[보고서]폐렴구균 pep27 변이주 접종에 의한 염증/패혈증으로부터 점막보호 기전 연구

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보고서 정보

주관연구기관

성균관대학교
SungKyunKwan University

연구책임자

이동권

보고서유형

최종보고서

발행국가

대한민국

언어

한국어

발행년월

2021-09

과제시작연도

2020

주관부처

과학기술정보통신부
Ministry of Science and ICT

등록번호

TRKO202200018940

과제고유번호

1711137576

사업명

개인기초연구(과기정통부)(R&D)

DB 구축일자

2023-03-14

키워드

폐렴구균.점막면역.폐렴/패혈증.면역저항/면역관용.염증성 장질환.Streptococcus pneumoniae.Mucosal immunity.Pneumonia/sepsis.Immune tolerance.Intestinal bowel diseases.

표 △pep27 접종으로 표피세포 유전자 발현 유도. Mice (n = 3) were immunized intranasally (i.n.) with Δpep27 (1 × 107 CFU) three times at two-week intervals without anesthization. Seven days after the last immunization, mRNA from lung or spleen was isolated. High-throughput sequencing identified up- and downregulated genes in the lung or spleen, especially, genes cluster related to epithelial cells
표 foxp3-gfp 생쥐에 △pep27 인후 접종에 의한 Treg 발현 확인. 폐와 간에서 Treg 발현이 유도됨
표 Δpep27 increases survival and attenuates IBD symptoms and inflammatory mediators and cell death in DIC via antioxidative mechanism. a Survival rate (%) in Δpep27 and lethal S. pneumoniae type 2 (D39) infection was evaluated in mice. b Macrophage viability was determined by MTT assay. c System biology analysis of lung transcriptome data predicts that Δpep27 mitigates both gastroenteritis and abnormalities of the large intestine. d, e Heat maps analysis revealed inhibition of inflammation-related genes, induction of the antioxidants SPRR family and repression of the caspase-14 by Δpep27 in lungs (d) and colon (e). f, g Progression of colitis was evaluated by disease activity index (DAI) (f) and colon length (g). h, j Colon sections were stained with H&E (20x magnification, scale bar, 200 μm) (h) with histological grading (j). i, k TUNEL assay (i) with statistical analysis of the TUNEL assay (k) (200x magnification, scale bar, 100 μm)
표 Δpep27 induces Tregs and inhibits proinflammatory cytokines expression and permeability. a-e Proinflammatory cytokines and caspase-14 transcripts were determined by qPCR. f-i Oxidative stress response as an assessment of reactive oxygen species (ROS) and reactive nitrogen species (RNS) was quantified by myeloperoxidase (MPO) and malondialdehyde (MDA) assays (f, g) and validated with antioxidant SPRR genes using qPCR analysis (h, i). j-l Treg transcription factor (J) and anti-inflammatory mediators (k, l) in colon samples were determined by qPCR. m, n Epithelial integrity was determined by Lor transcript and Evans blue dye assay. o Levels of various proteins were confirmed by western blot using β-actin as an internal control. p-s Immunohistochemistry analysis in lung samples from immunized and non-immunized mice before and after challenge with lethal pneumococcal strain D39 were stained with Loricrin with magnification at 200x and scale bar, 100 μm
표 Δpep27 restores healthy gut microbiota and species diversity. a-g Effects of Δpep27 on the microbial composition at various taxonomic levels in the DSS induced colitis (DIC) mice. Fecal pellets were analyzed by pyrosequencing of the 16S rRNA. Relative abundance (%) at phylum (a), family (b), and species level (c). Microbiome taxonomic profile revealed that the major phyla of A. mucinphila, B. valgatus, L. gasseri and L. murinus constitute approximately 40% of whole bacterial population at species level (c). d-g Diversity indices of gut microbiota were evaluated as (d) operational taxonomic unit (OUT) counts, (e) Chao1 (f) Shannon and (g) Simpson in the correspondent groups
표 Treg depletion aggravates inflammation and apoptosis. a-e Neutralization protocol of Tregs with anti-CD25 (a). After CD25 (Treg) neutralization using CD25 antibody (a), colon samples were H&E stained (b) or analyzed by TUNEL assay (c). b, d Representative photomicrographs of H&E staining at 20x magnification, scale bar = 200 μm and TUNEL assay (c, e) after anti-CD25-treatment. A representative image of the TUNEL assay after anti-CD25-treatment (200′ magnification, scale bar = 100 μm). f-l Transcripts in the colon samples were analyzed by qPCR. m Protein levels were confirmed by western blot
표 Δpep27 represses caspase-14 via Treg induction and alleviates DIC. a-e Blockade of caspase-14 expression via anti-caspase-14 neutralization assay (a). After caspase-14 neutralization using caspase-14 antibody (a), colon samples were H&E stained (b) and analyzed by TUNEL assay c. Representative photomicrographs of H&E staining at 20x magnification, scale bar = 200 μm and histological score (b, d) and (c, e) TUNEL assay (200′ magnification, scale bar = 100 μm) after anti-caspase-14-treatment. f-l Transcripts in the colon samples were analyzed by qPCR. m Protein levels were confirmed by western blot
