Nitric oxide production by a human osteoblast cell line stimulated with Aggregatibacter actinomycetemcomitans lipopolysaccharide

Background/aim:  Human osteoblasts induced by inflammatory stimuli express an inducible nitric oxide synthase (iNOS). The aim of the present study was to test the hypothesis that Aggregatibacter actinomycetemcomitans lipopolysaccharide stimulates the production of nitric oxide (NO) by a human osteoblast-like cell line (HOS cells).
Methods:  Cells were stimulated directly with A. actinomycetemcomitans lipopolysaccharide or pretreated with the following l -NIL (an iNOS inhibitor), anti-CD14, Toll-like receptor 2 (TLR2), or TLR4 antibody before stimulation with A. actinomycetemcomitans lipopolysaccharide. The role of the cyclic nucleotides was assessed by pretreating the cells with the following; ODQ (a guanylyl cyclase inhibitor); SQ22536 (an adenylyl cyclase inhibitor); db-cAMP (a cyclic adenosine monophosphate analog); br-cGMP (a cyclic guanosine monophosphate analog); forskolin (an adenylyl cyclase activator), IBMX [a non-specific phosphodiesterase (PDE) inhibitor], or KT5720 [a protein kinase A (PKA) inhibitor]. The cells were also preincubated with genistein [a protein tyrosine kinase (PTK) inhibitor], bisindolylmaleimide [a protein kinase C (PKC) inhibitor], BPB [a phospholipase A2 (PLA2) inhibitor], and NDGA (a lipoxygenase inhibitor). The iNOS activity and nitrite production in the cell cultures were determined spectrophotometrically.
Results:  The results showed that A. actinomycetemcomitans lipopolysaccharide stimulated both iNOS activity and nitrite production by HOS cells; this was reduced by l -NIL, anti-CD14, or anti-TLR4 antibody, SQ22536, KT5720, genistein, bisindolylmaleimde, BPB, and NDGA, but was enhanced by db-cAMP, IBMX, and forskolin.
Conclusion:  These results therefore suggest that A. actinomycetemcomitans lipopolysaccharide may induce the production of NO by HOS cells via a CD14–TLR4 molecule complex, a cAMP–PKA pathway, as well as by a PTK, PKC, PLA2, and lipoxygenase-dependent mechanism.     

Humoral immune response to Aggregatibacter actinomycetemcomitans leukotoxin

Brage M, Holmlund A, Johansson A. Humoral immune response toAggregatibacter actinomycetemcomitans leukotoxin. J Periodont Res 2011; 46: 170–175. © 2010 John Wiley & Sons A/S Background and Objective: Periodontal disease is an inflammatory condition caused by bacterial infections that result in loss of the tooth supporting tissue. The periodontal pathogens produce virulence factors with capacity to affect the host immune response. Aggregatibacter actinomycetemcomitans is a periodontal pathogen that produces a leukotoxin that specifically affects human leukocytes. The aims of the present study were to examine the presence and function of systemic antibodies to the leukotoxin. Material and Methods: One hundred and ninety‐seven middle‐aged (57 ± 5 years) Swedes with well‐documented periodontal status and medical factors related to cardiovascular diseases were studied. These data have been published previously. The serum samples were examined for the presence of leukotoxin antibodies by western blot and the capacity to neutralize leukotoxicity in an activity assay with leukotoxin and cultured leukemic cells. Results: The results showed a high prevalence (57%) of antibodies against A. actinomycetemcomitans leukotoxin in the analyzed population. These antibodies were correlated with leukotoxin neutralizing capacity as well as with the ELISA titers of A. actinomycetemcomitans‐specific IgA and IgG. In addition, high levels of leukotoxin antibodies were correlated with increasing age, but not with periodontal disease parameters or cardiovascular risk factors. Conclusion: Systemic antibodies against A. actionmycetemcomitans leukotoxin were common in this adult Swedish population. These antibodies might contribute to limit the systemic effects of the infection.     

