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1.  Therapeutic Effect of TSG-6 Engineered iPSC-Derived MSCs on Experimental Periodontitis in Rats: A Pilot Study 
PLoS ONE  2014;9(6):e100285.
We derived mesenchymal stem cells (MSCs) from rat induced pluripotent stem cells (iPSCs) and transduced them with tumor necrosis factor alpha-stimulated gene-6 (TSG-6), to test whether TSG-6 overexpression would boost the therapeutic effects of iPSC-derived MSCs in experimental periodontitis.
A total of 30 female Sprague-Dawley (SD) rats were randomly divided into four groups: healthy control group (Group-N, n = 5), untreated periodontitis group (Group-P, n = 5), iPS-MSCs-treated and iPSC-MSCs/TSG-6-treated periodontitis groups (Group-P1 and P2, n = 10 per group). Experimental periodontitis was established by ligature and infection with Porphyromonas gingivalis around the maxillae first molar bilaterally. MSC-like cells were generated from rat iPSCs, and transducted with TSG-6. iPSC-MSCs or iPSC-MSCs/TSG-6 were administrated to rats in Group-P1 or P2 intravenously and topically, once a week for three weeks. Blood samples were obtained one week post-injection for the analysis of serum pro-inflammatory cytokines. All animals were killed 3 months post-treatment; maxillae were then dissected for histological analysis, tartrate-resistant acid phosphatase (TRAP) staining, and morphological analysis of alveolar bone loss.
Administration of iPSC-MSC/TSG-6 significantly decreased serum levels of IL-1β and TNF-α in the Group-P2 rats (65.78 pg/ml and 0.56 pg/ml) compared with those in Group-P (168.31 pg/ml and 1.15 pg/ml respectively) (p<0.05). Both alveolar bone loss and the number of TRAP-positive osteoclasts showed a significant decrease in rats that received iPSC-MSC/TSG-6 treatment compared to untreated rats in Group-P (p<0.05),
We demonstrated that overexpression of TSG-6 in rat iPSC-derived MSCs were capable of decreasing inflammation in experimental periodontitis and inhibiting alveolar bone resorption. This may potentially serve as an alternative stem-cell-based approach in the treatment and regeneration of periodontal tissues.
PMCID: PMC4076279  PMID: 24979372
2.  Celecoxib Sensitizes Staphylococcus aureus to Antibiotics in Macrophages by Modulating SIRT1 
PLoS ONE  2014;9(6):e99285.
We have previously shown that celecoxib in combination with an antibiotic, increase the bacterial sensitivity to antibiotics. However, the underlying molecular mechanism remained elusive. Efficacy of the combinatorial treatment of celecoxib and ampicillin in vitro was evaluated on macrophage-phagocytosed S aureus. To elucidate the mechanism, signaling pathway of infection and inflammation involving TLR2, JNK, SIRT1 and NF-κB was studied by FACS, Western blot, ELISA and activity assays. Combinatorial treatment of ampicillin and celecoxib reduced the bacterial load in the macrophages. Further studies clearly suggested the activation of the master regulator of oxidative stress and inflammation SIRT1,, by celecoxib when used alone and/or in combination with ampicillin. Also, the results indicated that celecoxib inhibited JNK phosphorylation thereby stabilizing and activating SIRT1 protein that inhibited the COX-2 gene transcription with a significant decrease in the levels of protein inflammatory cytokines like IL-6, MIP-1α and IL-1β via inhibition of NF-κB. SIRT1 activation by celecoxib also resulted in increase of catalase and peroxidase activity with a decrease in Nitric oxide levels. In conclusion, we demonstrate a novel role of celecoxib in controlling inflammation as an enhancer of antibiotic activity against bacteria by modulating SIRT1.
PMCID: PMC4064976  PMID: 24950067
3.  Dental Caries, Prevalence and Risk Factors in Patients with Crohn’s Disease 
PLoS ONE  2014;9(3):e91059.
The present study tested the hypothesis that patients with Crohn’s disease (CD) have a higher prevalence and risk for caries compared to people without CD.
