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주관연구기관

성균관대학교
SungKyunKwan University

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2단계보고서

발행국가

대한민국

언어

한국어

발행년월

2013-07

과제시작연도

2012

주관부처

미래창조과학부
Ministry of Science, ICT and Future Planning

등록번호

TRKO202100004805

과제고유번호

1345176108

사업명

바이오·의료기술개발

DB 구축일자

2021-07-10

키워드

선천면역.대식세포.관용.유사 Toll 수용체.염증.innate immunity.macrophages.tolerance.TLR.inflammation.

표 Combination of islet transplantation and Pam3Cys, a TLR2 ligand, successfully treated already eatablished type 1 diabetes of NOD mice, while islet transplantation alone induced only transient remission of diabetes and immediate recurrence of diabetes (left). Insulin immunihistochemistry shows transplanted islets were not destroyed by host autoimmnity due to tolerance established by chronic treatment with Pam3Cys (right)
표 DPPIV inhibitor ameliorated high blood glucose after STZ treatment (left). Intraperitonela glucose tolerance test shows glucose tolerance was improved by adminstration of DPPIV inhibitor to diabetic mice after STZ treatment (right)
표 Administration of DPPIV inhibitor increased b-cell volume estimated by insulin immunihistochemistry in diabetic mice after STZ treatment to a small degree but significantly
표 Immunohistochemistry after BrdU injection into diabetic mice after STZ treatment showed that DPPIV inhibitor (DA) increased the number of BrdU+ proliferating b-cells
표 DPPIV inhibitors increased the number of small b-cell units comprising less than 4 insulin+ b-cells that represent b-cell neogenesis
표 DPPIV inhibitor induced PDX-1 expression in b-cells, which appears to be an important mechanism leading to b-cell differentiation and neogenesis
표 DPPIV inhibitor in conjuction with Pam3Cys induced significant amelioration of hyperglycemia in diabetic mice, while DPPIV inhibitor alone did not have such an effect
표 Three weeks after treatment with Pam3CSK and/or DA-1229, serum was obtained4 before and 15 min after 1 g/kg glucose injection i.p. to overnight fasted mice for measurement of insulin level by ELISA and calculation of insulinogenic index (left). Three weeks after treatment with Pam3CSK4 and/or DA-1229, fasting GLP-1 level was determined by ELISA (right)
표 Islet histology of diabetic NOD mice treated with Pam3CSK4 in combination with DA-1229. Pancreas tissue was harvested from diabetic NOD mice after treatment with Pam3CSK4 and/or DA-1229 for 3 weeks. Islet sections were also prepared from 15-week-old female prediabetic NOD mice and diabetic NOD mice untreated for 3 weeks (left). Distribution of islet size in 15-week-old prediabetic NOD mice, diabetic NOD mice treated with Pam3CSK4+DA-1229 for 3 weeks and diabetic NOD mice untreated for 3 weeks was determined (n=3 each) (right)
표 Mass and proliferation of beta cells in diabetic NOD mice treated with Pam3CSK4+DA-1229. Pancreas tissue was harvested from each group of mice for insulin IHC using specific Ab. Beta cells mass was assessed by point counting method as described in the Methods (right). Representative H&E sections are shown (left)
표 Sixteen h after i.p. injection of 100 mg/kg BrdU, pancreatic sections were prepared for double IHC using anti-insulin and -BrdU Abs to measure beta cell regeneration (right). Arrows indicate representative BrdU-incorporating beta cells (left)
표 Pancreatic sections were prepared as in a to determine the number of small beta cell units comprising 3 insulin+ cells at a maximum as a measure of beta cell neogenesis (right). Arrows indicate representative small beta cell units in NOD mice treated with Pam3CSK4+DA-1229 (left)
