MaFIA Mice Research Articles

Abstract Or112: Cardiac Macrophage are Key Immune Drivers of Cardiometabolic HFpEF

Heart failure with preserved ejection fraction (HFpEF) can develop with obesity, hyperinsulinemia, and glucose intolerance or type 2 diabetes, a phenogroup designated cardiometabolic HFpEF. While inflammation considered an important underlying mechanism, the role of innate immunity is poorly define. We investigated the role of macrophages in pathogenesis of cardiometabolic HFpEF. C57BL/6 mice were fed either high-fat diet (HFD, 45% kcal fat, 35% carbohydrate, 20% protein, 4.73 kcal/g) or control diet (CD, 10% kcal fat, 70% carbohydrate, 20% protein, 3.85 kcal/g) for 24-28 weeks. HFD mice exhibited significantly increased body weight (BW) at equivalent calorie consumption, increased white adipose tissue (WAT) mass, glucose intolerance, and hyperinsulinemia. HFD mice also exhibited HFpEF, evidenced by increased relative wall thickness (RWT), E/e’ ratio, LA area, LVEDP, and lung weight and decreased exercise tolerance with preserved EF. CD64 + MHCII + LyVE1 + CCR2 – resident and CD64 + MHCII + CCR2 + infiltrating macrophages expanded significantly in both cardiac and visceral fat tissue. To evaluate the role of macrophages in HFpEF, we used Macrophage Ablation Fas-Induced Apoptosis (MaFia) mice that allow for conditional depletion of mononuclear phagocytes with the Fas ligand dimerizer AP20187. AP20187 treatment after 24 weeks of HFD feeding in MaFia mice depleted macrophages and normalized BW, and reversed cardiac hypertrophy (decreased RWT, histological myocyte cross-sectional area [CSA]), and cardiac dysfunction (improved E/e’ ratio and global longitudinal strain [GLS]), as compared to vehicle-treated HFD-induced HFpEF MaFia mice. To establish sufficiency of cardiac macrophages, we FACS-sorted CD45 + CD64 + F4/80 + cardiac macrophages from HFD and CD mice, and adoptively transferred (10K cells/mouse) by intramyocardial injection into naïve recipient mice, with evaluation 4 w later. Mice with HFD-heart cardiac macrophage transfer evidenced significant increases in left atrial area (P=0.008, E/e’ ratio (P=0.016), cardiomyocyte CSA (P=0.015) and cardiac fibrosis (P<0.05), and reduced |GLS|, as compared to recipient mice with CD-heart cardiac macrophage transfer. There were no differences in body weight between the two groups. We conclude : cardiac macrophage expansion is central to the pathogenesis of cardiometabolic HFpEF, and are both necessary and sufficient for the cardiac phenotype. These results raise the potential for macrophage-targeted interventions to mitigate HFpEF.

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.

Macrophage depletion impairs neonatal tendon regeneration.

Tendons are dense connective tissues that transmit muscle forces to the skeleton. After adult injury, healing potential is generally poor and dominated by scar formation. Although the immune response is a key feature of healing, the specific immune cells and signals that drive tendon healing have not been fully defined. In particular, the immune regulators underlying tendon regeneration are almost completely unknown due to a paucity of tendon regeneration models. Using a mouse model of neonatal tendon regeneration, we screened for immune-related markers and identified upregulation of several genes associated with inflammation, macrophage chemotaxis, and TGFβ signaling after injury. Depletion of macrophages using AP20187 treatment of MaFIA mice resulted in impaired functional healing, reduced cell proliferation, reduced ScxGFP+ neo-tendon formation, and altered tendon gene expression. Collectively, these results show that inflammation is a key component of neonatal tendon regeneration and demonstrate a requirement for macrophages in effective functional healing.

