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1.  LITAF, a BCL6 target gene, regulates autophagy in mature B-cell lymphomas 
British journal of haematology  2013;162(5):621-630.
Summary
We have previously reported that LITAF is silenced by promoter hypermethylation in germinal center-derived B-cell lymphomas, but beyond these data the regulation and function of LITAF in B cells are unknown. Gene expression and immunohistochemical studies revealed that LITAF and BCL6 show opposite expression in tonsil B-cell subpopulations and B-cell lymphomas, suggesting that BCL6 may regulate LITAF expression. Accordingly, BCL6 silencing increased LITAF expression, and chromatin immunoprecipitation and luciferase reporter assays demonstrated a direct transcriptional repression of LITAF by BCL6. Gain- and loss-of-function experiments in different B-cell lymphoma cell lines revealed that, in contrast to its function in monocytes, LITAF does not induce LPS-mediated TNFα secretion in B cells. However, gene expression microarrays defined a LITAF-related transcriptional signature containing genes regulating autophagy, including MAP1LC3B (LC3B). In addition, immunofluorescence analysis co-localized LITAF with autophagosomes, further suggesting a possible role in autophagy modulation. Accordingly, ectopic LITAF expression in B-cell lymphoma cells enhanced autophagy responses to starvation, which were impaired upon LITAF silencing. Our results indicate that the BCL6-mediated transcriptional repression of LITAF may inhibit autophagy in B cells during the germinal center reaction, and suggest that constitutive repression of autophagy responses in BCL6-driven lymphomas may contribute to lymphomagenesis.
doi:10.1111/bjh.12440
PMCID: PMC4111142  PMID: 23795761
Non-Hogdkin Lymphoma; B cells; Transcription Factors; Germinal Center; Autophagy
2.  Novel Transcriptional Regulation of VEGF in inflammatory processes 
Vascular endothelial growth factor (VEGF) is a critical angiogenic factor affecting endothelial cells, inflammatory cells and neuronal cells. In addition to its well-defined positive role in wound healing, pathological roles for VEGF have been described in cancer and inflammatory diseases (i.e. atherosclerosis, rheumatoid arthritis, inflammatory bowel disease, and osteoarthritis). Recently we showed that transcription factors LITAF and STAT6B affected the inflammatory response. The present study builds upon our previous results in testing the role of mouse LITAF and STAT6B in the regulation of VEGF-mediated processes. Cells co-transfected with a series of VEGF promoter deletions along with truncated forms of mLITAF and/or mSTAT6B identified a DNA binding site (between −338 and −305 upstream of the transcription site) important in LITAF and/or STAT6B-mediated transcriptional regulation of VEGF. LITAF and STAT6B corresponding protein sites were identified. In addition, siRNA-mediated knockdown of mLITAF and/or mSTAT6B leads to significant reduction of VEGF mRNA levels and inhibits LPS-induced VEGF secretion in mouse RAW 264.7 cells and. Furthermore, VEGF treatment of mouse macrophage or endothelial cells induces LITAF/STAT6B nuclear translocation and cell migration. To translate these observations in vivo, VEGF164-soaked matrigel were implanted in whole-body LITAF-deficient animals (TamLITAF−/−), wild-type mice silenced for STAT6B, and in respective control animals. Vessel formation was found significantly reduced in TamLITAF−/− as well as in STAT6B-silenced wild-type animals compared to control animals. The present data demonstrates that VEGF regulation by LITAF and/or STAT6B is important in angiogenesis signaling pathways and may be a useful target in the treatment of VEGF diseases.
doi:10.1111/jcmm.12020
PMCID: PMC3612137  PMID: 23414097
regulation; VEGF; LITAF; STAT6B; interaction; angiogenesis
3.  Proteomic mapping of stimulus-specific signaling pathways involved in THP-1 cells exposed to Porphyromonas gingivalis or its purified components 
Journal of proteome research  2007;6(6):2211-2221.
Periodontitis is an inflammatory disease initiated by host-parasite interactions which contributes to connective tissue destruction and alveolar bone resorption. Porphyromonas gingivalis (P.g.), a black-pigmented Gram-negative anaerobic bacterium, is a major pathogen in the development and progression of periodontitis. To characterize the role that Porphyromonas gingivalis and its cell surface components play in disease processes, we investigated the differential expression of proteins induced by live P.g., P.g LPS and P.g FimA, using two dimensional gel electrophoresis in combination with mass spectrometry. We have tested whether, at the level of protein expression, unique signaling pathways are differentially induced by the bacterial components P.g. LPS and P.g. FimA, as compared to live P.g..
