NF-kappaB In Macrophages Research Articles

Abstract 2841: Inducible modulation of NF-kappaB in macrophages reveals that timing of intervention may be critical to therapeutic outcome during mammary tumorigenesis

Abstract Elevated levels of NF-kappaB signaling are associated with multiple cancers. Therefore, it is not surprising that inhibitors of NF-kappaB are being investigated as potential therapeutics. While extensive efforts are being made to identify inhibitors there is less focus on investigating the effects of inhibition in specific cell types and during defined stages of tumor progression. We have developed murine transgenic models that enable us to induce expression of an activator or inhibitor of NF-kappaB in macrophages, by adding doxycycline to the drinking water of mice. We have combined these with the Polyoma model of mammary cancer to investigate the effects of modulation of NF-kappaB signaling specifically within macrophages. Given several recent publications we were predicting that activation of NF-kappaB would have pro-tumor effects. However, using a tail vein injection model, our data identifies a stage during progression in which activation of NF-kappaB in macrophages significantly limits metastasis. Activation of NF-kappaB alters immune cell populations and results in rapid killing of tumor cells during the seeding phase. In this model inhibition of NF-kappaB in macrophages during this critical stage of metastasis is pro-tumor. This suggests that while inhibition of NF-kappaB at later stages of tumor progression may have therapeutic benefit, inhibition during the seeding phase my lead to the opposite effect. Our studies highlight the importance of continued investigation into aspects of cell specificity and timing that may change the potential outcome of a clinical trial using NF-kappaB inhibitors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2841. doi:10.1158/1538-7445.AM2011-2841

Intra-arterial Targeted Islet-specific Expression of Sirt1 Protects β Cells From Streptozotocin-induced Apoptosis in Mice

Gene therapy provides a promising approach to curing diabetes. However, an effective route for islet-specific targeting has yet to be established. Toward this end, the pancreatic blood circulation system in Balb/c mice was determined by the injection of rhodamine-containing beads. The efficiency of islet targeting was then measured by the injection of adenoviral vectors carrying a green fluorescence gene via the celiac trunk (C.T.). The results showed that >95% of islets and about 60% of β cells within the pancreatic body and tail could be labeled 3 days after surgery. α-Cell labeling was not as efficient, whereas labeling of nonendocrine tissues was barely detectable. For proof of principle, adenoviral vectors carrying a Sirtuin transgene were injected similarly to test the islet protection effect in the streptozotocin (STZ)-induced type 1 diabetic model. The results demonstrated that overexpression of Sirtuin in STZ-treated mice reduced the level of β-cell death and extent of glucose intolerance. This study reports on efficient islet-specific targeting by using adenoviral injection. This procedure could be invaluable to the treatment of diabetes and the study of islet biology.

Abstract 1352: Macrophage specific regulation reveals both pro- and anti-tumor effects of NF-kappaB during mammary tumor progression

Abstract An increased level of macrophages in human tumors is associated with a poorer prognosis. The transcription factor Nuclear Factor-kappaB (NF-kappaB) is an important regulator of gene expression within macrophages. Recent studies point to a pro-tumorigenic role for NF-kappaB activity in macrophages; however NF-kappaB also regulates expression of genes such as inducible nitric oxide synthase which are associated with anti-tumor effects. We believe that NF-kappaB activity in macrophages can have both anti- and pro-tumor effects depending on tumor stage. We have developed novel murine models to determine the effects of modulation of NF-kappaB activity specifically within macrophages during primary mammary tumor growth and metastasis. We have used the cfms promoter to specifically express the reverse tetracycline transactivator (rtTA) protein in macrophages. The rtTA can drive tet operon regulated gene expression in the presence of doxycyline (dox). We have used this with a dominant negative IkappaBalpha to inhibit NF-kappaB signaling in the Polyoma mouse mammary tumor model, this model is termed DNFP. We have also used the cfms-rtTA in combination with a constitutively active IKK2 transgene to activate NF-kappaB signaling, this model is termed IKFM. When NF-kappaB is inhibited in macrophages during primary tumor growth in our DNFP model there is a reduction in primary tumor growth accompanied by a reduced level of lung metastasis. This data suggests that NF-kappaB in macrophages is acting in a pro-tumor manner, in agreement with recently published data. In contrast, we have found that activation of NF-kappaB in macrophages by treating IKFM mice with dox for 1 week before and during two weeks following tail vein injection of polyoma mammary tumor cells leads to a significant reduction in surface lung tumor formation. In additional studies, IKFM mice were pretreated with dox for 1 week but treatment was stopped two days after tumor injection. A similar reduction in tumor number was observed, suggesting an inhibition of cell seeding. Real time PCR analysis of lungs from mice with activation of NF-kappaB in macrophages suggests an anti-tumor “M1” macrophage phenotype. In summary, our data indicates that NF-kappaB activity in macrophages can have both anti- and pro-tumor effects dependent on tumor stage. This data would suggest that cell specificity and timing of treatment are important considerations with regards to the use of NF-kappaB inhibitors in cancer treatment. 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 1352.