Intra-articular depletion of macrophages increases acute synovitis and alters macrophage polarity in the injured mouse knee

Acute synovial inflammation following joint trauma is associated with posttraumatic arthritis. Synovial macrophages have been implicated in degenerative changes. In this study, we sought to elucidate the role of intra-articular macrophages in the acute inflammatory response to fracture in the mouse knee. A closed articular fracture was induced in two models of synovial macrophage depletion: genetically-modified MaFIA mice administered AP20187 to induce programmed macrophage apoptosis, and wild-type C57BL/6 mice administered clodronate liposomes, both via intra-articular injection. Synovial inflammation, bone morphology, and levels of F4/80+ macrophages, NOS2+ M1 macrophages, and CD206+ M2 macrophages were quantified 7days after fracture using histology and micro-computed tomography. Intra-articular macrophage depletion with joint injury did not reduce acute synovitis or the number of synovial macrophages 7days after fracture in either macrophage-depleted MaFIA mice or in clodronate-treated C57BL/6 mice. In macrophage-depleted MaFIA mice, macrophage polarity shifted to a dominance of M1 macrophages and a reduction of M2 macrophages in the synovial stroma, indicating a shift in M1/M2 macrophage ratio in the joint following injury. Interestingly, MaFIA mice depleted 2days prior to fracture demonstrated increased synovitis (P=0.003), reduced bone mineral density (P=0.0004), higher levels of M1 macrophages (P=0.013), and lower levels of M2 macrophages (not statistically significant, P=0.084) compared to control-treated MaFIA mice. Our findings indicate that macrophages play a critical immunomodulatory role in the acute inflammatory response surrounding joint injury and suggest that inhibition of macrophage function can have prominent effects on joint inflammation and bone homeostasis after joint trauma.

CD45 expression discriminates waves of embryonic megakaryocytes in the mouse.

Embryonic megakaryopoiesis starts in the yolk sac on gestational day 7.5 as part of the primitive wave of hematopoiesis, and it continues in the fetal liver when this organ is colonized by hematopoietic progenitors between day 9.5 and 10.5, as the definitive hematopoiesis wave. We characterized the precise phenotype of embryo megakaryocytes in the liver at gestational day 11.5, identifying them as CD41++CD45-CD9++CD61+MPL+CD42c+ tetraploid cells that express megakaryocyte-specific transcripts and display differential traits when compared to those present in the yolk sac at the same age. In contrast to megakaryocytes from adult bone marrow, embryo megakaryocytes are CD45− until day 13.5 of gestation, as are both the megakaryocyte progenitors and megakaryocyte/erythroid-committed progenitors. At gestational day 11.5, liver and yolk sac also contain CD41+CD45+ and CD41+CD45− cells. These populations, and that of CD41++CD45−CD42c+ cells, isolated from liver, differentiate in culture into CD41++CD45−CD42c+ proplatelet-bearing megakaryocytes. Also present at this time are CD41−CD45++CD11b+ cells, which produce low numbers of CD41++CD45−CD42c+ megakaryocytes in vitro, as do fetal liver cells expressing the macrophage-specific Csf receptor-1 (Csf1r/CD115) from MaFIA transgenic mice, which give rise poorly to CD41++CD45−CD42c+ embryo megakaryocytes both in vivo and in vitro. In contrast, around 30% of adult megakaryocytes (CD41++CD45++CD9++CD42c+) from C57BL/6 and MaFIA mice express CD115. We propose that differential pathways operating in the mouse embryo liver at gestational day 11.5 beget CD41++CD45−CD42c+ embryo megakaryocytes that can be produced from CD41+CD45− or from CD41+CD45+ cells, at difference from those from bone marrow.

Resident corneal c-fms+ macrophages and dendritic cells mediate early cellular infiltration in adenovirus keratitis

The cornea contains a heterogeneous population of antigen-presenting cells with the capacity to contribute to immune responses. Adenovirus keratitis is a severe corneal infection with acute and chronic phases. The role of resident corneal antigen-presenting cells in adenovirus keratitis has not been studied. We utilized transgenic MaFIA mice in which c-fms expressing macrophages and dendritic cells can be induced to undergo apoptosis, in a mouse model of adenovirus keratitis. Clinical keratitis and recruitment of myeloperoxidase and CD45+ cells were diminished in c-fms depleted, adenovirus infected mice, as compared to controls, consistent with a role for myeloid-lineage cells in adenovirus keratitis.

