Reveals of quercetin's therapeutic effects on oral lichen planus based on network pharmacology approach and experimental validation.

Oral lichen planus (OLP) is a localized autoimmune disease of the oral mucosa, with an incidence of up to 2%. Although corticosteroids are the first-line treatment, they cause several adverse effects. Quercetin, a naturally occurring compound, has fewer side-effects and provides long-term benefits. Besides, it has powerful anti­inflammatory activities. Here, we combined network pharmacology with experimental verification to predict and verify the key targets of quercetin against OLP. First, 66 quercetin-OLP common targets were analyzed from various databases. The protein-protein interaction (PPI) network was constructed. Topology analysis and MCODE cluster analysis of common targets were conducted to identify 12 key targets including TP53, IL-6 and IFN-γ and their connections. Gene functions and key signaling pathways, including reactive oxygen species metabolism, IL-17 pathway and AGE-RAGE pathway, were enriched by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. Then, in vitro experiments showed that quercetin interfered with Th1/Th2 balance by acting on IL-6 and IFN-γ to modulate the immune system in treating OLP. Quercetin considerably affected the apoptosis and migration of T lymphocytes in OLP patients. Our study reveals the potential therapeutic targets and signaling pathways of quercetin associated with OLP, and establishes the groundwork for future clinical applications.

Adrenergic regulation of the vasculature impairs leukocyte interstitial migration and suppresses immune responses.

The sympathetic nervous system (SNS) controls various physiological functions via the neurotransmitter noradrenaline. Activation of the SNS in response to psychological or physical stress is frequently associated with weakened immunity. Here, we investigated how adrenoceptor signaling influences leukocyte behavior. Intravital two-photon imaging after injection of noradrenaline revealed transient inhibition of CD8+ and CD4+ T cell locomotion in tissues. Expression of ß-adrenergic receptor in hematopoietic cells was not required for NA-mediated inhibition of motility. Rather, chemogenetic activation of the SNS or treatment with adrenergic receptor agonists induced vasoconstriction and decreased local blood flow, resulting in abrupt hypoxia that triggered rapid calcium signaling in leukocytes and halted cell motility. Oxygen supplementation reversed these effects. Treatment with adrenergic receptor agonists impaired T cell responses induced in response to viral and parasitic infections, as well as anti-tumor responses. Thus, stimulation of the SNS impairs leukocyte mobility, providing a mechanistic understanding of the link between adrenergic receptors and compromised immunity.

Oligosaccharides from Polygonatum Cyrtonema Hua: Structural characterization and treatment of LPS-induced peritonitis in mice.

Fructooligosaccharide was isolated from Polygonatum Cyrtonema Hua (PFOS) for the first time. Structure characterized using FT-IR, MALDI-TOF-MS, NMR, AFM, and TEM, indicated that PFOS was graminan-type fructan with a degree of polymerization ranging from 5 to 10. A murine model of lipopolysaccharide (LPS)-induced peritonitis was used to evaluate the in vivo anti-inflammatory and lung protective efficacy of PFOS. The result shown that pretreatment with PFOS (1.0 mg/mL) in peritonitis-induced mice could significantly inhibit the level of pro-inflammatory cytokines (TNF-α, IL-1ß) in serum (P < 0.001), increase mice survival rate from 12.5 % to 54 % (P < 0.05), and alleviated lung injury through ameliorating the damage of the pulmonary cellular architecture and reducing inflammatory monocyte accumulation in lung tissue. This effect of oligosaccharides could explain the traditional usage of P. cyrtonema as a tonic medicine for respiratory problems and it could be used as a potential natural ingredient with anti-inflammatory activity.

Regional Hyperthermia Enhances Mesenchymal Stem Cell Recruitment to Tumor Stroma: Implications for Mesenchymal Stem Cell-Based Tumor Therapy.

