The TLR Expression Pattern on Monocyte-Derived Macrophages for Lipopolysaccharid Stimulation of Calves

In this paper, toll-like receptor expression pattern in monocytes-derived macrophages by lipopolysaccharid （LPS） stimulation was examined. Jugular venous blood samples from 4 Japanese calves were obtained and the peripheral blood mononuclear cells （PBMC） were isolated. The PBMC were cultured for 7 d so as to collect monocytes-derived macrophages in Repcell. The PBMC were stimulated by LPS for 24 h and the mRNA expression pattern of TLR and cytokines in monocytes-derived macrophages （Mod-Mφ） was analyzed. Results showed that LPS stimulation of Mod-Mφ could increase the mRNA levels of the genes of TNF-α, IL-6, and IL-8. In addition, the mRNA levels of the genes of TNF-α and IL-6 in the group of LPS stimulation were most significantly （P 〈 0.01） higher than those in control group and the mRNA levels of TLR1, 3, 5, 8, and 10 were significantly （P 〈 0.05） decreased after LPS stimulation. There was no difference in the mRNA expressions of TLR2, 4, 6, and 7 between the groups of the control and LPS stimulation. Besides, expression of TLR9 was not found. It suggested that monocytes-derived macrophages could respond to LPS and they might take an important role in the innate immunity. The important function of the cells might contribute to better disease treatment.

Tyrosine phosphorylation of monocyte-derived macrophage proteins in buffalo (Bubalus bubalis): A potential phenotype of natural resistance

The aim of this work was to explore the possibility of using the presence of tyrosine-phosphorylated macrophage proteins as a phenotype of natural resistance. Tyrosine-phosphorylation of macrophage proteins was investigated in 18 buffaloes, that carried either the resistant, or the non-resistant, Natural Resistance-Associated Macrophage Protein one (NRAMP1) genotype, that various authors have associated with susceptibility to intracellular bacterial diseases. Monocyte-derived macrophages were Interferon-gamma (IFN-γ) stimulated and tyrosine-phosphorylation was assessed by Western blotting. Evidence of phosphorylation after IFN-γ stimulation was shown by 75% of the buffaloes carriers of the resistant genotype, and by 20% of the carriers of the non-resistant genotype (Chisquare value between the groups = 5.44;P = 0.02). The study of the Proteoma of monocyte-derived macrophages might open the way to the genetic control of disease resistance.

Differential Responses Between Monocytes and Monocyte-Derived Macrophages for Lipopolysaccharide Stimulation of Calves

In this experiment Toll-like receptor expression pattern in monocytes and monocyte-derived macrophages by lipopolysaccharide （LPS） stimulation was examined. Jugular venous blood was collected from four Japanese calves, and the peripheral blood mononuclear cells （PBMCs） were isolated. The cells were directly used for collecting monocytes by magnetic cell sorting or cultured for 7 days to collect monocyte-derived macrophages in Repcell. Then we analyzed the mRNA expression pattern of TLRs and cytokines in monocytes and monocyte-derived macrophages after LPS stimulation for 24 h. LPS stimulation of both monocytes and monocyte-derived macrophages resulted in an increase in the levels of mRNA transcripts for TNF-α IL-6 and IL-8. Moreover, TNF-α and IL-6 mRNA expressions were significantly augmented by LPS stimulation in monocyte-derived macrophages. TLRs mRNA expressions were unchanged after LPS stimulation of monocytes, while TLRs mRNA expressions in monocyte-derived macrophages were complicated. TLR1, 3, 5, 8 and 10 were significantly decreased after LPS stimulation and there were no differences in the mRNA expressions of TLR2, 4, 6 and 7 between the groups of control and LPS stimulation. Besides, no expression of TLR9 was found. As antigen presenting cells, monocytes and monocyte-derived macrophages respond differently to LPS, so they may have different functions in the innate immune system.

