Yuying Liu

Clinical significance of increased CD4+CD25-Foxp3+T cells in patients with new-onset systemic lupus erythematosus

Objective: To investigate the expressions of Foxp3 and CD25 on CD4+ T cells from patients with new-onset systemic lupus erythematosus (SLE) and assess their clinical significance. Methods: 10 patients with active (systemic lupus erythematosus Disease Activity Index (SLEDAI) ⩾10) and 11 with inactive (SLEDAI ⩽5) new-onset SLE as well as 11 healthy volunteers were enrolled. The expressions of CD25, Foxp3 and CD127 on CD4+ T cells were analysed by flow cytometry. Proliferation assays were performed on isolated CD4+CD25+ or CD4+CD25− T cells, or both. Results: There was no significant difference in the number of CD4+CD25+Foxp3+ T cells in subjects with either active or inactive SLE compared with normal controls (p>0.05). Moreover, the suppressive capacity of CD4+CD25+ T cells in patients with new-onset lupus was not impaired as measured by the ability to inhibit proliferation of CD4+CD25− T cells. Interestingly, CD4+CD25−Foxp3+ T cells in new-onset lupus (2.97–10.94%) were significantly more frequent than in normal controls (1.01–3.62%) (p<0.01), and correlated positively with the titres of anti-dsDNA antibodies (p = 0.029). Few of these cells expressed CD127. Treatment with glucocorticoids and cyclophosphamide reduced CD4+CD25−Foxp3+ T cells in 8 of 10 patients with active disease. Conclusions: There was a significant increase in CD4+CD25−Foxp3+ T cells in patients with new-onset SLE that correlated with anti-dsDNA titres, whereas no alteration in either the percentage or function of CD4+CD25+Foxp3+ T cells was observed.

Reduction of forkhead box P3 levels in CD4+CD25high T cells in patients with new‐onset systemic lupus erythematosus

The aim of this study was to quantify and evaluate the forkhead box P3 (FoxP3) expression regulatory T cells in new‐onset systemic lupus erythematosus (SLE) patients before and after treatment. Forty‐four newly diagnosed and untreated SLE patients, including 24 with active disease (SLEDAI ≥ 10) and 20 with inactive disease (SLEDAI < 5), were enrolled in this study. Twenty‐one age‐ and sex‐matched healthy volunteers were also included as controls. Peripheral blood samples were collected and mononuclear cells isolated. The expression of CD25 and FoxP3 in CD4+ T cells were analysed with flow cytometry. CD4+CD25+ (3·95–13·04%) and CD4+CD25high (0·04–1·34%) T cells in peripheral blood in untreated patients with new‐onset active lupus were significantly lower than that in the patients with inactive lupus (7·27–24·48%, P < 0·05 and 0·14–3·07% P < 0·01 respectively) and that in healthy controls (5·84–14·84%, P < 0·05). Interestingly, the decrease in CD4+CD25high T cells was restored significantly in patients with active lupus after corticosteroid treatment. There was, however, a significantly higher percentage of CD4+FoxP3+ T cells in patients with active (5·30–23·00%) and inactive (7·46–17·38%) new‐onset lupus patients compared with healthy control subjects (2·51–12·94%) (P < 0·01). Intriguingly, CD25 expression in CD4+FoxP3+ T cells in patients with active lupus (25·24–62·47%) was significantly lower than that in those patients with inactive lupus (30·35–75·25%, P < 0·05) and healthy controls (54·83–86·38%, P < 0·01). Most strikingly, the levels of FoxP3 expression determined by mean fluorescence intensity in CD4+CD25high cells in patients with active SLE were significantly down‐regulated compared with healthy subjects (130 ± 22 versus 162 ± 21, P = 0·012). CD4+CD25high T cells are low in new‐onset patients with active SLE and restored after treatment. Despite that the percentage of CD4+FoxP3+ T cells appear high, the levels of FoxP3 expression in CD4+CD25high T cells are down‐regulated in untreated lupus patients. There is a disproportional expression between CD25high and FoxP3+ in new‐onset patients with active SLE.

