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Background Sepsis is a common indirect insult leading to acute respiratory distress syndrome (ARDS). Circulating extracellular vesicles (EVs) have been reported to participate in the pathogenesis of sepsis. However, the alteration of EV-bound S100A8/A9 during septic shock, along with the role of S100A8/A9 in driving acute lung injury, remains unexplored. Methods EVs were isolated from the plasma of patients upon admission with sepsis or septic shock, as well as from healthy controls. Levels of EV S100A8/A9 were assayed via ELISA. To examine the effects and underlying mechanisms of septic shock EVs in acute lung injury, these EVs were administered intratracheally into wild-type C57BL/6 mice or mice with a deficiency of advanced glycation end-products (RAGE). In addition, a mouse model of polymicrobial sepsis was introduced using cecal ligation and puncture (CLP). Results Levels of EV S100A8/A9 were significantly elevated in patients with sepsis or septic shock compared to healthy controls. Receiver operating characteristic (ROC) analysis demonstrated that EV S100A8/A9 effectively distinguished between septic shock and sepsis and had predictive potential for the development of ARDS. Notably, the levels of S100A8/A9 in EVs and alveolar macrophages from CLP mice were significantly higher than those in sham mice. Intratracheal administration of septic shock EVs directly induced acute lung injury and M1 macrophage polarization in a lipopolysaccharide-independent manner. Septic shock EVs were efficiently taken up by alveolar macrophages in vivo, leading to a significant increase in S100A8/A9 levels, which was inhibited by preincubating the EVs with an S100A8/A9 neutralizing antibody. Additionally, mice with deficiency in RAGE, a receptor for S100A8/A9, were partially protected from acute lung injury induced by septic shock EVs. In vitro, septic shock EVs prompted a proinflammatory response in bone marrow-derived macrophages. This response was blocked by preincubating the EVs with the S100A8/A9 neutralizing antibody. Conclusions Our results suggested that EV S100A8/A9 has potential value in distinguishing septic shock from sepsis and predicting the development of ARDS. Septic shock EVs-induced lung injury is at least partially mediated through S100A8/A9-RAGE pathway, involving the activation of alveolar macrophages.

The processes that drive fibrotic diseases are complex and include an influx of peripheral blood monocytes that can differentiate into fibroblast-like cells called fibrocytes. Monocytes can also differentiate into other cell types, such as tissue macrophages. The ability to discriminate between monocytes, macrophages, fibrocytes, and fibroblasts in fibrotic lesions could be beneficial in identifying therapies that target either stromal fibroblasts or fibrocytes. We have identified markers that discriminate between human peripheral blood monocytes, tissue macrophages, fibrocytes, and fibroblasts. Amongst these four cell types, only peripheral blood monocytes express the combination of CD45RO, CD93, and S100A8/A9; only macrophages express the combination of CD45RO, 25F9, S100A8/A9, and PM-2K; only fibrocytes express the combination of CD45RO, 25F9, and S100A8/A9, but not PM-2K; and only fibroblasts express the combination of CD90, cellular fibronectin, hyaluronan, and TE-7. These markers are effective both in vitro and in sections from human lung. We found that markers such as CD34, CD68, and collagen do not effectively discriminate between the four cell types. In addition, IL-4, IL-12, IL-13, IFN-gamma, and SAP differentially regulate the expression of CD32, CD163, CD172a, and CD206 on both macrophages and fibrocytes. Finally, CD49c (alpha3 integrin) expression identifies a subset of fibrocytes, and this subset increases with time in culture. These results suggest that discrimination of monocytes, macrophages, fibrocytes, and fibroblasts in fibrotic lesions is possible, and this may allow for an assessment of fibrocytes in fibrotic diseases.

