Increased Neutrophil H2O2 Production and Enhanced Pulmonary Clearance of Klebsiella pneumoniae in G6PD A- Mice
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PROVIDER: S-EPMC10980108 | biostudies-literature | 2024 Mar
REPOSITORIES: biostudies-literature
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Project description:BackgroundRecent trials demonstrate increased pneumonia risk in chronic obstructive pulmonary disease patients treated with the inhaled corticosteroid (ICS) fluticasone propionate (FP). There is limited work describing FP effects on host defenses against bacterial pneumonia.MethodsC57BL/6 mice received daily, nose-only exposure to nebulized FP or vehicle for 8 days, followed by pulmonary challenge with Klebsiella pneumoniae. Bacterial burden, phagocytosis, leukocyte recruitment, cytokine expression, nitric oxide release, and survival were measured.ResultsInhaled FP increased bacterial burden in lungs and blood 48 h after infection but affected neither in vivo phagocytosis of bacteria by alveolar macrophages (AM) nor alveolar neutrophil recruitment. AM from FP-treated mice showed impaired expression of infection induced TNF-alpha, IP-10 (CXCL-10), and interleukin 6 (IL-6), and AM also showed a trend towards impaired intracellular pathogen control following in vivo infection. In vitro FP treatment resulted in a dose-dependent impairment of cytokine expression by AM. Furthermore, infection-induced nitric oxide (but not hydrogen peroxide) production was impaired by FP in vivo and in vitro. FP decreased survival in this model.ConclusionsExposure to inhaled FP impairs pulmonary clearance of K. pneumoniae in mice, an effect associated with greater systemic bacteremia and death. Decreased AM cytokine and nitric oxide expression parallel the failure to control infection. These results support the study of ICS effects on human pulmonary host defenses.
| S-EPMC3426464 | biostudies-literature
Project description:Initial promising results with immune sera guided early human mAb approaches against Gram-negative sepsis to an LPS neutralization mechanism, but these efforts failed in human clinical trials. Emergence of multidrug resistance has renewed interest in pathogen-specific mAbs. We utilized a pair of antibodies targeting Klebsiella pneumoniae LPS, one that both neutralizes LPS/TLR4 signaling and mediates opsonophagocytic killing (OPK) (54H7) and one that only promotes OPK (KPE33), to better understand the contribution of each mechanism to mAb protection in an acutely lethal pneumonia model. Passive immunization 24 hours prior to infection with KPE33 protected against lethal infection significantly better than 54H7, while delivery of either mAb 1 hour after infection resulted in similar levels of protection. These data suggest that early neutralization of LPS-induced signaling limits protection afforded by these mAbs. LPS neutralization prevented increases in the numbers of γδT cells, a major producer of the antimicrobial cytokine IL-17A, the contribution of which was confirmed using il17a-knockout mice. We conclude that targeting LPS for OPK without LPS signaling neutralization has potential to combat Gram-negative infection by engaging host immune defenses, rather than inhibiting beneficial innate immune pathways.
| S-EPMC5414560 | biostudies-literature
Project description:Adaptive changes in the genome of a locally predominant clinical isolate of the multidrug-resistant Klebsiella pneumoniae ST258 (KP35) were identified and help to explain the selection of this strain as a successful pulmonary pathogen. The acquisition of 4 new ortholog groups, including an arginine transporter, enabled KP35 to outcompete related ST258 strains lacking these genes. KP35 infection elicited a monocytic response, dominated by Ly6Chi monocytic myeloid-derived suppressor cells that lacked phagocytic capabilities, expressed IL-10, arginase, and antiinflammatory surface markers. In comparison with other K. pneumoniae strains, KP35 induced global changes in the phagocytic response identified with proteomics, including evasion of Ca2+ and calpain activation necessary for phagocytic killing, confirmed in functional studies with neutrophils. This comprehensive analysis of an ST258 K. pneumoniae isolate reveals ongoing genetic adaptation to host microenvironments and innate immune clearance mechanisms that complements its repertoire of antimicrobial resistance genes and facilitates persistence in the lung.
| S-EPMC5070956 | biostudies-literature
Project description:Bacterial pneumonia is a burdensome, costly disease and increasingly challenging to treat due to antibiotic resistance. Complex host-pathogen interactions regulate protective immunity, and immune-directed therapies could yield novel treatments. Neutrophils play a central role in pulmonary bacterial immunity, and mechanistic understanding of neutrophil antibacterial functions in pneumonia has potential clinical and fundamental application. Nuclear receptor 4a1 (Nr4a1), a member of the nuclear orphan receptor family, has been described to regulate inflammation and immune development in a cell type-specific manner, but its role in pulmonary host defense is not well understood. This study demonstrates that Nr4a1 is essential for protection against K. pneumoniae as Nr4a1 -/-mice showed increased lung bacterial burden, dissemination, and reduced survival. Notably, neutrophil-specific Nr4a1 expression was critical for defense against K. pneumoniae infection by regulating the neutrophil transcriptome. These findings suggest targeting neutrophil-specific Nr4a1 may be beneficial for bacterial pneumonia treatment.
