reductions in sMLA-induced IκB kinase α/ß and IFN regulatory factor 3 activation and with restrained expression of their downstream targets, endothelin-1 and IFN-ß, respectively. Results of this study identify a pattern that is desirable in the context of vaccine adjuvant design: TRIF-biased sMLA can stimulate partial MyD88 activity, with MyD88-dependent SHIP1 helping to reduce proinflammatory signaling in DCs.

Endogenous Fms-like tyrosine kinase-3 ligand levels are not altered in mice after a severe burn and infection.

BACKGROUND: Fms-like tyrosine kinase-3 ligand (Flt3L) is a hemopoietic cytokine and dendritic cell (DC) growth factor that promotes the proliferation and differentiation of progenitor cells into DCs. We have previously found that treatment of severely burned mice with recombinant Flt3L significantly enhances DC production and bacterial clearance from infected burn wounds, and increases global immune cell activation and survival in response to a burn wound infection. These significant benefits of Flt3L treatment after burn injury have prompted the question of whether or not severe burn injury induces deficits in endogenous Flt3L levels that could affect DCs and subsequent responses to infection. RESULTS: To address this, male BALB/c mice received a 30% total body surface area scald burn. Blood, spleens, and wound-draining lymph nodes were harvested at various time-points after injury. Some mice received a wound inoculation with P. aeruginosa. Murine Flt3L and G-CSF levels were measured by ELISA. Burn injury had no significant effect on Flt3L levels at any post-burn time-point examined compared to normal Flt3L levels in the sera, spleen, or lymph nodes. Additionally, Flt3L levels in the sera, spleen, and lymph nodes were not significantly altered when wounds were inoculated on the day of burn injury or at post-burn time points examined. Alternatively, levels of G-CSF were increased in response to burn injury and burn wound infection. Additionally, DC numbers and functions were not altered following burn injury alone. There was no significant difference between the number of DCs in the spleens of sham-injured mice and mice at 5 days after burn injury. When naïve T cells from sham-injured mice were co-cultured with DCs from either sham- or burn-injured mice, IFN-gamma production was similar, however, IFN-gamma levels produced by T cells harvested from burn-injured mice were significantly lower than those produced by T cells from sham mice, regardless of which DC group, sham or burn, was used in the coculture. CONCLUSION: These data suggest that the beneficial effects of Flt3L treatments after burn injury are not due to correction of a burn-associated Flt3L deficiency but rather, are likely due to supplementary stimulation of DC production and immune responses to infection.

Cannabinoid treatment suppresses the T-helper cell-polarizing function of mouse dendritic cells stimulated with Legionella pneumophila infection.

Marijuana cannabinoids, such as delta-9-tetrahydrocannabinoid (THC), suppress type 1 T-helper 1 (Th1) immunity in a variety of models, including infection with the intracellular pathogen Legionella pneumophila (Lp). To examine the cellular mechanism of this effect, bone marrow-derived dendritic cells (DCs) were purified from BALB/c mice and studied following infection and drug treatment. DCs infected in vitro with Lp were able to protect mice when injected prior to a lethal Lp infection; however, the immunization potential of the Lp-loaded cells along with Th1 cytokine production was attenuated by THC treatment at the time of in vitro infection. In addition, THC-treated and Lp-loaded DCs were poorly stimulated in culture-primed splenic CD4(+) T cells to produce interferon-gamma; however, this stimulating deficiency was reversed by adding recombinant interleukin (IL)-12p40 protein to the cultures. Moreover, THC treatment inhibited the expression of DC maturation markers, such as major histocompatibility complex class II and costimulatory molecules CD86 and CD40 as determined by flow cytometry and suppressed the Notch ligand, Del-ta4, as determined by reverse transcription-polymerase chain reaction. However, THC treatment did not affect other DC functions, such as intracellular killing of Lp, determined by colony-forming unit counts of bacteria, and Lp-induced apoptosis, determined by annexin V staining. In conclusion, the data suggest that THC inhibits Th1 activation by targeting essential DC functions, such as IL-12p40 secretion, maturation, and expression of costimulatory and polarizing molecules.

Potentiation of antigen-stimulated V gamma 9V delta 2 T cell cytokine production by immature dendritic cells (DC) and reciprocal effect on DC maturation.

Vgamma9Vdelta2 T cells, a major gammadelta PBL subset in human adults, have been previously implicated in dendritic cell (DC) licensing, owing to their high frequency in peripheral tissues and their ability to produce inflammatory cytokines upon recognition of a broad array of conserved Ags. Although these observations implied efficient recognition of Ag-expressing immature DC (iDC) by Vgamma9Vdelta2 T cells, the role played by DC subsets in activation of these lymphocytes has not been carefully studied so far. We show that iDC, and to a lesser extent mature DC, potentiated Th1 and Th2 cytokine, but not cytolytic or proliferative responses, of established Vgamma9Vdelta2 T cell clones and ex vivo memory Vgamma9Vdelta2 PBL stimulated by synthetic agonists. The ability of iDC to potentiate Vgamma9Vdelta2 production of inflammatory cytokines required for their own maturation suggested that Vgamma9Vdelta2 T cells, despite their strong lytic activity, could promote efficient iDC licensing without killing at suboptimal Ag doses. Accordingly Vgamma9Vdelta2 cells induced accelerated maturation of Ag-expressing iDC but not "bystander" DC, even within mixed cell populations comprising both Ag-expressing and nonexpressing iDC. Furthermore Vgamma9Vdelta2 cells induced full differentiation into IL-12-producing cells of iDC infected by Vgamma9Vdelta2-stimulating mycobacteria that were otherwise unable to induce complete DC maturation. In conclusion the ability of iDC to selectively potentiate cytokine response of memory Vgamma9Vdelta2 T cells could underlie the adjuvant effect of these lymphocytes, and possibly other natural memory T cells, on conventional T cell responses.

Mechanisms of mouse spleen dendritic cell function in the generation of influenza-specific, cytolytic T lymphocytes.

We have evaluated the capacity of dendritic cells to function as antigen-presenting cells (APCs) for influenza and have examined their mechanism of action. Virus-pulsed dendritic cells were 100 times more efficient than bulk spleen cells in stimulating cytotoxic T lymphocyte (CTL) formation. The induction of CTLs required neither exogenous lymphokines nor APCs in the responding T cell population. Infectious virus entered dendritic cells through intracellular acidic vacuoles and directed the synthesis of several viral proteins. If ultraviolet (UV)-inactivated or bromelain-treated viruses were used, viral protein synthesis could not be detected, and there was poor induction of CTLs. This indicated that dendritic cells were not capable of processing noninfectious virus onto major histocompatibility complex (MHC) class I molecules. However, UV-inactivated and bromelain-treated viruses were presented efficiently to class II-restricted CD4+ T cells. The CD4+ T cells crossreacted with different strains of influenza and markedly amplified CTL formation. Cell lines that lacked MHC class II, and consequently the capacity to stimulate CD4+ T cells, failed to induce CTLs unless helper lymphokines were added. Similarly, dendritic cells pulsed with the MHC class I-restricted nucleoprotein 147-155 peptide were poor stimulators in the absence of exogenous helper factors. We conclude that the function of dendritic cells as APCs for the generation of virus-specific CTLs in vitro depends measurably upon: (a) charging class I molecules with peptides derived from endogenously synthesized viral antigens, and (b) stimulating a strong CD4+ helper T cell response.