Pneumonia In Mice Research Articles

Dayuan Yin alleviates symptoms of HCoV-229E-induced pneumonia and modulates the Ras/Raf1/MEK/ERK pathway

Viral pneumonia is characterized by inflammation in the lungs triggered by respiratory viruses. Dayuan Yin (DYY), a traditional Chinese medicine formula known for treating infectious diseases, is hypothesized to offer therapeutic benefits in treating viral pneumonia, although its specific molecular impacts remain understudied. This study aimed to evaluate the therapeutic effects of DYY in mitigating HCoV-229E virus-induced pneumonia in mice. This study employed an HCoV-229E virus-infected mouse model to investigate the therapeutic potential and underlying molecular mechanisms of DYY on virus-induced pneumonia. The respiratory function and organ indices post-treatment were assessed. Lung tissue and tracheal lesions were evaluated via immunohistochemistry. Spleen immune cell composition was analyzed using flow cytometry. Inflammatory cytokines and viral loads were quantified using hypersensitive multiplex electrochemiluminescence method and PCR analysis, respectively. The expression levels of MAS1, Ras, Raf1, MEK1/2, and ERK1/2 in lung tissues were determined through western blot analysis. DYY significantly improved respiratory function, and reduced organ pathology in infected mice. It effectively decreased viral loads and inflammatory cytokines such as IL-6, IL-1β, and TNF-α in lung tissues. Enhancements in immune response were evidenced by increased CD4/CD8 ratios in the spleen. DYY also notably upregulated MAS1 protein levels and suppressed the activation of the Ras/Raf1/MEK/ERK signaling pathway. DYY enhanced respiratory function and exerted significant antiviral and immunomodulatory effects in mice infected with the HCoV-229E virus, primarily by modulating MAS1 expression and inhibiting the Ras/Raf1/MEK/ERK pathway.Graphical

Targeted delivery of flagellin by nebulization offers optimized respiratory immunity and defense against pneumococcal pneumonia.

Novel therapeutic strategies are urgently needed to combat pneumonia caused by Streptococcus pneumoniae strains resistant to standard-of-care antibiotics. Previous studies have shown that targeted stimulation of lung innate immune defenses through intranasal administration of the Toll-like receptor 5 agonist flagellin improves the treatment of pneumonia when combined with antibiotics. To promote translation to the clinic application, this study assessed the direct delivery of flagellin to the airways through nebulization using a vibrating mesh nebulizer in mice. Intranasal delivery achieved approximately 40% lung deposition of the administered flagellin dose, whereas nebulization yielded less than 1%. Despite these differences, nebulized flagellin induced transient activation of lung innate immunity characterized by cytokine/chemokine production and neutrophil infiltration into airways analogous to intranasal administration. Furthermore, inhalation by nebulization resulted in an accelerated resolution of systemic pro-inflammatory responses. Lastly, adjunct therapy combining nebulized flagellin and amoxicillin proved effective against antibiotic-resistant pneumococcal pneumonia in mice. We posit that flagellin aerosol therapy represents a safe and promising approach to address bacterial pneumonia within the context of antimicrobial resistance.

H2S scavenger as a broad-spectrum strategy to deplete bacteria-derived H2S for antibacterial sensitization

Bacteria-derived H2S plays multifunctional protective roles against antibiotics insult, and the H2S biogenesis pathway is emerging as a viable target for the antibacterial adjuvant design. However, the development of a pan-inhibitor against H2S-synthesizing enzymes is challenging and underdeveloped. Herein, we propose an alternative strategy to downregulate the H2S levels in H2S-producing bacteria, which depletes the bacteria-derived H2S chemically by H2S scavengers without acting on the synthesizing enzymes. After the screening of chemically diversified scaffolds and a structural optimization campaign, a potent and specific H2S scavenger is successfully identified, which displays efficient H2S depletion in several H2S-producing bacteria, potentiates both bactericidal agents and photodynamic therapy, enhances the bacterial clearance of macrophages and polymorphonuclear neutrophils, disrupts the formation of bacterial biofilm and increases the sensitivity of bacterial persister cells to antibiotics. Most importantly, such an H2S scavenger exhibits sensitizing effects with gentamicin in Pseudomonas aeruginosa -infected pneumonia and skin wound female mouse models. In aggregate, our results not only provide an effective strategy to deplete bacteria-derived H2S and establish the H2S biogenesis pathway as a viable target for persisters and drug-resistant bacteria, but also deliver a promising antibacterial adjuvant for potential clinical translation.

