miRNA-381-3p Functions as a Tumor Suppressor to Inhibit Gastric Cancer by Targeting Fibroblast Growth Factor Receptor-2.

Objectives: MicroRNAs possess essential effects on gastric cancer (GC), whereas the underlying mechanisms have not been fully uncovered. The present work focused on investigating the role of miR-381-3p in GC cellular processes and the possible mechanisms. Materials and Methods: miR-381-3p levels within GC tissues and cells were measured through quantitative real-time polymerase chain reaction (qRT-PCR). This study measured cell proliferation, apoptosis, and metastasis through EdU, colony formation, flow cytometry, and Transwell assays separately. TargetScan was adopted to predict the miR-381-3p targets, whereas luciferase reporter assay was adopted for confirmation. Results: miR-381-3p levels were decreased, whereas fibroblast growth factor receptor-2 (FGFR2) expression was increased in GC. miR-381-3p upregulation inhibited proliferation, migration, and invasion and it promoted the apoptosis of GC cells. Further, FGFR2 overexpression partly reversed the miR-381-3p-mediated impacts on GC cellular processes. Conclusions: This study provides an experimental basis, suggesting the potential of using miR-381-3p as the novel marker for GC. Clinical Trial Registration number: 2020-05.

Associations between NKX2-5 gene polymorphisms and congenital heart disease in the Chinese Tibetan population.

BACKGROUND: The pathogenesis of congenital heart disease (CHD) has not been fully elucidated, and this study considers the interaction between inheritance and the environment as the main cause of CHD. Previous studies have found that the incidence of CHD in the Tibetan plateau population is significantly higher than in low-altitude populations. Numerous reports have confirmed that NKX2-5 gene mutations can lead to coronary heart disease, but the relationship between NKX2-5 and Tibetan nationality has not yet been reported. OBJECTIVE: To explore the relationship between NKX2-5 gene polymorphisms and CHD in Tibetan people. METHODS: Blood samples were collected retrospectively from Tibetan patients diagnosed with CHD as well as healthy Tibetans, and the exons of NKX2-5 were sequenced. The MassARRAY technique was used to detect and genotype candidate tag single nucleotide polymorphisms (SNPs) in the non-coding regions of NKX2-5. RESULTS: Exon sequencing revealed no difference in the coding regions of the NKX2-5 gene between the CHD and control groups. In the non-coding regions of NKX2-5, rs6882776 and rs2546741 differed significantly between the two groups. Strong linkage disequilibrium was found between the selected sites of NKX2-5. CONCLUSIONS: The NKX2-5 exons do not associate with CHD in Tibetans. Rs6882776 and rs2546741 in the non-coding regions of NKX2-5 may protect against CHD in Tibetans. The NKX2-5 haplotype associated with CHD occurrence in the Tibetan population.

Hsa-let-7d-5p Promotes Gastric Cancer Progression by Targeting PRDM5.

Gastric cancer (GC) is a common malignant tumor in the digestive system and a significant health burden worldwide. In this study, we found that hsa-let-7d-5p was upregulated in GC cells, promoted GC cell proliferation, migration, and invasion, and reduced apoptosis. Moreover, we found that the expression of PRDM5 (PR domain protein 5) was downregulated in GC cells and upregulated in GC cells treated with hsa-let-7d-5p inhibitor. Further investigation showed that hsa-let-7d-5p was the target of PRDM5, and the functions of hsa-let-7d-5p on GC progression were rescued by PRDM5 overexpression in GC cells. Collectively, our findings suggested that hsa-let-7d-5p promoted the development of GC by targeting PRDM5, indicating that hsa-let-7d-5p could be a promising therapeutic molecule for the treatment of gastric cancer.

Leptin: an entry point for the treatment of peripheral tissue fibrosis and related diseases.

