Inflammatory Factors In Macrophages Research Articles

Baricitinib inhibits the activation of innate immune cells and exerts therapeutic effects on acute peritonitis and systemic inflammatory response syndrome

Timely treatment of acute inflammatory diseases induced by bacteria or fungi is essential to prevent infectious damage. Baricitinib is an inhibitor of Janus kinases (JAKs) which was approved to treat rheumatoid arthritis, atoptic dermatitis, and alopecia areata. It is also known that JAKs play important roles in innate immunity and inflammatory response. In this study, we investigated the regulatory effects of baricitinib on the activation of macrophages and neutrophils and its therapeutic effects on acute peritonitis and systemic inflammatory response syndrome (SIRS). In addition, we also studied its anti-inflammatory mechanisms by transcriptome and immunoblotting analyses. The results showed that baricitinib inhibited the expression and secretion of multiple inflammatory factors in macrophages induced by multiple Toll-like receptor (TLR) agonists. Baricitinib also moderately suppressed superoxide release of neutrophils stimulated by TLR agonists. In zymosan-induced acute peritonitis, baricitinib significantly reduced the infiltration of neutrophils into peritoneal cavity and the production of inflammatory factors in peritoneal cavity. In addition, baricitinib also slightly decreased the production of inflammatory factors in SIRS. Transcriptome analysis revealed that baricitinib markedly inhibited the mRNA transcription of many interferon-inducible genes, antiviral immune-related genes, transcription factors in JAKs-STATs signaling pathways, inflammatory factors, chemokines, colony-stimulating factor, and immunoglobulin receptors in macrophages induced by lipopolysaccharide (LPS). Furthermore, immunoblotting analysis showed that baricitinib selectively inhibited the phosphorylation of STAT1 and STAT3 in macrophages stimulated by LPS, and almost completely blocked the phosphorylation of STAT1 and STAT3 induced by IFN-γ and IL-6. Collectively, baricitinib can moderately inhibit the activation of macrophages and superoxide production of neutrophils, and exert anti-inflammatory effects by blocking JAKs-STATs signaling pathways, and has potential to be developed into therapeutic drug for acute inflammatory diseases, especially local ones.

MOF-818 Nanozyme Suppresses Calcium Oxalate Kidney Stones by Alleviating Oxidative Stress and Inflammatory Injury.

There remains a lack of effective drugs to alleviate the kidney stones caused by oxidative stress and inflammatory damage. The MOF-818 nanozyme is utilized to lessen the generation of reactive oxygen species (ROS) effectively, restore the membrane potential of mitochondria, regulate the cell cycle, decrease cell death, hinder the recruitment of macrophages, and mitigate the release of inflammatory factors in macrophages. These effects are attributed to the nanozyme's ability to mimic the enzyme properties of catalase (CAT) and superoxide dismutase (SOD). It is demonstrated that this nanozyme can reduce kidney calcium oxalate crystal deposition by reducing the renal injury caused by high concentration oxalate, upregulate the expression levels of SOD and CAT in tissues, downregulate adhesion proteins and inflammatory factor IL-6 and TNF-α, and promote the polarization of macrophages from M1 to M2 phenotype in the rat model induced by ethylene glycol. Overall, MOF-818 has the potential to effectively suppress oxidative stress and inflammatory harm caused by high levels of oxalate, hence lowering the likelihood of stone formation. MOF-818 nanozyme is also expected to be used as an alternative drug for the treatment of calcium oxalate kidney stones and provide an experimental theoretical basis for the development of new nanomedicines.

Paeoniflorin mitigates MMP-12 inflammation in silicosis via Yang-Yin-Qing-Fei Decoction in murine models

BackgroundSilicosis presents a significant clinical challenges and economic burdens, with Traditional Chinese Medicine (TCM) emerging as a potential therapeutic avenue. However, the precise effects and mechanisms of TCM in treating silicosis remain uncertain and subject to debate. ObjectiveThe study aims to elucidate the therapeutic role and mechanisms of the Yang-Yin-Qing-Fei Decoction (YYQFD) and its key component, paeoniflorin, in silicosis using a murine model. MethodsSilicotic mice were treated with YYQFD, pirfenidone (PFD), or paeoniflorin. RAW264.7 cells and mouse lung fibroblasts (MLF) were stimulated with silica, matrix metalloproteinase-12 (MMP-12), or TGF-β1, followed by treatment with paeoniflorin, PFD, or relevant inhibitors. YYQFD constituents were characterized using High-Performance Liquid Chromatography (HPLC). Lung fibrosis severity was assessed via histopathological examination, micro-CT imaging, lung functions, and Western blot analysis. Transcriptome sequencing and bioinformatics analysis were employed to delineate the gene expression profile and target genes modulated by YYQFD in silicosis. ResultsTreatment with YYQFD ameliorated silica-induced lung fibrosis. Transcriptome sequencing identified MMP-12 as a potential common target of YYQFD and PFD. Additionally, a potential pro-inflammatory role of MMP-12, regulated by silica-induced TLR4 signaling pathways, was revealed. Paeoniflorin, one of the most distinctive compounds in YYQFD, attenuated silica-induced MMP-12 increase and its derived inflammatory factors in macrophages through a direct binding effect. Notably, paeoniflorin treatment exerted anti-fibrotic effects by inhibiting MMP-12-derived inflammatory factors and TGF-β1-induced myofibroblast differentiation in silica-exposed mice. ConclusionsThis study underscores paeoniflorin as one of the most principal bioactive compounds in YYQFD, highlighting its capacity to attenuate lung inflammation driven by macrophage-derived MMP-12 and reduce lung fibrosis both in vivo and in vitro.

