Neutrophilic Inflammation Research Articles

Deciphering the pancreatic cancer microbiome in Mainland China: Impact of Exiguobacterium/Bacillus ratio on tumor progression and prognostic significance

The existing body of research underscores the critical impact of intratumoral microbiomes on the progression of pancreatic ductal adenocarcinoma (PDAC), particularly in reshaping the tumor microenvironment and influencing gemcitabine resistance. However, peritumoral tissues’ microbiome, distinct from PDAC tumors, remain understudied, and Western-centric analyses overlooking potential variations in dietary-influenced microbiomes. Our study addresses this gap by 16 S rRNA sequencing of PDAC tumors and matched peritumoral tissues from Chinese Mainland patients. Our research has uncovered that the microbiome composition within tumors and paired peritumoral tissues exhibits a high degree of similarity, albeit with certain discrepancies. Notably, Exiguobacterium is found to be more abundant within the tumor tissues. Further investigations have revealed that a lower Exiguobacterium/Bacillus ratio in both the tumor and peritumoral tissues of PDAC patients is indicative of a more favorable prognosis. Further exploration utilizing an orthotopic tumor model demonstrates that the probiotic Bacillus Coagulans impedes PDAC progression, accompanied by an increased infiltration of inflammatory neutrophils in tumors. Additionally, in the subgroup with a low Exiguobacterium/Bacillus ratio, whole-exome sequencing reveals elevated missense mutations in ABL2 and MSH2. The elevated expression of ABL2 and MSH2 has been correlated with poorer prognostic outcomes in PDAC patients. Together, these insights shed light on risk factors influencing PDAC progression and unveil potential therapeutic targets, alongside probiotic intervention strategies.

Resveratrol has neuroprotective effects and plays an anti-inflammatory role through Dectin-1/p38 pathway in Aspergillus fumigatus keratitis

PurposeTo determine the antifungal, anti-inflammatory and neuroprotective effects of resveratrol (RES) in Aspergillus fumigatus (A. fumigatus) keratitis. MethodsCytotoxicity assay and Draize eye assay were performed to assess the toxicity of RES. The antifungal effect of RES was assessed by minimal inhibitory concentration, scanning or transmission electron microscopy, propidium iodide uptake assay, and Calcofluor white staining. Phosphorylation of p38 MAPK, mRNA and protein levels of Dectin-1 and related inflammatory factors were measured by qRT-PCR, ELISA and Western blot in vitro and in vivo. Clinical score, HE staining, plate count, and myeloperoxidase test were used to observe the progress of fungal keratitis. IF staining, qRT-PCR, and the Von Frey test were selected to assess the neuroprotective effects of RES. ResultsRES suppressed A. fumigatus hyphae growth and altered hyphae morphology in vitro. RES decreased the expression of Dectin-1, IL-1β and TNF-α, as well as p38 MAPK phosphorylation expression, and also decreased clinical scores, reduced inflammatory cell infiltration and neutrophil activity, and decreased fungal load. RES also protected corneal basal nerve fibers, down-regulated mechanosensitivity thresholds, and increased the mRNA levels of CGRP and TRPV-1.. ConclusionThese evidences revealed that RES could exert antifungal effects on A. fumigatus and ameliorate FK through suppressing the Dectin-1/p38 MAPK pathway to down-regulate IL-1β, IL-6, etc. expression and play protective effect on corneal nerves.

Small extracellular vesicles derived from human mesenchymal stem cells prevent Th17-dominant neutrophilic airway inflammation via immunoregulation on Th17 cells

Type 17 helper T cells (Th17)-dominant neutrophilic airway inflammation is critical in the pathogenesis of steroid-resistant airway inflammation such as severe asthma. Small extracellular vesicles (sEV) derived from human mesenchymal stem cells (MSCs) display extensive therapeutic effects and advantages in many diseases. However, the role of MSC-sEV in Th17-dominant neutrophilic airway inflammation and the related mechanisms are still poorly studied. Here we found that MSC-sEV significantly alleviated the infiltration of inflammatory cells in peribronchial interstitial tissues and reduced levels of inflammatory cells, especially neutrophils, in bronchoalveolar lavage fluids (BALF) of mice with neutrophilic airway inflammation. Consistently, MSC-sEV significantly decreased levels of IL-17A in BALF and Th17 in lung tissues. Furthermore, we found that labelled MSC-sEV were taken up by human CD4+ T cells most obviously at 12 h after incubation, and distributed mostly in mouse lungs. More importantly, potential signaling pathways involved in the MSC-sEV mediated inhibition of Th17 polarization were found using RNA sequencing. Using Western blot, JAK2-STAT3 pathway was identified as an important role in the inhibition of Th17 polarization by MSC-sEV. We found that proteins in MSC-sEV were mostly involved in the therapeutic effects of MSC-sEV. In total, our study suggested that MSC-sEV could be a potential therapeutic strategy for the treatment of neutrophilic airway inflammation.

