[Show abstract][Hide abstract] ABSTRACT: Fibromyalgia (FM) is defined as a widely distributed pain. While many rheumatologists and pain physicians have considered it to be a pain disorder, psychiatry, psychology, and general medicine have deemed it to be a syndrome (FMS) or psychosomatic disorder. The lack of concrete structural and/or pathological evidence has made patients suffer prejudice that FMS is a medically unexplained symptom, implying inauthenticity. Furthermore, FMS often exhibits comorbidity with rheumatoid arthritis (RA) or spondyloarthritis (SpA), both of which show similar indications. In this study, disease specific biomarkers were sought in blood samples from patients to facilitate objective diagnoses of FMS, and distinguish it from RA and SpA.
Peripheral blood mononuclear cells (PBMCs) from patients and healthy donors (HD) were subjected to multicolor flow cytometric analysis. The percentage of mucosal-associated invariant T (MAIT) cells in PBMCs and the mean fluorescent intensity (MFI) of cell surface antigen expression in MAIT cells were analyzed.
There was a decrease in the MAIT cell population in FMS, RA, and SpA compared with HD. Among the cell surface antigens in MAIT cells, three chemokine receptors, CCR4, CCR7, and CXCR1, a natural killer (NK) receptor, NKp80, a signaling lymphocyte associated molecule (SLAM) family, CD150, a degrunulation marker, CD107a, and a coreceptor, CD8β emerged as potential biomarkers for FMS to distinguish from HD. Additionally, a memory marker, CD44 and an inflammatory chemokine receptor, CXCR1 appeared possible markers for RA, while a homeostatic chemokine receptor, CXCR4 deserved for SpA to differentiate from FMS. Furthermore, the drug treatment interruption resulted in alternation of the expression of CCR4, CCR5, CXCR4, CD27, CD28, inducible costimulatory molecule (ICOS), CD127 (IL-7 receptor α), CD94, NKp80, an activation marker, CD69, an integrin family member, CD49d, and a dipeptidase, CD26, in FMS.
Combined with the currently available diagnostic procedures and criteria, analysis of MAIT cells offers a more objective standard for the diagnosis of FMS, RA, and SpA, which exhibit multifaceted and confusingly similar clinical manifestations.
PLoS ONE 04/2015; 10(4):e0121124. DOI:10.1371/journal.pone.0121124 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Mucosal-associated invariant T (MAIT) cells play an important physiological role in host pathogen defense and may also be involved in inflammatory disorders and multiple sclerosis. The rarity and inefficient expansion of these cells have hampered detailed analysis and application. Here, we report an induced pluripotent stem cell (iPSC)-based reprogramming approach for the expansion of functional MAIT cells. We found that human MAIT cells can be reprogrammed into iPSCs using a Sendai virus harboring standard reprogramming factors. Under T cell-permissive conditions, these iPSCs efficiently redifferentiate into MAIT-like lymphocytes expressing the T cell receptor Vα7.2, CD161, and interleukin-18 receptor chain α. Upon incubation with bacteria-fed monocytes, the derived MAIT cells show enhanced production of a broad range of cytokines. Following adoptive transfer into immunocompromised mice, these cells migrate to the bone marrow, liver, spleen, and intestine and protect against Mycobacterium abscessus. Our findings pave the way for further functional analysis of MAIT cells and determination of their therapeutic potential.
[Show abstract][Hide abstract] ABSTRACT: Abstract Somatic cell nuclear transfer allows the generation of cloned embryonic stem cells (ESC) and cloned mice from natural killer T (NKT) cells, an innate-type invariant T cell. The progeny of these cloned mice harboring a rearranged T cell receptor α loci specific for NKT cells, Vα14-Jα18, possess an increased number of NKT cells in the primary as well as in the secondary lymphoid organs. NKT cells in these mice are able to produce interferon-γ (IFN-γ) and interleukin-4 (IL-4) when stimulated with dendritic cells (DC) pulsed with an NKT cell agonist, α-galactosylceramide (α-GalCer). The directed differentiation of cloned ESC toward T cells results in quasi-exclusive generation of NKT cells. These NKT cells are functional as evidenced by the production of cytokines such as IFN-γ, IL-4, IL-10, and IL-13 in response to α-GalCer. Furthermore, they mature autonomously in vivo upon adoptive transfer, and exhibit an antigen-specific adjuvant effect, resulting in IFN-γ production from CD8+ T cells. This effect is evident when the growth of tumors is inhibited in an antigen-dependent manner upon tumor inoculation into the mouse. Unfortunately, in humans, NKT cells are rare, and there is no guarantee that the same technique will be applicable for use in the clinic. We, therefore, exploited a different type of T cells in humans. We established induced pluripotent stem cells (iPSC) from these T cells, and succeeded in directed differentiation of the iPSC into monoclonal T cells. The availability of iPSC-derived monoclonal T cells paves the way for their use in regenerative medicine. In addition, they will be useful for drug screening to target unmet medical needs such as autoimmunity, severe infection, allergy, and cancer.
