Eugene Kim

AIMP1/p43 Protein Induces the Maturation of Bone Marrow-Derived Dendritic Cells with T Helper Type 1-Polarizing Ability

AIMP1 (ARS-interacting multifunctional protein 1), previously known as p43, was initially identified as a factor associated with a macromolecular tRNA synthetase complex. Recently, we demonstrated that AIMP1 is also secreted and acts as a novel pleiotropic cytokine. In this study, we investigated whether AIMP1 induces the activation and maturation of murine bone marrow-derived dendritic cells (DCs). AIMP1-treated DCs exhibited up-regulated expression of cell-surface molecules, including CD40, CD86, and MHC class II. Additionally, microarray analysis and RT-PCR determinations indicated that the expression of known DC maturation genes also increased significantly following treatment with AIMP1. Treatment of DCs with AIMP1 resulted in a significant increase in IL-12 production and Ag-presenting capability, and it also stimulated the proliferation of allogeneic T cells. Importantly, AIMP1-treated DCs induced activation of Ag-specific Th type 1 (Th1) cells in vitro and in vivo. AIMP1-stimulated DCs significantly enhanced the IFN-γ production of cocultured CD4+ T cells. Immunization of mice with keyhole limpet hemocyanin-pulsed AIMP1 DCs efficiently led to Ag-specific Th1 cell responses, as determined by flow cytometry and ELISA. The addition of a neutralizing anti-IL-12 mAb to the cell cultures that had been treated with AIMP1 resulted in the decreased production of IFN-γ, thereby indicating that AIMP1-stimulated DCs may enhance the Th1 response through increased production of IL-12 by APCs. Taken together, these results indicate that AIMP1 protein induces the maturation and activation of DCs, which skew the immune response toward a Th1 response.

The Novel Cytokine p43 Induces IL-12 Production in Macrophages via NF-κB Activation, Leading to Enhanced IFN-γ Production in CD4+ T Cells

Recently, we determined that p43, an auxiliary factor of mammalian multiaminoacyl-tRNA synthetases, is secreted, and functions as a novel pleiotropic cytokine. In this study, we have attempted to characterize the effects of p43 on the generation of IL-12 in mouse macrophages. p43 was determined to induce significant IL-12 production from mouse macrophages in a dose-dependent manner. The stimulatory effect of p43 on the activation of IL-12p40 promoter was mapped to a region harboring an NF-κB binding site. The nuclear extracts from the p43-stimulated macrophages exhibited profound NF-κB DNA-binding activity, as determined by the EMSA. In addition, the p43-stimulated IL-12 induction and NF-κB DNA-binding activity were significantly suppressed by caffeic acid phenethyl ester and BAY11-7082, both inhibitors of NF-κB activation, indicating that p43 induced the production of IL-12 in macrophages mainly via the activation of NF-κB. Importantly, p43 increased the level of IFN-γ production in the Ag-primed lymph node cells, but had no effect on IL-4 levels. The addition of a neutralizing anti-IL-12p40 mAb to the cell cultures resulted in a decrease of the production of p43-enhanced IFN-γ by the keyhole limpet hemocyanin-primed lymph node cells. Furthermore, coincubation with p43-pretreated macrophages enhanced the production of IFN-γ by the keyhole limpet hemocyanin-primed CD4+ T cells, thereby indicating that p43 may enhance IFN-γ expression in CD4+ T cells via the induction of IL-12 production in macrophages. These results indicate that p43 may play an essential role in the development of the Th1 immune responses associated with cancer immunotherapy and protective immunity against intracellular pathogens.

Differential suppression of heat-killed lactobacilli isolated from kimchi, a Korean traditional food, on airway hyper-responsiveness in mice.

RationaleProbiotics have been shown to be effective in reducing allergic symptoms. However, there are few studies to evaluate the therapeutic effects of lactobacilli on allergen-induced airway inflammation.ObjectiveWe investigated whether three heat-killed lactobacilli, Lactobacillus plantarum, Lactobacillus curvatus and Lactobacillus sakei subsp. sakei, isolated from kimchi, exerted inhibitory effects on airway hyper-responsiveness in a murine asthma model.MethodsHeat-killed lactic acid bacteria were orally administered into BALB/c mice, followed by challenge with aerosolized ovalbumin, after which allergic symptoms were evaluated.ResultsAirway inflammation was suppressed in the L. plantarum- and L. curvatus-treated mice. Interleukin (IL)-4 and IL-5 levels were significantly lower in the L. plantarum- and L. curvatus-treated mice than in those treated with L. sakei subsp. sakei. Importantly, heat-killed L. plantarum administration induced Foxp3 expression in intestinal lamina propria cells, and heat-killed L. curvatus induced IL-10 as a way of inducing tolerance.ConclusionSpecific strains of lactobacilli isolated from kimchi can effectively suppress airway hyper-responsiveness.

Gene transfer of AIMP1 and B7.1 into epitope-loaded, fibroblasts induces tumor-specific CTL immunity, and prolongs the survival period of tumor-bearing mice.

