Recombinant Slit2 attenuates tracheal fibroblast activation in benign central airway obstruction by inhibiting the TGF-ß1/Smad3 signaling pathway.

Airway fibrosis is among the pathological manifestations of benign central airway obstruction noted in the absence of effective treatments and requires new drug targets to be developed. Slit guidance ligand 2-roundabout guidance receptor 1 (Slit2-Robo1) is involved in fibrosis and organ development. However, its significance in airway fibrosis has not yet been reported. The study explored how the recombinant protein Slit2 functions in transforming growth factor-ß1 (TGF-ß1)-mediated airway fibrosis in vivo and in vitro. In this study, Slit2 expression initially increased in the tracheal granulation tissues of patients with tracheobronchial stenosis but decreased in the fibrotic tissue. In primary rat tracheal fibroblasts (RTFs), recombinant Slit2 inhibited the expression of extracellular matrices such as Timp1, α-SMA, and COL1A2, whereas recombinant TGF-ß1 promoted the expression of Robo1, α-SMA, and COL1A2. Slit2 and TGF-ß1 played a mutual inhibitory role in RTFs. Slit2 supplementation and Robo1 downregulation inhibited excessive extracellular matrix (ECM) deposition induced by TGF-ß1 in RTFs via the TGF-ß1/Smad3 pathway. Ultimately, exogenous Slit2 and Robo1 knockdown-mediated attenuation of airway fibrosis were validated in a trauma-induced rat airway obstruction model. These findings demonstrate that recombinant Slit2 alleviated pathologic tracheobronchial healing by attenuating excessive ECM deposition. Slit2-Robo1 is an attractive target for further exploring the mechanisms and treatment of benign central airway obstruction.

Clinical Characteristics of Lung Abscess Caused by Streptococcus Constellatus Infection.

BACKGROUND: This study aimed to understand the clinical characteristics of pulmonary abscess caused by Streptococcus constellatus infection. METHODS: The clinical manifestations, laboratory examination, drug sensitivity, chest CT manifestations, and treatment and prognosis of patients with pulmonary abscess caused by Streptococcus constellatus infection were retrospectively collected and analyzed. RESULTS: A total of 9 cases of pulmonary abscess caused by Streptococcus constellatus infection were confirmed; one case was confirmed by traditional cultures, while metagenomic next-generation sequencing (mNGS) confirmed the other 8 cases. All of the 9 patients had different degrees of cough, sputum, fever, chest pain, and/or dyspnea, and the physical examination showed fast breathing, reduced respiratory sound, or moist rales on the affected side. In laboratory tests, 8 patients had elevated white blood cells and hypoproteinemia upon admission. Blood gas analysis showed an oxygenation index < 300. The antimicrobial susceptibility testing results in 1 patient with culture-confirmed pathogen diagnosis showed that Streptococcus constellatus was susceptible to ampicillin, penicillin G, cefotaxime, ceftriaxone, cefepime, meropenem, chloramphenicol, linezolid, levofloxacin, and vancomycin and resistant to tetracycline and clindamycin. Relevant antibiotic resistance genes were not detected by mNGS in the 8 patients with negative culture and positive mNGS results. A chest CT showed lung consolidation or cavity formation in 9 patients admitted to the hospital, and 5 patients had pleural effusion. 3 cases were admitted to the respiratory intensive care unit (RICU) and 6 cases were admitted to the general ward. There were 3 cases of nasal catheter oxygen inhalation, 1 case of mask oxygen inhalation, and 5 cases of non-invasive ventilator assisted ventilation. All patients received penicillin or respiratory quinolones anti-infection therapy, and 3 cases were treated with a thoracic closed drainage tube. All patients were discharged from the hospital after improvement, and the hospital stay was 15 - 23 days. CONCLUSIONS: Patients with pulmonary abscess caused by Streptococcus constellatus infection have an urgent condition and rapid progression. It is helpful to use mNGS combined with traditional culture as soon as possible to identify the pathogenic bacteria. Penicillin antibiotics should be the first choice for pulmonary abscess caused by a suspected Streptococcus constellatus infection. If a patient´s condition worsens during the treatment, especially for patients who have lesions involving the interlobar fissure or pleura, compressive atelectasis caused by pleural fluid formation or an increase in the amount of pleural effusion needs to be highly suspected.

GDF15 alleviates the progression of benign tracheobronchial stenosis by inhibiting epithelial-mesenchymal transition and inactivating fibroblasts.

