Liposome-encapsulated Clodronate Research Articles

Macrophage Depletion Improves Endothelial Insulin Resistance and Protects against Cardiovascular Injury in Salt-Sensitive Hypertension.

Vascular endothelial insulin signaling is critical for the maintenance of vascular and metabolic homeostasis. We have previously shown that in hypertensive Dahl rats, impaired vascular insulin action is linked to angiotensin II activation of the NFκB inflammatory pathway. Macrophage polarization (M1) has implicated in hypertensive and metabolic diseases. Here, we investigated the effect of macrophage depletion using liposome-encapsulated clodronate (LEC) on endothelial insulin resistance and cardiovascular remodeling in Dahl salt-sensitive (DS) rats. High salt intake (HS) for 5 weeks increased systolic blood pressure (SBP: 192 ± 5 vs. 144 ± 4 mmHg in NS, p < 0.05), aortic and cardiac hypertrophy, cardiac fibrosis, and impaired acetylcholine- and insulin-induced vasorelaxation, accompanied by impaired insulin activation of endothelial nitric oxide synthases (eNOS)/NO signaling. HS rats had a significant increase in CD68 (a monocyte/macrophage marker) expression in the aorta and the heart. LEC reduced SBP (168 ± 5 mmHg, p < 0.05) and cardiovascular injury and improved acetylcholine- and insulin-mediated vasorelaxation and insulin signaling molecules with a reduction in the macrophage infiltration in the aorta and the heart. HS rats also manifested an increase in the aortic expressions of inflammatory cytokines, including the ratio of phosphorylated inhibitory kappa B (Iκb)/Iκb, tumor necrosis factor α, and phosphorylated c-Jun N-terminal kinase (JNK) and oxidative stress, which were reduced in HS/LEC rats. Our results suggest that in salt-sensitive hypertension, macrophage may importantly contribute to endothelial insulin resistance, vascular inflammation, and injury. These findings support the idea that macrophages may be a new target for immunotherapy of vasculopathy in hypertensive and metabolic disorders.

GM-CSF induces noninflammatory proliferation of microglia and disturbs electrical neuronal network rhythms in situ

BackgroundThe granulocyte-macrophage colony-stimulating factor (GM-CSF) (or CSF-2) is involved in myeloid cell growth and differentiation, and, possibly, a major mediator of inflammation in body tissues. The role of GM-CSF in the activation of microglia (CNS resident macrophages) and the consequent impacts on neuronal survival, excitability, and synaptic transmission are widely unknown, however. Here, we focused on electrical neuronal network rhythms in the gamma frequency band (30–70 Hz). Gamma oscillations are fundamental to higher brain functions, such as perception, attention, and memory, and they are exquisitely sensitive to metabolic and oxidative stress.MethodsWe explored the effects of chronic GM-CSF exposure (72 h) on microglia in male rat organotypic hippocampal slice cultures (in situ), i.e., postnatal cortex tissue lacking leukocyte invasion (adaptive immunity). We applied extracellular electrophysiological recordings of local field potential, immunohistochemistry, design-based stereology, biochemical analysis, and pharmacological ablation of microglia.ResultsGM-CSF triggered substantial proliferation of microglia (microgliosis). By contrast, the release of proinflammatory cytokines (IL-6, TNF-α) and nitric oxide, the hippocampal cytoarchitecture as well as the morphology of parvalbumin-positive inhibitory interneurons were unaffected. Notably, GM-CSF induced concentration-dependent, long-lasting disturbances of gamma oscillations, such as slowing (beta frequency band) and neural burst firing (hyperexcitability), which were not mimicked by the T lymphocyte cytokine IL-17. These disturbances were attenuated by depletion of the microglial cell population with liposome-encapsulated clodronate. In contrast to priming with the cytokine IFN-γ (type II interferon), GM-CSF did not cause inflammatory neurodegeneration when paired with the TLR4 ligand LPS.ConclusionsGM-CSF has a unique role in the activation of microglia, including the potential to induce neuronal network dysfunction. These immunomodulatory properties might contribute to cognitive impairment and/or epileptic seizure development in disease featuring elevated GM-CSF levels, blood-brain barrier leakage, and/or T cell infiltration.

Neuronal gamma oscillations and activity-dependent potassium transients remain regular after depletion of microglia in postnatal cortex tissue.