Hormone molecular biology and clinical investigation 06/2012; 10(2):249-53. DOI:10.1515/hmbci-2012-0011
[Show abstract][Hide abstract] ABSTRACT: The metabolic syndrome is an important social problem affecting many people in developed countries. Obesity is a leading cause of this syndrome, hence understanding molecular mechanisms underlying obesity is of prime importance for preventive medicine to develop novel methods to alleviate the corresponding social cost as well as for pharmaceutical companies to develop antimetabolic drugs.
Since adipocytes play an important role in obesity, we explored the signaling pathways leading to differentiation of adipocytes. We used a preadipocyte cell line to monitor the differentiation of adipocytes, and virus-mediated gene transfer to assess the role of the transcription factor Stat5 in adipogenesis. Adipocyte differentiation was assessed by Northern blot and Western blot analyses as well as accumulation of fat droplets in cells. Promoter activity of the proadipogenic transcription factor peroxisome proliferator-activated receptor-gamma (PPARγ) was evaluated by luciferase assay.
Virus-mediated gene transfer of the constitutively active form of both Stat5A and Stat5B resulted in enhanced adipocyte differentiation in the absence of fetal bovine serum (FBS) as judged by expression of proadipogenic factors as well as accumulation of fat droplets in cells. Such a proadipogenic effect of Stat5 is, in part, mediated by its ability to enhance transcription of PPARγ, a master transcriptional regulator in adipogenesis.
The constitutively active form of Stat5A and Stat5B promoted adipocyte differentiation in the absence of FBS via induction of PPARγ.
Environmental Health and Preventive Medicine 12/2010; 16(4):247-52. DOI:10.1007/s12199-010-0193-7
[Show abstract][Hide abstract] ABSTRACT: Leptin, the product of the ob gene, plays important roles in the regulation of food intake and body weight through its receptor in the hypothalamus. To identify novel transcripts induced by leptin, we performed cDNA subtraction based on selective suppression of the polymerase chain reaction by using mRNA prepared from the forebrain of leptin-injected ob/ob mice. One of the genes isolated was a mouse homolog of human negative regulatory element-binding protein (NREBP). Its expression was markedly increased by leptin in the growth hormone secretagogue-receptor (GHS-R)-positive neurons of the arcuate nucleus and ventromedial hypothalamic nucleus. The promoter region of GHS-R contains one NREBP binding sequence, suggesting that NREBP regulates GHS-R transcription. Luciferase reporter assays showed that NREBP repressed GHS-R promoter activity in a hypothalamic neuronal cell line, GT1-7, and its repressive activity was abolished by the replacement of negative regulatory element in GHS-R promoter. Overexpression of NREBP reduced the protein expression of endogenous GHS-R without affecting the expression of ob-Rb in GT1-7 cells. To determine the functional importance of NREBP in the hypothalamus, we assessed the effects of NREBP on ghrelin action. Although phosphorylation of AMP-activated protein kinase α (AMPKα) was induced by ghrelin in GT1-7 cells, NREBP repressed ghrelin-induced AMPKα phosphorylation. These results suggest that leptin-induced NREBP is an important regulator of GHS-R expression in the hypothalamus and provides a novel molecular link between leptin and ghrelin signaling.
[Show abstract][Hide abstract] ABSTRACT: The recent discovery that natural killer T (NKT) cell nuclei are totipotent opens a novel avenue for further understanding NKT cell function in normal and diseased states. The progeny of a cloned mouse harboring the in-frame rearranged Valpha14-Jalpha18 T cell receptor in one allele showed a significant increase in NKT cell number compared with wild-type or littermate control mice that possessed a different TCR. Importantly, NKT cells from such progeny produced both interferon-gamma and interleukin-4, a hallmark of NKT cells. In these progeny, NKT cell development appeared to be instructively, rather than permissively, determined. Using embryonic stem cells prepared via the somatic cell nuclear transfer of NKT nuclei, relatively mature NKT cells were induced under conditions permissible for T cell induction. Furthermore, these NKT cells matured autonomously upon injection into mice, resulting in an antigen-specific adjuvant effect.
