Macrophage Ablation Research Articles

Identification of Sucrose Non-Fermenting–Related Kinase (SNRK) as a Suppressor of Adipocyte Inflammation

In this study, the role of sucrose non-fermenting–related kinase (SNRK) in white adipocyte biology was investigated. SNRK is abundantly expressed in adipose tissue, and the expression level is decreased in obese mice. SNRK expression is repressed by inflammatory signals but increased by insulin sensitizer in cultured adipocytes. In vivo, adipose tissue SNRK expression can be decreased by lipid injection but enhanced by macrophage ablation. Knocking down SNRK in cultured adipocytes activates both JNK and IKKβ pathways as well as promotes lipolysis. Insulin-stimulated Akt phosphorylation and glucose uptake are impaired in SNRK knockdown adipocytes. Phosphoproteomic analysis with SNRK knockdown adipocytes revealed significantly decreased phosphorylation of 49 proteins by 25% or more, which are involved in various aspects of adipocyte function with a clear indication of attenuated mTORC1 signaling. Phosphorylation of 43 proteins is significantly increased by onefold or higher, among which several proteins are known to be involved in inflammatory pathways. The inflammatory responses in SNRK knockdown adipocytes can be partially attributable to defective mTORC1 signaling, since rapamycin treatment activates IKKβ and induces lipolysis in adipocytes. In summary, SNRK may act as a suppressor of adipocyte inflammation and its presence is necessary for maintaining normal adipocyte function.

Abstract 192: Depletion of Inflammatory Monocytes Reduces Endothelial Nitric Oxide Synthase Uncoupling and iNOS Derived Nitrosative Stress in Angiotensin II-induced Vascular Dysfunction

Background Endothelial nitric oxide synthase (eNOS) uncoupling occurs in disease states of vascular dysfunction and amplifies vascular oxidative stress and loss of NO bioactivity. Inflammatory myelomonocytic cells as a major source of superoxide are critical for angiotensin-II induced vascular dysfunction; however their role in mediating eNOS uncopuling has not been defined yet. Methods and results Angiotensin II (1mg/kg/d, 7d) increased the number of CD11b + Gr-1 low iNOS + monocytes and macrophages in mouse aorta (verified by flow cytometry) in paralell to increasing nox4 and heme oxygenase 1 expression (assessed by Western blot analysis) and inducing eNOS uncoupling, measured by differential NOS dependent superoxide formation in the endothelial layer and glutathionylation of eNOS. Toxin mediated ablation of macrophages in LysM iDTR transgenic mice restored all of these parameters. Conversely, ablation of LysM + cells reduced iNOS expression and normalized vascular levels of tetrahydrobiopterin (measured by high-performance liquid chromatography) and expression of the GTP cyclohydrolase-1, essential for tetrahydrobiopterin synthesis, all of which were increased by angiotensin II in control mice. Conclusion Depletion of LysM + monocytes reduces angiotensin II induced eNOS uncopuling in vascular dysfunction, and simultaneously protects from iNOS mediated nitrosative stress: These findings highlight the role of vascular inflammatory cells in disturbed NO-bioactivity and vascular function

Macrophages Are Essential for the Early Wound Healing Response and the Formation of a Fibrovascular Scar

After wounding, multiple cell types interact to form a fibrovascular scar; the formation and cellular origins of these scars are incompletely understood. We used a laser-injury wound model of choroidal neovascularization in the eye to determine the spatiotemporal cellular events that lead to formation of a fibrovascular scar. After laser injury, F4/80(+) myeloid cells infiltrate the wound site and induce smooth muscle actin (SMA) expression in adjacent retinal pigment epithelial cells, with subsequent formation of a SMA(+)NG2(+) myofibroblastic scaffold, into which endothelial cells then infiltrate to form a fibrovascular lesion. Cells of the fibrovascular scaffold express the proangiogenic factor IL-1β strongly, whereas retinal pigment epithelial cells are the main source of VEGF-A. Subsequent choroidal neovascularization is limited to the area demarcated by this myofibroblastic scaffold and occurs independently of epithelial- or myeloid-derived VEGF-A. The SMA(+)NG2(+) myofibroblastic cells, F4/80(+) macrophages, and adjacent epithelial cells actively proliferate in the early phase of the wound healing response. Cell-lineage tracing experiments suggest that the SMA(+)NG2(+) myofibroblastic scaffold originates from choroidal pericyte-like cells. Targeted ablation of macrophages inhibits the formation of this fibrovascular scaffold, and expression analysis reveals that these macrophages are Arg1(+)YM1(+)F4/80(+) alternatively activated M2-like macrophages, which do not require IL-4/STAT6 or IL-10 signaling for their activation. Thus, macrophages are essential for the early wound healing response and the formation of a fibrovascular scar.

