Gene expression profiles and bioinformatics analysis in lung samples from ovalbumin-induced asthmatic mice.

BACKGROUND: Asthma is characterized by chronic inflammation and airway remodeling. However, limited study is conducted on the gene expression profiles of ovalbumin (OVA) induced asthma in mice. Here, we explored the gene expression profiles in lung tissues from mice with OVA-induced asthma using microarray and bioinformatics analysis. METHODS: For establishment of OVA-induced asthma model, mice first received intraperitoneal sensitization with OVA on day 0, 7 and 14, followed by atomizing inhalation of OVA 3 times a week for 8 weeks. The lung tissues were collected and subjected to microarray analysis, bioinformatics analysis and expression validation. RESULTS: Microarray data of lung tissues suggested that 3754 lncRNAs and 2976 mRNAs were differentially expressed in lung tissues between control and asthmatic mice, including 1647 up-regulated and 2106 down-regulated lncRNAs, and 1201 up-regulated and 1766 down-regulated mRNAs. GO analysis displayed that the up-regulated genes were enriched in inflammatory response, leukocyte migration involved in inflammatory response, and Notch signaling pathway. KEGG pathway analysis indicated that the enriched pathway terms of the up-regulated gene included Toll-like receptor signaling pathway and Th17 cell differentiation signaling pathway. Additionally, based on the previously published literatures on asthma and inflammation, we screened out down-regulated genes, such as Smg7, Sumo2, and Stat5a, and up-regulated genes, such as Myl9, Fos and Tlr4. According to the mRNA-lncRNA co-expression network, we selected lncRNAs associated with above genes, including the down-regulated lncRNAs of NONMMUT032848, NONMMUT008873, NONMMUT009478, and NONMMUT006807, and the up-regulated lncRNAs of NONMMUT052633, NONMMUT05340 and NONMMUT042325. The expression changes of the above genes were validated in lung tissues by real-time quantitaive PCR and Western blot. CONCLUSIONS: Overall, we performed gene microarray on lung samples from OVA-induced asthmatic mice and summarized core mRNAs and their related lncRNAs. This study may provide evidence for further research on the therapeutic targets of asthma.

DEK-targeting aptamer DTA-64 attenuates bronchial EMT-mediated airway remodelling by suppressing TGF-ß1/Smad, MAPK and PI3K signalling pathway in asthma.

This study is to investigate the inhibitory effects and mechanisms of DEK-targeting aptamer (DTA-64) on epithelial mesenchymaltransition (EMT)-mediated airway remodelling in mice and human bronchial epithelial cell line BEAS-2B. In the ovalbumin (OVA)-induced asthmatic mice, DTA-64 significantly reduced the infiltration of eosinophils and neutrophils in lung tissue, attenuated the airway resistance and the proliferation of goblet cells. In addition, DTA-64 reduced collagen deposition, transforming growth factor 1 (TGF-ß1) level in BALF and IgE levels in serum, balanced Th1/Th2/Th17 ratio, and decreased mesenchymal proteins (vimentin and α-SMA), as well as weekend matrix metalloproteinases (MMP-2 and MMP-9) and NF-κB p65 activity. In the in vitro experiments, we used TGF-ß1 to induce EMT in the human epithelial cell line BEAS-2B. DEK overexpression (ovDEK) or silencing (shDEK) up-regulated or down-regulated TGF-ß1 expression, respectively, on the contrary, TGF-ß1 exposure had no effect on DEK expression. Furthermore, ovDEK and TGF-ß1 synergistically promoted EMT, whereas shDEK significantly reduced mesenchymal markers and increased epithelial markers, thus inhibiting EMT. Additionally, shDEK inhibited key proteins in TGF-ß1-mediated signalling pathways, including Smad2/3, Smad4, p38 MAPK, ERK1/2, JNK and PI3K/AKT/mTOR. In conclusion, the effects of DTA-64 against EMT of asthmatic mice and BEAS-2B might partially be achieved through suppressing TGF-ß1/Smad, MAPK and PI3K signalling pathways. DTA-64 may be a new therapeutic option for the management of airway remodelling in asthma patients.

Imperatorin alleviates ROS-mediated airway remodeling by targeting the Nrf2/HO-1 signaling pathway.