We found that P.g. LPS stimulation of THP-1 up-regulated the expression of a set of proteins compared to control: deoxyribonuclease, actin, carbonic anhydrase 2, alpha enolase, adenylyl cyclase-associated protein (CAP1), protein disulfide isomerase (PDI), glucose regulated protein (grp78) and 70-kDa heat shock protein (HSP70), whereas FimA treatment did not result in statistically significant changes to protein levels versus the control. Live P.g. stimulation resulted in 12 differentially expressed proteins: CAP1, tubulin beta-2 chain, ATP synthase beta chain, tubulin alpha-6 chain, PDI, vimentin, 60-kDa heat shock protein and nucleolin were found to be up-regulated, while carbonic anhydrase II, beta-actin and HSP70 were down-regulated relative to control. These differential changes by the bacteria and its components are interpreted as preferential signal pathway activation in host immune/inflammatory responses to P.g. infection.
doi:10.1021/pr070031u
PMCID: PMC3925766  PMID: 17477557
Lipopolysaccharide; mass spectrometry; monocytes/macrophages; Porphyromonas gingivalis; Toll-like receptors; proteomics
4.  Metabolic Proximity in the Order of Colonization of a Microbial Community 
PLoS ONE  2013;8(10):e77617.
Microbial biofilms are often composed of multiple bacterial species that accumulate by adhering to a surface and to each other. Biofilms can be resistant to antibiotics and physical stresses, posing unresolved challenges in the fight against infectious diseases. It has been suggested that early colonizers of certain biofilms could cause local environmental changes, favoring the aggregation of subsequent organisms. Here we ask whether the enzyme content of different microbes in a well-characterized dental biofilm can be used to predict their order of colonization. We define a metabolic distance between different species, based on the overlap in their enzyme content. We next use this metric to quantify the average metabolic distance between neighboring organisms in the biofilm. We find that this distance is significantly smaller than the one observed for a random choice of prokaryotes, probably reflecting the environmental constraints on metabolic function of the community. More surprisingly, this metabolic metric is able to discriminate between observed and randomized orders of colonization of the biofilm, with the observed orders displaying smaller metabolic distance than randomized ones. By complementing these results with the analysis of individual vs. joint metabolic networks, we find that the tendency towards minimal metabolic distance may be counter-balanced by a propensity to pair organisms with maximal joint potential for synergistic interactions. The trade-off between these two tendencies may create a “sweet spot” of optimal inter-organism distance, with possible broad implications for our understanding of microbial community organization.
doi:10.1371/journal.pone.0077617
PMCID: PMC3813667  PMID: 24204896
5.  Macrophage polarization: An opportunity for improved outcomes in biomaterials and regenerative medicine✰ 
Biomaterials  2012;33(15):3792-3802.
The host response to biomaterials has been studied for decades. Largely, the interaction of host immune cells, macrophages in particular, with implanted materials has been considered to be a precursor to granulation tissue formation, the classic foreign body reaction, and eventual encapsulation with associated negative impacts upon device functionality. However, more recently, it has been shown that macrophages, depending upon context dependent polarization profiles, are capable of affecting both detrimental and beneficial outcomes in a number of disease processes and in tissue remodeling following injury. Herein, the diverse roles played by macrophages in these processes are discussed in addition to the potential manipulation of macrophage effector mechanisms as a strategy for promoting site-appropriate and constructive tissue remodeling as opposed to deleterious persistent inflammation and scar tissue formation.
doi:10.1016/j.biomaterials.2012.02.034
PMCID: PMC3727238  PMID: 22386919
Foreign body response; Leukocyte; Macrophage; Biocompatibility
6.  Partial Restoration of Macrophage Alteration from Diet-Induced Obesity in Response to Porphyromonas gingivalis Infection 
PLoS ONE  2013;8(7):e70320.
Obesity is a chronic inflammatory disease that weakens macrophage innate immune response to infections. Since M1 polarization is crucial during acute infectious diseases, we hypothesized that diet-induced obesity inhibits M1 polarization of macrophages in the response to bacterial infections. Bone marrow macrophages (BMMΦ) from lean and obese mice were exposed to live Porphyromonas gingivalis (P. gingivalis) for three incubation times (1 h, 4 h and 24 h). Flow cytometry analysis revealed that the M1 polarization was inhibited after P. gingivalis exposure in BMMΦ from obese mice when compared with BMMΦ from lean counterparts. Using a computational approach in conjunction with microarray data, we identified switching genes that may differentially control the behavior of response pathways in macrophages from lean and obese mice. The two most prominent switching genes were thrombospondin 1 and arginase 1. Protein expression levels of both genes were higher in obese BMMΦ than in lean BMMΦ after exposure to P. gingivalis. Inhibition of either thrombospondin 1 or arginase 1 by specific inhibitors recovered the M1 polarization of BMMΦ from obese mice after P. gingivalis exposure. These data indicate that thrombospondin 1 and arginase 1 are important bacterial response genes, whose regulation is altered in macrophages from obese mice.
