Pneumonia In Mice Research Articles

In vitro and in vivo efficacy of minocycline-based therapy for Elizabethkingia anophelis and the impact of reduced minocycline susceptibility

ObjectivesElizabethkingia anophelis is inherently resistant to multiple antibiotics, except minocycline. This study aimed to determine the in vitro and in vivo efficacy of minocycline monotherapy and combination therapy against susceptible strains and the impact of reduced minocycline susceptibility. MethodsThree clinical isolates and one laboratory-induced mutant with reduced minocycline susceptibility were included. Time-kill and checkerboard assays were used to assess in vitro efficacy and synergy, respectively. Galleria mellonella infection and mouse pneumonia models were used to assess in vivo efficacy, and a mouse thigh infection model was used to determine the bacterial load. ResultsMinocycline monotherapy exerted a modest inhibitory effect on three clinical minocycline-susceptible E. anophelis isolates in vitro, but delayed G. mellonella death and improved infected mouse survival; it also significantly reduced the in vivo bacterial load. Minocycline had decreased efficacy on G. mellonella and mice infected by the mutant with reduced minocycline susceptibility. Genome comparison revealed several spontaneous mutations associated with reduced minocycline susceptibility. Among eight antibiotics tested in combination with minocycline, rifampin consistently showed in vitro synergy. The addition of rifampin (1 mg/L) reduced the mutant prevention concentration of minocycline from 2–4 mg/L to < 0.5 mg/L. However, compared with monotherapy, the combination of rifampin and minocycline did not further reduce the bacterial load or improve the survival of G. mellonella or mice. ConclusionMinocycline monotherapy was in vivo effective against susceptible E. anophelis. Reduced minocycline susceptibility due to spontaneous mutation decreased its therapeutic efficacy. In combination with rifampin, it prevented the in vitro emergence of reduced susceptibility but did not provide additional in vivo survival benefit.

Green synthesised CuNPs using Alhagi maurorum extract and its ability to amelioration of Mycoplasma pneumoniae infected pneumonia mice model

In this study, copper nanoparticles were synthesised according to green chemistry rules using the aqueous extract of Alhagi maurorum. The green-synthesised CuNPs were characterised using different techniques such as EDX, FE-SEM, XRD and FT-IR. The FE-SEM results confirm spherical morphology for the nanoparticles with a size of 42.68–68.87 nm. In vivo, the Mycoplasma pneumonia injection was applied for inducing pneumonia in the BALB/c mice, also the treatment was with CuNPs. The serum parameters such as inflammatory mediators (IL-6, TNF-α, IL-8, TGF and IL-1), total cell counts, and total protein content levels were evaluated. CuNPs regulated the levels of the inflammatory mediators in the infected mice. The cellular arrangements at the histopathological images were ameliorated with the administration of CuNPs. At the antioxidant test, the CuNPs and BHT removed 50% of free radicals at the concentrations of 181 and 110 µg/ml. In conclusion, the results revealed the ameliorative property of CuNPs green-formulated by A. maurorum aqueous extract on M. pneumoniae infected pneumonia mice model.

Bacteriophage Effectively Rescues Pneumonia Caused by Prevalent Multidrug-Resistant Klebsiella pneumoniae in the Early Stage.

ABSTRACTPneumonia caused by multidrug-resistant (MDR) Klebsiella pneumoniae of sequence types ST11 and ST383 have highlighted the necessity for new therapies against these prevalent pathogens. Bacteriophages (phages) may be used as alternatives or complements to antibiotics for treating MDR bacteria because they show potential efficacy in mouse models and even individual clinical cases, and they also cause fewer side effects, such as microbiota-imbalance-induced diseases. In the present study, we screened two phages, pKp11 and pKp383, that targeted ST11 and ST383 MDR K. pneumoniae isolates collected from patients with pneumonia, and they exhibited a broad host range, high lytic activity, and high environmental adaptability. Both phages pKp11 and pKp383 provided an effective treatment for the early stage of pneumonia in a murine infection model without promoting obvious side effects, and cocktails consisting of the two phages were more effective for reducing bacterial loads, inflammation, and pathogenic injuries. Our findings support the application of phages as new medications for refractory ST11 and ST383 K. pneumoniae infections and emphasize the potential of enhancing phage therapy modalities through phage screening. These data provided important resources for assessing and optimizing phage therapies for MDR ST11 and ST383 infection treatment. However, substantial amounts of further work are needed before phage therapy can be translated to human therapeutics.IMPORTANCE K. pneumoniae is recognized as the most common pathogen of hospital- and community-acquired pneumonia across the world. The strains of ST11 and ST383 are frequently reported in patients with pneumonia. However, the efficacy of antibiotics toward K. pneumoniae is decreasing dramatically. As a new approach to combat MDR bacteria, phages have exhibited positive clinical effects and efficacy as synergetic or alternative strategies to antibiotics. Thus, we screened two phages that targeted ST11 and ST383 MDR K. pneumoniae, and they exhibited a broad host range, high lytic activity, and high environmental adaptability. Both phages provided an effective treatment for the early stage of pneumonia in mice, and cocktails consisting of the two phages were more effective in reducing bacterial loads, inflammation, and pathogenic injuries. Although these data suggest that phages are effective alternatives or complements to antibiotics, more research is needed before they can be translated into therapeutics for humans.

