Degree Of Neutrophil Infiltration Research Articles

Transcriptomics Reveals Effect of Pulsatilla Decoction Butanol Extract in Alleviating Vulvovaginal Candidiasis by Inhibiting Neutrophil Chemotaxis and Activation via TLR4 Signaling.

The Pulsatilla decoction is a well-known herbal remedy used in clinical settings for treating vulvovaginal candidiasis (VVC). However, the specific mechanism that makes it effective is still unclear. Recent studies have shown that in cases of VVC, neutrophils recruited to the vagina, influenced by heparan sulfate (HS), do not successfully engulf Candida albicans (C. albicans). Instead, they release many inflammatory factors that cause damage to the vaginal mucosa. This study aims to understand the molecular mechanism by which the n-butanol extract of Pulsatilla decoction (BEPD) treats VVC through transcriptomics. High-performance liquid chromatography was used to identify the primary active components of BEPD. A VVC mouse model was induced using an estrogen-dependent method and the mice were treated daily with BEPD (20 mg/kg, 40 mg/kg, and 80 mg/kg) for seven days. The vaginal lavage fluid of the mice was analyzed for various experimental indices, including fungal morphology, fungal burden, degree of neutrophil infiltration, and cytokines. Various assessments were then performed on mouse vaginal tissues, including pathological assessment, immunohistochemistry, immunofluorescence, Western blot (WB), quantitative real-time PCR, and transcriptome assays. Our results showed that BEPD reduced vaginal redness and swelling, decreased white discharge, inhibited C. albicans hyphae formation, reduced neutrophil infiltration and fungal burden, and attenuated vaginal tissue damage compared with the VVC model group. The high-dose BEPD group even restored the damaged vaginal tissue to normal levels. The medium- and high-dose groups of BEPD also significantly reduced the levels of IL-1β, IL-6, TNF-α, and LDH. Additionally, transcriptomic results showed that BEPD regulated several chemokine (CXCL1, CXCL3, and CXCL5) and S100 alarmin (S100A8 and S100A9) genes, suggesting that BEPD may treat VVC by affecting chemokine- and alarmin-mediated neutrophil chemotaxis. Finally, we verified that BEPD protects the vaginal mucosa of VVC mice by inhibiting neutrophil recruitment and chemotaxis in an animal model of VVC via the TLR4/MyD88/NF-κB pathway. This study provides further evidence to elucidate the mechanism of BEPD treatment of VVC.

Bronchial epithelial transcriptomics and experimental validation reveal asthma severity-related neutrophilc signatures and potential treatments

Airway epithelial transcriptome analysis of asthma patients with different severity was used to disentangle the immune infiltration mechanisms affecting asthma exacerbation, which may be advantageous to asthma treatment. Here we introduce various bioinformatics methods and develop two models: an OVA/CFA-induced neutrophil asthma mouse model and an LPS-induced human bronchial epithelial cell damage model. Our objective is to investigate the molecular mechanisms, potential targets, and therapeutic strategies associated with asthma severity. Multiple bioinformatics methods identify meaningful differences in the degree of neutrophil infiltration in asthma patients with different severity. Then, PTPRC, TLR2, MMP9, FCGR3B, TYROBP, CXCR1, S100A12, FPR1, CCR1 and CXCR2 are identified as the hub genes. Furthermore, the mRNA expression of 10 hub genes is determined in vivo and in vitro models. Reperixin is identified as a pivotal drug targeting CXCR1, CXCR2 and MMP9. We further test the potential efficiency of Reperixin in 16HBE cells, and conclude that Reperixin can attenuate LPS-induced cellular damage and inhibit the expression of them. In this study, we successfully identify and validate several neutrophilic signatures and targets associated with asthma severity. Notably, Reperixin displays the ability to target CXCR1, CXCR2, and MMP9, suggesting its potential therapeutic value for managing deteriorating asthma.

The inflammatory microenvironment of nasal polyps in patients with chronic rhinosinusitis and the relationship of this microenvironment with the nasal microbiome

BackgroundWe investigated the characteristics of the microbial community of the nasal sinuses in patients with chronic rhinosinusitis with nasal polyps (CRSwNP) and identified the correlations of the nasal microbiome with the inflammatory microenvironment of the nasal cavity. MethodologyWe collected matched nasal secretion and polyp tissue samples from 77 CRSwNP patients. Then, we extracted microbial DNA from cotton swabs, used high-throughput sequencing technology based on 16S ribosomal RNA (rRNA) to detect the bacterial community composition, and detected cytokines such as interleukin (IL)-5, IL-8, IL-17a, IL-17e, IL-18, IL-27 and interferon (INF)-gamma in the polyp tissue samples using Luminex. Eosinophils and neutrophils in the peripheral blood and polyp tissue were counted, and the relationships between inflammatory factors or inflammatory cell counts and nasal microbial diversity were analyzed. ResultsAmong the inflammatory factors evaluated, IL-5 had a positive rate of 32.47%, IFN-γ had a positive rate of 84.42%, IL-17A and IL-17E had positive rates of 75.32%, IL-18 had a positive rate of 94.81%, IL-27 had a positive rate of 68.83%, and IL-8 had a positive rate of 100%. IL-17a and IL-27 were negatively correlated with both Enterobacter and Anaerococcus, IL-8 was negatively correlated with both Enterobacter and Staphylococcus, IL-18 was positively correlated with Candidatus Arthromitus and negatively correlated with Haemophilus, and IL-27 was positively correlated with Faecalibaculum. Lactobacillus and Enterococcus were positively correlated with the degree of neutrophil infiltration in nasal polyp tissue. ConclusionsIn Southwest China, inflammation of the nasal polyps exhibits a variety of patterns. Enterobacteria and anaerobic bacteria may be correlated with the inflammatory pattern of nasal polyps. The neutrophil-mediated inflammatory response plays an important role in patients with CRSwNP in Southwest China.

