Molecular Mechanisms of IL18 in Disease.

Interleukin 18 (IL18) was originally identified as an inflammation-induced cytokine that is secreted by immune cells. An increasing number of studies have focused on its non-immunological functions, with demonstrated functions for IL18 in energy homeostasis and neural stability. IL18 is reportedly required for lipid metabolism in the liver and brown adipose tissue. Furthermore, IL18 (Il18) deficiency in mice leads to mitochondrial dysfunction in hippocampal cells, resulting in depressive-like symptoms and cognitive impairment. Microarray analyses of Il18-/- mice have revealed a set of genes with differential expression in liver, brown adipose tissue, and brain; however, the impact of IL18 deficiency in these tissues remains uncertain. In this review article, we discuss these genes, with a focus on their relationships with the phenotypic disease traits of Il18-/- mice.

Significant association between high serum CCL5 levels and better disease-free survival of patients with early breast cancer.

Analysis of anticancer immunity aids in assessing the prognosis of patients with breast cancer. From 250 operated breast cancers, we focused on serum levels of C-C motif chemokine ligand 5 (CCL5), which is involved in cancer immune reactions. Serum levels of CCL5 were measured using a cytometric bead-based immunoassay kit and CCL5 expression in cancer cells was determined using immunohistochemical staining. In addition, mRNA in cancer and stromal cells was analyzed by microdissection and comparison with the public dataset. Disease-free survival (DFS) of patients with high CCL5 levels (cut-off, 13.87 ng/mL; n = 192) was significantly better than those with low CCL5 levels (n = 58; hazard ratio, 0.20; 95% confidence interval, 0.10-0.39; P < .0001). An improved overall survival was observed in patients with high CCL5 levels compared to those with low CCL5 levels (P = .024). On the contrary, high immunohistochemical expression of CCL5 in cancer cells was significantly associated with decreased DFS. As serum CCL5 levels did not correlate with CCL5 expression in cancer cells and the relative expression of mRNA CCL5 was elevated in stromal cells in relation to cancer cells, serum CCL5 might be derived not from cancer cells, but from stromal cells. Expression of CCL5 in serum, but not in cancer cells, might contribute to improved patient prognosis mediating through not only immune reaction, but through other mechanisms. Determination of circulating CCL5 levels could be useful for predicting patient prognosis.

IL-12 regulates the expansion, phenotype, and function of murine NK cells activated by IL-15 and IL-18.

NK cells, which are composed of phenotypically and functionally heterogeneous subpopulations, play critical roles in immunity against cancer. The mechanism of generation of distinct subsets such as the effector and regulatory subtypes is unclear. Here, we show that this process comprises several steps, including generation of proliferating, highly cytotoxic cells activated by IL-15/IL-18 and differentiation into distinct cell populations induced with IL-12. Freshly prepared murine splenic NK cells expressed IL-15Rs and IL-18Rs and rapidly began to proliferate following stimulation with IL-15/IL-18. The proliferating NK cells highly expressed various activation markers such as B220, CD49b (DX5), lysosome-associated membrane glycoprotein 1 (LAMP-1), DNAX accessory molecule 1, perforin, and granzyme B and showed reduced expression of natural killer cell p46-related protein (NKp46) and IL-18Rα. These cells exerted strong cytotoxicity against YAC-1 cells, but did not secrete cytokines. IL-12 rapidly activated STAT4 in these cells, induced IFN-γ production, and then upregulated p21 and p27, leading to withdrawal from the cell cycle. In parallel, IL-12-stimulated cells gradually reduced cytotoxicity, decreased expression of activation markers, and instead increased expression of Sca-1, CD25, CD49a, and NKp46. Some IL-15/IL-18-induced cells strongly expressed PD-1, whereas NK cells induced with IL-15/IL-18 and IL-12 expressed high levels of T cell immunoglobulin mucin-3, LAG-3, and natural killer group 2 A. Furthermore, these cells spontaneously secreted IL-10 and TGF-ß following prolonged incubation. Thus, IL-12 regulates expansion of NK cells activated with IL-15/IL-18, influences the population size of highly cytotoxic cells, and induces differentiation to unique cells sharing some phenotypes of ILCs.

Impaired function of aorta and perivascular adipose tissue in IL-18-deficient mice.

