Apoptotic Macrophages Research Articles

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.

Multimodal Imaging-Assisted Intravascular Theranostic Photoactivation on Atherosclerotic Plaque.

Atherosclerosis is a chronic inflammatory disease causing a fatal plaque rupture, and its key aspect is a failure to resolve inflammation. We hypothesize that macrophage-targeted near-infrared fluorescence emitting photoactivation could simultaneously assess macrophage/lipid-rich plaques in vivo and facilitate inflammation resolution. We fabricated a Dectin-1-targeted photoactivatable theranostic agent through the chemical conjugation of the near-infrared fluorescence-emitting photosensitizer chlorin e6 and the Dectin-1 ligand laminarin (laminarin-chlorin e6 [LAM-Ce6]). Intravascular photoactivation by a customized fiber-based diffuser after administration of LAM-Ce6 effectively reduced inflammation in the targeted plaques of atherosclerotic rabbits in vivo as serially assessed by dual-modal optical coherence tomography-near-infrared fluorescence structural-molecular catheter imaging after 4 weeks. The number of apoptotic macrophages peaked at 1 day after laser irradiation and then resolved until 4 weeks. Autophagy was strongly augmented 1 hour after the light therapy, with the formation of autophagolysosomes. LAM-Ce6 photoactivation increased the terminal deoxynucleotidyl transferase dUTP (deoxyuridine triphosphate) nick end labeling/RAM11 (rabbit monocyte/macrophage antibody)- and MerTK (c-Mer tyrosine kinase)-positive cells in the plaques, suggesting enhanced efferocytosis. In line with inflammation resolution, photoactivation reduced the plaque burden through fibrotic replacement via the TGF (transforming growth factor)-β/CTGF (connective tissue growth factor) pathway. Optical coherence tomography-near-infrared fluorescence imaging-guided macrophage Dectin-1-targetable photoactivation could induce the transition of macrophage/lipid-rich plaques into collagen-rich lesions through autophagy-mediated inflammation resolution and TGF-β-dependent fibrotic replacement. This novel strategy offers a new opportunity for the catheter-based theranostic strategy.

Life cycle of macrophages in atherosclerotic inflammation progression and resolution: mediators and interventions (narrative review)

Abstract As one of the pathological causes of coronary heart disease, atherosclerosis poses a major threat to human health. Macrophages play an important role in regulating atherosclerotic disease progression. Specifically, atherosclerotic inflammation is initiated when low-density lipoproteins infiltrate the subcutaneous area and are phagocytosed by macrophages, leading to foam cell formation. The subsequent inflammation progression or resolution depends on the delicate balance between proinflammatory and anti-inflammatory mediators. In cases where proinflammatory factors dominate, macrophages tend to activate the pyroptosis and necrosis pathways, resulting in the release of intracellular damage-associated molecular patterns and promoting necrotic core formation and plaque progression. Conversely, when anti-inflammatory factors prevail, macrophages engage in autophagy-mediated intracellular lipid metabolism while inhibiting inflammation progression through the efferocytosis of apoptotic cells. The regulatory function of macrophages in atherosclerosis can also be understood from the perspective of their life cycles. Lipid retention within the arterial intima and its subsequent uptake by macrophages are the characteristic pathological hallmarks of atherosclerosis. As pivotal effector cells in this process, macrophages with their distinctive performances decisively determine the progression and resolution of atherosclerotic inflammation. The complete life cycle of macrophages in atherosclerotic plaques encompasses chemotaxis, infiltration, polarization, uptake of lipoproteins for metabolic efflux, foam cell formation, lipid overload, and various forms of programmed necrosis, including autophagy, pyroptosis, apoptosis, necrosis, and efferocytosis, to facilitate the removal of apoptotic macrophages and limit inflammation progression. The behavior of macrophages in atherosclerosis has rarely been comprehensively addressed in previous review articles. This article provides an extensive overview of the entire life cycle of macrophages following their response to atherosclerotic inflammation and the impact of regulatory factors on inflammation progression and resolution. Considering that macrophages play a pivotal role in the inflammatory response associated with atherosclerosis, targeting the regulation of their life cycle holds promise for therapeutic interventions against atherosclerosis-related cardiovascular diseases.