In this study, we investigated the role and mechanism of imperatorin (IMP) in chronic inflammation and airway remodeling. The levels of TNF-α, IL-1ß, IL-6, IL-8, VEGF, α-SMA, and ROS were detected by ELISA, immunohistochemistry (IHC), immunofluorescence, and Western blot. In addition, we evaluated the effect of IMP on MAPK, PI3K/Akt, NF-κB, and Nrf2/HO-1 signaling pathways. IMP treatment obviously attenuated the production of inflammatory cytokines and inflammatory cells in bronchoalveolar lavage fluid of OVA-induced airway remodeling model. Meanwhile, it significantly inhibited inflammatory cell infiltration, goblet cell hyperplasia, collagen deposition, VEGF production, α-SMA, and ROS expression. Our study has shown that IMP could regulate the signaling pathways including MAPK, PI3K/Akt, NF-κB, and Nrf2/HO-1 to release the inflammatory responses. IMP might attenuate airway remodeling by the down-regulation of Nrf2/HO-1/ROS/PI3K/Akt, Nrf2/HO-1/ROS/MAPK, and Nrf2/HO-1/ROS/NF-κB signaling pathways.

Recombinant pyrin domain protein attenuates allergic inflammation by suppressing NF-κB pathway in asthmatic mice.

Pyrin domain (PYD), a subclass of protein motif known as the death fold, is frequently involved in inflammation and immune responses. PYD modulates nuclear factor-kappa B (NF-κB) signalling pathway upon various stimuli. Herein, a novel recombinant pyrin domain protein (RPYD) was generated. Its role and mechanism in inflammatory response in an ovalbumin (OVA) induced asthma model was investigated. After OVA challenge, there was inflammatory cell infiltration in the lung, as well as airway hyper-responsiveness (AHR) to inhaled methacholine. In addition, eosinophils increased in the bronchoalveolar lavage fluids, alone with the elevated levels of Th-2 type cytokines [interleukin (IL)-4, IL-5 and IL-13], eotaxin, and adhesion molecules. However, the transnasal administration of RPYD before the OVA challenge significantly inhibited these asthmatic reactions. Moreover, RPYD markedly suppressed NF-κB translocation, reduced phosphorylation of p38 MAPK, and thus attenuated the expression of intercellular adhesion molecule 1 and IL-6 in the BEAS-2B cells stimulated by proinflammatory cytokines in vitro. These findings indicate that RPYD can protect asthma host from OVA-induced airway inflammation and AHR via down-regulation of NF-κB and p38 MAPK activities. RPYD may be used as a potential medicine for the treatment of asthma in clinic.

miR-128-3p alleviates airway inflammation in asthma by targeting SIX1 to regulate mitochondrial fission and fusion.

Bronchial asthma is known for airway inflammation, hyperresponsiveness, and remodeling.MicroRNAs (MiRNAs) have been involved in the development of asthma, whereas, the mechanism of various MiRNAs in asthma remains to be elucidated. In this study, we aim to explore the mechanism of miR-128-3p in asthma-related airway inflammation by targeting sine oculis homeobox homolog 1 (SIX1) to regulate the mitochondrial function. In an ovalbumin (OVA) asthma mouse model, miR-128-3p levels were found to be significantly diminished. Administration of miR-128-3p agomir decreased peribronchial inflammatory cell infiltration and improved airway inflammation. Afterwards, we used the luciferase reporter assay to predict and confirmed that SIX1 is a target gene of miR-128-3p. Overexpression of miR-128-3p attenuated IL-13-induced cellular inflammation and ROS production in bronchial epithelial cells (BEAS-2B). In vitro, overexpression of miR-128-3p and SIX1 knockdown mitigated mitochondrial fragmentation, reduced Drp1-mediated mitochondrial division, and upregulated mitochondrial membrane potential. Moreover, led to decreased production of ROS/mitochondrial ROS, P-Drp1(616) and Fis1 expression, while enhancing P-Drp1(637), MFN1, caspase-3/9, and Bax-mediated apoptosis. Our findings demonstrated that miR-128-3p could alleviate airway inflammation by downregulating SIX1 and improving mitochondrial function, positioning the miR-128-3p/SIX1/Drp1 signaling as a potential therapeutic target for asthma.

DEK deficiency suppresses mitophagy to protect against house dust mite-induced asthma.

