Prolonged NF-κB Activation Research Articles

Prolongation of carrageenan-induced inflammation in human colonic epithelial cells by activation of an NFκB‐BCL10 loop

Carrageenan, a sulfated polysaccharide that is widely used as a food additive, induces inflammatory responses in animal models and human cells. The carrageenan-induced inflammatory cascades involve toll-like receptor (TLR)4- and B-cell leukemia/lymphoma (BCL)10-dependent activation of NF-κB, leading to increased IL-8 production. Translocations involving BCL10 in the mucosa-associated lymphoid tissue (MALT) lymphomas are associated with constitutive activation of NF-κB. This report presents a mechanism by which carrageenan exposure leads to prolonged activation of both BCL10 and NF-κB in human colonic epithelial cells. Study findings demonstrate that nuclear RelA and RelB bind to an NF-κB binding motif in the BCL10 promoter in human colonic epithelial NCM460 and HT-29 cells. In vitro oligonucleotide binding assay, non-radioactive gel shift assay, and chromatin immunoprecipitation (ChIP) indicate binding of RelA and RelB to the BCL10 promoter. Prolonged inflammation follows activation of the BCL10-NFκB inflammatory loop in response to carrageenan, shown by increased BCL10, RelA, and IL-8 for 36 to 48h and increased RelB for 24h following withdrawal of carrageenan after 12h. In contrast, exposure to dextran sulfate sodium, which does not cause inflammation through TLR4 and BCL10 in the colonic epithelial cells, did not provoke prolonged activation of inflammation. The carrageenan-enhanced BCL10 promoter activity was blocked by caffeic acid phenethyl ester (CAPE) and MB-132 which inhibit NF-κB activation. These results indicate that NF-κB binding to the BCL10 promoter can lead to prolonged activation of the carrageenan-induced inflammatory cascade by a transcriptional mechanism involving an NF-κB‐BCL10 loop.

A20/TNFAIP3 inhibits NF-κB activation induced by the Kaposi's sarcoma-associated herpesvirus vFLIP oncoprotein.

Kaposi's sarcoma-associated herpesvirus (KSHV) K13/vFLIP (viral Flice-inhibitory protein) induces transcription of numerous genes through NF-κB activation, including pro-inflammatory cytokines, which contribute to the pathogenesis of Kaposi's sarcoma (KS). In this study, we report that KSHV vFLIP induces the expression of the NF-κB regulatory proteins A20, ABIN-1 and ABIN-3 (A20-binding NF-κB inhibitors) in primary human endothelial cells, and that KS spindle cells express A20 in KS tissue. In reporter assays, A20 strongly impaired vFLIP-induced NF-κB activation in 293T cells, but ABIN-1 and ABIN-3 did not. Mutational analysis established that the C-terminal domain (residues 427-790) is critical for A20 modulation of NF-κB, but the ubiquitin-editing OTU (ovarian tumor) domain is not. In functional assays, A20 inhibited vFLIP-induced expression of the chemokine IP-10, reduced vFLIP-induced cell proliferation and increased IKK1 protein levels. Thus, we demonstrate that A20 negatively regulates NF-κB activation directly induced by KSHV vFLIP. By attenuating excessive and prolonged vFLIP-induced NF-κB activation that could be harmful to KSHV-infected cells, A20 likely has an important role in the pathogenesis of KSHV-associated diseases, in which vFLIP is expressed.

Late Repression of NF-κB Activity by Invasive but Not Non-Invasive Meningococcal Isolates Is Required to Display Apoptosis of Epithelial Cells

Meningococcal invasive isolates of the ST-11 clonal complex are most frequently associated with disease and rarely found in carriers. Unlike carriage isolates, invasive isolates induce apoptosis in epithelial cells through the TNF-α signaling pathway. While invasive and non-invasive isolates are both able to trigger the TLR4/MyD88 pathway in lipooligosaccharide (LOS)-dependant manner, we show that only non-invasive isolates were able to induce sustained NF-κB activity in infected epithelial cells. ST-11 invasive isolates initially triggered a strong NF-κB activity in infected epithelial cells that was abolished after 9h of infection and was associated with sustained activation of JNK, increased levels of membrane TNFR1, and induction of apoptosis. In contrast, infection with carriage isolates lead to prolonged activation of NF-κB that was associated with a transient activation of JNK increased TACE/ADAM17-mediated shedding of TNFR1 and protection against apoptosis. Our data provide insights to understand the meningococcal duality between invasiveness and asymptomatic carriage.

