Mycobacterium vaccae alleviates allergic airway inflammation and airway hyper-responsiveness in asthmatic mice by altering intestinal microbiota.

Host immunity can influence the composition of the gut microbiota and consequently affect disease progression. Previously, we reported that a Mycobacterium vaccae vaccine could ameliorate allergic inflammation in asthmatic mice by regulating inflammatory immune processes. Here, we investigated the anti-inflammatory effects of M. vaccae on allergic asthma via gut microbiota modulation. An ovalbumin (OVA)-induced asthmatic murine model was established and treated with M. vaccae. Gut microbiota profiles were determined in 18 BALB/c mice using 16S rDNA gene sequencing and metabolomic profiling was performed using liquid chromatography quadrupole time-of-flight mass spectrometry. Mycobacterium vaccae alleviated airway hyper-reactivity and inflammatory infiltration in mice with OVA-induced allergic asthma. The microbiota of asthmatic mice is disrupted and that this can be reversed with M. vaccae. Additionally, a total of 24 differential metabolites were screened, and the abundance of PI(14:1(9Z)/18:0), a glycerophospholipid, was found to be correlated with macrophage numbers (r = 0.52, p = 0.039). These metabolites may affect chemokine (such as macrophage chemoattractant protein-1) concentrations in the serum, and ultimately affect pulmonary macrophage recruitment. Our data demonstrated that M. vaccae might alleviate airway inflammation and hyper-responsiveness in asthmatic mice by reversing imbalances in gut microbiota. These novel mechanistic insights are expected to pave the way for novel asthma therapeutic strategies.

Insight into the role of mitochondrion-related gene anchor signature in mitochondrial dysfunction of neutrophilic asthma.

OBJECTIVE: At present, targeting molecular-pharmacological therapy is still difficult in neutrophilic asthma. The investigation aims to identify and validate mitochondrion-related gene signatures for diagnosis and specific targeting therapeutics in neutrophilic asthma. METHODS: Bronchial biopsy samples of neutrophilic asthma and healthy people were identified from the GSE143303 dataset and then matched with human mitochondrial gene data to obtain mitochondria-related differential genes (MitoDEGs). Signature mitochondria-related diagnostic markers were jointly screened by support vector machine (SVM) analysis, least absolute shrinkage, and selection operator (LASSO) regression. The expression of marker MitoDEGs was evaluated by validation datasets GSE147878 and GSE43696. The diagnostic value was evaluated by receiver operating characteristic (ROC) curve analysis. Meanwhile, the infiltrating immune cells were analyzed by the CIBERSORT. Finally, oxidative stress level and mitochondrial functional morphology for asthmatic mice and BEAS-2B cells were evaluated. The expression of signature MitoDEGs was verified by qPCR. RESULTS: 67 MitoDEGs were identified. Five signature MitoDEGs (SOD2, MTHFD2, PPTC7, NME6, and SLC25A18) were further screened out. The area under the curve (AUC) of signature MitoDEGs presented a good diagnostic performance (more than 0.9). There were significant differences in the expression of signature MitoDEGs between neutrophilic asthma and non-neutrophilic asthma. In addition, the basic features of mitochondrial dysfunction were demonstrated by in vitro and in vivo experiments. The expression of signature MitoDEGs in the neutrophilic asthma mice presented a significant difference from the control group. CONCLUSIONS: These MitoDEGs signatures in neutrophilic asthma may hold potential as anchor diagnostic and therapeutic targets in neutrophilic asthma.

Aerosol inhalation of Mycobacterium vaccae ameliorates airway structural remodeling in chronic asthma mouse model.

