Histamine Regulates Actin Cytoskeleton in Human Toll-like Receptor 4-activated Monocyte-derived Dendritic Cells Tuning CD4+ T Lymphocyte Response.

Histamine, a major mediator in allergic diseases, differentially regulates the polarizing ability of dendritic cells after Toll-like receptor (TLR) stimulation, by not completely explained mechanisms. In this study we investigated the effects of histamine on innate immune reaction during the response of human monocyte-derived DCs (mDCs) to different TLR stimuli: LPS, specific for TLR4, and Pam3Cys, specific for heterodimer molecule TLR1/TLR2. We investigated actin remodeling induced by histamine together with mDCs phenotype, cytokine production, and the stimulatory and polarizing ability of Th0. By confocal microscopy and RT-PCR expression of Rac1/CdC42 Rho GTPases, responsible for actin remodeling, we show that histamine selectively modifies actin cytoskeleton organization induced by TLR4, but not TLR2 and this correlates with increased IL4 production and decreased IFNγ by primed T cells. We also demonstrate that histamine-induced cytoskeleton organization is at least in part mediated by down-regulation of small Rho GTPase CdC42 and the protein target PAK1, but not by down-regulation of Rac1. The presence and relative expression of histamine receptors HR1-4 and TLRs were determined as well. Independently of actin remodeling, histamine down-regulates IL12p70 and CXCL10 production in mDCs after TLR2 and TLR4 stimulation. We also observed a trend of IL10 up-regulation that, despite previous reports, did not reach statistical significance.

A novel allergen-adjuvant conjugate suitable for specific immunotherapy of respiratory allergy.

BACKGROUND: Several approaches to find a better adjuvant, focus immunomodulation, and reduce allergenicity are under investigation to improve the efficacy and safety of specific immunotherapy. OBJECTIVE: We performed an investigation of the in vitro and in vivo effects of a purified allergen chemically conjugated to a novel 8-OH modified adenine as an adjuvant. METHODS: Purified group 2 major allergen from house dust mite chemically conjugated to 4-(6-amino-9-benzyl-8-hydroxy-9H-purin-2-ylsulfanyl)-butyric acid succinimidyl ester was analyzed by using mass spectrometry. The adduct (nDer p 2-Conj) was assayed for Toll-like receptor activation on transfected HEK293 cells, stimulation of innate cells, and effects on the functional phenotype of specific T-cell lines and clones by means of flow cytometry, real-time PCR, and expression of TH-related transcription factors. Lung cells and sera of nDer p 2-Conj-sensitized C57Bl/6 mice were studied by means of cytology, histology, real-time PCR, and ELISA. RESULTS: nDer p 2-Conj stimulated IL-12 and IFN-α production from monocytes and plasmacytoid dendritic cells, respectively, retaining the ability to trigger Toll-like receptor 7 exclusively, and expanded human allergen-specific lymphocytes with reduced ability to produce T(H)2-related cytokines and increased IFN-γ levels, as based on GATA-3/T-bet expression. In vivo adduct-sensitized mice exhibited reduced eosinophil infiltration and IL-13 expression in the airways, IFN-γ upregulation together with IgE downregulation, and an increase in allergen-specific IgG(2a) levels in sera. The conjugate exhibited reduced ability to activate human FcεRI(+) cells without inducing T(H)17 cells or autoantibodies. CONCLUSIONS: The codelivery of an allergen with a modified adenine as a conjugate inducing modulatory cytokines from innate cells redirects in vitro and in vivo pathogenic TH2 responses without eliciting harmful effects.

Carbon nanotube scaffolds instruct human dendritic cells: modulating immune responses by contacts at the nanoscale.

