Manjunatha R Benakanakere

University of Pennsylvania, Philadelphia, Pennsylvania, United States

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Publications (34)78.89 Total impact

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    ABSTRACT: Periodontitis is a common chronic inflammatory disease that is initiated by a complex microbial biofilm that poses significant health and financial burdens globally. Porphyromonas gingivalis is a predominant pathogen that maintains chronic inflammatory periodontitis. Toll-like receptors (TLRs) play an important role in periodontitis by recognizing pathogens and maintaining tissue homeostasis. Deficiencies in TLR expression and downstream signaling may reduce the host's innate defenses against pathogens, leading to bacterial persistence and exacerbated inflammation, which are now being better appreciated in disease pathologies. In the case of periodontitis, gingival epithelial cells form the first line of defense against pathogens. Innate immune dysregulation in these cells relates to severe disease pathology. We recently identified a blunted TLR2 expression in certain gingival epithelial cells expressing diminished cytokine signaling upon P. gingivalis stimulation. Upon detailed analysis of the TLR2 promoter CpG Island, we noted higher CpG methylation in this dysregulated cell type. When these cells were treated with DNA methyltransferase inhibitor, TLR2 mRNA and cytokine expression were significantly increased. If TLR2 expression plasmid was ectopically expressed in dysfunctional cells prior to P. gingivalis stimulation, the cytokine expression was increased, confirming the requirement of TLR2 in the P. gingivalis-mediated inflammatory response. We designed a chronic in vitro infection model to test if P. gingivalis can induce DNA methylation in normal gingival epithelial cells that express higher TLR2 upon agonist stimulation. Chronic treatment of normal epithelial cells with P. gingivalis introduced de novo DNA methylation within the cells. In addition, increased DNA methylation was observed in the gingiva of mice infected with P. gingivalis in a periodontitis oral gavage model. Moreover, tissues obtained from periodontitis patients also exhibited differential TLR2 promoter methylation, as revealed by bisulfite DNA sequencing. Taken together, DNA methylation of TLR2 can modulate host innate defense mechanisms that may confer increased disease susceptibility.
    Journal of Dental Research 11/2014; · 4.14 Impact Factor
  • M. BENAKANAKERE, M. ABDOLHOSSEINI, D. KINANE
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    ABSTRACT: Epithelial recognition of P. gingivalis through TLR2 is crucial in mounting the innate immune response to counter bacterial attack in the human gingiva. However, in certain patients, gingival epithelial cells become dysfunctional creating abrogated epithelial inflammatory and antimicrobial response that may contribute to periodontal disease susceptibility. Objective: The aim was to show that P. gingivalis induces epigenetic reprogramming of TLR2 promoter in gingival epithelial cells that contributes to innate immune dysfunction. Method: DNA from epithelial cells was isolated and subjected to methylation specific qPCR array for TLR related gene network. For chronic infection model, the cells were stimulated with P. gingivalis (MOI: 5) for 30 minutes at 0, 4, 8 and 16 h time intervals. After 48 h of last exposure, the cells were split and repeated P. gingivalis infection cycle with or without 1 μM of decitabine. After which, TLR2 promoter CpG methylation was determined by methylation specific qPCR assay. To corroborate in vitro finding, DNA from healthy and periodontitis affected tissue was bisulfite sequenced to determine the extent of TLR2 promoter methylation. In vivo oral gavage mouse experiments were conducted and the gingiva was subjected TLR2 promoter methylation analysis using methylation specific PCR. Results: We identified TLR2 promoter CpG hypermethylation in ‘dysfunctional’ cells. This methylation status was reversed by the use of decitabine restoring the TLR2 gene and pro-inflammatory cytokine expression. We also show induction of TLR2 promoter CpG methylation by P. gingivalis in our in vitro chronic infection model. The gingival tissue from periodontal sites showed extensive TLR2 CpG methylation compared to healthy sites as revealed by bisulfite DNA sequencing. Moreover, mice inoculated with P. gingivalis in oral gavage mouse model showed increased TLR2 promoter methylation. Conclusion: Here we show specific epigenetic reprogramming of a promoter by P. gingivalis that may contribute to periodontal disease susceptibility.
