RESUMO
BACKGROUND: Environmental/occupational exposures cause significant lung diseases. Agricultural organic dust extracts (ODE) and bacterial component lipopolysaccharide (LPS) induce recruited, transitioning murine lung monocytes/macrophages, yet their cellular role remains unclear. METHODS: CCR2 RFP+ mice were intratracheally instilled with high concentration ODE (25%), LPS (10 µg), or gram-positive peptidoglycan (PGN, 100 µg) for monocyte/macrophage cell-trafficking studies. CCR2 knockout (KO) mice and administration of intravenous clodronate liposomes strategies were employed to reduce circulating monocytes available for lung recruitment following LPS exposure. Lung tissues and bronchoalveolar lavage fluid (BALF) were collected. Pro-inflammatory and/or pro-fibrotic cytokines, chemokines, and lung extracellular matrix mediators were quantitated by ELISA. Infiltrating lung cells including monocyte/macrophage subpopulations, neutrophils, and lymphocytes were characterized by flow cytometry. Lung histopathology, collagen content, vimentin, and post-translational protein citrullination and malondialdehyde acetaldehyde (MAA) modification were quantitated. Parametric statistical tests (one-way ANOVA, Tukey'smultiple comparison) and nonparametric statistical (Kruskal-Wallis, Dunn's multiple comparison) tests were used following Shapiro-Wilk testing for normality. RESULTS: Intratracheal instillation of ODE, LPS, or PGN robustly induced the recruitment of inflammatory CCR2+ CD11cintCD11bhi monocytes/macrophages and both CCR2+ and CCR2- CD11c-CD11bhi monocytes at 48 h. There were also increases in CCR2+ CD4+ and CD8+ T cells and NK cells. Despite reductions in LPS-induced lung infiltrating CD11cintCD11bhi cells (54% reduction), CCR2 knockout (KO) mice were not protected against LPS-induced inflammatory and pro-fibrotic consequences. Instead, compensatory increases in lung neutrophils and CCL2 and CCL7 release occurred. In contrast, the depletion of circulating monocytes through the administration of intravenous clodronate (vs. vehicle) liposomes 24 h prior to LPS exposure reduced LPS-induced infiltrating CD11cintCD11bhi monocyte-macrophage subpopulation by 59% without compensatory changes in other cell populations. Clodronate liposome pre-treatment significantly reduced LPS-induced IL-6 (66% reduction), matrix metalloproteinases (MMP)-3 (36%), MMP-8 (57%), tissue inhibitor of metalloproteinases (61%), fibronectin (38%), collagen content (22%), and vimentin (40%). LPS-induced lung protein citrullination and MAA modification, post-translational modifications implicated in lung disease, were reduced (39% and 48%) with clodronate vs. vehicle liposome. CONCLUSION: Highly concentrated environmental/occupational exposures induced the recruitment of CCR2+ and CCR2- transitioning monocyte-macrophage and monocyte subpopulations and targeting peripheral monocytes may reduce the adverse lung consequences resulting from exposures to LPS-enriched inhalants.
Assuntos
Pneumopatias , Monócitos , Camundongos , Animais , Monócitos/metabolismo , Lipossomos/metabolismo , Vimentina/metabolismo , Lipopolissacarídeos/farmacologia , Ácido Clodrônico/farmacologia , Ácido Clodrônico/metabolismo , Linfócitos T CD8-Positivos , Pulmão , Macrófagos/metabolismo , Pneumopatias/metabolismo , Exposição Ambiental , Colágeno/metabolismo , Camundongos Endogâmicos C57BLRESUMO
BACKGROUND: Renal fibrosis significantly contributes to the progressive loss of kidney function in chronic kidney disease (CKD), with alternatively activated M2 macrophages playing a crucial role in this progression. The serum succinate level is consistently elevated in individuals with diabetes and obesity, both of which are critical factors contributing to CKD. However, it remains unclear whether elevated succinate levels can mediate M2 polarization of macrophages and contribute to renal interstitial fibrosis. METHODS: Male C57/BL6 mice were administered water supplemented with 4% succinate for 12 weeks to assess its impact on renal interstitial fibrosis. Additionally, the significance of macrophages was confirmed in vivo by using clodronate liposomes to deplete them. Furthermore, we employed RAW 264.7 and NRK-49F cells to investigate the underlying molecular mechanisms. RESULTS: Succinate caused renal interstitial macrophage infiltration, activation of profibrotic M2 phenotype, upregulation of profibrotic factors, and interstitial fibrosis. Treatment of clodronate liposomes markedly depleted macrophages and prevented the succinate-induced increase in profibrotic factors and fibrosis. Mechanically, succinate promoted CTGF transcription via triggering SUCNR1-p-Akt/p-GSK3ß/ß-catenin signaling, which was inhibited by SUCNR1 siRNA. The knockdown of succinate receptor (SUCNR1) or pretreatment of anti-CTGF(connective tissue growth factor) antibody suppressed the stimulating effects of succinate on RAW 264.7 and NRK-49F cells. CONCLUSIONS: The causative effects of succinate on renal interstitial fibrosis were mediated by the activation of profibrotic M2 macrophages. Succinate-SUCNR1 played a role in activating p-Akt/p-GSK3ß/ß-catenin, CTGF expression, and facilitating crosstalk between macrophages and fibroblasts. Our findings suggest a promising strategy to prevent the progression of metabolic CKD by promoting the excretion of succinate in urine and/or using selective antagonists for SUCNR1.