2025-03-25 | GSE290097 | GEO
Project description:Klebsiella pneumoniae is a common respiratory pathogen, with some strains having developed broad resistance to clinically available antibiotics. Humans can become infected with many different K. pneumoniae strains that vary in genetic background, antibiotic susceptibility, capsule composition, and mucoid phenotype. Genome comparisons have revealed differences between K. pneumoniae strains, but the impact of genomic variability on immune-mediated clearance of pneumonia remains unclear. Experimental studies of pneumonia in mice have used the rodent-adapted 43816 strain of K. pneumoniae and demonstrated that neutrophils are essential for optimal host defense. It remains unclear, however, whether CCR2(+) monocytes contribute to K. pneumoniae clearance from the lung. We selectively depleted neutrophils, CCR2(+) monocytes, or both from immunocompetent mice and determined susceptibility to infection by the 43816 strain and 4 newly isolated clinical K. pneumoniae strains. The clinical K. pneumoniae strains, including one carbapenem-resistant ST258 strain, are less virulent than 43816. Optimal clearance of each of the 5 strains required either neutrophils or CCR2(+) monocytes. Selective neutrophil depletion markedly worsened infection with K. pneumoniae strain 43816 and three clinical isolates but did not increase susceptibility of mice to infection with the carbapenem-resistant K. pneumoniae ST258 strain. Depletion of CCR2(+) monocytes delayed recovery from infection with each of the 5 K. pneumoniae strains, revealing a contribution of these cells to bacterial clearance from the lung. Our findings demonstrate strain-dependent variation in the contributions of neutrophils and CCR2(+) monocytes to clearance of K. pneumoniae pulmonary infection.
| S-EPMC4534658 | biostudies-literature
Project description:IntroductionKlebsiella pneumoniae (Kp) is a common cause of hospital-acquired pneumonia. Although previous studies have suggested that evasion of phagocytic uptake is a virulence determinant of Kp, few studies have examined phagocytosis sensitivity in clinical Kp isolates.MethodsWe screened 19 clinical respiratory Kp isolates that were previously assessed for mucoviscosity for their sensitivity to macrophage phagocytic uptake, and evaluated phagocytosis as a functional correlate of in vivo Kp pathogenicity.ResultsThe respiratory Kp isolates displayed heterogeneity in the susceptibility to macrophage phagocytic uptake, with 14 out of 19 Kp isolates displaying relative phagocytosis-sensitivity compared to the reference Kp strain ATCC 43816, and 5 out of 19 Kp isolates displaying relative phagocytosis-resistance. Intratracheal infection with the non-mucoviscous phagocytosis-sensitive isolate S17 resulted in a significantly lower bacterial burden compared to infection with the mucoviscous phagocytosis-resistant isolate W42. In addition, infection with S17 was associated with a reduced inflammatory response, including reduced bronchoalveolar lavage fluid (BAL) polymorphonuclear (PMN) cell count, and reduced BAL TNF, IL-1β, and IL-12p40 levels. Importantly, host control of infection with the phagocytosis-sensitive S17 isolate was impaired in alveolar macrophage (AM)-depleted mice, whereas AM-depletion had no significant impact on host defense against infection with the phagocytosis-resistant W42 isolate.ConclusionAltogether, these findings show that phagocytosis is a primary determinant of pulmonary clearance of clinical Kp isolates.
| S-EPMC10086180 | biostudies-literature
Project description:BackgroundGlucose-6-phosphate dehydrogenase (G6PD) is essential to produce reduced nicotinamide adenine dinucleotide phosphate, which is required to protect cells against oxidative stress. G6PD deficiency is a genetic variation that may lead to hemolysis with potential consequences, such as kidney failure, and patients often experience low quality of life.ObjectivesTo establish a simple, efficient, and optimized method to produce a G6PDViangchan variant and characterize the phenotypes of recombinant human wild-type G6PD and G6PDViangchan.MethodsG6PD was amplified by polymerase chain reaction (PCR) from a human cDNA plasmid, and the gene for G6PDViangchan was amplified by initiating a mutation at location 871 (G>A) through site-directed mutagenesis. Protein expression and western blotting were conducted after successful cloning. The enzymatic activity of both proteins was assessed spectrophotometrically after purification.ResultsBoth amplicons were successfully cloned into a pET26b(+) expression vector and transformed into Escherichia coli BL21 (DE3) cells for overexpression as C-terminally histidine-tagged recombinant proteins. Western blotting confirmed that both proteins were successfully produced at similar levels. The enzymes were purified by immobilized metal (Co) affinity chromatography. Postpurification assay of enzyme activity revealed about 2-fold differences in the levels of specific activity between the wild-type G6PD (155.88 U/mg) and G6PDViangchan (81.85 U/mg), which is consistent with earlier reports. Analysis in silico showed that the coding change in G6PDViangchan has a substantial effect on protein folding structure.ConclusionsWe successfully cloned, expressed, and purified both wild-type G6PD and G6PDViangchan proteins. Such a protocol may be useful for creating a model system to study G6PD deficiency disease.
