Type I interferons and microbial metabolites of tryptophan modulate astrocyte activity and CNS inflammation via the aryl hydrocarbon receptor

Astrocytes play important roles in the central nervous system (CNS) during health and disease. Through genome-wide analyses we detected a transcriptional response to type I interferons (IFN-I) in astrocytes during experimental CNS autoimmunity and also in CNS lesions from multiple sclerosis (MS) patients. IFN-I signaling in astrocytes reduces inflammation and experimental autoimmune encephalomyelitis (EAE) disease scores via the ligand-activated transcription factor aryl hydrocarbon receptor (AhR) and suppressor of cytokine signaling 2 (SOCS2). The anti-inflammatory effects of nasally administered IFN-β are partly mediated by AhR. Dietary tryptophan is metabolized by the gut microbiota into AhR agonists that act on astrocytes to limit CNS inflammation. EAE scores were increased following ampicillin treatment during the recovery phase, and CNS inflammation was reduced in antibiotic-treated mice by supplementation with the tryptophan metabolites indole, indoxyl-3-sulfate (I3S), indole-3-propionic acid (IPA) and indole-3-aldehyde (IAld), or the bacterial enzyme tryptophanase. In individuals with MS, the circulating levels of AhR agonists were decreased. These findings suggest that IFN-I produced in the CNS act in combination with metabolites derived from dietary tryptophan by the gut flora to activate AhR signaling in astrocytes and suppress CNS inflammation

GATA3 and a dominant regulatory gene expression profile discriminate operational tolerance in human transplantation

Some organ-transplanted patients achieve a state of "operational tolerance" (01) in which graft function is maintained after the complete withdrawal of immunosuppressive drugs. We used a gene panel of regulatory/inflammatory molecules (FOXP3, GATA3, 100, TGFB1, TGFBR1/TBX21, TNF and IFNG) to investigate the gene expression profile in peripheral blood mononuclear cells of renal-transplanted individuals experiencing OT compared to transplanted individuals not displaying OT and healthy individuals (HI). OT subjects showed a predominant regulatory (REG) profile with higher gene expression of GATA3, FOXP3, TGFB1 and TGFB receptor 1 compared to the other groups. This predominant REG gene expression profile displayed stability over time. The significant GATA3 gene and protein expressions in OT individuals suggest that a Th2 deviation may be a relevant pathway to OT. Moreover, the capacity of the REG/INFLAMMA gene panel to discriminate OT by peripheral blood analysis indicates that this state has systemic repercussions. (C) 2011 Elsevier Inc. All rights reserved.FAPESPCNP

Tolerogenic nanoparticles inhibit T cell-mediated autoimmunity through SOCS2.

Type 1 diabetes (T1D) is a T cell-dependent autoimmune disease that is characterized by the destruction of insulin-producing β cells in the pancreas. The administration to patients of ex vivo-differentiated FoxP3(+) regulatory T (Treg) cells or tolerogenic dendritic cells (DCs) that promote Treg cell differentiation is considered a potential therapy for T1D; however, cell-based therapies cannot be easily translated into clinical practice. We engineered nanoparticles (NPs) to deliver both a tolerogenic molecule, the aryl hydrocarbon receptor (AhR) ligand 2-(1'H-indole-3'-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE), and the β cell antigen proinsulin (NPITE+Ins) to induce a tolerogenic phenotype in DCs and promote Treg cell generation in vivo. NPITE+Ins administration to 8-week-old nonobese diabetic mice suppressed autoimmune diabetes. NPITE+Ins induced a tolerogenic phenotype in DCs, which was characterized by a decreased ability to activate inflammatory effector T cells and was concomitant with the increased differentiation of FoxP3(+) Treg cells. The induction of a tolerogenic phenotype in DCs by NPs was mediated by the AhR-dependent induction of Socs2, which resulted in inhibition of nuclear factor κB activation and proinflammatory cytokine production (properties of tolerogenic DCs). Together, these data suggest that NPs constitute a potential tool to reestablish tolerance in T1D and potentially other autoimmune disorders

Preserving the B-Cell Compartment Favors Operational Tolerance in Human Renal Transplantation

Transplanted individuals in operational tolerance (OT) maintain long-term stable graft function after completely stopping immunosuppression. Understanding the mechanisms involved in OT can provide valuable information about pathways to human transplantation tolerance. Here we report that operationally tolerant individuals display quantitative and functional preservation of the B-c ell compartment in renal transplantation. OT exhibited normal numbers of circulating total B cells, naive, memory and regulatory B cells (Bregs) as well as preserved B-cell receptor repertoire, similar to healthy individuals. In addition, OT also displayed conserved capacity to activate the cluster of differentiation 40 (CD40)/signal transducer and activator of transcription 3 (STAT3) signaling pathway in Bregs, in contrast, with chronic rejection. Rather than expansion or higher activation, we show that the preservation of the B-cell compartment favors OT. Online address: http://www.molmed.org doi: 10.2119/molmed.2011.0028

