Characterization of the Transient Deficiency of PKC Isozyme Levels in Immature Cord Blood T Cells and Its Connection to Anti-Allergic Cytokine Profiles of the Matured Cells

Cord blood T cells (CBTC) from a proportion of newborns express low/deficient levels of some protein kinase C (PKC) isozymes, with low levels of PKCζ correlating with increased risk of developing allergy and associated decrease in interferon-gamma (IFN-γ) producing T cells. Interestingly, these lower levels of PKCζ were increased/normalized by supplementing women during pregnancy with n-3 polyunsaturated fatty acids. However, at present, we have little understanding of the transient nature of the deficiency in the neonate and how PKCζ relates to other PKC isozymes and whether their levels influence maturation into IFN-γ producing T cells. There is also no information on PKCζ isozyme levels in the T cell subpopulations, CD4⁺ and CD8⁺ cells. These issues were addressed in the present study using a classical culture model of neonatal T cell maturation, initiated with phytohaemagglutinin (PHA) and recombinant human interleukin-2 (rhIL-2). Of the isozymes evaluated, PKCζ, β2, δ, μ, ε, θ and λ/ι were low in CBTCs. The PKC isozyme deficiencies were also found in the CD4⁺ and CD8⁺ T cell subset levels of the PKC isozymes correlated between the two subpopulations. Examination of changes in the PKC isozymes in these deficient cells following addition of maturation signals showed a significant increase in expression within the first few hours for PKCζ, β2 and μ, and 1–2 days for PKCδ, ε, θ and λ/ι. Only CBTC PKCζ isozyme levels correlated with cytokine production, with a positive correlation with IFN-γ, interleukin (IL)-2 and tumour necrosis factor-alpha (TNF), and a negative association with IL-9 and IL-10. The findings reinforce the specificity in using CBTC PKCζ levels as a biomarker for risk of allergy development and identify a period in which this can be potentially ‘corrected’ after birth.     

A Novel Perspective and Approach to Intestinal Octreotide Absorption: Sinomenine-Mediated Reversible Tight Junction Opening and Its Molecular Mechanism

In this work, we assessed the effects of sinomenine (SN) on intestinal octreotide (OCT) absorption both in Caco-2 cell monolayers and in rats. We also investigated the molecular mechanisms of tight junction (TJ) disruption and recovery by SN-mediated changes in the claudin-1 and protein kinase C (PKC) signaling pathway. The data showed that exposure to SN resulted in a significant decrease in the expression of claudin-1, which represented TJ weakening and paracellular permeability enhancement. Then, the recovery of TJ after SN removal required an increase in claudin-1, which demonstrated the transient and reversible opening for TJ. Meanwhile, the SN-mediated translocation of PKC-α from the cytosol to the membrane was found to prove PKC activation. Finally, SN significantly improved the absolute OCT bioavailability in rats and the transport rate in Caco-2 cell monolayers. We conclude that SN has the ability to enhance intestinal OCT absorption and that these mechanisms are related at least in part to the important role of claudin-1 in SN-mediated, reversible TJ opening via PKC activation.     

Lunasin Sensitivity in Non-Small Cell Lung Cancer Cells Is Linked to Suppression of Integrin Signaling and Changes in Histone Acetylation

Lunasin is a plant derived bioactive peptide with both cancer chemopreventive and therapeutic activity. We recently showed lunasin inhibits non-small cell lung cancer (NSCLC) cell proliferation in a cell-line-specific manner. We now compared the effects of lunasin treatment of lunasin-sensitive (H661) and lunasin-insensitive (H1299) NSCLC cells with respect to lunasin uptake, histone acetylation and integrin signaling. Both cell lines exhibited changes in histone acetylation, with H661 cells showing a unique increase in H4K16 acetylation. Proximity ligation assays demonstrated lunasin interacted with integrins containing αv, α5, β1 and β3 subunits to a larger extent in the H661 compared to H1299 cells. Moreover, lunasin specifically disrupted the interaction of β1 and β3 subunits with the downstream signaling components phosphorylated Focal Adhesion Kinase (pFAK), Kindlin and Intergrin Linked Kinase in H661 cells. Immunoblot analyses demonstrated lunasin treatment of H661 resulted in reduced levels of pFAK, phosphorylated Akt and phosphorylated ERK1/2 whereas no changes were observed in H1299 cells. Silencing of αv expression in H661 cells confirmed signaling through integrins containing αv is essential for proliferation. Moreover, lunasin was unable to further inhibit proliferation in αv-silenced H661 cells. This indicates antagonism of integrin signaling via αv-containing integrins is an important component of lunasin’s mechanism of action.     

