Pro- and Anti-Inflammatory Cytokines in the Context of NK Cell–Trophoblast Interactions

During pregnancy, uterine NK cells interact with trophoblast cells. In addition to contact interactions, uterine NK cells are influenced by cytokines, which are secreted by the cells of the decidua microenvironment. Cytokines can affect the phenotypic characteristics of NK cells and change their functional activity. An imbalance of pro- and anti-inflammatory signals can lead to the development of reproductive pathology. The aim of this study was to assess the effects of cytokines on NK cells in the presence of trophoblast cells in an in vitro model. We used TNFα, IFNγ, TGFβ and IL-10; the NK-92 cell line; and peripheral blood NK cells (pNKs) from healthy, non-pregnant women. For trophoblast cells, the JEG-3 cell line was used. In the monoculture of NK-92 cells, TNFα caused a decrease in CD56 expression. In the coculture of NK cells with JEG-3 cells, TNFα increased the expression of NKG2C and NKG2A by NK-92 cells. Under the influence of TGFβ, the expression of CD56 increased and the expression of NKp30 decreased in the monoculture. After the preliminary cultivation of NK-92 cells in the presence of TGFβ, their cytotoxicity increased. In the case of adding TGFβ to the PBMC culture, as well as coculturing PBMCs and JEG-3 cells, the expression of CD56 and NKp44 by pNK cells was reduced. The differences in the effects of TGFβ in the model using NK-92 cells and pNK cells may be associated with the possible influence of monocytes or other lymphoid cells from the mononuclear fraction.     

Different Contribution of Monocyte- and Platelet-Derived Microvesicles to Endothelial Behavior

Several contributions of circulating microvesicles (MVs) to the endothelial dysfunction have been reported in the past; a head-to-head comparison of platelet- and monocyte–derived MVs has however never been performed. To this aim, we assessed the involvement of these MVs in vessel damage related processes, i.e., oxidative stress, inflammation, and leukocyte-endothelial adhesion. Platelets and monocytes isolated from healthy subjects (HS, n = 15) were stimulated with TRAP-6 and LPS to release MVs that were added to human vascular endothelial cell (hECV) culture to evaluate superoxide anion production, inflammatory markers (IL-6, TNFα, NF-κB mRNA expression), and hECV adhesiveness. The effects of the MVs-induced from HS were compared to those induced by MVs spontaneously released from cells of patients with ST-segment elevation myocardial infarction (STEMI, n = 7). MVs released by HS-activated cells triggered a threefold increase in oxidative burst in a concentration-dependent manner. Only MVs released from monocytes doubled IL-6, TNFα, and NF-κB mRNA expression and monocyte-endothelial adhesion. Interestingly, the effects of the MVs isolated from STEMI-monocytes were not superimposable to previous ones except for adhesion to hECV. Conversely, MVs released from STEMI-platelets sustained both redox state and inflammatory phenotype. These data provide evidence that MVs released from activated and/or pathologic platelets and monocytes differently affect endothelial behavior, highlighting platelet-MVs as causative factors of impaired endothelial function in the acute phase of STEMI.     

A Chimeric IL-15/IL-15Rα Molecule Expressed on NFκB-Activated Dendritic Cells Supports Their Capability to Activate Natural Killer Cells

Natural killer (NK) cells, members of the innate immune system, play an important role in the rejection of HLA class I negative tumor cells. Hence, a therapeutic vaccine, which can activate NK cells in addition to cells of the adaptive immune system might induce a more comprehensive cellular response, which could lead to increased tumor elimination. Dendritic cells (DCs) are capable of activating and expanding NK cells, especially when the NFκB pathway is activated in the DCs thereby leading to the secretion of the cytokine IL-12. Another prominent NK cell activator is IL-15, which can be bound by the IL-15 receptor alpha-chain (IL-15Rα) to be transpresented to the NK cells. However, monocyte-derived DCs do neither secrete IL-15, nor express the IL-15Rα. Hence, we designed a chimeric protein consisting of IL-15 and the IL-15Rα. Upon mRNA electroporation, the fusion protein was detectable on the surface of the DCs, and increased the potential of NFκB-activated, IL-12-producing DC to activate NK cells in an autologous cell culture system with ex vivo-generated cells from healthy donors. These data show that a chimeric IL-15/IL-15Rα molecule can be expressed by monocyte-derived DCs, is trafficked to the cell surface, and is functional regarding the activation of NK cells. These data represent an initial proof-of-concept for an additional possibility of further improving cellular DC-based immunotherapies of cancer.     

