Dachengqi decoction dispensing granule ameliorates LPS-induced acute lung injury by inhibiting PANoptosis in vivo and in vitro.

ETHNOPHARMACOLOGICAL RELEVANCE: Acute lung injury (ALI) is a serious health-threatening syndrome of intense inflammatory response in the lungs, with progression leading to acute respiratory distress syndrome (ARDS). Dachengqi decoction dispensing granule (DDG) has a pulmonary protective role, but its potential modulatory mechanism to alleviate ALI needs further excavation. AIM OF THE STUDY: This study aims to investigate the effect and potential mechanism of DDG on lipopolysaccharide (LPS)-induced ALI models in vivo and in vitro. MATERIALS AND METHODS: LPS-treated Balb/c mice and BEAS-2B cells were used to construct in vivo and in vitro ALI models, respectively. Hematoxylin-eosin (HE), Wet weight/Dry weight (W/D) calculation of lung tissue, and total protein and Lactic dehydrogenase (LDH) assays in BALF were performed to assess the extent of lung tissue injury and pulmonary edema. Enzyme-linked immunosorbent assay (ELISA) was used to detect the levels of tumor necrosis factor-alpha (TNF-α), interleukin-1ß (IL-1ß), and interleukin-18 (IL-18) in BALF, serum, and cell supernatant. The qRT-PCR was used to detect inflammatory factors, Z-DNA binding protein 1 (ZBP1), and receptor-interacting protein kinase 1 (RIPK1) expression in lung tissues and BEAS-2B cells. Double immunofluorescence staining and co-immunoprecipitation were used to detect the relative expression and co-localization of ZBP1 and RIPK1. The effects of LPS and DDG on BEAS-2B cell activity were detected by Cell Counting Kit-8 (CCK-8). Western blot (WB) was performed to analyze the expression of PANoptosis-related proteins in lung tissues and BEAS-2B cells. RESULTS: In vivo, DDG pretreatment could dose-dependently improve the pathological changes of lung tissue in ALI mice, and reduce the W/D ratio of lung, total protein concentration, and LDH content in BALF. In vitro, DDG reversed the inhibitory effect of LPS on BEAS-2B cell viability. Meanwhile, DDG significantly reduced the levels of inflammatory factors in vitro and in vivo. In addition, DDG could inhibit the expression levels of PANoptosis-related proteins, especially the upstream key regulatory molecules ZBP1 and RIPK1. CONCLUSION: DDG could inhibit excessive inflammation and PANoptosis to alleviate LPS-induced ALI, thus possessing good anti-inflammatory and lung-protective effects. This study establishes a theoretical basis for the further development of DDG and provides a new prospect for ALI treatment by targeting PANoptosis.

Shuangdan Jiedu Decoction improved LPS-induced acute lung injury by regulating both cGAS-STING pathway and inflammasome.

ETHNOPHARMACOLOGICAL RELEVANCE: Shuangdan Jiedu Decoction (SJD) is a formula composed of six Chinese herbs with heat-removing and detoxifying, antibacterial, and anti-inflammatory effects, which is clinically used in the therapy of various inflammatory diseases of the lungs including COVID-19, but the therapeutic material basis of its action as well as its molecular mechanism are still unclear. AIM OF THE STUDY: The study attempted to determine the therapeutic effect of SJD on LPS-induced acute lung injury (ALI), as well as to investigate its mechanism of action and assess its therapeutic potential for the cure of inflammation-related diseases in the clinical setting. MATERIALS AND METHODS: We established an ALI model by tracheal drip LPS, and after the administration of SJD, we collected the bronchoalveolar lavage fluid (BALF) and lung tissues of mice and examined the expression of inflammatory factors in them. In addition, we evaluated the effects of SJD on the cyclic guanosine monophosphate-adenosine monophosphate synthase -stimulator of interferon genes (cGAS-STING) and inflammasome by immunoblotting and real-time quantitative polymerase chain reaction (RT-qPCR). RESULTS: We demonstrated that SJD was effective in alleviating LPS-induced ALI by suppressing the levels of pro-inflammatory cytokines in the BALF, improving the level of lung histopathology and the number of neutrophils, as well as decreasing the inflammatory factor-associated gene expression. Importantly, we found that SJD could inhibit multiple stimulus-driven activation of cGAS-STING and inflammasome. Further studies showed that the Chinese herbal medicines in SJD had no influence on the cGAS-STING pathway and inflammasome alone at the formulated dose. By increasing the concentration of these herbs, we observed inhibitory effects on the cGAS-STING pathway and inflammasome, and the effect exerted was maximal when the six herbs were combined, indicating that the synergistic effects among these herbs plays a crucial role in the anti-inflammatory effects of SJD. CONCLUSIONS: Our research demonstrated that SJD has a favorable protective effect against ALI, and its mechanism of effect may be associated with the synergistic effect exerted between six Chinese medicines to inhibit the cGAS-STING and inflammasome abnormal activation. These results are favorable for the wide application of SJD in the clinic as well as for the development of drugs for ALI from herbal formulas.