ObjectiveInterleukin (IL)-36R signalling plays a proinflammatory role in different organs including the skin, but the expression of IL-36R ligands and their molecular function in intestinal inflammation are largely unknown.DesignWe studied the characteristics of IL-36R ligand expression in IBDs and experimental colitis. The functional role of IL-36R signalling in the intestine was addressed in experimental colitis and wound healing models in vivo by using mice with defective IL-36R signalling (IL-36R−/−) or Myd88, neutralising anti-IL-36R antibodies, recombinant IL-36R ligands and RNA-seq genome expression analysis.ResultsExpression of IL-36α and IL-36γ was significantly elevated in active human IBD and experimental colitis. While IL-36γ was predominantly detected in nuclei of the intestinal epithelium, IL-36α was mainly found in the cytoplasm of CD14+ inflammatory macrophages. Functional studies showed that defective IL-36R signalling causes high susceptibility to acute dextran sodium sulfate colitis and impairs wound healing. Mechanistically, IL-36R ligands released upon mucosal damage activated IL-36R+ colonic fibroblasts via Myd88 thereby inducing expression of chemokines, granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-6. Moreover, they induced proliferation of intestinal epithelial cells (IECs) and expression of the antimicrobial protein lipocalin 2. Finally, treatment of experimental intestinal wounds with IL-36R ligands significantly accelerated mucosal healing in vivo.ConclusionsIL-36R signalling is activated upon intestinal damage, stimulates IECs and fibroblasts and drives mucosal healing. Modulation of the IL-36R pathway emerges as a potential therapeutic strategy for induction of mucosal healing in IBD.

Background : Lactylation, a novel lactate-derived posttranslational modification, has been demonstrated to be linked with brain function. The present research is intended to explore the role of β-arrestin1 (ARRB1) lactylation post-subarachnoid hemorrhage (SAH). Methods : SAH models were established in mice via intravascular puncture and in primary neurons by oxyhemoglobin (oxyHb) stimulation. Lactylome analysis identified differentially lactylated proteins. Commercial kits measured lactate, mitochondrial membrane potential, reactive oxygen species (ROS), and ATP. Mitochondrial respiration was evaluated by detecting mitochondrial oxygen consumption rate. Cell viability and apoptosis were respectively determined by CCK-8 assay and flow cytometry/TUNEL assay. Protein interactions were assessed using co-immunoprecipitation and double-label immunofluorescence. Results : Elevated lactate and ARRB1 lactylation were observed in the brain of SAH mice. In primary neurons, reducing lactate with oxamate reversed mitochondrial dysfunction and apoptosis induced by oxyHb. Overexpression of ARRB1 exacerbated oxyHb-induced neuronal injury, yet this effect was absent with the ARRB1-lysine (K) 195 arginine (R) mutant. E1A binding protein P300 (P300) promoted ARRB1 lactylation to upregulate its protein expression. P300 knockdown inhibited oxyHb-induced neuronal injury, but this inhibitory effect was counteracted by ARRB1 overexpression. In oxyHb-stimulated neurons, ARRB1 lactylation upregulated S100 calcium binding protein A9 (S100A9) protein. Additionally, ARRB1 knockdown prevented mitochondrial respiratory dysfunction in neurons induced by oxyHb, which was antagonized by recombinant S100A9. ARRB1 silencing mitigated SAH injury in mice via suppressing S100A9-mediated mitochondrial dysfunction. Conclusion : P300 mediated ARRB1 lactylation, thereby increasing S100A9 to facilitate mitochondrial dysfunction and neuronal apoptosis in SAH. This study may provide prospective targets for improving SAH.

Background As primary immune sentinels of the central nervous system (CNS), microglia respond rapidly to acute brain injury and engage in dynamic crosstalk with infiltrating peripheral immune cells. This interplay critically shapes the neuroinflammatory microenvironment—a key determinant of secondary brain injury (SBI) following intracerebral hemorrhage (ICH). Fatty acid-binding protein 4 (FABP4), an adipokine associated with metabolic disorders, is recognized as a pivotal modulator of inflammatory responses; however, its role in ICH-induced SBI remains undefined. Objectives To investigate the pathogenic functions of FABP4 in microglia after ICH, elucidate its molecular mechanisms, and develop targeted therapeutic strategies. Methods Blood and brain tissue samples from ICH patients were analyzed to evaluate the relationships between FABP4 expression and prognosis. Behavioral tests, Nissl staining, and Golgi-Cox staining were used to quantify neuronal damage. Immunofluorescence and flow cytometry were used to assess microglial activation and immune cell infiltration. Transcriptomic, proteomic, co-immunoprecipitation, western blotting, and ChIP‒qPCR analyses were used to examine the FABP4 regulatory network. Brain-targeted nanoparticles were engineered to deliver FABP4-specific siRNA. Results Clinical analyses revealed microglia-specific FABP4 upregulation in ICH patients, correlating with poor neurological outcomes. Microglial Fabp4 knockout in mice attenuated neuronal loss, ameliorated cerebral edema, and enhanced functional recovery after ICH. Mechanistically, FABP4 promoted lipid droplet accumulation and inhibited the ubiquitin–proteasome-mediated degradation of S100A9 in microglia, synergistically amplifying neuroinflammation. Moreover, the activity of FABP4 in microglia facilitated neutrophil transendothelial migration into the brain parenchyma, exacerbating injury via the release of neutrophil extracellular traps (NETs). Finally, pharmacological FABP4 inhibition using brain-targeted nanoparticles conferred significant neuroprotective effects in ICH models. Conclusion This study establishes that FABP4 acts as a novel orchestrator of post-ICH neuroinflammation through dual enzymatic and nonenzymatic pathways. We also demonstrate a targeted nanotherapeutic strategy to suppress FABP4 and improve neurological outcomes.