doi:10.1371/journal.pone.0070320
PMCID: PMC3726386  PMID: 23922979
7.  Novel transcriptional regulation of VEGF in inflammatory processes 
Vascular endothelial growth factor (VEGF) is a critical angiogenic factor affecting endothelial cells, inflammatory cells and neuronal cells. In addition to its well-defined positive role in wound healing, pathological roles for VEGF have been described in cancer and inflammatory diseases (i.e. atherosclerosis, rheumatoid arthritis, inflammatory bowel disease and osteoarthritis). Recently, we showed that transcription factors LITAF and STAT6B affected the inflammatory response. This study builds upon our previous results in testing the role of mouse LITAF and STAT6B in the regulation of VEGF-mediated processes. Cells cotransfected with a series of VEGF promoter deletions along with truncated forms of mLITAF and/or mSTAT6B identified a DNA binding site (between −338 and −305 upstream of the transcription site) important in LITAF and/or STAT6B-mediated transcriptional regulation of VEGF. LITAF and STAT6B corresponding protein sites were identified. In addition, siRNA-mediated knockdown of mLITAF and/or mSTAT6B leads to significant reduction in VEGF mRNA levels and inhibits LPS-induced VEGF secretion in mouse RAW 264.7 cells. Furthermore, VEGF treatment of mouse macrophage or endothelial cells induces LITAF/STAT6B nuclear translocation and cell migration. To translate these observations in vivo, VEGF164-soaked matrigel were implanted in whole-body LITAF-deficient animals (TamLITAF−/−), wild-type mice silenced for STAT6B, and in respective control animals. Vessel formation was found significantly reduced in TamLITAF−/− as well as in STAT6B-silenced wild-type animals compared with control animals. The present data demonstrate that VEGF regulation by LITAF and/or STAT6B is important in angiogenesis signalling pathways and may be a useful target in the treatment of VEGF diseases.
doi:10.1111/jcmm.12020
PMCID: PMC3612137  PMID: 23414097
regulation; VEGF; LITAF; STAT6B; interaction; angiogenesis
8.  LITAF and TNFSF15, two downstream targets of AMPK, exert inhibitory effects on tumor growth 
Oncogene  2011;30(16):1892-1900.
LPS-induced TNFα factor (LITAF) is a multiple functional molecule whose sequence is identical to small integral membrane protein of the lysosome/late endosome (SIMPLE). LITAF was initially identified as a transcription factor that activates transcription of proinflammatory cytokine in macrophages in response to LPS. Mutations of the LITAF gene are associated with a genetic disease, called Charcot-Marie-Tooth syndrome. Recently we have reported that mRNA levels of LITAF and tumor necrosis factor superfamily member 15 (TNFSF15) are upregulated by AMPK. The present study further assesses their biological functions. Thus, we show that AICAR, a pharmacological activator of AMPK, increases the abundance of LITAF and TNFSF15 in the LNCaP and C4-2 prostate cancer cells, which is abrogated by shRNA or dominant negative mutant of AMPK α1 subunit. Our data further demonstrate that AMPK activation upregulates the transcription of LITAF. Intriguingly, silencing LITAF by shRNA enhances proliferation, anchorage-independent growth of these cancer cells, and tumor growth in xenograft model. In addition, our study reveals that LITAF mediates the effect of AMPK by binding to a specific sequence in the promoter region. Furthermore, we show that TNFSF15 remarkably inhibits the growth of prostate cancer cells and bovine aortic endothelial cells in vitro with a more potent effect toward the latter. In conjuncture, intratumor injection of TNFSF15 significantly reduces the size of tumors and number of blood vessels and induces changes characteristic of tumor cell differentiation. Therefore, our studies for the first time establish the regulatory axis of AMPK-LITAF-TNFSF15. They also suggest that LITAF may function as a tumor suppressor.
doi:10.1038/onc.2010.575
PMCID: PMC3431012  PMID: 21217782
AMPK; LITAF; TNFSF15; p53; tumor suppressor; tumorigenesis
9.  Deep Sequencing of the Oral Microbiome Reveals Signatures of Periodontal Disease 
PLoS ONE  2012;7(6):e37919.
The oral microbiome, the complex ecosystem of microbes inhabiting the human mouth, harbors several thousands of bacterial types. The proliferation of pathogenic bacteria within the mouth gives rise to periodontitis, an inflammatory disease known to also constitute a risk factor for cardiovascular disease. While much is known about individual species associated with pathogenesis, the system-level mechanisms underlying the transition from health to disease are still poorly understood. Through the sequencing of the 16S rRNA gene and of whole community DNA we provide a glimpse at the global genetic, metabolic, and ecological changes associated with periodontitis in 15 subgingival plaque samples, four from each of two periodontitis patients, and the remaining samples from three healthy individuals. We also demonstrate the power of whole-metagenome sequencing approaches in characterizing the genomes of key players in the oral microbiome, including an unculturable TM7 organism. We reveal the disease microbiome to be enriched in virulence factors, and adapted to a parasitic lifestyle that takes advantage of the disrupted host homeostasis. Furthermore, diseased samples share a common structure that was not found in completely healthy samples, suggesting that the disease state may occupy a narrow region within the space of possible configurations of the oral microbiome. Our pilot study demonstrates the power of high-throughput sequencing as a tool for understanding the role of the oral microbiome in periodontal disease. Despite a modest level of sequencing (∼2 lanes Illumina 76 bp PE) and high human DNA contamination (up to ∼90%) we were able to partially reconstruct several oral microbes and to preliminarily characterize some systems-level differences between the healthy and diseased oral microbiomes.
doi:10.1371/journal.pone.0037919
PMCID: PMC3366996  PMID: 22675498
10.  Controlling the Outcome of the Toll-Like Receptor Signaling Pathways 
PLoS ONE  2012;7(2):e31341.
