Girak Kim

Curcumin inhibits CD4(+) T cell activation, but augments CD69 expression and TGF-β1-mediated generation of regulatory T cells at late phase.

Background Curcumin is a promising candidate for a natural medicinal agent to treat chronic inflammatory diseases. Although CD4+ T cells have been implicated in the pathogenesis of chronic inflammation, whether curcumin directly regulates CD4+ T cells has not been definitively established. Here, we showed curcumin-mediated regulation of CD2/CD3/CD28-initiated CD4+ T cell activation in vitro. Methodology/Principal Findings Primary human CD4+ T cells were stimulated with anti-CD2/CD3/CD28 antibody-coated beads as an in vitro surrogate system for antigen presenting cell-T cell interaction and treated with curcumin. We found that curcumin suppresses CD2/CD3/CD28-initiated CD4+ T cell activation by inhibiting cell proliferation, differentiation and cytokine production. On the other hand, curcumin attenuated the spontaneous decline of CD69 expression and indirectly increased expression of CCR7, L-selectin and Transforming growth factor-β1 (TGF-β1) at the late phase of CD2/CD3/CD28-initiated T cell activation. Curcumin-mediated up-regulation of CD69 at late phase was associated with ERK1/2 signaling. Furthermore, TGF-β1 was involved in curcumin-mediated regulation of T cell activation and late-phase generation of regulatory T cells. Conclusions/Significance Curcumin not merely blocks, but regulates CD2/CD3/CD28-initiated CD4+ T cell activation by augmenting CD69, CCR7, L-selectin and TGF-β1 expression followed by regulatory T cell generation. These results suggest that curcumin could directly reduce T cell-dependent inflammatory stress by modulating CD4+ T cell activation at multiple levels.

Stress, Nutrition, and Intestinal Immune Responses in Pigs - A Review.

Modern livestock production became highly intensive and large scaled to increase production efficiency. This production environment could add stressors affecting the health and growth of animals. Major stressors can include environment (air quality and temperature), nutrition, and infection. These stressors can reduce growth performance and alter immune systems at systemic and local levels including the gastrointestinal tract. Heat stress increases the permeability, oxidative stress, and inflammatory responses in the gut. Nutritional stress from fasting, antinutritional compounds, and toxins induces the leakage and destruction of the tight junction proteins in the gut. Fasting is shown to suppress pro-inflammatory cytokines, whereas deoxynivalenol increases the recruitment of intestinal pro-inflammatory cytokines and the level of lymphocytes in the gut. Pathogenic and viral infections such as Enterotoxigenic E. coli (ETEC) and porcine epidemic diarrhea virus can lead to loosening the intestinal epithelial barrier. On the other hand, supplementation of Lactobacillus or Saccharaomyces reduced infectious stress by ETEC. It was noted that major stressors altered the permeability of intestinal barriers and profiles of genes and proteins of pro-inflammatory cytokines and chemokines in mucosal system in pigs. However, it is not sufficient to fully explain the mechanism of the gut immune system in pigs under stress conditions. Correlation and interaction of gut and systemic immune system under major stressors should be better defined to overcome aforementioned obstacles.

Exploring the Genetic Signature of Body Size in Yucatan Miniature Pig

Since being domesticated about 10,000–12,000 years ago, domestic pigs (Sus scrofa domesticus) have been selected for traits of economic importance, in particular large body size. However, Yucatan miniature pigs have been selected for small body size to withstand high temperature environment and for laboratory use. This renders the Yucatan miniature pig a valuable model for understanding the evolution of body size. We investigate the genetic signature for selection of body size in the Yucatan miniature pig. Phylogenetic distance of Yucatan miniature pig was compared to other large swine breeds (Yorkshire, Landrace, Duroc and wild boar). By estimating the XP-EHH statistic using re-sequencing data derived from 70 pigs, we were able to unravel the signatures of selection of body size. We found that both selections at the level of organism, and at the cellular level have occurred. Selection at the higher levels include feed intake, regulation of body weight and increase in mass while selection at the molecular level includes cell cycle and cell proliferation. Positively selected genes probed by XP-EHH may provide insight into the docile character and innate immunity as well as body size of Yucatan miniature pig.

Systemic administration of RANKL overcomes the bottleneck of oral vaccine delivery through microfold cells in ileum

