Elastase Research Articles

Clinical utility of the neutrophil elastase inhibitor sivelestat for the treatment of ALI/ARDS patients with COVID-19

BackgroundSivelestat, a neutrophil elastase inhibitor, is postulated to mitigate acute lung injury in patients following emergency surgery. However, its efficacy in patients with acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) induced by coronavirus disease 2019 (COVID-19) remains uncertain. This study aims to evaluate the pulmonary protective effects of sivelestat in COVID-19 patients with ALI/ARDS. MethodsA retrospective study was conducted involving 2454 COVID-19 patients between October 5, 2022, and February 1, 2023. Of these, 102 patients received sivelestat (0.2 mg/kg/h), while 2352 age- and sex-matched controls were identified. Propensity score matching (PSM) analysis was used to match sivelestat and non-sivelestat subgroups in ratios of 1:1 and 1:3 for sensitivity analysis. The primary outcome was a composite of effective outcomes, including 30-day mortality. Secondary outcomes included changes in partial pressure of arterial oxygen (PaO2), the ratio of PaO2 to the fraction of inspired oxygen (PaO2/FiO2), and various cytokine levels. Safety evaluations included assessments of liver function, kidney function, and leukopenia. ResultsIn the propensity score-matched analysis, the sivelestat group had a higher proportion of severe/critical patients (87.26 % vs. 51.02 %, P < 0.001), more ARDS patients (4.9 % vs. 0.43 %, P < 0.001), and more patients with interstitial lung disease (4.9 % vs. 1.49 %, P = 0.023), but fewer patients with stroke (17.65 % vs. 19.86 %, P < 0.001). Oxygen therapy rates were similar between the groups (79.41 % vs. 80.95 %, P = 0.9). The relative risk reduction in 30-day mortality was 88.45 % (95 % confidence interval [CI] 81.23%–93.21 %) for severe/critical COVID-19 patients treated with sivelestat. Sivelestat significantly decreased cytokine levels of interferon alpha (IFNα), interleukin-1 beta (IL-1β), and interleukin-2 (IL-2).In the sivelestat group, the mortality rate was significantly reduced with standard oxygenation and HFNC therapy(P < 0.05). The treatment with sivelestat did not increase side effects. ConclusionThe administration of the neutrophil elastase inhibitor sivelestat may improve clinical outcomes in COVID-19 patients with ALI/ARDS. These findings suggest that sivelestat could be considered an effective treatment option to alleviate pulmonary inflammatory injury caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

Exploration of novel human neutrophil elastase inhibitors from natural compounds: Virtual screening, in vitro, molecular dynamics simulation and in vivo study

BackgroundHuman neutrophil elastase (HNE) is an important contributor to lung diseases such as acute lung injury (ALI) or acute respiratory distress syndrome. Therefore, this study aimed to identify natural HNE inhibitors with anti-inflammatory activity through machine learning algorithms, in vitro assays, molecular dynamic simulation, and an in vivo ALI assay. MethodsBased on the optimized Discovery Studio two-dimensional molecular descriptors, combined with different molecular fingerprints, six machine learning models were established using the Naïve Bayesian (NB) method to identify HNE inhibitors. Subsequently, the optimal model was utilized to screen 6925 drug-like compounds obtained from the Traditional Chinese Medicine Systems Pharmacy Database and Analysis Platform (TCMSP), followed by ADMET analysis. Finally, 10 compounds with reported anti-inflammatory activity were selected to determine their inhibitory activities against HNE in vitro, and the compounds with the best activity were selected for a 100 ns molecular dynamics simulation and its anti-inflammatory effect was evaluated using Poly (I:C)-induced ALI model. ResultsThe evaluation of the in vitro HNE inhibition efficiency of the 10 selected compounds showed that the flavonoid tricetin had the strongest inhibitory effect on HNE. The molecular dynamics simulation indicated that the binding of tricetin to HNE was relatively stable throughout the simulation. Importantly, in vivo experiments indicated that tricetin treatment substantially improved the Poly (I:C)-induced ALI. ConclusionThe proposed NB model was proved valuable for exploring novel HNE inhibitors, and natural tricetin was screened out as a novel HNE inhibitor, which was confirmed by in vitro and in vivo assays for its inhibitory activities.