WNT/β-catenin-M2 macrophage interplay as a target for therapy against hepatocellular carcinoma:Role of Calculus bovis

Liver cancer,and in particular hepatocellular carcinoma(HCC)is a disease of rising prevalence and incidence.To date,definitive treatment options include either surgical excision or ablation of the affected area.With increasing research on several pathways that could be involved in the progression of HCC,new elements within these pathways emerge as potential targets for novel therapies.The WNT/β-catenin pathway favors the presence of M2 tumor-associated macrophages which in turn promote tumor growth and metastasis.The inhibition of this pathway is considered a good candidate for such targeted therapeutic interventions.Interestingly,as Huang et al show in their recently published article,Calculus bovis which is used in traditional Chinese medicine can exert an inhibitory effect on theβ-catenin pathway and become a potential candidate for targeted pharmacotherapy against liver cancer.

Role of macrophage polarization in diabetic foot ulcer healing:A bibliometric study

BACKGROUND Diabetic foot ulcers(DFUs)are a significant contributor to disability and mortality in diabetic patients.Macrophage polarization and functional regulation are promising areas of research and show therapeutic potential in the field of DFU healing.However,the complex mechanism,the difficulty in clinical translation,and the large heterogeneity present significant challenges.Hence,this study was to comprehensively analyze the publication status and trends of studies on macrophage polarization and DFU healing.AIM To examine the relevant literature on macrophage polarization in DFU healing.METHODS A bibliometric analysis was conducted using the Web of Science database.Relevant literature was retrieved from the Web of Science Core Collection database between 2013 to 2023 using literature visualization and analysis software(VOSviewer and CiteSpace)and bibliometric online platforms.The obtained literature was then subjected to visualization and analysis of different countries/regions,institutions,journals,authors,and keywords to reveal the research’s major trends and focus.RESULTS The number of publications on the role of macrophage polarization in DFU healing increased rapidly from 2013 to 2023,especially in the latter period.Chinese researchers were the most prolific in this field,with 217 publications,while American researchers had been engaged in this field for a longer period.Qian Tan of Nanjing Drum Tower Hospital and Qian Ding of Nanjing University were the first to publish in this field.Shanghai Jiao Tong University was the institution with the most publications(27).The keywords“bone marrow”,“adjustment,replacement,response,tissue repair”,and“activation,repair,differentiation”appeared more frequently.The study of macrophage polarization in DFU healing focused on the regulatory mechanism,gene expression,and other aspects.CONCLUSION This study through the bibliometric method reveals the research trends and development trends in this field of macrophage polarization in DFU healing from 2013 to 2023 in the Web of Science Core Collection database.The key hotspots in this field mainly include the regulation of macrophage activation,gene expression,wound tissue repair,and new wound materials.This study provides references for future research directions.

Dapagliflozin exerts anti-apoptotic effects by mitigating macrophage polarization via modulation of the phosphoinositide 3-kinase/protein kinase B signaling pathway