IL-18 is ubiquitously produced by both hematopoietic and non-hematopoietic cells. The present study examined the thoracic aorta, including the surrounding perivascular adipose tissue (PVAT), of IL-18KO mice from functional and histological perspectives. IL-18KO mice exhibited raised blood pressure compared with wild-type mice. Echocardiographic examination showed a thickened vascular wall and narrowed vascular diameter of the aorta. Examination by the Magnus test demonstrated dysfunction of endothelial cells (ECs) in the IL-18KO thoracic aorta and impairment of the anticontractile function of IL-18KO PVAT. Histological examination showed no inflammatory lesions in the aorta but indicated progressive fibrosis in the vessel and conversion of PVAT from brown adipose tissue-like features to white adipose tissue-like features. Electron microscopic observation suggested several deformed mitochondria in the aorta and vacuole-like structures in ECs from IL-18KO mice. In addition, activity of complex IV was lower and production of reactive oxygen species was augmented in the mitochondria of IL-18KO aorta. Although expression of LC3 B was higher, rapamycin-induced autophagy flux was impaired in the IL-18KO PVAT. Moreover, Western blot analysis revealed that LAMP 1/2 was increased in IL-18KO PVAT, and measurement of cathepsin-D activity indicated decreased levels in IL-18KO PVAT. The IL-18KO thoracic aorta thus showed defects in physiological functions related to histological alterations, and the inflammasome/IL-18 system was suggested to play a protective role in cardiovascular cells, probably through quality control of mitochondria via promotion of autophagosome/autophagolysosome formation.NEW & NOTEWORTHY IL-18 deficiency caused aortic abnormalities in terms of morphology and functions in parallel with an accumulation of damaged mitochondria and anomalous turnover of protein complexes, such as PGC-1 and LAMP1 and -2 in PVAT. These findings suggested that IL-18 plays roles in maintaining the homeostasis of vessels and PVAT around the aorta, possibly by promoting autophagy.

Low-intensity walking as mild medication for pressure control in prehypertensive and hypertensive subjects: how far shall we wander?

Successful prevention and treatment of hypertension depend on the appropriate combination of antihypertensive drug therapy and nondrug lifestyle modification. While most hypertension guidelines recommend moderate- to high-intensity exercise, we decided to explore a mild yet effective type of exercise to add to hypertension management, especially in populations with complications or frailty. After comparing the short-term cardiovascular effects of low-speed walking versus high-speed walking for 3 kilometers (km) (3 km/h versus 6 km/h) in young, healthy volunteers, we delivered low-speed walking (low-intensity walking, 2.5 metabolic equivalents of task, METs) as exercise therapy in 42 prehypertensive and 43 hypertensive subjects. We found that one session of 3 km low-intensity walking exerted a transient pressure-lowering effect as well as a mild negative chronotropic effect on heart rate in both the prehypertensive and hypertensive subjects; these short-term benefits on blood pressure and heart rate were accompanied by a brief increase in urine ß-endorphin output. Then we prescribed regular low-intensity walking with a target exercise dose (exercise volume) of 500-1000 METs·min/week (50-60 min/day and 5-7 times/week) in hypertensive subjects in addition to their daily activities. Regular low-intensity walking also showed mild but significant blood pressure-lowering and heart rate-reducing effects in 7 hypertensive subjects within two months. It is hypothesized that regular low-intensity exercise of the necessary dose could be taken as a pragmatic and supplementary medication for hypertension management.

Expansion of human γδ T cells for adoptive immunotherapy using a bisphosphonate prodrug.

Cancer immunotherapy with human γδ T cells expressing Vγ2Vδ2 T cell receptor (also termed Vγ9Vδ2) has shown promise because of their ability to recognize and kill most types of tumors in a major histocombatibility complex (MHC) -unrestricted fashion that is independent of the number of tumor mutations. In clinical trials, adoptive transfer of Vγ2Vδ2 T cells has been shown to be safe and does not require preconditioning. In this report, we describe a method for preparing highly enriched human Vγ2Vδ2 T cells using the bisphosphonate prodrug, tetrakis-pivaloyloxymethyl 2-(thiazole-2-ylamino)ethylidene-1,1-bisphosphonate (PTA). PTA stimulated the expansion of Vγ2Vδ2 cells to purities up to 99%. These levels were consistently higher than those observed after expansion with zoledronic acid, the most commonly used stimulator for clinical trials. Cell numbers also averaged more than those obtained with zoledronic acid and the expanded Vγ2Vδ2 cells exhibited high cytotoxicity against tumor cells. The high purity of Vγ2Vδ2 cells expanded by PTA increased engraftment success in immunodeficient NOG mice. Even low levels of contaminating αß T cells resulted in some mice with circulating human αß T cells rather than Vγ2Vδ2 cells. Vγ2Vδ2 cells from engrafted NOG mice upregulated CD25 and secreted tumor necrosis factor-α and interferon-γ in response to PTA-treated tumor cells. Thus, PTA expands Vγ2Vδ2 T cells to higher purity than zoledronic acid. The high purities allow the successful engraftment of immunodeficient mice without further purification and may speed up the development of allogeneic Vγ2Vδ2 T cell therapies derived from HLA-matched normal donors for patients with poor autologous Vγ2Vδ2 T cell responses.

