CSF-1R inhibitor PLX3397 attenuates peripheral and brain chronic GVHD and improves functional outcomes in mice.

Graft-versus-host disease (GVHD) is a serious complication of otherwise curative allogeneic haematopoietic stem cell transplants. Chronic GVHD induces pathological changes in peripheral organs as well as the brain and is a frequent cause of late morbidity and death after bone-marrow transplantation. In the periphery, bone-marrow-derived macrophages are key drivers of pathology, but recent evidence suggests that these cells also infiltrate into cGVHD-affected brains. Microglia are also persistently activated in the cGVHD-affected brain. To understand the involvement of these myeloid cell populations in the development and/or progression of cGVHD pathology, we here utilized the blood-brain-barrier permeable colony stimulating factor-1 receptor (CSF-1R) inhibitor PLX3397 (pexidartinib) at varying doses to pharmacologically deplete both cell types. We demonstrate that PLX3397 treatment during the development of cGVHD (i.e., 30 days post-transplant) improves disease symptoms, reducing both the clinical scores and histopathology of multiple cGVHD target organs, including the sequestration of T cells in cGVHD-affected skin tissue. Cognitive impairments associated with cGVHD and neuroinflammation were also attenuated by PLX3397 treatment. PLX3397 treatment prior to the onset of cGVHD (i.e., immediately post-transplant) did not change in clinical scores or histopathology. Overall, our data demonstrate significant benefits of using PLX3397 for the treatment of cGVHD and associated organ pathologies in both the periphery and brain, highlighting the therapeutic potential of pexidartinib for this condition.

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.

Macrophage depletion in stellate ganglia alleviates cardiac sympathetic overactivation and ventricular arrhythmogenesis by attenuating neuroinflammation in heart failure.

Cardiac sympathetic overactivation is involved in arrhythmogenesis in patients with chronic heart failure (CHF). Inflammatory infiltration in the stellate ganglion (SG) is a critical factor for cardiac sympathoexcitation in patients with ventricular arrhythmias. This study aims to investigate if macrophage depletion in SGs decreases cardiac sympathetic overactivation and ventricular arrhythmogenesis in CHF. Surgical ligation of the coronary artery was used for induction of CHF. Clodronate liposomes were microinjected into bilateral SGs of CHF rats for macrophage depletion. Using cytokine array, immunofluorescence staining, and Western blot analysis, we found that macrophage expansion and expression of TNFα and IL-1ß in SGs were markedly increased in CHF rats. Flow cytometry data confirmed that the percentage of macrophages in SGs was higher in CHF rats than that in sham rats. Clodronate liposomes significantly reduced CHF-elevated proinflammatory cytokine levels and macrophage expansion in SGs. Clodronate liposomes also reduced CHF-increased N-type Ca2+ currents and excitability of cardiac sympathetic postganglionic neurons and inhibited CHF-enhanced cardiac sympathetic nerve activity. ECG data from 24-h, continuous telemetry recording in conscious rats demonstrated that clodronate liposomes not only restored CHF-induced heterogeneity of ventricular electrical activities, but also decreased the incidence and duration of ventricular tachycardia/fibrillation in CHF. Macrophage depletion with clodronate liposomes attenuated CHF-induced cardiac sympathetic overactivation and ventricular arrhythmias through reduction of macrophage expansion and neuroinflammation in SGs.

Responses of perivascular macrophages to circulating lipopolysaccharides in the subfornical organ with special reference to endotoxin tolerance.