Near Infrared Imaging and Photothermal Ablation of Vascular Inflammation Using Single‐Walled Carbon Nanotubes

BackgroundMacrophages are critical contributors to atherosclerosis. Single-walled carbon nanotubes (SWNTs) show promising properties for cellular imaging and thermal therapy, which may have application to vascular macrophages.Methods and ResultsIn vitro uptake and photothermal destruction of mouse macrophage cells (RAW264.7) were performed with SWNTs (14.7 nmol/L) exposed to an 808-nm light source. SWNTs were taken up by 94±6% of macrophages, and light exposure induced 93±3% cell death. In vivo vascular macrophage uptake and ablation were then investigated in carotid-ligated FVB mice (n=33) after induction of hyperlipidemia and diabetes. Two weeks postligation, near-infrared fluorescence (NIRF) carotid imaging (n=12) was performed with SWNT-Cy5.5 (8 nmol of Cy5.5) given via the tail vein. Photothermal heating and macrophage apoptosis were evaluated on freshly excised carotid arteries (n=21). NIRF of SWNTs showed higher signal intensity in ligated carotids compared with sham, confirmed by both in situ and ex vivo NIRF imaging (P<0.05, ligation versus sham). Immunofluorescence staining showed colocalization of SWNT-Cy5.5 and macrophages in atherosclerotic lesions. Light (808 nm) exposure of freshly excised carotids showed heating and induction of macrophage apoptosis in ligated left carotid arteries with SWNTs, but not in control groups without SWNTs or without light exposure.ConclusionsCarbon nanotubes accumulate in atherosclerotic macrophages in vivo and provide a multifunctional platform for imaging and photothermal therapy of vascular inflammation.

Live Imaging Reveals Differing Roles of Macrophages and Neutrophils during Zebrafish Tail Fin Regeneration

Macrophages and neutrophils are the pivotal immune phagocytes that enter the wound after tissue injury to remove the cell debris and invaded microorganisms, which presumably facilitate the regrowth of injured tissues. Taking advantage of the regeneration abilities of zebrafish and the newly generated leukocyte-specific zebrafish lines with labeling of both leukocyte lineages, we assessed the behaviors and functions of neutrophils and macrophages during tail fin regeneration. Live imaging showed that within 6 hours post amputation, the inflammatory stage, neutrophils were the primary cells scavenging apoptotic bodies and small cell debris, although they had limited phagocytic capacity and quickly underwent apoptosis. From 6 hours post amputation on, the resolution and regeneration stage, macrophages became the dominant scavengers, efficiently resolving inflammation and facilitating tissue remodeling and regrowth. Ablation of macrophages but not neutrophils severely impaired the inflammatory resolution and tissue regeneration, resulting in the formation of large vacuoles in the regenerated fins. In contrast, removal of neutrophils slightly accelerates the regrowth of injured fin. Our study documents the differing behaviors and functions of macrophages and neutrophils during tissue regeneration.

