TOPK Affects Autophagy of Skin Squamous Cell Carcinoma by Regulating NF-KB Pathway through HDAC1.

Objective: To explore the effect and potential mechanism of T-LAK cell-originated protein kinase (TOPK) on autophagy in cutaneous squamous cell carcinoma (cSCC). Methods: Human cSCC cancer tissue and paracancerous tissue samples were collected clinically; immunohistochemistry was used to detect the expression of TOPK, nuclear factor κB p65 (NF-κB p65), phosphorylated nuclear factor κB inhibitor α (p-IκBα), Beclin-1, and microtubule-associated protein 1 light chain 3 (LC3) in cSCC tissue; and immunofluorescence was adopted to detect the coexpression of NF-κB p65, p-IκBα, and TOPK in cSCC tissue. After TOPK silencing or overexpression, SCL-1 and A431 cells were treated with PDTC and 3-MA, respectively. RT-qPCR and Western Blot were used to detect the mRNA and protein expressions of histone deacetylase 1 (HDAC1) in TOPK-silenced/overexpressing cells. Western Blot was performed to detect the protein expressions of NF-κB p65, p-p65, IκBα, p-IκBα, Beclin-1, and LC3 in each group. Transwell and scratch healing experiments were used to detect the ability of cells to invade and migrate. The formation of autophagosomes in each group was observed by TEM. Results: Compared with adjacent tissues, TOPK, NF-κB p65, p-IκBα, Beclin-1, and LC3 were highly expressed in cSCC cancer tissues; TOPK and NF-κB p65 were coexpressed; and TOPK and p-IκBα were expressed in cSCC cancer tissues both increased. The mRNA and protein levels of TOPK in human cSCC cells were significantly higher than those in human normal skin HaCaT cells. After TOPK knockout, the expression of HDAC1, p-IκBα/IκBα, NF-κB p65, p-p65, Beclin-1, LC3II/I proteins, cell invasion, and migration abilities were significantly reduced, and fewer autophagosomes were observed. Treatment with PDTC and 3-MA significantly downregulated NF-κB pathway protein activity and autophagy level and reduced cell migration and invasion ability. Conclusion: TOPK promotes the malignant progression of cSCC by upregulating HDAC1 to activate the NF-κB pathway and promote autophagy.

Geniposide attenuates Staphylococcus aureus-induced pneumonia in mice by inhibiting NF-κB activation.

Staphylococcus aureus (S. aureus) is a major cause of pneumonia that often affects young and immunocompetent patients. Inflammation plays an important role in the development of S. aureus-induced pneumonia. Geniposide, a major iridoid glucoside component of gardenia fruit, has been reported to have anti-inflammatory and anti-oxidative effects. The purpose of this study was to investigate the protective effects of geniposide on S. aureus-induced pneumonia in mice. Lung histopathological changes were detected by hematoxylin-eosin (H&E) staining. Lung myeloperoxidase (MPO) activity, wet-to-dry (W/D) ratio, and inflammatory cytokine levels in bronchoalveolar lavage fluid (BALF) were measured. The results showed that S. aureus-induced lung histopathological changes were attenuated by geniposide. S. aureus-induced MPO activity and lung W/D ratio were inhibited by treatment of geniposide. Furthermore, the levels of TNF-α and IL-1ß in the BALF were also suppressed by geniposide. In addition, geniposide significantly inhibited S. aureus-induced nuclear factor kappa B (NF-κB) activation. Taken together, these results showed that geniposide inhibited S. aureus-induced pneumonia in mice by inhibiting NF-κB signaling pathway. Geniposide might be used as a potential agent for the treatment of S. aureus-induced pneumonia.