[Show abstract][Hide abstract] ABSTRACT: The ectopic expression of the Notch receptor ligand delta-like 1 on stromal cells allows the induction of T cells from embryonic stem cells (ESCs). However, these in vitro-generated T cells are not transplantable because they are too immature to mount an immune response in an immunocompromised animal. We efficiently generated a subset of T cells called invariant natural killer T (iNKT) cells from ESCs derived from peripheral iNKT cells using somatic cell nuclear transfer (ntESCs). These iNKT cells matured autonomously in vivo and exhibited an adjuvant effect accompanying the production of interferon-gamma in an antigen-specific manner. This adjuvant effect culminated in the inhibition of inoculated tumor cell growth. Our results indicate that ntESC-derived iNKT cells are transplantable lymphocytes that will be beneficial for the induction of immune tolerance and the treatment of autoimmune diseases, tumors, and infections.
The FASEB Journal 08/2008; 22(7):2223-31. DOI:10.1096/fj.07-104687 · 5.48 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We have generated a novel mouse model harboring the in-frame rearranged TCRValpha specific for invariant NKT (iNKT) cells (Valpha14-Jalpha18) on one allele by crossing the mouse cloned from NKT cells with wild-type mice. This genomic configuration would ensure further rearrangement and expression of TCRValpha14-Jalpha18 under the endogenous promoters and enhancers. Mice harboring such an in-frame rearranged TCRValpha (Valpha14-Jalpha18 mouse) possessed an increase in iNKT cells in the thymus, liver, spleen, and bone marrow. Intriguingly, both Th1- and Th2-type cytokines were produced upon stimulation with alphaGalactosylceramide, an agonist of iNKT cells, and the IgE level in the serum remained unaffected in the Valpha14-Jalpha18 mouse. These features markedly distinguish the nature of iNKT cells present in the Valpha14-Jalpha18 mouse from that of iNKT cells found in the Valpha14-Jalpha18 transgenic mouse. Besides these, the expression of TCRVgammadelta cells remained intact, and the use of the TCRVbeta repertoire in iNKT cells was highly biased to TCRVbeta8 in the Valpha14-Jalpha18 mouse. Furthermore, alphaGalactosylceramide-CD1d dimer-reactive immature iNKT cells expressed less Rag2 as compared with the conventional immature T cells at the positive selection stage. Cell cycle analysis on the thymocytes revealed that no particular subset proliferated more vigorously than the others. Crossing the Valpha14-Jalpha18 mouse with the CD1d knockout mouse revealed a novel population of iNKT cells whose coreceptor expression profile was similar to that assigned to iNKT precursor cells. These mice will be useful for the study on the development of iNKT cells as well as on their functions in the immune system.
The Journal of Immunology 10/2007; 179(6):3888-95. DOI:10.4049/jimmunol.179.6.3888 · 5.36 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In mouse liver transplantation, tolerance is readily inducible. Recent studies have revealed that CD25+ CD4+ regulatory T cells play an important role in regulating various immune responses, including transplant tolerance. However, the contribution of these cells to tolerance in mouse liver transplantation has not been elucidated. We showed here that depletion of CD25+ CD4+ T cells increased proliferative response of CD4+ T cells and cytotoxic T lymphocyte induction of CD8+ T cells. Depletion of these cells in the recipient but not in the donor before liver transplantation caused rejection. Furthermore, the number of CD25+ CD4+ population and forkhead/winged helix transcription factor expression in liver mononuclear lymphocytes derived from tolerant mice were higher than those from grafts undergoing rejection. In conclusion, these results indicate that CD25+ CD4+ regulatory T cells in the recipient but not in the donor of liver transplantation are important for the tolerance induction.
