Cyclic GMP–AMP Synthase (cGAS) Deletion Reduces Severity in Bilateral Nephrectomy Mice through Changes in Neutrophil Extracellular Traps and Mitochondrial Respiration
Ontology highlight
ABSTRACT: Uremia-induced systemic inflammation is partly caused by the dissemination of microbial molecules such as lipopolysaccharide and bacterial double-stranded DNA from leaked gut damaged by immune cells in response to the microbial molecules. Cyclic GMP–AMP synthase (cGAS) can recognize fragmented DNA and induce cGAMP synthesis for the activation of the stimulator of interferon genes (STING) pathway. To study the effect of cGAS in uremia-induced systemic inflammation, we performed bilateral nephrectomy (BNx) in wild-type and cGAS knock-out mice and found that the gut leakage and blood uremia from both groups were similar. However, serum cytokines (TNF-α and IL-6) and neutrophil extracellular traps (NETs) decreased significantly in cGAS-/- neutrophils after stimulation with LPS or bacterial cell-free DNA. Transcriptomic analysis of LPS-stimulated cGAS-/- neutrophils also confirmed the down-regulation of neutrophil effector functions. The extracellular flux analysis showed that cGAS-/- neutrophils exhibited a higher respiratory rate than wild-type neutrophils despite having similar mitochondrial abundance and function. Our results suggest that cGAS may control effector functions and the mitochondrial respiration of neutrophils in response to LPS or bacterial DNA.
Project description:Polymorphonuclear cells (neutrophils) play an important role in the systemic inflammatory response syndrome and the development of sepsis. These cells are essential for the defense against microorganisms, but may also cause tissue damage. Therefore, neutrophil numbers and activity are considered to be tightly regulated. Previous studies have investigated gene transcription during experimental endotoxemia in whole blood and peripheral blood mononuclear cells. However, the gene transcription response of the circulating pool of neutrophils to systemic inflammatory stimulation in vivo is currently unclear. We examined neutrophil gene transcription kinetics in healthy human subjects (n=4) administered a single dose of endotoxin (LPS, 2 ng/kg iv). In addition, freshly isolated neutrophils were stimulated ex vivo with LPS, TNFM-NM-1, G-CSF and GM-CSF to identify stimulus-specific gene transcription responses. Whole transcriptome microarray analysis of circulating neutrophils at 2, 4 and 6 hours after LPS infusion revealed activation of inflammatory networks which are involved in signaling of TNFM-NM-1 and IL-1M-NM-1 and IL-1M-NM-2. The transcriptome profile of inflammatory activated neutrophils in vivo reflects extended survival and regulation of inflammatory responses. We show that these changes in neutrophil transcriptome are most likely due to a combination of early activation of circulating neutrophils by TNFM-NM-1 and G-CSF and a mobilization of young neutrophils from the bone marrow. After LPS infusion blood was taken at t=0, t=2, t=4 and t=6 hours. Neutrophils were isolated and gene expression of these cells was assessed. T=2, t=4 and t=6 were related to t=0 as control condition
Project description:During systemic inflammation, different neutrophil subsets are mobilized to the blood circulation. These neutrophil subsets can be distinguished from normal circulating neutrophils (CD16bright/CD62Lbright) based on either an immature CD16dim/CD62Lbright or a CD16bright/CD62Ldim phenotype. Interestingly, the latter neutrophil subset is known to suppress lymphocyte proliferation ex vivo, but the underlying mechanism is largely unknown. We performed transcriptome analysis on the different neutrophil subsets to identify changes that are relevant for their functions. Neutrophil subsets were isolated by FACS sorting from the blood of healthy volunteers who were administered a single dose of lipopolysaccharide (LPS). The transcriptome was determined by microarray. The mobilized neutrophil subsets were characterized by specific transcriptome profiles reflecting their phase in neutrophil lifespan. Interestingly, the CD16bright/CD62Ldim suppressive neutrophils showed an interferon-induced transcriptome profile. This was confirmed by stimulation of peripheral neutrophils with IFNgamma. These cells acquired the capacity to suppress lymphocyte proliferation through the expression of programmed death ligand 1 (PD-L1). These data demonstrate that the suppressive phenotype of the neutrophil subset is induced by IFNgamma. Specific stimulation of neutrophils might have a pivotal role in regulating lymphocyte-mediated inflammation and autoimmune disease. After LPS infusion, blood was taken at t=0 and t=4 hours. Neutrophils were FACS sorted based on CD16 and CD62L expression. Gene expression of neutrophil subsets was assessed relative to t=0 as control.
Project description:Neutrophil activation plays a critical role in the inflammatory response to gram-negative bacterial infections. Lipopolysaccharide (LPS) from gram-negative bacterial has been shown to be a major mediator of neutrophil activation to produce pro-inflammatory cytokines, chemokines and ROS which are important to tissue damage in LPS induced septic shock. We used microarrays to detail the global gene expression of neutrophils from miR-125a+/+ and miR-125a-/- mice after LPS stimulation.