Persistent neuroinflammation and functional deficits in a murine model of decompression sickness.

We hypothesized that early intra-central nervous system (CNS) responses in a murine model of decompression sickness (DCS) would be reflected by changes in the microparticles (MPs) that exit the brain via the glymphatic system, and due to systemic responses the MPs would cause inflammatory changes lasting for many days leading to functional neurological deficits. Elevations on the order of threefold of blood-borne inflammatory MPs, neutrophil activation, glymphatic flow, and neuroinflammation in cerebral cortex and hippocampus were found in mice at 12 days after exposure to 760 kPa of air for 2 h. Mice also exhibited a significant decline in memory and locomotor activity, as assessed by novel object recognition and rotarod testing. Similar inflammatory changes in blood, neuroinflammation, and functional impairments were initiated in naïve mice by injection of filamentous (F-) actin-positive MPs, but not F-actin-negative MPs, obtained from decompressed mice. We conclude that high pressure/decompression stress establishes a systemic inflammatory process that results in prolonged neuroinflammation and functional impairments in the mouse decompression model.NEW & NOTEWORTHY Elevated glymphatic flow due to astrocyte and microglial activation from high-pressure exposure triggers release of microparticles (MPs) to the circulation where neutrophil activation and production of filamentous (F)-actin expressing MPs result in a persistent feed-forward neuroinflammatory cycle and functional deficits lasting for at least 12 days.

Neutrophil Extracellular Traps-Inhibiting and Fouling-Resistant Polysulfoxides Potently Prevent Postoperative Adhesion, Tumor Recurrence, and Metastasis.

Peritoneal metastasis (PM) is considered one of the most dreaded forms of cancer metastases for both patients and physicians. Aggressive cytoreductive surgery (CRS) is the primary treatment for peritoneal metastasis. Unfortunately, this intensive treatment frequently causes clinical complications, such as postoperative recurrence, metastasis, and adhesion formation. Emerging evidence suggests that neutrophil extracellular traps (NETs) released by inflammatory neutrophils contribute to these complications. Effective NET-targeting strategies thus show considerable potential in counteracting these complications but remain challenging. Here, one type of sulfoxide-containing homopolymer, PMeSEA, with potent fouling-resistant and NET-inhibiting capabilities, is synthesized and screened. Hydrating sulfoxide groups endow PMeSEA with superior nonfouling ability, significantly inhibiting protein/cell adhesion. Besides, the polysulfoxides can be selectively oxidized by ClO- which is required to stabilize the NETs rather than H2O2, and ClO- scavenging effectively inhibits NETs formation without disturbing redox homeostasis in tumor cells and quiescent neutrophils. As a result, PMeSEA potently prevents postoperative adhesions, significantly suppresses peritoneal metastasis, and shows synergetic antitumor activity with chemotherapeutic 5-Fluorouracil. Moreover, coupling CRS with PMeSEA potently inhibits CRS-induced tumor metastatic relapse and postoperative adhesions. Notably, PMeSEA exhibits low in vivo acute and subacute toxicities, implying significant potential for clinical postoperative adjuvant treatment.

Single-Cell Analysis Uncovers Striking Cellular Heterogeneity of Lung-Infiltrating Regulatory T Cells during Eosinophilic versus Neutrophilic Allergic Airway Inflammation.