Physiological and molecular effects of interleukin-18 administration on the mouse kidney.

BACKGROUND: The cytokine interleukin-18 was originally identified as an interferon-γ-inducing proinflammatory factor; however, there is increasing evidence to suggest that it has non-immunological effects on physiological functions. We previously investigated the potential pathophysiological relationship between interleukin-18 and dyslipidemia, non-alcoholic fatty liver disease, and non-alcoholic steatohepatitis, and suggested interleukin-18 as a possible novel treatment for not only these diseases but also for cancer immunotherapy. Before clinical application, the effects of interleukin-18 on the kidney need to be determined. In the current study, we examined the kidney of interleukin-18 knockout (Il18-/-) mice and the effects of interleukin-18 on the kidney following intravenous administration of recombinant interleukin-18. METHODS: Il18-/- male mice were generated on the C57Bl/6 background and littermate C57Bl/6 Il18+/+ male mice were used as controls. To assess kidney damage, serum creatinine and blood urea nitrogen levels were measured and histopathological analysis was performed. For molecular analysis, microarray and quantitative reverse transcription PCR was performed using mice 6 and 12 weeks old. To evaluate the short- and long-term effects of interleukin-18 on the kidney, recombinant interleukin-18 was administered for 2 and 12 weeks, respectively. RESULTS: Compared with Il18+/+ mice, Il18-/- mice developed kidney failure in their youth-6 weeks of age, but the condition was observed to improve as the mice aged, even though dyslipidemia, arteriosclerosis, and higher insulin resistance occurred. Analyses of potential molecular mechanisms involved in the onset of early kidney failure in Il18-/- mice identified a number of associated genes, such as Itgam, Nov, and Ppard. Intravenous administration of recombinant interleukin-18 over both the short and long term showed no effects on the kidney despite significant improvement in metabolic diseases. CONCLUSIONS: Short- and long-term administration of interleukin-18 appeared to have no adverse effects on the kidney in these mice, suggesting that administration may be a safe and novel treatment for metabolic diseases and cancer.

Deficiency in interleukin-18 promotes differentiation of brown adipose tissue resulting in fat accumulation despite dyslipidemia.

BACKGROUND: The cytokine, interleukin-18 (IL-18), was originally identified as an interferon-γ-inducing proinflammatory factor; however, there is increasing evidence suggesting that it has non-immunological effects on physiological functions. We have previously investigated the potential pathophysiological relationship between IL-18 and dyslipidemia, non-alcoholic fatty liver disease and non-alcoholic steatohepatitis, which were mediated by lipid energy imbalance. Therefore, herein we focused on brown adipocytes (BAs) and brown adipose tissue (BAT) related to energy consumption as non-shivering thermogenesis. METHODS: Il18-/- male mice were generated on the C57Bl/6 background, and littermate C57Bl/6 Il18+/+ male mice were used as controls. To reveal the direct effect of IL-18, primary cell cultures derived from both mice were established. Moreover, for molecular analysis, microarray, quantitative reverse transcription PCR and western blotting were performed using 6 and 12 weeks old mice. To evaluate the short- and long-term effects of IL-18 on BAT, recombinant IL-18 was administered for 2 and 12 weeks, respectively. RESULTS: Compared with Il18+/+ mice, BAT of Il18-/- mice showed earlier differentiation and lipid accumulation. To examine the direct effect of IL-18 on BAT, BA cell cultures were established. Myogenic factor 5-expressing adipose precursor cells were extracted from Il18+/+ and Il18-/- mice. PR domain containing 16 (PRDM16), a differentiation inducer, was strongly expressed in Il18-/- BAs, and uncoupling protein 1, a thermogenic and differentiation marker, was upregulated, resulting in the promotion of BA differentiation. Moreover, PRDM16-dependent and independent molecules related to BAT function, such as fibroblast growth factor 21, were activated. These findings were confirmed by comparing Il18+/+ and Il18-/- mice at 6 and 12 weeks of age. Additional analyses of the molecular mechanisms influencing the 'Quantity of adipocytes' identified three associated genes, apolipoprotein C3 (Apoc3), insulin-induced gene 1 (Insig1) and vitamin D (1,25-dihydroxyvitamin D3) receptor (Vdr). Intravenous administration of IL-18 not only significantly improved the expression of some of these genes, but it also significantly decreased the adipocytes' size. CONCLUSIONS: This study demonstrated the critical function of IL-18 in differentiation and lipid metabolism in BAs. Furthermore, IL-18 may contribute to novel treatments by improving the energy imbalance.