Proinflammatory macrophage ablation in CD11b‐diphtheria toxin receptor (DTR) transgenic mice impairs skeletal muscle regeneration

Macrophages can be beneficial or detrimental to regeneration following muscle injury and macrophage polarization states are not well‐defined during this process. We previously demonstrated that macrophage ablation with diphtheria toxin (DT) given at day 1 after cardiotoxin injury increased residual necrotic myofibers and resulted in smaller regenerated myofibers in DTR mice compared to control mice that received mutant DT while mice receiving DT at day 4 were similar to controls. We used flow cytometry to evaluate the kinetics of macrophage surface markers to investigate the role of macrophage subtypes in muscle regeneration. In control mice, the majority of macrophages (CD11b+/F480+) were Ly6C+7/4+proinflammatory macrophages at day 1–2 after injury and switched to Ly6C−7/4−macrophages at day 4 after injury. M2 markers CD206+ or CD301+ were present on a small percentage of cells. Interestingly, the predominate macrophage population present from day 3–6 was negative for all 4 markers (Ly6C, 7/4, CD206 and CD301). Administration of DT at day 1 in DTR mice resulted in macrophage ablation at day 2–3 after injury with an increase in macrophages at day 4. Thus, the early recruitment of proinflammatory macrophages was critical for muscle regeneration; further studies are needed to define markers present on macrophages in the later stages of the regenerative process.Funding: Veterans Administration and NIH HL07423

The Glial Scar-Monocyte Interplay: A Pivotal Resolution Phase in Spinal Cord Repair

The inflammatory response in the injured spinal cord, an immune privileged site, has been mainly associated with the poor prognosis. However, recent data demonstrated that, in fact, some leukocytes, namely monocytes, are pivotal for repair due to their alternative anti-inflammatory phenotype. Given the pro-inflammatory milieu within the traumatized spinal cord, known to skew monocytes towards a classical phenotype, a pertinent question is how parenchymal-invading monocytes acquire resolving properties essential for healing, under such unfavorable conditions. In light of the spatial association between resolving (interleukin (IL)-10 producing) monocytes and the glial scar matrix chondroitin sulfate proteoglycan (CSPG), in this study we examined the mutual relationship between these two components. By inhibiting the de novo production of CSPG following spinal cord injury, we demonstrated that this extracellular matrix, mainly known for its ability to inhibit axonal growth, serves as a critical template skewing the entering monocytes towards the resolving phenotype. In vitro cell culture studies demonstrated that this matrix alone is sufficient to induce such monocyte polarization. Reciprocal conditional ablation of the monocyte-derived macrophages concentrated at the lesion margins, using diphtheria toxin, revealed that these cells have scar matrix-resolving properties. Replenishment of monocytic cell populations to the ablated mice demonstrated that this extracellular remodeling ability of the infiltrating monocytes requires their expression of the matrix-degrading enzyme, matrix metalloproteinase 13 (MMP-13), a property that was found here to be crucial for functional recovery. Altogether, this study demonstrates that the glial scar-matrix, a known obstacle to regeneration, is a critical component skewing the encountering monocytes towards a resolving phenotype. In an apparent feedback loop, monocytes were found to regulate scar resolution. This cross-regulation between the glial scar and monocytes primes the resolution of this interim phase of spinal cord repair, thereby providing a fundamental platform for the dynamic healing response.

P5 Pulmonary hypertension in a mouse model with reduced macrophage number (MacLow)

Introduction and ObjectivesPulmonary arterial hypertension (PAH) is a devastating condition with high morbidity and poor life expectancy. Pathologically PAH is characterised by the medial thickening of the small distal pulmonary...

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.

Macrophages are essential contributors to kidney injury in murine cryoglobulinemic membranoproliferative glomerulonephritis

