Using distinct molecular signatures of human monocytes and dendritic cells to predict adjuvant activity and pyrogenicity of TLR agonists

We present a systematic study that defines molecular profiles of adjuvanticity and pyrogenicity induced by agonists of human Toll-like receptor molecules in vitro. Using P3CSK4, Lipid A and Poly I:C as model adjuvants we show that all three molecules enhance the expansion of IFNγ+/CD4+ T cells from their naïve precursors following priming with allogeneic DC in vitro. In contrast, co-culture of naive CD4+ T cells with allogeneic monocytes and TLR2/TLR4 agonists only resulted in enhanced T cell proliferation. Distinct APC molecular signatures in response to each TLR agonist underline the dual effect observed on T cell responses. Using protein and gene expression assays, we show that TNF-α and CXCL10 represent DC-restricted molecular signatures of TLR2/TLR4 and TLR3 activation, respectively, in sharp contrast to IL-6 produced by monocytes upon stimulation with P3CSK4 and Lipid A. Furthermore, although all TLR agonists are able to up-regulate proIL-1β specific gene in both cell types, only monocyte activation with Lipid A results in detectable IL-1β release. These molecular profiles, provide a simple screen to select new immune enhancers of human Th1 responses suitable for clinical application

The host response to the probiotic Escherichia coli strain Nissle 1917: Specific up-regulation of the proinflammatory chemokine MCP-1

BACKGROUND: The use of live microorganisms to influence positively the course of intestinal disorders such as infectious diarrhea or chronic inflammatory conditions has recently gained increasing interest as a therapeutic alternative. In vitro and in vivo investigations have demonstrated that probiotic-host eukaryotic cell interactions evoke a large number of responses potentially responsible for the effects of probiotics. The aim of this study was to improve our understanding of the E. coli Nissle 1917-host interaction by analyzing the gene expression pattern initiated by this probiotic in human intestinal epithelial cells. METHODS: Gene expression profiles of Caco-2 cells treated with E. coli Nissle 1917 were analyzed with microarrays. A second human intestinal cell line and also pieces of small intestine from BALB/c mice were used to confirm regulatory data of selected genes by real-time RT-PCR and cytometric bead array (CBA) to detect secretion of corresponding proteins. RESULTS: Whole genome expression analysis revealed 126 genes specifically regulated after treatment of confluent Caco-2 cells with E. coli Nissle 1917. Among others, expression of genes encoding the proinflammatory molecules monocyte chemoattractant protein-1 ligand 2 (MCP-1), macrophage inflammatory protein-2 alpha (MIP-2α) and macrophage inflammatory protein-2 beta (MIP-2β) was increased up to 10 fold. Caco-2 cells cocultured with E. coli Nissle 1917 also secreted high amounts of MCP-1 protein. Elevated levels of MCP-1 and MIP-2α mRNA could be confirmed with Lovo cells. MCP-1 gene expression was also up-regulated in mouse intestinal tissue. CONCLUSION: Thus, probiotic E. coli Nissle 1917 specifically upregulates expression of proinflammatory genes and proteins in human and mouse intestinal epithelial cells

Pro-inflammatory profile of preeclamptic placental mesenchymal stromal cells: new insights into the etiopathogenesis of preeclampsia.

The objective of the present study was to evaluate whether placental mesenchymal stromal cells (PDMSCs) derived from normal and preeclamptic (PE) chorionic villous tissue presented differences in their cytokines expression profiles. Moreover, we investigated the effects of conditioned media from normal and PE-PDMSCs on the expression of pro-inflammatory Macrophage migration Inhibitory Factor (MIF), Vascular Endothelial Growth Factor (VEGF), soluble FMS-like tyrosine kinase-1 (sFlt-1) and free β-human Chorionic Gonadotropin (βhCG) by normal term villous explants. This information will help to understand whether anomalies in PE-PDMSCs could cause or contribute to the anomalies typical of preeclampsia. METHODS: Chorionic villous PDMSCs were isolated from severe preeclamptic (n = 12) and physiological control term (n = 12) placentae. Control and PE-PDMSCs’s cytokines expression profiles were determined by Cytokine Array. Control and PE-PDMSCs were plated for 72 h and conditioned media (CM) was collected. Physiological villous explants (n = 48) were treated with control or PE-PDMSCs CM for 72 h and processed for mRNA and protein isolation. MIF, VEGF and sFlt-1 mRNA and protein expression were analyzed by Real Time PCR and Western Blot respectively. Free βhCG was assessed by immunofluorescent. RESULTS: Cytokine array showed increased release of pro-inflammatory cytokines by PE relative to control PDMSCs. Physiological explants treated with PE-PDMSCs CM showed significantly increased MIF and sFlt-1 expression relative to untreated and control PDMSCs CM explants. Interestingly, both control and PE-PDMSCs media induced VEGF mRNA increase while only normal PDMSCs media promoted VEGF protein accumulation. PE-PDMSCs CM explants released significantly increased amounts of free βhCG relative to normal PDMSCs CM ones. CONCLUSIONS: Herein, we reported elevated production of pro-inflammatory cytokines by PE-PDMSCs. Importantly, PE PDMSCs induced a PE-like phenotype in physiological villous explants. Our data clearly depict chorionic mesenchymal stromal cells as central players in placental physiopathology, thus opening to new intriguing perspectives for the treatment of human placental-related disorders as preeclampsia