Leptin is a small peptide mainly secreted by adipocyte, which acts on the central nervous system of the hypothalamus to regulate the body's energy balance by inhibiting food intake, it also can directly act on specific cells through leptin receptors (for example, ObRa, which exists in the blood-brain barrier or kidneys), thereby affect cell metabolism. Excessive deposition of extracellular matrix (ECM) causes damage to normal tissues or destruction of organ structure, which will eventually lead to tissue or organ fibrosis. The sustainable development of fibrosis can lead to structural damage and functional decline of organs, and even exhaustion, which seriously threatens human health and life. In recent years, studies have found that leptin directly alleviates the fibrosis process of various tissues and organs in mammals. Therefore, we speculate that leptin may become a significant treatment for fibrosis of various tissues and organs in the future. So, the main purpose of this review is to explore the specific mechanism of leptin in the process of fibrosis in multiple tissues and organs, and to provide a theoretical basis for the treatment of various tissues and organs fibrosis and related diseases caused by it, which is of great significance in the future.

Lnc-hipk1 inhibits mouse adipocyte apoptosis as a sponge of miR-497.

Noncoding RNAs (ncRNAs) such as microRNAs (miRNAs), long noncoding RNAs (lncRNA), and circular RNAs are closely related to the biological processes related to obesity. As a miRNA that widely present in different cell types, miR497 is proved to be involved in cell development. However, research on the role of miR-497 as a key factor in regulating the development of adipocytes is still in gap. The role of miR-497 in the apoptosis and proliferation of mouse-derived adipocytes was detected by RNA-seq analysis, RT-qPCR, Western blot, immunofluorescence, and dual-luciferase reporter assay. Using miR-497 mimics to treat 3T3-L1 cells, we found that miR-497 targeted Bcl-2 to promote adipocyte apoptosis through the mitochondrial pathway, and this effect was consistent in the apoptosis model composed of palmitic acid (PA) and hydrogen peroxide (H2 O2 ). LncRNA homeodomain-interacting protein kinase 1 (lnc-hipk1) sponged miR-148b to weaken its silencing of Bcl-2, forming the competitive endogenous RNAs (CeRNAs) regulatory network. Furthermore, overexpression of lnc-hipk1 inhibited the apoptosis of adipocytes by targeting miR-497/Bcl-2. Co-treatment of miR-497 and lnc-hipk1 showed that lnc-hipk1 reversed the apoptosis of adipocytes caused by miR-497 overexpression. And in vivo experiments further confirmed that this effect was also achieved by the CeRNA system of lnc-hipk1/miR-497/Bcl-2. In summary, lnc-hipk1 targets miR-497/Bcl-2 to regulate adipocyte apoptosis through the mitochondrial pathway. This research enriches the research content of ncRNAs and CeRNA in adipocyte development, and provides new targets for the treatment of obesity and other metabolic syndromes.

The Mechanism of Leptin on Inhibiting Fibrosis and Promoting Browning of White Fat by Reducing ITGA5 in Mice.

Leptin is a small molecule protein secreted by adipocytes, which can promote white fat browning through activating the hypothalamic nervous system and inhibiting downstream signaling pathways. Moreover, white fat browning has been proven to alleviate fat tissue fibrosis. This study explores the mechanism of leptin in regulating adipose tissue fibrosis and white fat browning. After treating mice with leptin, we screened out the recombinant integrin alpha 5 (ITGA5) through proteomics sequencing, which may play a role in adipose tissue fibrosis. Through real-time quantitative PCR (qPCR), western blotting (WB), hematoxylin-eosin (HE) staining, Masson's trichrome, immunofluorescence, immunohistochemistry, etc., the results showed that after leptin treated adipocytes, the expression of fibrosis-related genes and ITGA5 was significantly down-regulated in adipocytes. We constructed fibrosis model through transforming growth factor-ß (TGF-ß) and a high-fat diet (HFD), and treated with ITGA5 overexpression vector and interference fragments. The results indicated the expression of fibrosis-related genes were significantly down-regulated after interfering with ITGA5. After treating adipocytes with wortmannin, fibrosis-related gene expression was inhibited after overexpression of ITGA5. Moreover, after injecting mice with leptin, we also found that leptin significantly up-regulated the expression of adipose tissue browning-related genes. Overall, our research shows that leptin can inhibit the activation of phosphatidylinositol 3 kinase (PI3K)-protein kinase B (AKT) signaling pathway by reducing the expression of ITGA5, which could alleviate adipose tissue fibrosis, and further promote white fat browning. Our research provides a theoretical basis for further research on the effect of leptin in fibrosis-related adipose tissue metabolism.