Host Immune Response to Clinical Hypervirulent Klebsiella pneumoniae Pulmonary Infections via Transcriptome Analysis.

Klebsiella pneumoniae (K. pneumoniae), especially those with hypervirulence, is becoming a global concern and posing great threat to human health. Studies on individual immune cells or cytokines have partially revealed the function of the host immune defense against K. pneumoniae pulmonary infection. However, systematic immune response against K. pneumoniae has not been fully elucidated. Herein, we report a transcriptome analysis of the lungs from a mouse pneumonia model infected with a newly isolated K. pneumoniae clinical strain YBQ. Total RNA was isolated from the lungs of mice 48 hours post infection to assess transcriptional alteration of genes. Transcriptome data were analyzed with KEGG, GO, and ICEPOP. Results indicated that upregulated transcription level of numerous cytokines and chemokines was coordinated with remarkably activated ribosome and several critical immune signaling pathways, including IL-17 and TNF signaling pathways. Notably, transcription of cysteine cathepsin inhibitor (stfa1, stfa2, and stfa3) and potential cysteine-type endopeptidase inhibitor (cstdc4, cstdc5, and cstdc6) were upregulated. Results of ICEPOP showed neutrophils functions as the most essential cell type against K. pneumoniae infection. Critical gene alterations were further validated by rt-PCR. Our findings provided a global transcriptional perspective on the mechanisms of host defense against K. pneumoniae infection and revealed some unique responding genes.

WYBQ-4: a New Bactericidal Agent against Methicillin-Resistant Staphylococcus aureus.

ABSTRACTMethicillin-resistant Staphylococcus aureus (MRSA) is a multidrug-resistant pathogen that currently poses a serious threat to global health. Novel antimicrobial agents against MRSA are urgently being developed. In this study, we investigated WYBQ-4, which is an effective antibacterial agent with potent bactericidal activity and bactericidal efficiency against MRSA USA300 and clinical isolate strains. In addition, WYBQ-4 exhibited low cytotoxicity without hemolytic activity according to a safety evaluation. Importantly, WYBQ-4 showed potent in vivo efficacy in an MRSA-induced mouse pneumonia model, systemic infection model, and intramuscular infection model. The efficacy of this new cephalosporin against MRSA was associated with a high affinity for penicillin-binding proteins (PBP1, PBP2, PBP3, PBP4, PBP2a) evaluated in a competition assay using bocillin as a reporter. In conclusion, WYBQ-4 has a significant bactericidal effect in vitro and in vivo, indicating that it is a promising compound to control MRSA infection.IMPORTANCE Antibiotic resistance is spreading faster than the introduction of new compounds into clinical practice, causing a public health crisis. Novel antimicrobial agents against MRSA are urgently being developed. In this study, we investigated WYBQ-4, which is an effective antibacterial agent with potent bacteriostatic activity and bactericidal efficiency against MRSA USA300 and clinical isolate strains. WYBQ-4 showed potent in vivo efficacy in MRSA-induced mouse models. Subsequently, we further revealed its antibacterial mechanism. In conclusion, WYBQ-4 has a significant bactericidal effect in vitro and in vivo, indicating that it is a promising compound to control MRSA infection.

Effect and mechanism of acupoint injection on influenza A virus induced pneumonia in mice