Background: Airway remodeling is accepted to be a determining component within the natural history of asthma. Nebulized inhalation of Mycobacterium vaccae (M. vaccae) has a protective effect on asthmatic mice. However, little is known regarding the effect of M. vaccae on airway structural remodeling in asthmatic mice. The purpose of this study was to explore the effect and the underlying mechanism of M. vaccae aerosol inhalation on airway structural remodeling in an asthma mouse model. Methods: Chronic asthma mouse models were established by ovalbumin induction. The number of inflammatory cells in bronchoalveolar lavage fluid (BALF), pathological alterations in lung tissue, and levels of associated cytokines (IL-5, IL-13, TNF-α, and ovalbumin-specific immunoglobulin E [OVA-sIgE]) were all assessed after M. vaccae therapy. The relative expression of interleukin (IL)-1ß, tumor necrosis factor-alpha (TNF-α), nuclear factor kappa B (NF-κB), and Wnt1-induced signaling protein 1 (WISP1) mRNA were detected. Western blotting and immunohistochemistry detected the expression of Wnt/ß-catenin pathway-related proteins in lung tissue. Results: M. vaccae aerosol inhalation relieved airway inflammation, airway hyper-responsiveness, and airway remodeling. M. vaccae reduced the levels of IL-5, IL-13, TNF-α, and OVA-sIgE in and downregulated the expression of IL-1ß, TNF-α, NF-κB, and WISP1 mRNA in the pulmonary. In addition, M. vaccae inhibited the expression of ß-catenin, WISP1, and Wnt1 protein and upregulated the expression of glycogen synthase kinase-3beta (GSK-3ß). Conclusion: Nebulized inhalation of M. vaccae can reduce airway remodeling during asthma.

Mycobacterium vaccae nebulization protects Balb/c mice against bronchial asthma through neural mechanisms.

OBJECTIVES: Bronchial asthma can be effectively controlled but not be cured, its etiology and pathogenesis are still unclear, and there are no effective preventive measures. The key characteristic of asthma is chronic airway inflammation, and recent research has found that airway neurogenic inflammation plays an important role in asthma. We previously found that Mycobacterium vaccae nebulization protects against asthma. Therefore, this objective of this study is to explore the effect of M. vaccae nebulization on asthmatic neural mechanisms. METHODS: A total 18 of female Balb/c mice were randomized into normal, asthma control, and M. vaccae nebulization (Neb.group) groups, and mice in the Neb.group were nebulized with M. vaccae one month before the asthmatic model was established. Then, 1 month later, the mice were sensitized and challenged with ovalbumin. Twenty-four hours after the last challenge, mouse airway responsiveness; pulmonary brain-derived neurotropic factor (BDNF), neurofilament-medium length (NF-M, using NF09 antibody), and acetylcholine expression; and nerve growth factor (NGF) mRNA level were determined. RESULTS: We found that the BDNF, NF09, acetylcholine expression, and NGF mRNA level were decreased in the Neb.group compared with levels in the asthma control group. CONCLUSION: M. vaccae nebulization may protected in Balb/c mice against bronchial asthma through neural mechanisms.

Effect of mild hypothermia on the expression of toll-like receptor 2 in lung tissues with experimental acute lung injury.

OBJECTIVE AND DESIGN: Our study aimed to determine the effect of mild hypothermia (MHT) on the expression of toll-like receptor 2 (TLR2) in lung tissue with acute lung injury. The animals were randomly divided into control, model and mild hypothermia groups. METHODS: A total of 40 rats was used in the study. Acute lung injury was induced by lipopolysaccharide and MHT was maintained at 32.5∼33.0 °C using body surface ice-bag placement combined with animal thermostat system. The ratio of PaO2/FiO2 was recorded. The mRNA and protein expressions of TLR2 were measured by real-time polymerase chain reaction and western blotting respectively. Moreover, enzyme linked immunosorbent assay were used for the quantification of TNF-α. RESULTS: The ratio of PaO2/FiO2 was increased by MHT. TLR2 and TNF-α were increased in the rat lung 1h and 8h in the rats with acute lung injury while they were significantly decreased by MHT. Histological examination revealed that MHT alleviated the degree of inflammation. CONCLUSION: Our study suggested that MHT might improve the lung function by inhibiting the inflammation via down-regulating the expressions of TLR2 in the acute injury lung tissues.

[Value of creatinine clearance rate estimated based on serum cystatin C in patients with acute kidney injury].