DEK protein is highly expressed in asthma. However, the mechanism of DEK on mitophagy in asthma has not been fully understood. This study aims to investigate the role and mechanism of DEK in asthmatic airway inflammation and in regulating PINK1-Parkin-mediated mitophagy, NLRP3 inflammasome activation, and apoptosis. PINK1-Parkin mitophagy, NLRP3 inflammasome, and apoptosis were examined after gene silencing or treatment with specific inhibitors (MitoTEMPO, MCC950, and Ac-DEVD-CHO) in house dust mite (HDM) or recombinant DEK (rmDEK)-induced WT and DEK-/- asthmatic mice and BEAS-2B cells. The regulatory role of DEK on ATAD3A was detected using ChIP-sequence and co-immunoprecipitation. rmDEK promoted eosinophil recruitment, and co-localization of TOM20 and LC3B, MFN1 and mitochondria, LC3B and VDAC, and ROS generation, reduced protein level of MnSOD in HDM induced-asthmatic mice. Moreover, rmDEK also increased DRP1 expression, PINK1-Parkin-mediated mitophagy, NLRP3 inflammasome activation, and apoptosis. These effects were partially reversed in DEK-/- mice. In BEAS-2B cells, siDEK diminished the Parkin, LC3B, and DRP1 translocation to mitochondria, mtROS, TOM20, and mtDNA. ChIP-sequence analysis showed that DEK was enriched on the ATAD3A promoter and could positively regulate ATAD3A expression. Additionally, ATAD3A was highly expressed in HDM-induced asthma models and interacted with DRP1, and siATAD3A could down-regulate DRP1 and mtDNA-mediated mitochondrial oxidative damage. Conclusively, DEK deficiency alleviates airway inflammation in asthma by down-regulating PINK1-Parkin mitophagy, NLRP3 inflammasome activation, and apoptosis. The mechanism may be through the DEK/ATAD3A/DRP1 signaling axis. Our findings may provide new potential therapeutic targets for asthma treatment.

MicroRNA-182-5p Attenuates Asthmatic Airway Inflammation by Targeting NOX4.

Bronchial asthma is characterized by chronic airway inflammation, airway hyperresponsiveness, and airway remodeling. MicroRNA (miRNA) has recently been implicated in the pathogenesis of asthma. However, the mechanisms of different miRNAs in asthma are complicated, and the mechanism of miRNA-182-5p in asthma is still unclear. Here, we aim to explore the mechanism of miRNA182-5p in asthma-related airway inflammation. Ovalbumin (OVA)-induced asthma model was established. MiRNA Microarray Analysis was performed to analyze the differentially expressed miRNAs in the asthma model. We found that the expression of miRNA-182-5p was significantly decreased in OVA-induced asthma. In vitro, IL-13 stimulation of BEAS-2B cells resulted in a significant up-regulation of NOX4 (nicotinamide adenine dinucleotide phosphate oxidase 4), accompanied by mitochondrial damage-induced apoptosis, NLRP3 (NOD-like receptor family pyrin domain-containing 3)/IL-1ß activation, and reduced miRNA-182-5p. In contrast, overexpression of miRNA-182-5p significantly inhibited epithelial cell apoptosis and NLRP3/IL-1ß activation. In addition, we found that miRNA-182-5p could bind to the 3' untranscripted region of NOX4 mRNA and inhibit epithelial cell inflammation by reducing oxidative stress and mitochondrial damage. In vivo, miRNA-182-5p agomir treatment significantly reduced the percentage of eosinophils in bronchoalveolar lavage fluid, and down-regulated Th2 inflammatory factors, including IL-4, IL-5, and OVA induced IL-13. Meanwhile, miRNA-182-5p agomir reduced the peribronchial inflammatory cell infiltration, goblet cell proliferation and collagen deposition. In summary, targeting miRNA-182-5p may provide a new strategy for the treatment of asthma.

[DEK-targeting aptamer DTA-64 inhibits epithelial-mesenchymal transition of airway epithelial cells in bronchial asthmatic mice via blocking TGF-ß1 signaling pathway].

