Microbiota-derived acetate activates intestinal innate immunity via the Tip60 histone acetyltransferase complex.

Microbe-derived acetate activates the Drosophila immunodeficiency (IMD) pathway in a subset of enteroendocrine cells (EECs) of the anterior midgut. In these cells, the IMD pathway co-regulates expression of antimicrobial and enteroendocrine peptides including tachykinin, a repressor of intestinal lipid synthesis. To determine whether acetate acts on a cell surface pattern recognition receptor or an intracellular target, we asked whether acetate import was essential for IMD signaling. Mutagenesis and RNA interference revealed that the putative monocarboxylic acid transporter Tarag was essential for enhancement of IMD signaling by dietary acetate. Interference with histone deacetylation in EECs augmented transcription of genes regulated by the steroid hormone ecdysone including IMD targets. Reduced expression of the histone acetyltransferase Tip60 decreased IMD signaling and blocked rescue by dietary acetate and other sources of intracellular acetyl-CoA. Thus, microbe-derived acetate induces chromatin remodeling within enteroendocrine cells, co-regulating host metabolism and intestinal innate immunity via a Tip60-steroid hormone axis that is conserved in mammals.

Alcohol Consumption in Rheumatoid Arthritis: A Path through the Immune System.

Benefits and harms of different components of human diet have been known for hundreds of years. Alcohol is one the highest consumed, abused, and addictive substances worldwide. Consequences of alcohol abuse are increased risks for diseases of the cardiovascular system, liver, and nervous system, as well as reduced immune system function. Paradoxically, alcohol has also been a consistent protective factor against the development of autoimmune diseases such as type 1 diabetes, multiple sclerosis, systemic lupus erythematosus, and rheumatoid arthritis (RA). Here, we focused on summarizing current findings on the effects of alcohol, as well as of its metabolites, acetaldehyde and acetate, on the immune system and RA. Heavy or moderate alcohol consumption can affect intestinal barrier integrity, as well as the microbiome, possibly contributing to RA. Additionally, systemic increase in acetate negatively affects humoral immune response, diminishing TFH cell as well as professional antigen-presenting cell (APC) function. Hence, alcohol consumption has profound effects on the efficacy of vaccinations, but also elicits protection against autoimmune diseases. The mechanism of alcohol's negative effects on the immune system is multivariate. Future studies addressing alcohol and its metabolite acetate's effect on individual components of the immune system remains crucial for our understanding and development of novel therapeutic pathways.

Memory CD8+ T Cells Balance Pro- and Anti-inflammatory Activity by Reprogramming Cellular Acetate Handling at Sites of Infection.

Serum acetate increases upon systemic infection. Acutely, assimilation of acetate expands the capacity of memory CD8+ T cells to produce IFN-γ. Whether acetate modulates memory CD8+ T cell metabolism and function during pathogen re-encounter remains unexplored. Here we show that at sites of infection, high acetate concentrations are being reached, yet memory CD8+ T cells shut down the acetate assimilating enzymes ACSS1 and ACSS2. Acetate, being thus largely excluded from incorporation into cellular metabolic pathways, now had different effects, namely (1) directly activating glutaminase, thereby augmenting glutaminolysis, cellular respiration, and survival, and (2) suppressing TCR-triggered calcium flux, and consequently cell activation and effector cell function. In vivo, high acetate abundance at sites of infection improved pathogen clearance while reducing immunopathology. This indicates that, during different stages of the immune response, the same metabolite-acetate-induces distinct immunometabolic programs within the same cell type.

Simultaneous detection of plant growth regulators jasmonic acid and methyl jasmonate in plant samples by a monoclonal antibody-based ELISA.

