FAP deficiency enhances thermogenesis and attenuates metabolic inflammation in diet-induced obesity.

OBJECTIVE: Fibroblast activation protein α (FAP) is expressed in normal adipose tissue and related to some pleiotropic metabolic regulators. However, the exact role and mechanism of FAP in obesity and related metabolic disorders are not well understood. METHODS: FAP knockout mice were fed a normal diet or a high-fat diet (HFD) for 12 weeks. FAP knockout mice or wild-type mice treated with an FAP inhibitor were subjected to cold stress for 5 days. RESULTS: FAP deficiency protected mice against HFD-induced obesity and obesity-associated metabolic dysfunction, including glucose intolerance, insulin resistance, hyperglycemia, hyperinsulinemia, and hyperlipidemia. Notably, FAP deficiency largely reversed obesity-induced adipose tissue macrophage accumulation and M1-M2 imbalance in white adipose tissue (WAT). Moreover, energy expenditure was significantly higher in FAP-deficient mice fed an HFD. Both FAP deficiency and inhibition increased cold tolerance through enhancing WAT beiging. CONCLUSIONS: This study demonstrated that FAP deficiency protects mice against diet-induced obesity and related metabolic dysfunction. Furthermore, the protective effects are probably mediated via the promotion of WAT beiging and suppression of inflammation.

Nanoparticle-based adjuvant for enhanced protective efficacy of DNA vaccine Ag85A-ESAT-6-IL-21 against Mycobacterium tuberculosis infection.

The goal of this study was to evaluate the protective efficacy of a cationic nanoparticle-based DNA vaccine expressing antigen 85A (Ag85A) and 6-kDa early secretory antigen target (ESAT-6) of Mycobacterium tuberculosis as well as cytokine interleukin-21 (IL-21) against M. tuberculosis infection. The results of this indicated that the anti-M. tuberculosis immune responses were induced in mice that had received the different DNA vaccines. More importantly, compared with using DNA vaccine Ag85A-ESAT-6-IL-21 alone, the nanoparticle-based DNA vaccine Ag85A-ESAT-6-IL-21 showed a statistically significant increase in the protective efficacy against M. tuberculosis infection in the immunized mice. We concluded that the nanoparticle-based DNA vaccine induced a strong immune response and markedly inhibited the growth of the M. tuberculosis in the mice. These findings highlighted the potential utility of Fe3O4-Glu-polyethyleneimine nanoparticles encapsulated with the DNA vaccine as a prophylactic vaccine in the M. tuberculosis-infected mouse model. FROM THE CLINICAL EDITOR: This study emphasizes the potential utility of Fe3O4-Glu-polyethyleneimine nanoparticles encapsulated with DNA vaccine against TB as a prophylactic vaccine. The authors demonstrated a strong immune response and marked growth inhibition of mycobacterium tuberculosis in the mice.

Elevated IL-35 level and iTr35 subset increase the bacterial burden and lung lesions in Mycobacterium tuberculosis-infected mice.

This study aimed to investigate the relationship between interleukin (IL)-35 level and IL-35-producing regulatory T cells (iTr35 subset) in Mycobacterium tuberculosis (Mtb)-infected mice. After the mice were injected with Mtb strain H37R via tail vein, the bacterial burden, lung lesions, and the impact of immune suppression on the infected mice were respectively assessed. The results, when compared with the control mice, showed that the mRNA expression levels of the p35 and Epstein-Barr virus-induced gene 3 of IL-35 were significantly increased in the Mtb-infected mouse spleen at 4 or 8 weeks post-infection and their protein expression levels were concurrently increased in the lungs of the mice, especially in 8 week infected mice. In addition, the levels of serum IL-35 and the iTr35 subset in the spleen of mice were also increased in 4 or 8 weeks post-infection compared with the control mice. Importantly, the high bacterial burden and lung lesions and the low mouse weight were found at 8 week post-infection. Therefore, the mice infected with Mtb resulted in elevating IL-35 level and iTr35 subset and increasing bacterial burden and lung lesions. The findings from the study suggest IL-35 and iTr35 cells may exert an immune suppression role in chronic Mtb-infected mice.

