Activation of the Melanocortin-4 receptor signaling by α-MSH stimulates nerve-dependent mouse digit regeneration.

BACKGROUND: Expression of Mc4r in peripheral organs indicates it has broader roles in organ homeostasis and regeneration. However, the expression and function of Mc4r in the mouse limb and digit has not been fully investigated. Our previous work showed that Mc4r-/- mice fail to regenerate the digit, but whether activation of MC4R signaling could rescue digit regeneration, or stimulate proximal digit regeneration is not clear. RESULTS: We analyzed the expression dynamics of Mc4r in the embryonic and postnatal mouse limb and digit using the Mc4r-gfp mice. We found that Mc4r-GFP is mainly expressed in the limb nerves, and in the limb muscles that are undergoing secondary myogenesis. Expression of Mc4r-GFP in the adult mouse digit is restricted to the nail matrix. We also examined the effect of α-MSH on mouse digit regeneration. We found that administration of α-MSH in the Mc4r+/- mice rescue the delayed regeneration of distal digit tip. α-MSH could rescue distal digit regeneration in denervated hindlimbs. In addition, α-MSH could stimulate regeneration of the proximally amputated digit, which is non-regenerative. CONCLUSIONS: Mc4r expression in the mouse limb and digit is closely related to nerve tissues, and α-MSH/MC4R signaling has a neurotrophic role in mouse digit tip regeneration.

Melanocortin Receptor 4 Signaling Regulates Vertebrate Limb Regeneration.

Melanocortin 4 receptor (Mc4r) plays a crucial role in the central control of energy homeostasis, but its role in peripheral organs has not been fully explored. We have investigated the roles of hypothalamus-mediated energy metabolism during Xenopus limb regeneration. We report that hypothalamus injury inhibits Xenopus tadpole limb regeneration. By loss-of-function and gain-of-function studies, we show that Mc4r signaling is required for limb regeneration in regeneration-competent tadpoles and stimulates limb regeneration in later-stage regeneration-defective tadpoles. It regulates limb regeneration through modulating energy homeostasis and ROS production. Even more interestingly, our results demonstrate that Mc4r signaling is regulated by innervation and α-MSH substitutes for the effect of nerves in limb regeneration. Mc4r signaling is also required for mouse digit regeneration. Thus, our findings link vertebrate limb regeneration with Mc4r-mediated energy homeostasis and provide a new avenue for understanding Mc4r signaling in the peripheral organs.

Generation of iPSC-derived limb progenitor-like cells for stimulating phalange regeneration in the adult mouse.

The capacity of digit tip regeneration observed both in rodents and humans establishes a foundation for promoting robust regeneration in mammals. However, stimulating regeneration at more proximal levels, such as the middle phalanges (P2) of the adult mouse, remains challenging. Having shown the effectiveness of transplantation of limb progenitor cells in stimulating limb regeneration in Xenopus, we are now applying the cell transplantation approach to the adult mouse. Here we report that both embryonic and induced pluripotent stem cell (iPSC)-derived limb progenitor-like cells can promote adult mouse P2 regeneration. We have established a simple and efficient protocol for deriving limb progenitor-like cells from mouse iPSCs. iPSCs are cultured as three-dimensional fibrin bodies, followed by treatment with combinations of Fgf8, CHIR99021, Purmorphamine and SB43542 during differentiation. These iPSC-derived limb progenitor-like cells resemble embryonic limb mesenchyme cells in their expression of limb-related genes. After transplantation, the limb progenitor-like cells can promote adult mouse P2 regeneration, as embryonic limb bud cells do. Our results provide a basis for further developing progenitor cell-based approaches for improving regeneration in the adult mouse limbs.

Overcoming HBV immune tolerance to eliminate HBsAg-positive hepatocytes via pre-administration of GM-CSF as a novel adjuvant for a hepatitis B vaccine in HBV transgenic mice.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is known to be a potential vaccine adjuvant despite contradictory results from animal and human studies. The discrepancies may be due to the different doses and regimens of GM-CSF that were used, given that either mature or immature dendritic cells (DCs) could be induced under different conditions. To test the hypothesis that GM-CSF can be used as a novel adjuvant for a hepatitis B virus (HBV) therapeutic vaccine, we administered GM-CSF once per day for three days prior to vaccination with recombinant HBV vaccine (rHBVvac) in mice. We observed greater DC maturation in these pre-treated animals at day 3 as compared to day 1 or day 2 of daily GM-CSF administration. This strategy was further investigated for its ability to break the immune tolerance established in hepatitis B surface antigen-transgenic (HBsAg-Tg) animals. We found that the levels of induced anti-HBsAg antibodies were significantly higher in animals following three days of GM-CSF pre-treatment before rHBV vaccination after the third immunization. In addition to the increase in anti-HBsAg antibody levels, cell-mediated anti-HBsAg responses, including delayed-type hypersensitivity, T-cell proliferation, interferon-γ production, and cytotoxic T lymphocytes, were dramatically enhanced in the three-day GM-CSF pre-treated group. After adoptive transfers of CD8+ T cells from immunized animals, antigen-specific CD8+ T cells were observed in the livers of recipient HBsAg-Tg animals. Moreover, the three-day pre-treatments with GM-CSF prior to rHBVvac vaccination could significantly eliminate HBsAg-positive hepatocytes, suggesting beneficial therapeutic effects. Therefore, this protocol utilizing GM-CSF as an adjuvant in combination with the rHBVvac vaccine has the potential to become a novel immunotherapy for chronic hepatitis B patients.