Methyl jasmonate (MeJA) and its free-acid form, jasmonic acid (JA), collectively referred to as jasmonates (JAs), are natural plant growth regulators that are widely present in higher plants. Simultaneous detection of JA and MeJA in plant samples is of significance and is a great challenging issue. In this study, coupling with two extraction methods, a sensitive monoclonal antibody (mAb) based enzyme-linked immunosorbent assay (ELISA) for simultaneous detection of JA and MeJA in plant samples was developed. The JA-bovine serum albumin (BSA) conjugate was used as an immunogen for the production of mAb. As the produced mAb exhibited higher recognition ability towards MeJA than towards JA, ELISA was established using MeJA as the standard. Under optimal experimental conditions, the IC50 and LOD values of ELISA for MeJA were 2.02 ng mL-1 and 0.20 ng mL-1, respectively. In the first extraction method, MeJA in plant samples was evaporated and only JA was extracted. In the second extraction method, both JA and MeJA were extracted. After methylation, JA in the extracts was converted into MeJA, and the whole MeJA in the extracts was measured by ELISA. Plant samples including the leaves of Salvia splendens, the flowers of Salvia splendens and the fruit of grapes were collected. JA and MeJA in these samples were detected by the proposed ELISA. It was found that the concentrations of JA in these three plant samples were about 3-5 times higher than those of MeJA in those samples. ELISA was also confirmed by HPLC. There was a good correlation between ELISA and HPLC.

Acetate coordinates neutrophil and ILC3 responses against C. difficile through FFAR2.

Antibiotic-induced dysbiosis is a key predisposing factor for Clostridium difficile infections (CDIs), which cause intestinal disease ranging from mild diarrhea to pseudomembranous colitis. Here, we examined the impact of a microbiota-derived metabolite, short-chain fatty acid acetate, on an acute mouse model of CDI. We found that administration of acetate is remarkably beneficial in ameliorating disease. Mechanistically, we show that acetate enhances innate immune responses by acting on both neutrophils and ILC3s through its cognate receptor free fatty acid receptor 2 (FFAR2). In neutrophils, acetate-FFAR2 signaling accelerates their recruitment to the inflammatory sites, facilitates inflammasome activation, and promotes the release of IL-1ß; in ILC3s, acetate-FFAR2 augments expression of the IL-1 receptor, which boosts IL-22 secretion in response to IL-1ß. We conclude that microbiota-derived acetate promotes host innate responses to C. difficile through coordinate action on neutrophils and ILC3s.

Decreased maternal serum acetate and impaired fetal thymic and regulatory T cell development in preeclampsia.

Maternal immune dysregulation seems to affect fetal or postnatal immune development. Preeclampsia is a pregnancy-associated disorder with an immune basis and is linked to atopic disorders in offspring. Here we show reduction of fetal thymic size, altered thymic architecture and reduced fetal thymic regulatory T (Treg) cell output in preeclamptic pregnancies, which persists up to 4 years of age in human offspring. In germ-free mice, fetal thymic CD4+ T cell and Treg cell development are compromised, but rescued by maternal supplementation with the intestinal bacterial metabolite short chain fatty acid (SCFA) acetate, which induces upregulation of the autoimmune regulator (AIRE), known to contribute to Treg cell generation. In our human cohorts, low maternal serum acetate is associated with subsequent preeclampsia, and correlates with serum acetate in the fetus. These findings suggest a potential role of acetate in the pathogenesis of preeclampsia and immune development in offspring.

Ethylene Perception Is Associated with Methyl-Jasmonate-Mediated Immune Response against Botrytis cinerea in Tomato Fruit.