표 Mononuclear cells were prepared from the spleen and PLNs of NOD mice treated prepared from BDC2.5/NOD mice by the negative-selection method were labelled with CFSE and transferred by tail vein injection into NODmice that were treated with Pam3CSK4+DA-1229 for 3 weeks or prediabetic NOD mice. CD4+ T cell proliferation in the PLNs was measured by analyzing CFSE dilution by flow cytometry gated on CD4+ and V4+ cells. Results pooled from 4 independent experiments are presented (right). Representative histograms are shown (left)
표 Mononuclear cells were prepared from the spleen and PLNs of NOD mice treated with Pam3CSK4+DA-1229 for 3 weeks or untreated prediabetic NOD mice in vivo. Cells were further incubated with Pam3CSK4 (gray filled graph) or PBS (black line) ex vivo for 1 day, and the expression of costimulatory molecules on DCs was determined by flow cytometry gated on CD11c+ cells (left). Changes of MFIs are shown (right)
표 Unaltered Treg cells in NOD mice treated with Pam3CSK4+DA-1229. a Mononuclear cells from the spleen, PLNs and or islets of NOD mice that were treated with Pam3CSK4+DA-1229 for 3 weeks or prediabetic NOD mice were stained using anti-CD4, -CD25 and -Foxp3 monoclonal Abs for triple-colored flow cytometry. The numbers in the left panel represent the percentage of CD4+Foxp3+ Treg cells (left). Those in the right panel denote the MFIs of CD25 on CD4+Foxp3+ cells (right)
표 Unaltered Th1/Th2/Th17 polarization in NOD mice treated with Pam3CSK4+DA-1229. Th1/Th2/Th17 skewing of mononuclear cells from the spleen or PLNs of NOD mice that were treated with Pam3CSK4+DA-1229 was examined. Results are representative of 3 ~ 4 independent experiments, showing similar tendencies
표 Pretreatment of peritoneal macrophages with iE-DAP, an Nod1 ligand, attenuated release of IL-6 or TNFa after Pam3Cys treatment, suggesting tolerance induction by non-TLR ligand
표 Administration of KF1B, a Nod1 ligand, at 50 ug dose, 3 times a weeks did not inhibit the development of diabetes in NOD mice
표 The effect of 100 ug dose of KF1B, 3 times a week, is being studied in NOD mice
표 Mouse primary islet에서 putative TLR2 endogenous ligand의 발현을 Western blot analysis 로 조사
표 Insulinoma cell extract를 potential TLR2 agonist 에 대한 antibody 로 immunodepletion 시킨 후 macrophage 로 부터의 cytokine release 를 측정함
표 Altered phenotype of lamina propria immune cells in obese mice
표 Increased apoptosis of lamina propria immune cells and enterocytes in obese mice
표 Decreased cell numbers and cytokine productions of subtypes of LPC in obese mice
표 Impaired lysozyme secretion and decreased expression levels of Paneth cell specific markers
표 Decreased granule contents of Paneth cells in obese mice (H&E)
표 Decreased granule contents of Paneth cells in obese mice (PAS-Alcian Blue)
표 Increased expression of the unfolded protein response-related genes in the intestines of obese mice
표 Decreased expression levels of gut tight-junction proteins in the intestines of obese mice
표 Increased permeability of the intestines in obese mice
표 Increased susceptibility of DSS-induced colitis owing to Paneth cell dysfunction in the intestines of obese mice
표 crypts isolated from NCD- or HFD-fed mice were stimulated with 10 uMcarbamylcholine (CCh) or 100 ng/ml LPS. Supernants were precipitated, resolved on SDS-PAGE, and detected by anti-lysozyme IgG (left). Small intestinal crypts were stimulated with CCh or LPS for 30 min, and supernatant were assayed for bactericidal activity against Salmonella typhimurium (middle). LAL assay was conducted using the liver lysates to measure endotoxin content (right)
표 Conoly count after bacterial culture of the spleen extract (left). Quantitation of whole bacteria was assayed by Q-PCR determination of 16S rRNA genes in the liver (right)
표 Mice were fed with NCD or HFD for 8 weeks and then were received mixture of antibiotics (Abs) or PBS by oral gavage for 6 weeks. LAL assay was conducted using serum from NCD-PBS, HFD-PBS or HFD-Abs group (left). Liver inflammation was examined by Q-PCR using specific primers for inflammatory markers (right)