Bone marrow macrophages support prostate cancer growth in bone

Resident macrophages in bone play important roles in bone remodeling, repair, and hematopoietic stem cell maintenance, yet their role in skeletal metastasis remains under investigated. The purpose of this study was to determine the role of macrophages in prostate cancer skeletal metastasis, using two in vivo mouse models of conditional macrophage depletion. RM-1 syngeneic tumor growth was analyzed in an inducible macrophage (CSF-1 receptor positive cells) ablation model (MAFIA mice). There was a significant reduction in tumor growth in the tibiae of macrophage-ablated mice, compared with control non-ablated mice. Similar results were observed when macrophage ablation was performed using liposome-encapsulated clodronate and human PC-3 prostate cancer cells where tumor-bearing long bones had increased numbers of tumor associated-macrophages. Although tumors were consistently smaller in macrophage-depleted mice, paradoxical results of macrophage depletion on bone were observed. Histomorphometric and micro-CT analyses demonstrated that clodronate-treated mice had increased bone volume, while MAFIA mice had reduced bone volume. These results suggest that the effect of macrophage depletion on tumor growth was independent of its effect on bone responses and that macrophages in bone may be more important to tumor growth than the bone itself. In conclusion, resident macrophages play a pivotal role in prostate cancer growth in bone.

The effect of conditional macrophage depletion on development of injury-induced knee OA in obese transgenic MAFIA mice

s / Osteoarthritis and Cartilage 23 (2015) A82eA416 A287 Conclusions: Contrary to our hypothesis, we found that systemic MF depletion in obese MAFIA mice did not mitigate cartilage degradation after joint injury; instead, it enhanced joint synovitis by increasing infiltration of CD3þ T cells and neutrophils into the operated joint. Our study is significant for elucidating the immunomodulatory activity of MFs in inflammation and joint injury in obesity. 451 MECHANICALLY AIDED TRANSPORT OF ANTIBODIES THROUGH ARTICULAR CARTILAGE C. DiDomenico, Z. Wang, L. Bonassar. Cornell Univ., Ithaca, NY, USA Purpose: Recently, significant advances in the clinical treatment of rheumatoid arthritis (RA) have been made using therapeutic antibodies that target and inhibit tumor necrosis factor alpha (TNF-a) found in the synovium of the joint. Additionally, TNF-a is also known to play a critical role in the progression of osteoarthritis (OA); however, TNF-a blocking antibodies are not currently used for OA therapy. Besides total joint replacement, there is no long-term effective treatment for OA. In contrast to RA, in which synovial inflammation is easily targeted by intravenous injection of therapeutics, TNF-a activity in OA occurs primarily within articular cartilage. Because of the high density of the cartilage extracellular matrix, it is not clear whether large proteins, like antibodies, will be able to sufficiently penetrate the cartilage tissue on a time scale that is clinically relevant. Previously, mechanical stimulation of cartilage tissue has been shown to aid in the penetration of proteins ( 100 kDa). The objective of this study is to characterize the effect of physiologically relevant mechanical loading on the transport of antibodies through cartilage. Methods: For each experiment, cylindrical articular cartilage plugs (4 mm diameter, 1.15 mm thick) were extracted from 1-3 day old bovids and randomly assigned to loaded and unloaded groups. Fluorescently labeled antibodies (MW: 150 kDa) in PBS were added to each groupspecific well plates while only PBS were added to negative controls. By compressing each sample 15% with an impermeable platen array, only the outside cylindrical (radial) surface was exposed to the antibody solution (Figure 1 inset). Each group is then placed into a 37 C incubator where only the loaded group undergo unconfined cyclic axial compressionwith a custom-built, displacement-controlled compressor. The transducer then applies a set dynamic strain to the loaded samples via the platen array for 3 hours at a set frequency. After loading, all plugs were bisected axially and the cut surface was viewed using confocal microscopy to characterize antibody penetration from the radial edges of the cut surface towards the center (Figure 1 inset). Average fluorescence intensity profiles, from each radial edge of the cut surface to the center, were fitted to a 1D diffusion model derived from Fick's 2nd law to obtain an average effective diffusivity for each sample (Figure 1). The diffusivities of loaded samples were then normalized to the average diffusivity of the unloaded samples for each experiment. These “transport enhancement ratios” were then analyzed across several loading amplitudes applied a 1 Hz (Figure 2). Results: Experiments showed that cyclic compression enhances transport of antibodies into articular cartilage. The 1D diffusion model accurately described the fluorescence profiles of both the unloaded and loaded samples (coefficient of variation between 5-20% and R2 between 0.980 and 0.995). For the unloaded samples, diffusivities ranged from 2.8 to 6.0 10-8 cm2/s, while loaded diffusivities ranged from 6.8 to 15.4 10-8 cm2/s. The convective enhancement ratios obtained ranged from 1.20 for the 0.25% strain (N.S. compared to an enhancement ratio of 1, i.e., no enhancement), 1.47 for 1.3% strain (p < 0.01), 1.80 for 2.5% strain (p < 0.001), and 2.61 for 5% strain (p < 0.001) (Figure 2). Further, for this range in dynamic strain amplitude there was linear trend of increasing effective diffusivity with strain amplitude (r2 1⁄4 0.98, p < 0.005). Conclusions: Overall, despite the small pore size of cartilage and the high MW of the antibody used, mechanical loading enhanced antibody transport to a large degree. More research will have to be done to determine how well antibodies diffuse across the articular surface and how other antibodies affect transport in more mature cartilage tissue. Given these results, there is promise that antibody therapy for OA could be enhanced by an exercise regimen or physical therapy, mimicking physiological loading conditions from these experiments. To our knowledge, this is the first study to test how mechanical stimulation affects the diffusion of antibodies in cartilage and suggest further study into how more efficient antibody therapy for OA can be developed. Figure 1. Normalized fluorescent intensity vs. radial depth for a 5% dynamic strain loaded sample (blue) and unloaded (red) sample to which ID diffusion models were fit (solid lines). (Inset): Experimental setup of an individual well and an example of a confocal radial antibody profile.