Comparing the diagnostic value of targeted with metagenomic next-generation sequencing in immunocompromised patients with lower respiratory tract infection.

BACKGROUND: Accurate identification of the etiology of lower respiratory tract infections (LRTI) is crucial, particularly for immunocompromised patients with more complex etiologies. The advent of next-generation sequencing (NGS) has enhanced the effectiveness of pathogen detection. However, assessments of the clinical diagnostic value of targeted NGS (tNGS) in immunocompromised patients with LRTI are limited. METHODS: To evaluate the diagnostic value of tNGS in immunocompromised patients with LRTI, a total of 88 patients, of whom 54 were immunocompromised, were enrolled. These patients underwent tNGS testing of bronchoalveolar lavage fluid (BALF). Results from both metagenomic next-generation sequencing (mNGS) and conventional microbiological tests (CMT) were also available for all participants. The performance of tNGS was assessed by comparing its findings against mNGS, CMT, and the clinical composite diagnosis. RESULTS: In the cohort of 88 patients, tNGS showed comparable diagnostic value to mNGS and was significantly superior to CMT. Compared to CMT and composite reference standard, tNGS showed sensitivity of 94.55% and 90.48%, respectively. In immunocompromised patients, despite a more diverse pathogen variety, tNGS maintained similar sensitivity to mNGS and outperformed CMT. tNGS positively influenced etiologic diagnosis and antibiotic decision-making in 72.72% of cases, leading to a change in antibiotic regimen in 17.05% of cases. We also compared the detection of microbial nucleic acids by tNGS with mNGS and found that tNGS could identify 87.99% of the microbial nucleic acids identified by mNGS. CONCLUSION: In summary, our study demonstrated that tNGS offers promising clinical diagnostic accuracy in immunocompromised patients, as evidenced by its favorable comparison with CMT, the composite reference standard, and mNGS.

Aconitate decarboxylase 1 mediates the acute airway inflammatory response to environmental exposures.

Background: Environmental lipopolysaccharide (LPS) and microbial component-enriched organic dusts cause significant lung disease. These environmental exposures induce the recruitment and activation of distinct lung monocyte/macrophage subpopulations involved in disease pathogenesis. Aconitate decarboxylase 1 (Acod1) was one of the most upregulated genes following LPS (vs. saline) exposure of murine whole lungs with transcriptomic profiling of sorted lung monocyte/macrophage subpopulations also highlighting its significance. Given monocyte/macrophage activation can be tightly linked to metabolism, the objective of these studies was to determine the role of the immunometabolic regulator ACOD1 in environmental exposure-induced lung inflammation. Methods: Wild-type (WT) mice were intratracheally (i.t.) instilled with 10 µg of LPS or saline. Whole lungs were profiled using bulk RNA sequencing or sorted to isolate monocyte/macrophage subpopulations. Sorted subpopulations were then characterized transcriptomically using a NanoString innate immunity multiplex array 48 h post-exposure. Next, WT and Acod1-/- mice were instilled with LPS, 25% organic dust extract (ODE), or saline, whereupon serum, bronchoalveolar lavage fluid (BALF), and lung tissues were collected. BALF metabolites of the tricarboxylic acid (TCA) cycle were quantified by mass spectrometry. Cytokines/chemokines and tissue remodeling mediators were quantitated by ELISA. Lung immune cells were characterized by flow cytometry. Invasive lung function testing was performed 3 h post-LPS with WT and Acod1-/- mice. Results: Acod1-/- mice treated with LPS demonstrated decreased BALF levels of itaconate, TCA cycle reprogramming, decreased BALF neutrophils, increased lung CD4+ T cells, decreased BALF and lung levels of TNF-α, and decreased BALF CXCL1 compared to WT animals. In comparison, Acod1-/- mice treated with ODE demonstrated decreased serum pentraxin-2, BALF levels of itaconate, lung total cell, neutrophil, monocyte, and B-cell infiltrates with decreased BALF levels of TNF-α and IL-6 and decreased lung CXCL1 vs. WT animals. Mediators of tissue remodeling (TIMP1, MMP-8, MMP-9) were also decreased in the LPS-exposed Acod1-/- mice, with MMP-9 also reduced in ODE-exposed Acod1-/- mice. Lung function assessments demonstrated a blunted response to LPS-induced airway hyperresponsiveness in Acod1-/- animals. Conclusion: Acod1 is robustly upregulated in the lungs following LPS exposure and encodes a key immunometabolic regulator. ACOD1 mediates the proinflammatory response to acute inhaled environmental LPS and organic dust exposure-induced lung inflammation.

[Changes of lung function and inflammatory factors in rat models of coal workers' pneumoconiosis].

