Macrophage Apoptosis Research Articles

Bidirectional roles of extracellular and intracellular HMGB2 in the development of atherosclerosis

Abstract Rationale: HMGB family plays an important role in homeostasis of immunity and regulation of inflammation. However, the role of HMGB2 in atherosclerosis remains uncharacterized. Objective The current study aimed to ascertain the relation of HMGB2 levels in macrophages of atherosclerotic plaques and in circulatory monocytes/macrophages with coronary artery disease (CAD), and to investigate the influence of HMGB2 on atherogenesis in mice and inflammatory reaction in vitro. Methods/Results We evaluated HMGB2 levels in macrophages of atherosclerotic plaques in coronary endarterectomy tissue from patients with angiographically documented severe coronary artery disease (CAD)(n=23), and in coronary artery specimens from autopsy cases with no or mild atherosclerosis (n=11) by immunohistochemistry. HMGB2 levels were significantly decreased in macrophages of progressive atherosclerotic plaques, as compared with those from mild plaques. Moreover, we also analyzed HMGB2 levels in peripheral blood monocytes from patients with CAD (n=44) or age- and sex-matched non-CAD controls (n=44). The results showed that decreased intracellular HMGB2 protein levels in monocytes were associated with CAD. In experiments, HMGB2 deficiency significantly promoted atherosclerosis in ApoE-/- mice, whereas such increment of atherosclerosis was markedly attenuated after transplantation of bone marrow derived macrophages from ApoE-/-mice. Mechanistically, intracellular HMGB2 repressed IL-1 transcription and induced autophagic degradation of NLRP3, thus suppressing NLRP3 inflammasome priming and activation. Extracellular HMGB2 levels increased in progressive atherosclerotic plaques with macrophage apoptosis. Recombinant HMGB2 protein promoted inflammation in vitro via RAGE and intraperitoneal administration of this protein exacerbated atherosclerosis in mice. Conclusion This study indicates that decreased HMGB2 level in macrophage is associated with inflammation and atherosclerosis in patients with CAD. Intracellular HMGB2 inhibits NLRP3 inflammasome and atherosclerosis in mice, whereas released HMGB2 promotes inflammation and atherosclerosis in mice.

Apoptotic vesicles from macrophages exacerbate periodontal bone resorption in periodontitis via delivering miR-143-3p targeting Igfbp5

AbstrctBackgroundApoptotic vesicles (ApoVs), which are extracellular vesicles released by apoptotic cells, have been reported to exhibit substantial therapeutic potential for inflammatory diseases and tissue regeneration. While extensive research has been dedicated to mesenchymal stem cells (MSCs), the investigation into immune cell-derived ApoVs remains limited, particularly regarding the function and fate of macrophage-derived ApoVs in the context of periodontitis (PD).ResultsOur study corroborates the occurrence and contribution of resident macrophage apoptosis in Porphyromonas gingivalis (Pg)-associated PD. The findings unveil the pivotal role played by apoptotic macrophages and their derived ApoVs in orchestrating periodontal bone remodeling. The enrichments of diverse functional miRNAs within these ApoVs are discerned through sequencing techniques. Moreover, our study elucidates that the macrophage-derived ApoVs predominantly deliver miR-143-3p, targeting insulin-like growth factor-binding protein 5 (IGFBP5), thereby disrupting periodontal bone homeostasis.ConclusionsOur study reveals that macrophages in Pg-associated PD undergo apoptosis and generate miR-143-3p-enriched ApoVs to silence IGFBP5, resulting in the perturbation of osteogenic-osteoclastic balance and the ensuing periodontal bone destruction. Accordingly, interventions targeting miR-143-3p in macrophages or employment of apoptosis inhibitor Z-VAD hold promise as effective therapeutic strategies for the management of PD.

IL-27 attenuated macrophage injury and inflammation induced by Mycobacterium tuberculosis by activating autophagy.