OBJECTIVE: To investigate diagnostic value of creatinine clearance rate (CCr) based on serum cystatin C (SCys C) in acute kidney injury (AKI), and whether it could predict the need for renal replacement therapy (RRT). METHODS: The patients enrolled with the length of intensive care unit (ICU) stay over 3 days were collected from August 2010 to May 2011. According to the diagnosis of AKI during the ICU stay, patients were divided into the AKI group (n=21) and non-AKI group (n=30). After patients were admitted, the level of SCys C and creatinine (SCr) were measured so as to count CCr based on SCys C (SCys C-CCr) or on SCr (SCr-CCr) respectively, meanwhile urine volume and acute physiology and chronic health evaluation II (APACHE II) score were monitored. The value of CCr counted by SCys C and SCr on predict AKI and the correlations between RRT were compared. RESULTS: SCr-CCr and SCys C-CCr in AKI group both were significantly lower than non-AKI group all the way through on admission, and 2 days and 1 day before AKI diagnosed and the day AKI diagnosed. The level of SCys C-CCr on 2 days prior to AKI diagnosed was significantly lower than the day admitted (70.6±8.4 ml×min(-1)×1.73 m(-2) vs. 114.8±15.8 ml×min(-1)×1.73 m(-2), P<0.01), whereas the level of SCr-CCr were not significantly changed (76.4±19.3 ml×min(-1)×1.73 m(-2) vs. 78.7±22.1 ml×min(-1)×1.73 m(-2), P>0.05). Receptor operative curve (ROC) analysis indicated that SCys C-CCr could predict AKI earlier than SCr-CCr, as the area under curve (AUC) of SCys C-CCr and SCr-CCr on 2 days prior to AKI diagnosed were 0.859 and 0.664, respectively, and the sensitivity were 90.5% and 47.6%, the specificity were 76.2% and 81.0%. In AKI group 6 patients were treated with RRT, the AKI patients receiving RRT had significantly higher APACHE II score on admission (29.6±4.5 vs. 17.0±5.6, P<0.05) and less urine volume within 24 hours (740±465 ml vs. 1780±1230 ml, P<0.05) than patients not received RRT, however, SCys C-CCr has no significant difference between the sub-group (50.4±11.2 ml×min(-1)×1.73 m(-2) vs. 53.0±8.4 ml×min(-1)×1.73 m(-2), P>0.05). SCys C-CCr did not predict the need of RRT on the day to diagnose AKI (AUC=0.65). CONCLUSIONS: The sensitivity of SCys C-CCr were high, but its specificity not. The SCys C-CCr may be helpful for excluding diagnose of AKI in high risk patients. However, it could not predict the need for renal replacement therapy on the day AKI diagnosed.

Transcriptomic Analysis Reveals a Link Between Hippo Signaling Pathway and Macrophages in Lungs of Mice with OVA-Induced Allergic Asthma.

PURPOSE: The Hippo signaling pathway participates in the restriction of cell proliferation and organ growth. Activated macrophages have been implicated in the pathogenesis of allergic asthma. Recent studies have shown that Hippo signaling pathway may also be involved in the regulation of asthma. However, the link between Hippo signaling pathway and macrophages in the context of allergic asthma has not been investigated. The purpose of this study was to explore the link between Hippo signaling pathway and macrophages using a mice model of OVA-induced allergic asthma. METHODS: Mice models of asthma were established. Lung tissues were collected from mice and pooled for mRNA sequencing and bioinformatics analysis. The relative mRNA expression of Hippo signalling pathway-related proteins Yap1, Lef1 and Ctgf was also measured. Double immunofluorescence staining was performed on lung tissues to evaluate macrophage marker F4/80 expression and Yap1/Lef1/Ctgf expression. RESULTS: Results of the RNA-Seq of lung tissues demonstrated that the Hippo signaling pathway was down-regulated in OVA-induced allergic asthma. Using the cytoHubba tool kits in Cytoscape, the following top 10 hub genes of Hippo signalling pathway were identified: Yap1, Lef1, Ctgf, Ccnd1, Axin2, Smad7, Wnt4, Wnt3a, Pard6b, and Wwc1. Using the seq-ImmuCC (http://218.4.234.74:3200/immune/), a negative correlation was found between macrophages and Hippo signaling pathway activity (R2 = 0.93). The mRNA expression levels of pulmonary Yap1, Lef1, and Ctgf were down-regulated in the mice model of OVA-induced allergic asthma. Moreover, double-stained immunofluorescence for F4/80 and Yap1, Lef1, Ctgf in mouse lung sections respectively revealed that macrophage proliferation was correlated with downregulation of the Hippo signaling pathway in the mice model of OVA-induced allergic asthma. CONCLUSION: These results demonstrated that the Hippo signaling pathway was down-regulated in asthma mice, and the proliferation of macrophages was associated with downregulation of the Hippo signaling pathway. These findings reveal novel insights into the pathogenesis and treatment of asthma.

Effects of Mycobacterium vaccae Aerosol Inhalation on Airway Inflammation in Asthma Mouse Model.