Targeted nuclear factor-kappaB suppression enhances gemcitabine response in human pancreatic tumor cell line murine xenografts.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is an almost uniformly fatal malignancy characterized by resistance to chemotherapy. Currently, gemcitabine is the agent used most commonly but demonstrates only a partial response. The transcription factor nuclear factor-kappaB (NF-κB), known to be involved in the inflammatory response, is constitutively activated in PDAC and further activated by gemcitabine. Our aim was to examine the effects of targeted NF-κB suppression on gemcitabine resistance using an in vivo tumor growth model. METHODS: To suppress the NF-κB pathway, the mutant IκBα super-repressor protein was stably expressed in PaCa-2 human PDAC cells. Athymic mice were injected subcutaneously with IκBα-super-repressor (SR) or vector-expressing PaCa-2 cells and randomized to receive phosphate-buffered saline (PBS) or 100 mg/kg gemcitabine(gem) for 4 weeks. RESULTS: The mean increase in tumor volume was 47 mm(3) (89%) and 196 mm(3) (326%) in gem/SR and gem/vector groups, respectively (P = .03). The PBS-treated groups demonstrated greater tumor growth, ∼340 mm(3) (850%) increase, in both PBS/vector and PBS/SR groups. Intratumoral NF-κB activity was decreased in gem/SR compared with the gem/vector group (P = .04). Decreased Ki-67 positivity was noted in gem/SR (49%) versus gem/vector tumors (73%) (P = .04), with no difference in apoptosis (apoptag, P = .3) or angiogenesis (CD31+, P = .9). CONCLUSION: Stable IκBα-SR expression in vivo potentiated the antitumor effects of gemcitabine, resulting in decreased tumor growth in association with decreased cell proliferation. Molecular suppression of the NF-κB pathway decreases successfully gemcitabine resistance in a relatively chemoresistant PDAC line. Thus, NF-κB-targeted agents may complement gemcitabine-based therapies and decrease chemoresistance in patients with PDAC.

Synergistic Reactivation of Latent HIV Expression by Ingenol-3-Angelate, PEP005, Targeted NF-kB Signaling in Combination with JQ1 Induced p-TEFb Activation.

Although anti-retroviral therapy (ART) is highly effective in suppressing HIV replication, it fails to eradicate the virus from HIV-infected individuals. Stable latent HIV reservoirs are rapidly established early after HIV infection. Therefore, effective strategies for eradication of the HIV reservoirs are urgently needed. We report that ingenol-3-angelate (PEP005), the only active component in a previously FDA approved drug (PICATO) for the topical treatment of precancerous actinic keratosis, can effectively reactivate latent HIV in vitro and ex vivo with relatively low cellular toxicity. Biochemical analysis showed that PEP005 reactivated latent HIV through the induction of the pS643/S676-PKCδ/θ-IκBα/ε-NF-κB signaling pathway. Importantly, PEP005 alone was sufficient to induce expression of fully elongated and processed HIV RNAs in primary CD4+ T cells from HIV infected individuals receiving suppressive ART. Furthermore, PEP005 and the P-TEFb agonist, JQ1, exhibited synergism in reactivation of latent HIV with a combined effect that is 7.5-fold higher than the effect of PEP005 alone. Conversely, PEP005 suppressed HIV infection of primary CD4+ T cells through down-modulation of cell surface expression of HIV co-receptors. This anti-cancer compound is a potential candidate for advancing HIV eradication strategies.

Nuclear factor kappa B inhibition improves conductance artery function in type 2 diabetic mice.

BACKGROUND: We previously reported that enhanced nuclear factor kappa B (NFκB) activity is responsible for resistance arteries dysfunction in type 2 diabetic mice. METHODS: In this study, we aimed to determine whether augmented NFκB activity also impairs conductance artery (thoracic aorta) function in type 2 diabetic mice. We treated type 2 diabetic (db(-) /db(-) ) and control (db(-) /db(+) ) mice with two NFκB inhibitors (dehydroxymethylepoxyquinomicin, 6 mg/kg, twice a week and IKK-NBD peptide, 500 µg/kg/day) for 4 weeks. RESULTS: As expected, the NFκB inhibition did not affect blood glucose level and body weight. Thoracic aorta vascular endothelium-dependent relaxation (EDR), determined by the wire myograph, was impaired in diabetic mice compared with control and was significantly improved after NFκB inhibition. Interestingly, thoracic EDR was also rescued in db(-) /db(-p50NFκB-/-) and db(-) /db(-PARP-1-/-) double knockout mice compared with db(-) /db(-) mice. Similarly, the acute in vitro down regulation of NFκB-p65 using p65 shRNA lentiviral particles in arteries from db(-) /db(-) mice also improved thoracic aorta EDR. Western blot analysis showed that the p65NFκB phosphorylation, cleaved PARP-1 and COX-2 expression were increased in thoracic aorta from diabetic mice, which were restored after NFκB inhibition and in db(-) /db(-p-50NFκB-/-) and db(-) /db(-PARP-1-/-) mice. CONCLUSIONS: The present results indicate that in male type 2 diabetic mice, the augmented NFκB activity also impairs conductance artery function through PARP-1 and COX-2-dependent mechanisms.