Interleukin-27 (IL-27) is a cytokine that is reported to be highly expressed in the peripheral blood of patients with pulmonary tuberculosis (PTB). IL-27-mediated signaling pathways, which exhibit anti- Mycobacterium tuberculosis (Mtb) properties, have also been demonstrated in macrophages infected with Mtb. However, the exact mechanism remains unclear. This study aimed to clarify the potential molecular mechanisms through which IL-27 enhances macrophage resistance to Mtb infection.Both normal and PTB patients provided bronchoalveolar lavage fluid (BALF). Peripheral blood mononuclear cells (PBMCs) were isolated from healthy individuals and stimulated with 50ng/mL macrophage-colony stimulating factor (M-CSF) to obtain monocyte-derived macrophages (MDMs). Using 100ng/mL phorbol 12-myristate 13-acetate (PMA), THP-1 cells were induced to differentiate into THP-1-derived macrophage-like cells (TDMs). Both MDMs and TDMs were subsequently infected with the Mtb strain H37Rv and treated with 50ng/mL IL-27 prior to infection. The damage and inflammation of macrophages were examined using flow cytometry, enzyme-linked immunosorbent assay (ELISA), and Western blotting.Patients with PTB had elevated levels of IL-27 in their BALF. Preconditioning with IL-27 was shown to reduce H37Rv-induced MDMs and TDMs apoptosis while also decreasing the levels of Cleaved Caspase-3, Bax and the proinflammatory cytokines TNF-α, IL-1β, and IL-6, promoting the expression of Bcl-2 and the anti-inflammatory factors IL-10 and IL-4. Silencing of the IL-27 receptor IL-27Ra increased macrophage damage and inflammation triggered by H37Rv. Mechanistically, IL-27 activates autophagy by inhibiting TLR4/NF-κB signaling and activating the PI3K/AKT signaling pathway, thereby inhibiting H37Rv-induced macrophage apoptosis and the inflammatory response.Our study suggests that IL-27 alleviates H37Rv-induced macrophage injury and the inflammatory response by activating autophagy and that IL-27 may be a new target for the treatment of PTB.

Enhancement of Autophagy in Macrophages via the p120-Catenin-Mediated mTOR Signaling Pathway.

Autophagy serves as a critical regulator of immune responses in sepsis. Macrophages are vital constituents of both innate and adaptive immunity. In this study, we delved into the intricate role of p120-catenin (p120) in orchestrating autophagy in macrophages in response to endotoxin stimulation. Depletion of p120 effectively suppressed LPS-induced autophagy in both J774A.1 macrophages and murine bone marrow-derived macrophages. LPS not only elevated the interaction between p120 and L chain 3 (LC3) I/II but also facilitated the association of p120 with mammalian target of rapamycin (mTOR). p120 depletion in macrophages by small interfering RNA reduced LPS-induced dissociation of mTOR and Unc-51-like kinase 1 (ULK1), leading to an increase in the phosphorylation of ULK1. p120 depletion also enhanced LPS-triggered macrophage apoptosis, as evidenced by increased levels of cleaved caspase 3, 7-aminoactinomycin D staining, and TUNEL assay. Notably, inhibiting autophagy reversed the decrease in apoptosis caused by LPS stimulation in macrophages overexpressing p120. Additionally, the ablation of p120 inhibited autophagy and accentuated apoptosis in alveolar macrophages in LPS-challenged mice. Collectively, our findings strongly suggest that p120 plays a pivotal role in fostering autophagy while concurrently hindering apoptosis in macrophages, achieved through modulation of the mTOR/ULK1 signaling pathway in sepsis. This underscores the potential of targeting macrophage p120 as an innovative therapeutic avenue for treating inflammatory disorders.

Novel, soluble 3-heteroaryl-substituted tanshinone mimics attenuate the inflammatory response in murine macrophages

The RNA binding protein Human Antigen R (HuR) has been identified as a main regulator of the innate immune response and its inhibition can lead to beneficial anti-inflammatory effects. To this aim, we previously synthesized a novel class of small molecules named Tanshinone Mimics (TMs) able to interfere with HuR-RNA binding, and that dampen the LPS-induced immune response. Herein, we present a novel series of TMs, encompassing thiophene 3/TM9 and 4/TM10, furan 5/TM11 and 6/TM12, pyrrole 7b/TM13, and pyrazole 8. The furan-containing 5(TM11) showed the greatest inhibitory effect of the series on HuR-RNA complex formation, as suggested by RNA Electromobility Shift Assay and Time-Resolved FRET. Molecular Dynamics Calculation of HuR − 5/TM11 interaction, quantum mechanics approaches and Surface Plasmon Resonance data, all indicates that, within the novel heteroaryl substituents, the furan ring better recapitulates the chemical features of the RNA bound to HuR. Compound 5/TM11 also showed improved aqueous solubility compared to previously reported TMs. Real-time monitoring of cell growth and flow cytometry analyses showed that 5/TM11 preferentially reduced cell proliferation rather than apoptosis in murine macrophages at immunomodulatory doses. We observed its effects on the innate immune response triggered by lipopolysaccharide (LPS) in macrophages, showing that 5/TM11 significantly reduced the expression of proinflammatory cytokines as Cxcl10 and Il1b.