Benign tracheobronchial stenosis (BTS) is a fatal and incurable disease. Epithelial repair and matrix reconstruction play an important role in the wound repair process. If the interstitial context is not restored and stabilized in time, it can lead to pathological fibrosis. Here we attempted to identify cytokines that are involved in promoting wound repair. Growth differentiation factor 15 (GDF15) is a cytokine secreted by tracheal epithelial cells, which is indispensable for the growth of epithelial cells and inhibits the overgrowth of fibroblasts. GDF15 can counteract transforming growth factor-ß (TGFß1) stimulation of epithelial-mesenchymal transition (EMT) in tracheal epithelial cells and inhibit fibroblast activation via the TGFß1-SMAD2/3 pathway. In a rat model of tracheal stenosis, GDF15 supplementation alleviated the degree of tracheal stenosis. These results suggest that GDF15 prevents fibroblast hyperactivation and promotes epithelial repair in injured trachea. GDF15 may be a potential therapy to improve benign tracheobronchial stenosis.

Characterization and classification of Bo4 as a cluster G mycobacteriophage that can infect and lyse M. tuberculosis.

Mycobacteriophage therapy is a potential alternative treatment for Mycobacterium tuberculosis infection. Here, we further characterized a mycobacteriophage, Bo4, and evaluated its ability to infect and kill M. tuberculosis. We first found that Bo4 can infect M. tuberculosis and Mycobacterium smegmatis. The observed clear plaques created by Bo4 infection indicated that Bo4 might be a lytic phage able to lyse mycobacterial strains, which was confirmed by phage antimicrobial activity. Bo4 formed clear zones in a medium with pH values of 7.4 or 5.0, suggesting the possibility that Bo4 could lyse mycobacteria, such as M. tuberculosis, in blood as well as in lysosomal macrophages. Further investigation into the Bo4 genome revealed that Bo4 had a dsDNA genome. Moreover, Bo4 contained ~39,318 bp comprised of 66.76 % G+C content. Complete genome sequencing showed high nucleotide identity with cluster G mycobacteriophages, thus classifying Bo4 as a member of the cluster G family. Additionally, annotation of the Bo4 genome indicated that it was a lytic bacteriophage and did not contain any harmful genes that increased mycobacterial virulence or decreased human immunity. Overall, the results of investigation indicate that the Bo4 possesses the potential to destroy M. tuberculosis, making it a potentially useful tool for diagnosing and treating tuberculosis.

Titer dynamic analysis of D29 within MTB-infected macrophages and effect on immune function of macrophages.

The use of mycobacteriophage D29 to treat Mycobacterium tuberculosis (MTB)-infected macrophages results in significant inhibitory activity. This study aims to explore the novel treatment strategy of intracellular mycobacterial infection from the point of view of phages. We investigated the dynamic phagocytosis and elimination of D29 by macrophages, measured the titer of D29 inside and outside MTB within macrophages by fluorescence quantitative PCR, and detected the levels of interleukin 12 (IL-12) and nitric oxide (NO) in the culture supernatants of D29-infected macrophages by ELISA. Results showed that the activity of D29 phagocytosed by macrophages was significantly lower than that of D29 phagocytosed by MTB-infected macrophages. The titer of D29 that infected intracellular MTB ranged from 10(9) pfu to 10(4) pfu. The titer of D29 inside and outside intracellular MTB transiently increased when MTB-infected macrophages were incubated with D29 for 40 and 50 min; then, a large number of D29 were eliminated by macrophages. The levels of IL-12 and NO had no significant differences versus the negative control but were significantly lower compared with the lipopolysaccharide (LPS) positive control. These results suggest D29 has no effect on the immune function of macrophages and that high phage titer must be administered repeatedly if D29 is applied to treat intracellular MTB infection.

IL-11 drives the phenotypic transformation of tracheal epithelial cells and fibroblasts to enhance abnormal repair after tracheal injury.