BACKGROUND Macrophages are central to the orchestration of immune responses,inflammatory processes,and the pathogenesis of diabetic complications.The dynamic polarization of macrophages into M1 and M2 phenotypes critically modulates inflammation and contributes to the progression of diabetic nephropathy.Sodiumglucose cotransporter 2 inhibitors such as dapagliflozin,which are acclaimed for their efficacy in diabetes management,may influence macrophage polarization,thereby ameliorating diabetic nephropathy.This investigation delves into these mechanistic pathways,aiming to elucidate novel therapeutic strategies for diabetes.AIM To investigate the inhibitory effect of dapagliflozin on macrophage M1 polarization and apoptosis and to explore its mechanism of action.METHODS We established a murine model of type 2 diabetes mellitus and harvested peritoneal macrophages following treatment with dapagliflozin.Concurrently,the human monocyte cell line cells were used for in vitro studies.Macrophage viability was assessed in a cell counting kit 8 assay,whereas apoptosis was evaluated by Annexin V/propidium iodide staining.Protein expression was examined through western blotting,and the expression levels of macrophage M1 surface immunosorbent assay,and quantitative real-time polymerase chain reaction analyses.RESULTS Dapagliflozin attenuated M1 macrophage polarization and mitigated apoptosis in the abdominal macrophages of diabetic mice,evidenced by the downregulation of proapoptotic genes(Caspase 3),inflammatory cytokines[interleukin(IL)-6,tumor necrosis factor-α,and IL-1β],and M1 surface markers(inducible nitric oxide synthase,and cluster of differentiation 86),as well as the upregulation of the antiapoptotic gene BCL2.Moreover,dapagliflozin suppressed the expression of proteins associated with the phosphoinositide 3-kinase(PI3K)/protein kinase B(AKT)signaling pathway(PI3K,AKT,phosphorylated protein kinase B).These observations were corroborated in vitro,where we found that the modulatory effects of dapagliflozin were abrogated by 740Y-P,an activator of the PI3K/AKT signaling pathway.CONCLUSION Dapagliflozin attenuates the polarization of macrophages toward the M1 phenotype,thereby mitigating inflammation and promoting macrophage apoptosis.These effects are likely mediated through the inhibition of the PI3K/AKT signaling pathway.

The transcriptome of MHV-infected RAW264.7 cells offers an alternative model for macrophage innate immunity research

Background:Macrophages are the primary innate immune cells encountered by the invading coronaviruses,and their abilities to initiate inflammatory reactions,to main-tain the immunity homeostasis by differential polarization,to train the innate immune system by epigenic modification have been reported in laboratory animal research.Methods:In the current in vitro research,murine macrophage RAW 264.7 cell were infected by mouse hepatitis virus,a coronavirus existed in mouse.At 3-,6-,12-,24-,and 48-h post infection(hpi.),the attached cells were washed with PBS and harvested in Trizol reagent.Then The harvest is subjected to transcriptome sequencing.Results:The transcriptome analysis showed the immediate(3 hpi.)up regulation of DEGs related to inflammation,like Il1b and Il6.DEGs related to M2 differential po-larization,like Irf4 showed up regulation at 24 hpi.,the late term after viral infection.In addition,DEGs related to metabolism and histone modification,like Ezh2 were de-tected,which might correlate with the trained immunity of macrophages.Conclusions:The current in vitro viral infection study showed the key innated im-munity character of macrophages,which suggested the replacement value of viral infection cells model,to reduce the animal usage in preclinical research.

Emodin promotes the recovery of rheumatoid arthritis by regulating the crosstalk between macrophage subsets and synovial fibroblast subsets

Background : To study the relationships among emodin, synovial fibroblasts (FLSs), and macrophages (STMs) to provide guidance for the use of emodin in rheumatoid arthritis (RA) treatment. Methods : RA clinical samples from patients with different pathological processes were collected, and the correlations between the subsets of FLSs and STMs and path- ological processes were analyzed via flow cytometry. In vitro experimental methods such as enzyme linked immunosorbent assay (ELISA), Western blotting, Transwell as- says, CCK- 8 assays and cell coculture were used to assess cell proliferation, migration and secretion of inflammatory factors. A collagen- induced arthritis mouse model was constructed to investigate the therapeutic potential of emodin in RA by flow cytom- etry, micro- CT and staining. Results : Unique subsets of FLSs and STMs, namely, FAPα ^(+)THY1 − FLSs, FAPα ^(+)THY1 ^(+)FLSs, and MerTK ^(pos) TREM2 ^(high) STMs, were identified in synovial tissues from RA patients. The number of MerTK ^(pos) TREM2 ^(high) STMs was negatively correlated with the degree of damage in RA, while the number of FAPα ^(+)THY1 − FLSs was positively correlated with damage. On the one hand, emodin promoted the aggregation of MerTKposTREM2high STMs. Moreover, MerTK pos TREM2 high STM- mediated secre- tion of exosomes was promoted, which can inhibit the secretion of pro- inflammatory factors by FAPα ^(+)THY1 ^(+)FLSs and promote the secretion of anti- inflammatory factors by FAPα ^(+)THY1 ^(+)FLSs, thereby inhibiting FAPα ^(+)THY1 − FLS proliferation and migration, improving the local immune microenvironment, and inhibiting RA damage. Conclusion : Emodin was shown to regulate the aggregation of STM subsets and exo- some secretion, affecting the secretion, proliferation and migration of inflammatory factors in FLS subsets, and ultimately achieving good therapeutic efficacy in RA pa- tients, suggesting that it has important clinical value.

