Inflammasome-Associated Nucleotide-Binding Domain, Leucine-Rich Repeat Proteins and Inflammatory Diseases

The nucleotide-binding domain, leucine-rich repeat (NLR) proteins are a recently discovered family of intracellular pathogen and danger signal sensors. NLRs have emerged as important contributors to innate immunity in animals. The physiological impact of these genes is increasingly evident, underscored by the genetic association of variant family members with an array of inflammatory diseases. The association of mutations in NLR genes with autoinflammatory diseases indicates an important function of these genes in inflammation in vivo. This review summarizes the role of the inflammasome NLR proteins in innate immunity and inflammatory diseases and explores the possible utility of some of these NLRs as pharmacological targets

The Histone Acetyltransferase Domains of CREB-binding Protein (CBP) and p300/CBP-associated Factor Are Not Necessary for Cooperativity with the Class II Transactivator

The class II transactivator (CIITA) is a transcriptional co-activator regulating the constitutive and interferon-gamma-inducible expression of class II major histocompatibility complex (MHC) and related genes. Promoter remodeling occurs following CIITA induction, suggesting the involvement of chromatin remodeling factors. Transcription of numerous genes requires the histone acetyltransferase (HAT) activities of CREB-binding protein (CBP), p300, and/or p300/CBP-associated factor (pCAF). These co-activators cooperate with CIITA and are hypothesized to promote class II major histocompatibility complex transcription through their HAT activity. To directly test this, we used HAT-defective CBP and pCAF. We demonstrate that cooperation between CIITA and CBP is independent of CBP HAT activity. Further, although pCAF enhances CIITA-mediated transcription, pCAF HAT domain dependence appears contingent upon the concentration of available CIITA. When HAT-defective CBP and pCAF are both present, cooperativity with CIITA is maintained. Consistent with a recent report, we show that nuclear localization of CIITA is enhanced by lysine 144, an in vitro target of pCAF-mediated HAT. Yet we find that neither mutation of lysine 144 nor deletion of residues 132-209 affects transcriptional cooperation with CBP or pCAF. Thus, acetylation of this residue may not be the primary mechanism for pCAF/CBP cooperation with CIITA. In conclusion, the HAT activities of the co-activators are not necessary for cooperation with CIITA

Inflammasome Inhibition as a Pathogenic Stealth Mechanism

The activation of inflammasomes containing NBD-LRR (NLRs) or non-NLRs is critical for effective host defense against microbial pathogens. Recent discoveries have uncovered a plethora of pathogenic strategies to inhibit inflammasome-mediated processing of IL-1β and IL-18. We review recent evidence for viral and bacterial manipulation of the inflammasome ranging from perturbation of caspase-1 activation to targeting of specific inflammasome components

MEK Inhibition Enhances Paclitaxel-induced Tumor Apoptosis

The anti-cancer drug paclitaxel (Taxol) alters microtubule assembly and activates pro-apoptotic signaling pathways. Previously, we and others found that paclitaxel activates endogenous JNK in tumor cells, and the activation of JNK contributes to tumor cell apoptosis. Here we find that paclitaxel activates the prosurvival MEK/ERK pathway, which conversely may compromise the efficacy of paclitaxel. Hence, a combination treatment of paclitaxel and MEK inhibitors was pursued to determine whether this treatment could lead to enhanced apoptosis. The inhibition of MEK/ERK with a pharmacologic inhibitor, U0126, together with paclitaxel resulted in a dramatic enhancement of apoptosis that is four times more than the additive value of the two drugs alone. Enhanced apoptosis was verified by the terminal transferase-mediated dUTP nick end labeling assay, by an enzyme-linked immunosorbent assay for histone-associated DNA fragments, and by flow cytometric analysis for DNA content. Specificity of the pharmacologic inhibitor was confirmed by the use of (a) a second MEK/ERK inhibitor and (b) a transdominant-negative MEK. Enhanced apoptosis was verified in breast, ovarian, and lung tumor cell lines, suggesting this effect is not cell type-specific. This is the first report of enhanced apoptosis detected in the presence of paclitaxel and MEK inhibition and suggests a new anticancer strategy

Cutting Edge: Inflammasome Activation by Alum and Alum's Adjuvant Effect Are Mediated by NLRP3