Neutrophil activation, acute lung injury and disease severity in Plasmodium knowlesi malaria.

The risk of severe malaria from the zoonotic parasite Plasmodium knowlesi approximates that from P. falciparum. In severe falciparum malaria, neutrophil activation contributes to inflammatory pathogenesis, including acute lung injury. The role of neutrophil activation in the pathogenesis of severe knowlesi malaria has not been examined. We evaluated 213 patients with P. knowlesi mono-infection (138 non-severe, 75 severe) and 49 Plasmodium-negative controls from Malaysia. Markers of neutrophil activation (soluble neutrophil elastase [NE], citrullinated histone [CitH3] and circulating neutrophil extracellular traps [NETs]) were quantified in peripheral blood by microscopy and immunoassays. Findings were correlated with malaria severity, ALI clinical criteria, biomarkers of parasite biomass, haemolysis, and endothelial activation. Neutrophil activation increased with disease severity, with median levels higher in severe than non-severe malaria and controls for NE (380[IQR:210-930]ng/mL, 236[139-448]ng/mL, 218[134-307]ng/mL, respectively) and CitH3 (8.72[IQR:3.0-23.1]ng/mL, 4.29[1.46-9.49]ng/mL, 1.53[0.6-2.59]ng/mL, respectively)[all p<0.01]. NETs were higher in severe malaria compared to controls (126/μL[IQR:49-323] vs 51[20-75]/μL, p<0.001). In non-severe malaria, neutrophil activation fell significantly upon discharge from hospital (p<0.03). In severe disease, NETs, NE, and CitH3 were correlated with parasitaemia, cell-free haemoglobin and angiopoietin-2 (all Pearson's r>0.24, p<0.05). Plasma NE and angiopoietin-2 were higher in knowlesi patients with ALI than those without (p<0.008); neutrophilia was associated with an increased risk of ALI (aOR 3.27, p<0.01). In conclusion, neutrophil activation is increased in ALI and in proportion to disease severity in knowlesi malaria, is associated with endothelial activation, and may contribute to disease pathogenesis. Trials of adjunctive therapies to regulate neutrophil activation are warranted in severe knowlesi malaria.

Evaluation of Novel Hydrazone‐Imide Hybrid Compound as HNE Inhibitor In Silico: Synthesis, XRD and DFT Analysis

AbstractInvestigation into the interactions between drugs and biomolecules can offer a precise understanding of their biological behaviours in vitro and in vivo, providing crucial insights for designing new drugs. Herein, we report the design, synthesis, and characterization of a bicyclic hydrazone derivative, along with its potential as an inhibitor of human neutrophil elastase (HNE), a critical enzyme in the treatment of acute and chronic lung injuries. We present an improved protocol for the synthesis of the compound using N‐amino‐bicyclo[2.2.1]hept‐5‐ene‐2,endo‐3‐endo‐dicarboximide and 4‐(trifluoromethyl)benzaldehyde through a facile, one‐pot, catalyst‐free synthesis in dry ethanol at reflux temperature. Within 12 hours, bicyclic‐hydrazone derivative was obtained with yields of up to 75 % and was characterized using various spectroscopic techniques and computational analyses based on the DFT approach. In silico ADME/T profile of the compound was evaluated and molecular docking simulation was utilized to predict its potential as a HNE inhibitor. The binding energy values of the THI ligand to the chosen PDB ID: 5ABW, 3Q77, and 2RG3 target proteins were determined as −8.41, −7.20, and −7.27 kcal/mol, respectively. In silico analysis findings indicate that the THI compound has promising drug‐likeness properties and has the potential to be evaluated in the development of new HNE inhibitors.

Elevation of circulating neutrophil extracellular traps in endometrial cancer: Poor prognostic value of cell-free double-stranded DNA