Jasmonic acid (JA)- and ethylene-mediated signaling pathways are reported to have synergistic effects on inhibiting gray mold. The present study aimed to explain the role of ethylene perception in methyl jasmonate (MeJA)-mediated immune responses. Results showed that exogenous MeJA enhanced disease resistance, accompanied by the induction of endogenous JA biosynthesis and ethylene production, which led to the activation of the phenolic metabolism pathway. Blocking ethylene perception using 1-methylcyclopropene (1-MCP) either before or after MeJA treatment could differently weaken the disease responses induced by MeJA, including suppressing the induction of ethylene production and JA contents and reducing activities of lipoxygenase and allene oxide synthase compared to MeJA treatment alone. Consequently, MeJA-induced elevations in the total phenolic content and the activities of phenylalanine ammonia-lyase, cinnamate 4-hydroxylase, 4-coumarate:coenzyme A ligase, and peroxidase were impaired by 1-MCP. These results suggested that ethylene perception participated in MeJA-mediated immune responses in tomato fruit.

Acetate Promotes T Cell Effector Function during Glucose Restriction.

Competition for nutrients like glucose can metabolically restrict T cells and contribute to their hyporesponsiveness during cancer. Metabolic adaptation to the surrounding microenvironment is therefore key for maintaining appropriate cell function. For instance, cancer cells use acetate as a substrate alternative to glucose to fuel metabolism and growth. Here, we show that acetate rescues effector function in glucose-restricted CD8+ T cells. Mechanistically, acetate promotes histone acetylation and chromatin accessibility and enhances IFN-γ gene transcription and cytokine production in an acetyl-CoA synthetase (ACSS)-dependent manner. Ex vivo acetate treatment increases IFN-γ production by exhausted T cells, whereas reducing ACSS expression in T cells impairs IFN-γ production by tumor-infiltrating lymphocytes and tumor clearance. Thus, hyporesponsive T cells can be epigenetically remodeled and reactivated by acetate, suggesting that pathways regulating the use of substrates alternative to glucose could be therapeutically targeted to promote T cell function during cancer.

Microbiota metabolite short-chain fatty acid acetate promotes intestinal IgA response to microbiota which is mediated by GPR43.

Intestinal IgA, which is regulated by gut microbiota, has a crucial role in maintenance of intestinal homeostasis and in protecting the intestines from inflammation. However, the means by which microbiota promotes intestinal IgA responses remain unclear. Emerging evidence suggests that the host can sense gut bacterial metabolites in addition to pathogen-associated molecular patterns and that recognition of these small molecules influences host immune response in the intestines and beyond. We reported here that microbiota metabolite short-chain fatty acid acetate promoted intestinal IgA responses, which was mediated by "metabolite-sensing" GPR43. GPR43-/- mice demonstrated lower levels of intestinal IgA and IgA+ gut bacteria compared with those in wild type (WT) mice. Feeding WT but not GPR43-/- mice acetate but not butyrate promoted intestinal IgA response independent of T cells. Acetate promoted B-cell IgA class switching and IgA production in vitro in the presence of WT but not GPR43-/- dendritic cells (DCs). Mechanistically, acetate-induced DC expression of Aldh1a2, which converts Vitamin A into its metabolite retinoic acid (RA). Moreover, blockade of RA signaling inhibited the acetate induction of B-cell IgA production. Our studies thus identified a new pathway by which microbiota promotes intestinal IgA response through its metabolites.

Antimodified protein antibody response pattern influences the risk for disease relapse in patients with rheumatoid arthritis tapering disease modifying antirheumatic drugs.

