Gustavo Monasterio

Variability of the dendritic cell response triggered by different serotypes of Aggregatibacter actinomycetemcomitans or Porphyromonas gingivalis is toll-like receptor 2 (TLR2) or TLR4 dependent.

BACKGROUND Different serotypes of Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis have been shown to induce differential dendritic cell (DC) responses. This study investigates whether cytokine and CC-chemokine receptor (CCR) production by DCs stimulated with different serotypes of A. actinomycetemcomitans or P. gingivalis is Toll-like receptor 2 (TLR2) and/or TLR4 dependent. METHODS DCs were obtained from healthy individuals and primed at a multiplicity of infection (MOI) of 10(2) with different A. actinomycetemcomitans or P. gingivalis serotypes in the presence or absence of anti-TLR2 or anti-TLR4 blocking antibodies. TLR2 and TLR4 expression, CCR5 and CCR6 expression, and interleukin (IL)-1β, IL-10, IL-12, and IL-23 expression and secretion were quantified by flow cytometry, real-time reverse-transcription polymerase chain reaction, and enzyme-linked immunosorbent assay. RESULTS When DCs were stimulated with serotype b of A. actinomycetemcomitans or serotype K1 of P. gingivalis, higher levels of TLR2 or TLR4, respectively, were detected compared to DCs stimulated with the other serotypes. Similarly, higher levels of cytokines and CCRs were detected in serotype b- or serotype K1-primed DCs compared to the others, and these increased levels positively correlated with levels of TLR2 or TLR4. When TLR2 signaling was blocked using a specific anti-TLR2 monoclonal antibody, serotype b-induced cytokine and CCR expression was inhibited; when TLR4 signaling was blocked, serotype K1-induced response was inhibited. CONCLUSIONS These results demonstrate that the variability of secretion of cytokines and expression of CCRs detected in DCs stimulated with different serotypes of A. actinomycetemcomitans or P. gingivalis is TLR2 or TLR4 dependent, respectively.

T‐lymphocyte phenotype and function triggered by Aggregatibacter actinomycetemcomitans is serotype‐dependent

BACKGROUND AND OBJECTIVE Based on lipopolysaccharide (LPS) antigenicity, different Aggregatibacter actinomycetemcomitans serotypes have been described. Serotype b strains have demonstrated a stronger capacity to trigger cytokine production on dendritic cells (DCs). As DCs regulate the development of T-lymphocyte lineages, the objective of this investigation was to study the response of T lymphocytes after being stimulated with autologous DCs primed with different bacterial strains belonging to the most prevalent serotypes of A. actinomycetemcomitans in humans: a-c. MATERIAL AND METHODS Human DCs were primed with increasing multiplicity of infection (10(-1) -10(2) ) or the purified LPS (10-50 ng/mL) of A. actinomycetemcomitans serotypes a-c and then used to stimulate autologous naïve CD4(+) T lymphocytes. The T-helper (Th) type 1, Th2, Th17 and T-regulatory transcription factors T-bet, GATA-3, RORC2 and Foxp3, which are the master-switch genes implied in their specific differentiation, as well as T-cell phenotype-specific cytokine patterns were quantified by real-time reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay. In addition, the intracellular expression of T-bet/interferon-γ, GATA-3/interleukin-4, RORC2/interleukin-17A and Foxp3/transforming growth factor-β1 was analysed by double staining and flow cytometry. RESULTS All the A. actinomycetemcomitans serotypes led to T-lymphocyte activation; however, when T lymphocytes were stimulated with DCs primed with the A. actinomycetemcomitans serotype b strain or their purified LPS, higher levels of Th1- and Th17-associated transcription factors and cytokines were detected compared with similar experiments with the other serotypes. CONCLUSION These results demonstrate that serotype b of A. actinomycetemcomitans has a higher capacity of trigger Th1 and Th17 phenotype and function and it was demonstrated that their LPS is a more potent immunogen compared with the other serotypes.

