isolation-characterization-functional-examination-gingival-immune

Isolation, Characterization and Functional Examination of the Gingival Immune Cell Network

Immune cell networks in tissues play a vital role in mediating local immunity and maintaining tissue homeostasis, yet little is known of the resident ...

Interleukin-21 (IL-21) controls the differentiation of T-helper Th17 cells and induces the production of IL-17 in this T-cell subtype. The aim of this study is to determine the relative expression of IL-21 in gingival tissues of chronic periodontitis patients and correlate/associate this expression with proinflammatory cytokines and clinical parameters of disease.

Interleukin-21 expression and its association with proinflammatory cytokines in untreated chronic periodontitis patients.

The goals of this study were to better understand the ecology of oral subgingival communities in health and periodontitis and elucidate the relationship between inflammation and the subgingival microbiome. Accordingly, we used 454-pyrosequencing of 16S rRNA gene libraries and quantitative PCR to characterize the subgingival microbiome of 22 subjects with chronic periodontitis. Each subject was sampled at two sites with similar periodontal destruction but differing in the presence of bleeding, a clinical indicator of increased inflammation. Communities in periodontitis were also compared with those from 10 healthy individuals. In periodontitis, presence of bleeding was not associated with different α-diversity or with a distinct microbiome, however, bleeding sites showed higher total bacterial load. In contrast, communities in health and periodontitis largely differed, with higher diversity and biomass in periodontitis. Shifts in community structure from health to periodontitis resembled ecological succession, with emergence of newly dominant taxa in periodontitis without replacement of primary health-associated species. That is, periodontitis communities had higher proportions of Spirochetes, Synergistetes, Firmicutes and Chloroflexi, among other taxa, while the proportions of Actinobacteria, particularly Actinomyces, were higher in health. Total Actinomyces load, however, remained constant from health to periodontitis. Moreover, an association existed between biomass and community structure in periodontitis, with the proportion of specific taxa correlating with bacterial load. Our study provides a global-scale framework for the ecological events in subgingival communities that underline the development of periodontitis. The association, in periodontitis, between inflammation, community biomass and community structure and their role in disease progression warrant further investigation.

The subgingival microbiome in health and periodontitis and its relationship with community biomass and inflammation.

Solid-organ transplant recipients rely on pharmacological immunosuppression to prevent allograft rejection. The effect of such chronic immunosuppression on the microflora at mucosal surfaces is not known. We evaluated the salivary bacterial microbiome of 20 transplant recipients and 19 nonimmunosuppressed controls via 454 pyrosequencing of 16S rRNA gene amplicons. Alpha-diversity and global community structure did not differ between transplant and control subjects. However, principal coordinate analysis showed differences in community membership. Taxa more prevalent in transplant subjects included operational taxonomic units (OTUs) of potentially opportunistic Gammaproteobacteria such as Klebsiella pneumoniae, Pseudomonas fluorescens, Acinetobacter species, Vibrio species, Enterobacteriaceae species, and the genera Acinetobacter and Klebsiella. Transplant subjects also had increased proportions of Pseudomonas aeruginosa, Acinetobacter species, Enterobacteriaceae species, and Enterococcus faecalis, among other OTUs, while genera with increased proportions included Klebsiella, Acinetobacter, Staphylococcus, and Enterococcus. Furthermore, in transplant subjects, the dose of the immunosuppressant prednisone positively correlated with bacterial richness, while prednisone and mycophenolate mofetil doses positively correlated with the prevalence and proportions of transplant-associated taxa. Correlation network analysis of OTU relative abundance revealed a cluster containing potentially opportunistic pathogens as transplant associated. This cluster positively correlated with serum levels of C-reactive protein, suggesting a link between the resident flora at mucosal compartments and systemic inflammation. Network connectivity analysis revealed opportunistic pathogens as highly connected to each other and to common oral commensals, pointing to bacterial interactions that may influence colonization. This work demonstrates that immunosuppression aimed at limiting T-cell-mediated responses creates a more permissive oral environment for potentially opportunistic pathogens without affecting other members of the salivary bacteriome.

Transplantation-associated long-term immunosuppression promotes oral colonization by potentially opportunistic pathogens without impacting other members of the salivary bacteriome.

Leukocyte adhesion deficiency type I (LAD-I), a disease syndrome associated with frequent microbial infections, is caused by mutations on the CD18 subunit of β₂ integrins. LAD-I is invariably associated with severe periodontal bone loss, which historically has been attributed to the lack of neutrophil surveillance of the periodontal infection. We provide an alternative mechanism by showing that the cytokine interleukin-17 (IL-17) plays a major role in the oral pathology of LAD-I. Defective neutrophil recruitment in LAD-I patients or in LFA-1 (CD11a/CD18)-deficient mice--which exhibit the LAD-I periodontal phenotype--was associated with excessive production of predominantly T cell-derived IL-17 in the periodontal tissue, although innate lymphoid cells also contributed to pathological IL-17 elevation in the LFA-1-deficient mice. Local treatment with antibodies to IL-17 or IL-23 in LFA-1-deficient mice not only blocked inflammatory periodontal bone loss but also caused a reduction in the total bacterial burden, suggesting that the IL-17-driven pathogenesis of LAD-I periodontitis leads to dysbiosis. Therefore, our findings support an IL-17-targeted therapy for periodontitis in LAD-I patients.