MicroRNA-19a-3p inhibits breast cancer progression and metastasis by inducing macrophage polarization through downregulated expression of Fra-1 proto-oncogene

One of the hallmarks of malignancy is the polarization of tumor-associated macrophages (TAMs) from a pro-immune (M1-like) phenotype to an immune-suppressive (M2-like) phenotype. However, the molecular basis of the process is still unclear. MicroRNA (miRNA) comprises a group of small, non-coding RNAs that are broadly expressed by a variety of organisms and are involved in cell behaviors such as suppression or promotion of tumorigenesis. Here, we demonstrate that miR-19a-3p, broadly conserved among vertebrates, was downregulated in RAW264.7 macrophage cells of the M2 phenotype in conditoned medium of 4T1 mouse breast tumor cells. This downregulation correlated with an increased expression of the Fra-1 gene, which was reported to act as a pro-oncogene by supporting the invasion and progression of breast tumors. We found significant upregulation of miR-19a-3p in RAW264.7 macrophages after transfection with a miR-19a-3p mimic that resulted in a significant suppression of the expression of this gene. In addition, we could measure the activity of binding between miR-19a-3p and Fra-1 with a psiCHECK luciferase reporter system. Further, transfection of RAW264.7 macrophage cells with the miR-19a-3p mimic decreased the expression of the Fra-1 downstream genes VEGF, STAT3 and pSTAT3. Most importantly, the capacity of 4T1 breast tumor cells to migrate and invade was impaired in vivo by the intratumoral injection of miR-19a-3p. Taken together, these findings indicate that miR-19a-3p is capable of downregulating the M2 phenotype in M2 macrophages and that the low expression of this miRNA has an important role in the upregulation of Fra-1 expression and induction of M2 macrophage polarization.

Glycogen synthase kinase-3β negatively regulates TGF-β1 and Angiotensin II-mediated cellular activity through interaction with Smad3

Glycogen synthase kinase-3beta (GSK3beta) is a major negative modulator of cardiac hypertrophy. Here we report that GSK3beta physically and functionally interacts with Smad3. The interaction between GSK3beta and Smad3 may participate in the negative regulation of transforming growth factor beta1 (TGF-beta1) and Angiotensin II-induced transcription and apoptosis. GSK3beta interacted directly with Smad3 to sequester it outside the nucleus and prevent its nuclear translocation. This resulted in the suppression of Smad3-mediated transcriptional activity and gene expression. GSK3beta counteracted the pro-apoptotic effect of Smad3 and attenuated Angiotensin II-induced apoptosis in cardiac myocytes. Furthermore, stimulation of these cells with TGF-beta1 and Angiotensin II led to the endogenous Smad3 disassociating from GSK3beta and inactivating GSK3beta by phosphorylation of its Ser9. These results uncovered a novel mechanism for the GSK3beta negative regulation of TGF-beta1/Smad3 and Angiotensin II/Smad3-mediated transcription and apoptosis by the identification of a crosstalk between GSK3beta and Smad3 signal pathway.

Blockade of IDO-kynurenine-AhR metabolic circuitry abrogates IFN-γ-induced immunologic dormancy of tumor-repopulating cells.

Interactions with the immune system may lead tumorigenic cells into dormancy. However, the underlying molecular mechanism is poorly understood. Using a 3D fibrin gel model, we show that IFN-γ induces tumour-repopulating cells (TRCs) to enter dormancy through an indolamine 2,3-dioxygenase 1 (IDO1)-kynurenine (Kyn)-aryl hydrocarbon receptor (AhR)-p27 dependent pathway. Mechanistically, IFN-γ signalling triggers differentiated tumour cell apoptosis via STAT1; however, when IDO1 and AhR are highly expressed as in TRCs, IFN-γ results in IDO1/AhR-dependent p27 induction that prevents STAT1 signalling, thus suppressing the process of cell death and activating the dormancy program. Blocking the IDO/AhR metabolic circuitry not only abrogates IFN-γ-induced dormancy but also results in enhanced repression of tumour growth by IFN-γ-induced apoptosis of TRCs both in vitro and in vivo. These data present a previously unrecognized mechanism of inducing TRC dormancy by IFN-γ, suggesting a potential effective cancer immunotherapeutic modality through the combination of IFN-γ and IDO/AhR inhibitors.

