Jeong-Hae Kie

Reactive Oxygen Species Prevent Imiquimod-Induced Psoriatic Dermatitis through Enhancing Regulatory T Cell Function

Psoriasis is a chronic inflammatory skin disease resulting from immune dysregulation. Regulatory T cells (Tregs) are important in the prevention of psoriasis. Traditionally, reactive oxygen species (ROS) are known to be implicated in the progression of inflammatory diseases, including psoriasis, but many recent studies suggested the protective role of ROS in immune-mediated diseases. In particular, severe cases of psoriasis vulgaris have been reported to be successfully treated by hyperbaric oxygen therapy (HBOT), which raises tissue level of ROS. Also it was reported that Treg function was closely associated with ROS level. However, it has been only investigated in lowered levels of ROS so far. Thus, in this study, to clarify the relationship between ROS level and Treg function, as well as their role in the pathogenesis of psoriasis, we investigated imiquimod-induced psoriatic dermatitis (PD) in association with Treg function both in elevated and lowered levels of ROS by using knockout mice, such as glutathione peroxidase-1−/− and neutrophil cytosolic factor-1−/− mice, as well as by using HBOT or chemicals, such as 2,3-dimethoxy-1,4-naphthoquinone and N-acetylcysteine. The results consistently showed Tregs were hyperfunctional in elevated levels of ROS, whereas hypofunctional in lowered levels of ROS. In addition, imiquimod-induced PD was attenuated in elevated levels of ROS, whereas aggravated in lowered levels of ROS. For the molecular mechanism that may link ROS level and Treg function, we investigated the expression of an immunoregulatory enzyme, indoleamine 2,3-dioxygenase (IDO) which is induced by ROS, in PD lesions. Taken together, it was implied that appropriately elevated levels of ROS might prevent psoriasis through enhancing IDO expression and Treg function.

Attenuation of Experimental Colitis in Glutathione Peroxidase 1 and Catalase Double Knockout Mice through Enhancing Regulatory T Cell Function

Reactive oxygen species (ROS) have been implicated in the progression of inflammatory diseases including inflammatory bowel diseases (IBD). Meanwhile, several studies suggested the protective role of ROS in immune-mediated inflammatory diseases, and it was recently reported that dextran sodium sulfate (DSS)-induced colitis was attenuated in mice with an elevated level of ROS due to deficiency of peroxiredoxin II. Regulatory T cells (Tregs) are critical in the prevention of IBD and Treg function was reported to be closely associated with ROS level, but it has been investigated only in lowered levels of ROS so far. In the present study, in order to clarify the relationship between ROS level and Treg function, and their role in the pathogenesis of IBD, we investigated mice with an elevated level of ROS due to deficiency of both glutathione peroxidase (GPx)-1 and catalase (Cat) for the susceptibility of DSS-induced colitis in association with Treg function. The results showed that DSS-induced colitis was attenuated and Tregs were hyperfunctional in GPx1−/− × Cat−/− mice. In vivo administration of N-acetylcysteine (NAC) aggravated DSS-induced colitis and decreased Treg function to the level comparable to WT mice. Attenuated Th17 cell differentiation from naïve CD4+ cells as well as impaired production of IL-6 and IL-17A by splenocytes upon stimulation suggested anti-inflammatory tendency of GPx1−/− × Cat−/− mice. Suppression of Stat3 activation in association with enhancement of indoleamine 2,3-dioxygenase and FoxP3 expression might be involved in the immunosuppressive mechanism of GPx1−/− × Cat−/− mice. Taken together, it is implied that ROS level is critical in the regulation of Treg function, and IBD may be attenuated in appropriately elevated levels of ROS.

Morphological and biochemical changes induced by arsenic trioxide in neuroblastoma cell lines.