What Do We Have to Know about PD-L1 Expression in Prostate Cancer? A Systematic Literature Review. Part 3: PD-L1, Intracellular Signaling Pathways and Tumor Microenvironment

The tumor microenvironment (TME) includes immune (T, B, NK, dendritic), stromal, mesenchymal, endothelial, adipocytic cells, extracellular matrix, and cytokines/chemokines/soluble factors regulating various intracellular signaling pathways (ISP) in tumor cells. TME influences the survival/progression of prostate cancer (PC), enabling tumor cell immune-evasion also through the activation of the PD-1/PD-L1 axis. We have performed a systematic literature review according to the PRISMA guidelines, to investigate how the PD-1/PD-L1 pathway is influenced by TME and ISPs. Tumor immune-escape mechanisms include suppression/exhaustion of tumor infiltrating cytotoxic T lymphocytes, inhibition of tumor suppressive NK cells, increase in immune-suppressive immune cells (regulatory T, M2 macrophagic, myeloid-derived suppressor, dendritic, stromal, and adipocytic cells). IFN-γ (the most investigated factor), TGF-β, TNF-α, IL-6, IL-17, IL-15, IL-27, complement factor C5a, and other soluble molecules secreted by TME components (and sometimes increased in patients’ serum), as well as and hypoxia, influenced the regulation of PD-L1. Experimental studies using human and mouse PC cell lines (derived from either androgen-sensitive or androgen-resistant tumors) revealed that the intracellular ERK/MEK, Akt-mTOR, NF-kB, WNT and JAK/STAT pathways were involved in PD-L1 upregulation in PC. Blocking the PD-1/PD-L1 signaling by using immunotherapy drugs can prevent tumor immune-escape, increasing the anti-tumor activity of immune cells.     

TAS-116, a Well-Tolerated Hsp90 Inhibitor,Prevents the Activation of the NLRP3 Inflammasome in Human Retinal Pigment Epithelial Cells

Chronic inflammation has been associated with several chronic diseases, such as age-related macular degeneration (AMD). The NLRP3 inflammasome is a central proinflammatory signaling complex that triggers caspase-1 activation leading to the maturation of IL-1β. We have previously shown that the inhibition of the chaperone protein, Hsp90, prevents NLRP3 activation in human retinal pigment epithelial (RPE) cells; these are cells which play a central role in the pathogenesis of AMD. In that study, we used a well-known Hsp90 inhibitor geldanamycin, but it cannot be used as a therapy due to its adverse effects, including ocular toxicity. Here, we have tested the effects of a novel Hsp90 inhibitor, TAS-116, on NLRP3 activation using geldanamycin as a reference compound. Using our existing protocol, inflammasome activation was induced in IL-1α-primed ARPE-19 cells with the proteasome and autophagy inhibitors MG-132 and bafilomycin A1, respectively. Intracellular caspase-1 activity was determined using a commercial caspase-1 activity kit and the FLICA assay. The levels of IL-1β were measured from cell culture medium samples by ELISA. Cell viability was monitored by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test and lactate dehydrogenase (LDH) measurements. Our findings show that TAS-116 could prevent the activation of caspase-1, subsequently reducing the release of mature IL-1β. TAS-116 has a better in vitro therapeutic index than geldanamycin. In summary, TAS-116 appears to be a well-tolerated Hsp90 inhibitor, with the capability to prevent the activation of the NLRP3 inflammasome in human RPE cells.     