Prim-O-glucosylcimifugin alleviates influenza virus-induced pneumonia in mice by inhibiting the TGF-β1/PI3KCD/MSK2/RELA signalling pathway.

Prim-O-glucosylcimifugin (POG) is a chromone derived primarily from Saposhnikovia divaricata (Turcz) Schischk and Cimicifuga simplex. Previous research has shown that POG possesses antibacterial, anticancer, anti-inflammatory, antioxidant, anticonvulsant, antipyretic, and analgesic properties. However, the specific impact of POG on influenza-virus-induced pneumonia is not well understood. In this study, we investigated the protective effects and underlying mechanisms of POG in pneumonia caused by influenza A virus (IAV). In vitro, POG was found to have a protective effect against infections caused by the respiratory viruses respiratory syncytial virus (RSV), human coronavirus OC43, and influenza A virus. POG inhibited A/FM/1/1947(H1N1) infection with an EC50 ranging from 3.01 to 10.43 in vitro. Intraperitoneal infection of mice with POG at a dose of 5 or 10 mg/kg resulted in a reduction in IAV-induced pneumonia, as evidenced by decreased pulmonary edema, improved lung histopathology, and reduced inflammatory cell accumulation. At the higher dose (10 mg/kg), POG treatment significantly increased survival rates, decreased viral titres in the lungs, improved lung histology, and reduced lung inflammation in IAV-infected mice. POG also effectively alleviated pulmonary fibrosis by reducing the levels of fibrotic markers (hydroxyproline [Hyp] and transforming growth factor β1 [TGF-β1]) and suppressing the expression of alpha smooth muscle actin (α-SMA), p focal adhesion kinase (p-FAK), and TGF-β1 in lung tissues. In addition, POG inhibited the expression of the RELA proto-oncogene (RELA), phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit delta (PIK3CD), and mitogen- and stress-activated protein kinase 2 (MSK2) in lung tissues. These results indicate that POG may have a protective effect against IAV-induced pneumonia by downregulating the TGF-β1/PI3KCD/MSK2/RELA signalling pathway in the lungs.

Therapeutic effects and mechanisms of Modified Ma-Xing-Shi-Gan Decoction on Klebsiella pneumoniae-induced pneumonia in mice assessed by Multi-omics

Ethnopharmacological relevanceModified Ma-Xing-Shi-Gan decoction (MMXSGD), a classic prescription from Treatise on Febrile Disease in China, is commonly used to treat Klebsiella pneumoniae (KP) infections in clinical settings. Materials and methodsThe aim of this study is to assess the efficacy of MMXSGD in the treatment of pneumonia and investigate its underlying mechanism of action. UHPLC-MS/MS was established to identify the main chemical components of serum after intragastric administration with MMXSGD. A mouse model of pneumonia caused by KP was used to evaluate the therapeutic potential of MMXSGD. The macrophage polarization was analyzed by immunohistochemistry. The cytokine profile was assessed using Luminex assay. Lung transcript and metabolite levels were assessed by transcriptomics and non-targeted metabolomics to analyze potential anti-pneumonia mechanisms and targets. Results22 major blood-entry components and 274 MMXSGD-pneumonia-related targets were identified. Compared with the model group, the mortality rate of mice in different dosage groups of MMXSGD was significantly reduced, and pathological lung damage was significantly alleviated. Among them, the low dose of MMXSGD treatment had the best protective effect. Further, MMXSGD treatment could regulates M1/M2 polarization in macrophages and inhibits the production of pro-inflammatory cytokines. The data from transcriptome and metabolome analysis indicate that MMXSGD could regulate inflammation-related pathways (PI3K/AKT, HIF-1, NF-κB pathway) and metabolites to modulate pulmonary inflammation. The results demonstrate that MMXSGD enhances the antibacterial effect in vivo by suppressing inflammation and regulating immunity rather than directly antibacterial effect. ConclusionThese findings provide a further assessment of MMXSGD, suggesting that MMXSGD has good therapeutic efficacy in bacterial infectious diseases.