Collagen XV Promotes ER Stress-Induced Inflammation through Activating Integrin ß1/FAK Signaling Pathway and M1 Macrophage Polarization in Adipose Tissue.

Collagen XV (Col XV), a basement membrane (BM) component, is highly expressed in adipose tissue, and studies have found that Col XV is related to extracellular matrix (ECM) remodeling involving in adipose tissue fibrosis and inflammation. Furthermore, the ECM is essential for maintaining normal development and tissue function. In this study, we found that Col XV is related to the endoplasmic reticulum stress (ERS) and inflammation of adipose tissue. Moreover, we found that overexpression of Col XV in mice could cause macrophages to infiltrate white adipose tissue (iWAT). At the same time, the expression of the ERS sensor IRE1α (Inositol-Requiring Enzyme-1α) was significantly up-regulated, which intensified the inflammation of adipose tissue and the polarization of M1 macrophages after the overexpression of Col XV in mice. In addition, after overexpression of Col XV, the intracellular Ca2+ concentration was significantly increased. Using focal adhesion kinase (FAK) inhibitor PF573228, we found that PF-573228 inhibited the phosphorylation of FAK and reversed the upward trend of Col XV-induced protein expression levels of IRE1α, C/EBP-homologous protein (CHOP), and 78 kDa glucose-regulated protein (GRP78). After treatment with IRE1α inhibitor STF-083010, the results showed that the expression of adipocyte inflammation-related genes interleukin 6 (IL-6) and tumor necrosis factor α (TNFα) significantly were decreased. Our results demonstrate that Col XV induces ER-stress in adipocytes by activating the Integrinß1/FAK pathway and disrupting the intracellular Ca2+ balance. At the same time, Col XV regulates the inflammation induced by ER stress in adipocytes by promoting IRE1α/XBP1 (X-Box binding protein 1) signaling. Our study provides new ideas for solving the problems of adipose tissue metabolism disorders caused by abnormal accumulation of ECM.

Metabolic syndrome and its relation to dietary patterns among a selected urbanised and semi-urbanised Tibetan population in transition from nomadic to settled living environment.

OBJECTIVE: To explore the scope of metabolic syndrome (MetS) and its relationship to the major dietary patterns among an urbanised and semi-urbanised Tibetan population in transition from nomadic to settled settings. DESIGN: Cross-sectional. SETTING: Community-based. PARTICIPANTS: Urbanised and semi-urbanised Tibetan adults (n 920, aged 18-90 years), who have moved from nomadic to settled living environments, answered questionnaires on food consumption frequency and lifestyle characteristics through structured face-to-face interviews and completed anthropometric measurement and metabolic biomarker tests. RESULTS: MetS prevalence was 30·1 % in males and 32·1 % in females. Low HDL-cholesterol and central obesity were the leading metabolic abnormalities (86·3 and 55·8 %, respectively). Three major dietary patterns - urban, western and pastoral - were identified. Beef/mutton was an important food group for all three identified dietary patterns. In addition, the urban dietary pattern was characterised by frequent consumption of vegetables, tubers/roots and refined carbohydrates; the western pattern was characterised by sweetened drinks, snacks and desserts; and the pastoral pattern featured tsamba (roasted Tibetan barley), Tibetan cheese, butter tea/milk tea and whole-fat dairy foods. Individuals in the highest quintile of urban dietary pattern scores were found to be at a higher risk of developing MetS (OR 2·43, 95 % CI 1·41, 4·18) and central obesity (OR 1·91, 95 % CI 1·16, 3·14) after controlling for potential confounders. CONCLUSIONS: MetS was common among urbanised and semi-urbanised Tibetan adult population in transition. The urban dietary pattern, in particular, was a risk factor for MetS. To prevent MetS, nutrition interventions need to be tailored to address the variety of local diet patterns to promote healthy eating.