Macrophage polarization in cardiac transplantation:Insights into immune modulation and therapeutic approaches

The role and regulatory mechanisms of macrophage polarization in cardiac transplantation have gained significant attention.Macrophages can polarize into either the M1(pro-inflammatory)or M2(anti-inflammatory)phenotype in response to environmental cues.M1 macrophages facilitate transplant rejection by releasing inflammatory mediators and activating T cells,whereas M2 macrophages support graft survival by secreting antiinflammatory factors and promoting tissue repair.Mitochondrial quality control regulation plays a crucial role in macrophage polarization,which may influence graft survival and immune responses.This review provides an overview of the current understanding of mitochondrial quality control-regulated macrophage polarization in cardiac transplantation,its effects on graft outcomes,and potential therapeutic strategies to modulate this process to enhance transplant success rates.The review was conducted by systematically analyzing recent studies and integrating findings from key research articles to synthesize a comprehensive understanding of this emerging field.

O-linked β-N-acetylglucosamine transferase regulates macrophage polarization in diabetic periodontitis: In vivo and in vitro study

BACKGROUND Periodontitis,when exacerbated by diabetes,is characterized by increased M1 macrophage polarization and decreased M2 polarization.O-linkedβ-N-acetylglucosamine(O-GlcNAcylation),catalyzed by O-GlcNAc transferase(OGT),promotes inflammatory responses in diabetic periodontitis(DP).Additionally,p38 mitogen-activated protein kinase regulates macrophage polarization.However,the interplay between OGT,macrophage polarization,and p38 signaling in the progression of DP remains unexplored.AIM To investigate the effect of OGT on macrophage polarization in DP and its role in mediating O-GlcNAcylation of p38.METHODS For in vivo experiments,mice were divided into four groups:Control,DP model,model+short hairpin(sh)RNAnegative control,and model+sh-OGT.Diabetes was induced by streptozotocin,followed by ligation and lipopolysaccharide(LPS)administration to induce periodontitis.The impact of OGT was assessed by injecting sh-OGT lentivirus.Maxillary bone destruction was evaluated using micro-computed tomography analysis and tartrateresistant acid phosphatase staining,while macrophage polarization was determined through quantitative real-time polymerase chain reaction(qPCR)and immunohistochemistry.For in vitro experiments,RAW264.7 cells were treated with LPS and high glucose(HG)(25 mmol/L D-glucose)to establish a cell model of DP.OGT was inhibited by OGT inhibitor(OSMI4)treatment and knocked down by sh-OGT transfection.M1/M2 polarization was analyzed using qPCR,immunofluorescence,and flow cytometry.Levels of O-GlcNAcylation were measured using immunoprecipitation and western blotting.RESULTS Our results demonstrated that M1 macrophage polarization led to maxillary bone loss in DP mice,associated with elevated O-GlcNAcylation and OGT levels.Knockdown of OGT promoted the shift from M1 to M2 macrophage polarization in both mouse periodontal tissues and LPS+HG-induced RAW264.7 cells.Furthermore,LPS+HG enhanced the O-GlcNAcylation of p38 in RAW264.7 cells.OGT interacted with p38 to promote its O-GlcNAcylation at residues A28,T241,and T347,as well as its phosphorylation at residue Y221.CONCLUSION Inhibition of OGT-mediated p38 O-GlcNAcylation deactivates the p38 pathway by suppressing its self-phosphorylation,thereby promoting M1 to M2 macrophage polarization and mitigating DP.These findings suggested that modulating macrophage polarization through regulation of O-GlcNAcylation may represent a novel therapeutic strategy for treating DP.