Material and Methods
Patients with CD were divided into groups; 71 patients (50.7±13.9 years) who had gone through resective intestinal surgery and 79 patients (42.0±14.4 years) who had not. The patients were compared to 75 controls (48.6±13.4 years) regarding DMF-T and DMF-S, Lactobacilli (LB), Streptococcus mutans (SM), salivary flow and dental plaque. Statistical methods including ANOVA or Chi-square test for calculation of demographic differences between groups, analysis of covariance (ANCOVA) to compare the clinical variable and Post hoc analyses were done with Fischers Least Significant Difference test or Chi-square. Non-parametric Spearman’s correlation matrix coefficient was estimated between clinical variables and disease duration.
CD patients who had been subjected to resective surgery had a higher DMF-S score (50.7 versus 36.5; p = 0.01) compared to the control group after adjusting for age, gender and smoking. These patients had higher counts of SM (1.5 versus 0.9; p = 0.04) and LB (10000.0 versus 1000.0; p = 0.01), and more dental plaque (53.7 versus 22.6; p = 0.001). CD patients reported a more frequent consumption of sweetened drinks between meals compared to controls (p = 0.001).
The present study shows that patients with CD who had undergone resective surgery had a higher DMFs score, and higher salivary counts of Lactobacilli and Streptococcus mutans compared to the control group.
PMCID: PMC3946651  PMID: 24608416
4.  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.
PMCID: PMC3612137  PMID: 23414097
regulation; VEGF; LITAF; STAT6B; interaction; angiogenesis
5.  The Transcervical Approach for Parapharyngeal Space Pleomorphic Adenomas: Indications and Technique 
PLoS ONE  2014;9(2):e90210.
Head and Neck Parapharyngeal space tumors are rare. Pleomorphic Adenomas are the most common Parapharyngeal space tumors. The purpose of this study was to define preoperative criteria for enabling full extirpation of parapharyngeal space pleomorphic adenomas via the transcervical approach while minimizing functional and cosmetic morbidity.
The surgical records and medical charts of 19 females and 10 males with parapharyngeal space pleomorphic adenomas operated between 1993 and 2012 were reviewed.
Fifteen patients were operated by a simple transcervical approach, 13 by a transparotid transcervical approach, and one by a transmandibular transcervical approach. Complications included facial nerve paralysis, infection, hemorrhage and first bite syndrome. There were three recurrences, but neither recurrence nor complications were associated with the type of surgical approach.
A simple transcervical approach is preferred for parapharyngeal space pleomorphic adenomas with narrow attachments to the deep lobe of the parotid gland and for pleomorphic adenomas originating in a minor salivary gland within the parapharyngeal space.
PMCID: PMC3937375  PMID: 24587286
6.  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.
PMCID: PMC3925766  PMID: 17477557
Lipopolysaccharide; mass spectrometry; monocytes/macrophages; Porphyromonas gingivalis; Toll-like receptors; proteomics
7.  Association between COX-2 rs 6681231 Genotype and Interleukin-6 in Periodontal Connective Tissue. A Pilot Study 
PLoS ONE  2014;9(2):e87023.
The aim of this pilot study was to investigate associations between IL-6 and COX-2 expression in gingival biopsies and both clinical diagnosis and genotypes in the IL-6 and COX-2 genes.
A case-control study included 41 gingival biopsies obtained from Caucasian patients grouped according to clinical diagnosis of gingival health (n = 10), gingivitis (n = 15) or chronic periodontitis (n = 16). Immunohistochemistry analyses were performed to determine COX-2 expression in lamina propria, IL-6 expression in lamina propria and gingival epithelium and level of inflammatory cell infiltrate. Individual DNA was extracted and genotyped by real-time PCR for IL6 SNPs rs 2069827 and rs 2069825 and for COX-2 rs 6681231.
The percentage of cellular COX-2 expression was associated with the extent of periodontal disease (Arbes index p = 0.026) and inflammatory infiltrate (p<0.0001). No association was observed between IL6 haplotypes and cells positive to IL-6 or COX-2 in gingival tissues. The COX-2 rs 6681231 was associated with cells positive to IL-6 in the connective tissue (p = 0.032).
COX-2 expression in gingival tissues may be a marker of periodontal disease severity. COX-2 rs 6681231 may be associated with IL-6 local production in gingival tissues.
PMCID: PMC3923747  PMID: 24551049
8.  Plasmacytoid Dendritic Cells Mediate the Regulation of Inflammatory Type T Cell Response for Optimal Immunity against Respiratory Chlamydia Pneumoniae Infection 
PLoS ONE  2013;8(12):e83463.