Abstract 17284: Selective Macrophage Ablation Attenuates Diet-Induced Obesity and Insulin Resistance and Reverses Diabetic Cardiomyopathy

Although activated macrophages infiltrate the heart and adipose tissue in type 2 diabetes (T2D), the role of these cells in the genesis of cardiomyopathic phenotype is unknown. We tested the hypothesis that activated resident macrophages are fundamental mediators of diet-induced diabetic cardiomyopathy. Transgenic mice expressing an AP20187-inducible Fas-dependent suicide gene in macrophages and dendritic cells (MAFIA mice) were fed a high fat diet (HFD, 45% kcal fat) or control diet (CD, 10% kcal fat) for 28 weeks to induce T2D, and subsequently given either vehicle or AP20187 (0.5mg/kg IP every 10 d) for one month to induce macrophage apoptosis and depletion. HFD-fed mice exhibited a significant (p &lt; 0.05) 43% increase in body weight when compared to CD fed mice (43±5 vs 35±1 g) and developed hyperglycemia (199±44 vs 121±10 mg/dL), hyperinsulinemia (1.27±0.6 vs 0.43±0.14 µg/L) and decreased insulin sensitivity indicating T2D. Echocardiography revealed concentric LV hypertrophy in HFD-fed mice with increased wall thickness (anterior wall 1.2±0.1 vs 0.9±0.1 mm), decreased LV end-diastolic diameter (LVEDD 3.9±0.2 vs 4.2±0.4 mm), and unchanged LV ejection fraction. AP20187 treatment significantly reduced the caloric intake (0.3±0.08 vs 0.4±0.05 kcal/g/day) and body weight of HFD-fed mice (36±2 vs 47±5 g), attenuated hyperglycemia (108±8 vs 195±14 mg/dL) and hyperinsulinemia (0.6±0.2 vs 1.9±0.5 µg/L), and improved insulin sensitivity when compared to vehicle treated HFD-fed mice. Importantly, the improvements in metabolic parameters were accompanied by reversal of LV remodeling with decreased wall thickness (anterior wall 0.8±0.1 vs 1.3±0.1 mm) and increased LVEDD (4.6±0.5 vs 3.9±0.2 mm). White adipose tissue weights in AP20187-treated HFD-fed mice were also significantly decreased when compared to vehicle treated HFD-fed mice [epididymal (0.6±0.2 vs 1.4±0.3 g); inguinal (0.6±0.2 vs 1.1±0.3 g); retroperitoneal (0.8±0.5 vs 1.4±0.4 g). We conclude that resident activated macrophages play central and causative roles in the development of diet-induced T2D and diabetic cardiomyopathy. Targeting systemic macrophage activation may represent a therapeutic approach to reverse pathological cardiac remodeling associated with T2D and obesity.