    AADR Annual Meeting & Exhibition 2014; 03/2014
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    ABSTRACT: Hydrastis canadensis L. also known as “golden seal” is used to treat skin infections. The use of this herb in oral infections particularly against periodontitis is being explored. Objective: The aim was to test the anti-microbial and anti-inflammatory properties of H. canadensis aqueous extract (HC) against Porphyromonas gingivalis (Pg) and primary human gingival epithelial cells (HGECs) stimulated with Pg, respectively. Method: HC root powder was boiled in endotoxin free water (100 mg/ml) for 4 hours, filtrate was dried at 60°C overnight and HC concentrate was dissolved in sterile endotoxin free water (100 µg/ml). The anti-bacterial effect was tested against Pg by inoculating 50 µl of mid-log phase grown bacteria in 2 ml of GAM broth in presence or absence of HC, incubated under anaerobic condition for 16h and OD600 was measured. The bactericidal activity was tested by inoculating 2 µl of HC in mid-log phase grown Pg and further incubated to test bacterial growth. The effect of HC on the production of pro-inflammatory cytokines, IL-1β, TNF and IL-8 were tested in HGECs stimulated with live Pg (MOI 1:10) using qPCR and ELISA. Result: HC was able to completely inhibit the growth of Pg in a dose dependent manner revealing its anti-bacterial property. HC inhibited Pg induced IL-1β, IL-8 and TNF by HGECs. Next, the time course experiment was performed by stimulating HGECs with Pg (MOI 1:10) for 0, 15, 30, 60, 90, 120 minutes and total protein was immunoblotted against phospho-IkBα, phospho-p38, phospho-Erk 1/2, phospho-JNK antibodies. Pg induced NF-kβ, p38 and JNK phosphorylation was down-regulated by HC except for ERK 1/2 in HC treated cells. Conclusion: HC was able to block pro-inflammatory cytokines via the inhibition of NF-kβ-p38-JNK pathway. Taken together, HC showed strong anti-bacterial and anti-inflammatory activities against Pg that can be exploited to develop mouth rinse against periodontitis.
    AADR Annual Meeting & Exhibition 2014; 03/2014
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    ABSTRACT: P. gingivalis is a prominent periodontal pathogen that has potent effects on host cells. In this study we challenged gingival epithelial cells with P. gingivalis with the aim of assessing how mRNA levels of key target genes were modulated by P. gingivalis via the transcription factors FOXO1 and FOXO3. Primary mono- and multi-layer cultures of gingival epithelial cells were challenged and barrier function was examined by fluorescent dextran and apoptosis was measured by cytoplasmic histone associated DNA. Gene expression levels were measured by real-time PCR with and without FOXO1 and FOXO3 siRNA compared to scrambled siRNA. P. gingivalis induced a loss of barrier function and stimulated gingival epithelial cell apoptosis in multilayer cultures that was in part gingipain dependent. P. gingivalis stimulated an increase in FOXO1 and FOXO3 mRNA, enhanced mRNA levels of genes associated with differentiated keratinocyte function (keratin-1, -10, -14, and involucrin), increased mRNA levels of apoptotic genes (BID and TRADD), reduced mRNA levels of genes that regulate inflammation (TLR-2 and -4) and reduced those associated with barrier function (integrin beta-1, -3 and -6). The ability of P. gingivalis to modulate these genes was predominantly FOXO1 and FOXO3 dependent. The results indicate that P. gingivalis has pronounced effects on gingival keratinocytes and modulates mRNA levels of genes that affect host response, differentiation, apoptosis and barrier function. Moreover, this modulation is dependent upon the transcription factors FOXO1 or FOXO3. In addition, a new function for FOXO1 was identified, that of suppressing TLR-2 and TLR-4 and maintaining integrin beta -1, beta -3 and beta -6 basal mRNA levels in keratinocytes.