Microglial cells (resident macrophages) feature rapid activation in CNS disease and can acquire multiple phenotypes exerting neuroprotection or neurotoxicity. The functional impact of surveying ("resting") microglia on neural excitability and neurotransmission in physiology is widely unknown, however. We addressed this issue in male rat hippocampal slice cultures (in situ) by pharmacological microglial ablation within days and by characterizing neuronal gamma-band oscillations (30-70Hz) that are highly sensitive to neuromodulators and disturbances in ion and energy regulation. Gamma oscillations support action potential timing and synaptic plasticity, associate with higher brain functions like perception and memory, and require precise communication between excitatory pyramidal cells and inhibitory (GABAergic) interneurons. The slice cultures featured well-preserved hippocampal cytoarchitecture and parvalbumin-positive interneuron networks, microglia with ramified morphology, and low basal levels of IL-6, TNF-α, and nitric oxide (NO). Stimulation of slice cultures with the pro-inflammatory cytokine IFN-γ or bacterial LPS serving as positive controls for microglial reactivity induced MHC-II expression and increased cytokine and NO release. Chronic exposure of slice cultures to liposome-encapsulated clodronate reduced the microglial cell population by about 96%, whereas neuronal structures, astrocyte GFAP expression, and basal levels of cytokines and NO were unchanged. Notably, the properties of gamma oscillations reflecting frequency, number and synchronization of synapse activity were regular after microglial depletion. Also, electrical stimulus-induced transients of the extracellular potassium concentration ([K+ ]o ) reflecting cellular K+ efflux, clearance and buffering were unchanged. This suggests that nonreactive microglia are dispensable for neuronal homeostasis and neuromodulation underlying network signaling and rhythm generation in cortical tissue.

Effects of immune cell-targeted treatments result from the suppression of neuronal oxidative stress and inflammation in experimental diabetic rats.

In this study, we hypothesized that reduction of immune cell activation as well as their oxidant or inflammatory mediators with minocycline (MCN), liposome-encapsulated clodronate (LEC), or anti-Ly6G treatments can be neuroprotective approaches in diabetic neuropathy. MCN (40mg/kg) for reduction of microglial activation, LEC (25mg/kg) for of macrophage inhibition, or anti-Ly6G (150μg/kg) for neutrophil suppression injected to streptozotocin (STZ)-induced diabetic rats twice, 3days, and 1week (half dose) after STZ. Animal mass and blood glucose levels were measured; thermal and mechanical sensitivities were tested for in pain sensations. The levels of chemokine C-X-C motif ligand 1 (CXCL1), CXCL8, and C-C motif ligand 2 (CCL2), CCL3, and total oxidant status (TOS) and total antioxidant status (TAS) were measured in the spinal cord and sciatic nerve tissues of rats. LEC significantly reduced the glucose level of diabetic rats compared with drug control. However, MCN or anti-LY6G did not change the glucose level. While diabetic rats showed a marked decrease in both thermal and mechanical sensations, all treatments alleviated these abnormal sensations. The levels of chemokines and oxidative stress parameters increased in diabetic rats. All drug treatments significantly decreased the CCL2, CXCL1, and CXCL8 levels of spinal cord tissues and ameliorated the neuronal oxidative stress compared with control treatments. Present findings suggest that the neuroprotective actions of MCN, LEC, or anti-Ly6G treatments may be due to the modulation of neuronal oxidative stress and/or inflammatory mediators of immune cells in diabetic rats with neuropathy.

Targeting alveolar macrophages shows better treatment response than deletion of interstitial macrophages in EGFR mutant lung adenocarcinoma.

IntroductionAlveolar macrophages (AMs) are critical in the development of lung adenocarcinoma driven by epidermal growth factor receptor (EGFR) mutations. Whether interstitial macrophages (IMs) are also involved in lung tumorigenesis is still unclear. Thus, the aim of this study is to evaluate the role of both AM and IM in the development of EGFR mutant driven lung adenocarcinoma.MethodsWe used the EGFR mutant doxycycline‐inducible mouse model of lung adenocarcinoma to deplete interstitial or AMs by clodronate‐encapsulated liposomes administered intravenously (IV) and intratracheally (IT), respectively. Tumor burden, AMs, and the tumor microenvironment were examined by immunohistochemistry, bronchoalveolar lavage fluid or flow cytometry.ResultsClodronate treatment resulted in a significant reduction of tumor burden compared with vehicle liposomes alone. Elimination of AMs resulted in a significant reduction of proliferation compared with IV treatment. However, both treatments resulted in a significantly higher number of Ki67 positive cells compared with control mice, suggesting that tumor cells still proliferate despite the treatment. The number of natural killer cells decreased during tumor development, and it remained low even after the elimination of AMs. We also observed that IT instillation of clodronate significantly increased the number of CD8+ T cells, which was higher compared with vehicle‐treated mice and mice where only IMs were depleted. The similar trend was observed in immunohistological analyses of CD8+ T cells.ConclusionsThese results suggest that the reduction of AMs has a stronger impact on restricting tumor progression compared with targeting IMs. The depletion of AMs leads to an elevated infiltration of CD8+ T cells into the lung that might be responsible for tumor growth impairment. Altogether, elimination of AMs is a better strategy to reduce EGFR mutant tumor growth and is less toxic, suggesting the selectively targeting of AMs to complement established therapies.