Mice transgenic for thymic stromal lymphopoietin (TSLP), under regulation of the lymphocyte-specific promoter Lck, develop cryoglobulinemia and membranoproliferative glomerulonephritis (MPGN) similar to the disease in patients. To determine whether infiltrating macrophages, a hallmark of this disease, are deleterious or beneficial in the injury process, we developed Lck-TSLP transgenic mice expressing the human diphtheria toxin receptor (DTR) under control of the monocyte/macrophage-restricted CD11b promoter (Lck-TSLP;CD11b-DTR). Treatment with DT resulted in a marked reduction of monocytes/macrophages in the peritoneal cavity of both CD11b-DTR and Lck-TSLP;CD11b-DTR mice and marked reduction of macrophage infiltration in glomeruli of Lck-TSLP;CD11b-DTR mice. Lck-TSLP;CD11b-DTR mice, with or without toxin treatment, had similar levels of cryoglobulinemia and glomerular immunoglobulin deposition as Lck-TSLP mice. Lck-TSLP;CD11b-DTR mice, treated with toxin, had reduced mesangial matrix expansion, glomerular collagen IV accumulation, expression of the activation marker α-smooth muscle actin and transforming growth factor-β1 in mesangial cells, and proteinuria compared with control mice. Thus, macrophage ablation confers protection in this model and indicates a predominately deleterious role for macrophages in the progression of kidney injury in cryoglobulinemic MPGN.

FlnA-null megakaryocytes prematurely release large and fragile platelets that circulate poorly

AbstractFilamin A (FlnA) is a large cytoplasmic protein that crosslinks actin filaments and anchors membrane receptors and signaling intermediates. FlnAloxP PF4-Cre mice that lack FlnA in the megakaryocyte (MK) lineage have a severe macrothrombocytopenia because of accelerated platelet clearance. Macrophage ablation by injection of clodronate-encapsulated liposomes increases blood platelet counts in FlnAloxP PF4-Cre mice and reveals the desintegration of FlnA-null platelets into microvesicles, a process that occurs spontaneously during storage. FlnAloxP PF4-Cre bone marrows and spleens have a 2.5- to 5-fold increase in MK numbers, indicating increased thrombopoiesis in vivo. Analysis of platelet production in vitro reveals that FlnA-null MKs prematurely convert their cytoplasm into large CD61+ platelet-sized particles, reminiscent of the large platelets observed in vivo. FlnA stabilizes the platelet von Willebrand factor receptor, as surface expression of von Willebrand factor receptor components is normal on FlnA-null MKs but decreased on FlnA-null platelets. Further, FlnA-null platelets contain multiple GPIbα degradation products and have increased expression of the ADAM17 and MMP9 metalloproteinases. Together, the findings indicate that FlnA-null MKs prematurely release large and fragile platelets that are removed rapidly from the circulation by macrophages.

The Natural Cytotoxicity Receptor 1 Contribution to Early Clearance of Streptococcus pneumoniae and to Natural Killer-Macrophage Cross Talk

Natural killer (NK) cells serve as a crucial first line of defense against tumors, viral and bacterial infections. We studied the involvement of a principal activating natural killer cell receptor, natural cytotoxicity receptor 1 (NCR1), in the innate immune response to S. pneumoniae infection. Our results demonstrate that the presence of the NCR1 receptor is imperative for the early clearance of S. pneumoniae. We tied the ends in vivo by showing that deficiency in NCR1 resulted in reduced lung NK cell activation and lung IFNγ production at the early stages of S. pneumoniae infection. NCR1 did not mediate direct recognition of S. pneumoniae. Therefore, we studied the involvement of lung macrophages and dendritic cells (DC) as the mediators of NK-expressed NCR1 involvement in response to S. pneumoniae. In vitro, wild type BM-derived macrophages and DC expressed ligands to NCR1 and co-incubation of S. pneumoniae-infected macrophages/DC with NCR1-deficient NK cells resulted in significantly lesser IFNγ levels compared to NCR1-expressing NK cells. In vivo, ablation of lung macrophages and DC was detrimental to the early clearance of S. pneumoniae. NCR1-expressing mice had more potent alveolar macrophages as compared to NCR1-deficient mice. This result correlated with the higher fraction of NCR1-ligandhigh lung macrophages, in NCR1-expressing mice, that had better phagocytic activity compared to NCR1-liganddull macrophages. Overall, our results point to the essential contribution of NK-expressed NCR1 in early response to S. pneumoniae infection and to NCR1-mediated interaction of NK and S. pneumoniae infected-macrophages and -DC.