Alum is the only adjuvant approved for routine use in humans, although the basis for its adjuvanticity remains poorly understood. We have recently shown that Alum activates caspase-1 and induces secretion of mature IL-1β and IL-18. Here we show that in human and mice macrophages, alum-induced IL-1β, IL-18, and IL-33 secretion is mediated by the NLR protein NLRP3 and its adaptor ASC, but not by NLRC4. Other particulate adjuvants, such as QuilA and chitosan, induce inflammasome activation in a NLRP3-dependent fashion, suggesting that activation of the NLRP3-inflammasome may be a common mechanism of action of particulate adjuvants. Importantly, we demonstrate that antigen-specific antibody production elicited by vaccines that contain alum is significantly impaired in NLRP3-deficient mice. Our results demonstrate for the first time a role for the NLRP3-inflammasome during development of the immune response elicited by alum-enhanced vaccination. and suggest that therapeutic intervention aimed at NLRP3 may improve adjuvant efficacy

Paclitaxel (Taxol)-induced Gene Expression and Cell Death Are Both Mediated by the Activation of c-Jun NH 2 -terminal Kinase (JNK/SAPK)

Paclitaxel (Taxol) is a novel anti-cancer drug that has shown efficacy toward several malignant tumors, particularly ovarian tumors. We reported previously that paclitaxel can induce interleukin (IL)-8 promoter activation in subgroups of ovarian cancer through the activation of both AP-1 and nuclear factor kappaB. Further analysis of paclitaxel analogs indicates that the degree of IL-8 induction by analysis correlates with the extent of cell death; however, IL-8 itself is not the cause of cell death. This suggests that pathways that lead to IL-8 and cell death may overlap, although IL-8 per se does not kill tumor cells. To decipher the upstream signals for paclitaxel-induced transcriptional activation and cell death, we studied the involvement of protein kinases that lead to the activation of AP-1, specifically the c-Jun NH2-terminal kinase (JNK1), p38, and the extracellular signal-regulated kinase 1 (ERK1). The role of IkappaB in paclitaxel-induced cell death was also analyzed. Paclitaxel activated JNK, and to a lesser degree p38, but not ERK1. Paclitaxel-induced IL-8 promoter activation was inhibited by dominant-inhibitory mutants of JNK, p38, and the super-repressor form of IkappaBalpha, but not by dominant-inhibitory forms of ERK1. Dominant-inhibitory mutants of JNK1 also greatly reduced paclitaxel-induced cell death, and the kinetics of JNK induction was closely followed by DNA fragmentation. These results indicate (i) that paclitaxel activates the JNK signaling pathway and (ii) that JNK activation is a common point of paclitaxel-induced gene induction and cell death

HIV-1 Infection Induces Interleukin-1β Production via TLR8 Protein-dependent and NLRP3 Inflammasome Mechanisms in Human Monocytes

The induction of inflammatory cytokines such as IL-1β is associated with the progression of human immunodeficiency virus, type 1 (HIV-1) disease or AIDS. Unlike most inflammatory cytokines that are regulated by NF-κB at the transcriptional level, production of mature IL-1β also depends on inflammasome activation. The mechanism by which HIV-1 induces pro-IL-1β expression and activates inflammasomes to cleave pro-IL-1β into its bioactive form is not clearly defined. We report here that HIV-1 infection in human monocytes efficiently induced IL-1β expression and inflammasome activation. Toll-like receptor 8 (TLR8) was required for inducing pro-IL-1β expression, whereas the NLRP3 inflammasome was required for IL-1β maturation and release. Furthermore, the lysosomal protease cathepsin B and HIV-1 induced production of reactive oxygen species were critical for HIV-induced inflammasome activation and IL-1β production. HIV-1 entry, reverse transcription, and integration were all required for both pro-IL-1β expression and inflammasome activation. Finally, we show that HIV-1-derived RNA was sufficient to induce both pro-IL-1β expression and inflammasome activation. We conclude that HIV-1 infection induced the expression of pro-IL-1β via TLR8-mediated mechanisms and activated caspase-1 through the NLRP3 inflammasome to cleave pro-IL-1β into bioactive IL-1β. These findings help to elucidate mechanisms of HIV-1 disease progression and identify novel targets for treating HIV-1 induced inflammation and immune activation

Regulation of Inosine-5′-monophosphate Dehydrogenase Type II Gene Expression in Human T Cells: ROLE FOR A NOVEL 5′ PALINDROMIC OCTAMER SEQUENCE

