Search Results (38)

Lysosomal acid lipase deficiency (LAL-D, OMIM #309900), caused by mutations of the LIPA gene, is involved in two continuum disease phenotypes: Wolman disease in infants and cholesteryl ester storage disease in adults. Their major phenotypes include steatosis, hepatomegaly, splenomegaly, and elevated liver enzymes. Enzyme replacement therapy has been used for the treatment of LAL-D. In this study, we examined the therapeutic effect of liposomal clodronate in an LAL-D mouse model. When liposomal clodronate was administered intraperitoneally by a single administration at 200 mg/kg to adult mice, organ cholesterol and triglyceride levels partially decreased. Furthermore, a significant correction of the relative organ weight of the liver and spleen as well as a reduction in splenic lipids were observed when liposomal clodronate was administered at 50 mg/kg to young mice intraperitoneally by multiple administrations. These results demonstrate that liposomal clodronate might have a therapeutic effect on the LAL-D mouse model. Full article

Background: Postoperative peritoneal adhesions arise from a dysregulated wound-healing response in which macrophages may have context-dependent effects. We investigated the contribution of macrophage-rich peritoneal and mesenteric compartments, the origin of macrophage-like cells in ischemic lesions, and the association between CCR2-dependent recruitment and postoperative inflammatory and reparative gene expression. Methods: Using a murine ischemic-button model, we assessed the effects of clodronate liposome treatment, bone marrow chimerism, and global CCR2 deficiency. Adhesion formation, F4/80+ cell accumulation, donor-marker expression, and selected inflammatory and wound-healing-associated transcripts were analyzed at predefined postoperative time points. Results: Clodronate liposome treatment was associated with reduced adhesion formation and substantial depletion of F4/80+ cells in peritoneal lavage and mesenteric tissue. However, F4/80+ cell numbers within ischemic buttons at postoperative day 3 were not significantly reduced, indicating that the depletion experiment does not establish selective depletion of all lesional macrophages. Bone marrow chimera experiments identified donor-marker-positive, F4/80+ cells within ischemic buttons, supporting recruitment of hematopoietic cells with a macrophage-like phenotype. CCR2 deficiency reduced F4/80+ cell accumulation in ischemic buttons and was associated with increased adhesion scores and altered expression of inflammatory and wound-healing-associated genes. These findings identify differential associations of clodronate-sensitive macrophage-rich compartments and CCR2-dependent hematopoietic recruitment with postoperative adhesion formation. Conclusions: Depletion of macrophage-rich peritoneal and mesenteric compartments was associated with reduced adhesion formation, whereas global CCR2 deficiency was associated with fewer lesional F4/80+ cells and greater adhesion severity. Because clodronate depletion, F4/80 staining, bone marrow chimerism, and global CCR2 deficiency do not provide cell-specific or fate-mapped resolution, these data do not establish distinct resident versus infiltrating macrophage functions or a reparative phenotype of CCR2-dependent cells. Cell-specific and temporally resolved validation is required to define the contributions and temporal relationships of individual macrophage subsets. Full article

Powassan virus (POWV) is an emerging tick-borne orthoflavivirus with consistently increasing annual prevalence. Though understood as a vector-transmitted virus associated with neurological disease, a 2016 fatal case of an immunocompromised 63-year-old man reported testicular pain with postmortem detection of viral antigen in the testes, suggesting an altered tropism. The extent to which POWV can infect testes remains unclear. We used a BALB/c mouse model of POWV to elucidate the potential of POWV to establish infection in testes and model a potential mechanism of testicular tropism. Here, we report that POWV productively infects the male reproductive tract of mice. We find that POWV consistently infects mouse testes and epididymides at 3 and 5 days post-infection (dpi) with peak titers measuring 6.1 log₁₀ PFU/gram of tissue in testes. Furthermore, we find that at 5 dpi, POWV-infected mice testes are significantly higher in mass than uninfected controls. Due to macrophages being implicated in driving testicular pathogenesis in Zika virus, a related orthoflavivirus, we depleted phagocytic cells using clodronate liposomes prior to POWV infection and compared them to control liposome-treated mice. Depletion of phagocytic cells was associated with higher and sustained viremia and significantly higher viral organ burden in the male reproductive tract of mice. Gene expression analysis revealed a robust antiviral, inflammatory, and chemokine response in the testes that was further amplified following phagocytic-cell depletion. These findings implicate the male reproductive tract as a previously underrecognized target of POWV disease and identify phagocytic cells as key in limiting viral infection of testes in mice. Full article