To investigate the effect and mechanism of acupoint injection with 0.1% vitamin C+vitamin B complex solution (VC+VBCo) at "Tiantu" (CV 22), "Quchi" (LI 11) and "Zusanli" (ST 36) in mouse model of pneumonia induced by influenza A virus (A/PR/8/34 [H1N1], PR8). Sixty male ICR mice were randomized into 6 groups, i.e. control group, model group, acupoint injection group, intraperitoneal injection group, non-target point group and ribavirin group, 10 mice in each one. Except the control group, the pneumonia models were induced by slow nasal dripping PR8 virus in the other groups. On the 2nd day of experiment, VC+VBCo solution, 40 μL was injected at "Tiantu" (CV 22), "Quchi" (LI 11, left) and "Zusanli" (ST 36, left) in the acupoint injection group; VC+VBCo solution, 120 μL was injected intraperitoneally in the intraperitoneal injection group; VC+VBCo solution, 40 μL was injected at non-target acupoints (0.5 cm away from "Tiantu" [CV 22] to the left side, "Quchi" [LI 11, left] and "Zusanli" [ST 36, left]) in the non-target point group; and ribavirin solution, 120 μL was injected intraperitoneally in the ribavirin group. The intervention was delivered once daily, for consecutive 7 days. Three parallel experiments were undertaken. The mean death rate and survival time were assessed in each group, the body mass and lung index were compared among groups. Using HE staining, the morphology of lung tissue was observed; and with real-time fluorescence quantitative PCR, viral load in lung tissue was detected. The concentrations of inflammatory factors (tumor necrosis factor α [TNF-α], interleukin [IL]-1β, IL-10) were detected in lung tissue of each group using ELISA; and those of oxidative stress markers (superoxide dismutase [SOD], glutathione peroxidase [GSH-Px], malondialdehyde [MDA]) were detected with chemiluminescence method. Compared with the control group, the body mass was decreased and lung index was increased in the model group (P<0.01). In comparison with the model group, body mass was increased in the acupoint injection group (P<0.05), lung index was reduced in the acupoint injection group the and ribavirin group (P<0.05); the mean death rate was decreased and the mean survival time prolonged in the mice of the acupoint injection group (P<0.01, P<0.05); and the mean death rate was reduced in the mice of the ribavirin group (P<0.05). In the model group, the alveolar structure was not integral, the alveolar septum was thickened, inflammatory cells were infiltrated and red blood cells exudated seriously (P<0.01). Compared with the model group, in the acupoint injection group and the ribavirin group, the alveolar structure was integral, the thickened alveolar septum was alleviated; and the infiltration of inflammatory cells and the exudation of red blood cells were reduced remarkably. The viral load was reduced in the mice of the ribavirin group when compared with the model group (P<0.01). Compared with the control group, the concentrations of TNF-α, IL-1β and MDA in lung tissue were increased and those of IL-10, SOD and GSH-Px were reduced in the model group (P<0.01). In the acupoint injection group and the ribavirin group, the concentrations of TNF-α, IL-1β and MDA were reduced in lung tissue and those of IL-10, SOD and GSH-Px were increased (P<0.05, P<0.01) when compared with the model group. Acupoint injection with VC+VBCo solution may alleviate inflammatory responses and oxidative stress in lung tissue of the PR8-induced pneumonia mice, improve survival rate and prolong the survival time in the case of no effect of the viral load.

Cross talk between glucose metabolism and immunosuppression in IFN-γ-primed mesenchymal stem cells.

The immunosuppressive function "licensed" by IFN-γ is a vital attribute of mesenchymal stem cells (MSCs) widely used in the treatment of inflammatory diseases. However, the mechanism and impact of metabolic reprogramming on MSC immunomodulatory plasticity remain unclear. Here, we explored the mechanism by which glucose metabolism affects the immunomodulatory reprogramming of MSCs "licensed" by IFN-γ. Our data showed that glucose metabolism regulates the immunosuppressive function of human umbilical cord MSCs (hUC-MSCs) challenged by IFN-γ through the Janus kinase-signal transducer and activator of transcription (JAK-STAT) pathway. Furthermore, ATP facilitated the cross talk between glucose metabolism and the JAK-STAT system, which stimulates the phosphorylation of JAK2 and STATs, as well as the expression of indoleamine 2, 3-dioxygenase and programmed cell death-1 ligand. Moreover, ATP synergistically enhanced the therapeutic efficacy of IFN-γ-primed hUC-MSCs against acute pneumonia in mice. These results indicate a novel cross talk between the immunosuppressive function, glucose metabolism, and mitochondrial oxidation and provide a novel targeting strategy to enhance the therapeutic efficacies of hUC-MSCs.

Multicomponent Pseudomonas aeruginosa Vaccines Eliciting Th17 Cells and Functional Antibody Responses Confer Enhanced Protection against Experimental Acute Pneumonia in Mice.