T helper type 1 (Th1) cell-mediated immune responses play various roles in cellular immunity, including inducing cytotoxic T lymphocytes (CTLs) and they have been shown to be crucial in cancer immunotherapy. Previously, we found that aminoacyl-tRNA synthetase-interacting multifunctional protein 1 (AIMP1) stimulated antigen-presenting cells to secrete IL-12, leading to enhanced Th1 cell responses. In this study, as a way of enhancing antigen-specific Th1 responses, mouse fibroblasts (H-2(b)) were genetically modified to express an AIMP1 and a costimulatory B7.1 (Fb/AIMP1/B7.1). Fb/AIMP1/B7.1 cells were then loaded with an ovalbumin epitope as a model antigen (Fb/AIMP1/B7.1/OVA), and tested to determine if they induced OVA-specific CTLs in C57BL/6 mice (H-2(b)). Immunization with Fb/AIMP1/B7.1/OVA cells induced strong cytotoxic activities against OVA-expressing EG7 tumor cells, but not against other H-2(b) tumor cells. The levels of the cytotoxic response in the immunized mice with Fb/AIMP1/B7.1/OVA cells were significantly higher than the responses in mice immunized with other cell constructs. CD8(+) T cells were a major cell-type of OVA-specific antitumor immunity induced by Fb/AIMP1/B7.1/OVA cells. Furthermore, treatment with Fb/AIMP1/B7.1/OVA cells significantly prolonged the survival period of EG7 tumor-bearing mice. These results indicate that AIMP1-secreting, epitope-loaded fibroblasts efficiently induce antigen-specific CTL responses in mice.

Interferon-α Is Involved in the Luteinizing Hormone-Induced Differentiation of Rat Preovulatory Granulosa Cells

A surge in luteinizing hormone (LH) triggers physiological changes within the ovarian follicles, including reprogramming to induce terminal differentiation of the granulosa cells (GCs). Cytokines are members of a large regulatory network that resides in the ovaries and are involved in the regulation of steroidogenesis and gamete production. Recently we found that interferon-alpha (IFN-alpha) was overexpressed in LH-treated preovulatory GCs, as determined by a microarray analysis. In this study, we evaluated the expression of IFN-alpha and its role in the differentiation of rat preovulatory GCs. Rat GCs were treated with LH in vitro or human chorionic gonadotropin (hCG) in vivo, both of which are well-known inducers of differentiation, and IFN-alpha production and cell differentiation were determined. Stimulation of rat primary GCs with LH or hCG increased expression of IFN-alpha. LH treatment led to increased phosphorylation of PI3-K and extracellular signal-regulated kinase (ERK), and specific inhibitors for PI3-K and ERK suppressed the LH-induced IFN-alpha expression in preovulatory GCs. Furthermore, treatment with anti-rat IFN-alpha blocking antibody delayed the LH-induced differentiation of GCs and suppressed the expression of ovulation-related genes, including progesterone receptor (PR) and steroidogenic acute regulatory protein (StAR). These results indicate that LH induces IFN-alpha expression in preovulatory GCs via a PI3-K/ERK signaling pathway and that interferon-alpha production may be involved in the LH-induced differentiation of preovulatory GCs in rats.

AIMP1 deficiency enhances airway hyperreactivity in mice via increased TH2 immune responses.

Aminoacyl tRNA synthetase complex-interacting multicomplex protein 1 (AIMP1) is known as a novel cytokine carrying out a variety of biological activities, including angiogenesis and wound repair. In our previous reports AIMP1 was demonstrated to induce TH1 polarization. However, the effects of AIMP1 deficiency in TH1 or TH2 immune disorders remain unclear. In this study, we characterized phenotypes of AIMP1-deficient mice and investigated the role of AIMP1 in TH2-biased airway hyperreactivity. Clinical signs of allergic airway inflammation were assessed in AIMP1-deficient mice and the effects of AIMP1 deficiency on production of TH2 cytokines were evaluated in T cells using AIMP1-specific siRNA. Additionally, the enhanced pause values and histologic analysis were assessed in mice receiving AIMP1-deficient CD4+ T cells with OVA challenge. Clinical signs of spontaneous airway inflammation were noted in AIMP1-deficienct mice. AIMP1-deficient mice showed strongly increased Penh values in response to methacholine without any allergen exposure. Adoptive transfer of AIMP1-deficient CD4+ T cells to OVA-sensitized C57BL/6 mice exacerbated OVA-induced airway inflammation and increased infiltration of inflammatory cells into the lung. Furthermore, lung DCs in AIMP1-deficient mice showed increased expression of surface molecules, and IL-12p40 level in sera significantly decreased in AIMP1-deficient mice compared to that of wild type mice. These results strongly indicate that AIMP1 plays a role in negatively regulating TH2 responses in vivo, and AIMP1 can be employed as a novel therapeutic agent against TH2-biased diseases, particularly asthma.