Interleukin-18 and testosterone levels in men with metabolic syndrome.

OBJECTIVE: Interleukin 18 (IL-18) is an adipokine associated with obesity. Data about the relationship of IL-18 to the metabolic syndrome (MS) are still scarce. Low testosterone (T) levels are common in men with MS, but we did not find data about the levels of IL-18 in men with low T. The aim of this study was to determine the levels of IL-18 in men with MS with or without low T. PATIENTS AND METHODS: A total of 251 men were included in the study. Of them 218 had MS (IDF 2005) and they were divided according to their morning total testosterone (TT) level (cutoff 10.4 nmol/l) into two groups: MS-low T (N = 84) and MS-normal T (N = 134). The control group consisted of 33 men without MS and low T. IL-18 was determined in serum using enzyme-linked immunosorbent assay. A small group of eight men with MS and low T levels received testosterone therapy for three months and physical and laboratory parameters were monitored at the end of that period. RESULTS: MS men were at mean age (±SD) = 53.77 ± 9.59 years; body mass index (BMI) = 34.0 ± 6.3 kg/m2; and TT = 12.59 ± 5.66 nmol/l. The control group was at age = 52.12 ± 5.2 years (NS); BMI = 25.6 ± 2.4 kg/m2 (p < .001); and TT = 17.8 ± 5.68 nmol/l (p < .001), respectively. The levels of IL-18 were higher in the MS group - 345 pg/ml compared to the control one - 264 pg/ml (p < .01). There was no significant difference between MS-low T (330.6 pg/ml) and MS-normal T (350.2 pg/ml) subgroups. The MS-normal T differed more significantly from the control group (p < .001). Significant correlation of testosterone with IL-18 levels was not found. IL-18 correlated with parameters of obesity, lipids, fasting blood sugar (p < .05) and the number of criteria for MS (p < .001). Three months on T treatment showed improvement in obesity parameters and only in one patient IL-18 had clear reduction while the rest showed no change. CONCLUSIONS: In this study, higher IL-18 levels were found in the presence of MS compared to healthy men, but they did not differ between men having MS with or without LOH.

Dysfunction of mitochondria and deformed gap junctions in the heart of IL-18-deficient mice.

Interleukin-18 (IL-18) was discovered as an interferon-γ-inducing factor and has been regarded as a proinflammatory cytokine. However, IL-18 is ubiquitously expressed both in immune/inflammatory cells and in nonimmune cells, and its biological roles have not been sufficiently elucidated. Here, we demonstrate that IL-18-deficient [IL-18 knockout (KO)] mice have heart abnormalities that may be related to impaired autophagy. In endurance running tests, IL-18KO mice ran significantly shorter distances compared with wild-type (WT) mice. Echocardiographs indicated disability in the systolic and diastolic functions of the IL-18KO mouse heart. Immunostaining of connexin 43 showed heterogeneous localization of gap junctions in the lateral membranes of the IL-18KO cardiac myocytes. Western blotting analysis revealed decreased phosphorylated connexin 43 in the IL-18KO heart. Electron microscopy revealed unusual localization of intercalated disks, swollen or damaged mitochondria, and broad, indistinct Z-lines in the IL-18KO heart. In accordance with the morphological observation, mitochondrial respiratory function, including that of complexes I and IV, was impaired, and production of reactive oxygen species was augmented in IL-18KO hearts. Notably, levels of LC3-II were markedly lower in the IL-18KO hearts than in WT hearts. In the culture of cardiac myocytes of IL-18KO neonates, exogenous IL-18 upregulated LC3-II and increased the number of intact mitochondria with high mitochondrial membrane potential. These results indicated that IL-18 has roles apart from those as a proinflammatory cytokine in cardiac myocytes and suggested that IL-18 contributes to the homeostatic maintenance of mitochondrial function and gap-junction turnover in cardiac myocytes, possibly by upregulating autophagy.