Effects of folate-chicory acid liposome on macrophage polarization and TLR4/NF-κB signaling pathway in ulcerative colitis mouse

BackgroundChichoric acid (CA) is a major active ingredient found in chicory and Echinacea. As a derivative of caffeic acid, it has various pharmacological effects. PurposeDue to the unclear etiology and disease mechanisms, effective treatment methods for ulcerative colitis (UC) are currently lacking. The study investigated the therapeutic effects of the folate-chicory acid liposome on both LPS-induced macrophage inflammation models and dextran sulfate sodium (DSS)-induced mouse UC models. MethodsFolate-chicory acid liposome was prepared using the double emulsion ultrasonic method with the aim of targeting folate receptors specifically expressed on macrophages. The study investigated the therapeutic effects of the folate-chicory acid liposome on both LPS-induced macrophage inflammation models and DSS -induced mouse UC models. Furthermore, the effects of the liposomes on macrophage polarization and their underlying mechanisms in UC were explored. ResultsThe average particle size of folate-chicory acid liposome was 120.4 ± 0.46 nm, with an encapsulation efficiency of 77.32 ± 3.19 %. The folate-chicory acid liposome could alleviate macrophage apoptosis induced by LPS, decrease the expression of inflammatory factors in macrophages, enhance the expression of anti-inflammatory factors, inhibit macrophage polarization towards the M1 phenotype, and mitigate cellular inflammation in vetro. In vivo test, folate-chicory acid liposome could attenuate clinical symptoms, increased colon length, reduced DAI scores, CMDI scores, and alleviated the severity of colonic histopathological damage in UC mice. Furthermore, it inhibited the polarization of macrophages towards the M1 phenotype in the colon and downregulated the TLR4/NF-κB signaling pathway, thereby ameliorating UC in mice. ConclusionFolate-chicory acid liposome exhibited a uniform particle size distribution and high encapsulation efficiency. It effectively treated UC mice by inhibiting the polarization of macrophages towards the M1 phenotype in the colon and downregulating the TLR4/NF-κB signaling pathway.

Oxidative Stress, inflammation and metallomatrix Proteinase-Regulated hydrogels for intervertebral disc regeneration

Intervertebral disc degeneration (IVDD) is still a challenge and the efficient treatment approaches are still lacking, due to the high oxidative stress and inflammation and matrix degradation. The utilization of bioactive hydrogels as carriers for therapeutic factors offers a powerful means to enhance IVDD treatment. However, the therapeutic efficacy of existing hydrogels heavily relies on the loaded therapeutic drugs, resulting in reduced treatment efficiency. Herein, we report a dual-component polymer network hydrogel (FPFG) based on gelatin methacrylate (GelMA) and poly (ferulic acid)-F127- poly (ferulic acid) (FPF). The FPFG hydrogel exhibited injectability, antioxidative properties, good biocompatibility, and biodegradability. FPFG hydrogel could efficiently suppress the intracellular reactive oxygen species (ROS) and metallomatrix proteinase in nucleus pulposus (NP) cells, inhibit the expression of inflammatory factors in macrophages and enhance the extracellular matrix formation. The rat puncture model revealed that the FPFG hydrogel effectively restored disc height, maintained disc hydration, and preserved tissue structure. By exerting anti-inflammatory and antioxidant activities, the FPFG hydrogel successfully ameliorated the hostile microenvironment commonly found in degenerated disc tissue. These findings highlight the potential application of FPFG hydrogel in enhancing IVDD treatment.

VX-702 Ameliorates the Severity of Sepsis-Associated Acute Kidney Injury by Downregulating Inflammatory Factors in Macrophages.