Pro-inflammatory macrophages coupled with glycolysis remodel adipose vasculature by producing platelet-derived growth factor-B in obesity

Adipose tissue macrophages (ATMs) play a central role in tissue remodeling and homeostasis. However, whether ATMs promote adipose angiogenesis in obesity remains unclear. We examined the impact of ATMs deletion on adipose angiogenesis and tissue expansion in the epididymal white adipose tissue (eWAT) of high-fat diet (HFD)-fed mice by using liposome-encapsulated clodronate. We further elucidated the induction mechanisms of platelet-derived growth factor (PDGF)-B in macrophages in response to obesity-associated metabolic stresses, since it plays a significant role in the regulation of pericyte behavior for the initiation of neoangiogenesis during tissue expansion. ATM depletion prevented adipose tissue expansion in HFD-fed mice by inhibiting pericyte detachment from vessels, resulting in less vasculature in eWAT. The lipopolysaccharide (LPS) stimulation and high glucose concentration augmented glucose incorporation and glycolytic capacity with the induction of Pdgfb mRNA. This effect was mediated through extracellular signal-regulated kinase (ERK) among mitogen-activated protein kinases coupled with glycolysis in RAW264.7 macrophages. The Pdgfb induction system was distinct from that of inflammatory cytokines mediated by mechanistic target of rapamycin complex 1 (mTORC1) and NFκB signaling. Thus, obesity-associated hyperglycemia and chronic inflammation fuels ERK signaling coupled with glycolysis in pro-inflammatory macrophages, which contribute to the expansion of eWAT through PDGF-B-dependent vascular remodeling.

ATP/P2X7r axis mediates the pathological process of allergic asthma by inducing M2 polarization of alveolar macrophages

Recent studies revealed that macrophages are polarized towards the M2 phenotype in an ovalbumin (OVA)-induced asthmatic model. Alveolar macrophages (AMs) are immune barriers in alveoli to various pathogens in the respiratory tract; AMs suppress Th2 cell proliferation, inhibit interleukin (IL)-4, IL-5, and IL-13 secretion, and protect against airway hyperresponsiveness in allergic asthma. However, the polarization status and effects of different types of AMs in the pathogenesis of asthma are not known. ATP/P2X7r, expressed mainly on macrophages and dendritic cells, is associated with acute and chronic asthmatic airway inflammation and Th2 immune responses in mice and humans and functions by activating the NLRP3 inflammasome complex and inducing proinflammatory cytokine release (IL-1β and IL-18). Therefore, we evaluated the association between the ATP/P2X7r axis and different types of AMs in the pathology of allergic asthma. A murine AM-depleted asthma model was established by administration of clodronate-encapsulated liposomes, and M1-or M2-AMs were adoptively transferred to confirm the effects of different AMs in allergic asthma. Brilliant Blue G and BzATP were administered to OVA/HDM-induced mice in vivo. Lipopolysaccharide + OVA, ATP, Brilliant Blue G, and BzATP were used to stimulate AMs isolated from control and asthmatic mice. We found that selective depletion of AMs aggravated lung inflammation in asthmatic mice. Further, M2-type AMs may play a key role in mediating asthmatic inflammatory responses via the adoptive transfer of M2-type AMs to AM-depleted asthmatic mice, and the phenotype of AMs differentiated to M2 type in asthma. P2X7r expression in M2-type AMs was higher than that in M1-type AMs. Activating P2X7r induced polarization of M2-type AMs and inhibited polarization of M1-type AMs, while blockage of P2X7r had the opposite effect. The ATP/P2X7r axis may participate in the pathogenesis of asthma by mediating the M2-type AM polarization.

Brain perivascular macrophages contribute to the development of hypertension in stroke-prone spontaneously hypertensive rats via sympathetic activation.

Hypertension is associated with systemic inflammation. The activation of the sympathetic nervous system is critically involved in the pathogenesis of hypertension. Brain perivascular macrophages (PVMs) can be affected by circulating inflammatory cytokines, and the contribution of brain PVMs to sympathoexcitation has been demonstrated in a heart failure model. We thus investigated whether brain PVMs contribute to the development of hypertension through sympathoexcitation. Stroke-prone spontaneously hypertensive rats (SHRSP) developed hypertension over an 8-week period from 4 to 12 weeks of age. The number of brain PVMs and plasma interleukin-1β levels significantly increased at the ages of 8 and 12 weeks in SHRSP compared with normotensive Wistar-Kyoto rats (WKY). To determine the contribution of brain PVMs to blood pressure elevation, we intracerebroventricularly injected liposome-encapsulated clodronate, which eliminates macrophages by inducing apoptosis, into 8-week-old rats; we then assessed its effects in 10-week-old rats. Clodronate treatment attenuated the increase in mean blood pressure in SHRSP but not in WKY. Clodronate treatment reduced the depressor effect of hexamethonium, an index of sympathetic activity; it also reduced neuronal activity in sympathetic regulatory nuclei such as the hypothalamic paraventricular nucleus and rostral ventrolateral medulla and reduced the expression of cyclooxygenase-2 and prostaglandin E2, a downstream pathway in activated macrophages, in SHRSP but not in WKY. Furthermore, clodronate treatment attenuated the increase in blood pressure and renal sympathetic nerve activity in response to an acute intravenous injection of interleukin-1β in WKY. In conclusion, brain PVMs contribute to the development of hypertension via sympathetic activation. PVMs may be activated by increased levels of circulating interleukin-1β.