Background:Mycobacterium vaccae vaccine, a composition of Mycobacterium proteins, has been known to have bidirectional immunomodulatory functions. Recent studies have shown that M. vaccae has a therapeutic potential for treating asthma. However, little is known regarding the effect of M. vaccae aerosol inhalation during allergen sensitization or challenge on asthma. The purpose of this study was to explore the effect and the underlying mechanism of M. vaccae aerosol inhalation during allergen sensitization or challenge on airway inflammation in an asthma mouse model. Methods: Asthma mouse models were established. Mice received aerosol inhalation with M. vaccae once daily during allergen sensitization or challenge for 5 days successively. Airway responsiveness, bronchoalveolar lavage fluid (BALF) cell count, histology, and cytokine concentrations (IL-4, IFN-γ, IL-10, and IL-17) were measured. The relative mRNA expression of ASC, caspase-1, TNF-α, and IL-1ß was also determined. Expression of pulmonary NLRP3 and nuclear factor kappa B (NF-κB) protein was measured using immunohistochemistry and Western blot. Results:M. vaccae aerosol inhalation suppressed airway hyperresponsiveness and inflammation, reduced levels of IL-4, upregulated expression of IFN-γ and IL-10 in BALF, inhibited mRNA expression of pulmonary ASC, caspase-1, TNF-α, and IL-1ß, and also inhibited expression of pulmonary NLRP3 and NF-κB protein during allergen sensitization or challenge. Conclusion:M. vaccae aerosol inhalation can suppress airway hyperresponsiveness and inflammation during allergen sensitization or challenge, and may be a promising approach for asthma therapy.

Mycobacterium vaccae Nebulization in the Treatment of COVID-19: A Randomized, Double-Blind, Placebo-Controlled Trial.

Background: Severe acute respiratory syndrome coronavirus 2 infection is associated with strong infectiousness and has no effective therapy. We aimed to explore the efficacy and safety of Mycobacterium vaccae nebulization in the treatment of Coronavirus Disease 2019 (COVID-19). Methods: In this randomized, double-blind, placebo-controlled clinical trial, we included 31 adult patients with moderate COVID-19 who were admitted to the Fourth People's Hospital of Nanning (Nanning, China) between January 22, 2020 and February 17, 2020. Patients were randomly divided into two groups: group A (standard care group) and group B (M. vaccae in combination with standard care group). The primary outcome was the time interval from admission to viral RNA negative conversion (oropharyngeal swabs were used in this study). Secondary outcomes included chest computed tomography (CT), mortality, length of hospital stay, complications during treatment, and so on. Patients were followed up to 4 weeks after discharge (reexamination of viral RNA, chest CT, etc.). Results: Nucleic acid test negative conversion time in group B was shorter than that in group A (2.9 days [2.7-8.7] vs. 6.8 days [3.3-13.8]; p = 0.045). No death and no conversion to severe or critical cases were observed in both groups. Two weeks after discharge, neither "relapse" nor "return to positive" cases were found. Four weeks after discharge, it was found that there was no case of " relapse " or "return to positive" in group B, and 1 patient in group A showed "return to positive", but there was no clinical manifestation and imaging progression. No adverse reactions related to M. vaccae were found during observation period. Conclusion:M. vaccae treatment might shorten the time interval from admission to viral RNA negative conversion, which might be beneficial to the prevention and treatment of COVID-19. Clinical Trial Registration: ChiCTR2000030016.

Identification of prognostic immune genes in laryngeal cancer.

OBJECTIVE: In this study, we aimed to identify prognostic immune-related genes and establish a prognostic model for laryngeal cancer based on these genes. METHODS: Transcriptome profiles and clinical data of patients with laryngeal cancer were downloaded from The Cancer Genome Atlas database. Integrated bioinformatics analyses were performed to identify genes associated with prognosis. RESULTS: Thirty prognostic immune-related genes for laryngeal cancer were identified. We constructed a regulatory network of prognosis comprising transcription factors and immune-related genes. Multivariate Cox regression analyses identified 15 immune-related genes in the network that were used to establish the prognostic model. The model exhibited excellent prognostic prediction ability with a high area under the curve value (0.916). The calculated risk score based on expression of the 15 immune-related genes was shown to be an independent prognostic factor for laryngeal cancer. CONCLUSION: We identified prognostic immune-related genes and established a prognostic model for laryngeal cancer, which might help identify novel predictive biomarkers and therapeutic targets of laryngeal cancer.