OBJECTIVE: To perform a detailed analysis of the autoantibody response against post-translationally modified proteins in patients with rheumatoid arthritis (RA) in sustained remission and to explore whether its composition influences the risk for disease relapse when tapering disease modifying antirheumatic drug (DMARD) therapy. METHODS: Immune responses against 10 citrullinated, homocitrullinated/carbamylated and acetylated peptides, as well as unmodified vimentin (control) and cyclic citrullinated peptide 2 (CCP2) were tested in baseline serum samples from 94 patients of the RETRO study. Patients were classified according to the number of autoantibody reactivities (0-1/10, 2-5/10 and >5/10) or specificity groups (citrullination, carbamylation and acetylation; 0-3) and tested for their risk to develop relapses after DMARD tapering. Demographic and disease-specific parameters were included in multivariate logistic regression analysis for defining the role of autoantibodies in predicting relapse. RESULTS: Patients varied in their antimodified protein antibody response with the extremes from recognition of no (0/10) to all antigens (10/10). Antibodies against citrullinated vimentin (51%), acetylated ornithine (46%) and acetylated lysine (37%) were the most frequently observed subspecificities. Relapse risk significantly (p=0.011) increased from 18% (0-1/10 reactivities) to 34% (2-5/10) and 55% (>5/10). With respect to specificity groups (0-3), relapse risk significantly (p=0.021) increased from 18% (no reactivity) to 28%, 36% and finally to 52% with one, two or three antibody specificity groups, respectively. CONCLUSIONS: The data suggest that the pattern of antimodified protein antibody response determines the risk of disease relapse in patients with RA tapering DMARD therapy. TRIAL REGISTRATION NUMBER: 2009-015740-42; Results.

Simultaneous induction of jasmonic acid and disease-responsive genes signifies tolerance of American elm to Dutch elm disease.

Dutch elm disease (DED), caused by three fungal species in the genus Ophiostoma, is the most devastating disease of both native European and North American elm trees. Although many tolerant cultivars have been identified and released, the tolerance mechanisms are not well understood and true resistance has not yet been achieved. Here we show that the expression of disease-responsive genes in reactions leading to tolerance or susceptibility is significantly differentiated within the first 144 hours post-inoculation (hpi). Analysis of the levels of endogenous plant defense molecules such as jasmonic acid (JA) and salicylic acid (SA) in tolerant and susceptible American elm saplings suggested SA and methyl-jasmonate as potential defense response elicitors, which was further confirmed by field observations. However, the tolerant phenotype can be best characterized by a concurrent induction of JA and disease-responsive genes at 96 hpi. Molecular investigations indicated that the expression of fungal genes (i.e. cerato ulmin) was also modulated by endogenous SA and JA and this response was unique among aggressive and non-aggressive fungal strains. The present study not only provides better understanding of tolerance mechanisms to DED, but also represents a first, verified template for examining simultaneous transcriptomic changes during American elm-fungus interactions.

Acetylated Trivalent Mannobioses: Chemical Modification, Structural Elucidation, and Biological Evaluation.

People suffering from allergies can be treated with repeated injections of increasing amounts of a specific allergen. This type of specific immunotherapy is currently the only way to treat the underlying pathological immune response associated with an allergy. The approach can afford long-lasting protection, but the process takes 3-5 years, can produce allergic reactions, and in severe cases treatment is often aborted due to anaphylaxis. However, treatment can be optimized with the use of specific adjuvants that modify the immune response, its duration, and that increase the production of the correct type of antibodies. In the pursuit of such adjuvants, two new trivalent acetylated ß-(1→2)-linked mannobioses based on a previously discovered lead molecule were prepared. The new molecules, along with the previously developed lead, were investigated by rigorous NMR and molecular modeling experiments in order to elucidate their behavior and preferred conformations in solution. Furthermore, the molecules were subjected to a biological investigation in which their immunostimulatory properties were evaluated by assessing their effect on the production of TH 2-type cytokine interleukin-4 (IL-4) and Treg pro-inflammatory cytokine tumor necrosis factor (TNF). Treatment of peripheral mononuclear blood cell cultures from patients suffering from birch allergy with birch allergen Bet v induced a strong IL-4 response, whereas the same treatment together with the trivalent acetylated mannobioses caused significant suppression of the induced IL-4.

Preparation of novel monoclonal antibodies against chelated cadmium ions.