ObjectiveNeutrophils produce neutrophil extracellular traps (NETs) by releasing nuclear contents into the extracellular environment. NETs are associated with systemic inflammation and cancer development and progression. We aimed to investigate whether NET markers are associated with the prognosis of endometrial cancer. MethodsCirculating levels of three NET markers (histone-DNA complex, cell-free double-stranded DNA (dsDNA), and neutrophil elastase) were measured in 98 patients with endometrial cancer who underwent surgery as primary treatment between January 2015 and June 2018 and 45 healthy women. Area under the receiver operating characteristic curve (AUC) analyses were conducted to investigate the diagnostic and prognostic utility of the markers for endometrial cancer. ResultsPatients with endometrial cancer showed significantly higher levels of the three NET markers than those in healthy controls. In discriminating endometrial cancer patients from healthy controls, the three NET markers showed AUC values in the following order: cell-free dsDNA (0.832; 95 % CI, 0.760–0.889), histone-DNA complex (0.740; 95 % CI, 0.660–0.809), and neutrophil elastase (0.689; 95 % CI, 0.607–0.764), comparable to those of CA-125 (0.741; 95 % CI, 0.659–0.813). Multivariate analysis adjusting for FIGO stage, histology, and lymphovascular space invasion, and lymph node involvement revealed that cell-free dsDNA level (cutoff: 95.2 ng/mL) was an independent prognostic marker for poor progression-free (adjusted HR, 2.75; 95 % CI, 1.096.92; P = 0.032) and overall survival (adjusted HR, 11.51; 95 % CI, 2.0664.22; P = 0.005) for patients with endometrial cancer. ConclusionHigh levels of circulating NET markers were observed in patients with endometrial cancer. Cell-free dsDNA levels may play a role as prognostic markers for endometrial cancer.

Evaluation of NETosis (Neutrophil Extracellular Traps) biomarkers in smokers and nonsmokers periodontitis patients

Background To evaluate the NETosis biomarkers citrullinated histone H3 (citH3), neutrophil elastase (ELA), calprotectin (CALPRO), and myeloperoxidase (MPO) as indicators of inflammation in the severe stages of periodontitis III and IV in both (smokers and nonsmokers) patients, and to determine the correlation between NETosis biomarkers and clinical periodontal parameters. Methods This study recruited male subjects with an age range of (20-60) years; 60 were stage III and stage IV periodontitis patients, 30 were cigarette smoker, and 30 were nonsmokers. After applying the inclusion and exclusion criteria to evaluate their eligibility for recruitment, 25 control subjects with a healthy periodontal status and good oral hygiene maintenance were included. Unstimulated saliva was obtained and evaluated using an enzyme-linked immunosorbent assay, and the following periodontal parameters were documented: [plaque index, bleeding on probing, periodontal pocket depth, and clinical attachment loss]. Results The mean levels of all salivary NETosis biomarkers citH3, ELA, CALPRO, and MPO were elevated in the periodontitis groups (smokers and nonsmokers) than in controls. Moreover, the mean NETosis biomarker‘s mean levels were significantly higher in smoker than in nonsmokers. In addition, the correlations were significant between CALPRO and CitH3 in smokers and between ELA and CitH3 in nonsmokers. Conclusions The results of this study showed that the chosen salivary biomarkers of NETosis revealed elevated clinical accuracy in differentiating the studied periodontitis groups (smokers and nonsmokers) from controls. In addition, cigarette smoking increases the risk of periodontitis, and neutrophils in smokers with periodontitis exhibited more susceptibility to form neutrophil extracellular traps when compared with nonsmokers.

S. aureus Eap is a polyvalent inhibitor of neutrophil serine proteases

Staphylococcus aureus expresses three high-affinity neutrophil serine protease (NSP) inhibitors known as the extracellular adherence protein domain (EAPs) proteins. Whereas EapH1 and EapH2 are comprised of a single EAP domain, the modular extracellular adherence protein (Eap) from S. aureus strain Mu50 consists of four EAP domains. We recently reported that EapH2 can simultaneously bind and inhibit cathepsin-G (CG) and neutrophil elastase (NE), which are the two most abundant NSPs. This unusual property of EapH2 arises from independent CG and NE-binding sites that lie on opposing faces of its EAP domain. Here we used X-ray crystallography and enzyme assays to show that all four individual domains of Eap (i.e. Eap1, Eap2, Eap3, and Eap4) exhibit an EapH2-like ability to form ternary complexes with CG and NE that inhibit both enzymes simultaneously. We found that Eap1, Eap2, and Eap3 have similar functional profiles insofar as NSP inhibition is concerned, but that Eap4 displays an unexpected ability to inhibit two NE enzymes simultaneously. Using X-ray crystallography, we determined that this second NE-binding site in Eap4 arises through the same region of its EAP domain that also comprises its CG-binding site. Interestingly, small angle X-ray scattering data showed that stable tail-to-tail dimers of the NE/Eap4/NE ternary complex exist in solution. This arrangement is compatible with NSP-binding at all available sites in a two-domain fragment of Eap. Together, our work implies that Eap is a polyvalent inhibitor of NSPs. It also raises the possibility that higher-order structures of NSP-bound Eap may have unique functional properties.

