Targeting transitioning lung monocytes/macrophages as treatment strategies in lung disease related to environmental exposures.

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.

Clodronate disodium does not produce measurable effects on bone metabolism in an exercising, juvenile, large animal model.

Bisphosphonates are commonly used to treat and prevent bone loss, but their effects in active, juvenile populations are unknown. This study examined the effects of intramuscular clodronate disodium (CLO) on bone turnover, serum bone biomarkers (SBB), bone mineral density (BMD), bone microstructure, biomechanical testing (BT), and cartilage glycosaminoglycan content (GAG) over 165 days. Forty juvenile sheep (253 ± 6 days of age) were divided into four groups: Control (saline), T0 (0.6 mg/kg CLO on day 0), T84 (0.6 mg/kg CLO on day 84), and T0+84 (0.6 mg/kg CLO on days 0 and 84). Sheep were exercised 4 days/week and underwent physical and lameness examinations every 14 days. Blood samples were collected for SBB every 28 days. Microstructure and BMD were calculated from tuber coxae (TC) biopsies (days 84 and 165) and bone healing was assessed by examining the prior biopsy site. BT and GAG were evaluated postmortem. Data, except lameness data, were analyzed using a mixed-effects model; lameness data were analyzed as ordinal data using a cumulative logistic model. CLO did not have any measurable effects on the skeleton of sheep. SBB showed changes over time (p ≤ 0.03), with increases in bone formation and decreases in some bone resorption markers. TC biopsies showed increasing bone volume fraction, trabecular spacing and thickness, and reduced trabecular number on day 165 versus day 84 (p ≤ 0.04). These changes may be attributed to exercise or growth. The absence of a treatment effect may be explained by the lower CLO dose used in large animals compared to humans. Further research is needed to examine whether low doses of bisphosphonates may be used in active juvenile populations for analgesia without evidence of bone changes.

Teriparatide and clodronate combination as a potential treatment for complex regional pain syndrome type I in delayed consolidation after foot surgery: a case report and review of the literature.

BACKGROUND: Complex regional pain syndrome type I is a pathological condition characterized by an exaggerated response of tissues to low or moderate pain stimuli. The exact pathogenesis and optimal medical treatment for complex regional pain syndrome type I are still not fully understood, although bisphosphonates have shown positive effects in reducing pain. Foot surgery can be complicated by the development of complex regional pain syndrome type I, leading to functional decline and difficulties in weight-bearing. CASE PRESENTATION: The authors present a clinical case involving complex regional pain syndrome type I that developed after surgical foot arthrodesis. The patient, a 42-year-old Caucasian male, did not respond to clodronate treatment but experienced successful outcomes upon the addition of teriparatide, which effectively stimulated the healing of arthrodesis. CONCLUSION: Teriparatide cannot be considered the primary treatment for complex regional pain syndrome due to insufficient solid clinical data. However, when complex regional pain syndrome is associated with or caused by delayed union, teriparatide can be used to address the underlying cause of complex regional pain syndrome.

Development of finely tuned liposome nanoplatform for macrophage depletion.

BACKGROUND: Immunotherapy with clodronate-encapsulated liposomes, which induce macrophage depletion, has been studied extensively. However, previously reported liposomal formulation-based drugs (Clodrosome® and m-Clodrosome®) are limited by their inconsistent size and therapeutic efficacy. Thus, we aimed to achieve consistent therapeutic effects by effectively depleting macrophages with uniform-sized liposomes. RESULTS: We developed four types of click chemistry-based liposome nanoplatforms that were uniformly sized and encapsulated with clodronate, for effective macrophage depletion, followed by conjugation with Man-N3 and radiolabeling. Functionalization with Man-N3 improves the specific targeting of M2 macrophages, and radioisotope labeling enables in vivo imaging of the liposome nanoplatforms. The functionalized liposome nanoplatforms are stable under physiological conditions. The difference in the biodistribution of the four liposome nanoplatforms in vivo were recorded using positron emission tomography imaging. Among the four platforms, the clodronate-encapsulated mannosylated liposome effectively depleted M2 macrophages in the normal liver and tumor microenvironment ex vivo compared to that by Clodrosome® and m-Clodrosome®. CONCLUSION: The newly-developed liposome nanoplatform, with finely tuned size control, high in vivo stability, and excellent ex vivo M2 macrophage targeting and depletion effects, is a promising macrophage-depleting agent.