[Show abstract][Hide abstract] ABSTRACT: Valpha14 NKT cells exhibit various immune regulatory properties in vivo, but their precise mechanisms remain to be solved. In this study, we demonstrate the mechanisms of generation of regulatory dendritic cells (DCs) by stimulation of Valpha14 NKT cells in vivo. After repeated injection of alpha-galactosylceramide (alpha-GalCer) into mice, splenic DCs acquired properties of regulatory DCs in IL-10-dependent fashion, such as nonmatured phenotypes and increased IL-10 but reduced IL-12 production. The unique cytokine profile in these DCs appears to be regulated by ERK1/2 and IkappaB(NS). These DCs also showed an ability to suppress the development of experimental allergic encephalomyelitis by generating IL-10-producing regulatory CD4 T cells in vivo. These findings contribute to explaining how Valpha14 NKT cells regulate the immune responses in vivo.
The Journal of Immunology 10/2005; 175(6):3648-55. DOI:10.4049/jimmunol.175.6.3648 · 5.36 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: It is well-documented that certain chemokines or their receptors play important roles in the graft rejection. However, the roles of chemokines and their receptors in the maintenance of transplantation tolerance remain unclear. In this study, we demonstrate that blocking of the interaction between the chemokine receptor, CXCR6, highly expressed on V alpha14+ NKT cells and its ligand, CXCL16, resulted in the failure to maintain graft tolerance and thus in the induction of acceleration of graft rejection. In a mouse transplant tolerance model, the expression of CXCL16 was up-regulated in the tolerated allografts, and anti-CXCL16 mAb inhibited intragraft accumulation of NKT cells. In vitro experiments further showed that blocking of CXCL16/CXCR6 interaction significantly affected not only chemotaxis but also cell adhesion of NKT cells. These results demonstrate the unique role of CXCL16 and CXCR6 molecules in the maintenance of cardiac allograft tolerance mediated by NKT cells.
The Journal of Immunology 09/2005; 175(4):2051-5. DOI:10.4049/jimmunol.175.4.2051 · 5.36 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Cloning mammals by nuclear transfer (NT) remains inefficient. One fundamental question is whether clones have really been derived from differentiated cells rather than from rare stem cells present in donor-cell samples. To date, cells, such as mature lymphocytes, with genetic differentiation markers have been cloned to generate mice only via a two-step NT involving embryonic stem (ES) cell generation and tetraploid complementation [1, 2 and 3]. Here, we show that the genome of a unique T-cell population, natural killer T (NKT) cells, can be fully reprogrammed by a single-step NT. The pups and their placentas possessed the rearranged TCR loci specific for NKT cells. The NKT-cell-cloned embryos had a high developmental potential in vitro: Most (71%) developed to the morula/blastocyst stage, in marked contrast to embryos from peripheral blood T cells (12%; p < 1 x 10(-25)). Furthermore, ES cell lines were efficiently established from these NKT-cell blastocysts. These findings clearly indicate a high level of plasticity in the NKT-cell genome. Thus, differentiation of the genome is not always a barrier to NT cloning for either reproductive or therapeutic purposes, so we can now postulate that at least some mammals cloned to date have indeed been derived from differentiated donor cells.
Current Biology 07/2005; 15(12):1114-8. DOI:10.1016/j.cub.2005.05.021 · 9.92 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Natural killer (NK) cells play a pivotal role in the immune reaction during the bone marrow allograft rejection. Little is known, however, about the molecular mechanisms underlying the NK cell-mediated allograft recognition and rejection. In this report, we assessed the role of a recently identified NK receptor, killer cell lectinlike receptor 1 (KLRE-1), by generating knock-out mice. KLRE-1-deficient mice were born at an expected frequency and showed no aberrant phenotype on growth and lymphoid development. Nevertheless, KLRE-1-deficient cells showed a severely compromised allogeneic cytotoxic activity compared with the wild-type cells. Furthermore, allogeneic bone marrow transfer culminated in colony formation in the spleen of KLRE-1-deficient mice, whereas no colony formation was observed in wild-type recipient mice. These results demonstrate that KLRE-1 is a receptor mediating recognition and rejection of allogeneic target cells in the host immune system.
[Show abstract][Hide abstract] ABSTRACT: Sarcoidosis is a systemic disorder associated with granuloma characterized by an abnormal T(h)1-type cytokine production and accumulation of T(h)1 CD4 T cells in the granuloma lesions, suggesting an importance of T(h)1 responses in sarcoidosis. However, the pathogenesis of sarcoidosis remains to be solved. Here, we investigated the nature of V(alpha)24 NKT cells with immunoregulatory functions in sarcoidosis. Patients with non-remitting sarcoidosis displayed a decrease in the number of V(alpha)24 NKT cells in peripheral blood, but an accumulation of these cells in granulomatous lesions. When stimulated with the specific glycolipid ligand, alpha-galactosylceramide, peripheral blood V(alpha)24 NKT cells from patients with non-remitting disease produced significantly less IFN-gamma than those from healthy volunteers, but normal levels of IL-4. The reduced IFN-gamma production was observed only in V(alpha)24 NKT cells and not conventional CD4 T cells, but was normal in patients with remitting disease, suggesting that non-remitting sarcoidosis involves an insufficient IFN-gamma production of V(alpha)24 NKT cells which is well correlated with disease activity. Thus, these results suggest that V(alpha)24 NKT cells play a crucial role in the disease status of sarcoidosis.