Sepsis-associated acute kidney injury (S-AKI) contributes to high mortality, but it is lack of specific treatments. We aimed to investigate the underlying mechanism of S-AKI and to identify target drugs to alleviate AKI. We establish a stable mouse model of S-AKI by Pseudomonas aeruginosa incision infection. Based on high-throughput sequencing and bioinformatics analysis, we investigated the underlying mechanism and selected the target drug (VX-702) for S-AKI. An in vitro model established by co-cultured of kidney tubular epithelial cell line (TCMK-1) cells with lipopolysaccharide (LPS)-induced leukemic monocyte/macrophage cells (RAW264.7), we explored the effect of VX-702 on S-AKI. The data showed interleukin (IL)-6 and IL-1β were the hub genes, and the mitogen-activated protein kinase (MAPK) signaling pathway was the main pathway involved in S-AKI. Administration of VX-702 by oral gavage decreased the elevated concentrations of IL-6, IL-1β, serum creatinine, and blood urea nitrogen in mice with S-AKI. Moreover, VX-702 reduced the number of apoptotic cells in damaged kidney tissues. Cell viability was decreased, and the number of apoptotic cells was increased in TCMK-1 cells co-cultured with LPS-induced RAW264.7 cells compared to LPS-induced TCMK-1 cells. VX-702 treatment reversed this effect. VX-702 treatment reduced the levels of phosphorylated p38 MAPK and proinflammatory cytokines in RAW264.7 cells and the supernatant. VX-702 could bind IL-6, IL-1β and MAPK, and affect the binding of IL-1β and its receptor, as demonstrated by molecular docking. VX-702 ameliorated S-AKI by inhibiting the release of proinflammatory cytokines from macrophages, indicating its potential as a novel therapeutic for S-AKI treatment.

Effect of Pp2cm Gene Silencing on Mouse Macrophage Resistance Against Staphylococcus aureus Infection via TLR Pathway

To investigate the effect of silencing protein phosphatase 2cm ( Pp2cm) gene on the expression of inflammatory factors in macrophages infected with Staphylococcus aureus ( S. aureus) and the mechanisms involved. The effects of Pp2cm knockdown on inflammatory factors, proliferation, apoptosis, and Toll-like receptor (TLR) signaling were analyzed in RAW 264.7 cells, a murine macrophage cell line, transfected with adenovirus (Ad). The cells were divided into four groups, including Ad-Ctrl group, Ad- Pp2cm group, Ad-Ctrl+ S. aureus group and Ad- Pp2cm+ S. aureus group. Cell transfection was achieved by separately introducing control adenovirus (Ad-Ctrl) or adenovirus targeting the Pp2cm gene (Ad- Pp2cm) and inflammation or the absence of inflammation was induced by applying or not applying S. aureus. The expression of tumor necrosis factor-alpha ( TNF-α), interleukin-1β ( IL-1 β), TLR2, TLR4, Toll-like receptor adaptor protein ( Tirap) and myeloid differentiation factor 88 ( Myd88) was determined by real-time fluorescent quantitative polymerase chain reaction (RT-qPCR). PP2Cm protein expression was determined by Western blot. Cell proliferation was determined by cell counting kit-8 (CCK-8) assay and cell apoptosis was measured by flow cytometry. The expression of Pp2cmgene and PP2Cm protein was downregulated in the Ad- Pp2cm group when compared to the Ad-Ctrl group, with the diference showing statistical significance ( P<0.05). When compared to those of the Ad-Ctrl+ S. aureus group, macrophages in the Ad- Pp2cm+ S. aureus group showed significantly increase in the TNF- α and IL-1 β gene levels ( P<0.01). Furthermore, the Ad- Pp2cm group demonstrated elevated gene expression levels of TLR2, TLR4, Tirap and Myd88 in macrophages when compared to the Ad-Ctrl group, with the difference showing statistical significance ( P<0.05). There were no statistically significant differences in cell apoptosis and proliferation between the Ad-Ctrl and Ad- Pp2cm groups. Silencing Pp2cm gene promotes the inflammatory response of macrophages to S. aureus infection. Moreover, the TLR pathway plays an important role in the inflammatory activation of macrophages.

Protective effects of ginsenosides on macrophages subjected to simulated weightlessness

ObjectiveTo investigate the evolution of inflammation under conditions and the effects of ginsenosides on macrophages subjected to the simulated weightlessness, with the aim of mitigating the inflammation. MethodsInitially, genes related to weightlessness, inflammation, and immunity were identified in the GeneCards database. Then, Search Tool for the Retrieval of Interaction Gene/Proteins (STRING) protein network analysis was conducted to determine the core targets involved in the weightlessness-induced inflammation. Subsequently, Label-Free Quantitative (LFQ) proteomics was carried out to discern the distinctive genes within ginsenoside-treated Tohoku Hospital Pediatrics-1 (THP-1) cells. Next, utilizing the outcomes of Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses, the biological processes and signaling pathways in which ginsenosides predominately engaged were scrutinized, and the primary targets of ginsenosides in combating weightlessness-induced inflammation were examined. Finally, enzyme-linked immunosorbent assay (ELISA) was performed to detect the secretion levels of interleukin (IL)-1β, IL-6, IL-8, and tumor necrosis factor (TNF)-α from lipopolysaccharide (LPS)-induced THP-1 cells under simulated weightlessness conditions, as well as during the weightlessness recovery period following treatment with ginsenosides. ResultsA total of 2 933 genes associated with inflammation, 425 genes linked to weightlessness, and 4 564 genes connected to immunity were retrieved from the GeneCards database. Protein-protein interaction (PPI) networks were generated to identify pivotal targets associated with weightlessness-induced inflammation such as IL-1β, IL-6, TNF, and albumin (ALB). It was found that ginsenosides primarily participated in the regulation of various inflammation-related signaling pathways and pathways related to pathogenic microorganism infections. Moreover, it has a significant impact on the expression of proteins such as cluster of differentiation 40 (CD40), IL-1β, and poly ADP-ribose polymerase 1 (PARP1). As revealed in the simulated weightlessness cell test, ginsenosides exhibited a remarkable capacity to attenuate the secretion of inflammatory factors, specifically IL-6 and TNF-α (P < 0.000 1), in THP-1 macrophages following induction by LPS under simulated weightlessness conditions. In addition, it reduced the secretion of IL-1β, IL-6, IL-8, and TNF-α (P < 0.000 1) during the weightlessness recovery phase. ConclusionWeightlessness can disrupt several inflammation-related signaling pathways, but ginsenosides were shown to mitigate the release of various inflammatory factors in macrophages subjected to simulated weightlessness, thereby exerting a protective role against inflammation. This study has laid a theoretical groundwork for further exploring the potential application of ginsenosides in safeguarding against LPS induced inflammation in a weightlessness environment.