Stimulation of neoplastic mouse lung cell proliferation by alveolar macrophage-derived, insulin-like growth factor-1 can be blocked by inhibiting MEK and PI3K activation

BackgroundWorldwide, lung cancer kills more people than breast, colon and prostate cancer combined. Alterations in macrophage number and function during lung tumorigenesis suggest that these immune effector cells stimulate lung cancer growth. Evidence from cancer models in other tissues suggests that cancer cells actively recruit growth factor-producing macrophages through a reciprocal signaling pathway. While the levels of lung macrophages increase during tumor progression in mouse models of lung cancer, and high pulmonary macrophage content correlates with a poor prognosis in human non-small cell lung cancer, the specific role of alveolar macrophages in lung tumorigenesis is not clear.MethodsAfter culturing either an immortalized lung macrophage cell line or primary murine alveolar macrophages from naïve and lung-tumor bearing mice with primary tumor isolates and immortalized cell lines, the effects on epithelial proliferation and cellular kinase activation were determined. Insulin-like growth factor-1 (IGF-1) was quantified by ELISA, and macrophage conditioned media IGF-1 levels manipulated by IL-4 treatment, immuno-depletion and siRNA transfection.ResultsPrimary macrophages from both naïve and lung-tumor bearing mice stimulated epithelial cell proliferation. The lungs of tumor-bearing mice contained 3.5-times more IGF-1 than naïve littermates, and media conditioned by freshly isolated tumor-educated macrophages contained more IGF-1 than media conditioned by naïve macrophages; IL-4 stimulated IGF-1 production by both macrophage subsets. The ability of macrophage conditioned media to stimulate neoplastic proliferation correlated with media IGF-1 levels, and recombinant IGF-1 alone was sufficient to induce epithelial proliferation in all cell lines evaluated. Macrophage-conditioned media and IGF-1 stimulated lung tumor cell growth in an additive manner, while EGF had no effect. Macrophage-derived factors increased p-Erk1/2, p-Akt and cyclin D1 levels in neoplastic cells, and the combined inhibition of both MEK and PI3K ablated macrophage-mediated increases in epithelial growth.ConclusionsMacrophages produce IGF-1 which directly stimulates neoplastic proliferation through Erk and Akt activation. This observation suggests that combining macrophage ablation therapy with IGF-1R, MEK and/or PI3K inhibition could improve therapeutic response in human lung cancer. Exploring macrophage-based intervention could be a fruitful avenue for future research.

Tumor-Associated Macrophages Recruit CCR6+ Regulatory T Cells and Promote the Development of Colorectal Cancer via Enhancing CCL20 Production in Mice

BackgroundTumor-associated macrophages (TAMs) remodel the colorectal cancer (CRC) microenvironment. Yet, findings on the role of TAMs in CRC seem to be contradictory compared with other cancers. FoxP3+ regulatory T (Treg)-cells dominantly infiltrate CRC. However, the underlying molecular mechanism in which TAMs may contribute to the trafficking of Treg-cells to the tumor mass remains unknown.Methodology/Principal FindingsCRC was either induced by N-methyl-N-nitrosourea (MNU) and H. pylori or established by subcutaneous injection of mouse colorectal tumor cell line (CMT93) in mice. CMT93 cells were co-cultured with primary macrophages in a transwell apparatus. Recruitment of FoxP3 green fluorescence protein positive (FoxP3GFP+) Treg-cells was assessed using the IVIS Imaging System or immunofluorescence staining. A role for macrophages in trafficking of Treg-cells and in the development of CRC was investigated in CD11b diphtheria toxin receptor (CD11b-DTR) transgenic C57BL/6J mice in which macrophages can be selectively depleted. Treg-cells remarkably infiltrated solid tumor, and predominantly expressed the homing chemokine receptor (CCR) 6 in the induced CRC model. Both CMT93 cancer cells and macrophages produced a large amount of CCL20, the sole ligand of CCR6 in vitro and in vivo. Injection of recombinant mouse CCL20 into tumor sites promoted its development with a marked recruitment of Treg-cells in the graft CRC model. Conditional macrophage ablation decreased CCL20 levels, blocked Treg-cell recruitment and inhibited tumor growth in CD11b-DTR mice grafted with CMT93.Conclusions/SignificanceTAMs recruit CCR6+ Treg-cells to tumor mass and promote its development via enhancing the production of CCL20 in a CRC mouse model.