Mechanism of n-butanol alcohol extract of Baitouweng Decoction in treatment of vulvovaginal candidiasis based on negative regulation of NLRP3 inflammasome via PKCδ/NLRC4/IL-1Ra axis

This study aimed to explore the mechanism of n-butanol alcohol extract of Baitouweng Decoction(BAEB) in the treatment of vulvovaginal candidiasis(VVC) in mice based on the negative regulation of NLRP3 inflammasome via PKCδ/NLRC4/IL-1Ra axis. In the experiment, female C57BL/6 mice were divided randomly into the following six groups: a blank control group, a VVC model group, high-, medium-, and low-dose BAEB groups(80, 40, and 20 mg·kg~(-1)), and a fluconazole group(20 mg·kg~(-1)). The VVC model was induced in mice except for those in the blank control group by the estrogen dependence method. After modeling, no treatment was carried out in the blank control group. The mice in the high-, medium-, and low-dose BAEB groups were treated with BAEB at 80, 40, and 20 mg·kg~(-1), respectively, and those in the fluconazole group were treated with fluconazole at 20 mg·kg~(-1). The mice in the VVC model group received the same volume of normal saline. The general state and body weight of mice in each group were observed every day, and the morphological changes of Candida albicans in the vaginal lavage of mice were examined by Gram staining. The fungal load in the vaginal lavage of mice was detected by microdilution assay. After the mice were killed, the degree of neutrophil infiltration in the vaginal lavage was detected by Papanicolaou staining. The content of inflammatory cytokines interleukin(IL)-1β, IL-18, and lactate dehydrogenase(LDH) in the vaginal lavage was tested by enzyme-linked immunosorbent assay(ELISA), and vaginal histopathology was analyzed by hematoxylin-eosin(HE) staining. The expression and distribution of NLRP3, PKCδ, pNLRC4, and IL-1Ra in vaginal tissues were measured by immunohistochemistry(IHC), and the expression and distribution of pNLRC4 and IL-1Ra in vaginal tissues were detected by immunofluorescence(IF). The protein expression of NLRP3, PKCδ, pNLRC4, and IL-1Ra was detected by Western blot(WB), and the mRNA expression of NLRP3, PKCδ, pNLRC4, and IL-1Ra was detected by qRT-PCR. The results showed that compared with the blank control group, the VVC model group showed redness, edema, and white secretions in the vagina. Compared with the VVC model group, the BAEB groups showed improved general state of VVC mice. As revealed by Gram staining, Papanicolaou staining, microdilution assay, and HE staining, compared with the blank control group, the VVC model group showed a large number of hyphae, neutrophils infiltration, and increased fungal load in the vaginal lavage, destroyed vaginal mucosa, and infiltration of a large number of inflammatory cells. BAEB could reduce the transformation of C. albicans from yeast to hyphae. High-dose BAEB could significantly reduce neutrophil infiltration and fungal load. Low-and medium-dose BAEB could reduce the da-mage to the vaginal tissue, while high-dose BAEB could restore the damaged vaginal tissues to normal levels. ELISA results showed that the content of inflammatory cytokines IL-1β, IL-18, and LDH in the VVC model group significantly increased compared with that in the blank control group, and the content of IL-1β, IL-18 and LDH in the medium-and high-dose BAEB groups was significantly reduced compared with that in the VVC model group. WB and qRT-PCR results showed that compared with the blank control group, the VVC model group showed reduced protein and mRNA expression of PKCδ, pNLRC4, and IL-1Ra in vaginal tissues of mice and increased protein and mRNA expression of NLRP3. Compared with the VVC model group, the medium-and high-dose BAEB groups showed up-regulated protein and mRNA expression of PKCδ, pNLRC4, and IL-1Ra in vaginal tissues and inhibited protein and mRNA expression of NLRP3 in vaginal tissues. This study indicated that the therapeutic effect of BAEB on VVC mice was presumably related to the negative regulation of NLRP3 inflammasome by promoting PKCδ/NLRC4/IL-1Ra axis.

Identifying neutrophil-associated subtypes in ulcerative colitis and confirming neutrophils promote colitis-associated colorectal cancer.

