Basophils balance healing after myocardial infarction via IL-4/IL-13

The inflammatory response after myocardial infarction (MI) is a precisely regulated process that greatly affects subsequent remodeling. Here, we show that basophil granulocytes infiltrated infarcted murine hearts, with a peak occurring between days 3 and 7. Antibody-mediated and genetic depletion of basophils deteriorated cardiac function and resulted in enhanced scar thinning after MI. Mechanistically, we found that basophil depletion was associated with a shift from reparative Ly6C^lo macrophages toward increased numbers of inflammatory Ly6C^hi monocytes in the infarcted myocardium. Restoration of basophils in basophil-deficient mice by adoptive transfer reversed this proinflammatory phenotype. Cellular alterations in the absence of basophils were accompanied by lower cardiac levels of IL-4 and IL-13, two major cytokines secreted by basophils. Mice with basophil-specific IL-4/IL-13 deficiency exhibited a similarly altered myeloid response with an increased fraction of Ly6C^hi monocytes and aggravated cardiac function after MI. In contrast, IL-4 induction in basophils via administration of the glycoprotein IPSE/α-1 led to improved post-MI healing. These results in mice were corroborated by the finding that initially low counts of blood basophils in patients with acute MI were associated with a worse cardiac outcome after 1 year, characterized by a larger scar size. In conclusion, we show that basophils promoted tissue repair after MI by increasing cardiac IL-4 and IL-13 levels.     

Myeloperoxidase-rich Ly-6C+ myeloid cells infiltrate allografts and contribute to an imaging signature of organ rejection in mice

Rates of graft rejection are high among recipients of heart transplants. The onset and progression of clinically significant heart transplant rejection are currently monitored by serial biopsy, but this approach is highly invasive and lacks sensitivity. Here, we have developed what we believe to be a new technique to measure organ rejection noninvasively that involves the exploration of tissue-infiltrating leukocytes as biomarker sources for diagnostic imaging. Specifically, we profiled the myeloid response in a murine model of heart transplantation with the aim of defining and validating an imaging signature of graft rejection. Ly-6C^hi monocytes, which promote inflammation, accumulated progressively in allografts but only transiently in isografts. Ly-6C^lo monocytes, which help resolve inflammation, did not accumulate, although they composed the majority of the few remaining monocytes in isografts. The persistence of Ly-6C^hi monocytes in allografts prompted us to screen for a Ly-6C^hi monocyte–associated imaging marker. Low-density array data revealed that Ly-6C^hi monocytes express 10-fold higher levels of myeloperoxidase (MPO) than Ly-6C^lo monocytes. Noninvasive magnetic resonance imaging of MPO with an MPO-activatable Gd-chelate revealed a spatially defined T1-weighted signal in rejected allografts but not in isografts or MPO-deficient allograft recipients. Flow cytometry, enzymography, and histology validated the approach by mapping MPO activity to Ly-6C^hi monocytes and neutrophils. Thus, MPO imaging represents a potential alternative to the current invasive clinical standard by which transplants are monitored.     

Healing and repair after myocardial infarction: the forgotten but resurgent basophil

The biphasic wound-healing response in the heart after myocardial infarction involves an initial inflammatory phase followed by a more prolonged period of inflammation resolution, tissue repair, and scar formation. Infiltrating proinflammatory Ly6C^hi monocytes and monocyte-derived macrophages are key drivers of the inflammatory phase and are also the source of the locally generated reparative macrophages that promote inflammation resolution. In this issue of the JCI, Sicklinger et al. from the Leuschner laboratory uncover a salutary role for cardiac basophils in this process. The authors demonstrated that basophils promote healing and proper scar formation and also limit late cardiac remodeling by augmenting reparative macrophages in the infarcted heart, in part via basophil-derived enhancement of cardiac IL-4 and IL-13 levels. These findings underscore the potentially disproportionate (relative to cell numbers) yet essential biological effects of immune cells of low abundance on cardiac repair and remodeling, related in part to amplification of downstream macrophage responses via secreted cytokines.     