Restoring conjunctival tolerance by topical nuclear factor-κB inhibitors reduces preservative-facilitated allergic conjunctivitis in mice.

PURPOSE: To evaluate the role of nuclear factor-κB (NF-κB) activation in eye drop preservative toxicity and the effect of topical NF-κB inhibitors on preservative-facilitated allergic conjunctivitis. METHODS: Balb/c mice were instilled ovalbumin (OVA) combined with benzalkonium chloride (BAK) and/or NF-κB inhibitors in both eyes. After immunization, T-cell responses and antigen-induced ocular inflammation were evaluated. Nuclear factor-κB activation and associated inflammatory changes also were assessed in murine eyes and in an epithelial cell line after BAK exposure. RESULTS: Benzalkonium chloride promoted allergic inflammation and leukocyte infiltration of the conjunctiva. Topical NF-κB inhibitors blocked the disruptive effect of BAK on conjunctival immunological tolerance and ameliorated subsequent ocular allergic reactions. In line with these findings, BAK induced NF-κB activation and the secretion of IL-6 and granulocyte-monocyte colony-stimulating factor in an epithelial cell line and in the conjunctiva of instilled mice. In addition, BAK favored major histocompatibility complex (MHC) II expression in cultured epithelial cells in an NF-κB-dependent fashion after interaction with T cells. CONCLUSIONS: Benzalkonium chloride triggers conjunctival epithelial NF-κB activation, which seems to mediate some of its immune side effects, such as proinflammatory cytokine release and increased MHC II expression. Breakdown of conjunctival tolerance by BAK favors allergic inflammation, and this effect can be prevented in mice by topical NF-κB inhibitors. These results suggest a new pharmacological target for preservative toxicity and highlight the importance of conjunctival tolerance in ocular surface homeostasis.

Effect of a novel nuclear factor-κB activation inhibitor on renal ischemia-reperfusion injury.

BACKGROUND: In kidney transplantation, the relationship between prolonged warm or cold ischemic storage of kidneys and a higher incidence of delayed graft function is previously known, and delayed graft function has been known to aggravate poor long-term graft survival. We investigated the effect of a novel nuclear factor-κB activation inhibitor, dehydroxymethylepoxyquinomicin (DHMEQ), on renal ischemia-reperfusion (I/R) injury. METHODS: DHMEQ was administered to Lewis rats once just before renal artery clamping (DHMEQ pretreatment group), and the effect on I/R injury was investigated. RESULTS: In the DHMEQ pretreatment group, the 24-hr urine volume on days 1 to 3 after I/R was significantly larger, and the protein concentration of the urine on days 2 to 7 was significantly smaller than in the untreated group. The serum creatinine level was significantly improved, and significantly lower levels of the inflammatory cells and inflammatory cytokines were present in the kidneys on day 1. The relative ratio of nuclear to cytoplasmic nuclear factor-κB and oxidative stress of kidney tissue on day 1 were significantly decreased. CONCLUSIONS: Treatment with DHMEQ before renal artery clamping may therefore be useful for renal I/R injury and application to renal transplantation is expected.

Targeted delivery of curcumin for treating type 2 diabetes.

