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Results for “RAW 264.7 Cells”

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At least 19 recordsLinked to original sources

PubMed1992

IFN-gamma mediates increased glucocorticoid receptor expression in murine macrophages.

Exposure of the murine macrophage cell line, RAW 264.7, to murine rIFN-gamma resulted in a significant increase in the number of glucocorticoid receptors (GcR). A doubling in the number of GcR was observed as early as 24 h after rIFN-gamma treatment, and receptor number was maximal by 36 h after rIFN-gamma treatment and represented approximately a fourfold increase. Scatchard analysis indicated that a twofold increase in GcR affinity was concomitant with the rIFN-gamma-induced increase in GcR number in RAW 264.7 cells. Increased GcR numbers were induced after exposure of RAW 264.7 cells to as little as 0.1 U/ml rIFN-gamma, and optimal expression was observed at 5 U/ml. Treatment of peritoneal exudate macrophages from C3H/OuJ mice and the LPS hyporesponsive mouse strain, C3H/HeJ, with rIFN-gamma induced an approximately twofold increase in the GcR with no concomitant change in receptor affinity. These results suggest that IFN-gamma may be essential not only for macrophage activation, but also for increasing macrophage sensitivity to feedback inhibition by glucocorticoids by increasing the number and/or affinity of available GcR.

Animals↗

PubMed1992

Lipopolysaccharide increases glucocorticoid receptor expression in murine macrophages. A possible mechanism for glucocorticoid-mediated suppression of endotoxicity.

In a previous study we demonstrated that IFN-gamma induced an increase in the number of glucocorticoid receptors (GR) in murine macrophages. To examine further the environmental signals involved in regulation of macrophage GR availability, we asked whether another classical macrophage-activating factor, LPS, would induce an increase in GR number in the macrophage cell line, RAW 264.7, and in primary macrophages from C3H mice. We report that treatment of RAW 264.7 cells and peritoneal exudate macrophages from C3H/OuJ mice with protein-free, phenol water-extracted LPS (PW-LPS) induced an increase in the number of GR. A significant increase in GR number was observed as early as 4 h after PW-LPS treatment, was maximal at 12 h, and remained heightened through 48 h. Optimal induction of the GR by PW-LPS was observed when murine macrophages were treated with 10 ng/ml of PW-LPS. The LPS-induced increase in macrophage GR number could be inhibited by polymyxin B. Macrophages obtained from the LPS hyporesponsive C3H/HeJ strain did not respond to PW-LPS, but did respond to protein-rich, butanol-extracted LPS with a modest increase in GR number after treatment with 2 micrograms/ml. Moreover, taxol, an antineoplastic agent with LPS mimetic activity, also increased GR number in murine macrophages. These results suggest that LPS is not only an important macrophage-activating signal, but may also be important in sensitizing the cell for negative regulatory events such as feedback inhibition by glucocorticoids.

Animals↗

PubMed1992

TNF-alpha differentially regulates Ia antigen expression and macrophage tumoricidal activity in two murine macrophage cell lines.

We have assessed tumor necrosis factor-alpha (TNF-alpha) production and its autocrine effects on activation in two murine macrophage cell lines which have distinct responses to the activation stimuli interferon-gamma (IFN-gamma) and bacterial lipopolysaccharide (LPS), and compared these responses to those observed in thioglycollate-elicited peritoneal macrophages. IFN-gamma induced TNF-alpha production in RAW 264.7 cells and this induction was regulated at the transcriptional level. IFN-gamma did not stimulate TNF-alpha production in either WEHI-3 cells or peritoneal macrophages, although MHC class II antigen expression was induced. LPS stimulated TNF-alpha production in the RAW 264.7 cell line and peritoneal macrophages; however, no TNF-alpha was detected in WEHI-3 cells activated with LPS. We also assessed the ability of endogenous TNF-alpha to serve as an autocrine regulator of two aspects of IFN-gamma-mediated macrophage activation, namely, induction of antibody-independent tumoricidal activity and induction of MHC class II antigen expression. These studies revealed that TNF-alpha could act synergistically or antagonistically with IFN-gamma in the regulation of these two functions, depending on both the macrophage population used and the function assessed. The results of our experiments suggest that the mechanism of induction of TNF-alpha production by IFN-gamma or LPS, and the ultimate autocrine contribution of such TNF-alpha to a given activation response, is dependent on the activated macrophage target population under analysis. The WEHI-3 and RAW 264.7 cell lines provide a model system for comparative exploration of the mechanistic basis of this differential regulation.