Class-switzched B cells display response to therapeutic B-cell depletion in rheumatoid arthritis

Introduction Reconstitution of peripheral blood (PB) B cells after therapeutic depletion with the chimeric anti-CD20 antibody rituximab (RTX) mimics lymphatic ontogeny. In this situation, the repletion kinetics and migratory properties of distinct developmental B-cell stages and their correlation to disease activity might facilitate our understanding of innate and adaptive B-cell functions in rheumatoid arthritis (RA). Methods Thirty-five 'RTX-naïve' RA patients with active arthritis were treated after failure of tumour necrosis factor blockade in an open-label study with two infusions of 1,000 mg RTX. Prednisone dose was tapered according to clinical improvement from a median of 10 mg at baseline to 5 mg at 9 and 12 months. Conventional disease-modifying antirheumatic drugs were kept stable. Subsets of CD19+ B cells were assessed by flow cytometry according to their IgD and CD27 surface expression. Their absolute number and relative frequency in PB were followed every 3 months and were determined in parallel in synovial tissue (n = 3) or synovial fluid (n = 3) in the case of florid arthritis. Results Six of 35 patients fulfilled the European League Against Rheumatism criteria for moderate clinical response, and 19 others for good clinical response. All PB B-cell fractions decreased significantly in number (P < 0.001) after the first infusion. Disease activity developed independently of the total B-cell number. B-cell repopulation was dominated in quantity by CD27-IgD+ 'naïve' B cells. The low number of CD27+IgD- class-switched memory B cells (MemB) in the blood, together with sustained reduction of rheumatoid factor serum concentrations, correlated with good clinical response. Class-switched MemB were found accumulated in flaring joints. Conclusions The present data support the hypothesis that control of adaptive immune processes involving germinal centre-derived, antigen, and T-cell-dependently matured B cells is essential for successful RTX treatment

Basophil histamine release and leukotriene production in response to anti-IgE and anti-IgE receptor antibodies: Comparison of normal subjects and patients with urticaria, atopic dermatitis or bronchial asthma

The IgE receptor-dependent in vitro mediator release in basophils is characterized by a large interindividual variability both in normal and atopic subjects. The mechanism and the clinical impact of this finding, however, is largely unclear. The aim of the present study was to examine the role of surface-bound IgE and of response-modifying cytokines such as interleukin 3 (IL-3) as possible factors determining basophil releasability in atopic patients and normal controls. Cells from 30 individuals (6 with urticaria, 7 with asthma, 7 with atopic dermatitis, and 30 healthy controls) were isolated and stimulated for mediator release by IL-3 and different triggering antibodies directed against IgE or IgE receptor. Our data suggest that serum IgE levels and basophil receptor occupancy with IgE are not involved in the mechanism of basophil releasability. Furthermore, IL-3-induced similar effects on mediator release in almost all individuals, rather excluding the possibility that releasability is regulated by cytokine priming of basophils. Interestingly, we found that patients with atopic disease have a reduced capacity of releasing mediators upon activation, the mechanism of which is unclear. In conclusion, our findings support the hypothesis that basophil releasability is dependent on cell-immanent mechanisms in basophils, which may be altered in selected atopic patients.link_to_subscribed_fulltex

Histamine releasability of basophils and skin mast cells in chronic urticaria

In order to clarify the pathogenetic role of basophils and mast cells in chronic urticaria, histamine and leukotriene (LT)C4 release was examined in washed mixed leukocytes (n = 8) and skin mast cells (n = 5) from patients with chronic urticaria and compared with the same cells from normal controls (n = 9). Anti-IgE-stimulated basophil histamine release was significantly reduced in urticaria patients (median 2.9% vs 15.1% in normal controls), whereas histamine release to A23187, FMLP, and PAF, as well as anti-IgE-induced LTC4 release, showed no differences in both groups. In contrast, anti-IgE-stimulated skin mast cells from urticaria patients reacted similarly to those of controls (median histamine release 11.4% vs 14.2% in normal controls). Pretreatment of the cells with interleukin (IL)-3 upregulated responsiveness of basophil histamine release to anti-IgE in urticaria patients (median histamine release 14.3%), but pretreatment with the H2-antagonist cimetidine showed no effect. These data show that reduced basophil histamine releasability in chronic urticaria is not H2 mediated. It is a stimulus-, mediator-, and cell type-restricted phenomenon that can, at least partially, be reversed in the presence of the cytokine IL-3