Zhenlin Hu

Investigation of HIFU-induced anti-tumor immunity in a murine tumor model

BackgroundHigh intensity focused ultrasound (HIFU) is an emerging non-invasive treatment modality for localized treatment of cancers. While current clinical strategies employ HIFU exclusively for thermal ablation of the target sites, biological responses associated with both thermal and mechanical damage from focused ultrasound have not been thoroughly investigated. In particular, endogenous danger signals from HIFU-damaged tumor cells may trigger the activation of dendritic cells. This response may play a critical role in a HIFU-elicited anti-tumor immune response which can be harnessed for more effective treatment.MethodsMice bearing MC-38 colon adenocarcinoma tumors were treated with thermal and mechanical HIFU exposure settings in order to independently observe HIFU-induced effects on the hosts immunological response. In vivo dendritic cell activity was assessed along with the hosts response to challenge tumor growth.ResultsThermal and mechanical HIFU were found to increase CD11c+ cells 3.1-fold and 4-fold, respectively, as compared to 1.5-fold observed for DC injection alone. In addition, thermal and mechanical HIFU increased CFSE+ DC accumulation in draining lymph nodes 5-fold and 10-fold, respectively. Moreover, focused ultrasound treatments not only caused a reduction in the growth of primary tumors, with tumor volume decreasing by 85% for thermal HIFU and 43% for mechanical HIFU, but they also provided protection against subcutaneous tumor re-challenge. Further immunological assays confirmed an enhanced CTL activity and increased tumor-specific IFN-γ-secreting cells in the mice treated by focused ultrasound, with cytotoxicity induced by mechanical HIFU reaching as high as 27% at a 10:1 effector:target ratio.ConclusionThese studies present initial encouraging results confirming that focused ultrasound treatment can elicit a systemic anti-tumor immune response, and they suggest that this immunity is closely related to dendritic cell activation. Because DC activation was more pronounced when tumor cells were mechanically lysed by focused ultrasound treatment, mechanical HIFU in particular may be employed as a potential strategy in combination with subsequent thermal ablations for increasing the efficacy of HIFU cancer treatment by enhancing the hosts anti-tumor immunity.

Berberine induces dendritic cell apoptosis and has therapeutic potential for rheumatoid arthritis

OBJECTIVE To investigate the effects of berberine on dendritic cell (DC) apoptosis and its potential as a therapeutic agent in rheumatoid arthritis (RA). METHODS Bone marrow (BM)-derived myeloid DCs (MDCs) and plasmacytoid DCs (PDCs) were generated by culturing BM cells with granulocyte-macrophage colony-stimulating factor/interleukin-4 or flt3L, respectively. Splenic DCs, T cells, and B cells were purified using a magnetic-activated cell sorting system. In vitro apoptosis was assessed by annexin V/propidium iodide or Hoechst 33258 staining. The in vivo effects of berberine were examined in mice with collagen-induced arthritis (CIA). Immune responses against type II collagen (CII) were determined by assaying serum antibody levels, lymphocyte proliferation, and cytokine production. The proportions of DCs and apoptosis of different immune cell subsets in spleens and lymph nodes were analyzed by flow cytometry and immunohistochemistry after subset-specific surface marker labeling and TUNEL staining. RESULTS Exposure of MDCs to berberine during BM cell differentiation reduced cell recovery by inducing apoptosis. Sensitivity to berberine-induced apoptosis was acquired starting on day 3 of DC differentiation, and mature DCs were more sensitive to berberine than immature DCs. Murine peritoneal macrophages, RAW 264.7 cells, and Jurkat cells were insensitive to berberine-induced apoptosis. Splenic DCs were more sensitive to berberine than T and B cells. Susceptibility of PDCs to berberine-induced apoptosis was similar to that of MDCs. In mice with CIA, berberine treatment ameliorated arthritis, suppressed CII-specific immune responses, and selectively increased the incidence of apoptosis in DCs within spleens and lymph nodes. CONCLUSION These findings show that berberine selectively induces apoptosis in DCs. Berberine may thus represent a novel therapeutic agent for RA.

Antifibrotic effects of luteolin on hepatic stellate cells and liver fibrosis by targeting AKT/mTOR/p70S6K and TGFβ/Smad signalling pathways

Luteolin has been reported to exert antifibrogenic effects in CCl4‐induced hepatic fibrosis in mice. However, limited information is available on the cellular and molecular events responsible for this effect. This study focused on the action of luteolin on hepatic stellate cells (HSCs) and the relevant signalling molecules and pathways as well as the antifibrotic efficacy in multiple models of fibrosis.