Activation of the glutaredoxin-1 gene by nuclear factor κB enhances signaling

The transcription factor nuclear factor κB (NF-κB) is a critical regulator of inflammation and immunity and is negatively regulated via S-glutathionylation. The inhibitory effect of S-glutathionylation is overcome by glutaredoxin-1 (Grx1), which under physiological conditions catalyzes deglutathionylation and enhances NF-κB activation. The mechanisms whereby expression of the Glrx1 gene is regulated remain unknown. Here we examined the role of NF-κB in regulating activation of Glrx1. Transgenic mice that express a doxycycline-inducible constitutively active version of inhibitory κB kinase-β (CA-IKKβ) demonstrate elevated expression of Grx1. Transient transfection of CA-IKKβ also resulted in significant induction of Grx1. A 2-kb region of the Glrx1 promoter that contains two putative NF-κB binding sites was activated by CA-IKKβ, RelA/p50, and lipopolysaccharide (LPS). Chromatin immunoprecipitation experiments confirmed binding of RelA to the promoter of Glrx1 in response to LPS. Stimulation of C10 lung epithelial cells with LPS caused transient increases in Grx1 mRNA expression and time-dependent increases in S-glutathionylation of IKKβ. Overexpression of Grx1 decreased S-glutathionylation of IKKβ, prolonged NF-κB activation, and increased levels of proinflammatory mediators. Collectively, this study demonstrates that the Glrx1 gene is positively regulated by NF-κB and suggests a feed-forward mechanism to promote NF-κB signaling by decreasing S-glutathionylation.

Multiple Facets of NF-κB in the Heart

The progression from cardiac injury to symptomatic heart failure has been intensely studied over the last decade, and is largely attributable to a loss of functional cardiac myocytes through necrosis, intrinsic and extrinsic apoptosis pathways and autophagy. Therefore, the molecular regulation of these cellular programs has been rigorously investigated in the hopes of identifying a potential cell target that could promote cell survival and/or inhibit cell death to avert, or at least prolong, the degeneration toward symptomatic heart failure. The nuclear factor (NF)-κB super family of transcription factors has been implicated in the regulation of immune cell maturation, cell survival, and inflammation in many cell types, including cardiac myocytes. Recent studies have shown that NF-κB is cardioprotective during acute hypoxia and reperfusion injury. However, prolonged activation of NF-κB appears to be detrimental and promotes heart failure by eliciting signals that trigger chronic inflammation through enhanced elaboration of cytokines including tumor necrosis factor α, interleukin-1, and interleukin-6, leading to endoplasmic reticulum stress responses and cell death. The underlying mechanisms that account for the multifaceted and differential outcomes of NF-κB on cardiac cell fate are presently unknown. Herein, we posit a novel paradigm in which the timing, duration of activation, and cellular context may explain mechanistically the differential outcomes of NF-κB signaling in the heart that may be essential for future development of novel therapeutic interventions designed to target NF-κB responses and heart failure following myocardial injury.

Secreted bacterial effectors that inhibit host protein synthesis are critical for induction of the innate immune response to virulent Legionella pneumophila.

The intracellular bacterial pathogen Legionella pneumophila causes an inflammatory pneumonia called Legionnaires' Disease. For virulence, L. pneumophila requires a Dot/Icm type IV secretion system that translocates bacterial effectors to the host cytosol. L. pneumophila lacking the Dot/Icm system is recognized by Toll-like receptors (TLRs), leading to a canonical NF-κB-dependent transcriptional response. In addition, L. pneumophila expressing a functional Dot/Icm system potently induces unique transcriptional targets, including proinflammatory genes such as Il23a and Csf2. Here we demonstrate that this Dot/Icm-dependent response, which we term the effector-triggered response (ETR), requires five translocated bacterial effectors that inhibit host protein synthesis. Upon infection of macrophages with virulent L. pneumophila, these five effectors caused a global decrease in host translation, thereby preventing synthesis of IκB, an inhibitor of the NF-κB transcription factor. Thus, macrophages infected with wildtype L. pneumophila exhibited prolonged activation of NF-κB, which was associated with transcription of ETR target genes such as Il23a and Csf2. L. pneumophila mutants lacking the five effectors still activated TLRs and NF-κB, but because the mutants permitted normal IκB synthesis, NF-κB activation was more transient and was not sufficient to fully induce the ETR. L. pneumophila mutants expressing enzymatically inactive effectors were also unable to fully induce the ETR, whereas multiple compounds or bacterial toxins that inhibit host protein synthesis via distinct mechanisms recapitulated the ETR when administered with TLR ligands. Previous studies have demonstrated that the host response to bacterial infection is induced primarily by specific microbial molecules that activate TLRs or cytosolic pattern recognition receptors. Our results add to this model by providing a striking illustration of how the host immune response to a virulent pathogen can also be shaped by pathogen-encoded activities, such as inhibition of host protein synthesis.