Arsenic trioxide has recently been shown to inhibit growth and induce apoptosis in a variety of hematologic malignancies, but very little is known about its effects on solid tumors and especially on neuroblastoma cells that have self-differentiating characteristics. To demonstrate the growth inhibition induced in neuroblastoma cells (the SH-SY5Y and SK-N-AS cell line) and acute promyelocytic leukemia cells (HL-60) by arsenic trioxide (As2O3), the viable cell numbers were counted after trypan blue staining. Apoptosis was assessed by the cell morphology, by flow cytometry with annexin-V staining, and by Western blot analysis for the apoptosis-related proteins (bcl-2 and PARP). To decide the dose for the clinical application of As2O3, normal peripheral blood lymphocytes were also examined. The growth and survival of the SH-SY5Y and SK-N-AS cells were markedly inhibited by As2O3 treatment at a 3 μM concentration before the changes of the normal lymphocytes were observed. The apoptotic cells showed a shrunken cell nucleus, and an increase in the number and balloon-like swelling of the mitochondria at 72 h after the As2O3 was added. Apoptosis of the annexin-V-positive cell proportion in the neuroblastoma cell lines was increased with increasing the exposure time and the concentration of As2O3, just like the HL-60 cells. Bcl-2 downregulation and PARP degradation were also noted all the cell lines, but these changes were not statistically significant among the 3 cell lines. Taken together, these results indicate that As2O3 is an excellent candidate as a therapeutic agent for the treatment of neuroblastoma.

In vitro differentiation of natural killer T cells from human cord blood CD34+ cells

Summary. Natural killer T (NKT) cells are involved in innate immune defence and also in the regulation of adaptive immune responses. However, the development of NKT cells in vitro has not been fully characterized and culture conditions have not been fully optimized. In the present study, we found that an NKT cell fraction developed during the in vitro culture of cord blood (CB) CD34+ cells, and this was subsequently characterized both phenotypically and morphologically. CD34+ cells purified from 10 human CB were cultured in the presence of several cytokines and analysed by flow cytometry, light microscopy and electron microscopy. The NKT cell fraction, defined phenotypically (CD3+CD16+CD56+CD94+) as expressing the invariant T‐cell receptor Vα24 and Vβ11, appeared in the CD56hi fractions. Intracytoplasmic staining demonstrated that interferon‐γ and interleukin 4 (IL‐4) were detected in the CD56hi fractions. IL‐15 was essential and, in combination with either flt3‐ligand (FL) or stem cell factor (SCF), was sufficient to induce the development of NKT cells. The phenotype of the NKT cell fraction was CD45RO+CD45RA– and CD4+CD8α+. Morphologically, they were very large, with either round or oval nuclei, moderately condensed chromatins, voluminous weakly basophilic cytoplasm and various cytoplasmic granules such as dense core granules, multivesicular bodies, and intermediate form granules. When CD34+ cells purified from bone marrow (BM) were compared with those from CB, the latter were consistently more efficient at generating CD56hi NKT cell fractions. In conclusion, IL‐15 in combination with FL and/or SCF can induce the differentiation of NKT cells from human CB CD34+ cells.

Ultrastructural and phenotypic analysis of in vitro erythropoiesis from human cord blood CD34 + cells

Erythropoietin (EPO) induces erythropoiesis in vitro as well as in vivo, and the process of erythroid differentiation has been explored phenotypically and morphologically. However, morphological analysis of in vitro erythropoiesis of human hematopoietic progenitor cells at the ultrastructural level has not been reported before. In the present study, we have traced the ultrastructural changes of erythroid differentiation during ex vivo expansion of human cord blood (CB) CD34+ cells in the presence of EPO by electron microscopy (EM), along with concurrent phenotypic analysis. CD34+ cells purified from ten CBs by immunomagnetic selection were cultured in serum-free essential media in the presence of a combination of the several cytokines including EPO, thrombopoietin, flt3-ligand (FL), stem cell factor (SCF), granulocyte colony-stimulating factor, interleukin (IL)-3 and/or IL-11. Phenotypic analysis was performed by flow cytometric analysis for erythroid markers, including glycophorin C (GPC), Kell-related, glycophorin A (GPA), band 3, Lub, and RhD. Ultrastructural analysis was performed by electron-microscopic examination of the cultured cells stained with uranyl acetate and lead citrate. Phenotypic analysis revealed that in the absence of EPO, genuine erythroid fraction expressing the typical pattern of erythroid markers did not appear. The order of the above markers expressed in the cultured cells in the presence of EPO was GPC, Kell-related, GPA, band 3, Lub, and RhD, irrespective of the type of cytokine added. Of the cytokines used in combination with EPO, FL + IL-3 was the most efficient in inducing erythroid differentiation, which was followed by SCF + IL-3. EM examination demonstrated complete process of erythroid development from pronormoblasts to reticulocytes with nuclei having been extruded and mature erythrocytes. These results suggest that morphologically intact erythrocytes could be produced by ex vivo expansion of CB CD34+ cells using EPO.