Multiple Na,K-ATPase Subunits Colocalize in the Brush Border of Mouse Choroid Plexus Epithelial Cells

(1) Background: The unusual accumulation of Na,K-ATPase complexes in the brush border membrane of choroid plexus epithelial cells have intrigued researchers for decades. However, the full range of the expressed Na,K-ATPase subunits and their relation to the microvillus cytoskeleton remains unknown. (2) Methods: RT-PCR analysis, co-immunoprecipitation, native PAGE, mass spectrometry, and differential centrifugation were combined with high-resolution immunofluorescence histochemistry, proximity ligase assays, and stimulated emission depletion (STED) microscopy on mouse choroid plexus cells or tissues in order to resolve these issues. (3) Results: The choroid plexus epithelium expresses Na,K-ATPase subunits α1, α2, β1, β2, β3, and phospholemman. The α1, α2, β1, and β2, subunits are all localized to the brush border membrane, where they appear to form a complex. The ATPase complexes may stabilize in the brush border membrane via anchoring to microvillar actin indirectly through ankyrin-3 or directly via other co-precipitated proteins. Aquaporin 1 (AQP1) may form part of the proposed multi-protein complexes in contrast to another membrane protein, the Na-K-2Cl cotransporter 1 (NKCC1). NKCC1 expression seems necessary for full brush border membrane accumulation of the Na,K-ATPase in the choroid plexus. (4) Conclusion: A multitude of Na,K-ATPase subunits form molecular complexes in the choroid plexus brush border, which may bind to the cytoskeleton by various alternative actin binding proteins.     

Strategies to Improve the Antitumor Effect of γδ T Cell Immunotherapy for Clinical Application

Human γδ T cells show potent cytotoxicity against various types of cancer cells in a major histocompatibility complex unrestricted manner. Phosphoantigens and nitrogen-containing bisphosphonates (N-bis) stimulate γδ T cells via interaction between the γδ T cell receptor (TCR) and butyrophilin subfamily 3 member A1 (BTN3A1) expressed on target cells. γδ T cell immunotherapy is classified as either in vivo or ex vivo according to the method of activation. Immunotherapy with activated γδ T cells is well tolerated; however, the clinical benefits are unsatisfactory. Therefore, the antitumor effects need to be increased. Administration of γδ T cells into local cavities might improve antitumor effects by increasing the effector-to-target cell ratio. Some anticancer and molecularly targeted agents increase the cytotoxicity of γδ T cells via mechanisms involving natural killer group 2 member D (NKG2D)-mediated recognition of target cells. Both the tumor microenvironment and cancer stem cells exert immunosuppressive effects via mechanisms that include inhibitory immune checkpoint molecules. Therefore, co-immunotherapy with γδ T cells plus immune checkpoint inhibitors is a strategy that may improve cytotoxicity. The use of a bispecific antibody and chimeric antigen receptor might be effective to overcome current therapeutic limitations. Such strategies should be tested in a clinical research setting.     

Receptor-Targeted,Magneto-Mechanical Stimulation of Osteogenic Differentiation of Human Bone Marrow-Derived Mesenchymal Stem Cells