ABSTRACTThe Gram-negative pathogen Pseudomonas aeruginosa is a common cause of pneumonia in hospitalized patients. Its increasing antibiotic resistance and widespread occurrence present a pressing need for vaccines. We previously showed that a P. aeruginosa type III secretion system protein, PopB, elicits a strong Th17 response in mice after intranasal (IN) immunization and confers antibody-independent protection against pneumonia in mice. In the current study, we evaluated the immunogenicity and protective efficacy in mice of the combination of PopB (purified with its chaperone protein PcrH) and OprF/I, an outer membrane hybrid fusion protein, compared with immunization with the proteins individually either by the intranasal (IN) or subcutaneous (SC) routes. Our results show that after vaccination, a Th17 recall response from splenocytes was detected only in mice vaccinated with PopB/PcrH, either alone or in combination with OprF/I. Mice immunized with the combination of PopB/PcrH and OprF/I had enhanced protection in an acute lethal P. aeruginosa pneumonia model, regardless of vaccine route, compared with mice vaccinated with either alone or adjuvant control. Immunization generated IgG titers against the vaccine proteins and whole P. aeruginosa cells. Interestingly, none of these antisera had opsonophagocytic killing activity, but antisera from mice immunized with vaccines containing OprF/I, had the ability to block IFN-γ binding to OprF/I, a known virulence mechanism. Hence, vaccines combining PopB/PcrH with OprF/I that elicit functional antibodies lead to a broadly and potently protective vaccine against P. aeruginosa pulmonary infections.

Intestinal microbiota metabolizing Houttuynia cordata polysaccharides in H1N1 induced pneumonia mice contributed to Th17/Treg rebalance in gut-lung axis

Influenza A virus is intricately linked to dysregulation of gut microbiota and host immunity. Previous study revealed that Houttuynia cordata polysaccharides (HCP) exert the therapeutic effect on influenza A virus inducing lung and intestine damage via regulating pulmonary and intestinal mucosal immunity. However, whether this result was due to the regulation of gut microbiota in the gut-lung axis remains unclear. Here, we firstly found that the elimination of gut microbiota using antibiotic cocktails led to both loss of the protective effect of HCP on intestine and lung injury, and reduction of the efficacy on regulating Th17/Treg balance in gut-lung axis. Fecal microbiota transplantation study confirmed that the gut microbiota fermented with HCP under pathological conditions (H1N1 infection) was responsible for reducing pulmonary and intestinal injury. Moreover, the interaction of HCP and gut microbiota under pathological conditions exhibited not only much more abundant gut microbial diversity, but also higher content of the acetate. Our results demonstrated that the underlying mechanism to ameliorate viral pneumonia in mice involving Th17/Treg rebalance via the gut microbiota and HCP metabolite (acetate) metabolized in pneumonia mice. Our results provided a new insight for macromolecular polysaccharides through targeting intestinal microenvironment reducing distant pulmonary infection.

MILD TRAUMATIC BRAIN INJURY ATTENUATES PNEUMONIA-INDUCED LUNG INJURY BY MODULATIONS OF ALVEOLAR MACROPHAGE BACTERICIDAL ACTIVITY AND M1 POLARIZATION.

Traumatic brain injury is one of the main causes of death and disability worldwide, and results in multisystem complications. However, the mechanism of mild traumatic brain injury (MTBI) on lung injury remains unclear. In this study, we used a murine model of MTBI and pneumonia ( Pseudomonas aeruginosa ;) to explore the relationship between these conditions and the underlying mechanism. Methods: Mice (n = 104) were divided into control, MTBI, pneumonia, and MTBI + pneumonia groups. MTBI was induced by the weight-drop method. Pneumonia was induced by intratracheal injection with P. aeruginosa Xen5 strain. Animals were killed 24 h after bacterial challenging. Histological, cellular, and molecular indices of brain and lung injury were assessed using various methods. Results: Mice in both the MTBI and pneumonia groups had more Fluoro-Jade C-positive neurons than did the controls ( P < 0.01), but mice in the MTBI + pneumonia group had fewer Fluoro-Jade C-positive cells than did the pneumonia group ( P < 0.01). The MTBI + pneumonia mice showed decreased bacterial load ( P < 0.05), reduced lung injury score and pulmonary permeability ( P < 0.01), less inflammatory cells, and lower levels of proinflammatory cytokines (TNF-α and IL-1β; P < 0.01) when compared with the pneumonia group. Molecular analysis indicated lower levels of phosphorylated nuclear factor-κB in the lung of MTBI + pneumonia mice compared with the pneumonia group ( P < 0.01). Furthermore, alveolar macrophages from MTBI mice exhibited enhanced bactericidal capacity compared with those from controls ( P < 0.01). Moreover, MTBI + pneumonia mice exhibited less CD86-positive M1 macrophages compared with the pneumonia group ( P < 0.01). Conclusions: MTBI attenuates pneumonia-induced acute lung injury through the modulation of alveolar macrophage bactericidal capacity and M1 polarization in bacterial pneumonia model.