Manipulating Tumor Associated Macrophages (TAM) in a Mouse Model of B-Cell Non-Hodgkin Lymphoma (NHL)

Abstract 1657 Introduction:In vitro stimulation of macrophages can produce classically activated (M1) or alternatively activated (M2) phenotypes. In various solid tumors, tumor associated macrophages (TAM) have been described as approximating an M2 phenotype and are potentially pro-tumoral. We are testing the hypothesis that TAM play an important role in the progression of NHL, and might therefore constitute a rational and effective therapeutic target.Our aim is to investigate tumor-macrophage interactions by manipulating macrophage numbers and phenotype in a transplantable mouse lymphoma. Experimental approach:We used as a model system B-cell lymphoma arising in Em-myc/bcl-2 transgenic mice, which, when intravenously injected into C57BL/6 mice produces disseminated lymphoma.Lymphoma growth was assessed by measuring lymph node weight, and cross-sectional tumor area in tissue sections, as well as by flow cytometry. Gene expression changes were determined by real-time PCR in whole lymph nodes or sorted macrophages in control mice or mice with lymphoma, and following interventions to manipulate macrophage populations.Changes to the cellular composition of the immune microenvironment were assessed by flow cytometry of lymph nodes and blood, and by immunohistochemistry. Methods and results:The consequences of widespread monocyte and macrophage depletion were first investigated using intravenous injection of the liposome-encapsulated bisphosphonate, sodium clodronate (Liposomal Clodronate, LC).Intravenous LC successfully depleted macrophages in the bone marrow, lymph nodes, liver and spleen, and correlated with significantly reduced lymphoma mass by weight and flow cytometry, compared to controls (p=0.0058).Next we examined MaFIA mice (Macrophage Fas Induced Apoptosis, Csf1r-EGFP-NGFR/FKBP1A/TNFRSF6) that combine the expression of an enhanced green fluorescent protein on cells with the macrophage-specific csf-1 receptor, with a drug (dimerizer, AP20187) inducible suicide gene leading to Fas-mediated apoptosis in resting and cycling cells of the macrophage lineage.We observed the effects of this non-pharmacological method of macrophage depletion on lymphoma progression.Lymph node macrophage numbers are not reduced in normal healthy mice treated with the dimerizer, but were significantly reduced in the infiltrated lymph nodes of mice with lymphoma given the dimerizer. This was associated with a reduced lymphoma mass (p=0.044).Subsequent studies employed adoptive transfer of syngeneic bone marrow derived macrophages (BMDM), in vitro polarized to M1, M2 and model-specific TAM phenotypes; the purpose being to test whether supplementing the TAM population might invigorate lymphoma growth, and whether administering M1 phenotype macrophages might lead to anti-tumor effects.Adoptive transfer of M1-polarized BMDM attenuated lymphoma growth, and transfer of TAM augmented lymphoma growth (p=0.0003).Having gathered evidence that reducing TAM numbers might slow lymphoma growth, we explored a novel approach to targeting this macrophage sub-population in lymphoma, with translational potential for human studies. More specific targeting of TAM, and relative sparing of physiological monocytes and resident tissue macrophages, was attempted by targeting the CSF1 receptor with an orally available highly selective small molecule inhibitor. Current literature suggests this might reduce both monocyte trafficking to areas of malignancy, and maturation of monocytes to TAM. This strategy was used against growing lymphomas, resulting in an attenuated TAM population (p=0.0009) and reduced lymphoma mass (p=0.0036), whilst not depleting the circulating monocyte pool.In vitro cytotoxicity studies confirmed that neither LC, nor the CSF1R inhibitor nor AP20187 dimerizer caused significant direct lymphoma cell death, confirming that the observed in vivo attenuation of lymphoma growth likely resulted from indirect phenomena, such as TAM depletion. Conclusion:By evaluating a variety of pharmacological, genetic and cellular therapy strategies we have generated a strong body of evidence supporting a causal relationship between TAM abundance and lymphoma progression in this model. These data support the hypothesis that targeting TAM may well provide an attractive therapeutic opportunity to improve outcome in human B-cell lymphomas. Disclosures:Hagemann:Astra Zeneca and the Medical Research Council: In receipt of a joint studentship with Astra Zeneca and the UK Medical Research Council. Gribben:Roche: Honoraria; Celgene: Honoraria; GSK: Honoraria; Mundipharma: Honoraria; Gilead: Honoraria; Pharmacyclics: Honoraria.