| S-EPMC10373393 | biostudies-literature
Project description:Increasing antibiotic resistance among bacterial pathogens has rendered some infections untreatable with available antibiotics. Klebsiella pneumoniae, a bacterial pathogen that has acquired high-level antibiotic resistance, is a common cause of pulmonary infections. Optimal clearance of K. pneumoniae from the host lung requires TNF and IL-17A. Herein, we demonstrate that inflammatory monocytes are rapidly recruited to the lungs of K. pneumoniae-infected mice and produce TNF, which markedly increases the frequency of IL-17-producing innate lymphoid cells. While pulmonary clearance of K. pneumoniae is preserved in neutrophil-depleted mice, monocyte depletion or TNF deficiency impairs IL-17A-dependent resolution of pneumonia. Monocyte-mediated bacterial uptake and killing is enhanced by ILC production of IL-17A, indicating that innate lymphocytes engage in a positive-feedback loop with monocytes that promotes clearance of pneumonia. Innate immune defense against a highly antibiotic-resistant bacterial pathogen depends on crosstalk between inflammatory monocytes and innate lymphocytes that is mediated by TNF and IL-17A.
| S-EPMC4842125 | biostudies-literature
Project description:The wavelength of light is a critical determinant of light's capacity to entrain adaptive biological mechanisms, such as enhanced immune surveillance, that precede and prepare us for the active circadian day, a time when the risk of encountering pathogen is highest. Light rich in the shorter wavelength visible blue spectrum maximally entrains these circadian rhythms. We hypothesized that exposure to blue light during sepsis will augment immunity and improve outcome. Using a clinically relevant Klebsiella pneumoniae acute lower respiratory tract infection model, we show that blue spectrum light shifts autonomic tone toward parasympathetic predominance and enhances immune competence, as characterized by accelerated pathogen clearance that is accompanied by reduced alveolar neutrophil influx, inflammation, and improved survival. Blue light functioned through an optic-cholinergic pathway and expansion of splenic Ccr2+ monocytes to increase control of the infection and improve survival. The "keystone" mediating these effects is the circadian clock protein Rev-Erbα, and biochemical activation with Rev-Erbα agonist SR9009 enhanced mononuclear cell phagocytosis in vitro and recapitulated the enhanced pathogen elimination in vivo observed with blue light. These findings underscore the potential therapeutic value of blue light and modulating Rev-Erbα to enhance host immunity against infection.
| S-EPMC7456460 | biostudies-literature
Project description:IntroductionBacterial pneumonia is a burdensome, costly disease and increasingly challenging to treat due to antibiotic resistance. Complex host-pathogen interactions regulate protective immunity. Neutrophils play a central role in pulmonary bacterial immunity, and mechanistic understanding of neutrophil functions in bacterial pneumonia has potential clinical and fundamental application. Nuclear receptor 4a1 (Nr4a1), a member of the nuclear orphan receptor family, has been described to regulate inflammation and immune development in a cell type-specific manner, but its role in pulmonary host defense is not well understood.MethodsWild-type (WT) and Nr4a1-/- mice, as well as bone marrow chimeric and Gr-1+ antibody depleted mice, were infected with Klebsiella pneumoniae and assessed for bacterial burden in the lung and spleen, gene transcription, protein levels, histology and cellular abundance by flow cytometry in the lung. WT and Nr4a1-/- neutrophils were exposed to live Klebsiella pneumoniae to quantify bacterial killing, as well as bulk RNA sequencing to assess transcriptomic differences.ResultsNr4a1-deficient mice are highly susceptible to Klebsiella pneumoniae pneumonia, which was mediated by Nr4a1 expression in immune cells. Gr-1+ antibody depletion ameliorated the Nr4a1-dependent phenotype. Ex vivo, Nr4a1-deficient neutrophils had impaired bactericidal capacity, and transcriptomic analysis identified an Nr4a1-dependent host defense program in neutrophils.DiscussionNeutrophil Nr4a1 expression is critical for defense against K. pneumoniae infection by regulating the neutrophil transcriptome. These findings suggest targeting Nr4a1 signaling pathways in neutrophils may be useful for bacterial pneumonia treatment.
| S-EPMC11922696 | biostudies-literature