Boosting high-intensity focused ultrasound-induced anti-tumor immunity using a sparse-scan strategy that can more effectively promote dendritic cell maturation

BackgroundThe conventional treatment protocol in high-intensity focused ultrasound (HIFU) therapy utilizes a dense-scan strategy to produce closely packed thermal lesions aiming at eradicating as much tumor mass as possible. However, this strategy is not most effective in terms of inducing a systemic anti-tumor immunity so that it cannot provide efficient micro-metastatic control and long-term tumor resistance. We have previously provided evidence that HIFU may enhance systemic anti-tumor immunity by in situ activation of dendritic cells (DCs) inside HIFU-treated tumor tissue. The present study was conducted to test the feasibility of a sparse-scan strategy to boost HIFU-induced anti-tumor immune response by more effectively promoting DC maturation.MethodsAn experimental HIFU system was set up to perform tumor ablation experiments in subcutaneous implanted MC-38 and B16 tumor with dense- or sparse-scan strategy to produce closely-packed or separated thermal lesions. DCs infiltration into HIFU-treated tumor tissues was detected by immunohistochemistry and flow cytometry. DCs maturation was evaluated by IL-12/IL-10 production and CD80/CD86 expression after co-culture with tumor cells treated with different HIFU. HIFU-induced anti-tumor immune response was evaluated by detecting growth-retarding effects on distant re-challenged tumor and tumor-specific IFN-γ-secreting cells in HIFU-treated mice.ResultsHIFU exposure raised temperature up to 80 degrees centigrade at beam focus within 4 s in experimental tumors and led to formation of a well-defined thermal lesion. The infiltrated DCs were recruited to the periphery of lesion, where the peak temperature was only 55 degrees centigrade during HIFU exposure. Tumor cells heated to 55 degrees centigrade in 4-s HIFU exposure were more effective to stimulate co-cultured DCs to mature. Sparse-scan HIFU, which can reserve 55 degrees-heated tumor cells surrounding the separated lesions, elicited an enhanced anti-tumor immune response than dense-scan HIFU, while their suppressive effects on the treated primary tumor were maintained at the same level. Flow cytometry analysis showed that sparse-scan HIFU was more effective than dense-scan HIFU in enhancing DC infiltration into tumor tissues and promoting their maturation in situ.ConclusionOptimizing scan strategy is a feasible way to boost HIFU-induced anti-tumor immunity by more effectively promoting DC maturation.

Bioactive compound reveals a novel function for ribosomal protein S5 in hepatic stellate cell activation and hepatic fibrosis

Liver fibrosis and its endstage, cirrhosis, represent a major public health problem worldwide. Activation of hepatic stellate cells (HSCs) is a central event in hepatic fibrosis. However, the proteins that control HSC activation are incompletely understood. Here we show that (6aS, 10S, 11aR, 11bR, 11cS)‐10‐methylamino‐dodecahydro‐3a, 7a‐diaza‐benzo [de]anthracene‐8‐thione (MASM) exhibits potent inhibitory activity against liver fibrosis in vitro and in vivo associated with the reduction of Akt phosphorylation. Furthermore, ribosomal protein S5 (RPS5) was identified as a direct target of MASM, which stabilized RPS5 in cultured HSCs and in the liver of experimental animals after dimethylnitrosamine (DMN) or bile duct ligation (BDL). Functional studies revealed that RPS5 could prevent HSC activation. RPS5 overexpression in HSCs resulted in Akt dephosphorylation at both Ser473 and Thr308, and led to subsequent dephosphorylation of GSK3β or P70S6K. Progression of DMN‐ and BDL‐induced hepatic fibrosis was aggravated by Rps5 knockdown and alleviated by RPS5 overexpression, which correlated with the modulation of Akt phosphorylation and HSC number in the fibrotic livers. Moreover, RPS5 was substantially reduced in the transdifferentiated HSCs, experimental fibrotic livers, and human cirrhosis samples. Conclusion: These results demonstrate that RPS5 is implicated in hepatic fibrogenesis and may represent a promising target for potential therapeutic intervention in liver fibrotic diseases. (Hepatology 2014;60:648–660)