Type 2 diabetes is a chronic condition in which cells have reduced insulin signalling, leading to hyperglycemia and long-term complications, including heart, kidney and liver disease. Macrophages activated by dying or stressed cells, induce the transcription factor nuclear factor kappa-B leading to the production of pro-inflammatory cytokines including TNF and IL-6. These inflammatory macrophages in liver and adipose tissue promote insulin resistance, and medications which reduce inflammation and enhance insulin signalling improve glucose control. Curcumin is an anti-oxidant and nuclear factor kappa-B inhibitor derived from turmeric. A number of studies have shown that dietary curcumin reduces inflammation and delays or prevents obesity-induced insulin resistance and associated complications, including atherosclerosis and immune mediate liver disease. Unfortunately dietary curcumin is poorly absorbed by the digestive system and undergoes glucuronidation and excretion rather than being released into the serum and systemically distributed. This confounds understanding of how dietary curcumin exerts its beneficial effects in type 2 diabetes and associated diseases. New improved methods of delivering curcumin are being developed including nanoparticles and lipid/liposome formulations that increase absorption and bioavailability of curcumin. Development and refinement of these technologies will enable cell-directed targeting of curcumin and improved therapeutic outcome.

NF-κB activation fails to protect cells to TNFα-induced apoptosis in the absence of Bcl-xL, but not Mcl-1, Bcl-2 or Bcl-w.

TNFα can promote either cell survival or cell death. The activation of NF-κB plays a central role in cell survival while its inhibition makes TNFα-triggered cytotoxicity possible. Here, we report that the overexpression of a non-degradable mutant of the inhibitor of NF-κB (super-repressor (SR)-IκBα) sensitizes HeLa cells towards TNFα-induced apoptosis, involving caspases activation and cytocrome C release from the mitochondria. Interestingly, we describe that the specific knockdown of Bcl-xL, but not that of Bcl-2, Bcl-w or Mcl-1, renders cells sensitive to TNFα-induced apoptosis. This cytotoxic effect occurs without altering the activation of NF-κB. Then, the activation of the NF-κB pathway is not sufficient to protect Bcl-xL-downregulated cells from TNFα-induced cell death, meaning that TNFα is not able to promote cell survival in the absence of Bcl-xL. In addition, Bcl-xL silencing does not potentiate the cytotoxicity afforded by the cytokine in SR-IκBα-overexpressing cells. This indicates that TNFα-induced apoptosis in SR-IκBα-overexpressing cells relies on the protein levels of Bcl-xL. We have corroborated these findings using RD and DU-145 cells, which also become sensitive to TNFα-induced apoptosis after Bcl-xL knockdown despite that NF-κB remains activated. Altogether, our results point out that the impairment of the anti-apoptotic function of Bcl-xL should make cells sensitive towards external insults circumventing the TNFα-triggered NF-κB-mediated cytoprotective effect. Hence, the specific inhibition of Bcl-xL could be envisaged as a promising alternative strategy against NF-κB-dependent highly chemoresistant proliferative malignancies.

CpG ODN-induced matrix metalloproteinase-13 expression is mediated via activation of the ERK and NF-κB signalling pathways in odontoblast cells.

AIM: To investigate the effects of CpG ODN (CpG oligodeoxynucleotides) to model the action of bacterial challenge on pulpal matrix metalloproteinase-13 (MMP-13) expression and elucidate the associated intracellular signalling pathways. METHODOLOGY: Real-time PCR was used to detect the effects of CpG ODN on MMP-13 mRNA expression levels in a murine odontoblast-lineage cell line (OLCs). The possible involvement of TLR9/MyD88, NF-κB or MAPK pathways involved in the CpG ODN-induced MMP-13 expression was examined by real-time PCR, transient transfection, luciferase activity assay and ELISA. Western blotting was performed to assay the phosphorylation of ERK at a range of time points. RESULTS: MMP-13 was constitutively expressed in OLCs, and their exposure to CpG ODN significantly increased MMP-13 expression. Pre-treatment of OLCs with the inhibitory peptide MyD88, or chloroquine, attenuated the CpG ODN-induced expression of MMP-13. Treatment of the OLCs with CpG ODN increased NF-κB-luciferase activity. This activity was decreased by the over-expression of a nondegrading mutant of IκBα (IκBαSR), although enhanced by the over-expression of NF-κB p65. MMP-13 expression induced by CpG ODN was markedly suppressed by NF-κB inhibitors (pyrrolidine dithiocarbamate, PDTC), IκBα phosphorylation inhibitors (Bay 117082) or IκB protease inhibitor (L-1-tosylamido-2-phenylethyl chloromethyl ketone, TPCK). The inhibitor of ERK1/2, U0126, but not inhibitors of p38 MAPK and JNK, SB203580 and SP600125, decreased CpG ODN-mediated MMP-13 expression. CONCLUSION: The CpG ODN-induced MMP-13 expression in OLCs is mediated through TLR9, NF-κB and the ERK pathway indicating that potentially the recognition of CpG ODN by TLR9 on odontoblasts may regulate the remodelling of injured dental pulp and hard tissues by inducing MMP-13 expression.

