Kaurenoic Acid from Sphagneticola trilobata Inhibits Inflammatory Pain: Effect on Cytokine Production and Activation of the NO-Cyclic GMP-Protein Kinase G-ATP-Sensitive Potassium Channel Signaling Pathway

Kaurenoic acid [ent-kaur-16-en-19-oic acid (1)] is a diterpene present in several plants including Sphagneticola trilobata. The only documented evidence for its antinociceptive effect is that it inhibits the writhing response induced by acetic acid in mice. Therefore, the analgesic effect of 1 in different models of pain and its mechanisms in mice were investigated further. Intraperitoneal and oral treatment with 1 dose-dependently inhibited inflammatory nociception induced by acetic acid. Oral treatment with 1 also inhibited overt nociception-like behavior induced by phenyl-p-benzoquinone, complete Freund's adjuvant (CFA), and both phases of the formalin test. Compound 1 also inhibited acute carrageenin- and PGE(2)-induced and chronic CFA-induced inflammatory mechanical hyperalgesia. Mechanistically, 1 inhibited the production of the hyperalgesic cytokines TNF-alpha and IL-1 beta. Furthermore, the analgesic effect of 1 was inhibited by L-NAME, ODQ, KT5823, and glybenclamide treatment, demonstrating that such activity also depends on activation of the NO-cyclic GMP-protein kinase G-ATP-sensitive potassium channel signaling pathway, respectively. These results demonstrate that 1 exhibits an analgesic effect in a consistent manner and that its mechanisms involve the inhibition of cytokine production and activation of the NO-cyclic GMP-protein lcinase G-ATP-sensitive potassium channel signaling pathway.

Local inflammatory events induced by Bothrops atrox snake venom and the release of distinct classes of inflammatory mediators

Bothrops atrox is responsible for most accidents involving snakes in the Brazilian Amazon and its venom induces serious systemic and local effects. The local effects are not neutralized effectively by commercial antivenoms, resulting in serious sequelae in individuals bitten by this species. This study investigates the local inflammatory events induced in mice by B. atrox venom (Bay), such as vascular permeability, leukocyte influx and the release of important inflammatory mediators such as cytokines, eicosanoids and the chemokine CCL-2, at the injection site. The effect of Bay on cyclooxygenase (COX-1 and COX-2) expression was also investigated. The results showed that intraperitoneal (i.p.) injection of BaV promoted a rapid and significant increase in vascular permeability, which reached a peak 1 h after venom administration. Furthermore, BaV caused leukocyte infiltration into the peritoneal cavity between 1 and 8 h after i.p. injection, with mononuclear leukocytes (MNs) predominating in the first 4 h, and polymorphonuclear leukocytes (PMNs) in the last 4 h. Increased protein expression of COX-2, but not of COX-1, was detected in leukocytes recruited in the first and fourth hours after injection of BaV. The venom caused the release of eicosanoids PGD(2), PGE(2), TXA(2) and LTB4, cytokines TNF-alpha, IL-6, IL-10 and IL-12p70, but not IFN-gamma, and chemokine CCL-2 at different times. The results show that Bay is able to induce an early increase in vascular permeability and a leukocyte influx to the injection site consisting mainly of MNs initially and PMNs during the later stages. These phenomena are associated with the production of cytokines, the chemokine CCL-2 and eicosanoids derived from COX-1 and COX-2. (c) 2012 Elsevier Ltd. All rights reserved.

Fas-activated serine/threonine phosphoprotein promotes immune-mediated pulmonary inflammation

We generated Fas-activated serine threonine phosphoprotein (FAST)-deficient mice (FAST(-/-)) to study the in vivo role of FAST in immune system function. In a model of house dust mite-induced allergic pulmonary inflammation, wild type mice develop a mixed cellular infiltrate composed of eosinophils, lymphocytes, and neutrophils. FAST(-/-) mice develop airway inflammation that is distinguished by the near absence of neutrophils. Similarly, LPS-induced alveolar neutrophil recruitment is markedly reduced in FAST(-/-) mice compared with wild type controls. This is accompanied by reduced concentrations of cytokines (TNF-alpha and IL-6 and -23) and chemoattractants (MIP-2 and keratinocyte chemoattractant) in bronchoalveolar lavage fluids. Because FAST(-/-) neutrophils exhibit normal chemotaxis and survival, impaired neutrophil recruitment is likely to be due to reduced production of chemoattractants within the pulmonary parenchyma. Studies using bone marrow chimeras implicate lung resident hematopoietic cells (e.g., pulmonary dendritic cells and/or alveolar macrophages) in this process. In conclusion, our results introduce FAST as a proinflammatory factor that modulates the function of lung resident hematopoietic cells to promote neutrophil recruitment and pulmonary inflammation.