Immunization with DNA vaccine expressing herpes simplex virus type 1 gD and IL-21 protects against mouse herpes keratitis.

The development of novel vaccines to eradicate herpes simplex virus (HSV) is a global public health priority. In this study, we developed a DNA vaccine expressing HSV-1 glycoprotein D (gD) and mouse interleukin-21(IL-21) and intramuscularly inoculated mice 3 times at 2-week intervals with a total of 300 ?g/mouse. Two weeks after the last immunization the specific antibody, splenocyte proliferative response to gD, IFN-? and IL-4 as well as the cytotoxic activities of splenocytes and natural killer (NK) cells were assayed. Immune protection against herpes keratitis was concurrently evaluated in the immunized mice after HSV-1 challenge of the mouse cornea. The results showed that the DNA vaccine pRSC-gD-IL-21 generated higher levels of antibody, IFN-? and IL-4, and enhanced the splenocyte proliferative response to gD as well as the cytotoxic activity of splenocytes and NK cells to target cells compared with the response in either the pRSC-gD or mock plasmid pRSC immunized mice. Importantly, the pRSC-gD-IL-21 ameliorated herpes keratitis severity and time course after corneal infection with HSV-1. The findings suggest that the DNA vaccine pRSC-gD-IL-21 may induce an immune response that can limit HSV-1 infection and development of herpes keratitis in the immunized mice.

Human umbilical blood mononuclear cell-derived mesenchymal stem cells serve as interleukin-21 gene delivery vehicles for epithelial ovarian cancer therapy in nude mice.

Ovarian cancer causes more deaths than any other cancer of the female reproductive system, and its overall cure rate remains low. The present study investigated human umbilical blood mononuclear cell (UBMC)-derived mesenchymal stem cells (UBMC-MSCs) as interleukin-21 (IL-21) gene delivery vehicles for ovarian cancer therapy in nude mice. MSCs were isolated from UBMCs and the expanded cells were phenotyped by flow cytometry. Cultured UBMCs were differentiated into osteocytes and adipocytes using appropriate media and then the UBMC-MSCs were transfected with recombinant pIRES2-IL-21-enhancement green fluorescent protein. UBMC-MSCs expressing IL-21 were named as UBMC-MSC-IL-21. Mice with A2780 ovarian cancer were treated with UBMC-MSC-IL-21 intravenously, and the therapeutic efficacy was evaluated by the tumor volume and mouse survival. To address the mechanism of UBMC-MSC-IL-21 against ovarian cancer, the expression of IL-21, natural killer glucoprotein 2 domain and major histocompatibility complex class I chain-related molecules A/B were detected in UBMC-MSC-IL-21 and in the tumor sites. Interferon-γ-secreting splenocyte numbers and natural killer cytotoxicity were significantly increased in the UBMC-MSC-IL-21-treated mice as compared with the UBMC-MSCs or the UBMC-MSC-mock plasmid-treated mice. Most notably, tumor growth was delayed and survival was prolonged in ovarian-cancer-bearing mice treated with UBMC-MSC-IL-21. Our data provide important evidence that UBMC-MSCs can serve as vehicles for IL-21 gene delivery and inhibit the established tumor.

Antitumor efficacy of viable tumor vaccine modified by heterogenetic ESAT-6 antigen and cytokine IL-21 in melanomatous mouse.