The tropism of mesenchymal stem cells (MSCs) for tumors forms the basis for their use as delivery vehicles for the tumor-specific transport of therapeutic genes, such as the theranostic sodium iodide symporter (NIS). Hyperthermia is used as an adjuvant for various tumor therapies and has been proposed to enhance leukocyte recruitment. Here, we describe the enhanced recruitment of adoptively applied NIS-expressing MSCs to tumors in response to regional hyperthermia. Hyperthermia (41°C, 1 h) of human hepatocellular carcinoma cells (HuH7) led to transiently increased production of immunomodulatory factors. MSCs showed enhanced chemotaxis to supernatants derived from heat-treated cells in a 3D live-cell tracking assay and was validated in vivo in subcutaneous HuH7 mouse xenografts. Cytomegalovirus (CMV)-NIS-MSCs were applied 6-48 h after or 24-48 h before hyperthermia treatment. Using 123I-scintigraphy, thermo-stimulation (41°C, 1 h) 24 h after CMV-NIS-MSC injection resulted in a significantly increased uptake of 123I in heat-treated tumors compared with controls. Immunohistochemical staining and real-time PCR confirmed tumor-selective, temperature-dependent MSC migration. Therapeutic efficacy was significantly enhanced by combining CMV-NIS-MSC-mediated 131I therapy with regional hyperthermia. We demonstrate here for the first time that hyperthermia can significantly boost tumoral MSC recruitment, thereby significantly enhancing therapeutic efficacy of MSC-mediated NIS gene therapy.

Inhibition of macrophage migration in zebrafish larvae demonstrates in vivo efficacy of human CCR2 inhibitors.

The chemokine signaling axes CCR2-CCL2 and CXCR3-CXCL11 participate in the inflammatory response by recruiting leukocytes to damaged tissue or sites of infection and are, therefore, potential pharmacological targets to treat inflammatory disorders. Although multiple CCR2 orthosteric and allosteric inhibitors have been developed, none of these compounds has been approved for clinical use, highlighting the need for a fast, simple and robust preclinical test system to determine the in vivo efficacy of CCR2 inhibitors. Herein we show that human CCL2 and CXCL11 drive macrophage recruitment in zebrafish larvae and that CCR2 inhibitors designed for humans also limit macrophage recruitment in this model organism due to the high conservation of the chemokine system. We demonstrated anti-inflammatory activities of three orthosteric and two allosteric CCR2 inhibitors using macrophage recruitment to injury as a functional read-out of their efficiency, while simultaneously evaluating toxicity. These results provide proof-of-principle for screening CCR2 inhibitors in the zebrafish model.

Screening of a Panel of Low Molecular Weight Compounds That Inhibit Synovial Fibroblast Invasion in Rheumatoid Arthritis.

Increased invasion of synovial fibroblasts and their involvement in cartilage damage are characteristic phenotypes of rheumatoid arthritis (RA). To identify low molecular weight compounds that suppress synovial fibroblast invasion, a panel of inhibitors (n = 330) was initially screened using a real-time cell analysis system for human synovial fibroblasts that were enzymatically isolated from surgical samples of RA patients. To evaluate the effects of the inhibitors identified in the screen, synovial fibroblast migration was measured using a wound-healing assay, and phosphorylation of intracellular signaling molecules was determined by immunoblots. Several candidate inhibitors were identified in the screen, including inhibitors against platelet-derived growth factor receptor (PDGFR), Akt, PI3K, and glycogen kinase synthetase 3 (GSK-3). These inhibitors strongly suppressed synovial fibroblast migration after 72 h and downregulated phosphorylation of Akt (Ser473) at 48 h. When the inhibitors were removed from the culture conditions, both migration and phosphorylated Akt (Ser473) levels were restored. Furthermore, all the categories of inhibitors except for PDGFR inhibitor IV decreased cell proliferation as well as IL-6 production in synovial fibroblasts. Interestingly, GSK-3 inhibitors increased anti-inflammatory cytokine IL-10 production but suppressed IL-23 production from LPS-primed macrophages obtained from healthy donors. In conclusion, blocking PDGFR, PI3K, or GSK-3 could have therapeutic value as an RA treatment that targets the invasion/migration of synovial fibroblasts.