Robustaflavone as a novel scaffold for inhibitors of native and auto-proteolysed human neutrophil elastase

ABSTRACT Human neutrophil elastase (HNE) plays a key role in initiating inflammation in the cardiopulmonary and systemic contexts. Pathological auto-proteolysed two-chain (tc) HNE exhibits reduced binding affinity with inhibitors. Using AutoDock Vina v1.2.0, 66 flavonoid inhibitors, sivelestat and alvelestat were docked with single-chain (sc) HNE and tcHNE. Schrodinger PHASE v13.4.132 was used to generate a 3D-QSAR model. Molecular dynamics (MD) simulations were conducted with AMBER v18. The 3D-QSAR model for flavonoids with scHNE showed r 2 = 0.95 and q 2 = 0.91. High-activity compounds had hydrophobic A/A2 and C/C2 rings in the S1 subsite, with hydrogen bond donors at C5 and C7 positions of the A/A2 ring, and the C4’ position of the B/B1 ring. All flavonoids except robustaflavone occupied the S1’-S2’ subsites of tcHNE with decreased AutoDock binding affinities. During MD simulations, robustaflavone remained highly stable with both HNE forms. Principal Component Analysis suggested that robustaflavone binding induced structural stability in both HNE forms. Cluster analysis and free energy landscape plots showed that robustaflavone remained within the sc and tcHNE binding site throughout the 100 ns MD simulation. The robustaflavone scaffold likely inhibits both tcHNE and scHNE. It is potentially superior to sivelestat and alvelestat and can aid in developing therapeutics targeting both forms of HNE.

In-silico screening of phytochemicals from Tridax procumbens. Linn against human neutrophil elastase targeting chronic obstructive pulmonary disease

In-silico screening of phytochemicals from Tridax procumbens. Linn against human neutrophil elastase targeting chronic obstructive pulmonary disease

CPHEN-017: Comprehensive phenotyping of neutrophil extracellular traps (NETs) on peripheral human neutrophils.

With the recent discovery of their ability to produce neutrophil extracellular traps (NETs), neutrophils are increasingly appreciated as active participants in infection and inflammation. NETs are characterized as large, web-like networks of DNA and proteins extruded from neutrophils, and there is considerable interest in how these structures drive disease in humans. Advancing research in this field is contingent on developing novel tools for quantifying NETosis. To this end, we have developed a 7-marker flow cytometry panel for analyzing NETosis on human peripheral neutrophils following in vitro stimulation, and in fresh circulating neutrophils under inflammatory conditions. This panel was optimized on neutrophils isolated from whole blood and analyzed fresh or in vitro stimulated with phorbol 12-myristate 13-acetate (PMA) or ionomycin, two known NET-inducing agonists. Neutrophils were identified as SSChighFSChighCD15+CD66b+. Neutrophils positive for amine residues and 7-Aminoactinomycin D (7-AAD), our DNA dye of choice, were deemed necrotic (Zombie-NIR+7-AAD+) and were removed from downstream analysis. Exclusion of Zombie-NIR and positivity for 7-AAD (Zombie-NIRdim7-AAD+) was used here as a marker of neutrophil-appendant DNA, a key feature of NETs. The presence of two NET-associated proteins - myeloperoxidase (MPO) and neutrophil elastase (NE) - were utilized to identify neutrophil-appendant NET events (SSChighFSChighCD15+CD66b+Zombie NIRdim7-AAD+MPO+NE+). We also demonstrate that NETotic neutrophils express citrullinated histone H3 (H3cit), are concentration-dependently induced by in vitro PMA and ionomycin stimulation but are disassembled with DNase treatment, and are present in both chronic and acute inflammation. This 7-color flow cytometry panel provides a novel tool for examining NETosis in humans.

Novel cathepsin C inhibitor, BI 1291583, intended for treatment of bronchiectasis: Phase I characterization in healthy volunteers.