Chlamydia pneumoniae (Cpn) infection is a leading cause for a variety of respiratory diseases and has been implicated in the pathogenesis of chronic inflammatory diseases. The regulatory mechanisms in host defense against Cpn infection are less understood. In this study, we investigated the role of plasmacytoid dendritic cells (pDCs) in immune regulation in Cpn respiratory tract infection. We found that in vivo depletion of pDCs increased the severity of infection and lung pathology. Mice depleted of pDC had greater body weight loss, higher lung bacterial burden and excessive tissue inflammation compared to the control mice. Analysis of specific T cell cytokine production pattern in the lung following Cpn infection revealed that pDC depleted mice produced significantly higher amounts of inflammatory cytokines, especially TNF-α, but lower IL-10 compared to the controls. In particular, pDC depleted mice showed pathogenic T cell responses characterized by inflammatory type-1 (CD8 and CD4) and inflammatory Th2 cell responses. Moreover, pDC depletion dramatically reduced CD4 regulatory T cells (Tregs) in the lungs and draining lymph nodes. Furthermore, pDC-T cell co-culture experiments showed that pDCs isolated from Cpn infected mice were potent in inducing IL-10 producing CD4 Tregs. Together, these findings provide in vivo evidence for a critical role of pDCs in homeostatic regulation of immunity during Cpn infection. Our findings highlight the importance of a ‘balanced’ immune response for host protective immunity and preventing detrimental immunopathology during microbial infections.
PMCID: PMC3873288  PMID: 24386207
9.  Cranialization of the Frontal Sinus for Secondary Mucocele Prevention following Open Surgery for Benign Frontal Lesions 
PLoS ONE  2013;8(12):e83820.
To compare frontal sinus cranialization to obliteration for future prevention of secondary mucocele formation following open surgery for benign lesions of the frontal sinus.
Study Design
Retrospective case series.
Tertiary academic medical center.
Sixty-nine patients operated for benign frontal sinus pathology between 1994 and 2011.
Open excision of benign frontal sinus pathology followed by either frontal obliteration (n = 41, 59%) or frontal cranialization (n = 28, 41%).
Main Outcome Measures
The prevalence of post-surgical complications and secondary mucocele formation were compiled.
Pathologies included osteoma (n = 34, 49%), mucocele (n = 27, 39%), fibrous dysplasia (n = 6, 9%), and encephalocele (n = 2, 3%). Complications included skin infections (n = 6), postoperative cutaneous fistula (n = 1), telecanthus (n = 4), diplopia (n = 3), nasal deformity (n = 2) and epiphora (n = 1). None of the patients suffered from postoperative CSF leak, meningitis or pneumocephalus. Six patients, all of whom had previously undergone frontal sinus obliteration, required revision surgery due to secondary mucocele formation. Statistical analysis using non-inferiority test reveal that cranialization of the frontal sinus is non-inferior to obliteration for preventing secondary mucocele formation (P<0.0001).
Cranialization of the frontal sinus appears to be a good option for prevention of secondary mucocele development after open excision of benign frontal sinus lesions.
PMCID: PMC3871679  PMID: 24376760
10.  Heat Shock Modulates Neutrophil Motility in Zebrafish 
PLoS ONE  2013;8(12):e84436.
Heat shock is a routine method used for inducible gene expression in animal models including zebrafish. Environmental temperature plays an important role in the immune system and infection progression of ectotherms. In this study, we analyzed the impact of short-term heat shock on neutrophil function using zebrafish (Danio rerio) as an animal model. Short-term heat shock decreased neutrophil recruitment to localized Streptococcus iniae infection and tail fin wounding. Heat shock also increased random neutrophil motility transiently and increased the number of circulating neutrophils. With the use of the translating ribosome affinity purification (TRAP) method for RNA isolation from specific cell types such as neutrophils, macrophages and epithelial cells, we found that heat shock induced the immediate expression of heat shock protein 70 (hsp70) and a prolonged expression of heat shock protein 27 (hsp27). Heat shock also induced cell stress as detected by the splicing of X-box binding protein 1 (xbp1) mRNA, a marker for endoplasmic reticulum (ER) stress. Exogenous expression of Hsp70, Hsp27 and spliced Xbp1 in neutrophils or epithelial cells did not reproduce the heat shock induced effects on neutrophil recruitment. The effect of heat shock on neutrophils is likely due to a combination of complex changes, including, but not limited to changes in gene expression. Our results indicate that routine heat shock can alter neutrophil function in zebrafish. The findings suggest that caution should be taken when employing a heat shock-dependent inducible system to study the innate immune response.