Increased levels of the T-helper 22-associated cytokine (interleukin-22) and transcription factor (aryl hydrocarbon receptor) in patients with periodontitis are associated with osteoclast resorptive activity and severity of the disease

BACKGROUND AND OBJECTIVE Two new T-helper (Th) phenotypes have been recently described and named Th9 and Th22 lymphocytes; however, their role in the pathogenesis of periodontitis remains unclear. This study was aimed to assess whether Th9 and Th22 lymphocytes, through interleukin (IL)-9 and IL-22 production, respectively, are associated with the severity of periodontitis and bone resorption. MATERIAL AND METHODS Gingival crevicular fluid samples and biopsies were obtained from patients with moderate-to-advanced chronic periodontitis and gingivitis, and healthy controls. The levels for the Th9 and Th22-associated cytokines and master-switch transcription factors Spi-B and aryl hydrocarbon receptor (AhR) were quantified by enzyme-linked immunosorbent assay, real-time reverse-transcription quantitative polymerase chain reaction and flow cytometry. In addition, the osteoclast activity in response to tissue homogenates from periodontitis and healthy samples was analyzed quantifying the number of TRAP-positive cells and areas of bone resorption pits produced, in the presence or absence of recombinant human IL-22 and anti-IL-22 neutralization antibody. RESULTS Higher levels of IL-22 and AhR were detected in patients with periodontitis compared with gingivitis and healthy individuals. In addition, higher levels of IL-9 and Spi-B were detected in gingivitis patients compared with periodontitis and healthy individuals. In patients with periodontitis, a significant positive correlation was detected between secreted levels of IL-22 and clinical attachment level of the sampled periodontal pockets. When osteoclasts were exposed to tissue homogenates obtained from patients with periodontitis, higher levels of resorptive activity were observed as compared with the same cells exposed to tissue homogenates obtained from healthy individuals, and this increment was dependent on the presence and neutralization of IL-22. CONCLUSION Increased levels of IL-22 produced by Th22 lymphocytes are associated with the pathogenesis of periodontitis, in particular, with osteoclast resorptive activity and severity of disease.

Differential human Th22-lymphocyte response triggered by Aggregatibacter actinomycetemcomitans serotypes

OBJECTIVE In Aggregatibacter actinomycetemcomitans, different serotypes have been described based on lipopolysaccharide (LPS) antigenicity. When T lymphocytes were stimulated with these serotypes, different patterns of T-helper (Th)1 and Th17-type of immune responses were reported. Recently, two new Th phenotypes have been described and named Th9 and Th22 lymphocytes; however, their role in the pathogenesis of periodontitis remains unclear. This study aimed to investigate the potential Th9 and/or Th22 lymphocyte responses when stimulated with autologous dendritic cells infected with different A. actinomycetemcomitans serotypes. METHODS Monocyte-derived dendritic cells and naïve CD4+ T lymphocytes were obtained from healthy donors and stimulated with different serotypes of A. actinomycetemcomitans at a multiplicity of infection MOI=102 or their purified LPS (10-50ng/ml). The levels for the Th9 and Th22-associated cytokines, as well as the transcription factor master-switch genes implied in their differentiation Spi-B and AhR, were quantified by qPCR and ELISA. RESULTS When stimulated with the serotype b of A. actinomycetemcomitans, higher levels of interleukin (IL)-6 and tumor necrosis factor (TNF)-α were detected in dendritic cells, as well as higher levels of IL-22 and AhR were detected in T lymphocytes, when compared with stimulation with the other serotypes. CONCLUSIONS The serotype b of A. actinomycetemcomitans has a higher capacity of trigger Th22-type of immune response in both dendritic cells and T lymphocytes. These data allow us to suggest that, when the serotype b of A. actinomycetemcomitans is a significant part of the subgingival biofilm, the Th22 polarization might be triggered within the periodontal lesion.