Role of PI3K/Akt/mTOR pathway-mediated macrophage autophagy in affecting the phenotype transformation of lung fibroblasts induced by silica dust exposure.

The phosphoinositide 3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/Akt/mTOR) pathway is one of the main signaling pathways related to autophagy. Autophagy plays a key role in the formation of silicosis fibrosis. The phenotypic transformation of lung fibroblasts into myofibroblasts is a hallmark of the transition from the inflammatory phase to the fibrotic phase in silicosis. This study aims to investigate whether the PI3K/Akt/mTOR pathway affects the phenotypic transformation of silicosis-induced lung fibroblasts into myofibroblasts via mediating macrophage autophagy. The human monocytic leukemia cell line THP-1 cells were differentiated into macrophages by treating with 100 ng/mL of phorbol ester for 24 h. Macrophages were exposed to different concentrations (0, 25, 50, 100, 200, 400 μg/mL) and different times (0, 6, 12, 24, 48 h) of SiO2 dust suspension. The survival rate of macrophages was measured by cell counting kit-8 (CCK-8) method. Enzyme linked immunosorbent assay (ELISA) was used to measure the contents of transforming growth factor-β1 (TGF-β1) and tumor necrosis factor-α (TNF-α) in the cell supernatant. The co-culture system of macrophages and HFL-1 cells was established by transwell. A blank control group, a SiO2 group, a LY294002 group, a SC79 group, a LY294002+SiO2 group, and a SC79+SiO2 group were set up in this experiment. Macrophages in the LY294002+SiO2 group were pretreated with LY294002 (PI3K inhibitor) for 18 hours, and macrophages in the SC79+SiO2 group were pretreated with SC79 (Akt activator) for 24 hours, and then exposed to SiO2 (100 μg/mL) dust suspension for 12 hours. The expression of microtubule-associated protein 1 light chain 3 (LC3) protein in macrophages was detected by the immunofluorescence method. The protein expressions of PI3K, Akt, mTOR, Beclin-1, LC3 in macrophages, and collagen III (Col III), α-smooth muscle actin (α-SMA), fibronectin (FN), matrix metalloproteinase-1 (MMP-1), tissue metalloproteinase inhibitor-1 (TIMP-1) in HFL-1 cells were measured by Western blotting. After the macrophages were exposed to SiO2 dust suspension of different concentrations for 12 h, the survival rates of macrophages were gradually decreased with the increase of SiO2 concentration. Compared with the 0 μg/mL group, the survival rates of macrophages in the 100, 200, and 400 μg/mL groups were significantly decreased, and the concentrations of TGF-β1 and TNF-α in the cell supernatant were obviously increased (all P<0.05). When 100 μg/mL SiO2 dust suspension was applied to macrophages, the survival rates of macrophages were decreased with the prolonged exposure time. Compared with the 0 h group, the survival rates of macrophages were significantly decreased (all P<0.05), the concentrations of TGF-β1 and TNF-α in the cell supernatant were significantly increased, and the protein expression levels of Beclin-1 and LC3II were increased markedly in the 6, 12, 24, and 48 h groups (all P<0.05). Immunofluorescence results demonstrated that after exposure to SiO2 (100 μg/mL) dust for 12 h, LC3 exhibited punctate aggregation and significantly higher fluorescence intensity compared to the blank control group (P<0.05). Compared with the blank control group, the protein expressions of Col III, FN, α-SMA, MMP-1, and TIMP-1 in HFL-1 cells were up-regulated in the SiO2 group (all P<0.05). Compared with the SiO2 group, the protein expressions of PI3K, Akt, and mTOR were down-regulated and the protein expressions of LC3II and Beclin-1 were up-regulated in macrophages (all P<0.05), the contents of TNF-α and TGF-β1 in the cell supernatant were decreased (both P<0.01), and the protein expressions of Col III, FN, α-SMA, MMP-1, and TIMP-1 in HFL-1 cells were down-regulated (all P<0.05) in the LY294002+SiO2 group. Compared with the SiO2 group, the protein expressions of PI3K, Akt, and mTOR were up-regulated and the protein expressions of LC3II and Beclin-1 were down-regulated in macrophages (all P<0.05), the contents of TNF-α and TGF-β1 in the cell supernatant were increased (both P<0.01), and the protein expressions of Col III, FN, α-SMA, MMP-1, and TIMP-1 in HFL-1 cells were up-regulated (all P<0.05) in the SC79+SiO2 group. Silica dust exposure inhibits the PI3K/Akt/mTOR pathway, increases autophagy and concentration of inflammatory factors in macrophages, and promotes the phenotype transformation of HFL-1 cells into myofibroblasts. The regulation of the PI3K/Akt/mTOR pathway can affect the autophagy induction and the concentration of inflammatory factors of macrophages by silica dust exposure, and then affect the phenotype transformation of HFL-1 cells into myofibroblasts induced by silica dust exposure.