Background Seronegative joint diseases are characterized by a lack of well-defined biomarkers since autoantibodies are not elevated. Calprotectin (S100A8/A9) is a damage-associated molecular pattern (DAMP) which is released by activated phagocytes, and high levels are found in seronegative arthritides. In this study, we investigated the biomarker potential of systemic and local levels of these S100 proteins to assess joint inflammation and joint destruction in an experimental model for seronegative arthritis. Methods Serum levels of S100A8/A9 and various cytokines were monitored during disease development in interleukin-1 receptor antagonist (IL-1Ra) –/– mice using ELISA and multiplex bead-based immunoassay, and were correlated to macroscopic and microscopic parameters for joint inflammation, bone erosion, and cartilage damage. Local expression of S100A8 and S100A9 and matrix metalloproteinase (MMP)-mediated cartilage damage in the ankle joints were investigated by immunohistochemistry. In addition, local S100A8 and activated MMPs were monitored in vivo by optical imaging using anti-S100A8-Cy7 and AF489-Cy5.5, a specific tracer for activated MMPs. Results Serum levels of S100A8/A9 were significantly increased in IL-1Ra –/– mice and correlated with macroscopic joint swelling and histological inflammation, while serum levels of pro-inflammatory cytokines did not correlate with joint swelling. In addition, early serum S100A8/A9 levels were prognostic for disease outcome at a later stage. The increased serum S100A8/A9 levels were reflected by an increased expression of S100A8 and S100A9 within the ankle joint, as visualized by molecular imaging. Next to inflammatory processes, serum S100A8/A9 also correlated with histological parameters for bone erosion and cartilage damage. In addition, arthritic IL-1Ra –/– mice with increased synovial S100A8 and S100A9 expression showed increased cartilage damage that coincided with MMP-mediated neoepitope expression and in vivo imaging of activated MMPs. Conclusions Expression of S100A8 and S100A9 in IL-1Ra –/– mice strongly correlates with synovial inflammation, bone erosion, and cartilage damage, underlining the potential of S100A8/A9 as a systemic and local biomarker in seronegative arthritis not only for assessing inflammation but also for assessing severity of inflammatory joint destruction.

Background S100 calcium binding protein A9 (S100A9) is a pro-inflammatory alarmin associated with several inflammation-related diseases. However, the role of S100A9 in lung injury in sepsis has not been fully investigated. Therefore, the present study aimed to determine the role of S100A9 in a lipopolysaccharide (LPS)-induced lung injury murine model and its underlying molecular mechanisms. Methods LPS was utilized to induce sepsis and lung injury in C57BL/6 or NOD-like receptor family pyrin domain containing 3 (NLRP3) −/− mice. To investigate the effects of S100A9 blockade, mice were treated with a specific inhibitor of S100A9. Subsequently, lung injury and inflammation were evaluated by histology and enzyme‑linked immunosorbent assay (ELISA), respectively. Furthermore, western blot analysis and RT-qPCR were carried out to investigate the molecular mechanisms underlying the effects of S100A9. Results S100A9 was upregulated in the lung tissues of LPS-treated mice. However, inhibition of S100A9 alleviated LPS-induced lung injury. Additionally, S100A9 blockade also attenuated the inflammatory responses and apoptosis in the lungs of LPS-challenged mice. Furthermore, the increased expression of NLRP3 was also suppressed by S100A9 blockade, while S100A9 blockade had no effect on NLRP3 −/− mice. In vitro, S100A9 downregulation mitigated LPS-induced inflammation. Interestingly, these effects were blunted by NLRP3 overexpression. Conclusion The results of the current study suggested that inhibition of S100A9 could protect against LPS-induced lung injury via inhibiting the NLRP3 pathway. Therefore, S100A9 blockade could be considered as a novel therapeutic strategy for lung injury in sepsis.