Dual in vitro perfusion of an isolated cotyledon as a model to study the implication of changes in the third trimester placenta on preeclampsia

In the current study perfusions of an isolated cotyledon of term placenta using standard medium were compared to medium containing xanthine plus xanthine oxidase (X+XO), which generates reactive oxygen species (ROS). A time-dependant increase in the levels of different cytokines (TNF-alpha, IL-1ss, IL-6, IL-8 and IL-10) was observed between 1 and 7h with more than 90% of the total recovered from the maternal compartment with no significant difference between the 2 groups. For 8-iso-PGF2alpha 90% of the total was found in the fetal compartment and a significantly higher total release was seen in the X+XO group. Microparticles (MPs) isolated from the maternal circuit were identified by flow cytometry as trophoblastic sheddings, whereas MPs from the fetal circuit were predominantly derived from endothelial cells. More than 90% of the total of MPs was found in the maternal circuit. The absolute amount of the total as well as the maternal fraction were significantly higher in the X+XO group. Immunohistochemistry (IHC) of the perfused tissue revealed staining for IL-1beta of villous stroma cells, which became clearly more pronounced in experiments with X+XO. Western blot of tissue homogenate revealed 2 isoforms of IL-1beta at 17 and 31kD. In X+XO experiments there was a tendency for increased expression of antioxidant enzymes in the tissue. Western blot of MPs from the maternal circuit showed increased expression of antioxidant enzymes in the X+XO group and for IL-1beta only the 17kD band was detected. In vitro reperfusion of human placental tissue results in mild tissue injury suggestive of oxidative stress. In view of the increased generation of ROS in perfused tissue with further increase under the influence of X+XO, the overall manifestation of oxidative stress remained rather mild. Preservation of antioxidant capacity of human placental tissue could be a sign of integrity of structure and function being maintained in vitro by dual perfusion of an isolated cotyledon. The observed changes resemble findings seen in placentae from preeclampsia.

Platelet chemokines and their receptors: what is their relevance to platelet storage and transfusion practice?

The role of platelets as inflammatory cells is demonstrated by the fact that they can release many growth factors and inflammatory mediators, including chemokines, when they are activated. The best known platelet chemokine family members are platelet factor 4 (PF4) and beta-thromboglobulin (beta-TG), which are synthesized in megakaryocytes, stored as preformed proteins in alpha-granules and released from activated platelets. However, platelets also contain many other chemokines such as interleukin-8 (IL-8), growth-regulating oncogene-alpha(GRO-alpha), epithelial neutrophil-activating protein 78 (ENA-78), regulated on activation normal T expressed and secreted (RANTES), macrophage inflammatory protein-1alpha (MIP-1alpha), and monocyte chemotactic protein-3 (MCP-3). They also express chemokine receptors such as CCR4, CXCR4, CCR1 and CCR3. Platelet activation is a feature of many inflammatory diseases such as heparin-induced thrombocytopenia, acquired immunodeficiency syndrome, and congestive heart failure. Substantial amounts of PF4, beta-TG and RANTES are released from platelets on activation, which may occur during storage. Although very few data are available on the in vivo effects of transfused chemokines, it has been suggested that the high incidence of adverse reactions often observed after platelet transfusions may be attributed to the chemokines present in the plasma of stored platelet concentrates.

Functional expression of CCR1, CCR3, CCR4, and CXCR4 chemokine receptors on human platelets