Correlation between DNA methylation and Thymic Stromal Lymphopoietin expression in asthmatic airway epithelial cells.

BACKGROUND: The overexpression of TSLP and DNA methylation in asthma were both risk factors the relationship was not clear. OBJECTIVE: This study aimed to investigate the relationship between methylation status of TSLP promoter and mRNA/protein expression in asthmatic airway epithelial cells. METHODS: Human bronchial epithelial cells were cultured in vitro and divided into: Control group, treated with PBS, model group, sensitized with LPS (10 µg/mL) for 12 h (37 °C, 5% CO2). Other groups were cultured with the pCMV3 plasmid (M + NC/pCMV), pGPH1 plasmid (M + NC/pGPH), DNMT1/pCMV3 plasmid (M + DNMT1/pCMV), and DNMT1/pGPH1 plasmid (M + DNMT1/pGPH) for 48 h. The expression of DNA methyltransferase 1 and TSLP were measured using real-time PCR and western blotting. RESULTS: Compared with the control group, TSLP mRNA (1.00 ± 0.00 vs. 2.82 ± 0.81 vs. 1, P < 0.001) and protein (1.07 ± 0.04 vs. 1.46 ± 0.11, P < 0.01) were significantly greater, and the methylation of promoter was lower (92.75 ± 1.26 vs. 58.57 ± 3.34, P < 0.05) in the model group. Compared with the model group, TSLP mRNA (2.82 ± 0.81 vs. 1.17 ± 0.10, P < 0.001) decreased, but TSLP promoter methylation increased (58.57 ± 3.34 vs. 92.58 ± 7.30, P < 0.05) in M + DNMT1/pCMV. TSLP mRNA and protein were higher (2.82 ± 0.81 vs. 5.32 ± 0.21, P < 0.001; 1.46 ± 0.11 vs. 1.94 ± 0.11, respectively, P < 0.01), TSLP promoter methylation was lower (58.57 ± 3.34 vs. 33.57 ± 4.29, P < 0.05) in M + DNMT1/pGPH. CONCLUSIONS: Overexpression of TSLP in asthmatic airway epithelial cells may be regulated by DNA demethylation.

One-Week Nebulization of Mycobacterium vaccae Can Protect Against Pulmonary Respiratory Syncytial Virus Infection in Balb/c Mice.

Background: Respiratory syncytial virus (RSV) infection is the most common cause of acute lower respiratory tract infection in children, leading to their death. Currently, no effective prevention and treatment methods for RSV infection are available. RSV and many other unknown viruses pose a serious threat to human health. Our previous study demonstrated that Mycobacterium vaccae nebulization can protect against allergic asthma. As RSV infection and asthma are closely related, we hypothesized that M. vaccae could protect against pulmonary RSV infection. Therefore, we evaluated the effect of M. vaccae on RSV infection in Balb/c mice. Methods: The mice were randomized into three groups: normal, RSV, and M. vaccae. One week before the RSV infection model was established, the mice in the M. vaccae group were nebulized with M. vaccae. On the fourth day after RSV infection, airway responsiveness, airway inflammation, pulmonary RSV infection, mRNA levels of pulmonary toll-like receptor (TLR) 7 and TLR8, and pulmonary NF09, acetylcholine, and epidermal growth factor regulator (EGFR) expression levels in all mice were measured. Results: The airway inflammation in the M. vaccae group was alleviated compared with that in the RSV group. In the M. vaccae group, the pulmonary mRNA level of RSV and the pulmonary expression levels of NF09, acetylcholine, and EGFR were decreased considerably, whereas the mRNA levels of TLR7 and TLR8 were increased significantly. Conclusions: One-week nebulization of M. vaccae can protect against RSV infection in Balb/c mice. The mechanism involves the regulation of neurotransmitters and expression of TLR7, TLR8, and EGFR.

Inhalation of nebulized Mycobacterium vaccae can protect against allergic bronchial asthma in mice by regulating the TGF-ß/Smad signal transduction pathway.