Effect of inhibition of inducible nitric oxide synthase (iNOS) on the murine splenic immune response induced by Aggregatibacter (Actinobacillus) actinomycetemcomitans lipopolysaccharide

Animal studies suggest that inducible nitric oxide synthase (iNOS) may be associated with destructive periodontal disease. l- N ⁶-(1-Iminoethyl)-lysine ( l -NIL) has been shown to inhibit iNOS in a selective manner, and hence the aim of the present study was to test the hypothesis that treatment with l -NIL may induce a T-cell helper 1 (Th1)-like immune response by Aggregatibacter ( Actinobacillus ) actinomycetemcomitans lipopolysaccharide (LPS)-stimulated murine spleen cells in vitro . BALB/c mice were either sham-immunized or immunized with A. actinomycetemcomitans LPS. Spleen cells were stimulated with A. actinomycetemcomitans LPS in the presence or absence of l -NIL. Nitric oxide (NO), iNOS activity, specific IgG subclass antibodies, interferon- γ (IFN- γ ), and interleukin-4 (IL-4) levels and cell proliferation were determined. The results showed that treatment with l -NIL suppressed both NO production and iNOS activity but enhanced specific IgG2a, IFN- γ levels, and increased cell proliferation following stimulation with A. actinomycetemcomitans LPS-stimulated cells. The results of the present study suggest that inhibition of iNOS activity by l -NIL may skew the A. actinomycetemcomitans LPS-stimulated murine splenic immune response towards the Th1-like immune profile in vitro .     

Adherence of Aggregatibacter actinomycetemcomitans via serotype-specific polysaccharide antigens in lipopolysaccharides

Introduction:  Gram-negative Aggregatibacter actinomycetemcomitans is recognized as an important periodontal pathogen. A striking property of this bacterium is its ability to form a tenacious biofilm adhering to abiotic surfaces. Both fimbrial and non-fimbrial adhesins are believed to be responsible for this ability. In our study, specific markerless mutants in the biosynthesis genes of cell surface polysaccharides were constructed with the Cre- loxP recombination system to identify non-fimbrial adhesin(s).
Methods:  Non-fimbriated A. actinomycetemcomitans strain ATCC29523 (serotype a) was used to construct a deletion mutant of serotype-a specific polysaccharide antigen (SPA-a) in lipopolysaccharide (LPS). The LPS was purified through a polymyxin B column following phenol extraction, and verified by silver staining following sodium dodecyl sulfate–polyacrylamide gel electrophoresis and by immunoblot analysis using rabbit antisera raised against SPA-a. Strains were grown in broth for 2 days and examined for the adherence of bacterial cells on the glass surface.
Results:  Strain ATCC29523 formed a thin film of bacterial growth on the glass surface. The deletion of SPA-a affected its ability to form this thin film. When this mutant was rescued with the wild-type SPA-a gene cluster, its adherence-positive phenotype was restored.
Conclusion:  SPA-a in the LPS molecule appears to promote the adherence of A. actinomycetemcomitans cells to abiotic surfaces.     

Effect of L-N6-(1-iminoethyl)-lysine, an inducible nitric oxide synthase inhibitor, on murine immune response induced by Actinobacillus actinomycetemcomitans lipopolysaccharide

BACKGROUND AND OBJECTIVES: Inducible nitric oxide synthase (iNOS) activity is known to regulate the immune response. The present study was carried out to determine the effect of L-N6-(1-iminoethyl)-lysine (L-NIL), an iNOS inhibitor, on the induction of immune response to Actinobacillus actinomycetemcomitans lipopolysaccharide in mice. MATERIAL AND METHODS: BALB/c mice were sham-immunized (group I), immunized with A. actinomycetemcomitans lipopolysaccharide (group II) or treated with L-NIL and immunized with A. actinomycetemcomitans lipopolysaccharide (group III). All animals were then challenged with viable A. actinomycetemcomitans. The levels of serum nitric oxide (NO), specific immunoglobulin G (IgG) isotypes and both interferon-gamma and interleukin-4, as well as spleen cell-derived iNOS activity, before and after bacterial challenge, were assessed. The diameter of skin lesions was also determined. Serum and spleen cells from the above groups were adoptively transferred to the recipients that were then subsequently challenged with live bacteria. RESULTS: Treatment with L-NIL suppressed serum NO and splenic iNOS activity, but enhanced serum-specific IgG2a antibody and interferon-gamma levels. The lesions in L-NIL-treated mice healed much more rapidly. Transfer with serum and cells from L-NIL-treated and A. actinomycetemcomitans lipopolysaccharide-immunized donors resulted in rapid healing of the lesions in the recipients. CONCLUSION: It is suggested that treatment with L-NIL in mice immunized with A. actinomycetemcomitans lipopolysaccharide may shift the immune response towards a protective T helper 1-like immunity against A. actinomycetemcomitans-induced infection.     

