Cytokines differentially regulate CXCL10 production by interferon-gamma-stimulated or tumor necrosis factor-alpha-stimulated human gingival fibroblasts

Background and Objective: CXC chemokine 10 (CXCL10) activates CXC chemokine receptor 3 (CXCR3) and attracts activated T‐helper 1 cells. In this study we examined the effects of cytokines on CXCL10 production by human gingival fibroblasts. Material and Methods: Human gingival fibroblasts were exposed to pro‐inflammatory cytokines (interleukin‐1β, tumor necrosis factor‐α), a T‐helper 1 cytokine (interferon‐γ), T‐helper 2 cytokines (interleukin‐4, interleukin‐13), T‐helper 17 cytokines (interleukin‐17A, interleukin‐22) and regulatory T‐cell cytokines (interleukin‐10, transforming growth factor‐β1) for 24 h. CXCL10 production by human gingival fibroblasts was examined by enzyme‐linked immunosorbent assay. Results: Human gingival fibroblasts produced CXCL10 protein upon stimulation with interleukin‐1β, tumor necrosis factor‐α and interferon‐γ. Treatment of human gingival fibroblasts with interferon‐γ in combination with tumor necrosis factor‐α or interleukin‐1β resulted in a synergistic production of CXCL10. However, interleukin‐4 and interleukin‐13 inhibited CXCL10 production by interferon‐γ‐stimulated or tumor necrosis factor‐α‐stimulated‐human gingival fibroblasts. On the other hand, interleukin‐17A and interleukin‐22 enhanced CXCL10 production by human gingival fibroblasts treated with interferon‐γ and inhibited CXCL10 production by tumor necrosis factor‐α‐stimulated human gingival fibroblasts. Furthermore, the anti‐inflammatory cytokine, interleukin‐10, inhibited CXCL10 production by both interferon‐γ‐ and tumor necrosis factor‐α‐stimulated human gingival fibroblasts, but transforming growth factor‐β1 enhanced interferon‐γ‐mediated CXCL10 production by human gingival fibroblasts. Conclusion: These results mean that the balance of cytokines in periodontally diseased tissue may be essential for the control of CXCL10 production by human gingival fibroblasts, and the production of CXCL10 might be important for the regulation of T‐helper 1 cell infiltration in periodontally diseased tissue.     

Tumor Necrosis Factor‐α Inhibits Transforming Growth Factor‐β–Stimulated Myofibroblastic Differentiation and Extracellular Matrix Production in Human Gingival Fibroblasts

Background: Fibroblasts play a critical role during wound healing and chronic inflammation through the synthesis and assembly of extracellular matrix (ECM) molecules. These responses may be modulated by soluble cytokines and growth factors present in tissues. In the present study, we evaluate whether transforming growth factor‐β1 (TGF‐β1) and tumor necrosis factor‐α (TNF‐α) modulate myofibroblastic differentiation and the production of ECM components. Methods: Primary cultures of human gingival fibroblasts (HGFs) were stimulated with recombinant TGF‐β1 and TNF‐α. Protein levels of α‐smooth muscle actin (α‐SMA), type I collagen, heat shock protein‐47 (HSP‐47), fibronectin (FN), ED‐A‐FN, and periostin and activation of the Smad pathway were evaluated through Western blot analysis. α‐SMA and actin fibers were identified by immunofluorescence. TGF‐β1, TNF‐α, and α‐SMA were identified by immunohistochemistry in biopsies of inflamed human gingival tissues. TGF‐β1 activity was evaluated using a plasminogen activator inhibitor‐1 (PAI‐1) reporter transfected in HGFs. Results: TGF‐β1 stimulated the differentiation of myofibroblasts as evidenced by strong expression of α‐SMA and ED‐A‐FN. Moreover, TGF‐β1 induced the production of type I collagen, HSP‐47, FN, and periostin. Costimulation with TNF‐α and TGF‐β1 significantly reduced the expression of all the above‐mentioned proteins. TNF‐α also inhibited the activation of the Smad2/3 pathway and the activity of the PAI‐1 reporter. Conclusions: TNF‐α inhibits several cell responses induced by TGF‐β1, including the differentiation of myofibroblasts, the activation of the Smad signaling pathway, and the production of key molecules involved in tissue repair, such as type I collagen, FN, and periostin. The interaction between cytokines may explain the delayed tissue repair observed in chronic inflammation of gingival tissues.     