The impact of telmisartan on angiotensin converting enzyme 2 mRNA expression in monocyte-derived macrophages of diabetic hypertensive patients

Objective To investigate the effects of telmisartan on the expression of angiotensin converting enzyme 2(ACE2) mRNA in monocyte-derived macrophages of hypertensive patients accompanied with diabetes. Methods 62 essential hypertensive patients accompanied

Effects of sulforaphane on Toll-like receptor 4/myeloid differentiation factor 88 pathway of monocyte-derived macrophages from patients with chronic obstructive pulmonary disease

Objective To explore the effects of sulforaphane on Toll-like receptor 4(TLR4)/myeloid differentiation factor88(MyD88)pathway and its downstream inflammatory cytokines in patients with chronic obstructive pulmonary disease(COPD).Methods From Jan.2012 to Mar.2013,thirty-two stable COPD patients and thirty healthy donors(non-COPD group)from the First Hospital of Lanzhou University were recruited.The peripheral

Global trends in publications regarding macrophages-related diabetic foot ulcers in the last two decades

BACKGROUND Diabetic foot ulcers(DFUs)are one of the most severe and popular complications of diabetes.The persistent non-healing of DFUs is the leading cause of amputation,which causes significant mental and financial stress to patients and their families.Macrophages are critical cells in wound healing and perform essential roles in all phases of wound healing.However,no studies have been carried out to systematically illustrate this area from a scientometric point of view.Although there have been some bibliometric studies on diabetes,reports focusing on the investigation of macrophages in DFUs are lacking.AIM To perform a bibliometric analysis to systematically assess the current state of research on macrophage-related DFUs.METHODS The publications of macrophage-related DFUs from January 1,2004,to December 31,2023,were retrieved from the Web of Science Core Collection on January 9,2024.Four different analytical tools:VOSviewer(v1.6.19),CiteSpace(v6.2.R4),HistCite(v12.03.07),and Excel 2021 were used for the scientometric research.RESULTS A total of 330 articles on macrophage-related DFUs were retrieved.The most published countries,institutions,journals,and authors in this field were China,Shanghai Jiao Tong University of China,Wound Repair and Regeneration,and Aristidis Veves.Through the analysis of keyword co-occurrence networks,historical direct citation networks,thematic maps,and trend topics maps,we synthesized the prevailing research hotspots and emerging trends in this field.CONCLUSION Our bibliometric analysis provides a comprehensive overview of macrophage-related DFUs research and insights into promising upcoming research.

Calculus bovis inhibits M2 tumor-associated macrophage polarization via Wnt/β-catenin pathway modulation to suppress liver cancer

BACKGROUND Calculus bovis(CB),used in traditional Chinese medicine,exhibits anti-tumor effects in various cancer models.It also constitutes an integral component of a compound formulation known as Pien Tze Huang,which is indicated for the treatment of liver cancer.However,its impact on the liver cancer tumor microenvironment,particularly on tumor-associated macrophages(TAMs),is not well understood.AIM To elucidate the anti-liver cancer effect of CB by inhibiting M2-TAM polarization via Wnt/β-catenin pathway modulation.METHODS This study identified the active components of CB using UPLC-Q-TOF-MS,evaluated its anti-neoplastic effects in a nude mouse model,and elucidated the underlying mechanisms via network pharmacology,transcriptomics,and molecular docking.In vitro assays were used to investigate the effects of CB-containing serum on HepG2 cells and M2-TAMs,and Wnt pathway modulation was validated by real-time reverse transcriptase-polymerase chain reaction and Western blot analysis.RESULTS This study identified 22 active components in CB,11 of which were detected in the bloodstream.Preclinical investigations have demonstrated the ability of CB to effectively inhibit liver tumor growth.An integrated approach employing network pharmacology,transcriptomics,and molecular docking implicated the Wnt signaling pathway as a target of the antineoplastic activity of CB by suppressing M2-TAM polarization.In vitro and in vivo experiments further confirmed that CB significantly hinders M2-TAM polarization and suppresses Wnt/β-catenin pathway activation.The inhibitory effect of CB on M2-TAMs was reversed when treated with the Wnt agonist SKL2001,confirming its pathway specificity.CONCLUSION This study demonstrated that CB mediates inhibition of M2-TAM polarization through the Wnt/β-catenin pathway,contributing to the suppression of liver cancer growth.