Adherence of Aggregatibacter actinomycetemcomitans via serotype‐specific polysaccharide antigens in lipopolysaccharides

Introduction: Gram‐negative Aggregatibacter actinomycetemcomitans is recognized as an important periodontal pathogen. A striking property of this bacterium is its ability to form a tenacious biofilm adhering to abiotic surfaces. Both fimbrial and non‐fimbrial adhesins are believed to be responsible for this ability. In our study, specific markerless mutants in the biosynthesis genes of cell surface polysaccharides were constructed with the Cre‐loxP recombination system to identify non‐fimbrial adhesin(s). Methods: Non‐fimbriated A. actinomycetemcomitans strain ATCC29523 (serotype a) was used to construct a deletion mutant of serotype‐a specific polysaccharide antigen (SPA‐a) in lipopolysaccharide (LPS). The LPS was purified through a polymyxin B column following phenol extraction, and verified by silver staining following sodium dodecyl sulfate–polyacrylamide gel electrophoresis and by immunoblot analysis using rabbit antisera raised against SPA‐a. Strains were grown in broth for 2 days and examined for the adherence of bacterial cells on the glass surface. Results: Strain ATCC29523 formed a thin film of bacterial growth on the glass surface. The deletion of SPA‐a affected its ability to form this thin film. When this mutant was rescued with the wild‐type SPA‐a gene cluster, its adherence‐positive phenotype was restored. Conclusion: SPA‐a in the LPS molecule appears to promote the adherence of A. actinomycetemcomitans cells to abiotic surfaces.     

伴放线放线杆菌形态变化对白细胞毒素分泌影响的研究

目的观察伴放线放线杆菌形态变化对白细胞毒素分泌的影响。方法选择粗糙型和光滑型伴放线放线杆菌各8株,应用聚丙烯酰胺凝胶电泳,检测液体培养12、24、48、60、72h的菌体及培养上清液中116kDa大小白细胞毒素蛋白条带的情况,应用超滤法分离纯化培养上清液蛋白,应用台盼蓝染色排除法检测上清液蛋白白细胞毒素活性。结果粗糙型伴放线放线杆菌菌株液体培养12、24、48、60、72h菌体蛋白电泳均可见116kDa大小的蛋白条带,培养上清液蛋白电泳结果显示116kDa大小的蛋白条带均出现于培养24和48h;光滑型伴放线放线杆菌菌株液体培养12、24、48、60、72h菌体蛋白电泳结果均缺少116kDa大小的蛋白条带,培养上清液蛋白电泳结果显示116kDa大小的蛋白条带出现于培养12和24h;实验菌株培养上清液提取蛋白均具有白细胞毒素活性。结论伴放线放线杆菌粗糙型和光滑型菌株均可分泌具有直接杀灭人多形核白细胞活性的白细胞毒素,但粗糙型菌株分泌白细胞毒素的时间晚于光滑型。     

Macrophage tolerance response to Aggregatibacter actinomycetemcomitans lipopolysaccharide induces differential regulation of tumor necrosis factor-alpha, interleukin-1 beta and matrix metalloproteinase 9 secretion

Background and Objective: The lipopolysaccharide of Aggregatibacter actinomycetemcomitans, a potent stimulator of the immune system, induces the secretion of inflammatory mediators that modulate periodontal tissue destruction. In this study, we investigated the tolerance response of human macrophages to stimulation with A. actinomycetemcomitans lipopolysaccharide. Material and Methods: U937 monocytes were differentiated into adherent macrophage‐like cells by treatment with phorbol myristic acid. Macrophage‐like cells were then pretreated for 24 h with either 0.01 or 0.1 μg/mL LPS A. actinomycetemcomitans. Culture medium supernatants were removed and cells were restimulated with LPS at 1 μg/mL. Cell‐free supernatants were collected after 24 h of stimulation and analyzed by ELISA for TNF‐α, IL‐1β, IL‐6, IL‐8, PGE₂ and MMP‐9. Results: Phorbol myristic acid‐differentiated U937 macrophages treated with low doses of lipopolysaccharide developed tolerance to subsequent lipopolysaccharide treatments, resulting in significantly reduced secretion of tumor necrosis factor‐α. However, this tolerance response was associated with increased secretion of interleukin‐1β and matrix metalloproteinase 9, whereas the secretion of interleukin‐6, interleukin‐8 and prostaglandin E₂ was unaffected. Phosphatidylinositol‐3′‐kinase inhibitors added during the tolerance‐induction period markedly attenuated the increase in interleukin‐1β secretion but had no effect on tumor necrosis factor‐α. Conclusion: This study showed that A. actinomycetemcomitans lipopolysaccharide can induce a tolerance response in macrophages that alters the secretion of two important inflammatory mediators as well as of the tissue‐degrading enzyme matrix metalloproteinase‐9. This phenomenon may play a role in modulating the host inflammatory response and the progression of periodontitis.     