The Toll-Like Receptors (TLRs) are proteins involved in the immune system that increase cytokine levels when triggered. While cytokines coordinate the response to infection, they appear to be detrimental to the host when reaching too high levels. Several studies have shown that the deletion of specific TLRs was beneficial for the host, as cytokine levels were decreased consequently. It is not clear, however, how targeting other components of the TLR pathways can improve the responses to infections. We applied the concept of Minimal Cut Sets (MCS) to the ihsTLR v1.0 model of the TLR pathways to determine sets of reactions whose knockouts disrupt these pathways. We decomposed the TLR network into 34 modules and determined signatures for each MCS, i.e. the list of targeted modules. We uncovered 2,669 MCS organized in 68 signatures. Very few MCS targeted directly the TLRs, indicating that they may not be efficient targets for controlling these pathways. We mapped the species of the TLR network to genes in human and mouse, and determined more than 10,000 Essential Gene Sets (EGS). Each EGS provides genes whose deletion suppresses the network's outputs.
doi:10.1371/journal.pone.0031341
PMCID: PMC3282698  PMID: 22363624
11.  Periodontal Innate Immune Mechanisms Relevant to Atherosclerosis and Obesity 
Periodontology 2000  2010;54(1):207-221.
doi:10.1111/j.1600-0757.2010.00358.x
PMCID: PMC2928057  PMID: 20712641
12.  LITAF Mediation of Increased TNF-α Secretion from Inflamed Colonic Lamina Propria Macrophages 
PLoS ONE  2011;6(9):e25849.
Dysregulation of TNF-α in lamina propria macrophages (LPM) is a feature of inflammatory bowel diseases (IBD). LPS-Induced-TNF-Alpha-Factor (LITAF) is a transcription factor that mediates TNF-α expression. To determine whether LITAF participates in the mediation of TNF-α expression in acutely inflamed colonic tissues, we first established the TNBS-induced colonic inflammation model in C57BL/6 mice. LPM were harvested from non-inflamed and inflamed colonic tissue and inflammatory parameters TNF-α and LITAF mRNA and protein levels were measured ex-vivo. LPM from TNBS-treated mice secreted significantly more TNF-α at basal state and in response to LPS than LPM from untreated mice (p<0.05). LITAF mRNA and protein levels were elevated in LPM from TNBS compared with untreated animals and LPS further increased LITAF protein levels in LPM from inflamed tissue (P<0.05). To further confirm the role of LITAF in acutely inflamed colonic tissues, TNBS-induced colonic inflammation was produced in LITAF macrophage specific knockout mice (LITAF mac -/- mice) and compared to wild type (WT) C57BL/6. Twenty four hours following TNBS administration, colonic tissue from LITAF mac -/- mice had less MPO activity and reduced colonic TNF-α mRNA then WT C57BL/6 mice (p<0.05). LPM harvested from LITAF mac -/- secreted significantly less TNF-α in response to LPS than wild type (WT) C57BL/6 (p<0.05). This study provides evidence that LITAF contributes to the regulation of TNF-α in LPM harvested following acute inflammation or LPS treatment paving the way for future work focusing on LITAF inhibitors in the treatment of TNF-α-mediated inflammatory conditions.
doi:10.1371/journal.pone.0025849
PMCID: PMC3184169  PMID: 21984950
13.  Novel Regulation of CCL2 Gene Expression by Murine LITAF and STAT6B 
PLoS ONE  2011;6(9):e25083.
Inflammation is a multifaceted process: beneficial as a defense mechanism but also detrimental depending on its severity and duration. At the site of injury, inflammatory cells are activated by a cascade of mediators, one of which is LITAF, a transcription regulator known to upregulate TNF-α. We previously showed that human LITAF forms a complex with human STAT6B, which translocates into the nucleus to upregulate cytokine transcription. To dissect the molecular implications of this complex, a murine model was developed and interactions between mouse STAT6B (mSTAT6B) and mouse LITAF (mLITAF) were analyzed. Both mLITAF and mSTAT6B expression were MyD88- and TLR ligand-dependent. Furthermore, mLITAF was found to mediate LPS-induced CCL2 gene transcription with the cooperation of mSTAT6B leading to CCL2 protein expression. In LITAF-deficient mice, mLITAF-mediated CCL2 production in macrophages was significantly reduced compared to the wild-type control animals. Mice knockdown for mSTAT6B by 6BsiRNA1 tail vein injection resulted in a decrease in serum TNF-α and CCL2 production. mLITAF/mSTAT6B complex is proposed to play a role in LPS-induced CCL2 expression and possibly other cytokines.
doi:10.1371/journal.pone.0025083
PMCID: PMC3182193  PMID: 21980379
14.  Bioinformatics Analysis of Macrophages Exposed to Porphyromonas gingivalis: Implications in Acute vs. Chronic Infections 
PLoS ONE  2010;5(12):e15613.
Background
Periodontitis is the most common human infection affecting tooth-supporting structures. It was shown to play a role in aggravating atherosclerosis. To deepen our understanding of the pathogenesis of this disease, we exposed human macrophages to an oral bacteria, Porphyromonas gingivalis (P. gingivalis), either as live bacteria or its LPS or fimbria. Microarray data from treated macrophages or control cells were analyzed to define molecular signatures. Changes in genes identified in relevant pathways were validated by RT-PCR.