The goal of this study was to investigate whether glycosylphosphatidylinositol (GPI)-anchored 6 kDa early secreted antigenic target (ESAT-6) and IL-21-producing B16F10/ESAT-6-GPI-IL-21 viable vaccine would induce antitumor efficacy. Mice were immunized with B16F10/ESAT-6-GPI-IL-21 vaccine and challenged by B16F10 cells 2 weeks later. Antitumor efficacy and mechanisms of the vaccine were analyzed. Vaccination with the viable B16F10/ESAT-6-GPI-IL-21 vaccine resulted in an increase of IFN-γ level and the CD8(+)CTL cytotoxicity, a decrease in TGF-ß generation and increase in the expression of miR-200c that serves as melanoma suppressor by directly targeting zinc-finger E-box binding homeobox 1 to inhibit epithelial-mesenchymal transition and block tumor metastasis. The vaccine significantly inhibited the melanoma growth, reduced the lung melanoma nodules, and prolonged the mouse survival compared with the controls. These findings highlighted IL-21 as an immune adjuvant in an engineered viable tumor vaccine to reinforce heterogenetic antigen ESAT-6 immune tolerance break to induce powerful antitumor efficacy in mice.

Antitumor efficacy induced by a B16F10 tumor cell vaccine treated with mitoxantrone alone or in combination with reserpine and verapamil in mice.

An apoptotic tumor cell serves as a potential potent trigger for the initiation of naturally occurring tumor immunity. In the present study, a B16F10 tumor cell vaccine treated with mitoxantrone (MIT) was developed, and its antitumor effect on mice was evaluated. The results indicated that the B16F10 tumor cell vaccine treated with MIT alone or in combination with reserpine (RP) and verapamil (VP) for 12 h triggered apoptosis, and that the expression of CD80, the MHC II class molecule, NKG2D and its ligand were significantly increased compared to the expression levels in the control group. The tumor vaccine immunogenicity was significantly enhanced in the vaccinated mice, resulting in augmented cytotoxicity of splenocytes and NK cells as well as the splenocyte proliferative response compared to the control group mice. Notably, the mice vaccinated with the B16F10 tumor cell vaccine treated with MIT, RP and VP did not generate tumors only after 60 days into the observation, but the mice also generated a powerful immune prophylactic efficiency against the B16F10 tumor cell challenge. These findings demonstrated the safety and efficacy of the B16F10 tumor cell vaccine treated with MIT alone or in combination with RP and VP in the murine model, and suggest that an apoptotic tumor cell vaccine modeled on naturally occurring tumor immune responses in vivo may provide a safe and immunogenic tumor vaccine for potential applications in humans.

An ocular mucosal administration of nanoparticles containing DNA vaccine pRSC-gD-IL-21 confers protection against mucosal challenge with herpes simplex virus type 1 in mice.

Herpes stromal keratitis (HSK) is a chronic inflammatory process caused by the infection of herpes simplex virus type 1 (HSV-1). Development of a HSV-1 vaccine is a priority because these infections are common and cannot be well prevented. It appears that the potential of nanocarriers in DNA vaccination will be required to augment the immune response to DNA vaccines. Therefore, in the study, nanoparticles Fe(3)O(4) coated with glutamic acid, DNA vaccine pRSC-gD-IL-21 and polyethylenimine were prepared and immunized in the mice by ocular mucosal administration. The immune responses and protection efficiency against HSV-1 challenge were also tested. The results showed that the nanoparticles containing DNA vaccine pRSC-gD-IL-21 induced mice to generate higher levels of specific neutralizing antibody, sIgA in tears, and IFN-γ, IL-4 in serum, and to enhance the cytotoxicities of NK cells and splenocytes as well as splenocyte proliferative response to glycoprotein D compared with those of the control mice. More importantly, the mice immunized with the experimental vaccine showed less HSK degree than that of the control mice after HSV-1 challenge of the murine ocular mucosa. In conclusion, an ocular mucosal administration of nanoparticles containing DNA vaccine confers strong specific immune responses and effective inhibition of HSK in a HSV-1 infected murine model.

Augmenting therapy of ovarian cancer efficacy by secreting IL-21 human umbilical cord blood stem cells in nude mice.