BACKGROUND: Circulating endotoxins including lipopolysaccharides (LPS) cause brain responses such as fever and decrease of food and water intake, while pre-injection of endotoxins attenuates these responses. This phenomenon is called endotoxin tolerance, but the mechanisms underlying it remain unclear. The subfornical organ (SFO) rapidly produces proinflammatory cytokines including interleukin-1ß (IL-1ß) in response to peripherally injected LPS, and repeated LPS injection attenuates IL-1ß production in the SFO, indicating that the SFO is involved in endotoxin tolerance. The purpose of this study is to investigate features of the IL-1ß source cells in the SFO of LPS-non-tolerant and LPS-tolerant mice. METHODS: We first established the endotoxin-tolerant mouse model by injecting LPS into adult male mice (C57BL/6J). Immunohistochemistry was performed to characterize IL-1ß-expressing cells, which were perivascular macrophages in the SFO. We depleted perivascular macrophages using clodronate liposomes to confirm the contribution of IL-1ß production. To assess the effect of LPS pre-injection on perivascular macrophages, we transferred bone marrow-derived cells obtained from male mice (C57BL/6-Tg (CAG-EGFP)) to male recipient mice (C57BL/6N). Finally, we examined the effect of a second LPS injection on IL-1ß expression in the SFO perivascular macrophages. RESULTS: We report that perivascular macrophages but not parenchymal microglia rapidly produced the proinflammatory cytokine IL-1ß in response to LPS. We found that peripherally injected LPS localized in the SFO perivascular space. Depletion of macrophages by injection of clodronate liposomes attenuated LPS-induced IL-1ß expression in the SFO. When tolerance developed to LPS-induced sickness behavior in mice, the SFO perivascular macrophages ceased producing IL-1ß, although bone marrow-derived perivascular macrophages increased in number in the SFO and peripherally injected LPS reached the SFO perivascular space. CONCLUSIONS: The current data indicate that perivascular macrophages enable the SFO to produce IL-1ß in response to circulating LPS and that its hyporesponsiveness may be the cause of endotoxin tolerance.

Targeting Macrophages as a Potential Therapeutic Intervention: Impact on Inflammatory Diseases and Cancer.

Macrophages, cells belonging to the innate immune system, present a high plasticity grade, being able to change their phenotype in response to environmental stimuli. They play central roles during development, homeostatic tissue processes, tissue repair, and immunity. Furthermore, it is recognized that macrophages are involved in chronic inflammation and that they play central roles in inflammatory diseases and cancer. Due to their large involvement in the pathogenesis of several types of human diseases, macrophages are considered to be relevant therapeutic targets. Nanotechnology-based systems have attracted a lot of attention in this field, gaining a pivotal role as useful moieties to target macrophages in diseased tissues. Among the different approaches that can target macrophages, the most radical is represented by their depletion, commonly obtained by means of clodronate-containing liposomal formulations and/or depleting antibodies. These strategies have produced encouraging results in experimental mouse models. In this review, we focus on macrophage targeting, based on the results so far obtained in preclinical models of inflammatory diseases and cancer. Pros and cons of these therapeutic interventions will be highlighted.

Neonatal Pulmonary Macrophage Depletion Coupled to Defective Mucus Clearance Increases Susceptibility to Pneumonia and Alters Pulmonary Immune Responses.

Resident immune cells (e.g., macrophages [MΦs]) and airway mucus clearance both contribute to a healthy lung environment. To investigate interactions between pulmonary MΦ function and defective mucus clearance, a genetic model of lysozyme M (LysM) promoter-mediated MΦ depletion was generated, characterized, and crossed with the sodium channel ß subunit transgenic (Scnn1b-Tg) mouse model of defective mucus clearance. Diphtheria toxin A-mediated depletion of LysM(+) pulmonary MΦs in wild-type mice with normal mucus clearance resulted in lethal pneumonia in 24% of neonates. The pneumonias were dominated by Pasteurella pneumotropica and accompanied by emaciation, neutrophilic inflammation, and elevated Th1 cytokines. The incidence of emaciation and pneumonia reached 51% when LysM(+) MΦ depletion was superimposed on the airway mucus clearance defect of Scnn1b-Tg mice. In LysM(+) MΦ-depleted Scnn1b-Tg mice, pneumonias were associated with a broader spectrum of bacterial species and a significant reduction in airway mucus plugging. Bacterial burden (CFUs) was comparable between Scnn1b-Tg and nonpneumonic LysM(+) MΦ-depleted Scnn1b-Tg mice. However, the nonpneumonic LysM(+) MΦ-depleted Scnn1b-Tg mice exhibited increased airway inflammation, the presence of neutrophilic infiltration, and increased levels of inflammatory cytokines in bronchoalveolar lavage fluid compared with Scnn1b-Tg mice. Collectively, these data identify key MΦ-mucus clearance interactions with respect to both infectious and inflammatory components of muco-obstructive lung disease.