An additional long-term mechanism of NF-κB regulation after cytokine treatment in a human hepatoma cell line

The eukaryotic transcription factor nuclear factor-κB (NF-κB) is mainly involved in the regulation of immune response and inflammation. A prolonged activation of NF-κB has been reported in context with chronic diseases. What leads to a prolongation of NF-κB activity is not well understood. Here, an increase in total intracellular NF-κB protein and mRNA levels as well as a temporary colocalization of NF-κB with proteasomes in human hepatocytes after treatment with TNF-α or IL-1β is reported. This indicates that beside an instantaneous activation of NF-κB and partly autoregulated inactivation by breakdown and synthesis of the inhibitors of NF-κB (IκBs), there are also mechanisms for a long-term regulation by de novo-synthesis and degradation of NF-κB protein.

Anti-inflammatory and antiosteoclastogenesis properties of endogenous melanocortin receptor type 3 in experimental arthritis

The development of biological therapies has improved management of rheumatoid arthritis. However, costs and unresponsiveness to therapy in a sizeable proportion of patients limit their use, making it imperative to identify new targets for drug development programs. Here we investigated the melanocortin-receptor type 3 (MC(3)) pathway. Gene-deficient mice were subjected to a model of serum-transfer-induced arthritis and joints analyzed for gene expression (cytokines, MCs) and morphology. Pharmacological analyses were also conducted in this model. Osteoclastogenesis was studied from bone marrow cells. Mc(3)(-/-) mice displayed an exacerbated inflammatory arthritis, associated with prominent bone erosion and higher articular expression of Rankl. Osteoclastogenesis studied from Mc(3)(-/-) bone marrow cells revealed a higher degree of responsiveness to Rankl, linked to prolonged NF-κB activation compared to wild types. Up-regulation of a discrete set of inflammatory genes, including Il-1β, Il-6, and Nos2, was measured in Mc(3)(-/-) mice, and a marked up-regulation of joint Mc(3) accompanied arthritis resolution in wild-type mice. Administration of an MC(3) agonist, D[Trp8]-γ-MSH, attenuated disease incidence and severity in wild-type but not Mc(3)(-/-) mice. Overall, these findings identify MC(3)-mediated signaling as a beneficial pathway in experimental arthritis; hence this receptor is a novel target for the development of therapeutics for arthritis.

Oxidative stress enhances IL-8 and inhibits CCL20 production from intestinal epithelial cells in response to bacterial flagellin

Intestinal epithelial cells act as innate immune sentinels, as the first cells that encounter diarrheal pathogens. They use pattern recognition molecules such as the Toll-like receptors (TLRs) to identify molecular signals found on microbes but not host cells or food components. TLRs cannot generally distinguish the molecular signals on pathogenic bacteria from those found in commensals, yet under healthy conditions epithelial immune responses are kept in check. We hypothesized that, in the setting of tissue damage or stress, intestinal epithelial cells would upregulate their responses to TLR ligands to reflect the greater need for immediate protection against pathogens. We treated Caco-2 cells with the TLR5 agonist flagellin in the presence or absence of H(2)O(2) and measured chemokine production and intracellular signaling pathways. H(2)O(2) increased flagellin-induced IL-8 (CXCL8) production in a dose-dependent manner. This was associated with synergistic phosphorylation of p38 MAP kinase and with prolonged I-kappaB degradation and NF-kappaB activation. The H(2)O(2)-mediated potentiation of IL-8 production required the activity of p38, tyrosine kinases, phospholipase Cgamma, and intracellular calcium, but not protein kinase C or protein kinase D. H(2)O(2) prolonged and augmented NF-kappaB activation by flagellin. In contrast to IL-8, CCL20 (MIP3alpha) production by flagellin was reduced by H(2)O(2), and this effect was not calcium dependent. Oxidative stress biases intestinal epithelial responses to flagellin, leading to increased production of IL-8 and decreased production of CCL20. This suggests that epithelial cells are capable of sensing the extracellular environment and adjusting their antimicrobial responses accordingly.