The relationships among Leishmania infantum and phyllostomid bats assessed by histopathological and molecular assays

Bats have been reported as reservoir host of Leishmania spp. worldwide, mostly by molecular detection. However, it is still unclear whether bats act as reservoirs of Leishmania infantum to sandflies vectors. In this sense, the investigation of amastigotes forms in the target organs, and the characterization of their associated inflammation, may help to clarify the epidemiological importance of bats in endemic areas for leishmaniasis. The aim of this work was to investigate the host-parasite relationships under microscopic evaluation and predict the epidemiological role of two phyllostomid bats species naturally infected by L. infantum in an endemic area for human leishmaniasis. Fragments of skin, liver and spleen of L. infantum positive and negative bats (Artibeus planirostris and Carollia perspicillata) by qPCR, were studied by histological and immunohistochemical techniques. Both groups, positive and negative, did not show differences in the histopathological study, presenting only discrete tissue changes. Liver and skin showed mild inflammatory reactions. Findings on spleen consisted of reactivity of the lymphoid follicles, expressive presence of apoptotic cells and macrophages containing abundant phagocytic cells debris. We did not find amastigote forms in tissues by histological and IHC techniques in positive qPCR bats. Our results allow us to hypothesize that phyllostomid bats seem to have an important role in reducing the risk of transmission, possibly acting as dead-end host.

Quercetin ameliorates Helicobacter pylori-induced gastric epithelial cell injury by regulating specificity protein 1/lipocalin 2 axis in gastritis.

Helicobacter pylori (HP) infection is the main cause of most cases of gastritis. Quercetin has been shown to have anti-inflammatory, anti-bacterial, and antiviral activities and has been demonstrated to be involved in HP-induced gastric mucosa injury. Moreover, the secretory protein lipocalin-2 (LCN2) was elevated in HP-infected gastric mucosa. Thus, this work aimed to study the interaction between quercetin and LCN2 in HP-triggered gastric injury during gastritis. Human gastric epithelial cell line GES-1 cells were exposed to HP for functional experiments. Cell viability, apoptosis, and inflammation were evaluated by cell counting kit-8, flow cytometry, and enzyme-linked immunosorbent assay, respectively. Levels of genes and proteins were tested using quantitative reverse transcription polymerase chain reaction and western blotting analyses. The interaction between LCN2 and specificity protein 1 (SP1) was validated using chromatin immunoprecipitation assay and dual-luciferase reporter assay. Thereafter, we found quercetin treatment suppressed HP-induced GES-1 cell apoptotic and inflammatory injury and macrophage M1 polarization. LCN2 was highly expressed in HP-infected gastritis patients and HP-infected GES-1 cells, while quercetin reduced LCN2 expression in HP-infected GES-1 cells; moreover, LCN2 knockdown reversed HP-induced GES-1 cell injury and macrophage M1 polarization, and forced expression of LCN2 abolished the protective effects of quercetin on GES-1 cells under HP infection. Mechanistically, SP1 bound to LCN2 promoter and promoted its transcription. Also, SP1 overexpression counteracted the functions of quercetin on HP-stimulated GES-1 cells. In all, quercetin ameliorated HP-induced gastric epithelial cell apoptotic and inflammatory injuries, and macrophage M1 polarization via the SP1/LCN2 axis.

Abstract B033: Multiplex analysis of the tumor immune microenvironment during treatment with atezolizumab/pelareorep/letrozole reveals novel immune-tumor interactions

Abstract Background: Pelareorep (pela) is a non-modified intravenously administered oncolytic reovirus exhibiting effective tumor suppression through both innate and adaptive immune responses, as well as direct tumor lysis. Findings from the AWARE-1 window of opportunity study previously demonstrated the synergistic potential of combining pela with atezolizumab (atezo), showcasing promising immunological reactions within tumors of early breast cancer (eBC) patients. To gain deeper insights into the complex tumor immune microenvironment (TiME) pre- and post-treatment, we employed imaging mass cytometry (IMC) to conduct high-dimensional, single-cell analysis of tissue samples. Methods: Patients (n=10) received pela (days 1, 2 and 8, 9), atezolizumab (day 3), and letrozole (day 1 to 21). Tumor biopsies (FFPE samples) were collected pre-treatment on day 3 (prior to atezolizumab administration) and on day ~21 (surgical excision). Samples from 8 out of 10 patients were subjected to IMC with a comprehensive panel of 37 antibodies for single cell investigation of TiME changes. Tumor, proliferation, T cell, macrophage, NK, and immunoregulatory markers were included in the panel. Results: Our first pass analysis of IMC images, showed an increase in proliferating (Ki67+) cytotoxic T cells adjacent to apoptotic (caspase 3+) tumor cells post-treatment (both D3 and D21) samples indicating immunogenic cell death. Conversely, we observed a decrease in Ki67+ and ER-positive tumor cells after treatment indicating decreased tumor cell proliferation and hormone expression. In 3 out of 8 patients, treatment was associated with a shift in monocyte/macrophages from an M1 (CD68+/CD163-) to M2 (CD68+/CD163+) phenotype associated with significant tumor infiltration. Surprisingly, in these patients we also noted a significant increase in apoptotic M2 macrophages on D21. Consistent with the known immune priming effects of pela, both PD-1 and PD-L1 increased on D3. Subsequently, on day 21, post atezo, tumor PD-L1 decreased on tumor cells while PD-1 expression persisted. Pela induced IDO expression on both tumor and monocyte/macrophage cells. Unlike its effect on PD-L1, atezo did not appear to attenuate IDO expression on day 21. We were also able to detect a treatment related increase in immune cell perivascular localization on D3 providing further evidence of TiME priming. Conclusions: The immune system plays a crucial role in regulating cancer progression; however, our understanding of immune interactions with tumors remains limited. Our study utilizing IMC revealed complex immune-tumor interactions changes during treatment with atezo/pela/letrozole. These findings provide valuable insights into the intricate dynamics of the TiME and may inform new therapeutic combination approaches in breast cancer treatment. Citation Format: Homa Dadrastoussi, Julian Olea, Eduardo Fernandez Hernandez, Hugo Lara Martinez, Kaijin Wu, Houra Loghmani, Thomas Heineman, Richard Trauger, Matt Coffey, Akil Merchant, Kevin Kelly. Multiplex analysis of the tumor immune microenvironment during treatment with atezolizumab/pelareorep/letrozole reveals novel immune-tumor interactions [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2023 Oct 1-4; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Immunol Res 2023;11(12 Suppl):Abstract nr B033.