A coimmunization vaccine of Aß42 ameliorates cognitive deficits without brain inflammation in an Alzheimer's disease model.

INTRODUCTION: Vaccination against amyloid-ß protein (Aß42) induces high levels of antibody, making it a promising strategy for treating Alzheimer's disease (AD). One drawback in the past was that clinical trial approval was withheld because of speculation that the Aß42 vaccine induces CD4(+) T cell infiltrations into the central nervous system. To reduce T-cell activation while concomitantly maintaining high anti-Aß42 titers is a great challenge in immunology. METHODS: We aimed to demonstrate that coimmunization with Aß42 protein and expression plasmid can be beneficial in a mouse AD model and can prevent inflammation. We immunized the AD mice with the coimmunization vaccine and assessed behavior change and Aß42 deposition. Furthermore, to determine the safety of the coimmunization vaccine, we used an induced Aß42-EAE model to mimic the meningoencephalitis that happened in the AN-1792 vaccine clinical phase II trial and tested whether the coimmunization vaccine could ameliorate T-cell-mediated brain inflammation. RESULTS: The coimmunization vaccination reduced Aß plaques and significantly ameliorated cognitive deficit while inhibiting T-cell-mediated brain inflammation and infiltration. These studies demonstrate that the coimmunization strategy that we describe in this article can ameliorate AD pathology without notable adverse effects in mice. CONCLUSIONS: A coimmunization strategy leading to the development of a safe immunotherapeutic/preventive protocol against AD in humans is warranted.

Transgenic Eimeria tenella expressing enhanced yellow fluorescent protein targeted to different cellular compartments stimulated dichotomic immune responses in chickens.

Eimeria tenella, one of the seven species of chicken coccidia, elicits protective immunity against challenge infection with both homologous and heterologous strains. We endeavor to use recombinant E. tenella as a vaccine vehicle for expressing and delivering pathogen Ags and investigate immune responses against these foreign Ags. In this study, two lines of transgenic E. tenella expressing a model Ag, enhanced yellow fluorescent protein (EYFP), targeted to the micronemes and to the cytoplasm of the recombinant parasites were constructed to study the impact of Ag compartmentalization on immunogenicity. The MTT assay, intracellular cytokine staining, and real-time PCR were performed to detect the EYFP-specific proliferation and effector functions of splenic lymphocytes of immunized chickens. ELISA was used to measure anti-EYFP IgG and IgA responses. The results showed that both lines of transgenic parasites stimulated EYFP-specific lymphocyte proliferation and IFN-γ expression in CD4 and CD8 T cells, whereas a higher level of Ag-specific lymphocyte proliferation was elicited by the transgenic line expressing microneme-targeted EYFP. Furthermore, this line stimulated stronger IgA response than the one expressing cytoplasm-targeted EYFP after the second immunization. Our findings are encouraging for further investigation of the effect of Ag compartmentalization in transgenic Eimeria on immunogenicity and for the development of a eukaryotic vaccine vector using genetically modified Apicomplexa parasites.

Increasing a robust antigen-specific cytotoxic T lymphocyte response by FMDV DNA vaccination with IL-9 expressing construct.

Various chemokines and cytokines as adjuvants can be used to improve efficacy of DNA vaccination. In this study, we sought to investigate if a DNA construct expressing IL-9 (designed as proV-IL9) as a molecular adjuvant enhance antigen specific immune responses elicited by the pcD-VP1 DNA vaccination. Mice immunized with pcD-VP1 combined with proV-IL9 developed a strong humoral response. In addition, the coinoculation induced significant higher level of antigen-specific cell proliferation and cytotoxic response. This agreed well with higher expression level of IFN-gamma and perforin in CD8+ T cells, but not with IL-17 in these T cells. The results indicate that IL-9 induces the development of IFN-gamma-producing CD8+ T cells (Tc1), but not the IL-17-producing CD8+ T cells (Tc17). Up-regulated expressions of BCL-2 and BCL-XL were exhibited in these Tc1 cells, suggesting that IL-9 may trigger antiapoptosis mechanism in these cells. Together, these results demonstrated that IL-9 used as molecular adjuvant could enhance the immunogenicity of DNA vaccination, in augmenting humoral and cellular responses and particularly promoting Tc1 activations. Thus, the IL-9 may be utilized as a potent Tc1 adjuvant for DNA vaccines.

CD40-expressing plasmid induces anti-CD40 antibody and enhances immune responses to DNA vaccination.

BACKGROUND: Various approaches have been used to improve the efficacy of DNA vaccination, including the incorporation of molecular adjuvants. Because the CD40 ligand-CD40 interaction plays a major role in initiating immune responses, we sought to develop a molecular adjuvant targeting this interaction. METHODS AND RESULTS: We immunized mice with a foot-and-mouth disease virus DNA vaccine, pcD-VP1, together with a CD40-expressing plasmid, pcD-CD40. We found that pcD-CD40 induced anti-CD40 antibodies, which temporally correlated with the augmented production of anti-VP1 antibody. pcD-CD40 similarly augmented the humoral response of another DNA vaccine that targets hepatitis B virus, and passive transfer of anti-CD40 antisera also showed a similar effect. Furthermore, the pcD-CD40-elicited anti-CD40 antibodies were able to activate the CD40 signal pathway in antigen-presenting cells in vitro, which led to the maturation of dendritic cells (DCs) and DC-mediated T cell activation. Thus, pcD-CD40 augments DNA vaccination by inducing anti-CD40 antibodies, which in turn promotes T cell activation. CONCLUSIONS: This is the first reported 'proadjuvant' that augments DNA vaccination indirectly by eliciting agonistic antibodies.