Animals↗

PubMed1992

A collection of mRNA species that are inducible in the RAW 264.7 mouse macrophage cell line by gamma interferon and other agents.

To identify genes induced during macrophage activation, a cDNA library was prepared from cultures of the RAW 264.7 mouse macrophage cell line that had been treated with conditioned medium from mitogen-stimulated spleen cells, and the cDNA library was screened by differential plaque hybridization. Eleven cDNA clones, designated CRG-1 through CRG-11, corresponding to mRNA species inducible in RAW 264.7 cells by the spleen cell conditioned medium, were isolated. Inductions were not blocked by cycloheximide. All of the mRNAs were inducible by gamma interferon, and some were also inducible by alpha and beta interferons, by lipopolysaccharide, by phorbol 12-myristate 13-acetate, and by the calcium ionophore A23187. Sequencing of the cDNAs revealed that CRG-1, CRG-3, and CRG-5 are cDNAs of recently identified transcription factors IRF-1, zif/268, and LRF-1 respectively. As previously reported, CRG-2 and CRG-10 (MIG) encode new members of the platelet factor 4 family of cytokines. CRG-6 corresponds to a new member of a family of interferon-inducible genes clustered on mouse chromosome 1, CRG-9 corresponds to a prostaglandin synthase homolog, CRG-8 corresponds to beta 2-microglobulin, and CRG-4 corresponds to metallothionein II. CRG-11 contains sequences of a truncated L1Md repetitive element as well as nonrepetitive sequences. The nonrepetitive sequence of CRG-11 as well as the sequences of CRG-7 are not closely related to published sequences. The CRG genes and proteins are of interest because of their involvement in macrophage activation, because of their roles as mediators of the effects of gamma interferon and other pleiotropic agents, and because of their usefulness as tools for studying the signal pathways through which gamma interferon and other inducers exert their effects on gene and protein expression.

Animals↗

PubMed2025

Elucidation of the immunotoxicity of PEDOT: PSS on RAW264.7 macrophages by oxidative stress, inflammatory response, and NF-κB pathway activation.

Poly(3,4-ethylenedioxythiophene): poly(styrenesulfonate) (PEDOT: PSS) nanoparticles, widely used conductive polymers, pose environmental and health risks due to their nanoscale dispersion. However, the characteristics of PEDOT: PSS in aquatic systems and the underlying mechanisms of its toxicity in animal and cell models remain poorly understood. This study aimed to investigate the toxicological effects of PEDOT: PSS nanoparticles on macrophages, with a focus on RAW 264.7 cells. After an acute exposure to PEDOT: PSS nanoparticles at different concentrations (5, 10, 20 μg/mL), we observed significant impairments in cell viability, proliferation, migration, adhesion, and phagocytosis, as well as morphological alterations. Concurrently, there was a marked upregulation of inflammatory markers, including reactive oxygen species (ROS), tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), and interleukin-1 beta (IL-1β), indicating the induction of oxidative stress and inflammation. Mechanistically, PEDOT: PSS nanoparticles activated the nuclear factor kappa B (NF-κB) signaling pathway, a key regulator of inflammatory responses, suggesting that they may mediate inflammatory responses and cell damage via activation of the NF-κB signaling pathway. These findings reveal the toxic mechanism of PEDOT: PSS nanoparticles in macrophages and provide new insights into their biological safety implications.

Animals↗

PubMed1992

Killing of Legionella pneumophila by nitric oxide in gamma-interferon-activated macrophages.