Assuntos
Insuficiência Renal Crônica , beta Catenina , Masculino , Camundongos , Animais , beta Catenina/metabolismo , Ácido Succínico/metabolismo , Lipossomos/metabolismo , Ácido Clodrônico/metabolismo , Glicogênio Sintase Quinase 3 beta/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Insuficiência Renal Crônica/metabolismo , Fibrose , Macrófagos/metabolismoRESUMO
Pathological conditions in cochlea, such as ototoxicity, acoustic trauma, and age-related cochlear degeneration, induce cell death in the organ of Corti and degeneration of the spiral ganglion neurons (SGNs). Although macrophages play an essential role after cochlear injury, its role in the SGNs is limitedly understood. We analyzed the status of macrophage activation and neuronal damage in the spiral ganglion after kanamycin-induced unilateral hearing loss in mice. The number of ionized calcium-binding adapter molecule 1 (Iba1)-positive macrophages increased 3 days after unilateral kanamycin injection. Macrophages showed larger cell bodies, suggesting activation status. Interestingly, the number of activating transcription factor 3 (ATF3)-positive-neurons, an indicator of early neuronal damage, also increased at the same timing. In the later stages, the number of macrophages decreased, and the cell bodies became smaller, although the number of neuronal deaths increased. To understand their role in neuronal damage, macrophages were depleted via intraperitoneal injection of clodronate liposome 24 h after kanamycin injection. Macrophage depletion decreased the number of ATF3-positive neurons at day 3 and neuronal death at day 28 in the spiral ganglion following kanamycin injection. Our results suggest that suppression of inflammation by clodronate at early timing can protect spiral ganglion damage following cochlear insult.
Assuntos
Perda Auditiva Unilateral , Gânglio Espiral da Cóclea , Camundongos , Animais , Gânglio Espiral da Cóclea/metabolismo , Canamicina/toxicidade , Perda Auditiva Unilateral/patologia , Ácido Clodrônico/metabolismo , Células Ciliadas Auditivas/metabolismo , Cóclea , Neurônios , MacrófagosRESUMO
Hemophagocytic lymphohistiocytosis (HLH), a hyperinflammatory syndrome, is caused by the incessant activation of lymphocytes and macrophages, resulting in damage to organs, including hematopoietic organs. Recently, we demonstrated that repeated lipopolysaccharide (LPS) treatment induces HLH-like features in senescence-accelerated (SAMP1/TA-1) mice but not in senescence-resistant control (SAMR1) mice. Hematopoietic failure in LPS-treated SAMP1/TA-1 mice was attributed to hematopoietic microenvironment dysfunction, concomitant with severely imbalanced M1 and M2 macrophage polarization. Macrophages are a major component of the bone marrow (BM) hematopoietic microenvironment. Clodronate liposomes are useful tools for in vivo macrophage depletion. In this study, we depleted macrophages using clodronate liposomes to determine their role in the hematopoietic microenvironment in SAMP1/TA-1 and SAMR1 mice. Under clodronate liposome treatment, the response between SAMR1 and SAMP1/TA-1 mice differed as follows: (1) increase in the number of activated M1 and M2 macrophages derived from newly generated macrophages and M2-dominant and imbalanced M1 and M2 macrophage polarization in the BM and spleen; (2) severe anemia and thrombocytopenia; (3) high mortality rate; (4) decrease in erythroid progenitors and B cell progenitors in the BM; and (5) decrease in the mRNA expression of erythroid-positive regulators such as erythropoietin and increase in that of erythroid- and B lymphoid-negative regulators such as interferon-γ in the BM. Depletion of residual macrophages in SAMP1/TA-1 mice impaired hematopoietic homeostasis, particularly erythropoiesis and B lymphopoiesis, owing to functional impairment of the hematopoietic microenvironment accompanied by persistently imbalanced M1/M2 polarization. Thus, macrophages play a vital role in regulating the hematopoietic microenvironment to maintain homeostasis.
Assuntos
Linfo-Histiocitose Hemofagocítica , Camundongos , Animais , Linfo-Histiocitose Hemofagocítica/metabolismo , Lipossomos/metabolismo , Ácido Clodrônico/farmacologia , Ácido Clodrônico/metabolismo , Lipopolissacarídeos , Macrófagos/metabolismoRESUMO
BACKGROUND: Brain perivascular macrophages (PVMs) are potential treatment targets for subarachnoid hemorrhage (SAH), and previous studies revealed that their depletion by clodronate (CLD) improved outcomes after experimental SAH. However, the underlying mechanisms are not well understood. Therefore, we investigated whether reducing PVMs by CLD pretreatment improves SAH prognosis by inhibiting posthemorrhagic impairment of cerebral blood flow (CBF). METHODS: In total, 80 male Sprague-Dawley rats received an intracerebroventricular injection of the vehicle (liposomes) or CLD. Subsequently, the rats were categorized into the prechiasmatic saline injection (sham) and blood injection (SAH) groups after 72 h. We assessed its effects on weak and severe SAH, which were induced by 200- and 300-µL arterial blood injections, respectively. In addition, neurological function at 72 h and CBF changes from before the intervention to 5 min after were assessed in rats after sham/SAH induction as the primary and secondary end points, respectively. RESULTS: CLD significantly reduced PVMs before SAH induction. Although pretreatment with CLD in the weak SAH group provided no additive effects on the primary end point, rats in the severe SAH group showed significant improvement in the rotarod test. In the severe SAH group, CLD inhibited acute reduction of CBF and tended to decrease hypoxia-inducible factor 1α expression. Furthermore, CLD reduced the number of PVMs in rats subjected to sham and SAH surgery, although no effects were observed in oxidative stress and inflammation. CONCLUSIONS: Our study proposes that pretreatment with CLD-targeting PVMs can improve the prognosis of severe SAH through a candidate mechanism of inhibition of posthemorrhagic CBF reduction.