Identifying specific miRNAs and associated mRNAs in CD44 and CD90 cancer stem cell subtypes in gastric cancer cell line SNU-5.

Cancer stem cells (CSCs) are capable of generating multiple types of cells and play a vital role in promoting gastric cancer (GC) progression. Our previous research indicated that gastric CSCs with surface markers of CD44+ were more invasive compared to CD44- CD90+ CSCs (CD90+ CSCs), whereas CD90+ CSCs exhibited higher levels of proliferation than CD44+ CSCs. However, the mechanism and characteristics of marker-positive gastric CSCs are poorly understood. In this study, we profiled expression of miRNAs and mRNAs in CD44+ CSCs, CD90+ CSCs, and CD44- CD90- cell subtype (control) from SNU-5 cells by microarray analysis. Our results suggested some specially expressed miRNA-mRNA pairs in CD44+ and CD90+ CSCs. We performed Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses to analyze the correlation and function of those pairs. We also validated the pairs that may play roles in metastasis by qRT-PCR. In CD44+ CSCs, we observed hsa-miR-15b-5p was up-regulated and its target genes AMOT, USP31, KALRN, EPB41L4B, ATP2B2, and EMC4 were down-regulated, which may relate to invasion and migration. In CD90+ CSCs, we observed hsa-miR-3631-3p is up-regulated, while its target genes QKI, TRIM67 and HMGA2 are down-regulated, which is associated with proliferation. We also found that hsa-miR-1910-5p is up-regulated while its target gene QKI and HMGA2 are down-regulated in CD90+ CSCs. The screened miRNA-mRNA pairs give us new insight into the mechanism of different phenotypes and biomarkers capable of identifying and isolating metastatic and tumorigenic CSCs. Those miRNA-mRNA pairs may also act as treatment for GC.

Job Burnout and Occupational Stressors among Chinese Healthcare Professionals at County-Level Health Alliances.

BACKGROUND: This study aimed to examine the degrees of job burnout and occupational stressors and their associations among healthcare professionals from county-level health alliances in Qinghai-Tibet Plateau, China. METHODS: A cross-sectional study was conducted in county-level health alliances in Qinghai Province, China, in November 2018. The Maslach Burnout Inventory-General Survey and the 38-item Chinese version of the "Scale for occupational stressors on clinicians" were used. Medical staff in four health alliances from two counties were invited to complete the questionnaire. RESULTS: A total of 1052 (age: 34.06 ± 9.22 years, 79.1% females) healthcare professionals were included, 68.2% (95% CI: 65.2-71.0%) of the participants had job burnout symptoms. Occupational stressors had positive associations with moderate (OR = 1.06, 95% CI: 1.05-1.07) and serious (OR = 1.15, 95% CI: 1.13-1.19) level of job burnout. Stressors from vocational interest produced the greatest magnitude of odds ratio (OR = 1.76, 95% CI: 1.62-1.92) for serious degree of burnout, followed by doctor-patient relationship, interpersonal relationship as well as other domains of occupational stressors. CONCLUSIONS: Job burnout was very common among healthcare professionals working in Chinese county-level health alliances, different occupational stressors had associations with job burnout. Appropriate and effective policies and measures should be developed and implemented.

Major dietary patterns and their relationship to obesity among urbanized adult Tibetan pastoralists.