Macrophages are essential for maintaining a M2 protective response early after ischemic brain injury.

Resident microglia and recruited macrophages are major contributors to the post-ischemic inflammatory response. Initially considered functionally homogeneous populations, data now suggest distinct but still controversial roles after brain injury. Using a model of conditional monocyte/macrophage depletion we studied the contribution of these myeloid cells to brain lesion progression after ischemia, and their influence on the ischemic inflammatory environment. Male CD11b-DTR transgenic mice, expressing the human diphtheria toxin receptor under the control of the CD11b promoter, were treated with diphtheria toxin to induce monocyte/macrophage depletion. Twenty four hours later the middle cerebral artery was permanently occluded. The ischemic lesion was measured 24h after injury. At the same time microglia and macrophage activation and polarization were assessed by quantitative immunohistochemistry and confocal microscopy for CD45high, CD11b, CD68, CD16/32, iNOS, Arg1, Ym1, and CD206, and gene expression was investigated on CD11b+ sorted cells. Depletion of monocytes/macrophages worsened the ischemic lesion within 24h after the ischemic insult. This effect was associated with higher M1/M2 polarization ratio in the ischemic lesion. Moreover, depletion increased the expression of M1 phenotypic markers on CD11b positive cells. Gene expression on CD11b+ sorted cells indicated a selective increase of iNOS and lower Arg1 mRNA expression than in non depleted mice. Depletion of monocytes/macrophages increases the ischemic lesion, an effect accompanied by an increase in the M1/M2 polarization ratio of microglia and macrophages in the ischemic area. Thus in ischemic injury recruited monocytes/macrophages may control an excessive M1 pro-inflammatory response, suggesting their ability to drive M2 protective polarization.

Tumor-Associated Macrophage-Mediated Targeted Therapy of Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) is the most aggressive form of breast cancer. TNBC is often infiltrated with a large number of macrophages, which in turn promote tumor growth and metastasis. In this study, tumor-associated macrophages (TAMs) were exploited as a target to deliver doxorubicin (DOX), a chemotherapeutic agent, to TNBC using nanoparticles surface-functionalized by (i) acid-sensitive sheddable PEGylation and (ii) modifying with mannose (i.e., DOX-AS-M-PLGA-NPs). In mice with orthotopic M-Wnt triple-negative mammary tumors, a single intravenous injection of DOX-AS-M-PLGA-NPs significantly reduced macrophage population in tumors within 2 days, and the density of the macrophages recovered slowly. Repeated injections of DOX-AS-M-PLGA-NPs can help maintain the population of the macrophages at a lower level. In M-Wnt tumor-bearing mice that were pretreated with zoledronic acid to nonselectively deplete macrophages, the TAM-targeting DOX-AS-M-PLGA-NPs were not more effective than the DOX-AS-PLGA-NPs that were not surface-modified with mannose and thus do not target TAMs in controlling tumor growth. However, in M-Wnt tumor-bearing mice that were not pretreated with zoledronic acid, the TAM-targeting DOX-AS-M-PLGA-NPs were significantly more effective than the nontargeting DOX-AS-PLGA-NPs in controlling the tumor growth. The AS-M-PLGA-NPs or other nanoparticles surface-functionalized similarly, when loaded with a chemotherapeutic agent commonly used in adjuvant therapy of TNBC, may be developed into targeted therapy for TNBC.

MyD88 mediates in vivo effector functions of alveolar macrophages in acute lung inflammatory responses to carbon nanotube exposure.