MyD88 is essential for alveolar bone loss induced by Aggregatibacter actinomycetemcomitans lipopolysaccharide in mice

Aggregatibacter actinomycetemcomitans is a Gram‐negative bacteria highly associated with localized aggressive periodontitis. The recognition of microbial factors, such as lipopolysaccharide from A. actinomycetemcomitans (_AaLPS), in the oral environment is made mainly by surface receptors known as Toll‐like receptors (TLR). TLR4 is the major LPS receptor. This interaction leads to the production of inflammatory cytokines by myeloid differentiation primary‐response protein 88 (MyD88) ‐dependent and ‐independent pathways, which may involve the adaptor Toll/interleukin‐1 receptor‐domain‐containing adaptor inducing interferon‐β (TRIF). The aim of this study was to assess the involvement of MyD88 in alveolar bone loss induced by _AaLPS in mice. C57BL6/J wild‐type (WT) mice, MyD88, TRIF or TRIF/MyD88 knockout mice received 10 injections of _AaLPS strain FDC Y4 (5 μg in 3 μl), in the palatal gingival tissue of the right first molar, every 48 h. Phosphate‐buffered saline was injected in the opposite side and used as control. Animals were sacrificed 24 h after the 10th injection and the maxillae were removed for macroscopic and biochemical analyses. The injections of _AaLPS induced significant alveolar bone loss in WT mice. In the absence of MyD88 or TRIF/MyD88 no bone loss induced by _AaLPS was observed. In contrast, responses in TRIF^?/? mice were similar to those in WT mice. Diminished bone loss in the absence of MyD88 was associated with fewer TRAP‐positive cells and increased expression of osteoblast markers, RUNX2 and osteopontin. There was also reduced tumor necrosis factor‐α production in MyD88^?/? mice. There was less osteoclast differentiation of hematopoietic bone marrow cells from MyD88^?/? mice after _AaLPS stimulation. Hence, the signaling through MyD88 is pivotal for _AaLPS‐induced osteoclast formation and alveolar bone loss.     

Nitric oxide production by a murine macrophage cell line (RAW264.7) stimulated with lipopolysaccharide from Actinobacillus actinomycetemcomitans

The aim of this study was to determine whether Actinobacillus actinomycetemcomitans lipopolysaccharide (LPS-A. actinomycetemcomitans) could stimulate a murine macrophage cell line (RAW264.7 cells) to produce nitric oxide (NO). The cells were treated with LPS-A. actinomycetemcomitans or Escherichia coli LPS (LPS-Ec) for 24 h. The effects of N(G)-monomethyl-L-arginine (NMMA), polymyxin B and cytokines (IFN-gamma, TNF-alpha, IL-4 and IL-12) on the production of NO were also determined. The role of protein tyrosine kinase, protein kinase C and microtubulin organization on NO production were assessed by incubating RAW264.7 cells with genistein, bisindolylmaleide and colchicine prior to LPS-A. actinomycetemcomitans stimulation, respectively. NO levels from the culture supernatants were determined by the Griess reaction. The results showed that LPS-A. actinomycetemcomitans stimulated NO production by RAW264.7 cells in a dose-dependent manner, but was slightly less potent than LPS-Ec. NMMA and polymyxin B blocked the production of NO. IFN-gamma and IL-12 potentiated but IL-4 depressed NO production by LPS-A. actinomycetemcomitans-stimulated RAW264.7 cells. TNF-alpha had no effects on NO production. Genistein and bisindolylmalemaide, but not colchicine, reduced the production of NO in a dose-dependent mechanism. The results of the present study suggest that A. actinomycetemcomitans LPS, via the activation of protein tyrosine kinase and protein kinase C and the regulatory control of cytokines, stimulates NO production by murine macrophages.     