Assuntos
Ácido Clodrônico , Hemorragia Subaracnóidea , Ratos , Masculino , Animais , Ratos Sprague-Dawley , Ácido Clodrônico/farmacologia , Ácido Clodrônico/metabolismo , Hemorragia Subaracnóidea/complicações , Encéfalo/metabolismo , Circulação Cerebrovascular/fisiologia , Modelos Animais de DoençasRESUMO
Both monocyte-derived macrophages (MDMs) and brain resident microglia participate in hematoma resolution after intracerebral hemorrhage (ICH). Here, we utilized a transgenic mouse line with enhanced green fluorescent protein (EGFP) labeled microglia (Tmem119-EGFP mice) combined with a F4/80 immunohistochemistry (a pan-macrophage marker) to visualize changes in MDMs and microglia after ICH. A murine model of ICH was used in which autologous blood was stereotactically injected into the right basal ganglia. The autologous blood was co-injected with CD47 blocking antibodies to enhance phagocytosis or clodronate liposomes for phagocyte depletion. In addition, Tmem119-EGFP mice were injected with the blood components peroxiredoxin 2 (Prx2) or thrombin. MDMs entered the brain and formed a peri-hematoma cell layer by day 3 after ICH and giant phagocytes engulfed red blood cells were found. CD47 blocking antibody increased the number of MDMs around and inside the hematoma and extended MDM phagocytic activity to day 7. Both MDMs and microglia could be diminished by clodronate liposomes. Intracerebral injection of Prx2 but not thrombin attracted MDMs into brain parenchyma. In conclusion, MDMs play an important role in phagocytosis after ICH which can be enhanced by CD47 blocking antibody, suggesting the modulation of MDMs after ICH could be a future therapeutic target.
Assuntos
Antígeno CD47 , Microglia , Camundongos , Animais , Microglia/metabolismo , Antígeno CD47/metabolismo , Antígeno CD47/uso terapêutico , Ácido Clodrônico/farmacologia , Ácido Clodrônico/metabolismo , Ácido Clodrônico/uso terapêutico , Lipossomos/metabolismo , Macrófagos/metabolismo , Hemorragia Cerebral/metabolismo , Camundongos Transgênicos , Hematoma/metabolismoRESUMO
OBJECTIVE: To determine the effects of clodronate disodium (CLO) on control and recombinant equine interleukin-1ß (IL-1ß)-treated equine joint tissues. STUDY DESIGN: In vitro experimental study. SAMPLE POPULATION: Cartilage explants, chondrocytes, and synoviocytes (n = 3 horses). METHODS: Monolayer cultures of chondrocytes and synoviocytes from three horses were subjected to: control media (CON), 5 ng/ml CLO (C/low), 50 ng/ml CLO (C/med), 100 ng/ml CLO (C/high), with and without IL-1ß, and 10 ng/ml IL-1ß (IL) alone for 72 hours. Cartilage explants from three horses were subjected to CON, IL, C/low, and C/med with and without IL-1ß for 72 hours. Culture media was analyzed for prostaglandin-E2 (PGE2 ), interleukin-6 (IL-6), and nitric oxide (NO). Explant media was analyzed for glycosaminoglycan (GAG) content and NO. At 72 hours, explant and monolayer culture viability were assessed, and explant GAG content was measured. RESULTS: IL-1ß treatment resulted in higher media concentrations of GAG, NO, PGE2 , and IL-6 compared to the CON treatment (p < .05), demonstrating a catabolic effect of IL-1ß on explants and monolayer cultures. CLO treatments did not increase media concentrations of GAG, NO, PGE2 , or IL-6 compared to CON, indicating no cytotoxic effect. Nevertheless, CLO treatments administered to IL-1ß-treated monolayer cultures and explants did not significantly reduce the inflammatory response regardless of concentration. CONCLUSION: CLO did not demonstrate cytotoxic nor cytoprotective effects in normal and IL-1ß-stimulated chondrocytes, synoviocytes or explants in culture. CLINICAL SIGNIFICANCE: This study does not support the use of CLO as an anti-inflammatory treatment. Further research is necessary to confirm any anti-inflammatory effects of CLO on joint tissues.