Astaxanthin prevents osteoarthritis by blocking Rspo2-mediated Wnt/β-catenin signaling in chondrocytes and abolishing Rspo2-related inflammatory factors in macrophages.

Chondrocyte degeneration and classically activated macrophage (AM)-related inflammation play critical roles in osteoarthritis (OA). Here, we explored the effects of astaxanthin and Rspo2 on OA in vitro and in vivo. We observed that the Rspo2 gene was markedly elevated in synovial tissues of OA patients compared with healthy controls. In 2D cultures, Rspo2 and inflammatory factors were enhanced in AMs compared with nonactivated macrophages (NMs), and the protein expression levels of Rspo2, β-catenin, and inflammatory factors were increased, and anabolic markers were reduced in osteoarthritic chondrocytes (OACs) compared to normal chondrocytes (NCs). Astaxanthin reversed these changes in AMs and OACs. Furthermore, Rspo2 shRNA significantly abolished inflammatory factors and elevated anabolic markers in OACs. In NCs cocultured with AM, and in OACs cocultured with AMs or NMs, astaxanthin reversed these changes in these coculture systems and promoted secretion of Rspo2, β-catenin and inflammatory factors and suppressed anabolic markers compared to NCs or OACs cultured alone. In AMs, coculture with NCs resulted in a slight elevation of Rspo2 and AM-related genes, but not protein expression, compared to culture alone, but when cocultured with OACs, these inflammatory mediators were significantly enhanced at both the gene and protein levels. Astaxanthin reversed these changes in all the groups. In vivo, we observed a deterioration in cartilage quality after intra-articular injection of Rspo2 associated with medial meniscus (DMM)-induced instability in the OA group, and astaxanthin was protective in these groups. Our results collectively revealed that astaxanthin attenuated the process of OA by abolishing Rspo2 both in vitro and in vivo.

Hydrogen sulfide attenuates TMAO‑induced macrophage inflammation through increased SIRT1 sulfhydration.

Chronic inflammation is a key factor that accelerates the progression of inflammatory vascular disease. Hydrogen sulfide (H2S) has potent anti‑inflammatory effects; however, its underlying mechanism of action has not been fully elucidated. The present study aimed to investigate the potential effect of H2S on sirtuin 1 (SIRT1) sulfhydration in trimethylamine N‑oxide (TMAO)‑induced macrophage inflammation, and its underlying mechanism. Pro‑inflammatory M1 cytokines (MCP‑1, IL‑1β, and IL‑6) and anti‑inflammatory M2 cytokines (IL‑4 and IL‑10) were detected by RT‑qPCR. CSE, p65 NF‑κB, p‑p65 NF‑κB, IL‑1β, IL‑6 and TNF‑α levels were measured by Western blot. The results revealed that cystathionine γ‑lyase protein expression was negatively associated with TMAO‑induced inflammation. Sodium hydrosulfide (a donor of H2S) increased SIRT1 expression and inhibited the expression of inflammatory cytokines in TMAO‑stimulated macrophages. Furthermore, nicotinamide, a SIRT1 inhibitor, antagonized the protective effect of H2S, which contributed to P65 NF‑κB phosphorylation and upregulated the expression of inflammatory factors in macrophages. H2S ameliorated TMAO‑induced activation of the NF‑κB signaling pathway via SIRT1 sulfhydration. Moreover, the antagonistic effect of H2S on inflammatory activation was largely eliminated by the desulfhydration reagent dithiothreitol. These results indicated that H2S may prevent TMAO‑induced macrophage inflammation by reducing P65 NF‑κB phosphorylation via the upregulation and sulfhydration of SIRT1, suggesting that H2S may be used to treat inflammatory vascular diseases.

Ibrutinib suppresses the activation of neutrophils and macrophages and exerts therapeutic effect on acute peritonitis induced by zymosan.