BACKGROUND AND OBJECTIVES: This study investigated major dietary patterns and their relationship to obesity among urbanized Tibetan pastoralists. METHODS AND STUDY DESIGN: Using a cross-sectional design, this study assessed 782 urbanized Tibetan pastoralists aged 18-84 y. A food frequency questionnaire and anthropometric measurements were conducted in 2018. Principal component analysis was used to identify dietary patterns. Logistic regression was applied to compare the risks for overweight (BMI >=24 kg/m2), obesity (BMI >=28 kg/m2), and central obesity (waist circumference >=80 cm for women and >=85 cm for men) across quintiles of dietary pattern scores after controlling for gender, age, education, medical insurance, smoking status, alcohol consumption and physical activity. RESULTS: This study identified three major dietary patterns: an urban pattern characterized by high intake of vegetables, tubers/roots, and refined carbohydrates; a western pattern characterized by sugary drinks, snacks, and desserts; and a pastoral pattern characterized by tsamba (roasted Tibetan barley), Tibetan cheese, and buttered/milk tea. Subjects in the highest quintile of urban pattern scores were more likely to be overweight (OR=2.58, 95% CI 1.48-4.49) (p-for-trend=0.001), obese (2.94, 1.57-5.49) (p-for-trend=0.001), and centrally obese (1.94, 1.12-3.36) (p-for-trend=0.019) compared to those in the lowest quintile with confounders controlled. The western dietary pattern was positively associated with overweight (p-for-trend=0.037). No clear association was observed for the pastoral dietary pattern. CONCLUSIONS: Urban and western dietary patterns independently predict the likelihood of being overweight. Improved nutrition education may contribute to healthier eating behaviors, thus reducing or preventing obesity.

Curcumin upregulates the Nrf2 system by repressing inflammatory signaling-mediated Keap1 expression in insulin-resistant conditions.

Both oxidative stress and inflammation contribute to the development of insulin resistance (IR). Curcumin (Cur) not only has an anti-inflammatory effect but also has an antioxidative stress effect via the activation of NF-E2-related factor 2 (Nrf2). Since there is close cross-communication between inflammation and oxidative stress, we examined whether Cur could modulate Nrf2 function via its anti-inflammatory ability and investigated its underlying mechanism. In this study, we show that Cur inhibits inflammatory signaling and Kelch-like ECH-associated protein 1 (Keap1) expression, which is accompanied by the activation of the Nrf2 system. We further identified that the proinflammatory cytokine tumor necrosis factor alpha (TNFα) could stimulate Keap1 synthesis and increase Nrf2 polyubiquitination, but these effects could be significantly inhibited by Cur treatment. This study demonstrates that Cur-induced Nrf2 activation occurs through the inhibition of inflammatory signaling-mediated upregulation of Keap1, contributing to its beneficial effects on redox homeostasis and insulin sensitivity.

Sequencing the exons of human glucocorticoid receptor (NR3C1) gene in Han Chinese with high-altitude pulmonary edema.

BACKGROUND: High-altitude pulmonary edema (HAPE) is a serious acute mountain sickness that mainly occurs in non-acclimatized individuals after rapid ascent to high altitude. The precise etiology of HAPE remains unclear. This study aimed to investigate whether NR3C1 gene polymorphism is associated with the susceptibility to HAPE. METHODS: The exons of NR3C1 gene were sequenced by a ABI 3730 DNA analyzer in 133 HAPE patients and matched 135 healthy Han Chinese controls from the Yushu area in Qinghai (the altitude greater than 3500 m). RESULTS: DNA sequencing showed the heterozygous substitutions at codon 588 (rs6194) in exon 6 and 766 (rs6196) in exon 9 of NR3C1 gene. The genotypic distributions and allelic frequencies of NR3C1 SNP rs6194 showed significant differences in two groups (P < 0.05). The frequencies of the C allele were significantly higher in the HAPE group than in the control group (P < 0.05) with an odds ratio of 3.009 (95% CI = 1.250-7.244). There were no differences in genotypic and allelic frequencies in rs6196 polymorphism between the two groups. CONCLUSIONS: NR3C1 gene rs6194 polymorphism is correlated with HAPE susceptibility. CC genotype and C allele of rs6194 polymorphism might increase the risk of HAPE in Han Chinese.