The role of cyclic-AMP on arginase activity by a murine macrophage cell line (RAW264.7) stimulated with lipopolysaccharide from Actinobacillus actinomycetemcomitans

AIMS: The aim of the present study was to determine the role of cyclic adenosine monophosphate (cAMP) on arginase activity in a murine macrophage cell line (RAW264.7 cells) stimulated with lipopolysaccharide (LPS) from Actinobacillus actinomycetemcomitans. MATERIALS AND METHODS: The cells were treated with A. actinomycetemcomitans LPS for 24 h. The effects of SQ22536 (an adenylyl cyclase inhibitor), ODQ (a guanylyl cyclase inhibitor), dibutyryl cAMP (a cAMP analog), 8-bromo cyclic guanosine monophosphate (a cGMP analog), forskolin (an adenylyl cylase activator), and cycloheximide (a protein synthesis inhibitor) on arginase activity in A. actinomycetemcomitans LPS-stimulated RAW264.7 cells were also determined. Arginase activity was assessed in LPS-stimulated cells in the presence of 3-isobutyl-1-methylxanthine (IBMX), siguazodan and rolipram [phosphodiesterase (PDE) inhibitors] as well as KT5720 [a protein kinase A (PKA) inhibitor]. RESULTS: Arginase activity in A. actinomycetemcomitans LPS-stimulated RAW264.7 cells was suppressed by SQ22536 but not ODQ. Enhancement of arginase activity was observed in the presence of cAMP analog or forskolin but not cGMP analog. Cycloheximide blocked arginase activity in the cells in the presence of cAMP analog or forskolin with or without A. actinomycetemcomitans LPS. IBMX augmented arginase activity in A. actinomycetemcomitans LPS-stimulated cells. Rolipram (a PDE4 inhibitor) increased the levels of arginase activity higher than siguazodan (a PDE3 inhibitor) in the antigen-stimulated cells. The effect of cAMP analog or forskolin on arginase activity in the presence or absence of A. actinomycetemcomitans LPS was blocked by the PKA inhibitor (KT5720). CONCLUSION: The results of the present study suggest that A. actinomycetemcomitans LPS may stimulate arginase activity in murine macrophages (RAW264.7 cells) in a cAMP-PKA-dependent pathway.     

Adoptive transfer of suppressor T cells induced by Actinobacillus actinomycetemcomitans regulates immune response

Purified splenic T cells from C3H/HeN mice primed with immunosuppressive fraction (ISF) from A. actinomycetemcomitans were adoptively transferred to syngeneic mice with SRBC. The transfer of splenic T cells from mice, primed with various amounts of ISF for 6 days, resulted in the dose-dependent inhibition of IgM anti-sheep red blood cells (SRBC) plaque-forming cells (PFC) compared with normal and BSA-primed splenic T cells. Furthermore, the transfer of T cells from mice primed with 100 μg of ISF for 6 days to syngeneic and CD4-depleted mice caused the highest inhibition. Immune suppression did not depend on the B cell population in spleen from donor mice primed with ISF, nor on haplotypes as recipient. The immunosuppressive function of these ISF-primed T cells was abrogated by 1000 rad irradiation. Splenic T cells from ISF-treated mice could suppress the T cell-dependent proliferative responses of cocultured normal spleen cells in vitro . Analysis of T cell subsets of spleen cells from ISF-treated mice showed that the suppressor cell is susceptible to treatment with anti-CD8 and complement (C). ISF-sensitized suppressor T cells also suppressed secondary IgG anti-SRBC-PFC response after immunization with SRBC in vivo depending on sensitized periods induced by ISF. Treatment of T cells from mice which primed with ISF for 8 days, with goat anti-mouse CD8 antibody and C abrogated their suppressive effects, and secondary IgG response occurred. These results indicate that the adoptive transfer of ISF-induced T cells, which increased suppressor function, caused the perfect blocking of immune response, allowing promotion of secondary infection.     