    PLoS ONE 11/2013; 8(11):e78541. · 3.53 Impact Factor
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    ABSTRACT: Porphyromonas gingivalis is a Gram –ve anaerobe that initiates periodontitis. As a first line of defense against pathogen attack, human gingival epithelial cells (HGECs) secrete antimicrobial peptides, chemokines and pro-inflammatory cytokines including Interleukin-7. IL-7 is a growth factor cytokine and crucial in lymphocyte homeostasis during infection. Yet, the underlying mechanism of P. gingivalis induced epithelial IL-7 in modulating T-cell responses is unknown. Objective: We noted up-regulation of IL-7 cytokine by P. gingivalis in HGECs. In the present study, we aimed at describing the role of epithelial IL-7 in modulating primary human naïve T-cell responses. Method: We stimulated primary HGECs with P. gingivalis for 0, 30, 60, 120 and 240 minutes and supernatant was subjected to dot blot using human inflammation antibody array III (Raybiotech, GA). We setup a transwell co-culture experiment with primary HGECs on the bottom well (106 cells/well) and Naïve human T-cells (0.5 x 106 cells/well) on transwell insert. We then carefully stimulated epithelial cells with P. gingivalis (MOI:10) without disturbing T-cells for 4 and 24 h. Separately, T-cells were stimulated with recombinant human IL-7 (2 ng/mL). After stimulation, epithelial cells were used for transcript profiling and T-cells were stained with CD4 and CD8 antibodies for FACS analysis. The T-cells were also used to determine Foxp3 and IL-10 expression by real-time PCR. Results: We observed a time dependent increase in IL-7 cytokine by P. gingivalis in HGECs. P. gingivalis stimulation in HGECs modulated T-cell response by increasing CD8 and decreasing CD4 surface markers. P. gingivalis didn’t induce the same effect on T-cells in the absence of HGECs. Interestingly, Foxp3 and IL-10 expressions were significantly up-regulated in the presence of HGECs compared to T-cells alone. Conclusion: Taken together, our data suggests that gingival epithelial cells play an important role in the maintenance of T-cell homeostasis by inducing IL-7 expression.
    IADR/AADR/CADR General Session and Exhibition 2013; 03/2013
  • M.A. ALSARHAN, M. BENAKANAKERE, D. KINANE, J. KOROSTOFF
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    ABSTRACT: Objective: B Lymphocyte Stimulator (BLYS) and ‪A PRoliferation-Inducing Ligand (APRIL) are members of the TNF-alpha superfamily that are involved in the induction of B cell proliferation/differentiation and maintenance of plasma cell survival, respectively. The predominance of plasma cells in chronic periodontitis lesions suggests that these molecules are involved in the pathogenesis of the disease. The goal of this study is to test the hypothesis that expression of BLYS and APRIL is upregulated in gingival tissue from patients with chronic periodontitis relative to samples from healthy individuals. Method: Normally discarded gingival tissue was procured from patients undergoing pocket reduction surgery to treat chronic periodontitis (n=20), and clinically healthy subjects who required crown lengthening surgery (n=20) as control. mRNA was prepared and analyzed by RealTime-PCR to determine relative levels of BLYS, APRIL and RANKL expression between the two groups. Student’s t-test was used to determine whether the difference were statistically significant. Additional samples from the diseased group were processed and evaluated via immunohistochemistry to identify the cellular sources of the molecules. Result: BLYS , APRIL and RANKL expression was significantly higher in the diseased samples relative to the controls (p≤0.05). Immunohistochemistry analysis showed significant expression of APRIL within the deeper layers of the gingival epithelium in proximity to the basement membrane. Conclusion: These findings suggest that increased expression of BLYS and APRIL contribute to the pathogenesis of chronic periodontitis. These molecules likely contribute to the survival of RANKL-expressing B cells and plasma cells that facilitate osteoclastogenesis leading to alveolar bone resorption. Furthermore, the results indicate that gingival epithelial cells contribute to disease progression via enhanced expression of APRIL. Acknowledgement:Discretionary funds allocated to J.Korostoff.
    IADR/AADR/CADR General Session and Exhibition 2013; 03/2013
  • M. BENAKANAKERE, M. ABDOLHOSSEINI, D. KINANE
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    ABSTRACT: Pathogen mediated gain of epigenetic reprogramming in humans is recently being implicated in cancer and immune mediated diseases. Periodontitis is an immune mediated disease instigated by a biofilm pathogen Porphyromonas gingivalis. The host innate immune system recognizes P. gingivalis through TLR2/4. Nevertheless, P. gingivalis induced epigenetic reprogramming remains to be elucidated. Objective: We aimed to characterize P. gingivalis induced epigenetic reprogramming in human gingival epithelial cells (HGECs) because we hypothesized that these modifications may render periodontal disease susceptibility. Method: We have isolated HGECs from patients and characterized on their inflammatory responses to P. gingivlalis as ‘normal’ (normal cytokine responders) ‘hypo-responsive’ (diminished cytokine responders). DNA from these cells was subjected to methylation specific qPCR array for the TLR gene network. The ‘normal’ cells were stimulated with P. gingivalis (MOI:5) for 30 minutes at 0, 4, 8 and 16 h time intervals. After 48 h of last stimulation, the cells were split and repeated stimulation cycle with or without 1 mM of 5-Aza-2’-deoxycytidine (decitabine). After which, TLR2 promoter CpG methylation was determined by methylation specific qPCR assay. DNA from healthy and periodontitis affected tissue was subjected bisulfite sequencing to determine CpG methylation. Time course P. gingivalis stimulation was done and protein was subjected to immunoblot against histone antibodies. Results: We identified TLR2 promoter CpG hypermethylation in ‘hypo-responsive’ cells. This methylation status was reversed by the use of decitabine restoring the TLR2 and pro-inflammatory cytokine expression. We observed the induction of TLR2 CpG methylation by P. gingivalis in HGECs. Moreover, the tissue from periodontal sites showed sporadic TLR2 CpG methylation compared to healthy sites as revealed by bisulfite sequencing. We also observed rapid histone modifications upon P. gingivalis stimulation in HGECs. Conclusion: Here we show previously unknown specific epigenetic reprogramming of epithelial chromatin by P. gingivalis that may contribute to periodontal disease susceptibility.