Ulcerative colitis (UC) is a chronic inflammatory disease of the intestinal mucosa, the incidence of which has increased worldwide. There is still a lack of clear understanding of the pathogenesis of ulcerative colitis that ultimately leads to colitis-associated colorectal cancer. We download UC transcriptome data from the GEO database and pass the limma package in order to identify differentially expressed genes. Gene Set Enrichment Analysis (GSEA) was used to identify potential biological pathways. We identified immune cells associated with UC by CIBERSORT and Weighted co-expression network analysis (WGCNA). We used validation cohorts and mouse models to verify the expression of the hub genes and the role of neutrophils. We identified 65 differentially expressed genes in UC samples and healthy controls. GSEA, KEGG, and GO analyses displayed that DEGs were enriched in immune-related pathways. CIBERSORT analysis revealed increased infiltration of neutrophils in UC tissues. The red module, obtained by WGCNA analysis, was considered to be the most relevant module for neutrophils.Based on neutrophil-associated differentially expressed genes, UC patients were classified into two subtypes of neutrophil infiltration. We discovered that the highly neutrophil-infiltrated subtype B of UC patients had a higher risk of developing CAC. Five genes were identified as biomarkers by searching for DEGs between distinct subtypes. Finally, using the mouse model, we determined the expression of these five genes in the control, DSS, and AOM/DSS groups. The degree of neutrophil infiltration in mice and the percentage of MPO and pSTAT3 expression in neutrophils were analyzed by flow cytometry. In the AOM/DSS model, MPO and pSTAT3 expressions were significantly increased. These findings suggested neutrophils might promote the conversion of UC into CAC. These findings improve our understanding of the pathogenesis of CAC and provide new and more effective insights into the prevention and treatment of CAC.

Acute and chronic inflammation alter immunometabolism in a cutaneous delayed-type hypersensitivity reaction (DTHR) mouse model

T-cell-driven immune responses are responsible for several autoimmune disorders, such as psoriasis vulgaris and rheumatoid arthritis. Identification of metabolic signatures in inflamed tissues is needed to facilitate novel and individualised therapeutic developments. Here we show the temporal metabolic dynamics of T-cell-driven inflammation characterised by nuclear magnetic resonance spectroscopy-based metabolomics, histopathology and immunohistochemistry in acute and chronic cutaneous delayed-type hypersensitivity reaction (DTHR). During acute DTHR, an increase in glutathione and glutathione disulfide is consistent with the ear swelling response and degree of neutrophilic infiltration, while taurine and ascorbate dominate the chronic phase, suggesting a switch in redox metabolism. Lowered amino acids, an increase in cell membrane repair-related metabolites and infiltration of T cells and macrophages further characterise chronic DTHR. Acute and chronic cutaneous DTHR can be distinguished by characteristic metabolic patterns associated with individual inflammatory pathways providing knowledge that will aid target discovery of specialised therapeutics.

The Relationship of MMP-3 Levels with the Degree of Helicobacter Pylori Gastritis

Gastritis is an inflammation in the stomach tissue that can occur either acutely or gradually as a chronic condition. Incidence of gastritis in Indonesia is high, which is 274,396 cases out of 238,452,952 inhabitants. One of the most common causes of gastritis is Helicobacter pylori (H. pylori). Matrix metalloproteinase (MMP) is a dependent proteinase that is able to break down almost all extracellular matrix proteins. It is known from several studies that MMP is closely related to gastric ulcer disease and cancer. At present, in vivo and in vitro studies have established the activation of several MMPs associated with H. Pylori infection. There were 80 samples obtained through consecutive sampling in March-June 2019. Gastritis was ensured by endoscopy (Olympus, Tokyo, Japan). H.pylori is established through a change in color from yellow to red, magenta, pink, or orange in the examination of Campylobacter Like Organism test (CLO). MMP-3 level examination using a serum MMP3 was measured using RayBio Human MMP-3 and MICA ELISA kits (Norcross, GA). Data were collected and analyzed using SPSS version 22. There was a significant relationship between MMP-3 levels and the severity of H. pylori gastritis, namely between the degree of neutrophil infiltration (p = 0.016), atrophy (p = 0.001), and intestinal metaplasia (p <0.001). Keywords: Gastritis, Helicobacter Pylori, MMP-3.