Cathepsin X Activity Does Not Affect NK-Target Cell Synapse but Is Rather Distributed to Cytotoxic Granules

Cathepsin X is a lysosomal peptidase that is involved in tumour progression and represents a potential target for therapeutic interventions. In addition, it regulates important functions of immune cells and is implicated in the modulation of tumour cell–immune cell crosstalk. Selective cathepsin X inhibitors have been proposed as prospective antitumour agents to prevent cancer progression; however, their impact on the antitumour immune response has been overlooked. Previous studies indicate that the migration and adhesion of T cells and dendritic cells are affected by diminished cathepsin X activity. Meanwhile, the influence of cathepsin X inhibition on natural killer (NK) cell function has not yet been explored. Here, we examined the localization patterns of cathepsin X and the role of its inhibitors on the cytotoxicity of cell line NK-92, which is used for adoptive cellular immunotherapy in cancer patients. NK-92 cells depend on lymphocyte function-associated antigen 1 (LFA-1) to form stable immunoconjugates with target cells, providing, in this way, optimal cytotoxicity. Since LFA-1 is a substrate for cathepsin X activity in other types of cells, we hypothesized that cathepsin X could disturb the formation of NK-92 immunoconjugates. Thus, we employed cathepsin X reversible and irreversible inhibitors and evaluated their effects on the NK-92 cell interactions with target cells and on the NK-92 cell cytotoxicity. We show that cathepsin X inhibition does not impair stable conjugate formation or the lytic activity of NK-92 cells. Similarly, the conjugate formation between Jurkat T cells and target cells was not affected by cathepsin X activity. Unlike in previous migration and adhesion studies on T cells, in NK-92 cells cathepsin X was not co-localized with LFA-1 at the plasma membrane but was, rather, redistributed to the cytotoxic granules and secreted during degranulation.     

MAO-A Inhibition by Metaxalone Reverts IL-1β-Induced Inflammatory Phenotype in Microglial Cells

Experimental and clinical studies have suggested that several neurological disorders are associated with the occurrence of central nervous system neuroinflammation. Metaxalone is an FDA-approved muscle relaxant that has been reported to inhibit monoamine oxidase A (MAO-A). The aim of this study was to investigate whether metaxalone might exert antioxidant and anti-inflammatory effects in HMC3 microglial cells. An inflammatory phenotype was induced in HMC3 microglial cells through stimulation with interleukin-1β (IL-1β). Control cells and IL-1β-stimulated cells were subsequently treated with metaxalone (10, 20, and 40 µM) for six hours. IL-1β stimulated the release of the pro-inflammatory cytokines tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6), but reduced the anti-inflammatory cytokine interleukin-13 (IL-13). The upstream signal consisted of an increased priming of nuclear factor-kB (NF-kB), blunted peroxisome proliferator-activated receptor gamma (PPARγ), and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) expression. IL-1β also augmented MAO-A expression/activity and malondialdehyde levels and decreased Nrf2 mRNA expression and protein levels. Metaxalone decreased MAO-A activity and expression, reduced NF-kB, TNF-α, and IL-6, enhanced IL-13, and also increased PPARγ, PGC-1α, and Nrf2 expression. The present experimental study suggests that metaxalone has potential for the treatment of several neurological disorders associated with neuroinflammation.     

Peritoneal Fluid from Patients with Ovarian Endometriosis Displays Immunosuppressive Potential and Stimulates Th2 Response