The host cellular mechanisms determining whether Dirofilaria immitis (canine heartworm) larvae establish infection remain poorly understood. The Mongolian gerbil (Meriones unguiculatus), a naturally nonpermissive host, mounts a rapid macrophage-dominated peritoneal cellular response to intraperitoneal (IP) larval challenge. To test whether macrophages mediate larval clearance, peritoneal macrophages were selectively depleted with IP clodronate liposomes (100 µL/10 g body weight; days −4 and −1 relative to infection) before challenge with approximately 40 D. immitis third-stage larvae (L3). A rat anti-mouse anti-F4/80 antibody was validated as a cross-reactive macrophage marker in jird peritoneal exudate cells (PECs) by standard and imaging flow cytometry, and depletion efficacy was confirmed prior to infection. Significantly more L3 were recovered from macrophage-depleted jirds (mean = 17.7%; SEM = 6.0%) than from PBS liposome controls (mean = 3.8%; SEM = 1.9%) at 1-day post-infection (p < 0.03). Cytology and flow cytometry confirmed near-complete macrophage depletion, and a significant compensatory neutrophilia was observed in depleted animals (64.5% ± 5.7% vs. 19.6% ± 5.1% in controls; p < 0.0001). These findings establish peritoneal macrophages as the primary innate cellular mediators of D. immitis L3 clearance in a nonpermissive host, providing a validated in vivo framework for mechanistic dissection of filarial host specificity. Full article

Kynurenic acid (KynA), a tryptophan metabolite that regulates immune homeostasis via G protein-coupled receptor 35 (GPR35), has an undefined role in post-myocardial infarction (MI) immune responses. To clarify this role, we established a murine MI model and administered KynA intraperitoneally to evaluate cardiac function and ventricular remodeling. Macrophage infiltration was assessed, and macrophages were depleted via clodronate liposomes to confirm their contribution to KynA-mediated cardioprotection. In bone marrow-derived macrophages (BMDMs), GPR35-targeted siRNA verified the receptor-dependent action of KynA. KynA improved cardiac function, reduced infarct scarring and fibrosis, and suppressed pro-inflammatory macrophage infiltration in MI mice, with these cardioprotective effects abrogated by macrophage depletion. Mechanistically, KynA inhibited voltage-dependent anion channel 1 oligomerization, prevented mitochondrial DNA leakage, and downregulated the cGAS/STING/TBK1/IκBα/P65 pathway in macrophages, while exogenous mitochondrial DNA counteracted this inhibition. Collectively, the KynA/GPR35 axis exerts cardioprotective effects against MI by attenuating macrophage pro-inflammatory responses, highlighting its potential as a novel therapeutic target. Full article