A dynamic spectrum of monocytes arising from the in situ reprogramming of CCR2+ monocytes at a site of sterile injury

Monocytes are recruited from the blood to sites of inflammation, where they contribute to wound healing and tissue repair. There are at least two subsets of monocytes: classical or proinflammatory (CCR2^hiCX₃CR1^low) and nonclassical, patrolling, or alternative (CCR2^lowCX₃CR1^hi) monocytes. Using spinning-disk confocal intravital microscopy and mice with fluorescent reporters for each of these subsets, we were able to track the dynamic spectrum of monocytes that enter a site of sterile hepatic injury in vivo. We observed that the CCR2^hiCX₃CR1^low monocytes were recruited early and persisted for at least 48 h, forming a ringlike structure around the injured area. These monocytes transitioned, in situ, from CCR2^hiCx₃CR1^low to CX₃CR1^hiCCR2^low within the ringlike structure and then entered the injury site. This phenotypic conversion was essential for optimal repair. These results demonstrate a local, cytokine driven reprogramming of classic, proinflammatory monocytes into nonclassical or alternative monocytes to facilitate proper wound-healing.Effective responses to infection and injury have been directed by millions of years of evolutionary pressure. A response to sterile injury such as trauma has been optimized to include essential components of repair while excluding unwanted or disruptive influences. Understanding evolution-driven responses to sterile injury will be critical to understanding and modulating iatrogenic or lifestyle-driven inflammatory disease. The initial immune response to sterile traumatic injury, classically defined as tissue injury in the absence of infection, is most notably characterized by early neutrophil swarming into the site, a process that has been extensively studied and well documented, highlighting sequential phases of cellular recruitment (Chtanova et al., 2008; McDonald et al., 2010; Lämmermann et al., 2013).Less is known about monocyte recruitment. Monocytes had been thought to enter at a delayed time point although this view has been challenged (Auffray et al., 2007). Moreover, monocytes have been demonstrated to possess impressive plasticity and have been implicated not only as proinflammatory but also as reparative cells (Geissmann et al., 2003; Gautier et al., 2012; Gordon, 2012). Literature to date suggests that they are endogenously present as at least two distinct populations: (1) as a classical proinflammatory cell circulating in blood and expressing high levels of CCR2 and Ly6C and low levels of CX₃CR1 (CCR2^hiCX₃CR1^low) and (2) as a locally patrolling alternative nonclassical monocyte with high levels of CX₃CR1 and low or absent CCR2 (CX₃CR1^hiCCR2^low) and Ly6C (Geissmann et al., 2003; Sunderkötter et al., 2004). The CCR2^hiCX₃CR1^low monocytes are believed to be selectively recruited to inflamed tissues and produce high levels of inflammatory cytokines during infection or tissue damage and may become tissue macrophages (Palframan et al., 2001; Geissmann et al., 2003; Sunderkötter et al., 2004; Varga et al., 2013; Hilgendorf et al., 2014), whereas the distinct CX₃CR1^hiCCR2^low monocytes are thought to be primarily reparative, producing IL-10 in inflammatory milieus. They may become alternative macrophages (Arnold et al., 2007; Auffray et al., 2007).We used spinning-disk fluorescent confocal intravital microscopy (SD-IVM) to track neutrophils and the two subsets of monocytes in an effort to examine their dynamic behavior in a bona fide sterile tissue injury. We have identified that only CCR2^hiCX₃CR1^low monocytes are recruited to the injured liver followed by a cytokine-driven switch to CCR2^lowCX₃CR1^hi monocytes in the proximity of the lesion. This suggests local education and regulation of monocyte subtypes during the repair process.     

Immature monocytes acquire antigens from other cells in the bone marrow and present them to T cells after maturing in the periphery

Monocytes are circulating precursors for tissue macrophages and dendritic cells (DCs) but are not recognized to directly participate in antigen presentation. We developed techniques to label mouse monocyte subsets with particulate tracers in vivo. Gr-1^lo but not Gr-1^hi monocytes were stably labeled by intravenous injection of 0.5-μm microspheres. Gr-1^hi monocytes could be labeled when the microspheres were injected after systemic depletion of blood monocytes and spleen macrophages. In this condition, the phagocytic tracer was transferred to immature bone marrow monocytes by neutrophils and B cells that first carried the particles to the bone marrow. Moreover, antigens from B cells or proteins conjugated to the tracer particles were processed for presentation by monocytes and could induce T cell responses in the periphery. Cell-associated antigen taken up by bone marrow monocytes was retained intracellularly for presentation of the antigen days later when monocyte-derived DCs migrated to lymph nodes or in vitro after differentiation with granulocyte/macrophage colony-stimulating factor. These data reveal that immature monocytes unexpectedly sample antigen from the bone marrow environment and that they can present these antigens after they leave the bone marrow.     