Megakaryothrombopoiesis during ex vivo expansion of human cord blood CD34+ cells using thrombopoietin.

Thrombopoietin (TPO) is one of the most promising stimulants for ex vivo expansion of haematopoietic stem cells. Previously, we have found that TPO induces a characteristic pattern of apoptosis during ex vivo expansion of human cord blood (CB) CD34+ cells and that the TPO‐induced apoptotic cells belong to megakaryocyte (MK) lineage. In this study, we have examined the maturation of MK and platelet production in association with the TPO‐induced apoptosis. CD34+ cells, purified from human CB, were expanded in serum‐free conditions stimulated with TPO. Apoptosis was confirmed by terminal deoxynucleotidyl transferase‐mediated deoxyuridine triphosphate‐biotin nick end labelling (TUNEL) assay and electron microscopy (EM). Simultaneous measurement of DNA content and immunophenotyping revealed that the cells with higher DNA content (>8u2003N) constituted less than 5% of the CD41+ fractions until day 14, implying premature apoptosis of MKs before full polyploidization. Nevertheless, EM observation showed not only platelet territories but also newly produced platelets in which granules and microfilaments could be identified. Furthermore, flow cytometry demonstrated that the platelet fraction expressed P‐selectin and an activation motif on GPIIb/IIIa recognized by monoclonal antibody PAC‐1 upon stimulation with adenosine diphosphate (ADP). In addition, periodic acid‐Schiff (PAS)‐positive materials and nonspecific esterase activities could be demonstrated. Therefore, it is suggested that platelet production and the accompanying processes, rather than apoptosis only, be hastened during the ex vivo expansion of CB CD34+ cells when using TPO.

CD4+CD25+ regulatory T cells from MRL/lpr mice were functionally more active in vitro but did not prevent spontaneous as well as adriamycin-induced nephropathy in vivo

OBJECTIVEnThe frequency and function of Tregs are important in the pathogenesis of SLE. Nonetheless, the function of Tregs is still controversial in SLE patients and lupus mouse models. In the present study, we investigated the suppressive function of Tregs from MRL/lpr mice in vitro and in vivo by using an alternative quantitative assay.nnnMETHODSnWe assessed the suppressive function of CD4(+)CD25(+) Tregs, the proliferative activity of CD4(+)CD25(-) effector T cells (Teffs) and the feeder activity of CD11c(+) dendritic cells (DCs), isolated from the spleens of MRL/lpr mice and wild-type (WT) MRL/+ mice, by carboxyfluorescein diacetate succinimidyl ester dilution assay stimulated with two distinct types of signals, weak and strong. In order to assess the protective function of Tregs from an immune-mediated disease in vivo, we induced renal damage by injecting adriamycin (ADN) into the mice.nnnRESULTSnThe in vitro assay showed enhanced suppressive activity of Tregs and feeder activity of DCs, but far less proliferative activity of Teffs from MRL/lpr mice, compared with those from the WT mice. The in vivo study showed more severe ADN-induced nephropathy in MRL/lpr mice than in the WT mice, while mild interstitial nephritis had already begun spontaneously by 16 weeks in MRL/lpr mice.nnnCONCLUSIONnIt was suggested that Tregs from MRL/lpr mice were functionally competent and intrinsically more active in vitro, but they were not capable of preventing the ADN-induced as well as the spontaneously developing nephropathy in vivo.

Hyperoxygenation Attenuated a Murine Model of Atopic Dermatitis through Raising Skin Level of ROS

Atopic dermatitis (AD) is a chronic inflammatory skin disease resulting from excessive stimulation of immune cells. Traditionally, reactive oxygen species (ROS) have been implicated in the progression of inflammatory diseases, but several opposing observations suggest the protective role of ROS in inflammatory disease. Recently, we demonstrated ROS prevented imiquimod-induced psoriatic dermatitis through enhancing regulatory T cell function. Thus, we hypothesized AD might also be attenuated in elevated levels of ROS through tissue hyperoxygenation, such as by hyperbaric oxygen therapy (HBOT) or applying an oxygen-carrying chemical, perfluorodecalin (PFD). Elevated levels of ROS in the skin have been demonstrated directly by staining with dihydroethidum as well as indirectly by immunohistochemistry (IHC) for indoleamine 2,3-dioxygenase (IDO). A murine model of AD was developed by repeated application of a chemical irritant (1% 2,4-dinitrochlorobenzene) and house dust mite (Dermatophagoide farinae) extract on one ear of BALB/c mice. The results showed treatment with HBOT or PFD significantly attenuated AD, comparably with 0.1% prednicarbate without any signs of side effects, such as telangiectasia. The expressions of interleukin-17A and interferon-γ were also decreased in the AD lesions by treatment with HBOT or PFD. Enhanced expression of IDO and reduced level of hypoxia-inducible factor-1α, in association with increased frequency of FoxP3+ regulatory T cells in the AD lesions, might be involved in the underlying mechanism of oxygen therapy. Taken together, it was suggested that tissue hyperoxygenation, by HBOT or treatment with PFD, might attenuate AD through enhancing skin ROS level.