Abstract 5734: Prolonged activation of NFκB (RelA and RelB), BCL10, and IL-8 following exposure to common food additive carrageenan in human colonic epithelial cells may be attributable to NFκB binding site in BCL10 promoter

Abstract The common food additive carrageenan (CGN) is a sulfated polysaccharide that resembles the naturally occurring sulfated glycosaminoglycans (sGAG), particularly chondroitin sulfate and keratan sulfate. Unlike the naturally occurring sGAG, CGN contains alpha-1,3-galactosidic linkages that are immunogenic in humans. Previously, we reported that CGN activation of a TLR4-MyD88 mediated inflammatory cascade proceeds through BCL10 and NFκB (p65), leading to increased secretion of IL-8 in human colonic epithelial cells. Translocations involving BCL10 and MALT1 are found in mucosa-associated lymphoid tissue (MALT) lymphomas, associated with constitutive activation of NFκB. Since CGN has been widely used to induce colitis in animal models of inflammatory bowel disease, we addressed whether CGN exposure can lead to sustained activation of BCL10 and NFκB, consistent with a model of constitutive activation of inflammation and possible pre-malignant changes. Experiments were performed with NCM460 cells, a human colonic epithelial cell line derived from normal colon, in M3:10A medium (Incell) with λCGN (1μg/ml). Triplicate cell preparations and spent media from untreated control cell preparations, cells treated with CGN for only the initial 12 hour time interval, and cells treated for six 12-hour periods were harvested every 12 hours over 72 hours, and media, with or without CGN was replenished. IL-8 in the spent media, and BCL10 in the cytosolic preparations and NFκB [RelA (p65) and RelB] in the nuclear preparations were measured at successive 12-hour periods. IL-8 remained significantly increased for an additional 36 hours following withdrawal of CGN. BCL10 and RelA remained significantly increased for an additional 24 hours, and RelB was significantly increased for an additional 12 hours. These findings are consistent with previous data that demonstrate that CGN activates both canonical and non-canonical pathways of NFκB. The presence of a sequence with high homology to the established NFκB consensus oligonucleotide sequence in the BCL10 promoter may be responsible for an ongoing process of activation following withdrawal of the CGN exposure. In vitro testing of binding of RelA and RelB to the NFκB consensus oligonucleotide, the homologous BCL10 promoter sequence, a mutated BCL10 promoter sequence, and a scrambled NFκB consensus sequence indicates over 50% binding of either RelA or RelB to the BCL10 promoter sequence, compared to 100% to the established NFκB consensus sequence. Hence, the study findings demonstrate that an environmental exposure to a common food additive (CGN) can trigger prolonged activation of an inflammatory loop involving BCL10, RelA, RelB, and IL-8 in human colonic epithelial cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5734.

Kinetic of RelA Activation Controls Magnitude of TLR-Mediated IL-12p40 Induction

IL-12 is a crucial cytokine for dendritic cell-mediated induction of Th 1 cell differentiation. TLR ligands induce IL-12 to differing extents. Stimulation of dendritic cells allowed for the differentiation of three groups of TLRs; potency to induce IL-12 decreased in the order of TLR7/9, TLR3/4, and TLR1/2/6 stimulation. The MAPK, PI3K, and IRF (IFN regulatory factor) signaling pathways could be ruled out to be the cause for the differences in IL-12p40 induction. However, we observed that stimulation of dendritic cells with different TLR ligands resulted in striking differences in the kinetics of NF-kappaB activation. LPS induced a rapid but short-lived activation of RelA, whereas CpG-DNA stimulation resulted in prolonged RelA activity at the IL-12p40 promoter. Only TLR2 and TLR4 ligands were capable of inducing S536 phosphorylation of RelA, which has been proposed to be responsible for early termination of NF-kappaB activation. It is suggested that differences in the kinetics of a common TLR signaling module affect the biological response patterns of various TLRs, with IL-12p40 being a gene that needs prolonged NF-kappaB activation.