Methodology/Principal Findings
We focused our analysis on three important groups of genes. Group PG (genes differentially expressed by live bacteria only); Group LFG (genes differentially expressed in response to exposure to LPS and/or FimA); Group CG (core gene set jointly activated by all 3 stimulants). A total of 842 macrophage genes were differentially expressed in at least one of the three conditions compared to naïve cells. Using pathway analysis, we found that group CG activates the initial phagocytosis process and induces genes relevant to immune response, whereas group PG can de-activate the phagocytosis process associated with phagosome-lysosome fusion. LFG mostly affected RIG-I-like receptor signaling pathway.
Conclusion/Significance
In light of the fact that acute infections involve live bacteria while chronic infections involve a combination of live bacteria and their byproducts, group PG could represent acute P. gingivalis infection while group LFG could represent chronic P. gingivalis infection. Group CG may be associated with core immune pathways, triggered irrespective of the specific stimulants and indispensable to mount an appropriate immune response. Implications in acute vs. chronic infection are discussed.
doi:10.1371/journal.pone.0015613
PMCID: PMC3009741  PMID: 21203416
15.  A PTP4A3 Peptide PIMAP39 Modulates TNF-Alpha Levels and Endotoxic Shock 
Journal of Innate Immunity  2009;2(1):43-55.
Lipopolysaccharide (LPS) stimulation of macrophages initiates intracellular signaling pathways leading to activation of MAPK and its subsequent influence on cytokine production. We recently identified a LITAF-STAT6(B) complex regulated by p38 MAPK in response to LPS stimulation. However, the LPS-induced cascade in the p38/LITAF/TNF signaling pathway remains unclear. Here, we identified PTP4A3, a protein tyrosine phosphotase, as a novel negative regulator of LPS-induced LITAF/TNF-α production. PTP4A3 exerts its negative role by dephosphorylating p38α MAPK in response to LPS stimulation of primary macrophages. PTP4A3 expression is upregulated in primary macrophages. Further structure-function analysis revealed that a unique short peptide (PIMAP39) derived from PTP4A3 is capable of mimicking the functionality of full-length PTP4A3 to selectively dephosphorylate p38α and indirectly suppress LPS-induced LITAF-STAT6B complex when it is translocated from the cytoplasmic region to the nucleus of the cell. Treatment of mice with PIMAP39 significantly attenuates the severity of adverse host responses to LPS stimulation, and in some cases provides complete resistance to a lethal dose of LPS due to suppression of TNF-α production. All together, these results reveal a previously unrecognized role for the PTP4A3 pathway in response to LPS.
doi:10.1159/000235685
PMCID: PMC2943518  PMID: 20375622
Immunology; In vitro assays; Inflammation; Kinases; Regulation of lethal toxin; MAP kinases; Phosphatases; Signal transduction; LITAF; Phosphorylation; Peptide; PIMAP39; TNF
16.  Beneficial Dysregulation of the Time Course of Inflammatory Mediators in Lipopolysaccharide-Induced Tumor Necrosis Factor Alpha Factor-Deficient Mice▿  
To begin to understand the surprising survival of macrophage-specific lipopolysaccharide-induced tumor necrosis factor alpha factor-deficient (macLITAF−/−) animals after a lethal dose of lipopolysaccharide (LPS), as reported earlier, the present follow-up study focuses on the role of LITAF in the regulation of inflammatory cytokines secreted in response to lethal or sublethal doses of LPS administered to wild-type (WT) and macLITAF−/− mice. A time course study of kinase expression in peritoneal macrophages revealed increased phosphorylation of prosurvival kinases Akt, Erk1/2, and ribosomal S6 kinase (RSK) in macLITAF−/− mice compared to that in WT mice (n = 8), confirming their role in LPS-mediated diseases. macLITAF−/− mice (n = 8) survived a lethal dose of LPS plus d-galactosamine (d-GalN), expressing lower serum levels of pro- and anti-inflammatory cytokines than the WT levels. To extend our knowledge on LPS-induced inflammatory events, an effective sublethal dose of LPS was administered to the animals (n = 14). WT animals exhibited an acute inflammatory response that decreased after 4 h. Interestingly, macLITAF−/− mice exhibited an initial delay in the secretion of proinflammatory cytokines that peaked after 8 h and reached WT levels after 18 h. Anti-inflammatory cytokine secretions were initially delayed but increased after 4 h and remained elevated compared to WT levels, even after 18 h. Our results demonstrate that LITAF deficiency in vivo affects cytokines other than TNF-α and influences the balance between the pro- and anti-inflammatory cytokines, which protects the animals from the deleterious effects of an LPS-induced inflammatory response, resulting in a beneficial host regulation of inflammatory cytokines and in enhanced survival. Therapeutic intervention aimed at reducing LITAF via kinase modulators may prove useful in preventing LPS-induced mortality.
doi:10.1128/CVI.00510-09
PMCID: PMC2863386  PMID: 20219876
17.  Identification and characterization of Kava-derived compounds mediating TNF-α suppression 
Chemical biology & drug design  2009;74(2):121-128.