Carbon nanotubes (CNTs) are rapidly emerging as high-priority occupational toxicants. CNT powders contain fibrous particles that aerosolize readily in places of manufacture and handling, posing an inhalation risk for workers. Studies using animal models indicate that lung exposure to CNTs causes prolonged inflammatory responses and diffuse alveolar injury. The mechanisms governing CNT-induced lung inflammation are not fully understood but have been suggested to involve alveolar macrophages (AMs). In the current study, we sought to systematically assess the effector role of AMs in vivo in the induction of lung inflammatory responses to CNT exposures and investigate their cell type-specific mechanisms. Multi-wall CNTs characterized for various physicochemical attributes were used as the CNT type. Using an AM-specific depletion and repopulation approach in a mouse model, we unambiguously demonstrated that AMs are major effector cells necessary for the in vivo elaboration of CNT-induced lung inflammation. We further investigated in vitro AM responses and identified molecular targets which proved critical to pro-inflammatory responses in this model, namely MyD88 as well as MAPKs and Ca(2+)/CamKII. We further demonstrated that MyD88 inhibition in donor AMs abrogated their capacity to reconstitute CNT-induced inflammation when adoptively transferred into AM-depleted mice. Taken together, this is the first in vivo demonstration that AMs act as critical effector cell types in CNT-induced lung inflammation and that MyD88 is required for this in vivo effector function. AMs and their cell type-specific mechanisms may therefore represent potential targets for future therapeutic intervention of CNT-related lung injury.

The role of hepatic and splenic macrophages in E. coli-induced memory impairments in aged rats.

Bi-directional communication between the peripheral and central nervous systems has been extensively demonstrated. Aged rats exhibit a prolonged proinflammatory response in the hippocampus region of the brain following a peripheral bacterial infection, and this response in turn causes robust memory declines. Here we aimed to determine whether hepatic or splenic macrophages play a role in the maintenance of this central response. Proinflammatory cytokines measured in liver and spleen four days following an Escherichia coli infection revealed a potentiated proinflammatory response in liver, and to a lesser extent in spleen, in aged relative to young rats. To determine whether this potentiated response was caused by impaired bacterial clearance in these organs, E. coli colony forming units in liver and spleen were measured 4 days after infection, and there were no difference between young and aged rats in either organ. No E. coli was detected in the hippocampus, eliminating the possibility that the aged blood brain barrier allowed E. coli to enter the brain. Depletion of hepatic and splenic macrophages with clodronate-encapsulated liposomes effectively eliminated the proinflammatory response to E. coli at four days in both organs. However, this treatment failed to reduce the proinflammatory response in the hippocampus. Moreover, depletion of peripheral macrophages from liver and spleen did not prevent E. coli-induced memory impairment. These data strongly suggest that hepatic and splenic macrophages do not play a major role in the long-lasting maintenance of the proinflammatory response in the hippocampus of aged rats following a bacterial infection, or the memory declines that this response produces.

Biodistribution and in vivo activities of tumor-associated macrophage-targeting nanoparticles incorporated with doxorubicin.

Tumor-associated macrophages (TAMs) are increasingly considered a viable target for tumor imaging and therapy. Previously, we reported that innovative surface-functionalization of nanoparticles may help target them to TAMs. In this report, using poly(lactic-co-glycolic) acid (PLGA) nanoparticles incorporated with doxorubicin (DOX) (DOX-NPs), we studied the effect of surface-modification of the nanoparticles with mannose and/or acid-sensitive sheddable polyethylene glycol (PEG) on the biodistribution of DOX and the uptake of DOX by TAMs in tumor-bearing mice. We demonstrated that surface-modification of the DOX-NPs with both mannose and acid-sensitive sheddable PEG significantly increased the accumulation of DOX in tumors, enhanced the uptake of the DOX by TAMs, but decreased the distribution of DOX in mononuclear phagocyte system (MPS), such as liver. We also confirmed that the acid-sensitive sheddable PEGylated, mannose-modified DOX-nanoparticles (DOX-AS-M-NPs) targeted TAMs because depletion of TAMs in tumor-bearing mice significantly decreased the accumulation of DOX in tumor tissues. Furthermore, in a B16-F10 tumor-bearing mouse model, we showed that the DOX-AS-M-NPs were significantly more effective than free DOX in controlling tumor growth but had only minimum effect on the macrophage population in mouse liver and spleen. The AS-M-NPs are promising in targeting cytotoxic or macrophage-modulating agents into tumors to improve tumor therapy.

Non-resolving neuroinflammation regulates axon regeneration in chronic spinal cord injury.