Roles of galectin­3 in the tumor microenvironment and tumor metabolism (Review).

Galectin­3 is expressed in various tissues and plays an important role in the tumor microenvironment (TME). Galectin­3 has been found to be overexpressed in a variety of cancers and is associated with tumor progression and metastasis. Over the past decades, emerging evidence has suggested that the TME may induce galectin­3 expression to maintain cellular homeostasis and promote cell survival. Furthermore, galectin­3 regulates immune cell function to promote tumor­driven immunosuppression through several mechanisms. In the TME, intracellular and extracellular galectin­3 has different functions. In addition, it has been reported that galectin­3 is associated with glycolysis and mitochondrial metabolism in tumors, and it is involved in the regulation of relevant signaling pathways, thus promoting cancer cell survival via adapting to the TME. The aim of the present review was to summarize the current knowledge on galectin­3 production and its function in the TME, its effect on TME immunosuppression, its association with tumor metabolism and relevant signaling pathways, and to report common types of cancer in which galectin­3 is highly expressed, in order to ensure a comprehensive understanding of the critical effects of galectin­3 on tumor progression and metastasis.

Shikonin blocks human lung adenocarcinoma cell migration and invasion in the inflammatory microenvironment via the IL­6/STAT3 signaling pathway.

Increasing evidence indicates that the inflammatory tumor microenvironment can lead to cancer cell metastasis. Shikonin, which is extracted from the Chinese herb Zicao (the dried root of Lithospermum erythrorhizon), possesses various pharmacological effects, but its effect on tumor metastasis in the inflammatory microenvironment remains unknown. In the present study, we aimed to investigate the potential effect of shikonin on tumor metastasis in an inflammatory microenvironment as well as the underlying molecular mechanisms. It was found that, in the inflammatory microenvironment simulated by THP­1 cell conditioned medium (THP­1­CM) in vitro, shikonin significantly inhibited the epithelial­mesenchymal transition (EMT), migration and invasion of human lung adenocarcinoma cell lines A549 and H1299. In addition, we found that interleukin­6 (IL­6), which is expressed in THP­1­CM, promoted the EMT of lung adenocarcinoma cells, and shikonin markedly inhibited IL­6­induced EMT and cell motility. Moreover, shikonin inhibited IL­6­induced phosphorylation of signal transducer and activator of transcription 3 (STAT3), prevented phosphorylated STAT3 (p­STAT3) translocation into the nucleus, and suppressed p­STAT3 transactivation activity. Additionally, it was found that shikonin inhibited lung metastasis, EMT and expression of p­STAT3 of A549 cells in vivo. Furthermore, IL­6 levels in human lung adenocarcinoma tissues were significantly associated with tumor­node­metastasis stage and lymph node metastasis, and its expression was correlated with tumor­associated macrophage (TAM) infiltration. Together, these results suggest that shikonin suppresses the migration and invasion of human lung adenocarcinoma cells in an inflammatory microenvironment involving the IL­6/STAT3 signaling pathway.

Molecular iodine exerts antineoplastic effects by diminishing proliferation and invasive potential and activating the immune response in mammary cancer xenografts.