A prognostic model of non small cell lung cancer based on TCGA and ImmPort databases

Bioinformatics methods are used to construct an immune gene prognosis assessment model for patients with non-small cell lung cancer (NSCLC), and to screen biomarkers that affect the occurrence and prognosis of NSCLC. The transcriptomic data and clinicopathological data of NSCLC and cancer-adjacent normal tissues were downloaded from the Cancer Genome Atlas (TCGA) database and the immune-related genes were obtained from the IMMPORT database (http://www.immport.org/); then, the differentially expressed immune genes were screened out. Based on these genes, an immune gene prognosis model was constructed. The Cox proportional hazards regression model was used for univariate and multivariate analyses. Further, the correlations among the risk score, clinicopathological characteristics, tumor microenvironment, and the prognosis of NSCLC were analyzed. A total of 193 differentially expressed immune genes related to NSCLC were screened based on the "wilcox.test" in R language, and Cox single factor analysis showed that 19 differentially expressed immune genes were associated with the prognosis of NSCLC (P < 0.05). After including 19 differentially expressed immune genes with P < 0.05 into the Cox multivariate analysis, an immune gene prognosis model of NSCLC was constructed (it included 13 differentially expressed immune genes). Based on the risk score, the samples were divided into the high-risk and low-risk groups. The Kaplan–Meier survival curve results showed that the 5-year overall survival rate in the high-risk group was 32.4%, and the 5-year overall survival rate in the low-risk group was 53.7%. The receiver operating characteristic model curve confirmed that the prediction model had a certain accuracy (AUC = 0.673). After incorporating multiple variables into the Cox regression analysis, the results showed that the immune gene prognostic risk score was an independent predictor of the prognosis of NSCLC patients. There was a certain correlation between the risk score and degree of neutrophil infiltration in the tumor microenvironment. The NSCLC immune gene prognosis assessment model was constructed based on bioinformatics methods, and it can be used to calculate the prognostic risk score of NSCLC patients. Further, this model is expected to provide help for clinical judgment of the prognosis of NSCLC patients.

Protective Effect of Xiao-Xu-Ming Decoction-Mediated Inhibition of ROS/NLRP3 Axis on Lipopolysaccharide-Induced Acute Lung Injury In Vitro and In Vivo.

Background As a traditional Chinese medicine prescription, Xiao-Xu-Ming decoction (XXMD) could reduce the incidence of lung infection of patients with cerebral infarction. Nonetheless, the therapeutic mechanisms of XXMD in acute lung injury (ALI) remain to be elucidated. Our study was aimed to assess the effects of XXMD protects against ALI. Methods ALI model was induced by intraperitoneal injection of lipopolysaccharide (LPS) in vivo. In vitro, human pulmonary alveolar epithelial cells (HPAEpiC) were treated with XXMD and were followed by LPS treatment. The levels of ZO-1, CLDN4, NLRP3, and caspase 1 were detected by Western blot, and the content of IL-1 and IL-18 was determined by ELISA. Transepithelial electrical resistance was used to detect the cell permeability. The reactive oxygen species (ROS) levels within the cells were evaluated by flow cytometry. Results Our results showed that XXMD attenuated LPS-induced oxidative stress, barrier dysfunction, and the activation of NLRP3 inflammasome in vitro, as evidenced by enhanced ROS production, TEER levels, expression of NLRP3 and caspase 1 (p20) and release of IL-1β and IL-18, and weakened cell permeability. In addition, XXMD could counteract the effects of NLRP3 overexpression on HPAEpiC and vice versa. XXMD treatment also ameliorated the degree of neutrophil infiltration, barrier dysfunction, and the activation of NLRP3 in LPS-induced ALI lung tissues in vivo. Conclusion The findings showed that XXMD could alleviate LPS-induced ALI injury and inhibit inflammation and suppress ROS/NLRP3 signaling pathway, which were involved in these protective effects.

Algal Oil Rich in n-3 PUFA Alleviates DSS-Induced Colitis via Regulation of Gut Microbiota and Restoration of Intestinal Barrier.

Algal oil is rich in docosahexaenoic acid (DHA) and has various health benefits against human metabolic disorders and disease. This study aimed to investigate the effects of DHA algal oil on colonic inflammation and intestinal microbiota in dextran sulfate sodium (DSS)-induced colitis mice model. Male C57BL/6 mice was induced colitis by 2.5% DSS and followed by 2 weeks of treatment with algal oil (250 or 500 mg/kg/day). The colonic inflammation was assessed by colon macroscopic damage scores, and the degree of neutrophil infiltration was evaluated by measuring tissue-associated myeloperoxidase (MPO) activity in colonic mucosa. Tight junction proteins in the colonic tissue were measured by real-time PCR and western blot. Moreover, the intestinal microbiota and shot chain fatty acids (SCFAs) were estimated by bioinformatic analysis and GC, respectively. Colonic damage due to DSS treatment was significantly ameliorated by algal oil supplementation. In addition, algal oil significantly inhibited the increases of malondialdehyde (MDA) content, MPO activity, pro-inflammatory cytokines level and tight junction proteins expression in DSS-treated mice. Furthermore, supplementation of algal oil modulated the intestinal microbiota structure in DSS induced colitis mice by increasing the proportion of the unidentified_S24_7 and decreasing the relative abundance of unidentified_Ruminococcaceae, Clostridium and Roseburia. On the analysis of SCFAs, the caecal content of acetic acid, propionic acid, isobutyric acid, buturic, and the total SCFAs showed a significant increase in algal oil-administered mice. Together, these results suggested that algal oil rich in DHA inhibited the progress of DSS-induced colitis in mice by modulating the intestinal microbiota and metabolites and repairing the intestinal barrier, which may be applied in the development of therapeutics for intestinal inflammation.