Assuntos
Antineoplásicos , Cartilagem Articular , Animais , Cavalos , Interleucina-1beta/farmacologia , Interleucina-1beta/metabolismo , Ácido Clodrônico/farmacologia , Ácido Clodrônico/metabolismo , Interleucina-6/metabolismo , Interleucina-6/farmacologia , Condrócitos , Glicosaminoglicanos/farmacologia , Glicosaminoglicanos/metabolismo , Antineoplásicos/farmacologiaRESUMO
Studies have shown that epigenetic enzymes such as histone deacetylase (HDAC) are closely related to cancers and that several HDAC inhibitors exert antitumor effects. Studies have further suggested that class IIa HDAC inhibitors are related to immune functions, including immune responses and the expression of chemokines and complement pathway components. TMP195, a selective class IIa HDAC inhibitor, has been reported to be effective against breast cancer. However, the role and mechanism of TMP195 in colorectal cancer remain unknown. In this study, we found that TMP195 significantly reduced the tumor burden in two mouse models of colitis-associated colorectal cancer (CAC) and subcutaneous tumor. Mechanistically, TMP195 decreased the proportion of total macrophages but increased the proportion of M1 macrophages by promoting polarization, resulting in the increased release of inflammatory cytokines. TMP195 had no direct effect on the proliferation of colorectal cancer cells, and its antitumor effect on the colorectal cancer disappeared when macrophages were partly depleted by clodronate liposomes. In addition, TMP195 enhanced the efficacy of PD-1 blockade. The present study revealed that the combination of TMP195 and PD-1 blockade may provide a therapeutic strategy for colorectal cancer.
Assuntos
Neoplasias Colorretais , Inibidores de Histona Desacetilases , Camundongos , Animais , Inibidores de Histona Desacetilases/farmacologia , Receptor de Morte Celular Programada 1/metabolismo , Ácido Clodrônico/metabolismo , Ácido Clodrônico/farmacologia , Lipossomos/metabolismo , Lipossomos/farmacologia , Macrófagos/metabolismo , Histona Desacetilases/metabolismo , Citocinas/metabolismo , Neoplasias Colorretais/metabolismo , Linhagem Celular TumoralRESUMO
BACKGROUND: Malnutrition affects various physiological functions, including immune defenses. However, it remains unclear how malnutrition reduces immune responses. AIM: To elucidate mechanisms underlying malnutrition-induced immunodeficiency, we focused on the spleen, which plays an essential role in the immune system, and examined the impacts of malnutrition on the spleen. MAIN METHODS: The impact of malnutrition on the spleen was assessed using dietary-restricted mice as a model. Weights of the spleen were measured and normalized to body weights. Macrophage maker protein expression was observed using fluorescent immunostaining. Clodronate-containing liposomes were injected into the mice to test whether macrophages are involved in splenic changes induced by dietary restriction. KEY FINDINGS: The spleen of dietary-restricted mice involuted with significant reductions in the relative weight of the spleen to the body weight and ratio of the red pulp in the spleen. Then, we examined whether macrophages mediate dietary restriction-induced splenic involution. The IBA1/AIF1 protein level was increased in the marginal zone, which is the interface between the red and white pulps of the spleen, by dietary restriction. We tested whether macrophages are needed for dietary restriction-induced splenic involution. The increase in IBA1/AIF1 expression in the marginal zone and splenic involution were suppressed by clodronate liposome administration. These results indicate that the macrophages in the splenic marginal zone were activated by dietary restriction and were required for dietary restriction-induced splenic involution. SIGNIFICANCE: Our study proposes macrophage-mediated splenic involution as a novel mechanism linking malnutrition to immunodeficiency.
Assuntos
Desnutrição , Baço , Camundongos , Animais , Baço/metabolismo , Ácido Clodrônico/farmacologia , Ácido Clodrônico/metabolismo , Macrófagos/metabolismo , Lipossomos , Desnutrição/metabolismoRESUMO
BACKGROUND: Emerging evidence provides mechanistic insights into the pathogenesis of pulmonary fibrosis (PF), and rare anti-PF therapeutic method has promising effect in its treatment. Rho-associated coiled-coil kinases (ROCK) inhibition significantly ameliorates bleomycin-induced PF and decreases macrophage infiltration, but the mechanism remains unclear. We established bleomycin and radiation-induced PF to identify the activity of WXWH0265, a newly designed unselective ROCK inhibitor in regulating macrophages. METHODS: Bleomycin-induced PF was induced by intratracheal instillation and radiation-induced PF was induced by bilateral thoracic irradiation. Histopathological techniques (haematoxylin and eosin, Masson's trichrome and immunohistochemistry) and hydroxyproline were used to evaluate PF severity. Western blot, quantitative real-time reverse transcription-polymerase chain reaction and flow cytometry were performed to explore the underlying mechanisms. Bone marrow-derived macrophages (BMDMs) were used to verify their therapeutic effect. Clodronate liposomes were applied to deplete macrophages and to identify the therapeutic effect of WXWH0265. RESULTS: Therapeutic administration of ROCK inhibitor ameliorates bleomycin-induced PF by inhibiting M2 macrophages polarisation. ROCK inhibitor showed no significant anti-fibrotic effect in macrophages-depleted mice. Treatment with WXWH0265 demonstrated superior protection effect in bleomycin-induced PF compared with positive drugs. In radiation-induced PF, ROCK inhibitor effectively ameliorated PF. Fibroblasts co-cultured with supernatant from various M2 macrophages phenotypes revealed that M2 macrophages stimulated by interleukin-4 promoted extracellular matrix production. Polarisation of M2 macrophages was inhibited by ROCK inhibitor treatment in vitro. The p-signal transducer and activator of transcription 3 (STAT3) in lung tissue and BMDMs was significantly decreased in PF in vivo and vitro after treated with ROCK inhibitors. CONCLUSION: Inhibiting ROCK could significantly attenuate bleomycin- and radiation-induced PF by regulating the macrophages polarisation via phosphorylation of STAT3. WXWH0265 is a kind of efficient unselective ROCK inhibitor in ameliorating PF. Furthermore, the results provide empirical evidence that ROCK inhibitor, WXWH0265 is a potential drug to prevent the development of PF.