Objective To investigate the inhibitory effect of DEK targeting aptamer 64 (DTA-64) on airway inflammation and epithelial to mesenchymal transition (EMT) induced by ovalbumin (OVA) in asthmatic mice. Methods Thirty-two female BALB/c mice (8 weeks old) were randomly divided into PBS group, OVA model group, DTA-64 group (1 µg/mouse), and control aptamer group, with 8 in each. HE staining of lung tissues was used to detect inflammatory cell infiltration around the airways; immunohistochemical staining was used to detect DEK expression around the airways; ELISA was used to detect serum IgE, and Th2-type cytokines IL-4, IL-5, IL-13 and Th1-type cytokine IFN-γ in bronchoalveolar lavage fluid (BALF); Western blot was applied to detect the EMT-related proteins α-SMA, Snail+Slug, vimentin, and E-cadherin, and TGF-ß1/Smad, MAPK, PI3K, AKT, as well as mTOR in lung; and flow cytometry was used to observe the α-SMA expression in the lung single cell suspensions. Results DEK protein was highly expressed in the lung tissues of OVA group mice and decreased in the DTA-64 group mice; DTA-64 reduced the infiltration of eosinophils and neutrophils around the airways, down-regulated serum OVA-specific IgE and IL-4, IL-5, IL-13 in BALF, and up-regulated IFN-γ; DTA-64 also reduced the expressions of vimentin, α-SMA, Snail+Slug in the lung tissue, and up-regulated epithelial marker E-cadherin. DTA-64 inhibited the expressions of TGF-ß1 and its downstream canonical pathways Smad2/3 and Smad4, as well as the phosphorylation of non-canonical TGF-ß1 pathways ERK1/2, p38 MAPK, JNK and PI3K/AKT/mTOR. Conclusion DTA-64 may inhibit the airway inflammation and EMT induced by OVA in asthmatic mice via blocking TGF-ß1/Smad, MAPK and PI3K signaling pathways, thereby alleviating airway remodeling in asthma.

Sesamin Alleviates Asthma Airway Inflammation by Regulating Mitophagy and Mitochondrial Apoptosis.

Bronchial asthma poses a considerable burden on both individual patients and public health. Sesamin is a natural lignan that relieves asthma. However, the potential regulatory mechanism has not been fully validated. In this study, we revealed the mechanism of sesamin in inhibiting airway inflammation of asthma. In cockroach extract (CRE)-induced asthmatic mice, sesamin efficiently inhibited inflammatory cell infiltration, expressions of total and CRE-specific IgE in serum, and inflammatory cytokines (including IL-4, 5, 13) in bronchoalveolar lavage fluid. Further study revealed that sesamin inhibited Th2 cells in the mediastinal lymph nodes and spleen, the expression of PTEN-induced putative kinase 1 (PINK1) and Parkin, and apoptosis of lung airway epithelial cells. In vitro, sesamin had no significant cytotoxicity to BEAS-2B cells. Sesamin significantly increased TNF-α/IL-4-induced superoxide dismutase (SOD), catalase (CAT), heme oxygenase 1 (HO-1), and nuclear factor erythroid 2 related factor 2 (Nrf2), and decreased malondialdehyde. Sesamin also inhibited TNF-α/IL-4-induced mitochondrial reactive oxygen species, increased mitochondrial membrane potential, and reduced cell apoptosis as well as PINK1/Parkin expression and translocation to mitochondria. Conclusively, sesamin may relieve asthma airway inflammation by inhibiting mitophagy and mitochondrial apoptosis. Thus, sesamin may become a potential therapeutic agent for asthma.

miR-181-5p attenuates neutrophilic inflammation in asthma by targeting DEK.

We investigated the regulatory role of miR-181b-5p in neutrophilic asthma and its mechanisms by targeting DEK. DEK, matrix metalloproteinase (MMP)-2, and MMP-9 were overexpressed and the miR-181b-5p was decreased in mice with neutrophilic asthma. DEK was a direct target of miR-181b-5p. In mouse model, miR-181b-5p agomir had an inhibitory effect on airway inflammation and remodeling. miR-181b-5p inhibited DEK/p-GSK-3ßSer9/ß-catenin/MMP-9 pathway activation by regulating Wnt ligands in BEAS-2B and 16HBE cells. The ability of supernatants from human bronchial epithelial cells (hBECs) co-stimulated with CXCL8 (IL-8) and miR-181b-5p to induce NETs was weaker than that of IL-8 alone. Moreover, DEK overexpression led to excessive mitochondrial dysfunction, including DRP1 up-regulation, p-DRP1ser637 and MFN2 down-regulation, mitochondrial membrane potential loss, excessive mtROS generation and mitochondrial incompleteness. Interestingly, all these phenotypes were rescued by Wnt inhibitor DKK-1 and miR-181b-5p agomir. Additionally, inhibition of DRP1 with Mdivi-1 decreased MMP-9 on BEAS-2B cells. Overall, miR-181b-5p could attenuate neutrophilic asthma through inhibition of NETs release, DEK/p-GSK-3ßSer9/ß-catenin/MMP-9 pathway, DEK/Wnt/DRP1/MMP-9 and mitochondria damage. It may become a new therapeutic target for neutrophilic asthma.

Attenuation of antigen-induced airway hyperresponsiveness and inflammation in CXCR3 knockout mice.