BACKGROUND: Mycobacterium vaccae nebulization imparted protective effect against allergic asthma in a mouse model. The TGF-ß/Smad signal transduction pathway plays an important role in allergic bronchial asthma. However, the effect of M. vaccae nebulization on the TGF-ß/Smad signal transduction pathway in mouse models of allergic asthma remains unclear. This study investigated the preventive effect of M. vaccae nebulization during bronchial asthma in a mouse model and elucidate the implication of TGF-ß/Smad signal transduction pathway in the process. METHODS: In total, 24 female Balb/c mice were randomized to normal control (group A), asthma control (group B), and M. vaccae nebulization (group C) groups. Both groups B and C were sensitized using ovalbumin for establishment of the asthmatic model; group A received phosphate-buffered solution. Prior to the establishment of asthma, Group C was nebulized with M. vaccae. Airway responsiveness was measured in all the groups, using a noninvasive lung function machine before and 24 h after establishment of the asthmatic model. The animals were then harvested, and bronchoalveolar lavage fluid (BALF) and lung tissue were collected. The total cell counts in BALF was estimated. Protein expression of TGF-ß1, TßR1, Smad1, and Smad7 was detected by immunohistochemistry. The population of CD3 + γδT, IL-13 + CD3 + T, TGF-ß + CD3 + T, IL-13 + CD3 + γδT, and TGF-ß+ CD3+ γδT cells were detected by flow cytometry. One-way analysis of variance for within-group comparisons, the least significant difference t-test or Student-Newman-Keuls test for intergroup comparisons, and the nonparametric rank sum test for analysis of airway inflammation scores were used in the study. RESULTS: The eosinophil count; protein expression of TGF-ß1, TßR1, and Smad1; and percentages of CD3 + γδT and IL-13 + CD3 + T cells were significantly lower in the M. vaccae nebulization group than in the asthma control group (P < 0.01). There were significant intergroup differences in the percentages of TGF-ß + CD3 + T and IL-13 + CD3 + γδT cells (P < 0.05). CONCLUSIONS: Mycobacterium vaccae nebulization could confer protection against allergic bronchial asthma by reducing airway responsiveness and alleviating airway inflammation in mice. The underlying mechanism might be attributed its effect on the deregulated expression of TGF-ß1, TßR1, Smad1, and Smad7 of the TGF-ß/Smad signal transduction pathway.

Regulation of γδT17 cells by Mycobacterium vaccae through interference with Notch/Jagged1 signaling pathway.

The objective of this study was to investigate the effect of Mycobacterium vaccae on Jagged 1 and gamma delta T17 (γδT17) cells in asthmatic mice. An asthma mouse model was established through immunization with ovalbumin (OVA). Gamma-secretase inhibitor (DAPT) was used to block the Notch signaling pathway. M. vaccae was used to treat asthma, and related indicators were measured. Blocking Notch signaling inhibited the production of γδT17 cells and secretion of cytokine interleukin (IL)-17, which was accompanied by a decrease in Jagged1 mRNA and protein expression in the treated asthma group compared with the untreated asthma group. Similarly, treatment with M. vaccae inhibited Jagged1 expression and γδT17 cell production, which was associated with decreased airway inflammation and reactivity. The Notch signaling pathway may play a role in the pathogenesis of asthma through the induction of Jagged1 receptor. On the other hand, the inhibitory effect of M. vaccae on Jagged1 receptor in γδT17 cells could be used for the prevention and treatment of asthma.

A parameterized shift-splitting preconditioner for saddle point problems.

Recently, Chen and Ma [A generalized shift-splitting preconditioner for saddle point problems, Applied Mathematics Letters, 43 (2015) 49-55] introduced a generalized shift-splitting preconditioner for saddle point problems with symmetric positive definite (1,1)-block. In this paper, I establish a parameterized shift-splitting preconditioner for solving the large sparse augmented systems of linear equations. Furthermore, the preconditioner is based on the parameterized shift-splitting of the saddle point matrix, resulting in an unconditional convergent fixed-point iteration, which has the intersection with the generalized shift-splitting preconditioner. In final, one example is provided to confirm the effectiveness.

Nrf2 Activator RTA-408 Protects Against Ozone-Induced Acute Asthma Exacerbation by Suppressing ROS and γδT17 Cells.