    IADR/AADR/CADR General Session and Exhibition 2013; 03/2013
  • J.A. KINANE, M. BENAKANAKERE, J. ZHAO, K. HOSUR, D. KINANE
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    ABSTRACT: Objective: Porphyromonas gingivalis, a Gram-negative oral pathogen has been shown to induce apoptosis in human gingival epithelial cells, yet the underlining cellular mechanisms controlling this process are poorly understood. We hypothesized that ‘P. gingivalis induced apoptosis’ is mediated through degradation of actin by gingipains inducing cytoskeleton collapse and leading ultimately to cell death. Method: To test this hypothesis, we utilized primary human gingival epithelial cells (HGECs) isolated from healthy young donors. These cells were stimulated with 1:100 MOI of P. gingivalis 33277, W50 and mutants lacking either arginine gingipain or lysine gingipain or both. After stimulation, the protein extracts were prepared from 0 h to 48 h time point samples. These samples are immunoblotted against human β-actin. Purified gingipains and actin from rabbit smooth muscle were used to confirm the substrate specificity. Result: In this study, stimulation of human gingival epithelial cells with P. gingivalis strains 33277 and W50 at MOI:100 induced β-actin cleavage as early as 1 hour and human serum inhibited this effect may be by inactivating gingipains. By using gingipain deficient mutants of P. gingivalis and purified gingipains, we demonstrate that lysine gingipain is involved in actin hydrolysis in a dose and time dependent manner. Use of Jasplakinolide and cytochalasin D revealed that P. gingivalis internalization may be necessary for actin cleavage. Conclusion: Taken together, we have identified actin as a substrate for lysine gingipain and demonstrated a novel mechanism involved in host cell invasion and apoptosis.
    IADR General Session 2012; 06/2012
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    ABSTRACT: Objective: Systemic inflammation is common in obese individuals with elevated levels of inflammatory cytokines such as TNF-a and IL-6 accompanied by high levels of acute phase proteins such as CRP. These inflammatory mediators are commonly considered as sequelae to periodontal disease. This project is part of a larger study aimed at determining relations between periodontitis and obesity in subjects pre and post bariatric surgery. Method: 16 subjects (25 to 66 years of age: 3 M, 13 F) were recruited from patients undergoing bariatric surgery. Inclusion criteria: BMI ≥30 and weight >220 lbs. Subjects underwent periodontal examination 1-2 weeks before surgery and at 3 and 6 months post-surgery without any periodontal intervention. Blood and saliva samples were collected at baseline, 3 and 6 months post-operation. HbA1c was analyzed using chromatographic method and the ‘HbA1c now kit’. Results: This reports the initial findings on the feasibility of the use of bariatric surgery to recover a non-diabetic state. Banding, sleeve gastrectromy and roux-en-Y bypass were performed and after three months mean change in weight 42lbs (range 11-68, stdev +/- 19lbs) was noted. Significant reduction in HbA1c was observed with an average of 6.2 to 5.6. 6 patients were above the diabetic cut-off point of 6.5 and three months after surgery all 16 subjects were below this level. Statistically significant correlation (p<0.01) was noted with an r-value of 46% for the relationship between weight loss and HbA1C levels. Conclusion: The larger study constitutes a clinical model that should permit us to study the effects of hyperglycemia, or diabetic state, on dental health within the same subject over a relatively short time frame. The study has considerable real life potential confounders which need to be considered, none-the-less should permit a within subject comparison of periodontal status and microbial flora between hyperglycemia and normal glycemia states.