Administration of Lactobacillus plantarum GUANKE alleviates SARS-CoV-2-induced pneumonia in mice.

Most otherwise healthy individuals develop only mild or moderate symptoms of coronavirus disease 2019 (COVID-19) caused by current strains of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and virus replication is mainly confined to the upper respiratory tract; however, the virus can infect the lower respiratory tract and promote inflammation. Probiotic supplementation has been shown to reduce nasopharyngeal SARS-CoV-2 viral load, reduce the overall number and duration of symptoms, and attenuate inflammation in clinical trials. We showed that a novel L. plantarum GUANKE strain alleviated SARS-CoV-2-induced pneumonia in mice. The transcription and production of inflammatory cytokines were suppressed, and GUANKE moderately reduced the viral load. L. plantarum GUANKE has the potential to become a candidate drug for the treatment of COVID-19 or other viral respiratory infections.

Essential role of proline synthesis and the one-carbon metabolism pathways for systemic virulence of Streptococcus pneumoniae.

Virulence screens have indicated potential roles during Streptococcus pneumoniae infection for the one-carbon metabolism pathway component Fhs and proline synthesis mediated by ProABC. To define how these metabolic pathways affect S. pneumoniae virulence, we have investigated the phenotypes, transcription, and metabolic profiles of Δfhs and ΔproABC mutants. S. pneumoniae capsular serotype 6B BHN418 Δfhs and ΔproABC mutant strains had strongly reduced virulence in mouse sepsis and pneumonia models but could colonize the nasopharynx. Both mutant strains grew normally in complete media but had markedly impaired growth in chemically defined medium, human serum, and human cerebrospinal fluid. The BHN418 ΔproABC strain also had impaired growth under conditions of osmotic and oxidative stress. The virulence role of proABC was strain specific, as the D39 ΔproABC strain could still cause septicemia and grow in serum. Compared to culture in broth, in serum, the BHN418 Δfhs and ΔproABC strains showed considerable derangement in global gene transcription that affected multiple but different metabolic pathways for each mutant strain. Metabolic data suggested that Δfhs had an impaired stringent response, and when cultured in sera, BHN418 Δfhs and ΔproABC were under increased oxidative stress and had altered lipid profiles. Loss of proABC also affected carbohydrate metabolism and the accumulation of peptidoglycan synthesis precursors in the BHN418 but not the D39 background, linking this phenotype to the conditional virulence phenotype. These data identify the S. pneumoniae metabolic functions affected by S. pneumoniae one-carbon metabolism and proline biosynthesis, and the role of these genetic loci for establishing systemic infection.IMPORTANCERapid adaptation to grow within the physiological conditions found in the host environment is an essential but poorly understood virulence requirement for systemic pathogens such as Streptococcus pneumoniae. We have now demonstrated an essential role for the one-carbon metabolism pathway and a conditional role depending on strain background for proline biosynthesis for S. pneumoniae growth in serum or cerebrospinal fluid, and therefore for systemic virulence. RNAseq and metabolomic data demonstrated that the loss of one-carbon metabolism or proline biosynthesis has profound but differing effects on S. pneumoniae metabolism in human serum, identifying the metabolic processes dependent on each pathway during systemic infection. These data provide a more detailed understanding of the adaptations required by systemic bacterial pathogens in order to cause infection and demonstrate that the requirement for some of these adaptations varies between strains from the same species and could therefore underpin strain variations in virulence potential.

In vitro and in vivo activities of scutellarein, a novel polyphosphate kinase 1 inhibitor against Acinetobacter baumannii infection

BackgroundInorganic polyphosphate (polyP)-targeted polyphosphate kinase 1 (PPK1) has attracted much attention by virtue of its importance in bacterial pathogenicity and persistence, as well as its exclusive presence in microorganisms. However, only very few drugs have been found to be efficacious in inhibiting the Acinetobacter baumannii (A. baumannii) PPK1 protein.ResultsIn this study, we identified Scutellarein (Scu), a potent PPK1 inhibitor that could significantly influence PPK1-regulated motility, biofilm formation, and bacterial persistence, which was further validated by the results of transcriptome analysis. Mechanistic explorations revealed that Scu achieved its enzyme inhibitory activity predominantly through direct engagement with the active center of PPK1. Moreover, the survival rate of Galleria mellonella larvae was increased by about 35% with 20 mg/kg of Scu treatment. The remarkable therapeutic benefits of Scu were also observed in the mouse pneumonia model, shown mainly by reduced bacterial colonization, pathological lesions, and inflammatory factors.ConclusionOur results revealed that Scu could attenuate the pathogenicity and persistence of A. baumannii by interfering with its important kinase PPK1.

