Macrophage Depletion via Clodronate Pretreatment Reduces Transgene Expression from AAV Vectors In Vivo

Adeno-associated virus is a popular gene delivery vehicle for gene therapy studies. A potential roadblock to widespread clinical adoption is the high vector doses required for efficient transduction in vivo, and the potential for subsequent immune responses that may limit prolonged transgene expression. We hypothesized that the depletion of macrophages via systemic delivery of liposome-encapsulated clodronate would improve transgene expression if given prior to systemic AAV vector administration, as has been shown to be the case with adenoviral vectors. Contrary to our expectations, clodronate liposome pretreatment resulted in significantly reduced transgene expression in the liver and heart, but permitted moderate transduction of the white pulp of the spleen. There was a remarkable localization of transgene expression from the red pulp to the center of the white pulp in clodronate-treated mice compared to untreated mice. Similarly, a greater proportion of transgene expression could be observed in the medulla located in the center of the lymph node in mice treated with clodronate-containing liposomes as compared to untreated mice where transgene expression was localized primarily to the cortex. These results underscore the highly significant role that the immune system plays in influencing the distribution and relative numbers of transduced cells in the context of AAV-mediated gene delivery.

Repeated clodronate-liposome treatment results in neutrophilia and is not effective in limiting obesity-linked metabolic impairments

Depletion of macrophages is thought to be a therapeutic option for obesity-induced inflammation and metabolic dysfunction. However, whether the therapeutic effect is a direct result of reduced macrophage-derived inflammation or secondary to decreases in fat mass is controversial, as macrophage depletion has been shown to disrupt energy homeostasis. This study was designed to determine if macrophage depletion via clodronate-liposome (CLD) treatment could serve as an effective intervention to reduce obesity-driven inflammatory and metabolic impairments independent of changes in energy intake. After 16 wk on a high-fat diet (HFD) or the AIN-76A control (low-fat) diet (LFD) ( n = 30/diet treatment), male C57BL/6J mice were assigned to a CLD- or PBS-liposome treatment ( n = 15/group) for 4 wk. Liposomes were administered biweekly via intraperitoneal injections (8 administrations in total). PBS-liposome-treated groups were pair-fed to their CLD-treated dietary counterparts. Metabolic function was assessed before and after liposome treatment. Adipose tissue, as well as the liver, was investigated for macrophage infiltration and the presence of inflammatory mediators. Additionally, a complete blood count was performed. CLD treatment reduced energy intake. When controlling for energy intake, CLD treatment was unable to regress metabolic dysfunction or nonalcoholic fatty liver disease and impaired adipose tissue insulin action. Moreover, repeated CLD treatment induced neutrophilia and anemia, increased adipose tissue mRNA expression of the proinflammatory cytokines IL-6 and IL-1β, and augmented circulating IL-6 and monocyte chemoattractant protein-1 concentrations ( P < 0.05). This study suggests that repeated intraperitoneal administration of CLD to deplete macrophages attenuates obesity by limiting energy intake. Moreover, after controlling for the benefits of weight loss, the accompanying detrimental side effects limit regular CLD treatment as an effective therapeutic strategy.

Abstract 341: The Role of Nucleotidase in Arterial Thrombosis

Objective: To determine how leukocyte nucleotidase affects arterial thrombosis. Approach and Results: Ectonucleoside triphosphate diphosphohydrolase-1 (CD39) is expressed on circulating cells, endothelium and smooth muscle cells where it hydrolyzes extracellular ATP or ADP to AMP. We have demonstrated that transgenic mice with a global overexpression of human CD39 (hCD39-Tg) are protected against ferric chloride-induced carotid artery thrombosis. Furthermore, transplant of hCD39-Tg bone marrow into WT recipient mice increases the time to thrombosis when compared to recipient mice (wild-type or hCD39-Tg) receiving wild-type bone marrow. Based upon these data and previously published work, we hypothesized that CD39 expression on leukocytes is responsible for the prolongation of the time to thrombosis measured in hCD39-Tg mice. To test this hypothesis, we first performed ex vivo mixing experiments. Addition of hCD39-Tg monocytes to WT blood inhibits the expression of activated glycoprotein IIb/IIIa on platelets in response to ADP as measured by FACS analysis (Baseline: 1224 ± 94.9 MFI vs hCD39-Tg monocytes: 663.5 ± 61.5 activated glycoprotein IIb/IIIa MFI: n=4; p< 0.001). Subsequently, in vivo we demonstrated that monocytes with increased CD39 contribute to extending the time to thrombosis. Clodronate liposome depletion of monocytes (WT: 69% decrease; hCD39-Tg: 63% decrease) resulted in a normalization of the time to thrombosis in hCD39-Tg mice (8.0 ± 1.07 minutes, n = 10) when compared to control loaded liposomes (120.0 ± 0.0, n = 14). No changes in the time to thrombosis were detected in wild-type mice treated with clodronate (8.6 ± 1.35 minutes, n = 8) or control liposomes (7.8 ± 0.80 minutes, n=8). Conclusion: Increased expression of CD39 on monocytes can inhibit platelet activation and extend the time to thrombosis following ferric chloride-induced carotid artery injury.