There is a substantial unmet need for new classes of drugs that block TNF-α-mediated inflammation, and particularly for small molecule agents that can be taken orally. We have screened a library of natural products against an assay measuring TNF-α secretion in lipopolysaccharide (LPS)-stimulated THP-1 cells, seeking compounds capable of interfering with the TNF-α inducing transcription factor Lipopolysaccharide Induced TNF Alpha Factor (LITAF). Among the active compounds were several produced by the kava plant (Piper mysticum), extracts of which have previously been linked to a range of therapeutic effects. When tested in vivo, a representative of these compounds, kavain, was found to render mice immune to lethal doses of LPS. Kavain displays promising pharmaceutical properties, including good solubility and high cell permeability, but pharmacokinetic experiments in mice showed relatively rapid clearance. A small set of kavain analogs was synthesized, resulting in compounds of similar or greater potency in vitro compared to kavain. Interestingly, a ring-opened analog of kavain inhibited TNF-α secretion in the cell based assay and suppressed LITAF expression in the same cells, whereas the other compounds inhibited TNF-α secretion without affecting LITAF levels, indicating a potential divergence in mechanism of action.
doi:10.1111/j.1747-0285.2009.00838.x
PMCID: PMC2754715  PMID: 19538508
TNF-α; inflammation; kava root extract; kavain; medicinal chemistry; optimization; LITAF
18.  Amelioration of emphysema in mice through lentiviral transduction of long-lived pulmonary alveolar macrophages 
Directed gene transfer into specific cell lineages in vivo is an attractive approach for both modulating gene expression and correcting inherited mutations such as emphysema caused by human α1 antitrypsin (hAAT) deficiency. However, somatic tissues are mainly comprised of heterogeneous, differentiated cell lineages that can be short lived and difficult to specifically transfect. Here, we describe an intratracheally instilled lentiviral system able to deliver genes selectively to as many as 70% of alveolar macrophages (AMs) in the mouse lung. Following a single in vivo lentiviral transduction, genetically tagged AMs persisted in lung alveoli and expressed transferred genes for the lifetime of the adult mouse. A prolonged macrophage lifespan, rather than precursor cell proliferation, accounted for the surprisingly sustained presence of transduced AMs. We utilized this long-lived population to achieve localized secretion of therapeutic levels of hAAT protein in lung epithelial lining fluid. In an established mouse model of emphysema, lentivirally delivered hAAT ameliorated the progression of emphysema, as evidenced by attenuation of increased lung compliance and alveolar size. After 24 weeks of sustained gene expression, no humoral or cellular immune responses to hAAT protein were detected. Our results challenge the dogma that AMs are short lived and suggest that these differentiated cells may be a possible target cell population for in vivo gene therapy applications, including the sustained correction of hAAT deficiency.
doi:10.1172/JCI36666
PMCID: PMC2798672  PMID: 20038801
19.  Metabolic Network Model of a Human Oral Pathogen▿ ‡  
Journal of Bacteriology  2008;191(1):74-90.
The microbial community present in the human mouth is engaged in a complex network of diverse metabolic activities. In addition to serving as energy and building-block sources, metabolites are key players in interspecies and host-pathogen interactions. Metabolites are also implicated in triggering the local inflammatory response, which can affect systemic conditions such as atherosclerosis, obesity, and diabetes. While the genome of several oral pathogens has been sequenced, quantitative understanding of the metabolic functions of any oral pathogen at the system level has not been explored yet. Here we pursue the computational construction and analysis of the genome-scale metabolic network of Porphyromonas gingivalis, a gram-negative anaerobe that is endemic in the human population and largely responsible for adult periodontitis. Integrating information from the genome, online databases, and literature screening, we built a stoichiometric model that encompasses 679 metabolic reactions. By using flux balance approaches and automated network visualization, we analyze the growth capacity under amino-acid-rich medium and provide evidence that amino acid preference and cytotoxic by-product secretion rates are suitably reproduced by the model. To provide further insight into the basic metabolic functions of P. gingivalis and suggest potential drug targets, we study systematically how the network responds to any reaction knockout. We focus specifically on the lipopolysaccharide biosynthesis pathway and identify eight putative targets, one of which has been recently verified experimentally. The current model, which is amenable to further experimental testing and refinements, could prove useful in evaluating the oral microbiome dynamics and in the development of novel biomedical applications.
doi:10.1128/JB.01123-08
PMCID: PMC2612419  PMID: 18931137
20.  Atheroprotective Role of Interleukin-6 in Diet- and/or Pathogen-associated Atherosclerosis using an ApoE Heterozygote Murine Model 
Atherosclerosis  2007;197(2):504-514.
Background
Pathogens have been implicated in the pathogenesis of inflammatory atherosclerosis. Given the pleiotropic role of Interleukin-6 in the regulation of cytokines, lipid homeostasis, vascular remodeling, and apoptosis we hypothesized that IL-6 plays an important role in development and progression to inflammatory atherosclerosis.