Interaction between mesenchymal stromal cells and tuberculous mycobacteria in vitro

The objective: in an in vitro experiment, we compared phagocytic parameters of mesenchymal stromal cells (MSCs) and macrophages to tuberculous mycobacteria, assessed the ability of MSCs and macrophages to lyse mycobacteria or maintain their intracellular growth, their effect on formation of phenotypic drug resistance of mycobacteria, as well as the effect of tuberculous mycobacteria on the type of MSCs cell death.Subjects and Methods. Balb/c male mice, aged 6 to 8 weeks, were used in the experiment. Bone marrow MSCs were obtained from femurs and tibias by further cultivation, peritoneal macrophages were elicited with 4% alpha-glucan. The intracellular content of mycobacteria was counted using a confocal microscope with x 400 magnification. Susceptibility of mycobacteria to isoniazid and development of phenotypic drug resistance after culturing MSCs and macrophages with MTB on Lowenstein–Jensen medium was assessed by counting CFU. In 5 days after the infection, the number of apoptotic and necrotic MSCs and macrophages was determined by a flow cytometer.Results. On Day 1, the total number of phagocytosed MTB, as well as the number of phagocytic-active macrophages, exceeds the corresponding figures for MSCs more than twice. MSCs phagocytize tuberculous mycobacteria in a smaller amount, but MTB reproduces in them more actively: the number of CFU after 7 days of cell cultivation with MTB exceeded the corresponding parameter by almost 50 times after 24 hours of cultivation. In cultures of infected MSCs cultivated for 7 days, regardless of the presence of isoniazid, there was a rapid growth of tuberculous mycobacteria. On Day 5 after infection of macrophage culture with tuberculous mycobacteria, the number of necrotic cells was 2.7 times greater than that of uninfected necrotic macrophages, but the number of apototic cells in these groups differed slightly. In the culture of MSCs, there were 8.5 times more infected nectrotic cells versus uninfected necrotic MSCs, and the number of necrotic MSCs was 4.5 times higher than the number of MSCs with apoptosis, while in the culture of infected macrophages, the number of necrotic cells was the same as number of apoptotic cells. Unlike macrophages, treatment of MSCs with isoniazid did not inhibit the intracellular proliferation of MTB.Conclusion. MSCs have the ability to phagocytose mycobacteria, but they do it less actively than macrophages and, unlike macrophages, they are not able to restrain the reproduction of tuberculous mycobacteria. Mycobacteria have phenotypic drug resistance in MSCs. In MSCs, when infected with tuberculous mycobacteria, there is a pronounced shift towards necrosis in the type of cell death, which can lead to dissemination of MTB and development of local destructive changes.

Defective efferocytosis by aged macrophages promotes STING signaling mediated inflammatory liver injury

Aged livers have shown aggravated liver ischemia and reperfusion (IR) injury. Timely efferocytosis of apoptotic cells is a key mechanism for avoiding excessive inflammation and tissue injury. Here, we investigated the alteration of efferocytosis by aged macrophages and its role in regulating macrophage STING (stimulator of interferon genes) signaling and liver IR injury. Aged and young mice were subjected to liver partial IR model. Liver injury and inflammation were measured. Efferocytosis by aged macrophages and the underlying regulatory mechanism were analyzed as well. Aged macrophages exhibited impaired efferocytosis with decreased MerTK (c-mer proto-oncogene tyrosine kinase) activation, which was reversed by treatment of the MerTK CRISPR activation plasmid. Increased MerTK cleavage by ADAM17 (a disintegrin and metalloproteinase 17) due to enhanced ROS (reactive oxygen species) levels contributed to defective efferocytosis by aged macrophages. MerTK activation by suppressing ADAM17 or ROS improved aged macrophage efferocytosis, leading to reduced inflammatory liver injury. Moreover, increased apoptotic hepatocytes, DNA accumulation, and macrophage STING activation were observed in aged ischemic livers. Improvement in efferocytosis by aged macrophages via MerTK activation suppressed STING activation and inflammatory liver injury. Our study demonstrates that aging suppresses MerTK- mediated macrophage efferocytosis to promote macrophage STING activation and inflammatory liver IR injury, suggesting a new mechanism and potential therapy to promote inflammation resolution and efferocytosis in aged livers.