Comparative pathogenicity of influenza virus-induced pneumonia mouse model following intranasal and aerosolized intratracheal inoculation

BackgroundInfection of mice with mouse-adapted strains of influenza virus has been widely used to establish mouse pneumonia models. Intranasal inoculation is the traditional route for constructing an influenza virus-induced pneumonia mouse model, while intratracheal inoculation has been gradually applied in recent years. In this article, the pathogenicity of influenza virus-induced pneumonia mouse models following intranasal and aerosolized intratracheal inoculation were compared.MethodsBy comparing the two ways of influenza inoculation, intranasal and intratracheal, a variety of indices such as survival rate, body weight change, viral titer and load, pathological change, lung wet/dry ratio, and inflammatory factors were investigated. Meanwhile, the transcriptome was applied for the initial exploration of the mechanism underlying the variations in the results between the two inoculation methods.ResultsThe findings suggest that aerosolized intratracheal infection leads to more severe lung injury and higher viral loads in the lungs compared to intranasal infection, which may be influenced by the initial site of infection, sialic acid receptor distribution, and host innate immunity.ConclusionIntratracheal inoculation is a better method for modelling severe pneumonia in mice than intranasal infection.

Study on the activity and mechanism of herbal formula anti-infection powder (AIP) against influenza-virus-induced pneumonia through genetic susceptibility genes

Anti-infection powder (AIP), a patented Chinese herbal formulation, is used traditionally in the treatment of upper respiratory tract infections. In this study, an ethanol extract of AIP was demonstrated to inhibit influenza A virus (IAV) infection and IAV-induced pneumonia (IVP), both in vitro and in vivo, highlighting its potential mechanism of action. To determine the anti-IAV activity of AIP and to explore the possible mechanisms of inhibiting IAV-induced pneumonia. An ethanol extract was extracted from AIP and its major ingredients were determined by high-performance liquid chromatography (HPLC). An IAV-infected A549 cell model and an IAV-induced mouse pneumonia model were established to evaluate the therapeutic effects of AIP on IVP in vivo and in vitro. The mice were respectively administered AIP at high- and low-dose in different groups. The anti-IAV activity of AIP was evaluated by detecting viral load, lung lesion, lung index, suvival time, inflammatory cytokines and transcriptomic analysis in the lung tissue. The potential pathways and targets that involved in AIP against IVP were predicted by network pharmacology. Mendelian randomization (MR), colocalization analysis, and molecular docking were employed to identify novel therapeutic targets for IVP. Polymerase chain reaction (PCR) and Western blot (WB) techniques were used to confirm the effect of AIP on the expression of risk target genes in the lungs of IVP mice. In A549 cell line, AIP effectively inhibited IAV infection with IC50 values of 65.49 μg/mL. The anti-IAV activity of AIP was mainly determined by chlorogenic acid, forsythiarin, puerarin, paeoniflorin and prim-o-glucosylcimigin. Moreover, AIP inhibited the neuraminidase activity and the M gene expression in vitro. In vivo, oral administration of AIP significantly reduced viral load and improved lung tissue lesions. AIP decreased the concentration of pro-inflammatory factors such as IL-1β, TNF-α, and IFN-γ, and significantly increased the concentration of the anti-inflammatory factor IL-4. According to network pharmacology analysis, toll-like receptor signaling pathway, chemokine signaling pathway, and TNF signaling pathway may be the possible mechanisms by which AIP inhibits IVP and regulates excessive inflammatory response.Two new genes, LRG1 and PSMA4, associated with genetic susceptibility to influenza and pneumonia, predicted as potential IVP drug target genes by MR and colocalization analysis. The antiviral mechanism of AIP may be to inhibit the expression levels of LRG1 and PSMA4 in lungs of mouse IVP. AIP exhibited anti-IAV activities both in vitro and in vivo. AIP had a protective effect against pneumonia caused by influenza virus and can inhibit the progression of inflammation. This effect may be associated with its ability to inhibit the expression levels of genetic susceptibility genes (LRG1 and PSMA4) in lungs of mouse IVP. The findings of this study enhance our understanding of the role and mechanisms of AIP in the treatment of IVP.