Novel treatments are needed to reduce inflammation, improve symptoms, address exacerbations, and slow disease progression in bronchiectasis. Cathepsin C (CatC) inhibition promises to achieve this through reduction of neutrophil-derived serine protease (including neutrophil elastase [NE] and proteinase 3 [PR3]) activation. Here, we present the phase I characterization of the novel CatC inhibitor, BI 1291583. Five phase I trials of BI 1291583 in healthy subjects are presented: a single-rising-dose study (NCT03414008) and two multiple-rising-dose studies (NCT03868540 and NCT04866160) assessing the safety, tolerability, pharmacodynamics, and pharmacokinetics of BI 1291583; a food effect study (NCT03837964); and a drug-drug interaction study (NCT03890887) of BI 1291583 and itraconazole. BI 1291583 was safe and well tolerated across the doses tested in these trials. Most adverse events (AEs) were mild or moderate in intensity, with no serious AEs, AEs of special interest or deaths reported in any trial. Drug-related skin exfoliation was not reported more frequently in subjects treated with BI 1291583 compared with placebo. BI 1291583 was readily absorbed, and pharmacokinetics were supra-proportional over the dose ranges assessed. Additionally, BI 1291583 inhibited CatC in a dose-dependent manner, inhibited downstream NE activity, and decreased PR3 levels. No food effect was observed. Co-administration of multiple doses of itraconazole increased BI 1291583 exposure approximately twofold. Due to these promising phase I results, a multinational phase II program of BI 1291583 in adults with bronchiectasis is ongoing (Airleaf™ [NCT05238675], Clairafly™ [NCT05865886], and Clairleaf™ [NCT05846230]).

Altered neutrophil extracellular traps formation among medical residents with sleep deprivation

Resident physicians on long-term night shifts often face sleep deprivation, affecting the immune response, notably neutrophils, vital to innate defense mechanisms. Sleep-deprived residents exhibit altered neutrophil counts and reduced phagocytosis and NADPH oxidase activity, critical to combating infections. Our study focused on neutrophil extracellular traps (NETs), a defense process against pathogens not previously linked to sleep loss. Results revealed that sleep-deprived residents exhibited a 19.8 % reduction in NET formation compared to hospital workers with regular sleep patterns (P < 0.01). Additionally, key NETs proteins, Neutrophil Elastase and Myeloperoxidase, were less active in sleep-deprived individuals (1.53mU; P < 0.01 and 0.95U; P < 0.001 decrease, accordingly). Interestingly, the ability to form NETs resumed to normal levels three months post-residency among pediatric residents. The causal relationship between reduced NETs due to sleep deprivation and the increased susceptibility to infections, as well as its implications for infection severity, is a critical area for further investigation.

Deficiency of hasB accelerated the clearance of Streptococcus equi subsp. Zooepidemicus through gasdermin d-dependent neutrophil extracellular traps

Streptococcus equi subsp. zooepidemicus (S. zooepidemicus, SEZ) is an essential zoonotic bacterial pathogen that can cause various inflammation, such as meningitis, endocarditis, and pneumonia. UDP-glucose dehydrogenase (hasB) is indispensable in synthesizing SEZ virulence factor hyaluronan capsules. Our study investigated the infection of hasB on mice response to SEZ by employing a constructed capsule-deficient mutant strain designated as the ΔhasB strain. This deficiency was associated with a reduced SEZ bacterial load in the mice’s blood and peritoneal lavage fluid (PLF) post-infection. Besides, the ΔhasB SEZ strain exhibited a higher propensity for neutrophil infiltration and release of cell-free DNA (cfDNA) in vivo compared to the wild-type (WT) SEZ strain. In vitro experiments further revealed that ΔhasB SEZ more effectively induced the formation of neutrophil extracellular traps (NETs) containing histone 3 (H3), neutrophil elastase (NE), and DNA, than its WT counterpart. Moreover, the release of NETs was determined to be gasdermin D (GSDMD)-dependent during the infection process. Taken together, these findings underscore that the deficiency of the hasB gene in SEZ leads to enhanced GSDMD-dependent NET release from neutrophils, thereby reducing SEZ’s capacity to resist NETs-mediated eradication during infection. Our finding paves the way for the development of innovative therapeutic strategies against SEZ.