Platelets are known to contain platelet factor 4 and beta-thromboglobulin, alpha-chemokines containing the CXC motif, but recent studies extended the range to the beta-family characterized by the CC motif, including RANTES and Gro-alpha. There is also evidence for expression of chemokine receptors CCR4 and CXCR4 in platelets. This study shows that platelets have functional CCR1, CCR3, CCR4, and CXCR4 chemokine receptors. Polymerase chain reaction detected chemokine receptor messenger RNA in platelet RNA. CCR1, CCR3, and especially CCR4 gave strong signals; CXCR1 and CXCR4 were weakly positive. Flow cytometry with specific antibodies showed the presence of a clear signal for CXCR4 and weak signals for CCR1 and CCR3, whereas CXCR1, CXCR2, CXCR3, and CCR5 were all negative. Immunoprecipitation and Western blotting with polyclonal antibodies to cytoplasmic peptides clearly showed the presence of CCR1 and CCR4 in platelets in amounts comparable to monocytes and CCR4 transfected cells, respectively. Chemokines specific for these receptors, including monocyte chemotactic protein 1, macrophage inflammatory peptide 1alpha, eotaxin, RANTES, TARC, macrophage-derived chemokine, and stromal cell-derived factor 1, activate platelets to give Ca(++) signals, aggregation, and release of granule contents. Platelet aggregation was dependent on release of adenosine diphosphate (ADP) and its interaction with platelet ADP receptors. Part, but not all, of the Ca(++) signal was due to ADP release feeding back to its receptors. Platelet activation also involved heparan or chondroitin sulfate associated with the platelet surface and was inhibited by cleavage of these glycosaminoglycans or by heparin or low molecular weight heparin. These platelet receptors may be involved in inflammatory or allergic responses or in platelet activation in human immunodeficiency virus infection.

Different functions for the interleukin 8 receptors (IL-8R) of human neutrophil leukocytes: NADPH oxidase and phospholipase D are activated through IL-8R1 but not IL-8R2.

Two monoclonal antibodies, anti-IL8R1 and anti-IL8R2, raised against both interleukin 8 receptors (IL-8R) of human neutrophils, IL-8R1 and IL-8R2, were used to study individual receptor functions after stimulation with IL-8, GRO alpha, or NAP-2. Efficacy and selectivity of the antibodies were tested in Jurkat cells transfected with cDNA coding for one or the other receptor. The binding of 125 I labeled IL-8 and IL-8-induced changes of the cytosolic free Ca2+ concentration were inhibited by anti-IL8RI in cells expressing IL-8R1 and by anti-IL8R2 in cells expressing IL-8R2. In human neutrophils, release of elastase was observed after stimulation with IL-8 or GRO alpha. The response to IL-8 was inhibited slightly by anti-IL8R1 and more substantially when both monoclonal antibodies were present, while the response to GRO alpha was inhibited by anti-IL8R2 but was not affected by anti-IL8R1. These results indicate that both IL-8 receptors can signal independently for granule enzyme release. Superoxide production, a measure of the respiratory burst, was obtained with increasing concentrations of IL-8 with maximum effects at 25 to 50 nM, but no response was observed upon challenge with GRO alpha or NAP-2 up to 1000 nM. The superoxide production induced by IL-8 was inhibited by anti-IL8R1, but was not affected by anti-IL8R2. Stimulation of neutrophils with IL-8, in contrast to GRO alpha or NAP-2, also elicited phospholipase D activity. The effect of IL-8 was again inhibited by anti-IL-8R1 but not by anti-IL8R2, indicating that this response, like the respiratory burst, was mediated by IL-8R1. Taken together, our results show that IL-8R1 and IL-8R2 are functionally different. Responses, such as cytosolic free Ca2+ changes and the release of granule enzymes, are mediated through both receptors, whereas the respiratory burst and the activation of phospholipase D depend exclusively on stimulation through IL-8R1.

Effects of 15d-PGJ(2)-loaded poly(D,L-lactide-co-glycolide) nanocapsules on inflammation

BACKGROUND and PURPOSEThe PPAR-gamma agonist 15d-PGJ(2) is a potent anti-inflammatory agent but only at high doses. To improve the efficiency of 15d-PGJ(2), we used poly(D,L-lactide-co-glycolide) nanocapsules to encapsulate it, and function as a drug carrier system. The effects of these loaded nanocapsules (15d-PGJ(2)-NC) on inflammation induced by different stimuli were compared with those of free 15d-PGJ(2).EXPERIMENTAL APPROACHMice were pretreated (s.c.) with either 15d-PGJ(2)-NC or unloaded 15d-PGJ(2) (3, 10 or 30 mu g center dot kg-1), before induction of an inflammatory response by i.p. injection of either endotoxin (LPS), carrageenan (Cg) or mBSA (immune response).KEY RESULTSThe 15d-PGJ(2)-NC complex did not display changes in physico-chemical parameters or drug association efficiency over time, and was stable for up to 60 days of storage. Neutrophil migration induced by i.p. administration of LPS, Cg or mBSA was inhibited by 15d-PGJ(2)-NC, but not by unloaded 15d-PGJ(2). In the Cg model, 15d-PGJ(2)-NC markedly inhibited serum levels of the pro-inflammatory cytokines TNF-alpha, IL-1 beta and IL-12p70. Importantly, 15d-PGJ(2)-NC released high amounts of 15d-PGJ(2), reaching a peak between 2 and 8 h after administration. 15d-PGJ(2) was detected in mouse serum after 24 h, indicating sustained release from the carrier. When the same concentration of unloaded 15d-PGJ(2) was administered, only small amounts of 15d-PGJ(2) were found in the serum after a few hours.CONCLUSIONS and IMPLICATIONSThe present findings clearly indicate the potential of the novel anti-inflammatory 15d-PGJ(2) carrier formulation, administered systemically. The formulation enables the use of a much smaller drug dose, and is significantly more effective compared with unloaded 15d-PGJ(2).