The role of nitric oxide (NO) radicals in killing the intracellular bacterial pathogen Legionella pneumophila (Lp) was examined in infected macrophages. Murine (RAW 264.7) and human (HL-60) cell monolayers were treated with 100 U/ml gamma-interferon (IFN) and cocultured with Lp in the presence and absence of NGMMA, a specific inhibitor of NO production. Viable Lp in IFN-treated RAW 264.7 cells decreased from 3.8 to 0.7 +/- 0.12 log CFU/ml after 24 h incubation, whereas in IFN+NGMMA-treated RAW 264.7 cells, viable Lp persisted at 2.2 +/- 0.2 log CFU/ml after 24 h. This increased survival corresponded with an inhibition of NO production (5.65 +/- 2.99 microM with NGMMA vs. 58.6 +/- 5.36 microM without NGMMA). Viable Lp were susceptible to killing, in a dose-dependent fashion, by 0, 2.5, and 5.0 mM sodium nitroprusside, a source of NO radicals. IFN-treated RAW 264.7 cells also had significantly decreased levels of intracellular iron (below assay limit) when compared to IFN+NGMMA-treated cells (72.0 +/- 0.78% of control). Normally permissive HL-60 cells treated with IFN were bacteriostatic rather than bactericidal, and NO production was not detected above background. Thus, NO radicals play a critical role in the bactericidal activity against Lp by IFN-treated RAW 264.7 cells, but the absence of NO production limits IFN-treated HL-60 cells to bacteriostasis.

Animals↗

PubMed2022

Multiplexed microfluidic chip for cell co-culture.

Paracrine signaling is challenging to study in vitro, as conventional culture tools dilute soluble factors and offer little to no spatiotemporal control over signaling. Microfluidic chips offer potential to address both of these issues. However, few solutions offer both control over onset and duration of cell-cell communication, and high throughput. We have developed a microfluidic chip designed to culture cells in adjacent chambers, separated by valves to selectively allow or prevent exchange of paracrine signals. The chip features 16 fluidic inputs and 128 individually-addressable chambers arranged in 32 sets of 4 chambers. Media can be continuously perfused or delivered by diffusion, which we model under different culture conditions to ensure normal cell viability. Immunocytochemistry assays can be performed in the chip, which we modeled and fine-tuned to reduce total assay time to 1 h. Finally, we validate the use of the chip for co-culture studies by showing that HEK293Ta cells respond to signals secreted by RAW 264.7 immune cells in adjacent chambers, only when the valve between the chambers is opened.

Microfluidics↗

PubMed2026

Fasudil induces anti-inflammatory transcriptomic changes and increased proliferation in human trisomy 21 neural progenitor cells.

Down syndrome (DS) results from trisomy for human chromosome 21 and is the most frequent genetic cause of intellectual disability. No effective treatments currently exist that improve neurodevelopment and cognition. Atypical brain development in individuals with DS is apparent before birth, which suggests that the optimal time to begin administration of therapies is prenatally. Human neural progenitor cell (NPC) cultures provide a tractable in vitro model system to examine the effects of trisomy 21 (T21) on neurodevelopment and to measure the effects of pharmacological interventions. Here, we report the results of preclinical studies evaluating 24 candidate therapies. RNA sequencing analyses found that euploid and T21 NPCs showed different transcriptomic responses to five candidate pharmacotherapies. The Rho-associated coiled-coil kinase inhibitor fasudil increased proliferation of T21 NPCs, reduced expression of inflammatory pathway genes in T21 NPCs, and reduced markers of inflammation in LPS-stimulated microglial model systems. These results demonstrate that fasudil can alter multiple T21-associated abnormalities in a beneficial manner, suggesting that fasudil warrants further study as a candidate prenatal pharmacotherapy for DS.

Down Syndrome↗

PubMed1992

Poly(A) removal is the kinase-regulated step in tumor necrosis factor mRNA decay.