Gut colonization with antibiotic-resistant Escherichia coli pathobionts leads to disease severity in ulcerative colitis

Escherichia coli is a Gram-negative commensal of human gut. Surprisingly, the role of E. coli in the pathogenesis of ulcerative colitis (UC) has not been explored until now. Human gut microbiota composition and meta-gut resistome were evaluated using metagenomics. Antibiotic susceptibility of E. coli isolates against different class of antibiotics was investigated. Further, the genome sequence analysis of E. coli isolates was performed to gain insight into the antimicrobial resistance (AMR) mechanism and virulence factors. Gut proteome of UC and non-UC was examined to understand the effect of resistant bacteria on host physiology. In UC patients, meta-gut resistome was found to be dominated by AMR genes (829) compared to healthy controls (HC) [518]. The metagenome study revealed a higher prevalence of AMR genes in the rural population (378 in HC; 607 in UC) compared to the urban (340 in HC; 578 in UC). Approximately, 40% of all E. coli isolates were multi-drug resistant (MDR), with higher prevalence in UC (43.75%) compared to HC (33.33%). Up-regulated expression of antimicrobial human proteins (lactotransferrin, azurocidin, cathepsin G, neutrophil elastase, and neutrophil defensin 3) and inflammatory mediator (Protein S100-A9 and Protein S100-A8) suggest microbial infection in UC gut. In addition to the conventional culturomics method, a multi-omics strategy provides deeper insights into the disease etiology, emergence of MDR pathobionts, and their roles in the disruption of the healthy gut environment in UC patients.

NQO1 Triggers Neutrophil Recruitment and NET Formation to Drive Lung Metastasis of Invasive Breast Cancer.

Metastasis to the lungs is a leading cause of death for patients with breast cancer. Therefore, effective therapies are urgently needed to prevent and treat lung metastasis. In this study, we uncovered a mechanism by which NAD(P)H:quinone oxidoreductase 1 (NQO1) orchestrates lung metastasis. NQO1 stabilized and upregulated peptidyl-prolyl cis-trans isomerase A (PPIA), a chaperone that regulates protein conformation and activity, by preventing its oxidation at a critical cysteine residue C161. PPIA subsequently activated CD147, a membrane protein that facilitates cell invasion. Moreover, NQO1-induced secretion of PPIA modulated the immune landscape of both primary and lung metastatic sites. Secreted PPIA engaged CD147 on neutrophils and triggered the release of neutrophil extracellular traps (NET) and neutrophil elastase, which enhanced tumor progression, invasiveness, and lung colonization. Pharmacological targeting of PPIA effectively inhibited NQO1-mediated breast cancer lung metastasis. These findings reveal a previously unrecognized NQO1-PPIA-CD147-NET axis that drives breast cancer lung metastasis. Inhibiting this axis is a potential therapeutic strategy to limit lung metastasis in patients with breast cancer. Significance: NQO1 stabilizes and promotes the secretion of PPIA to activate CD147 in neutrophils and stimulate NET formation, promoting breast cancer lung metastasis and providing therapeutic targets for this fatal condition.

PD-L1 siRNA hitched polyethyleneimine-elastase constituting nanovesicle induces tumor immunogenicity and PD-L1 silencing for synergistic antitumor immunotherapy

BackgroundPD-1/PD-L1 blockade has become a powerful method to treat malignant tumors. However, a large proportion of patients still do not benefit from this treatment, due to low tumor immunogenicity and low tumor penetration of the agents. Recently, neutrophil elastase has been shown to induce robust tumor immunogenicity, while the insufficient enzyme activity at the tumor site restricted its anti-tumor application. Here, we designed polyethyleneimine-modified neutrophil elastase (PEI-elastase) loaded with PD-L1small interfering RNA (PD-L1 siRNA) for improving enzymatic activity and delivering siRNA to tumor, which was expected to solve the above-mentioned problems.ResultsWe first demonstrated that PEI-elastase possessed high enzymatic activity, which was also identified as an excellent gene-delivery material. Then, we synthesized anti-tumor lipopolymer (P-E/S Lip) by encapsulating PEI-elastase and PD-L1siRNA with pH-responsive anionic liposomes. The P-E/S Lip could be rapidly cleaved in tumor acidic environment, leading to exposure of the PEI-elastase/PD-L1 siRNA. Consequently, PEI-elastase induced powerful tumor immunogenicity upon direct tumor killing with minimal toxicity to normal cells. In parallel, PEI-elastase delivered PD-L1siRNA into the tumor and reduced PD-L1 expression. Orthotopic tumor administration of P-E/S Lip not only attenuated primary tumor growth, but also produced systemic anti-tumor immune response to inhibit growth of distant tumors and metastasis. Moreover, intravenous administration of P-E/S Lip into mice bearing subcutaneous tumors leaded to an effective inhibition of established B16-F10 tumor and 4T1 tumor, with histological analyses indicating an absence of detectable toxicity.ConclusionsIn our study, a protease-based nanoplatform was used to cooperatively provoke robust tumor immunogenicity and down-regulate PD-L1 expression, which exhibited great potential as a combination therapy for precisely treating solid tumors.Graphical