Jian-Ti-Kang-Yi decoction alleviates poly(I:C)-induced pneumonia by inhibiting inflammatory response, reducing oxidative stress, and modulating host metabolism.

Jian-Ti-Kang-Yi decoction (JTKY) is widely used in the treatment of COVID-19. However, the protective mechanisms of JTKY against pneumonia remain unknown. In this study, polyinosinic-polycytidylic acid (poly(I:C)), a mimic of viral dsRNA, was used to induce pneumonia in mice; the therapeutic effects of JTKY on poly(I:C)-induced pneumonia model mice were evaluated. In addition, the anti-inflammatory and anti-oxidative potentials of JTKY were also investigated. Lastly, the metabolic regulatory effects of JTKY in poly(I:C)-induced pneumonia model mice were studied using untargeted metabolomics. Our results showed that JTKY treatment decreased the wet-to-dry ratio in the lung tissue, total protein concentration, and total cell count of the bronchoalveolar lavage fluid (BALF). Hematoxylin and Eosin (HE) and Masson staining indicated that the JTKY treatment alleviated the pathological changes and decreased the fibrotic contents in the lungs. JTKY treatment also decreased the expression of pro-inflammatory cytokines [interleukin (IL)-1β, IL-6, and tumor necrosis factor-alpha (TNF-α)] and increased the levels of immunomodulatory cytokines (IL-4 and IL-10) in the BALF and serum. Flow cytometry analysis showed that the JTKY treatment lowered the ratio of CD86+/CD206+ macrophages in the BALF, decreased inducible nitric oxide synthase (iNOS) level, and increased arginase 1 (Arg-1) level in lung. JTKY also lowered CD11b+Ly6G+ neutrophils in BALF and decreased myeloperoxidase (MPO) activity in lung. Moreover, it also elevated superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities and decreased methane dicarboxylic aldehyde (MDA) level in lung. Untargeted metabolomic analysis showed that the JTKY treatment could affect 19 metabolites in lung, such as L-adrenaline, L-asparagine, ornithine, and alpha-ketoglutaric acid. These metabolites are associated with the synthesis and degradation of ketone bodies, butanoate, alanine, aspartate, and glutamate metabolism, and tricarboxylic acid (TCA) cycle processes. In conclusion, our study demonstrated that treatment with JTKY ameliorated poly(I:C)-induced pneumonia. The mechanism of action of JTKY may be associated with the inhibition of the inflammatory response, the reduction of oxidative stress, and the regulation of the synthesis and degradation of ketone bodies, TCA cycle, and metabolism of alanine, aspartate, glutamate, and butanoate processes in lung.

The Effects of H2S and Recombinant Human Hsp70 on Inflammation Induced by SARS and Other Agents In Vitro and In Vivo.

The ongoing epidemic caused by SARS-CoV-2 infection led to the search for fundamentally new ways and means to combat inflammation and other pathologies caused by this virus. Using a cellular model of lipopolysaccharide (LPS)-induced sepsis (human promonocytes), we showed that both a hydrogen sulfide donor (sodium thiosulfate, STS) and a recombinant Heat shock protein 70 (rHsp70) effectively block all major inflammatory mediators when administrated before and after LPS challenge. The protective anti-inflammatory effect of rHsp70 and H2S was also confirmed in vivo using various animal models of pneumonia. Specifically, it was found that rHsp70 injections prevented the development of the acute respiratory distress syndrome in highly pathogenic pneumonia in mice, increased animal survival, and reduced the number of Programmed death-1 (PD-1)-positive T-lymphocytes in peripheral blood. Based on our model experiments we developed a combined two-phase therapeutic approach for the treatment of COVID-19 patients. This procedure includes the inhalation of hot helium–oxygen mixtures for induction of endogenous Hsp70 in the first phase and STS inhalation in the second phase. The use of this approach has yielded positive results in COVID-19 patients, reducing the area of lung lesions, restoring parameters of innate immunity and T-cell immune response against coronavirus infection, and preventing the development of pulmonary fibrosis and immune exhaustion syndrome.

Intestinal Klebsiella pneumoniae Contributes to Pneumonia by Synthesizing Glutamine in Multiple Myeloma.