The pathogenic role of succinate-SUCNR1: a critical function that induces renal fibrosis via M2 macrophage.

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.

Non-purulent myositis caused by direct invasion of skeletal muscle tissue by Leptospira in a hamster model.

Myalgia is a common symptom of Leptospira infection in humans. Autopsies have reported that muscle tissue shows degeneration and necrosis of the myofibers and infiltration of inflammatory cells composed mainly of macrophages and lymphocytes. It remains unclear whether Leptospira directly infects the muscle and how the infiltrating inflammatory cells are involved in muscle fiber destruction. This study evaluated the relationship between histopathological changes and leptospiral localization in the muscle tissue of a hamster model. The influence of macrophages in skeletal muscle injury was also investigated, using selective depletion of macrophages by administration of liposomal clodronate. Hamsters infected subcutaneously with Leptospira interrogans serovar Manilae strain UP-MMC-SM showed myositis of the thighs adjacent to the inoculated area beginning at 6 days post-infection. The myositis was non-purulent and showed sporadic degeneration and necrosis of muscle fibers. The degeneration of myofibers was accompanied by aggregations of macrophages. Immunofluorescence staining revealed leptospires surrounding the damaged muscle fibers. Subcutaneous injection of formalin-killed Leptospira or intraperitoneal injection of live Leptospira caused no myositis in hamster thighs. Liposomal clodronate treatment in infected hamsters reduced macrophage infiltration in muscle tissue without impacting bacterial clearance. Muscle necrosis was still observed in the infected hamsters treated with liposomal clodronate, and there was no significant change in serum creatine kinase levels compared to those in animals treated with liposomes alone. Our findings suggest that leptospiral invasion of muscle tissue from an inoculation site leads to the destruction of muscle fibers and causes non-purulent myositis, whereas the infiltrating macrophages contribute less to muscle destruction.

Depletion of muscularis macrophages ameliorates inflammation-driven dysmotility in murine colitis model.

Previously, the presence of a blood-myenteric plexus barrier and its disruption was reported in experimentally induced colitis via a macrophage-dependent process. The aim of this study is to reveal how myenteric barrier disruption and subsequent neuronal injury affects gut motility in vivo in a murine colitis model. We induced colitis with dextran sulfate sodium (DSS), with the co-administration of liposome-encapsulated clodronate (L-clodronate) to simultaneously deplete blood monocytes contributing to macrophage infiltration in the inflamed muscularis of experimental mice. DSS-treated animals receiving concurrent L-clodronate injection showed significantly decreased blood monocyte numbers and colon muscularis macrophage (MM) density compared to DSS-treated control (DSS-vehicle). DSS-clodronate-treated mice exhibited significantly slower whole gut transit time than DSS-vehicle-treated animals and comparable to that of controls. Experiments with oral gavage-fed Evans-blue dye showed similar whole gut transit times in DSS-clodronate-treated mice as in control animals. Furthermore, qPCR-analysis and immunofluorescence on colon muscularis samples revealed that factors associated with neuroinflammation and neurodegeneration, including Bax1, Hdac4, IL-18, Casp8 and Hif1a are overexpressed after DSS-treatment, but not in the case of concurrent L-clodronate administration. Our findings highlight that MM-infiltration in the muscularis layer is responsible for colitis-associated dysmotility and enteric neuronal dysfunction along with the release of mediators associated with neurodegeneration in a murine experimental model.

Microglia Depletion Attenuates the Pro-Resolving Activity of the Formyl Peptide Receptor 2 Agonist AMS21 Related to Inhibition of Inflammasome NLRP3 Signalling Pathway: A Study of Organotypic Hippocampal Cultures.