BACKGROUND: The immune system is a crucial component in cancer progression or regression. Molecular iodine (I2) exerts significant antineoplastic effects, acting as a differentiation inductor and immune modulator, but its effects in antitumor immune response are not elucidated. METHODS: The present work analyzed the effect of I2 in human breast cancer cell lines with low (MCF-7) and high (MDA-MB231) metastatic potential under both in vitro (cell proliferation and invasion assay) and in vivo (xenografts of athymic nude mice) conditions. RESULTS: In vitro analysis showed that the 200 µM I2 supplement decreases the proliferation rate in both cell lines and diminishes the epithelial-mesenchymal transition (EMT) profile and the invasive capacity in MDA-MB231. In immunosuppressed mice, the I2 supplement impairs implantation (incidence), tumoral growth, and proliferation of both types of cells. Xenografts of the animals treated with I2 decrease the expression of invasion markers like CD44, vimentin, urokinase plasminogen activator and its receptor, and vascular endothelial growth factor; and increase peroxisome proliferator-activated receptor gamma. Moreover, in mice with xenografts, the I2 supplement increases the circulating level of leukocytes and the number of intratumoral infiltrating lymphocytes, some of them activated as CD8+, suggesting the activation of antitumor immune responses. CONCLUSIONS: I2 decreases the invasive potential of a triple negative basal cancer cell line, and under in vivo conditions the oral supplement of this halogen activates the antitumor immune response, preventing progression of xenografts from laminal and basal mammary cancer cells. These effects allow us to propose iodine supplementation as a possible adjuvant in breast cancer therapy.

Dendritic cell trafficking in tumor-bearing mice.

Prostate cancer is one of the leading causes of cancer deaths, with no curative treatments once it spreads. Alternative therapies, including immunotherapy, have shown limited efficacy. Dendritic cells (DC) have been widely used in the treatment of various malignancies. DC capture antigens and move to the lymphoid organs where they prime naive T cells. Interaction between DC and T cells are most active in lymph nodes and suppression of DC trafficking to lymph nodes impairs the immune response. In this work, we aimed to study trafficking of DC in vivo via various routes of delivery, to optimize the effectiveness of DC-based therapy. A DC labeling system was developed using 1,1'-dioctadecyltetramethyl indotricarbocyanine Iodine for in vivo fluorescent imaging. DC harvested from C57B/6 mice were matured, labeled, and injected intravenously, subcutaneously, or intratumorally, with or without antigen loading with whole tumor lysate, into C57B/6 mice inoculated with RM-1 murine prostate tumor cells. Signal intensity was measured in vivo and ex vivo. Signal intensity at the tumor site increased over time, suggesting trafficking of DC to the tumor with all modes of injection. Subcutaneous injection showed preferential trafficking to lymph nodes and tumor. Intravenous injection showed trafficking to lungs, intestines, and spleen. Subcutaneous injection of DC pulsed with whole tumor lysate resulted in the highest increase in signal intensity at the tumor site and lymph nodes, suggesting subcutaneous injection of primed DC leads to highest preferential trafficking of DC to the immunocompetent organs.

Modulation the crosstalk between tumor-associated macrophages and non-small cell lung cancer to inhibit tumor migration and invasion by ginsenoside Rh2.