Synthetic response of stimulated respiratory epithelium: modulation by prednisolone and iKK2 inhibition.

BACKGROUND: The airway epithelium plays a central role in wound repair and host defense and is implicated in the immunopathogenesis of asthma. Whether there are intrinsic differences between the synthetic capacity of epithelial cells derived from subjects with asthma and healthy control subjects and how this mediator release is modulated by antiinflammatory therapy remains uncertain. We sought to examine the synthetic function of epithelial cells from different locations in the airway tree from subjects with and without asthma and to determine the effects of antiinflammatory therapies upon this synthetic capacity. METHODS: Primary epithelial cells were derived from 17 subjects with asthma and 16 control subjects. The release of 13 cytokines and chemokines from nasal, bronchial basal, and air-liquid interface differentiated epithelial cells before and after stimulation with IL-1ß, IL-1ß and interferon-γ, or Poly-IC (Toll-like receptor 3 agonist) was measured using MesoScale discovery or enzyme-linked immunosorbent assay, and the effects of prednisolone and an inhibitor of nuclear factor κ-B2 (IKK2i) were determined. RESULTS: The pattern of release of cytokines and chemokines was significantly different between nasal, bronchial basal, and differentiated epithelial cells but not between health and disease. Stimulation of the epithelial cells caused marked upregulation of most mediators, which were broadly corticosteroid unresponsive but attenuated by IKK2i. CONCLUSION: Synthetic capacity of primary airway epithelial cells varied between location and degree of differentiation but was not disease specific. Activation of epithelial cells by proinflammatory cytokines and toll-like receptor 3 agonism is attenuated by IKK2i, but not corticosteroids, suggesting that IKK2i may represent an important novel therapy for asthma.

Caffeine suppresses lipopolysaccharide-stimulated BV2 microglial cells by suppressing Akt-mediated NF-κB activation and ERK phosphorylation.

Since the anti-inflammatory effect of caffeine is unclear in microglial cells, we performed whether caffeine attenuates the expression of pro-inflammatory mediators in lipopolysaccharide (LPS)-stimulated BV2 microglial cells. Caffeine substantially suppressed the LPS-induced pro-inflammatory mediators nitric oxide (NO), prostaglandin E(2) (PGE(2)) and tumor necrosis factor-α (TNF-α) in BV2 microglial cells. These effects resulted from the inhibition of their regulatory genes inducible NO synthase (iNOS), cycloxygenase-2 (COX-2) and TNF-α. In addition, caffeine significantly decreased LPS-induced DNA-binding activity of nuclear factor-κB (NF-κB) by suppressing the nuclear translocation of p50 and p65 subunits. A specific NF-κB inhibitor, pyrrolidine dithiocarbamate (PDTC), attenuated the LPS-induced expression of iNOS, COX-2 and TNF-α genes. In addition, we elucidated that inhibition of Akt phosphorylation plays a crucial role in caffeine-mediated NF-κB regulation in LPS-stimulated BV2 microglial cells. Caffeine also attenuated the LPS-induced phosphorylation of extracellular signal-regulated kinase (ERK) and a specific inhibitor of ERK, PD98059, subsequently downregulated the expression of the pro-inflammatory genes iNOS, COX-2 and TNF-α. Taken together, our data indicate that caffeine suppresses the generation of pro-inflammatory mediators, such as NO, PGE(2) and TNF-α as well as their regulatory genes in LPS-stimulated BV2 microglial cells by inhibiting Akt-dependent NF-κB activation and the ERK signaling pathway.

Activation of nuclear factor-κB pathway is responsible for tumor necrosis factor-α-induced up-regulation of endothelin B2 receptor expression in vascular smooth muscle cells in vitro.