Tumor necrosis factor (TNF) is a pleiotropic biomodulator and an important inducer of certain pathophysiologic immune reactions such as granuloma formation, cachexia, and septic shock. The production of TNF by astrocytes, which may figure prominently in the development of immune responses within the central nervous system, is subject to post-transcriptional regulation. We have previously shown that in virus-stimulated astrocytes, inhibition of protein kinase C results in a specific, 10-fold decrease in TNF mRNA half-life. Here we show that the decay of TNF messages induced in the macrophage-like cell line RAW 264.7 by either virus or lipopolysaccharide was subject to similar regulation, and that this pathway influenced the amount of TNF protein released by stimulated cells. Using a modified RNase protection assay, we demonstrate that inhibition of protein kinase C significantly enhanced the rate of poly(A) removal from TNF mRNA, thus facilitating an early event in the process of mRNA degradation.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed1992

Endotoxin induces the expression of macrophage inflammatory protein 1 alpha mRNA by rat alveolar and bone marrow-derived macrophages.

Macrophage inflammatory protein 1 alpha (MIP-1 alpha) is a newly described cytokine that is present in large amounts in the culture supernatant of an endotoxin-stimulated murine macrophage-like cell line (RAW 264.7). There is increasing information that suggests that this cytokine mediates acute neutrophilic inflammation, although the mechanism of mediation is unknown. Data examining the production and regulation of MIP-1 alpha by primary rat macrophages are lacking, and MIP-1 alpha has not been studied previously in an animal model of endotoxin-induced neutrophilic alveolitis. In this study, we performed Northern analysis of steady-state rat MIP-1 alpha mRNA using an oligonucleotide probe complementary to amino acids 4-13 of murine MIP-1 alpha. Our data demonstrate that rat alveolar and bone marrow-derived macrophages can be induced by in vitro endotoxin treatment to express a 1.1-kb MIP-1 alpha mRNA. Expression of the mRNA could be elicited by treatment with 0.1 to 10.0 micrograms/ml of endotoxin in vitro with peak steady-state levels detectable up to 9 h after adding endotoxin to the media. Alveolar macrophages recovered by whole lung lavage from endotoxin-treated rats expressed increased amounts of the mRNA homologous to MIP-1 alpha mRNA when treated in vitro with endotoxin. We also found that rat neutrophils could be induced by endotoxin in vitro to express the MIP-1 alpha mRNA. We were able to identify MIP-1 alpha in culture supernatant from endotoxin-stimulated rat alveolar and bone marrow-derived macrophages by immunoprecipitation with a specific goat anti-murine MIP-1 alpha.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed2025

Panduratin A Induces Autophagy Through AMPK Activation Independent of mTOR Inhibition and Restricts Mycobacterium tuberculosis in Host Macrophages.

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), remains a major global health burden, especially with the increasing prevalence of drug-resistant strains. There is an urgent need for new therapeutics that act via alternative mechanisms. Autophagy, a vital cell-autonomous defense process, allows macrophages to degrade intracellular pathogens such as Mtb and has gained attention as a potential target for host-directed therapy. In this study, we conducted a high-content imaging screen of herb-derived compounds to identify autophagy inducers in RAW264.7 macrophages. Panduratin A (NPA), a natural compound from Boesenbergia rotunda, was found to potently induce autophagy. NPA promoted autophagic vacuole formation in a dose-dependent fashion at low micromolar levels. Its autophagy-inducing effect was validated using RFP-GFP-LC3 dual fluorescence assays and immunoblotting in the presence of bafilomycin A1. Further mechanistic analysis revealed that NPA activates autophagy through AMPK activation, independent of mTOR inhibition. Importantly, NPA significantly promoted intracellular Mtb clearance and increased colocalization of Mtb with autophagosomes and lysosomes, in a manner dependent on Beclin-1. These findings highlight NPA as a potent enhancer of macrophage antimicrobial responses via autophagy, supporting its potential as a candidate for host-directed adjunctive therapy against TB.

Autophagy↗

PubMed2026

Proteomic and phosphoproteomic profiles of time-dependent dynamic changes in LPS-induced macrophage polarization.