Objective: To observe the changes of lung function and inflammatory factors in rat models of coal workers' pneumoconiosis at different time points. Methods: In June 2021, 96 healthy male SD rats with SPF grade were divided into 1, 3, and 6-month control group and dust staining group (coal dust group, coal silica dust group, quartz group) according to random number table method, with 8 rats in each group. After one week of adaptive feeding, a one-time non-exposed tracheal perfusion method (1 ml/ piece) was used. The dust dyeing group was given 50 g/L coal dust, coal silica mixed dust and quartz dust suspension, respectively, and the control group was given 0.9% normal saline solution. At 1, 3 and 6 months after perfusion, lung function was detected by animal lung function apparatus, then all lung tissues and alveolar lavage fluid were killed, and lung histopathological morphological changes were observed by HE staining, and the contents of interleukin (IL-1ß), IL-18, IL-4 and IL-10 in alveolar lavage fluid were detected by ELISA. One-way analysis of variance was used to compare groups. Two factors (inter-group treatment factor (4 levels) and observation time factor (3 levels) ) were used in the analysis of the effects of inter-group treatment and treatment time on related indicators. Results: HE staining results showed that coal spot appeared in the lung tissue of coal dust group, coal spot and coal silicon nodule appeared in the lung tissue of coal dust group, and silicon nodule appeared in the lung tissue of quartz group. Compared with the control group, the forced vital capacity (FVC) and forced expiratory volume at 0.2 second (FEV(0.2)) of rats in the dust staining group had interaction between the treatment and treatment time (P<0.05). With the increase of dust dyeing time, FVC and FEV(0.2) decreased significantly at 3-6 months of dust dyeing, and the maximum gas volume per minute (MVV) decreased significantly at 1-3 months of dust dyeing (P<0.05). The lowest lung function index was in quartz group, followed by coal-silica group and coal-dust group. There were statistically significant differences in the main effect and interaction effect of the pro-inflammatory factor IL-18 among all groups in treatment and treatment time (IL-18: F=70.79, 45.97, 5.90, P<0.001), and interaction existed. The highest content of inflammatory factors in alveolar lavage fluid of all dust groups was quartz group, followed by coal silica group and coal dust group. There were significant differences in the main effect and interaction effect of anti-inflammatory factors between groups and treatment time (IL-4: F=41.55, 33.01, 5.23, P<0.001, <0.001, <0.001; IL-10: F=7.46, 20.80, 2.91, P=0.002, <0.001, 0.024), and there was interaction. The highest content of anti-inflammatory factor was in quartz group, followed by coal silica group and coal dust group. Conclusion: Lung function decreased and levels of inflammatory fators increased in rat models of coal workers' pneumoconiosis, with the quartz group being the most severely damaged. Lung function is mainly impaired in thrid-six months, and the content of inflammatory factors begins to change in first-thrid months. MVV are the earliest and most obvious in lung function. IL-18 is suitable for monitoring changes in the pro-inflammatory response of coal workers' pneumoconiosis, and IL-10 is suitable for monitoring changes in anti-inflammatory response.

The storage mite Tyrophagus putrescentiae induces greater lung inflammation than house dust mites in mice.

Exposure to storage mite (SM) and house dust mite (HDM) allergens is a risk factor for sensitization and asthma development; however, the related immune responses and their pathology have not been fully investigated. The HDMs Dermatophagoides farinae and Dermatophagoides pteronyssinus and SM Tyrophagus putrescentiae are potent allergens that induce asthma. Most SM-related studies have focused on the allergic reactions of individuals by measuring their immunoglobulin (Ig)E expression. Considering the limited research on this topic, the present study aims to investigate the differences in the immune responses induced by HDMs and SMs and histologically analyze lung tissues in a mouse asthma model to understand the differential effects of HDM and SM. The results revealed that all mite species induced airway inflammation. Mice challenged with T. putrescentiae had the highest airway resistance and total cell, eosinophil, and neutrophil counts in the bronchoalveolar lavage fluid (BALF). The SM-sensitized groups showed more severe lesions and mucus hypersecretions than the HDM-sensitized groups. Although the degree of HDM and SM exposure was the same, the damage to the respiratory lung tissue was more severe in SM-exposed mice, which resulted in excessive mucin secretion and increased fibrosis. Furthermore, these findings suggest that SM sensitization induces a more significant hypersensitivity response in mucosal immunity than HDM sensitization in asthma models.

Metagenomic next-generation sequencing targeted and metagenomic next-generation sequencing for pulmonary infection in HIV-infected and non-HIV-infected individuals.