Timely treatment of acute inflammatory reactions induced by fungi or bacteria is essential to prevent infectious damage. Ibrutinib is a Bruton's tyrosine kinase (BTK) inhibitor which is used to treat various lymphoid cancers. It is also known that BTK plays important roles in innate immunity and inflammatory response. In the present study, we investigated the regulatory effects of Ibrutinib on the activation of neutrophils and macrophages and its therapeutic effects on acute peritonitis. In addition, we also studied its anti-inflammatory mechanisms. The results showed that Ibrutinib inhibited the expression and secretion of inflammatory factors in macrophages induced by multiple Toll-like receptor (TLR) agonists. In the study of neutrophils, Ibrutinib selectively suppressed the activation, superoxide release, and calcium influx of neutrophils stimulated by zymosan. Furthermore, in zymosan-induced mice acute peritonitis, Ibrutinib significantly reduced the infiltration of neutrophils into peritoneal cavity, the release of myeloperoxidase (MPO) and β-glucuronidase as well as the production of inflammatory factors in peritoneal cavity. In mechanism study, Ibrutinib selectively inhibited the phosphorylation of PLCγ2, PKCδ, and ERK1/2 in neutrophils induced by zymosan. Collectively, Ibrutinib can significantly inhibit the activation of neutrophils and macrophages by inhibiting BTK-PLCγ2-PKC signaling pathway, and has great potential to be developed into therapeutic drug for acute inflammatory diseases.

PACAP Improves Macrophage M1 Polarization and Inflammatory Response by Regulating NF-κB and MAPKs Pathways

Inflammation is an important defense mechanism evolved by the host to defend against external threats. But an excessive inflammatory response can lead to a variety of inflammatory diseases. Macrophage M1 polarization is a key factor in inducing inflammatory response, and inhibiting macrophage M1 polarization is of great significance for regulating inflammatory diseases. PACAP is an anti-inflammatory neuropeptide, but its mechanism of action on macrophage polarization and inflammatory responses is unclear. The macrophage M1 polarization and inflammation model was constructed using lipopolysaccharide (LPS), and then treated with PACAP. The results showed that PACAP significantly inhibited the LPS-induced expression of M1 polarization marker genes and inflammatory factors in macrophages, and down-regulated the phosphorylation of IKK2/IκBα/NF-κB, while PACAP also inhibited the phosphorylation of JNK/p38. Thus， PACAP improved LPS-induced macrophage M1 polarization and inflammatory response by regulating NF-κB and MAPKs signaling pathways.

Zn-dipicolylamine-based reactive oxygen species-responsive lipids for siRNA delivery and in vivo colitis treatment

The reduction of reactive oxygen species (ROS) and inflammatory factor levels plays an important role in the treatment of colitis. A series of ROS-responsive lipids (ZnDPA-R) based on the thioketal structure were designed and synthesized. It was expected that the lipidic materials could combine ROS consumption and siRNA delivery capacity to achieve synergistic treatment of colitis. The target liposomes could combine with the phosphate backbone of siRNA to form lipoplexes of size ∼100 nm and could deliver siRNA cargo into the cell. The results of in vitro anti-inflammatory experiments showed that the lipids may effectively consume ROS in the cells. The lipoplexes significantly reduced the expression levels of TNF-α mRNA and related inflammatory factors in macrophages. After PEGylation, the lipoplex was used for treating mouse colitis, and the biodistribution results proved that the lipoplexes effectively aggregated in the intestine. The delivery system could not only respond to the high ROS level in colitis through breaking of thioketal structure, but it could also assist in treating inflammation by ROS consumption. The treatment results revealed that the levels of TNF-α mRNA and related inflammatory factors in the colon lesion were largely reduced, and the inflammatory symptoms were significantly relieved. Hematology test results indicated that the treatment was safe and induced no side effects in mice. The present study may shed light on the synergistic treatment of colitis through anti-inflammatory siRNA delivery and ROS depletion strategies. Statement of significanceDownregulation of inflammatory factors and reactive oxygen species (ROS) levels is critical in treating colitis. In the present study, a series of ROS-responsive lipid molecules based on the Zn-DPA headgroup and thioketal linkage were synthesized for delivering TNF-α siRNA and for treating colitis. In addition to silencing the expression of TNF-α mRNA and the related inflammatory factors, the material also achieved synergistic treatment by simultaneous consumption of ROS in the colon lesion. In vitro cell experiments and in vivo colitis treatment in mice showed that the lipoplex exerted a satisfactory therapeutic effect on colitis, and the symptoms of colitis in mice were significantly alleviated. The present study may shed light on the synergistic treatment for colitis through anti-inflammatory siRNA delivery and ROS depletion strategies.

Ferulic acid exhibits anti-inflammatory effects by inducing autophagy and blocking NLRP3 inflammasome activation.