Assuntos
Fibrose Pulmonar , Fator de Transcrição STAT3 , Quinases Associadas a rho , Animais , Bleomicina/efeitos adversos , Ácido Clodrônico/metabolismo , Interleucina-4/metabolismo , Lipossomos/metabolismo , Macrófagos/metabolismo , Camundongos , Fosforilação , Fibrose Pulmonar/induzido quimicamente , Fibrose Pulmonar/tratamento farmacológico , Fibrose Pulmonar/patologia , Fator de Transcrição STAT3/metabolismo , Quinases Associadas a rho/antagonistas & inibidoresRESUMO
Macrophages are important mediators of skeletal muscle function in both healthy and diseased states. In vivo specific depletion of macrophages provides an experimental method to understand physiological and pathophysiological effects of macrophages. Systemic depletion of macrophages can deplete skeletal muscle macrophages but also alters systemic inflammatory responses and metabolism, which confounds the muscle specific effects of macrophage depletion. The primary aim of this manuscript is to evaluate two methods of murine intramuscular macrophage depletion in an acute lung injury-associated indirect skeletal muscle wasting mouse model. Adult C57BL/6 (WT) and Macrophage Fas-Induced Apoptosis (MaFIA, C57BL/6-Tg) mice received clodronate liposomes or the dimerization drug AP20187 through intramuscular injection of the tibialis anterior muscle compartment, respectively. Vehicle control was injected in the contralateral muscle. We demonstrate intramuscular AP20187 in the MaFIA mouse depletes macrophages but causes an infiltration of CD45 intermediate neutrophils. In contrast, intramuscular clodronate liposomes successfully depletes macrophages without an associated increase in CD45 intermediate cells. In conclusion, intramuscular clodronate is effective for selective depletion of muscle macrophages without eliciting acute inflammation seen with AP20187 in MaFIA mice. This technique is an important tool to study the functional roles of macrophages in skeletal muscle.
Assuntos
Ácido Clodrônico , Lipossomos , Animais , Ácido Clodrônico/metabolismo , Ácido Clodrônico/farmacologia , Lipossomos/metabolismo , Macrófagos , Camundongos , Camundongos Endogâmicos C57BL , Músculo Esquelético/metabolismoRESUMO
INTRODUCTION: Intracerebral hemorrhage (ICH) causes devastating morbidity and mortality, and studies have shown that the toxic components of hematomas play key roles in brain damage after ICH. Recent studies have found that TLR9 participates in regulating the phagocytosis of peripheral macrophages. The current study examined the role of TLR9 in macrophage/microglial (M/M) function after ICH. METHODS: RAW264.7 (macrophage), BV2 (microglia), and HT22# (neurons) cell lines were transfected with lentivirus for TLR9 overexpression. Whole blood from C57BL/6 or EGFPTg/+ mice was infused for phagocytosis and injury experiments, and brusatol was used for the experiments. Intraperitoneal injection of the TLR9 agonist ODN1826 or control ODN2138 was performed on days 1, 3, 5, 7, and 28 after ICH to study the effects of TLR9 in mice. In addition, clodronate was coinjected in M/M elimination experiments. The brains were collected for histological and protein experiments at different time points after ICH induction. Cellular and histological methods were used to measure hematoma/iron residual, M/Ms variation, neural injury, and brain tissue loss. Behavioral tests were performed premodeling and on days 1, 3, 7, and 28 post-ICH. RESULTS: Overexpression of TLR9 facilitated M/M phagocytosis and protected neurons from blood-derived hazards in vitro. Furthermore, ODN1826 boosted M/M activation and phagocytic function, facilitated hematoma/iron resolution, reduced brain injury, and improved neurological function recovery in ICH mice, which were abolished by clodronate injection. The experimental results indicated that the Nrf2/CD204 pathway participated in TLR9-induced M/M phagocytosis after ICH. CONCLUSION: Our study suggests a protective role for TLR9-enhanced M/M phagocytosis via the Nrf2/CD204 pathway after ICH. Our findings may serve as potential targets for ICH treatment.