A combined analysis of bulk RNA-seq and scRNA-seq was performed to investigate the molecular mechanisms associated with the occurrence of myocardial infarction

BackgroundMyocardial infarction (MI) induces complex transcriptional changes across diverse cardiac cell types. Single-cell RNA sequencing (scRNA-seq) provides an unparalleled ability to discern cellular diversity during infarction, yet the veracity of these discoveries necessitates confirmation. This investigation sought to elucidate MI mechanisms by integrating scRNA-seq and bulk RNA-seq data.MethodsPublicly available scRNA-seq (GSE136088) and bulk RNA-seq (GSE153485) data from mice MI models were analyzed. Cell types were annotated, and differential expression analysis conducted. Bulk RNA-seq underwent quality control, principal component analysis, and differential expression analysis.ResultsIn scRNA-seq data, the comparison between MI and sham groups unveiled a reduction in endothelial cell populations, but macrophages and monocytes increased. Within fibroblast subgroups, three distinct categories were discerned, with two exhibiting upregulation in MI. Notably, endothelial cells exhibited an elevated expression of genes associated with apoptosis and ferroptosis. In bulk RNA-seq analysis, distinct patterns emerged when comparing MI and sham groups. Specifically, six genes linked to endothelial ferroptosis exhibited heightened expression in MI group, thereby corroborating the scRNA-seq findings. Moreover, the examination of isolated cardiac macrophages from mice MI model revealed increased expression of Spp1, Col1a2, Col3a1, Ctsd, and Lgals3 compared to sham group, thus substantiating the dysregulation of macrophage apoptosis-related proteins following MI.ConclusionMI altered the transcriptomic landscapes of cardiac cells with increased expression of apoptotic genes. Moreover, the upregulation of macrophage apoptosis marker was confirmed within MI models. The presence of endothelial cell depletion and ferroptosis in MI has been demonstrated.

Regulation of Bacillus Calmette-Guérin-induced macrophage autophagy and apoptosis by the AMPK–mTOR–ULK1 pathway

Tuberculosis (TB) is a chronic wasting infectious disease caused by Mycobacterium tuberculosis (MTB) or Mycobacterium bovis that can be transmitted among people and domestic animals. During the development ofTB, macrophages of the innate immune system can act against MTB via autophagy and apoptosis to prevent the spread of the disease. Among the many autophagy regulatory pathways, the adenosine monophosphate (AMP)–activated protein kinase (AMPK)–mammalian rapamycin target protein(mTOR)–Unc-51-like kinase 1(ULK1) pathway has received considerable attention. This study investigates the regulatory role of the AMPK–mTOR–ULK1 pathway in attenuating M. bovis Bacillus Calmette–Guérin (BCG)-induced autophagy and apoptosis in murine monocyte macrophages (RAW264.7). Changes in macrophage autophagy and apoptosis were analyzed using the AMPK activator AICAR and inhibitor Compound C to interfere with the AMPK–mTOR–ULK1 pathway and siRNA to silence the pathway. Consequently, BCG stimulation of macrophages significantly activated the AMPK–mTOR–ULK1 pathway while BCG-induced macrophage AMPK activation promoted macrophage autophagy and apoptosis. Activation of the AMPK–mTOR–ULK1 pathway by AICAR significantly improved autophagy occurrence in BCG-induced macrophages and increased apoptosis while Compound C with siRNA produced opposing effects by attenuating autophagy and apoptosis in BCG-induced macrophages. Thus, the AMPK–mTOR–ULK1 pathway has a dual regulatory role in BCG-induced macrophage autophagy and apoptosis and may have synergistic effects. This study analyzes the mechanism of resistance of host cells to MTB and provides a theoretical basis for new therapeutic strategies and related drug development.

High plasma levels of fortilin are associated with cardiovascular events in patients undergoing coronary angiography.