Oral infection with periodontal pathogens induced chronic obstructive pulmonary disease-like lung changes in mice

BackgroundEpidemiological studies have demonstrated that periodontitis is an independent risk factor for chronic obstructive pulmonary disease (COPD). However, the mechanism underlying the association between these two diseases remains unclear. The lung microbiota shares similarities with the oral microbiota, and there is growing evidence to suggest that the lung microbiome could play a role in the pathogenesis of COPD. This study aimed to investigate whether periodontal pathogens could contribute to the pathogenesis of COPD in a mouse model.MethodsWe established mouse models with oral infection by typical periodontal pathogens, porphyromonas gingivalis (Pg group) or fusobacterium nucleatum (Fn group), over a three-month period. Mice that did not receive oral infection were set as the control group (C group). We assessed the level of alveolar bone resorption, lung function, and histological changes in the lungs of the mice. Additionally, we measured the levels of inflammatory factors and tissue damage associated factors in the lung tissues.ResultsLung function indices, including airway resistance, peak inspiratory/expiratory flow and expiratory flow-50%, were significantly reduced in the Fn group compared to the C group. Additionally, histological examination revealed an increased number of inflammatory cells and bullae formation in the lung tissue sections of the Fn group. Meanwhile, levels of inflammatory factors such as IL-1β, IL-6, IFN-γ, and TNF-α, as well as tissue damage associated factors like matrix metalloproteinase-8 and neutrophil elastase, were significantly elevated in the lung tissue of the Fn group in comparison to the C group. The Pg group also showed similar but milder lung changes compared to the Fn group. Pg or Fn could be detected in the lungs of both oral infected groups.ConclusionThe results indicated that oral periodontal pathogens infection could induce COPD-like lung changes in mice, and they may play a biological role in the association between periodontitis and COPD.

Progesterone modulates the immune microenvironment to suppress ovalbumin-induced airway inflammation by inhibiting NETosis

Studies have demonstrated that prior to puberty, girls have a lower incidence and severity of asthma symptoms compared to boys. This study aimed to explore the role of progesterone (P4), a sex hormone, in reducing inflammation and altering the immune microenvironment in a mouse model of allergic asthma induced by OVA. Female BALB/c mice with or without ovariectomy to remove the influence of sex hormones were used for the investigations. Serum, bronchoalveolar lavage fluid (BALF), and lung tissue samples were collected for analysis. The results indicated that P4 treatment was effective in decreasing inflammation and mucus secretion in the lungs of OVA-induced allergic asthma mice. P4 treatment also reduced the influx of inflammatory cells into the BALF and increased the levels of Th1 and Th17 cytokines while decreasing the levels of Th2 and Treg cytokines in both BALF and lung microenvironment CD45+ T cells. Furthermore, P4 inhibited the infiltration of inflammatory cells into the lungs, suppressed NETosis, and reduced the number of pulmonary CD4+ T cells while increasing the number of regulatory T cells. The neutrophil elastase inhibitor GW311616A also suppressed airway inflammation and mucus production and modified the secretion of immune Th1, Th2, Th17, and Treg cytokines in lung CD45+ immune cells. These changes led to an alteration of the immunological milieu with increased Th1 and Th17 cells, accompanied by decreased Th2, Treg, and CD44+ T cells, similar to the effects of P4 treatment. Treatment with P4 inhibited NETosis by suppressing the p38 pathway activation, leading to reduced reactive oxygen species production. Moreover, P4 treatment hindered the release of double-stranded DNA during NETosis, thereby influencing the immune microenvironment in the lungs. These findings suggest that P4 treatment may be beneficial in reducing inflammation associated with allergic asthma by modulating the immune microenvironment. In conclusion, this research indicates the potential of P4 as a therapeutic agent for ameliorating inflammation in OVA-induced allergic asthma mice.