Mechanical cues are employed to promote stem cell differentiation and functional tissue formation in tissue engineering and regenerative medicine. We have developed a Magnetic Force Bioreactor (MFB) that delivers highly targeted local forces to cells at a pico-newton level, utilizing magnetic micro- and nano-particles to target cell surface receptors. In this study, we investigated the effects of magnetically targeting and actuating specific two mechanical-sensitive cell membrane receptors—platelet-derived growth factor receptor α (PDGFRα) and integrin α_νβ₃. It was found that a higher mineral-to-matrix ratio was obtained after three weeks of magneto-mechanical stimulation coupled with osteogenic medium culture by initially targeting PDGFRα compared with targeting integrin α_νβ₃ and non-treated controls. Moreover, different initiation sites caused a differentiated response profile when using a 2-day-lagged magneto-mechanical stimulation over culture periods of 7 and 12 days). However, both resulted in statistically higher osteogenic marker genes expression compared with immediate magneto-mechanical stimulation. These results provide insights into important parameters for designing appropriate protocols for ex vivo induced bone formation via magneto-mechanical actuation.     

Epithelial Regeneration Ability of Crohn’s Disease Assessed Using Patient-Derived Intestinal Organoids

Little is known about the ability for epithelial regeneration and wound healing in patients with inflammatory bowel diseases. We evaluated the epithelial proliferation and wound healing ability of patients with Crohn’s disease (CD) using patient-derived intestinal organoids. Human intestinal organoids were constructed in a three-dimensional intestinal crypt culture of enteroscopic biopsy samples from controls and CD patients. The organoid-forming efficiency of ileal crypts derived from CD patients was reduced compared with those from control subjects (p < 0.001). Long-term cultured organoids (≥6 passages) derived from controls and CD patients showed an indistinguishable microscopic appearance and culturing behavior. Under TNFα-enriched conditions (30 ng/mL), the organoid reconstitution rate and cell viability of CD patient-derived organoids were significantly lower than those of the control organoids (p < 0.05 for each). The number of EdU+ cells was significantly lower in TNFα-treated organoids derived from CD patients than in TNFα-treated control organoids (p < 0.05). In a wound healing assay, the unhealed area in TNFα-treated CD patient-derived organoids was significantly larger than that of TNFα-treated control organoids (p < 0.001). The wound healing ability of CD patient-derived organoids is reduced in TNFα-enriched conditions, due to reduced cell proliferation. Epithelial regeneration ability may be impaired in patients with CD.     

Human Caspase 12 Enhances NF-κB Activity through Activation of IKK in Nasopharyngeal Carcinoma Cells

Human nasopharyngeal carcinoma (NPC) is a highly invasive cancer associated with proinflammation. Caspase-12 (Casp12), an inflammatory caspase, is implicated in the regulation of NF-κB-mediated cellular invasion via the modulation of the IκBα protein in NPC cells. However, the effect mechanisms of Casp12 need to be elucidated. NPC cells were transfected with the full length of human Casp12 cDNA (pC12) and the effect of human Casp12 (hCasp12) on the NF-κB activity was investigated. We found ectopic expression of hCasp12 increased the NF-κB activity accompanied by an increased p-IκBα expression and a decreased IκBα expression. Treatment of BMS, a specific IKK inhibitor, and pC12-transfected cells markedly decreased the NF-κB activity and ameliorated the expression level of IκBα reduced by hCasp12. Co-immunoprecipitation assays validated the physical interaction of hCasp12 with IKKα/β, but not with NEMO. Furthermore, the NF-κB activity of ΔCasp12-Q (a mutated catalytic of hCasp12) transfected cells was concentration-dependently induced, but lower than that of hCasp12-transfected cells. Importantly, the hCasp12-mediated NF-kB activity was enhanced by TNFα stimulation. That indicated a role of the catalytic motif of hCasp12 in the regulation of the NF-κB activity. This study indicated hCasp12 activated the NF-κB pathway through the activation of IKK in human NPC cells.     