Simple SummaryMultiple myeloma (MM) is characterized by the presence of systemic clinical manifestations. Among them, pneumonia accounts for a significant cause of morbidity and mortality, highlighting an urgent need to explore possible susceptibility and potential mechanisms of pneumonia in MM. Recently, the gut–lung axis has emphasized the relevance of gut microbiota and pneumonia, indicating that the compromised function of gut microbiota may be susceptible to pneumonia. It has been previously shown that intestinal Klebsiella pneumonia enriches in MM patients and promotes MM progression in our previous work. In addition, host metabolism has been identified as a key regulator of MM. However, what role the altered gut microbiota plays in MM with pneumonia remains unknown. In this study, we explored the association between gut microbiota and MM with pneumonia. This is the first study to identify and elucidate the function of gut microbiota involved in MM with pneumonia.Pneumonia accounts for a significant cause of morbidity and mortality in multiple myeloma (MM) patients. It has been previously shown that intestinal Klebsiella pneumonia (K. pneumonia) enriches in MM and promotes MM progression. However, what role the altered gut microbiota plays in MM with pneumonia remains unknown. Here, we show that intestinal K. pneumonia is significantly enriched in MM with pneumonia. This enriched intestinal K. pneumonia links to the incidence of pneumonia in MM, and intestinal colonization of K. pneumonia contributes to pneumonia in a 5TGM1 MM mice model. Further targeted metabolomic assays reveal the elevated level of glutamine, which is consistently increased with the enrichment of K. pneumonia in MM mice and patients, is synthesized by K. pneumonia, and leads to the elevated secretion of TNF-α in the lung normal fibroblast cells for the higher incidence of pneumonia. Inhibiting glutamine synthesis by establishing glnA-mutated K. pneumonia alleviates the incidence of pneumonia in the 5TGM1 MM mice model. Overall, our work proposes that intestinal K. pneumonia indirectly contributes to pneumonia in MM by synthesizing glutamine. Altogether, we unveil a gut–lung axis in MM with pneumonia and establish a novel mechanism and a possible intervention strategy for MM with pneumonia.

Aloe polymeric acemannan inhibits the cytokine storm in mouse pneumonia models by modulating macrophage metabolism

The cytokine storm is highly associated with inflammatory-type disease severity and patients' survival. Plant polysaccharides, the main natural phytomedicine source, have a great potential to be an effective drug to treat cytokine storm. Herein we found that a polymeric acemannan (ABPA1) isolated from Aloe Vera Barbadensis extract C (AVBEC) exerted prominent inhibitory effects on inflammation-induced cytokine storm. The results displayed that ABPA1 effectively suppressed LPS-induced proinflammatory cytokines release in vitro. Moreover, ABPA1 treatment alleviated the cytokine storm and tissue damage in LPS- and IAV-induced mouse pneumonia models, and altered the phenotypic balance of macrophages in lung tissues. Functionally, ABPA1 enhanced macrophage M2 polarization and phagocytosis in RAW264.7 cells and inhibited LPS-induced M1 polarization. Mechanistically, ABPA1 enhanced mitochondrial metabolism and OXPHOS through activated PI3K/Akt/GSK-3β signalling pathway. Overall, our findings suggest that ABPA1 may modulate macrophage activation and mitochondrial metabolism by targeting PI3K/Akt/GSK-3β signalling pathway, thereby alleviating cytokine storm and inflammation.

2,3-Dehydrokievitone combats methicillin-resistant Staphylococcus aureus infection by reducing alpha-hemolysin expression

Due to powerful drug resistance and fatal toxicity of methicillin-resistant Staphylococcus aureus (MRSA), therapeutic strategies against virulence factors present obvious advantages since no evolutionary pressure will induce bacterial resistance. Alpha-hemolysin (Hla) is an extracellular toxin secreted by Staphylococcus aureus and contributes to bacterial pathogenicity. Herein, we identified a natural product 2,3-dehydrokievitone (2,3-DHKV) for inhibiting Hla activity of MRSA strain USA300 but not affecting bacteria growth. 2,3-DHKV significantly decreased hemolysin expression in a dose-dependent manner, but it did not potently neutralize hemolysin activity. Subsequently, cellular thermal shift and heptamer formation assays confirmed that 2,3-DHK affects hemolytic activity through indirect binding to Hla. RT-qPCR and western blot revealed that 2,3-DHKV suppressed Hla expression at the mRNA and protein levels, and further decreased accessory gene regulator A (agrA) transcription levels. We also observed that 2,3-DHK significantly attenuated the damage of A549 cells by S. aureus and reduced the release of lactate dehydrogenase (LDH). Moreover, in the MRSA-induced pneumonia mouse model, 2,3-DHK treatment prolonged the life span of mice and reduced the bacterial load in the lungs, which significantly alleviated the damage to the lungs. In summary, this study proved that 2,3-DHK as a Hla inhibitor is a potential antivirulence agent against MRSA infection.