    IADR General Session 2012; 06/2012
  • M. BENAKANAKERE, J. ZHAO, K. HOSUR, D. KINANE
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    ABSTRACT: Objective: Having identified TLR3 as one of the important receptors in primary gingival epithelial cells inflammation, we hypothesized that its downstream signalling may control other TLRs expression and require Myd88 for proinflammatory cytokines expression. Method: To test this hypothesis, we utilized primary human gingival epithelial cells (HGECs) isolated from healthy young donors. These cells were stimulated with 5 µg/ml Polyinosinic:polycytidylic acid (Poly I:C). After stimulation, the mRNA was subjected to qPCR array analysis. The supernatants were used to measure ELISA for IL-6 and TNFα production. We used RNA interference of TLR2, TLR3 and Myd88 to study the effect of Poly I:C on gingival epithelial cells in Pro-inflammatory cytokine production. Results: In this study, stimulation of human gingival epithelial cells with Poly I:C led to robust increase in pro-inflammatory cytokines and other TLRs in particular TLR2 expression. siRNA knock-down of TLR3 significantly down-regulated TLR2 and Myd88 mRNA and pro-inflammatory cytokine expression. siRNA against Myd88 down-regulated TLR2 and pro-inflammatory cytokine expression upon Poly I:C treatment. Conclusion: We have identified a previously unknown interactive TLR3 signalling pathway underlying the importance of TLRs interplay in human gingival epithelial cells inflammation.
    06/2012
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    ABSTRACT: Porphyromonas gingivalis, a Gram-negative oral pathogen, has been shown to induce apoptosis in human gingival epithelial cells, yet the underlining cellular mechanisms controlling this process are poorly understood. We have previously shown that the P. gingivalis proteases arginine and lysine gingipains, are necessary and sufficient to induce host cell apoptosis. In the present study, we demonstrate that 'P. gingivalis-induced apoptosis' is mediated through degradation of actin leading to cytoskeleton collapse. Stimulation of human gingival epithelial cells with P. gingivalis strains 33277 and W50 at moi:100 induced β-actin cleavage as early as 1 h and human serum inhibited this effect. By using gingipain-deficient mutants of P. gingivalis and purified gingipains, we demonstrate that lysine gingipain is involved in actin hydrolysis in a dose and time-dependent manner. Use of Jasplakinolide and cytochalasin D revealed that P. gingivalis internalization is necessary for actin cleavage. Further, we also show that lysine gingipain from P. gingivalis can cleave active caspase 3. Taken together, we have identified actin as a substrate for lysine gingipain and demonstrated a novel mechanism involved in microbial host cell invasion and apoptosis.
    Cellular Microbiology 03/2012; 14(7):1085-96. · 4.82 Impact Factor
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    ABSTRACT: Recent studies implicate the mammalian target of rapamycin (mTOR) pathway in the control of inflammatory responses following Toll-like receptors (TLRs) stimulation in myeloid cells but its role in non-myeloid cells such as human gingival epithelial cells is unknown. Objective: Our objective was to unravel the role of mTOR and its signaling in non-myeloid human gingival epithelial cells and to tap the possibility of mTOR inhibitors as anti-inflammatory molecules in the treatment of periodontitis. Methods: We utilized primary human gingival epithelial cells isolated from subjects who underwent 3rd molar extraction and tested the pro-inflammatory cytokine secretion in vitro by treating the cells with TLR2, TLR3 and TLR4 ligands by inhibiting mTOR with rapamycin. Results: Our data demonstrate that TLR3 signaling can induce robust cytokine secretion including IL-1β, TNFα, IL-12p70 and interferon beta (IFN-β), and our data reveal for the first time that inhibiting mTOR with rapamycin, suppresses these TLR3 induced cytokines but actually enhances bioactive IL-12p70 production in human. Rapamycin inhibited the phosphorylation of the 70-kDa ribosomal protein S6 kinase (p70S6K) and the 4E binding protein 1 (4EBP-1), and suppressed the MAPK pathway by decreasing phosphorylation of c-Jun N-terminal kinase (JNK). We also show that TLR3 induces interferon regulatory factor 3 (IRF3) activation by Akt via an mTOR-p70S6K-4EBP1 pathway. Furthermore, We provide evidence that Poly I:C induced expression of IL-1β, TNFα, IL-12p70 and IFN-β was blocked by JNK inhibitor SP600125. TLR3 preferentially phosphorylated IKKβ through mTOR to activate NF-kB in human gingival epithelial cells. Conclusion: Taken together, these data demonstrate p70S6K, p4EBP1, JNK, NF-kB and IRF3 are involved in the regulation of inflammatory mediators by TLR3 via the mTOR pathway. mTOR is a novel pathway modulating TLR3 induced inflammatory and antiviral responses in human gingival epithelial cells. Supported by NIH-NIDCR grant DE017384