The detection of cadmium ions using enzyme-linked immunosorbent assays (ELISA) has been reported by several research groups. Because cadmium ions are too small to stimulate the immune system, high molecular weight immunogens of cadmium are constructed using bifunctional chelators. At present, the most commonly used bifunctional chelator for the preparation of antigens for heavy metal ions is 1-(4-isothiocyanobenzyl) ethylenediamine N,N,N',N'-tetraacetic acid (ITCBE). However, the price of ITCBE is high. So we are interested in a cheaper bifunctional chelator, 1-(4-aminobenzyl) ethylenediamine N,N,N',N'-tetraacetic acid (aminobenzyl-EDTA). Here, cadmium ions were conjugated to carrier proteins using aminobenzyl-EDTA to make artificial antigens. Then, several mice were immunized with the antigen. And monoclonal antibodies (MAbs) against cadmium were produced. Spleen cells of immunized mice were fused with myeloma cells. The resulting hybridomas were screened using protein conjugates which were covalently bound to metal-free EDTA or cadmium. Three hybridoma cell lines (A3, E4 and B5) that produced MAbs with high selectivity and sensitivity were expanded for further study. Cross-reactivities with other metals were below 1 %. These antibodies were used to construct competitive ELISAs. The IC50 for A3 was 8.4 µg/l. The detection range and the lowest detection limit using the antibody A3 was 0.394-64.39 and 0.051 µg/l, respectively. Spike-recovery studies in tap water showed that the antibody A3 could be used for cadmium detection in drinking water.

Montelukast treatment (cysteinyl leukotriene receptor antagonist) in a model of food allergy: modifications in lymphatic cell population from rectal mucosa

Objective: the aim is to determine immunopathological modifications in rectal mucosa from rabbits after local challenge in ovalbumin (OVA) sensitized animals previously treated with montelukast. Material and methods: experimental design: thirty two rabbits divided into four groups: G1: normal; G2: subcutaneously OVA sensitized; G3: sensitized, locally OVA challenged and sampled 4 hours after challenge; and G4: sensitized, locally OVA challenged and treated 4 hours before challenge with montelukast (0.15 mg/kg). Specific anti-OVA IgE levels were evaluated by passive cutaneous anaphylaxis test (PCA). In each group 200 high microscopical power fields (HPF) were counted. Results were expressed as arithmetic mean and SE. Anti -CD4, CD5, micro chain monoclonal antibodies were used. Avidin biotin horseradish peroxidase system was used. Results: CD 4: G1: 8.3 ± 0.06; G2: 13.4 ± 0.08, G3: 8.25 ± 0.06, G4: 11.8 ± 0.02. CD 5: G1: 7.3 ± 0.05; G2: 9.4 ± 0.05, G3: 11.3 ± 0.06, G4: 8.1 ± 0.06. ì chain: G1: 10.4 ± 0.06; G2: 3.8 ± 0.02, G3: 6.0 ± 0.10, G4: 2.2 ± 0.10. In all cases, experimental groups (G3 vs. G4) presented statistical significant differences (p < 0.05). CD4+, CD5+ cells and micro chain+ decrease in experimental group (G4), probably due to lymphocyte migration inhibition to challenged mucosa. micro chain+ cell decrease could be based on B cell activation and expression of different surface immunoglobulins. Cells expressing micro chain decreased in G2 and G3 likely due to activation of B cells and subsequent expression of other immunoglobulin chains in cell surface. Conclusions: we conclude that obtained data are important to elucidate immunopathology of local anaphylactic reaction in rectal mucosa from systemic sensitized animals after treatment with montelukast(AU)

Ulvan, a sulfated polysaccharide from green algae, activates plant immunity through the jasmonic acid signaling pathway.