Endometriosis is a common gynaecological disorder characterized by the ectopic growth of endometrial tissue outside the uterine cavity. It is associated with chronic pelvic inflammation and autoimmune reactivity manifesting by autoantibody production and abrogated cellular immune responses. Endometriotic peritoneal fluid contains various infiltrating leucocyte populations and a bulk of proinflammatory and immunoregulatory cytokines. However, the nature and significance of the peritoneal milieu in women with endometriosis still remains obscure. Therefore, the aim of the present study was to investigate the immunoregulatory activity of the peritoneal fluid (PF) from women with endometriosis. The peritoneal fluid samples were collected during laparoscopic surgery from 30 women with and without endometriosis. Immunoregulatory cytokines (IL-2, IL-4, IL-6, IL-10, IL-17A, IFN-γ and TNF) and chemokines (CCL2, CCL5, CXCL8 and CXCL9) were evaluated in PF and culture supernatants generated by unstimulated and CD3/CD28/IL-2-stimulated CD4⁺ T cells cultured in the presence of PF. The effect of PF on the generation of Treg and Th17 cells in CD4⁺ T cell cultures, as well as the natural cytotoxic activity of peripheral blood mononuclear cells, was also investigated. Concentrations of IL-6, IL-10, CCL2, CXCL8 and CXCL9 were significantly upregulated in the PF from women with endometriosis when compared to control women, whereas concentrations of other cytokines and chemokines were unaffected. The culturing of unstimulated and CD3/CD28/IL-2-stimulated CD4⁺ T cells in the presence of endometriotic PF resulted in the downregulation of their IL-2, IFN-γ, IL-17A and TNF production as compared to culture medium alone. On the other side, endometriotic PF significantly stimulated the production of IL-4 and IL-10. Endometriotic PF also stimulated the release of CCL2 and CXCL8, whereas the production of CCL5 and CXCL9 was downregulated. Endometriotic PF stimulated the generation of Treg cells and had an inhibitory effect on the generation of Th17 cells in cultures of CD4⁺ T cells. It also inhibited the NK cell cytotoxic activity of the peripheral blood lymphocytes. These results strongly imply that the PF from patients with endometriosis has immunoregulatory/immunosuppressive activity and shifts the Th1/Th2 cytokine balance toward the Th2 response, which may account for deviation of local and systemic immune responses. However, a similar trend, albeit not a statistically significant one, was also observed in case of PF from women without endometriosis, thus suggesting that peritoneal milieu may in general display some immunoregulatory/immunosuppressive properties. It should be stressed, however, that our present observations were made on a relatively small number of PF samples and further studies are needed to reveal possible mechanism(s) responsible for this phenomenon.     

In Vitro Conditioning of Adipose-Derived Mesenchymal Stem Cells by the Endothelial Microenvironment: Modeling Cell Responsiveness towards Non-Genetic Correction of Haemophilia A

In recent decades, the use of adult multipotent stem cells has paved the way for the identification of new therapeutic approaches for the treatment of monogenic diseases such as Haemophilia A. Being already studied for regenerative purposes, adipose-derived mesenchymal stem cells (Ad-MSCs) are still poorly considered for Haemophilia A cell therapy and their capacity to produce coagulation factor VIII (FVIII) after proper stimulation and without resorting to gene transfection. In this work, Ad-MSCs were in vitro conditioned towards the endothelial lineage, considered to be responsible for coagulation factor production. The cells were cultured in an inductive medium enriched with endothelial growth factors for up to 21 days. In addition to significantly responding to the chemotactic endothelial stimuli, the cell populations started to form capillary-like structures and up-regulated the expression of specific endothelial markers (CD34, PDGFRα, VEGFR2, VE-cadherin, CD31, and vWF). A dot blot protein study detected the presence of FVIII in culture media collected from both unstimulated and stimulated Ad-MSCs. Remarkably, the activated partial thromboplastin time test demonstrated that the clot formation was accelerated, and FVIII activity was enhanced when FVIII deficient plasma was mixed with culture media from the untreated/stimulated Ad-MSCs. Overall, the collected evidence supported a possible Ad-MSC contribution to HA correction via specific stimulation by the endothelial microenvironment and without any need for gene transfection.     

Escherichia coli Maltose-Binding Protein Induces M1 Polarity of RAW264.7 Macrophage Cells via a TLR2- and TLR4-Dependent Manner