Nanomaterials interact with cells and modify their function and biology. Manufacturing this ability can provide tissue-engineering scaffolds with nanostructures able to influence tissue growth and performance. Carbon nanotube compatibility with biomolecules motivated ongoing interest in the development of biosensors and devices including such materials. More recently, carbon nanotubes have been applied in several areas of nerve tissue engineering to study cell behavior or to instruct the growth and organization of neural networks. To gather further knowledge on the true potential of future constructs, in particular to assess their immune-modulatory action, we evaluate carbon nanotubes interactions with human dendritic cells (DCs). DCs are professional antigen-presenting cells and their behavior can predict immune responses triggered by adhesion-dependent signaling. Here, we incorporate DC cultures to carbon nanotubes and we show by phenotype, microscopy, and transcriptional analysis that in vitro differentiated and activated DCs show when interfaced to carbon nanotubes a lower immunogenic profile.

Etanercept downregulates the Th17 pathway and decreases the IL-17+/IL-10+ cell ratio in patients with psoriasis vulgaris.

PURPOSE: To evaluate circulating and lesional CD4(+) and CD8(+) cells belonging to Th1, Th2, and Th17 patterns as well as IL-10(+) cells before and after a 12-week lasting course with etanercept or acitretin in patients with psoriasis. METHODS: 15 patients were given etanercept 50 mg twice weekly and 15 patients acitretin 0,4 mg/kg/day, both for 12 weeks. At the baseline and at the end of the treatment, blood and skin samples were taken to investigate IL-4, IL-8, IL-10, IL-17, and IFN-γ-producing CD4(+) and CD8(+) cells. As controls, 10 healthy controls (HC) and 6 atopic dermatitis (AD) patients were included into the study. RESULTS: Psoriasis patients showed augmented IL-17- and IL-8-producing CD4(+) cells in the blood than HC and AD patients. In the skin lesions, IL-17(+) cells were more represented in psoriasis than in AD, while the number of IL-4-producing cells was reduced in psoriasis patients than in AD ones. Etanercept was able to significantly reduce the number of IL-17- and IL-8-producing CD4(+) and CD8(+) cells both in skin and blood, as well as to augment the proportion of IL-10-producing CD4(+) cells in the skin of psoriatic patients, while acitretin was not. CONCLUSIONS: Our results confirmed the role of Th17 cells in the pathogenesis of psoriasis. Etanercept, but not acitretin, was able to downregulate the Th17 pathway and to increase the percentages of IL-10-producing cells in the skin.

Modified adenine (9-benzyl-2-butoxy-8-hydroxyadenine) redirects Th2-mediated murine lung inflammation by triggering TLR7.

Substitute adenine (SA)-2, a synthetic heterocycle chemically related to adenine with substitutions in positions 9-, 2-, and 8- (i.e., 9-benzyl-2-butoxy-8-hydroxyadenine), induces in vitro immunodeviation of Th2 cells to a Th0/Th1 phenotype. In this article, we evaluate the in vivo ability of SA-2 to affect Th2-mediated lung inflammation and its safety. TLR triggering and NF-kappaB activation by SA-2 were analyzed on TLR-transfected HEK293 cells and on purified bone marrow dendritic cells. The in vivo effect of SA-2 on experimental airway inflammation was evaluated in both prepriming and prechallenge protocols by analyzing lung inflammation, including tissue eosinophilia and goblet cell hyperplasia, bronchoalveolar lavage fluid cell types, and the functional profile of Ag-specific T cells from draining lymph nodes and spleens. SA-2 induced mRNA expression and production of proinflammatory (IL-6, IL-12, and IL-27) and regulatory (IL-10) cytokines and chemokines (CXCL10) in dendritic cells but down-regulated TGF-beta. Prepriming administration of SA-2 inhibited OVA-specific Abs and Th2-driven lung inflammation, including tissue eosinophilia and goblet cells, with a prevalent Foxp3-independent regulatory mechanism. Prechallenge treatment with SA-2 reduced the lung inflammation through the induction of a prevalent Th1-related mechanism. In this model the activity of SA-2 was route-independent, but adjuvant- and Ag dose-dependent. SA-2-treated mice did not develop any increase of serum antinuclear autoantibodies. In conclusion, critical substitutions in the adenine backbone creates a novel synthetic TLR7 ligand that shows the ability to ameliorate Th2-mediated airway inflammation by a complex mechanism, involving Th1 redirection and cytokine-mediated regulation, which prevents autoreactivity.