An inhalable nanoparticle enabling virulence factor elimination and antibiotics delivery for pneumococcal pneumonia therapy

Streptococcus pneumoniae (S. pneumoniae) is a major cause of community-acquired pneumonia. Current standard clinical therapies mainly focus on combating S. pneumoniae through antibiotics. However, the limited delivery of antibiotics and the undetoxified hydrogen peroxide (H2O2) virulence factor secreted by S. pneumoniae impede the therapeutic outcomes. Here we report an inhalable catalase (CAT)-tannic acid (TA) nanoassembly for local antibiotic (levofloxacin) delivery and simultaneously neutralizing the secreted H2O2 virulence factors to treat pneumococcal pneumonia. After aerosol inhalation, the inhalable formulation (denoted as CT@LVX) effectively accumulates in lung tissues through TA-mediated mucoadhesion. CAT can reduce alveolar epithelial cells apoptosis by catalyzing the decomposition of accumulated H2O2 in the infected lung tissues. In synergy with antibiotic LVX-mediated S. pneumoniae elimination, CT@LVX significantly decreases lung injury companied with reduced inflammatory, resulting in 100 % survival of mice with pneumonia. In a clinically isolated S. pneumoniae strain-induced pneumonia mouse model, CT@LVX also shows superior outcomes compared to the traditional antibiotic treatment, highlighting its potential clinical application prospects.

Investigating the active components and mechanistic effects of Forsythia suspensa Leaf against RSV via the PI3K/Akt-NLRP3 pathway

BackgroundPulmonary infections resulting from respiratory syncytial virus (RSV) continue to pose a significant threat to the well-being of infants and the elderly, but there is no safe, effective and specific treatment except symptomatic treatment. Forsythia Suspensa Leaf (FSL) is cold in nature and bitter in taste, and has the efficacy of clearing away heat and toxic materials. Previous research by our research group showed that the active components in FSL have the pharmacological effect of anti-RSV. Based on that, this study aims further to clarify the anti-RSV active components and mechanism of FSL. MethodsFirstly, we established the BALB/c mouse model of RSV infection, assessed the in vivo anti-RSV efficacy, and determined the optimal dosage of FSL and its active components. Evaluation parameters included body weight changes, organ indices, lung tissue pathological sections, lung tissue viral load, and inflammatory factors. Additionally, we used RT-PCR, Western Blot and other molecular biology techniques to determine the expression changes of key factors such as Nrf2 and NLRP3 in PI3K/Akt-NLRP3 pathway, and revealed the anti-RSV mechanism of FSL and its active components. ResultsPharmacodynamic experiments in animals showed that the FSL Low (0.4 g/kg·d), RosA Low (100 mg/kg·d) and Phillyrin Medium (100 mg/kg·d) groups could effectively improve the pathological conditions of mice with RSV pneumonia, such as weight loss, the level of pulmonary inflammatory factors and the increase of viral load. In addition, oral administration of Phillyrin at a dose of 100 mg/kg d to RSV-infected mice can effectively control the trend that the expression of Nrf2 protein decreases and the expression of NLRP3 protein increases in RSV pneumonia mice. ConclusionPhillyrin, the active component in FSL, can not only directly inhibit the replication of RSV, but also effectively control the inflammatory reaction caused by RSV infection and improve lung injury, which is expected to become a potential drug against RSV pneumonia.

Near-infrared in vivo imaging system for dynamic visualization of lung-colonizing bacteria in mouse pneumonia.

Conventional animal models of infectious diseases have traditionally relied upon average assessments involving numerous individuals, meaning they do not directly reflect changes in the pathology of an individual. Moreover, in recent years, ethical concerns have resulted in the demand to reduce the number of animals used in such models. Although in vivo imaging offers an effective approach for longitudinally evaluating the pathogenesis of infectious diseases in individual animals, a standardized method has not yet been established. To our knowledge, this study is the first to develop a highly versatile in vivo pulmonary bacterial quantification system utilizing near-infrared luminescence, plasmid-mediated expression of firefly luciferase in bacteria, and a scientific complementary metal-oxide semiconductor camera. Our research holds promise as a useful tool for assessing the efficacy of therapeutic drugs and pathogenesis of infectious diseases.