Knockout of C6orf120 in Rats Alleviates Concanavalin A-induced Autoimmune Hepatitis by Regulating Macrophage Polarization

Objective The effect of the functionally unknown gene C6orf120 on autoimmune hepatitis was investigated on C6orf120 knockout rats(C6orf120^(-/-))and THP-1 cells.Method Six–eight-week-old C6orf120^(-/-)and wild-type(WT)SD rats were injected with Con A(16 mg/kg),and euthanized after 24 h.The sera,livers,and spleens were collected.THP-1 cells and the recombinant protein(rC6ORF120)were used to explore the mechanism in vitro.The frequency of M1 and M2 macrophages was analyzed using flow cytometry.Western blotting and PCR were used to detect macrophage polarization-associated factors.Results C6orf120 knockout attenuated Con A-induced autoimmune hepatitis.Flow cytometry indicated that the proportion of CD68^(+)CD86^(+)M1 macrophages from the liver and spleen in the C6orf120^(-/-)rats decreased.C6orf120 knockout induced downregulation of CD86 protein and the mRNA levels of related inflammatory factors TNF-α,IL-1β,and IL-6 in the liver.C6orf120 knockout did not affect the polarization of THP-1 cells.However,rC6ORF120 promoted the THP-1 cells toward CD68^(+)CD80^(+)M1 macrophages and inhibited the CD68^(+)CD206^(+)M2 phenotype.Conclusion C6orf120 knockout alleviates Con A-induced autoimmune hepatitis by inhibiting macrophage polarization toward M1 macrophages and reducing the expression of related inflammatory factors in C6orf120^(-/-)rats.

Myricetin induces M2 macrophage polarization to alleviate renal tubulointerstitial fibrosis in diabetic nephropathy via PI3K/Akt pathway

BACKGROUND Development of end-stage renal disease is predominantly attributed to diabetic nephropathy(DN).Previous studies have indicated that myricetin possesses the potential to mitigate the pathological alterations observed in renal tissue.Never-theless,the precise molecular mechanism through which myricetin influences the progression of DN remains uncertain.AIM To investigate the effects of myricetin on DN and explore its potential therapeutic mechanism.METHODS Db/db mice were administered myricetin intragastrically on a daily basis at doses of 50 mg/kg or 100 mg/kg for a duration of 12 wk.Subsequently,blood and urine indexes were assessed,along with examination of renal tissue pathology.Kidney morphology and fibrosis were evaluated using various staining techniques including hematoxylin and eosin,periodic acid–Schiff,Masson’s trichrome,and Sirius-red.Additionally,high-glucose culturing was conducted on the RAW 264.7 cell line,treated with 25 mM myricetin or co-administered with the PI3K/Akt inhibitor LY294002 for a period of 24 h.In both in vivo and in vitro settings,quantification of inflammation factor levels was conducted using western blotting,real-time qPCR and ELISA.RESULTS In db/db mice,administration of myricetin led to a mitigating effect on DN-induced renal dysfunction and fibrosis.Notably,we observed a significant reduction in expressions of the kidney injury markers kidney injury molecule-1 and neutrophil gelatinase associated lipocalin,along with a decrease in expressions of inflammatory cytokine-related factors.Furthermore,myricetin treatment effectively inhibited the up-regulation of tumor necrosis factor-alpha,interleukin-6,and interluekin-1βinduced by high glucose in RAW 264.7 cells.Additionally,myricetin modulated the M1-type polarization of the RAW 264.7 cells.Molecular docking and bioinformatic analyses revealed Akt as the target of myricetin.The protective effect of myricetin was nullified upon blocking the polarization of RAW 264.7 via inhibition of PI3K/Akt activation using LY294002.CONCLUSION This study demonstrated that myricetin effectively mitigates kidney injury in DN mice through the regulation of macrophage polarization via the PI3K/Akt signaling pathway.