The temporal proteomic and phosphoproteomic reprogramming during early M1 macrophage polarization (0-6 h) remains poorly understood. We performed time-resolved proteomic and phosphoproteomic analyses of LPS-stimulated RAW264.7 macrophages at seven time points within 6 h. Time-clustering of differentially expressed molecules revealed two patterns: initial change with partial recovery, and sustained dysregulation. Upregulated proteins and phosphorylation sites were enriched in the Rho GTPase signaling pathway, T-cell receptor signaling pathway, NF-κB cascade, osteoclast differentiation pathway, and antiviral immune pathway. Downregulated pathways were associated with cell cycle regulation, chromatin remodeling, RNA metabolism, and mRNA processing, indicating resource reallocation to prioritize acute inflammatory responses. Kinase-substrate network analysis confirmed the mitogen-activated protein kinase (MAPK), cyclin-dependent kinase (CDK), protein kinase B (AKT), and ribosomal S6 kinase (RSK) families as core upstream phosphorylation regulators. Integrated analysis revealed synergistic and antagonistic relationships between proteomic and phosphoproteomic changes. This study provides a temporal molecular atlas of M1 polarization, delineating inflammatory signaling dynamics and offering a basis for therapeutic target discovery in inflammatory diseases. SIGNIFICANCE: Macrophage M1 polarization is a central event in innate immune defense against pathogenic invasion, yet its dysregulation is a pivotal driver of the onset and progression of a broad spectrum of inflammation-associated disorders, spanning autoimmune diseases, infectious conditions and inflammatory bone diseases, making the dissection of its molecular regulatory mechanisms an urgent research priority in immunology and translational medicine. Dynamic molecular events within 0-6 h after LPS stimulation are critical for initiating and shaping M1 inflammatory activation, yet systematic time-resolved proteomic and phosphoproteomic profiling remains insufficient.In this study, we comprehensively characterized temporal proteome and phosphoproteome changes at seven consecutive time points during macrophage polarization, clarified two distinct dynamic molecular patterns, identified core signaling pathways and key kinase regulators involved in inflammatory reprogramming, and uncovered the leading role of post-translational phosphorylation modifications in initiating polarization. This work delineates the time-series molecular atlas of early macrophage activation, provides novel insights into the temporal regulatory mechanism of inflammatory signaling networks, and lays a solid experimental foundation for exploring new intervention targets and regulatory nodes in clinical translational research.

Lipopolysaccharides↗

PubMed2026

11-O-galloylbergenin alleviates LPS-stimulated inflammation in RAW 264.7 macrophages by targeting Grb2, RhoA, and Cdc42 in the RAS signaling pathway.

OBJECTIVE: This study aimed to explore the anti-inflammatory mechanism of 11-O-galloylbergenin in macrophages. METHODS: Lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages were treated with 11-O-galloylbergenin. Cytotoxicity was assessed by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, and cytokine secretion was measured by enzyme-linked immunosorbent (ELISA) assay. Data-independent acquisition (DIA)-based proteomics, Gene Ontology (GO)/Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, western blotting, and molecular docking were also performed. RESULTS: 11-O-Galloylbergenin (≤50 μg/mL) was non-cytotoxic and significantly suppressed tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) secretion. Proteomics analysis identified 209 differentially expressed proteins (DEPs) that showed opposite expression trends before and after 11-O-galloylbergenin treatment. Seven of these DEPs were enriched in the RAS pathway: RhoA, Cdc42, Grb2, RalB, Calm3, Gnb2, and Pla2g4a. Western blotting confirmed that 11-O-galloylbergenin downregulated RhoA, Cdc42, and Grb2 expression. Molecular docking revealed good binding affinity of 11-O-galloylbergenin to RhoA, Cdc42, and Grb2. CONCLUSION: 11-O-Galloylbergenin alleviates LPS-stimulated inflammation in RAW 264.7 macrophages by inhibiting the RAS signaling pathway.

Animals↗

PubMed2026

ATF4-histone 2-hydroxyisobutyrylation feedback loop drives sepsis-induced inflammation.