Background/Objectives: Microglia are the primary immune cells in the central nervous system (CNS) and are known as “resident” macrophages. The aim of this study was to determine the effect of acute ethanol (EtOH) on the microglia state and monocyte infiltration into the CNS, with particular attention to the role of peripheral and central dopamine (DA) D2 receptors (D2Rs) in mediating EtOH effects on peripheral and central substrates. We hypothesize that EtOH interacts with peripheral immune mediators via D2Rs including monocyte-derived macrophages (MDMs) to modulate midbrain neurons, DA transmission in the mesolimbic pathway from the ventral tegmental area (VTA) to nucleus accumbens (NAc), and the intoxicating effects of acute EtOH. Methods: Using the Macrophage FAS-Induced Apoptosis (MaFIA) mouse model (GFP+ on Csf1r promoter), we assessed the effects of three intraperitoneal (IP) doses of EtOH (1, 2, and 4 g/kg) at three time points (0.5, 1, and 2 h after injection) on D2R expression in blood leukocytes and microglia, as well as midbrain neuronal activity, DA release, and behavior. Results: Acute EtOH significantly enhanced lymphocyte and monocyte D2R expression at 1.0 g/kg by 2 h after injection in vivo but decreased D2R expression in vitro. Ethanol enhanced microglia D2R expression in the NAc, while not altering D2R expression in the VTA, but altered the microglia state in these areas, shifting them toward an inflammatory phenotype. Acute EtOH induced prolonged and progressive hypersensitivity of D2R activation of VTA GABA neurons. Intravenous injection of the macrophage depleter liposomal clodronate significantly reduced blood macrophages by 55.3% and blocked the typical inhibition of VTA GABA neurons by EtOH, as well as the enhancement of DA levels in the NAc, and the locomotor indices of intoxication produced by acute EtOH, but not choice place preference. Conclusions: These findings strongly suggest a neuroimmune peripheral connection for acute low-dose EtOH use and challenge the dogma that central actions of EtOH exclusively mediate its effect on DA neuronal activity and release. Full article

Since the initial report indicating that LPA₁ signaling plays a key role in initiating nerve injury-induced neuropathic pain (NeuP), subsequent studies using knockout mice and LPA_1/3 antagonists have demonstrated that LPA₁ and LPA₃ signaling impact NeuP and fibromyalgia (FM) models. In the present study, we identified hyperalgesia sexual dimorphism involving LPA_1/3 signaling in the intermittent psychological stress induced-related FM-like model called intermittent psychological stress (IPS)-induced generalized pain (IPGP) model where the hyperalgesia in IPGP mice was abolished in LPA₁- and LPA₃-knock-out mice. Pharmacological intervention by intraperitoneal (i.p.) treatments with the LPA_1/3 antagonist Ki16425 consistently prevented hyperalgesia. However, intracerebroventricular treatments with Ki16425 abolished hyperalgesia in male, but not female, mice. Notably, intrathecal treatments of Ki16425 did not prevent hyperalgesia. Further studies revealed that splenocytes derived from female IPGP mice could initiate hyperalgesia via adoptive transfer in naïve mice, and this effect was abolished when donor mice were pre-treated with Ki16425 (i.p.). Thus, these studies identify male-specific LPA_1/3-mediated mechanisms in the brain underlying IPGP, as well as distinct LPA-LPA_1/3-mediated peripheral immune mechanisms. Full article

Background: Iron is an important micronutrient under physiological conditions, including pregnancy. On the other hand, excessive iron intake is also associated with adverse pregnancy outcomes. Macrophages are crucial in regulating iron homeostasis and pregnancy conditions. However, the role of macrophages in iron metabolism during pregnancy is unclear. Therefore, we used mouse models to investigate whether maternal iron overload induces pregnancy complications and their interactions with macrophages. Methods and Results: Administration of high-dose iron (iron dextran) by intraperitoneal injection to pregnant mice induced pregnancy complications such as fetal death, but low-dose iron did not affect fetal weight. In the placenta, the amount of iron was significantly increased and levels of macrophages were decreased by iron administration. In the liver, iron administration dramatically increased the amount of iron, with increased inflammatory cytokines tumor necrosis factor-α (TNFα) and interleukin-6. Macrophages were observed to surround deposited iron in the liver. In an in vitro experiment, treatment with iron stimulated TNFα secretion with cell death in macrophages, but not in liver cells. To investigate the importance of macrophages during pregnancy, clodronate liposomes were administered to reduce macrophages in pregnant mice. The macrophage reduction in pregnant mice resulted in an increased absorption rate and fetal growth restriction, together with higher iron accumulation and inflammatory cytokines in the liver. Conclusions: Maternal excess iron may induce inflammatory conditions with macrophage dysfunction in the liver, resulting in pregnancy complications. The reduction in macrophages also induced higher iron levels and adverse effects during pregnancy, suggesting a vicious cycle between excessive iron and macrophage dysfunction during pregnancy. Full article