Excessive apoptosis and its insufficient clearance is characteristic of atherosclerotic plaques. Fortilin has potent antiapoptotic property and is abundantly expressed in atherosclerotic plaques. Fortilin-deficient mice had less atherosclerosis with more macrophage apoptosis. Recently, we reported that plasma fortilin levels were high in patients with coronary artery disease (CAD). However, its prognostic value has not been elucidated. We investigated plasma fortilin levels and major adverse cardiovascular events (MACE) in 404 patients (mean age 68 ± 12 years; 276 males) undergoing coronary angiography for suspected CAD. MACE was defined as cardiovascular death, myocardial infarction, unstable angina, heart failure, stroke, or coronary revascularization. Of the 404 patients, 218 (54%) had CAD. Plasma fortilin levels were higher in patients with CAD than without CAD (median 74.9 vs. 70.9 pg/mL, p < 0.05). During a mean follow-up of 5.7 ± 4.2 years, MACE was observed in 59 (15%) patients. Notably, patients with MACE had higher fortilin levels (median 83.0 vs. 71.4 pg/mL) and more often had fortilin level > 80.0 pg/mL (54% vs. 36%) than those without MACE (p < 0.025). A Kaplan-Meier analysis showed lower event-free survival in patients with fortilin > 80.0 pg/mL than in those with ≤ 80.0 pg/mL (p < 0.001). In multivariate Cox proportional hazards analysis, fortilin level (> 80.0 pg/mL) was an independent predictor of MACE (hazard ratio: 2.29, 95%CI: 1.36-3.85, p < 0.002). Among the 218 patients with CAD, fortilin level was also a significant predictor of MACE (hazard ratio: 2.48; 95%CI: 1.34-4.61, p < 0.005). Thus, high plasma fortilin levels were found to be associated with cardiovascular events in patients with CAD as well as those undergoing coronary angiography.

Two-Pronged Attack: Dual Activation of Fat Reduction Using Near-Infrared-Responsive Nanosandwich for Targeted Anti-Obesity Treatment.

Excessive fat accumulation and chronic inflammation are two typical characteristics of obesity. AMP-activated protein kinase (AMPK), a master regulator of energy metabolism, is involved in adipogenesis, lipogenesis, and inflammation modulation in adipose tissue (AT). Thus, effective lipid reduction and anti-inflammation through AMPK regulation play vital roles in treating obesity. Herein, an anti-obesity nanosandwich is fabricated through attaching polymetformin (PolyMet) onto photothermal agent black phosphorus nanosheets (BP). PolyMet activates AMPK to inhibit adipogenesis, promote browning, and mitigate AT inflammation by decreasing macrophage infiltration, repolarizing macrophage phenotype, and downregulating pro-inflammatory cytokines. Additionally, BP induces lipolysis and apoptosis of adipocytes and macrophages through a photothermal effect. By further functionalization using hyaluronic acid (HA) and MMP2 substrate-linking P3 peptide-modified HA (P3-HA), an enhanced anti-obesity effect is obtained by dual-targeting of P3 and HA, and HA-mediated CD44 poly-clustering after MMP2 cleavage. Upon laser irradiation, the designed nanosandwich (P3-HA/PM@BP) effectively inhibits obesity development in obese mice, increases M2/M1 ratio in AT, reduces the serum levels of cholesterol/triglyceride and improves insulin sensitivity, exhibiting promising research potential to facilitate the clinical development of modern anti-obesity therapies.

Mycophenolate mofetil directly modulates myeloid viability and pro-fibrotic activation of human macrophages.

Mycophenolate mofetil (MMF) is an immunosuppressant used to treat rheumatological diseases, including Systemic Sclerosis (SSc). While MMF is an established inhibitor of lymphocyte proliferation, recent evidence suggests MMF also mediates effects on other cell types. The goal of this study was to determine the effect of MMF on monocytes and macrophages, which have been implicated in SSc pathogenesis. Human monocyte-derived macrophages were cultured with the active MMF metabolite, mycophenolic acid (MPA), and assessed for changes in viability and immuno-phenotype. Guanosine supplementation studies were performed to determine whether MPA-mediated effects were dependent on de novo purine synthesis. The ability of MPA-treated macrophages to induce fibroblast activation was evaluated, and dermal myeloid expression signatures were analyzed in MMF-treated SSc patients. MPA reduced viability and induced apoptosis in monocytes and macrophages at doses (average IC50 = 1.15 µg/ml) within the target serum concentration of MMF patients (1-3µg/ml). These effects were reversed by guanosine supplementation. Low-dose MPA (0.5 µg/ml) attenuated IL-4 or SSc plasma-mediated macrophage activation, and inhibited the ability of SSc plasma-activated macrophages to induce SSc fibroblast activation. Gene expression studies demonstrated significant reductions in dermal myeloid signatures in MMF-responsive SSc patients. For the first time, we demonstrate that MMF inhibits the viability and pro-fibrotic activation of human monocytes and macrophages, which is dependent on de novo purine synthesis. Coupled with myeloid gene expression attenuation following MMF treatment in patients, these results suggest that fibrotic inhibition observed with MMF may be attributable, at least in part, to direct effects on myeloid cells.