Relationship Between Chemokines and Dendritic Cells in Human Chronic Periodontitis

Background: The purpose of this study was to evaluate the relationship between chemokines and dendritic cells (DCs) in human chronic periodontitis (CP). Methods: Gingival samples were obtained from 23 individuals with CP, and six samples of normal mucosa (NM) overlapping the third molar were used to control for the chemokine levels. Periodontal examination was conducted. Immunohistochemistry was performed for Factor XIIIa⁺ and cluster of differentiation (CD)1a⁺ immature DCs and CD83⁺ mature DCs. Levels of the CC chemokine ligand (CCL)2, CCL3, CCL5, CCL19, CCL20, and CXC chemokine ligand (CXCL)8 were measured in gingival tissues using enzyme‐linked immunosorbent assay. Inflammatory infiltrate, DCs, chemokines, classification of human CP, and clinical parameters were correlated and compared. Results: The expression of CCL2 and CCL20 was positively correlated with increased densities of CD1a⁺ DCs. CCL3 and CXCL8 were positively related to the clinical attachment level. CCL3, CCL5, CCL19, and CXCL8 levels increased in the gingival samples of patients with CP compared with NM, whereas CCL20 levels increased in advanced CP compared with mild–moderate CP. Conclusions: More CD1a⁺ immature DCs are related to CCL2 and CCL20. CCL3 and CXCL8 chemokines are related to a greater severity of human CP.     

Toll‐Like Receptor 2 Knockdown Modulates Interleukin (IL)‐6 and IL‐8 but not Stromal Derived Factor‐1 (SDF‐1/CXCL12) in Human Periodontal Ligament and Gingival Fibroblasts

Background: Fibroblasts are now seen as active components of the immune response because these cells express Toll‐like receptors (TLRs), recognize pathogen‐associated molecular patterns, and mediate the production of cytokines and chemokines during inflammation. The innate host response to lipopolysaccharide (LPS) from Porphyromonas gingivalis is unusual inasmuch as different studies have reported that it can be an agonist for Toll‐like receptor 2 (TLR2) and an antagonist or agonist for Toll‐like receptor 4 (TLR4). This study investigates and compares whether signaling through TLR2 or TLR4 could affect the secretion of interleukin (IL)‐6, IL‐8, and stromal derived factor‐1 (SDF‐1/CXCL12) in both human gingival fibroblasts (HGF) and human periodontal ligament fibroblasts (HPDLF). Methods: After small interfering RNA‐mediated silencing of TLR2 and TLR4, HGF and HPDLF from the same donors were stimulated with P. gingivalis LPS or with two synthetic ligands of TLR2, Pam2CSK4 and Pam3CSK4, for 6 hours. IL‐6, IL‐8, and CXCL12 mRNA expression and protein secretion were evaluated by quantitative polymerase chain reaction and enzyme‐linked immunosorbent assay, respectively. Results: TLR2 mRNA expression was upregulated in HGF but not in HPDLF by all the stimuli applied. Knockdown of TLR2 decreased IL‐6 and IL‐8 in response to P. gingivalis LPS, or Pam2CSK4 and Pam3CSK4, in a similar manner in both fibroblasts subpopulations. Conversely, CXCL12 remained unchanged by TLR2 or TLR4 silencing. Conclusion: These results suggest that signaling through TLR2 by gingival and periodontal ligament fibroblasts can control the secretion of IL‐6 and IL‐8, which contribute to periodontal pathogenesis, but do not interfere with CXCL12 levels, an important chemokine in the repair process.     

Role of the junctional epithelium in periodontal innate defense and homeostasis

Tsukamoto Y, Usui M, Yamamoto G, Takagi Y, Tachikawa T, Yamamoto M, Nakamura M. Role of the junctional epithelium in periodontal innate defense and homeostasis. J Periodont Res 2012; 47: 750–757. © 2012 John Wiley & Sons A/S Background and Objective: The junctional epithelium provides the front‐line defense against periodontal bacterial infection. The migration of neutrophils into the junctional epithelium might represent a protective reaction against bacterial infections. However, neutrophils penetrate into the junctional epithelium even under sterile conditions. In this study, we analyzed and compared the number of neutrophils and the cytokine expression related to neutrophil migration in the junctional epithelium in conventional and germ‐free mice. Material and Methods: Germ‐free and conventional ICR mice were used at 12 wk of age. Frozen sections were used for the detection of Gr‐1, macrophage inflammatory protein‐2 (MIP‐2/CXCL2) and proliferating cell nuclear antigen‐positive cells in the two groups of mice. Laser capture microdissection and RT‐PCR analysis were used to evaluate the expression of keratinocyte‐derived chemokine (KC/CXCL1), MIP‐2, interleukin‐1β (IL‐1β) and tumor necrosis factor‐α (TNF‐α) mRNAs in the two groups of mice. Results: Morphometric examination indicated an increase in the area of the junctional epithelium upon bacterial infection. Immunohistochemical studies also detected an increased number of neutrophils in the junctional epithelium upon bacterial infection. Higher up‐regulation of KC and MIP‐2 were detected in the junctional epithelium of conventional mice than in germ‐free mice, whereas the expression of Il‐1β and Tnfα mRNAs was not affected. Conclusion: Junctional epithelium cells constitutively expressed several types of chemokines and cytokines and the expression of chemokines was augmented by bacterial infection. Therefore, the constitutive expression of cytokines in junctional epithelium might be related to the morphological and functional homeostasis of the junctional epithelium in addition to the defense against the bacterial infection.     