BackgroundInflammation is involved in the healing process; however, when inflammation is overactivated, multiple diseases can occur. The continued discovery of new anti-inflammatory drugs is crucial in the treatment of inflammation-linked diseases.ObjectivesFerulic acid (FA), a precursor necessary for lignan synthesis, is widely distributed in plant-based whole foods and is a strong antioxidant. However, the effect of FA on the expression level of inflammatory factors in macrophages has not been fully clarified. The current study aimed to explore the anti-inflammatory effect and mechanism of ferulic acid.ResultsThe results showed that THP-1 cells were induced to differentiate into macrophages by Phorbol-12-myristate-13-acetate (PMA), and THP-1-derived macrophages were stimulated by LPS to establish an inflammatory cell model. Compared with the control group, low (5 μmol·mL−1), medium (10 μmol·mL−1), and high (20 μmol·mL−1) concentration ferulic acid groups have decreased cell viability and increased apoptosis rate in a dose-dependent manner. FA reduced the transcriptional levels of Interleukin-1β (IL-1β), Interleukin-6 (IL-6) and tumor necrosis factor α (TNF-α). Importantly, FA-induced autophagy and inhibited NLRP3 inflammasome activation. 3-MA (a widely used autophagy inhibitor) enhanced the secretion of TNF-α, IL-6 and IL-1β. Moreover, autophagy inhibition by 3-MA resulted in increased proteins expression associated with NLRP3 inflammasome signaling pathway. Besides, the inhibition of inflammasome activation by MCC950 reduced the expression of TNF-α, IL-6 and IL-1β.ConclusionIt is concluded that FA enhanced autophagy, inhibited the activation of NLRP3 inflammasome and reduced the expression and release of inflammatory factors.

Protective Effect of Rifampicin Loaded by HPMA-PLA Nanopolymer on Macrophages Infected with Mycobacterium Tuberculosis.

Purpose This research was designed to investigate the protective effect of rifampicin (RIF) loaded by N-(2-hydroxypropyl) methylacrylamide- (HPMA-) polylactic acid (PLA) nanopolymer on macrophages infected with Mycobacterium tuberculosis (MTB). Methods We first induced H37Rv to infect macrophages to build a cell model. Then, the HPMA-PLA nanopolymer loaded with RIF was prepared to treat MTB-infected macrophages. The macrophage activity was tested by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, the nitric oxide (NO) in cells was measured through Griess reagent, and the bacterial activity of MTB was observed via the colony-forming unit (CFU) assay. The inflammation-related factors in cells were detected via the enzyme-linked immunosorbent assay (ELISA), the apoptosis of macrophages was examined via flow cytometry, and the expression of apoptosis-related proteins was determined by western blot (WB). Results HPMA-PLA had no obvious toxicity to macrophages. The expression of NO and inflammatory factors in macrophages infected with MTB increased significantly, but the apoptosis rate was not significantly different from that of uninfected cells. However, after treatment with HPMA-PLA-RIF or free RIF, the inflammatory reaction of infected cells was inhibited, the expression of NO was decreased, the apoptosis rate was increased, and the bacterial activity in cells was decreased, with statistically significant differences; moreover, HPMA-PLA-RIF was more effective than free RIF. Conclusions HPMA-PLA-RIF has a high protective effect on macrophages infected with MTB, with high safety. Its protective mechanism is at least partly through inhibiting the production of NO and inflammatory response, which can inhibit bacterial activity and induce cell apoptosis.

Deficiency of IKKα in Macrophages Mitigates Fibrosis Progression in the Kidney after Renal Ischemia-Reperfusion Injury

Aims. Acute kidney injury (AKI) can lead to chronic kidney disease (CKD), and macrophages play a key role in this process. The aim of this study was to discover the role of IκB kinase α (IKKα) in macrophages in the process of AKI-to-CKD transition. Main Methods. We crossed lyz2-Cre mice with IKKα-floxed mice to generate mice with IKKα ablation in macrophages (Mac IKKα-/-). A mouse renal ischemia/reperfusion injury (IRI) model was induced by clamping the renal artery for 45 minutes. Treated mice were evaluated for blood biochemistry, tissue histopathology, and fibrosis markers. Macrophages were isolated from the peritoneal cavity for coculturing with tubular epithelial cells (TECs) and flow cytometry analysis. Key Findings. We found that fibrosis and kidney function loss after IRI were significantly alleviated in Mac IKKα-/- mice compared with wild-type (WT) mice. The expression of fibrosis markers and the infiltration of M2 macrophages were decreased in the kidneys of Mac IKKα-/- mice after IRI. The in vitro experiment showed that the IRI TECs cocultured with IKKα-/- macrophages (KO MΦs) downregulated the fibrosis markers accompanied by a downregulation of Wnt/β-catenin signaling. Significance. These data support the hypothesis that IKKα is involved in mediating macrophage polarization and increasing the expression of fibrosis-promoting inflammatory factors in macrophages. Therefore, knockdown of IKKα in macrophages may be a potential method that can be used to alleviate the AKI-to-CKD transition after IRI.

Untargeted metabolomics identifies succinate as a biomarker and therapeutic target in aortic aneurysm and dissection.