Toll-like receptors 3 and 4 are expressed by human bone marrow-derived mesenchymal stem cells and can inhibit their T-cell modulatory activity by impairing Notch signaling.

Bone marrow (BM)-derived mesenchymal stem cells (MSCs) are multipotent, nonhemopoietic progenitors that also possess regulatory activity on immune effector cells through different mechanisms. We demonstrate that human BM-derived MSCs expressed high levels of Toll-like receptors (TLRs) 3 and 4, which are both functional, as shown by the ability of their ligands to induce nuclear factor kappaB (NF-kappaB) activity, as well as the production of interleukin (IL)-6, IL-8, and CXCL10. Of note, ligation of TLR3 and TLR4 on MSCs also inhibited the ability of these cells to suppress the proliferation of T cells, without influencing their immunophenotype or differentiation potential. The TLR triggering effects appeared to be related to the impairment of MSC signaling to Notch receptors in T cells. Indeed, MSCs expressed the Notch ligand Jagged-1, and TLR3 or TLR4 ligation resulted in its strong downregulation. Moreover, anti-Jagged-1 neutralizing antibody and N[N-(3,5-difluorophenacetyl-l-alanyl)]-S-phenylglycine t-butyl ester (DAPT), an inhibitor of Notch signaling, hampered the suppressive activity of MSCs on T-cell proliferation. These data suggest that TLR3 and TLR4 expression on MSCs may provide an effective mechanism to block the immunosuppressive activity of MSCs and therefore to restore an efficient T-cell response in the course of dangerous infections, such as those sustained by double-stranded RNA viruses or Gram-negative bacteria, respectively.

Differences in mesenchymal stem cell cytokine profiles between MS patients and healthy donors: implication for assessment of disease activity and treatment.

MSCs have been proposed as possible treatment in MS: In this study MSCs obtained from 10 MS patients and 6 healthy donors (HD) were compared in terms of phenotypical and functional characteristics. We show that MSCs isolated from MS and HD differ significantly for IP10 production. Therefore, although MSCs isolated from MS patients exhibit the same properties of HD MSCs in terms of proliferation, phenotype, in vitro differentiation, TLR expression, immunosuppressive ability, inhibition of DC differentiation and activation, the use of autologous MSCs in cell therapy of autoimmune diseases should be submitted to attentive evaluation and treatment.

Increased CXCL10 expression in MS MSCs and monocytes is unaffected by AHSCT.

OBJECTIVE: To confirm CXCL10 over production in bone marrow mesenchymal stem cells (MSCs) and circulating monocytes isolated from multiple sclerosis patients (MS) and identify predate cell molecular signature; to extend this analysis after autologous hematopoietic stem cell transplantation (AHSCT) to test if therapy has modifying effects on MSCs and circulating monocytes. METHODS: MSCs and monocytes were isolated from 19 MS patients who undergone AHSCT before and seven of them at least 3 years after transplant. CXCL10 production was detected after LPS/IFN-γ stimulation. TLR4 signaling pathways were investigated by means of transcription factors phosphorylation/activation level. RT-PCR of activated transcription factors was performed to quantify their expression. All experiments were conducted in parallel with 24 matched healthy donors (HD). RESULTS: CXCL10 expression was significantly increased in both peripheral circulating monocytes and BM MSCs compared to HD. We showed that CXCL10 production is determined by an altered signaling pathway downstream TLR4, with the involvement of STAT-1, NF-κB, p38, JNK, and CREB. All upregulated transcription factors are more phosphorylated in MS patient sample. These features are not modified after AHSCT. INTERPRETATION: We demonstrated that in MS two different cell lineages are characterized by significantly increased production of CXCL10, due to altered signaling pathways of innate immune reaction mediated by TLR4, probably associated with disease phenotype. This characteristic is not modified by AHSCT, suggesting that when T and B lymphocytes are reset, other possible components of MS pathology, such as CXCL10 over production, do not determine therapy outcome.