Assuntos
Lesões Encefálicas , Microglia , Animais , Lesões Encefálicas/patologia , Hemorragia Cerebral/metabolismo , Ácido Clodrônico/metabolismo , Hematoma/metabolismo , Ferro/metabolismo , Macrófagos/patologia , Camundongos , Camundongos Endogâmicos C57BL , Microglia/patologia , Fator 2 Relacionado a NF-E2/metabolismo , Fagocitose , Receptor Toll-Like 9/metabolismoRESUMO
Background: Tumor-associated macrophages (TAMs) are known to generate an immune-suppressive tumor microenvironment (TME) and promote tumor progression. Hepatocellular carcinoma (HCC) is a devastating disease that evolves in the background of chronic inflammatory liver damage. In this study, we aimed to uncover the mechanism by which HCC cells recruit macrophages into the TME. Methods: Bioinformatic analysis was performed to identify differentially expressed genes related to macrophage infiltration. An orthotopic HCC xenograft model was used to determine the role of macrophages in HCC tumor growth. Clodronate liposomes were used to delete macrophages. Western blotting analysis, quantitative real-time PCR, and enzyme-linked immunosorbent assay were performed to determine the underlying mechanisms. Results: The high mobility group A1 (HMGA1) gene was identified as a putative modulator of macrophage infiltration in HCC. Deletion of macrophages with clodronate liposomes significantly abrogated the tumor-promoting effects of HMGA1 on HCC growth. Mechanistically, HMGA1 can regulate the expression of C-C Motif Chemokine Ligand 2 (CCL2), also referred to as monocyte chemoattractant protein 1 (MCP1), which is responsible for macrophage recruitment. Moreover, NF-κB was required for HMGA1-mediated CCL2 expression. Pharmacological or genetic inhibition of NF-κB largely blocked CCL2 levels in HMGA1-overexpressing HCC cells. Conclusions: This study reveals HMGA1 as a crucial regulator of macrophage recruitment by activating NF-κB-CCL2 signaling, proves that HMGA1-induced HCC aggressiveness dependents on the macrophage, and provide an attractive target for therapeutic interventions in HCC.
Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Animais , Carcinoma Hepatocelular/patologia , Quimiocina CCL2/genética , Quimiocina CCL2/metabolismo , Ácido Clodrônico/metabolismo , Ácido Clodrônico/farmacologia , Ácido Clodrônico/uso terapêutico , Proteína HMGA1a/metabolismo , Proteína HMGA1a/uso terapêutico , Humanos , Ligantes , Lipossomos , Neoplasias Hepáticas/patologia , Macrófagos/metabolismo , NF-kappa B/metabolismo , Microambiente TumoralRESUMO
BACKGROUND: Acute compartment syndrome (ACS), a well-known complication of musculoskeletal injury, results in muscle necrosis and cell death. Embryonic stem cell-derived mesenchymal stem cells (ESC-MSCs) have been shown to be a promising therapy for ACS. However, their effectiveness and potentially protective mechanism remain unknown. The present study was designed to investigate the efficacy and underlying mechanism of ESC-MSCs in ACS-induced skeletal muscle injury. METHOD: A total of 168 male Sprague-Dawley (SD) rats underwent 2 h of intracompartmental pressure elevation by saline infusion into the anterior compartment of the left hindlimb to establish the ACS model. ESC-MSCs were differentiated from the human embryonic stem cell (ESC) line H9. A dose of 1.2 × 106 of ESC-MSCs was intravenously injected during fasciotomy. Post-ACS assessments included skeletal edema index, serum indicators, histological analysis, apoptosis, fibrosis, regeneration, and functional recovery of skeletal muscle. Then, fluorescence microscopy was used to observe the distribution of labeled ESC-MSCs in vivo, and western blotting and immunofluorescence analyses were performed to examine macrophages infiltration in skeletal muscle. Finally, we used liposomal clodronate to deplete macrophages and reassess skeletal muscle injury in response to ESC-MSC therapy. RESULT: ESC-MSCs significantly reduced systemic inflammatory responses, ACS-induced skeletal muscle edema, and cell apoptosis. In addition, ESC-MSCs inhibited skeletal muscle fibrosis and increased regeneration and functional recovery of skeletal muscle after ACS. The beneficial effects of ESC-MSCs on ACS-induced skeletal muscle injury were accompanied by a decrease in CD86-positive M1 macrophage polarization and an increase in CD206-positive M2 macrophage polarization. After depleting macrophages with liposomal clodronate, the beneficial effects of ESC-MSCs were attenuated. CONCLUSION: Our findings suggest that embryonic stem cell-derived mesenchymal stem cells infusion could effectively alleviate ACS-induced skeletal muscle injury, in which the beneficial effects were related to the regulation of macrophages polarization.