Evaluation of the humoral immune response to the cytolethal distending toxin of Aggregatibacter actinomycetemcomitans Y4 in subjects with localized aggressive periodontitis

Introduction: Cytolethal distending toxin (Cdt) is potentially one of several virulence factors of Aggregatibacter actinomycetemcomitans, the prime etiological agent of localized aggressive periodontitis (LAP). Little is known regarding the Cdt‐specific antibody response in humans. The current study is a quantitative and qualitative evaluation of the toxin‐specific antibody response in a cohort of LAP patients and age‐, race‐ and sex‐matched controls. Methods: Ninety‐five subjects provided a total of 692 serum samples. Sera were analysed by enzyme‐linked immunosorbent assays to determine the titers of antibody against the intact Cdt holotoxin as well as the individual subunit proteins (CdtA, CdtB, and CdtC). Neutralization of growth inhibition mediated by Cdt was evaluated in a modified colony‐forming assay using Chinese hamster ovary cells. Results: Fourteen of the 95 subjects exhibited significant serum Cdt‐binding activity. There were no differences in the percentages of seropositive individuals or in the mean antibody titers between the control and LAP groups. Binding activity was detected against each of the three Cdt subunit proteins in all of the positive samples. Neutralization of Cdt‐mediated growth inhibition was detected in samples from all of the seropositive subjects (range 20–75%). Conclusions: Cdt, a recently identified A. actinomycetemcomitans virulence factor, is capable of inducing a neutralizing antibody response indicating that the toxin is produced during natural infection of humans. The failure of a vast majority (20 of 23) of the LAP subjects to mount a significant anti‐Cdt response may in part explain their relative susceptibility to the disease.     

The role of cyclic‐AMP on arginase activity by a murine macrophage cell line (RAW264.7) stimulated with lipopolysaccharide from Actinobacillus actinomycetemcomitans

Aims: The aim of the present study was to determine the role of cyclic adenosine monophosphate (cAMP) on arginase activity in a murine macrophage cell line (RAW264.7 cells) stimulated with lipopolysaccharide (LPS) from Actinobacillus actinomycetemcomitans. Materials and methods: The cells were treated with A. actinomycetemcomitans LPS for 24 h. The effects of SQ22536 (an adenylyl cyclase inhibitor), ODQ (a guanylyl cyclase inhibitor), dibutyryl cAMP (a cAMP analog), 8‐bromo cyclic guanosine monophosphate (a cGMP analog), forskolin (an adenylyl cylase activator), and cycloheximide (a protein synthesis inhibitor) on arginase activity in A. actinomycetemcomitans LPS‐stimulated RAW264.7 cells were also determined. Arginase activity was assessed in LPS‐stimulated cells in the presence of 3‐isobutyl‐1‐methylxanthine (IBMX), siguazodan and rolipram [phosphodiesterase (PDE) inhibitors] as well as KT5720 [a protein kinase A (PKA) inhibitor]. Results: Arginase activity in A. actinomycetemcomitans LPS‐stimulated RAW264.7 cells was suppressed by SQ22536 but not ODQ. Enhancement of arginase activity was observed in the presence of cAMP analog or forskolin but not cGMP analog. Cycloheximide blocked arginase activity in the cells in the presence of cAMP analog or forskolin with or without A. actinomycetemcomitans LPS. IBMX augmented arginase activity in A. actinomycetemcomitans LPS‐stimulated cells. Rolipram (a PDE4 inhibitor) increased the levels of arginase activity higher than siguazodan (a PDE3 inhibitor) in the antigen‐stimulated cells. The effect of cAMP analog or forskolin on arginase activity in the presence or absence of A. actinomycetemcomitans LPS was blocked by the PKA inhibitor (KT5720). Conclusion: The results of the present study suggest that A. actinomycetemcomitans LPS may stimulate arginase activity in murine macrophages (RAW264.7 cells) in a cAMP‐PKA‐dependent pathway.     

Alpha‐amylase is a human salivary protein with affinity to lipopolysaccharide of Aggregatibacter actinomycetemcomitans