Abstract 5279: MCPIP1 is an Inducible Corepressor of NF-KappaB

The transcription factor NF-κB is a central regulator of host immune and inflammatory response. However, excessive and prolonged activation of NF-κB can cause massive damage to host tissues and can result in human inflammatory diseases, such as atherosclerosis and arthritis. Thus, NF-κB activation must be terminated at the appropriate time points. MCPIP1 is a recent identified CCCH-type zinc finger containing protein, which is significantly induced by MCP-1 treatment of human monocytes and thus designated as MCP-induced protein1 (MCPIP1). Here we report that MCPIP1 function as an inducible corepressor of NF-κB to feedback control NF-κB activation. In the transfection experiments, MCPIP1 dose-dependently inhibits inflammatory stimuli-induced NF-κB reporter activity. Overexpression of MCPIP1 does not affect TNFα-initiated IKK activation, NF-κB translocation and DNA binding. Further study reveals that MCPIP1 can directly bind to p65 of NF-κB and recruit NCoR and HDAC1 to form repressing complex, which blocks NF-κB target gene expression. Interestingly, the same stimuli that induce NF-κB activation also can induce MCPIP1 expression and translocation into nucleus. Taken together, our results suggest that MCPIP1 is a novel inducible corepressor of NF-κB and may be a critical player in host immune and inflammatory response. This research has received full or partial funding support from the American Heart Association, AHA Greater Southeast Affiliate (Alabama, Florida, Georgia, Louisiana, Mississippi, Puerto Rico & Tennessee).

A regulatory network of canonical nuclear factor-κB activation predicts high-grade glioma outcome

2007 Background: The mechanism for decision between cell death and survival is under fine regulation. Nuclear factor-κB is a transcription regulator that is known to promote survival in many cellular events. Excessive and prolonged activation of NF-κB has been established as a principal mechanism of tumor chemoresistance, which is primarily mediated by its antiapoptotic activity. The pathway leading to the activation of NF-κB involves a complicated network that includes multiple modulatory signaling molecules. We have discovered alterations in several NF-κB pathway modulators in glioblastoma cells, which may act synergistically in activating NF-κB during resistance formation to O6-alkylating agents, including the TNFAIP3, NFKBIA, and TNIP1 genes. Methods: We have retrospectively evaluated the outcome relationship for these and additional endogenous inhibitors of canonical NF-κB activation in three independent high-grade glioma cohorts comprising 175 tumors. Results: We here confirmed the putative importance of NF-κB pathway activation status in predicting high-grade glioma outcome. Our data indicate an increasing complexity and linkage of several modulatory molecules (TNFAIP3, NFKBIA, TNIP1, and TNIP2) to patient outcome that interact physical and functionally in a cooperative fashion to regulate NF-κB activation. We found that for many of these molecules combined predictor models outperform the predictive power of the individual molecules. This observation is consistent with recent evidence suggesting the cooperation of these endogenous inhibitors in a negative feedback regulation of NF-κB activation and a mutual facilitation of their repressive ability. In terms of outcome prediction, we found several of these inhibitors to outperform established clinical and morphological prognostic variables such as patient age and tumor grade (III vs. IV), as well as the O6-methylguanine DNA methyltransferase (MGMT) gene, the currently most established outcome marker in glioblastomas. Conclusions: These findings raise the hope for this regulatory network as an amenable target to modulate NF-κB-mediated resistance in glioblastoma cells, with the ultimate goal of increasing the efficacy of chemotherapy in patients harboring these challenging tumors. No significant financial relationships to disclose.

NF-κB inhibition by an adenovirus expressed aptamer sensitizes TNFα-induced apoptosis

Prolonged activation of NF-κB is involved in the pathogenesis of chronic inflammatory diseases and associated cancers. NF-κB activation is considered to be a main mechanism opposing TNFα-induced apoptosis. We investigated whether inhibition of NF-κB could sensitize tumor and endothelial cells to TNFα-induced apoptosis. As such, we developed a novel H1 RNA polymerase III promoter driven adenoviral vector to express an RNA aptamer, Ad-A-p50, which selectively inhibits NF-κB activation in the nucleus. This event sensitizes human lung adenocarcinoma cells (A549) and human endothelial cells (HUVEC) to TNFα-induced apoptosis through the multiple pathways regulated by NF-κB, including Bcl-XL, HIF-1α, and VEGF. Our findings also suggest a new mechanism of HIF-1α regulation by NF-κB in the normoxic environment. RNA aptamer inhibition of NF-κB offers exciting opportunities for sensitizing inflammatory and tumor cells to TNFα-induced apoptosis.