Abstract 74: Osteoblasts efferocytosis in bone marrow microenvironment induces osteoblasts’ senescence and apoptosis

Abstract Increased cellular apoptosis is commonly seen in myelodysplastic syndrome (MDS), but how clearance of apoptotic cells, especially neutrophils, impacts disease progression is unknown. Moreover, while dysfunction of bone marrow (BM) stromal cell populations has been described in patients with MDS, the mechanisms inducing stromal defects are not well understood. Our laboratory found that BM macrophages are defective in a murine model of MDS (Vav-Nup98 HoxD13, aka NHD13), with decreased efferocytotic rates and increased inflammatory mediators. The increased apoptotic burden and defective macrophages would be expected to recruit non-professional phagocytes (osteoblasts) in the bone and bone marrow. Based on these data, we hypothesize that OBs (osteoblasts) participate in the clearance of apoptotic cells in the bone marrow microenvironment (BMME) to maintain BM homeostasis. However, the increased osteoblastic efferocytosis burden in MDS contributes to OBs’ dysfunction as a mechanism of MDS-dependent BMME disruption that may lead to MDS progression. We isolated from wild-type (B6) mice bone-associated cells (BAC), which are enriched with OBs. In vitro co-culture with labeled human end-stage neutrophils at 1:1 and 1:10 (OB: PMN) increased OB apoptosis, while senescence was increased only in high-dose PMN co-culture. We found that these changes were cell-autonomous. In vivo injection of neutrophils into WT mice showed around 5-10% efferocytosis rate in OBs. In efferocytotic OBs, apoptosis rates were significantly increased. Together, these data show that OBs participate in efferocytosis and that efferocytosis disrupts OBs, causing apoptosis and senescence. To mimic the MDS microenvironment, we used a genetic model where one of the Isocitrate dehydrogenase 2 (IDH2) mutations found in MDS (R140Q) is targeted to hematopoietic cells via the Vav promoter (IDH2R140Q mice) as well as NHD13 mice, representing early and late MDS BMME respectively. We observed that 6-to-8 months old NHD13 mice experienced more end-stage neutrophils[CL1] in the BMME (4.6% rate of end-stage neutrophils in WT compared to 9.5% rate in NHD13 mice), where OB apoptosis was also increased in vivo. In IDH2R140Q mice at 3 months of age, we confirmed the presence of mutated IDH2 in bone marrow cells by increased intracellular levels of the oncometabolite 2-hydroxyglutarate. At this early time point, IDH2R140Q mice did not have increased rates of apoptosis in CD45+ cells, and we found no increase in OB apoptosis. Analysis of IDH2R140Q mice at later time points is ongoing. Our data identify novel mechanisms to explain the relationship between defective myeloid cells and OB apoptosis in MDS that may contribute to the inflammatory MDS microenvironment, impact disease progression, and serve as future therapeutic approaches for MDS patients. Citation Format: Chunmo Chen, Emily R. Quarato, Yuko Kawano, Noah A. Salama, Hiroki Kawano, Michael W. Becker, Jeevisha Bajaj, Laura Calvi. Osteoblasts efferocytosis in bone marrow microenvironment induces osteoblasts’ senescence and apoptosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 74.

Desipramine enhances the stability of atherosclerotic plaque in rabbits monitored with molecular imaging.

