The stimulatory effect of α‐melanotropin on progesterone release from rat granulosa cells is inhibited by interleukin‐1β and by tumour necrosis factor‐α

Aim: Several studies have shown that a variety of peptides and cytokines are involved in ovarian regulatory mechanisms; however, their exact function is still unclear. In this work we study whether the administration of peptide α‐melanotropin and the cytokines interleukin‐1β (IL‐1β) and tumour necrosis factor‐α (TNF‐α) on their own modify the release of progesterone in cultured granulosa cells (GC) from pro‐oestrous rats. We also investigate an interaction between these cytokines and α‐melanotropin in the modulation of progesterone secretion. Methods: Granulosa cells were collected from the ovaries of female Wistar rats and cultured for up to 24 h in the presence of different concentrations of α‐melanotropin, cytokines or a combination of both. Progesterone concentration was measured by radioimmunoassay. Results: The addition of α‐melanotropin in a dose of 0.01 and 0.1 mm had no effect on progesterone release, whereas a dose of 1 mm significantly increased progesterone release (P < 0.01) compared with the control culture. Progesterone release was not modified when different concentrations of interleukin‐1β or TNF‐α were added to the cell cultures. However, when interleukin‐1β or TNF‐α were added simultaneously with 1 μm α‐melanotropin, a significant reduction (P < 0.01 for interleukin‐1β and P < 0.05 for TNF‐α) of the steroid release was found with respect to the α‐melanotropin‐treated group. Conclusions: These results lead us to suggest that, although α‐melanotropin stimulates progesterone release in pre‐ovulatory GC, this effect is blocked by the presence of interleukin‐1β or TNF‐α.     

IL‐17‐producing γδ T cells

IL‐17 is produced not only by CD4⁺ αβ T cells, but also CD8⁺ αβ T cells, NKT cells, and γδ T cells, plus some non‐T cells, including macrophages and neutrophils. The ability of IL‐17 to deploy neutrophils to sites of inflammation imparts this cytokine with a key role in diseases of several types. Surprisingly, γδ T cells are responsible for much of the IL‐17 produced in several disease models, particularly early on.     

Significant involvement of nuclear factor‐κB‐inducing kinase in proper differentiation of αβ and γδ T cells

Nuclear factor‐κB‐inducing kinase (NIK) is known to play a critical role in maintaining proper immune function. This is exemplified in the spontaneous mutant mouse lacking functional NIK, alymphoplasia (aly), which is simultaneously immune‐compromised and autoimmune‐prone. To investigate the role of NIK in αβ T‐cell repertoire formation, we analysed T‐cell development in aly/aly mice bearing a transgenic T‐cell receptor (TCR). Although there were no apparent abnormalities in the mature αβ T cells of non‐transgenic aly/aly mice, the maturation efficiency of idiotype^high+ T cells in the TCR‐transgenic mice was lower in aly/aly mice compared with those found in aly/+ mice, suggesting that the mature αβ T‐cell repertoire could be altered by the absence of functional NIK. In one strain of TCR‐transgenic aly/aly mice with a negatively selecting H‐2 background, the proportion of CD8^low+ idiotype^high+ cells, which are thought to potentially represent the γδ lineage of T cells, was markedly decreased. When the γδ T cells in non‐transgenic aly/aly mice were investigated, the proportion of γδ T cells in the peripheral organs of aly/aly mice was found to be one‐half to one‐fifth of those in aly/+ mice. Analyses of bone marrow chimera mice indicated that NIK in host cells, rather than in donor cells was important for generating a normal number of peripheral γδ T cells. Collectively, these results suggest that NIK could be involved in thymic positive selection of some αβ T cells and that NIK in non‐haematopoietic cells is important for the optimal development and/or maintenance of γδ T cells.     

Synthesis of New Homopolyester and Copolyesters by Anionic Ring‐opening Polymerization of α,α′,β‐Trisubstituted β‐Lactones

Summary: Novel polyester and copolyesters have been prepared by anionic ring‐opening polymerization of racemic 4‐alkyloxycarbonyl‐3,3‐dimethyl‐2‐oxetanones that had been synthesized in five steps from diethyl oxalpropionate used as chemical precursor. The anionic polymerizations, realized in bulk or in solution with tetraethylammonium benzoate as initiator, led to a homopolymer and copolymers with high molecular weights and polydispersity indices close to unity. These features can be explained by the presence of two methyl groups on the same carbon atom in the lactone, preventing transfer reactions to the monomer. Preparation of seeds and re‐initiation by addition of fresh monomer confirmed a living process. The hydrolysis of poly[(R,S)‐3,3‐dimethylmalic acid] under physiological conditions yielded (R,S)‐3,3‐dimethylmalic acid. A terpolymer was also prepared for biological studies related to its use as biodegradable materials for tissue regeneration.

