Project description:Severe systemic inflammatory reactions, including sepsis, often lead to shock, organ failure and death, in part through an acute release of cytokines that promote vascular dysfunction. However, little is known about the vascular endothelial signaling pathways regulating the transcriptional profile in failing organs. Among all the cytokines altered during a cytokine storm, IL-6 levels are particularly informative, as they correlate with systemic disease severity and are highly predictive of mortality. Here, we show that loss of endothelial expression of the IL-6 pathway inhibitor, SOCS3, promotes a type I interferon (IFNI)-like gene signature in response to endotoxemia in mouse kidneys and brains. In cultured primary human endothelial cells, IL-6 induces a transient IFNI-like gene expression in a non-canonical, interferon-independent fashion. We further show that STAT3, which we had previously shown to control IL-6-driven endothelial barrier function, is dispensable for this activity. Instead, IL-6 promotes a transient increase in cytosolic mitochondrial DNA and requires STAT1, cGAS, STING, and the IRFs 1, 3, and 4. Inhibition of this pathway in endothelial-specific STING knockout mice or global STAT1 knockout mice leads to reduced severity of an acute endotoxemic challenge and prevents the endotoxin-induced IFNI-like gene signature. These results suggest that permeability and DNA sensing responses are driven by parallel pathways downstream of this cytokine, provide new insights into the complex response to acute inflammatory responses, and offer the possibility of novel therapeutic strategies for independently controlling the intracellular responses to IL-6 in order to tailor the inflammatory response.
Project description:RNA-seq analysis of kidney endothelium and whole kidney from mice 15 hours after an intraperitoneal challenge with 250 micrograms of LPS per mouse.
Project description:The transcription factor Signal Transducer and Activator of Transcription (STAT) 1 is activated by Interferon gamma (IFNγ) but also Lipopolysaccharide (LPS) is the trigger of inflammation. STAT1 together with downstream activated Interferon Regulatory Factors (IRF) create a platform for signal integration between IFNγ and the Toll-Like Receptor (TLR) 4 ligand in immune cells. Little is known about the role of STAT1 and IRFs on potential synergism between LPS- and INFγ-signaling in cells from the vasculature. We investigated whether vascular cells can promote inflammatory signaling by the STAT1-IRFs pathway.
Project description:Transcription profiles of BV2 microglial cell lines: unstimulated, stimulated with LPS or transfected with constitutively active Stat1 and Stat3.
Project description:Since its discovery over three decades ago, signal transducer and activator of transcription 1 (STAT1) has been extensively studied as a central mediator for interferons (IFNs) signaling and antiviral defense. Here, using genetic and biochemical assays, we unveil Thr748 as a conserved IFN-independent phosphorylation switch in Stat1, which restricts IFN signaling and promotes innate inflammatory responses following the recognition of the bacterial-derived toxin lipopolysaccharide (LPS). Genetically-engineered mice expressing phospho-deficient threonine748–to-alanine (T748A) mutant Stat1 are resistant to LPS–induced lethality. Of note, T748A mice exhibited undisturbed IFN signaling, as well as total expression of Stat1. Further, the T748A point-mutation of Stat1 recapitulates the safeguard effect of the genetic ablation of Stat1 following LPS-induced lethality, indicating that the Thr748 phosphorylation contributes inflammatory functionalities of Stat1. Mechanistically, LPS-induced Toll-like receptor 4 endocytosis activates a cell-intrinsic IκB kinase (IKK)–mediated Thr748 phosphorylation of Stat1, which promotes macrophages inflammatory response while restricting the IFN and anti-inflammatory responses. Depletion of macrophages restores the sensitivity of the T748A mice to LPS-induced lethality. Together, our study indicates a phosphorylation-dependent functional dichotomy of Stat1 in innate immune responses: IFN phospho-tyrosine dependent, and inflammatory phospho-threonine dependent. Better understanding of the Thr748 phosphorylation of Stat1 may uncover novel pharmacologically targetable molecules and offer better treatment modalities for sepsis, a disease that claims millions of lives annually.
Project description:Analysis of gene expression profile in peritoneal macrophage extracted from LPS or PBS challenged DUSP3-/- and WT mice. DUSP3 deletion protects mice from sepsis and endotoxemia. We performed a microarray analysis to get insights into the differentially regulated pathways between WT and KO under inflammatory conditions.
Project description:24 hours following transfection with either a control mix, a construct overexpressing GAM or siRNAs directed against GAM (siGAM), THP-1 cells were challenged with LPS 100 ng/ml. RNAs were analyzed after 6 hours of LPS challenge
Project description:Mice intraperitoneally administered with LPS and Stx exhibit HUS-like pathology. While mouse and human Gb3 localization is different, LPS and Stx induced kidney injury models in mice have been used to confirm responsiveness to various stx-related inflammatory pathways and treatments. In order for this mouse model to apply tHUS in humans, more detailed and exhaustive comprehension of this animal model is needed. Although molecular studies have been conducted on this mouse model before, we consider that there is still scope for further investigation of molecular pathways and studies on kidney damage segments. Overall, Biological pathways, upstream regulators, and downstream biological activities occurring in the kidney after LPS/Stx administration were identified through Ingenuity Pathway Analysis ™ using the result of microarray. In addition, we identified the detailed damaged site in the renal tubule from the down-regulation gene revealed by microarray.
Project description:Neutrophils play important roles in inflammatory airway diseases. Here, we assessed whether apolipoprotein A-I (apoA-I) modifies neutrophil heterogeneity as part of the mechanism by which it attenuates acute airway inflammation. Neutrophilic airway inflammation was induced by daily intranasal administration of LPS plus house dust mite (LPS+HDM) to Apoa1-/- and Apoa1+/+ mice for 3 days. Single cell RNA sequencing was performed on cells recovered in bronchoalveolar lavage fluid (BALF) on day 4. Unsupervised profiling identified 10 clusters of neutrophils in BALF from Apoa1-/- and Apoa1+/+ mice. LPS+HDM-challenged Apoa1-/- mice had an increased proportion of the Neu4 neutrophil cluster that expressed S100a8, S100a9, and Mmp8, and had high maturation, aggregation, and TLR4 binding scores. There was also an increase in the Neu6 cluster of immature neutrophils, whereas neutrophil clusters expressing interferon-stimulated genes were decreased. An unsupervised trajectory analysis showed that Neu4 represented a distinct lineage in Apoa1-/- mice. LPS+HDM-challenged Apoa1-/- mice also had an increased proportion of recruited airspace macrophages, which was associated with a reciprocal reduction in resident airspace macrophages. Increased expression of a common set of pro-inflammatory genes, S100a8, S100a9, and Lcn2, was present in all neutrophils and airspace macrophages from LPS+HDM-challenged Apoa1-/- mice. Apoa1-/- mice have increases in specific neutrophil and macrophage clusters in the lung during acute inflammation mediated by LPS+HDM, as well as enhanced expression of a common set of pro-inflammatory genes. This suggests that modifications in neutrophil and macrophage heterogeneity contribute to the mechanism by which apoA-I attenuates acute airway inflammation.