Allergic airway inflammation results from uncontrolled immune responses to environmental Ags. Although it is well established that allergic immune responses exhibit a high degree of diversity, driven by primary effector cell types such as eosinophils, neutrophils, or CD4 T cells with distinct effector signatures, the mechanisms responsible for such pathogenesis remain elusive. Foxp3+ regulatory T cells (Tregs) are essential immune regulators during chronic inflammation, including allergic airway inflammation. Emerging evidence suggests that Tregs infiltrating inflamed tissues exhibit distinct phenotypes dependent on the specific tissue sites and can display heterogeneity and tissue residency. Whether diverse allergic airway inflammatory responses influence infiltrating Treg heterogeneity or Treg lung residency has not been explored. We employed an unbiased single-cell RNA sequencing approach to investigate lung-infiltrating Tregs in models of eosinophilic and neutrophilic airway inflammation. We found that lung-infiltrating Tregs are highly heterogeneous, and that Tregs displaying lung-resident phenotypes are significantly different depending on the types of inflammation. Treg expression of ST2, a receptor for alarmin IL-33, was predominantly associated with eosinophilic inflammation and tissue residency. Nevertheless, Treg-specific ST2 deficiency did not affect the development of eosinophilic allergic inflammation or the generation of lung-resident Tregs. These results uncover a stark heterogeneity among Tregs infiltrating the lungs during allergic airway inflammation. The results indicate that varying types of inflammation may give rise to phenotypically distinct lung-resident Tregs, underscoring a (to our knowledge) novel mechanism by which inflammatory cues may shape the composition of infiltrating Tregs, allowing them to regulate inflammatory responses through tissue-adapted mechanisms.

Bacterial interactome disturbance in chronic obstructive pulmonary disease clinical stability and exacerbations

RationaleOur understanding of airway dysbiosis in chronic obstructive pulmonary disease (COPD) remains incomplete, which may be improved by unraveling the complexity in microbial interactome.ObjectivesTo characterize reproducible features of airway bacterial interactome in COPD at clinical stability and during exacerbation, and evaluate their associations with disease phenotypes.MethodsWe performed weighted ensemble-based co-occurrence network analysis of 1742 sputum microbiomes from published and new microbiome datasets, comprising two case-control studies of stable COPD versus healthy control, two studies of COPD stability versus exacerbation, and one study with exacerbation-recovery time series data.ResultsPatients with COPD had reproducibly lower degree of negative bacterial interactions, i.e. total number of negative interactions as a proportion of total interactions, in their airway microbiome compared with healthy controls. Evaluation of the Haemophilus interactome showed that the antagonistic interaction networks of this established pathogen rather than its abundance consistently changed in COPD. Interactome dynamic analysis revealed reproducibly reduced antagonistic interactions but not diversity loss during COPD exacerbation, which recovered after treatment. In phenotypic analysis, unsupervised network clustering showed that loss of antagonistic interactions was associated with worse clinical symptoms (dyspnea), poorer lung function, exaggerated neutrophilic inflammation, and higher exacerbation risk. Furthermore, the frequent exacerbators (≥ 2 exacerbations per year) had significantly reduced antagonistic bacterial interactions while exhibiting subtle compositional changes in their airway microbiota.ConclusionsBacterial interactome disturbance characterized by reduced antagonistic interactions, rather than change in pathogen abundance or diversity, is a reproducible feature of airway dysbiosis in COPD clinical stability and exacerbations, which suggests that we may target interactome rather than pathogen alone for disease treatment.

Anti-inflammatory effect of Anadenanthera colubrina var. cebil (Griseb.) Altschul in experimental elastase-induced pulmonary emphysema in rats