IFN-γ Protects Cerulein-Induced Acute Pancreatitis by Repressing NF-κB Activation

We explored the pathophysiological roles of IFN-gamma in cerulein-induced acute pancreatitis. In wild-type (WT) mice, cerulein injection caused acute pancreatitis as evidenced by increased serum amylase levels and pathological changes such as interstitial edema, vacuolization, acinar cell necrosis, and neutrophil infiltration in pancreas. Concomitantly, cerulein treatment augmented intrapancreatic gene expression of TNF-alpha, KC/CXCL1, MIP-2/CXCL2, cyclooxygenase-2 (COX-2), and IFN-gamma in WT mice. In situ hybridization combined with immunofluorescence analyses demonstrated that infiltrating neutrophils expressed IFN-gamma mRNA. Unexpectedly, IFN-gamma(-/-) mice exhibited exacerbated cerulein-induced pancreatic injury, with enhanced neutrophil recruitment. Moreover, intrapancreatic gene expression of TNF-alpha, KC/CXCL1, MIP-2/CXCL2, and COX-2 were significantly exaggerated in IFN-gamma(-/-) mice, compared with WT mice. Cerulein activated NF-kappaB, an indispensable transcription factor for gene transcription of TNF-alpha, KC/CXCL1, MIP-2/CXCL2, and COX-2, in pancreas of cerulein-treated WT mice as evidenced by the increases in nuclear amount and DNA-binding activity of NF-kappaB p65. In comparison with WT mice, IFN-gamma(-/-) mice exhibited exaggerated and prolonged NF-kappaB activation, probably due to reduced acetylation of Stat1, a main signal transducer of IFN-gamma, because acetylated Stat1 can inhibit NF-kappaB activation. Indeed, IFN-gamma acetylated Stat1 and reciprocally reduced NF-kappaB activation and COX-2 expression in neutrophils. Finally, even when administered 4 h after the first cerulein injection, IFN-gamma remarkably attenuated acute pancreatitis in both WT and IFN-gamma(-/-) mice, with reduced NF-kappaB activation and COX-2 expression. Thus, IFN-gamma can have anti-inflammatory effects on acute pancreatitis by depressing the proinflammatory consequences of NF-kappaB activation.

Histone Deacetylase Inhibitor Trichostatin A Sustains Sodium Pervanadate-induced NF-κB Activation by Delaying IκBα mRNA Resynthesis

Histone Deacetylase Inhibitor Trichostatin A Sustains Sodium Pervanadate-induced NF-κB Activation by Delaying IκBα mRNA Resynthesis

Prolonged poly(ADP-ribose) polymerase-1 activity regulates JP-8-induced sustained cytokine expression in alveolar macrophages

Environmental pollutants inducing oxidative stress stimulate chronic inflammatory responses in the lung leading to pulmonary tissue dysfunction. In response to oxidative stress, alveolar macrophages produce both reactive oxygen species and reactive nitrogen species, which induce the expression of a wide variety of immune-response genes. We found that a prolonged exposure of alveolar macrophages to a nonlethal dose (8 μg/ml) of JP-8, the kerosene-based hydrocarbon jet fuel, induced the persistent expression of IL-1, iNOS, and COX-2, as well as cell adhesion molecules (ICAM-1 and VCAM-1). Because poly(ADP-ribose) polymerase (PARP-1), a coactivator of NF-κB, regulates inflammatory responses and associated disorders in the airways, we determined whether JP-8 induces the poly(ADP-ribosyl)ation automodification of PARP-1 in alveolar macrophages. We observed that PARP-1 is activated in a time-dependent manner, which was temporally coincident with the prolonged activation of NF-κB and with the augmented expression of the proinflammatory factors described above. The 4 μg/ml dilution of JP-8 also increased the activity of PARP-1 as well as the expression of iNOS and COX-2, indicating that lower doses of JP-8 also affect the regulation of proinflammatory factors in pulmonary macrophages. Together, these results demonstrate that an extensive induction of PARP-1 might coordinate the persistent expression of proinflammatory mediators in alveolar macrophages activated by aromatic hydrocarbons that can result in lung injury from occupational exposure.

