Pro-inflammatory cytokine and chemokine responses mediated by lipopolysaccharide (LPS) activation of Toll-like receptor 4 (TLR4) play critical roles in host defense and endotoxin shock upon infection with gram-negative bacteria (GNB). Cell wall components derived from GNB are recognized by cytosolic nucleotide-binding oligomerization domain 1 (NOD1). In this study, we explored whether prior activation of NOD1 regulates TLR4-mediated pro-inflammatory cytokine and chemokine responses. Mice were pre-treated with an intraperitoneal injection of a NOD1 ligand, with or without LPS, followed by a second intraperitoneal LPS injection. Serum concentrations of C-C motif chemokine ligand 2, C-X-C motif chemokine ligand 10, and interleukin-6 following the second LPS ‍injection were markedly higher in mice pre-treated with the NOD1 ligand than in those pre-treated with the NOD2 ligand or phosphate-buffered saline. Prior activation of NOD1 enhanced TLR4-mediated pro-inflammatory cytokine and chemokine responses through the activation of signaling pathways mediated by receptor-interacting serine/threonine kinase 2, nuclear factor-κB, and type I interferons. Furthermore, NOD1-deficient mice were protected from abdominal sepsis induced by cecal ligation and puncture. Collectively, these findings indicate that sequential activation of NOD1 and TLR4 may be involved in pro-inflammatory cytokine and chemokine responses in GNB infection.

This study investigated the protective effects of Ginkgolide A (GA) against sepsis-induced lung injury and its underlying mechanisms. Human pulmonary alveolar epithelial cells (HPAEpiC) were stimulated with lipopolysaccharide (LPS) to establish an in vitro model of alveolar epithelial cell injury. The cells were treated with different concentrations of GA, after which cell viability was assessed using the CCK-8 assay, the levels of inflammatory cytokines were measured by ELISA and qPCR, oxidative stress markers were detected by fluorescence microscopy and biochemical detection kits, and apoptosis was evaluated using flow cytometry and Western blotting. The protein expression levels of NOX4, Nrf2, and HO-1 were further measured by Western blotting to clarify the involvement of the NOX4/Nrf2 pathway. GA significantly enhanced the viability of HPAEpiC cells exposed to LPS and markedly reduced the LPS-induced expression of pro-inflammatory factors. In addition, GA suppressed LPS-induced oxidative stress, inhibited apoptosis in alveolar epithelial cells, and regulated the NOX4/Nrf2 signaling pathway, as reflected by changes in the protein expression levels of NOX4, Nrf2, and HO-1. These findings indicate that GA can alleviate LPS-induced inflammatory injury, oxidative stress and apoptosis in alveolar epithelial cells by regulating the NOX4/Nrf2 signaling, suggesting that GA may exert a protective effect against sepsis-induced lung injury.

Inflammation severity predicts mortality risk in intensive care unit (ICU) patients. Hyperphenylalaninemia (HPA) is associated with inflammation. We investigated whether a phenylalanine (PHE)-free diet (PFD) reduces PHE, downstream metabolite, and proinflammatory cytokine levels in patients with HPA. This study enrolled ICU patients with PHE levels of ≥95 µM. They were divided into PFD and usual diet (UD) groups. Changes in PHE, downstream metabolite, and proinflammatory cytokine levels were assessed over a 5-day period. We included 66 propensity score–matched patients (PFD: 33; UD: 33). Reductions in PHE levels were greater in the PFD group (from 136±44.7 to 97.0±41.7 µM) than in the UD group (from 125±24.5 to 108±30.9 µM; p=0.008). PFD group exhibited greater reductions in phenylpyruvate, hydroxyl-phenyllactate, interleukin-6, interleukin-10, tumor necrosis factor-α, and macrophage inflammatory protein-1β levels and a smaller increase in interferon-inducible T-cell α chemoattractant level than did the UD group (all p<0.05). Lower PHE intake was associated with lower cytokine levels. PFD intake was associated with a trend of lowering 15-day mortality (p=0.072). Our findings suggest that PFD intake reduces the levels of PHE and proinflammatory cytokines in ICU patients with HPA. Randomized placebo-controlled trials are required to elucidate the benefits of PFDs.

Trial registration: Registered on clinicaltrials.gov (NCT04896281 on April 26, 2021. https://clinicaltrials.gov/study/NCT04896281).

Adipose tissue plays a central role in metabolic homeostasis, and its dysfunction caused by macrophage-mediated inflammation contributes to vascular disease. This study investigated the role of fumarate in adipocyte–macrophage interactions and its involvement in foam cell formation. Serum levels of S-(2-succinyl)cysteine, a marker of protein modification by fumarate, were elevated in individuals with atherosclerosis and in apolipoprotein E-deficient mice. To investigate the involvement of adipocyte–macrophage interactions in fumarate accumulation, adipocytes and macrophages were co-cultured. Fumarate production and S-(2-succinyl)cysteine formation were increased in co-cultures compared with monocultures. Increased fumarate production was accompanied by elevated expression of monocyte chemoattractant protein-1, tumor necrosis factor-alpha, and transforming growth factor-beta in macrophages. Tumor necrosis factor-alpha promoted fumarate secretion from adipocytes and induced accumulation of metabolic intermediates from glycolysis to the tricarboxylic acid cycle, together with reduced expression of tricarboxylic acid cycle enzymes. Furthermore, fumarate enhanced tumor necrosis factor-alpha production in macrophages and promoted lipid accumulation from low-density lipoprotein, resulting in foam cell formation. These findings provide evidence that fumarate mediates adipocyte–macrophage interactions, establishing a chronic inflammatory cycle that drives foam cell formation and atherogenesis.