Aminoacyl-tRNA synthetase-interacting multifunctional protein 1 suppresses tumor growth in breast cancer-bearing mice by negatively regulating myeloid-derived suppressor cell functions

Myeloid-derived suppressor cells (MDSCs) are one of the most important cell types that contribute to negative regulation of immune responses in the tumor microenvironment. Recently, aminoacyl-tRNA synthetase-interacting multifunctional protein 1 (AIMP1), a novel pleiotropic cytokine, was identified as an antitumor protein that inhibits angiogenesis and induces antitumor responses. However, the effect of AIMP1 on MDSCs in the tumor environment remains unclear. In the present study, we demonstrated that AIMP1 significantly inhibited tumor growth in 4T1 breast cancer-bearing mice and reduced MDSCs population of tumor sites and spleens of tumor-bearing mice. AIMP1 reduced expansion of MDSCs from bone marrow-derived cells in the tumor-conditioned media. AIMP1 also negatively regulated suppressive activities of MDSCs by inhibiting IL-6 and NO production, and Arg-1 expression. Furthermore, treatment of breast cancer-bearing mice with AIMP1 decreased the capacity of MDSCs to suppress T cell proliferation and Treg cell induction. Western blot and inhibition experiments showed that downregulation of MDSCs functions by AIMP1 may result from attenuated activation of STATs, Akt, and ERK. These findings indicate that AIMP1 plays an essential role in negative regulation of suppressive functions of MDSCs. Therefore, it has a significant potential as a therapeutic agent for cancer treatment.

Enhancement of Toll-like receptor 2-mediated immune responses by AIMP1, a novel cytokine, in mouse dendritic cells

Aminoacyl tRNA synthetase‐interacting protein 1 (AIMP1) is a novel pleiotropic cytokine that was identified initially from Meth A‐induced fibrosarcoma. It is expressed in the salivary glands, small intestine and large intestine, and is associated with the innate immune system. Previously, we demonstrated that AIMP1 might function as a regulator of innate immune responses by inducing the maturation and activation of bone‐marrow‐derived dendritic cells (BM‐DCs). Toll‐like receptors (TLRs) are major pathogen‐recognition receptors that are constitutively expressed on DCs. In this study, we attempted to determine whether AIMP1 is capable of regulating the expression of TLRs, and also capable of affecting the TLR‐mediated activation of DCs. Expression of TLR1, ‐2, ‐3 and ‐7 was highly induced by AIMP1 treatment in BM‐DCs, whereas the expression of other TLRs was either down‐regulated or remained unchanged. In particular, the expression of the TLR2 protein was up‐regulated by AIMP1 in a time‐dependent and dose‐dependent manner, and was suppressed upon the addition of BAY11‐7082, an inhibitor of nuclear factor‐κB. AIMP1 was also shown to increase nuclear factor‐κB binding activity. Importantly, AIMP1 enhanced the production of interleukin‐6 and interleukin‐12, and the expression of co‐stimulatory molecules on BM‐DCs when combined with lipoteichoic acid or Pam3Cys, two well‐known TLR2 agonists. Collectively, these results demonstrate that the AIMP1 protein enhances TLR2‐mediated immune responses via the up‐regulation of TLR2 expression.

A DNA adjuvant encoding a fusion protein between anti-CD3 single-chain Fv and AIMP1 enhances T helper type 1 cell-mediated immune responses in antigen-sensitized mice

T helper type 1 (Th1) cell‐mediated immune responses contribute to host defences against intracellular pathogen infections and cancer. Previously, we found that aminoacyl tRNA synthetase‐interacting multifunctional protein 1 (AIMP1) activated macrophages and dendritic cells to enhance Th1 responses. Herein, we manipulated this property to improve Th1 immune responses in vivo by constructing a mammalian expression plasmid (pAnti‐CD3sFv/AIMP1) encoding AIMP1 fused to the anti‐CD3 single‐chain Fv (sFv), the smallest unit of the antibody that interacts with the CD3ε region of the T‐cell receptor. Intramuscular injection of ovalbumin (OVA)‐sensitized BALB/c mice with pAnti‐CD3sFv/AIMP1 DNA adjuvant increased the OVA‐specific, interferon‐γ production by their CD4+ T cells and the levels of anti‐OVA immunoglobulin G2a (IgG2a) isotype in their sera. Furthermore, the pAnti‐CD3sFv/AIMP1 DNA adjuvant decreased interleukin‐4 production and anti‐OVA IgE levels in the OVA‐injected mice. Importantly, the pAnti‐CD3sFv/AIMP1 was more efficient than a mixture of pAnti‐CD3sFv and pAIMP1 in inducing OVA‐specific Th1 immune responses and also in inhibiting OVA‐specific Th2 responses during antigen priming. These studies indicated that the pAnti‐CD3sFv/AIMP1 fusion DNA adjuvant enhanced Th1 immune responses in antigen‐sensitized mice.