Ethnopharmacological relevanceMedicinal plants have shown promise in the search for new treatments of pulmonary emphysema. Anadenanthera colubrina, a species native to the Caatinga biome in northeastern Brazil, is widely recognized and traditionally employed in the treatment of pulmonary diseases. Many studies corroborate popular knowledge about the medicinal applications of A. colubrina, which has demonstrated a remarkable variety of pharmacological properties, however, its anti-inflammatory and antioxidant properties are highlighted. Aim of the studyThe objective of this study was to investigate the anti-inflammatory potential of the crude hydroethanolic extract of A. colubrina var. cebil (Griseb.) Altschul on pulmonary emphysema in rats as well as to determine its potential genotoxic and cytotoxic effects using the micronucleus assay. Materials and methodsThe stem bark of the plant was collected in Pimenteiras-PI and sample was extracted by maceration using 70% ethanol. A portion of the extract underwent phytochemical analyses using TLC and HPLC. In this study, 8-week-old, male Wistar rats weighing approximately ±200 g was utilized following approval by local ethics committee for animal experimentation (No. 718/2022). Pulmonary emphysema was induced through orotracheal instillation of elastase, and treatment with A. colubrina extract or dexamethasone (positive control) concomitantly during induction. Twenty-eight days after the initiation of the protocol, plasma was used for cytokine measurement. Bronchoalveolar lavage (BAL) was used for leukocyte count. After euthanasia, lung samples were processed for histological analysis and quantification of oxidative stress markers. The micronucleus test was performed by evaluating the number of polychromatic erythrocytes (PCE) with micronuclei (MNPCE) to verify potential genotoxic effects of A. colubrina. A differential count of PCE and normochromatic erythrocytes (NCE) was performed to verify the potential cytotoxicity of the extract. Parametric data were subjected to normality analysis and subsequently to analysis of variance and Tukey or Dunnett post-test, non-parametric data were treated using the Kruskal-Wallis test with Dunn's post-test for unpaired samples. P value < 0.05 were considered significant. ResultsThe A. colubrina extract did not show a significant increase in the number of MNPCE (p > 0.05), demonstrating low genotoxicity. No changes were observed in the PCE/NCE ratio of treated animals, compared with the vehicle, suggesting low cytotoxic potential of the extract. A significant reduction (p < 0.05) in neutrophilic inflammation was observed in the lungs of rats treated with the extract, evidenced by presence of these cells in both the tissue and BAL. The extract also demonstrated pulmonary antioxidant activity, with a significant decrease (p < 0.05) in myeloperoxidase, malondialdehyde, and nitrite levels. TNFα, IL-1β, and IL-6 levels, as well as alveolar damage, were significantly reduced in animals treated with A. colubrina extract. Phytochemical analyses identified the presence of phenolic compounds and hydrolysable tannins in the A. colubrina extract. ConclusionsThe findings of this study highlights the safety of the hydroethanolic extract of Anadenanthera colubrina, and demonstrates its potential as a therapeutic approach in the treatment of emphysema. The observed properties of this medicinal plant provide an optimistic outlook in the development of therapies for the treatment of pulmonary emphysema.

Corilagin Inhibits Neutrophil Extracellular Trap Formation and Protects against Hydrochloric Acid/Lipopolysaccharide-Induced Acute Lung Injury in Mice by Suppressing the STAT3 and NOX2 Signaling Pathways.

Acute lung injury (ALI) and its severe manifestation, acute respiratory distress syndrome (ARDS), are characterized by uncontrolled inflammatory responses, neutrophil activation and infiltration, damage to the alveolar capillary membrane, and diffuse alveolar injury. Neutrophil extracellular traps (NETs), formed by activated neutrophils, contribute significantly to various inflammatory disorders and can lead to tissue damage and organ dysfunction. Corilagin, a compound found in Phyllanthus urinaria, possesses antioxidative and anti-inflammatory properties. In this study, we investigated the protective effects and underlying mechanisms of corilagin in hydrochloric acid (HCl)/lipopolysaccharide (LPS)-induced lung injury. Mice received intraperitoneal administration of corilagin (2.5, 5, or 10 mg/kg) or an equal volume of saline 30 min after intratracheal HCl/LPS administration. After 20 h, lung tissues were collected for analysis. Corilagin treatment significantly mitigated lung injury, as evidenced by reduced inflammatory cell infiltration, decreased production of proinflammatory cytokines, and alleviated oxidative stress. Furthermore, corilagin treatment suppressed neutrophil elastase expression, reduced NET formation, and inhibited the expression of ERK, p38, AKT, STAT3, and NOX2. Our findings suggest that corilagin inhibits NET formation and protects against HCl/LPS-induced ALI in mice by modulating the STAT3 and NOX2 signaling pathways.

8-Iso-prostaglandin F2α as a biomarker of type 2 low airway inflammation and remodeling in adult asthma