Acid sphingomyelinase (ASM) promotes atherogenesis and acute cardiovascular events. We previously demonstrated ASM inhibitor desipramine attenuated oxidized-LDL-induced macrophage apoptosis in vitro. Here, we aim to determine whether ASM-mediated apoptosis in plaque improves stability in vivo. In this study, rabbits with abdominal aorta balloon injury and a 12-week high-cholesterol diet (HCD) were used to simulate an atherosclerotic plaque model. Atherosclerotic rabbits received oral administration of saline (Control group), atorvastatin (Ator group), or desipramine (DES group). ASM activity and ceramide level were measured by ultra-performance liquid chromatography (UPLC). Plaque morphology was assessed by histochemistry and immunohistochemistry. Apoptosis was evaluated by SPECT/CT imaging of 99mTc-duramycin uptake and TUNEL. We found that increasing ASM activity and ceramide level in atherosclerotic rabbits was abated by additional atorvastatin and desipramine treatment. Meanwhile, the DES and Ator groups were similar in plaque stability, with smaller plaque size, areas of macrophages, higher smooth muscle cell content, and decreased apoptosis and matrix metalloproteinase (MMP) activities relative to the Control group. 99mTc-duramycin uptake of rabbit aorta was significantly higher in Control than in the Normal group, while it was reduced by desipramine and atorvastatin administration. Moreover, the uptake of 99mTc-duramycin positively correlated with apoptotic cell number, macrophage infiltration, and plaque instability. The present study demonstrated that desipramine exerted plaque-stabilizing effects partially by suppressing apoptosis and MMP activity in a rabbit model. And 99mTc-duramycin SPECT/CT imaging allowed noninvasively monitoring of atherosclerotic disease and evaluation of anti-atherosclerotic therapy.

Malaria-derived exosomes exacerbate liver injury during blood stage of Plasmodium berghei infection

Liver injury is a common clinical feature of Plasmodium spp. infection and contributes to multi-organ failure of severe malaria. Malaria-derived exosomes (MD-Exos) have recently engaged as key mediators in parasite-host interactions, modulating the subsequent pathogenic process. However, the role of MD-Exos in malaria-related liver injury and the underlying mechanisms remain unclear. Herein, exosomes from C57BL/6 mice infected with or without P. berghei ANKA serum (namely inf-Exos or un-Exos) were isolated and characterized by transmission electron microscopy, western blotting, and nanoparticle tracking analysis. The miRNAs profiling between inf-Exos and un-Exos were generated using RNA-seq and qPCR. The functions of inf-Exos on liver injury were investigated after two types of exosomes injected into mice intravenously (i.v.), by examining histopathological and apoptotic changes, macrophage polarization, and pro-inflammatory response. The infected red blood cells-stimulated mouse Raw264.7 macrophage cells targeted by inf-Exos or un-Exos were cultured for further study and verification the potential mechanisms. We found that both inf-Exos and un-Exos displayed a typical cup-shaped structure with a diameter of 60–200 nm, and had a positive expression of exosomal markers (e.g., CD9, CD63, and CD81). Compared with infected control mice, the treatment of inf-Exos but not un-Exos dramatically enhanced peripheral blood parasitemia and ECM incidence, exacerbated liver histopathological damage, elevated numbers of liver apoptotic cells, CD68+and CD86+ macrophages. The CD68+-TREM-1+ macrophages in liver tissues and the mRNA levels of pro-inflammatory cytokines (e.g., iNOS, TNF-α, IL-1β, and IL-6) were increased by inf-Exos treatment in vivo. Meanwhile, the treatment of inf-Exos resulted in a substantial increase of the mRNA levels of CD86, iNOS, TNF-α, IL-1β, and IL-6, but led to a remarkable decrease of Bcl-6 and SOCS-1 in Raw264.7 cells stimulated with iRBC in vitro. Notably, compared to un-Exos, five types of miRNAs (including miR-10a-5p, miR-10b-5p, miR-155–5p, miR-205–5p, and miR-21a-5p), that were previously reported to target Bcl-6 or SOCS-1, present higher abundance on inf-Exos, as demonstrated by RNA-seq and qPCR. Collectively, our data suggest that inf-Exos exacerbate malaria-induced liver pathology via triggering excessive pro-inflammatory response and promoting macrophage M1 polarization. Our findings will provide new insights into the roles of inf-Exos in malaria parasite-host interaction and pathogenesis of liver injury.

Abstract 14622: Macrophage Dectin-1 Targeted Photoactivation Promotes Inflammation Resolution With Stabilization of the High-Risk Atheroma as Serially Assessed by Intravascular OCT-NIRF Imaging

Backgrounds: Atherosclerosis is a chronic inflammatory disease causing a fatal plaque rupture and its key aspect is a failure to resolve inflammation. We hypothesized that macrophage targeted near-infrared fluorescence (NIRF) emitting photoactivation could simultaneously in vivo assess the inflamed high-risk plaques and facilitate the inflammation resolution. Method and results: We newly synthesized a Dectin-1 targeted photoactivatable theranostic agent by the chemical conjugation of photosensitizer chlorin e6 (Ce6) and Dectin-1 ligand laminarin (LAM-Ce6). Intravascular photoactivation through a customized fiber-based diffuser effectively reduced inflammation in the targeted plaques 4 weeks after LAM-Ce6 administration as serially assessed by dual-modal optical coherence tomography (OCT)-NIRF catheter imaging. The number of TUNEL-positive apoptotic macrophages peaked at 1 day after laser irradiation, and then resolved until 4 weeks. One hour after the light therapy, autophagy was strongly augmented with the formation of autophagolysosomes revealed by co-localization of enhanced microtubule-associated protein light-chain 3 (LC3) and lysosome-associated membrane protein 2 (LAMP2) expressions in the plaques (Figure). LAM-Ce6 photoactivation increased the TUNEL/RAM11- and MerTK-positive cells in the plaques, suggestive of enhanced efferocytosis. In line with inflammation resolution, photoactivation reduced the plaque burden with collagen-rich fibrotic replacement via TGF-β pathway confirmed by corroborative immunostainings ( p <0.01). Conclusions: OCT-NIRF imaging-guided macrophage Dectin-1 targetable photoactivation could stabilize the inflamed high-risk plaques by autophagy-mediated inflammation resolution and TGF-β dependent fibrotic replacement. This novel targeted photoactivation will offer new opportunity for the catheter based theranostic strategy.