BACKGROUND: CD8+ T cells participate in airway hyperresponsiveness (AHR) and allergic pulmonary inflammation that are characteristics of asthma. CXCL10 by binding to CXCR3 expressed preferentially on activated CD8+ T cells, attracts T cells homing to the lung. We studied the contribution and limitation of CXCR3 to AHR and airway inflammation induced by ovalbumin (OVA) using CXCR3 knockout (KO) mice. METHODS: Mice were sensitized and challenged with OVA. Lung histopathological changes, AHR, cellular composition and levels of inflammatory mediators in bronchoalveolar lavage (BAL) fluid, and lungs at mRNA and protein levels, were compared between CXCR3 KO mice and wild type (WT) mice. RESULTS: Compared with the WT controls, CXCR3 KO mice showed less OVA-induced infiltration of inflammatory cells around airways and vessels, and less mucus production. CXCR3 KO mice failed to develop significant AHR. They also demonstrated significantly fewer CD8+ T and CD4+ T cells in BAL fluid, lower levels of TNFα and IL-4 in lung tissue measured by real-time RT-PCR and in BAL fluid by ELISA, with significant elevation of IFNγ mRNA and protein expression levels. CONCLUSIONS: We conclude that CXCR3 is crucial for AHR and airway inflammation by promoting recruitment of more CD8+ T cells, as well as CD4+ T cells, and initiating release of proinflammatory mediators following OVA sensitization and challenge. CXCR3 may represent a novel therapeutic target for asthma.

Current status and preventive strategies of chronic obstructive pulmonary disease in China: a literature review.

Chronic obstructive pulmonary disease (COPD) is a common respiratory disease that seriously threatens human health and wellbeing, thereby representing an important public health problem. At present, it is the fourth leading cause of death worldwide, and is estimated to become the third greatest cause of death by 2030. In China, the prevalence of COPD is increasing, secondary to an increase in smoking, air pollution and an aging population, resulting in a current the mortality of COPD in China which is higher than the global average. Moreover, the disability-adjusted life year (DALY) rate of COPD in China is still relatively high, with an associated heavy economic burden to patients, their families and society. Unfortunately, current measures for treatment and prevention of COPD in China are not optimal. This primarily results from limited public awareness of COPD and pulmonary function tests amongst residents of China, and the generally poor disease-specific knowledge of primary care doctors. In recent years, a series of preventative strategies have been introduced in China across at the level of national policy, societies and associations, and scientific research. This review focuses upon both the epidemiology of COPD and the current status of preventative and treatment strategies in China.

Glaucocalyxin A Attenuates Allergic Responses by Inhibiting Mast Cell Degranulation through p38MAPK/NrF2/HO-1 and HMGB1/TLR4/NF-κB Signaling Pathways.

Glaucocalyxin A (GLA) has various pharmacological effects like antioxidation, immune regulation, and antiatherosclerosis. Here, in this study, the effect and mechanism of GLA on mast cell degranulation were studied. The results of the anti-DNP IgE-mediated passive cutaneous anaphylaxis (PCA) showed that GLA dramatically inhibited PCA in vivo, as evidenced by reduced Evans blue extravasation and decreased ear thickness. In addition, GLA significantly reduced the release of histamine and ß-hexosaminidase, calcium influx, cytokine (IL-4, TNF-α, IL-1ß, IL-13, and IL-8) production in the RBL-2H3 (rat basophilic leukemia cells), and RPMCs (peritoneal mast cells) in vitro. Moreover, we further investigated the regulatory mechanism of GLA on antigen-induced mast cells by Western blot, which showed that GLA inhibited FcεRI-mediated signal transduction and invalidated the phosphorylation of Syk, Fyn, Lyn, Gab2, and PLC-γ1. In addition, GLA inhibited the recombinant mouse high mobility group protein B1- (HMGB1-) induced mast cell degranulation through limiting nuclear translocation of NF-κBp65. Treatment of mast cells with siRNA-HMGB1 significantly inhibited HMGB1 levels, as well as MyD88 and TLR4, decreased intracellular calcium levels, and suppressed the release of ß-hexosaminidase. Meanwhile, GLA increased NrF2 and HO-1 levels by activating p38MAPK phosphorylation. Consequently, these data suggest that GLA regulates the NrF2/HO-1 signaling pathway through p38MAPK phosphorylation and inhibits HMGB1/TLR4/NF-κB signaling pathway to reduce mast cell degranulation and allergic inflammation. Our findings could be used as a promising therapeutic drug against allergic inflammatory disease.

Resistance mechanisms of epidermal growth factor receptor tyrosine kinase inhibitors in non-small cell lung cancer patients: A meta-analysis.