Modulating dendritic cells (DC) from immunogenic to tolerogenic responses: a novel mechanism of AZA/6-MP.

Azathioprine (Aza), 6-Mercaptopurine (6-MP) and 6-Thioguanine (6-TG) are thiopurine drugs widely used as immunosuppressants/anti-inflammatory agents in organ transplantation and chemotherapy. Aza is well tolerated and effective in modifying the course of MS. Here we investigated the action of 6-MP on human dendritic cells (DCs). We described for the first time that 6-MP impairs in vitro differentiation of DCs, has an inhibitory effect during DC activation processes inducing a functionally less immunogenic phenotype. Moreover, 6-MP significantly reduces DC IL-23 production and CCR7 expression, at the same time induces IL-10 augmentation. All these findings add a novel action mechanism in Aza immune modulation.

Redirection of allergen-specific TH2 responses by a modified adenine through Toll-like receptor 7 interaction and IL-12/IFN release.

BACKGROUND: Natural or synthetic ligands of Toll-like receptors (TLRs), such as CpG-containing oligodeoxynucleotides and imidazoquinolines, affect the functional phenotype of antigen-specific human T lymphocytes by inducing cytokine release by cells of the innate immunity. OBJECTIVE: In vitro investigation of the ability of substitute adenines (SAs) to affect antigen-presenting cells and shift the functional phenotype of specific human T(H)2 cells was performed. METHODS: The functional profile of hapten- and allergen-specific T-cell lines obtained in the absence or presence of modified adenines was assessed by means of quantitative real-time PCR, flow cytometry, and ELISAs. Activation of TLRs was evaluated by means of nucleofection of HEK293 cells. RESULTS: The synthetic heterocycle, chemically related to adenine with substitution in positions 2-, 8-, and 9- (SA-2), but not its related derivative lacking 2- and 8- substitutions, stimulated the production of high amounts of IL-12, IL-10, TNF-alpha, and IL-6 by CD14(+) cells and IFN-alpha and CXCL10 by blood dendritic cell antigen (BDCA)-4(+) plasmacytoid dendritic cells. A nuclear factor kappaB-dependent signaling pathway mediated by SA-2 ligation of TLR7 was responsible for these effects. SA-2 also redirected the in vitro differentiation of either Dermatophagoides pteronyssinus group 1 or amoxicillin-specific T(H)2 cells toward the T(H)1/T(H)0 phenotype, with parallel downregulation of GATA-3 and upregulation of T-box expressed in T cells transcription factors. CONCLUSION: Critical substitutions of the adenine backbone confer the ability to activate TLR7, inducing the production of modulatory cytokines able to shift human allergen-specific T(H)2 cells to a T(H)1/T(H)0 phenotype. CLINICAL IMPLICATIONS: Appropriately modified adenines might be used as effective adjuvants for the development of novel immunotherapeutic strategies of allergic disorders.

Vascular endothelial growth factor receptor-2 and low affinity VEGF binding sites on human glomerular endothelial cells: Biological effects and advanced glycosilation end products modulation.