Severe Heterotopic Ossification in the Skeletal Muscle and Endothelial Cells Recruitment to Chondrogenesis Are Enhanced by Monocyte/Macrophage Depletion.

Altered macrophage infiltration upon tissue damage results in inadequate healing due to inappropriate remodeling and stem cell recruitment and differentiation. We investigated in vivo whether cells of endothelial origin phenotypically change upon heterotopic ossification induction and whether infiltration of innate immunity cells influences their commitment and alters the ectopic bone formation. Liposome-encapsulated clodronate was used to assess macrophage impact on endothelial cells in the skeletal muscle upon acute damage in the ECs specific lineage-tracing Cdh5CreERT2:R26REYFP/dtTomato transgenic mice. Macrophage depletion in the injured skeletal muscle partially shifts the fate of ECs toward endochondral differentiation. Upon ectopic stimulation of BMP signaling, monocyte depletion leads to an enhanced contribution of ECs chondrogenesis and to ectopic bone formation, with increased bone volume and density, that is reversed by ACVR1/SMAD pathway inhibitor dipyridamole. This suggests that macrophages contribute to preserve endothelial fate and to limit the bone lesion in a BMP/injury-induced mouse model of heterotopic ossification. Therefore, alterations of the macrophage-endothelial axis may represent a novel target for molecular intervention in heterotopic ossification.

Macrophages Guard Endothelial Lineage by Hindering Endothelial-to-Mesenchymal Transition: Implications for the Pathogenesis of Systemic Sclerosis.

The signals that control endothelial plasticity in inflamed tissues have only been partially characterized. For example, it has been shown that inadequate vasculogenesis in systemic sclerosis (SSc) has been associated with an endothelial defect. We used a genetic lineage tracing model to investigate whether endothelial cells die or change phenotypically after fibrosis induction and whether signals released by cells of the innate immune system and in the blood of patients influence their commitment. We observed that in the lineage-tracing transgenic mice Cdh5-CreERT2::R26R-EYFP, endothelial-derived cells (EdCs) underwent fibrosis after treatment with bleomycin, and EdCs retrieved from the lung showed expression of endothelial-to-mesenchymal transition (EndoMT) markers. Liposome-encapsulated clodronate was used to assess macrophage impact on EdCs. Clodronate treatment affected the number of alternatively activated macrophages in the lung, with upregulated expression of EndoMT markers in lung EdCs. Endothelial fate and function were investigated in vitro upon challenge with serum signals from SSc patients or released by activated macrophages. Sera of SSc patients with anti-Scl70 Abs, at higher risk of visceral organ fibrosis, induced EndoMT and jeopardized endothelial function. In conclusion, EdCs in SSc might be defective because of commitment to a mesenchymal fate, which is sustained by soluble signals in the patient's blood. Macrophages contribute to preserve the endothelial identity of precursor cells. Altered macrophage-dependent plasticity of EdCs could contribute to link vasculopathy with fibrosis.

Alveolar Macrophage Apoptosis-associated Bacterial Killing Helps Prevent Murine Pneumonia.

Rationale: Antimicrobial resistance challenges therapy of pneumonia. Enhancing macrophage microbicidal responses would combat this problem but is limited by our understanding of how alveolar macrophages (AMs) kill bacteria. Objectives: To define the role and mechanism of AM apoptosis-associated bacterial killing in the lung. Methods: We generated a unique CD68.hMcl-1 transgenic mouse with macrophage-specific overexpression of the human antiapoptotic Mcl-1 protein, a factor upregulated in AMs from patients at increased risk of community-acquired pneumonia, to address the requirement for apoptosis-associated killing. Measurements and Main Results: Wild-type and transgenic macrophages demonstrated comparable ingestion and initial phagolysosomal killing of bacteria. Continued ingestion (for ≥12 h) overwhelmed initial killing, and a second, late-phase microbicidal response killed viable bacteria in wild-type macrophages, but this response was blunted in CD68.hMcl-1 transgenic macrophages. The late phase of bacterial killing required both caspase-induced generation of mitochondrial reactive oxygen species and nitric oxide, the peak generation of which coincided with the late phase of killing. The CD68.hMcl-1 transgene prevented mitochondrial reactive oxygen species but not nitric oxide generation. Apoptosis-associated killing enhanced pulmonary clearance of Streptococcus pneumoniae and Haemophilus influenzae in wild-type mice but not CD68.hMcl-1 transgenic mice. Bacterial clearance was enhanced in vivo in CD68.hMcl-1 transgenic mice by reconstitution of apoptosis with BH3 mimetics or clodronate-encapsulated liposomes. Apoptosis-associated killing was not activated during Staphylococcus aureus lung infection. Conclusions: Mcl-1 upregulation prevents macrophage apoptosis-associated killing and establishes that apoptosis-associated killing is required to allow AMs to clear ingested bacteria. Engagement of macrophage apoptosis should be investigated as a novel, host-based antimicrobial strategy.