Background: When individuals infected with human immunodeficiency virus (HIV) experience pulmonary infections, they often exhibit severe symptoms and face a grim prognosis. Consequently, early, rapid, and accurate pathogen diagnosis is vital for informing effective treatment strategies. This study aimed to use metagenomic next-generation sequencing (mNGS) and targeted mNGS (tNGS) to elucidate the characteristics of pulmonary infections in HIV and non-HIV individuals. Methods: This study enrolled 90 patients with pulmonary infection at the Department of Infectious Diseases of The First Hospital of Jilin University from June 2022 to May 2023, and they were divided into HIV (n=46) and non-HIV (n=44) infection groups. Their bronchoalveolar lavage fluid (BALF) was collected for mNGS analysis to evaluate the differences in pulmonary infection pathogens, and tNGS detection was performed on BALF samples from 15 HIV-infected patients. Results: A total of 37 pathogens were identified in this study, including 21 bacteria, 5 fungi, 5 viruses, 5 mycobacteria, and 1 mycoplasma. The sensitivity of mNGS was 78.9% (71/90), which is significantly higher than that of conventional methods (CTM) (39/90, P=1.5E-8). The combination of mNGS with CTM can greatly enhance the sensitivity of pathogen detection. The prevalence of Pneumocystis jirovecii (82.6% vs. 9.1%), cytomegalovirus (CMV) (58.7% vs. 0%), and Epstein-Barr virus (EBV) (17.4% vs. 2.3%) was significantly higher in the HIV infection group than in the non-HIV infection group (P<0.05). Although no statistically significant difference was observed, the detection rate of Mycobacteria was higher in HIV-infected patients (17.4%) than in the non-HIV group (6.8%). Furthermore, the tNGS results of BALF from 15 HIV-infected patients were not entirely consistent with the mNGS results., and the concordance rate of tNGS for the detection of main pathogens reached 86.7% (13/15). Conclusion: Next-generation sequencing (NGS) can accurately detect pathogens in the BALF of patients with pulmonary infection. The sensitivity of tNGS is comparable to that of mNGS. Therefore, this technique should be promoted in the clinic for better patient outcomes.

SARS-CoV-2 not Detected in the Nasopharyngeal Sample but in Bronchoalveolar Lavage Fluid.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection typically relies on reverse transcription-polymerase chain reaction (RT-PCR) technology. However, there is a certain rate of missed detection of SARS-CoV-2 in nasopharyngeal samples, particularly among immunosuppressed individuals. METHODS: In this case, SARS-CoV-2 was detected in nasopharyngeal swabs and bronchoalveolar lavage fluid (BALF) using RT-PCR. Pulmonary imaging was performed using computed tomography (CT). Patient clinical data were retrieved from the Laboratory Information System (LIS). RESULTS: SARS-CoV-2 was negative in two nasopharyngeal tests of the patient, but was finally detected in BALF, confirming that the lung lesions were infected by SARS-CoV-2. CONCLUSIONS: In the post-epidemic era, it is necessary to use BALF to identify SARS-CoV-2 infection in cases where other factors have been ruled out in immunosuppressed individuals with pulmonary infections, especially when the nasopharyngeal test yields a negative result.

Impact of time intervals on drug efficacy and phenotypic outcomes in acute respiratory distress syndrome in mice.

Acute respiratory distress syndrome is a severe lung condition resulting from various causes, with life-threatening consequences that necessitate intensive care. The phenomenon can be modeled in preclinical models, notably through the use of lipopolysaccharide (LPS) instillation in mice. The phenotype induced closely recapitulates the human syndrome, including pulmonary edema, leukocyte infiltration, acute inflammation, impaired pulmonary function, and histological damage. However, the experimental designs using LPS instillations are extremely diverse in the literature. This highly complicates the interpretation of the induced phenotype chronology for future study design and hinders the proper identification of the optimal time frame to assess different readouts. Therefore, the definition of the treatment window in relation to the beginning of the disease onset also presents a significant challenge to address questions or test compound efficacy. In this context, the temporality of the different readouts usually measured in the model was evaluated in both normal and neutrophil-depleted male C57bl/6 mice using LPS-induction to assess the best window for proper readout evaluation with an optimal dynamic response range. Ventilation parameters were evaluated by whole-body plethysmography and neutrophil recruitment were evaluated in bronchoalveolar lavage fluids and in lung tissues directly. Imaging evaluation of myeloperoxidase along with activity in lung lysates and fluids were compared, along with inflammatory cytokines and lung extravasation by enzyme-linked immunoassays. Moreover, dexamethasone, the gold standard positive control in this model, was also administered at different times before and after phenotype induction to assess how kinetics affected each parameter. Overall, our data demonstrate that each readout evaluated in this study has a singular kinetic and highlights the key importance of the timing between ARDS phenotype and treatment administration and/or analysis. These findings also strongly suggest that analyzes, both in-life and post-mortem should be conducted at multiple time points to properly capture the dynamic phenotype of the LPS-ARDS model and response to treatment.

Nuclear Factor Erythroid 2-Related Factor 2 Intervenes the Release of Neutrophil Extracellular Traps during Lipopolysaccharide-Induced Acute Lung Injury in Mice.