Microglial cells have been demonstrated to be significant resident immune cells that maintain homeostasis under physiological conditions. However, prolonged or excessive microglial activation leads to disturbances in the resolution of inflammation (RoI). Formyl peptide receptor 2 (FPR2) is a crucial player in the RoI, interacting with various ligands to induce distinct conformational changes and, consequently, diverse biological effects. Due to the poor pharmacokinetic properties of endogenous FPR2 ligands, the aim of our study was to evaluate the pro-resolving effects of a new ureidopropanamide agonist, compound AMS21, in hippocampal organotypic cultures (OHCs) stimulated with lipopolysaccharide (LPS). Moreover, to assess whether AMS21 exerts its action via FPR2 specifically located on microglial cells, we conducted a set of experiments in OHCs depleted of microglial cells using clodronate. We demonstrated that the protective and anti-inflammatory activity of AMS21 manifested as decreased levels of lactate dehydrogenase (LDH), nitric oxide (NO), and proinflammatory cytokines IL-1ß and IL-6 release evoked by LPS in OHCs. Moreover, in LPS-stimulated OHCs, AMS21 treatment downregulated NLRP3 inflammasome-related factors (CASP1, NLRP3, PYCARD) and this effect was mediated through FPR2 because it was blocked by the FPR2 antagonist WRW4 pre-treatment. Importantly this beneficial effect of AMS21 was only observed in the presence of microglial FPR2, and absent in OHCs depleted with microglial cells using clodronate. Our results strongly suggest that the compound AMS21 exerts, at nanomolar doses, protective and anti-inflammatory properties and an FPR2 receptor located specifically on microglial cells mediates the anti-inflammatory response of AMS21. Therefore, microglial FPR2 represents a promising target for the enhancement of RoI.

Myeloid Cell Derived IL1ß Contributes to Pulmonary Hypertension in HFpEF.

BACKGROUND: Pulmonary hypertension (PH) in heart failure with preserved ejection fraction (HFpEF) is a common and highly morbid syndrome, but mechanisms driving PH-HFpEF are poorly understood. We sought to determine whether a well-accepted murine model of HFpEF also displays features of PH, and we sought to identify pathways that might drive early remodeling of the pulmonary vasculature in HFpEF. METHODS: Eight-week-old male and female C57BL/6J mice received either Nγ-nitro-L-arginine methyl ester and high-fat diet or control water and diet for 2, 5, and 12 weeks. The db/db mice were studied as a second model of HFpEF. Early pathways regulating PH were identified by bulk and single-cell RNA sequencing. Findings were confirmed by immunostain in lungs of mice or lung slides from clinically performed autopsies of patients with PH-HFpEF. ELISA was used to verify IL-1ß (interleukin-1 beta) in mouse lung, mouse plasma, and also human plasma from patients with PH-HFpEF obtained at the time of right heart catheterization. Clodronate liposomes and an anti-IL-1ß antibody were utilized to deplete macrophages and IL-1ß, respectively, to assess their impact on pulmonary vascular remodeling in HFpEF in mouse models. RESULTS: Nγ-nitro-L-arginine methyl ester/high-fat diet-treated mice developed PH, small vessel muscularization, and right heart dysfunction. Inflammation-related gene ontologies were overrepresented in bulk RNA sequencing analysis of whole lungs, with an increase in CD68+ cells in both murine and human PH-HFpEF lungs. Cytokine profiling showed an increase in IL-1ß in mouse and human plasma. Finally, clodronate liposome treatment in mice prevented PH in Nγ-nitro-L-arginine methyl ester/high-fat diet-treated mice, and IL-1ß depletion also attenuated PH in Nγ-nitro-L-arginine methyl ester/high-fat diet-treated mice. CONCLUSIONS: We report a novel model for the study of PH and right heart remodeling in HFpEF, and we identify myeloid cell-derived IL-1ß as an important contributor to PH in HFpEF.

Early macrophage-mediated Bdnf expression in white adipose tissue during high-fat diet feeding.