Early Assessment of Atherosclerotic Lesions and Vulnerable Plaques in vivo by Targeting Apoptotic Macrophages with AV Nanobubbles.

BackgroundThe early detection of atherosclerotic lesions is particularly important for risk prediction of acute cardiovascular events. Macrophages apoptosis was significantly associated with the degree of AS lesions and especially contributed to plaque vulnerability. In this research, we mainly sought to explore the feasibility of a home-made AV-nanobubbles (NBAV) for visualization of apoptotic macrophages and assessment of atherosclerosis (AS) lesions by contrast-enhanced ultrasound (CEUS) imaging.MethodsNBAV were prepared by “Optimized Thin-Film Hydration” and “Biotin-Avidin-Biotin” methods. Then, the characterization and echogenicity of NBAV were measured and analyzed in vitro. The targeting ability of NBAV to ox-LDL–induced apoptotic macrophages was observed by laser scanning confocal microscope. The ApoE−/− mice mode fed with high fat diet were observed by high-frequency ultrasound, microanatomy and oil red O staining. CEUS imaging in vivo was performed on AS plaques with NBAV and NBCtrl injection through the tail vein in turn in ApoE−/− mice. After CEUS imaging, the plaques were confirmed and analyzed by histopathological and immunological assessment.ResultsThe prepared NBAV had a nano-scale size distribution with a low PDI and a negative zeta potential. Moreover, NBAV showed an excellent stability and exhibited a significantly echogenic signal than saline in vitro. In addition, we found that NBAV could target apoptotic macrophages induced by ox-LDL. Compared with NBCtrl, CEUS imaging of NBAV showed strong and sustained echo enhancement in plaque area of aortic arch in vivo. Further research showed that NBAV sensitive plaques presented more significant pathological changes with several vulnerable plaque features and abundant TUNEL-positive area.ConclusionNBAV displayed a sensitive indicator to evaluate apoptotic macrophages, indicating a promising CEUS molecular probe for AS lesions and vulnerable plaques identification.

Infiltration of Apoptotic M2 Macrophage Subpopulation Is Negatively Correlated with the Immunotherapy Response in Colorectal Cancer.

The polarization of tumor-associated macrophages (TAMs) plays a key role in tumor development and immunotherapy in colorectal cancer (CRC) patients. However, the impact of apoptosis on TAM polarization and immunotherapy efficacy in patients with different mismatch repair statuses (MMR) remains unclear. Here, we constructed an atlas of macrophage and found a higher rate of infiltration of M2-like TAM subpopulation in pMMR CRC tumor tissues compared with that in dMMR CRC tumor tissues. Importantly, a lower infiltration rate of M2c-like TAMs was associated with immunotherapy response. The M2 polarization trajectory revealed the apoptosis of M2c-like TAMs in dMMR while the differentiation of M2c-like TAMs in pMMR, implying a higher polarization level of M2 in pMMR. Furthermore, we found that a high expression of S100A6 induces the apoptosis of M2c-like TAMs in dMMR. In conclusion, we identified apoptotic TAM subpopulations in the M2 polarization trajectory and found that apoptosis caused by the high expression of S100A6 reduces their infiltration in tumors as well as the level of M2 polarization and contributes to a favorable immunotherapy response. These findings provide new insights into the potential role of apoptosis in suppressing tumors and enhancing immunotherapeutic efficacy.