TLR4 and TLR5 on Corneal Macrophages Regulate Pseudomonas aeruginosa Keratitis by Signaling through MyD88-Dependent and -Independent Pathways

Pseudomonas aeruginosa is a major cause of blindness and visual impairment in the United States and worldwide. Using a murine model of keratitis in which abraded corneas are infected with P. aeruginosa parent and ΔfliC (aflagellar) strains 19660 and PAO1, we found that F4/80(+) macrophages were the predominant cell type in the cornea expressing TLR2, TLR4, and TLR5. Depletion of macrophages and dendritic cells using transgenic Mafia mice, in which Fas ligand is selectively activated in these cells, resulted in diminished cytokine production and cellular infiltration to the corneal stroma and unimpaired bacterial growth. TLR4(-/-) mice showed a similar phenotype postinfection with ΔfliC strains, whereas TLR4/5(-/-) mice were susceptible to corneal infection with parent strains. Bone marrow-derived macrophages stimulated with ΔfliC bacteria induced Toll/IL-1R intracellular domain (TIR)-containing adaptor inducing IFN-β (TRIF)-dependent phosphorylation of IFN regulatory factor 3 in addition to TIR-containing adaptor protein/MyD88-dependent phosphorylation of IκB and nuclear translocation of the p65 subunit of NFκB. Furthermore, TRIF(-/-) mice showed a similar phenotype as TLR4(-/-) mice in regulating only ΔfliC bacteria, whereas MyD88(-/-) mice were unable to clear parent or ΔfliC bacteria. Finally, IL-1R1(-/-) and IL-1α/β(-/-) mice were highly susceptible to infection. Taken together, these findings indicate that P. aeruginosa activates TLR4/5 on resident corneal macrophages, which signal through TRIF and TIR-containing adaptor protein/MyD88 pathways, leading to NF-κB translocation to the nucleus, transcription of CXCL1 and other CXC chemokines, recruitment of neutrophils to the corneal stroma, and subsequent bacterial killing and tissue damage. IL-1α and IL-1β are also produced, which activate an IL-1R1/MyD88-positive feedback loop in macrophages and IL-1R on other resident cells in the cornea.

Distinct roles for Dectin-1 and TLR4 in the pathogenesis of Aspergillus fumigatus keratitis.

Aspergillus species are a major worldwide cause of corneal ulcers, resulting in visual impairment and blindness in immunocompetent individuals. To enhance our understanding of the pathogenesis of Aspergillus keratitis, we developed a murine model in which red fluorescent protein (RFP)-expressing A. fumigatus (Af293.1RFP) conidia are injected into the corneal stroma, and disease progression and fungal survival are tracked over time. Using Mafia mice in which c-fms expressing macrophages and dendritic cells can be induced to undergo apoptosis, we demonstrated that the presence of resident corneal macrophages is essential for production of IL-1β and CXCL1/KC, and for recruitment of neutrophils and mononuclear cells into the corneal stroma. We found that β-glucan was highly expressed on germinating conidia and hyphae in the cornea stroma, and that both Dectin-1 and phospho-Syk were up-regulated in infected corneas. Additionally, we show that infected Dectin-1−/− corneas have impaired IL-1β and CXCL1/KC production, resulting in diminished cellular infiltration and fungal clearance compared with control mice, especially during infection with clinical isolates expressing high β-glucan. In contrast to Dectin 1−/− mice, cellular infiltration into infected TLR2−/−, TLR4−/−, and MD-2−/− mice corneas was unimpaired, indicating no role for these receptors in cell recruitment; however, fungal killing was significantly reduced in TLR4−/− mice, but not TLR2−/− or MD-2−/− mice. We also found that TRIF−/− and TIRAP−/− mice exhibited no fungal-killing defects, but that MyD88−/− and IL-1R1−/− mice were unable to regulate fungal growth. In conclusion, these data are consistent with a model in which β-glucan on A.fumigatus germinating conidia activates Dectin-1 on corneal macrophages to produce IL-1β, and CXCL1, which together with IL-1R1/MyD88-dependent activation, results in recruitment of neutrophils to the corneal stroma and TLR4-dependent fungal killing.