Abstract 5553: The effect of PLAG on the PD-L1 immune-checkpoint drug therapy in the MB49 bladder cancer syngeneic model

Abstract Background: The development of immune-checkpoint drugs, a fourth-generation anticancer drug, is a significant breakthrough in anticancer research. Cancer therapy using immune-checkpoint drugs are still enlarged. To improve the efficacy of immune-checkpoint drugs, modulation of the tumor microenvironment is essentially required. In this experiment, we examined the elevated anti-cancer efficacy of 1-Palmitoyl-2-Linoleoyl-3-Acetyl-rac-Glycerol (PLAG), which has been demonstrated to attenuate tumor infiltrated neutrophils (TINs) in the tumor, along with the PD-L1 immune checkpoint inhibitor treatment. Methods: The syngeneic model was used (n=6) to investigate the enhanced anti-tumor effect of PD-L1 antibody with the addition of PLAG. MB49 murine bladder cancer cells were implanted into the C57BL/6 mice subcutaneously and bred for 5 weeks. After a week from tumor implantation, PLAG at different dosages (25/50/100 mpk) were daily administered orally for another 4 weeks with or without 5 mpk PD-L1 antibody (10F.9G2). PD-L1 antibody was delivered via IP injection twice a week. Tumor growth was calculated in 3-day intervals. Neutrophil chemotaxis-related chemokines and neutrophil-to-lymphocyte rate (NLR) were also evaluated with a 2-week interval. The degree of neutrophil infiltration in the tumor on the day of sacrifice and the degree of infiltration between lymphocytes and neutrophils were analyzed. Results: The PLAG treatment groups demonstrated that the tumor burden decreased in a concentration-dependent manner. In 50 and 100 mpk of PLAG treated mice, the tumor burden was decreased to a significant value compared to a positive control (p &amp;lt; 0.05). In the group treated with PD-L1 antibody alone, the growth rate of the tumor decreased until about 2 weeks. In the group treated simultaneously with PLAG and PD-L1 antibody, the growth of the tumor was significantly reduced compared to the group treated with PD-L1 antibody alone. As a result of calculating neutrophils and lymphocytes every two weeks, NLR level in the group treated with PLAG and PD-L1 was significantly decreased compared to that treated with PD-L1 antibody alone. Besides, the number of TINs were effectively reduced by PLAG treatment alone. The interruption of tumor growth and reduced chemokine secretion were also observed in the PLAG treated groups. In summary, our data suggest that PLAG provides an enhanced PD-L1 antibody effect on the regression of tumor burden in the syngeneic mice model via reducing the number of TINs. Conclusion: PLAG may be utilized for improving the efficacy of PD-L1 antibody on reducing the tumor burden at the devastating tumor microenvironment. Citation Format: Guen Tae Kim, Sun Young Yoon, Ji Sun Park, Ki-Young Sohn, Jae Wha Kim. The effect of PLAG on the PD-L1 immune-checkpoint drug therapy in the MB49 bladder cancer syngeneic model [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5553.

Reduced water quality associated with higher stocking density disturbs the intestinal barrier functions of Atlantic salmon (Salmo salar L.)

The stocking density of fish in aquaculture is of major importance as it may have profound effects on water quality resulting in impact on fish health and possibly affect the external barriers that protect against pathogens There are many husbandry conditions, including stocking density, that may affect the primary protective barriers, i.e. the skin, intestine and gills, against invading pathogens and other harmful substances. It is well known that increased fish density will lead to decreased dissolved oxygen (DO) levels and affect other water quality parameters such as carbon dioxide, pH and ammonia. It is not known if such changes in the rearing environment affect the intestinal primary barriers of Atlantic salmon. Groups of Atlantic salmon post-smolts were kept for 57 days in tanks supplied with seawater at a constant flow at stocking densities of 10, 30, 50 and 70 kg m−3; reduced water quality was associated with higher stocking density. Repeated sampling for plasma cortisol and water cortisol release rate indicate that the highest stocking density elicited a primary stress response in the fish, which decreased with time. The physical intestinal barrier was assessed using paracellular permeability measurements, i.e. transepithelial electrical resistance (TER) and diffusion rate of 14C-mannitol, in combination with the translocation rate of heat-inactivated Aeromonas salmonicida. The physical intestinal barrier decreased with increasing density, both when measured as decreased transepithelial electrical resistance and as elevated paracellular permeability for 14C-mannitol. As this was observed at a time point when no differences could be seen in plasma cortisol or cortisol release rate, it suggests that intestinal paracellular permeability can be a useful marker for chronic stress in salmon. The status of the intestinal immune system was assessed as degree of neutrophil infiltration as well as the mRNA expression of the pro-inflammatory cytokines, interferon γ (IFNγ), interleukin (IL) 1β and tumor necrosis factor (TNF) α, anti-inflammatory cytokines, IL-10 and transforming growth factor β (TGFβ) and other immune related-genes, IL-8 and the inhibitor of the transcription factor nuclear factor κB (IκB). The intestinal immune system was affected at the highest stocking density as observed by a decreased expression of IFNγ in parallel with higher infiltration of neutrophils. In conclusion, high stocking density associated with reduced water quality is chronically stressful to the fish as it elicits a primary stress response as well as a weakened physical and disturbed immunological primary barrier.