International Immunology 03/2004; 16(2):215-22. · 3.18 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: NKT cells expressing the invariant Valpha14 antigen receptor constitute a novel lymphocyte subpopulation with immunoregulatory functions. Stimulation via their invariant Valpha14 receptor with anti-CD3 or a ligand, alpha-galactosylceramide (alpha-GalCer), triggers activation of Valpha14 NKT cells, resulting in a rapid cytokine production such as IFN-gamma and IL-4. Soon after their receptor activation, Valpha14 NKT cells disappeared as judged by staining with CD1d tetramer loaded with alpha-GalCer (alpha-GalCer/CD1d tetramer), which has been believed to be due to apoptotic cell death. Here we show that such a disappearance was largely attributed to down-regulation of the Valpha14 receptor. In fact, Valpha14 NKT cells were relatively resistant to apoptosis compared to the conventional T cells as evidenced by less staining with Annexin-V, a limited DNA fragmentation, and their preferential expression of anti-apoptotic genes such as NAIP and MyD118. Furthermore, they did not become tolerant, and maintained their proliferative capacity and cytokine production even after their receptor down-regulation. These as yet unrecognized facets of Valpha14 NKT cells are discussed in relation to their regulatory functions.
International Immunology 03/2004; 16(2):241-7. · 3.18 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: NKT cells expressing the invariant Va14 antigen receptor constitute a novel lymphocyte subpopulation with immunoregulatory functions. Stimulation via their invariant Va14 receptor with anti-CD3 or a ligand, a-galactosylceramide (a-GalCer), triggers activation of Va14 NKT cells, resulting in a rapid cytokine production such as IFN-g and IL-4. Soon after their receptor activation, Va14 NKT cells disappeared as judged by staining with CD1d tetramer loaded with a-GalCer (a- GalCer/CD1d tetramer), which has been believed to be due to apoptotic cell death. Here we show that such a disappearance was largely attributed to down-regulation of the Va14 receptor. In fact, Va14 NKT cells were relatively resistant to apoptosis compared to the conventional T cells as evidenced by less staining with Annexin-V, a limited DNA fragmentation, and their preferential expression of anti-apoptotic genes such as NAIP and MyD118. Furthermore, they did not become tolerant, and maintained their proliferative capacity and cytokine production even after their receptor down-regulation. These as yet unrecognized facets of Va14 NKT cells are discussed in relation to their regulatory functions.
International Immunology 02/2004; 16(2):241-247. DOI:10.1093/intimm/dxh023 · 3.18 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Natural killer (NK) cells mediate bone marrow allograft rejection. However, the molecular mechanisms underlying such a rejection remain elusive. In previous analyses, it has been shown that NK cells recognize allogeneic target cells through Ly-49s and CD94/NKG2 heterodimers. Here, we describe identification and characterization of a novel murine NK receptor, NKG2I, belonging to the NKG2 family. NKG2I, which was composed of 226 amino acids, showed approximately 40% homology to the murine NKG2D and CD94 in the C-type lectin domain. Flow cytometric analysis with anti-NKG2I monoclonal antibody (mAb) revealed that expression of NKG2I was largely confined to NK and NKT cells, but was not seen in T cells. Furthermore, anti-NKG2I mAb inhibited NK cell-mediated cytotoxicity, whereas cross-linking of NKG2I enhanced interleukin 2- and interleukin 12-dependent interferon-gamma production. Similarly, the injection of anti-NKG2I mAb before the allogeneic bone marrow transfer in vivo impinged on the function of NKG2I, resulting in the enhanced colony formation in the spleen. NKG2I is a novel activating receptor mediating recognition and rejection of allogeneic target cells.
Journal of Experimental Medicine 02/2004; 199(1):137-44. DOI:10.1084/jem.20030851 · 13.91 Impact Factor