The industrial use of elicitors as alternative tools for disease control needs the identification of abundant sources of them. We report on an elicitor obtained from the green algae Ulva spp. A fraction containing most exclusively the sulfated polysaccharide known as ulvan-induced expression of a GUS gene placed under the control of a lipoxygenase gene promoter. Gene expression profiling was performed upon ulvan treatments on Medicago truncatula and compared to phytohormone effects. Ulvan induced a gene expression signature similar to that observed upon methyl jasmonate treatment (MeJA). Involvement of jasmonic acid (JA) in ulvan response was confirmed by detecting induction of protease inhibitory activity and by hormonal profiling of JA, salicylic acid (SA) and abscisic acid (ABA). Ulvan activity on the hormonal pathway was further consolidated by using Arabidopsis hormonal mutants. Altogether, our results demonstrate that green algae are a potential reservoir of ulvan elicitor which acts through the JA pathway.

Single-dose desloratadine and montelukast and allergen-induced late airway responses.

Montelukast and desloratadine synergistically inhibit the allergen-induced early asthmatic response. Montelukast also suppresses the allergen-induced late asthmatic response, but there are no reports on the effect of desloratadine or the combination on the allergen-induced late asthmatic response. Atopic asthmatics (n = 10) completed a multicentric randomised double-blind crossover study comparing single-dose placebo, 5 mg desloratadine, 10 mg montelukast and the combination administered 2 h prior to allergen inhalation challenge. Methacholine challenges were performed 24 h before and after allergen challenge. Exhaled nitric oxide measurements and sputum inflammatory cell counts were also carried out. All active treatments significantly decreased the late asthmatic response area under the curve. Combination therapy provided the greatest inhibition compared to desloratadine and montelukast. Montelukast was nonsignificantly better than desloratadine but not as effective as the combination. There was a trend towards a decrease in airway responsiveness following montelukast and combination. Montelukast, but not desloratadine or the combination, decreased exhaled NO levels 24 h after allergen. The allergen-induced increase in sputum eosinophil numbers was significantly suppressed at 7 h with desloratadine and combination therapy, and at 24 h with montelukast and combination therapy. Single-dose co-administration of desloratadine and montelukast 2 h prior to allergen inhalation clinically abolished the late asthmatic response and eosinophil recruitment.

Hapten synthesis and monoclonal antibody-based immunoassay development for detection of the fungicide trifloxystrobin.

High-affinity and selective monoclonal antibodies have been produced against the strobilurin fungicide trifloxystrobin. A battery of functionalized haptens has been synthesized, and conjugate-coated enzyme-linked immunosorbent assays following different procedures have been developed. On the one hand, a two-step conjugate-coated immunoassay was optimized using extended or short incubation times, with limits of detection of 0.10 ng/mL for the extended assay and 0.17 ng/mL for the rapid assay. On the other hand, an immunoassay in the conjugate-coated format was optimized following a procedure consisting of just one incubation step. This one-step assay had a limit of detection of 0.21 ng/mL. All of these assays showed detection limits for trifloxystrobin in the low parts per billion range, well below the common maximum residue limits for this pesticide in foodstuffs (50 microg/kg).

Montelukast treatment in children with moderately severe atopic dermatitis.

BACKGROUND: Moderately severe atopic dermatitis makes up nearly one-fifth of children with atopic dermatitis. OBJECTIVE: To determine the clinical and laboratory effects of montelukast in moderately severe atopic dermatitis. METHODS: Randomized, double-blind, placebo-controlled, crossover trial with washout period, conducted from May 2002 to February 2006. The study involved 25 patients, 2-16 years old with dermatitis. Patients received oral montelukast (9 patients, Group B) or placebo (16 patients, Group A) in phase 1, and were crossed over to placebo or montelukast, respectively, for phase 2. Patients included if > 10% of skin was involved and failed response to 2 week conventional treatment. Itching, sleep disturbance, frequency of use of oral antihistamines & topical steroids, severity scores were serially assessed. In addition, eosinophil and serum IgE were serially collected. RESULTS: Most of patients were 6-10 years of age. Both groups had comparable gender distribution. The patients in Group B were more likely to have a history of bronchial asthma (55.6%) or allergic rhinitis (33.3%) than patients in Group A, but were less likely to have a positive history of atopy. While on montelukast, there was a reduction of mean score for itching in phase 2, for sleep disturbance in phase 2, for antihistamines in phase 1, for extent-of-disease in phase 1 and 2, and for severity score in phase 2 and blood eosinophil & IgE in phase 2. CONCLUSION: Montelukast reduces itching, sleep disturbance, disease extent and severity, blood eosinophil count and serum IgE.