Bone marrow-derived macrophages (BMMs) exhibit dynamic behavior and functional capabilities in response to specific microenvironmental stimuli. Recent investigations have proved that BMMs play crucial roles in promoting necrotic lesion resolution. Despite substantial advancements in understanding their activation and interaction with injured livers, researchers face challenges to develop effective treatments based on manipulating BMMs function. Caveolin-1 (Cav-1) is the major structural protein on the plasma membrane. We previously reported that Cav-1 knockout (KO) mice exhibited less functional damage and necrosis in carbon tetrachloride (CCl₄)-induced liver injury. We hypothesize that the activation and recruitment of BMMs are involved in the resolution of necrotic lesions in Cav-1 KO mice. Wild-type (WT) and Cav-1 KO mice were injected with CCl₄ (10% v/v) to induce acute liver injury model. Blood samples and hepatic tissues were harvested for serum alanine transaminase (ALT) activity assessment, histopathological examination through hematoxylin–eosin (H&E) staining, and BMMs subpopulation analysis via flow cytometry. Then, primary BMMs were isolated and cultured to investigate the effect of Cav-1 on BMMs polarization, migration, and activation of STAT3 signal pathway. Validation of hepatic macrophage depletion was induced by administrating clodronate liposomes (CLs), and BMMs reconstitution was evaluated by EGFP labelled BMMs. Following this, hepatic macrophages were depleted by CLs, BMMs were isolated from Cav-1 KO, and WT mice were cultured and administrated to evaluate the protective role of Cav-1-deleted BMMs on the resolution of hepatocellular necrosis and apoptosis in acute liver injury. The BMMs ratio significantly increased from 2.12% (1D), 4.38% (1W), and 5.38% (2W) in oil control mice to 7.17%, 14.90%, and 19.30% in CCl₄-treated mice (p < 0.01 or p < 0.001). Concurrently, Cav-1 positive BMMs exhibited a marked elevation from 6.41% at 1D to 24.90% by 2W (p = 0.0228). Cav-1 KO exerted protective effects by reducing serum ALT by 26% (p = 0.0265) and necrotic areas by 28% (p = 0.0220) and enhancing BMMs infiltration by 60% (p = 0.0059). In vitro, Cav-1 KO BMMs showed a decrease in CD206 fluorescence intensity (p < 0.001), a time-dependent upregulation of arginase-1 mRNA (p < 0.05 or p < 0.01), a 1.22-fold increase in phosphorylated STAT3 (p = 0.0036), and impaired wound healing from 12 to 24 h (p < 0.001). The macrophage-depleting action in livers by CL injection persists for a minimum of 48 h. Administrated EGFP⁺ BMMs emerged as the predominant population following CL injection for a duration of 48 h. Following clodronate liposome-mediated hepatic macrophage depletion, the adoptive transfer of Cav-1 KO BMMs demonstrated therapeutic efficacy in CCl₄-induced acute liver injury. In CCl₄-induced acute liver injury, the adoptive transfer of Cav-1 KO BMMs reduced necrosis by 12.8% (p = 0.0105), apoptosis by 25.2% (p = 0.0127), doubled macrophages infiltration (p = 0.0269), and suppressed CXCL9/10 mRNA expression (p = 0.0044 or p = 0.0385). BMMs play a key role in the resolution of liver necrotic lesions in CCl₄-induced acute liver injury. Cav-1 depletion attenuates hepatocellular necrosis and apoptosis by accelerating BMMs recruitment and M2 polarization. Cav-1 in macrophages may represent a potential therapeutic target for acute liver injury. Full article