    03/2011
  • M. BENAKANAKERE, J. ZHAO, K. HOSUR, D.F. KINANE
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    ABSTRACT: Porphyromonas gingivalis is a Gram-negative anaerobe of dental plaque and a putative pathogen in chronic periodontitis. Recently our group demonstrated the induction of apoptosis by P. gingivalis in human gingival epithelial cells (HGECs) or oral keratinocytes through gingipain dependent manner (Stathopoulou et. al. BMC Microbiol. 2009 May 27;9:107). However, it is still unclear about molecular mechanisms that host cells adopt to undergo apoptosis. Objective: To investigate the molecular mechanism involved in the induction of apoptosis in HGECs by P. gingivalis. Methods: We utilized live P. gingivalis (strain ATCC 33277) to treat HGECs and carried out the global miRNA expression profiling. Results: Our data showed 8 miRNAs differentially up-regulated upon live P. gingivalis challenge and we examined the role of these miRNAs by overexpression analysis and determined that hsa-miR-663 induces apoptosis in HGECs. We then validated live P. gingivalis specific miRNA expression in HGECs using real time PCR and northern blot analysis. Here we demonstrate the molecular mechanism by which hsa-miR-663 induces apoptosis in HGECs. Bioinformatics using Microcosm, Targetscan and mRNA secondary structure analyses showed Apoptosis Antagonizing Transcription Factor (AATF) as a putative target for miR-663. Interestingly, live P. gingivalis challenge in HGECs completely abolished the protein expression of AATF. When miR-663 is overexpressed in HGECs and OBA-9 cell line, the protein expression of AATF was knocked down with increased cell death revealed by apoptosis assay. Conclusion: Bioinformatics and In vitro data clearly shows AATF as a target for hsa-miR-663 demonstrating one of the cellular mechanisms adopted by host cells to undertake apoptosis. Supported by United States Public Health Service, National Institutes of Health, NIDCR grant DE017384
    IADR General Session 2011; 03/2011
  • J. ZHAO, K. HOSUR, M. BENAKANAKERE, D.F. KINANE
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    ABSTRACT: Toll like receptors are germ line encoded receptors that play a key role in innate immunity. Human Gingival Epithelial Cells (HGECs) forms first line of defense against plaque bacteria and viruses in the oral cavity. Analyzing the expression of TLRs and its signaling networks in HGECs becomes important in understanding periodontal disease pathogenesis. Objective: Our objective was to screen and determine the expression levels of different TLRs and their signaling pattern in primary HGECs by qPCR gene expression profiling and cytokine protein expression to compare the signaling pathways modulating the innate immune responses. Method: We utilized HGECs from healthy and periodontitis subjects and determined the gene expression of TLRs. The cells were also challenged with TLR ligands such as heat killed P.gingivalis (TLR2/4 agonist), Pam3CSK4 (TLR1/2 agonist), P.gingivalis LPS (TLR2 agonist), Poly I:C (TLR3 agonist), E. coli LPS (TLR4 agonist), Flagellin (TLR5 agonist), FSL1 (TLR2/6 agonist), Imiquimod (TLR7 agonist), ssRNA40 (TLR8 agonist), and ODN2006 (TLR9 agonist). After challenge, the cDNA was analyzed for innate immune responses using qPCR arrays and cytokine expression by ELISA. Results: The expressions of TLRs were distinct among HGECs of healthy and periodontitis subjects. The intracellular TLR3 and TLR9 induced robust inflammatory gene and cytokine expression upon ligand challenge. TLR4 expression was low in HGECs of healthy and periodontitis subjects and the inflammatory response to E. coli LPS was minimal. Triggering TLR2/6 in HGECs induced higher inflammatory gene expression when compared to TLR1/2. This may be due to higher level of TLR6 expression compared to TLR1. Conclusion: Here we show the gene expression of TLRs in HGECs of healthy and periodontitis subjects with distinct cellular signaling pattern and cytokine induction. Our data shows TLR4 as a weak receptor in HGECs compared to TLR2 and does not contribute to interferon expression. Supported by NIH-NIDCR grant DE017384
    IADR General Session 2011; 03/2011