Transcriptomics and metabolomics analyses of graphene oxide toxicity on porcine alveolar macrophages

Graphene oxide (GO) is a type of nanomaterial widely used in tissue engineering, photocatalysis, and biomedicine. GO has been found to produce adverse effects on a broad range of cells and tissues. However, the molecular mechanisms underlying GO toxicity still remain to be explored. In this study, using porcine alveolar macrophages as a study model, we explored the toxic effects of GO and performed genome-wide detection of genes and metabolites associated with GO exposure using RNA-seq and liquid chromatograph mass spectrometer techniques. GO exposure significantly inhibited cell viability and induced apoptosis and oxidative stress in porcine alveolar macrophages. Further, GO exposure promoted cellular inflammation by upregulating the expression of pro-inflammatory cytokines (IL-6, IL-8, and IL-12). Transcriptomic analysis of GO-exposed cells revealed 424 differentially expressed genes. Functional enrichment analysis showed that the differentially expressed genes were significantly enriched in the pathways of Ribosome and oxidative phosphorylation (OXPHOS). In addition, metabolic analysis identified 203 differential metabolites, and these metabolites were significantly enriched in biosynthesis of cofactors, purine metabolism, and nucleotide metabolism. Integrative analyses of transcriptome and metabolome showed that OXPHOS was the most significantly enriched pathway and the involved genes were downregulated. This study revealed the toxic effects of GO on porcine alveolar macrophages and provided global insights to the metabolomic and transcriptomic alterations related to GO exposure. The results contributed to our understanding of the molecular mechanism of GO, and may further promote the detection of biomarkers for the prediction and control of GO toxicity.

Mmu-let-7a-5p inhibits macrophage apoptosis by targeting CASP3 to increase bacterial load and facilities mycobacterium survival.

We have been trying to find a miRNA that can specifically regulate the function of mycobacterial host cells to achieve the purpose of eliminating Mycobacterium tuberculosis. The purpose of this study is to investigate the regulation of mmu-let-7a-5p on macrophages apoptosis and its effect on intracellular BCG clearance. After a series of in vitro experiments, we found that mmu-let-7a-5p could negatively regulate the apoptosis of macrophages by targeting Caspase-3. The extrinsic apoptosis signal axis TNFR1/FADD/Caspase-8/Caspase-3 was inhibited after BCG infection. Up-regulated the expression level of mmu-let-7a-5p increase the cell proliferation viability and inhibit apoptosis rate of macrophages, but down-regulated its level could apparently reduce the bacterial load of intracellular Mycobacteria and accelerate the clearance of residual Mycobacteria effectively. Mmu-let-7a-5p has great potential to be utilized as an optimal candidate exosomal loaded miRNA for anti-tuberculosis immunotherapy in our subsequent research.

Macrophage targeted graphene oxide nanosystem synergize antibiotic killing and host immune defense for Tuberculosis Therapy

