Microbial analysis of subgingival plaque samples compared to that of whole saliva in patients with periodontitis.

BACKGROUND: The detection of special bacterial species in patients with periodontitis is considered to be useful for clinical diagnosis and treatment. The collection of subgingival plaque samples is the common way for the determination of periodontopathic bacteria. However, recently, salivary analysis has been discussed as an advantageous future diagnostic method for periodontitis because it offers simple quantitative sampling and the possibility to assess various bacteria. The aim of this cross-sectional study is to investigate whether there is a correlation between the results of different bacterial species in saliva and subgingival plaque samples from individuals with aggressive periodontitis (AgP) and chronic periodontitis (CP). METHODS: Whole saliva and subgingival plaque samples from the deepest pocket of each quadrant were collected from 43 patients with CP and 33 patients with AgP. Twenty different bacterial species from both samplings were determined by the 16S ribosomal RNA-based polymerase chain reaction with microarray technique. RESULTS: All bacterial species were detected in salivary and subgingival plaque samples. For Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia, as well as Actinomyces viscosus, Campylobacter rectus/showae, Prevotella intermedia, Parvimonas micra, Eubacterium nodatum, and Campylobacter gracilis, a significant positive correlation between salivary and subgingival plaque samples was detected in patients with both types of periodontitis. There were no significant differences in bacteria in salivary and subgingival plaque samples between AgP and CP. CONCLUSION: Salivary analysis might be discussed as a potential alternative to subgingival plaque sampling for microbiologic analysis in both AgP and CP.

The B-cell superantigen Finegoldia magna protein L causes pulmonary inflammation by a mechanism dependent on MyD88 but not B cells or immunoglobulins.

OBJECTIVE AND DESIGN: To determine whether Finegoldia magna protein L (PL) causes lung inflammation and, if so, whether the response is dependent on its immunoglobulin (Ig)-binding B-cell superantigenic property. MATERIAL: Pulmonary inflammatory reactions were analyzed at various time points after intratracheal administration of PL to various strains of mice. RESULTS: PL caused peribronchial and perivascular inflammation that peaked at 18-24 h. Polymorphonuclear cells (PMNs) began to accumulate in bronchoalveolar lavage fluid (BALF) of PL-challenged mice by 4 h and accounted for >90% of leukocytes by 18-24 h. Inflammation was marked by the appearance of MIP-2, KC, TNF-α, and IL-6 in the BALF with peak levels attained 4 h after PL administration. PL-induced pulmonary inflammation was associated with increased airway hyper-reactivity following inhalation of methacholine. The inflammatory reaction was unabated in mice lacking B cells and immunoglobulins. In contrast, PL-induced inflammation was abrogated in MyD88-deficient mice. PL-induced responses required alveolar macrophages. CONCLUSIONS: These results strongly suggest that PL-induced lung inflammation is dependent on an innate MyD88-dependent pathway rather than the Ig-binding properties of this microbial B cell superantigen. We propose that this pulmonary inflammatory reaction is caused by the interaction of PL with a Toll-like receptor expressed on alveolar macrophages.

Social stress enhances allergen-induced airway inflammation in mice and inhibits corticosteroid responsiveness of cytokine production.

Chronic psychosocial stress exacerbates asthma, but the underlying mechanisms remain poorly understood. We hypothesized that psychosocial stress aggravates allergic airway inflammation by altering innate immune cell function. The effects of stress on airway inflammation, lung function, and glucocorticoid responsiveness were studied in a novel in vivo murine model of combined social disruption stress and allergic sensitization. The effects of corticosterone were assessed on cytokine profile and glucocorticoid receptor activation in LPS-stimulated spleen cell cultures in vitro. Airway inflammation resolved 48 h after a single allergen provocation in sensitized control mice, but not in animals that were repeatedly exposed to stress before allergen challenge. The enhanced eosinophilic airway inflammation 48 h after allergen challenge in these mice was associated with increased levels of IL-5, GM-CSF, IgG1, thymus-activated and regulatory chemokine, TNF-alpha, and IL-6 in the airways and a diminished inhibition of these mediators by corticosterone in LPS-stimulated splenocyte cultures in vitro. Stress-induced reduction of the corticosteroid effects paralleled increased p65 expression and a decreased DNA-binding capability of the glucocorticoid receptor in vitro. Furthermore, glucocorticoid receptor mRNA and protein expression in the lungs of mice exposed to both stress and allergen was markedly reduced in comparison with that in either condition alone or in naive mice. Thus, exposure to repeated social stress before allergen inhalation enhances and prolongs airway inflammation and alters corticosterone responsiveness. We speculate that these effects were mediated at least in part by impaired glucocorticoid receptor expression and function.

Essential role of IFNbeta and CD38 in TNFalpha-induced airway smooth muscle hyper-responsiveness.