Macrophage-derived apoptotic vesicles regulate fate commitment of mesenchymal stem cells via miR155

BackgroundIn tissue engineering, mesenchymal stem cells (MSCs) are common seed cells because of abundant sources, strong proliferation ability and immunomodulatory function. Numerous researches have demonstrated that MSC-macrophage crosstalk played a key role in the tissue engineering. Macrophages could regulate the differentiation of MSCs via different molecular mechanisms, including extracellular vesicles. Apoptotic macrophages could generate large amounts of apoptotic vesicles (apoVs). ApoVs are rich in proteins, RNA (microRNAs, mRNAs, ncRNAs, etc.) and lipids, and are a key intercellular communication mediator that can exert different regulatory effects on recipient cells. MiRNAs account for about half of the total RNAs of extracellular vesicles, and play important roles in biological processes such as cell proliferation and differentiation, whereas the functions of macrophage-derived apoVs remain largely unknown. There was no research to clarify the role of macrophage-derived apoVs in MSC fate choices. In this study, we aimed to characterize macrophage-derived apoVs, and investigate the roles of macrophage-derived apoVs in the fate commitment of MSCs.MethodsWe characterized macrophage-derived apoVs, and investigated their role in MSC osteogenesis and adipogenesis in vitro and in vivo. Furthermore, we performed microRNA loss- and gain-of-function experiments and western blot to determine the molecular mechanism.ResultsMacrophages could produce a large number of apoVs after apoptosis. MSCs could uptake apoVs. Then, we found that macrophage-derived apoVs inhibited osteogenesis and promoted adipogenesis of MSCs in vitro and in vivo. In mechanism, apoVs were enriched for microRNA155 (miR155), and apoVs regulated osteogenesis and adipogenesis of MSCs by delivering miR155. Besides, miR155 regulated osteogenesis and adipogenesis of MSCs cultured with macrophage-derived apoVs via the SMAD2 signaling pathway.ConclusionsMacrophage-derived apoVs could regulate the osteogenesis and adipogenesis of MSCs through delivering miR155, which provided novel insights for MSC-mediated tissue engineering.

Protective role of Cytoglobin and Neuroglobin against the Lipopolysaccharide (LPS)-induced inflammation in Leydig cells ex vivo

Leydig cells are responsible for testosterone production in male testis upon stimulation by luteinizing hormone. Inflammation and oxidative stress related Leydig cell dysfunction is one of the major causes of male infertility. Cytoglobin (CYGB) and Neuroglobin (NGB) are two globin family member proteins which protect cells against oxidative stress.In the current study, we established a Lipopolysaccharide (LPS)-induced inflammation model in TM3 Leydig cell culture to study the function of CYGB and NGB proteins under inflammatory conditions. CYGB and NGB were downregulated using siRNA and shRNA based experimental strategies. Overexpression was conducted using lentiviral pLenti-III-CYGB-2A-GFP, and pLenti-III-NGB-2A-GFP vector systems. As testicular macrophages regulate immune function upon inflammation and steroidogenesis of Leydig cells, we generated direct/indirect co-culture systems of TM3 and mouse macrophage (RAW264.7) cells ex vivo.Downregulation of CYGB and NGB induced nitride oxide (NO) release, blocked cell cycle progression, reduced testosterone production and increased inflammatory and apoptotic pathway gene expression in the presence and absence of LPS. On the other hand, CYGB and NGB overexpression reduced TNFα and COX-2 protein expressions and increased the expression of testosterone biogenesis pathway genes upon LPS stimulation. In addition, CYGB and NGB overexpression upregulated testosterone production. The present study successfully established an inflammatory interaction model of TM3 and RAW264.7 cells. Suppression of CYGB and NGB in TM3 cells changed macrophage morphology, enhanced macrophage cell number and NO release in co-culture experiments upon LPS exposure.In summary, these results demonstrate that globin family members might control LPS induced inflammation by regulating apoptotic mechanisms and macrophage response.

Hereditary Mucin Deficiency Caused by Biallelic Loss of Function of MUC5B.

Rationale: Mucin homeostasis is fundamental to airway health. Upregulation of airway mucus glycoprotein MUC5B is observed in diverse common lung diseases and represents a potential therapeutic target. In mice, Muc5b is required for mucociliary clearance and for controlling inflammation after microbial exposure. The consequences of its loss in humans are unclear. Objectives: The goal of this study was to identify and characterize a family with congenital absence of MUC5B protein. Methods: We performed whole-genome sequencing in an adult proband with unexplained bronchiectasis, impaired pulmonary function, and repeated Staphylococcus aureus infection. Deep phenotyping over a 12-year period included assessments of pulmonary radioaerosol mucociliary clearance. Genotyping with reverse phenotyping was organized for eight family members. Extensive experiments, including immunofluorescence staining and mass spectrometry for mucins, were performed across accessible sample types. Measurements and Main Results: The proband, and her symptomatic sibling who also had extensive sinus disease with nasal polyps, were homozygous for a novel splicing variant in the MUC5B gene (NM_002458.2: c.1938 + 1G>A). MUC5B was absent from saliva, sputum, and nasal samples. Mucociliary clearance was impaired in the proband, and large numbers of apoptotic macrophages were present in sputum. Three siblings heterozygous for the familial MUC5B variant were asymptomatic but had a shared pattern of mild lung function impairments. Conclusions: Congenital absence of MUC5B defines a new category of genetic respiratory disease. The human phenotype is highly concordant with that of the Muc5b-/- murine model. Further study of individuals with decreased MUC5B production could provide unique mechanistic insights into airway mucus biology.