Baicalein Selectively Induces Apoptosis in Activated Lymphocytes and Ameliorates Concanavalin A-Induced Hepatitis in Mice

Background Insufficient apoptosis in activated lymphocytes contributes to the development of autoimmune hepatitis (AIH). Baicalein (BE), a flavonoid originally isolated from the root of Scutellaria baicalensis Georgi, possesses anti-inflammatory properties. However, whether BE can selectively induce apoptosis in activated lymphocytes and exert therapeutic effect on AIH has not been studied. Methodology/Principal Findings The pro-apoptotic properties of BE were evaluated in vitro on different types of immune cells, and in vivo effects of BE were examined in a murine model of Concanavalin A (Con A)-induced hepatitis. In vitro treatment with BE resulted in a higher increase in the level of apoptosis in Con A-stimulated murine splenocytes, Con A-stimulated CD3+ splenocytes, lipopolysaccharide (LPS)-stimulated CD19+ splenocytes, and phorbol 12-myristate 13-acetate/ionomycin-stimulated Jurkat T cells, compared with that in unstimulated naïve ones. Murine bone marrow-derived dentritic cells, peritoneal macrophages, and RAW264.7 cells, either stimulated with LPS or unstimulated, were all insensitive to the BE-induced apoptosis. BE treatment also led to a loss of mitochondrial membrane potential, an increase of cytochrome c release from mitochondria to the cytosol, a decrease in the ratio of Bcl-2/Bax, and activation of caspase-9,-3 in Con A-stimulated CD3+ splenocytes and LPS-stimulated CD19+ splenocytes, while showing no impact on Fas/FasL expressions and caspase-8 activation. In vivo administration of BE alleviated Con A-induced liver injury, suppressed serum level of TNF-α and IFN-γ, and reduced liver infiltration of mononuclear cells (MNCs). Furthermore, BE treatment increased the incidences of apoptosis in liver-infiltrating MNCs and splenocytes, as well as in CD3+ and CD19+ splenocytes. When liver MNCs and splenocytes from BE-treated mice were cultured in vitro for 24 h, they exhibited marked increase in apoptosis compared to vehicle-treated control. Conclusions/Significance The present study demonstrates the ability of BE to promote apoptosis in activated lymphocytes through mitochondrial pathway and its potential use in the treatment of AIH.

An HNF1α-regulated feedback circuit modulates hepatic fibrogenesis via the crosstalk between hepatocytes and hepatic stellate cells

Hepatocytes are critical for the maintenance of liver homeostasis, but its involvement in hepatic fibrogenesis remains elusive. Hepatocyte nuclear factor 1α (HNF1α) is a liver-enriched transcription factor that plays a key role in hepatocyte function. Our previous study revealed a significant inhibitory effect of HNF1α on hepatocellular carcinoma. In this study, we report that the expression of HNF1α is significantly repressed in both human and rat fibrotic liver. Knockdown of HNF1α in the liver significantly aggravates hepatic fibrogenesis in either dimethylnitrosamine (DMN) or bile duct ligation (BDL) model in rats. In contrast, forced expression of HNF1α markedly alleviates hepatic fibrosis. HNF1α regulates the transcriptional expression of SH2 domain-containing phosphatase-1 (SHP-1) via directly binding to SHP-1 promoter in hepatocytes. Inhibition of SHP-1 expression abrogates the anti-fibrotic effect of HNF1α in DMN-treated rats. Moreover, HNF1α repression in primary hepatocytes leads to the activation of NF-κB and JAK/STAT pathways and initiates an inflammatory feedback circuit consisting of HNF1α, SHP-1, STAT3, p65, miR-21 and miR-146a, which sustains the deregulation of HNF1α in hepatocytes. More interestingly, a coordinated crosstalk between hepatocytes and hepatic stellate cells (HSCs) participates in this positive feedback circuit and facilitates the progression of hepatocellular damage. Our findings demonstrate that impaired hepatocytes play an active role in hepatic fibrogenesis. Early intervention of HNF1α-regulated inflammatory feedback loop in hepatocytes may have beneficial effects in the treatment of chronic liver diseases.