Ezh2 phosphorylation state determines its capacity to maintain CD8+ T memory precursors for antitumor immunity.

Memory T cells sustain effector T-cell production while self-renewing in reaction to persistent antigen; yet, excessive expansion reduces memory potential and impairs antitumor immunity. Epigenetic mechanisms are thought to be important for balancing effector and memory differentiation; however, the epigenetic regulator(s) underpinning this process remains unknown. Herein, we show that the histone methyltransferase Ezh2 controls CD8+ T memory precursor formation and antitumor activity. Ezh2 activates Id3 while silencing Id2, Prdm1 and Eomes, promoting the expansion of memory precursor cells and their differentiation into functional memory cells. Akt activation phosphorylates Ezh2 and decreases its control of these transcriptional programs, causing enhanced effector differentiation at the expense of T memory precursors. Engineering T cells with an Akt-insensitive Ezh2 mutant markedly improves their memory potential and capability of controlling tumor growth compared to transiently inhibiting Akt. These findings establish Akt-mediated phosphorylation of Ezh2 as a critical target to potentiate antitumor immunotherapeutic strategies.

Preliminary investigation for effects of hypothalamic Leptin/Ghrelin and arcuate nucleus pro-opiomelanocortin system on regulation of high-altitude acclimatization.

This study aims to investigate the mechanism of hypothalamic Leptin/Ghrelin and arcuate nucleus pro-opiomelanocortin (POMC) system in the regulation of high-altitude acclimatization. SD rats (male) were divided into two groups and separately fed at the 2260m and 4700m altitude. Tow groups contained 5 small groups separately, including 1 d, 3 d, 7 d, 15 d and 30 d, and 8 rats in each group. Blood, cerebrospinal fluid and tissues were taken at setting time. Leptin and Ghrelin were detected by using radioactivity immuno-assay. RNA expression of NPY and POMC were detected by using RT-PCR assay. The number of NPY positive neurons was detected by using immunofluorescence (IF) and cell counting. Other rats were sent to the 4300m and fed in animal room with regular diet and drinking. The results indicated that after being sent to high altitude region, Leptin levels at the 3rd and 7th day were significantly higher than the 1st day, while decreased at 15th, and the level at 30th day was closed to the 1st day. Ghrelin levels decreased at the 3rd, 7th and 15th day, and were lower at the 30th day. Comparing to the 1st day, NPY transcription levels increased at the 7th day, while decreased at the 30th. POMC transcription level decreased at the 7th day, while increased at the 30th gradually. The feeding of the rats fed at the 4300m decreased at the 3rd and the 5th, while increased at the 7th, 15th and 30th day. The weight of the rats changed as the feeding changing. In conclusion, after being sent to the high region, the rats were adaptive to the hypoxia environment gradually, and the steady of neuro-endocrine regulation recovered or established.

Hsp90 inhibition destabilizes Ezh2 protein in alloreactive T cells and reduces graft-versus-host disease in mice.