Shuanghuanglian volatile oil exerts antipyretic, anti-inflammatory, and antibacterial synergistic effects through multiple pathways

Ethnopharmacological relevanceTraditional Chinese Medicine (TCM) has a rich history spanning 2000 years. Shuanghuanglian, a traditional Chinese herbal formula composed of three botanicals, is primarily used to treat colds, respiratory infections (including bacterial pneumonia), and pharyngitis. Previous research has found that the volatile oil of Shuanghuanglian is crucial for its efficacy. However, there is a lack of studies investigating its mechanisms. Aim of the studyThis study aims to explore the antibacterial and anti-inflammatory mechanisms of Shuanghuanglian volatile oil and its potential to enhance the antibacterial effects when used in conjunction with antibiotics. MethodsDetermination of the GC-MS fingerprint of SVO using Gas Chromatography-Mass Spectrometry (GC-MS), The antibacterial effects of SVO on multidrug-resistant Klebsiella pneumoniae (MDR-KP) were assessed by detecting MIC, checkerboard method assay, time-kill curves, resistance growth curves, transcriptome sequencing analysis, scanning electron microscopy(SEM), purification, and quantitative analysis of extracellular polysaccharides(EPS). In vivo part, an MDR-KP induced mouse pneumonia model was established to evaluate the mitigating effects of SVO on mouse pneumonia, using comprehensive network pharmacology and bioinformatics to identify genes related to bacterial pneumonia and potential targets of SVO. Validation was performed through molecular docking, qPCR, and ELISA tests. ResultsSVO modulates the expression of MDR-KP mRNA for wecB, wecC, murA, murD, murE, murF, inhibiting the synthesis of O-antigen polysaccharides and peptidoglycans, thereby compromising bacterial cell wall integrity and affecting the synthesis of biofilms. These actions not only exhibit antibacterial effects but also enhance antibacterial activity, restoring the sensitivity of CEF to MDR-KP. SVO suppresses the biological activity of PTGS2, reducing the production of Prostaglandin E2 (PGE2), thereby exerting antipyretic and anti-inflammatory effects, providing new insights for the development of natural non-steroidal anti-inflammatory drugs (NSAIDs). ConclusionsOur research indicates that SVO exerts antipyretic, anti-inflammatory, and antibacterial synergistic effects through multiple pathways.

Blocking HXA3-mediated neutrophil elastase release during S. pneumoniae lung infection limits pulmonary epithelial barrier disruption and bacteremia.

Streptococcus pneumoniae (Sp), a leading cause of community-acquired pneumonia, can spread from the lung into the bloodstream to cause septicemia and meningitis, with a concomitant threefold increase in mortality. Limitations in vaccine efficacy and a rise in antimicrobial resistance have spurred searches for host-directed therapies that target pathogenic immune processes. Polymorphonuclear leukocytes (PMNs) are essential for infection control but can also promote tissue damage and pathogen spread. The major Sp virulence factor, pneumolysin, triggers acute inflammation by stimulating the 12-lipoxygenase (12-LOX) eicosanoid synthesis pathway in epithelial cells. This pathway is required for systemic spread in a mouse pneumonia model and produces a number of bioactive lipids, including hepoxilin A3 (HXA3), a hydroxy epoxide PMN chemoattractant that has been hypothesized to facilitate breach of mucosal barriers. To understand how 12-LOX-dependent inflammation promotes dissemination during Sp lung infection and dissemination, we utilized bronchial stem cell-derived air-liquid interface cultures that lack this enzyme to show that HXA3 methyl ester (HXA3-ME) is sufficient to promote basolateral-to-apical PMN transmigration, monolayer disruption, and concomitant Sp barrier breach. In contrast, PMN transmigration in response to the non-eicosanoid chemoattractant N-formyl-L-methionyl-L-leucyl-phenylalanine (fMLP) did not lead to epithelial disruption or bacterial translocation. Correspondingly, HXA3-ME but not fMLP increased the release of neutrophil elastase (NE) from Sp-infected PMNs. Pharmacologic blockade of NE secretion or activity diminished epithelial barrier disruption and bacteremia after pulmonary challenge of mice. Thus, HXA3 promotes barrier-disrupting PMN transmigration and NE release, pathological events that can be targeted to curtail systemic disease following pneumococcal pneumonia.IMPORTANCEStreptococcus pneumoniae (Sp), a leading cause of pneumonia, can spread from the lung into the bloodstream to cause systemic disease. Limitations in vaccine efficacy and a rise in antimicrobial resistance have spurred searches for host-directed therapies that limit pathologic host immune responses to Sp. Excessive polymorphonuclear leukocyte (PMN) infiltration into Sp-infected airways promotes systemic disease. Using stem cell-derived respiratory cultures that reflect bona fide lung epithelium, we identified eicosanoid hepoxilin A3 as a critical pulmonary PMN chemoattractant that is sufficient to drive PMN-mediated epithelial damage by inducing the release of neutrophil elastase. Inhibition of the release or activity of this protease in mice limited epithelial barrier disruption and bacterial dissemination, suggesting a new host-directed treatment for Sp lung infection.