Tuberculosis (TB), a deadly disease caused by Mycobacterium tuberculosis (Mtb) infection, remains one of the top killers among infectious diseases worldwide. How to increase targeting effects of current anti-TB chemotherapeutics and enhance anti-TB immunological responses remains a big challenge in TB and drug-resistant TB treatment. Here, mannose functionalized and polyetherimide protected graphene oxide system (GO-PEI-MAN) was designed for macrophage-targeted antibiotic (rifampicin) and autophagy inducer (carbamazepine) delivery to achieve more effective Mtb killings by combining targeted drug killing and host immunological clearance. GO-PEI-MAN system demonstrated selective uptake by in vitro macrophages and ex vivo macrophages from macaques. The endocytosed GO-PEI-MAN system would be transported into lysosomes, where the drug loaded Rif@Car@GO-PEI-MAN system would undergo accelerated drug release in acidic lysosomal conditions. Rif@Car@GO-PEI-MAN could significantly promote autophagy and apoptosis in Mtb infected macrophages, as well as induce anti-bacterial M1 polarization of Mtb infected macrophages to increase anti-bacterial IFN-γ and nitric oxide production. Collectively, Rif@Car@GO-PEI-MAN demonstrated effectively enhanced intracellular Mtb killing effects than rifampicin, carbamazepine or GO-PEI-MAN alone in Mtb infected macrophages, and could significantly reduce mycobacterial burdens in the lung of infected mice with alleviated pathology and inflammation without systemic toxicity. This macrophage targeted nanosystem synergizing increased drug killing efficiency and enhanced host immunological defense may be served as more effective therapeutics against TB and drug-resistant TB.

Blockade of IKK signaling induces RIPK1-independent apoptosis in human macrophages.

Regulated cell death in response to microbial infection plays an important role in immune defense and is triggered by pathogen disruption of essential cellular pathways. Gram-negative bacterial pathogens in the Yersinia genus disrupt NF-κB signaling via translocated effectors injected by a type III secretion system, thereby preventing induction of cytokine production and antimicrobial defense. In murine models of infection, Yersinia blockade of NF-κB signaling triggers cell-extrinsic apoptosis through Receptor Interacting Serine-Threonine Protein Kinase 1 (RIPK1) and caspase-8, which is required for bacterial clearance and host survival. Unexpectedly, we find that human macrophages undergo apoptosis independently of RIPK1 in response to Yersinia or chemical blockade of IKKβ. Instead, IKK blockade led to decreased cFLIP expression, and overexpression of cFLIP contributed to protection from IKK blockade-induced apoptosis in human macrophages. We found that IKK blockade also induces RIPK1 kinase activity-independent apoptosis in human T cells and human pancreatic cells. Altogether, our data indicate that, in contrast to murine cells, blockade of IKK activity in human cells triggers a distinct apoptosis pathway that is independent of RIPK1 kinase activity. These findings have implications for the contribution of RIPK1 to cell death in human cells and the efficacy of RIPK1 inhibition in human diseases.

MiR-155 enhances apoptosis of macrophage through suppressing PI3K-AKT activation in Pseudomonas aeruginosa keratitis

Keratitis induced by Pseudomonas aeruginosa (P. aeruginosa) is an acute and serious corneal inflammation. As a family of gene regulators, miRNAs play a crucial role in modulating host response after microbial invasion. However, their functions in P. aeruginosa keratitis remain largely unclear. In the present study, we demonstrated that miR-155 expression was significantly increased in macrophages and corneal tissue after P. aeruginosa infection. In vivo studies demonstrated that mice with miR-155 knockdown displayed more resistance to P. aeruginosa keratitis, with a lower bacterial burden. In addition, in vitro and in vivo studies indicated that miR-155 enhanced apoptosis of macrophages after P. aeruginosa infection, and resulted in a susceptible phenotype of P. aeruginosa keratitis. Moreover, miR-155 induced apoptosis through reducing activation of PI3K-Akt signaling pathway. Our data provided evidence of miR-155 mediated apoptosis of macrophage in P. aeruginosa keratitis, which may be an underlying target for the therapy of P. aeruginosa keratitis and other infectious diseases.

Reactive Oxygen Species-Responsive Nanoparticle Delivery of Small Interfering Ribonucleic Acid Targeting Olfactory Receptor 2 for Atherosclerosis Theranostics.