Interleukin-18 deficiency protects against renal interstitial fibrosis in aldosterone/salt-treated mice.

Interleukin (IL)-18 is a member of the IL-1 family of cytokines and was described originally as an interferon γ-inducing factor. Aldosterone plays a central role in the regulation of sodium and potassium homoeostasis by binding to the mineralocorticoid receptor and contributes to kidney and cardiovascular damage. Aldosterone has been reported to induce IL-18, resulting in cardiac fibrosis with induced IL-18-mediated osteopontin (OPN). We therefore hypothesized that aldosterone-induced renal fibrosis via OPN may be mediated by IL-18. To verify this hypothesis, we compared mice deficient in IL-18 and wild-type (WT) mice in a model of aldosterone/salt-induced hypertension. IL-18(-/-) and C57BL/6 WT mice were used for the uninephrectomized aldosterone/salt hypertensive model, whereas NRK-52E cells (rat kidney epithelial cells) were used in an in vitro model. In the present in vivo study, IL-18 protein expression was localized in medullary tubules in the WT mice, whereas in aldosterone-infused WT mice this expression was up-regulated markedly in the proximal tubules, especially in injured and dilated tubules. This renal damage caused by aldosterone was attenuated significantly by IL-18 knockout with down-regulation of OPN expression. In the present in vitro study, aldosterone directly induced IL-18 gene expression in renal tubular epithelial cells in a concentration- and time-dependent manner. These effects were inhibited completely by spironolactone. IL-18 may be a key mediator of aldosterone-induced renal fibrosis by inducing OPN, thereby exacerbating renal interstitial fibrosis. Inhibition of IL-18 may therefore provide a potential target for therapeutic intervention aimed at preventing the progression of renal injury.

Stress-Induced Glucocorticoid Release Upregulates Uncoupling Protein-2 Expression and Enhances Resistance to Endotoxin-Induced Lethality.

OBJECTIVE: Although psychological and/or physiological stress has been well documented to influence immune responses, the precise mechanism for immunomodulation remains to be elucidated. The present work describes the role of the hypothalamic-pituitary-adrenal (HPA) axis in the mechanism of stress-mediated enhanced-resistance to lethality after lipopolysaccharide (LPS) injection. METHODS/RESULTS: Preconditioning with restraint stress (RS) resulted in enhanced activation of the HPA axis in response to LPS injection and suppressed LPS-induced release of proinflammatory cytokines and nitric oxide metabolites. Melanocortin 2 receptor-deficient mice (MC2R(-/-)) failed to increase plasma levels of glucocorticoids in response to LPS injection, and exhibited high sensitivity to LPS-induced lethality with enhanced release of proinflammatory cytokines as compared with MC2R(+/-) mice. Real-time PCR analysis revealed that RS induced upregulation of uncoupling protein-2 (UCP2) in macrophages in the lung and the liver of MC2R(+/-), but not of MC2R(-/-), mice. In addition, RS increased UCP2-dependent uncoupling activity of isolated mitochondria from the liver of MC2R(+/-), but not of MC2R(-/-), mice. In vitro study revealed that corticosterone and dexamethasone directly increased UCP2 expression in mouse RAW 264.7 macrophages and suppressed the generation of LPS-induced mitochondrial reactive oxygen species (ROS) and TNF-α production. Knockdown of UCP2 by small interfering RNA blunted the dexamethasone action for suppressing LPS-induced mitochondrial ROS and TNF-α production. CONCLUSION: The present work suggests that RS enhances activation of the HPA axis to release glucocorticoids and upregulation of UCP2 in macrophages, thereby increasing the resistance to endotoxin-induced systemic inflammation and death.

Estrogen decreases the expression of claudin-5 in vascular endothelial cells in the murine uterus.