BACKGROUND: Differences in the resistance mechanisms of epidermal growth factor receptor tyrosine kinase inhibitors in patients with non-small cell lung cancer (NSCLC) harboring epidermal growth factor receptor mutations are unknown. This meta-analysis aimed to clarify the differences in resistance mechanisms after treatment with various epidermal growth factor receptor tyrosine kinase inhibitors. METHODS: We systematically searched PubMed, Cochrane, and Web of Science on July 29, 2020, for relevant studies on acquired resistance mechanisms against epidermal growth factor receptor tyrosine kinase inhibitors. The primary outcome measure was differences in the resistance mechanism between individual or generations of epidermal growth factor receptor tyrosine kinase inhibitors. RESULTS: In total, 33 trials involving 2418 individuals were included and analyzed. T790M was significantly less frequent after afatinib treatment (40.2%, 95% confidence interval [CI]: 31.7%-48.7%) than after gefitinib and erlotinib treatments (52.5%, 95% CI: 48.7%-56.3%, p = 0.005). There were no significant differences between Asian and non-Asian patients in the incidence of T790M after gefitinib, erlotinib, and afatinib treatments. Regarding epidermal growth factor receptor pathway-independent resistant mechanisms, the incidences of small cell lung cancer transformation (osimertinib: 7.9%, 95% CI: 3.6%-12.2%, others: 2.3%, 95% CI: 0.8%-3.8%) and Kirsten rat sarcoma (KRAS) viral oncogene homolog mutation (osimertinib: 4.6%, 95% CI: 1.5%-7.7%, others: 0.2%, 95% CI: 0.0%-1.7%) were significantly higher following osimertinib treatment than with others. CONCLUSIONS: Significant differences in the incidence of resistance mechanisms among epidermal growth factor receptor tyrosine kinase inhibitors exist, which should be taken into consideration when choosing the treatment strategy.

T-cell response to phytohemagglutinin in the interferon-γ release assay as a potential biomarker for the response to immune checkpoint inhibitors in patients with non-small cell lung cancer.

BACKGROUND: Immune checkpoint inhibitors are a standard treatment for advanced lung cancer, although it remains important to identify biomarkers that can accurately predict treatment response. Immune checkpoint inhibitors enhance the antitumor T-cell response, and interferon-γ plays an important role in this process. Therefore, this study evaluated whether the number of interferon-γ-releasing peripheral T cells after phytohemagglutinin stimulation in the interferon-γ release assay might act as a biomarker for the response of non-small cell lung cancer to immune checkpoint inhibitor treatment. METHODS: Data were retrospectively collected regarding 74 patients with non-small cell lung cancer who had received immune checkpoint inhibitors. Pretreatment screening tests had been performed using the T-SPOT.TB assay, which quantifies the number of interferon-γ-releasing T cells (as immunospots) in response to phytohemagglutinin and tuberculosis-specific antigen stimulation. Clinical factors and the number of spots in the T-SPOT fields were evaluated for associations with patient outcomes. The median number of spots was used to categorize patients as having high or low values, and the two groups were compared. RESULTS: Relative to patients with a low ratio, patients with a high ratio of phytohemagglutinin/tuberculosis-specific antigen spots (i.e. more responsive T cells) had significantly better progression-free survival after immune checkpoint inhibitor treatment. When we only considered patients with negative T-SPOT results, a high number of phytohemagglutinin-stimulated spots corresponded to significantly longer progression-free survival. CONCLUSION: The T-SPOT.TB assay can be used to quantify the number of immunospots in response to antigen stimulation, which may predict the response to immune checkpoint inhibitors in patients with non-small cell lung cancer.

Recombinant Pyrin Domain Protein Attenuates Airway Inflammation and Alleviates Epithelial-Mesenchymal Transition by Inhibiting Crosstalk Between TGFß1 and Notch1 Signaling in Chronic Asthmatic Mice.