The pathogenesis of acute lung injury is complex. Studies have demonstrated the role of neutrophil extracellular traps (NETs) in the process of lipopolysaccharide (LPS)-induced acute lung injury (ALI). However, the underlying mechanism remains unclear. In this study, the regulation of Nrf2 in the formation of NETs, which was pathogenic in LPS-induced ALI, was identified by analyzing the levels of Cit-H3, lung function, lung tissue pathology, lung wet/dry ratio, the inflammatory cells, cytokines and proteins in the bronchoalveolar lavage fluid (BALF) and in addition, the activity of lung myeloperoxidase (MPO) was also measured. Results showed that the levels of Cit-H3 measured by western blot in Nrf2-knockout (KO) mice were higher compared with the WT mice after LPS stimulation. To further investigate the NETs formation was pathogenic during LPS-induced ALI, the Nrf2-KO mice were treated with DNase I. Results showed that DNase I improved lung function and lung tissue pathology and significantly reduced lung wet/dry ratio and proteins in the BALF. Besides, DNase I also attenuated the infiltration of inflammatory cells and the cytokines (TNF-α, IL-1ß) production in the BALF and the activity of lung MPO. Therefore, these results together indicate that Nrf2 may intervene in the release of NETs during LPS-induced ALI in mice.

Association between equine asthma and fungal elements in the tracheal wash: An environment-matched case-control study.

The presence of fungi in tracheal wash (TW) of horses was recently linked to mild-moderate equine asthma, indicating a possible causal role; however, increased numbers of fungi may also stem from asthma-related alteration of tracheal mucus clearance or from environmental exposure. Our objective was to elucidate the association between the presence of fungi in TW and asthma status while controlling for relevant confounders. We conducted a retrospective case-control study involving 73 horses, including 34 controls and 39 asthmatic cases. Each asthmatic horse was matched with a control from the same barn to account for the influence of environmental exposure. All horses underwent respiratory clinical scoring, endoscopy, TW, and bronchoalveolar lavage (BAL). The association between asthma status and presence of TW fungi was tested with multivariable logistic regression modelling, accounting for selected management factors, tracheal mucus accumulation, and selected TW and BAL cytological characteristics, including multinucleated giant cells (MGCs) in the TW. Given the variability in MGC definitions in the literature, particularly concerning their morphology and number of nuclei, we constructed two distinct models for each outcome (asthma status or presence of fungi in TW): one considering MGCs as cells with ≥ 3 nuclei, and another using a criterion of ≥ 10 nuclei. Horses with a tracheal mucus score ≥ 2 exhibited 3.6 to 4.3 higher odds of being asthmatic, depending on the MGC definition. None of the other variables examined were associated with either asthma status or TW fungi detection. Notably, the presence of fungal elements in the TW was not associated with equine asthma.

Aspergillus fumigatus antigen-reactive Th17 cells are enriched in bronchoalveolar lavage fluid in severe equine asthma.

Introduction: Equine asthma (EA) is a common disease of adult horses with chronic respiratory pathology and common neutrophilic airway inflammation. It presents with hyperreactivity to hay dust components such as molds, and underlying dysregulated T cell responses have been suggested. Thus far, T cells have been analysed in EA with conflicting results and the antigen reactivity of T cells has not been demonstrated. Serological and epidemiological data point to the relevance of Aspergillus fumigatus as an antigen source in EA. Here, we aimed to identify and characterise Aspergillus antigen-reactive T cells in EA. Methods: Cryopreserved bronchoalveolar lavage cells (BALC) and peripheral blood mononuclear cells (PBMC) from healthy horses (HE, n=9) and those with mild-moderate (MEA, n=3) or severe asthma (SEA, n=8) were stimulated in vitro with the recombinant A. fumigatus antigens Asp f 1, or Asp f 7 combined with Asp f 8, to assess antigen reactivity, and with phorbol-12-myristat-13-acetate and ionomycin (P/i) to assess overall T cell reactivity. Stimulated cells were analysed by flow cytometry for CD4, CD8, IL-17, IL-4, and IFN-γ. Cytokine expression in all lymphocytes, and in CD4+ or CD8+ T cells, was quantified and compared between the groups. In BAL fluid (BALF), soluble cytokines and chemokines were quantified by bead-based assays. Results: Antigen restimulation of BALC with Asp f 1 or Asp f 7/8 provoked higher frequencies of IL-17+ lymphocytes, CD4+IL-17+ Th17 cells, and CD4+IL-4+ Th2 cells in SEA than in HE, whereas MEA and HE were similar. Antigen stimulation of PBMC did not result in group differences. P/i stimulation of BALC resulted in increased IL-17+ lymphocyte and CD4+IL-17+ Th17 cell frequencies in MEA compared with HE but the limited number of horses with MEA must be considered. P/i-stimulated PBMC from MEA or SEA contained more IL-17+ lymphocytes compared with HE. Cytokines were hardly detected in BALF and similar between the groups but CCL2 and CCL5 concentrations were increased in BALF from SEA or MEA, respectively, compared with HE. Conclusion: Horses with SEA have increased Aspergillus antigen-reactive Th17 cells in their airways, emphasising local T cell responses to this mold, which were quantified in EA for the first time here.

Self-report underestimates the frequency of the acute respiratory exacerbations of COPD but is associated with BAL neutrophilia and lymphocytosis: an observational study.

