Notch signaling pathway mediates anti-inflammatory effects of vagus nerve stimulation during lipopolysaccharide-induced acute kidney injury.

Calycosin attenuates LPS-induced microglia inflammatory responses and microglia-mediated synaptic impairment via modulation of TLR4/MyD88/NF-κB signaling pathway

Objective Neuroinflammation plays an important role in the initiation and progression of central nervous system (CNS) disorders. Calycosin (Caly) has been reported to exert anti-inflammatory and neuroprotective effects. However, little is known about its potential role in microglia-mediated neurotoxicity and the underlying mechanism. The aim of the present study is to investigate whether Caly attenuates LPS-induced inflammatory response in BV2 microglial cells and microglia-mediated neuronal injuries in HT-22 cells. Methods BV2 cells were stimulated with LPS (1 μg/mL, 24 h) and treated with Caly (5, 10, or 20 μM) or Dex (2 μM). Molecular docking, CETSA, ITDRFCETSA and DARTS assays were used to evaluate the potential target engagement between Caly and TLR4. Cell viability, microglial activation, inflammatory mediator expression, secreted cytokine levels, and TLR4/MyD88/NF-κB signaling pathway were examined. HT-22 cells were cultured with conditioned medium (CM) derived from LPS-stimulated or Caly-treated BV2 cells, cell viability and expression of synaptic plasticity-related proteins were evaluated. Supplementary validation was also performed in HMC3 human microglial cells and SH-SY5Y human neuronal cells. Results Stimulation with LPS could induce BV2 microglial activation, accompanied by the increased expression and secretion of pro-inflammatory mediators, and dysregulation of TLR4/MyD88/NF-κB signaling pathway. CM derived from LPS-stimulated BV2 cells decreased HT-22 cell viability and reduced synaptic plasticity-related protein expression levels. Caly treatment attenuated LPS-induced cytotoxicity and inflammatory responses, and reversed imbalanced expression of TLR4/MyD88/NF-κB signaling pathway in LPS-challenged BV2 cells, as indicated by the reduced Iba-1 fluorescence intensity, decreased mRNA expression levels and secretion of IL-1β, IL-6, and TNF-α, and decreased proteins expression of IL-17, TLR4, MyD88, p-NF-κB/NF-κB and p-IKB/IKB. Caly also increased the expression of the anti-inflammatory phenotype-associated markers CD206 and Arg1, while reducing the pro-inflammatory markers CD86 and iNOS. Moreover, CM derived from Caly-treated BV2 cells partially restored HT-22 cell viability and protein expression of β-Tubulin, PSD95, and Synapsin I. Supplementary experiments in HMC3 and SH-SY5Y cells showed similar anti-inflammatory and synaptic protein-preserving effects. Conclusion Caly could exert potential effects on suppressing LPS-induced microglia activation and inflammatory responses in immortalized BV2 cells by regulating TLR4/MyD88/NF-κB signaling pathway, and attenuating microglia-mediated neuronal injury and synaptic impairment in immortalized HT-22 cells.

Acetylcholine protects against inflammatory cytokine induced epithelial barrier dysfunction

Glucose Metabolism Mediates Feedback Control of Innate Immune Signaling in Human Macrophages

Glucose metabolism is pivotal in regulating innate immune responses in primary human monocyte-derived macrophages (MDMs). Lipopolysaccharide (LPS) stimulation induces both inflammatory and antiviral programs; however, despite the established importance of glucose metabolism in these responses, its precise role in coordinating them remains poorly defined. Here, we identify the STAT1/NF-κB/IRF5 signaling axis as a key mediator linking glucose metabolism to inflammatory responses through the upregulation of the rate-limiting glycolytic enzyme PFKFB3. We found that LPS triggered delayed expression and activation of NF-κB p65, accompanied by increased expression of inflammatory target genes, including CD38 and CD40. Using complementary pharmacological and genetic approaches, we demonstrate that glycolysis and PFKFB3 activity are required for NF-κB p65 expression and activation. Strikingly, inhibition of PFKFB3 also suppressed LPS-induced STAT1 activation and nuclear translocation, revealing a glucose-dependent amplification loop that potentiates STAT1-mediated antiviral and NF-κB p65-mediated inflammatory responses. Collectively, these findings establish a mechanistic link between glycolytic metabolism and STAT1/IRF5- and NF-κB-dependent transcriptional programs in human MDMs responding to LPS, highlighting potential therapeutic targets for modulating innate immune responses in inflammatory disease.

4-1BBL Suppresses Anti-Inflammatory Responses by Regulating Metabolic Reprogramming of Macrophages.