BackgroundAlthough 8-iso-prostaglandin F2a has been proposed as a potential biomarker for oxidative stress in airway diseases, its specific role in asthma remains poorly understood. ObjectiveTo evaluate the diagnostic potential of 8-iso-prostaglandin F2a in assessing airway inflammation, airway remodeling, airway hyperresponsiveness, and oxidative stress in asthma. MethodsBlood and urine concentrations of 8-iso-prostaglandin F2a were quantified using liquid chromatography–tandem mass spectrometry in 128 adults with asthma who had maintained antiasthma medications. Their correlations with clinical data, sputum cell counts, lung function parameters, and serum markers of epithelial/neutrophil activity and airway remodeling were then analyzed. ResultsThe urinary 8-iso-prostaglandin F2a concentrations were significantly higher in patients with noneosinophilic asthma than in those with eosinophilic asthma (P < .05). The area under the curve was 0.678, indicating moderate diagnostic accuracy for noneosinophilic asthma. There were significant correlations with neutrophilic inflammation markers and airway remodeling markers (all P < .05). Negative correlations were observed with forced expiratory volume in 1 second (%), forced expiratory volume in 1 second/forced vital capacity, forced expiratory flow at 25% to 75% of forced vital capacity, and serum club cell protein 16 levels (all P < .05). High 8-iso-prostaglandin F2a concentrations were also noted in obese and smoking subgroups (all P < .05). However, the serum 8-iso-prostaglandin F2a concentrations were not correlated with these asthma-related parameters. ConclusionUrinary 8-iso-prostaglandin F2a concentrations are a potential biomarker for phenotyping severe asthma, particularly noneosinophilic asthma, offering oxidative stress-induced epithelial inflammation/remodeling as an additional target in asthma management.

Unconventional Activation of IRE1 Enhances Th17 Responses and Promotes Airway Neutrophilia.

Heightened unfolded protein responses (UPRs) are associated with the risk for asthma, including severe asthma. Treatment-refractory severe asthma manifests a neutrophilic phenotype with T helper (Th)17 responses. However, how UPRs participate in the deregulation of Th17 cells leading to neutrophilic asthma remains elusive. This study found that the UPR sensor IRE1 is induced in the murine lung with fungal asthma and is highly expressed in Th17 cells relative to naive CD4+ T cells. Cytokine (e.g., IL-23) signals induce the IRE1-XBP1s axis in a JAK2-dependent manner. This noncanonical activation of the IRE1-XBP1s pathway promotes UPRs and cytokine secretion by both human and mouse Th17 cells. Ern1 (encoding IRE1) deficiency decreases the expression of endoplasmic reticulum stress factors and impairs the differentiation and cytokine secretion of Th17 cells. Genetic ablation of Ern1 leads to alleviated Th17 responses and airway neutrophilia in a fungal airway inflammation model. Consistently, IL-23 activates the JAK2-IRE1-XBP1s pathway invivo and enhances Th17 responses and neutrophilic infiltration into the airway. Taken together, our data indicate that IRE1, noncanonically activated by cytokine signals, promotes neutrophilic airway inflammation through the UPR-mediated secretory function of Th17 cells. The findings provide a novel insight into the fundamental understanding of IRE1 in Th17-biased TH2-low asthma.

Insulin eye drops improve corneal wound healing in STZ-induced diabetic mice by regulating corneal inflammation and neuropeptide release

IntroductionIn recent years, insulin eye drops have attracted increasing attention from researchers and ophthalmologists. The aim of this study was to investigate the efficacy and possible mechanism of action of insulin eye drops in diabetic mice with corneal wounds.MethodsA type 1 diabetes model was induced, and a corneal epithelial injury model of 2.5 mm was established. We used corneal fluorescein staining, hematoxylin-eosin (H-E) staining and the Cochet-Bonnet esthesiometer to examine the process of wound healing. Subsequently, the expression levels of Ki-67, IL-1β, β3-tubulin and neuropeptides, including substance P (SP) and calcitonin gene-related peptide (CGRP), were examined at 72 h after corneal injury.ResultsFluorescein staining demonstrated an acceleration of the recovery of corneal epithelial injury in diabetic mice compared with the saline treatment, which was further evidenced by the overexpression of Ki-67. Moreover, 72 h of insulin application attenuated the expression of inflammatory cytokines and neutrophil infiltration. Remarkably, the results demonstrated that topical insulin treatment enhanced the density of corneal epithelial nerves, as well as neuropeptide SP and CGRP release, in the healing cornea via immunofluorescence staining.ConclusionsOur results indicated that insulin eye drops may accelerate corneal wound healing and decrease inflammatory responses in diabetic mice by promoting nerve regeneration and increasing levels of neuropeptides SP and CGRP.

Disruption of post-thymic tolerance in skin-reactive TCR transgenic mice through the interaction of lymphopenia and intestinal microbiota.