RATIONALE: Research studies typically quantify acute respiratory exacerbation episodes (AECOPD) among people with chronic obstructive pulmonary disease (COPD) based on self-report elicited by survey questionnaire. However, AECOPD quantification by self-report could be inaccurate, potentially rendering it an imprecise tool for identification of those with exacerbation tendency. OBJECTIVE: Determine the agreement between self-reported and health records-documented quantification of AECOPD and their association with airway inflammation. METHODS: We administered a questionnaire to elicit the incidence and severity of respiratory exacerbations in the three years preceding the survey among current or former heavy smokers with or without diagnosis of COPD. We then examined electronic health records (EHR) of those with COPD and those without (tobacco-exposed persons with preserved spirometry or TEPS) to determine whether the documentation of the three-year incidence of moderate to very severe respiratory exacerbations was consistent with self-report using Kappa Interrater statistic. A subgroup of participants also underwent bronchoalveolar lavage (BAL) to quantify their airway inflammatory cells. We further used multivariable regressions analysis to estimate the association between respiratory exacerbations and BAL inflammatory cell composition with adjustment for covariates including age, sex, height, weight, smoking status (current versus former) and burden (pack-years). RESULTS: Overall, a total of 511 participants completed the questionnaire, from whom 487 had EHR available for review. Among the 222 participants with COPD (70 ± 7 years-old; 96% male; 70 ± 38 pack-years smoking; 42% current smoking), 57 (26%) reported having any moderate to very severe AECOPD (m/s-AECOPD) while 66 (30%) had EHR documentation of m/s-AECOPD. However, 42% of those with EHR-identified m/s-AECOPD had none by self-report, and 33% of those who reported m/s-AECOPD had none by EHR, suggesting only moderate agreement (Cohen's Kappa = 0.47 ± 0.07; P < 0.001). Nevertheless, self-reported and EHR-identified m/s-AECOPD events were both associated with higher BAL neutrophils (ß ± SEM: 3.0 ± 1.1 and 1.3 ± 0.5 per 10% neutrophil increase; P ≤ 0.018) and lymphocytes (0.9 ± 0.4 and 0.7 ± 0.3 per 10% lymphocyte increase; P ≤ 0.041). Exacerbation by either measure combined was associated with a larger estimated effect (3.7 ± 1.2 and 1.0 ± 0.5 per 10% increase in neutrophils and lymphocytes, respectively) but was not statistically significantly different compared to the self-report only approach. Among the 184 TEPS participants, there were fewer moderate to very severe respiratory exacerbations by self-report (n = 15 or 8%) or EHR-documentation (n = 9 or 5%), but a similar level of agreement as those with COPD was observed (Cohen's Kappa = 0.38 ± 0.07; P < 0.001). DISCUSSION: While there is modest agreement between self-reported and EHR-identified m/s-AECOPD, events are missed by relying on either method alone. However, m/s-AECOPD quantified by self-report or health records is associated with BAL neutrophilia and lymphocytosis.

Bronchoalveolar lavage fluid analysis in patients with checkpoint inhibitor pneumonitis.

BACKGROUND: Checkpoint inhibitor pneumonitis (CIP) is a relatively uncommon but potentially life-threatening immune-related adverse event (irAE). Lung biopsies have not been commonly performed for CIP patients. Bronchoalveolar lavage fluid (BALF) analysis is a useful diagnostic approach for interstitial lung disease. However, BALF features were inconsistent across different studies. METHODS: We retrospectively reviewed the medical records of 154 patients with pathologically confirmed malignancies and suffering from CIPs between July 2018 and December 2022. Patients who had bronchoalveolar lavage (BAL) data available were enrolled in our study. Patient clinical, laboratory, radiological and follow-up data were reviewed and analyzed. RESULTS: The BALF differential cell count and lymphocyte subset analysis were performed for 42 CIP patients. There were 32 males (76.2%). The mean age at diagnosis of CIP was 62.0 ± 10.4 (range: 31-78) years. The median time to onset of CIP was 98.5 days after the start of immunotherapy. There were 18 patients (42.9%) with low-grade CIPs and 24 patients (57.1%) with high-grade CIPs. The mean lymphocyte percentage was 36.7 ± 22.5%. There were 34 (81%) CIP patients with a lymphocytic cellular pattern. The median ratio of CD3+CD4+/CD3+CD8+ lymphocytes was 0.5 (0.3, 1.0). The ratio was less than 1.0 for 31 CIP patients (73.8%). However, there was no significant difference in the BALF features between patients with low-grade CIPs and those with high-grade CIPs. CONCLUSIONS: The CD3+CD8+ lymphocytosis pattern was the main inflammatory profile in the BALF of CIP patients in this cohort. Targeting CD3+CD8+ lymphocytes might be a treatment option for CIPs.

Pulmonary Administration of TLR2/6 Agonist after Allergic Sensitization Inhibits Airway Hyper-Responsiveness and Recruits Natural Killer Cells in Lung Parenchyma.