CD74 in Apoptotic Macrophages Is Associated with Inflammation, Plaque Progression and Clinical Manifestations in Human Atherosclerotic Lesions.

The aim of this study was to investigate whether CD74 levels in atherosclerotic lesions are associated with inflammation, apoptosis, plaque severity, and clinical symptoms among patients with carotid atherosclerosis. We further studied whether CD74 expression is associated with apoptosis in macrophages induced by 7ketocholesterol (7keto). Sixty-one carotid samples (39 males and 22 females) were immunostained with macrophages, smooth muscle cells, CD74, ferritin, TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end labeling), and thrombin receptors. Double immunocytochemistry of CD74 and caspase 3 or CD74 and Annexin V was performed on THP-1 macrophages exposed to 7keto. In human carotid plaques, CD74 expression is lesion-dependently increased and is associated with necrotic core formation and plaque rupture, clinical symptoms, macrophage apoptosis, ferritin, and thrombin receptors. CD74 levels were inversely correlated to high-density lipoproteins and statin treatment, and positively correlated to triglycerides. In THP-1 macrophages, 7keto induced a significant increase in levels of CD74, ferritin, and apoptotic cell death. This study suggests that CD74 in apoptotic macrophages is linked to inflammation and thrombosis in progression of human atherosclerotic plaques, lipid metabolism, and clinical manifestation in atherosclerosis. Surface CD74 in apoptotic macrophages and ferritin production induced by oxidized lipids may contribute to inflammation and plaque vulnerability in atherosclerosis.

Cyclophilin A Impairs Efferocytosis and Accelerates Atherosclerosis by Overexpressing CD 47 and Down-Regulating Calreticulin.

Impairment of efferocytosis in apoptotic macrophages is a known determinant of the severity of atherosclerosis and the vulnerability of plaques to rupture. The precise mechanisms involved in impaired efferocytosis are unclear. Given the well-recognized role of the inflammatory cytokine cyclophilin A (Cyp A) in modulating several atherogenic mechanisms in high-glucose primed monocytes, we investigated the role of Cyp A in macrophage efferocytosis. The efficiency of efferocytosis in RAW 264.7 macrophages grown in vitro and primed with cyclophilin A was assessed using flow cytometry and confocal assays. Cholesterol content in cells was measured using cell-based cholesterol efflux assay. Proteomic analysis and bioinformatics tools were employed to decipher the link between cyclophilin A and the known ligand receptors involved in efferocytosis. Cyclophilin A was found to impair efferocytosis in apoptotic macrophages by reducing ABCA1-mediated cholesterol efflux in foam cells derived from macrophages. Cyclophilin A-primed macrophages showed an increase in expression of the don’t-eat-me signal CD 47 and a decrease in the expression of the eat-me signal, calreticulin. Phagocytosis was restored upon silencing of cyclophilin A. New Zealand white rabbits were fed a high-fat diet, and lesions in their aortae were analyzed histologically for evidence of atherosclerosis and the expression of Cyp A, CD 47 and calreticulin, the ligand receptor involved in efferocytosis. Gene and protein expressions in aortae and macrophages were analyzed by real-time PCR and Western blotting. Cyclophilin A, via its effects on the expression of CD 47 and calreticulin, impairs efferocytosis in apoptotic macrophages. Together with its impact on cholesterol efflux from macrophages, these effects can amplify other mechanisms of Cyp A in accelerating the progression of atherosclerosis.

Association between serum PGE2 levels and degree of acid-fast bacilli positivity in sputum of pulmonary tuberculosis patients

BackgroundMycobacterium tuberculosis that infected apoptotic macrophages is triggered by PGE2. Apoptosis suppresses the growth of Mycobacterium tuberculosis bacteria, which is shown in the results of acid-fast bacilli (AFB) in the sputum that becomes a marker of the number of bacteria. ObjectiveAnalyzing the association between serum PGE2 levels and the positivity of AFB in the sputum of tuberculosis patients. MethodsA cross-sectional study was carried out from August 2019–July 2020. Serum PGE2 levels and AFB levels in sputum were collected from participants. Data analysis used the Chi-square test and Spearman's correlation with p < 0.05. ResultsThe average participants’ serum PGE2 levels were 446.37 ± 510.27 pg/ml, with a median value of 216.95 pg/ml. Most participants had normal serum PGE2 levels (62.9%). Most participants had a high positivity of AFB in sputum (58.1%). Analysis of the association between serum PGE2 levels and the degree of AFB positivity in sputum obtained r = −0.036 and p-value = 0.780. ConclusionThere is a weak negative association between serum PGE2 levels and the degree of AFB positivity in sputum but not statistically significant.