鞘氨醇1-磷酸盐受体拮抗剂（FTY720）抑制皮肤中分泌白细胞介素17的γδT细胞治疗银屑病的机制研究

Objective To evaluate therapeutic effect of the sphingosine-1-phosphate receptor antagonist FTY720 on the imiquimod-induced psoriasis-like mouse model, and to explore its molecular mechanism. Methods A total of 8 specific pathogen-free (SPF) female C57BL/6 mice were used in this study, and divided into experimental group (n=3) and control group (n=5) : each mouse ear was topically treated with 10 mg/d imiquimod for 7 consecutive days, and the experimental group and control group were intraperitoneally injected with FTY720 and phosphate buffer saline (PBS) respectively on days 2, 4 and 6. Ear skin thickness was measured every day. These mice were sacrificed on day 8, and histopathological changes of the ear skin were observed. The ear tissues and draining cervical lymph node cells were obtained. Flow cytometry was performed to analyze changes in the proportion of interleukin (IL) -17+ γδT cells in the mouse skin and draining lymph node tissues between the two above groups, and to determine the expression of sphingosine-1-phosphate receptor 1 (S1P1) and cutaneous lymphocyte antigen (CLA) on the surface of the γδT cells. The means of two independent samples were compared by using t test. Results After the 2-day topical application of imiquimod, the mouse ear thickness was significantly lower in the experimental group (0.217 mm ± 0.003 mm) than in the control group (0.232 mm ± 0.002 mm, t=4.23, P < 0.01) on day 3, and the significant difference existed till day 8 (all P < 0.01) . Histopathological examination of the ear skin revealed that the epidermal thickness was significantly lower in the experimental group (18.62 μm ± 0.19 μm) than in the control group (27.79 μm ± 1.58 μm, t=4.35, P < 0.01) . Immunofluorescence staining showed that the degree of neutrophil infiltration in the mouse ear tissue was lower in the experimental group than in the control group. As flow cytometry showed, the proportion of neutrophils was significantly lower in the experimental group (1.57% ± 0.12%) than in the control group (3.03% ± 0.33%, t=3.31, P=0.016) . In the ear tissues, the experimental group showed significantly decreased proportion of γδT cells or IL-17+ γδT cells (4.88% ± 0.42%, 40.53% ± 1.76% respectively) compared with the control group (9.45% ± 1.22%, 56.56% ± 0.66% respectively; t=2.75, 10.27 respectively, both P < 0.05) . In the draining lymph nodes, the experimental group showed significantly increased proportion of γδT cells or IL-17+ γδT cells compared with the control group (t=5.781, 4.140 respectively, both P < 0.05) , and the fluorescence intensity of S1P1 and CLA in the γδT cells was significantly lower in the experimental group than in the control group (P < 0.05) . Conclusion FTY720 can alleviate imiquimod-induced psoriasis-like manifestations in mouse models, likely by down-regulating the expression of S1P1 and CLA in γδT cells, increasing the proportion of γδT cells in the draining lymph nodes followed by the decrease of their proportion in the skin, and decreasing the production of IL-17 in skin tissues. Key words: Psoriasis; Mice, inbred C57BL; Receptors, antigen, T-cell, gamma-delta; Interleukin-17; Sphingosine-1-phosphate receptors

Attenuation of intestinal inflammation of polysaccharides from the seeds ofPlantago asiaticaL. as affected by ultrasonication

Ultrasound treatment was applied to modify the physicochemical properties of polysaccharides from the seeds of Plantago asiatica L. The initial polysaccharides concentration of 10 mg/mL, ultrasonic power of 800 W, and different treatment time (15 s, 30 s, 1 min, 2 min) were used. After irradiation, the average molecular weight (Mw) and polydispersity index of relative molecular mass (PID) of polysaccharides decreased. The apparent viscosity of degraded polysaccharides dropped compared with native polysaccharides. By using dextran sodium sulphate (DSS) colitis mouse model, enhanced intestinal anti‐inflammatory activity of degraded polysaccharides was proved by a significant reduction in the degree of neutrophil infiltration, indicated by decreased macrophage inflammatory protein‐2 secretion, and levels of inflammatory cytokines (TNF‐α and IL‐4) in inflamed colon. These results suggested that ultrasound treatment is a viable modification technology for high Mw polymer materials with high anti‐inflammatory activity. PRACTICAL APPLICATIONS: Ulcerative colitis (UC) is a chronic and nonspecific inflammatory disorder of the colonic mucosa, and assumed to be an autoimmune disease. PLCP was found to be one of the active ingredients responsible for many of the observed bioactivities including anti‐inflammation, anticomplement and facilitating defecation. However, it's extremely complex structure with high molecular weights and high viscosity hampered its applications. In this study, ultrasound irradiation was used to depolymerize PLCP, and its attenuation of intestinal inflammation was also studied.