2000-P: Intracellular Metabolism-Dependent PDGF-B Induction in Inflammatory Macrophages Contributes to Adipose Tissue Expansion with Obesity

White adipose tissue (WAT) expands with angiogenesis. We have demonstrated that adipose-tissue-macrophages (ATMs) highly express platelet-derived growth factor (PDGF)-B in obesity, and exposure of PDGF-B stimulates detachment of pericytes (PCs) from vessels. Since vessel-attached PCs inhibit endothelial cell growth, the detachment restarts angiogenesis. In the present study, we aimed to clarify the significance of ATMs on adipose angiogenesis. Intraperitoneal injection of liposome-encapsulated clodronate (Clod) for 6 weeks achieved depletion of macrophages and decreased expression of Pdgfb mRNA in WAT of HFD-fed mice. Clod-treated mice showed smaller WAT with vessels well covered with PCs compared to control mice. We next investigated underlying mechanism of Pdgfb induction. LPS but not IL-4 stimulated Pdgfb expression in peritoneal macrophages. In RAW 264.7 macrophages, Pdgfb expression induced by high glucose plus LPS stimulation was completely blocked by pretreatment with 2-deoxyglucose (2-DG), a hexokinase inhibitor or heptelidic acid, a GAPDH inhibitor, whereas it was not affected by treatment with 6-aminonicotinamide, an inhibitor of glucose-6-phosphate dehydrogenase acting as the rate-limiting enzyme of the pentose phosphate pathway. In addition, high glucose plus LPS induced phosphorylations of S6 kinase, ERK, and JNK but not p38 MAP kinase. These phosphorylations were significantly attenuated by treatment with 2DG. Importantly, enhanced expression of Pdgfb by high glucose plus LPS was effectively suppressed by inhibitor or knockdown of Erk but not by rapamycin. The expression was also attenuated by inhibition of NFκb pathway. In conclusion, hyperglycemia and chronic inflammation reprogram metabolic pathways toward glycolysis, and subsequent activation of NFκB plays a pivotal role in the production of PDGF-B in ATMs during obesity development. Disclosure T. Wada: None. Y. Onogi: None. A. Okekawa: None. T. Matsuzawa: None. E. Watanabe: None. G. Komatsu: None. H. Tsuneki: None. T. Sasaoka: None. Funding Japan Society for the Promotion of Science (JP18K08469)

Momordicoside G Regulates Macrophage Phenotypes to Stimulate Efficient Repair of Lung Injury and Prevent Urethane-Induced Lung Carcinoma Lesions.

Momordicoside G is a bioactive component from Momordica charantia, this study explores the contributions of macrophages to the effects of momordicoside G on lung injury and carcinoma lesion. In vitro, when administered at the dose that has no effect on cell viability in M2-like macrophages, momordicoside G decreased ROS and promoted autophagy and thus induced apoptosis in M1-like macrophages with the morphological changes. In the urethane-induced lung carcinogenic model, prior to lung carcinoma lesions, urethane induced obvious lung injury accompanied by the increased macrophage infiltration. The lung carcinoma lesions were positively correlated with lung tissue injury and macrophage infiltration in alveolar cavities in the control group, these macrophages showed mainly a M1-like (iNOS+/CD68+) phenotype. ELISA showed that the levels of IL-6 and IL-12 were increased and the levels of IL-10 and TGF-β1 were reduced in the control group. After momordicoside G treatment, lung tissue injury and carcinoma lesions were ameliorated with the decreased M1-like macrophages and the increased M2-like (arginase+/CD68+) macrophages, whereas macrophage depletion by liposome-encapsulated clodronate (LEC) decreased significantly lung tissue injury and carcinoma lesions and also attenuated the protective efficacy of momordicoside G. The M2 macrophage dependent efficacy of momordicoside G was confirmed in a LPS-induced lung injury model in which epithelial closure was promoted by the transfer of M2-like macrophages and delayed by the transfer of M1-like macrophages. To acquire further insight into the underlying molecular mechanisms by which momordicoside G regulates M1 macrophages, we conduct a comprehensive bioinformatics analysis of momordicoside G relevant targets and pathways involved in M1 macrophage phenotype. This study suggests a function of momordicoside G, whereby it selectively suppresses M1 macrophages to stimulate M2-associated lung injury repair and prevent inflammation-associated lung carcinoma lesions.

Microglial Depletion with Clodronate Liposomes Increases Proinflammatory Cytokine Levels, Induces Astrocyte Activation, and Damages Blood Vessel Integrity.