BACKGROUND: Tumor-associated macrophages (TAMs) play a critical role in modulating the tumor microenvironment and promote tumor metastases. Our studies have demonstrated that ginsenoside Rh2 (G-Rh2), a monomeric compound extracted from ginseng, is a promising anti-tumor agent in lung cancer cells. However, it remains unclear whetherG-Rh2 can modulate the differentiation of TAMs and its interaction with tumor microenvironment. In this study, we investigated how G-Rh2 regulates the phenotype of macrophages and affects the migration of non-small cell lung cancer (NSCLC) cells. METHODS: Murine macrophage-like RAW264.7 cells and human THP-1 monocyte were differentiated into M1 and M2 subsets of macrophages with different cytokines combination, which were further identified by flow cytometry with specific biomarkers. M2 macrophages were sorted out to co-culture with NSCLC cell lines, A549 and H1299. Wound healing assay was performed to examine the cell migration. Expression levels of matrix metalloproteinases 2 and 9 (MMP-2, - 9) and vascular endothelial growth factor-C (VEGF-C) were measured by RT-qPCR and western blot, and the release of VEGF in the supernatant was measured by a VEGF ELISA kit. Finally, modulation of TAMs phenotype and VEGF expression by G-Rh2 was examined in vivo. RESULTS: We demonstrated that M2 subset of macrophages alternatively differentiated from RAW264.7 or THP-1cells promote migration of NSCLC cells. Further examinations revealed that NSCLC significantly increased the release of VEGF to the media and elevated the expression levels of VEGF at mRNA and protein levels after being co-cultured with M2 macrophages. Similar alterations in MMP-2 and MMP-9 were observed in NSCLC after being co-cultured. Of note,G-Rh2 had a potential to effectively convert M2 phenotype to M1 subset of macrophages. Importantly, G-Rh2 had a preference to decrease the expression levels of VEGF, MMP2, and MMP9 in co-cultured lung cancer cells, over than those in lung cancer cells without co-culturing. Consistently, G-Rh2 reduced M2 macrophage marker CD206 and VEGF expression levels in vivo. CONCLUSIONS: All of these results suggested that M2 subset macrophages drive lung cancer cells with more aggressive phenotypes. G-Rh2 has a potential to convert TAMs from M2 subset to M1 in the microenvironment and prevents lung cancer cell migration, suggesting the therapeutic effects of G-Rh2onlung cancer.

An Aliphatic Ester Diisopropyl Sebacate Exhibited an Adjuvant Effect on Fluorescein Isothiocyanate-Induced Contact Hypersensitivity Mouse Models.

Alternative plasticizers have become more popular due to health concerns about phthalate esters. We demonstrated that phthalate esters enhanced skin sensitization to fluorescein isothiocyanate (FITC) in mouse contact hypersensitivity models. Alternative plasticizers have not been well studied as to their effect on the immune system. We previously found that diisopropyl adipate (DIPA), an aliphatic dicarboxylic acid ester, enhanced skin sensitization to FITC. Sebacate esters are also widely used as alternative plasticizers. Here we tested diisopropyl sebacate (DIPS), which has the same alcohol with an aliphatic dicarboxylic acid of longer chain, using BALB/c mice. The results showed that DIPS facilitated skin sensitization to FITC and increased FITC-presenting dendritic cell trafficking from the skin to draining lymph nodes. Furthermore, DIPS activated transient receptor potential ankyrin 1 (TRPA1). The latter feature has been commonly observed for phthalate esters and DIPA, which have adjuvant effects. In summary, the adjuvant effect of a sebacate ester was demonstrated in a mouse model.

The Alpha-Tocopherol Form of Vitamin E Boosts Elastase Activity of Human PMNs and Their Ability to Kill Streptococcus pneumoniae.

Despite the availability of vaccines, Streptococcus pneumoniae remains a leading cause of life-threatening infections, such as pneumonia, bacteremia and meningitis. Polymorphonuclear leukocytes (PMNs) are a key determinant of disease course, because optimal host defense requires an initial robust pulmonary PMN response to control bacterial numbers followed by modulation of this response later in infection. The elderly, who manifest a general decline in immune function and higher basal levels of inflammation, are at increased risk of developing pneumococcal pneumonia. Using an aged mouse infection model, we previously showed that oral supplementation with the alpha-tocopherol form of vitamin E (α-Toc) decreases pulmonary inflammation, in part by modulating neutrophil migration across lung epithelium into alveolar spaces, and reverses the age-associated decline in resistance to pneumococcal pneumonia. The objective of this study was to test the effect of α-Toc on the ability of neutrophils isolated from young (22-35 years) or elderly (65-69 years) individuals to migrate across epithelial cell monolayers in response to S. pneumoniae and to kill complement-opsonized pneumococci. We found that basal levels of pneumococcal-induced transepithelial migration by PMNs from young or elderly donors were indistinguishable, suggesting that the age-associated exacerbation of pulmonary inflammation is not due to intrinsic properties of PMNs of elderly individuals but rather may reflect the inflammatory milieu of the aged lung. Consistent with its anti-inflammatory activity, α-Toc treatment diminished PMN migration regardless of donor age. Unexpectedly, unlike previous studies showing poor killing of antibody-opsonized bacteria, we found that PMNs of elderly donors were more efficient at killing complement-opsonized bacteria ex vivo than their younger counterparts. We also found that the heightened antimicrobial activity in PMNs from older donors correlated with increased activity of neutrophil elastase, a serine protease that is required to kill pneumococci. Notably, incubation with α-Toc increased PMN elastase activity from young donors and boosted their ability to kill complement-opsonized pneumococci. These findings demonstrate that α-Toc is a potent modulator of PMN responses and is a potential nutritional intervention to combat pneumococcal infection.