Ozone is a strong oxidant in air pollution that exacerbates respiratory disorders and is a major risk factor for acute asthma exacerbation. Ozone can induce reactive oxygen species (ROS) and airway neutrophilic inflammation. In addition, γδT17 cells contribute to IL-17A production upon ozone challenge, resulting in neutrophilic inflammation. It is known, however, that Nrf2 can ameliorate oxidative stress. We therefore investigated whether RTA-408, an Nrf2 activator, can attenuate airway inflammation and inhibit ROS production and whether this effect involves γδT17 cells. Balb/c mice were sensitized/challenged with ovalbumin (OVA) and followed by ozone exposure. We investigated the effect of Nrf2 activator RTA-408 on airway hyperresponsiveness, neutrophilic airway inflammation, cytokine/chemokine production, and OVA-specific IgE level in a mouse model of O3 induced asthma exacerbation. Furthermore, malondialdehyde (MDA) and glutathione (GSH) levels in lung and intracellular ROS were measured. IL-17+ γδT cell percentage by flow cytometer was determined. Nrf2 protein expression by western blot was also examined. We observed that RTA-408 attenuated ROS release during ozone-induced asthma exacerbation and suppressed neutrophil lung infiltration. RTA-408 decreased pro-inflammatory cytokine production and reduced the percentage of IL-17+ γδT cells. Thus, our results suggest that RTA-408 does attenuate airway inflammation in a murine model of ozone-induced asthma exacerbation.

Imbalance of γδT17/γδTreg cells in the pathogenesis of allergic asthma induced by ovalbumin.

We aimed to explore the imbalance between the T helper 17 γδT cells (γδT17) and the regulatory γδT cells (γδTreg) in asthmatic mice. Male Balb/c mice were randomly divided into the normal control group and the asthmatic model group. The asthmatic model group mice were intraperitoneally injected with the mixture of ovalbumin (OVA)/Al(OH)3 and then activated by exposure of the animals to OVA atomization. Airway hyperresponsiveness (AHR) was determined by a non-invasive lung function machine. Hematoxylin and eosin and Alcian blue-periodic acid Schiff staining were done for histopathological analysis. Interleukin (IL)-17 and IL-35 levels in bronchoalveolar lavage fluid were detected by ELISA. The percentage of IL-17+ γδT cells and Foxp3+ γδT cells in spleen cells suspension were detected and the transcription levels of RORγt and Foxp3 in the lung tissue were determined. Compared with the normal control, the severity of airway inflammation and AHR were higher in the asthmatic mice. Furthermore, mice in the asthmatic group displayed significant increases of IL-17+ γδT cells, expression of IL-17A, and RORγt, whereas control mice displayed marked decreases of Foxp3+ γδT cells, expression of IL-35, and transcription factor Foxp3. In addition, the mRNA expression of RORγt was positively correlated with the percentage of IL-17+γδT cells, and the mRNA level of Foxp3 was positively correlated with the percentage of Foxp3+ γδT cells. The imbalance of γδT17/γδTreg in the asthmatic mice may contribute to the pathogenesis of OVA-induced asthma.

Imbalance of Th17/Treg in the Pathogenesis of Mice with Paraquat-induced Acute Lung Injury.

Recent studies suggest that imbalances in the ratios of CD4+ T helper cell subsets, T helper-17 (Th17) and regulatory T (Treg) cells play a crucial role in the pathogenesis of acute lung injury (ALI). However, studies of the imbalance of Th17/Treg in paraquat (PQ)-induced ALI have not been reported. Therefore, we investigated whether the ratio of Th17/Treg cells in a mouse model of PQ-induced ALI contributes to pathogenesis of ALI. Male Kunming mice were randomly treated with saline (control group) or PQ (PQ-poisoned (PQP) group); mice were sacrificed at either 12 hours (PQP-12h) or 24 hours (PQP-24h and control) post-treatment. Hematoxylin-eosin and TUNEL staining procedures were performed to examine inflammation and apoptosis. The presence of Th17 and Treg cells was measured by flow cytometry; the expression of putative Th17 cytokines and transcription factors was measured by ELISA and western blot analysis. Compared with control mice, lung inflammation and apoptosis were dramatically increased in PQP mice at 12 and 24 hours after poisoning. In addition, poisoned mice displayed significant increases in the presence of CD4+IL-17+ T cells (Th17) and in the expression of IL-17A and IL-17, as measured by flow cytometry and western blot assays. This increase was most notable after 24 hours of PQ exposure. Furthermore, poisoned mice displayed marked decreases in the presence of CD4+CD25+Foxp3+ T cells (Treg) and in the expression of IL-35 and the transcription factor Foxp3. These results suggest that an imbalanced ratio of Th17/Treg cells may contribute to the pathogenesis of PQ-induced ALI.

Effect of partial splenic embolization on the immune function of cirrhosis patients with hypersplenism.