Investigators are increasingly interested in using microglial depletion to study the role of microglia under pathologic conditions. Liposome-encapsulated clodronate is commonly used to eliminate macrophage populations because it causes functionally irreversible inhibition and apoptosis once phagocytized by macrophages. Recent studies have shown that microglia can be depleted in disease models by injecting clodronate liposomes into the brain parenchyma. However, it is unclear whether intracerebral administration of clodronate liposomes is a practical method of eliminating microglia under physiologic conditions or whether microglial depletion induces damage to other brain cells. In this study, injecting 1μL of clodronate liposomes (7μg/μL) into the striatum of mice caused ablation of microglia at 1day that persisted for 3days. Microglia reappeared in the boundary regions of microglia elimination after 5days. Importantly, we observed an increase in proinflammatory cytokine levels and an increase in neural/glial antigen 2 and glial fibrillary acidic protein expression in the perilesional region. In contrast, expression levels of myelin basic protein, microtubule-associated protein 2, and postsynaptic protein-95 decreased in the periphery of regions where microglia were depleted. Moreover, clodronate liposome administration decreased the density and integrity of blood vessels in the perilesional regions. In cultured primary neurons, clodronate liposome exposure also attenuated ATP synthesis. Together, these findings suggest that intracerebral administration of clodronate liposomes into brain parenchyma can deplete microglia, but can also damage other brain cells and blood vessel integrity.

Abstract A165: Depletion of macrophages in the tumor-draining lymph node enhances dendritic cell-induced antitumor immunity and survival

Abstract Effective antitumor immunity involves successful priming of tumor-specific T-cells by dendritic cells (DC) in the lymph node (LN), followed by trafficking, infiltration and sustained elimination of tumor cells by T-cells. Immunotherapies aim to facilitate or further invigorate these processes, but at which anatomic sites these therapies act and in which specific patients remains unknown. We and others have identified macrophages as key mediators of immune suppression in the tumor microenvironment (TME). Tumor-associated macrophages (TAMs) are capable of negating the effectiveness of multiple conventional- and immune-targeted anticancer therapies. Several current therapeutic strategies aim to deplete or reprogram macrophages; however, as these drugs act systemically, their precise mechanism and site of action remains unclear. Besides TAMs, macrophages in the LN have been identified as potent immune modulatory cells in diverse settings. Using a novel method that allows for the specific interrogation of LN-macrophages, we aimed to investigate the role of LN macrophages in regulating DC-induced anti-tumor immunity. To determine the immune modulatory functions of the tumor on the LN, we extensively characterized the immune contexture and phenotype of the tumor-draining lymph node (TDLN) compared to a distant non-tumor draining lymph node (non-TDLN) using multicolor flow- and histo-cytometry, in an orthotopic mouse model of peritoneal mesothelioma. In addition, the effects of systemic macrophage depletion using an CSF1R-kinase inhibitor or clodronate encapsulated liposomes (CEL) were evaluated. We developed a method to specifically deplete TDLN-macrophages while leaving TAMs and other tissue macrophages intact, by intrapleural (i.pl) injection of low-dose CEL. Using this model, we investigated the immune-regulatory properties of LN-macrophages following adoptive transfer of activated, tumor-loaded DCs in the TDLN by both flow- and histo-cytometry. Comparison of the TDLN and non-TDLN immune contexture revealed prominent shifts in immune cell frequencies and phenotypes, including a decrease in T-cell frequencies and a marked increase in CD169+ LN-macrophages in the TDLN. Systemic macrophage targeting using CSF1R-kinase inhibition or CEL effectively minimized LN-macrophage subsets as well as TAMs, therefore preventing the specific interrogation of LN-macrophage biology during tumor growth and in the context of immune activation. Conversely, titrating CEL doses down to 5% of the total dosing volume injected i.pl. allowed for the specific depletion of LN-macrophages while leaving systemic macrophages undisturbed. LN-macrophages limited the presence of migratory ex vivo activated and tumor-loaded DCs, as indicated by increased counts of CFSE+ DCs in the in the TDLN following adoptive transfer in CEL-pretreated mice. Depletion of TDLN-macrophages increased LN- and blood frequencies and activation status of DC-induced CD8+ T-cells and CD4+ T-helper cells, indicative of enhanced antitumor immunity. Furthermore, whereas DC-induced immune activation alone was insufficient to impede tumor growth and prolong survival, elimination of LN-macrophages prior to DC-administration enabled the successful impediment of tumor growth. Interestingly, interrogation of tumor-infiltrating T-cell (TIL) phenotype and distribution showed a robust increase in CD4+ T-helper TILs and a concurrent decrease in CD8+ TIL- exhaustion phenotype, as evidenced by diminished PD-1 and LAG-3 expression. Altogether, these results indicate an important immune inhibitory role for LN-macrophages in settings of immune activation in the solid tumor setting. These findings further increase our knowledge of where and how macrophages exert their immune-inhibitory functions and allow for better insight into the underpinnings of immunotherapy resistance. Citation Format: Floris F. Dammeijer, Mandy van Gulijk, Melanie M. Lukkes, Menno van Nimwegen, Rudi W. Hendriks, Thorbald T. van Hall, Heleen H. Vroman, Joachim J.G.J.V. Aerts. Depletion of macrophages in the tumor-draining lymph node enhances dendritic cell-induced antitumor immunity and survival [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A165.