The endothelin B2 (ET(B2)) receptors are induced in vascular smooth muscle cells (VSMCs) in cardiovascular diseases. We tested if in vitro short-term exposure to the pro-inflammatory cytokine tumor necrosis factor-α (TNF-α) could up-regulate ET(B2) receptors in rat mesenteric arteries, and if this effect is through activation of intracellular nuclear factor-κB (NF-κB) pathway. The mesenteric arteries were dissected from male Sprague-Dawley rats and the endothelium was removed. The arteries were co-incubated with TNF-α in serum-free Dulbecco's modified Eagle's medium. Real-time reverse transcription-PCR, Western blot and immunohistochemical staining were employed to assess the mRNA/protein expression of ET(B2) receptors and activation of NF-κB pathway. The results showed that, during organ culture, TNF-α concentration-dependently enhanced ET(B2) receptors expression at both mRNA and protein levels, paralleled with activation of NF-κB pathway in VSMC. The up-regulated ET(B2) receptor expression and NF-κB activation could be effectively suppressed by general transcriptional inhibitor actinomycin D, or either of the selective IκB kinase inhibitors wedelolactone and IMD-0354. Conclusively, the activation of intracellular NF-κB pathway is responsible for the up-regulation of ET(B2) receptors induced by short-term exposure to TNF-α. This could partly explain the toxic effects of TNF-α on VSMCs that account for cardiovascular diseases.

Medicinal mushroom Lingzhi or Reishi, Ganoderma lucidum (W.Curt.:Fr.) P. Karst., beta-glucan induces Toll-like receptors and fails to induce inflammatory cytokines in NF-kappaB inhibitor-treated macrophages.

Beta-Glucan of medicinal Lingzhi or Reishi mushroom, Ganoderma lucidum (BGG), possesses immunostimulatory and anti-tumor activities. Innate immune cells are activated by the binding of beta-glucan to the dectin-1 receptor. The present study investigated the immunostimulating activities of BGG, including binding to dectin-1, secretion of cytokines and reactive oxygen species, and induction of Toll-like receptors (TLRs) in RAW264.7 mouse macrophages. Reverse transcription-polymerase chain reaction and flow cytometry were used for the cytokine and TLR analyses. A mouse inflammation antibody array was used for protein-level cytokine analysis. BGG bound to dectin-1 and induced RAW264.7 cell secretion of several cytokines, including granulocyte colony-stimulating factor, interleukin (IL)-6, regulated upon activation normal T cell expressed and secreted (RANTES), tissue inhibitor of metalloproteinase-1, and tumor necrosis factor-alpha. The secretion of these cytokines was further increased by the addition of lipopolysaccharide (LPS). BGG also induced both nitric oxide and inducible nitric oxide synthase (iNOS). Treatment with an inhibitor of nuclear factor-kappa B (NF-kappaB) reduced the induction of IL-1, IL-6, and iNOS in a concentration-dependent manner. Expressions of TLR2, TLR4, and TLR6 were increased by BGG treatment, and addition of LPS induced further induction of TLR4 and TLR6. Our result indicates that BGG induces macrophage secretion of inflammatory cytokines, which can be potentiated by the presence of LPS, likely by binding to dectin-1 and TLR-2/6 receptors, which activate NF-kappaB and prompt the secretion of cytokines.

Hot water extract of the sclerotium of Polyporus rhinocerus Cooke enhances the immune functions of murine macrophages.

Treatment of hot water extract of the sclerotium of Polyporus rhinocerus (PRW) with murine macrophages including RAW 264.7 cell line and primary macrophages (PMs) could enhance their functional activities. These include a significant up-regulation of pinocytosis; an increase in the production of reactive oxygen species (ROS) and nitric oxide (NO); an increase in tumor necrosis factor alpha (TNF-alpha) production and inducible nitric oxide synthase (iNOS) expression in both RAW 264.7 cells and PMs. Cell surface receptors for yeast-derived beta-glucan, including Dectin-1, CR3, and TLR2, were determined by flow cytometry, and the expression of Dectin-1+ cells on the cell surface decreased in the responses of PMs to PRW. PRW increased phosphorylation of IkappaBalpha, which could trigger the nuclear factor kappa B (NF-kappaB) signal pathway for macrophage activation in RAW 264.7 cells. Therefore, the immunomodulatory effect of PRW could be mediated by macrophage activation via the NF-kappaB signal pathway.