We recently identified autocrine interferon (IFN)beta as a novel mechanism mediating tumor necrosis factor (TNF)alpha-induced expression of inflammatory genes in airway smooth muscle (ASM) cells, including CD38, known to regulate calcium signaling. Here, we investigated the putative involvement of IFNbeta in regulating TNFalpha-induced airway hyper-responsiveness (AHR), a defining feature of asthma. Using our pharmacodynamic model to assess ex vivo AHR isolated murine tracheal rings, we found that TNFalpha-induced enhanced contractile responses to carbachol and bradykinin was abrogated by neutralizing anti-IFNbeta antibody or in tracheal rings deficient in CD38. In cultured human ASM cells, where CD38 has been involved in TNFalpha-induced enhanced calcium signals to carbachol and bradykinin, we found that neutralizing anti-IFNbeta prevented TNFalpha enhancing action only on carbachol responses but not to that induced by bradykinin. In a well-characterized model of allergic asthma (mice sensitized and challenged with Aspergillus fumigatus (Af)), we found heightened expression of both IFNbeta and CD38 in the airways. Furthermore, allergen-associated AHR to methacholine, assessed by lung resistance and dynamic compliance, was completely suppressed in CD38-deficient mice, despite the preservation of airway inflammation. These data provide the first evidence that ASM-derived IFNbeta and CD38 may play a significant role in the development of TNFalpha-associated AHR.

Surfactant protein D inhibits TNF-alpha production by macrophages and dendritic cells in mice.

BACKGROUND: Surfactant protein (SP) D shares target cells with the proinflammatory cytokine TNF-alpha, an important autocrine stimulator of dendritic cells and macrophages in the airways. OBJECTIVE: We sought to study the mechanisms by which TNF-alpha and SP-D can affect cellular components of the pulmonary innate immune system. METHODS: Cytokine and SP-D protein and mRNA expression was assessed by means of ELISA, Western blotting, and real-time PCR, respectively, by using in vivo models of allergic airway sensitization. Macrophage and dendritic cell phenotypes were analyzed by means of FACS analysis. Maturation of bone marrow-derived dendritic cells was investigated in vitro. RESULTS: TNF-alpha, elicited either by allergen exposure or pulmonary overexpression, induced SP-D, IL-13, and mononuclear cell influx in the lung. Recombinant IL-13 by itself was also capable of enhancing SP-D in vivo and in vitro, and the SP-D response to allergen challenge was impaired in IL-13-deficient mice. Allergen-induced increase of SP-D in the airways coincided with resolution of TNF-alpha release and cell influx. SP-D-deficient mice had constitutively high numbers of alveolar mononuclear cells expressing TNF-alpha, MHC class II, CD86, and CD11b, characteristics of proinflammatory, myeloid dendritic cells. Recombinant SP-D significantly suppressed all of these molecules in bone marrow-derived dendritic cell cultures. CONCLUSIONS: TNF-alpha can contribute to enhanced SP-D production in the lung indirectly through inducing IL-13. SP-D, on the other hand, can antagonize the proinflammatory effects of TNF-alpha on macrophages and dendritic cells, at least partly, by inhibiting production of this cytokine.

Chronic obstructive pulmonary disease and inhaled steroids alter surfactant protein D (SP-D) levels: a cross-sectional study.

BACKGROUND: Surfactant protein D (SP-D), an innate immune molecule, plays an important protective role during airway inflammation. Deficiency of this molecule induces emphysematous changes in murine lungs, but its significance in human COPD remains unclear. METHODS: We collected bronchoalveolar lavage fluid from 20 subjects with varying degrees of COPD (8 former smokers and 12 current smokers) and 15 asymptomatic healthy control subjects (5 never smokers, 3 remote former smokers, and 7 current smokers). All subjects underwent a complete medical history and pulmonary function testing. SP-D was measured by Enzyme-Linked ImmunoSorbent Assay. Statistical analysis was performed using nonparametric methods and multivariable linear regression for control of confounding. The effect of corticosteroid treatment on SP-D synthesis was studied in vitro using an established model of isolated type II alveolar epithelial cell culture. RESULTS: Among former smokers, those with COPD had significantly lower SP-D levels than healthy subjects (median 502 and 1067 ng/mL, respectively, p = 0.01). In a multivariable linear regression model controlling for age, sex, race, and pack-years of tobacco, COPD was independently associated with lower SP-D levels (model coefficient -539, p = 0.04) and inhaled corticosteroid use was independently associated with higher SP-D levels (398, p = 0.046). To support the hypothesis that corticosteroids increase SP-D production we used type II alveolar epithelial cells isolated from adult rat lungs. These cells responded to dexamethasone treatment by a significant increase of SP-D mRNA (p = 0.041) and protein (p = 0.037) production after 4 days of culture. CONCLUSION: Among former smokers, COPD is associated with lower levels of SP-D and inhaled corticosteroid use is associated with higher levels of SP-D in the lung. Dexamethasone induced SP-D mRNA and protein expression in isolated epithelial cells in vitro. Given the importance of this molecule as a modulator of innate immunity and inflammation in the lung, low levels may play a role in the pathogenesis and/or progression of COPD. Further, we speculate that inhaled steroids may induce SP-D expression and that this mechanism may contribute to their beneficial effects in COPD. Larger, prospective studies are warranted to further elucidate the role of surfactant protein D in modulating pulmonary inflammation and COPD pathogenesis.