PARP‐1 activation mediates expression of NF‐κB‐regulated pro‐inflammatory mediators in response to JP‐8 jet fuel in alveolar macrophages

Lung alveolar macrophages are critical in the regulation of airway inflammation in response to environmental pollutants. In response to oxidative stress, macrophages produce both reactive oxygen species (ROS) and nitrogen species (RNS), which induce the expression of a wide variety of immune-response genes. We found that a prolonged exposure of rat alveolar macrophages to a non-lethal dose (8 μg/ml) of JP-8 (a hydrocarbon mix) during several time courses induced the persistent expression of IL-1, iNOS, COX-2, as well as cell adhesion molecules (ICAM-1 and VCAM-1). We determined that JP-8 induced the poly(ADP-ribosyl)ation automodification of PARP-1, a coactivator of NF-κB, in alveolar macrophages. PARP-1 is activated in a time dependent manner, which was temporally coincident with the prolonged activation of NF-κB, and with the augmented expression of the pro-inflammatory factors described above. Together, these results demonstrated that an extensive induction of PARP-1 might coordinate the persistent expression pro-inflammatory mediators in alveolar macrophages activated by aromatic hydrocarbons that can result in lung injury of occupational exposure. Supported by the AFOSR Grant FA9550-04-1-0395.

Escherichia coliPrevents Phagocytosis-Induced Death of Macrophages via Classical NF-κB Signaling, a Link to T-Cell Activation

NF-kappaB is a crucial mediator of macrophage inflammatory responses, but its role in the context of pathogen-induced adaptive immune responses has yet to be elucidated. Here, we demonstrate that classical NF-kappaB activation delays phagocytosis-induced cell death (PICD) in Raw 264.7 and bone marrow-derived macrophages (BMDMs) upon ingestion of bacteria from the Escherichia coli laboratory strain Top10. By expression of a nondegradable form of IkappaBalpha (superrepressor) and pyrrolidine dithiocarbamate treatment, prolonged activation of NF-kappaB upon bacterial coculture is suppressed, whereas initial induction is only partially inhibited. This activation pattern results in partial inhibition of cellular activation and reduced expression of costimulatory CD86. Notably, suppression of classical NF-kappaB activation does not influence bacterial uptake rates but is followed by increased production of oxygen radicals and enhanced intracellular killing in Raw macrophages. This is associated with reduced expression of NF-kappaB-dependent antiapoptotic c-IAP-2 and a loss of the mitochondrial transmembrane potential. Accordingly, NF-kappaB inhibition in Raw cells and BMDMs causes increased apoptotic rates within 12 h of bacterial ingestion. Interestingly, accelerated eradication of E. coli in NF-kappaB-inhibited macrophages is associated with reduced antigen-specific T-cell activation in macrophage-lymphocyte cocultures. These data suggest that E. coli inhibits PICD of macrophages via classical, antiapoptotic NF-kappaB activation and thus facilitates signaling to T cells. Subsequently, a proper adaptive immune response is likely to be generated. Conclusively, therapeutic inhibition of classical NF-kappaB activation in macrophages may hamper the initiation of adaptive immunity.

Prolonged activation of NF-κB by human cytomegalovirus promotes efficient viral replication and late gene expression

Infection of fibroblasts by human cytomegalovirus (HCMV) rapidly activates the NF-κB signaling pathway, which we documented promotes efficient transactivation of the major immediate-early promoter (DeMeritt, I.B., Milford, L.E., Yurochko, A.D. (2004). Activation of the NF-κB pathway in human cytomegalovirus-infected cells is necessary for efficient transactivation of the major immediate-early promoter. J. Virol. 78, 4498–4507). Because a second, sustained increase in NF-κB activity following the initial phase of NF-κB activation was also observed, we investigated the role that this prolonged NF-κB activation played in viral replication and late gene expression. We first investigated HCMV replication in cells in which NF-κB activation was blocked by pretreatment with NF-κB inhibitors: HCMV replication was significantly decreased in these cultures. A decrease in replication was also observed when NF-κB was inhibited up to 48 h post-infection, suggesting a previously unidentified role for NF-κB in the regulation of the later class of viral genes.