Influence of Interferon-beta on expression profile of memory T-cells
ABSTRACT: Inflammasomes are multi-protein complexes that control the production of pro-inflammatory cytokines such as IL-1beta. Inflammasomes play an important role in the control of immunity to tumors and infections, and also in autoimmune diseases, but the mechanisms controlling the activation of human inflammasomes are largely unknown. We found that human activated CD4+CD45RO+ memory T-cells specifically suppress P2X7R-mediated NLRP3 inflammasome activation, without affecting P2X7R-independent NLRP3 or NLRP1 inflammasome activation. The concomitant increase in pro-IL-1β production induced by activated memory T-cells concealed this effect. Priming with IFNβ decreased pro-IL-1β production in addition to NLRP3 inflammasome inhibition and thus unmasked the inhibitory effect on NLRP3 inflammasome activation. IFNβ did not suppress NLRP3 inflammasome activation by acting directly on monocytes. The inhibition of pro-IL-1β production and suppression of NLRP3 inflammasome activation by IFNβ-primed human CD4+CD45RO+ memory T-cells is partly mediated by soluble FasL and is associated with down-regulated P2X7R mRNA expression and reduced response to ATP in monocytes. CD4+CD45RO+ memory T-cells from multiple sclerosis (MS) patients showed a reduced ability to suppress NLRP3 inflammasome activation, however their suppressive ability was recovered following in vivo treatment with IFNβ. Thus, our data demonstrate that human P2X7R-mediated NLRP3 inflammasome activation is regulated by activated CD4+CD45RO+ memory T cells, and provide new information on the mechanisms mediating the therapeutic effects of IFNβ in MS. Memory T-cells were cultured in the presence of monocytes with and without Interferon-beta, resorted and expression profile was determined
Project description:Inflammasomes are multiprotein platforms of caspase-1 activation. The NLRP3 inflammasome is composed of NLRP3, ASC, and procaspase-1. This inflammasome is activated by various endogenous and exogenous stimuli, including pneumococci, and protects from a variety of microbial pathogens. In the present study, we examined the role of NLRP3 inflammasome components in gene expression in the lung during pneumococcal pneumina. Overall design: Gene expression was measured in the lung of WT, Casp1-/-, Asc-/-, and Nlrp3-/- mice 24 h after intranasal infection with Streptococcus pneuminiae.
Project description:Peripheral Blood Mononuclear Cells (PBMCs) were isolated from a buffy coat (Australian Blood Bank) using Ficoll methodology. CD4+ T cells were isolated using Dynal Beads kit. Pure CD4+ T cells were then stained using a cocktail of monoclonal antobodies (mAbs), including: anti-CD4PE, CD45RO ECD, CD62L APC-Cy7, CD25 APC, CD127 Pacific Blue. After incubation, cells were washed twice in PBS/FCS (0.2%), and sorted into five different cell subsets: CD4+CD25+CD127low CD62L+CD45RO- (naive regulatory T cells), CD4+CD25+CD127low CD62L+/- CD45RO+ (activated regulatory T cells), CD4+CD25+CD127hi CD62L+/- CD45RO+ (memory T cells), CD4+CD25-CD127low CD62L+/- CD45RO+ (effector T cells) and CD4+CD25-CD127hi CD62L+ CD45RO- (naive T cells).
Project description:The interleukin-23 (IL-23) pathway plays a critical role in the pathogenesis of multiple chronic inflammatory disorders, however, inter-individual variability in IL-23-induced signal transduction in circulating human lymphocytes has not been well-defined. In this study, we observed marked, reproducible inter-individual differences in IL-23 responsiveness (measured by STAT3 phosphorylation) in peripheral blood CD8+CD45RO+ memory T and CD3+CD56+ NKT cells. To define mechanisms that might be contributing to the differential IL-23-induced STAT3 activation between individuals, we examined mRNA expression differences in CD8+CD45RO+ memory T cells between IL-23 responsive and non-responsive individuals. We analyzed unstimulated and IL-23 stimulated FACS sorted CD8+CD45RO+ memory T cells from two individuals demonstrating robust IL-23 responsiveness, and two individuals demonstrating low IL-23 responsiveness, using the Affymetrix Human Exon 1.0 ST platform. Array data was processed by Affymetrix Expression Console software. No techinical replicates were performed.
Project description:Inflammasome activation is critical for host defense against various microbial infections. Activation of the NLRC4 inflammasome requires detection of flagellin or type III secretion system (T3SS) components by NLR family apoptosis inhibitory proteins (NAIPs); yet how this pathway is regulated is unknown. Here we found that interferon regulatory factor 8 (IRF8) is required for optimal activation of the NLRC4 inflammasome in bone marrow-derived macrophages infected with Salmonella Typhimurium, Burkholderia thailandensis, or Pseudomonas aeruginosa but is dispensable for activation of the canonical and non-canonical NLRP3, AIM2, and Pyrin inflammasomes. IRF8 governs the transcription of Naips to allow detection of flagellin or T3SS proteins to mediate NLRC4 inflammasome activation. Furthermore, we found that IRF8 confers protection against bacterial infection in vivo, owing to its role in inflammasome-dependent cytokine production and pyroptosis. Altogether, our findings suggest that IRF8 is a critical regulator of NAIPs and NLRC4 inflammasome activation for defense against bacterial infection. Overall design: We used microarrays to explore the gene expression profiles differentially expressed in Salmonella-infected bone marrow derived macrophages (BMDM) isolated from IRF8-/- and wild-type mice.