Project description:Polymorphonuclear cells (neutrophils) play an important role in the systemic inflammatory response syndrome and the development of sepsis. These cells are essential for the defense against microorganisms, but may also cause tissue damage. Therefore, neutrophil numbers and activity are considered to be tightly regulated. Previous studies have investigated gene transcription during experimental endotoxemia in whole blood and peripheral blood mononuclear cells. However, the gene transcription response of the circulating pool of neutrophils to systemic inflammatory stimulation in vivo is currently unclear. We examined neutrophil gene transcription kinetics in healthy human subjects (n=4) administered a single dose of endotoxin (LPS, 2 ng/kg iv). In addition, freshly isolated neutrophils were stimulated ex vivo with LPS, TNFα, G-CSF and GM-CSF to identify stimulus-specific gene transcription responses. Whole transcriptome microarray analysis of circulating neutrophils at 2, 4 and 6 hours after LPS infusion revealed activation of inflammatory networks which are involved in signaling of TNFα and IL-1α and IL-1β. The transcriptome profile of inflammatory activated neutrophils in vivo reflects extended survival and regulation of inflammatory responses. We show that these changes in neutrophil transcriptome are most likely due to a combination of early activation of circulating neutrophils by TNFα and G-CSF and a mobilization of young neutrophils from the bone marrow.
Project description:In this study, we used chromatin immunoprecipitation sequencing (ChIP-Seq) analysis of histone H3K4me3-marked, to identify various TSSs associated with LPS-, TNF-alfa- and IL-10-stimulated neutrophils from healthy individuals, as well as neutrophils derived from the patients with sepsis (systemic septic inflammation with LPS-stimulated neutrophils), NMOSD (aseptic inflammation with neutrophils pre-activated by TNF-alfa and periodontitis (localized self-limiting septic inflammation with IL-10-positive neutrophils). We provided comprehensive epigenomic analysis within H3K4me3- marked histone that allowed us to identify human neutrophil regulators affecting their plasticity during inflammation as well as suppression.
Project description:Neutrophil Extracellular Traps (NETs) are structures consisting of chromatin and antimicrobial molecules that are released by neutrophils during a form of regulated cell death called NETosis. NETs trap invading pathogens, promote coagulation and activate myeloid cells to produce Type I interferons (type I IFN), proinflammatory cytokines that regulate the immune system. The mechanism of NET recognition by myeloid cells is not yet clearly identified. Here we show that macrophages and other myeloid cells phagocytose NETs. Once in phagosomes, NETs translocate to the cytosol, where they activate the DNA sensor cyclic GMP-AMP synthase (cGAS) and induce type I IFN expression. cGAS recognizes the DNA backbone of NETs. Interestingly, the NET associated serine protease Neutrophil Elastase (NE) mediates the activation of the pathway. We confirmed that NETs activate cGAS in vivo. Thus, our findings identify cGAS as a major sensor of NETs, mediating the immune activation during infection and in auto-immune diseases.
Project description:Infection by the Gram-negative bacterium Pseudomonas aeruginosa is common in hospitalized immunocompromised as well as immunocompetent ventilated patients and is often life-threatening because of resistance of the bacteria to antibiotics. This prompts the question whether the host’s immune system can be educated to combat this bacterium. Bacterial lipopolysaccharide (LPS) is known to promote host resistance to bacterial infections although an understanding of the underlying mechanism is lacking. Here we show that prior exposure to a single low dose of LPS protects mice from a lethal infection by P. aeruginosa. These mice displayed expansion of a neutrophil and an interstitial macrophage population that were barely detectable in mice that did not receive LPS prior to infection. Both cell populations were distinguishable from other immune cell populations by being enriched in gene sets that included phagocytosis- and cell-killing-associated genes. The cell killing gene set in the neutrophil population uniquely expressed Lgals3, which encodes the multifunctional antibacterial protein, galectin-3. Intravital imaging of neutrophils for bacterial phagocytosis, assessment of bacterial killing and neutrophil-associated galectin-3 protein levels together with use of galectin-3-deficient mice collectively highlight neutrophils and galectin-3 as central players in LPS-mediated protection. Interrogating the relevance of these findings in hospitalized patients with acute respiratory failure revealed significantly higher galectin-3 levels in endotracheal aspirates (ETAs) of survivors compared to non-survivors, galectin-3 levels strongly correlating with a neutrophil signature in the ETAs. Taken together, our study provides impetus to harnessing the potential of galectin-3-expressing neutrophils to protect the lung from lethal infections and respiratory failure.
