Carla Alvarez

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.

Serotype-dependent response of human dendritic cells stimulated with Aggregatibacter actinomycetemcomitans

AIM Different serotypes of Aggregatibacter actinomycetemcomitans have been described based on the lipopolysaccharide (LPS)-O-polysaccharide antigenicity. In turn, a distinct effect of A. actinomycetemcomitans serotypes has been described on cell proliferation and pro-inflammatory cytokine production in different human cells. This study was aimed to investigate the differential dendritic cell (DC) response when stimulated with different bacterial strains belonging to the most prevalent serotypes of A. actinomycetemcomitans (a-c). MATERIALS AND METHODS Dendritic cells were obtained from healthy subjects and stimulated with increasing multiplicity of infection (MOI = 10(-1) -10(2)) of A. actinomycetemcomitans, serotypes a-c, or their lipopolysaccharide (10-50 ng/ml). The levels for interferon (IFN)-γ, tumour necrosis factor (TNF)-α, interleukin (IL)-1β, IL-5, IL-6, IL-10, IL-12 and IL-23 were quantified by real-time RT-PCR and ELISA. RESULTS Variable DC responses were detected when stimulated with the different strains of A. actinomycetemcomitans. DCs stimulated with A. actinomycetemcomitans strains belonging to the serotype b or their purified LPS expressed higher levels of IL-1β, IL-6, IL-12, IL-23, IFN-γ and TNF-α than DCs stimulated with the other serotypes. CONCLUSIONS Aggregatibacter actinomycetemcomitans strains belonging to the serotype b demonstrated a higher capacity to trigger Th1 and Th17-type cytokine production on DCs. These increased potential is likely explained by a higher immunogenicity of their LPS.

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.

High levels of CXC Ligand 12/stromal cell-derived factor 1 in apical lesions of endodontic origin associated with mast cell infiltration

INTRODUCTION CXC ligand 12/stromal-derived factor-1 (CXCL12/SDF-1) is a pleiotropic chemokine that regulates the influx of a wide range of leukocytes. The aim of this study was to characterize CXCL12/SDF-1 in apical lesions (ALs) of endodontic origin, with special emphasis in associated immune cell populations. METHODS In this case-control study, 29 individuals with chronic apical periodontitis and 21 healthy volunteers were enrolled. ALs and healthy periodontal ligament samples were obtained for tissue homogenization, immune Western blotting, and enzyme-linked immunosorbent assay to determine CXCL12/SDF-1 forms and levels. Anatomopathologic diagnosis, immunostaining for CXCL12/SDF-1, CD117-CXCL12/SDF-1, and toluidine blue were also performed to identify tissue and cell localization. Finally, a set of tissue samples were digested and analyzed by flow cytometry to identify CXCL12/SDF-1 in different immune cell populations. Data were analyzed with Stata v11 and WinDi 2.9 software, and significance was considered if P < .05. RESULTS CXCL12/SDF-1 was predominantly identified as monomers; levels of CXCL12/SDF-1 were significantly higher in ALs compared with controls, and it was primarily localized to inflammatory infiltrates. Expression of CXCL12/SDF-1 was colocalized to mast cells in tissue sections. Furthermore, CD117(+) mast cells were the second most frequent infiltrating cells and the main CXCL12/SDF-1 expressing cells, followed by CD4(+) lymphocytes, monocytes/macrophages, neutrophils, and dendritic cells. CONCLUSIONS ALs of endodontic origin demonstrated higher levels of CXCL12/SDF-1 compared with controls. CXCL12/SDF-1 was identified in immune cell populations, whereas mast cells represented the major CXCL12/SDF-1 expressing cells, suggesting that this chemokine might play a central role in apical tissue destruction, most probably inducing persistent recruitment of immune cells, particularly of mast cells.

Differential expression of CC chemokines (CCLs) and receptors (CCRs) by human T lymphocytes in response to different Aggregatibacter actinomycetemcomitans serotypes

In Aggregatibacter actinomycetemcomitans, different serotypes have been described based on LPS antigenicity. Recently, our research group has reported a differential immunogenicity when T lymphocytes were stimulated with these different serotypes. In particular, it was demonstrated that the serotype b of A. actinomycetemcomitans has a stronger capacity to trigger Th1- and Th17-type cytokine production. Objective This study aimed to quantify the expression of different CC chemokines (CCLs) and receptors (CCRs) in T lymphocytes stimulated with the different A. actinomycetemcomitans serotypes. In addition, the expression of the transcription factors T-bet, GATA-3, RORC2, and Foxp3, master-switch genes implied in the Th1, Th2, Th17, and T-regulatory differentiation, respectively, was analysed in order to determine T-cell phenotype-specific patterns of CCL and CCR expression upon A. actinomycetemcomitans stimulation. Material and Methods Human naïve CD4+ T lymphocytes were obtained from healthy subjects and stimulated with autologous dendritic cells primed with the different A. actinomycetemcomitans serotypes. The expression levels for the chemokines CCL1, CCL2, CCL3, CCL5, CCL11, CCL17, CCL20, CCL21, CCL25, and CCL28, as well as the chemokine receptors CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, and CCR10 were quantified by qPCR. Similarly, the expression levels for the transcription factors T-bet, GATA-3, RORC2, and Foxp3 were quantified and correlated with the CCL and CCR expression levels. Results Higher expression levels of CCL2, CCL3, CCL5, CCL20, CCL21, CCL28, CCR1, CCR2, CCR5, CCR6, CCR7, and CCR9 were detected in T lymphocytes stimulated with the serotype b of A. actinomycetemcomitans compared with the other serotypes. In addition, these higher expression levels of CCLs and CCRs positively correlated with the increased levels of T-bet and RORC2 when T lymphocytes were stimulated with the serotype b. Conclusion A T-lymphocyte response biased towards a Th1- and Th17-pattern of CCL and CCR expression was detected under stimulation with the serotype b of A. actinomycetemcomitans.

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.

Variability in the response of canine and human dendritic cells stimulated with Brucella canis

Brucella canis is a small intracellular Gram-negative bacterium whose primary host is the dog, but it also can cause mild human brucellosis. One of the main causes of an inefficient immune response against other species of Brucella is their interaction with dendritic cells (DCs), which affects antigen presentation and impairs the development of an effective Th1 immune response. This study analysed the cytokine pattern production, by RT-qPCR and ELISA, in human and canine DCs against whole B. canis or its purified LPS. Human and canine DCs produced different patterns of cytokines after stimulation with B. canis. In particular, while human DCs produced a Th1-pattern of cytokines (IL-1β, IL-12, and TNF-α), canine cells produced both Th1 and Th17-related cytokines (IL-6, IL-12, IL-17, and IFN-γ). Thus, differences in susceptibility and pathogenicity between these two hosts could be explained, at least partly, by the distinct cytokine patterns observed in this study, where we propose that human DCs induce an effective Th1 immune response to control the infection, while canine DCs lead to a less effective immune response, with the activation of Th17-related response ineffective to control the B. canis infection.

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.