IKKβ Enforces a LIN28B/TCF7L2 Positive Feedback Loop That Promotes Cancer Cell Stemness and Metastasis

Considerable evidence suggests that proinflammatory pathways drive self-renewal of cancer stem-like cells (CSC), but the underlying mechanisms remain mainly undefined. Here we report that the let7 repressor LIN28B and its regulator IKBKB (IKKβ) sustain cancer cell stemness by interacting with the Wnt/TCF7L2 (TCF4) signaling pathway to promote cancer progression. We found that LIN28B expression correlated with clinical progression and stemness marker expression in breast cancer patients. Functional studies demonstrated that the stemness properties of LIN28B-expressing human breast and lung cancer cells were enhanced by IKKβ, whereas loss of LIN28B abolished stemness properties in these settings. These phenomena were driven through interactions with TCF7L2, which enhanced LIN28B expression by direct binding to intron 1 of the LIN28B gene, which in turn promoted TCF7L2 mRNA translation through a positive feedback loop. Notably, RNAi-mediated silencing of LIN28B or pharmacologic inhibition of IKKβ was sufficient to suppress primary and metastatic tumor growth in vivo. Together, our results establish the LIN28B/TCF7L2 interaction loop as a central mediator of cancer stemness driven by proinflammatory processes during progression and metastasis, possibly offering a new therapeutic target for generalized interventions in advanced cancers.

[Effect of chlorogenic acid on disordered glucose and lipid metabolism in db/db mice and its mechanism].

OBJECTIVE To explore the effect of chlorogenic acid on disordered glucose and lipid metabolism in db/db mice and its mechanism. METHODS Thirteen 5-6-week-old male db/db mice were randomly divided into db/db-CGA group (n=7) and db/db-CON group (n=6), and thirteen 5-6-week-old male db/m mice were randomly divided into db/m-CGA group (n=6) and db/m-CON group (n=7). Mice in the CGA groups were administrated with CGA 80 mg/(kg·d)by gavage, and mice in the CON groups were administrated with PBS in the same volume by gavage. Twelve weeks later, the level of biomedical parameters in plasma, liver, and skeletal muscle were determined, the concentrations of adiponectin and visfatin in visceral adipose, and the mRNA expression of glucose-6-phosphatase (G-6-Pase) and peroxisome proliferators-activated receptor-α (PPAR-α) as well as the protein level of PPAR-α in liver were detected. RESULTS Twelve weeks after CGA administration, the levels of triglycerides in plasma, liver, and skeletal muscle and the fasting plasma glucose in db/db-CGA group were significantly lower than those in db/db-CON group(P<0.05). The muscle glycogen level was significantly higher than that in db/db-CON group (P<0.05), and the adiponectin concentration was significantly higher than that in db/db-CON group ( P<0.01) and lower than that in db/m-CGA group(P<0.05). The visfatin concentration in db/db-CGA group was significantly lower than that in db/db-CON group (P<0.01) and significantly higher than that in db/m-CGA group(P<0.05). The mRNA expression level of G-6-Pase was significantly down-regulated in db/db-CGA group when compared with db/db-CON group (P<0.05). Both the mRNA and the protein expression levels of PPAR-α were significantly up-regulated in db/db-CGA group(P<0.05) compared with in db/db-CON group. CONCLUSION CGA improves the disordered glucose/lipid metabolism in db/db mice, which is speculated to be related with its role in modulating the adipokines secretion, up-regulating hepatic PPAR-α, and inhibiting G-6-Pase expression.