Cellular senescence is a hallmark of aging and contributes to age-related diseases, including diabetic nephropathy (DN). Additionally, macrophage-mediated inflammation has been linked with DKD. Therefore, we investigated the effect of macrophage depletion on kidney cell senescence in DN, focusing on the relationship between the GDF-15 and Klotho signaling pathways. Wistar albino rats (n = 24) were divided into four groups: healthy control, liposomal clodronate (LC)-treated healthy, diabetic, and LC-treated diabetic groups. Rats in the LC-treated healthy, diabetic, and LC-treated diabetic groups were intravenously administered LC once a week for 4 weeks. Rat models of type 2 diabetes were successfully established via the administration of streptozotocin and a high-fat diet, as evidenced by increased blood glucose levels, kidney weight to body weight (KW/BW) ratio, serum albumin, creatinine, and urea levels, kidney damage, and oxidative stress. However, LC-mediated macrophage depletion reduced the KW/BW ratio, improved serum and oxidative parameters, decreased inflammatory markers (IL-6 and TNF-α), and ameliorated oxidative stress. Additionally, LC treatment promoted macrophage polarization towards the anti-inflammatory phenotype, downregulated GDF-15 expression, upregulated Klotho expression, and ameliorated kidney damage. In conclusion, macrophage depletion combats kidney senescence by modulating Klotho and GDF-15, indicating their potential as novel targets in DN treatment. Full article

Obesity is concurrent with immunological dysregulation, resulting in chronic low-grade inflammation and cellular dysfunction. In pancreatic islets, this loss of function has been correlated with mature β-cells dedifferentiating into a precursor-like state through constant exposure to inflammatory stressors. As mature adipocytes likewise have the capability to dedifferentiate in vitro and in vivo, we wanted to analyze this cellular change in relation to adipose tissue (AT) inflammation and adipose tissue macrophage (ATM) activity. Using our organotypic AT explant culture method combined with a double-reporter mouse model for labeling ATMs and mature adipocytes, we were able to visualize and quantify dedifferentiated fat (DFAT) cells in AT explants. Preliminary testing showed increased dedifferentiation after tamoxifen (TAM) stimulation, making TAM-dependent lineage-tracing models unsuitable for quantification of naturally occurring DFAT cells. The regulatory role of ATMs in adipocyte dedifferentiation was shown through macrophage depletion using Plexxicon 5622 or clodronate liposomes, which significantly increased DFAT cell levels. Subsequent bulk RNA sequencing of macrophage-depleted explants revealed enrichment of the tumor necrosis factor α (TNFα) signaling pathway as well as downregulation of associated genes. Direct stimulation with TNFα decreased adipocyte dedifferentiation, while application of a TNFα-neutralizing antibody did not significantly alter DFAT cell levels. Our findings suggest a regulatory role of resident ATMs in maintaining the mature adipocyte phenotype and preventing excessive adipocyte dedifferentiation. The specific regulatory pathways as well as the impact that DFAT cells might have on ATMs, and vice versa, are subject to further investigation. Full article

Cardiovascular diseases (CVD) and cancer are the top deadly diseases in the world. Both CVD and cancer have common risk factors; therefore, with the advances in treatment and life span, both diseases may occur simultaneously in patients. It is becoming evident that CVD and cancer are highly connected, establishing a novel discipline known as cardio-oncology. This includes the cardiomyocyte death following any anti-tumor therapy known as cardiotoxicity as well the intricate interplay between heart failure and cancer. Recent studies, using various mouse models, showed that heart failure promotes tumor growth and metastasis spread. Indeed, patients with heart failure were found to be at higher risk of developing malignant diseases. While the effect of heart failure on cancer is well established, little is known regarding the effect of tumors on heart failure. A recent study from our lab has demonstrated that tumor growth and metastasis ameliorate cardiac remodeling in a pressure-overload mouse model. Nevertheless, this study was inconclusive regarding whether tumor growth solely suppresses cardiac remodeling or is able to reverse existing heart failure outcomes as well. Here, we used a regulable transgenic mouse model for cardiac hypertrophy and fibrosis. Cancer cell implantation suppressed cardiac dysfunction and fibrosis as shown using echocardiography, qRT-PCR and fibrosis staining. In addition, tumor growth resulted in an M1 to M2 macrophage switch, which is correlated with cardiac repair. Macrophage depletion using clodronate liposomes completely abrogated the tumors’ beneficial effect. This study highly suggests that harnessing tumor paradigms may lead to the development of novel therapeutic strategies for CVDs and fibrosis. Full article