Expression of the gene encoding human inosine- 5'-monophosphate dehydrogenase (IMPDH) type II, an enzyme catalyzing the rate-limiting step in the generation of guanine nucleotides, is increased more than 10-fold in activated peripheral blood T lymphocytes and is required for T cell activation. We have examined the 5'-regulatory sequences that are important for the transcriptional regulation of this gene in T cells. DNase I mapping of genomic DNA identified a hypersensitive element near the transcription initiation site. Fine mapping by in vivo footprinting demonstrated five transcription factor binding sites that are occupied in both resting and activated peripheral blood T lymphocytes; these are tandem CRE motifs, a Sp1 site, an overlapping Egr-1/Sp1 site, and a novel palindromic octamer sequence (POS). The tandem CRE and POS sites are of major functional importance as judged by mutational and electrophoretic mobility shift analyses. These data provide evidence that expression of the human IMPDH type II gene is predominantly regulated by the nuclear factors ATF-2 and an as yet unidentified POS-binding protein. Additional major protein-DNA interactions do not occur within the promoter region after T lymphocyte activation, indicating a requirement for additional protein-protein interactions and/or post-translational modifications of pre-bound transcription factors to account for the observed increase in IMPDH type II gene expression

Functional Genomic Analysis of Remyelination Reveals Importance of Inflammation in Oligodendrocyte Regeneration

Tumor necrosis factor alpha (TNFalpha), a proinflammatory cytokine, was shown previously to promote remyelination and oligodendrocyte precursor proliferation in a murine model for demyelination and remyelination. We used Affymetrix microarrays in this study to identify (1) changes in gene expression that accompany demyelination versus remyelination and (2) changes in gene expression during the successful remyelination of wild-type mice versus the unsuccessful attempts in mice lacking TNFalpha. Alterations in inflammatory genes represented the most prominent changes, with major histocompatibility complex (MHC) genes dramatically enhanced in microglia and astrocytes during demyelination, remyelination, and as a consequence of TNFalpha stimulation. Studies to examine the roles of these genes in remyelination were then performed using mice lacking specific genes identified by the microarray. Analysis of MHC-II-null mice showed delayed remyelination and regeneration of oligodendrocytes, whereas removal of MHC-I had little effect. These data point to the induction of MHC-II by TNFalpha as an important regulatory event in remyelination and emphasize the active inflammatory response in regeneration after pathology in the brain

Coordinate regulation of the human TAP1 and LMP2 genes from a shared bidirectional promoter

Recently, four genes (TAP1, TAP2, LMP2, LMP7) involved or potentially involved in the processing and transport of major histocompatibility complex class I-associated antigen to the endoplasmic reticulum have been identified. We now report the initial characterization of the bidirectional promoter for the human transporter associated with antigen processing 1 (TAP1) and low molecular mass polypeptide 2 (LMP2) genes. These genes are divergently transcribed from a central promoter region of only 593 bp. Functional analysis using a bidirectional reporter system demonstrates the minimal 593-bp promoter is sufficient for concurrent expression in both directions. There is no TATA box homology at either end but there is a prevalence of GC boxes. Transcription is initiated at multiple sites for each gene without any of the TAP1 transcripts overlapping with the LMP2 transcripts. The region proximal to the TAP1 gene is required for maximal basal level expression of not only TAP1 but also LMP2. Furthermore, this region is necessary for tumor necrosis factor alpha (TNF-alpha) induction of both genes. Site-specific mutations of an NF-kappa B element in the TAP1 proximal region blocked induction by TNF-alpha in both the TAP1 and LMP2 directions. An adjacent GC box was required for basal expression of both genes as well as augmenting the TNF-alpha induction of the distal LMP2 gene. In vivo genomic foot-printing of this region revealed strong protein/DNA interactions at the NF-kappa B and GC box consensus sequences. In vitro binding studies confirmed the capacity of the NF- kappa B site to bind p50/p65 and p52/p65 heterodimers and of the GC box to bind Sp1. Thus, the promoter elements proximal to the TAP1 gene play a significant role in regulating basal and induced expression of both TAP1 and LMP2. The findings presented in this report clearly link LMP2 expression with TAP1 expression and provide additional suggestive evidence linking LMP2 to class I antigen presentation