Immunostimulatory Effects ofCordyceps militarison Macrophages through the Enhanced Production of Cytokines via the Activation of NF-κB

BackgroundCordyceps militaris has been used in traditional medicine to treat numerous diseases and has been reported to possess both antitumor and immunomodulatory activities in vitro and in vivo. However, the pharmacological and biochemical mechanisms of Cordyceps militaris extract (CME) on macrophages have not been clearly elucidated. In the present study, we examined how CME induces the production of proinflammatory cytokines, transcription factor, and the expression of co-stimulatory molecules.MethodsWe confirmed the mRNA and protein levels of proinflammatory cytokines through RT-PCR and western blot analysis, followed by a FACS analysis for surface molecules.ResultsCME dose dependently increased the production of NO and proinflammatory cytokines such as IL-1β, IL-6, TNF-α, and PGE2, and it induced the protein levels of iNOS, COX-2, and proinflammatory cytokines in a concentration-dependent manner, as determined by western blot and RT-PCR analysis, respectively. The expression of co-stimulatory molecules such as ICAM-1, B7-1, and B7-2 was also enhanced by CME. Furthermore, the activation of the nuclear transcription factor, NF-κB in macrophages was stimulated by CME.ConclusionBased on these observations, CME increased proinflammatory cytokines through the activation of NF-κB, further suggesting that CME may prove useful as an immune-enhancing agent in the treatment of immunological disease.

Temporal resolution of two-tracked NF-κB activation byLegionella pneumophila

The intracellular pathogen Legionella pneumophila activates the transcription factor NF-kappaB in macrophages and human epithelial cells, contributing to cytokine production and anti-apoptosis. The former is important for the innate immune response to infection, the latter for intracellular replication by securing host cell survival. Here, we demonstrate biphasic activation of NF-kappaB by L. pneumophila in human epithelial cells, using a p65-GFP expressing variant of A549 cells. Early in infection, a strong but transient nuclear translocation of p65 was observed. Only flagellin-deficient (DeltafliA and DeltaflaA) mutants could not induce this first, TLR5 and MyD88-dependent activation. The second p65 translocation event, however, is a long-term activation, independent of flagellin, TLR5 and MyD88, and marked by permanent nuclear localization of p65-GFP without oscillation for 30 h. Persistent p65 translocation also involved degradation of IkappaBalpha and upregulation of anti-apoptotic genes. L. pneumophila mutants lacking a functional Dot/Icm secretion system (DeltadotA; DeltaicmB/dotO), Dot/Icm effectors (DeltasdbA; DeltalubX) and two bacterial effector mutants (DeltaenhC; DeltaptsP) could not induce persistent p65 translocation. Strikingly, all these mutants were deficient in intracellular replication in A549 cells. Our data underline the strong connection between NF-kappaB activation and intracellular replication and hints at an active interference of NF-kappaB signalling by L. pneumophila.