Sulfated Cyclocarya paliurus polysaccharides exert immunomodulatory potential on macrophages via Toll-like receptor 4 mediated MAPK/NF-κB signaling pathways

The biological activity of plant polysaccharides can be enhanced by sulfated modification.In this study,the immunomodulatory effect of sulfated Cyclocarya paliurus polysaccharides(SCP3)on macrophages RAW264.7 and its potential molecular mechanism were investigated.Results showed that SCP3 at 25-100μg/m L increased viability and improved phagocytosis of RAW264.7 cells.Meanwhile,SCP3 could activate mitogen-activated protein kinase(MAPK)and nuclear factor kappa B(NF-κB)signaling pathways,which increased the phosphorylation of Erk1/2,JNK,p38 and NF-κB p65,promoting secretion of cytokines tumor necrosis factorα(TNF-α),interleukin 6(IL-6)and nitric oxide(NO)as well as the production of reactive oxygen species(ROS).In addition,Toll-like receptor 4(TLR4)receptor inhibitors were able to block the production of NO and TNF-αby SCP3-stimulated macrophages.Based on Western blot analysis and validation using specific inhibitors against MAPK and NF-κB signaling pathways,the results demonstrated that SCP3 induced macrophages activation and enhanced TNF-αand NO production via TLR4-mediated MAPK and NF-κB pathways.In summary,SCP3 has significant immunomodulatory potential.The underlying molecular mechanism was that SCP3 activates macrophages via TLR4 receptors to promote ROS production,which in turn activates the downstream MAPK/NF-κB signaling pathway and then increases the secretion levels of cytokines and NO.

Fasudil-modified macrophages reduce inflammation and regulate the immune response in experimental autoimmune encephalomyelitis

Multiple sclerosis is characterized by demyelination and neuronal loss caused by inflammatory cell activation and infiltration into the central nervous system.Macrophage polarization plays an important role in the pathogenesis of experimental autoimmune encephalomyelitis,a traditional experimental model of multiple sclerosis.This study investigated the effect of Fasudil on macrophages and examined the therapeutic potential of Fasudil-modified macrophages in experimental autoimmune encephalomyelitis.We found that Fasudil induced the conversion of macrophages from the pro-inflammatory M1 type to the anti-inflammatory M2 type,as shown by reduced expression of inducible nitric oxide synthase/nitric oxide,interleukin-12,and CD16/32 and increased expression of arginase-1,interleukin-10,CD14,and CD206,which was linked to inhibition of Rho kinase activity,decreased expression of toll-like receptors,nuclear factor-κB,and components of the mitogen-activated protein kinase signaling pathway,and generation of the pro-inflammatory cytokines tumor necrosis factor-α,interleukin-1β,and interleukin-6.Crucially,Fasudil-modified macrophages effectively decreased the impact of experimental autoimmune encephalomyelitis,resulting in later onset of disease,lower symptom scores,less weight loss,and reduced demyelination compared with unmodified macrophages.In addition,Fasudil-modified macrophages decreased interleukin-17 expression on CD4^(+)T cells and CD16/32,inducible nitric oxide synthase,and interleukin-12 expression on F4/80^(+)macrophages,as well as increasing interleukin-10 expression on CD4^(+)T cells and arginase-1,CD206,and interleukin-10 expression on F4/80^(+)macrophages,which improved immune regulation and reduced inflammation.These findings suggest that Fasudil-modified macrophages may help treat experimental autoimmune encephalomyelitis by inducing M2 macrophage polarization and inhibiting the inflammatory response,thereby providing new insight into cell immunotherapy for multiple sclerosis.