Structure of poly[(R,S)‐3,3‐dimethylmalic acid].     

Inhibition of murine γδ lymphocyte expansion and effector function by regulatory αβ T cells is cell‐contact‐dependent and sensitive to GITR modulation

γδ T cells are highly cytolytic lymphocytes that produce large amounts of pro‐inflammatory cytokines during immune responses to multiple pathogens. Furthermore, their ability to kill tumor cells has fueled the development of γδ‐T‐cell‐based cancer therapies. Thus, the regulation of γδ‐T‐cell activity is of great biological and clinical relevance. Here, we show that murine CD4⁺CD25⁺ αβ T cells, the vast majority of which express the Treg marker, Foxp3, abolish key effector functions of γδ T cells, namely the production of the pro‐inflammatory cytokines, IFN‐γ and IL‐17, cytotoxicity, and lymphocyte proliferation in vitro and in vivo. We further show that suppression is dependent on cellular contact between Treg and γδ T cells, results in the induction of an anergic state in γδ lymphocytes, and can be partially reversed by manipulating glucocorticoid‐induced TNF receptor‐related protein (GITR) signals. Our data collectively dissect a novel mechanism by which the expansion and pro‐inflammatory functions of γδ T cells are regulated.     

ERRα augments HIF‐1 signalling by directly interacting with HIF‐1α in normoxic and hypoxic prostate cancer cells

Adaptation of cancer cells to a hypoxic microenvironment is important for their facilitated malignant growth and advanced development. One major mechanism mediating the hypoxic response involves up‐regulation of hypoxia‐inducible factor 1 (HIF‐1) expression, which controls reprogramming of energy metabolism and angiogenesis. Oestrogen‐related receptor‐α (ERRα) is a pivotal regulator of cellular energy metabolism and many biosynthetic pathways, and has also been proposed to be an important factor promoting the Warburg effect in advanced cancer. We and others have previously shown that ERRα expression is increased in prostate cancer and is also a prognostic marker. Here we show that ERRα is oncogenic in prostate cancer and also a key hypoxic growth regulator. ERRα‐over‐expressing prostate cancer cells were more resistant to hypoxia and showed enhanced HIF‐1α protein expression and HIF‐1 signalling. These effects could also be observed in ERRα‐over‐expressing cells grown under normoxia, suggesting that ERRα could function to pre‐adapt cancer cells to meet hypoxia stress. Immunoprecipitation and FRET assays indicated that ERRα could physically interact with HIF‐1α via its AF‐2 domain. A ubiquitination assay showed that this ERRα–HIF‐1α interaction could inhibit ubiquitination of HIF‐1α and thus reduce its degradation. Such ERRα–HIF‐1α interaction could be attenuated by XCT790, an ERRα‐specific inverse agonist, resulting in reduced HIF‐1α levels. In summary, we show that ERRα can promote the hypoxic growth adaptation of prostate cancer cells via a protective interaction with HIF‐1α, suggesting ERRα as a potential therapeutic target for cancer treatment. Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Frequency and clonality of peripheral γδ T cells in psoriasis patients receiving anti‐tumour necrosis factor‐α therapy

Hepatosplenic γδ T cell lymphoma (HSTCL) has been observed in patients with Crohn's disease (CD) who received anti‐tumour necrosis factor (TNF)‐α agents and thiopurines, but only one case was reported in a psoriasis patient worldwide. This difference could be due to differences in either the nature of the inflammatory diseases or in the use of immunomodulators. We investigated the impact of anti‐TNF‐α agents on the level and repertoire of γδ T cells in peripheral blood from psoriasis patients. Forty‐five men and 10 women who were treated with anti‐TNF‐α agents for psoriasis were monitored for a median 11 months for the level and clonality of γδ T cells via flow cytometry and polymerase chain reaction (PCR) analysis of T cell receptor gamma (TCR‐γ) gene rearrangements. Seventeen men had a repeated analysis within 48 h of the infliximab infusion to reveal a possible expansion of γδ T cells, as observed previously in CD patients. Ten psoriasis patients who were never exposed to biologicals and 20 healthy individuals served as controls. In the majority of psoriasis patients, the level and clonal pattern of γδ T cells was remarkably stable during infliximab treatment. A single male patient repeatedly experienced a significant increase in the level of γδ T cells after infliximab infusions. A monoclonal γδ T cell repertoire in a polyclonal background tended to be more frequent in anti‐TNF‐α‐treated patients than naive patients, suggesting that anti‐TNF‐α therapy may promote the clonal selection of γδ T cells in psoriasis patients.     