PMCID: PMC3868611  PMID: 24367659
11.  Inadequate Exercise as a Risk Factor for Sepsis Mortality 
PLoS ONE  2013;8(12):e79344.
Test whether inadequate exercise is related to sepsis mortality.
Research Design and Methods
Mortality surveillance of an epidemiological cohort of 155,484 National Walkers' and Runners' Health Study participants residing in the United States. Deaths were monitored for an average of 11.6-years using the National Death index through December 31, 2008. Cox proportional hazard analyses were used to compare sepsis mortality (ICD-10 A40-41) to inadequate exercise (<1.07 METh/d run or walked) as measured on their baseline questionnaires. Deaths occurring within one year of the baseline survey were excluded.
Sepsis was the underlying cause in 54 deaths (sepsisunderlying) and a contributing cause in 184 deaths (sepsiscontributing), or 238 total sepsis-related deaths (sepsistotal). Inadequate exercise was associated with 2.24-fold increased risk for sepsisunderlying (95%CI: 1.21 to 4.07-fold, P = 0.01), 2.11-fold increased risk for sepsiscontributing (95%CI: 1.51- to 2.92-fold, P<10−4), and 2.13-fold increased risk for sepsistotal (95%CI: 1.59- to 2.84-fold, P<10−6) when adjusted for age, sex, race, and cohort. The risk increase did not differ significantly between runners and walkers, by sex, or by age. Sepsistotal risk was greater in diabetics (P = 10−5), cancer survivors (P = 0.0001), and heart attack survivors (P = 0.003) and increased with waist circumference (P = 0.0004). The sepsistotal risk associated with inadequate exercise persisted when further adjusted for diabetes, prior cancer, prior heart attack and waist circumference, and when excluding deaths with cancer, or cardiovascular, respiratory, or genitourinary disease as the underlying cause. Inadequate exercise also increased sepsistotal risk in 2163 baseline diabetics (4.78-fold, 95%CI: 2.1- to 13.8-fold, P = 0.0001) when adjusted, which was significantly greater (P = 0.03) than the adjusted risk increase in non-diabetics (1.80-fold, 95%CI: 1.30- to 2.46-fold, P = 0.0006).
Inadequate exercise is a risk factor for sepsis mortality, particular in diabetics.
PMCID: PMC3850902  PMID: 24324580
12.  Activation of Olfactory Receptors on Mouse Pulmonary Macrophages Promotes Monocyte Chemotactic Protein-1 Production 
PLoS ONE  2013;8(11):e80148.
Emerging evidence suggests that non-olfactory tissues and cells can express olfactory receptors (ORs), however, the exact function of ectopic OR expression remains unknown. We have previously shown in mouse models that a unique cooperation between interferon-γ (IFN-γ) and lipopolysaccharide (LPS) drives the activation of pulmonary macrophages and leads to the induction of pathogenic responses in the respiratory tract. Further, through gene array studies, we have shown that activation of macrophages by these molecules results in the selective expression of a number of ORs. In this study, we validated the expression of these ORs in mouse airway and pulmonary macrophages in response to IFN-γ and LPS (γ/LPS) stimulation, and further explored the effect of odorant stimulation on macrophage function.
Methodology/Principal Findings
OR expression in airway and pulmonary macrophages in response to IFN-γ, LPS or γ/LPS treatments was assessed by microarray and validated by q-PCR. OR expression (e.g. OR622) on macrophages was confirmed by visualization in immunofluoresence assays. Functional responses to odorants were assessed by quantifying inflammatory cytokine and chemokine expression using q-PCR and cell migration was assessed by a modified Boyden chamber migration assay. Our results demonstrate that eight ORs are expressed at basal levels in both airway and pulmonary macrophages, and that γ/LPS stimulation cooperatively increased this expression. Pulmonary macrophages exposed to the combined treatment of γ/LPS+octanal (an odorant) exhibited a 3-fold increase in MCP-1 protein production, compared to cells treated with γ/LPS alone. Supernatants from γ/LPS+octanal exposed macrophages also increased macrophage migration in vitro.