Abstract PL03-01: Macrophage diversity promotes tumor progression and metastasis

Abstract The tumor microenvironment represents a complex and changing ecology of many different cell types whose interactions determines the overall malignancy of the tumor. This environment consists of tumor cells harboring oncogenic and tumor suppressor mutations together with normal cells that include resident ones and those recruited from the bone marrow. In this latter class myeloid cells and especially macrophages, the subject of our studies, are particularly well represented. Associative clinical studies have indicated that the abundance of macrophages or overexpression of their signaling pathways is often an indicator of poor prognosis in a wide variety of cancers. These data suggests a pro-tumoral role for tumor-associated macrophages. This hypothesis that macrophages promoted malignancy is supported by a significant number of studies using genetic or pharmacological ablation of macrophages or their signaling pathways. In our studies, we used genetic ablation of macrophages in the model of breast cancer caused by the mammary epithelial expression of the Polyoma Middle T oncoprotein (PyMT). This ablation resulted in a slowing of tumor progression to malignancy and an inhibition of metastasis. We and our collaborators have identified several traits that macrophages confer in the primary tumor that account for their enhancement of malignancy. These are in the promotion of angiogenesis, stimulation of tumor cell invasion, migration and intravasation. At the metastatic site we have also shown that macrophages promote tumor cell extravasation and subsequent growth. Macrophage isolation and characterization from PyMT tumors and their metastatic sites suggest that each of these activities is regulated by a distinct subsets of macrophages that nevertheless express canonical macrophage markers. Each of these macrophage populations have defining cell surface markers and unique transcriptional profiles that suggest individual functions (Condeelis and Pollard, 2006; Qian and Pollard, 2010). Metastasis is the major cause of the failure of therapy and therefore death in cancer patients. Therefore we have focused our recent research on this process. We showed that macrophages are rapidly recruited to tumor cells as they metastasize to the lung. Ablation of these metastasis-associated macrophages (MAMs) using either genetic or pharmacological means results in an inhibition of tumor cell seeding and subsequent growth. Importantly ablation of these macrophages after the tumor cells have become established also inhibited subsequent metastatic growth suggesting that these cells represent therapeutic targets. Using ex vivo imaging methods we showed that a major action of these macrophages was on the promotion of tumor cell extravasation with subsequent effects on viability. This effect of macrophage on tumor cells could be recapitulated in an ex vivo extravasation assay thus enabling the interrogation of specific signaling molecules that may have function in promoting extravasation. To analyze the source of these MAMs, we performed adoptive transfers of the two different populations of monocytes that are defined by the presence or absence of the surface antigen Ly6C. Interestingly there was preferential recruitment of the Ly6C+ population to the metastatic sites whether in an experimental model of metastasis or into spontaneous metastases from PyMT tumors. Tracking of these monocytes showed that they rapidly differentiated into MAMs in the lung. A similar monocytic population (CD14+CD16-) circulating in human peripheral blood was recruited to human breast cancer metastases following adoptive transfer into immunocompromised mice. Furthermore, in mice we found that the Ly6C+ monocytes are preferentially recruited to bone metastases. In contrast to these data, Ly6C negative monocytes are recruited to the spontaneous tumors and to liver metastases there is a reduction in the recruitment of both populations of monocytes. Inhibition of the recruitment of these Ly6C+ monocytes inhibited tumor cell extravasation and thus metastases in bone and lung but not liver. These data suggest that alternative and tissue-specific mechanisms are operative at different metastatic sites. The dynamic acquisition of monocytic populations to primary and metastatic tumors and their differentiation into distinct macrophage populations represents the potential to target aspects of the tumor microenvironment. Such novel therapeutics in combination with conventional chemotherapeutic and/or radiation therapy may provide synergistic benefits to the patient. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr PL03-01. doi:10.1158/1538-7445.AM2011-PL03-01