Aggregatibacter actinomycetemcomitans lipopolysaccharide (Aa.LPS) is a major virulence factor associated with aggressive periodontitis. Although the recognition of Aa.LPS is potentially initiated by salivary proteins in the oral cavity, Aa.LPS‐binding proteins (Aa.LPS‐BPs) in saliva are poorly characterized. The purpose of this study was to capture and identify Aa.LPS‐BPs in human saliva using a LTQ‐Orbitrap hybrid Fourier transform mass spectrometry. Aa.LPS conjugated onto N‐hydroxysuccinimidyl‐Sepharose^® 4 Fast Flow beads (Aa.LPS‐beads) activated Toll‐like receptor 4 and produced nitric oxide and Interferon gamma‐inducible protein‐10, implying that the conjugation process did not alter the biological properties of Aa.LPS. Aa.LPS‐BPs were subsequently isolated from the nine human saliva samples from healthy individuals with the Aa.LPS‐beads followed by identification with the mass spectrometry. Aa.LPS‐BPs include α‐amylase, serum albumin, cystatin, lysozyme C, submaxillary gland androgen‐regulated protein 3B, immunoglobulin subunits, polymeric immunoglobulin receptor, deleted in malignant brain tumors 1, prolactin‐inducible protein, lipocalin‐1, and basic salivary proline‐rich protein 2. Specific binding was validated using a pull‐down assay with α‐amylase which was captured at the highest frequency. Alpha‐amylase demonstrated to interfere with the adherence and biofilm formation of A. actinomycetemcomitans. Even heat‐inactivated α‐amylase showed the interference to the same extent. Conclusively, we identified unique Aa.LPS‐BPs that provide useful information to understand bacterial pathogenesis and host innate immunity in the oral cavity.     

Azithromycin Enhances Phagocytic Killing of Aggregatibacter actinomycetemcomitans Y4 by Human Neutrophils

Background: Aggregatibacter actinomycetemcomitans resists killing by neutrophils and is inhibited by azithromycin (AZM) and amoxicillin (AMX). AZM actively concentrates inside host cells, whereas AMX enters by diffusion. The present study is conducted to determine whether AZM is more effective than AMX at enhancing phagocytic killing of A. actinomycetemcomitans by neutrophils. Methods: Killing assays were conducted in the presence of either 2 μg/mL AZM or 16 μg/mL AMX (equipotent against A. actinomycetemcomitans). Neutrophils were loaded by incubation with the appropriate antibiotic. Opsonized A. actinomycetemcomitans strain Y4 was incubated with the indicated antibiotic alone, with loaded neutrophils and antibiotic, or with control neutrophils (without antibiotic) at multiplicities of infection (MOIs) of 30 and 90 bacteria per neutrophil. Results: Neutrophil incubation with 2 μg/mL AZM yielded an intracellular concentration of 10 μg/mL. At an MOI of 30, neutrophils loaded with AZM failed to kill significantly more bacteria than control neutrophils during the 60‐ and 90‐minute assay periods. At an MOI of 90, neutrophils loaded with AZM killed significantly more bacteria than either AZM alone or control neutrophils during 60‐ and 90‐minute incubations (P <0.05), and killed significantly more bacteria after 90 minutes than the sum of the killing produced by AZM alone or neutrophils alone. Neutrophils incubated with AMX under identical conditions also killed significantly more bacteria than either AMX alone or control neutrophils, but there was no evidence of synergism between AMX and neutrophils. Conclusions: Neutrophils possess a concentrative transport system for AZM that may enhance killing of A. actinomycetemcomitans. Its effects are most pronounced when neutrophils are greatly outnumbered by bacteria.     

L-arginine-dependent nitric oxide production of a murine macrophage-like RAW 264.7 cell line stimulated with Porphyromonas gingivalis lipopolysaccharide

The aim of this study was to determine nitric oxide (NO) production of a murine macrophage cell line (RAW 264.7 cells) when stimulated with Porphyromonas gingivalis lipopolysaccharides (Pg-LPS). RAW 264.7 cells were incubated with i) various concentrations of Pg-LPS or Salmonella typhosa LPS (St-LPS), ii) Pg-LPS with or without L-arginine and/or NG-monomethyl-L-arginine (NMMA), an arginine analog or iii) Pg-LPS and interferon-gamma (IFN-gamma) with or without anti-IFN-gamma antibodies or interleukin-10 (IL-10). Tissue culture supernatants were assayed for NO levels after 24 h in culture. NO was not observed in tissue culture supernatants of RAW 264.7 cells following stimulation with Pg-LPS, but was observed after stimulation with St-LPS. Exogenous L-arginine restored the ability of Pg-LPS to induce NO production; however, the increase in NO levels of cells stimulated with Pg-LPS with exogenous L-arginine was abolished by NMMA. IFN-gamma induced independent NO production by Pg-LPS-stimulated macrophages and this stimulatory effect of IFN-gamma could be completely suppressed by anti-IFN-gamma antibodies and IL-10. These results suggest that Pg-LPS is able to stimulate NO production in the RAW 264.7 macrophage cell model in an L-arginine-dependent mechanism which is itself independent of the action of IFN-gamma.