Cyanidin chloride protects mice from methicillin-resistant Staphylococcus aureus-induced pneumonia by targeting Sortase A

ABSTRACT Methicillin-resistant Staphylococcus aureus (MRSA) has been developing rapidly in recent years. It poses a severe peril to global health care, and the new strategies to against the MRSA is urgently needed. Sortase A (SrtA) regulates the anchoring of many surface proteins. Compounds repress Staphylococcus aureus (S. aureus) cysteine transpeptidase SrtA are considered adequate potent virulence inhibitors. Then, we describe the identification of an effective SrtA inhibitor, cyanidin chloride, a bioflavonoid compound isolated from various plants. It has a reversible inhibitory effect on SrtA activity at an IC50 of 21.91 μg/mL. As a SrtA inhibitor, cyanidin chloride antagonizes SrtA-related virulence phenotypes due to its breadth and specificity, including fibrinogen adhesion, A549 cell invasion, biofilm formation, and surface protein (SpA) anchoring. Subsequently, molecular docking and fluorescence quenching revealed that SrtA and cyanidin chloride had robust mutual affinity. Further mechanistic studies revealed that Arg-197, Gly-167, and Sep-116 were the key-binding sites mediating the interaction between SrtA and cyanidin chloride. Notably, a significant therapeutic effect of cyanidin chloride in vivo was also observed on the mouse pneumonia model induced by MRSA. In conclusion, our study indicates that cyanidin chloride potentially represents a new candidate SrtA inhibitor for S. aureus and potentially be developed as a new antivirulence agent.

A Novel Triple Combination To Combat Serious Infections with Carbapenem-Resistant Acinetobacter baumannii in a Mouse Pneumonia Model.

ABSTRACTThe ongoing crisis of antimicrobial resistance demands novel combinations between antimicrobials and nonantimicrobials to manage infections caused by highly resistant pathogens. This study aimed to evaluate the effect of combining sodium ascorbate and/or apo-transferrin with imipenem, forming double and triple combinations, against 20 multiple-carbapenemase-producing Acinetobacter baumannii strains using the checkerboard test, time-kill assay, and disc diffusion test. The results of the checkerboard assay revealed that all double combinations showed indifference, while only triple combination recorded a synergistic effect (fractional inhibitory concentration index [FICI] < 0.8) in 95% the test isolates. Moreover, the MIC of imipenem (MICimp) was strongly reduced (up to 128-fold reduction) after treatment with the triple combination against highly resistant isolates and reached the susceptible range. The time-kill assay revealed that the triple combination led to a 4-log10 reduction in the CFU at 8 h compared with the initial bacterial count, and no viable count was recorded at 10 h. The mouse pneumonia model showed restoration of lung function and structure, with mild to moderate residual inflammation and moderately congested vessels observed 8 h following administration of the triple rescue therapy. Additionally, normal lungs with normal patent alveoli were detected 72 h following treatment. Accordingly, sodium ascorbate and apo-transferrin are promising adjunct biological agents with the potential to restore the effectiveness of critically essential antibiotics like imipenem, commonly used for the treatment of A. baumannii infections.IMPORTANCE Combination therapy provides a perspective to threat multidrug-resistant (MDR) strains. The present study sheds light on a novel and effective triple combination against carbapenem-resistant A. baumannii. Our in vitro results showed that combining imipenem with apo-transferrin and sodium ascorbate yielded synergism in 95% of test isolates, and this was associated with a marked reduction in imipenem MIC, shifting it below the breakpoint. Furthermore, a bactericidal effect was recorded, with no viable count detected at 10 h. An in vivo murine model of pneumonia was induced to mimic human disease. The triple combination therapy restored lung function and structure, with mild to moderate residual inflammation and moderately congested vessels observed 8 h following the initiation of therapy. Therefore, our findings suggest novel insights about a promising new combination therapy against highly resistant carbapenemase-producing A. baumannii to restore the effectiveness of imipenem.

Oridonin attenuates lung inflammation and fibrosis in silicosis via covalent targeting iNOS

Silicosis, the most common type of pneumoconiosis, exhibits a high incidence in workers who are chronically exposed to crystalline silica (CS). No specific remedy for cure as yet. The terpenoid oridonin exerts multiple modulatory functions in neoplasms and inflammations as a natural compound. In this study, we explored the effect of oridonin on silicosis and revealed the underlying molecular mechanism. An experimental silicosis mouse model was established to evaluate the effects of oridonin on pneumonia and pulmonary fibrosis. In addition, the impact of oridonin on alveolar macrophages (AMs) was examined in the MH-S cell line. Its molecular target, inducible nitric oxide synthase (iNOS), was identified by chemobiological means, and virus-mediated gene overexpression systems confirmed that oridonin directly restrained iNOS protein levels. Oridonin alleviated pneumonia and pulmonary fibrosis in silicosis mice with no obvious systemic toxicity. These effects were partially related to oridonin inhibition of CS-induced AMs injury and inflammation. Furthermore, oridonin suppressed iNOS enzymatic expression and activity by covalently binding to the Thr109 residue of the iNOS target. Thus, our results indicate oridonin as a potential iNOS enzymatic suppressor in experimental silicosis that attenuates pneumonia and pulmonary fibrosis progression, which provides a therapeutic avenue for silicosis prevention and treatment.