Protective Effects of Huang-Lian-Jie-Du-Tang against Polymicrobial Sepsis Induced by Cecal Ligation and Puncture in Rats

Huang-Lian-Jie-Du-Tang (HLJDT) is a traditional formula that has long been used for treatment of inflammatory diseases in Traditional Chinese Medicine. In this study, we examined its protective effect against sepsis in an experimental septic model induced by cecal ligation and puncture (CLP) in rats. The results demonstrated that prophylactic administration of HLJDT protected rats from CLP-induced lethality and ameliorated CLP-induced liver and lung injury. HLJDT treatment suppressed the production of proinflammatory cytokines, including TNF-α, IL-1, IL-6, and IL-17A, indicating HLJDT could limit excessive inflammatory responses in septic condition. In addition, HLJDT facilitated bacterial clearance by increasing phagocytic activities of peritoneal macrophages. Furthermore, HLJDT treatment reversed CLP-induced suppression of IFN-γ expression and blocked CLP-induced increase in IL-4 expression in spleens of rats at 24 h after CLP, indicating that HLJDT could reverse the shift from Th1 to Th2 response and promote Th1/Th2 balance toward Th1 predominance in septic rats. Moreover, HLJDT also inhibited the expression of IL-17A and ROR-γt in spleens of septic rats, indicating HLJDT is able to inhibit Th17 activation in septic condition. In conclusion, the present study demonstrated the protective effects of HLJDT against sepsis and highlighted the potential of HLJDT as a medication for septic patients.

Matrine Inhibits Infiltration of the Inflammatory Gr1(hi) Monocyte Subset in Injured Mouse Liver through Inhibition of Monocyte Chemoattractant Protein-1.

Matrine (Mat) is a major alkaloid extracted from Sophora flavescens Ait, an herb which is used in the traditional Chinese medicine for treatment of inflammation, cancer, and other diseases. The present study examined the impact of Mat on the CCl4-induced hepatic infiltration of Gr1hi monocytes to explore the possible mechanisms underlying its anti-inflammatory and antifibrotic effects. The results indicated that Mat protected mice from acute liver injury induced by single intraperitoneal injection of CCl4 and attenuated liver fibrosis induced by repeated CCl4 injection. Meanwhile, the infiltrations of Gr1hi monocytes in both acute and chronic injured livers were all inhibited, and the enhanced hepatic expression of MCP-1 was suppressed. Cellular experiments demonstrated that Mat directly inhibited MCP-1 production in both nonparenchymal cells and hepatic stellate cells derived from CCl4-injured livers. Transwell chemotaxis assays showed that Mat significantly inhibited the chemotactic activity of MCP-1. These results suggest that the anti-inflammatory and antifibrotic effects of Mat could be contributed, at least in part, to its prevention of Gr1hi monocyte infiltration into the injured livers and inhibition of MCP-1 production and activity. These findings extend our understanding of the mechanisms underlying the anti-inflammatory and antifibrotic effects of Mat.

Effects of esculentoside A on autoimmune syndrome induced by Campylobacter jejuni in mice and its modulation on T-lymphocyte proliferation and apoptosis

Esculentoside A (EsA), a saponin isolated from the root of Phytolacca esculenta, has been reported to exert anti-inflammatory effects in several animal models of acute and chronic inflammation by inhibiting the production and activity of pro-inflammatory cytokines in macrophages and epithelial cells. However, little is known about its modulation on T cells. In the present study, we further investigated its potential in treatment of autoimmune disease and its modulation on T cells, using an experimental autoimmune model established through immunizing mice with Campylobacterjejuni strain CJ-S(131) in Freunds complete adjuvant. Our results demonstrated that EsA administration markedly alleviated the inflammatory injury in liver and kidney of model mice, decreased the anti-CD3/CD28-stimulated proliferation of splenocytes and lymph node cells, and reduced the percentage of CD3+, CD4+, and CD8+ lymphocytes in peripheral blood. Furthermore, we demonstrated that EsA induced apoptosis in ConA-activated thymocytes but not in non-activated thymocytes. Gene expression analysis revealed that EsA up-regulated the expression of a group of pro-apoptotic genes more profoundly in Con A-activated thymocytes than in non-activated thymocytes. EsA-affected pro-apoptotic genes included those involved in Fas induction, p53 activation, redox metabolism, calcium- and glucocorticoid-induced apoptosis signals, suggesting that EsA may modulate multiple apoptotic signal pathways in activated T cells. Taken together, our findings suggest that EsA may be useful for the treatment of autoimmune disease through modulation on T cell-mediated adaptive immunity.