Effect of scaling and root planing on gingival crevicular fluid cytokine/chemokine levels in smokers with chronic periodontitis: A systematic review

In the present study, the impact of scaling and root planing (SRP) on gingival crevicular fluid (GCF) cytokine/chemokine levels in smokers with chronic periodontitis was assessed. The PICO (population, intervention, comparison, outcome) question was: In smokers with chronic periodontitis (population), what is the effect of SRP (intervention) in comparison to SRP in non‐smokers with chronic periodontitis (comparison) on the GCF cytokine/chemokine level (outcome)? Indexed databases were searched up to September 2017. Of 4330 titles, nine studies reporting the levels of 13 different cytokines/chemokines were included. Eight studies had a moderate risk of bias, while one study had a high risk of bias. Almost all cytokines/chemokines were pro‐inflammatory cytokines. Five cytokines/chemokines studied in four clinical studies were decreased in the smoker‐chronic periodontitis group following SRP. One study observed that the GCF levels of interleukin‐17 increased, while anti‐inflammatory osteoprotegerin was reduced in both the SCP and non‐smoker‐chronic periodontitis groups at follow up. However, the majority of cytokines/chemokines did not change in the SCP groups at follow up. The current weight of evidence is not sufficient to prove that SRP has an impact on GCF cytokine/chemokine profile in smokers with chronic periodontitis. Evaluation of wide panels of pro‐inflammatory cytokines/chemokines related to collagen degradation and alveolar bone destruction in future studies are warranted.     

Hypertension favors the inflammatory process in rats with experimentally induced periodontitis

Bonato CF, do‐Amaral CCF, Belini L, Salzedas LMP, Oliveira SHP. Hypertension favors the inflammatory process in rats with experimentally induced periodontitis. J Periodont Res 2012; 47: 783–792. © 2012 John Wiley & Sons A/S Background and Objective: Cardiovascular diseases are significantly correlated with chronic periodontitis. The aim of this study was to evaluate bone‐loss level, neutrophil migration, CXCL2/CINC‐2α, CXCL5/LIX, CCL20/MIP‐3α and tumor necrosis factor‐α (TNF‐α) production, inducible nitric oxide synthase (iNOS) expression and C‐reactive protein (CRP) release in spontaneously hypertensive rats (SHRs) and normotensive (WTK) rats after experimental induction of periodontal disease. Material and Methods: Periodontitis was induced by placement of silk yarn ligatures around the first molar counterparts. The levels of CRP, CCL20/MIP‐3α and CXCL5/LIX were evaluated in the peripheral blood, and bone‐loss level, neutrophil recruitment, the production of myeloperoxidase, CXCL2, CXCL5, CCL20 and TNF‐α, and the expression of iNOS were evaluated in the gingival tissue. Histological sections were taken to evaluate and measure bone resorption and neutrophil recruitment in the furcation region. Results: Rats with periodontitis had alveolar bone resorption. SHRs with periodontitis showed marked bone loss and increased neutrophil infiltration in comparison with WTK rats. SHRs with periodontitis showed increased levels of TNF‐α and CXCL2, and a slight tendency for increased levels of CXCL5, in the gingival tissue but no increase in the level of CCL20. In SHRs, even without periodontitis, the levels of TNF‐α, CXCL2, CXCL5 and CCL20 showed a slight tendency to increase. In the WTK rats, TNF‐α, CXCL2 and CXCL5 levels were increased with periodontitis, but the level of CCL20 was not. iNOS was expressed in the gingival tissue of WTK rats and SHRs with periodontitis; however, SHRs appeared to express a higher level of iNOS than did WKT rats. The CRP level was elevated in both types of rats with periodontitis; however, the CRP level was higher in SHRs with periodontitis than in WTK rats with periodontitis. Conclusion: In SHRs, the hypertensive condition per se seems to favor the inflammatory processes that become potentiated with periodontitis, when compared with WKT rats.     