표 Δpep27 suppresses noncanonical Wnt and attenuates DIC. a, b Noncanonical Wnt expressions were analyzed by qPCR. c Blockade of Wnt5a and Wnt11 expressions using (ctrl, VEET) and NFAT inhibitor (VIVIT). d-f Disease activity index (DAI) was assessed by body weight loss (d), DAI score (e) and colon length measurement (f). g-m mRNA expression level was analyzed by qPCR and shown as mean ± SEM using GAPDH as an internal control
표 Δpep27-elicited-Treg restores mucosal immune response in DIC. DIC depletes Tregs and subsequently induces proinflammatory cytokines, noncanonical Wnt and caspase-14, resulting in inflammation and apoptosis as well as gut microbiota disruption. In contrast, Δpep27-dependent M2 macrophage and Treg induction elicits robust anti-inflammatory mucosal tolerance and maintains the barrier integrity for intestinal immune homeostasis and subsequent restoration of gut microbiota
표 Δpep27 relieves AR symptoms, and represses total and OVA-specific IgE in an OVA-induced AR model. (A) Schematic diagram of the OVA-induced AR experiment using Δpep27 immunization. (B) The frequency of nasal rubbing and sneezing after the final challenge was assessed by counting for 10 min. (C) The total IgE and OVA-specific IgE levels in serum were determined by ELISA. Three independent experiments were performed, and the data are presented as the mean ± SEM, * p < 0.05, ** p < 0.01, *** p < 0.001, ns; not significant. A representative of 3 independent experiments was analyzed with one-way ANOVA (Bonferroni’s Multiple Comparison Test)
표 Δpep27 immunization suppresses eosinophil infiltration into the nasal mucosa. (A) H scale bar= 20μm. (B) The number of eosinophils in the nasal mucosa. P-value was calculated by one-way ANOVA and expressed as mean ± SEM, * p < 0.05, ** p < 0.01, *** p < 0.001, ns; not significant (Bonferroni’s Multiple Comparison Test)
표 Δpep27 immunization inhibits Th2-dependent cytokines in the nasal mucosa. (A) Th2 cytokines in the nasal mucosa were determined by qPCR. (B) Production of Th2 cytokines in splenocyte supernatant was analyzed by ELISA. Values are presented as the mean ± SEMs (n= 3 per group). P-value was calculated by one-way ANOVA and expressed as mean ± SEM, * p < 0.05, ** p < 0.01, *** p < 0.001, ns; not significant (Tukey’s Multiple Comparison Test)
표 Δpep27 immunization represses the TLR pathway in the nasal mucosa. (A) mRNA and (B, C) protein levels were detected by qPCR and western blot , immunization represses the TLR pathway in the nasal mucosa. (A) mRNA and (B, C) protein levels were detected by qPCR and western blot analysis, respectively. Values are presented as the mean ± SEMs (n= 3 per group), * p < 0.05, ** p < 0.01, *** p < 0.001, ns; not significant (Tukey’s Multiple Comparison Test)
표 Δpep27 immunization inhibits NLRP3 inflammasome activation. (A) mRNA levels of NLRP3 and IL-1β in nasal mucosa were measured by qPCR. (B) Protein levels of NLRP3, caspase-1, cleaved caspase-1, p-IκB-α, and IL-1β in nasal mucosa were measured by Western blot (n= 3 per group). P-value was calculated by one-way ANOVA and expressed as mean ± SEM *P < 0.05, **P < 0.01 and ***P < 0.001, N.S; not significant (Tukey’s Multiple Comparison Test)
표 Δpep27 immunization negatively regulates NLRP3 inflammasome activation. NF-kB is translocated to the nucleus of immune cells through activation of TLR during AR and then activates transcription of NLRP3. Immunization with Δpep27 in the nasal mucosa reduces the Th2 response
표 Δpep27 immunization reduces ear inflammation in AD model. (a) Mice were immunized intranasally (i.n.) with Δpep27 three times and seven days after the last immunization, mice were sensitized with 1 % and 0.2 % OXA to the inner and outer surface of both ears. (b) The ear of the mouse was photographed after 24 h of the last sensitization. (c) Mice ear thickness was measured with micrometre calliper (n=10). (d, e) Histological analysis of epidermal and dermal indices and infiltration of the ear cells by H normal = 0, 2 times greater than normal = 1, 3 times greater than normal = 2, 3 times more than normal = 3) (n=2). Microscopic photographs of H&E staining for inflammation were expressed as x 200 magnification
표 Δpep27 immunization mitigates the Th2 response and increases Treg activity in AD. Twenty-four hours after the last oxazolone challenge, serum and ear tissues were collected. (a) The mRNA levels of Th2 and Treg related markers on mouse ear tissues were measured by qPCR (n=6). (b) The cytokines IL-4, IL-5, and IL-10 level in serum were measured by ELISA (n=5). (c) The tota lIgE level in serum were measured by ELISA (n=5). Each value is expressed as the average ± standard error of the mean (SEM). Statistical comparison among groups was performed using one-way ANOVA and Tukey’s test (*P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001, ns, not significant)