Methods and Results
To explore the role of IL-6 in inflammation- and infection-associated atherosclerosis, 10 week-old ApoE+/−-IL-6+/− and ApoE+/−-IL-6−/− mice fed either high fat diet or regular chow diet were inoculated intravenously, once per week for 14 or 24 consecutive weeks with 50µl live Porphyromonas gingivalis (P.g.) (107CFU) or vehicle (normal saline). Animals were euthanized at 24 weeks of age (14 weeks injection) or 34 weeks of age (24 weeks injection). Histomorphometric analysis of atheromatous lesions, en face analysis over the aortic tree, immunohistochemistry for macrophages and smooth muscle cell, TUNEL staining for apoptotic cells, serum amyloid A (SAA) levels, serum lipids and glucose level, serum cytokines were obtained. ApoE+/−-IL-6−/− mice showed a significant increase in atheromatous lesions in proximal aorta and aortic tree compared to ApoE+/−-IL-6+/− mice for all conditions (chow diet and P.g-inoculated, high fat diet and P.g-inoculated, high fat diet and vehicle-inoculated) at 14 weeks and greater at 24 weeks. SAA levels from ApoE+/−-IL-6−/− mice were significantly higher than ApoE+/−-IL-6+/− mice. IL-6 deficiency led to profound changes in plaque composition evidenced by increased macrophage infiltration, apoptosis, lipid content and decreased smooth muscle cell mass reflecting an unstable plaque phenotype. Array analysis revealed increased levels of proinflammatory cytokines in ApoE+/−-IL-6−/− mice compared to ApoE+/−-IL-6+/− mice, irrespective of diet or inoculation.
Conclusion
The genetic deficiency of IL-6 was found to enhance the formation of diet- and/or pathogen associated atherosclerotic plaques and suggests that IL-6 may play an atheroprotective role.
doi:10.1016/j.atherosclerosis.2007.02.023
PMCID: PMC2430020  PMID: 17412346
Atherosclerosis; Cytokine; Infection; Inflammation; Porphyromonas gingivalis
21.  Toll-Like Receptor-2 Mediates Diet and/or Pathogen Associated Atherosclerosis: Proteomic Findings 
PLoS ONE  2008;3(9):e3204.
Background
Accumulating evidence implicates a fundamental link between the immune system and atherosclerosis. Toll-like receptors are principal sensors of the innate immune system. Here we report an assessment of the role of the TLR2 pathway in atherosclerosis associated with a high-fat diet and/or bacteria in ApoE+/− mice.
Methods and Results
To explore the role of TLR2 in inflammation- and infection-associated atherosclerosis, 10 week-old ApoE+/−-TLR2+/+, ApoE+/−-TLR2+/− and ApoE+/−-TLR2−/− mice were fed either a high fat diet or a regular chow diet. All mice were inoculated intravenously, once per week for 24 consecutive weeks, with 50 µl live Porphyromonas gingivalis (P.g) (107 CFU) or vehicle (normal saline). Animals were euthanized 24 weeks after the first inoculation. ApoE+/−-TLR2+/+ mice showed a significant increase in atheromatous lesions in proximal aorta and aortic tree compared to ApoE+/−-TLR2+/− and ApoE+/−-TLR2−/− mice for all diet conditions. They also displayed profound changes in plaque composition, as evidenced by increased macrophage infiltration and apoptosis, increased lipid content, and decreased smooth muscle cell mass, all reflecting an unstable plaque phenotype. SAA levels from ApoE+/−-TLR2+/+ mice were significantly higher than from ApoE+/−-TLR2+/− and ApoE+/−-TLR2−/− mice. Serum cytokine analysis revealed increased levels of pro-inflammatory cytokines in ApoE+/−-TLR2+/+ mice compared to ApoE+/−-TLR2+/− and TLR2−/− mice, irrespective of diet or bacterial challenge. ApoE+/−-TLR2+/+ mice injected weekly for 24 weeks with FSL-1 (a TLR2 agonist) also demonstrated significant increases in atherosclerotic lesions, SAA and serum cytokine levels compared to ApoE+/−-TLR2−/− mice under same treatment condition. Finally, mass-spectrometry (MALDI-TOF-MS) of aortic samples analyzed by 2-dimentional gel electrophoresis differential display, identified 6 proteins upregulated greater than 2-fold in ApoE+/−-TLR2+/+ mice fed the high fat diet and inoculated with P.g compared to any other group.
Conclusion
Genetic deficiency of TLR2 reduces diet- and/or pathogen-associated atherosclerosis in ApoE+/− mice, along with differences in plaque composition suggesting greater structural stability while TLR-2 ligand-specific activation triggers atherosclerosis. The present data offers new insights into the pathophysiological pathways involved in atherosclerosis and paves the way for new pharmacological interventions aimed at reducing atherosclerosis.
doi:10.1371/journal.pone.0003204
PMCID: PMC2527517  PMID: 18787704
22.  Periodontal disease and systemic conditions: a bidirectional relationship 
For decades, physicians and dentists have paid close attention to their own respective fields, specializing in medicine pertaining to the body and the oral cavity, respectively. However, recent findings have strongly suggested that oral health may be indicative of systemic health. Currently, this gap between allopathic medicine and dental medicine is quickly closing, due to significant findings supporting the association between periodontal disease and systemic conditions such as cardiovascular disease, type 2 diabetes mellitus, adverse pregnancy outcomes, and osteoporosis. Significant effort has brought numerous advances in revealing the etiological and pathological links between this chronic inflammatory dental disease and these other conditions. Therefore, there is reason to hope that the strong evidence from these studies may guide researchers towards greatly improved treatment of periodontal infection that would also ameliorate these systemic illnesses. Hence, researchers must continue not only to uncover more information about the correlations between periodontal and systemic diseases but also to focus on positive associations that may result from treating periodontal disease as a means of ameliorating systemic diseases.