Chronic spinal cord injury (SCI) lesions retain increased densities of microglia and macrophages. In acute SCI, macrophages induce growth cone collapse, facilitate axon retraction away from lesion boundaries, as well as play a key role in orchestrating the growth-inhibitory glial scar. Little is known about the role of sustained inflammation in chronic SCI, or whether chronic inflammation affects repair and regeneration. We performed transcriptional analysis using the Nanostring Neuropathology panel to characterize the resolution of inflammation into chronic SCI, to characterize the chronic SCI microenvironment, as well as to identify spinal cord responses to macrophage depletion and repopulation using the CSF1R inhibitor, PLX-5622. We determined the ability for macrophage depletion and repopulation to augment axon growth into chronic lesions both with and without regenerative stimulation using neuronal-specific PTEN knockout (PTEN-KO). PTEN-KO was delivered with spinal injections of retrogradely transported adeno associated viruses (AAVrg's). Both transcriptional analyses and immunohistochemistry revealed the ability for PLX-5622 to significantly deplete inflammation around and within chronic SCI lesions, with a return to pre-depleted inflammatory densities after treatment removal. Neuronal-specific transcripts were significantly elevated in mice after inflammatory repopulation, but no significant effects were observed with macrophage depletion alone. Axon densities significantly increased within the lesion after PLX-5622 treatment with a more consistent effect observed in mice with inflammatory repopulation. PTEN-KO did not further increase axon densities within the lesion beyond effects induced by PLX-5622. We identified that PLX-5622 increased axon densities within the lesion that are histologically identified as 5-HT+and CGRP+, both of which are not robustly transduced by AAVrg's. Our work identified that increased macrophage/microglia densities in the chronic SCI environment may be actively retained by homeostatic mechanisms likely affiliated with a sustained elevated expression of CSF1 and other chemokines. Finally, we identify a novel role of sustained inflammation as a prospective barrier to axon regeneration in chronic SCI.

Induction of M2-macrophages by tumour cells and tumour growth promotion by M2-macrophages: a quid pro quo in pancreatic cancer.

INTRODUCTION: More effective therapies are required to improve survival of pancreatic cancer. Possible immunologic targets include tumour associated macrophages (TAMs), generally consisting of M1- and M2-macrophages. We have analysed the impact of TAMS on pancreatic cancer in a syngeneic orthotopic murine model. METHODS: 6606PDA murine pancreatic cancer cells were orthotopically injected into C57BL6 mice. Tumour growth was monitored using MRI. Macrophages were depleted by clodronate liposomes. Tumours including microvessel density were evaluated using immunohistochemistry, immunofluorescence and/or cytometric beads assays. Naïve macrophages were generated employing peritoneal macrophages. In vitro experiments included culturing of macrophages in tumour supernatants as well as tumour cells cultured in macrophage supernatants using arginase as well as Griess assays. RESULTS: Clodronate treatment depleted macrophages by 80% in livers (p = 0.0051) and by 60% in pancreatic tumours (p = 0.0169). MRI revealed tumour growth inhibition from 221.8 mm(3) to 92.3 mm(3) (p = 0.0216). Micro vessel densities were decreased by 44% (p = 0.0315). Yet, MCP-1-, IL-4- and IL-10-levels within pancreatic tumours were unchanged. 6606PDA culture supernatants led to a shift from naïve macrophages towards an M2-phenotype after a 36 h treatment (p < 0.0001), reducing M1-macrophages at the same time (p < 0.037). In vivo, M2-macrophages represented 85% of all TAMs (p < 0.0001). Finally, culture supernatants of M2-macrophages induced tumour growth in vitro by 63.2% (p = 0.0034). CONCLUSIONS: This quid pro quo of tumour cells and M2-macrophages could serve as a new target for future immunotherapies that interrupt tumour promoting activities of TAMs and change the iNOS-arginase balance towards their tumoricidal capacities.

Alveolar Macrophages Participate in the Promotion of Influenza Virus Infection by Aflatoxin B1 at an Early Stage.