표 Mice were fed with NCD or HFD for 8 weeks and then were received mixture of antibiotics (Abs) or PBS by oral gavage for 6 weeks as in Fig. 15. Intraperitoneal glucose tolerance test was done (left) and the results were converted to the values of area under glucose curves (right)
표 Metformin 또는 Akkermansia 투여 후 glucose tolerance test에서 area under the curve (AUC) 가 감소하여 glucose tolerance가 호전됨을 관찰
$Metformin 또는 Akkermansia 투여 후 adipose tissue 의 stromal vascular fraction (SVF)에서 Treg 의 비율 (A), number (B) 가 증가함을 관찰. Representative FACS analysis 결과를 (C) 에서 제시함$ 표 Metformin 또는 Akkermansia 투여 후 adipose tissue 의 stromal vascular fraction (SVF)에서 Treg 의 비율 (A), number (B) 가 증가함을 관찰. Representative FACS analysis 결과를 (C) 에서 제시함
표 The effects of LCN2 protein expressed in mammalian cells (mLCN2) on the chemokine gene expression and cell migration in astrocytes. Astrocytes were treated with the NSO murine melanoma cell-derived mouse LCN2 protein (10 mg/ml; mLCN2) for 8 hr, and the total RNA was isolated for traditional RT-PCR. Cells were also treated for 8 hr with LPS (100 ng/ml) plus IFN-g (50 units/ml) for comparison purposes. The mRNA levels of CXCL10 and PIAS3 were determined by traditional RT-PCR (A). b-actin was used as an internal control. The results are one representative of more than three independent experiments. Astrocytes (1 x 104cells/upper well) were exposed to the melanoma cell-expressed LCN2 protein (10 mg/ml; mLCN2)-stimulated astrocyte-conditioned media (ACM-mLCN2). Astrocytes placed in the Boyden chambers were then incubated at 37°C for 24-72 hr to evaluate cell migration (B). A representative microscopic image for each condition was shown (magnification, x 100) (upper). ACM-None, untreated ACM; ACM-mLCN2, mLCN2-treated ACM. The quantification of cell migration was done by enumerating the migrated cells as described in the Materials and Methods section (lower). The results are mean ± SD (n=3). *p<0.05 compared with ACM-None at the same time point
표 The effect of LCN2-treated ACM on the morphology of astrocytes and microglia. ACM was prepared after the treatment of primary astrocytes with LCN2 (10 mg/ml) for 24 hr. The addition of LCN2-treated ACM (ACM-LCN2) induced morphological changes in primary astrocytes and primary microglia cultures after 24hr (A). Primary astrocytes were stained with GFAP antibody (original magnification, x 400), followed by the incubation with anti-mouse IgG-fluorescein isothiocyanate (FITC)-conjugated secondary antibody. Primary microglia were stained with the peroxidase-labeled isolectin B4 (original magnification, x 100), followed by incubation with diaminobenzidine tetrahydrochloride (DAB). Scale bar, 25 mm. The length of the longest process in each astrocyte or the percentage of ramified microglia was assessed by examining several randomly chosen microscopic fields (B). The results are mean ± SD (n=3). *p<0.05;compared with the untreated ACM control (ACM-None)
표 Tissue distribution of LCN2 expression. Western blot analysis was performed on protein extracts from several adult mouse tissues in LCN2-wild-type (LCN2+/+), LCN2 heterozygous (LCN2+/-), and LCN2-deficient (LCN2-/-)mice using LCN2-specific antibody. a-tubulin was also detected to confirm the equal loading of the samples
표 Essential role of LCN2 in astrocyte migration and CXCL10 induction in LPS-induced mouse neuroinflammation model and cortical stab wound injury. LCN2-wild-type (LCN2+/+) or LCN2-deficient mice (LCN2-/-) were injected with LPS intracortically (A)or icv (B). After 2 day post-injury (dpi), mice were sacrificed and cryosections were immunostained with antibodies against LCN2 (green, upper) or GFAP (red, middle). (Nuclei; blue, lower). The mRNA levels of lcn2, CXCL10, and GFAP in LCN2+/+and LCN2-/- mice were examined by Real-Time PCR of cortical tissue around the injection site or hippocampus at 2 days after