Comparative Characteristics of Inflammatory Markers in Alzheimer’s Disease in Males and Females

Background: the role of the sex factor in the differences between the manifestations of normality and pathology is not limited to the phenomenon of sexual dimorphism. It is known that the prevalence of certain diseases in males and females is different, in particular, there is a multiple increase in the incidence of dementia in females compared to males in Alzheimer’s disease (AD). Taking into account the role of neuroinflammation in the pathogenesis of neurodegenerative diseases, there is reason to assume gender differences in inflammation indicators at different stages of dementia in AD. The aim of the study was to conduct comparative analysis of indicators of the inflammatory system in the blood plasma of males and females at different stages of Alzheimer’s disease. Patients, control group and methods: a total of 210 patients with AD (101 males and 109 females) aged 49 to 94 years (average age 72.3 ± 8.2) with varying degrees of dementia severity, i.e. mild, moderate, severe, were examined. In peripheral blood plasma, the enzymatic activity of leukocyte elastase (LE) and the functional activity of α1-proteinase inhibitor (α1-PI) were determined by the spectrophotometric method, and the level of C-reactive protein (CRP) and IL-6 were determined by the enzyme-linked immunosorbent method (ELISA). The control group consisted of 52 healthy people, who did not differ from the patients in age and gender. Results and discussion: in the blood of patients with varying severity of dementia in AD, a statistically significant increase in α1-PI activity was observed compared to controls (p &lt; 0.0001), regardless of gender. For all subgroups of patients with AD, the indicators of LE enzymatic activity were within the control range or beyond its lower limit. Low LE activity was observed in males compared to females both in the general group and in moderate dementia (p = 0.005105, p = 0.028672, respectively). In severe dementia, a significant decrease in LE activity compared to the controls did not depend on gender. Low LE activity in the blood of patients with AD, along with elevated levels of other inflammatory markers, may reflect a critical violation of the permeability of the blood-brain barrier and/or functional exhaustion of neutrophils due to a long-term inflammatory process. In males, compared with females, an increase in the level of the pro-inflammatory cytokine IL-6 was detected in the general group and in moderate dementia (p = 0.021238, p = 0.027894, respectively). A highly significant increase in CRP levels was only detected in males in subgroups with different severity of dementia. CRP levels in males were significantly higher than in females at the stage of moderate and severe dementia (p = 0.000906, p = 0.000049, respectively). Conclusion: distinctive features of inflammatory markers spectrum were identified, depending on gender and severity of dementia in AD. These results can be used to develop sex-specific preventive or therapeutic strategies for patients with mild cognitive impairment to determine risk and resistance to developing dementia.

Human Neutrophil Elastase: Characterization of Intra- vs. Extracellular Inhibition.

Neutrophil elastase (HNE), like other members of the so-called GASPIDs (Granule-Associated Serine Peptidases of Immune Defense), is activated during protein biosynthesis in myeloid precursors and stored enzymatically active in cytoplasmic granules of resting neutrophils until secreted at sites of host defense and inflammation. Inhibitors thus could bind to the fully formed active site of the protease intracellularly in immature progenitors, in circulating neutrophils, or to HNE secreted into the extracellular space. Here, we have compared the ability of a panel of diverse inhibitors to inhibit HNE in the U937 progenitor cell line, in human blood-derived neutrophils, and in solution. Most synthetic inhibitors and, surprisingly, even a small naturally occurring proteinaceous inhibitor inhibit HNE intracellularly, but the extent and dynamics differ markedly from classical enzyme kinetics describing extracellular inhibition. Intracellular inhibition of HNE potentially affects neutrophil functions and has side effects, but it avoids competition of inhibitors with extracellular substrates that limit its efficacy. As both intra- and extracellular inhibition have advantages and disadvantages, the quantification of intracellular inhibition, in addition to classical enzyme kinetics, will aid the design of novel, clinically applicable HNE inhibitors with targeted sites of action.