Modulating T-cell alloreactivity has been a main strategy to reduce graft-versus-host disease (GVHD), a life-threatening complication after allogeneic hematopoietic stem-cell transplantation (HSCT). Genetic deletion of T-cell Ezh2, which catalyzes trimethylation of histone H3 at lysine 27 (H3K27me3), inhibits GVHD. Therefore, reducing Ezh2-mediated H3K27me3 is thought to be essential for inhibiting GVHD. We tested this hypothesis in mouse GVHD models. Unexpectedly, administration of the Ezh2 inhibitor GSK126, which specifically decreases H3K27me3 without affecting Ezh2 protein, failed to prevent the disease. In contrast, destabilizing T-cell Ezh2 protein by inhibiting Hsp90 using its specific inhibitor AUY922 reduced GVHD in mice undergoing allogeneic HSCT. In vivo administration of AUY922 selectively induced apoptosis of activated T cells and decreased the production of effector cells producing interferon γ and tumor necrosis factor α, similar to genetic deletion of Ezh2. Introduction of Ezh2 into alloreactive T cells restored their expansion and production of effector cytokines upon AUY922 treatment, suggesting that impaired T-cell alloreactivity by inhibiting Hsp90 is achieved mainly through depleting Ezh2. Mechanistic analysis revealed that the enzymatic SET domain of Ezh2 directly interacted with Hsp90 to prevent Ezh2 from rapid degradation in activated T cells. Importantly, pharmacological inhibition of Hsp90 preserved antileukemia activity of donor T cells, leading to improved overall survival of recipient mice after allogeneic HSCT. Our findings identify the Ezh2-Hsp90 interaction as a previously unrecognized mechanism essential for T-cell responses and an effective target for controlling GVHD.

miRNA-181a/b Regulates Phenotypes of Vessel Smooth Muscle Cells Through Serum Response Factor.

The phenotypic modulation of vessel smooth muscle cells (VSMCs) plays a crucial role in the physiological and pathological conditions of vasculature in response to local environmental changes. The phenotypic transition of VSMCs is largely modulated by the serum response factor (SRF). miR-181a and miR-181b are members of the well-studied miR-181 family and both have complementary sequence in the 3' untranslated region (UTR) of SRF gene. In this article, evidence insinuates that miR-181a/b was involved in VSMCs differentiation through upregulating synthetic marker genes and downregulating contractile ones, respectively. We also confirmed the roles of the miR-181a/b in promoting SMC proliferation, migration, and synthetic phenotype transformation. In addition, miR-181a/b was indicated as directly targeting at 3' UTR of SRF by dual-luciferase assay and Western blot assay. In a word, miR-181a/b is one of the factors involved in VSMC differentiation toward a synthetic phenotype through targeting at SRF. These findings may provide a potential therapeutic approach that miR-181a/b took part in regulating the vessel disorders.

Programming of donor T cells using allogeneic δ-like ligand 4-positive dendritic cells to reduce GVHD in mice.

Alloreactive T cells play a critical role in eliminating hematopoietic malignant cells but are also the mediators of graft-versus-host disease (GVHD), a major complication that subverts the success of allogeneic hematopoietic stem cell transplantation (HSCT). However, induction of alloreactive T cells does not necessarily lead to GVHD. Here we report the development of a cellular programming approach to render alloreactive T cells incapable of causing severe GVHD in both major histocompatibility complex (MHC)-mismatched and MHC-identical but minor histocompatibility antigen-mismatched mouse models. We established a novel platform that produced δ-like ligand 4-positive dendritic cells (Dll4(hi)DCs) from murine bone marrow using Flt3 ligand and Toll-like receptor agonists. Upon allogeneic Dll4(hi)DC stimulation, CD4(+) naïve T cells underwent effector differentiation and produced high levels of interferon γ (IFN-γ) and interleukin-17 in vitro, depending on Dll4 activation of Notch signaling. Following transfer, allogeneic Dll4(hi)DC-induced T cells were unable to mediate severe GVHD but preserved antileukemic activity, significantly improving the survival of leukemic mice undergoing allogeneic HSCT. This effect of Dll4(hi)DC-induced T cells was associated with their impaired expansion in GVHD target tissues. IFN-γ was important for Dll4(hi)DC programming to reduce GVHD toxicities of alloreactive T cells. Absence of T-cell IFN-γ led to improved survival and expansion of Dll4(hi)DC-induced CD4(+) T cells in transplant recipients and caused lethal GVHD. Our findings demonstrate that Dll4(hi)DC programming can overcome GVHD toxicity of donor T cells and produce leukemia-reactive T cells for effective immunotherapy.