OBJECTIVE: To discover the effect of partial splenic embolization on the immune function of cirrhotic patients with hypersplenism. METHODS: Patients involved in the study were enrolled and divided into three groups, including control group, experimental group, and complication group. Numbers of CD3(+), CD4(+) and CD8(+) T cells and CD4(+)CD25(+)CDl27(low/-) Treg cells in the peripheral blood of patients before surgery, 1 month, 6 months, 1 year, and 2 years after surgery were analyzed by fluorescence active cell sorting (FACS). Contents of immunoglobulins (IgA, IgG and IgM) were analyzed by auto immunoassay analyzer. RESULTS: In the peripheral blood of patients from experimental group, numbers of CD3(+), CD4(+) and CD8(+) T cells initially declined, but afterwards increased to normal level; in the peripheral blood of patients from complication group, CD3(+) and CD8(+) T cells showed the same trend, but the number of CD4(+) T cells was below normal level at all detection times. Furthermore, CD3(+), CD4(+) and CD8(+) T cells in the peripheral blood of patients from complication group were initially less than those in experimental group, and afterwards were comparable between two groups. In patients from both experimental group and complication group, the number of CD4(+) CD25(+) CDl27(low/-)Treg cells increased 1 month and 6 months after surgery, and gradually restored to normal level. CD4(+)CD25(+)CDl27(low/-) Treg cell counts in patients from complication group were initially more than those in patients from experimental group 1 month and 6 months after surgery, but then they were comparable. Furthermore, contents of immunoglobulins (IgA, IgG and IgM) were comparable in three groups at all detection times. CONCLUSION: Partial splenic embolization influenced the immune function of cirrhotic patients with hypersplenism in the short term but the immune function could afterwards gradually restore to normal. Our results implicated that measures that prevent infection and improve immune function were necessary in early stage after undergoing PSE in order to reduce complications.

[Role of nitric oxide-nuclear factor kappaB signal pathway in functional expression of T lymphocytes from asthmatic patients].

OBJECTIVE: To investigate the regulatory role of nitric oxide (NO)-nuclear factor kappaB (NF-kappaB) signal pathway in cytokine expressions, cell proliferation and apoptosis of T lymphocytes from asthmatic patients. METHODS: We observed the expressions and secretions of IL-4, IL-5 and IFNgamma from T lymphocytes and measured the proliferation and apoptosis of these cells by using in situ hybridization, electrophoresis mobility shift assay (EMSA), immunofluorescence, flow cytometry, two antibodies sandwich enzyme linked immuno-sorbent assay and MTT, and by administering NO donor sodium nitroprusside (SNP) and NF-kappaB inhibitor pyrrolidine dithio-carbamic acid (PTDC). RESULTS: (1) The mRNA expression and protein secretion of IL-4 and IL-5 by, as well as the proliferation rate of T lymphocytes from asthmatic patients were augmented, while the mRNA expression and protein secretion of IFNgamma by, as well as the apoptosis rate of T lymphocytes from asthmatic patients were decrease as compared with the values of the control groups (P < 0.05). (2) Low dose of SNP (10 micromol/L) up-regulated the expression of these three cytokines, promoted the proliferation rate and suppressed the apoptosis rate of T lymphocytes. However, middle or high dose of SNP (100 micromol/L, 1 mmol/L, 10 mmol/L) dose-dependently down-regulated cytokine expressions and cell proliferation rate and promoted apoptosis rate. (3) PDTC inhibited the higher expressions of IL-5 and IFNgamma induced by 10 micromol/L SNP and strengthened the inhibitory effect of 1 mmol/L SNP on the expressions of IL-5 and IFNgamma. At the same time, PDTC weakened the promoting effect of 10 micromol/L SNP and strengthened the inhibitory effect of 1 mmol/L SNP on the proliferation of T lymphocytes. (4) The percentage of NF-kappaB-activated cells and NF-kappaB activity were significantly increased in T lymphocytes treated with low dose of SNP (P < 0.05); whereas these parameters were decreased in cells treated with middle or high dose of SNP (P < 0.05). CONCLUSIONS: The hyper-expression of inflammatory cytokines and the increased proliferation and decreased apoptosis of T lymphocytes from asthmatic patients are related to the abnormal over-activation of NF-kappaB. NO has bi-phasic effects on the expressions of IL-5 and IFNgamma and on the proliferation and apoptosis of T lymphocytes from asthmatic patients via its bi-phasic regulations on the activity of NF-kappaB.