Autoimmune diseases often arise from conditions where the immune system is compromised. While lymphopenia-induced proliferation (LIP) is crucial for immune system development and maturation, it is also caused by environmental insults, such as infection, and becomes a risk factor for autoimmunity in adults. We used Dsg3H1 TCR transgenic mice, whose T cells are designed to recognize desmogrein-3, a skin antigen, to explore the impact of lymphopenia on post-thymic tolerance. Dsg3H1 mice are known to delete the most highly autoreactive T cells in the thymus, and develop only subtle immune-mediated pathology in the steady state. However, we found that transient lymphopenia induced by total body irradiation (TBI) or cyclophosphamide (CY) results in massive dermatitis in Dsg3H1 mice. The symptoms included expansion and development of self-reactive T cells, their differentiation into CD44high IL-17-producing helper T cells, and severe neutrophilic inflammation. Repopulation of FOXP3+ T regulatory cells after lymphopenia normally occurred, suggesting escape of skin-reactive conventional T cells from control by regulatory T cells. Furthermore, we found that a depletion of the intestinal microbiota by antibiotics prevents CY-induced dermatitis, indicating roles of the commensal intestinal microbiota in LIP and Th17 development in vivo. The current data suggested that post-thymic tolerance of Dsg3H1 mice is established on a fragile balance in the lymphoreplete immune environment and broken by the interplay between lymphopenia and intestinal microbiota. The dynamic phenotypes observed in Dsg3H1 mice prompt a re-evaluation of opportunistic lymphopenia together with the microbiota as pivotal environmental factors, impacting individuals with genetic predispositions for autoimmune diseases.

Effects of Potential Prebiotics from Codium fragile on Intestinal Diseases

This study examined the effects of an extract of the green algae Codium fragile (hereafter referred to as CFE) on dextran sulfate sodium (DSS)-induced colitis. As the administration of CFE increased, the proliferation of Akkermansia muciniphila, which is a key player in metabolic and gastrointestinal disorders, also increased. After CFE administration for 10 weeks, acetic acid was identified as the major metabolite in mouse cecum and β-glucuronidase activity in mouse fecesdecreased. Further, CFE significantly alleviated the acute intestinal injury induced by DSS administration, including DAI score, colon length, and histological score. The experimental group also displayed indications of significantly lower neutrophil activity and inflammation. In conclusion, the protective effect of CFE against DSS colitis suggests its clinical use by IBD patients.

Neutrophil-mediated innate immune resistance to bacterial pneumonia is dependent on Tet2 function.

Individuals with clonal hematopoiesis of indeterminate potential (CHIP) are at increased risk of aging related health conditions and all-cause mortality, but whether CHIP affects risk of infection is much less clear. Using UK Biobank data, we revealed a positive association between CHIP and incident pneumonia in 438,421 individuals. We show that inflammation enhanced pneumonia risk, as CHIP carriers with a hypomorphic IL6 receptor polymorphism were protected. To better characterize the pathways of susceptibility, we challenged hematopoietic Tet Methylcytosine Dioxygenase 2-knockout (Tet2-/-) and floxed control mice (Tet2fl/fl) with Streptococcus pneumoniae. As with human CHIP carriers, Tet2-/- mice had hematopoietic abnormalities resulting in the expansion of inflammatory monocytes and neutrophils in peripheral blood. Yet, these cells were insufficient in defending against S. pneumoniae and resulted in increased pathology, impaired bacterial clearance, and higher mortality in Tet2-/- mice. We delineated the transcriptional landscape of Tet2-/- neutrophils and found that, while inflammation-related pathways were upregulated in Tet2-/- neutrophils, migration and motility pathways were compromised. Using live-imaging techniques, we demonstrated impairments in motility, pathogen uptake, and neutrophil extracellular trap (NET) formation by Tet2-/- neutrophils. Collectively, we show that CHIP is a risk factor for bacterial pneumonia related to innate immune impairments.

Altered circular RNA expressions in extracellular vesicles from bronchoalveolar lavage fluids in mice after bacterial infections.