Vascular endothelial (VE)-cadherin and claudin-5 are major components of the adherens and tight junctions of vascular endothelial cells, respectively, and decreases in their expression are associated with increases in endothelial paracellular permeability. In the uterus, estrogen induces endometrial edema. However, the in vivo effect of estrogen on endothelial paracellular permeability is unknown. Therefore, we studied the expression of VE-cadherin and claudin-5 in vascular endothelial cells in murine uteri stimulated by estrogen or progesterone. Ovariectomized mature mice were injected with estradiol-17ß (1 µg/mouse) or progesterone (1 mg/mouse) at intervals of 24 hours for 6 days. The frozen transverse sections of the uteri of these mice and untreated mice were stained for CD31 (vascular endothelial cell marker) plus VE-cadherin or claudin-5 using a double-immunofluorescence method. Then, the percentages of VE-cadherin- or claudin-5-positive vessels among CD31-positive vessels were examined in the uterine endometria. VE-cadherin and claudin-5 were expressed in most CD31-positive vessels in the endometria of the untreated mice. Progesterone did not affect the expression of both VE-cadherin and claudin-5 and estradiol-17ß also did not affect the VE-cadherin expression, but estradiol-17ß significantly decreased the claudin-5 expression. This decreasing effect of estradiol-17ß was detected from 24 hours later when the water content per a uterus significantly increased. The present study indicates that estrogen, but not progesterone, decreases the expression of claudin-5 in vascular endothelial cells in the murine uterine endometrium from 24 hours later, suggesting that the decrease in the claudin-5 expression contributes to the endometrial edema late after the estrogen stimulation.

Chymase inhibition attenuates lipopolysaccharide/ d-galactosamine-induced acute liver failure in hamsters.

BACKGROUND/AIMS: Chymase inhibition has been shown to attenuate matrix metalloproteinase (MMP)-9 and tumor necrosis factor (TNF)-α, both of which are associated with the pathogenesis of acute liver failure (ALF). This study investigated the effects of the chymase inhibitor TY-51469 on lipopolysaccharide (LPS)/D-galactosamine (GalN)-induced ALF in hamsters. METHODS: TY-51469 (10 or 30 mg/kg) or placebo was administered 1 h before the LPS (160 µg/kg)/GalN (400 mg/kg) injection. RESULTS: Hepatic chymase activity was significantly increased after the LPS/GalN injection, but the significant increase was dose-dependently and significantly attenuated by treatment with TY-51469. Significant increases in hepatic MMP-9 activity and TNF-α concentration were observed after the LPS/GalN injection, but these increases were also attenuated by treatment with TY-51469. Plasma aspartate aminotransferase and alanine aminotransferase activities were significantly increased after LPS/GalN injection in the placebo-treated group, but the increases were significantly attenuated in the TY-51469-treated group. The area of hepatic necrotic after LPS/GalN injection was significantly reduced by treatment with TY-51469. Treatment with TY-51469 resulted in significant reductions in the hepatic malondialdehyde concentration, mast cell numbers, and gene expressions of interleukin-1ß and myeloperoxidase. DISCUSSION: Chymase inhibition could be a useful strategy to attenuate LPS/GalN-induced ALF in hamsters.

Development of Innate-Immune-Cell-Based Immunotherapy for Adult T-Cell Leukemia-Lymphoma.

γδ T cells and natural killer (NK) cells have attracted much attention as promising effector cell subsets for adoptive transfer for use in the treatment of malignant and infectious diseases, because they exhibit potent cytotoxic activity against a variety of malignant tumors, as well as virus-infected cells, in a major histocompatibility complex (MHC)-unrestricted manner. In addition, γδ T cells and NK cells express a high level of CD16, a receptor required for antibody-dependent cellular cytotoxicity. Adult T-cell leukemia-lymphoma (ATL) is caused by human T-lymphotropic virus type I (HTLV-1) and is characterized by the proliferation of malignant peripheral CD4+ T cells. Although several treatments, such as chemotherapy, monoclonal antibodies, and allogeneic hematopoietic stem cell transplantation, are currently available, their efficacy is limited. In order to develop alternative therapeutic modalities, we considered the possibility of infusion therapy harnessing γδ T cells and NK cells expanded using a novel nitrogen-containing bisphosphonate prodrug (PTA) and interleukin (IL)-2/IL-18, and we examined the efficacy of the cell-based therapy for ATL in vitro. Peripheral blood samples were collected from 55 patients with ATL and peripheral blood mononuclear cells (PBMCs) were stimulated with PTA and IL-2/IL-18 for 11 days to expand γδ T cells and NK cells. To expand NK cells alone, CD3+ T-cell-depleted PBMCs were cultured with IL-2/IL-18 for 10 days. Subsequently, the expanded cells were examined for cytotoxicity against ATL cell lines in vitro. The proportion of γδ T cells in PBMCs was markedly low in elderly ATL patients. The median expansion rate of the γδ T cells was 1998-fold, and it was 12-fold for the NK cells, indicating that γδ T cells derived from ATL patients were efficiently expanded ex vivo, irrespective of aging and HTLV-1 infection status. Anti-CCR4 antibodies enhanced the cytotoxic activity of the γδ T cells and NK cells against HTLV-1-infected CCR4-expressing CD4+ T cells in an antibody concentration-dependent manner. Taken together, the adoptive transfer of γδ T cells and NK cells expanded with PTA/IL-2/IL-18 is a promising alternative therapy for ATL.