Tracheal stenosis (TS) is a multifactorial and heterogeneous disease that can easily lead to respiratory failure and even death. Interleukin-11 (IL-11) has recently received increased attention as a fibrogenic factor, but its function in TS is uncertain. This study aimed to investigate the role of IL-11 in TS regulation based on clinical samples from patients with TS and a rat model of TS produced by nylon brush scraping. Using lentiviral vectors expressing shRNA (lentivirus-shRNA) targeting the IL-11 receptor (IL-11Rα), we lowered IL-11Rα levels in the rat trachea. Histological and immunostaining methods were used to evaluate the effects of IL-11Rα knockdown on tracheal injury, molecular phenotype, and fibrosis in TS rats. We show that IL-11 was significantly elevated in circulating serum and granulation tissue in patients with TS. In vitro, TGFß1 dose-dependently stimulated IL-11 secretion from human tracheal epithelial cells (Beas-2b) and primary rat tracheal fibroblasts (PRTF). IL-11 transformed the epithelial cell phenotype to the mesenchymal cell phenotype by activating the ß-catenin pathway. Furthermore, IL-11 activated the atypical ERK signaling pathway, stimulated fibroblasts proliferation, and transformed fibroblasts into alpha-smooth muscle actin (α-SMA) positive myofibroblasts. IL-11-neutralizing antibodies (IL-11NAb) or ERK inhibitors (U0126) inhibited IL-11 activity and downregulated fibrotic responses involving TGFß/SMAD signaling. In vivo, IL-11Rα knockdown rats showed unobstructed tracheal lumen, relatively intact epithelial structure, and significantly reduced granulation tissue proliferation and collagen fiber deposition. Our findings confirm that IL-11 may be a target for future drug prevention and treatment of tracheal stenosis.

Estradiol inhibits autophagy of Mycobacterium tuberculosis­infected 16HBE cells and controls the proliferation of intracellular Mycobacterium tuberculosis.

Tracheobronchial tuberculosis (TBTB) is most common in young, middle­aged females. Despite adequate anti­tuberculosis therapy, >90% of patients develop tracheobronchial stenosis, which has a high rate of resulting in disability. The present study aimed to explore the effect of estradiol on the development of TBTB. Estrogen receptor (ER) expression in granulomatous tissue was assessed via immunofluorescence. In order to determine whether estrogen affected the proliferation of intracellular Mycobacterium tuberculosis (Mtb), 16HBE cells were infected with Mtb in vitro, followed by estradiol treatment. Intracellular Mtb was quantified via colony counting. The effect of estradiol on autophagy of infected 16HBE cells was determined via western blotting and transmission electron microscopy. Necrosis assays of infected 16HBE cells were analyzed using propidium iodide staining and assessing lactate dehydrogenase (LDH) release. To determine how estradiol affects autophagy, infected 16HBE cells were treated with ER­specific and non­specific modulators. Reactive oxygen species (ROS) levels were analyzed via flow cytometry. Additionally, the protein expression levels of autophagy­associated proteins were determined via western blotting. Mtb could enter human lobar bronchial goblet cells and ciliated cells in patients with TBTB. The results also demonstrated that ERα was expressed in granulomatous tissue from patients with TBTB. Administration of 10­6 M estradiol reduced the number of intracellular Mtb colony­forming units in vitro in the 16HBE human bronchial epithelial cell line at day 3 after infection. Furthermore, cells treated with estradiol and infected with Mtb released less LDH at 72 h and exhibited reduced necrosis levels at 24 h compared with the untreated cells. In addition, autophagy of infected 16HBE cells was inhibited by estradiol. Estradiol and the specific ERα agonist had similar effects on autophagy in infected 16HBE cells. Additionally, treatment with the ERα antagonist abolished the inhibition of autophagy by estradiol in infected 16BHE cells. Compared with the untreated infected 16HBE cells, the ROS levels in the infected 16HBE cells treated with estradiol and the ERα agonist significantly decreased. The levels of phosphorylated (p)­mTOR and p­AKT notably increased in estradiol­ and ERα agonist­treated infected 16HBE cells. In summary, estradiol may serve a key role in the development of TBTB through binding to ERα.

Knockdown of SOX9 alleviates tracheal fibrosis through the Wnt/ß-catenin signaling pathway.