Colorectal Cancer Apoptosis Induced by Dietary δ-Valerobetaine Involves PINK1/Parkin Dependent-Mitophagy and SIRT3

Understanding the mechanisms of colorectal cancer progression is crucial in the setting of strategies for its prevention. δ-Valerobetaine (δVB) is an emerging dietary metabolite showing cytotoxic activity in colon cancer cells via autophagy and apoptosis. Here, we aimed to deepen current knowledge on the mechanism of δVB-induced colon cancer cell death by investigating the apoptotic cascade in colorectal adenocarcinoma SW480 and SW620 cells and evaluating the molecular players of mitochondrial dysfunction. Results indicated that δVB reduced cell viability in a time-dependent manner, reaching IC50 after 72 h of incubation with δVB 1.5 mM, and caused a G2/M cell cycle arrest with upregulation of cyclin A and cyclin B protein levels. The increased apoptotic cell rate occurred via caspase-3 activation with a concomitant loss in mitochondrial membrane potential and SIRT3 downregulation. Functional studies indicated that δVB activated mitochondrial apoptosis through PINK1/Parkin pathways, as upregulation of PINK1, Parkin, and LC3B protein levels was observed (p < 0.0001). Together, these findings support a critical role of PINK1/Parkin-mediated mitophagy in mitochondrial dysfunction and apoptosis induced by δVB in SW480 and SW620 colon cancer cells.     

Geranylgeraniol Inhibits Lipopolysaccharide-Induced Inflammation in Mouse-Derived MG6 Microglial Cells via NF-κB Signaling Modulation

Persistent inflammatory reactions in microglial cells are strongly associated with neurodegenerative pathogenesis. Additionally, geranylgeraniol (GGOH), a plant-derived isoprenoid, has been found to improve inflammatory conditions in several animal models. It has also been observed that its chemical structure is similar to that of the side chain of menaquinone-4, which is a vitamin K₂ sub-type that suppresses inflammation in mouse-derived microglial cells. In this study, we investigated whether GGOH has a similar anti-inflammatory effect in activated microglial cells. Particularly, mouse-derived MG6 cells pre-treated with GGOH were exposed to lipopolysaccharide (LPS). Thereafter, the mRNA levels of pro-inflammatory cytokines were determined via qRT-PCR, while protein expression levels, especially the expression of NF-κB signaling cascade-related proteins, were determined via Western blot analysis. The distribution of NF-κB p65 protein was also analyzed via fluorescence microscopy. Thus, it was observed that GGOH dose-dependently suppressed the LPS-induced increase in the mRNA levels of Il-1β, Tnf-α, Il-6, and Cox-2. Furthermore, GGOH inhibited the phosphorylation of TAK1, IKKα/β, and NF-κB p65 proteins as well as NF-κB nuclear translocation induced by LPS while maintaining IκBα expression. We showed that GGOH, similar to menaquinone-4, could alleviate LPS-induced microglial inflammation by targeting the NF-kB signaling pathway.     

Preconceptional Immunization Can Modulate Offspring Intrathymic IL-17-Producing γδT Cells with Epigenetic Implications Mediated by microRNAs

The mechanisms through which maternal immunization can modulate offspring thymic maturation of lymphocytes are not fully understood. Here, we aimed to evaluate whether maternal OVA-immunization can inhibit the maturation of IL-17-producing γδT cells in offspring thymus, and if this mechanism has epigenetic implications mediated by microRNAs (miRNAs) expression. Wild-type (WT) C57BL/6 females were immunized with OVA in Alum or Alum alone and were mated with normal WT males. Evaluating their offspring thymus at 3 or 20 days old (d.o.), we observed that maternal OVA immunization could inhibit the thymic frequency of offspring CD27- and IL-17⁺ γδT cells at the neonatal and until 20 days old. Furthermore, we evaluated the expression of function-related γ and δ variable γδTCR chains (Vγ1, Vγ2, Vγ3, Vδ4, and Vδ6.3), observing that maternal OVA-immunization inhibits Vγ2 chains expression. The small RNAs (sRNAs), particularly miRNAs, and messenger RNAs (mRNA) expression profiles by pools of thymus tissue samples (from 9 to 11 mice) from offspring OVA-immunized or Alum-immunized mothers were analyzed via Illumina sequencing platform and bioinformatics approaches. Using a fold change >4, our results showed that seven miRNAs (mmu-miR-126a-3p, 101a-3p, 744-3p,142-5p, 15a-5p, 532-5p, and 98-5p) were differentially expressed between both groups. Ten target genes were predicted to interact with the seven selected miRNAs. There were no enriched categories of gene ontology functional annotation and pathway enrichment analysis for the target genes. Interestingly, four of the identified miRNAs (mmu-miR-15a, mmu-miR-101 mmu-miR-126, and mmu-miR-142) are related to IL-17 production. Our data is of significance because we demonstrate that maternal immunization can modulate offspring thymic maturation of IL-17-producing γδT cells possibly by an epigenetic mechanism mediated by miRNAs.     