Integration of intracellular signalling and metabolic reprogramming is critical for macrophage polarisation and the induction of pro- or anti-inflammatory responses. However, the molecular switches that govern these processes remain incompletely defined. While 4-1BB ligand (4-1BBL), a member of the TNF superfamily, is known to promote sustained pro-inflammatory responses in macrophages, its role in anti-inflammatory macrophage responses has not been fully elucidated. This study identifies that 4-1BBL serves as a negative regulator of anti-inflammatory macrophage polarisation. Genetic deletion or pharmacological inhibition of 4-1BBL significantly enhanced the expression of anti-inflammatory cytokines and markers in mouse macrophages. In IL-4R signalling, 4-1BBL restrained Janus kinase 1 (JAK1) and signal transducer and activator of transcription 6 (STAT6) phosphorylation, thereby modulating transcriptional programmes associated with anti-inflammatory macrophage activation. Consistently, 4-1BBL deficiency elevated mitochondrial oxidative phosphorylation and fatty acid oxidation, accompanied by increased expression of metabolic genes in anti-inflammatory macrophages. Transcriptomic analysis further revealed a shift towards anti-inflammatory and oxidative metabolic gene signatures in IL-4-treated 4-1BBL-deficient macrophages. Importantly, the inhibition of 4-1BBL signalling also augmented anti-inflammatory responses in human monocytes and facilitated the transition from pro-inflammatory to anti-inflammatory phenotypes. Collectively, these findings establish 4-1BBL as a crucial molecular switch that regulates macrophage polarisation by integrating inflammatory signalling with metabolic reprogramming, highlighting 4-1BBL as a potential therapeutic target for promoting inflammation resolution.

Receptor-interacting Protein Kinase 1 Regulates Microglial Polarization via the Csf3-JAK2/STAT3 Axis in Lipopolysaccharide-Induced inflammation and Lysolecithin-Induced Demyelination.

In numerous demyelinating diseases, brain tissue exhibits excessive inflammatory responses that promote the activation of immune cells, thereby exacerbating cellular damage and amplifying inflammatory cascades. Emerging evidence highlights receptor-interacting protein kinase 1 (RIPK1) is a pivotal regulator of neuroinflammation, and its dysregulation contributes to the pathogenesis of various central nervous system (CNS) disorders. While our previous work established that RIPK1 kinase inhibition promotes remyelination in acute demyelinating models, however, the precise mechanisms of RIPK1 in microglial activation remain unclear. Here, to investigate RIPK1's role in microglial polarization, we employed lipopolysaccharide (LPS)-stimulated BV2 cells and the lysolecithin (LPC)-induced demyelination mouse model. By combining RIPK1D138N kinase-dead knock-in mice and the pharmacological inhibitor Nec-1s，we performed experiments in both in vitro and in vivo models. We demonstrated that RIPK1 inhibition significantly attenuates M1 microglial polarization and pro-inflammatory cytokine production. Mechanistically, we identified that RIPK1 orchestrates the expression of colony-stimulating factor 3 (Csf3), which subsequently activates the JAK2/STAT3 signaling pathway. Pharmacological inhibition of RIPK1 decreased Csf3 expression, leading to reduced phosphorylation of JAK2/STAT3 and subsequent suppression of M1 polarization. Our findings reveal a novel RIPK1-Csf3-JAK2/STAT3 signaling axis governing microglial polarization and highlight its potential as a therapeutic target for demyelinating diseases.

Oxysterol-Mediated Modulation of NFï•«B -Linked Systemic Inflammatory Response in a Neonatal Mouse Model of Intestinal Perforation 2259826

Neonatal intestinal perforations are a major complication of preterm birth, strongly associated with mortality and neurodevelopmental impairment (NDI). Our prior work links intestinal perforation to NFκB-mediated inflammatory injury in the subventricular zone (SVZ), a neural stem cell niche critical for brain development. We hypothesize that the choroid plexus (ChP) mediates SVZ injury through NFκB-activated monocyte/macrophage trafficking after modeled intestinal perforation (MIP), and that modulation of this inflammatory cascade by 20α-hydroxycholesterol (20HC), a breast milk—derived oxysterol with anti-inflammatory activity, protects the SVZ. Using single-nucleus RNA sequencing (snRNA-seq) and spatial transcriptomics, we observed peripheral monocyte/macrophage transit through the ChP and colonization of the SVZ after MIP, resulting in cytokine exposure, ependymal activation, astroglial scarring, and reduced progenitor production. For transcriptomic analyses, ChP samples were obtained from control, MIP-induced, and 20HC+MIP-treated mice. Neonatal mice received daily subcutaneous 20HC (100 mg/kg/day in MCT oil) from postnatal day 3 (P3); MIP was induced on P5, and ChP harvested 24 h later for scRNA-seq to characterize immune activation. Complementary in vitro studies used 20HC-pretreated macrophage and microglia stimulated with lipopolysaccharide (LPS) to assess NFκB-linked cytokine expression by qPCR. MIP induction in P5 mice caused bacterial seeding in brain, lungs, and spleen within 48 hours and elevated NFκB-linked cytokines (TNFα, IL-1β, IL-6) across organs. 20HC treatment significantly reduced cytokine expression, preserved SVZ integrity, and decreased inflammatory pathway enrichment in ChP macrophage clusters. In vitro, 20HC suppressed IL-1β and IL-6 via NFκB inhibition. These findings identify the ChP as a mediator of inflammation-induced SVZ injury and highlight 20HC as a promising therapy to protect the developing brain after neonatal intestinal perforation. Pathogenic Mechanisms of Inflammatory Subventricular Zone Injury in Preterm Infants-National Institutes of Health (5R01-NS140419-02) Neuroimmunology (NEUR)