Phloretin Targets Polyphosphate Kinase 1 to Attenuate Acinetobacter baumannii Virulence and Persistence In Vitro and In Vivo

Acinetobacter baumannii (A. baumannii) is well known for its virulence and persistence, particularly in intensive care units. Therefore, new strategies and candidates to treat A. baumannii infection are urgently needed considering the emergence of drug-resistant bacteria. Polyphosphate kinase 1 (PPK1) is required for bacterial survival as it is involved in maintaining antibiotic resistance or tolerance, pathogenesis, and adversity resistance. Multiple phenotypic assays related to virulence and persistence were performed in this study, and phloretin was shown to attenuate A. baumannii virulence and persistence by inhibiting PPK1 activity. Phloretin hampered mobility, interfered with biofilm formation and decreased resistance to ampicillin, heat, and hydrogen peroxide stress in A. baumannii. The therapeutic effect was also examined in a mouse pneumonia infection model. Molecular simulation and site-directed mutagenesis revealed that ARG-22, MET-622, ASN57, and ARG-65 were the sites of phloretin action against PPK1. Phloretin treatment led to changes in metabolic pathways associated with A. baumannii virulence and persistence, including glycerophospholipid metabolism and fatty acid biosynthesis. Furthermore, phloretin alleviated pneumonic injury in a mouse pneumonia infection model in vivo, indicating that phloretin is a promising compound for preventing A. baumannii infection resistance by targeting PPK1.

Modeling "Two-Hit" Severe Pneumonia in Mice: Pathological Characteristics and Mechanistic Studies.

Severe pneumonia is one of the most common critical diseases in clinical practice. Existing models for severe pneumonia have limitations, leading to limited clinical translation. In this study, a two-hit severe pneumonia mouse model was established by inducing primary pneumonia through intratracheal instillation of 800μg lipopolysaccharide (LPS), followed by intraperitoneal injection of 10mg/kg LPS. The effectiveness of various inflammatory indicators and the lung tissue damage during the time course of this model were confirmed and evaluated. At 3h post two-hit, the IL-6, TNF-α levels in peripheral blood and bronchoalveolar lavage fluid (BALF), and the white blood cells, neutrophils, and lymphocytes in BALF notably exhibited the most pronounced elevation. At 12h post two-hit, the white blood cells and neutrophils in peripheral blood significantly increased, accompanied by notable alterations in splenic immune cells and worsened pulmonary histopathological damage. Transcriptomics of lung tissue, microbiota analysis of lung and gut, as well as plasma metabolomics analyses further indicated changes in transcriptional profiles, microbial composition, and metabolites due to the two-hit modeling. These results validate that the two-hit model mimics the clinical presentation of severe pneumonia and serves as a robust experimental tool for studying the pathogenesis of severe pneumonia and developing and assessing treatment strategies.