Trachealfibrosis is an important cause of tracheal stenosis without effective treatments, and new drug targets need to be developed. The role of SOX9 in the injury and repair of the trachea is unknown; this study aims to investigate the role of SOX9 in the regulation of tracheal fibrosis based on clinical samples from patients with tracheal injury and a model of tracheal fibrosis produced by tracheal brushing in rats. The results showed that the expressions of SOX9 and mesenchymal and ECM-related indicators were increased in the injury and fibrosis of the trachea in patients and rats. Serum SOX9 levels exhibited a sensitivity of 83.87% and specificity of 90% in distinguishing patients with tracheal fibrosis from healthy volunteers when the cut­off value was 13.24 ng/ml. Knockdown SOX9 can markedly inhibit granulation tissue proliferation, reduce inflammation and ECM deposition, promote epithelial regeneration and granulation tissue apoptosis, and attenuate the tracheal fibrosis after injury. Additionally, RNA sequencing showed that the proliferation, migration, and ECM deposition of tracheal granulation tissue were related to the activation of Wnt pathway, activation of the ß-catenin, and p-GSK3ß after injury can be inhibited by the knockdown of SOX9. In summary, SOX9 is upregulated in tracheas fibrosis and may be a novel factor to promote tracheal fibrosis progression. Inhibiting SOX9 may be used to prevent and treat tracheal fibrosis in the future. KEY MESSAGE : The expression of SOX9 is upregulated the process of injury and repair of the tracheal fibrosis. Knocking down SOX9 can attenuate tracheal fibrosis after injury by inhibiting inflammation response, granulation tissue proliferation, ECM deposition, and promoting granulation tissue apoptosis. The Wnt/ß-catenin-SOX9 axis is activated during tracheal injury and fibrosis, and inhibition of SOX9 can partially alleviate tracheal fibrosis. SOX9 may act as a new diagnostic and therapeutic target in patients with tracheal fibrosis in the future.

Clinical significance of serum transforming growth factor-ß1 and procollagen type I N-propeptide in post-tuberculosis tracheobronchial stenosis.

Non-invasive strategies for monitoring post-tuberculosis (TB) tracheobronchial stenosis (PTTS) are clinically important but currently lacking. Transforming growth factor-ß1 (TGF-ß1) and procollagen type I N-propeptide (PINP) have been identified as markers of fibrosis. The present study aimed to investigate the clinical significance of serum TGF-ß1 and PINP in PTTS. Serum samples were collected from 119 patients with tracheobronchial TB after the condition was treated for at least 6 months (59 patients with airway stenosis and 60 patients with no stenosis). Serum TGF-ß1 and PINP levels were measured using ELISA and compared between the groups. Relationships between serum TGF-ß1 and PINP levels and clinical characteristics, interventional bronchoscopy and outcomes of airway stenosis were analysed. The correlation between TGF-ß1 and PINP, and their diagnostic efficacy for airway stenosis were also analysed. The TGF-ß1 and PINP levels in the airway stenosis group were higher than those in the non-stenosis group. Furthermore, airway stenosis with atelectasis or mucus plugging was associated with higher TGF-ß1 levels, and airway stenosis with atelectasis, mucus plugging, right main bronchus stenosis or severe airway tracheal stenosis was associated with higher PINP levels. In addition, TGF-ß1 and PINP levels increased after interventional bronchoscopy therapy and airway stenosis with recurrent stenosis was associated with higher baseline levels of both markers. Finally, TGF-ß1 levels were positively correlated with PINP levels in patients with airway stenosis. The area under the receiver operating characteristic curve of TGF-ß1 and PINP for distinguishing airway stenosis from non-stenosis cases was 0.824 (95% CI: 0.748-0.900) and 0.863 (95% CI: 0.796-0.930), respectively. Therefore, TGF-ß1 and PINP are potential biomarkers that may be useful for diagnosing and monitoring PTTS.

Exogenous intrapleural injection of interleukin-27 may improves outcome and prognosis in patients with tuberculous pleural effusion.

We hypothesize that exogenous intrapleural injection of interleukin-27 may improve outcome and prognosis in patients with tuberculous pleural effusion (TPE). Studies have found that the balance of Th1/Th2 determines the development trend of TPE. High concentrations of IFN-γ and TNF-α in pleural effusion are associated with pleural adhesion in patients with TPE. Interleukin-27 is a member of the IL-12 family, and IL-27 has a dual regulatory effect on Th1 immunity. On one hand, IL-27 can promote the initial CD4+ T cell proliferation by inducing the expression of T-bet, IL-12Rß2 and ICAM-1 in the initial CD4+ T cells, and also promote its differentiation into Th1 cells and IFN-γ production in the early infection. On the other hand, in the case of high Th1 polarization, IL-27 induced STAT3 phosphorylation and inhibited TNF and IL-12 production in activated peritoneal macrophages, indicating a novel feedback mechanism by which IL-27 can modulate excessive inflammation, thereby preventing damage to the body caused by excessive immune response. Studies haves confirmed that after stimulation of antigen by mononuclear cells in TPE, the Th1 and Th2 cell subsets and Th1/Th2 ratio markedly increase, and the increase of Th1 is more obvious than that of Th2. Therefore, compared to patients with TPE in the high-level IL-27 group, we hypothesized that pleural effusion is absorbed more slowly, pleural thickening is more obvious, pleural adhesions are more extensive, and the incidence of thoracic collapse is higher in the low-level IL-27 group under the same conditions of anti-tuberculosis treatment. However, exogenous intrapleural injection of IL-27 may induce Stat3 phosphorylation and inhibit TNF and IL-12 production, finally reduces the secretion of IFN-γ and TNF-α. This negative regulation inhibits the excessive inflammatory reaction caused by tuberculosis infection, reduces pleural adhesion, pleural thickening and local pleural tissue damage, thereby improving the prognosis of patients.

