Kyeong Eun Hyung

Therapeutic potential of Lactobacillus plantarum CJLP133 for house-dust mite-induced dermatitis in NC/Nga mice.

Lactobacillus plantarum CJLP133 was isolated from Kimchi, a Korean fermented food, and its potential to improve mouse atopic dermatitis after onset was studied. Dermatitis was developed through house dust-mite extract application onto NC/Nga mice, and then CJLP133 feeding was started. CJLP133 suppressed dermatitis-like skin lesions and decreased high serum IgE levels through balancing between IL-4 and IFN-γ in serum. CJLP133 diminished skin thickening, mast cell accumulation into inflamed site, and lymph node enlargement. In lymph node cells, CJLP133 repressed secretion of T cell cytokines such as IFN-γ, IL-4, IL-5, and IL-10. However, CJLP133 decreased ratios of IFN-γ and IL-5 to IL-10 in lymph node cells, while it did not decrease ratios of IL-4 and IL-5 to IFN-γ. Conclusively, CJLP133 exhibited therapeutic potential for atopic dermatitis in mice through orderly increment of type 1 helper T cell activation and regulatory T cell activation. These results suggest that CJLP133 could treat human atopic dermatitis.

Peroxiredoxin‐1, a possible target in modulating inflammatory cytokine production in macrophage like cell line RAW264.7

Peroxiredoxin (PRX), a scavenger of H2O2 and alkyl hydroperoxides in living organisms, protects cells from oxidative stress. Contrary to its known anti‐oxidant roles, the involvement of PRX‐1 in the regulation of lipopolysaccharide (LPS) signaling is poorly understood, possible immunological functions of PRX‐1 having been uncovered only recently. In the present study, it was discovered that the PRX‐1 deficient macrophage like cell line (RAW264.7) has anti‐inflammatory activity when stimulated by LPS. Treatment with LPS for 3 hrs resulted in increased gene expression of an anti‐inflammatory cytokine, interleukin‐10 (IL‐10), in PRX‐1 knock down RAW264.7 cells. Gene expression of pro‐inflammatory cytokines IL‐1β and tumor necrosis factor‐ α (TNF‐α) did not show notable changes under the same conditions. However, production of these cytokines significantly decreased in PRX‐1 knock down RAW264.7 cells with 12 hrs of stimulation. Production of IL‐10 was also increased in PRX‐1 knock down RAW264.7 cells with 12 hrs of stimulation. We predicted that higher concentrations of IL‐10 would result in decreased expression of IL‐1β and TNF‐α in PRX‐1 knock‐down cells. This was confirmed by blocking IL‐10, which reestablished IL‐1β and TNF‐α secretion. We also observed that increased concentrations of IL‐10 do not affect the NF‐κB pathway. Interestingly, STAT3 phosphorylation by LPS stimulation was significantly increased in PRX‐1 knockdown RAW264.7 cells. Up‐regulation of IL‐10 in PRX‐1 knockdown cells and the resulting downregulation of proinflammatory cytokine production seem to involve the STAT3 pathway in macrophages. Thus, down‐regulation of PRX‐1 may contribute to the suppression of adverse effects caused by excessive activation of macrophages through affecting the STAT3 signaling pathway.

SUMO2 overexpression enhances the generation and function of interleukin-17-producing CD8⁺ T cells in mice.

Small ubiquitin-like modifier (SUMO) 2 is a small protein that controls the activity and stability of other proteins by SUMOylation. In this study, T cell-specific SUMO2 overexpressing transgenic mice were generated to study the effect of SUMO2 on T lymphocytes. SUMO2 overexpression promoted differentiation of interleukin (IL)-17-producing CD8(+) T cells, and significantly suppressed the growth of EL4 tumor cells in vivo. Moreover, the tumor tissue from SUMO2-overexpressing mice had higher interferon (IFN)-γ and granzyme B mRNA levels. Although SUMO2 overexpression did not increase IFN-γ or granzyme B production in cytotoxic T lymphocytes, IL-12 treatment restored and increased IFN-γ secretion in IL-17-producing CD8(+) T cells. SUMO2 overexpression also increased gene expression of chemokines, CCL4, and CXCL10, which attract cytotoxic T lymphocytes to tumor tissues. Additionally, SUMO2-overexpressing T cells exhibited increased STAT3 phosphorylation, implying a SUMO2 target which up-regulates STAT3 activity governing IL-17A-producing CD8(+) T cell differentiation and antitumor immune responses.