The expression of brain-derived neurotrophic factor (BDNF) is observed not only in the brain, but also in peripheral tissues including white adipose tissues (WATs). Here, we showed that the mRNA expression of Bdnf in inguinal WAT (iWAT) and epididymal WAT (eWAT) increased within 2 weeks of feeding mice with a high-fat diet (HFD). In mice on a 2-week HFD, the induction of Bdnf expression in WATs was significantly correlated with increases in body weight, suggesting that Bdnf expression may increase at an early stage of obesity. The mRNA expression of hypoxia-inducible factor 1α and platelet-derived growth factor, which are involved in neovascularization and the subsequent expansion of adipose tissues, increased in the iWAT of mice on the 2-week HFD. We also found that the expression of macrophage marker F4/80 in iWAT increased under the HFD. Interestingly, HFD-induced Bdnf expression in iWAT was not observed when macrophages were removed by the administration of clodronate liposomes. Accordingly, mice receiving clodronate liposomes also exhibited a significant reduction in the HFD-induced increase in body weight. In conclusion, increased body weight in HFD-induced obese model mice was accompanied by the induction of Bdnf expression in iWAT and was probably mediated by macrophages. Our findings imply a novel function for BDNF in iWAT at an early stage of obesity.

Macrophage depletion attenuates degeneration of spiral ganglion neurons in kanamycin-induced unilateral hearing loss model.

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.

Monocyte-derived macrophages contribute to the deterioration of immunological liver injury in mice.

BACKGROUND & AIMS: Autoimmune hepatitis (AIH) is characterized by hepatocyte destruction, leading to lymphocyte and macrophage accumulation in the liver. However, the specific mechanisms of how macrophages participate in the occurrence and development of AIH are still unclear. In this study, we investigated the effect of monocyte-derived macrophages on Con A-induced immunological liver injury in mice and we hypothesized that inhibition of CCR2 with the dual CCR2/5 inhibitor, cenicriviroc (CVC), would attenuate Con A-induced hepatitis in mice by reducing the recruitment of monocytes into the liver. METHODS: Murine experimental AIH was established by concanavalin A (Con A) injection intravenously. Macrophages were depleted by injection of clodronate liposomes in Con A-treated mice. Moreover, inhibition of the CCR2/5 signaling pathway in Con A mice is achieved by CVC. Liver injury and infiltration of monocyte-derived macrophages were assessed by serum transaminase levels, histopathology, immunohistochemistry, flow cytometry, RT-qPCR, ELISA, TUNEL assay and dihydroethidium staining. RESULTS: The number of macrophages in the mouse livers increased in the Con A-induced hepatitis mouse model, and flow cytometry showed a significant increase in the proportion of F4/80loCD11bhi monocyte-derived macrophages, while there was no significant change in the proportion of F4/80hiCD11blo Kupffer cells. After the depletion of liver macrophages by clodronate liposomes, the levels of serum ALT and AST, and the degree of liver tissue damage were alleviated in Con A-treated mice. Furthermore, Con A leaded an increase in the expression of a group of CC chemokines in mouse livers, and the elevation of CCL2 was prevented with the depletion of macrophages. Additionally, CVC reduced macrophage infiltration in the liver and ameliorated Con A-induced liver injury. Meanwhile, CVC reduced the apoptosis and oxidative damage of hepatocytes caused by Con A. CONCLUSIONS: Our research demonstrates that there is an increase in monocyte-derived macrophages in the livers due to the monocyte infiltration resulted from the activation of the CCL2-CCR2 axis in Con A-induced liver injury mouse model. Pharmacological inhibition of CCR2 monocyte recruitment by CVC efficiently ameliorates the hepatic inflammation, indicating the therapeutic potential of CVC in patients with AIH.

Retrospective evaluation of acute kidney injury in horses treated with nonnitrogenous bisphosphonates (2013-2020): 8 cases.