Tumor-associated macrophages recruit CCR6+ regulatory T cells and promote the development of colorectal cancer via enhancing CCL20 production in mice (66.33)

Abstract Background: Tumor-associated macrophages (TAMs) remodel the colorectal cancer (CRC) microenvironment. Yet, findings on the role of TAMs in CRC seem to be contradictory compared with other cancers. Regulatory T (Treg)-cells dominantly infiltrate CRC. However, the underlying molecular mechanism in which TAMs may contribute to the trafficking of Treg-cells to the tumor mass remains unknown. Methodology/Principal Findings: CRC was either induced by MNU and H. pylori or established by subcutaneous injection of mouse colorectal tumor cell line (CMT93) in mice. Recruitment of FoxP3GFP+ Treg-cells was assessed using the IVIS Imaging System. A role for macrophages in trafficking of Treg-cells and in the development of CRC was investigated in CD11b diphtheria toxin receptor (CD11b-DTR) transgenic mice in which macrophages can be selectively depleted. Treg-cells remarkably infiltrated solid tumor, and predominantly expressed the homing chemokine receptor (CCR) 6 in the induced CRC model. Macrophages produced a large amount of CCL20, the sole ligand of CCR6 when co-cultured with CRC cells. Injection of recombinant CCL20 into tumor sites promoted its development with a marked recruitment of Treg-cells. Conditional macrophage ablation decreased CCL20 levels, blocked Treg-cell recruitment and inhibited tumor growth in CD11b-DTR mice. Conclusions/Significance: TAMs recruit CCR6+ Treg-cells to tumor mass and promote its development via enhancing the production of CCL20 in a CRC mouse model.

Conditional ablation of macrophages disrupts ovarian vasculature.

Macrophages are the most abundant immune cell within the ovary. Their dynamic distribution throughout the ovarian cycle and heterogenic array of functions suggest the involvement in various ovarian processes, but their functional role has yet to be fully established. The aim was to induce conditional macrophage ablation to elucidate the putative role of macrophages in maintaining the integrity of ovarian vasculature. Using the CD11b-diphtheria toxin receptor (DTR) mouse, in which expression of human DTR is under the control of the macrophage-specific promoter sequence CD11b, ovarian macrophages were specifically ablated in adult females by injections of diphtheria toxin (DT). CD11b-DTR mice were given DT treatment or vehicle and ovaries collected at 2, 8, 16, 24 and 48 h. Histochemical stains were employed to characterise morphological changes, immunohistochemistry for F4/80 to identify macrophages and the endothelial cell marker CD31 used to quantify vascular changes. In normal ovaries, macrophages were detected in corpora lutea and in the theca layer of healthy and atretic follicles. As macrophage ablation progressed, increasing amounts of ovarian haemorrhage were observed affecting both luteal and thecal tissue associated with significant endothelial cell depletion, increased erythrocyte accumulation and increased follicular atresia by 16 h. These events were followed by necrosis and profound structural damage. Changes were limited to the ovary, as DT treatment does not disrupt the vasculature of other tissues likely reflecting the unique cyclical nature of the ovarian vasculature and heterogeneity between macrophages within different tissues. These results show that macrophages play a critical role in maintaining ovarian vascular integrity.

Macrophage Activation Is Responsible for Loss of Anticontractile Function in Inflamed Perivascular Fat