Asthma is a chronic lung disease with persistent airway inflammation, bronchial hyper-reactivity, mucus overproduction, and airway remodeling. Antagonizing T2 responses by triggering the immune system with microbial components such as Toll-like receptors (TLRs) has been suggested as a therapeutic concept for allergic asthma. The aim of this study was to evaluate the effect of a TLR2/6 agonist, FSL-1 (Pam2CGDPKHPKSF), administered by intranasal instillation after an allergic airway reaction was established in the ovalbumin (OVA) mouse model and to analyze the role of natural killer (NK) cells in this effect. We showed that FSL-1 decreased established OVA-induced airway hyper-responsiveness and eosinophilic inflammation but did not reduce the T2 or T17 response. FSL-1 increased the recruitment and activation of NK cells in the lung parenchyma and modified the repartition of NK cell subsets in lung compartments. Finally, the transfer or depletion of NK cells did not modify airway hyper-responsiveness and eosinophilia after OVA and/or FSL-1 treatment. Thus, the administration of FSL-1 reduces airway hyper-responsiveness and bronchoalveolar lavage eosinophilia. However, despite modifications of their functions following OVA sensitization, NK cells play no role in OVA-induced asthma and its inhibition by FSL-1. Therefore, the significance of NK cell functions and localization in the airways remains to be unraveled in asthma.

Metagenomic versus targeted next-generation sequencing for detection of microorganisms in bronchoalveolar lavage fluid among renal transplantation recipients.

Background: Metagenomic next-generation sequencing (mNGS), which provides untargeted and unbiased pathogens detection, has been extensively applied to improve diagnosis of pulmonary infection. This study aimed to compare the clinical performance between mNGS and targeted NGS (tNGS) for microbial detection and identification in bronchoalveolar lavage fluid (BALF) from kidney transplantation recipients (KTRs). Methods: BALF samples with microbiological results from mNGS and conventional microbiological test (CMT) were included. For tNGS, samples were extracted, amplified by polymerase chain reaction with pathogen-specific primers, and sequenced on an Illumina Nextseq. Results: A total of 99 BALF from 99 KTRs, among which 93 were diagnosed as pulmonary infection, were analyzed. Compared with CMT, both mNGS and tNGS showed higher positive rate and sensitivity (p<0.001) for overall, bacterial and fungal detection. Although the positive rate for mNGS and tNGS was comparable, mNGS significantly outperformed tNGS in sensitivity (100% vs. 93.55%, p<0.05), particularly for bacteria and virus (p<0.001). Moreover, the true positive rate for detected microbes of mNGS was superior over that of tNGS (73.97% vs. 63.15%, p<0.05), and the difference was also significant when specific for bacteria (94.59% vs. 64.81%, p<0.001) and fungi (93.85% vs. 72.58%, p<0.01). Additionally, we found that, unlike most microbes such as SARS-CoV-2, Aspergillus, and EBV, which were predominantly detected from recipients who underwent surgery over 3 years, Torque teno virus (TTV) were principally detected from recipients within 1-year post-transplant, and as post-transplantation time increased, the percentage of TTV positivity declined. Conclusion: Although tNGS was inferior to mNGS owing to lower sensitivity and true positive rate in identifying respiratory pathogens among KTRs, both considerably outperformed CMT.

Deficiency and dysfunctional roles of natural killer T cells in patients with ARDS.

Objective: Acute respiratory distress syndrome (ARDS) presents a global health challenge, characterized by significant morbidity and mortality. However, the role of natural killer T (NKT) cells in human ARDS remains poorly understood. Therefore, this study explored the numerical and functional status of NKT cells in patients with ARDS, examining their clinical relevance and interactions with macrophages and fibroblasts during various stages of the syndrome. Methods: Peripheral blood from 40 ARDS patients and 30 healthy controls was analyzed, with paired samples of peripheral blood and bronchoalveolar lavage fluid (BALF) from seven ARDS patients. We measured levels of NKT cells, cytokines, CD69, programmed death-1 (PD-1), and annexin-V using flow cytometry, and extracellular matrix (ECM) protein expression using real-time PCR. Results: ARDS patients exhibited decreased circulating NKT cells with elevated CD69 expression and enhanced IL-17 production. The reduction in NKT cells correlated with PaO2/FiO2 ratio, albumin, and C-reactive protein levels. Proliferative responses to α-galactosylceramide (α-GalCer) were impaired, and co-culturing NKT cells with monocytes or T cells from ARDS patients resulted in a reduced α-GalCer response. Increased and activated NKT cells in BALF induced proinflammatory cytokine release by macrophages and ECM protein expression in fibroblasts. Conclusion: ARDS is associated with a numerical deficiency but functional activation of circulating NKT cells, showing impaired responses to α-GalCer and altered interactions with immune cells. The increase in NKT cells within BALF suggests their role in inducing inflammation and remodeling/fibrosis, highlighting the potential of targeting NKT cells as a therapeutic approach for ARDS.

Comparative analysis of metagenomic and targeted next-generation sequencing for pathogens diagnosis in bronchoalveolar lavage fluid specimens.