OBJECTIVE: To describe a population of horses with acute kidney injury (AKI) following administration of bisphosphonates including clinical signs, clinicopathologic data, treatment, and outcome. DESIGN: Retrospective study from August 2013 to July 2020. SETTING: Veterinary university teaching hospital. ANIMALS: Eight adult horses with AKI following administration of nonnitrogenous bisphosphonates. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Five horses received intramuscular clodronate (5/8; 62.5%) and 3 horses received intravenous tiludronate (3/8; 37.5%). Six horses (6/8; 75%) received concurrent nonsteroidal anti-inflammatory drugs. The most common initial presenting complaint was poor appetite (6/8; 75%), followed by abnormal urination (2/8; 25%). At the time of initial evaluation, the mean serum or plasma creatinine was 451.72 ± 190.06 µmol/L (5.11 ± 2.15 mg/dL) and BUN was 18.84 ± 8.85 mmol/L (52.75 ± 24.77 mg/dL). Five horses (5/6; 83.3%) had either an increased number of red blood cells (n = 4) or hemoprotein (n = 1) in the urine. All horses were treated with IV isotonic, balanced crystalloids either as a bolus, continuous rate infusion, or a combination of the 2. Seven horses (7/8; 87.5%) survived the initial episode of AKI and 1 horse (1/8; 12.5%) was euthanized. Of the 7 surviving horses, 2 horses (2/7; 28.5%) went on to develop chronic renal dysfunction. Warmblood breeds were overrepresented in the AKI group (P = 0.008; odds ratio: 11.5, 95% confidence interval: 1.8-72.1), when compared to horses that received bisphosphonates during the study period and did not develop AKI. CONCLUSIONS: Bisphosphonate administration, with or without concurrent nonsteroidal anti-inflammatory drugs, can be associated with AKI in horses. Serum creatinine should be monitored prior to and following bisphosphonate treatment to minimize this risk. Further evaluation of renal function is warranted in horses that develop clinical signs of poor appetite, lethargy, or altered urination in the days following bisphosphonate treatment.

Macrophage depletion using clodronate liposomes reveals latent dysfunction of the hematopoietic microenvironment associated with persistently imbalanced M1/M2 macrophage polarization in a mouse model of hemophagocytic lymphohistiocytosis.

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.

Preparation and PET/CT imaging of implant directed 68Ga-labeled magnetic nanoporous silica nanoparticles.

BACKGROUND: Implant infections caused by biofilm forming bacteria are a major threat in orthopedic surgery. Delivering antibiotics directly to an implant affected by a bacterial biofilm via superparamagnetic nanoporous silica nanoparticles could present a promising approach. Nevertheless, short blood circulation half-life because of rapid interactions of nanoparticles with the host's immune system hinder them from being clinically used. The aim of this study was to determine the temporal in vivo resolution of magnetic nanoporous silica nanoparticle (MNPSNP) distribution and the effect of PEGylation and clodronate application using PET/CT imaging and gamma counting in an implant mouse model. METHODS: PEGylated and non-PEGylated MNPSNPs were radiolabeled with gallium-68 (68Ga), implementing the chelator tris(hydroxypyridinone). 36 mice were included in the study, 24 mice received a magnetic implant subcutaneously on the left and a titanium implant on the right hind leg. MNPSNP pharmacokinetics and implant accumulation was analyzed in dependence on PEGylation and additional clodronate application. Subsequently gamma counting was performed for further final analysis. RESULTS: The pharmacokinetics and biodistribution of all radiolabeled nanoparticles could clearly be visualized and followed by dynamic PET/CT imaging. Both variants of 68Ga-labeled MNPSNP accumulated mainly in liver and spleen. PEGylation of the nanoparticles already resulted in lower liver uptakes. Combination with macrophage depletion led to a highly significant effect whereas macrophage depletion alone could not reveal significant differences. Although MNPSNP accumulation around implants was low in comparison to the inner organs in PET/CT imaging, gamma counting displayed a significantly higher %I.D./g for the tissue surrounding the magnetic implants compared to the titanium control. Additional PEGylation and/or macrophage depletion revealed no significant differences regarding nanoparticle accumulation at the implantation site. CONCLUSION: Tracking of 68Ga-labeled nanoparticles in a mouse model in the first critical hours post-injection by PET/CT imaging provided a better understanding of MNPSNP distribution, elimination and accumulation. Although PEGylation increases circulation time, nanoparticle accumulation at the implantation site was still insufficient for infection treatment and additional efforts are needed to increase local accumulation.

Clodronate is not protective in lethal viral encephalitis despite substantially reducing inflammatory monocyte infiltration in the CNS.