A successful delivery of antigen through oral route requires to overcome several barriers, such as enzymatic barrier of gastrointestinal tract and epithelial barrier that constitutes of microfold cells (M cells) for antigen uptake. Although each barrier represents a critical step in determining the final efficiency of antigen delivery, the transcytosis of antigen by M cells in the follicle-associated epithelium (FAE) to Peyers patches appears to be a major bottleneck. Considering the systemic administration of receptor activator of nuclear factor (NF)-ĸB ligand (RANKL) induces differentiation of receptor activator of nuclear factor (NF)-ĸB (RANK)-expressing enterocytes into M cells, here, we illustrated a promising approach of antigen delivery using full length transmembrane RANKL (mRANKL). The results showed that the intraperitoneal injection of mRANKL increased the population of dendritic cells and macrophages in mesenteric lymph nodes and spleen. Subsequently, systemic administration of mRANKL resulted in significantly higher number of functional GP2(+) M cells leading higher transcytosis of fluorescent beads through them. To corroborate the effect of mRANKL in antigen delivery through M cells, we orally delivered microparticulate antigen to mice treated with mRANKL. Oral immunization induced strong protective IgA and systemic IgG antibody responses against orally delivered antigen in mRANKL-treated mice. The higher antibody responses are attributed to the higher transcytosis of antigens through M cells. Ultimately, the higher memory B cells and effector memory CD4 T cells after oral immunization in RANKL-treated mice confirmed potency of RANKL-mediated antigen delivery. To the best of our knowledge, this is the first study to demonstrate significant induction of mucosal and humoral immune responses to M cell targeted oral vaccines after the systemic administration of RANKL.

A Bacterial Metabolite, Compound K, Induces Programmed Necrosis in MCF-7 Cells via GSK3β.

Ginsenosides, the major active component of ginseng, are traditionally used to treat various diseases, including cancer, inflammation, and obesity. Among these, compound K (CK), an intestinal bacterial metabolite of the ginsenosides Rb1, Rb2, and Rc from Bacteroides JY-6, is reported to inhibit cancer cell growth by inducing cell-cycle arrest or cell death, including apoptosis and necrosis. However, the precise effect of CK on breast cancer cells remains unclear. MCF-7 cells were treated with CK (0-70 micrometer) for 24 or 48 h. Cell proliferation and death were evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and flow cytometry assays, respectively. Changes in downstream signaling molecules involved in cell death, including glycogen synthase kinase 3β (GSK3β), GSK3β, β-catenin, and cyclin D1, were analyzed by western blot assay. To block GSK3β signaling, MCF-7 cells were pretreated with GSK3β inhibitors 1 h prior to CK treatment. Cell death and the expression of β-catenin and cyclin D1 were then examined. CK dose- and time-dependently inhibited MCF-7 cell proliferation. Interestingly, CK induced programmed necrosis, but not apoptosis, via the GSK3β signaling pathway in MCF-7 cells. CK inhibited GSK3β phosphorylation, thereby suppressing the expression of β-catenin and cyclin D1. Our results suggest that CK induces programmed necrosis in MCF-7 breast cancer cells via the GSK3β signaling pathway.

H9N2-specific IgG and CD4+CD25+ T cells in broilers fed a diet supplemented with organic acids

&NA; Organic acids have long been known for their beneficial effects on growth performance in domestic animals. However, their impact on immune responses against viral antigens in chickens is unclear. The present study aimed to investigate immunological parameters in broilers immunized with a H9N2 vaccine and/or fed a diet containing organic acids (citric, formic, and lactic acids). We allotted 1‐day‐old broilers into 4 groups: control (C), fed a diet supplemented with organic acids (O), administered a H9N2 vaccine (V), and fed a diet supplemented with organic acids and administered a H9N2 vaccine (OV). Blood and spleen samples were taken at 2, 7 and 14 d post vaccination (DPV). At 14 DPV, total and H9N2‐specific IgG levels were significantly lower in the OV group than in the V group. However, it was intriguing to observe that at 2 DPV, the percentage of CD4+CD25+ T cells was significantly higher in the OV group than in the other groups, indicating the potential induction of regulatory T cells by organic acids. In contrast, at 2 DPV, the percentage of CD4+CD28+ T cells were significantly lower in the OV group than in the other groups, suggesting that CD28 molecules are down‐regulated by the treatment. The expression of CD28 on CD4+ T cells, up‐regulated by the stimulation with phorbol 12‐myristate 13‐acetate (PMA) and ionomycin (Iono), was inhibited upon organic acid treatment in OV group. In addition, the proliferation of lymphocytes, stimulated with formalin‐inactivated H9N2, was significantly higher in the V group than in the OV group. Alpha 1‐acid glycoprotein (AGP) production was significantly lower in the OV group than in the V group, suggesting that the organic acids inhibited the inflammation caused by the vaccination. Overall, induction of regulatory CD4+CD25+ T cells, coinciding with the decrease of H9N2‐specific antibodies, was observed in broilers fed organic acids.

Regulation of CD4 + CD8 − CD25 + and CD4 + CD8 + CD25 + T cells by gut microbiota in chicken

The gut microbiota in chicken has long been studied, mostly from the perspective of growth performance. However, there are some immunological studies regarding gut homeostasis in chicken. Although CD4+CD25+ T cells are reported to act as regulatory T cells (Tregs) in chicken, there have been no studies showing the relationship between gut microbiota and Tregs. Therefore, we established a model for ‘antibiotics (ABX)-treated chickens’ through administration of an antibiotic cocktail consisting of ampicillin, gentamycin, neomycin, metronidazole, and vancomycin in water for 7 days. CD4+CD8−CD25+ and CD4+CD8+CD25+ T cells in cecal tonsils were significantly decreased in this model. Gram-positive bacteria, especially Clostridia, was responsible for the changes in CD4+CD8−CD25+ or CD4+CD8+CD25+ T cells in cecal tonsils. Feeding ABX-treated chickens with acetate recovered CD4+CD8−CD25+ and CD4+CD8+CD25+ T cells in cecal tonsils. GPR43, a receptor for acetate, was highly expressed in CD4+CD8−CD25+ T cells. In conclusion, our study demonstrated that the gut microbiota can regulate the population of CD4+CD8−CD25+ and CD4+CD8+CD25+ T cells, and that acetate is responsible for the induction of CD4+CD8−CD25+ T cells in cecal tonsils via GPR43.