Background: Although the emerging NGS-based assays, metagenomic next-generation sequencing (mNGS) and targeted next-generation sequencing (tNGS), have been extensively utilized for the identification of pathogens in pulmonary infections, there have been limited studies systematically evaluating differences in the efficacy of mNGS and multiplex PCR-based tNGS in bronchoalveolar lavage fluid (BALF) specimens. Methods: In this study, 85 suspected infectious BALF specimens were collected. Parallel mNGS and tNGS workflows to each sample were performed; then, we comparatively compared their consistency in detecting pathogens. The differential results for clinically key pathogens were confirmed using PCR. Results: The microbial detection rates of BALF specimens by the mNGS and tNGS workflows were 95.18% (79/83) and 92.77% (77/83), respectively, with no significant difference. mNGS identified 55 different microorganisms, whereas tNGS detected 49 pathogens. The comparative analysis of mNGS and tNGS revealed that 86.75% (72/83) of the specimens were complete or partial concordance. Particularly, mNGS and tNGS differed significantly in detection rates for some of the human herpesviruses only, including Human gammaherpesvirus 4 (P<0.001), Human betaherpesvirus 7 (P<0.001), Human betaherpesvirus 5 (P<0.05) and Human betaherpesvirus 6 (P<0.01), in which tNGS always had higher detection rates. Orthogonal testing of clinically critical pathogens showed a total coincidence rate of 50% for mNGS and PCR, as well as for tNGS and PCR. Conclusions: Overall, the performance of mNGS and multiplex PCR-based tNGS assays was similar for bacteria and fungi, and tNGS may be superior to mNGS for the detection of DNA viruses. No significant differences were seen between the two NGS assays compared to PCR.

Allergo-immunopathology mechanism of thymol-inhibiting airway remodeling in asthmatic mice by regulating TGF-ß/Smad3 pathway.

Allergic asthma is an important public health problem and is a complicated respiratory sickness that is characterized by bronchial inflammation, bronchoconstriction, and breathlessness. Asthma is orchestrated by type 2 immune response and remodeling is one of the important outputted problem in chronic asthma. Thymol is a naturally occurring monocyclic phenolic, it has a series of biological properties, and its immunomodulatory and anti-remodeling effects on allergic asthma were evaluated. The OVA-LPS-induced asthmatic mice were treated with thymol. Methacholine challenge test, eosinophil count, and levels of IL-4, IL-5, IL-13, and IL-33 in bronchoalveolar lavage fluid, total and OVA-specific IgE levels in serum, remodeling factors, gene expression of TGF-ß, Smad2, Smad3, and lung histopathology were done. Treatment with thymol could control AHR, eosinophil percentage levels of Th2 cytokines and Igs, remodeling factors, expression of TGF-ß, Smad2 and Smad3 genes, inflammation, goblet cell hyperplasia, and mucus production in asthmatic mice. Thymol can control asthma pathogens and related remodeling and fibrosis bio-factors and can be a potential treatment of asthma.

[Establishment of Nano-ITO induced rat model of indium lung disease and exploration of its pathological characteristics].

Objective: To study the dynamic pathological characteristics of lung tissue in a Nano-ITO induced rat model of indium lung disease and to guide clinical and basic scientific research to further explore the mechanisms of pulmonary interstitial injury and pulmonary alveolar proteinosis (PAP). Methods: Dose-response (three divided doses) and time-course studies (six exposure periods) were performed to investigate the pulmonary toxicity induced by Nano-ITO. At the end of the experiment, cytokine levels and oxidative stress were analyzed in the bronchoalveolar lavage fluid. Rat lung tissues were also collected for staining with H&E, PAS, Masson's, Oil Red O, and Sirius Red. Ultrastructure of lung tissue cells was observed by transmission electron microscopy. Expression of IL-1ß, HO-1, SP-A was observed by immunohistochemistry, and the expression of α-SMA was observed by immunofluorescence. Results: Nano-ITO intratracheal instillation caused pulmonary toxicity by inducing acute inflammation at 3 days, granuloma (nodule) formation and collagen hyperplasia at 14 days, and alveolar proteinosis at 56 days post-exposure. Pathological features of lung tissue included typical alveolar exudates, cellular fibrous nodules, enlarged alveolar fat droplet fusion, cholesterol crystal granuloma and pulmonary alveolar proteinosis. The intra-alveolar eosinophilic material (multilamellated, lattice-shaped, and myelin-like structure) showed abnormal lamellar bodies (features of alveolar type Ⅱ epithelial cells) and abundant rough endoplasmic reticulum and mitochondria (features of fibroblasts) on transmission electron microscopy of the lung tissue from rats exposed to Nano-ITO on the 84th day. Cellular pathology revealed that a large amount of amorphous PAS stain-positive substances appear in BALF at 28 days post-exposure, and pink granular protein-like substances can be seen in alveolar macrophages. Conclusions: There are three characteristic developmental stages in Nano-ITO induced pulmonary injury in rats, acute inflammation, granuloma (nodule) formation and collagen proliferation, and pulmonary alveolar proteinosis, which provide a reference feature model for the pathogenesis of indium lung disease.