Aortic aneurysm and dissection (AAD) are high-risk cardiovascular diseases with no effective cure. Macrophages play an important role in the development of AAD. As succinate triggers inflammatory changes in macrophages, we investigated the significance of succinate in the pathogenesis of AAD and its clinical relevance. We used untargeted metabolomics and mass spectrometry to determine plasma succinate concentrations in 40 and 1665 individuals of the discovery and validation cohorts, respectively. Three different murine AAD models were used to determine the role of succinate in AAD development. We further examined the role of oxoglutarate dehydrogenase (OGDH) and its transcription factor cyclic adenosine monophosphate-responsive element-binding protein 1 (CREB) in the context of macrophage-mediated inflammation and established p38αMKOApoe-/- mice. Succinate was the most upregulated metabolite in the discovery cohort; this was confirmed in the validation cohort. Plasma succinate concentrations were higher in patients with AAD compared with those in healthy controls, patients with acute myocardial infarction (AMI), and patients with pulmonary embolism (PE). Moreover, succinate administration aggravated angiotensin II-induced AAD and vascular inflammation in mice. In contrast, knockdown of OGDH reduced the expression of inflammatory factors in macrophages. The conditional deletion of p38α decreased CREB phosphorylation, OGDH expression, and succinate concentrations. Conditional deletion of p38α in macrophages reduced angiotensin II-induced AAD. Plasma succinate concentrations allow to distinguish patients with AAD from both healthy controls and patients with AMI or PE. Succinate concentrations are regulated by the p38α-CREB-OGDH axis in macrophages.

SCFAs promote intestinal double-negative T cells to regulate the inflammatory response mediated by NLRP3 inflammasome.

Short-chain fatty acids (SCFAs) are a product of intestinal bacteria metabolism. Our previous study has found that intestinal bacteria in patients with Alzheimer’s disease (AD) can promote the activation of NLRP3 inflammasome and mediate neuroinflammation. In this study, we mainly explored the regulation of intestinal microenvironmental immunity by intestinal bacterial metabolite SCFAs and the mechanism of NLRP3 activation. First, wild-type (WT) and APP/PS1 mice were intervened with SCFAs. As a result, the proportion of double-negative T cells (CD3+CD4−CD8−, DNTs) in the intestine was increased, SCFAs could promote the expression of intestinal NLRP3 and inflammatory factors (IL-18, IL-6 and TNF-α). Moreover, SCAFs could also promote the level of inflammatory factors in the cerebrospinal fluid (CSF) of mice and aggravate the cognitive impairment in AD mice. CD3+ T cells isolated from the spleen were pre-treated with SCFAs, followed by detection of the proportion of DNTs. Consequently, SCFAs could promote the formation of DNTs, activate OX40 signal and simultaneously up-regulate the protein expression of Bcl-2, Bcl-xl and Survivin. Knockdown of OX40 could inhibit SCFAs-induced differentiation of DNTs. The co-culture of DNTs and intestinal macrophages showed that DNTs could activate Fas/FasL-TNF-α signal and induce the activation of NLRP3 inflammasome. In AD mouse models, treatment with Fas and TNFR1 inhibitors could significantly inhibit SCFAs-induced NLRP3 activation and inflammatory factors, while attenuate the inflammatory response in the brain tissue of mice and improve the cognitive ability of mice, however, without significant effect on the level of DNTs.The present study showed that SCFAs can promote the formation of DNTs through OX40. DNTs could induce the activation of NLRP3 inflammasome and the release of inflammatory factors in macrophages through Fas/FasL-TNF-α signals, thereby increasing the level of inflammatory factors in the central nervous system. When Fas and TNFR1 were inhibited by suppressing the functions of DNTs and macrophages, the activation of NLRP3 was inhibited. DNTs are affected by SCFAs, which is a new mechanism of neuroinflammation in AD.

Conditioned Media from Ultrasound‐treated C2C12 Myotubes Regulate Macrophage Inflammatory Responses

Background Macrophages play important roles in innate immunity and regulate inflammatory responses. It is necessary to tightly regulate inflammation to avoid excessive tissue damage. Therefore, the therapeutic approaches to regulate the inflammatory responses of macrophages are essential to maintain cell functions. Skeletal muscle is the largest organ of the human body and releases anti-inflammatory/immune-modulatory factors. The facilitatory effects of ultrasound (US) on the release of extracellular vesicles (EVs) have been elucidated in cultured myotubes in our previous study. Purpose The aim of this study was to investigate the effects of EV-containing conditioned media from US-treated myotubes on inflammatory responses in macrophages. Methods Conditioned media from C2C12 myotubes with/without US irradiation were applied to murine bone marrow-derived macrophages for 1.5 hours. The mRNA expressions of pro- and anti-inflammatory factors were measured by quantitative real-time PCR. Results Conditioned media from both US-treated and untreated myotubes significantly suppressed the expression of IL-1β in macrophages without causing cell damage. US irradiation significantly increased the concentration level of EVs in conditioned media. In addition, the US-treated conditioned media decreased the expression levels of IL-1β and IL-6 by 23% and 28% compared to the US-untreated media, respectively. Furthermore, US treatment increased the expression level of Mgl, an anti-inflammatory marker, by 12% compared to US-untreated media. Discussion Ultrasound increased EV release from myotubes and fortified the anti-inflammatory effects of the conditioned media. These results suggest that ultrasound stimulation to myotubes could promote the anti-inflammatory effects via the enhancements in EV release. Conclusion Ultrasound-induced EVs in conditioned media from C2C12 myotubes could suppress the expression of the inflammatory factors in macrophages.