Circular RNAs (circRNAs) are a class of transcripts that often are generated by back-splicing that covalently connects the 3'end of the exon to the 5'end. CircRNAs are more resistant to nuclease and more stable than their linear counterparts. One of the well-recognized roles of circRNAs is the miRNA sponging effects that potentially lead to the regulation of downstream proteins. Despite that circRNAs have been reported to be involved in a wide range of human diseases, including cancers, cardiovascular, and neurological diseases, they have not been studied in inflammatory lung responses. Here, we analyzed the circRNA profiles detected in extracellular vesicles (EVs) obtained from the broncho-alveolar lavage fluids (BALF) in response to LPS or acid instillation in mice. Next, we validated two specific circRNAs in the BALF-EVs and BALF cells in response to endotoxin by RT-qPCR, using specific primers targeting the circular form of RNAs rather than the linear host RNAs. The expression of these selected circRNAs in the BALF inflammatory cells, alveolar macrophages (AMs), neutrophils, and lung tissue were analyzed. We further predicted the potential miRNAs that interact with these circRNAs. Our study is the first report to show that circRNAs are detectable in BALF EVs obtained from mice. The EV-cargo circRNAs are significantly altered by the noxious stimuli. The circRNAs identified using microarrays may be validated by RT-qPCR using primers specific to the circular but not the linear form. Future studies to investigate circRNA expression and function including miRNA sponging in lung inflammation potentially uncover novel strategies to develop diagnostic/therapeutic targets.

Impaired local Vitamin D3 metabolism contributes to IL-36g overproduction in epithelial cells in chronic rhinosinusitis with nasal polyps.

Vitamin D (VD) possesses immunomodulatory properties, but its role in chronic rhinosinusitis with nasal polyps (CRSwNP) remains poorly studied. Herein, we aim to explore the regulation and function of VD3 in CRSwNP. 25-hydroxyvitamin D3 (25VD3) levels in serum and tissue lysates were detected by ELISA. The expression of VD receptor (VDR) and cytochrome P450 family 27 subfamily B member 1 (CYP27B1), the enzyme that converts 25VD3 to the active 1,25-hydroxyvitamin D3 (1,25VD3), and their expression regulation in human nasal epithelial cells (HNECs) were studied by RT-PCR, western blotting, immunofluorescence, and flow cytometry. RNA sequencing was performed to identify genes regulated by 1,25VD3 in HNECs. HNECs and polyp tissue explants were treated with 1,25VD3, 25VD3, and dexamethasone. 25VD3 levels in serum and nasal tissue lysates were decreased in patients with eosinophilic and noneosinophilic CRSwNP than control subjects. The expression of VDR and CYP27B1 were reduced in eosinophilic and noneosinophilic CRSwNP, particularly in nasal epithelial cells. VDR and CYP27B1 expression in HNECs were downregulated by interferon y and poly (I:C). Polyp-derived epithelial cells demonstrated an impaired ability to convert 25VD3 to 1,25VD3 than control tissues. 1,25VD3 and 25VD3 suppressed IL-36y production in HNECs and polyp tissues, and the effect of 25VD3 was abolished by siCYP27B1 treatment. Tissue 25VD3 levels negatively correlated with IL-36y expression and neutrophilic inflammation in CRSwNP. Reduced systemic 25VD3 level, local 1,25VD3 generation and VDR expression result in impaired VD3 signaling activation in nasal epithelial cells, thereby exaggerating IL-36y production and neutrophilic inflammation in CRSwNP.

Clinical criteria accurately diagnose severe but not moderate alcohol-associated hepatitis: A systematic review and meta-analysis.

The precision of clinical criteria and the utility of liver biopsy for diagnosis or prognosis remain unclear in patients with alcohol-associated hepatitis (AH). We systematically reviewed the literature to answer these questions. Four databases were searched for studies describing the precision of clinical criteria (National Institute on Alcohol Abuse and Alcoholism, European Association for Study of Liver, or classical) and the role of histology in AH. The precision(positive predictive value) of criteria was pooled through random-effects meta-analysis, and its variation was investigated through subgroups and meta-regression of study-level factors with their percent contribution to variation (R2). The risk of bias among studies was evaluated through the QUADAS2 tool (PROSPERO-ID-CRD4203457250). Of 4320 studies, 18 in the systematic review and 15 (10/5: low/high risk of bias, N=1639) were included in the meta-analysis. The pooled precision of clinical criteria was 80.2% (95% CI: 69.7-89.7, I2:93%, p < 0.01), higher in studies with severe AH (mean-Model for End-Stage Liver Disease > 20) versus moderate AH (mean-Model for End-Stage Liver Disease < 20): 92% versus 67.1%, p < 0.01, and in studies with serum bilirubin cutoff 5 versus 3 mg/dL (88.5% vs.78.8%, p = 0.01). The factors contributing to variation in precision were Model for End-Stage Liver Disease (R2:72.7%), upper gastrointestinal bleed (R2:56.3%), aspartate aminotransferase:aspartate aminotransferase ratio (R2:100%), clinical criteria (R2:40.9%), bilirubin (R2:22.5%), and Mallory body on histology (R2:19.1%).The net inter-pathologist agreement for histologic findings of AH was variable (0.33-0.97), best among 2 studies describing AH through simple and uniform criteria, including steatosis, ballooning, and neutrophilic inflammation. Few studies reported the utility of histology in estimating steroid responsiveness (N = 1) and patient prognosis (N = 4); however, very broad septa, pericellular fibrosis, and cholestasis were associated with mortality. Bilirubinostasis was associated with infection in 1 study. Clinical criteria are reasonably precise for diagnosing severe AH, while there is an unmet need for better criteria for diagnosing moderate AH. Histologic diagnosis of AH should be simple and uniform.