Anti-inflammatory properties of Liposome-encapsulated clodronate or Anti-Ly6G can be modulated by peripheral or central inflammatory markers in carrageenan-induced inflammation model.

Overproduction of inflammatory markers by immune cells, such as macrophages and neutrophils, is one of the main reasons for many inflammatory conditions and inhibiting or suppressing of their production by cell depletion may provide new therapeutic targets or approaches to prevent a variety of inflammatory conditions. In this study, we examined the possible effects of anti-Ly6G-mediated systemic neutrophil depletion and liposome-encapsulated clodronate (LEC)-mediated systemic macrophage depletion on the inflammatory signs (thermal hyperalgesia, mechanical allodynia, oedema and fever) and measured the levels of various inflammation markers (tumour necrosis factor-α (TNF-α), interleukins (IL)-1β, IL-4, IL-10, macrophage inflammatory protein-1 alpha (MIP-1α/CCL3) and myeloperoxidase (MPO) in paw and spinal cord tissues in carrageenan (CG)-induced hindpaw inflammation model in rats. CG injection into the paw caused inflammation characterized by redness, swelling, heat and pain hypersensitivities. Anti-Ly6G or LEC significantly ameliorated the pain behaviours, and decreased the oedema and fever. Efficacies of anti-Ly6G or LEC on inflammatory responses changed depend on the degree of inhibition in inflammatory markers of inflamed paw or spinal cord. Anti-inflammatory properties of anti-Ly6G or LEC suggest that macrophages and/or neutrophil-mediated inflammatory cascade in inflamed site and spinal cord which can play key roles in inflammatory pain responses. These systemic or peripheral inflammatory mediators may be therapeutic targets in the treatment of many inflammatory conditions and related various diseases.

Macrophages mediate corticotomy-accelerated orthodontic tooth movement

Clinical evidence has suggested that surgical corticotomy of the alveolar bone can accelerate local orthodontic tooth movement (OTM), but the underlying cell and molecular mechanisms remain largely unclear. The present study examined the role of macrophages played in corticotomy-assisted OTM. Orthodontic nickel-titanium springs were applied to the left maxillary first molars of rats or mice to induce OTM with or without corticotomy. Corticotomy enhanced OTM distance by accelerating movement through induction of local osteoclastogenesis and macrophage infiltration during OTM. Further analysis showed that macrophages were polarized toward an M1-like phenotype immediately after corticotomy and then switched to an M2-like phenotype during OTM. The microenvironment of corticotomy induced macrophage infiltration and polarization through the production of TNF-α. More importantly, the amount of OTM induced by corticotomy was significantly decreased after mice were depleted of monocyte/macrophages by injection of liposome-encapsulated clodronate. Further experiments by incubating cultured macrophages with fresh tissue suspension obtained from post-corticotomy gingiva switched the cells to an M1 phenotype through activation of the nuclear factor-κB (NF-κB) signaling pathway, and to an M2 phenotype through activation of the JAK/STAT3 signaling pathway. Our results suggest that corticotomy induces macrophage polarization first by activating the NF-κB signaling pathway and later by activating the JAK/STAT3 signaling pathway, and that these processes contribute to OTM by triggering production of inflammatory cytokines and osteoclastogenesis.

Monocyte depletion attenuates the development of posttraumatic hydrocephalus and preserves white matter integrity after traumatic brain injury.

Monocytes are amongst the first cells recruited into the brain after traumatic brain injury (TBI). We have shown monocyte depletion 24 hours prior to TBI reduces brain edema, decreases neutrophil infiltration and improves behavioral outcomes. Additionally, both lesion and ventricle size correlate with poor neurologic outcome after TBI. Therefore, we aimed to determine the association between monocyte infiltration, lesion size, and ventricle volume. We hypothesized that monocyte depletion would attenuate lesion size, decrease ventricle enlargement, and preserve white matter in mice after TBI. C57BL/6 mice underwent pan monocyte depletion via intravenous injection of liposome-encapsulated clodronate. Control mice were injected with liposome-encapsulated PBS. TBI was induced via an open-head, controlled cortical impact. Mice were imaged using magnetic resonance imaging (MRI) at 1, 7, and 14 days post-injury to evaluate progression of lesion and to detect morphological changes associated with injury (3D T1-weighted MRI) including regional alterations in white matter patterns (multi-direction diffusion MRI). Lesion size and ventricle volume were measured using semi-automatic segmentation and active contour methods with the software program ITK-SNAP. Data was analyzed with the statistical software program PRISM. No significant effect of monocyte depletion on lesion size was detected using MRI following TBI (p = 0.4). However, progressive ventricle enlargement following TBI was observed to be attenuated in the monocyte-depleted cohort (5.3 ± 0.9mm3) as compared to the sham-depleted cohort (13.2 ± 3.1mm3; p = 0.02). Global white matter integrity and regional patterns were evaluated and quantified for each mouse after extracting fractional anisotropy maps from the multi-direction diffusion-MRI data using Siemens Syngo DTI analysis package. Fractional anisotropy (FA) values were preserved in the monocyte-depleted cohort (123.0 ± 4.4mm3) as compared to sham-depleted mice (94.9 ± 4.6mm3; p = 0.025) by 14 days post-TBI. All TBI mice exhibited FA values lower than those from a representative naïve control group with intact white matter tracts and FA~200 mm3). The MRI derived assessment of injury progression suggests that monocyte depletion at the time of injury may be a novel therapeutic strategy in the treatment of TBI. Furthermore, non-invasive longitudinal imaging allows for the evaluation of both TBI progression as well as therapeutic response over the course of injury.