Regulatory T Lymphocytes in Periodontitis: A Translational View

Periodontitis is a chronic immuno-inflammatory disease in which the disruption of the balance between host and microbiota interactions is key to the onset and progression of the disease. The immune homeostasis associated with periodontal health requires a regulated immuno-inflammatory response, during which the presence of regulatory T cells (Tregs) is essential to ensure a controlled response that minimizes collateral tissue damage. Since Tregs modulate both innate and adaptive immunity, pathological conditions that may resolve by the acquisition of immuno-tolerance, such as periodontitis, may benefit by the use of Treg immunotherapy. In recent years, many strategies have been proposed to take advantage of the immuno-suppressive capabilities of Tregs as immunotherapy, including the ex vivo and in vivo manipulation of the Treg compartment. Ongoing research in both basic and translational studies let us gain a better understanding of the diversity of Treg subsets, their phenotypic plasticity, and suppressive functions, which can be used as a substrate for new immunotherapies. Certainly, as our knowledge of Treg biology increases, we will be capable to develop new therapies designed to enhance the stability and function of Tregs during periodontitis.

Th1/Th17/Th22 immune response and their association with joint pain, imagenological bone loss, RANKL expression and osteoclast activity in temporomandibular joint osteoarthritis: A preliminary report

It is well accepted that the presence of cytokines belonging to the Th1/Th17/Th22 axis of immuno-inflammatory response in the joint environment, such as IL-1β, IL-17 and IL-22, respectively, are associated with pathogenesis of several synovial joint degenerative disorders. During temporomandibular joint osteoarthritis (TMJ-OA), IL-1β and IL-17 have been implicated in the inflammation and resorption of sub-chondral bone; however, the role of Th22 response in the TMJ-OA pathophysiology has not been established. This study aimed to compare the expression of Th1/Th17/Th22-type cytokines, chemokines and chemokine receptors in synovial fluid samples obtained from TMJ-OA or disk displacement with reduction (DDWR) patients. In addition, it aimed to associate these levels with joint pain, imagenological signs of bone degeneration, RANKL production, osteoclastogenesis and osteoclast-induced bone resorption. Higher levels of IL-1β, IL-17 and IL-22 were expressed in TMJ-OA compared with DDWR subjects, and these increased levels significantly correlated with RANKL expression, joint pain and articular bone degeneration. Higher levels of CCR5, CCR6 and CCR7, as well as their respective ligands CCL5 and CCL20, responsible for recruitment of IL-1β, IL-17 and IL-22-producing cells, were over-expressed in TMJ-OA compared with DDWR subjects. Osteoclastogenesis and osteoclast-induced bone resorption were significantly greater in presence of synovial fluid from TMJ-OA compared with DDWR subjects. These data demonstrate that cytokines, CCLs and CCRs associated with the Th1/Th17/Th22 axis of immuno-inflammatory response are involved in TMJ-OA pathogenesis. These findings suggest that IL-22 is involved in the RANKL expression in TMJ-OA, which in turn induces differentiation of osteoclasts and subsequent resorption of sub-chondral bone.

Immunostimulatory activity of low-molecular-weight hyaluronan on dendritic cells stimulated with Aggregatibacter actinomycetemcomitans or Porphyromonas gingivalis

ObjectivesPeriodontitis is a chronic inflammatory disease characterized by tooth-supporting tissue destruction, which is elicited by the host’s immune response triggered against periodonto-pathogen bacteria. During periodontal tissue destruction, extracellular matrix components are metabolized and fragmented. Some extracellular matrix component-derived fragments, such as low-molecular-weight hyaluronan (LMW-HA), have potent immunogenic potential, playing a role as damage-associated molecular patterns (DAMPs) during activation of immune cells. Dendritic cells (DCs) play a central role in the host’s immune response displayed during periodontitis; thus, this study aimed to analyze whether LMW-HA has an immunostimulatory activity on DCs when stimulated with periodonto-pathogen bacteria.Materials and methodsLMW-HA-treated and non-treated DCs were stimulated with Aggregatibacter actinomycetemcomitans or Porphyromonas gingivalis and the mRNA expression for cytokines tumor necrosis factor-α (TNF-alpha), interleukin-1β (IL-1B), interleukin-6 (IL-6), and interleukin-23 (IL-23A) was quantified by RT-qPCR. In addition, transcription factors interferon regulatory factor 4 (IRF4), interferon regulatory factor 8 (IRF8), neurogenic locus notch homolog protein 2 (NOTCH2), and basic leucine zipper ATF-like transcription factor 3 (BATF3), involved in DC activation, were analyzed.ResultsHigher expression levels of TNF-alpha, IL-1B, IL-6, and IL-23A were detected in LMW-HA-treated DCs after bacterial infection, as compared with non-treated DCs. When LMW-HA-treated DCs were infected with A. actinomycetemcomitans, higher levels of IRF4, NOTCH2, and BATF3 were detected compared with non-treated cells; whereas against P. gingivalis infection, increased levels of IRF4 and NOTCH2 were detected.ConclusionLMW-HA plays an immunostimulatory role on the immune response triggered by DCs during infection with A. actinomycetemcomitans or P. gingivalis.Clinical relevanceDetection of extracellular matrix component-derived fragments produced during periodontal tissue destruction, such as LMW-HA, could explain at least partly unsuccessful periodontal treatment and the chronicity of the disease.