Quantitative and enantioselective analyses of non-extractable residues of the fungicide metalaxyl in soil

Epigenetic modifications refer to a number of biological processes which alter the structure of chromatin and its transcriptional activity such as DNA methylation and histone post-translational processing. Studies have tried to elucidate how the viral genome and its products are affected by epigenetic modifications imposed by cell machinery and how it affects the ability of the virus to either, replicate and produce a viable progeny or be driven to latency. the purpose of this study was to evaluate epigenetic modifications in PBMCs and CD4(+) cells after HIV-1 infection analyzing three approaches: (i) global DNA-methylation; (ii) qPCR array and (iii) western blot. HIV-1 infection led to methylation increases in the cellular DNA regardless the activation status of PBMCs. the analysis of H3K9me3 and H3K27me3 suggested a trend towards transcriptional repression in activated cells after HIV-1 infection. Using a qPCR array, we detected genes related to epigenetic processes highly modulated in activated HIV-1 infected cells. SETDB2 and RSK2 transcripts showed highest up-regulation levels. SETDB2 signaling is related to transcriptional silencing while RSK2 is related to either silencing or activation of gene expression depending on the signaling pathway triggered down-stream. in addition, activated cells infected by HIV-1 showed lower CD69 expression and a decrease of IL-2, IFN-gamma and metabolism-related factors transcripts indicating a possible functional consequence towards global transcriptional repression found in HIV-1 infected cells. Conversely, based on epigenetic markers studied here, non-stimulated cells infected by HIV-1, showed signs of global transcriptional activation. Our results suggest that HIV-1 infection exerts epigenetic modulations in activated cells that may lead these cells to transcriptional repression with important functional consequences. Moreover, non-stimulated cells seem to increase gene transcription after HIV-1 infection. Based on these observations, it is possible to speculate that the outcome of viral infections may be influenced by the cellular activation status at the moment of infection

Modulation in the functional markers of activated CD4+ T cells after HIV-1 infection.

<p><b>(A)</b> Flow cytometry of purified CD4⁺ T cells at 36h post HIV-1 infection—dot plots of cell populations (gated on CD4⁺CD3⁺ cells) analyzed for the T cell early activation markers CD25, CD69 (percentages are shown in each quadrant) and graphical representation of the percentages of CD25⁺CD69⁺ cells (gated on CD4⁺CD3⁺ cells). <b>(B)</b> IL2 and IFN- γ mRNA relative expression in CD4+ T cells at 24h after HIV-1 infection. <b>(C)</b> GLUT-1 and ENO-1 mRNA relative expression in CD4+ T cells at 24h after HIV-1 infection. GAPDH was used as a normalizer of all reactions to calculate the relative expression by 2^-ΔΔCt method. Data are shown as mean ± SD of triplicates and are representative of three independent experiments using cells of three different healthy donors. Two-tailed Student’s t-test: *, p < 0.05. (<b>NI</b>) non-infected cells, (<b>I</b>) HIV-1 infected cells.</p

Classical markers of epigenetic transcriptional silencing and activation in activated PBMCs after HIV-1 infection.

<p><b>(A)</b> Graphical representation of protein ratios of epigenetic transcriptional silencing marker H3K27me3 over the total H3. <b>(B)</b> Graphical representation of protein ratios of epigenetic transcriptional silencing marker and H3K9m3 over the total H3. <b>(C)</b> Graphical representation of protein ratios of epigenetic transcriptional activation marker H3K4m3 over the total H3. Protein levels of each marker were calculated by the ratio of band intensities between specific markers (H3K27me3, H3K9me3 or H3K4me3) over the total H3 (normalizer) using the software ImageJ v. 1.45s (Public domain, NIH, USA). Dark bars—HIV-1 infected cells; White bars—non-infected cells (control group). <b>(D)</b> Representative Western blot image for each epigenetic marker (H3K27me3—upper panel, H3K9me3—middle panel, H3K4me3—lower panel and the total H3 as normalizer. The data represent the mean of three different measurements of the same experiment and the error bars indicate the differences between two independent experiments. 2way ANOVA: *** p< 0.001, ** p < 0.01 and *, p < 0.05. (<b>NI</b>) non-infected cells, (<b>I</b>) HIV-1 infected cells.</p