Macrophages and Galectin 3 Control Bacterial Burden in Acute and Subacute Murine Leptospirosis That Determines Chronic Kidney Fibrosis.

Previous studies have suggested that macrophages may contribute to acute Leptospira dissemination, as well as having a major role in kidney fibrosis. Our aim was to characterize the role of macrophages and galectin 3 (Gal-3) on the survival, clinical course, bacterial burden, interstitial nephritis, and chronic kidney fibrosis in Leptospira interrogans serovar Copenhageni (LIC)-induced experimental murine leptospirosis. C57BL/6J mice depleted of macrophages by liposome-encapsulated clodronate treatment and infected with LIC presented a higher bacterial burden, had reduced subacute nephritis and enhanced chronic kidney fibrosis relative to untreated, infected mice. Moreover, LIC infection in mice whose Gal-3 was disrupted (Lgals3−/–) had a higher bacterial burden and enhanced subacute nephritis and chronic kidney fibrosis when compared to C57BL/6J wild-type mice. Chronic fibrosis did not correlate with higher transcription levels of TGF-β1 or IL-13 in the kidneys. Kidney fibrosis was found in chronically infected rats as well as in wild infected rats. On the other hand, human fibroblast cultures exhibited enhanced differentiation to myofibroblasts after treatment with LIC. Our results demonstrate that macrophages and Gal-3 play a critical role in controlling the LIC burden but has a minor role in subsequent fibrosis. Instead, kidney fibrosis was better correlated with bacterial burden. Taken together, our results do not support a role for macrophages to disseminate leptospires during acute infection, nor in chronic kidney fibrosis.

Glycolytic Activation Promotes PDGF-B Production in Adipose Tissue Macrophages during Obesity Development

The vascular network develops with remodeling in adipose tissue (AT) during obesity. We have found that PDGF-B detaches pericytes from the vasculature, which plays a crucial role in angiogenesis during obesity. The expression of PDGF-B was significantly elevated in obesity-associated infiltrated CD11c-positive (+) AT macrophages (ATMs). Interestingly, the expression was low in both CD11c+ ATMs and CD206+ ATMs of lean mice. Since glycolytic and oxidative metabolism are key metabolic programs that regulate ATMs’ polarity and function, we hypothesized that hyperglycemia and inflammation synergistically drive PDGF-B production by reprogramming metabolic pathways in ATMs. The expression of PDGF-B was increased in the AT of high-fat diet (HFD)-fed mice and STZ-induced hyperglycemic mice. In accordance, exposure to high glucose led to increase the expression of PDGF-B in association with that of Itgax (CD11c) in RAW264.7 macrophages. In peritoneal macrophages, LPS but not IL-4 provoked PDGF-B expression, which was completely blocked by pretreatment with 2-DG, a glycolysis inhibitor. Moreover, LPS effectively augmented high glucose-induced its expression, and that was abolished by MG132, an NFκB inhibitor. Since macrophages highly accumulated in obese AT, where pericytes were detached from vasculature, we examined directly whether PDGF-B from macrophage involves in AT expansion during obesity. To this end, we intraperitoneally injected liposome-encapsulated clodronate (CL) to mice twice a week for 6 weeks during HFD feeding. ATMs were effectively depleted by CL with reduced PDGF-B expression in AT of HFD-induced obese mice. Importantly, HFD-induced obesity was effectively prevented by CL. In conclusion, PDGF-B from ATMs has a crucial function in AT expansion during obesity. Hyperglycemia and chronic inflammation reprogram metabolic pathways toward glycolysis, and subsequent activation of NFκB plays a pivotal role in the production of PDGF-B in ATMs during obesity. Disclosure Y. Onogi: None. T. Wada: Research Support; Self; Sanwa Kagaku Kenkyusho Co., Ltd.. T. Matsuzawa: None. A. Okekawa: None. E. Watanabe: None. H. Tsuneki: None. T. Sasaoka: None.