Atherosclerosis (AS) is a chronic inflammatory disorder characterized by arterial intimal lipid plaques. Small interfering ribonucleic acid (siRNA)-based therapies, with their ability to suppress specific genes with high targeting precision and minimal side effects, have shown great potential for AS treatment. However, targets of siRNA therapies based on macrophages for AS treatment are still limited. Olfactory receptor 2 (Olfr2), a potential target for plaque formation, was discovered recently. Herein, anti-Olfr2 siRNA (si-Olfr2) targeting macrophages was designed, and the theranostic platform encapsulating si-Olfr2 to target macrophages within atherosclerotic lesions was also developed, with the aim of downregulating Olfr2, as well as diagnosing AS through photoacoustic imaging (PAI) in the second near-infrared (NIR-II) window with high resolution. By utilization of a reactive oxygen species (ROS)-responsive nanocarrier system, the expression of Olfr2 on macrophages within atherosclerotic plaques was effectively downregulated, leading to the inhibition of NLR family pyrin domain containing 3 (NLRP3) inflammasome activation and interleukin-1 β (IL-1β) secretion, thereby reducing the formation of atherosclerotic plaques. As manifested by decreased Olfr2 expression, the lesions exhibited a significantly alleviated inflammatory response that led to reduced lipid deposition, macrophage apoptosis, and a noticeable decrease in the necrotic areas. This study provides a proof of concept for evaluating the theranostic nanoplatform to specifically deliver si-Olfr2 to lesional macrophages for AS diagnosis and treatment.

Mycoplasma hyopneumoniae inhibits the unfolded protein response to prevent host macrophage apoptosis and M2 polarization.

Enzootic pneumonia caused by Mycoplasma hyopneumoniae (M. hyopneumoniae) has inflicted substantial economic losses on the global pig industry. The progression of M. hyopneumoniae induced-pneumonia is associated with lung immune cell infiltration and extensive proinflammatory cytokine secretion. Our previous study established that M. hyopneumoniae disrupts the host unfolded protein response (UPR), a process vital for the survival and immune function of macrophages. In this study, we demonstrated that M. hyopneumoniae targets the UPR- and caspase-12-mediated endoplasmic reticulum (ER)-associated classical intrinsic apoptotic pathway to interfere with host cell apoptosis signaling, thereby preserving the survival of host tracheal epithelial cells (PTECs) and alveolar macrophages (PAMs) during the early stages of infection. Even in the presence of apoptosis inducers, host cells infected with M. hyopneumoniae exhibited an anti-apoptotic potential. Further analyses revealed that M. hyopneumoniae suppresses the three UPR branches and their induced apoptosis. Interestingly, while UPR activation typically drives host macrophages toward an M2 polarization phenotype, M. hyopneumoniae specifically obstructs this process to maintain a proinflammatory phenotype in the host macrophages. Overall, our findings propose that M. hyopneumoniae inhibits the host UPR to sustain macrophage survival and a proinflammatory phenotype, which may be implicated in its pathogenesis in inducing host pneumonia.

Abstract Tu135: FYCO1 ameliorates cardiac remodeling in response to ischemia by amplifying autophagic flux

Background: Dysregulation of autophagy is known to have detrimental effects on cardiac remodeling. FYCO1, a novel regulator of autophagy mediates the transport of autophagosomes, thereby amplifying autophagic flux. Here, we investigate the impact of FYCO1 on myocardial healing via autophagy modulation. Methods: FYCO1-Tg mice crossed with RFP-EGFP-LC3 reporter mice (FYCO1-TGxRGFP) were utilized to analyze autophagic flux via confocal microscopy following permanent ligation of the left anterior descending artery (LAD) to induce myocardial ischemia. Autophagy activity was evaluated via Western blot of p62 and LC3 II. Macrophage infiltration and apoptosis was assessed by immunofluorescence microscopy, while Masson's Trichrome staining detected fibrosis and infarct size. Cardiac function was determined by echocardiography. Results: FYCO1-TGxRGFP mice displayed significantly enhanced autophagic flux post MI, as evidenced by increased p62 (WT LAD: 2,9±0,4; TG LAD:4,0±0,5) and LC3 II (WT LAD: 1,6±0,2; TG LAD:14,0±1,4) protein levels. Confocal microscopy revealed an accumulation of autolysosomes in WT mice 30 days post MI (WT autophagosomes: 1±0,4; WT autolysosomes: 38,6±3,8), contrasting with the sustained ratio of autophagosomes to autolysosomes in FYCO1-TGxRGFP mice (TG autophagosomes:43,3±5,1; TG autolysosomes: 73,5±12,4), implying the preservation of adequate autophagic flux. FYCO1 overexpression reduced macrophage infiltration into the border zone post MI (WT LAD: 132,4 ±14,3; TG LAD: 18,91±3,7 and inhibited apoptosis induction shown by cleaved caspase7 activity (WT LAD: 64,6±10,1; TG LAD: 9,2±2,1). Moreover, FYCO1-TGxRGFP mice exhibited significantly reduced fibrosis (WT:19,6±1,8%; TG:10,6±1,6%) and infarct size (WT:34,5 ±3,9%; TG:5,5±2%) in the left ventricle (LV), accompanied by marked improvements in global heart function post MI (EF WT: 32,7±3,2%; TG: 56,4±3,9%) compared to WT mice. Conclusion: In conclusion, FYCO1 mitigates adverse consequences of ischemic injury via induction of autophagy and a previously unrecognized role in modulating inflammation in response to myocardial infarction.