In the present study, CD34(+) human umbilical cord blood stem cells (UCBSCs) were engineered to express interleukin-21 (IL-21) and then were transplanted into A2780 ovarian cancer xenograft-bearing Balb/c nude mice. The therapeutic efficacy of this procedure on ovarian cancer was evaluated. The findings from the study indicated that UCBSCs did not form gross or histological teratomas until up to 70 days postinjection. The CD34(+) UCBSC-IL-21 therapy showed a consistent effect in the ovarian cancer of the treated mice, delaying the tumor appearance, reducing the tumor sizes, and extending life expectancy. The efficacy was attributable to keeping CD34(+) UCBSC-IL-21 in the neoplastic tissues for more than 21 days. The secreted IL-21 not only increased the quantity of CD11a(+) and CD56(+) NK cells but also increased NK cell cytotoxicities to YAC-1 cells and A2780 cells, respectively. The efficacy was also associated with enhancing the levels of IFN-γ, IL-4, and TNF-α in the mice as well as the high expressions of the NKG2D and MIC A/B molecules in the tumor tissues. This study suggested that transferring CD34(+) UCBSC-IL-21 into the nude mice was safe and feasible in ovarian cancer therapy, and that the method would be a promising new strategy for clinical treatment of ovarian cancer.

Enhancing therapy of B16F10 melanoma efficacy through tumor vaccine expressing GPI-anchored IL-21 and secreting GM-CSF in mouse model.

In the present study, we developed the tumor vaccine expressing IL-21 in the GPI-anchored form together with secreting GM-CSFs and investigated its antitumor efficacy in C57BL/6 mouse model. The fusion genes containing IL-21 and the GPI anchor signal sequence were acquired by overlaping PCR, inserted into the downstream of two multi-clone sites in recombinant plasmid pRSC/GM-CSFs to form pRSC/IL-21-gpi-GM-CSFs that was transfected into the B16F10 cells. The tumor cell vaccine B16F10/IL-21-gpi-GM-CSFs was identified by reverse transcription PCR, IFA and FCM, respectively. The results showed that the pRSC/IL-21-gpi-GM-CSFs had no cell cycle and proliferative state impact on the B16F10 cells after transfected, and that the tumor vaccine B16F10/IL-21-gpi-GM-CSFs increased the cytotoxicities of NK cells and CD8(+)CTL, enhanced the level of serum IFN-gamma, augmented therapy of tumor effect and prolonged survival time in the tumor-bearing mice immunized with the tumor vaccine B16F10/IL-21-gpi-GM-CSFs. The data that we presented here provided a rationale and practical platform for clinical testing of enhancing cell therapy of B16F10 melanoma efficacy by modified tumor vaccine expressing GPI-anchored IL-21 and secreting GM-CSF.

Investigation of immunogenic effect of the BCG priming and Ag85A- GM-CSF boosting in Balb/c mice model.

Mycobacterium tuberculosis (M. tuberculosis) has once again become a major public health threat owing to the combined effects of a worldwide anti-tuberculosis drug resistance and the emergence of the human immunodeficiency virus pandemic. The Bacille Calmette-Guérin (BCG)-based vaccine has displayed inconsistent efficacy in different trials although it continues to be used to prevent tuberculosis in many countries. In current work, we have developed DNA vaccines expressing M. tuberculosis antigen 85A (Ag85A) and cytokine granulocyte macrophage colony stimulating factor (GM-CSF) and aimed to investigate the immune effect in mice based on BCG priming and DNA vaccine boosting and immune protection against M. tuberculosis challenge. Our results showed that the activity of cytotoxic T lymphocyte and spleen cell proliferative responses to Ag85A and IFN-gamma level as well as the specific antibody titer against Ag85A were significantly increased in mice immunized with prime-boost strategy in comparison with the mice immunized with BCG or DNA vaccine expressing Ag85A and GM-CSF alone. Meanwhile, the immune strategy of BCG-prime and DNA vaccine boost induced mice to generate efficient immune protection against M. tuberculosis challenge. Our data demonstrate that BCG-prime and DNA vaccine expressing Ag85A and GM-CSF boost provides a rational strategy for further development of DNA vaccine against M. tuberculosis infection.