Low levels of microRNA-21 in neutrophil-derived exosomes may contribute to airway smooth muscle hyperproliferation in horses with severe asthma.

Neutrophilic inflammation is associated with the degree of airway obstruction in severe equine asthma (SEA), but the contribution of these leukocytes to bronchial remodeling remains ill defined. Neutrophils could cause structural alterations of the airways by the release of exosomes, a type of cell-derived nanoparticles that can modify the biology of local and distant cells. Neutrophil-derived exosomes have been shown to increase airway smooth muscle (ASM) cell proliferation in humans and horses. Therefore, this study aimed to identify neutrophil exosomal microRNAs (miRs) implicated in the regulation of ASM biology in SEA. 6 horses with SEA and 6 healthy controls. The expression of selected miRs in exosomes from peripheral neutrophils was studied by quantitative PCR. The effects of miR-21 transfection in ASM cells were evaluated by gene expression analysis and proliferation studies. The miR-21 was downregulated in neutrophil exosomes from SEA horses, and it attenuated the proliferation of ASM cells stimulated with lipopolysaccharide. The lower level of miR-21 in neutrophil-derived exosomes could contribute to ASM hyperproliferation, which could, in turn, promote the thickening of the bronchial wall in SEA.

The Efficacy & Molecular Mechanisms of a Terpenoid Compound Ganoderic Acid C1 on Corticosteroid-Resistant Neutrophilic Airway Inflammation: In vivo and in vitro Validation.

Neutrophil predominant airway inflammation is associated with severe and steroid-resistant asthma clusters. Previously, we reported efficacy of ASHMI, a three-herb TCM asthma formula in a steroid-resistant neutrophil-dominant murine asthma model and further identified Ganoderic Acid C1 (GAC1) as a key ASHMI active compound in vitro. The objective of this study is to investigate GAC1 effect on neutrophil-dominant, steroid-resistant asthma in a murine model. In this study, Balb/c mice were systematically sensitized with ragweed (RW) and alum and intranasally challenged with ragweed. Unsensitized/PBS challenged mice served as normal controls. Post sensitization, mice were given 4 weeks of oral treatment with GAC1 or acute dexamethasone (Dex) treatment at 48 hours prior to challenge. Pulmonary cytokines were measured by ELISA, and lung sections were processed for histology by H&E staining. Furthermore, GAC1 effect on MUC5AC expression and on reactive oxygen species (ROS) production in human lung epithelial cell line (NCI-H292) was determined by qRT-PCR and ROS assay kit, respectively. Computational analysis was applied to select potential targets of GAC1 in steroid-resistant neutrophil-dominant asthma. Molecular docking was performed to predict binding modes between GAC1 and Dex with TNF-α. The result of the study showed that chronic GAC1 treatment, significantly reduced pulmonary inflammation (P < 0.01-0.001 vs Sham) and airway neutrophilia (P < 0.01 vs Sham), inhibited TNF-α, IL-4 and IL-5 levels (P < 0.05-0.001 vs Sham). Acute Dex treatment reduced eosinophilic inflammation and IL-4, IL-5 levels, but had no effect on neutrophilia and TNF-α production. GAC1 treated H292 cells showed decreased MUC5AC gene expression and production of ROS (P < 0.001 vs stimulated/untreated cells). Molecular docking results showed binding energy of complex GAC1-TNF was -10.8 kcal/mol. GAC1 may be a promising anti-asthma botanical drug for treatment of steroid-resistant asthma.