Autoimmune response in MRL+/+ mice following treatment with dichloroacetyl chloride or dichloroacetic anhydride.

Dichloroacetyl chloride (DCAC) is formed from trichloroethene (TCE), which is implicated in inducing/accelerating autoimmune response. Due to its potent acylating activity, DCAC may convert proteins to neo-antigens and thus could induce autoimmune responses. Dichloroacetic anhydride (DCAA), which is a similar acylating agent, might also induce autoimmune responses. To evaluate if chloroacylation plays a role in the induction of autoimmunity, we have measured the autoimmune responses following treatment with DCAC or DCAA in autoimmune-prone MRL+/+ mice. Five-week-old female mice were injected intraperitoneally (twice weekly) with 0.2 mmol/kg of DCAC or DCAA in corn oil for 6 weeks. Total serum IgG, IgG1, and IgE levels were significantly increased in DCAC-treated mice as compared to controls. These increases corresponded with increases in DCAC-specific IgG and IgG1 levels. Total serum IgM was decreased in both DCAC- and DCAA-treated mice. Antinuclear antibodies, measured as an indication of systemic autoimmune responses, were increased in both DCAC- and DCAA-treated mice. Of eight Th1/Th2 cytokines measured in the serum, only IL-5 was significantly decreased in both treatment groups. The cytokine secretion patterns of splenic lymphocytes after stimulation with antibodies against CD3 (T cell receptor-mediated signal) and CD28 (costimulatory signal) differed between treatment and control groups. Levels of IL-1, IL-3, IL-6, IFN-gamma, G-CSF, and KC were higher in cultures of stimulated splenocytes from either DCAC- or DCAA-treated mice than from controls. The level of IL-17 was only increased in cultures from DCAC-treated mice. Increased lymphocytic populations were found in the red pulp of spleens following treatment with either DCAC or DCAA. In addition, thickening of the alveolar septa in the lungs of DCAC- or DCAA-treated mice was observed. The lung histopathology in exposed mice was consistent with the symptomology observed in welders exposed to DCAC/phosgene. Thickening was more pronounced in DCAC-treated mice. Our data suggest that DCAC and DCAA elicit autoimmune responses in MRL+/+ mice that might be reflective of their chloroacylation potential in vivo.

Assessing acetylation of NF-kappaB.

To achieve its full biological activity, NF-kappaB must undergo a variety of post-translational modifications, including acetylation. Acetylation plays a prominent role in regulating the nuclear action of NF-kappaB. The RelA subunit of NF-kappaB forms the major target of acetylation at several different sites. Acetylation of discrete lysine residues in RelA modulates distinct functions of NF-kappaB, including transcriptional activation, DNA binding, and assembly with its inhibitor IkappaBalpha. Here, we describe the experimental methods that have allowed the detection and functional analysis of acetylated forms of NF-kappaB. Acetylation of NF-kappaB can be studied both in vivo and in vitro. In vivo [3H]acetate labeling assays provides a useful, albeit rather insensitive, method for initial verification of acetylation of either over-expressed or endogenous subunits of NF-kappaB. A second valuable in vivo approach involves the use of anti-acetylated lysine antibodies for immunoblotting. However, the success of this approach varies with the specific antibody employed and the target protein studied. In vitro acetylation assays provide a rapid and sensitive method to validate the involvement of candidate histone acetyltransferases and to map the sites of acetylation. Anti-RelA antibodies that selectively react with site-specific acetylated forms of RelA are a singularly powerful tool for the study of NF-kappaB acetylation both in vivo and in vitro.