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    ABSTRACT: The ability of IFN-β to induce IL-10 production from innate immune cells is important for its anti-inflammatory properties and is believed to contribute to its therapeutic value in treating multiple sclerosis patients. In this study, we identified that IFN-β stimulates IL-10 production by activating the JAK1- and PI3K-signaling pathways. JAK1 activity was required for IFN-β to activate PI3K and Akt1 that resulted in repression of glycogen synthase kinase 3 (GSK3)-β activity. IFN-β-mediated suppression of GSK3-β promoted IL-10, because IL-10 production by IFN-β-stimulated dendritic cells (DC) expressing an active GSK3-β knockin was severely reduced, whereas pharmacological or genetic inhibition of GSK3-β augmented IL-10 production. IFN-β increased the phosphorylated levels of CREB and STAT3 but only CREB levels were affected by PI3K. Also, a knockdown in CREB, but not STAT3, affected the capacity of IFN-β to induce IL-10 from DC. IL-10 production by IFN-β-stimulated DC was shown to suppress IFN-γ and IL-17 production by myelin oligodendrocyte glycoprotein-specific CD4(+) T cells, and this IL-10-dependent anti-inflammatory effect was enhanced by directly targeting GSK3 in DC. These findings highlight how IFN-β induces IL-10 production and the importance that IL-10 plays in its anti-inflammatory properties, as well as identify a therapeutic target that could be used to increase the IL-10-dependent anti-inflammatory properties of IFN-β.
    The Journal of Immunology 01/2011; 186(2):675-84. · 5.36 Impact Factor
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    ABSTRACT: Recent studies implicate the mammalian target of rapamycin (mTOR) pathway in the control of inflammatory responses following Toll-like receptor (TLR) stimulation in myeloid cells but its role in non-myeloid cells such as human keratinocytes is unknown. Here we show that TLR3 signaling can induce robust cytokine secretion including interleukin 1 beta (IL-1β), tumor necrosis factor alpha (TNFα), IL-12p70 and interferon beta (IFN-β), and our data reveal for the first time that inhibiting mTOR with rapamycin, suppresses these TLR3 induced responses but actually enhances bioactive IL-12p70 production in human oral keratinocytes. Rapamycin inhibited the phosphorylation of the 70-kDa ribosomal protein S6 kinase (p70S6K) and the 4E binding protein 1 (4EBP-1), and suppressed the mitogen activated protein kinase (MAPK) pathway by decreasing phosphorylation of c-Jun N-terminal kinase (JNK). We also show that TLR3 induces interferon regulatory factor 3 (IRF3) activation by Akt via an mTOR-p70S6K-4EBP1 pathway. Furthermore, we provide evidence that Poly I:C induced expression of IL-1β, TNFα, IL-12p70 and IFN-β was blocked by JNK inhibitor SP600125. TLR3 preferentially phosphorylated IKKα through mTOR to activate nuclear factor kappa beta (NF-κB) in human oral keratinocytes. Taken together, these data demonstrate p70S6K, p4EBP1, JNK, NF-κB and IRF3 are involved in the regulation of inflammatory mediators by TLR3 via the mTOR pathway. mTOR is a novel pathway modulating TLR3 induced inflammatory and antiviral responses in human oral keratinocytes.
    Molecular Immunology 11/2010; 48(1-3):294-304. · 3.00 Impact Factor
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    ABSTRACT: Host defense against invading pathogens is triggered by various receptors including toll-like receptors (TLRs). Activation of TLRs is a pivotal step in the initiation of innate, inflammatory, and antimicrobial defense mechanisms. Human beta-defensin 2 (HBD-2) is a cationic antimicrobial peptide secreted upon gram-negative bacterial perturbation in many cells. Stimulation of various TLRs has been shown to induce HBD-2 in oral keratinocytes, yet the underlying cellular mechanisms of this induction are poorly understood. Here we demonstrate that HBD-2 induction is mediated by the Sphingosine kinase-1 (Sphk-1) and augmented by the inhibition of Glycogen Synthase Kinase-3beta (GSK-3beta) via the Phosphoinositide 3-kinase (PI3K) dependent pathway. HBD-2 secretion was dose dependently inhibited by a pharmacological inhibitor of Sphk-1. Interestingly, inhibition of GSK-3beta by SB 216763 or by RNA interference, augmented HBD-2 induction. Overexpression of Sphk-1 with concomitant inhibition of GSK-3beta enhanced the induction of beta-defensin-2 in oral keratinocytes. Ectopic expression of constitutively active GSK-3beta (S9A) abrogated HBD-2 whereas kinase inactive GSK-3beta (R85A) induced higher amounts of HBD-2. These data implicate Sphk-1 in HBD-2 regulation in oral keratinocytes which also involves the activation of PI3K, AKT, GSK-3beta and ERK 1/2. Thus we reveal the intricate relationship and pathways of toll-signaling molecules regulating HBD-2 which may have therapeutic potential.