Assuntos
Síndromes Compartimentais , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais , Animais , Ácido Clodrônico/metabolismo , Ácido Clodrônico/farmacologia , Síndromes Compartimentais/metabolismo , Síndromes Compartimentais/terapia , Células-Tronco Embrionárias , Fibrose , Humanos , Masculino , Transplante de Células-Tronco Mesenquimais/métodos , Células-Tronco Mesenquimais/metabolismo , Músculo Esquelético , Ratos , Ratos Sprague-DawleyRESUMO
BACKGROUND: Toll-like receptor 4 (TLR4) activation causes excessive production of proinflammatory mediators and an increased expression of costimulatory molecules that leads to neuroinflammation after subarachnoid hemorrhage (SAH). Although TLR4-mediated inflammatory pathways have long been studied in neuroinflammation, the specific glia implicated in initiation and propagation of neuroinflammation in SAH have not been well elucidated. In this study, we investigated the involvement of glial TLR4 including microglia and astrocytes in brain damage and poor neurological outcome. METHODS: In this study, global TLR4 knockout, cell-specific TLR4 knockout, and floxxed control male and female mice were used. The mice were injected with 60 µl autologous blood near the mesencephalon to induce SAH; animals were euthanized on postoperative day 7 for immunohistochemistry of glia and apoptotic cells. Microglial morphology was evaluated by using immunofluorescence density quantification to determine correlations between morphology and neuroinflammation. Microglial depletion was accomplished with the intracerebroventricular administration of clodronate liposomes. Cognitive function was assessed with Barnes maze. RESULTS: On postoperative day 7 after SAH induction, neuronal apoptosis was markedly reduced in the clodronate liposome group compared with phosphate-buffered saline control liposomes, and cognitive performance in the clodronate group was improved, as well. Differences in microglial activation, assessed by morphometric analysis, and neuronal apoptosis were significantly greater in wildtype knockouts compared with cell-specific and global TLR4 knockouts. The mice lacking TLR4 on astrocytes and neurons showed no differences compared with wildtype mice on any end points. CONCLUSIONS: Our data suggest that microglial depletion with the intracerebroventricular administration of clodronate can improve the cognitive function in an SAH mouse model, and TLR4 is critical for microglial activation and neuronal injury. Only microglial TLR4 is necessary for brain damage and poor cognitive outcome rather than astrocyte or neuronal TLR4. Thus, microglial TLR4 could be a potent therapeutic target to treat SAH-associated neuronal injury and protect against cognitive dysfunction.
Assuntos
Lesões Encefálicas , Disfunção Cognitiva , Hemorragia Subaracnóidea , Feminino , Masculino , Camundongos , Animais , Microglia/metabolismo , Receptor 4 Toll-Like/metabolismo , Hemorragia Subaracnóidea/complicações , Hemorragia Subaracnóidea/metabolismo , Ácido Clodrônico/metabolismo , Lipossomos/metabolismo , Camundongos Endogâmicos C57BL , Neurônios/metabolismo , Lesões Encefálicas/etiologia , Modelos Animais de Doenças , Disfunção Cognitiva/etiologiaRESUMO
Exhaustive exercise is known to induce acute renal damage. However, the precise mechanisms remain unclear. We investigated the effects of macrophage depletion on exhaustive exercise-induced acute renal damage. Male C57BL/6 J mice were divided into four groups: sedentary with control liposome (n=8), sedentary with clodronate liposome (n=8), exhaustive exercise with control liposome (n=8), and exhaustive exercise with clodronate liposome (n=8). Mice were treated with clodronate liposomes or control liposomes intraperitoneally for 48 h before undergoing exhaustive exercise. Renal function and renal histology were tested at 24 h. The expression levels of kidney injury molecule (KIM)-1 and inflammatory cytokines in kidney tissues were measured by quantitative RT-PCR, and KIM-1 concentration was semi-quantified by immunostaining. As a result, exhaustive exercise increased macrophage infiltration into the kidney. However, clodronate reduced it. Although exhaustive exercise resulted in an increase in KIM-1 mRNA expression levels and concentration, injection of clodronate liposome reduced it. In addition, TUNEL positive apoptotic cells were increased after exercise, but significantly reduced by clodronate. Clodronate liposome treatment also decreased the mRNA expression levels of inflammatory cytokines (TNF-α, IL-1ß, and IL-6) in the kidney after exhaustive exercise. These results suggest that macrophages play a critical role in increasing renal damage by regulating inflammation.
Assuntos
Ácido Clodrônico , Lipossomos , Animais , Ácido Clodrônico/metabolismo , Ácido Clodrônico/farmacologia , Citocinas/genética , Citocinas/metabolismo , Interleucina-6/metabolismo , Rim/fisiologia , Lipossomos/metabolismo , Lipossomos/farmacologia , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , RNA Mensageiro/metabolismo , Fator de Necrose Tumoral alfaRESUMO
Adeno-associated virus is a popular gene delivery vehicle for gene therapy studies. A potential roadblock to widespread clinical adoption is the high vector doses required for efficient transduction in vivo, and the potential for subsequent immune responses that may limit prolonged transgene expression. We hypothesized that the depletion of macrophages via systemic delivery of liposome-encapsulated clodronate would improve transgene expression if given prior to systemic AAV vector administration, as has been shown to be the case with adenoviral vectors. Contrary to our expectations, clodronate liposome pretreatment resulted in significantly reduced transgene expression in the liver and heart, but permitted moderate transduction of the white pulp of the spleen. There was a remarkable localization of transgene expression from the red pulp to the center of the white pulp in clodronate-treated mice compared to untreated mice. Similarly, a greater proportion of transgene expression could be observed in the medulla located in the center of the lymph node in mice treated with clodronate-containing liposomes as compared to untreated mice where transgene expression was localized primarily to the cortex. These results underscore the highly significant role that the immune system plays in influencing the distribution and relative numbers of transduced cells in the context of AAV-mediated gene delivery.