Osteomacs are critical for optimal intramembranous bone formation in a tibial defect model of bone healing

Osteal tissues contain a resident population of macrophages (OsteoMacs) that regulate osteoblast mineralisation in vitro. Osteo-Macs form a canopy structure covering osteoblast bone forming surfaces (ObS) suggesting OsteoMacs regulate bone anabolic responses. Using the Mafia (Macrophage-Fas-Induced Apoptosis) transgenic mouse, in which the csf1r promoter drives the expression of AP20187 (www.ariad.com/regulationkits) ligand-inducible Fas-based suicide gene in macrophage/myeloid cells, we demonstrated that OsteoMacs are required for maintenance of ObS at sites of bone modelling. To definitively examine OsteoMacs contribution to in vivo bone formation a tibial defect model was employed in which the defect site is filled with woven bone via intramembranous ossification 7 days post surgery. Immunohistochemistry demonstrated that F4/80+ OsteoMacs accumulated within the defect site, forming the canopy structure covering collagen type I (CTI)+ osteoblasts on woven bone surfaces. Osteotomies were performed in Mafia mice and a single injection of ligand or vehicle administered intra-defect at surgery. Healing was examined 7 days post surgery. Flow cytometry confirmed a 46±7% decrease in bone marrow F4/80+ macrophages in the contralateral limb of ligand treated mice. In ligand treated animals there was a striking reduction in the number of CT1+ osteoblasts and F4/80+ OsteoMac canopy within the defect. Quantitative immunohistochemical analysis of intra-defect CTI+ matrix area demonstrated a significant reduction (p=0.03) in bone formation in ligand (20±6%) compared to vehicle (44±5%) treated mice. Osteoclasts express csf1r and therefore may be susceptible to apoptosis in ligand treated Mafia mice. To rule out that the decreased bone formation was indirectly due to inhibition of osteoclasts, osteotomies were performed in C57/Bl6 mice that were treated with OPG (1 mg/kg, initial intra-defect administration and subsequently subcutaneous injection every second day) to specifically inhibit osteoclasts. Immunohistochemical analysis of OPG treated animals demonstrated minimal change in intra-defect CTI+ woven bone deposition or defect healing. Distribution of F4/80+ OsteoMacs within the defect was unaffected by OPG treatment. These observations support that OsteoMacs, and not osteoclasts, play a critical role in driving and maintaining osteoblast function at sites of intramembranous bone deposition during growth and bone healing. Therefore OsteoMacs are novel participants in bone formation in vivo.

OsteoMacs maintain the endosteal hematopoietic stem cell niche and participate in mobilization

OsteoMacs are tissue macrophages that reside within bone lining tissues. OsteoMacs enhance osteoblast function in vitro and maintain osteoblast bone forming surface in vivo. Given the endosteum is a preferred niche for hematopoietic stem cells (HSC), we investigated whether OsteoMacs participate in HSC mobilization induced by granulocyte colony-stimulating factor (G-CSF). Immunohistochemistry (IHC) using the F4/80 antibody demonstrated that mobilization induced by G-CSF resulted in a sharp reduction in the number of F4/80+ OsteoMacs within the endosteal niche from days 2–6 post initiation of G-CSF administration. At day 2, clustering of F4/80+ cells in the central marrow and in particular around endothelial sinuses was evident. In contract, localization and maintenance of OsteoMacs within the periosteum was unaffected by G-CSF administration. Flow cytometry demonstrated increased numbers of F4/80+ macrophages in blood from days 1–6, indicating egress of macrophages from bone marrow to blood. Static and dynamic histomorphemtry and IHC for osteocalcin expression demonstrated a concomitant significant reduction in endosteal osteoblast number and function as well as osteoid. Notably, the kinetics of OsteoMac loss strikingly parallels, if not precedes, that of endosteal osteoblasts loss and mobilization of HSC to blood. In contrast, osteoclast numbers were not significantly altered by G-CSF administration and treatment with the bisphosphonate zoledronate increased HSC mobilization in response to G-CSF. F4/80+ macrophages repopulation of bone marrow and endosteal surfaces initiated at day 8, 2 days after cessation of G-CSF administration, and was quickly followed at day 10 by recovery of OsteoMac canopy covering cubiodal osteoblast-like cells. Prior experimentation has indicated that mobilization induced by G-CSF acts through an intermediary G-CSF receptor expressing myeloid population. Finally, we demonstrate a pivotal role of macrophages in maintaining endosteal HSC niches in transgenic MAFIA mice in which macrophages can be specifically deleted in vivo using AP20187. At day 5 following macrophages deletion, we observed a collapse in the number of osteoblasts lining the endosteum and robust mobilization of HSC in peripheral blood and spleen. Given macrophages can express the G-CSF receptor and the observations reported here, we propose that G-CSF induces HSC mobilization through OsteoMac-directed collapse of the endosteal HSC niches.