Nicotine reduces TNF-α expression through a α7 nAChR/MyD88/NF-ĸB pathway in HBE16 airway epithelial cells.

AIMS: To explore the signaling mechanism associated with the inhibitory effect of nicotine on tumor necrosis factor (TNF)- α expression in human airway epithelial cells. METHODS: HBE16 airway epithelial cells were cultured and incubated with either nicotine or cigarette smoke extract (CE). Cells were then transfected with α1, α5, or α7 nicotinic acetylcholine receptor (nAChR)-specific small interfering RNAs (siRNAs). The effects of nicotine on the production of proinflammatory factors TNF-α, in transfected cells were analyzed. Furthermore, we assayed the expression levels of myeloid differentiation primary response gene 88 (MyD88) protein, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) p65 protein, NF-κB activity and NF-κB inhibitor alpha (I-κBα) expression in cells after treatment with nicotine or α7 nAChR inhibitor, α -bungarotoxin (α-BTX). RESULTS: The production of TNF-α was lower in cells pretreated with nicotine before lipopolysaccharide (LPS) stimulation, compared with LPS-only-treated cells. In contrast, in α7 siRNA-transfected cells incubated with nicotine and LPS, TNF-α expression was higher than that in non-transfected cells or in α1 or α5 siRNA-transfected cells. Addition of MyD88 siRNA or the NF-κB inhibitor pyridine-2,6-dithiocarboxylic acid (PDTC) also reduced TNF-α expression. Furthermore, we found that nicotine decreased MyD88 protein, NF-κB p65 protein, NF-κB activity and phospho-I-κBα expression induced by CE or LPS. The inhibitor α-BTX could reverse these effects. CONCLUSION: Nicotine reduces TNF-α expression in HBE16 airway epithelial cells, mainly through an α7 nAChR/MyD88/NF-κB pathway.

Osteopontin enhances HIV replication and is increased in the brain and cerebrospinal fluid of HIV-infected individuals.

Despite effective and widely available suppressive anti-HIV therapy, the prevalence of mild neurocognitive dysfunction continues to increase. HIV-associated neurocognitive disorder (HAND) is a multifactorial disease with sustained central nervous system inflammation and immune activation as prominent features. Inflammatory macrophages, HIV-infected and uninfected, play a central role in the development of HIV dementia. There is a critical need to identify biomarkers and to better understand the molecular mechanisms leading to cognitive dysfunction in HAND. In this regard, we identified through a subtractive hybridization strategy osteopontin (OPN, SPP1, gene) an inflammatory marker, as an upregulated gene in HIV-infected primary human monocyte-derived macrophages. Knockdown of OPN in primary macrophages resulted in a threefold decrease in HIV-1 replication. Ectopic expression of OPN in the TZM-bl cell line significantly enhanced HIV infectivity and replication. A significant increase in the degradation of the NF-κB inhibitor, IκBα and an increase in the nuclear-to-cytoplasmic ratio of NF-κB were found in HIV-infected cells expressing OPN compared to controls. Moreover, mutation of the NF-κB binding domain in the HIV-LTR abrogated enhanced promoter activity stimulated by OPN. Interestingly, compared to cerebrospinal fluid from normal and multiple sclerosis controls, OPN levels were significantly higher in HIV-infected individuals both with and without neurocognitive disorder. OPN levels were highest in HIV-infected individuals with moderate to severe cognitive impairment. Moreover, OPN was significantly elevated in brain tissue from HIV-infected individuals with cognitive disorder versus those without impairment. Collectively, these data suggest that OPN stimulates HIV-1 replication and that high levels of OPN are present in the CNS compartment of HIV-infected individuals, reflecting ongoing inflammatory processes at this site despite anti-HIV therapy.

Two homologues of inhibitor of NF-kappa B (IκB) are involved in the immune defense of the Pacific oyster, Crassostrea gigas.