This article aims to investigate the effects of recombinant pyrin domain (RPYD) on airway inflammation and remodeling in mice with chronic asthma. The chronic asthma BALB/c mouse model was first sensitized by ovalbumin (OVA) and then challenged by OVA nebulization. RPYD or dexamethasone was given before OVA challenge. Our results showed that RPYD significantly inhibited the increase of total cell number, eosinophils, neutrophils and lymphocytes in bronchoalveolar lavage fluid (BALF) induced by OVA, and reduced the infiltration of inflammatory cells, the proliferation of goblet cells and collagen deposition. In addition, RPYD inhibited the mRNA and protein levels of α-smooth muscle actin (α-SMA), transforming growth factor (TGF)-ß1, Jagged1, Notch1, Hes1 and Smad3, as well as Smad3 phosphorylation. TGFß1 down-regulated the level of E-cadherin and promoted the expression of α-SMA, thus inducing epithelial-mesenchymal transition (EMT) in bronchial epithelial cells. We found that RPYD reduced EMT by inhibiting TGFß1/smad3 and Jagged1/Notch1 signaling pathways. Further overexpression of NICD showed that under the stimulation of TGFß1, NICD enhanced the phosphorylated Smad3 and nuclear Smad3, accompanied by the increased expression of Notch1 target gene Hes1. In contrast, after treatment with smad3 siRNA, the expression of Hes1 was down regulated as the decrease of Smad3, which indicates that there is crosstalk between smad3 and NICD on Hes1 expression. In conclusion, RPYD reduces airway inflammation, improves airway remodeling and reduces EMT in chronic asthmatic mice by inhibiting the crosstalk between TGFß1/smad3 and Jagged1/Notch1 signaling pathways.

[Recombinant pyrin domain protein attenuates airway inflammation and airway remodeling through TGF-ß1/SMAD and Jagged1/Notch1 signaling pathways in chronic bronchial asthma mice].

Objective To investigate whether the recombinant pyrin domain protein can alleviate the airway inflammation and airway remodeling of OVA-induced mice with chronic asthma by inhibiting transforming growth factor ß1(TGF-ß1)/SMAD and Jagged1/Notch1 signaling pathways. Methods Thirty-two male BALB/c mice were selected and divided into 4 groups with 8 mice in each group. The four groups were the control group, OVA model group, recombinant pyrin domain protein treatment group (100 µg/kg), and the dexamethasone treatment group (1 mg/kg). Enzyme-linked immunosorbent assay (ELISA) was used to determine the expression of inflammatory factors in bronchoalveolar lavage fluid (BALF) of mice in each group. hematoxylin-eosin staining (HE) was used to observe the inflammatory infiltration of bronchus in mice. The changes of goblet cells were observed by periodic acid-Schiff (PAS) staining and collagen fibers by Masson staining. Immunohistochemical staining (IHC) was performed to observe the expression distribution of α smooth muscle actin (α-SMA), TGF-ß1 and Notch1 proteins in lung tissues. Western blotting was used to detect the protein levels of α-SMA, E-cadherin, TGF-ß1, SMAD2/3, SMAD7, Jagged1, Notch1 and Hes1 in lung tissues. Results The recombinant pyrin domain protein not only improved the airway inflammatory response of the OVA-induced mice with bronchial asthma, but also inhibited the hyperplasia of goblet cells and collagen fiber deposition, reduced the tumor necrosis factor α (TNF-α) in BALF, interleukin 1ß (IL-1ß), IL-4, IL-13 levels, and inhibited the protein expression of TGF-ß1, SMAD2/3, Jagged1, Notch1, Hes1 and α-SMA in lung tissues. Conclusion The recombinant pyrin domain protein can reduce the airway inflammation and airway remodeling of asthmatic mice by inhibiting TGF-ß1/SMAD and Jagged1/Notch1 signaling pathways.

Class A CpG oligodeoxynucleotide inhibits IFN-γ-induced signaling and apoptosis in lung cancer.

BACKGROUND: Currently, anticancer immunotherapy based on PD-1/PD-L1 blockade with immune checkpoint inhibitors (ICIs) is being used as a standard therapy for non-small cell lung cancer (NSCLC). However, more effective treatments are required as these tumors are often resistant and refractory. Here, we aimed to determine the effects of immunomodulatory oligodeoxynucleotides (ODNs) in terms of the presence or absence of CpG motifs and the number of consecutive guanosines. METHODS: Western blots were used to measure the molecules which regulate the expression of PD-L1 in human lung cancer cell lines after incubation with several cytokines and ODNs. The expression of PD-L1 and ß2-microglobulin (ß2-MG) on A549 cells, and IFN-γ-induced apoptosis with ODNs were examined by flow cytometry. The relationship between IFN-γ receptor and ODN was analyzed by ELISA and immunofluorescence chemistry. RESULTS: Our results verified that A-CpG ODNs suppress the upregulation of IFN-γ-induced PD-L1 and ß2-MG expression. In addition, we found that ODNs with six or more consecutive guanosines (ODNs with poly-G sequences) may competitively inhibit the IFN-γ receptor and abolish the effect of IFN-γ, thereby suppressing apoptosis and indoleamine 2,3-dioxygenase 1 expression in human lung cancer cells. The tumor microenvironment regulates whether this action will promote or suppress tumor immunity. Thus, in immunotherapy with CpG ODNs, it is essential to consider the effect of ODNs with poly-G sequences. CONCLUSIONS: This study suggests that ODNs containing six or more consecutive guanosines may inhibit the binding of IFN-γ to IFN-γ receptor. However, it does not directly show that ODNs containing six or more consecutive guanosines competitively inhibit the IFN-γ receptor, and further studies are warranted to confirm this finding. KEY POINTS: Significant findings of the study: Oligodeoxynucleotides with a contiguous sequence of six or more guanosines may competitively inhibit the IFN-γ receptor and abolish the action of IFN-γ. This may suppress IFN-γ-induced apoptosis and indoleamine-2,3-dioxygenase-1 expression in human lung cancer cells. WHAT THIS STUDY ADDS: A-CpG and poly-G ODN may overcome tolerance if the cause of ICI tolerance is high IDO expression. However, IFN-γ also has the effect of suppressing apoptosis of cancer cells, and it is necessary to identify the cause of resistance.