Spi1 regulates the microglial/macrophage inflammatory response via the PI3K/AKT/mTOR signaling pathway after intracerebral hemorrhage

Preclinical and clinical studies have shown that microglia and macrophages participate in a multiphasic brain damage repair process following intracerebral hemorrhage.The E26 transformation-specific sequence-related transcription factor Spi1 regulates microglial/macrophage commitment and maturation.However,the effect of Spi1 on intracerebral hemorrhage remains unclear.In this study,we found that Spi1 may regulate recovery from the neuroinflammation and neurofunctional damage caused by intracerebral hemorrhage by modulating the microglial/macrophage transcriptome.We showed that high Spi1expression in microglia/macrophages after intracerebral hemorrhage is associated with the activation of many pathways that promote phagocytosis,glycolysis,and autophagy,as well as debris clearance and sustained remyelination.Notably,microglia with higher levels of Soil expression were chara cterized by activation of pathways associated with a variety of hemorrhage-related cellular processes,such as complement activation,angiogenesis,and coagulation.In conclusion,our results suggest that Spi1 plays a vital role in the microglial/macrophage inflammatory response following intracerebral hemorrhage.This new insight into the regulation of Spi1 and its target genes may advance our understanding of neuroinflammation in intracerebral hemorrhage and provide therapeutic targets for patients with intracerebral hemorrhage.

Atherosis-associated lnc_000048 activates PKR to enhance STAT1-mediated polarization of THP-1 macrophages to M1 phenotype

Our previous study has demonstrated that lnc_000048 is upregulated in large-artery atherosclerotic stroke and promotes atherosclerosis in ApoE^(-/-)mice.However,little is known about the role of lnc_000048 in classically activated macrophage(M1)polarization.In this study,we established THP-1-derived testing state macrophages(M0),M1 macrophages,and alternately activated macrophages(M2).Real-time fluorescence quantitative PCR was used to verify the expression of marker genes and the expression of lnc_000048 in macrophages.Flow cytometry was used to detect phenotypic proteins(CD11b,CD38,CD80).We generated cell lines with lentivirus-mediated upregulation or downregulation of lnc_000048.Flow cytometry,western blot,and real-time fluorescence quantitative PCR results showed that down-regulation of lnc_000048 reduced M1 macrophage polarization and the inflammation response,while over-expression of lnc_000048 led to the opposite effect.Western blot results indicated that lnc_000048 enhanced the activation of the STAT1 pathway and mediated the M1 macrophage polarization.Moreover,catRAPID prediction,RNA-pull down,and mass spectrometry were used to identify and screen the protein kinase RNA-activated(PKR),then catRAPID and RPIseq were used to predict the binding ability of lnc_000048 to PKR.Immunofluorescence(IF)-RNA fluorescence in situ hybridization(FISH)double labeling was performed to verify the subcellular colocalization of lnc_000048 and PKR in the cytoplasm of M1 macrophage.We speculate that lnc_000048 may form stem-loop structure-specific binding and activate PKR by inducing its phosphorylation,leading to activation of STAT1 phosphorylation and thereby enhancing STAT1 pathway-mediated polarization of THP-1 macrophages to M1 and inflammatory factor expression.Taken together,these results reveal that the lnc_000048/PKR/STAT1 axis plays a crucial role in the polarization of M1 macrophages and may be a novel therapeutic target for atherosclerosis alleviation in stroke.