The Notch Ligand DLL4 Defines a Capability of Human Dendritic Cells in Regulating Th1 and Th17 Differentiation.

Notch signaling regulates multiple helper CD4(+) T cell programs. We have recently demonstrated that dendritic cells (DCs) expressing the Notch ligand DLL4 are critical for eliciting alloreactive T cell responses and induction of graft-versus-host disease in mice. However, the human counterpart of murine DLL4(+) DCs has yet to be examined. We report the identification of human DLL4(+) DCs and their critical role in regulating Th1 and Th17 differentiation. CD1c(+) DCs and plasmacytoid DCs (pDCs) from the peripheral blood (PB) of healthy donors did not express DLL4. In contrast, patients undergoing allogeneic hematopoietic stem cell transplantation had a 16-fold more DLL4(+)CD1c(+) DCs than healthy donors. Upon activation of TLR signaling, healthy donor-derived CD1c(+) DCs dramatically upregulated DLL4, as did pDCs to a lesser extent. Activated DLL4(+) DCs were better able to promote Th1 and Th17 differentiation than unstimulated PB DCs. Blocking DLL4 using a neutralizing Ab decreased Notch signaling in T cells stimulated with DLL4(+) DCs, and it reduced the generation of Th1 and Th17 cells. Both NF-κB and STAT3 were crucial for inducing DLL4 in human DCs. Interestingly, STAT3 directly activated DLL4 transcription and inhibiting STAT3 alone was sufficient to reduce DLL4 in activated PB DCs. Thus, DLL4 is a unique functional molecule of human circulating DCs critical for directing Th1 and Th17 differentiation. These findings identify a pathway for therapeutic intervention for inflammatory disorders in humans, such as graft-versus-host disease after allogeneic hematopoietic stem cell transplantation, autoimmunity, and tumor immunity.

Ezh2 regulates transcriptional and posttranslational expression of T-bet and promotes Th1 cell responses mediating aplastic anemia in mice.

Acquired aplastic anemia (AA) is a potentially fatal bone marrow (BM) failure syndrome. IFN-γ-producing Th1 CD4(+) T cells mediate the immune destruction of hematopoietic cells, and they are central to the pathogenesis. However, the molecular events that control the development of BM-destructive Th1 cells remain largely unknown. Ezh2 is a chromatin-modifying enzyme that regulates multiple cellular processes primarily by silencing gene expression. We recently reported that Ezh2 is crucial for inflammatory T cell responses after allogeneic BM transplantation. To elucidate whether Ezh2 mediates pathogenic Th1 responses in AA and the mechanism of Ezh2 action in regulating Th1 cells, we studied the effects of Ezh2 inhibition in CD4(+) T cells using a mouse model of human AA. Conditionally deleting Ezh2 in mature T cells dramatically reduced the production of BM-destructive Th1 cells in vivo, decreased BM-infiltrating Th1 cells, and rescued mice from BM failure. Ezh2 inhibition resulted in significant decrease in the expression of Tbx21 and Stat4, which encode transcription factors T-bet and STAT4, respectively. Introduction of T-bet but not STAT4 into Ezh2-deficient T cells fully rescued their differentiation into Th1 cells mediating AA. Ezh2 bound to the Tbx21 promoter in Th1 cells and directly activated Tbx21 transcription. Unexpectedly, Ezh2 was also required to prevent proteasome-mediated degradation of T-bet protein in Th1 cells. Our results demonstrate that Ezh2 promotes the generation of BM-destructive Th1 cells through a mechanism of transcriptional and posttranscriptional regulation of T-bet. These results also highlight the therapeutic potential of Ezh2 inhibition in reducing AA and other autoimmune diseases.