표 Δpep27 immunization decreases apoptotic cell death in preventive AD model in mouse ear tissue. (a, c) TUNEL assay on dermis samples and the percentage of the plot are shown. Photomicrographs for 2 mice/group of ear tissue degree of apoptosis. (b, d) Caspase-3 level in ear skin was measured by Immunohistochemistry. IHC analysis shows Caspase-3 protein expressions in the mouse ear skins of each group (n = 2). (e) Bax and Bcl2 mRNA levels on mouse ear tissues were measured by qPCR (n = 3). Data are expressed as mean ± standard error of mean (SEM). Statistical comparison among groups was performed using one-way ANOVA and Tukey’s test (*P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001, ns, not significant)
표 Δpep27 immunization represses Th2 cytokines, and TSLP, and induced TGF-β and IL-10 in therapeutic AD model. The mice were immunized three times with Δpep27 during oxazolone-induced AD skin lesion. (a) The mRNA expression levels on mouse ear tissues were analyzed by RT-PCR (n=3). (b) The total serum IL-4, IL-5 and IL-17A measured by ELISA (n=3). (c) Serum IgE level measured by ELISA (n=3). Each value is expressed as mean ± standard error of mean (SEM). Statistical comparison among groups was performed using one-way ANOVA and Tukey’s test (*P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001, ns, not significant)
표 Δpep27 immunization alleviates AD symptoms through the regulation of TSLP and Tregs. (a) After Δpep27 immunization as in Fig. 1a, Treg inactivation or TSLP neutralization was performed by intraperitoneal injection of either CD25 or TSLP antibody. (b) Photos of mouse ears 24 hours after the last OXA challenge. (c) Mice ear thickness was measured with micrometre calliper on the days shown. The right panel is the ear thickness at day 15 (n=10). (d) Analysis of epidermal and dermal section after H & E staining. The top pictures were taken at x 200 and the bottom pictures at x 400 magnification (n=3). (e) Number of eosinophils in the dermal layer and epidermal thickness (n=9) were measured. (f) TUNEL assay results were converted into relative percentage of the control. Each value is expressed as the average ± standard error of the mean (SEM). Statistical comparison among groups was performed using one-way ANOVA and Tukey’s test (*P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001, ns, not significant)
표 Δpep27 immunization inhibits the production of TSLP, which increases the activity of Tregs, thereby reducing AD symptoms. (a) TSLP mRNA expression in mouse ear tissue and serum TSLP measurement by ELISA (n=6). (b) The mRNA levels of representative markers of Th2 and Treg were measured by RT-PCR (n=6). (c) The cytokines IL-4, IL-5, IL-13, IL-1β and IL-10 level in serum were analyzed by multiplex cytokine assay (n=5). (d) The total IgE level in serum were measured by ELISA (n=5). Each value is expressed as the average ± standard error of the mean (SEM). Statistical comparison among groups was performed using one-way ANOVA and Tukey’s test (*P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001, ns, not significant)
표 Δpep27 immunization induces only Tregs differentiation and does not induce any pathological changes. (a, d) mRNA expression levels of TSLP, Treg (Foxp3, IL-10), and Th2 cell specific makers (Gata-3, and IL-4) were analyzed by qPCR in mice ear after antibody treatment (n=5). (b) Graph of the number of eosinophils found in the dermal layer (n=5). (c) The percentage of TUNEL-positive cells from 4 random field from each mouse ear section (n=4). (e) The IL-10 and IL-4 cytokine levels in serum were measured by ELISA (n=5). Data are expressed as mean ± standard error of mean (SEM). Statistical comparison among groups was performed using one-way ANOVA and Tukey’s test (*P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001)
표 Δpep27 immunization restored Th2/Treg balance on preventive and therapeutic model. (a) mRNA expression levels of Th cell specific makers (Gata-3, T-bet, Foxp3 and RORγt) were analyzed by qPCR in therapeutic model (n=3). (b) Th1(T-bet and TNF-α) and Th17-related markers (RORγt and IL-17) were analyzed by qPCR in preventive model (n=5). Data are expressed as mean ± standard error of mean (SEM). Statistical comparison among groups was performed using one-way ANOVA and Tukey’s test (*P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001)
표 Δpep27 immunization restored Th2/Treg balance on preventive and therapeutic model

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폐렴구균 pep27 변이주 접종에 의한 염증/패혈증으로부터 점막보호 기전 연구
Elucidation of mucosa protection from inflammation/sepsis by the pneumococcal pep27 mutant immunization 원문보기

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폐렴구균 pep27 변이주 접종에 의한 염증/패혈증으로부터 점막보호 기전 연구 Elucidation of mucosa protection from inflammation/sepsis by the pneumococcal pep27 mutant immunization 원문보기

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폐렴구균 pep27 변이주 접종에 의한 염증/패혈증으로부터 점막보호 기전 연구
Elucidation of mucosa protection from inflammation/sepsis by the pneumococcal pep27 mutant immunization 원문보기

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