Serotype a of Aggregatibacter actinomycetemcomitans down-regulates the increased serotype b -induced cytokine and chemokine production in dendritic cells

OBJECTIVE In Aggregatibacter actinomycetemcomitans, different serotypes have been described based on LPS antigenicity. Mixed infection with the different A. actinomycetemcomitans serotypes is frequent in periodontitis patients; accordingly, the role of this bacterial species in the pathogenesis of periodontitis may differ depending whether patients or periodontal lesions harbour one or more of the A. actinomycetemcomitans serotypes. We hypothesized that different combinations of these serotypes could be associated with distinct host responses and hence different inflammatory patterns. This investigation was aimed to assess whether the increased immuno-stimulatory potential attributed to the serotype b of A. actinomycetemcomitans on immune cells is able to be modified during co-infection with other A. actinomycetemcomitans serotypes. METHODS Dendritic cells (DCs) were obtained from healthy subjects and stimulated with the different A. actinomycetemcomitans serotypes or their purified LPS using the following stimulatory conditions: serotype a, b, or c, and the combinations a+b, a+c, b+c, or a+b+c. The cytokine, CCR, and CCL levels were quantified by qPCR and ELISA. RESULTS Higher levels of cytokines, CCRs, and CCLs were induced when DCs were stimulated with the serotype b of A. actinomycetemcomitans compared with the same cells stimulated with the other serotypes. When DCs were co-infected, these levels decreased in comparison with the serotype b-stimulation alone, in particular when the serotype a was present in the mixed infection. CONCLUSIONS The increased immuno-stimulatory potential attributed to the serotype b was modified when DCs were co-infected with other A. actinomycetemcomitans serotypes, in particular, when the serotype a was present, the DC response diminished.

Variabilidad de la síntesis de citoquinas por células dendríticas humanas estimuladas con los distintos serotipos de Aggregatibacter actinomycetecomitans

Resumen es: Objetivo: Sobre la base de la antigenicidad del polisacarido O del LPS, en A. actinomycetemcomitans se describen distintos serotipos bacterianos y entre ...

Variability in the cytokine synthesis by human dendritic cells in response to different Aggregatibacter actinomycetemcomitans serotypes

-1 -10 -2 con los serotipos a, b y c de A. Actinomycetemcomitans. Mediante PCR y ELISA se evaluaron los niveles de expresion y secrecion de citoquinas. Resultados: En las celulas dendriticas, la produccion de citoquinas fue diferente ante los distintos serotipos de A. actinomycetemcomitans, con mayores niveles de secrecion de IL-1β, IL-6, IL-12, IL-23, IFN-γ y TNF-α cuando el microorganismo estimulante fue la cepa ATCC ® 43718™ (serotipo b). Conclusion: El serotipo b de A. actinomycetemcomitans posee un mayor potencial inmuno-estimulador de celulas dendriticas comparado con los otros serotipos bacterianos y potencialmente contribuiria a inducir un patron de respuesta inmune tipo Th1 y/o Th17 durante las periodontitis. Rev. Clin. Periodoncia Implantol. Rehabil. Oral Vol. 6(2); 57-62, 2013. Palabras clave: Celulas dendriticas, citoquinas, Aggregatibacter actinomycetemcomitans.