Pinocembrin Relieves Mycoplasma pneumoniae Infection‑Induced Pneumonia in Mice Through the Inhibition of Oxidative Stress and Inflammatory Response.

Pneumonia is a serious infectious disease with increased morbidity and mortality worldwide. The M. pneumoniae is a major airway pathogen that mainly affects respiratory tract and ultimately leads to the development of pneumonia. The current exploration was aimed to uncover the beneficial properties of pinocembrin against the M. pneumoniae-triggered pneumonia in mice via its anti-inflammatory property. The pneumonia was stimulated to the BALB/c mice via infecting them with M. pneumoniae (100µl) for 2days through nasal drops and concomitantly treated with pinocembrin (10mg/kg) for 3days. The azithromycin (100mg/kg) was used as a standard drug. Then the lung weight, nitric oxide, and myeloperoxidase (MPO) activity was assessed. The content of MDA, GSH, and SOD activity was scrutinized using kits. The total cells and DNA amount present in the bronchoalveolar lavage fluid (BALF) was assessed by standard methods. The IL-1, IL-6, IL-8, TNF-α, and TGF contents in the BALF samples and NF-κB level in the lung tissues were assessed using kits. The lung histopathology was assessed microscopically to detect the histological alterations. The 10mg/kg of pinocembrin treatment substantially decreased the lung weight, nitric oxide (NO) level, and MPO activity. The MDA level was decreased, and GSH content and SOD activity were improved by the pinocembrin treatment. The pinocembrin administered pneumonia animals also demonstrated the decreased total cells, DNA amount, IL-1, IL-6, IL-8, TNF-α, and TGF in the BALF and NF-κB level. The findings of histological studies also witnessed the beneficial role of pinocembrin against M. pneumoniae-infected pneumonia. In conclusion, our findings confirmed that the pinocembrin effectively ameliorated the M. pneumoniae-provoked inflammation and oxidative stress in the pneumonia mice model. Hence, it could be a hopeful therapeutic agent to treat the pneumonia in the future.

The Epidemiology of Invasive, Multipleantibiotic-resistant Klebsiella pneumoniae Infection in a Breeding Colony of Immunocompromised NSG Mice.

Klebsiella pneumoniae (Kp) is a gram-negative opportunistic pathogen that causes severe pneumonia, pyelonephritis, and sepsis in immunocompromised hosts. During a 4-mo interval, several NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) breeders and pups in our facilities were diagnosed with Kp infections. An initial 6 adult and 1 juvenile NSG mice were submitted for necropsy and histologic examination because of acute onset of diarrhea and death. The evaluation revealed typhlocolitis in 2 of the mice and tritrichomoniasis in all 7. Escherichia coli positive for polyketide synthase (pks+) and Kp were isolated from the intestines. Given a history of sepsis due to pks+ E. coli in NSG mice in our facilities and determination of its antimicrobial susceptibility, trimethoprim-sulfamethoxazole (TMP-SMX) was administered to the colony in the drinking water for 4 wk. After this intervention, an additional 21 mice became ill or died; 11 of these mice had suppurative pneumonia, meningoencephalitis, hepatitis, metritis, pyelonephritis, or sepsis. Kp was cultured from pulmonary abscesses or blood of 10 of the mice. Whole-genome sequencing (WGS) indicated that the Kp isolates contained genes associated with phenotypes found in pore-forming Kp isolates cultured from humans with ulcerative colitis and primary sclerosing cholangitis. None of the Kp isolates exhibited a hyperviscous phenotype, but 13 of 14 were resistant to TMP-SMX. Antimicrobial susceptibility testing indicated sensitivity of the Kp to enrofloxacin, which was administered in the drinking water. Antibiotic sensitivity profiles were confirmed by WGS of the Kp strains; key virulence and resistance genes to quaternary ammonia compounds were also identified. Enrofloxacin treatment resulted in a marked reduction in mortality, and the study using the NSG mice was completed successfully. Our findings implicate intestinal translocation of Kp as the cause of pneumonia and systemic infections in NSG mice and highlight the importance of identification of enteric microbial pathogens and targeted antibiotic selection when treating bacterial infections in immunocompromised mice.