Ameliorative effect of a rarely occurring C-methylrotenoid on HMGB1-induced septic responses in vitro and in vivo.

The ubiquitous nuclear protein, high mobility group box-1 (HMGB1) functions as a late mediator of sepsis. A new rarely occurring C-methylrotenoid, named boeravinone X (comp 1), was isolated and identified from Abronia nana suspension cultures during our continuous works on the discovery of anti-septic natural products. Here, we investigated the antiseptic effects and underlying mechanisms of comp 1 against HMGB1-mediated septic responses. According to the results, comp 1 effectively inhibited lipopolysaccharide (LPS)-induced release of HMGB1, and suppressed HMGB1-mediated septic responses, such as hyperpermeability, adhesion and migration of leukocytes, and expression of cell adhesion molecules. And, comp 1 suppressed the production of tumor necrosis factor alpha (TNF-α) and interleukin 6 (IL-6), and the activation of nuclear factor-κB (NF-κB) and extracellular signal-regulated kinases 1 and 2 (ERK1/2) by HMGB1. Collectively, these results indicate that comp 1 could be potential therapeutic agents for the treatment of various severe vascular inflammatory diseases via inhibition of the HMGB1 signaling pathway.

UV-inactivated HSV-1 potently activates NK cell killing of leukemic cells.

Herein we demonstrate that oncolytic herpes simplex virus-1 (HSV-1) potently activates human peripheral blood mononuclear cells (PBMCs) to lyse leukemic cell lines and primary acute myeloid leukemia samples, but not healthy allogeneic lymphocytes. Intriguingly, we found that UV light-inactivated HSV-1 (UV-HSV-1) is equally effective in promoting PBMC cytolysis of leukemic cells and is 1000- to 10 000-fold more potent at stimulating innate antileukemic responses than UV-inactivated cytomegalovirus, vesicular stomatitis virus, reovirus, or adenovirus. Mechanistically, UV-HSV-1 stimulates PBMC cytolysis of leukemic cells, partly via Toll-like receptor-2/protein kinase C/nuclear factor-κB signaling, and potently stimulates expression of CD69, degranulation, migration, and cytokine production in natural killer (NK) cells, suggesting that surface components of UV-HSV-1 directly activate NK cells. Importantly, UV-HSV-1 synergizes with interleukin-15 (IL-15) and IL-2 in inducing activation and cytolytic activity of NK cells. Additionally, UV-HSV-1 stimulates glycolysis and fatty acid oxidation-dependent oxygen consumption in NK cells, but only glycolysis is required for their enhanced antileukemic activity. Last, we demonstrate that T cell-depleted human PBMCs exposed to UV-HSV-1 provide a survival benefit in a murine xenograft model of human acute myeloid leukemia (AML). Taken together, our results support the preclinical development of UV-HSV-1 as an adjuvant, alone or in combination with IL-15, for allogeneic donor mononuclear cell infusions to treat AML.

Interaction of dendritic cells and T lymphocytes for the therapeutic effect of Dangguiliuhuang decoction to autoimmune diabetes.