Aflatoxin B1 (AFB1), one of the most common environmental mycotoxin contaminations in food and feed, poses significant threats to human and animal health. Our previous study indicated that even non-toxic AFB1 concentrations could promote influenza virus replication and induce influenza virus-infected alveolar macrophages polarizing from M1 (immunostimulatory phenotype) to M2 (immunosuppressive phenotype) over time. However, whether AFB1 promotes influenza replication via modulating the polarization of alveolar macrophages is unknown. Here, we specifically depleted alveolar macrophages using clodronate-containing liposomes in swine influenza virus (SIV)-infected mice to explore the mechanism the promotion of SIV replication by AFB1. The results show that the depletion of alveolar macrophages significantly alleviated the AFB1-induced weight loss, inflammatory responses, and lung and immune organ damage of the SIV-infected mice after 14 days and greatly diminished the AFB1-promoted SIV replication. In contrast, the depletion of alveolar macrophages did not alleviate the AFB1-induced weight loss, and lung and immune organ damage of the SIV-infected mice after 28 days and slightly diminished the AFB1-promoted SIV replication. Collectively, the data indicate that alveolar macrophages play a crucial role the promotion of SIV infection by AFB1 in the early rather than late stage, and AFB1 can promote SIV replication by inducing alveolar macrophages to polarize towards M1 macrophages. This research provides novel targets for reducing the risk of AFB1-promoted influenza virus infection.

Novel strategy of combined interstitial macrophage depletion with intravenous targeted therapy to ameliorate pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a severe interstitial lung disease with poor prognosis and high mortality rate. In the process of IPF, inflammatory dysregulation of macrophages and massive fibroblast aggregation and proliferation destroy alveoli, which cause pulmonary dysfunction, and ultimately lead to death due to respiratory failure. In the treatment of IPF, crossing biological barriers and delivering drugs to lung interstitium are the major challenges. In order to avoid the side effect of macrophages proliferation, we proposed, designed, and evaluated the strategy which combined macrophage depletion by intervaginal space injection and intravenous targeted therapy on bleomycin mouse model. We found that it inhibited pulmonary macrophages, reduced macrophage depletion in non-target organs, improved pulmonary drug targeting, impeded the progression of pulmonary fibrosis, and accelerated the recovery of pulmonary function. This combination therapeutic strategy shows good biosafety and efficacy, induces a targeted response, and is promising as a practical new clinical approach towards the treatment of pulmonary fibrosis.

Kazachstania pintolopesii in Blood and Intestinal Wall of Macrophage-Depleted Mice with Cecal Ligation and Puncture, the Control of Fungi by Macrophages during Sepsis.

Although macrophage depletion is a possible emerging therapeutic strategy for osteoporosis and melanoma, the lack of macrophage functions can lead to inappropriate microbial control, especially the regulation of intestinal microbiota. Cecal ligation and puncture (CLP) sepsis was performed in regular mice and in mice with clodronate-induced macrophage depletion. Macrophage depletion significantly increased the mortality and severity of sepsis-CLP mice, partly through the increased fecal Ascomycota, especially Kazachstania pintolopesii, with polymicrobialbacteremia (Klebsiella pneumoniae, Enterococcus faecalis, and Acinetobacter radioresistens). Indeed, macrophage depletion with sepsis facilitated gut dysbiosis that directly affected gut permeability as yeast cells were located and hidden in the colon crypts. To determine the interactions of fungal molecules on bacterial abundance, the heat-kill lysate of fungi (K. pintolopesii and C. albicans) and purified (1→3)-ß-d-glucan (BG; a major component of the fungal cell wall) were incubated with bacteria that were isolated from the blood of macrophage-depleted mice. There was enhanced cytokine production of enterocytes (Caco-2) after the incubation of the lysate of K. pintolopesii (isolated from sepsis mice), the lysate of C. albicans (extracted from sepsis patients), and BG, together with bacterial lysate. These data support a possible influence of fungi in worsening sepsis severity. In conclusion, macrophage depletion enhanced K. pintolopesii in feces, causing the overgrowth of fecal pathogenic bacteria and inducing a gut permeability defect that additively worsened sepsis severity. Hence, the fecal fungus could be spontaneously elevated and altered in response to macrophage-depleted therapy, which might be associated with sepsis severity.