Targeted counteracting of overactive macrophages by melittin stable-loaded solid lipid nanoparticles alleviates cytokine storm and acute inflammatory injury

The continuous activation of macrophages play a critical role in the pathogenesis of cytokine storm (CS). Considering that CS results from the participation of multiple cytokines, the therapeutic effect of a single cytokine or its receptor-targeted blockade therapy remains uncertain. Melittin, which can systematically suppress the overexpression of proinflammatory mediators via inhibiting the mitogen-activated protein kinase and nuclear factor kappa-B pathways in activated macrophages, shows great potential in alleviating CS and acute inflammatory injury (AII). However, its clinical application is limited by its hemolytic activity, non-specific cytotoxicity and lack of targeting. In this study, a folic acid-modified and melittin stable-loaded solid lipid nanoparticle (Fa-MpG@LNP) with a core–shell structure was developed for CS control via targeted inhibition of the overproduction of proinflammatory mediators in activated macrophages with specific expression of folate receptor-β. The resultant Fa-MpG@LNP showed ideal physicochemical properties and stability, low hemolytic activity and non-specific cytotoxicity, and it can specifically bind to lipopolysaccharide (LPS)-stimulated macrophages and effectively reduce the elevated levels of proinflammatory mediators. After intravenous administration, the Fa-MpG@LNP accumulated at inflamed tissue and significantly downregulate the overproduction of proinflammatory cytokines in tissue-infiltrated macrophages, resulting in a significant decrease of cytokine concentration in inflamed tissue and serum in LPS-induced acute pneumonia mice, and finally alleviate AII with undetectable toxic side effects. These results indicate the clinical application potential of Fa-MpG@LNP in alleviating CS and its related symptoms.

Discovery of novel dihydropyrrolidone-thiadiazole compound crosstalk between the YycG/F two-component regulatory pathway and cell membrane homeostasis to combat methicillin-resistant Staphylococcus aureus

The rapid emergence and spread of multidrug-resistant (MDR) Gram-positive pathogens present a significant challenge to global healthcare. Methicillin-resistant Staphylococcus aureus (MRSA) is a particular concern because of its high resistance to most antibiotics. Based on our previously reported chemical structure of compound 62, a series of novel derivatives were synthesized and evaluated for their antibacterial activities. We found that some of these derivatives displayed effective antibacterial activity against Gram-positive pathogens, with minimal cytotoxicity (CC50>100 μM) and hemolytic activity (HC50>200 μM). Among these derivatives, the minimum inhibitory concentration (MIC) of 62-7c against Gram-positive bacterial isolates ranged from 6.25 to 25 μM. This derivative also exhibited significant synergistic antibacterial effects with daptomycin both in vitro and in vivo, with an ability to eradicate planktonic and persister cells of MRSA. Additionally, 62-7c inhibited biofilm formation and eradicated mature biofilms of MRSA. Mechanistic studies revealed that 62-7c inhibited the YycG kinase activity and disrupted the cell membrane by binding to cardiolipin (CL), leading to cell death. Importantly, no development of drug resistance was observed even after 20 serial passages. Furthermore, 62-7c exhibited high biosafety and potent effectiveness in combating infections in both mouse pneumonia and mouse wound models infected with MRSA. Thus, our study revealed that 62-7c has the potential to serve as a novel antibacterial agent for treating MRSA infections.

Sinigrin reduces the virulence of Staphylococcus aureus by targeting coagulase

Multi-resistant Staphylococcus aureus (S. aureus) infection is a significant global health concern owing to its high mortality and morbidity rates. Coagulase (Coa), a key enzyme that activates prothrombin to initiate host coagulation, has emerged as a promising target for anti-infective therapeutic approaches. This study identified sinigrin as a potent Coa inhibitor that significantly inhibited S. aureus-induced coagulation at concentration as low as 32 mg/L. Additionally, at a higher concentration of 128 mg/L, sinigrin disrupted the self-protection mechanism of S. aureus. Thermal shift and fluorescence-quenching assays confirmed the direct binding of sinigrin to the Coa protein. Molecular docking analysis predicted specific binding sites for sinigrin in the Coa molecule, and point mutation experiments highlighted the importance of Arg-187 and Asp-222 as critical binding sites for both Coa and sinigrin. In vivo studies demonstrated that the combination of sinigrin with oxacillin exhibited greater antibacterial efficacy than oxacillin alone in the treatment of S. aureus-induced pneumonia in mice. Furthermore, sinigrin was shown to reduce bacterial counts and inflammatory cytokine levels in the lung tissues of S. aureus-infected mice. In summary, sinigrin was shown to directly target Coa, resulting in the attenuation of S. aureus virulence, which suggests the potential of sinigrin as an adjuvant for future antimicrobial therapies.