Therapeutic effects of orally administered CJLP55 for atopic dermatitis via the regulation of immune response

Atopic dermatitis (AD) is an inflammatory skin condition accompanied by symptoms such as edema and hemorrhage. Kimchi is a traditional fermented Korean dish consisting of various probiotics. In this study, the therapeutic effect of Lactobacillus plantarum CJLP55 isolated from Kimchi was studied in AD-induced mice. Orally administered Lactobacillus strain, CJLP55, suppressed AD symptoms and high serum IgE levels. CJLP55 administration reduced the thickness of the epidermis, infiltration of mast cells and eosinophils into the skin lesion, enlargement of axillary lymph nodes, and increase in cell population in axillary lymph nodes. CJLP55 treatment decreased the production of type 2 cytokines, such as interleukin (IL)-4, IL-5, IL-10, IL-12, interferon (IFN)-γ, and IL-6,which were stimulated by house dust mite extracts, in the axillary lymph node cells. Orally administered CJLP55 exhibited a therapeutic effect on house dust mite-induced AD in NC/Nga mice after onset of the disease by altering immune cell activation. The Lactobacillus strain, CJLP55, isolated from Kimchi, suppressed AD. Our results suggest its possible use as a potential candidate for management of AD.

Biaryl amide compounds reduce the inflammatory response in macrophages by regulating Dectin-1.

Macrophages are archetypal innate immune cells that play crucial roles in the recognition and phagocytosis of invading pathogens, which they identify using pattern recognition receptors (PRRs). Dectin-1 is essential for antifungal immune responses, recognizing the fungal cellular component β-glucan, and its role as a PRR has been of increasing interest. Previously, we discovered and characterized a novel biaryl amide compound, MPS 03, capable of inhibiting macrophage phagocytosis of zymosan. Therefore, in this study we aimed to identify other biaryl amide compounds with greater effectiveness than MPS 03, and elucidate their cellular mechanisms. Several MPS 03 derivatives were screened, four of which reduced zymosan phagocytosis in a similar manner to MPS 03. To establish whether such phagocytosis inhibition influenced the production of inflammatory mediators, pro-inflammatory cytokine and nitric oxide (NO) levels were measured. The production of TNF-α, IL-6, IL-12, and NO was significantly reduced in a dose-dependent manner. Moreover, the inflammation-associated MAPK signaling pathway was also affected by biaryl amide compounds. To investigate the underlying cellular mechanism, PRR expression was measured. MPS 03 and its derivatives were found to inhibit zymosan phagocytosis by decreasing Dectin-1 expression. Furthermore, when macrophages were stimulated by zymosan after pretreatment with biaryl amide compounds, downstream transcription factors such as NFAT, AP-1, and NF-κB were downregulated. In conclusion, biaryl amide compounds reduce zymosan-induced inflammatory responses by downregulating Dectin-1 expression. Therefore, such compounds could be used to inhibit Dectin-1 in immunological experiments and possibly regulate excessive inflammatory responses.

IL-6 induced proliferation and cytotoxic activity of CD8(+) T cells is elevated by SUMO2 overexpression.

T cells play an important role in adaptive immune responses that destroy pathogens or infected cells. Therefore, regulation of T cell activity is important in various diseases, such as autoimmune diseases, hypersensitivity, and cancer. The conjugation of small ubiquitin-related modifier (SUMO) is a post-translational protein modification that regulates activity, stability, and subcellular translocation of target proteins. In this study, CD8+ T cells overexpressing SUMO2 showed greater proliferation and cytotoxic activity against tumor cells in the presence of IL-6 than wild-type CD8+ T cells in vitro. These CD8+ T cell functions were suppressed during treatment with MEK1 or PI3K-specific inhibitors. Therefore, our findings suggest that IL-6-derived signaling pathways, including the MEK1 and PI3K pathways, are upregulated by SUMO2 overexpression. However, transgenic expression of SUMO2 in T cells did not modulate Th1/2 balance. Collectively, our results showed that SUMO2-Tg promotes cytotoxic activity against tumor cells by increasing the proliferation and cytotoxicity of CD8+ T cells.