IL-18 primes T cells with an antigen-inexperienced memory phenotype for proliferation and differentiation into effector cells through Notch signaling.

Recent studies have revealed that a subset of CD8+ T cells exhibit innate features and can be activated by cytokines. However, the precise mechanisms underlying the proliferation and differentiation of these cells remain unclear. Here, we demonstrated that CD44highCD8+ T cells in the mouse spleen express functional interleukin-18 (IL-18) receptors, whereas CD44lowCD8+ T cells do not. In response to IL-18 stimulation, these cells activated various metabolic pathways, upregulated the expression of surface molecules, such as c-Kit (CD117), CD25, and PD-1, and induced progression through the G1/S phase in the cell cycle. IL-18-primed cells, expressing a high-affinity receptor for IL-2, exhibited robust proliferation in response to IL-2 and underwent differentiation into effector cells. The splenic CD44highCD8+ T cells exhibited high expression levels of CD122, CD62L, CCR7, and CXCR3, along with CD5, indicating their potential for migration to the lymph nodes, where they could undergo expansion and terminal differentiation into effector cells. Additionally, in a tumor model, administration of IL-18 increased the accumulation of CD8+ T cells in both the lymph nodes and tumors. It is noteworthy that stimulation of CD44highCD8+ T cells with IL-18 upregulated the Notch-1 receptor and c-Myc. Moreover, inclusion of γ-secretase inhibitors attenuated the effect of IL-18 on both proliferation and interferon-γ production in the cells. These results demonstrate that IL-18 primes CD44highCD122highCXCR3highCD62LhighCD8+ T cells for expansion and differentiation into effector cells in a Notch signaling-dependent manner.

Meteorin-like Protein and Zonulin in Polycystic Ovary Syndrome: Exploring Associations with Obesity, Metabolic Parameters, and Inflammation.

OBJECTIVE: Polycystic ovary syndrome (PCOS) is a prevalent hormonal and metabolic disorder, wherein the adipose tissue and gut microbiome have been demonstrated to contribute to its pathogenesis. This study aims to assess the concentrations of the adipokine, meteorin-like protein (Metrnl) and the protein, zonulin, related to intestine permeability, in individuals with PCOS with a particular emphasis on their relationship with obesity, clinical manifestations, hormonal profiles, and metabolic parameters. METHODS: A cohort comprising 58 women with PCOS, classified according to the Rotterdam criteria, was enrolled. The study also considered age, body mass index (BMI), and ethnicity-matched controls (n = 30). Comprehensive anthropometric and clinical evaluations, hormonal assays, and biochemical analyses were conducted during the follicular phase. Subsequent subgroup analyses were executed within the PCOS cohort based on waist-to-height ratio (WHtR), insulin resistance (IR), and free androgen index (FAI). Serum concentrations of Metrnl and zonulin were quantified via the enzyme-linked immunosorbent assay (ELISA) technique. RESULTS: The Metrnl and zonulin levels exhibited no significant disparity between PCOS patients and controls. Nevertheless, within the entire participant cohort and the PCOS group exclusively, overweight/obese participants demonstrated higher Metrnl concentrations relative to their normal-weight counterparts (p < 0.001, p = 0.001, respectively). Furthermore, higher Metrnl concentrations were identified in subgroups characterized by high WHtR and IR in comparison to those with low WHtR (p = 0.001) and without IR (p = 0.001), respectively. A correlation emerged between Metrnl levels and various anthropometric and metabolic parameters, as well as sex-hormone-binding globulin (SHBG) and interleukin-18 (IL-18) within the PCOS group. Multiple linear regression analysis identified HOMA-IR as the sole independent predictor of Metrnl levels. CONCLUSION: While Metrnl and zonulin levels do not serve as diagnostic indicators of PCOS, elevated Metrnl concentrations exhibited robust associations with proinflammatory and metabolic irregularities within the PCOS population.

Analysis of the Effector Functions of Vδ2 γδ T Cells and NK Cells against Cholangiocarcinoma Cells.