Roles of the Immune Cytokine Environment on Clonal Growth of Murine and Human Tet2 Mutant Bone Marrow Progenitors: Implications for Myelodysplastic Syndromes

TET2 is a tumor suppressor gene that has been implicated in the epigenetic regulation of gene expression. Inactivating TET2 mutations are common in MDS. These mutations may contribute to early clonal dominance and myeloid transformation, although the exact mechanisms remain to be elucidated. Common to the environment of MDS are elevations in cytokines, such as TNFα and IFN-γ. It was hypothesized that inflammatory cytokines TNF-α and IFN-γ may promote clonal expansion of TET2 mutant progenitors. Adult (10-14 weeks-old) Tet2 wild type (+/+) and Tet2 mutant (-/-) C57BL/6 mice strains were chosen as a model system. Lineage negative cells (Lin-), enriched for hematopoietic stem and progenitor cells, were isolated from Tet2 +/+ and -/- bone marrow and cultured in the absence or presence of varying concentrations of TNFα or IFN-γ in methylcellulose colony formation assays and long term cell culture assays, over a period of 12 and 30 days respectively, and their colony growth, cell count, immunophenotype and resistance to apoptosis were examined. Where indicated, serial re-plating was performed. Expression of apoptotic regulators was assessed by qRT-PCR. In the triplicate experiments, starting with equal densities of Tet2 +/+ and -/- Lin- cells, Tet2 -/- Lin- cells displayed increased resistance to cytokine-induced growth suppression and superior colony forming ability over +/+ in the serial re-plating assays under stress of increasing TNFα or IFN γ. Tet2 -/- progenitors also displayed a lower apoptotic index compared to +/+ under stress of increasing TNFα, suggesting increased resistance to TNFα induced apoptosis. Transcriptional data showed low expression of Tnfr1, Fas and caspase 8, as well as a high expression of Bcl-2 and Iap1 in Tet2 -/- compared to +/+ under stress of TNFα. Tet2-/- also showed increased basal expression of endogenous TNFα mRNA compared to +/+. In the human colony growth assay, the clonal growth of TET2 mutant CFU-GM progenitors was enhanced at low TNFα concentrations. Conclusion: Mutations that promote resistance to environmental stem cell stressors are a known mechanism of clonal selection in aplastic anaemia and JAK2-mutant MPN and our findings suggest that this mechanism may be critical to clonal selection and dominance in MDS.

Expression of proinflammatory cytokines tumor necrosis factor-alpha and interleukin-1beta in the brain during experimental group B streptococcal meningitis

We performed mRNA in situ hybridization for TNF-alpha and IL-1beta from infant rats with group B streptococcal meningitis. Induction of both cytokines was seen in the ependyma and the meninges at 4 h. Both cytokines were expressed in the brain parenchyma at 12 h. Induction of IL-1beta mRNA was seen in vessels within the brain cortex. Neutrophilic infiltrate at all time points examined was minimal and could not account for the observed cytokine expression.

Cytokine synthesis by intestinal intraepithelial lymphocytes. Both gamma/delta T cell receptor-positive and alpha/beta T cell receptor-positive T cells in the G1 phase of cell cycle produce IFN-gamma and IL-5