In traditional Chinese medicine (TCM), Dangguiliuhuang decoction (DGLHD) is an effective treatment of autoimmune diabetes. Here, we studied potential anti-diabetic mechanisms of DGLHD in a non-obese diabetic (NOD) mouse model. In vitro, DGLHD and individual active ingredients enhanced glucose uptake in HepG2 cells, inhibited T lymphocyte proliferation, and suppressed dendritic cells (DCs) function. In vivo, DGLHD significantly inhibited insulitis, delayed the onset and development of diabetes, promoted insulin secretion and sensitivity, and balanced partially normalized Th1 and Th2 cytokines in NOD mice. In addition, DGLHD increased α1-antitrypsin (AAT-1), Bcl-2, and CyclinD1, and decreased Bax levels in pancreas, spleen, thymus, DCs, and a NIT-1 cell line, all consistent with protecting and repairing islet ß cell. More detailed studies indicated that DGLHD regulated the maturation and function of DCs, decreased the percentage of merocytic dendritic cells (mcDCs) subset, and increased programmed death ligand-1 (PD-L1) expression in DCs. DGLHD also impeded T lymphocyte proliferation and promoted regulatory T cells (T(regs)) differentiation in vivo. A JAK2-STAT3-dependent pathway was involved in the suppression by DGLHD of interactions between DCs and T lymphocyte. The experiments implicated five active ingredients in specific anti-diabetic actions of DGLHD. The results demonstrated the reasonable composition of the formula.

Critical role for syndecan-4 in dendritic cell migration during development of allergic airway inflammation.

Syndecan-4 (SDC4), expressed on dendritic cells (DCs) and activated T cells, plays a crucial role in DC motility and has been shown as a potential target for activated T-cell-driven diseases. In the present study, we investigate the role of SDC4 in the development of T-helper 2 cell-mediated allergic asthma. Using SDC4-deficient mice or an anti-SDC4 antibody we show that the absence or blocking of SDC4 signalling in ovalbumin-sensitized mice results in a reduced asthma phenotype compared with control animals. Most importantly, even established asthma is significantly decreased using the anti-SDC4 antibody. The disturbed SDC4 signalling leads to an impaired motility and directional migration of antigen-presenting DCs and therefore, to a modified sensitization leading to diminished airway inflammation. Our results demonstrate that SDC4 plays an important role in asthma induction and indicate SDC4 as possible target for therapeutic intervention in this disease.

Serial low doses of sorafenib enhance therapeutic efficacy of adoptive T cell therapy in a murine model by improving tumor microenvironment.

Requirements of large numbers of transferred T cells and various immunosuppressive factors and cells in the tumor microenvironment limit the applications of adoptive T cells therapy (ACT) in clinic. Accumulating evidences show that chemotherapeutic drugs could act as immune supportive instead of immunosuppressive agents when proper dosage is used, and combined with immunotherapy often results in better treatment outcomes than monotherapy. Controversial immunomodulation effects of sorafenib, a multi-kinases inhibitor, at high and low doses have been reported in several types of cancer. However, what is the range of the low-dose sorafenib will influence the host immunity and responses of ACT is still ambiguous. Here we used a well-established E.G7/OT-1 murine model to understand the effects of serial low doses of sorafenib on both tumor microenvironment and transferred CD8+ T cells and the underlying mechanisms. Sorafenib lowered the expressions of immunosuppressive factors, and enhanced functions and migrations of transferred CD8+ T cells through inhibition of STAT3 and other immunosuppressive factors. CD8+ T cells were transduced with granzyme B promoter for driving imaging reporters to visualize the activation and distribution of transferred CD8+ T cells prior to adoptive transfer. Better activations of CD8+ T cells and tumor inhibitions were found in the combinational group compared with CD8+ T cells or sorafenib alone groups. Not only immunosuppressive factors but myeloid derived suppressive cells (MDSCs) and regulatory T cells (Tregs) were decreased in sorafenib-treated group, indicating that augmentation of tumor inhibition and function of CD8+ T cells by serial low doses of sorafenib were via reversing the immunosuppressive microenvironment. These results revealed that the tumor inhibitions of sorafenib not only through eradicating tumor cells but modifying tumor microenvironment, which helps outcomes of ACT significantly.