Project description:Inflammasome, activated by pathogen-derived and host-derived danger signals, constitutes a multimolecular signaling complex that serves as a platform for caspase-1 (CASP1) activation and interleukin-1beta (IL1B) maturation. The activation of NLRP3 inflammasome requires two-step signals. The first “priming” signal (Signal 1) enhances gene expression of inflammasome components. The second “activation” signal (Signal 2) promotes the assembly of inflammasome components. Deregulated activation of NLRP3 inflammasome contributes to the pathological processes of Alzheimer’s disease (AD) and multiple sclerosis (MS). However, at present, the precise mechanism regulating NLRP3 inflammasome activation and deactivation remains largely unknown. By genome-wide gene expression profiling, we studied the molecular network of NLRP3 inflammasome activation-responsive genes in a human monocyte cell line THP-1 sequentially given two-step signals. We identified the set of 83 NLRP3 inflammasome activation-responsive genes. Among them, we found the NR4A nuclear receptor family NR4A1, NR4A2, and NR4A3, the EGR family EGR1, EGR2, and EGR3, the IkappaB family NFKBIZ, NFKBID, and NFKBIA as a key group of the genes that possibly constitute a negative feedback loop for shutting down inflammation following NLRP3 inflammasome activation. By molecular network analysis, we identified a complex network of NLRP3 inflammasome activation-responsive genes involved in cellular development and death, and immune and inflammatory responses, where transcription factors AP-1, NR4A, and EGR serve as a hub. Thus, NLRP3 inflammasome activation-responsive genes constitute the molecular network composed of a set of negative feedback regulators for prompt resolution of inflammation. To load the Signal 1 (S1), THP-1 cells were incubated for 3 hours in the culture medium with or without inclusion of 0.2 microgram/ml lipopolysaccharide (LPS). To load the Signal 2 (S2), they were incubated further for 2 hours in the culture medium with inclusion of 10 microM nigericin sodium salt dissolved in ethanol or the equal v/v% concentration of ethanol (vehicle), followed by processing for microarray analysis on Human Gene 1.0 ST Array (Affymetrix).
Project description:NLRP3 inflammasome assembles in response to stress or danger signals and leads to unconventional secretion of proinflammatory IL-1. FADD is an NLRP3 inflammasome component. Here we found that classical NLRP3 inflammasome activation in human monocytes/macrophages induced FADD secretion, which required potassium efflux, functional NLRP3 sensor, ASC adaptor and caspase-1 scaffold molecule. FADD is a leaderless protein unconventionally secreted through plasma membrane-derived microvesicles. Blood-derived monocytes from rheumatoid arthritis (RA) patients secreted more FADD following NLRP3 inflammasome activation than those from healthy donors, and we found increased levels of FADD in the sera (ESPOIR cohort) and synovial fluids from RA patients. Levels of synovial FADD correlated with the inflammatory status of the joint. These data reveal that FADD secretion occurs during inflammatory disease in vivo.
Project description:To compare human memory CD4+ T cell subsets in peripheral blood (PB) and bone marrow (BM) of healthy individuals at transcriptional level, we analyzed the global gene expression of ex vivo PB CD69- as well as BM CD69- and CD69+ memory CD4+ T cells from 4 paired PB and BM samples. The gene expression of these subsets was additionally compared to the transcriptional profile of 8 PB samples taken ex vivo or stimulated with phorbol myristate acetate (PMA) and Ionomycin for 3 hours. Three ex vivo memory CD4+ T cell subsets (CD4+CD45RO+CD69+ and CD4+CD45RO+CD69- cells from bone marrow; CD4+CD45RO+CD69- cells from peripheral blood) were isolated from 4 paired bone marrow and blood samples (two males and two females) or from a different cohort of 8 blood samples (3 males and five females). The purity of sorted cells was higher than 95% as assessed by FACS. Subsequently, a fraction of purified cells from the 8 blood samples were stimulated with phorbol-myristic-acid/ionomycin (PMA/iono) for 3 h and used as high controls. Total RNA of each cell subset was extracted using a NucleoSpin RNA XS Kit (Macherey-Nagel) or RNeasy Mini kit (Qiagen). The integrity and amount of isolated RNA was assessed for each sample using an Agilent 2100 Bioanalyzer (Agilent, Waldbronn, Germany) and a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE). Double-stranded complementary RNA was synthesized from 1 µg total RNA using Message AmpII Biotin (Ambion, USA). Fifteen micrograms of fragmented cRNA of each sample were hybridized to 28 HG-U133A plus 2.0 GeneChips (Affymetrix). Hybridization was performed in a Hybridization Oven 640, and chips were washed and stained in the Fluidics Station 400 (both Affymetrix). Finally, the arrays were scanned with a GeneChip Scanner 3000 using the GCOS software, version 1.4, both Affymetrix. All relevant GCOS data of quality checked microarrays were analyzed with High Performance Chip Data Analysis (HPCDA, unpublished), using the BioRetis database (www.bioretis-analysis.de), as described and validated previously.