Attenuation of obesity and related metabolic disorders by the individual or combination treatment with IL-2/anti-IL-2 complex and hyperbaric oxygen

Obesity is the disease accumulating excessive fat in the body. The prevalence of obesity and related metabolic disorders is increasing every year worldwide. Immunologically, obesity is a chronic low-grade inflammatory state with the increase of M1 macrophages and decrease of regulatory T cells (Tregs). IL-2/anti-IL-2 complex (IL-2C) and hyperbaric oxygen (HBO) are known to expand Tregs in vivo and suppress inflammation. Therefore, in this study, IL-2C and HBO were investigated for the preventive effect of obesity and related metabolic disorders. Male C57BL/6 mice were fed with a high-fat diet (HFD) for 16 weeks, and counterparts were fed with a low-fat diet (LFD). At the end of the experiment, the body weight gain and impaired glucose metabolism, elevated levels of insulin and total cholesterol induced by HFD were improved by the individual or combination treatment with Il-2C and HBO. Histological examination of the epididymal white adipose tissue showed adipocyte hypertrophy and many crown-like structures in the HFD control groups. In addition, the liver showed the progression of non-alcoholic fatty liver disease (NAFLD) in the HFD control groups, but it was significantly improved by the individual or combination treatment with IL-2C and HBO. As for the underlying mechanism, inflammation induced by obesity was decreased, and HIF-1α expression by adipocyte hypertrophy was also reduced by the individual or combination treatment with IL-2C and HBO. In addition, adipose tissue browning was activated in brown and inguinal adipose tissue, and the expression of UCP-1 involved in the thermogenesis was increased by the individual or combination treatment with IL-2C and HBO. Overall, these results suggested that IL-2C and HBO might be a new promising immunotherapy for the treatment of obesity and related metabolic disorders by regulation of inflammation and activation of adipose tissue browning.

Attenuation of collagen-induced arthritis by hyperbaric oxygen therapy through altering immune balance in favor of regulatory T cells.

Hyperbaric oxygen (HBO₂) therapy is currently used for the treatment of chronic wounds, radiation-induced soft tissue necrosis, several oxygen-deficiency conditions and decompression sickness. In addition to the current indications, much empirical and experimental data suggest that HBO₂ therapy may benefit autoimmune diseases by suppressing immunity, but the underlying mechanism is not well understood. Therefore, in the present study, we investigated whether HBO₂ prevents the development of collagen-induced arthritis (CIA) in association with alteration of the immune balance between pro-inflammatory Th17 and anti-inflammatory regulatory T cells (Tregs). Arthritis was induced in DBA/1 mice by intradermal injection of type II collagen. Animals received either no treatment or 90 minutes of HBO₂ (100% oxygen, at 2.0 ATA) daily beginning three days prior to the injection and were monitored for the development of arthritis. Six weeks later, joint tissues and spleens were analyzed for the alteration of immune balance between Th17 and Tregs by immunohistochemistry (IHC) or Western blot. Injection of collagen-induced extensive arthritis and extramedullary hematopoiesis in the spleens. Meanwhile, joint swelling and inflammatory tissue damages as well as extramedullary hematopoiesis were significantly less severe in the mice treated with HBO₂. Both IHC and Western blot showed a decrease of FOXP3 and an increase of pSTAT3 expressions in the joints and spleens of the mice injected with collagen. This suggested that the systemic immune balance was biased toward Th17 cells, which was reversed by HBO₂ therapy. These results suggested acute CIA associated with an immune balance favoring Th17 was attenuated by HBO₂ in parallel with restoration of the immune balance to favor Tregs.