Vascular Endothelial Growth Factor (VEGF), binding to its receptor in endothelial cells, seems to modulate the increased blood flow in the early phase of diabetic renal disease. The aim of the study was to evaluate, in a diabetic milieu, the expression, biological function and modulation of VEGF binding sites in human glomerular endothelial cells (GENC). We demonstrated the presence of VEGF binding sites with high (VEGFR-2) and low (heparan sulfate proteoglycans, HSPG) affinity. VEGF165 and VEGF121 working through VEGFR-2 stimulated nitric oxide (NO) production at low doses (0.1-1 nM), whereas only VEGF165 at high doses (10-100 nM) increased thymidine incorporation. 1 nM VEGF165 and VEGF121 induced in GENC a significant peak of inducible NO synthase (iNOS) production and, at a lower level, of endothelial NOS (eNOS). The copresence of VEGF165 with aminoguanidine (iNOS inhibitor) determined an increase of eNOS and a significant increase in thymidine incorporation. Advanced glycation end products (AGEs) working through specific receptors (RAGE) up-regulated the expression of VEGFR-2, decreased the expression of HSPG sites and reduced GENC growth. These results identify in GENC VEGFR-2 as a mediator of iNOS and eNOS release under control of VEGF, whereas HSPG binding sites seem to mediate the weak growth effect. The presence of AGEs, up-regulating the VEGFR-2 and decreasing HSPG sites might participate to the block of glomerular angiogenesis addressing the VEGF effects on glomerular permeability.

AT1 and AT2 receptors in human glomerular endothelial cells at different passages.

In human adult kidney angiotensin II (AngII) effects are mediated by the AT1 receptor, while the functions of AT2 receptors are mostly unknown. Since AngII regulates endothelial cell growth by AT1 and AT2 receptors, we analysed their functional aspects at different passages in human glomerular endothelial cells (GENC). Semiquantitative reverse transcription-polymerase chain reaction revealed the presence of AT1 and AT2 receptors between 2p and 15p cell passages with different levels of expression. In fact, binding studies of different families of displacement curves using AngII, DUP753 (AT1 antagonist), and PD123177 (AT2 antagonist) showed the presence of AT1a and AT2 receptors at 4p-9p while in GENC 2p only the presence of AT2. In terms of mitogenic activity, AngII was unable to stimulate GENC 2p growth. On the contrary, in GENC 4p-9p and 15p a significant thymidine incorporation was observed. This stimulatory effect seemed to be induced also by the concomitant release of PDGF-BB AT1a mediated. In conclusion, AT1a and AT2 receptors are represented in GENC with a different ratio depending upon the cell passage. AngII regulates the mitogenic effect through AT1a receptors (in later cell passages 4p-15p) involving the release of PDGF-BB, while AT2 (in early cell passage 2p) showed a predominant negative growth control.

The novel synthetic immune response modifier R-848 (Resiquimod) shifts human allergen-specific CD4+ TH2 lymphocytes into IFN-gamma-producing cells.

BACKGROUND: In experimental models, imidazoquinolines exhibit several immunomodulatory activities via Toll-like receptor signaling on cells of the innate immunity. OBJECTIVE: The aim of our study was to investigate whether R-848 (Resiquimod), a small-molecular-weight synthetic compound belonging to the imidazoquinoline family and known for its ability to substantially delay the onset of recurrent genital herpes lesions in both animals and human beings, could influence, at least in vitro, the cytokine production profile of human hapten- or allergen-specific T cells. METHODS: Ampicillin- and Der p 1-specific T-cell lines were derived from peripheral blood of allergic donors in the absence or presence of R-848 and assessed by flow cytometry at the single-cell level for their ability to produce IL-4 and/or IFN-gamma. RESULTS: R-848 induced both hapten- and allergen-specific circulating T cells, including T(H)2 effectors, to produce IFN-gamma and even to lose the ability to produce IL-4, thus shifting their phenotype of cytokine production to a type 0 (T(H)0) or even T(H)1 profile. This effect was associated with an increase in the production of IL-12, IFN-alpha, IL-18, TNF-alpha, IL-10, and IL-15 by CD14(+) cells, as well as an increase in the proportions of IFN-gamma-producing CD3(-)CD16(+) (natural killer) cells. CONCLUSIONS: These results suggest that R-848, and probably other imidazoquinolines, might be used as adjuvants in view of novel allergen-specific immunotherapeutic regimens for the treatment of allergic disorders.