Maltose-binding protein (MBP) is a critical player of the maltose/maltodextrin transport system in Escherichia coli. Our previous studies have revealed that MBP nonspecifically induces T helper type 1 (Th1) cell activation and activates peritoneal macrophages obtained from mouse. In the present study, we reported a direct stimulatory effect of MBP on RAW264.7 cells, a murine macrophage cell line. When stimulated with MBP, the production of nitric oxide (NO), IL-1β, IL-6 and IL-12p70, and the expressions of CD80, MHC class II and inducible nitric oxide synthase (iNOS) were all increased in RAW264.7 cells, indicating the activation and polarization of RAW264.7 cells into M1 macrophages induced by MBP. Further study showed that MBP stimulation upregulated the expression of TLR2 and TLR4 on RAW264.7 cells, which was accompanied by subsequent phosphorylation of IκB-α and p38 MAPK. Pretreatment with anti-TLR2 or anti-TLR4 antibodies largely inhibited the phosphorylation of IκB-α and p38 MAPK, and greatly reduced MBP-induced NO and IL-12p70 production, suggesting that the MBP-induced macrophage activation and polarization were mediated by TLR2 and TLR4 signaling pathways. The observed results were independent of lipopolysaccharide contamination. Our study provides a new insight into a mechanism by which MBP enhances immune responses and warrants the potential application of MBP as an immune adjuvant in immune therapies.     

IL-33 Enhances IFNγ and TNFα Production by Human MAIT Cells: A New Pro-Th1 Effect of IL-33

Mucosal-associated invariant T (MAIT) cells represent a distinct T cell population restricted by the MHC-class-I-related molecule, MR1, which recognizes microbial-derived vitamin B2 (riboflavin) metabolites. Their abundance in humans, together with their ability to promptly produce distinct cytokines including interferon γ (IFNγ) and tumor necrosis factor α (TNFα), are consistent with regulatory functions in innate as well as adaptive immunity. Here, we tested whether the alarmin interleukin 33 (IL-33), which is secreted following inflammation or cell damage, could activate human MAIT cells. We found that MAIT cells stimulated with IL-33 produced high levels of IFNγ, TNFα and Granzyme B (GrzB). The action of IL-33 required IL-12 but was independent of T cell receptor (TCR) cross-linking. MAIT cells expressed the IL-33 receptor ST2 (suppression of tumorigenicity 2) and upregulated Tbet (T-box expressed in T cells) in response to IL-12 or IL-33. Electronically sorted MAIT cells also upregulated the expression of CCL3 (Chemokine C-C motif ligand 3), CD40L (CD40 Ligand), CSF-1 (Colony Stimulating Factor 1), LTA (Lymphotoxin-alpha) and IL-2RA (IL-2 receptor alpha chain) mRNAs in response to IL-33 plus IL-12. In conclusion, IL-33 combined with IL-12 can directly target MAIT cells to induce their activation and cytokine production. This novel mechanism of IL-33 activation provides insight into the mode of action by which human MAIT cells can promote inflammatory responses in a TCR-independent manner.     

Extract from the Coriolus versicolor Fungus as an Anti-Inflammatory Agent with Cytotoxic Properties against Endothelial Cells and Breast Cancer Cells

Chronic inflammation is a well-recognised tumour-enabling component, which includes bioactive molecules from cells infiltrating the tumour microenvironment and increases the risk of cancer progression. Since long-term use of the currently available anti-inflammatory drugs used in cancer therapy causes numerous side effects, the aim of this study was to investigate the effect of an extract isolated from the Coriolus versicolor fungus (CV extract) on HUVEC endothelial cells and MCF-7 breast cancer cells in a pro-inflammatory microenvironment mimicked by lipopolysaccharide (LPS). The cells were simultaneously stimulated with the LPS and CV extract. After co-treatment, the cell viability, generation of reactive oxygen species (ROS), wound-healing assay, production of the pro-inflammatory and pro-angiogenic factors (interleukin (IL) 6, IL-8, and metalloproteinase (MMP) 9)), as well as expression of Toll-like receptor (TLR) 4 and phosphorylated IκB (p-IκB) were evaluated. The results showed that the CV extract inhibited IL-6, IL-8, and MMP-9 production by the LPS-stimulated cells. This effect was accompanied by a decrease in TLR4 and p-IκB expression. The CV extract also had anti-migratory properties and induced a cytotoxic effect on the cells that was enhanced in the presence of LPS. The observed cytotoxicity was associated with an increase in ROS generation. We conclude that the CV extract possesses cytotoxic activity against cancer cells and endothelial cells and has the ability to inhibit the expression of the pro-tumorigenic factors associated with inflammation.     