Eight different ORs are expressed at basal levels in pulmonary macrophages and expression is upregulated by the synergistic action of γ/LPS. Octanal stimulation further increased MCP-1 production and the motility of macrophages. Our results suggest that ORs may mediate macrophage function by regulating MCP-1 production and cell migration.
PMCID: PMC3836993  PMID: 24278251
13.  Relation of Leptin, Ghrelin and Inflammatory Cytokines with Body Mass Index in Pulmonary Tuberculosis Patients with and without Type 2 Diabetes Mellitus 
PLoS ONE  2013;8(11):e80122.
Pulmonary tuberculosis (TB) patients often suffer from anorexia and poor nutrition, causing weight loss. The peptide hormones leptin and its counterpart ghrelin, acting in the regulation of food intake and fat utilization, play an important role in nutritional balance. This study aimed to investigate the association of blood concentrations of leptin, ghrelin and inflammatory cytokines with body mass index (BMI) in TB patients with and without type 2 diabetes mellitus (T2DM).
BMI, biochemical parameters and plasma levels of leptin, ghrelin and inflammatory cytokines were measured before the start of treatment in 27 incident TB patients with T2DM, 21 TB patients and 23 healthy subjects enrolled in this study.
The levels of leptin were significantly higher in TB patients (35.2±19.1 ng/ml) than TB+T2DM (12.6±6.1 ng/ml) and control (16.1±11.1 ng/ml) groups. The level of ghrelin was significantly lower in TB (119.9±46.1 pg/ml) and non-significantly lower in TB+T2DM (127.7±38.6 pg/ml) groups than control (191.6±86.5 pg/ml) group. The levels of TNF-α were higher, while IFN-γ and IL-6 levels were lower in patients than in the control group. Leptin showed a negative correlation with BMI in TB (r=-0.622, p<0.05) and TB+T2DM (r= -0.654, p<0.05) groups, but a positive correlation with BMI in the control group (r=0.521, p<0.05). Contrary ghrelin showed a positive correlation with BMI in TB (r=0.695, p<0.05) and TB+T2DM (r= 0.199, p>0.05) groups, but negative correlation with BMI in the control (r=-0.693, p<0.05) group. Inflammatory cytokines were poorly correlated with BMI in this study. Only IFN-γ showed a significant negative correlation with BMI in the control group (r=-0.545, p<0.05).
This study may suggest that possible abnormalities in ghrelin and leptin regulation (high levels of leptin and low levels of ghrelin) may be associated with low BMI and may account for the poor nutrition associated with TB and TB+T2DM.
PMCID: PMC3832650  PMID: 24260344
14.  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.
PMCID: PMC3813667  PMID: 24204896
15.  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.
PMCID: PMC3727238  PMID: 22386919
Foreign body response; Leukocyte; Macrophage; Biocompatibility
16.  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.
PMCID: PMC3726386  PMID: 23922979
17.  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.
PMCID: PMC3431012  PMID: 21217782
AMPK; LITAF; TNFSF15; p53; tumor suppressor; tumorigenesis
18.  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.
PMCID: PMC3366996  PMID: 22675498
19.  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.
PMCID: PMC3282698  PMID: 22363624
20.  Periodontal Innate Immune Mechanisms Relevant to Atherosclerosis and Obesity 
Periodontology 2000  2010;54(1):207-221.
PMCID: PMC2928057  PMID: 20712641
21.  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.
PMCID: PMC3184169  PMID: 21984950
22.  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.
PMCID: PMC3182193  PMID: 21980379
23.  Bioinformatics Analysis of Macrophages Exposed to Porphyromonas gingivalis: Implications in Acute vs. Chronic Infections 
PLoS ONE  2010;5(12):e15613.
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.
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.
PMCID: PMC3009741  PMID: 21203416
24.  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.
PMCID: PMC2943518  PMID: 20375622
Immunology; In vitro assays; Inflammation; Kinases; Regulation of lethal toxin; MAP kinases; Phosphatases; Signal transduction; LITAF; Phosphorylation; Peptide; PIMAP39; TNF
25.  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.
PMCID: PMC2863386  PMID: 20219876

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