Abstract W MP47: Roles of Macrophage PPARγ on Intracranial Aneurysmal Rupture

Background and Purpose: Peroxisome proliferator-activated receptor gamma (PPARγ) plays a role in regulating not only lipid and glucose metabolism but also atherosclerosis and inflammation which are associated with intracranial aneurysmal rupture. It has been suggested that PPARγ is present in various kinds of cell types including macrophages which we reported to contribute to intracranial aneurysm formation. In addition, macrophage PPARγ regulates inflammatory cytokines negatively. Therefore we hypothesized that macrophage PPARγ is protective against intracranial aneurysmal rupture. We tested this hypothesis by using our own established mouse model of intracranial aneurysm. Methods: Intracranial aneurysms were induced in male mice using a combination of a single injection of elastase into the cerebrospinal fluid and the deoxycorticosterone acetate (DOCA) salt hypertension. Six days after aneurysm induction, we started 2-week treatment with vehicle or PPARγ agonist (pioglitazone:PGZ) or clodronate liposome. PPARγ antagonist (GW9662) was added to PGZ treatment. We induced aneurysms to macrophage-specific PPARγ knockout mice and treated them with vehicle or PGZ with the same protocol mentioned above. Aneurysmal rupture was detected by neurological symptoms and confirmed by the presence of intracranial aneurysms with subarachnoid hemorrhage. Results: Both PGZ and depletion of macrophage by clodronate liposome reduced the incidence of ruptured aneurysms and rupture rate. PGZ treatment decreased the inflammatory cytokines that were increased in cerebral arteries of mice with aneurysm induction compared to mice without aneurysm induction. To examine whether the macrophage PPARγ is associated with intracranial aneurysmal rupture, we administered PGZ to macrophage-specific PPARγ knock out mice. PGZ did not have protective effect against aneurysmal rupture in macrophage-specific PPARγ knock out mice although it exerted protective effects in wild type mice. Conclusion: PPARγ activation is protective against intracranial aneurysmal rupture by suppressing inflammatory cytokines and macrophage PPARγ is associated with this protective effects.

Abstract W P253: Repeated Remote Ischemic Conditioning (RIC) after Intracerebral Hemorrhage Regulates Macrophage Polarization and CD36 Expression to Promote Hematoma Resolution

Background: RIC-therapy during sub-arachnoid hemorrhage in humans was found well tolerated. We tested the hypothesis that repeated daily RIC-therapy would improve post-ICH outcomes in mice. Methods: ICH was induced by collagenase injection into the brain of CD1 male mice (4-mo old). Mice were randomized for either once daily RIC-therapy or related sham-procedure starting 2-hours post-ICH until sacrifice. RIC-therapy post-ICH was also tested in mice pre-treated with clodronate-liposome consequently deficient in macrophage population. Laser speckle imager (LSCI) was used to detect changes in peripheral ischemia resulting from space occupying hematoma. SWI/ FLASH (T2*W) MRI was used to estimate hematoma size. Behavioral outcomes were assessed by focal deficit score and beam walk challenge. Hemoglobin content in the brain tissue by spectrophotometry, macrophage polarization with CD36 expression in peripheral blood by flowcytometry, and histopathological analyses in the brain, were also performed. Statistical significance was determined at p<0.05. Results: Even after 5-days post-ICH, peripheral cerebral ischemia was evident, which was significantly attenuated by RIC-therapy due to reduced hematoma size. Effect of RIC-therapy on hematoma resolution was abolished in the clodronate-liposome treated mice. Higher focal deficit score and impaired fine motor coordination were significantly evident in ICH mice, which was attenuated by RIC-therapy. Post-ICH increased hemoglobin content at day 5 was significantly reduced by RIC-therapy. Interestingly, repeated RIC-therapy promoted macrophage polarization towards M2 (anti-inflammatory) phenotype with increased CD36 expression. At 2-weeks post ICH, cresyl violet and Luxol-Fast blue staining showed significantly increased cell death and white matter degeneration (WMD), respectively, in the ICH mice. RIC-therapy after ICH significantly reduced the cell death and WMD in a 2-weeks follow up. Conclusion: Human subjects deficient in CD36 have impaired capability of hematoma resolution. Long-term RIC-therapy might be helpful in spontaneous hematoma resolution via increased M2-type macrophage and CD36 expression.

Macrophages Are the Determinant of Resistance to and Outcome of Nonlethal Babesia microti Infection in Mice

In the present study, we examined the contributions of macrophages to the outcome of infection withBabesia microti, the etiological agent of human and rodent babesiosis, in BALB/c mice. Mice were treated with clodronate liposome at different times during the course ofB. microtiinfection in order to deplete the macrophages. Notably, a depletion of host macrophages at the early and acute phases of infection caused a significant elevation of parasitemia associated with remarkable mortality in the mice. The depletion of macrophages at the resolving and latent phases of infection resulted in an immediate and temporal exacerbation of parasitemia coupled with mortality in mice. Reconstituting clodronate liposome-treated mice at the acute phase of infection with macrophages from naive mice resulted in a slight reduction in parasitemia with improved survival compared to that of mice that received the drug alone. These results indicate that macrophages play a crucial role in the control of and resistance toB. microtiinfection in mice. Moreover, analyses of host immune responses revealed that macrophage-depleted mice diminished their production of Th1 cell cytokines, including gamma interferon (IFN-γ) and tumor necrosis factor alpha (TNF-α). Furthermore, depletion of macrophages at different times exaggerated the pathogenesis of the infection in deficient IFN-γ−/−and severe combined immunodeficiency (SCID) mice. Collectively, our data provide important clues about the role of macrophages in the resistance and control ofB. microtiand imply that the severity of the infection in immunocompromised patients might be due to impairment of macrophage function.