Murine intestinal intraepithelial lymphocytes (IEL) have been shown to contain subsets of alpha/beta TCR+ and gamma/delta TCR+ T cells that spontaneously produce cytokines such as IFN-gamma and IL-5. We have now determined the nature and cell cycle stage of these cytokine-producing T lymphocytes in EIL by using IFN-gamma- and IL-5-specific ELISPOT assay, cytokine-specific mRNA-cDNA dot-blot hybridization and polymerase chain reaction, and flow cytometry (FACS) for DNA analysis. When CD3+ T cells from IEL of normal C3H/HeN mice were separated into low and high density fractions by discontinuous Percoll gradients, IFN-gamma and IL-5 spot-forming cells were only found in the former population. Analysis of mRNA for these cytokines by both IFN-gamma- and IL-5-specific dot-blot hybridization and polymerase chain reaction revealed that higher levels of message for IFN-gamma and IL-5 were also seen in the low density fraction. However, cell cycle analysis of these two fractions by FACS using propidium iodide showed a similar pattern of cell cycle stages in both low and high density populations (G0 + G1 approximately 96 to 98% and S/G2 + M approximately 2 to 4%). Finally, mRNA from gamma/delta TCR+ and alpha/beta TCR+ T cells in both low and high density fractions of IEL were analyzed for IFN-gamma and IL-5 message by polymerase chain reaction. After 35 cycles of amplification, both gamma/delta TCR+ and alpha/beta TCR+ T cells in the low density fraction expressed higher levels of message for these two cytokines when compared with the high density population. These results have now shown that both gamma/delta and alpha/beta TCR+ IEL can be separated into low and high density subsets and both fractions possess a similar stage of cell cycle. However, only the low density cells (in G1 phase) of both gamma/delta and alpha/beta TCR types possess increased cytokine-specific mRNA and produce the cytokines IFN-gamma and IL-5. Our results suggest that alpha/beta TCR+ and gamma/delta TCR+ IEL can produce cytokines without cell proliferation.

Body temperature and its effect on leukocyte mobilization, cytokines and markers of neutrophil activation during and after exercise

We investigated the influence of rectal temperature on the immune system during and after exercise. Ten well-trained male cyclists completed exercise trials (90 min cycling at 60% VO(2max) + 16.1 - km time trial) on three separate occasions: once in 18 degrees C and twice in 32 degrees C. Twenty minutes after the trials in 32 degrees C, the cyclists sat for approximately 20 min in cold water (14 degrees C) on one occasion, whereas on another occasion they sat at room temperature. Rectal temperature increased significantly during cycling in both conditions, and was significantly higher after cycling in 32 degrees C than in 18 degrees C (P < 0.05). Leukocyte counts increased significantly during cycling but did not differ between the conditions. The concentrations of serum interleukin (IL)-6, IL-8 and IL-10, plasma catecholamines, granulocyte-colony stimulating factor, myeloperoxidase and calprotectin increased significantly following cycling in both conditions. The concentrations of serum IL-8 (25%), IL-10 (120%), IL-1 receptor antagonist (70%), tumour necrosis factor-alpha (17%), plasma myeloperoxidase (26%) and norepinephrine (130%) were significantly higher after cycling in 32 degrees C than in 18 degrees C. During recovery from exercise in 32 degrees C, rectal temperature was significantly lower in response to sitting in cold water than at room temperature. However, immune changes during 90 min of recovery did not differ significantly between sitting in cold water and at room temperature. The greater rise in rectal temperature during exercise in 32 degrees C increased the concentrations of serum IL-8, IL-10, IL-1ra, TNF-alpha and plasma myeloperoxidase, whereas the greater decline in rectal temperature during cold water immersion after exercise did not affect immune responses.

Preliminary characterisation of tumor necrosis factor alpha and interleukin-10 responses to chlamydia pecorum infection in the koala (Phascolarctos cinereus)

Debilitating infectious diseases caused by Chlamydia are major contributors to the decline of Australia's iconic native marsupial species, the koala (Phascolarctos cinereus). An understanding of koala chlamydial disease pathogenesis and the development of effective strategies to control infections continue to be hindered by an almost complete lack of species-specific immunological reagents. The cell-mediated immune response has been shown to play an influential role in the response to chlamydial infection in other hosts. The objective of this study, hence, was to provide preliminary data on the role of two key cytokines, pro-inflammatory tumour necrosis factor alpha (TNFα) and anti-inflammatory interleukin 10 (IL10), in the koala Chlamydia pecorum response. Utilising sequence homology between the cytokine sequences obtained from several recently sequenced marsupial genomes, this report describes the first mRNA sequences of any koala cytokine and the development of koala specific TNFα and IL10 real-time PCR assays to measure the expression of these genes from koala samples. In preliminary studies comparing wild koalas with overt chlamydial disease, previous evidence of C. pecorum infection or no signs of C. pecorum infection, we revealed strong but variable expression of TNFα and IL10 in wild koalas with current signs of chlamydiosis. The description of these assays and the preliminary data on the cell-mediated immune response of koalas to chlamydial infection paves the way for future studies characterising the koala immune response to a range of its pathogens while providing reagents to assist with measuring the efficacy of ongoing attempts to develop a koala chlamydial vaccine.