BACKGROUND AND PURPOSE: The role and mechanisms of lysine 2-hydroxyisobutyrylation (Khib) in the acute inflammatory phase of sepsis remain unclear. We investigated the function and underlying mechanisms of histone H4 lysine 5 2-hydroxyisobutyrylation (H4K5-hib) in sepsis-induced inflammation in vivo and in vitro. EXPERIMENTAL APPROACH: Acute sepsis was induced by caecal ligation and puncture (CLP) in mice, and inflammatory responses were modelled in lipopolysaccharide (LPS)-stimulated macrophages. CUT&Tag-seq was used to identify genomic targets associated with H4K5-hib and activating transcription factor 4 (ATF4). Immunofluorescence, Western blotting, qPCR, dual-luciferase assays, and ELISA were performed to investigate the underlying mechanisms. KEY RESULTS: H4K5-hib levels were increased in macrophages during the acute inflammatory phase of sepsis. LPS stimulation enhanced H4K5-hib enrichment at the ATF4 promoter, thereby promoting ATF4 transcription. Inhibition of EP300-mediated 2-hydroxyisobutyrylation or mutation of H4K5 abolished ATF4 activation. Increased H4K5-hib activated the ATF4/NLRP3 signalling axis, promoting inflammasome assembly and amplifying inflammatory responses. ATF4 directly bound to the EP300 promoter and enhanced its transcription, forming a positive feedback loop that further increased H4K5-hib levels. In CLP-induced sepsis, pharmacological inhibition of EP300 or ATF4 reduced H4K5-hib levels and suppressed NLRP3 inflammasome activation. CONCLUSION AND IMPLICATIONS: These findings reveal a previously unrecognized epigenetic mechanism underlying sepsis-induced inflammation and identify the EP300/ATF4/H4K5-hib positive feedback loop as a potential therapeutic target for sepsis.

Animals↗

PubMed1992

Human IL-1 receptor antagonist promoter. Cell type-specific activity and identification of regulatory regions.

To study the molecular mechanisms involved in transcriptional regulation of the human IL-1R antagonist (IL-1ra) we have isolated 1680-bp of 5'-flanking region DNA from the IL-1ra gene. This region of DNA was sequenced and cloned into the luciferase expression vector pA3Luc (pRA-1680.Luc) for use in gene transfer studies aimed at determining the cis-acting DNA elements required for IL-1ra expression. Sequence analysis of the IL-1ra promoter revealed a TATAA box at -26, with consensus sequences for possible NF-kB-, NFIL-1 beta A-, AP-1-, and CRE-binding sites located further upstream. When transfected into a variety of human and murine cell lines, the cloned IL-1ra promoter was preferentially active in those cell lines in which expression of the endogenous IL-1ra gene could be detected. The cloned promoter and the endogenous IL-1ra promoter utilized the same transcriptional start site. This promoter activity was LPS-inducible in the RAW 264.7 murine macrophage cell line. In the human monocytic cell line U937, IL-1ra promoter activity was inducible by LPS or PMA treatment, but the combination of LPS and PMA led to the greatest increase in promoter activity, identical to the pattern of expression of endogenous IL-1ra mRNA as detected by polymerase chain reaction analysis. A series of 5'-truncated promoter constructs having a common 3'-end at +27 were created to map potential cis-acting transcriptional elements important for full IL-1ra promoter activity. Removal of sequences between -294 and -148 led to a greater than 90% decrease in both unstimulated and LPS-induced promoter activity; further deletion to -85 led to an almost complete abrogation of promoter activity. These studies demonstrate that the cloned IL-1ra promoter behaves in a manner consistent with that of the endogenous gene. Two regions within the IL-1ra promoter are identified which are required for full promoter activity.

Animals↗

PubMed1992

The role of protein kinase C in the induction of nitric oxide synthesis by murine macrophages.

The role of protein kinase C (PKC) in the induction of nitric oxide synthesis by interferon-gamma (IFN-gamma) was investigated using two murine macrophage cell lines, J774 and RAW 264.7. Nitric oxide (NO) production was markedly reduced by a PKC inhibitor, Ro31-8220 in a dose-dependent manner. Incubation of cells with IFN-gamma resulted in translocation of PKC to the cell membrane. Prolonged incubation of cells with a high concentration of phorbol ester, which down-regulated PKC activity, also reduced nitric oxide production. These findings provide evidence that PKC is involved in the induction of nitric oxide synthesis by IFN-gamma.

Animals↗

PubMed2026

Transcriptomic Profiling Reveals NF-κB-Associated Immune Regulatory Signatures Underlying the Regenerative Effects of Hypoxia-Preconditioned Tendon Stem Cell-Derived Extracellular Vesicles.