Osteoarthritis (OA) is a degenerative disease characterized by the destruction of cartilage. The greatest risk factors for the development of OA include age and obesity. Recent studies suggest the role of inflammation in the pathogenesis of OA. The two most common locations for OA to occur are in the knee and hip joints. The knee joint experiences more mechanical stress, cartilage degeneration, and inflammation than the hip joint. This could contribute to the increased incidence of OA in the knee joint. Damage-associated molecular patterns (DAMPs), including high-mobility group box-1, receptor for advanced glycation end products, and alarmins (S100A8 and S100A9), are released in the joint in response to stress-mediated chondrocyte and cartilage damage. This facilitates increased cartilage degradation and inflammation in the joint. Studies have documented the role of DAMPs in the pathogenesis of OA; however, the comparison of DAMPs and its influence on OA has not been discussed. In this study, we compared the DAMPs between OA knee and hip joints and found a significant difference in the levels of DAMPs expressed in the knee joint compared to the hip joint. The increased levels of DAMPs suggest a difference in the underlying pathogenesis of OA in the knee and the hip and highlights DAMPs as potential therapeutic targets for OA in the future.

Calprotectin, a heterodimer of S100A8 and S100A9 subunits, is associated with inflammatory disorders such as rheumatoid arthritis and cystic fibrosis. Although calprotectin levels are increased significantly in the gingival crevicular fluid (GCF) of periodontitis patients, its effects on periodontal ligament cells (PDLCs) remain largely unknown. The aim of this study was to evaluate calprotectin levels in the GCF of generalized aggressive periodontitis (AgP) patients and to investigate the effects of recombinant human calprotectin (rhS100A8/A9) and its subunits (rhS100A8 and rhS100A9) in PDLCs. Both the concentration and amount of crevicular calprotectin were significantly higher in the AgP group compared with healthy controls. In addition, the GCF calprotectin levels were correlated positively with clinical periodontal parameters including bleeding index, probing depth, and clinical attachment loss. rhS100A8/A9 promoted cell apoptosis, whereas rhS100A8 and rhS100A9 individually exerted little effect on apoptosis in PDLCs. rhS100A9 and rhS100A8/A9 increased the activation of nuclear factor-κB (NF-κB) by promoting the nuclear translocation of p65 in PDLCs, subsequently inducing expression of the pro-inflammatory cytokines IL-6, IL-8, TNFα, and COX2. Treatment with an NF-κB inhibitor partially reversed the rhS100A9- and rhS100A8/A9-induced upregulation of the pro-inflammatory cytokines. rhS100A9, and not rhS100A8, was mainly responsible for the pro-inflammatory role of calprotectin. Collectively, our results suggest that calprotectin promotes apoptosis and the inflammatory response in PDLCs via rhS100A9. These findings might help identify novel treatments for periodontitis.

Phthalates are used as plasticizers in the manufacture of flexible vinyl, which is used in food contact applications. Phthalates have been demonstrated to have an adverse impact on human health, particularly in terms of cancer development. In the present study, we showed for the first time that benzyl butyl phthalate (BBP) potentiates the effect of tumor-associated dendritic cells (TADCs) on the chemoresistance of breast cancer. Specific knockdown analysis revealed that S100A9 is the major factor responsible for the chemoresistance of doxorubicin/cyclophosphamide induced by BBP-stimulated TADCs in breast cancer. BBP exposure also increased tumor infiltrating myeloid-derived suppressor cell (MDSC) secretion of S100A8/A9, thereby exacerbating the resistance of breast cancer to doxorubicin with cyclophosphamide. In addition, BBP also stimulated the production of CXCL1/GROα by TADCs, which increased the angiogenesis of breast cancer in a mouse model. Inhibition of CXCL1/GROα by a neutralizing antibody, decreased the BBP-induced angiogenesis induced by BBP after chemotherapy in the mouse model. These results, for the first time, provide evidence that BBP influences the efficacy of chemotherapy by remodeling the tumor microenvironment of breast cancer.