Assuntos
Ácido Clodrônico/farmacologia , Dependovirus/genética , Terapia Genética/métodos , Adenoviridae/genética , Animais , Ácido Clodrônico/metabolismo , Técnicas de Transferência de Genes , Vetores Genéticos/genética , Células HEK293 , Humanos , Fígado/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Baço/metabolismo , Transgenes/genéticaRESUMO
Vesicular nucleotide transporter (VNUT) is the last identified member of the SLC17 organic anion transporter family, which plays a central role in vesicular storage in ATP-secreting cells. The discovery of VNUT demonstrated that, despite having been neglected for a long time, vesicular ATP release represents a major pathway for purinergic chemical transmission, which had been mainly attributed to ATP permeation channels. This article summarizes recent advances in our understanding of the mechanism of VNUT and its physiopathological roles as well as the development of inhibitors. Regulating the activity and/or the expression of VNUT represents a new and promising therapeutic strategy for the treatment of multiple diseases.
Assuntos
Trifosfato de Adenosina/metabolismo , Proteínas de Transporte de Nucleotídeos/metabolismo , Animais , Ritmo Circadiano , Ácido Clodrônico/metabolismo , Humanos , Inflamação/metabolismo , Inflamação/patologia , Neurônios/metabolismo , Proteínas de Transporte de Nucleotídeos/antagonistas & inibidores , Proteínas de Transporte de Nucleotídeos/genética , Percepção da Dor/fisiologia , Poroceratose/genética , Poroceratose/patologiaRESUMO
Bispecific T cell engagers have demonstrated clinical efficacy; however, their use can be accompanied by severe toxicity. Mechanistic understanding of these toxicities is limited by a lack of suitable immunocompetent preclinical models. In this study, we describe an immunocompetent mouse tumor model that exhibits bispecific T cell engager-induced toxicity and recapitulates key features similar to those in human cytokine release syndrome. In this study, toxicity occurred between the second and fourth injections of an NK Group 2D bispecific T cell engager protein. Symptoms were transient, peaking 3-4 h after treatment and resolving by 8 h. Mice developed weight loss, elevated plasma cytokines, a significant reduction in spleen white pulp, and lymphocyte infiltration in the liver. Systemic cellular immune changes also occurred; notably, an increase in CD8+ T cell activation, an increase in myeloid cells in the blood, and a population of Ly-6Cint monocytes (CD11b+Ly-6G-F4/80-) emerged in the liver and spleens of bispecific protein-treated mice. IFN-γ was primarily produced by CD8+ T cells in the spleen and was required for the observed changes in both T cell and myeloid populations. Rag deficiency, IFN-γ deficiency, or depletion of either CD4+ or CD8+ T cells prevented toxicity, whereas perforin deficiency, GM-CSF deficiency, or modulation of the myeloid population through clodronate-mediated depletion showed a partial abrogation of toxicity. Together, these findings reveal that T cell activation by a bispecific T cell engager leads to changes in the host myeloid cell population, both of which contribute to treatment induced toxicity in immunocompetent mice.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Neoplasias do Colo/imunologia , Subfamília K de Receptores Semelhantes a Lectina de Células NK/metabolismo , Receptores de Antígenos Quiméricos/metabolismo , Animais , Complexo CD3 , Linhagem Celular Tumoral , Ácido Clodrônico/metabolismo , Neoplasias do Colo/terapia , Síndrome da Liberação de Citocina/etiologia , Modelos Animais de Doenças , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos , Humanos , Interferon gama/metabolismo , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células Mieloides/imunologia , Subfamília K de Receptores Semelhantes a Lectina de Células NK/genética , Receptores de Antígenos Quiméricos/genética , Especificidade do Receptor de Antígeno de Linfócitos TRESUMO
Since the first report in 2003, bisphosphonate-related osteonecrosis of the jaw (BRONJ) has been increasing, without effective clinical strategies. Osteoporosis is common in elderly women, and bisphosphonates (BPs) are typical and widely used anti-osteoporotic or anti-bone-resorptive drugs. BRONJ is now a serious concern in dentistry. As BPs are pyrophosphate analogues and bind strongly to bone hydroxyapatite, and the P-C-P structure of BPs is non-hydrolysable, they accumulate in bones upon repeated administration. During bone-resorption, BPs are taken into osteoclasts and exhibit cytotoxicity, producing a long-lasting anti-bone-resorptive effect. BPs are divided into nitrogen-containing BPs (N-BPs) and non-nitrogen-containing BPs (non-N-BPs). N-BPs have far stronger anti-bone-resorptive effects than non-N-BPs, and BRONJ is caused by N-BPs. Our murine experiments have revealed the following. N-BPs, but not non-N-BPs, exhibit direct and potent inflammatory/necrotic effects on soft-tissues. These effects are augmented by lipopolysaccharide (the inflammatory component of bacterial cell-walls) and the accumulation of N-BPs in jawbones is augmented by inflammation. N-BPs are taken into soft-tissue cells via phosphate-transporters, while the non-N-BPs etidronate and clodronate inhibit this transportation. Etidronate, but not clodronate, has the effect of expelling N-BPs that have accumulated in bones. Moreover, etidronate and clodronate each have an analgesic effect, while clodronate has an anti-inflammatory effect via inhibition of phosphate-transporters. These findings suggest that BRONJ may be induced by phosphate-transporter-mediated and infection-promoted mechanisms, and that etidronate and clodronate may be useful for preventing and treating BRONJ. Our clinical trials support etidronate being useful for treating BRONJ, although additional clinical trials of etidronate and clodronate are needed.