IL-33/ST2 plays a critical role in endothelial cell activation and microglia-mediated neuroinflammation modulation

BackgroundInterleukin-33 (IL-33) is increasingly being recognized as a key immunomodulatory cytokine in many neurological diseases.MethodsIn the present study, wild-type (WT) and IL-33−/− mice received intracerebroventricular (i.c.v.) injection of lipopolysaccharide (LPS) to induce neuroinflammation. Intravital microscopy was employed to examine leukocyte–endothelial interactions in the brain vasculature. The degree of neutrophil infiltration was determined by myeloperoxidase (MPO) staining. Real-time PCR and western blotting were used to detect endothelial activation. Enzyme-linked immunosorbent assay and quantitative PCR were conducted to detect pro-inflammatory cytokine levels in the brain.ResultsIn IL-33−/− mice, neutrophil infiltration in the brain cortex and leukocyte–endothelial cell interactions in the cerebral microvessels were significantly decreased as compared to WT mice after LPS injection. In addition, IL-33−/− mice showed reduced activation of microglia and cerebral endothelial cells. In vitro results indicated that IL-33 directly activated cerebral endothelial cells and promoted pro-inflammatory cytokine production in LPS-stimulated microglia.ConclusionsOur study indicated that IL-33/ST2 signaling plays an important role in the activation of microglia and cerebral endothelial cells and, therefore, is essential in leukocyte recruitment in brain inflammation.Graphical abstractThe role of IL-33/ST2 in LPS induced neuroinflammation

Analysis of bronchial biopsies in chronic cough

BackgroundChronic cough is commonly associated with asthma, gastro-oesophageal reflux disease and postnasal drip, but in a significant proportion, no associated cause can be found. We determined whether examination of bronchial biopsies would be useful in determining the cause associated with chronic cough. Methods100 consecutive patients referred to a specialist cough clinic underwent a systematic assessment including a fiberoptic bronchoscopy for bronchial biopsies. ResultsIn 38 patients, treatment of associated causes led to amelioration of cough (‘explained’) and in 62, there was no association or improvement (‘idiopathic’). The latter group had a longer duration of cough, a lower FeNO levels and a more sensitive capsaicin cough response, with an increase in basement membrane thickness with no differences in goblet cell hyperplasia and seromucinous hyperplasia, and in lymphocyte, neutrophil and eosinophil counts. The duration of cough was inversely correlated with the degree of neutrophil infiltration. ConclusionWe conclude that pathological examination of bronchial biopsies is unlikely to be useful in the diagnosis of chronic cough in non-smokers.

Complement component C3a plays a critical role in endothelial activation and leukocyte recruitment into the brain.

BackgroundThe complement system is becoming increasingly recognized as a key participant in many neurodegenerative diseases of the brain. Complement-deficient animals exhibit reduced neuroinflammation.MethodsIn the present study, we administered intracerebroventricularly lipopolysaccharide (LPS) to mimic local infection of the brain and investigated the role of key complement component C3 in brain vasculature endothelial activation and leukocyte recruitment. The degree of neutrophil infiltration was determined by esterase staining. Leukocyte-endothelial interactions were measured using intravital microscopy. Cerebral endothelial activation was evaluated using real-time PCR and Western blotting.ResultsNeutrophil infiltration into the brain cortex and hippocampus was significantly reduced in C3−/− mice and C3aR−/− mice but not in C6−/− mice. We detected markedly attenuated leukocyte-endothelial interactions in the brain microvasculature of C3−/− mice. Accordingly, in response to LPS administration, the brain microvasculature in these mice had decreased expression of P-selectin, E-selectin, intercellular cell adhesion molecule 1 (ICAM-1), and vascular cell adhesion molecule 1 (VCAM-1). Depletion of C3 from the circulation also caused reduction in VCAM-1 and E-selectin expression and leukocyte recruitment, suggesting that C3 in the circulation contributed to brain endothelial activation. Furthermore, C3−/− mice exhibited decreased leukocyte recruitment into the brain upon tumor necrosis factor-α (TNF-α) stimulation. C3a activated the phosphorylation of p38 mitogen-activated protein kinase (MAPK) and nuclear factor-κB (NF-κB) and induced the upregulation of VCAM-1 and ICAM-1 expression in murine primary cerebral endothelial cells in vitro.ConclusionsOur study provides the first evidence that C3a plays a critical role in cerebral endothelial activation and leukocyte recruitment during inflammation in the brain.Electronic supplementary materialThe online version of this article (doi:10.1186/s12974-016-0485-y) contains supplementary material, which is available to authorized users.

Retrotransposition of long interspersed nucleotide element-1 is associated with colitis but not tumors in a murine colitic cancer model.