The Wnt/ß-catenin pathway attenuates experimental allergic airway disease.

Signaling via the Wnt/ß-catenin pathway plays crucial roles in embryogenesis and homeostasis of adult tissues. In the lung, the canonical Wnt/ß-catenin pathway has been implicated in remodeling processes, development of emphysema, and fibrosis. However, its relevance for the modulation of allergic responses in the lung remains unclear. Using genetically modified mice with lung-specific inducible (doxycycline) Wnt-1 expression (CCSP-rtTA × tetO-Wnt1), the impact of Wnt on the development of allergic airway disease was analyzed. Overexpression of Wnt during the allergen challenge phase attenuated the development of airway inflammation in an acute model, as well as in a more therapeutic model of secondary challenge. These findings were further supported by treatment of allergen-sensitized mice with LiCl during challenge. Similar to Wnt, LiCl prevented the degradation of ß-catenin and, thus, attenuated allergic airway inflammation and hyperresponsiveness. Migration studies revealed that lung-specific expression of Wnt reduced the migration of Ag-loaded dendritic cells (DCs) into the draining lymph nodes following allergen challenge. Administration of in vitro allergen-loaded DCs overcame Wnt-mediated suppression of airway inflammation. Furthermore, in vitro studies confirmed that DC-dependent T cell activation is impaired by blocking ß-catenin degradation. These results demonstrate an important role for the canonical Wnt/ß-catenin pathway in the DC-mediated regulation of allergic responses in the lung.

The immunosuppressant Protosappanin A diminished recipient T cell migration into allograft via inhibition of IP-10 in rat heart transplant.

The immunosuppressant Protosappanin A (PrA), isolated from the medicinal herb, promotes cardiac allograft survival, diminishes inflammatory cell infiltration, and inhibits interferon γ-induced protein 10 kDa (IP-10) mRNA expression in rats cardiac grafts. Binding of the chemokine IP-10 to its cognate receptor, CXCR3, plays crucial roles in allograft immunity, especially by mediating the recruitment of effector T cells to allografted tissues. In this study, we attempted to determine whether PrA-mediated inhibition of IP-10 contributes to the effect of reduced T cell infiltration into cardiac allograft within a rat model. Administration of PrA (25 mg/kg daily) via oral gavage following heart transplantation significantly reduced the increase of IP-10 mRNA level in allograft and prevented IP-10 secretion by peripheral blood mononuclear cells (PBMC) isolated from recipient rats seven days posttransplantation. Furthermore, in vitro experiments demonstrated that PrA addition to control PBMC prevented IP-10 secretion. Chemotactic migration assays were utilized to evaluate recipient T cell migration towards PBMC supernatant. PrA administration impaired PBMC supernatant-induced T cell migration. Additional in vitro experiments revealed that PrA slightly reduced naïve T cell migration towards chemokines. The presence of IP-10 in PBMC supernatant prevented PrA from reducing T cell migration in PrA-treated recipients. Neither CXCR3 chemokine ligand Mig nor non-CXCR3 chemokine ligand SDF-1 had any effect on T cell migration in PrA-treated recipients. The addition of anti-CXCR3 antibody restored PrA-mediated inhibition of T cell migration. Immunofluorescence microscopy showed that IP-10 was expressed mainly in CD68 positive infiltrating monocytes. Furthermore, PrA consistently reduced CXCR3+T cell infiltration into cardiac allografts. The reduced intensity of CXCR3 staining in PrA-treated allografts contributed to the previously depressed naïve T cell migrating activity induced by PrA. Collectively, these data indicate that PrA inhibition of IP-10 activity reduced recipient T cell migration and infiltration of cardiac allografts, thus partially explaining the immunosuppressive effect of PrA.