IL-33 Is Involved in the Anti-Inflammatory Effects of Butyrate and Propionate on TNFα-Activated Endothelial Cells

Short-chain fatty acids (e.g., butyrate and propionate) are able to diminish endothelial cell activation. The aim of this study was to investigate whether intracellular IL-33 mediates the effects of butyrate and propionate on TNFα-induced IL-8 production and vascular cell adhesion molecule-1 (VCAM-1) expression. In addition, it was investigated whether regulating NF-κB and MAPK signaling pathways are involved. Intracellular IL-33 was measured in human endothelial cells (HUVECs) pre-incubated for 24 h with butyrate (0.1 mM or 5 mM), propionate (0.3 mM or 10 mM), or trichostatin A (TSA, 0.5 μM) prior to TNFα (1 ng/mL) stimulation (24 h). The effects of butyrate, propionate, and TSA on TNFα-induced IL-8, vascular cell adhesion molecule-1 (VCAM-1), NF-κB, and MAPK signaling pathways in normal HUVECs and IL-33 siRNA (siIL-33)-transfected HUVECs were compared to study the role of IL-33 in the protective effects of butyrate and propionate. Endogenous IL-33 was highly expressed in the perinuclear in HUVECs, which was significantly reduced by TNFα stimulation. The TNFα-induced reduction in IL-33 was prevented by pre-incubation with butyrate or propionate. Butyrate (0.1 mM), propionate (0.3 mM), and TSA inhibited the IL-8 production and activation of NF-κB. Interestingly, this effect was not observed in siIL-33-transfected HUVECs. The effects of butyrate (5 mM), propionate (10 mM), and TSA (0.5 μM) on VCAM-1 expression and activation of MAPK signaling pathways were not affected by siIL-33 transfection. In conclusion, we showed that the inhibitory effects of butyrate and propionate on TNFα-induced IL-8 production were mediated by the HDACs/IL-33/NF-κB pathway, while their effects on VCAM-1 expression might be associated with the HDACs/MAPK signaling pathway, independently of IL-33.     

Interleukin-32θ Triggers Cellular Senescence and Reduces Sensitivity to Doxorubicin-Mediated Cytotoxicity in MDA-MB-231 Cells

The recently discovered interleukin (IL)- 32 isoform IL-32θ exerts anti-metastatic effects in the breast tumor microenvironment. However, the involvement of IL-32θ in breast cancer cell proliferation is not yet fully understood; therefore, the current study aimed to determine how IL-32θ affects cancer cell growth and evaluated the responses of IL-32θ-expressing cells to other cancer therapy. We compared the functions of IL-32θ in triple-negative breast cancer MDA-MB-231 cells that stably express IL-32θ, with MDA-MB-231 cells transfected with a mock vector. Slower growth was observed in cells expressing IL-32θ than in control cells, and changes were noted in nuclear morphology, mitotic division, and nucleolar size between the two groups of cells. Interleukin-32θ significantly reduced the colony-forming ability of MDA-MB-231 cells and induced permanent cell cycle arrest at the G1 phase. Long-term IL-32θ accumulation triggered permanent senescence and chromosomal instability in MDA-MB-231 cells. Genotoxic drug doxorubicin (DR) reduced the viability of MDA-MB-231 cells not expressing IL-32θ more than in cells expressing IL-32θ. Overall, these findings suggest that IL-32θ exerts antiproliferative effects in breast cancer cells and initiates senescence, which may cause DR resistance. Therefore, targeting IL-32θ in combination with DR treatment may not be suitable for treating metastatic breast cancer.     

Tonabersat Inhibits Connexin43 Hemichannel Opening and Inflammasome Activation in an In Vitro Retinal Epithelial Cell Model of Diabetic Retinopathy

This study was undertaken to evaluate the connexin hemichannel blocker tonabersat for the inhibition of inflammasome activation and use as a potential treatment for diabetic retinopathy. Human retinal pigment epithelial cells (ARPE-19) were stimulated with hyperglycemia and the inflammatory cytokines IL-1β and TNFα in order to mimic diabetic retinopathy molecular signs in vitro. Immunohistochemistry was used to evaluate the effect of tonabersat treatment on NLRP3, NLRP1, and cleaved caspase-1 expression and distribution. A Luminex cytokine release assay was performed to determine whether tonabersat affected proinflammatory cytokine release. NLRP1 was not activated in ARPE-19 cells, and IL-18 was not produced under disease conditions. However, NLRP3 and cleaved caspase-1 complex formation increased with hyperglycemia and cytokine challenge but was inhibited by tonabersat treatment. It also prevented the release of proinflammatory cytokines IL-1β, VEGF, and IL-6. Tonabersat therefore has the potential to reduce inflammasome-mediated inflammation in diabetic retinopathy.     