Nuclear factor-kappa B in intestinal protection and destruction

Nuclear factor-kappa B (NF-kappaB) is a key transcriptional regulator of innate and adaptive immunity. This review highlights new insights into the functions of NF-kappaB in normal homeostasis and specific disease processes in the intestinal tract. Inflammatory bowel disease and experimental intestinal inflammation are characterized by NF-kappaB activation and increased expression of proinflammatory NF-kappaB target genes. Accordingly, NF-kappaB inhibition protects against chronic intestinal inflammation and necrotizing enterocolitis in animal models. However, recent findings suggest that NF-kappaB has not only proinflammatory but also tissue-protective functions. Thus, genetic ablation of the regulatory subunit, IkappaB kinase (IKK)gamma, of the central kinase complex required for NF-kappaB activation, IKK, or of both kinase subunits, IKKalpha and IKKbeta, in intestinal epithelial cells causes spontaneous murine colitis. Pharmacological inhibition of IKKbeta, and loss of IKKbeta or NF-kappaB p65 in the epithelium, sensitizes mice to acute inflammatory and injurious challenges. Deficiency in Toll-like receptor 5, a strong activator of NF-kappaB, results in spontaneous colitis and exacerbates mucosal inflammatory responses to Salmonella infection. Conversely, Toll-like receptor 5 stimulation confers radioprotection in the intestine. NF-kappaB has multiple, often opposing functions in the intestine. Antiapoptotic actions of NF-kappaB in intestinal epithelial cells dominate tissue responses to many acute inflammatory and injurious challenges, whereas proinflammatory and cell survival functions of NF-kappaB in macrophages and T cells govern chronic intestinal inflammation.

Upregulation of Phagocyte-Derived Catecholamines Augments the Acute Inflammatory Response

Following our recent report that phagocytic cells (neutrophils, PMNs, and macrophages) are newly discovered sources of catecholamines, we now show that both epinephrine and norepinephrine directly activate NFκB in macrophages, causing enhanced release of proinflammatory cytokines (TNFα, IL-1β, IL-6). Both adrenal-intact (AD+) and adrenalectomized (ADX) rodents were used, because ADX animals had greatly enhanced catecholamine release from phagocytes, facilitating our efforts to understand the role of catecholamines released from phagocytes. Phagocytes isolated from adrenalectomized rats displayed enhanced expression of tyrosine-hydroxylase and dopamine-β-hydroxylase, two key enzymes for catecholamine production and exhibited higher baseline secretion of norepinephrine and epinephrine. The effects of upregulation of phagocyte-derived catecholamines were investigated in two models of acute lung injury (ALI). Increased levels of phagocyte-derived catecholamines were associated with intensification of the acute inflammatory response, as assessed by increased plasma leak of albumin, enhanced myeloperoxidase content in lungs, augmented levels of proinflammatory mediators in bronchoalveolar lavage fluids, and elevated expression of pulmonary ICAM-1 and VCAM-1. In adrenalectomized rats, development of ALI was enhanced and related to α2-adrenoceptors engagement but not to involvement of mineralocorticoid or glucocorticoid receptors. Collectively, these data demonstrate that catecholamines are potent inflammatory activators of macrophages, upregulating NFκB and further downstream cytokine production of these cells. In adrenalectomized animals, which have been used to further assess the role of catecholamines, there appears to be a compensatory increase in catecholamine generating enzymes and catecholamines in macrophages, resulting in amplification of the acute inflammatory response via engagement of α2-adrenoceptors.

Evidence for a Pathogenic Determinant in HIV-1 Nef Involved in B Cell Dysfunction in HIV/AIDS

B lymphocyte hyperactivation and elevated immunoglobulin levels (hypergammaglobulinemia) are pathogenic manifestations of HIV-1 infection. Here we provide evidence that these hallmarks are caused by a soluble factor whose production by infected macrophages is induced by the HIV-1 Nef protein. In vitro, HIV-1-infected macrophages or macrophages expressing Nef promoted B cell activation and differentiation to immunoglobulin-secreting cells. Nef-mediated activation of NF-kappaB in macrophages induced secretion of the acute-phase protein ferritin, and ferritin was necessary and sufficient for the observed Nef-dependent B cell changes. The extent of hypergammaglobulinemia in HIV-1-infected individuals correlated directly with plasma ferritin levels and with viral load. Furthermore, the induction of ferritin production and hypergammaglobulinemia was recapitulated when Nef was specifically expressed in macrophages and T cells of transgenic mice. Collectively, these results indicate that the HIV-1 Nef protein carries a pathogenic determinant that governs B cell defects in HIV-1 infection.