JDF promotes the apoptosis of M2 macrophages and reduces epithelial-mesenchymal transition and migration of liver cancer cells by inhibiting CSF-1/PI3K/AKT signaling pathway

BackgroundThe interaction between cancer cells and the tumor microenvironment is of critical importance in liver cancer. Jiedu Granule formula (JDF) has been shown to minimize the risk of recurrence and metastasis following liver cancer resection. Investigating the mechanism underlying the therapeutic effects of JDF can extend its field of application and develop novel treatment approaches. MethodsWe established a rat liver orthotopic transplantation tumor model, and recorded the prognostic effects of JDF adjuvant therapy on the recurrence and metastasis of liver cancer. Liver and lung tissues were collected for immunofluorescence staining and H&E staining, respectively. In addition, THP-1 cells were incubated with PMA and IL-4 to induce them to differentiate into M2 macrophages. CSF-1 expression was knocked down using lentivirus to determine the function of CSF-1. Liver cancer cells were cultured with a conditioned medium (CM) or co-cultured with macrophages. Cell viability was determined using the MTT assay. The levels of CSF-1, CSF-1R, E-cadherin, N-cadherin, PI3K, AKT, and cleaved caspase-3 were detected using ELISA, Western blotting and qPCR. The ability of cells to migrate was assessed using cell scratch and transwell assays. Apoptosis was evaluated using flow cytometry. ResultsThe JDF treatment decreased the risk of liver cancer metastasis after surgery and the infiltration of CD206/CD68 cells in liver cancer tissue. In cell experiments, JDF showed effects in suppressing M2 macrophages activity and downregulating the expression of CSF-1 and CSF-1R. The concentration of CSF-1 in the supernatant was also lower in the JDF-treated group. Futhermore, M2-CM was found to promote cancer cell migration and epithelial-mesenchymal transition (EMT); however, these effects were weakened after administering JDF. Knocking down endogenous CSF-1 in M2 macrophages resulted in a comparable suppression of cancer cell migration and EMT. Additionally, JDF treatment inhibited activation of the PI3K/AKT pathway, thus promoting the apoptosis of M2 macrophages. ConclusionsTreatment with JDF reduced the EMT and migratory capacity of liver cancer cells, which might be attributed to the inhibition of M2 macrophage infiltration and interruption of the CSF-1/PI3K/AKT signaling pathway. This mechanism may hold significant implications for mitigating the risk of metastatic spread in the aftermath of hepatic surgery.

Administration of an antibody against apoptosis inhibitor of macrophage prevents aortic aneurysm progression in mice

Apoptosis inhibitor of macrophage (AIM) is known to induce apoptosis resistance in macrophages and to exacerbate chronic inflammation, leading to arteriosclerosis. The role of AIM in aortic aneurysm (AA) remains unknown. This study examined the effects of an anti-AIM antibody in preventing AA formation and progression. In apolipoprotein E-deficient mice, AA was induced by subcutaneous angiotensin II infusion. Mice were randomly divided into two groups: (i) AIM group; weekly anti-murine AIM monoclonal antibody injection (n = 10), and (ii) IgG group; anti-murine IgG antibody injection as control (n = 14). The AIM group, compared with the IgG group, exhibited reduced AA enlargement (aortic diameter at 4 weeks: 2.1 vs. 2.7 mm, respectively, p = 0.012); decreased loss of elastic lamellae construction; reduced expression levels of IL-6, TNF-α, and MCP-1; decreased numbers of AIM-positive cells and inflammatory M1 macrophages (AIM: 1.4 vs. 8.0%, respectively, p = 0.004; M1 macrophages: 24.5 vs. 55.7%, respectively, p = 0.017); and higher expression of caspase-3 in the aortic wall (22.8 vs. 10.5%, respectively, p = 0.019). Our results suggest that administration of an anti-AIM antibody mitigated AA progression by alleviating inflammation and promoting M1 macrophage apoptosis.