Cord Blood T Cells Expressing High and Low PKCζ Levels Develop into Cells with a Propensity to Display Th1 and Th9 Cytokine Profiles,Respectively

Low Protein Kinase C zeta (PKCζ) levels in cord blood T cells (CBTC) have been shown to correlate with the development of allergic sensitization in childhood. However, little is known about the mechanisms responsible. We have examined the relationship between the expression of different levels of PKCζ in CBTC and their development into mature T cell cytokine producers that relate to allergy or anti-allergy promoting cells. Maturation of naïve CBTC was initiated with anti-CD3/-CD28 antibodies and recombinant human interleukin-2 (rhIL-2). To stimulate lymphocyte proliferation and cytokine production the cells were treated with Phytohaemagglutinin (PHA) and Phorbol myristate acetate (PMA). Irrespective of the PKCζ levels expressed, immature CBTC showed no difference in lymphocyte proliferation and the production of T helper 2 (Th2) cytokine interleukin-4 (IL-4) and Th1 cytokine, interferon-gamma (IFN-γ), and influenced neither their maturation from CD45RA⁺ to CD45RO⁺ cells nor cell viability/apoptosis. However, upon maturation the low PKCζ expressing cells produced low levels of the Th1 cytokines, IFN-γ, IL-2 and tumour necrosis factor-alpha (TNF), no changes to levels of the Th2 cytokines, IL-4, IL-5 and IL-13, and an increase in the Th9 cytokine, IL-9. Other cytokines, lymphotoxin-α (LT-α), IL-10, IL-17, IL-21, IL-22 and Transforming growth factor-beta (TGF-β) were not significantly different. The findings support the view that low CBTC PKCζ levels relate to the increased risk of developing allergic diseases.     

Platelet-Rich Fibrin Reduces IL-1β Release from Macrophages Undergoing Pyroptosis

Background: Pyroptosis is a catabolic process relevant to periodontal disorders for which interleukin-1β (IL-1β) inflammation is central to the pathophysiology of the disease. Despite platelet-rich fibrin (PRF) anti-inflammatory properties and its application to support periodontal regeneration, the capacity of PRF to modulate pyroptosis, specifically the production and release of IL-1β, remains unknown. The question arises whether PRF could regulate IL-1β release from macrophages in vitro. Methods: To answer this question, RAW 264.7 macrophages and primary macrophages obtained from murine bone marrow were primed with PRF before being challenged by lipopolysaccharide (LPS). Cells were then analysed for the pyroptosis signalling components by gene expression analyses and IL-1β secretion at the protein level. The release of mitochondrial reactive oxygen species (ROS) was also detected. Results: PRF lowered the LPS-induced expression of IL-1β and NLRP3 inflammasome, caspase-11 and IL-18 in primary macrophages, and IL-1β and caspase-11 in RAW 264.7 cells. Additionally, PRF diminished the secretion of IL-1β at the protein level in LPS-induced RAW 264.7 cells. This was shown through immunoassays performed with the supernatant and further confirmed by analysing the lysates of permeabilised cells. Furthermore, PRF reduced the ROS release provoked by LPS in RAW 264.7 cells. Finally, to enhance IL-1β release from the LPS-primed macrophages, we introduced a second signal with adenosine triphosphate (ATP). In this setting, PRF significantly reduced IL-1β release in RAW 264.7 cells and a trend to diminish IL-1β release in primary macrophages. Conclusion: These findings suggest that PRF can reduce IL-1β release and, at least in part, inhibit pyroptosis-related factors in LPS-challenged macrophages.