Bone marrow (BM)-derived monocytes induce inflammation and tissue damage in a range of pathologies. In particular, in a mouse model of West Nile virus (WNV) encephalitis (WNE), nitric oxide-producing, Ly6Chi inflammatory monocytes from the BM are recruited to the central nervous system (CNS) and contribute to lethal immune pathology. Reducing the migration of these cells into the CNS using monoclonal antibody blockade, immune-modifying particles or CSF-1R inhibitors reduces neuroinflammation, improving survival and/or clinical outcomes. Macrophages can also be targeted more broadly by administration of clodronate-encapsulated liposomes, which induce apoptosis in phagocytes. In this study, clodronate reduced the inflammatory infiltrate by 70% in WNE, however, surprisingly, this had no effect on disease outcome. More detailed analysis demonstrated a compensatory increase in neutrophils and enhanced activation status of microglia in the brain. In addition, we observed increased numbers of Ly6Chi BM monocytes with an increased proliferative capacity and expression of SCA-1 and CD16/32, potentially indicating output of immature cells from the BM. Once in the brain, these cells were more phagocytic and had a reduced expression of antigen-presenting molecules. Lastly, we show that clodronate also reduces non-myeloid cells in the spleen and BM, as well as ablating red blood cells and their proliferation. These factors likely impeded the therapeutic potential of clodronate in WNE. Thus, while clodronate provides an excellent system to deplete macrophages in the body, it has larger and broader effects on the phagocytic and non-phagocytic system, which must be considered in the interpretation of data.

Pharmacokinetics and plasma protein binding of a single dose of clodronate disodium are similar for juvenile sheep and horses.

OBJECTIVE: To determine the single-dose pharmacokinetics of clodronate disodium (CLO) in juvenile sheep and the plasma protein binding (PPB) of CLO in juvenile sheep and horses. ANIMALS: 11 juvenile crossbred sheep (252 ± 6 days) for the pharmacokinetic study. Three juvenile crossbred sheep (281 ± 4 days) and 3 juvenile Quarter Horses (599 ± 25 days) for PPB analysis. METHODS: CLO concentrations were determined using liquid chromatography-mass spectrometry. Pharmacokinetic parameters were calculated by noncompartmental analysis from plasma samples obtained at 0, 0.5, 1, 3, 6, 12, 24, 48, and 72 hours after CLO administered IM at 0.6 mg/kg. PPB was determined using equine and ovine plasma in a single-use rapid equilibrium dialysis system. RESULTS: The mean and range for maximum plasma concentration (Cmax: 5,596; 2,396-8,613 ng/mL), time of maximal concentration (Tmax: 0.5; 0.5-1.0 h), and area under the curve (AUCall: 12,831; 7,590-17,593 h X ng/mL) were similar to those previously reported in horses. PPB in sheep and horses was moderate to high, with unbound fractions of 26.1 ± 5.1% in sheep and 18.7 ± 7.5% in horses, showing less than a 1.4-fold difference. CLINICAL RELEVANCE: The pharmacokinetic parameters and PPB of CLO in juvenile sheep were similar to those previously reported in horses. The results suggest that juvenile sheep can be utilized as an animal model for studying the potential risks and/or benefits of bisphosphonate use in juvenile horses.

Comparative Study of Nanoparticle Blood Circulation after Forced Clearance of Own Erythrocytes (Mononuclear Phagocyte System-Cytoblockade) or Administration of Cytotoxic Doxorubicin- or Clodronate-Loaded Liposomes.

Recent developments in the field of nanomedicine have introduced a wide variety of nanomaterials that are capable of recognizing and killing tumor cells with increased specificity. A major limitation preventing the widespread introduction of nanomaterials into the clinical setting is their fast clearance from the bloodstream via the mononuclear phagocyte system (MPS). One of the most promising methods used to overcome this limitation is the MPS-cytoblockade, which forces the MPS to intensify the clearance of erythrocytes by injecting allogeneic anti-erythrocyte antibodies and, thus, significantly prolongs the circulation of nanoagents in the blood. However, on the way to the clinical application of this approach, the question arises whether the induced suppression of macrophage phagocytosis via the MPS-cytoblockade could pose health risks. Here, we show that highly cytotoxic doxorubicin- or clodronate-loaded liposomes, which are widely used for cancer therapy and biomedical research, induce a similar increase in the nanoparticle blood circulation half-life in mice as the MPS-cytoblockade, which only gently and temporarily saturates the macrophages with the organism's own erythrocytes. This result suggests that from the point of view of in vivo macrophage suppression, the MPS-cytoblockade should be less detrimental than the liposomal anti-cancer drugs that are already approved for clinical application while allowing for the substantial improvement in the nanoagent effectiveness.