doi:10.1007/s10266-006-0060-6
PMCID: PMC2443711  PMID: 16998613
Periodontal diseases; Systemic diseases; Cardiovascular diseases; Diabetes; Adverse pregnancy outcomes; Osteoporosis
24.  Identification of Proteins Differentially Expressed in Human Monocytes Exposed to Porphyromonas gingivalis and Its Purified Components by High-Throughput Immunoblotting  
Infection and Immunity  2006;74(2):1204-1214.
To characterize the roles of Porphyromonas gingivalis and its components in disease processes, we investigated the cytokine profiles induced by live P. gingivalis, its lipopolysaccharide (LPS), and its major fimbrial protein, fimbrillin (FimA). A cytokine antibody array revealed that human monocyte-derived macrophages were induced to produce chemokines (e.g., monocyte chemoattractant protein 1, macrophage inflammatory protein 1β [MIP-1β], and MIP-3α) as early as 1 h after exposure to P. gingivalis, with production declining after 24 h of exposure. As expected, an extensive repertoire of inflammatory mediators increased subsequent to infection, most predominantly tumor necrosis factor alpha (TNF-α), interleukin 1β (IL-1β), IL-6, IL-10, and granulocyte-macrophage colony-stimulating factor. The induction of cytokines by P. gingivalis was not triggered simply by bacterial cell surface components, since purified P. gingivalis LPS and FimA induced similar patterns of cytokines, while the pattern of cytokines induced by live P. gingivalis was significantly different, indicating that the host defense system senses live bacteria differently than it does the cell surface components LPS and FimA. To further understand the mechanisms by which live P. gingivalis and its components exert their effects, we used a high-throughput immunoblot screening approach (Becton-Dickinson PowerBlot) to analyze intracellular proteins involved in P. gingivalis infection in human macrophages. Exposure of human macrophages to either live P. gingivalis, its LPS, or its FimA protein led to the up-regulation of 12, 8, and 10 proteins and the down-regulation of 15, 8, and 17 proteins, respectively. The expression of proteins involved in gene transcription (e.g., monocyte enhancer factor 2D [MEF2D], signal transducer and activator of transcription 1 [STAT1], STAT3, STAT6, and IL enhancer binding factors [ILF3]), of protein kinases (e.g., mitogen-activated protein kinase 3 [MAPK3], MAP3K8, double-stranded RNA-activated protein kinase [PRKR], and MAP2K4), and of proteins involved in immune responses (e.g., TNF super family member 6 [TNFSF6] and interferon-induced protein with tetratricopeptide repeat 4 [IFIT4]), apoptosis (e.g., genes associated with retinoid interferon-induced mortality 19 [GRIM19]), and other fundamental cellular processes (e.g., clathrin heavy-chain polypeptide, culreticulin, and Ras-associated protein RAB27A) was found to be modulated differentially by P. gingivalis, LPS, and FimA. These differential changes are interpreted as preferential signal pathway activation in host immune/inflammatory responses to P. gingivalis infection.
doi:10.1128/IAI.74.2.1204-1214.2006
PMCID: PMC1360359  PMID: 16428770
25.  Immunization Enhances Inflammation and Tissue Destruction in Response to Porphyromonas gingivalis  
Infection and Immunity  2006;74(4):2286-2292.
It is well established that host-bacterium interactions play a critical role in the initiation and progression of periodontal diseases. By the use of inhibitors, it has been shown that mediators associated with the innate immune response significantly contribute to the disease process. Less is known regarding the role of the acquired immune response. To investigate mechanisms by which the acquired immune response to Porphyromonas gingivalis could affect connective tissue, we used a well-documented calvarial model to study host-bacterium interactions. Injection of P. gingivalis stimulated gamma interferon, interleukin 6, macrophage inflammatory protein 2, and monocyte chemoattractant protein 1 expression as determined by real-time PCR. Prior immunization against P. gingivalis significantly enhanced the mRNA levels of these cytokines and chemokines. Similarly, immunization significantly increased and prolonged the formation of a polymorphonuclear leukocyte and mononuclear cell infiltrate (P < 0.05). In addition, the area of connective tissue destruction, osteoclastogenesis, bone loss, mRNA expression of proapoptotic genes, and degree of fibroblast apoptosis were increased in immunized mice (P < 0.05). These results indicate that activation of the acquired immunity by P. gingivalis increases the inflammatory and destructive responses which occur in part through up-regulating the innate immune response and enhancing osteoclastogenesis and fibroblast apoptosis.
doi:10.1128/IAI.74.4.2286-2292.2006
PMCID: PMC1418897  PMID: 16552059

Results 1-25 (32)