Chloroquine modulates antitumor immune response by resetting tumor-associated macrophages toward M1 phenotype

Resetting tumor-associated macrophages (TAMs) is a promising strategy to ameliorate the immunosuppressive tumor microenvironment and improve innate and adaptive antitumor immunity. Here we show that chloroquine (CQ), a proven anti-malarial drug, can function as an antitumor immune modulator that switches TAMs from M2 to tumor-killing M1 phenotype. Mechanistically, CQ increases macrophage lysosomal pH, causing Ca2+ release via the lysosomal Ca2+ channel mucolipin-1 (Mcoln1), which induces the activation of p38 and NF-κB, thus polarizing TAMs to M1 phenotype. In parallel, the released Ca2+ activates transcription factor EB (TFEB), which reprograms the metabolism of TAMs from oxidative phosphorylation to glycolysis. As a result, CQ-reset macrophages ameliorate tumor immune microenvironment by decreasing immunosuppressive infiltration of myeloid-derived suppressor cells and Treg cells, thus enhancing antitumor T-cell immunity. These data illuminate a previously unrecognized antitumor mechanism of CQ, suggesting a potential new macrophage-based tumor immunotherapeutic modality.Tumour-associated macrophages (TAMs) display an M2 phenotype that promote tumour immune escape. Here the authors show that Chloroquine (CQ), a lysosome inhibitor used against malaria, inhibits tumour growth by switching TAMs into an M1 tumor-killing phenotype by repolarizing macrophages metabolism.

Pre-instillation of tumor microparticles enhances intravesical chemotherapy of nonmuscle-invasive bladder cancer through a lysosomal pathway

Nonmuscle-invasive bladder cancer (NMIBC) is treated with transurethral resection followed by intravesical chemotherapy. However, drug-resistant tumorigenic cells cannot be eliminated, leading to half of the treated cancers recur with increased stage and grade. Innovative approaches to enhance drug sensitivity and eradicate tumorigenic cells in NMIBC treatment are urgently needed. Here, we show that pre-instillation of tumor cell-derived microparticles (T-MP) as natural biomaterials markedly enhance the inhibitory effects of intravesical chemotherapy on growth and hematuria occurrence of orthotropic bladder cancer in mice. We provide evidence that T-MPs enter and increase the pH value of lysosomes from 4.6 to 5.6, leading to the migration of drug-loaded lysosomes along microtubule tracks toward the nucleus and discharging the drugs whereby for the entry of the nucleus. We propose that T-MPs may function as a potent sensitizer for augmenting NMIBC chemotherapy with unprecedented clinical benefits.

STAT3/p53 pathway activation disrupts IFN-β–induced dormancy in tumor-repopulating cells

Dynamic interaction with the immune system profoundly regulates tumor cell dormancy. However, it is unclear how immunological cues trigger cancer cell–intrinsic signaling pathways for entering into dormancy. Here, we show that IFN-&bgr; treatment induced tumor-repopulating cells (TRC) to enter dormancy through an indolamine 2,3-dioxygenase/kynurenine/aryl hydrocarbon receptor/p27–dependent (IDO/Kyn/AhR/p27-dependent) pathway. Strategies to block this metabolic circuitry did not relieve dormancy, but led to apoptosis of dormant TRCs in murine and human melanoma models. Specifically, blocking AhR redirected IFN-&bgr; signaling to STAT3 phosphorylation through both tyrosine and serine sites, which subsequently facilitated STAT3 nuclear translocation and subsequent binding to the p53 promoter in the nucleus. Upregulation of p53 in turn disrupted the pentose phosphate pathway, leading to excessive ROS production and dormant TRC death. Additionally, in melanoma patients, high expression of IFN-&bgr; correlated with tumor cell dormancy. Identification of this mechanism for controlling TRC dormancy by IFN-&bgr; provides deeper insights into cancer-immune interaction and potential new cancer immunotherapeutic modalities.