Project description:MicroRNAs (miRNAs) have emerged as important players in the regulation of T-cell functionality. However, comprehensive insight into the extent of age-related miRNA changes in T cells is lacking. We established miRNA expression patterns of CD45RO- naïve and CD45RO+ memory T-cell subsets isolated from peripheral blood cells from young and elderly individuals. Unsupervised clustering of the miRNA expression data revealed an age-related clustering in the CD45RO- T cells, while CD45RO+ T cells clustered based on expression of CD4 and CD8. Seventeen miRNAs showed an at least 2-fold up- or downregulation in CD45RO- T cells obtained from young as compared to old donors. Validation on the same and independent samples revealed a statistically significant age-related upregulation of miR-21, miR-223 and miR-15a. In a T-cell subset analysis focusing on known age-related phenotypic changes, we showed significantly higher miR-21 and miR-223 levels in CD8+CD45RO-CCR7- TEMRA compared to CD45RO-CCR7+ TNAIVE-cells. Moreover, miR-21 but not miR-223 levels were significantly increased in CD45RO-CD31- post-thymic TNAIVE cells as compared to thymic CD45RO-CD31+ TNAIVE cells. Upon activation of CD45RO- TNAIVE cells we observed a significant induction of miR-21 especially in CD4+ T cells, while miR-223 levels significantly decreased only in CD4+ T cells. Besides composition and activation, we showed a borderline significant increase in miR-21 levels upon an increasing number of population doublings in CD4+ T-cell clones. Together, our results show that ageing related changes in miRNA expression are dominant in the CD45RO- T-cell compartment. The differential expression patterns can be explained by age related changes in T-cell composition, i.e. accumulation of CD8+ TEMRA and CD4+ post thymic expanded CD31- T cells and by cellular ageing, as demonstrated in a longitudinal clonal culture model. MicroRNA profiling was performed in eight T cell subsets: CD4 naive (CD3+CD4+CD45RO-), CD8 naive (CD3+CD4-CD45RO-), CD4 memory (CD3+CD4+CD45RO+) and CD8 memory (CD3+CD4-CD45RO+) T cells derived from 5 healthy young and 5 healthy old participants.
Project description:Type I interferon (IFN) is a family of 15 cytokines (in human 13α, 1β,1ω) which exert several cellular functions through the binding to a common receptor. Despite the initial activation of the same Jak/Stat signalling pathway, the cellular response may be different depending on the type I IFN subtype. We investigated the activity of different type I IFN subtypes - IFNα1, α2, α8, α21, ω and β- on the differentiation of DC. Transcriptome analyses identified two distinct groups, the IFNα/ω-DC and the IFNβ-DC. 78 genes, 7 chemokines and expression levels of cell surface markers characteristic of DC distinguished IFNα-DC and IFNβ-DC. These differences are unlikely to impact the efficacy of T cell functional response since IFNα2-DC and IFNβ-DC were equipotent in inducing the proliferation and the polarization of allogenic naïve CD4 T cells into Th1 cells and in stimulating autologous memory CD4 or CD8 T cells. In contrast, IFNα2-DC were found to be more efficient than IFNβ-DC in the phagocytic uptake of dead cells. Human blood monocytes were differentiated in DC by using 5 differents IFN type I (IFNα2, α1, α8, α21 and β). After 3 days of differentiation RNA were extracted and analyzed by affymetrix microarray.
Project description:The adaptor protein ASC contributes to innate immunity through the assembly of caspase-1-activating inflammasome complexes. We demonstrate that ASC plays an inflammasome-independent cell-intrinsic role in adaptive immune cells. Asc-/- mice displayed defective antigen presentation by dendritic cells and lymphocyte migration due to impaired Rac-mediated actin polymerization. Genome-wide analysis showed that ASC, but not Nlrp3 or caspase-1, controls mRNA stability and expression of DOCK2, a guanine nucleotide exchange factor that mediates Rac-dependent signaling in immune cells. DOCK2-deficient dendritic cells showed similar defective antigen uptake as Asc-/- cells. Ectopic expression of DOCK2 in ASC-deficient cells restored Rac-mediated actin polymerization, antigen uptake and chemotaxis. Thus, ASC shapes adaptive immunity independently of inflammasomes by modulating DOCK2-dependent Rac activation and F-actin polymerization in dendritic cells and lymphocytes. Three replicates of naïve WT and three replicates of Asc-/- bone marrow derived dendritic cells were analyzed on the Affymetrix HT MG-430 PM plate array.