Protective functions of peroxiredoxin-1 against cytokine-induced MIN6 pancreatic β-cell line death

Pancreatic β-cells play a crucial role in glucose homeostasis, and the failure of these cells to function results in the development of type 1 diabetes (T1D). The MIN6 cell line, which closely resembles pancreatic β-cells, was used to unravel the relationship between pancreatic β-cell function and the antioxidant enzyme PRX-1. PRX-1 was knocked down in MIN6 cells using a shPRX-1 lentiviral construct, and a mixture of inflammatory cytokines was administered to challenge the MIN6 cells. Nitric oxide (NO) production and inducible NO synthase (iNOS) expression were elevated in shPRX-1 compared with the control. Also, shPRX-1 transduced cells showed higher levels of NF-κB nuclear translocation, suggesting that PRX-1 has a regulatory role in NF-κB nuclear translocation and iNOS expression. In correlation with NO levels, decreased anti-apoptotic gene Bcl-xl level and elevated pro-apoptotic gene Bim levels were observed in shPRX-1 cells compared with scramble, and cell viability decreased accordingly. A rescue experiment was performed subsequently using an iNOS inhibitor to confirm NO as the cause of cell death. Overall, the results of this study suggest possible protective roles of the antioxidant enzyme PRX-1 in the insulinoma cell line MIN6 and possibly in pancreatic β-cells under T1D conditions.

Bcl-2 Knockdown Accelerates T Cell Receptor-Triggered Activation-Induced Cell Death in Jurkat T Cells

Cell death and survival are tightly controlled through the highly coordinated activation/inhibition of diverse signal transduction pathways to insure normal development and physiology. Imbalance between cell death and survival often leads to autoimmune diseases and cancer. Death receptors sense extracellular signals to induce caspase-mediated apoptosis. Acting upstream of CED-3 family proteases, such as caspase-3, Bcl-2 prevents apoptosis. Using short hairpin RNAs (shRNAs), we suppressed Bcl-2 expression in Jurkat T cells, and this increased TCR-triggered AICD and enhanced TNFR gene expression. Also, knockdown of Bcl-2 in Jurkat T cells suppressed the gene expression of FLIP, TNF receptor-associated factors 3 (TRAF3) and TRAF4. Furthermore, suppressed Bcl-2 expression increased caspase-3 and diminished nuclear factor kappa B (NF-κB) translocation.

Effect of PRX-1 Downregulation in the Type 1 Diabetes Microenvironment

Type 1 diabetes (T1D) is caused by dysregulation of the immune system in the pancreatic islets, which eventually leads to insulin-producing pancreatic β-cell death and destabilization of glucose homeostasis. One of the major characteristics of T1D pathogenesis is the production of inflammatory mediators by macrophages that result in destruction or damage of pancreatic β-cells. In this study the inflammatory microenvironment of T1D was simulated with RAW264.7 cells and MIN6 cells, acting as macrophages and pancreatic β-cells respectably. In this setting, peroxiredoxin-1, an anti-oxidant enzyme was knocked down to observe its functions in the pathogenesis of T1D. RAW264.7 cells were primed with lipopolysaccharide and co-cultured with MIN6 cells while PRX-1 was knocked down in one or both cell types. Our results suggest that hindrance of PRX-1 activity or the deficiency of this enzyme in inflammatory conditions negatively affects pancreatic β-cell survival. The observed decrease in viability of MIN6 cells seems to be caused by nitric oxide production. Additionally, it seems that PRX-1 affects previously reported protective activity of IL-6 in pancreatic β cells as well. These results signify new, undiscovered roles for PRX-1 in inflammatory conditions and may contribute toward our understanding of autoimmunity.

CTLA-4-Tg/CD-28-KO Mice Exhibit Reduced T Cell Proliferation in vivo Compared to CD-28-KO Mice in a Graft-versus-host Disease Model

Activated T cells express inhibitory receptors such as CTLA-4 that can downregulate immune responses. Blockade of or genetic deficiency in CTLA-4 can result in autoimmunity. Therefore, strategies to increase the inhibitory function of CTLA-4 may be attractive in settings of undesirable T cell responses such as autoimmunity or transplant rejection. We have tested the hypothesis that transgenic constitutive expression of CTLA-4 can further attenuate immune responses when compared with normal inducible expression. Our results indicate that transgenic expression of CTLA-4 in mouse T cells (CTLA-4-Tg T cells) results in reduced cell cycle progression and increased apoptosis of TCR-stimulated T cells. CTLA-4-Tg T cells display reduced T cell proliferation in an in vivo model of graft versus host disease (GVHD). These results further our understanding of how CTLA-4 can be manipulated to inhibit immune responses and may help development of new therapeutic strategies for clinical settings of autoimmunity and transplantation.