We previously demonstrated that neural stem/progenitor cells (NSPCs) were induced within and around the ischemic areas in a mouse model of ischemic stroke. These injury/ischemia-induced NSPCs (iNSPCs) differentiated to electrophysiologically functional neurons in vitro, indicating the presence of a self-repair system following injury. However, during the healing process after stroke, ischemic areas were gradually occupied by inflammatory cells, mainly microglial cells/macrophages (MGs/MΦs), and neurogenesis rarely occurred within and around the ischemic areas. Therefore, to achieve neural regeneration by utilizing endogenous iNSPCs, regulation of MGs/MΦs after an ischemic stroke might be necessary. To test this hypothesis, we used iNSPCs isolated from the ischemic areas after a stroke in our mouse model to investigate the role of MGs/MΦs in iNSPC regulation. In coculture experiments, we show that the presence of MGs/MΦs significantly reduces not only the proliferation but also the differentiation of iNSPCs toward neuronal cells, thereby preventing neurogenesis. These effects, however, are mitigated by MG/MΦ depletion using clodronate encapsulated in liposomes. Additionally, gene ontology analysis reveals that proliferation and neuronal differentiation are negatively regulated in iNSPCs cocultured with MGs/MΦs. These results indicate that MGs/MΦs negatively impact neurogenesis via iNSPCs, suggesting that the regulation of MGs/MΦs is essential to achieve iNSPC-based neural regeneration following an ischemic stroke. Full article

Psychological stress is a significant contributor to various chronic diseases and affects multiple physiological processes including erythropoiesis. This study aimed to examine the tissue-specific contributions of macrophages and extracellular ATP, as a signal of disturbed tissue homeostasis, to erythropoiesis under conditions of repeated psychological stress. Adult male BALB/c mice were subjected to 2 h daily restraint stress for seven consecutive days. Clodronate-liposomes were used to deplete resident macrophages from the bone marrow and spleen two days prior to the first restraint procedure, as well as newly recruited macrophages, every third day for the duration of the experiment. Repeated stress induced a considerable increase in the number of erythroid progenitor cells as well as in the percentage of CD71+/Ter119+ and CD71−/Ter119+ cells in the bone marrow and spleen. Macrophage depletion completely abolished the stimulative effect of repeated stress on immature erythroid cells, and prevented stress-induced increases in ATP levels, P2X7 receptor (P2X7R) expression, and ectonucleotidase CD39 activity and expression in the bone marrow and spleen. The obtained results demonstrate the stimulative effects of repeated stress on erythroid cells, extracellular ATP levels, P2X7R expression, CD39 activity and expression within the bone marrow and spleen, as well as the essential role of macrophages in stress-induced changes. Full article

After amputation, granular hemocytes infiltrate the blastema of regenerating cephalic tentacles of the freshwater snail Pomacea canaliculata. Here, the circulating phagocytic hemocytes were chemically depleted by injecting the snails with clodronate liposomes, and the effects on the cephalic tentacle regeneration onset and on Pc-Hemocyanin, Pc-transglutaminase (Pc-TG) and Pc-Allograft Inflammatory Factor-1 (Pc-AIF-1) gene expressions were investigated. Flow cytometry analysis demonstrated that clodronate liposomes targeted large circulating hemocytes, resulting in a transient decrease in their number. Corresponding with the phagocyte depletion, tentacle regeneration onset was halted, and it resumed at the expected pace when clodronate liposome effects were no longer visible. In addition to the regeneration progress, the expressions of Pc-Hemocyanin, Pc-TG, and Pc-AIF-1, which are markers of hemocyte-mediated functions like oxygen transport and immunity, clotting, and inflammation, were modified. After the injection of clodronate liposomes, a specific computer-assisted image analysis protocol still evidenced the presence of granular hemocytes in the tentacle blastema. This is consistent with reports indicating the large and agranular hemocyte population as the most represented among the professional phagocytes of P. canaliculata and with the hypothesis that different hemocyte morphologies could exert diverse biological functions, as it has been observed in other invertebrates. Full article