Cholangiocarcinoma (CCA) is a rare disease characterized by malignant cells derived from the epithelial cells of the biliary duct system. Despite extensive treatments, the prognosis for CCA remains poor, emphasizing the critical need for the development of novel treatments. Considerable attention has been directed towards innate immune effector cells, which can recognize tumor cells independently of the major histocompatibility complex, laying the foundation for the development of off-the-shelf drugs. In this study, we cultured innate immune cells obtained from the peripheral blood of healthy adults and conducted a comparative analysis of the effector functions against CCA cell lines by Vδ2 γδ T cells and NK cells. This analysis was performed using standard short- and long-term cytotoxicity assays, as well as ELISA for IFN-γ. Vδ2 γδ T cells demonstrated cytotoxicity and IFN-γ production in response to CCA cells in a TCR-dependent manner, particularly in the presence of tetrakis-pivaloyloxymethyl 2-(thiazole-2-ylamino)ethylidene-1,1-bisphosphonate, a bisphosphonate prodrug. In contrast, direct killing and antibody-dependent cellular cytotoxicity were relatively slow and weak. Conversely, NK cells displayed potent, direct cytotoxicity against CCA cells. In summary, both Vδ2 γδ T cells and NK cells show promise as innate immune effector cells for adoptive transfer therapy in the context of CCA.

Enhanced antitumor efficacy of fiber-modified, midkine promoter-regulated oncolytic adenovirus in human malignant mesothelioma.

Oncolytic virotherapy using adenoviruses has potential for therapeutic benefits in malignant mesothelioma. However, the downregulation of coxsackie virus/adenovirus receptor (CAR) expression is frequently a critical rate-limiting factor that impedes the effectiveness of adenovirus serotype 5 (Ad5)-based vectors in many cancer types. We evaluated CAR (Ad5 receptor) and CD46 (adenovirus serotype 35 [Ad35] receptor) expression in six human malignant mesothelioma cell lines. Very low CAR expression was observed in MSTO-211H and NCI-H2052 cells, whereas the other cell lines showed strong expression. In contrast, CD46 was highly expressed in all mesothelioma cell lines. On this basis, we replaced the CAR binding sequence of Ad5 with the CD46 binding sequence of Ad35 in the replication-defective adenoviruses and the tumor-specific midkine promoter-regulated oncolytic adenoviruses. By this fiber modification, the infectivity, virus progeny production, and in vitro cytocidal effects of the adenoviruses were significantly enhanced in low CAR-expressing MSTO-211H and NCI-H2052 cells, also resulting in similar or even higher levels in high CAR-expressing mesothelioma cell lines. In MSTO-211H xenograft models, the fiber-modified oncolytic adenovirus significantly enhanced antitumor effect compared to its equivalent Ad5-based vector. Our data demonstrate that Ad35 fiber modification of binding tropism in a midkine promoter-regulated oncolytic Ad5 vector confers transductional targeting to oncolytic adenoviruses, thereby facilitating more effective treatment of malignant mesothelioma.

Comparison of γδ T cell responses and farnesyl diphosphate synthase inhibition in tumor cells pretreated with zoledronic acid.

Exposing human tumor cells to nitrogen-containing bisphosphonates, such as zoledronic acid (Zol), greatly increases their susceptibility to killing by γδ T cells. Based on this finding and other studies, cancer immunotherapy using γδ T cells and nitrogen-containing bisphosphonates has been studied in pilot clinical trials and has shown benefits. Although Zol treatment can render a wide variety of human tumor cells susceptible to γδ T cell killing, there has not been a systematic investigation to determine which types of tumor cells are the most susceptible to γδ T cell-mediated cytotoxicity. In this study, we determined the Zol concentrations required to stimulate half maximal tumor necrosis factor-α production by γδ T cells cultured with various tumor cell lines pretreated with Zol and compared these concentrations with those required for half maximal inhibition of farnesyl diphosphate synthase (FPPS) in the same tumor cell lines. The inhibition of tumor cell growth by Zol was also assessed. We found that FPPS inhibition strongly correlated with γδ T cell activation, confirming that the mechanism underlying γδ T cell activation by Zol is isopentenyl diphosphate (IPP) accumulation due to FPPS blockade. In addition, we showed that γδ T-cell receptor-mediated signaling correlated with γδ T cell tumor necrosis factor-α production and cytotoxicity. Some lymphoma, myeloid leukemia, and mammary carcinoma cell lines were relatively resistant to Zol treatment, suggesting that assessing tumor sensitivity to Zol may help select those patients most likely to benefit from immunotherapy with γδ T cells.