Defective neutrophil recruitment in leukocyte adhesion deficiency type I disease causes local IL-17-driven inflammatory bone loss.

The advent of next-generation sequencing has significantly facilitated characterization of the oral microbiome. Despite great efforts in streamlining the processes of sequencing and data curation, upstream steps required for amplicon library generation could still influence 16S rRNA gene-based microbial profiles. Among upstream processes, DNA extraction is a critical step that could represent a great source of bias. Accounting for bias introduced by extraction procedures is important when comparing studies that use different methods. Identifying the method that best portrays communities is also desirable. Accordingly, the aim of this study was to evaluate bias introduced by different DNA extraction procedures on oral microbiome profiles.

Influence of DNA extraction on oral microbial profiles obtained via 16S rRNA gene sequencing.

Leukocyte Adhesion Deficiency I (LAD-I) is a primary immunodeficiency caused by single gene mutations in the CD18 subunit of β2 integrins which result in defective transmigration of neutrophils into the tissues. Affected patients suffer from recurrent life threatening infections and severe oral disease (periodontitis). Microbial communities in the local environment (subgingival plaque) are thought to be the triggers for inflammatory periodontitis, yet little is known regarding the microbial communities associated with LAD-I periodontitis. Here we present the first comprehensive characterization of the subgingival communities in LAD-I, using a 16S rRNA gene-based microarray, and investigate the relationship of this tooth adherent microbiome to the local immunopathology of periodontitis. We show that the LAD subgingival microbiome is distinct from that of health and Localized Aggressive Periodontitits. Select periodontitis-associated species in the LAD microbiome included Parvimonas micra, Porphyromonas endodontalis, Eubacterium brachy and Treponema species. Pseudomonas aeruginosa, a bacterium not typically found in subgingival plaque is detected in LAD-I. We suggest that microbial products from LAD-associated communities may have a role in stimulating the local inflammatory response. We demonstrate that bacterial LPS translocates into the lesions of LAD-periodontitis potentially triggering immunopathology. We also show in in vitro assays with human macrophages and in vivo in animal models that microbial products from LAD-associated subgingival plaque trigger IL-23-related immune responses, which have been shown to dominate in patient lesions. In conclusion, our current study characterizes the subgingival microbial communities in LAD-periodontitis and supports their role as triggers of disease pathogenesis.

Subgingival microbial communities in Leukocyte Adhesion Deficiency and their relationship with local immunopathology.

Periodontal diseases usually refer to common inflammatory disorders known as gingivitis and periodontitis, which are caused by a pathogenic microbiota in the subgingival biofilm, including Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, Tannerella forsythia and Treponema denticola that trigger innate, inflammatory, and adaptive immune responses. These processes result in the destruction of the tissues surrounding and supporting the teeth, and eventually in tissue, bone and finally, tooth loss. The innate immune response constitutes a homeostatic system, which is the first line of defense, and is able to recognize invading microorganisms as non-self, triggering immune responses to eliminate them. In addition to the innate immunity, adaptive immunity cells and characteristic cytokines have been described as important players in the periodontal disease pathogenesis scenario, with a special attention to CD4+ T-cells (T-helper cells). Interestingly, the T cell-mediated adaptive immunity development is highly dependent on innate immunity-associated antigen presenting cells, which after antigen capture undergo into a maturation process and migrate towards the lymph nodes, where they produce distinct patterns of cytokines that will contribute to the subsequent polarization and activation of specific T CD4+ lymphocytes. Skeletal homeostasis depends on a dynamic balance between the activities of the bone-forming osteoblasts (OBLs) and bone-resorbing osteoclasts (OCLs). This balance is tightly controlled by various regulatory systems, such as the endocrine system, and is influenced by the immune system, an osteoimmunological regulation depending on lymphocyte- and macrophage-derived cytokines. All these cytokines and inflammatory mediators are capable of acting alone or in concert, to stimulate periodontal breakdown and collagen destruction via tissue-derived matrix metalloproteinases, a characterization of the progression of periodontitis as a stage that presents a significantly host immune and inflammatory response to the microbial challenge that determine of susceptibility to develop the destructive/progressive periodontitis under the influence of multiple behavioral, environmental and genetic factors.

Article	Year
Interleukin-21 expression and its association with proinflammatory cytokines in untreated chronic periodontitis patients. Journal of periodontology\| PubMed ID: 22181687	2012
The subgingival microbiome in health and periodontitis and its relationship with community biomass and inflammation. The ISME journal\| PubMed ID: 23303375	2013
Transplantation-associated long-term immunosuppression promotes oral colonization by potentially opportunistic pathogens without impacting other members of the salivary bacteriome. Clinical and vaccine immunology : CVI\| PubMed ID: 23616410	2013
Defective neutrophil recruitment in leukocyte adhesion deficiency type I disease causes local IL-17-driven inflammatory bone loss. Science translational medicine\| PubMed ID: 24670684	2014
Influence of DNA extraction on oral microbial profiles obtained via 16S rRNA gene sequencing. Journal of oral microbiology\| PubMed ID: 24778776	2014
Subgingival microbial communities in Leukocyte Adhesion Deficiency and their relationship with local immunopathology. PLoS pathogens\| PubMed ID: 25741691	2015
Host response mechanisms in periodontal diseases. Journal of applied oral science : revista FOB\| PubMed ID: 26221929	2015

Loreto Abusleme

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