Project description:Neutrophil gene transcription following lipopolysaccharide exposure. Microarray analysis of lipopolysaccharide-treated human neutrophils. Neutrophils respond to infection by degranulation, release of reactive oxygen intermediates, and secretion of chemokines and cytokines; however, activation of neutrophil transcriptional machinery has been little appreciated. Recent findings suggest that gene expression may represent an additional neutrophil function after exposure to lipopolysaccharide (LPS). We performed microarray gene expression analysis of 4,608 mostly nonredundant genes on LPS-stimulated human neutrophils. Analysis of three donors indicated some variability but also a high degree of reproducibility in gene expression. Twenty-eight verifiable, distinct genes were induced by 4 h of LPS treatment, and 13 genes were repressed. Genes other than cytokines and chemokines are regulated; interestingly, genes involved in cell growth regulation and survival, transcriptional regulation, and interferon response are among those induced, whereas genes involved in cytoskeletal regulation are predominantly repressed. In addition, we identified monocyte chemoattractant protein-1 as a novel LPS-regulated chemokine in neutrophils. Included in these lists are five clones with no defined function. These data suggest molecular mechanisms by which neutrophils respond to infection and indicate that the transcriptional potential of neutrophils is greater than previously thought.
Project description:Host defense against bacterial and fungal infections diminishes with age. In humans, this is thought to be caused in part by a decline in neutrophil responses. However, it remains unclear whether a similar decline in neutrophil function occurs in mice. Here, we show that old mice have a reduced capacity to clear pathogenic E. coli during septic peritonitis. Neutrophil recruitment to the peritoneum was elevated during lipopolysaccharide (LPS)-induced septic peritonitis but not aseptic peritonitis. Neutrophils from old mice showed reduced chemoattractant-induced reactive oxygen species (ROS) production upon priming with LPS but not GM-CSF/TNF. Phagocytosis and degranulation were reduced in a partially LPS-dependent manner, whereas NETosis was impaired independently of LPS. The chemoattractant-stimulated production of PIP3 was reduced upon priming with LPS but not GM-CSF/TNF, whereas PI(4,5)P2 levels were constitutively low. Unexpectedly, chemotaxis was normal regardless of priming pathway, as were the chemoattractant-stimulated activities of Rac1 and Rac2. The expression of 5% of neutrophil proteins was deregulated in old age. Granule proteins, particularly cathepsins and serpins, as well as toll-like receptor (TLR) pathway proteins and membrane receptors were upregulated, whereas chromatin and RNA regulators were downregulated. Upregulation of Cd180 and downregulation of MyD88 may contribute to the impaired LPS priming and LPS-dependent PIP3 production. In summary, all major neutrophil responses except chemotaxis decline with age in mice, particularly upon LPS priming. This LPS-TLR4 pathway dependence resolves some of the controversy regarding the effects of age on murine neutrophils and confirms mice are an appropriate model for the declining human neutrophil function.
Project description:Leber2015 - Mucosal immunity and gut
microbiome interaction during C. difficile infection
This model is described in the article:
Systems Modeling of
Interactions between Mucosal Immunity and the Gut Microbiome
during Clostridium difficile Infection.
Leber A, Viladomiu M, Hontecillas R,
Abedi V, Philipson C, Hoops S, Howard B, Bassaganya-Riera
J.
PLoS ONE 2015; 10(7): e0134849
Abstract:
Clostridium difficile infections are associated with the use
of broad-spectrum antibiotics and result in an exuberant
inflammatory response, leading to nosocomial diarrhea, colitis
and even death. To better understand the dynamics of mucosal
immunity during C. difficile infection from initiation through
expansion to resolution, we built a computational model of the
mucosal immune response to the bacterium. The model was
calibrated using data from a mouse model of C. difficile
infection. The model demonstrates a crucial role of T helper 17
(Th17) effector responses in the colonic lamina propria and
luminal commensal bacteria populations in the clearance of C.
difficile and colonic pathology, whereas regulatory T (Treg)
cells responses are associated with the recovery phase. In
addition, the production of anti-microbial peptides by inflamed
epithelial cells and activated neutrophils in response to C.
difficile infection inhibit the re-growth of beneficial
commensal bacterial species. Computational simulations suggest
that the removal of neutrophil and epithelial cell derived
anti-microbial inhibitions, separately and together, on
commensal bacterial regrowth promote recovery and minimize
colonic inflammatory pathology. Simulation results predict a
decrease in colonic inflammatory markers, such as neutrophilic
influx and Th17 cells in the colonic lamina propria, and length
of infection with accelerated commensal bacteria re-growth
through altered anti-microbial inhibition. Computational
modeling provides novel insights on the therapeutic value of
repopulating the colonic microbiome and inducing regulatory
mucosal immune responses during C. difficile infection. Thus,
modeling mucosal immunity-gut microbiota interactions has the
potential to guide the development of targeted fecal
transplantation therapies in the context of precision medicine
interventions.
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