A novel homologue of IκB was cloned from a hemocyte cDNA of Crassostrea gigas (designed as CgIκB2). The complete cDNA of CgIκB2 includes an open reading frame (ORF) of 1032 bp, and 3' and 5'untranslated regions (UTR's) of 141 bp and 279 bp, respectively. The ORF encodes a putative protein of 343 amino acids with a calculated molecular weight of approximately 37.8 kDa. Alignment analysis reveals that CgIκB2 contains a conserved degradation motif and six ankyrin repeats. A phylogenetic analysis suggests that a gene duplication event prior to the gastropod-bivalve divergence resulted in the emergence of two IκB homologues in C. gigas. Distinct maximal expression patterns of CgIκB1 in hemocytes and CgIκB2 in the gonad were observed. CgIκB1 and CgIκB2 expression in response to bacterial challenge is similar and inducible. Moreover, both CgIκB1 and CgIκB2 are able to inhibit NF-κb/Rel activating transcription in S2 or HEK293 cells. Our findings demonstrate that both CgIκB1 and CgIκB2 are involved in immune defense in C. gigas through regulation of NF-κB/Rel activity.

Activity of antimicrobial peptide mimetics in the oral cavity: II. Activity against periopathogenic biofilms and anti-inflammatory activity.

Whereas periodontal disease is ultimately of bacterial etiology, from multispecies biofilms of gram-negative anaerobic microorganisms, much of the deleterious effects are caused by the resultant epithelial inflammatory response. Hence, development of a treatment that combines anti-biofilm antibiotic activity with anti-inflammatory activity would be of great utility. Antimicrobial peptides (AMPs) such as defensins are naturally occurring peptides that exhibit broad-spectrum activity as well as a variety of immunomodulatory activities. Furthermore, bacteria do not readily develop resistance to these agents. However, clinical studies have suggested that they do not represent optimal candidates for exogenous therapeutic agents. Small-molecule mimetics of these AMPs exhibit similar activities to the parent peptides, in addition to having low toxicity, high stability and low cost. To determine whether AMP mimetics have the potential for treatment of periodontal disease, we examined the activity of one mimetic, mPE, against biofilm cultures of Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis. Metabolic assays as well as culture and biomass measurement assays demonstrated that mPE exhibits potent activity against biofilm cultures of both species. Furthermore, as little as 2 µg ml(-1) mPE was sufficient to inhibit interleukin-1ß-induced secretion of interleukin-8 in both gingival epithelial cells and THP-1 cells. This anti-inflammatory activity is associated with a reduction in activation of nuclear factor-κB, suggesting that mPE can act both as an anti-biofilm agent in an anaerobic environment and as an anti-inflammatory agent in infected tissues.

Fibroblast expression of an IκB dominant-negative transgene attenuates renal fibrosis.

It is not clear whether interstitial fibroblasts or tubular epithelial cells are primarily responsible for the profibrotic effects of NF-κB activation during renal fibrogenesis. Here, we crossed mice carrying a conditional IκB dominant-negative transgene (IκBdN) with mice transgenic for cell-specific FSP1.Cre (FSP1(+) fibroblasts) or γGT.Cre (proximal tubular epithelia) and challenged all progeny with unilateral ureteral obstruction. We determined NF-κB activation by nuclear localization of phosphorylated p65 ((p)p65) in renal tissues after 7 days. We observed inhibition of NF-κB activation in interstitial cells and tubular epithelia in obstructed kidneys of FSP1.Cre;IκBdN and γGT.Cre;IκBdN mice, respectively, compared with IκBdN controls (P < 0.05). Deposition of extracellular matrix, however, was significantly lower in the obstructed kidneys of FSP1.Cre;IκBdN mice but not in γGT.Cre;IκBdN mice (P < 0.05). In addition, levels of mRNA encoding the profibrotic PAI-1, fibronectin-EIIIA, and type I (α1) procollagen were significantly lower in obstructed kidneys of FSP1.Cre;IκBdN mice compared with γGT.Cre;IκBdN mice (P < 0.05). Taken together, these data support a profibrotic role for fibroblasts, but not proximal tubular epithelial cells, in modulating NF-κB activation during renal fibrogenesis.