Expression of cytokines in the gingival crevicular fluid and serum from patients with inflammatory bowel disease and untreated chronic periodontitis

Figueredo CM, Brito F, Barros FC, Menegat JSB, Pedreira RR, Fischer RG, Gustafsson A. Expression of cytokines in the gingival crevicular fluid and serum from patients with inflammatory bowel disease and untreated chronic periodontitis. J Periodont Res 2011; 46: 141–146.© 2010 John Wiley & Sons A/S Background and Objective: Previous studies have reported an increased prevalence/severity of chronic periodontitis in patients with inflammatory bowel disease. However, the pathogenesis of periodontal lesions in such patients has not been characterized. The aim of this pilot study was to characterize the pattern of expression of cytokines in the gingival crevicular fluid and serum from patients with untreated chronic periodontitis and Crohn’s disease, ulcerative colitis and systemically healthy controls. Material and Methods: Fifteen patients with Crohn’s disease, 15 patients with ulcerative colitis and 15 controls participated in the study. All subjects had been diagnosed with untreated chronic periodontitis. The clinical parameters evaluated were clinical attachment loss, bleeding on probing and percentage of plaque. The gingival crevicular fluid was sampled from four shallow and four deep periodontal sites of each patient. The concentrations of the cytokines interleukin (IL)‐1β, IL‐4, IL‐6, IL‐10, IL‐12p40, IL‐12p70, interferon‐γ and tumor necrosis factor‐α were measured using a commercially available Lincoplex kit and the concentration of IL‐18 was measured using an ELISA. Results: Multiple comparisons analysis showed that clinical attachment loss, bleeding on probing, percentage of plaque and volume of gingival crevicular fluid were similar across the groups. The concentration of IL‐4 in the gingival crevicular fluid differed significantly between groups in shallow sites (p = 0.046), with higher values found for the controls. In serum, the concentration of IL‐18 was also significantly different between groups, with lower values found for controls (p = 0.018). Conclusion: This study showed a higher concentration of IL‐18 in serum, but not in the gingival crevicular fluid, from periodontitis patients with Crohn’s disease or ulcerative colitis compared with controls. The expression of cytokines was similar in the gingival crevicular fluid from patients with untreated chronic periodontitis who also had Crohn’s disease or ulcerative colitis and in systemically healthy controls with untreated chronic periodontitis.     

Expression levels of adiponectin receptors and periodontitis

Yamaguchi N, Hamachi T, Kamio N, Akifusa S, Masuda K, Nakamura Y, Nonaka K, Maeda K, Hanazawa S, Yamashita Y. Expression levels of adiponectin receptors and periodontitis. J Periodont Res 2010; 45: 296–300. © 2010 John Wiley & Sons A/S Background and Objective: We recently showed that adiponectin, an adipocyte‐derived cytokine, may function as a negative regulator of the Toll‐like receptor signaling pathway and of osteoclast formation in periodontal disease. In this study, we investigated whether the expression levels of adiponectin receptors (AdipoR1 and AdipoR2) are related to the presence of periodontitis. Material and Methods: We initially examined, using RT‐PCR, the expression of the AdipoR1 and AdipoR2 genes at the mRNA level in several oral tissues of C57BL mice. Next, we investigated (using real‐time PCR assays) whether inflammatory cytokines, such as tumor necrosis factor‐α, could affect the expression levels of these genes in human gingival fibroblasts. Lastly, we compared the expression levels of these receptor proteins in gingival tissues between two healthy subjects and five patients with severe periodontal disease using western blotting analysis. Results: The AdipoR1 and AdipoR2 receptors were ubiquitously expressed in the oral tissues of mice. We observed that treatment with tumor necrosis factor‐α could significantly reduce the expression levels of both AdipoR1 and AdipoR2 genes in human gingival fibroblasts. Moreover, we found that the expression of both receptors was lower in periodontal tissues from patients with severe periodontitis than in patients with healthy periodontal tissues. Conclusion: These observations suggest that adiponectin may not function efficiently in sites of periodontal disease because of a decrease in the number of its receptors, and this probable dysfunction may play a role in worsening periodontitis in patients.