Absence of bacterially induced RELMbeta reduces injury in the dextran sodium sulfate model of colitis.

Although inflammatory bowel disease (IBD) is the result of a dysregulated immune response to commensal gut bacteria in genetically predisposed individuals, the mechanism(s) by which bacteria lead to the development of IBD are unknown. Interestingly, deletion of intestinal goblet cells protects against intestinal injury, suggesting that this epithelial cell lineage may produce molecules that exacerbate IBD. We previously reported that resistin-like molecule beta (RELMbeta; also known as FIZZ2) is an intestinal goblet cell-specific protein that is induced upon bacterial colonization whereupon it is expressed in the ileum and colon, regions of the gut most often involved in IBD. Herein, we show that disruption of this gene reduces the severity of colitis in the dextran sodium sulfate (DSS) model of murine colonic injury. Although RELMbeta does not alter colonic epithelial proliferation or barrier function, we show that recombinant protein activates macrophages to produce TNF-alpha both in vitro and in vivo. RELMbeta expression is also strongly induced in the terminal ileum of the SAMP1/Fc model of IBD. These results suggest a model whereby the loss of epithelial barrier function by DSS results in the activation of the innate mucosal response by RELMbeta located in the lumen, supporting the hypothesis that this protein is a link among goblet cells, commensal bacteria, and the pathogenesis of IBD.

IL-4 and IL-13 form a negative feedback circuit with surfactant protein-D in the allergic airway response.

The innate immune molecule surfactant protein-D (SP-D) plays an important regulatory role in the allergic airway response. In this study, we demonstrate that mice sensitized and challenged with either Aspergillus fumigatus (Af) or OVA have increased SP-D levels in their lung. SP-D mRNA and protein levels in the lung also increased in response to either rIL-4 or rIL-13 treatment. Type II alveolar epithelial cell expression of IL-4Rs in mice sensitized and challenged with Af, and in vitro induction of SP-D mRNA and protein by IL-4 and IL-13, but not IFN-gamma, suggested a direct role of IL-4R-mediated events. The regulatory function of IL-4 and IL-13 was further supported in STAT-6-deficient mice as well as in IL-4/IL-13 double knockout mice that failed to increase SP-D production upon allergen challenge. Interestingly, addition of rSP-D significantly inhibited Af-driven Th2 cell activation in vitro whereas mice lacking SP-D had increased numbers of CD4(+) cells with elevated IL-13 and thymus- and activation-regulated chemokine levels in the lung and showed exaggerated production of IgE and IgG1 following allergic sensitization. We propose that allergen exposure induces elevation in SP-D protein levels in an IL-4/IL-13-dependent manner, which in turn, prevents further activation of sensitized T cells. This negative feedback regulatory circuit could be essential in protecting the airways from inflammatory damage after allergen inhalation.

Surfactant protein-A inhibits Aspergillus fumigatus-induced allergic T-cell responses.

BACKGROUND: The pulmonary surfactant protein (SP)-A has potent immunomodulatory activities but its role and regulation during allergic airway inflammation is unknown. METHODS: We studied changes in SP-A expression in the bronchoalveolar lavage (BAL) using a murine model of single Aspergillus fumigatus (Af) challenge of sensitized animals. RESULTS: SP-A protein levels in the BAL fluid showed a rapid, transient decline that reached the lowest values (25% of controls) 12 h after intranasal Af provocation of sensitized mice. Decrease of SP-A was associated with influx of inflammatory cells and increase of IL-4 and IL-5 mRNA and protein levels. Since levels of SP-A showed a significant negative correlation with these BAL cytokines (but not with IFN-gamma), we hypothesized that SP-A exerts an inhibitory effect on Th2-type immune responses. To study this hypothesis, we used an in vitro Af-rechallenge model. Af-induced lymphocyte proliferation of cells isolated from sensitized mice was inhibited in a dose-dependent manner by addition of purified human SP-A (0.1-10 microg/ml). Flow cytometric studies on Af-stimulated lymphocytes indicated that the numbers of CD4+ (but not CD8+) T cells were significantly increased in the parental population and decreased in the third and fourth generation in the presence of SP-A. Further, addition of SP-A to the tissue culture inhibited Af-induced IL-4 and IL-5 production suggesting that SP-A directly suppressed allergen-stimulated CD4+ T cell function. CONCLUSION: We speculate that a transient lack of this lung collectin following allergen exposure of the airways may significantly contribute to the development of a T-cell dependent allergic immune response.