TNFalpha induces ABCA1 through NF-kappaB in macrophages and in phagocytes ingesting apoptotic cells.

Recent evidence suggests that tumor necrosis factor alpha (TNFalpha) signaling in vascular cells can have antiatherogenic consequences, but the mechanisms are poorly understood. TNFalpha is released by free cholesterol-loaded apoptotic macrophages, and the clearance of these cells by phagocytic macrophages may help to limit plaque development. Macrophage cholesterol uptake induces ATP-binding cassette (ABC) transporter ABCA1 promoting cholesterol efflux to apolipoprotein A-I and reducing atherosclerosis. We show that TNFalpha induces ABCA1 mRNA and protein in control and cholesterol-loaded macrophages and enhances cholesterol efflux to apolipoprotein A-I. The induction of ABCA1 by TNFalpha is reduced by 65% in IkappaB kinase beta-deficient macrophages and by 30% in p38alpha-deficient macrophages, but not in jun kinase 1 (JNK1)- or JNK2-deficient macrophages. To evaluate the potential pathophysiological significance of these observations, we fed TNFalpha-secreting free cholesterol-loaded apoptotic macrophages to a healthy macrophage monolayer (phagocytes). ABCA1 mRNA and protein were markedly induced in the phagocytes, a response that was mediated both by TNFalpha signaling and by liver X receptor activation. Thus, TNFalpha signals primarily through NF-kappaB to induce ABCA1 expression in macrophages. In atherosclerotic plaques, this process may help phagocytic macrophages to efflux excess lipids derived from the ingestion of cholesterol-rich apoptotic corpses.

A20 is required for the negative regulation of Lps-induced Nf-kappab in macrophages

A20 is required for the negative regulation of Lps-induced Nf-kappab in macrophages

Macrophages require constitutive NF-kappaB activation to maintain A1 expression and mitochondrial homeostasis.

NF-kappaB is a critical mediator of macrophage inflammatory responses, but its role in regulating macrophage survival has yet to be elucidated. Here, we demonstrate that constitutive NF-kappaB activation is essential for macrophage survival. Blocking the constitutive activation of NF-kappaB with pyrrolidine dithiocarbamate or expression of IkappaBalpha induced apoptosis in macrophagelike RAW 264.7 cells and primary human macrophages. This apoptosis was independent of additional death-inducing stimuli, including Fas ligation. Suppression of NF-kappaB activation induced a time-dependent loss of mitochondrial transmembrane potential (DeltaPsi(m)) and DNA fragmentation. Examination of initiator caspases revealed the cleavage of caspase 9 but not caspase 8 or the effector caspase 3. Addition of a general caspase inhibitor, z-VAD. fmk, or a specific caspase 9 inhibitor reduced DNA fragmentation but had no effect on DeltaPsi(m) collapse, indicating this event was caspase independent. To determine the pathway leading to mitochondrial dysfunction, analysis of Bcl-2 family members established that only A1 mRNA levels were reduced prior to DeltaPsi(m) loss and that ectopic expression of A1 protected against cell death following inactivation of NF-kappaB. These data suggest that inhibition of NF-kappaB in macrophages initiates caspase 3-independent apoptosis through reduced A1 expression and mitochondrial dysfunction. Thus, constitutive NF-kappaB activation preserves macrophage viability by maintaining A1 expression and mitochondrial homeostasis.