    PLoS ONE 07/2010; 5(7):e11512. · 3.53 Impact Factor
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    ABSTRACT: The dental plaque is comprised of numerous bacterial species, which may or may not be pathogenic. Human gingival epithelial cells (HGECs) respond to perturbation by various bacteria of the dental plaque by production of different levels of inflammatory cytokines, which is a putative reflection of their virulence. The aim of the current study was to determine responses in terms of interleukin (IL)-1beta, IL-6, IL-8 and IL-10 secretion induced by Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, Fusobacterium nucleatum and Streptococcus gordonii in order to gauge their virulence potential. HGECs were challenged with the four bacterial species, live or heat killed, at various multiplicity of infections and the elicited IL-1beta, IL-6, IL-8 and IL-10 responses were assayed by enzyme-linked immunosorbent assay. Primary HGECs challenged with live P. gingivalis produced high levels of IL-1beta, while challenge with live A. actinomycetemcomitans gave high levels of IL-8. The opportunistic pathogen F. nucleatum induces the highest levels of pro-inflammatory cytokines, while the commensal S. gordonii is the least stimulatory. We conclude that various dental plaque biofilm bacteria induce different cytokine response profiles in primary HGECs that may reflect their individual virulence or commensal status.
    Journal Of Clinical Periodontology 01/2010; 37(1):24-9. · 3.61 Impact Factor
  • PLoS ONE 01/2010; 5(7). · 3.53 Impact Factor
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    ABSTRACT: IL-12p70 is an immunoregulatory cytokine that has been shown to induce IL-10 production from CD4+ T cells, yet the underlying cellular mechanisms controlling this process are poorly understood. In the present study, we demonstrate that IL-12p70 induces IL-10 production from human memory CD4+ T cells via a PI3K-dependent signaling mechanism. Specifically, stimulation of human memory CD4+ T cells in the presence of IL-12p70 lead to increased PI3K activity and the subsequent phosphorylation and inactivation of the downstream constitutively active serine/threonine kinase, glycogen synthase kinase-3beta (GSK3beta). Inhibition of PI3K prevented the inactivation of GSK3beta by IL-12p70, as well as the subsequent ability of IL-12p70 to augment IL-10 levels by memory CD4+ T cells. Moreover, ectopic expression of a constitutively active form of GSK3beta abrogated the ability of IL-12p70 to increase IL-10 production by TCR-stimulated CD4+ T cells. In contrast, direct inhibition of GSK3 mimicked the effect of IL-12p70 on IL-10 production by memory CD4+ T cells. Analysis of downstream transcription factors identified that the ability of IL-12p70 to inactivate GSK3beta lead to increased levels of c-jun. The ability of IL-12p70 to inactivate GSK3beta and induce c-jun levels was required for IL-12 to augment IL-10 production by human memory CD4+ T cells, since small interfering RNA-mediated gene silencing of c-jun abrogated this process. These studies identify the cellular mechanism by which IL-12 induces IL-10 production from human memory CD4+ T cells.
    The Journal of Immunology 10/2009; 183(7):4475-82. · 5.36 Impact Factor

Publication Stats

339 Citations
78.89 Total Impact Points

Institutions

  • 2010–2014
    • University of Pennsylvania
      • • Department of Periodontics
      • • Department of Pathology
      Philadelphia, Pennsylvania, United States
  • 2008–2011
    • University of Louisville
      • • Department of Microbiology and Immunology
      • • Center for Oral Health and Systemic Disease
      Louisville, KY, United States
  • 2009
    • University of Zurich
      • Institut für Orale Biologie
      Zürich, ZH, Switzerland