Characterization of cytokines, matrix metalloproteinases and toll-like receptors in human periodontal tissue destruction

Periodontal Disease affects the supporting structures of the teeth and is initiated by a microbial biofilm called dental plaque. Severity ranges from superficial inflammation of the gingiva (gingivitis) to extensive destruction of connective tissue and bone leading to tooth loss (periodontitis). In periodontitis the destruction of tissue is caused by a cascade of microbial and host factors together with proteolytic enzymes. Matrix metalloproteinases (MMPs) are known to be central mediators of the pathologic destruction in periodontitis. Initially plaque bacteria provide pathogen-associated molecular patterns (PAMPs) which are sensed by Toll-like receptors (TLRs), and initiate intracellular signaling cascades leading to host inflammation. Our aim was to characterize TNF-α (tumor necrosis factor-alpha) and its type I and II receptors in periodontal tissues, as well as, the effects of TNF-α, IL-1β (interleukin-1beta) and IL-17 on the production and/or activation of MMP-3, MMP-8 and MMP-9. Furthermore we mapped the TLRs in periodontal tissues and assessed how some of the PAMPs binding to the key TLRs found in periodontal tissues affect production of TNF-α and IL-1β by gingival epithelial cells with or without combination of IL-17. TNF-α and its receptors were detected in pericoronitis. Furthermore, increased expression of interleukin-1β and vascular cell adhesion molecule-1 was found as a biological indicator of TNF-α ligand-receptor interaction. MMP-3, -8, and 9 were investigated in periodontitis affected human gingival crevicular fluid and gingival fibroblasts produced pro-MMP-3. Following that, the effect of IL-17 was studied on MMP and pro-inflammatory cytokine production. IL-17 was increased in periodontitis and up-regulated IL-1β, TNF-α, MMP-1 and MMP-3. We continued by demonstrating TLRs in gingival tissues, in which significant differences between patients with periodontitis and healthy controls were found. Finally, enzyme-linked immunosorbent assays were performed to show that the gingival cells response to inflammatory responses in a TLR-dependent manner. Briefly, this thesis demonstrates that TLRs are present in periodontal tissues and present differences in periodontitis compared to healthy controls. The cells of gingival tissues respond to inflammatory process in a TLR-dependent manner by producing pro-inflammatory cytokines. During the destruction of periodontal tissues, the release (IL-1β and TNF-α) and co-operation with other pro-inflammatory cytokines (IL-17), which in turn increase the inflammation and thus be more harmful to the host with the increased presence of MMPs (MMP-1, MMP-3, MMP-8, MMP-9) in diseased over healthy sites.

Proinflammatory cytokines regulate cementogenic differentiation of periodontal ligament cells by Wnt/Ca2+ signaling pathway

Periodontal inflammation can inhibit cell differentiation of periodontal ligament cells (PDLCs), resulting in decreased bone/cementum regeneration ability. The Wnt signaling pathway, including canonical Wnt/β-catenin signaling and noncanonical Wnt/Ca2+ signaling, plays essential roles in cell proliferation and differentiation during tooth development. However, little is still known whether noncanonical Wnt/Ca2+ signaling cascade could regulate cementogenic/osteogenic differentiation capability of PDLCs within an inflammatory environment. Therefore, in this study, human PDLCs (hPDLCs) and their cementogenic differentiation potential were investigated in the presence of cytokines. The data demonstrated that both cytokines interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-α) inhibited cell proliferation, relative alkaline phosphatase activity, bone/cementum-related gene/protein expression, and canonical Wnt pathway-related gene/protein expression in hPDLCs. Interestingly, both cytokines upregulated the noncanonical Wnt/Ca2+ signaling-related gene and protein expression in hPDLCs. When the Wnt/Ca2+ pathway was blocked by Ca2+/calmodulin-dependent protein kinase II inhibitor KN93, even in the presence of IL-6 and TNF-α, cementogenesis could be stimulated in hPDLCs. Our data indicate that the Wnt/Ca2+ pathway plays an inhibitory role on PDLC cementogenic differentiation in inflammatory microenvironments. Therefore, targeting the Wnt/Ca2+ pathway may provide a novel therapeutic approach to improve periodontal regeneration for periodontal diseases.