Adoptive transfer of nuclear factor-κB–inactive macrophages to the glomerulus

Macrophages have been regarded as "blackguards" in the generation of glomerular injury. However, it is still unclear what kind of cellular machinery is responsible for their pathogenic actions. To explore this issue, this investigation aims at developing a novel strategy using adoptive transfer of "loss-of-function" macrophages to the glomerulus. As a prototypal investigation, this study examines a role for nuclear factor-kappaB (NF-kappaB) in effector actions of macrophages within the glomerular microenvironment. NF-kappaB-inactive macrophages, NIKMACNR, were created by transduction of NR8383 rat macrophages with retrovirus encoding a super-repressor mutant of IkappaBalpha, IkappaBalphaM. The effector functions of NIKMACNR cells on resident cells were evaluated by coculture, cross-feeding, and in vivo macrophage transfer. Rat mesangial cells cocultured with control macrophages showed abundant expression of activation markers, including monocyte chemoattractant protein-1, stromelysin, and gelatinase B. In contrast, coculture with NIKMACNR macrophages induced only modest gene expression. Similarly, culture medium conditioned by activated, control macrophages triggered mesangial cells and isolated glomeruli to express the activation markers, whereas the stimulatory effect was not observed in medium conditioned by NIKMACNR macrophages. To evaluate effector actions of NIKMACNR macrophages in the glomerulus, control macrophages and NIKMACNR cells were transferred into normal rat glomeruli via renal artery injection. After the transfer of control macrophages, substantial induction of the activation marker stromelysin was observed in resident glomerular cells. This induction was dramatically diminished in the glomeruli transferred with NIKMACNR macrophages. Inactivation of NF-kappaB in macrophages effectively disrupted paracrine, stimulatory loops from macrophages to resident glomerular cells. A combination of "loss-of-function" strategies with the technique for adoptive cell transfer is thus useful to explore pathophysiologic roles for certain machinery of macrophages within the glomerulus.

Antisense oligonucleotides to NF-kappaB improve survival in bleomycin-induced pneumopathy of the mouse.

We examined the effect of antisense oligonucleotides to the p65 subunit of NF-kappaB on the survival of bleomycin (BLM)-induced pneumonitis in C57BL/6 mice (female, 8 wk of age, 17 to 20 g body weight). Fifty-three percent and all control mice died within 6 to 9 d after intravenous administration of 150 and 300 mg/kg BLM alone, respectively. The intravenous administration of the antisense oligonucleotides (900 microgram/animal dissolved in 200 microliter saline, 6 h before and 5 d after BLM administration) significantly improved the survival rate to 100 and 40% in 150- and 300-mg/kg-treated animals, respectively. The antisense oligonucleotides also significantly improved the loss of body weight, the increase in lung hydroxyproline, and histologic changes by BLM, whereas the antisense oligonucleotides themselves did not produce any significant changes in the behavior or lung histology. Both peripheral blood monocytes and alveolar macrophages were confirmed to contain large amounts of intracellular antisense oligonucleotides after BLM injection by FITC-labeled fluorescent microscopy. Further, Western blotting confirmed the inhibition of NF-kappaB in macrophages by the antisense oligonucleotides. These findings suggest that the antisense oligonucleotides are incorporated into activated alveolar macrophages and ameleriorate the lung injury and pneumonitis/fibrosis, thereby improving the survival of BLM-induced pneumopathy in mice.

Nitric oxide up-regulates DNA-binding activity of nuclear factor-kappaB in macrophages stimulated with silica and inflammatory stimulants.