Protective role of γ/δ T cells and α/β T cells in tuberculosis

Tuberculosis is a chronic infectious disease which causes major health problems globally. Although acquired resistance crucially depends on α/β lymphocytes, circumstantial evidence suggests that, in addition, γ/δ T lymphocytes contribute to protection against tuberculosis. We have studied Mycobacterium tuberculosis infection in TcR-δ-/- or TcR-β-/- gene deletion mutants which completely lack γ/δ T cells or α/β T cells, respectively. Low inocula of M. tuberculosis led to death of TcR-β-/- mice and transient disease exacerbation in TcR-δ-/- mutants. Infection with higher inocula caused rapid death of TcR-δ-/- mice. The development of and bacterial containment in granulomatous lesions was markedly impaired in TcR-β-/-, and less severly affected in TcR-δ-/- mutants. Mycobacteria-induced IFN-γ production by spleen cells in vitro was almost abolished in TcR-β-/- and virtually unaffected in TcR-δ-/- mice. Our data confirm the crucial role of α/β T cells in protection against established tuberculosis and formally prove a protective role of γ/δ T cells in early tuberculosis.     

γδ T cells are secondary participants in acute graft-versus-host reactions initiated by CD4+ αβT cells

To examine the role of T cell subpopulations in an acute graft-versus-host (GVH) reaction, γδ T cells and αβ T cells expressing one of the two prototypic Vβ gene families were negatively isolated from adult blood samples and injected into allogeneic chick embryos. CD4⁺ αβ T cells expressing either Vβ1 or Vβ2 receptors were equally capable of inducing acute GVH reactions, consistent with the idea that αβ T cell alloreactivity is determined by CDR3 variability. By themselves, the γδ T cells were incapable of inducing GVH reactions. However, host γδ T cells were recruited into the donor αβ T cell-initiated lesions, where they were activated and induced to proliferate. The data suggest that γβ T cells may play a secondary role in GVH reactions.     

γδ T cells restrain extrathymic development of Foxp3+‐inducible regulatory T cells via IFN‐γ

Inducible Treg (iTreg) cells generated from Ag‐stimulated naïve CD4⁺ T cells in the periphery play an important role in regulating immune responses. TGF‐β is a key cytokine that promotes this conversion process; however, how this process is regulated in vivo remains unclear. Here, we report that γδ T cells play a crucial role in controlling iTreg generation and suppressor function. Ag‐induced iTreg generation was significantly enhanced in C57BL/6 mice in the absence of γδ T cells. Inhibition of iTreg conversion was mediated by IFN‐γ produced by activated γδ T cells but not by activated CD4⁺ T cells. BM chimera experiments further confirmed γδ‐derived IFN‐γ‐dependent mechanism in regulating iTreg generation in vivo. Lastly, human peripheral blood γδ T cells also interfere with iTreg conversion via IFN‐γ. Our results suggest a novel function of γδ T cells in limiting the generation of iTreg cells, potentially balancing immunity and tolerance.     

DL4‐mediated Notch signaling is required for the development of fetal αβ and γδ T cells

T‐cell development depends upon interactions between thymocytes and thymic epithelial cells (TECs). The engagement of delta‐like 4 (DL4) on TECs by Notch1 expressed by blood‐borne BM‐derived precursors is essential for T‐cell commitment in the adult thymus. In contrast to the adult, the earliest T‐cell progenitors in the embryo originate in the fetal liver and migrate to the nonvascularized fetal thymus via chemokine signals. Within the fetal thymus, some T‐cell precursors undergo programmed TCRγ and TCRδ rearrangement and selection, giving rise to unique γδ T cells. Despite these fundamental differences between fetal and adult T‐cell lymphopoiesis, we show here that DL4‐mediated Notch signaling is essential for the development of both αβ and γδ T‐cell lineages in the embryo. Deletion of the DL4 gene in fetal TECs results in an early block in αβ T‐cell development and a dramatic reduction of all γδ T‐cell subsets in the fetal thymus. In contrast to the adult, no dramatic deviation of T‐cell precursors to alternative fates was observed in the fetal thymus in the absence of Notch signaling. Taken together, our data reveal a common requirement for DL4‐mediated Notch signaling in fetal and adult thymopoiesis.     