Distinct pattern of immune tolerance in dendritic cells treated with lipopolysaccharide or lipoteichoic acid

HighlightsRepeated LTA stimulation suppressed TNF‐&agr; and IL‐6 production in BMDCs.BMDCs treated with repeated LTA down‐regulated IL‐10 and enhanced LAP.BMDCs treated with LTA showed sustained expression of TOLLIP and IDO.LPS exposure to BMDCs induced cross tolerance to LTA while LTA did not to LPS. &NA; Cytokine induction is often critical for the host defense during acute immune responses while, if not tightly regulated, it may cause an immunological pathology coincident with tissue damage. Despite the fact that gram‐positive bacterial infection has become increasingly prevalent, immune modulation induced by lipoteichoic acid (LTA), the major cell wall component of gram‐positive bacteria has not been studied thoroughly at the cellular level. In the current study, tolerance induction in mouse bone marrow‐derived dendritic cells (BMDCs) treated with single or repeated stimulation of Staphylococcus aureus LTA was compared with those of Escherichia coli lipopolysaccharide (LPS). The results showed that repeated LTA stimulation significantly suppressed pro‐inflammatory cytokine (TNF‐&agr; and IL‐6) production in BMDCs, comparable to that of LPS, but with less extent, down‐regulated IL‐10 and enhanced the inhibitory molecule, LAG‐3‐associated protein (LAP). Furthermore, we observed a sustained expression of unique negative regulators, Toll interacting protein (TOLLIP) and Indoleamine 2,3‐dioxygenase (IDO), in BMDCs treated with LTA. A transient hyporesponsiveness period appeared when DCs were treated repeatedly with LTA or LPS showing a distinctive pattern. Intriguingly, LPS exposure induced cross tolerance to LTA while LTA exposure did not to LPS, implicating that a distinct signaling components are involved in response to LTA. Collectively, a distinct immune regulation appeared to be responsible for the LPS‐ and LTA‐induced tolerance on cytokine production, expression of surface markers and intracellular proteins.

A new way of producing pediocin in Pediococcus acidilactici through intracellular stimulation by internalized inulin nanoparticles

One of the most challenging aspects of probiotics as a replacement for antibiotics is to enhance their antimicrobial activity against pathogens. Given that prebiotics stimulate the growth and/or activity of probiotics, we developed phthalyl inulin nanoparticles (PINs) as prebiotics and observed their effects on the cellular and antimicrobial activities of Pediococcus acidilactici (PA). First, we assessed the internalization of PINs into PA. The internalization of PINs was largely regulated by glucose transporters in PA, and the process was energy-dependent. Once internalized, PINs induced PA to produce substantial amounts of antimicrobial peptide (pediocin), which is effective against both Gram-positive (Salmonella Gallinarum) and Gram-negative (Listeria monocytogenes) pathogens. When treated with small-sized PINs, PA witnessed a nine-fold increase in antimicrobial activity. The rise in pediocin activity in PA treated with PINs was accompanied by enhanced expression of stress response genes (groEL, groES, dnaK) and pediocin biosynthesis genes (pedA, pedD). Although the mechanism is not clear, it appears that the internalization of PINs by PA causes mild stress to activate the PA defense system, leading to increased production of pediocin. Overall, we identified a prebiotic in nanoparticle form for intracellular stimulation of probiotics, demonstrating a new avenue for the biological production of antimicrobial peptides.

Transcription Factor KLF10 Constrains IL-17-Committed Vγ4+ γδ T Cells

γδ T cells, known to be an important source of innate IL-17 in mice, provide critical contributions to host immune responses. Development and function of γδ T cells are directed by networks of diverse transcription factors (TFs). Here, we examine the role of the zinc finger TFs, Kruppel-like factor 10 (KLF10), in the regulation of IL-17-committed CD27− γδ T (γδ27−-17) cells. We found selective augmentation of Vγ4+ γδ27− cells with higher IL-17 production in KLF10-deficient mice. Surprisingly, KLF10-deficient CD127hi Vγ4+ γδ27−-17 cells expressed higher levels of CD5 than their wild-type counterparts, with hyper-responsiveness to cytokine, but not T-cell receptor, stimuli. Thymic maturation of Vγ4+ γδ27− cells was enhanced in newborn mice deficient in KLF10. Finally, a mixed bone marrow chimera study indicates that intrinsic KLF10 signaling is requisite to limit Vγ4+ γδ27−-17 cells. Collectively, these findings demonstrate that KLF10 regulates thymic development of Vγ4+ γδ27− cells and their peripheral homeostasis at steady state.