Nitric oxide (NO), a reactive nitrogen species, plays an important role in inflammatory lung damage. In the present study, we investigated the role of NO in DNA-binding activity of NF-kappaB in macrophages stimulated with silica or other inflammatory stimulants. Treatment of mouse macrophages (RAW264.7 cells) with a selective inhibitor of inducible nitric oxide synthase (iNOS), L-N6-(1-iminoethyl) lysine (L-NIL), or a nonselective iNOS inhibitor, N omega-nitro-L-arginine methylester (L-NAME), resulted in inhibition of silica-induced nitric oxide production as well as silica-induced NF-kappaB activation. L-NIL also effectively inhibited NF-kappaB activation induced by other inflammatory stimulants, such as lipopolysaccharide (LPS) or muramyl dipeptide (MDP). These inhibitory effects of L-NIL and L-NAME on silica- or LPS-induced NF-kappaB activation were also observed in primary rat alveolar macrophages. Furthermore, NO generating compounds, such as sodium nitroprusside (SNP) and 3-morpholinosydnonimine (SIN-1), caused a dose-dependent increase in NF-kappaB activation, which was positively correlated with the level of NO production. Specific inhibitors of protein tyrosine kinase, such as genistein and AG494, prevented NF-kappaB activation in SNP- or SIN-1 treated cells, suggesting involvement of tyrosine kinase in the NO signaling pathway leading to NF-kappaB activation. In contrast, inhibitors of protein kinase C or A, such as staurosporine or H89, had no inhibitory effect on SIN-1 induced NF-kappaB activation. Metalloporphyrins, such as tetrakis (N-methyl-4'-pyridyl) porphyrinato iron (III) (Fe-TMPyP) and Zn-TMPyP which are known to alter NO-dependent activity, markedly inhibited silica- and LPS-induced NF-kappaB activation. The results suggest that NF-kappaB activation in macrophages can be induced under certain conditions by nitric oxide and that nitric oxide produced by phagocytes exposed to inflammatory agents may up-regulate the activation of NF-kappaB.

Resistance to endotoxic shock as a consequence of defective NF-kappaB activation in poly (ADP-ribose) polymerase-1 deficient mice.

Poly (ADP-ribose) polymerase-1 is a nuclear DNA-binding protein that participates in the DNA base excision repair pathway in response to genotoxic stress in mammalian cells. Here we show that PARP-1-deficient cells are defective in NF-kappaB-dependent transcription activation, but not in its nuclear translocation, in response to TNF-alpha. Treating mice with lipopolysaccharide (LPS) resulted in the rapid activation of NF-kappaB in macrophages from PARP-1(+/+) but not from PARP-1(-/-) mice. PARP-1-deficient mice were extremely resistant to LPS-induced endotoxic shock. The molecular basis for this resistance relies on an almost complete abrogation of NF-kappaB-dependent accumulation of TNF-alpha in the serum and a down-regulation of inducible nitric oxide synthase (iNOS), leading to decreased NO synthesis, which is the main source of free radical generation during inflammation. These results demonstrate a functional association in vivo between PARP-1 and NF-kappaB, with consequences for the transcriptional activation of NF-kappaB and a systemic inflammatory process.

Yersinia enterocolitica impairs activation of transcription factor NF-kappaB: involvement in the induction of programmed cell death and in the suppression of the macrophage tumor necrosis factor alpha production.

In this study, we investigated the activity of transcription factor NF-kappaB in macrophages infected with Yersinia enterocolitica. Although triggering initially a weak NF-kappaB signal, Y. enterocolitica inhibited NF-kappaB activation in murine J774A.1 and peritoneal macrophages within 60 to 90 min. Simultaneously, Y. enterocolitica prevented prolonged degradation of the inhibitory proteins IkappaB-alpha and IkappaB-beta observed by treatment with lipopolysaccharide (LPS) or nonvirulent, plasmid-cured yersiniae. Analysis of different Y. enterocolitica mutants revealed a striking correlation between the abilities of these strains to inhibit NF-kappaB and to suppress the tumor necrosis factor alpha (TNF-alpha) production as well as to trigger macrophage apoptosis. When NF-kappaB activation was prevented by the proteasome inhibitor MG-132, nonvirulent yersiniae as well as LPS became able to trigger J774A.1 cell apoptosis and inhibition of the TNF-alpha secretion. Y. enterocolitica also impaired the activity of NF-kappaB in epithelial HeLa cells. Although neither Y. enterocolitica nor TNF-alpha could induce HeLa cell apoptosis alone, TNF-alpha provoked apoptosis when activation of NF-kappaB was inhibited by Yersinia infection or by the proteasome inhibitor MG-132. Together, these data demonstrate that Y. enterocolitica suppresses cellular activation of NF-kappaB, which inhibits TNF-alpha release and triggers apoptosis in macrophages. Our results also suggest that Yersinia infection confers susceptibility to programmed cell death to other cell types, provided that the appropriate death signal is delivered.