Long interspersed element-1 (L1) is a transposable element that can move within the genome, potentially leading to genome diversity and modified gene function. Although L1 activity in somatic cells is normally suppressed through DNA methylation, some L1s are activated in tumors including colorectal carcinoma. However, how L1-retrotransposition (L1-RTP) is involved in gastrointestinal disorders remains to be elucidated. We hypothesized that L1-RTP in somatic cells might contribute to colitis-associated cancer (CAC). To address this, we employed an experimental model of CAC using transgenic L1-reporter mice carrying a human L1-EGFP reporter gene. Mice were subjected to repeated cycles of colitis induced by administration of dextran sodium sulfate (DSS) in drinking water with injection of carcinogen azoxymethane (AOM). L1-RTP levels were measured by a quantitative polymerase chain reaction targeting the newly inserted reporter EGFP in various tissues and cell types, including samples obtained by laser microdissection and cell sorting with flow cytometry. DNA methylation levels of the human L1 promoter were analyzed by bisulfite pyrosequencing. AOM+DSS-treated mice exhibited significantly higher levels of L1-RTP in whole colon tissue during the acute phase of colitis when compared with control naïve mice. L1-RTP levels in whole colon tissue were positively correlated with the histological severity of colitis and degree of neutrophil infiltration into the lamina propria (LP), but not with tumor development in the colon. L1-RTP was enriched in LP mesenchymal cells rather than epithelial cells (ECs), myeloid, or lymphoid cells. DNA methylation levels of the human L1 promoter region showed a negative correlation with L1-RTP levels. L1-RTP was absent from most tumors found in 22-week-old mice. In conclusion, we demonstrated that L1-RTP was induced in the mouse CAC mucosa in accordance with the acute inflammatory response; however, retrotransposition appears not to have direct relevance to colitis-induced cancer initiation.

A Histologic Scoring System for Prognosis of Patients With Alcoholic Hepatitis

There is no histologic classification system to determine prognoses of patients with alcoholic hepatitis (AH). We identified histologic features associated with disease severity and created a histologic scoring system to predict short-term (90-day) mortality. We analyzed data from 121 patients admitted to the Liver Unit (Hospital Clinic, Barcelona, Spain) from January 2000 to January 2008 with features of AH and developed a histologic scoring system to determine the risk of death using logistic regression. The system was tested and updated in a test set of 96 patients from 5 academic centers in the United States and Europe, and a semiquantitative scoring system called the Alcoholic Hepatitis Histologic Score (AHHS) was developed. The system was validated in an independent set of 109 patients. Interobserver agreement was evaluated by weighted κ statistical analysis. The degree of fibrosis, degree of neutrophil infiltration, type of bilirubinostasis, and presence of megamitochondria were independently associated with 90-day mortality. We used these 4 parameters to develop the AHHS to identify patients with a low (0-3 points), moderate (4-5 points), or high (6-9 points) risk of death within 90 days (3%, 19%, and 51%, respectively; P < .0001). The AHHS estimated 90-day mortality in the training and test sets with an area under the receiver operating characteristic value of 0.77 (95% confidence interval, 0.71-0.83). Interrater agreement values were 0.65 for fibrosis, 0.86 for bilirubinostasis, 0.60 for neutrophil infiltration, and 0.46 for megamitochondria. Interestingly, the type of bilirubinostasis predicted the development of bacterial infections. We identified histologic features associated with the severity of AH and developed a patient classification system that might be used in clinical decision making.

Anti-inflammatory intestinal activity of Arctium lappa L. (Asteraceae) in TNBS colitis model

Ethnopharmacological relevanceIn Brazilian traditional medicine, Arctium lappa (Asteraceae), has been reported to relieve gastrointestinal symptoms. Aim of the studyIn the present study, we investigated the effects of the lactone sesquiterpene onopordopicrin enriched fraction (ONP fraction) from Arctium lappa in an experimental colitis model induced by 2,4,6 trinitrobenzene sulfonic acid and performed experiments to elucidate the underlying action mechanisms involved in that effect. Materials and methodsONP fraction (25 and 50mg/kg/day) was orally administered 48, 24 and 1h prior to the induction of colitis and 24h after. The inflammatory response was assessed by gross appearance, myeloperoxidase (MPO) activity, tumor necrosis factor alpha (TNF-α) levels and a histological study of the lesions. We determined cyclooxygenase (COX)-1 and -2 protein expressions by western blotting and immunohistochemistry assays. ResultsTNBS group was characterized by increased colonic wall thickness, edema, diffuse inflammatory cell infiltration, increased MPO activity and TNF-α levels. On the contrary, ONP fraction (25 and 50mg/kg) treatment significantly reduced the macroscopic inflammation scores (p<0.05 and p<0.01, respectively) and morphological alterations associated with an increase in the mucus secretion. Similarly, the degree of neutrophil infiltration and the cytokine levels were significantly ameliorated. Moreover, COX-2 expression was up regulated in TNBS-treated rats. In contrast, ONP fraction (50mg/kg) administration reduced COX-2 overexpression. ConclusionsWe have shown that the ONP fraction obtained from Arctium lappa exert marked protective effects in acute experimental colitis, confirming and justifying, at least in part, the popular use of this plant to treat gastrointestinal diseases.