Bone Marrow Chimeras and c-fms Conditional Ablation (Mafia) Mice Reveal an Essential Role for Resident Myeloid Cells in Lipopolysaccharide/TLR4-Induced Corneal Inflammation

The mammalian cornea contains an extensive network of resident macrophages and dendritic cells. To determine the role of these cells in LPS-induced corneal inflammation, TLR4(-/-) mice were sublethally irradiated and reconstituted with bone marrow cells from either enhanced GFP (eGFP)(+)/C57BL/6 or eGFP(+)/TLR4(-/-) mice. The corneal epithelium was abraded, LPS was added topically, and cellular infiltration to the corneal stroma and development of corneal haze were examined after 24 h. TLR4(-/-) mice reconstituted with C57BL/6, but not TLR4(-/-) bone marrow cells donor cells were found to cause infiltration of eGFP(+) cells to the cornea, including neutrophils, and also increased corneal haze compared with saline-treated corneas. In a second experimental approach, corneas of transgenic macrophage Fas induced apoptosis (Mafia) mice were stimulated with LPS. These mice express eGFP and a suicide gene under control of the c-fms promoter, and systemic treatment with the FK506 dimerizer (AP20187) causes Fas-mediated apoptosis of monocytic cells. AP20187-treated mice had significantly fewer eGFP(+) cells in the cornea than untreated mice. After stimulation with LPS neutrophil recruitment and development of corneal haze were impaired in AP20187-treated mice compared with untreated controls. Furthermore, LPS induced CXCL1/KC and IL-1alpha production within 4 h in corneas of untreated Mafia mice, which is before cellular infiltration; however, cytokine production was impaired after AP20187 treatment. Together, results from both experimental approaches demonstrate an essential role for resident corneal monocytic lineage cells (macrophages and dendritic cells) in development of corneal inflammation.

Development of Peritoneal Adhesions in Macrophage Depleted Mice

We present a new mouse model for the study of peritoneal adhesions using macrophage Fas-induced apoptosis (Mafia) transgenic mice expressing a Fas-FKBP construct under control of the murine c-fms promoter. Mafia mice allow systemic macrophage depletion by dimerization of Fas with a synthetic dimerizer, AP20187. Results demonstrate that macrophage depletion in Mafia mice induces peritoneal adhesion formation when the peritoneal cavity is also exposed to an irritant. The Mafia mouse model presents a reproducible, non-surgical approach for research in adhesion formation and prevention. Mafia mice were treated with AP20187 using an intravenous (i.v.) or intraperitoneal (i.p.) injection. Control groups included mock-treated Mafia mice and both AP20187 and mock-treated wild type mice. Seven days after treatment, mice were observed for the presence of adhesions. After i.p. injection with AP20187, 76% of Mafia mice developed adhesions whereas none of the mock-treated Mafia or wild-type mice developed adhesions, and only one AP20187-treated wild-type mouse (5.8%) developed a mild adhesion. Mafia mice treated with AP20187 i.v. exhibited macrophage depletion not significantly different than i.p. treated mice, but did not develop adhesions. In contrast, Mafia mice treated with AP20187 i.v. developed adhesions when diluent was also injected into the peritoneal cavity, whereas i.p diluent alone had no effect. Macrophage depletion, combined with a peritoneal irritant, results in peritoneal adhesion formation in transgenic Mafia mice. Macrophages appear to play a protective role in the development and/or repair of peritoneal adhesions.