The dormant cells of Mycobacterium tuberculosis may be resuscitated by targeting-expression system of recombinant mycobacteriophage-Rpf: implication of shorter course of TB chemotherapy in the future.

Here we hypothesized that dormant cells of Mycobacterium tuberculosis (M. tuberculosis) may be resuscitated by a new expression system of recombinant mycobacteriophage-resuscitation-promoting factor (Rpf). In this system, gene of targeted Rpf was cloned into mycobacteriophage genome, since mycobacteriophages possess several characteristics, including automatic identification and specific infection of M. tuberculosis. Thus the targeted delivery and endogenous expression of Rpf to the infected area of M. tuberculosis can be realized, followed by resuscitating the dormant cells of M. tuberculosis. Finally, these resuscitated M. tuberculosis can be thoroughly killed by a strong short-term subsequent chemotherapy, which makes the course of TB chemotherapy much shorter in the future compared to simple chemotherapy. Early studies have confirmed that dormant cells of M. tuberculosis can be resuscitated by Rpf in vitro, but so far, there is no report that Rpf can succeed in resuscitating dormant cells of M. tuberculosis in vivo, the reason may be that it is difficult for purified Rpf to remain active in vivo, especially to achieve targeted delivery of exogenous Rpf to the infected area of dormant cells of M. tuberculosis. Mycobacteriophage is a virus, capable of specifically identifying and infecting mycobacterium, such as M. tuberculosis. Several studies show that motif 3-containing proteins have peptidoglycan-hydrolysing activity and that while this activity is not required for mycobacteriophage viability, it facilitates efficient infection and DNA injection of mycobacteriophage (including motif 3 protein) into stationary phase cells. Thus this expression system can achieve targeted delivery and endogenous expression of Rpf to infected area of dormant cells of M. tuberculosis. Finally, we discuss the implication of this recombinant expression system for shortening the course of TB chemotherapy.

Controlling strategy of dormant Mycobacterium tuberculosis.

OBJECTIVE: This study aimed to review the available literatures on control of latent tuberculosis (TB) infection and propose a new control strategy to shorten the course of TB chemotherapy. DATA SOURCES: The data used in this review were mainly obtained from articles listed in PubMed. The search terms were "therapy (treatment) of tuberculosis," "therapy (treatment) of latent TB infection," and "vaccine of TB." STUDY SELECTION: Articles regarding treatment and vaccine of TB were selected and reviewed. RESULTS: The most crucial reason causing the prolonged course of TB chemotherapy is the dormant state of Mycobacterium tuberculosis (M. tuberculosis). Nevertheless, there are, to date, no effective drugs that can directly kill the dormant cells of M. tuberculosis in clinical therapy. In accordance with the growth cycle of dormant M. tuberculosis in the body, the methods for controlling dormant M. tuberculosis include direct killing with drugs, prevention of dormant M. tuberculosis resuscitation with vaccines, and resuscitating dormant M. tuberculosis with preparations or drugs and then thoroughly killing these resuscitated M. tuberculosis by using anti-TB therapy. CONCLUSIONS: The comprehensive analysis of the above three methods suggests that the drugs directly killing dormant cells are in clinical trials, TMC207 is the most beneficial for controlling TB. Because the side effect of vaccines is less and their action period is long, prevention of dormant cells resuscitation with vaccines is promising. The last control method makes it probable that when a huge number of active cells of M. tuberculosis have been killed and eradicated after 1-month short chemotherapy, only a strong short-term subsequent chemotherapy can completely kill and eradicate the remaining M. tuberculosis. This control strategy is expected to significantly shorten the course of TB chemotherapy and bring a new change and breakthrough in TB treatment.