Alpha‐defensins (α‐Defs) in Crohn's disease: decrease of ileal α‐Def 5 via permanent methylation and increase in plasma α‐Def 1‐3 concentrations offering biomarker utility

An impaired expression of α‐defensins (α‐Defs) in the ileal mucosa and, conversely, increased levels in plasma, have been reported in Crohn's disease (CD). However, the specificity and correlation of these findings with the degree of inflammation are unclear. We aimed to characterize the concentration and utility of ileal and plasma α‐Defs in CD and to analyse a potential epigenetic mechanism of α‐Def expression. Peripheral blood samples and ileal biopsies were obtained from patients at disease onset (aCD), from those who achieved remission (iCD) and from two control groups (healthy controls and non‐CD‐aetiology ileitis patients). Plasma α‐Defs 1–3 and 4 were detected by enzyme‐linked immunosorbent assay (ELISA); α‐Def 5 by immunolocalization. Methylation analysis of the α‐Def 5 gene was performed using the MassARRAY EpiTYPER system. Plasma α‐Defs 1–3 concentrations were significantly higher in aCD with ileal involvement (L1, L3) versus iCD or the control groups. The α‐Defs 1–3 concentrations were also similar to healthy controls in patients with non‐CD ileitis. There was a significant positive correlation between plasma α‐Defs 1–3 levels in aCD and the endoscopic index, as well as with C‐reactive protein (CRP) levels. The immunopositivity scoring showed significantly reduced α‐Def 5 expression in ileal inflamed (aCD) versus non‐inflamed mucosa (iCD and healthy controls). The α‐Def 5 gene showed a higher methylation status in CD patients than controls, regardless of the inflammation. Plasma α‐Defs 1–3 concentrations correlate with the degree of inflammation and appear to be specific biomarkers of ileal‐CD at diagnosis. Ileal α‐Def 5 expression is down‐regulated permanently by methylation.     

Molecular Motions in the Supramolecular Complexes between Poly(ε‐caprolactone)‐Poly(ethylene oxide)‐Poly(ε‐caprolactone) and α‐ and γ‐Cyclodextrins

The structure and molecular motions of the triblock copolymer PCL‐PEO‐PCL and its inclusion complexes with α‐ and γ‐cyclodextrins (α‐ and γ‐CDs) have been studied by solid‐state NMR. Different cross‐polarization dynamics have been observed for the guest polymer and host CDs. Guest–host magnetization exchange has been observed by proton spin lattice relaxation T₁, proton spin lattice frame relaxation T_1ρ and 2D heteronuclear correlation experiments. A homogeneous phase has been observed for these complexes. Conventional relaxation experiments and 2D wide‐line separation NMR with windowless isotropic mixing have been used to measure the chain dynamics. The results show that for localized molecular motion in the megahertz regime, the included PCL block chains are much more mobile than the crystalline PCL blocks in the bulk triblock copolymer. However, the mobility of the included PEO block chains is not very different from the amorphous PEO blocks of the bulk sample. The cooperative, long chain motions in the mid‐kilohertz regime for pairs of PCL‐PEO‐PCL chains in their γ‐CD channels seem more restricted than for the single PCL‐PEO‐PCL chains in the α‐CD channels, however, they are not influencing the more localized, higher frequency megahertz motions.     

STS‐1 promotes IFN‐α induced autophagy by activating the JAK1‐STAT1 signaling pathway in B cells

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the overexpression of IFN‐α. IFN‐α induces autophagy via the JAK1‐STAT1 signaling pathway, contributing to the pathogenesis of SLE. Recent studies reported that B cells from patients with SLE and NZB/W F1 mice had enhanced autophagy activity; however, the mechanism still remains unknown. Here, we show that the protein tyrosine phosphatase STS‐1 (suppressor of T‐cell receptor signaling 1) was significantly overexpressed in B cells from patients with SLE and MRL/lpr mice. Notably, STS‐1 promoted IFN‐α‐induced autophagy in B cells by enhancing the JAK1‐STAT1 signaling activation. STS‐1 inhibited the phosphorylation of the E3 ubiquitin protein ligase c‐cbl, and subsequently promoted IFN‐α‐induced phosphorylation of tyrosine kinase 2, leading to JAK1‐STAT1 signaling activation. Furthermore, STAT1 and JAK1 inhibitors blocked the IFN‐α‐induced autophagy promoted by STS‐1, indicating that STS‐1 promotes IFN‐α‐induced autophagy via the JAK1‐STAT1 signaling. Our results demonstrate the importance of STS‐1 in regulating IFN‐α‐induced autophagy in B cells, and this could be used as a therapeutic approach to treat SLE.