Identification of a novel biomarker based on lymphocyte count, albumin level, and TBAg/PHA ratio for differentiation between active and latent tuberculosis infection in Japan.

Data from China have shown that the ratio of Mycobacterium tuberculosis-specific antigen (TBAg) spots obtained from the T-SPOT.TB test to the number of positive control phytohemagglutinin (PHA) spots (TBAg/PHA ratio) could help distinguish between active tuberculosis infection (ATBI) and latent tuberculosis infection (LTBI). As the applicability of the T-SPOT.TB test may differ according to region and race, we retrospectively verified the utility of the TBAg/PHA ratio in distinguishing between ATBI and LTBI in Japan. The TBAg/PHA ratio was significantly lower in the LTBI group than in the ATBI group. Area under the receiver operating characteristic curve (AUC) analysis between ATBI and LTBI according to the TBAg/PHA ratio was 0.76, with a sensitivity of 65.8% and a specificity of 75.6%. The best AUC was obtained when the TBAg/PHA ratio was divided by both lymphocyte count and albumin levels. Our results demonstrate that, in Japan, the TBAg/PHA ratio is superior to TBAg alone for distinguishing between ATBI and LTBI. In addition, the sensitivity and specificity were improved by combining the TBAg/PHA ratio with lymphocyte count and albumin levels.

MicroRNA-200b is a potential biomarker of the expression of PD-L1 in patients with lung cancer.

BACKGROUND: Advanced non-small cell lung cancer (NSCLC) has a high mortality rate and poor prognosis. However, outcomes have gradually improved after the introduction of novel immunotherapies, including immune checkpoint inhibitors (ICIs). Although programmed death-ligand 1 (PD-L1) expression in tumor tissues is a known biomarker for guiding ICI treatment of NSCLC, challenges such as difficulty of liquid biopsy and heterogeneous results during treatment persist. This study evaluated the potential of miR200b as a surrogate biomarker for PD-L1 expression. METHODS: We used the human lung cancer cell lines H226, H460, H520, A549, and H1975. miR200b expression in blood and bronchoscopy specimens of NSCLC patients was evaluated using reverse-transcription-quantitative PCR. Using flow cytometry, PD-L1 expression in vitro, as well as in tumor tissues, was evaluated after transfection with a mimic miR200b or siRNA. RESULTS: miR200b expression negatively correlated with PD-L1 expression in all cell lines. The induction or knockdown of miR200b also altered PD-L1 expression in vitro. The patient group with a PD-L1 tumor proportion score ≥ 50% had significantly lower miR200b expression in the bronchoscopy specimens (P = 0.025) and serum-derived exosomes (P = 0.022) than that with PD-L1 tumor proportion score < 50%. CONCLUSIONS: miR200b can regulate PD-L1 expression in lung cancer cells, and miR200b expression in clinical specimens negatively correlated with PD-L1 expression. Thus, miR200b may be a useful surrogate biomarker for PD-L1 expression in lung cancer patients. KEY POINTS: SIGNIFICANT FINDINGS OF THE STUDY: High PD-L1 expression was linked to low miR200b expression, whereas low PD-L1 expression was linked to high miR200b expression in human lung cancer patients. Thus, miR200b overexpression or silencing can control PD-L1 expression in cancer cells. What this study adds We demonstrated the potential of miR200b as a surrogate biomarker for PD-L1 expression in lung cancer patients.