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.

Skeletal muscle macrophage ablation in mice.

Macrophages are important mediators of skeletal muscle function in both healthy and diseased states. In vivo specific depletion of macrophages provides an experimental method to understand physiological and pathophysiological effects of macrophages. Systemic depletion of macrophages can deplete skeletal muscle macrophages but also alters systemic inflammatory responses and metabolism, which confounds the muscle specific effects of macrophage depletion. The primary aim of this manuscript is to evaluate two methods of murine intramuscular macrophage depletion in an acute lung injury-associated indirect skeletal muscle wasting mouse model. Adult C57BL/6 (WT) and Macrophage Fas-Induced Apoptosis (MaFIA, C57BL/6-Tg) mice received clodronate liposomes or the dimerization drug AP20187 through intramuscular injection of the tibialis anterior muscle compartment, respectively. Vehicle control was injected in the contralateral muscle. We demonstrate intramuscular AP20187 in the MaFIA mouse depletes macrophages but causes an infiltration of CD45 intermediate neutrophils. In contrast, intramuscular clodronate liposomes successfully depletes macrophages without an associated increase in CD45 intermediate cells. In conclusion, intramuscular clodronate is effective for selective depletion of muscle macrophages without eliciting acute inflammation seen with AP20187 in MaFIA mice. This technique is an important tool to study the functional roles of macrophages in skeletal muscle.

Clodronate.

Two early observations about the first generation bisphosphonate, clodronate, suggested that it would likely have clinical utility; specifically, it was a more potent anti-resorptive but a less potent inhibitor of mineralisation than its predecessor etidronate. The known mechanism of action differs from that of the later nitrogen-containing bisphosphonates, as clodronate is metabolised intracellularly to a toxic analog of adenosine triphosphate, AppCCl2p, which causes mitochondrial dysfunction, impaired cellular energy metabolism and osteoclast apoptosis. For pre-clinical studies in a variety of disease models, liposomal clodronate has become the agent of choice for macrophage depletion, for example in a recent study to enhance haematopoietic chimerism and donor-specific skin allograft tolerance in a mouse model. For clinical use, clodronate was developed in oral and injectable formulations; while poorly absorbed from the gastro-intestinal tract, its absorption at 1-3% of the administered dose is approximately three-fold higher than for nitrogen-containing bisphosphonates. Following an early setback due to an erroneous association with toxic adverse events, a number of successful clinical studies have established clodronate, predominantly in its oral formulations, as a highly successful treatment in Paget's disease, hypercalcaemia (benign and malignant), multiple myeloma, and early or metastatic breast cancer. Novel uses in other disease areas, including veterinary use, continue to be explored.

Liposomal Clodronate-mediated Macrophage Depletion in the Zebrafish Model.

The ability to conduct in vivo macrophage-specific depletion remains an effective means to uncover functions of macrophages in a wide range of physiological contexts. Compared to the murine model, zebrafish offer superior imaging capabilities due to their optical transparency starting from a single-cell stage to throughout larval development. These qualities become important for in vivo cell specific depletions so that the elimination of the targeted cells can be tracked and validated in real time through microscopy. Multiple methods to deplete macrophages in zebrafish are available, including genetic (such as an irf8 knockout), chemogenetic (such as the nitroreductase/metronidazole system), and toxin-based depletions (such as using clodronate liposomes). The use of clodronate-containing liposomes to induce macrophage apoptosis after phagocytosing the liposomes is effective in depleting macrophages as well as testing their ability to phagocytose. Here we describe a detailed protocol for the systemic depletion of macrophages in zebrafish larvae by intravenous injection of liposomal clodronate supplemented with fluorescent dextran conjugates. Co-injection with the fluorescent dextran allows tracking of macrophage depletion in real time starting with verifying the successful intravenous injection to macrophage uptake of molecules and their eventual death. To verify a high degree of macrophage depletion, the level of brain macrophage (microglia) elimination can be determined by a rapid neutral red vital dye staining when clodronate injection is performed at early larval stages. Graphical abstract: Experimental workflow for in vivo macrophage-specific depletion by liposomal clodronate in larval zebrafish.