intracortical or icv injection with LPS, respectively. LCN2-/- mice exhibited markedly lower levels of CXCL10 and GFAP as compared with LCN2+/+littermates. The mRNA levels of lcn2 (upper) and CXCL10 (lower)in LCN2+/+and LCN2-/- mice were examined at 2 days after cortical stab wound injury (C). RNA was isolated from the injury site in the cortex and subjected to Real-Time PCR. At 2 days after icv injection of LCN2+/+mice with LPS, hippocampus was immunostained with antibodies against LCN2 (green) or GFAP (red) (D). Nuclei were counterstained with DAPI (blue). Merged image is shown in the lower right panel. Arrowheads indicate co-localization of LCN2 and GFAP (yellow). Cell bodies and processes of astrocytes in hippocampus were stained with LCN2 antibody
표 LCN2 induction of M1-related gene expression profile in microglia. Primary microglia were treated with the NSO murine melanoma cell-derived mouse LCN2 protein (10 mg/ml; mLCN2) or GST protein (10 mg/ml) for 8 hr, and the total RNA was isolated. The mRNA levels of cytokines (IL-12, IL-23, IL-10, iNOS, and TNF-a), chemokines (CXCL10, CCL17, and CCL22), and surface receptors (ARG and MRC1) were determined by Real-Time PCR (A, B). BV-2 cells with an increased or decreased lcn2 expression were established by stable transfection of lcn2 sense or antisense cDNA (C). BV-2 cells were incubated with the LPS (100 ng/ml) plus IFN-g (50 units/ml), IL-4 (10 ng/ml), or IL-10 (10 ng/ml) for 8 hr, and the total RNA was isolated. The mRNA levels of cytokines (IL-12, IL-23, IL-10, IL-6, IL-1b, iNOS, and TNF-a) and chemokines (CXCL10) were determined by traditional RT-PCR (D)
표 LCN2 inhibition of IL-4-induced STAT6 activation and M2 gene expression in microglia. After primary microglia were treated with the recombinant LCN2 protein (10 mg/ml) or leflunomide (10 mM) in the presence or absence of IL-4 (10 ng/ml) for 24 hr, ARG1 or MRC1 surface expression was detected by flow cytometric analysis using monoclonal antibodies specific for ARG1 or MRC1. Histograms from specific staining are compared with background staining
표 LCN2 is essential for M1, but not M2, polarization of microglia. Primary microglia prepared from LCN2+/+or LCN2-/-mice. LCN2+/+ or LCN2-/- primary microglia were treated with the LPS (100 ng/ml) plus IFN-g (50 units/ml), IL-4 (10 ng/ml), immune complexes plus LPS (100 ng/ml), or IL-10 (10 ng/ml) for 8 hr, and the total RNA was isolated. Cells also were treated in the presence of LPS for 20 hr, washed, and maintained in DMEM medium for 2 hr and then retreated with LPS for 4 hr. (LPS//LPS), and the total RNA was isolated. The mRNA levels of cytokines (IL-12, IL-23, IL-10, iNOS, and TNF-a), chemokines (CXCL10, CCL17, and CCL22), and surface receptors (ARG1 and MRC1) were determined by Real-Time PCR (A, B). The expression of representative M1 genes (A)and M2 genes(B). GAPDH was used as an internal control. The results are mean ± SD (n=3)
표 LCN2 deficiency inhibits microglial M1 polarization in a mouse neuroinflammation model. LCN2 wild type (LCN2+/+) or LCN2-deficient (LCN2-/-) mice were anesthetized using enflurane, and received an intraperitoneal injection of saline and LPS (5 mg/kg) for 4 hr, and the RNAs from half of whole brain and primary adult microglia cultures were isolated. The mRNA levels of cytokines (IL-12, IL-23, IL-10, iNOS, and TNF-a), chemokines (CXCL10, CCL17, and CCL22), surface receptors (ARG1 and MRC1), and lcn2 were determined by Real-Time PCR (A, B). The expression of representative M1 genes (A) and M2 genes (B). The expression of representative whole brain (open bar) and primary adult microglia cultures (filled bar)
표 LCN2 can contribute to tissue impairment in neuroinflammation model. Brain