The aim of this study was to determine whether macrophages dispersed throughout perivascular fat are crucial to the loss of anticontractile function when healthy adipose tissue becomes inflamed and to gain an understanding of the mechanisms involved. Pharmacological studies on in vitro small arterial segments from a mouse model of inducible macrophage ablation and on wild-type animals were carried out with and without perivascular fat using 2 physiological stimuli of inflammation: aldosterone and hypoxia. Both inflammatory insults caused a similar loss of anticontractile capacity of perivascular fat and increased macrophage activation. Aldosterone receptor antagonism and free radical scavengers were able to restore this capacity and reduce macrophage activation. However, in a mouse deficient of macrophages CD11b-diptheria toxin receptor (CD11b-DTR), there was no increase in contractility of arteries following aldosterone incubation or hypoxia. The presence and activation of macrophages in adipose tissue is the key modulator of the increase in contractility in arteries with perivascular fat following induction of inflammation. Despite multiple factors that may be involved in bringing about the vascular consequences of obesity, the ability of eplerenone to ameliorate the inflammatory effects of both aldosterone and hypoxia may be of potential therapeutic interest.

CD4+CD25+ regulatory T cells attenuate cisplatin-induced nephrotoxicity in mice

Nephrotoxicity limits the use of cisplatin, a widely used chemotherapeutic agent for treatment of various malignancies. Overall, CD4+ T cells mediate cisplatin-induced renal injury; however, the CD4+CD25+ regulatory T-cell subset (CD4+CD25+ Treg) has broad suppressive effects on many different cell types. In this study, we determined whether CD4+CD25+ Treg cells had protective effects against cisplatin-induced acute renal injury in nu/nu mice that lack mature T cells. In these mice, there was marked attenuation of the decreased survival, renal dysfunction and tubular injury, renal tumor necrosis factor-α, and interleukin-1β cytokine levels. Furthermore, renal macrophage accumulation was reduced in CD4+CD25+ Treg cell-adoptive transferred nu/nu mice compared with control mice. Infusion of CD4+CD25+Treg cells into wild-type Balb/c mice reduced serum blood urea nitrogen and creatinine levels equivalent to those in nu/nu mice and extended their survival time after cisplatin injection. In contrast, depletion of CD4+CD25+ Treg cells in wild-type mice exacerbated kidney injury after cisplatin administration. Transcription factor Foxp3-positive cells (Treg cells) were detected in the kidneys of nu/nu mice after cisplatin injection. Our results suggest that CD4+CD25+ Treg cells directly affect cisplatin nephrotoxicity and their modulation represents an additional treatment strategy.

Macrophages Are Essential for Maintenance of Corpus Luteum Function in Early Pregnancy.

Macrophages are abundant within the ovary, and have been identified in the corpus luteum (CL) of most species studied, including in rodents and human. Through their secretory products, macrophages are thought to be involved in ovarian tissue remodelling, including luteinization, and in regulating steroidogenesis. Macrophages co-cultured with granulosa or luteal cells act to stimulate progesterone secretion in vitro. To determine the impact of macrophage ablation during early pregnancy, we utilised CD11b-DTR transgenic mice to elicit transient systemic ablation of macrophages by administration of diphtheria toxin (DT). The effects of macrophage ablation during the pre-implantation phase of pregnancy were evaluated. Ablation of macrophages on day 1 pc or day 4 pc caused complete pregnancy loss in all DT-treated CD11b-DTR mice, while DT-treated wild-type mice or PBS-treated CD11b-DTR mice maintained viable pregnancies. Macrophage ablation on day 3 pc significantly reduced serum progesterone (P4) levels to 40% of control levels when measured 24 h later. Administration of exogenous P4 on each of day 4-7 pc prevented fetal loss in DT-treated CD11b-DTR mice, and pregnancy progressed with viable pups at late gestation, while no pregnancies remained viable in DT-treated mice administered vehicle only. Immunohistochemical evaluation with anti-CD31 mAb showed that macrophage ablation in the CD11b-DTR mice resulted in disappearance of endothelial cells from within the CL. This suggests that macrophages are essential for support of endothelial cells within the CL, either by provision of trophic support and/or by macrophage transdifferentiation into endothelial cells. In conclusion, these data indicate a critical role for macrophages in corpus luteum development and steroidogenic function in early pregnancy. Given that various nutritional and immune stressors can profoundly affect macrophage numbers and behaviour, it seems reasonable to speculate that macrophage-regulated CL development is a vulnerable event and may contribute to some forms of unexplained infertility. (platform)