Investigation of the Antiproliferative Properties of Natural Sesquiterpenes from Artemisia asiatica and Onopordum acanthium on HL-60 Cells in Vitro

Plants and plant extracts play a crucial role in the research into novel antineoplastic agents. Four sesquiterpene lactones, artecanin (1), 3β-chloro-4α,10α-dihydroxy-1α,2α-epoxy-5α,7αH-guaia-11(13)-en-12,6α-olide (2), iso-seco-tanapartholide 3-O-methyl ether (3) and 4β,15-dihydro-3-dehydrozaluzanin C (4), were isolated from two traditionally used Asteraceae species (Onopordum acanthium and Artemisia asiatica). When tested for antiproliferative action on HL-60 leukemia cells, these compounds exhibited reasonable IC₅₀ values in the range 3.6–13.5 μM. Treatment with the tested compounds resulted in a cell cycle disturbance characterized by increases in the G1 and G2/M populations, while there was a decrease in the S phase. Additionally, 1–3 elicited increases in the hypodiploid (subG1) population. The compounds elicited concentration-dependent chromatin condensation and disruption of the membrane integrity, as revealed by Hoechst 33258–propidium staining. Treatment for 24 h resulted in significant increases in activity of caspases-3 and -9, indicating that the tested sesquiterpenes induced the mitochondrial pathway of apoptosis. The proapoptotic properties of the sesquiterpene lactones were additionally demonstrated withannexin V staining. Compounds 1 and 2 increased the Bax/Bcl-2 expression and decreased the expressions of CDK1 and cyclin B2, as determined at the mRNA level by means of RT-PCR. These experimental results indicate that sesquiterpene lactones may be regarded as potential starting structures for the development of novel anticancer agents.     

The CD200 Regulates Inflammation in Mice Independently of TNF-α Production

Inflammatory bowel disease is characterized by the infiltration of immune cells and chronic inflammation. The immune inhibitory receptor, CD200R, is involved in the downregulation of the activation of immune cells to prevent excessive inflammation. We aimed to define the role of CD200R ligand-CD200 in the experimental model of intestinal inflammation in conventionally-reared mice. Mice were given a dextran sodium sulfate solution in drinking water. Bodyweight loss was monitored daily and the disease activity index was calculated, and a histological evaluation of the colon was performed. TNF-α production was measured in the culture of small fragments of the distal colon or bone marrow-derived macrophages (BMDMs) cocultured with CD200⁺ cells. We found that Cd200^−/− mice displayed diminished severity of colitis when compared to WT mice. Inflammation significantly diminished CD200 expression in WT mice, particularly on vascular endothelial cells and immune cells. The co-culture of BMDMs with CD200⁺ cells inhibited TNF-α secretion. In vivo, acute colitis induced by DSS significantly increased TNF-α secretion in colon tissue in comparison to untreated controls. However, Cd200^−/− mice secreted a similar level of TNF-α to WT mice in vivo. CD200 regulates the severity of DSS-induced colitis in conventionally-reared mice. The presence of CD200⁺ cells decreases TNF-α production by macrophages in vitro. However, during DDS-induced intestinal inflammation secretion of TNF-α is independent of CD200 expression.     

Cortisol Metabolism in Carp Macrophages: A Role for Macrophage-Derived Cortisol in M1/M2 Polarization