표 polarization, respectively, in vivo. LCN2 wild-type (LCN2+/+; n = 3) or LCN2-deficient (LCN2-/-; n = 3) mice received an intracerebroventricular injection of saline, LPS (5 mg), or IL-4 (10 mg). After 24 hr, primary adult microglia cultures prepared from the brain. The mRNA levels of M1-related genes (A) or M2-related genes (B) were determined by Real-Time RT-PCR. GAPDH was used as an internal control. The results are one representative of more than three independent experiments. *p < 0.05; significantly different from the saline-treated control
표 sections were double-stained by immunohistochemistry for Iba-I (microglia, green), NeuN (neuron, red), with DAPI (blue). LPS treatment induced microglial activation in cortex, hippocampus and cerebellum of LCN2+/+ mice. Particularly, LPS administration seriously inflicted an injury to hippocampus in LPS+/+ mice. In the LPS-administered LCN2-/- mice, the tissue impairment was significantly improved, and activated microglia was reduced
표 Intracerebroventricular injection of LPS or IL-4 induces microglial M1 or M2 polarization, respectively, in vivo. LCN2 wild-type (LCN2+/+; n = 3) or LCN2-deficient (LCN2-/-; n = 3) mice received an intracerebroventricular injection of saline, LPS (5 mg), or IL-4 (10 mg). After 24 hr, primary adult microglia cultures prepared from the brain. The mRNA levels of M1-related genes (A) or M2-related genes (B) were determined by Real-Time RT-PCR. GAPDH was used as an internal control. The results are one representative of more than three independent experiments. *p < 0.05; significantly different from the saline-treated control
표 Transgenic expression of lcn2 affected neuronal migration in the spinal cord of zebrafish embryos. The her4:egfp-injected control embryo (A) or her4:lcn2:egfp-injected embryo (B) was labeled with an anti-Hu antibody to detect neurons (red fluorescence) at 24 hpf. Arrowheads indicate neurons near the egfp-expressing cells (A) or lcn2:egfp-expressing cells (B)(green fluorescence). Dotted lines indicate midline of the spinal cord. The wild-type embryo (C) or her4:lcn2:egfp-injected transgenic embryo (D) was labeled with an anti-Hu antibody to detect neurons (red fluorescence) at 24 hpf. Arrowheads indicate neurons near the lcn2:egfp-expressing cells (green fluorescence). Dotted lines indicate a lateral margin (a) and medial position (b) of the spinal cord. Values indicate percentage of neuronal cells in each region. All images are transverse sections of zebrafish spinal cord, dorsal to top. The results are mean ± SD
표 The Effect of small molecule compounds on NO production in LPS-Stimulated BV-2 microglial cells. BV-2 microglial cells were treated with LPS (100 ng/ml) in the absence or presence of the compounds (5 mM) for 24 h. Nitrite content in the culture media was measured using the Griess reaction and the results were expressed as a concentration of released NO from LPS-stimulated BV-2 cells (A; left). Cytotoxicity of each compound was assessed by MTT assays and the results were expressed as a percentage of surviving cells over control cells (A; right). Among the compounds tested, A01 most strongly reduced LPS-induced NO production in BV-2 cells. A01 reduced NO production in a dose-dependent manner without cytotoxicity (B)
표 A01 inhibited microglial activation in a mouse neuroinflammation model. C57BL/6 mice were injected intraperitoneally with vehicle (48% PEG containing 2% DMSO) or A01 (diluted in 48% PEG) once daily at 2 or 10 mg/kg for 4 days. At 24 h after the first injection of vehicle or A01, mice were intraperitoneally injected with 5 mg/kg LPS. Mice were anesthetized with 23% urethane and transcardially perfused with ice-cold saline 72 h after the LPS injection. Brains were removed and sections were stained with Iba-1 (a marker for microglia). Iba-1-positive cells were observed in substantia nigra, hippocampus, and cortex region of vehicle + LPS- or A01 + LPS-injected mouse brains

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