Remodeling of the immune microenvironment is a critical determinant of tissue regeneration, yet the molecular programs associated with the enhanced therapeutic activity of hypoxia-preconditioned extracellular vesicles remain incompletely defined. In this study, we investigated the regenerative and immunomodulatory effects of hypoxia-preconditioned tendon stem cell-derived extracellular vesicles (Hypo-EVs) and employed transcriptomic profiling to identify molecular signatures associated with their biological activity. The therapeutic effects of Hypo-EVs were evaluated using a rat patellar tendon defect model and lipopolysaccharide-stimulated RAW 264.7 macrophages. Histological analysis, immunostaining, biomechanical testing, and reverse transcription-quantitative polymerase chain reaction were performed to assess tendon healing and macrophage polarization, while RNA sequencing was conducted in macrophages treated with Hypo-EVs or normoxia-derived EVs, followed by Gene Set Enrichment Analysis, Gene Ontology, and Kyoto Encyclopaedia of Genes and Genomes pathway analyses. Hypo-EVs significantly alleviated local inflammatory responses, improved collagen organization and biomechanical properties of repaired tendons, and promoted macrophage polarization toward a reparative M2 phenotype both in vivo and in vitro. Consistent with these biological effects, transcriptomic profiling revealed extensive remodeling of inflammation-related gene expression programs, including significant suppression of NF-κB, TNF, IL-17, and cytokine-cytokine receptor interaction pathways. Integrative bioinformatic analyses identified an NF-κB-associated immune-regulatory signature that distinguished Hypo-EV-treated macrophages from those receiving normoxic EVs. Mechanistically, Hypo-EVs attenuated NF-κB activation, as evidenced by reduced phosphorylation of p65 and IκBα, whereas TNF-α-mediated NF-κB activation partially diminished their macrophage-repolarizing effects. Collectively, these findings demonstrate that hypoxic preconditioning enhances the immunomodulatory and regenerative functions of tendon stem cell-derived EVs. Transcriptomic analyses identified an NF-κB-associated immune-regulatory signature linked to the biological activity of Hypo-EVs, providing a molecular framework for understanding EV-mediated immune modulation and supporting the development of transcriptome-guided molecular signatures for regenerative therapies targeting tendon immune homeostasis.

Animals↗

PubMed2026

Proteomic insights into the immunomodulatory effects of Ca/Sr co-doped sol-gel coatings for titanium implants.

Ionic functionalization of biomaterial coatings has emerged as a powerful strategy to regulate early host responses at the implant interface. However, how combined Ca/Sr incorporation governs the adsorbed proteome and downstream immune signaling remains poorly understood. This study analyses, employing in vitro tests and proteomics, the effect of adding Sr and Ca to Si-based coatings designed to bioactivate Ti implants. Hybrid Si-based coatings were synthesized by the sol-gel route with a fixed Ca content (0.5 wt%) and increasing Sr contents (0.5, 1.0, 1.5 wt%), and their physicochemical properties, ion release kinetics, and hydrolytic stability were characterized. The coatings remained highly crosslinked despite Ca/Sr incorporation, whereas the highest Sr content increased hydrolytic degradation to around 70% after 56 days. Proteomic analysis identified 183 adsorbed proteins, of which 56 were differentially adsorbed on Ca/Sr-coatings, mainly associated with immune and coagulation pathways. In vitro, RAW 264.7 showed increased gene expression of TNF-α and TGF-β; with an enhanced TNF-α secretion by the addition of Ca and Sr. In parallel, MC3T3-E1 indicated that Ca/Sr-coatings were not cytotoxic and did not impair cell proliferation. However, ALP activity was reduced in the co-doped groups, indicating that the immunomodulatory effects induced by Ca/Sr incorporation were not accompanied by enhanced early osteogenic differentiation. The Ca/Sr combination induced alterations in the adsorption of immune-related proteins, which correlated with the in vitro findings. The deeper insight into how Ca/Sr mixtures modulate protein adsorption on biomaterial surfaces may be key to understanding the immunomodulatory capacity of these bioactive cations.

Animals↗