Macrophages are crucial not only for initiation of inflammation and pathogen eradication (classically polarized M1 macrophages), but also for inflammation inhibition and tissue regeneration (alternatively polarized M2 macrophages). Their polarization toward the M1 population occurs under the influence of interferon-γ + lipopolysaccharide (IFN-γ + LPS), while alternatively polarized M2 macrophages evolve upon, e.g., interlukin 4 (IL-4) or cortisol stimulation. This in vitro study focused on a possible role for macrophage-derived cortisol in M1/M2 polarization in common carp. We studied the expression of molecules involved in cortisol synthesis/conversion from and to cortisone like 11β-hydroxysteroid dehydrogenase type 2 and 3. (11β-HSD2 and 3) and 11β-hydroxylase (CYP11b), as well as the expression of glucocorticoid receptors (GRs) and proliferator-activated receptor gamma (PPARγ) in M1 and M2 macrophages. Lastly, we analyzed how inhibition of these molecules affect macrophage polarization. In M1 cells, upregulation of gene expression of GRs and 11β-HSD3 was found, while, in M2 macrophages, expression of 11β-hsd2 was upregulated. Moreover, blocking of cortisol synthesis/conversion and GRs or PPARγ induced changes in expression of anti-inflammatory interleukin 10 (IL-10). Consequently, our data show that carp monocytes/macrophages can convert cortisol. The results strongly suggest that cortisol, via intracrine interaction with GRs, is important for IL-10-dependent control of the activity of macrophages and for the regulation of M1/M2 polarization to finally determine the outcome of an infection.     

Protein Kinase C as a Therapeutic Target in Non-Small Cell Lung Cancer

Driver-directed therapeutics have revolutionized cancer treatment, presenting similar or better efficacy compared to traditional chemotherapy and substantially improving quality of life. Despite significant advances, targeted therapy is greatly limited by resistance acquisition, which emerges in nearly all patients receiving treatment. As a result, identifying the molecular modulators of resistance is of great interest. Recent work has implicated protein kinase C (PKC) isozymes as mediators of drug resistance in non-small cell lung cancer (NSCLC). Importantly, previous findings on PKC have implicated this family of enzymes in both tumor-promotive and tumor-suppressive biology in various tissues. Here, we review the biological role of PKC isozymes in NSCLC through extensive analysis of cell-line-based studies to better understand the rationale for PKC inhibition. PKC isoforms α, ε, η, ι, ζ upregulation has been reported in lung cancer, and overexpression correlates with worse prognosis in NSCLC patients. Most importantly, PKC isozymes have been established as mediators of resistance to tyrosine kinase inhibitors in NSCLC. Unfortunately, however, PKC-directed therapeutics have yielded unsatisfactory results, likely due to a lack of specific evaluation for PKC. To achieve satisfactory results in clinical trials, predictive biomarkers of PKC activity must be established and screened for prior to patient enrollment. Furthermore, tandem inhibition of PKC and molecular drivers may be a potential therapeutic strategy to prevent the emergence of resistance in NSCLC.     

Gene Doping with Peroxisome-Proliferator-Activated Receptor Beta/Delta Agonists Alters Immunity but Exercise Training Mitigates the Detection of Effects in Blood Samples

Synthetic ligands of peroxisome-proliferator-activated receptor beta/delta (PPARβ/δ) are being used as performance-enhancing drugs by athletes. Since we previously showed that PPARβ/δ activation affects T cell biology, we wanted to investigate whether a specific blood T cell signature could be employed as a method to detect the use of PPARβ/δ agonists. We analyzed in primary human T cells the in vitro effect of PPARβ/δ activation on fatty acid oxidation (FAO) and on their differentiation into regulatory T cells (Tregs). Furthermore, we conducted studies in mice assigned to groups according to an 8-week exercise training program and/or a 6-week treatment with 3 mg/kg/day of GW0742, a PPARβ/δ agonist, in order to (1) determine the immune impact of the treatment on secondary lymphoid organs and to (2) validate a blood signature. Our results show that PPARβ/δ activation increases FAO potential in human and mouse T cells and mouse secondary lymphoid organs. This was accompanied by increased Treg polarization of human primary T cells. Moreover, Treg prevalence in mouse lymph nodes was increased when PPARβ/δ activation was combined with exercise training. Lastly, PPARβ/δ activation increased FAO potential in mouse blood T cells. Unfortunately, this signature was masked by training in mice. In conclusion, beyond the fact that it is unlikely that this signature could be used as a doping-control strategy, our results suggest that the use of PPARβ/δ agonists could have potential detrimental immune effects that may not be detectable in blood samples.