The hepatic GABAergic system promotes liver macrophage M2 polarization and mediates HBV replication in mice.

Macrophages display functional phenotypic plasticity. Hepatitis B virus (HBV) infection induces polarizations of liver macrophages either to M1-like pro-inflammatory phenotype or to M2-like anti-inflammatory phenotype. Gamma-aminobutyric acid (GABA) signaling exists in various non-neuronal cells including hepatocytes and some immune cells. Here we report that macrophages express functional GABAergic signaling components and activation of type A GABA receptors (GABAARs) promotes M2-polarization thus advancing HBV replication. Notably, intraperitoneal injection of GABA or the GABAAR agonist muscimol increased HBV replication in HBV-carrier mice that were generated by hydrodynamical injection of adeno-associated virus/HBV1.2 plasmids (pAAV/HBV1.2). The GABA-augmented HBV replication in HBV-carrier mice was significantly reduced by the GABAAR inhibitor picrotoxin although picrotoxin had no significant effect on serum HBsAg levels in control HBV-carrier mice. Depletion of liver macrophages by liposomal clodronate treatment also significantly reduced the GABA-augmented HBV replication. Yet adoptive transfer of liver macrophages isolated from GABA-treated donor HBV-carrier mice into the liposomal clodronate-pretreated recipient HBV-carrier mice restored HBV replication. Moreover, GABA or muscimol treatment increased the expression of "M2" cytokines in macrophages, but had no direct effect on HBV replication in the HepG2.2.15 cells, HBV1.3-transfected Huh7, HepG2, or HepaRG cells, or HBV-infected Huh7-NTCP cells. Taken together, these results suggest that increasing GABA signaling in the liver promotes HBV replication in HBV-carrier mice by suppressing the immunity of liver macrophages, but not by increasing the susceptibility of hepatocytes to HBV infection. Our study shows that a previously unknown GABAergic system in liver macrophage has an essential role in HBV replication.

Perivascular Macrophages Mediate Microvasospasms After Experimental Subarachnoid Hemorrhage.

BACKGROUND: Subarachnoid hemorrhage (SAH) is characterized by acute and delayed reductions of cerebral blood flow (CBF) caused, among others, by spasms of cerebral arteries and arterioles. Recently, the inactivation of perivascular macrophages (PVM) has been demonstrated to improve neurological outcomes after experimental SAH, but the underlying mechanisms of protection remain unclear. The aim of our exploratory study was, therefore, to investigate the role of PVM in the formation of acute microvasospasms after experimental SAH. METHODS: PVMs were depleted in 8- to 10-week-old male C57BL/6 mice (n=8/group) by intracerebroventricular application of clodronate-loaded liposomes and compared with mice with vehicle liposome injections. Seven days later, SAH was induced by filament perforation under continuous monitoring of CBF and intracranial pressure. Results were compared with sham-operated animals and animals who underwent SAH induction but no liposome injection (n=4/group each). Six hours after SAH induction or sham surgery, numbers of microvasospasms per volume of interest and % of affected pial and penetrating arterioles were examined in 9 standardized regions of interest per animal by in vivo 2-photon microscopy. Depletion of PVMs was proven by quantification of PVMs/mm3 identified by immunohistochemical staining for CD206 and Collagen IV. Statistical significance was tested with t tests for parametric data and Mann-Whitney U test for nonparametric data. RESULTS: PVMs were located around pial and intraparenchymal arterioles and were effectively depleted by clodronate from 671±28 to 46±14 PVMs/mm3 (P<0.001). After SAH, microvasospasms was observed in pial arteries and penetrating and precapillary arterioles and were accompanied by an increase to 1405±142 PVMs/mm3. PVM depletion significantly reduced the number of microvasospasms from 9 IQR 5 to 3 IQR 3 (P<0.001). CONCLUSIONS: Our results suggest that PVMs contribute to the formation of microvasospasms after experimental SAH.