An adventitial IL-6/MCP1 amplification loop accelerates macrophage-mediated vascular inflammation leading to aortic dissection in mice

Vascular inflammation contributes to cardiovascular diseases such as aortic aneurysm and dissection. However, the precise inflammatory pathways involved have not been clearly defined. We have shown here that subcutaneous infusion of Ang II, a vasopressor known to promote vascular inflammation, into older C57BL/6J mice induced aortic production of the proinflammatory cytokine IL-6 and the monocyte chemoattractant MCP-1. Production of these factors occurred predominantly in the tunica adventitia, along with macrophage recruitment, adventitial expansion, and development of thoracic and suprarenal aortic dissections. In contrast, a reduced incidence of dissections was observed after Ang II infusion into mice lacking either IL-6 or the MCP-1 receptor CCR2. Further analysis revealed that Ang II induced CCR2⁺CD14^hiCD11b^hiF4/80^– macrophage accumulation selectively in aortic dissections and not in aortas from Il6^–/– mice. Adoptive transfer of Ccr2^+/+ monocytes into Ccr2^–/– mice resulted in selective monocyte uptake into the ascending and suprarenal aorta in regions of enhanced ROS stress, with restoration of IL-6 secretion and increased incidence of dissection. In vitro, coculture of monocytes and aortic adventitial fibroblasts produced MCP-1– and IL-6–enriched conditioned medium that promoted differentiation of monocytes into macrophages, induced CD14 and CD11b upregulation, and induced MCP-1 and MMP-9 expression. These results suggest that leukocyte-fibroblast interactions in the aortic adventitia potentiate IL-6 production, inducing local monocyte recruitment and activation, thereby promoting MCP-1 secretion, vascular inflammation, ECM remodeling, and aortic destabilization.     

TREM-1 orchestrates angiotensin II–induced monocyte trafficking and promotes experimental abdominal aortic aneurysm

The triggering receptor expressed on myeloid cells 1 (TREM-1) drives inflammatory responses in several cardiovascular diseases but its role in abdominal aortic aneurysm (AAA) remains unknown. Our objective was to explore the role of TREM-1 in a mouse model of angiotensin II–induced (AngII–induced) AAA. TREM-1 expression was detected in mouse aortic aneurysm and colocalized with macrophages. Trem1 gene deletion (Apoe^–/–Trem1^–/–), as well as TREM-1 pharmacological blockade with LR-12 peptide, limited both AAA development and severity. Trem1 gene deletion attenuated the inflammatory response in the aorta, with a reduction of Il1b, Tnfa, Mmp2, and Mmp9 mRNA expression, and led to a decreased macrophage content due to a reduction of Ly6C^hi classical monocyte trafficking. Conversely, antibody-mediated TREM-1 stimulation exacerbated Ly6C^hi monocyte aorta infiltration after AngII infusion through CD62L upregulation and promoted proinflammatory signature in the aorta, resulting in worsening AAA severity. AngII infusion stimulated TREM-1 expression and activation on Ly6C^hi monocytes through AngII receptor type I (AT₁R). In human AAA, TREM-1 was detected and TREM1 mRNA expression correlated with SELL mRNA expression. Finally, circulating levels of sTREM-1 were increased in patients with AAA when compared with patients without AAA. In conclusion, TREM-1 is involved in AAA pathophysiology and may represent a promising therapeutic target in humans.     

GM-CSF regulates intimal cell proliferation in nascent atherosclerotic lesions

The contribution of intimal cell proliferation to the formation of early atherosclerotic lesions is poorly understood. We combined 5-bromo-2′-deoxyuridine pulse labeling with sensitive en face immunoconfocal microscopy analysis, and quantified intimal cell proliferation and Ly-6C^high monocyte recruitment in low density lipoprotein receptor–null mice. Cell proliferation begins in nascent lesions preferentially at their periphery, and proliferating cells accumulate in lesions over time. Although intimal cell proliferation increases in parallel to monocyte recruitment as lesions grow, proliferation continues when monocyte recruitment is inhibited. The majority of proliferating intimal cells are dendritic cells expressing CD11c and major histocompatibility complex class II and 33D1, but not CD11b. Systemic injection of granulocyte/macrophage colony-stimulating factor (GM-CSF) markedly increased cell proliferation in early lesions, whereas function-blocking anti–GM-CSF antibody inhibited proliferation. These findings establish GM-CSF as a key regulator of intimal cell proliferation in lesions, and demonstrate that both proliferation and monocyte recruitment contribute to the inception of atherosclerosis.In atherosclerosis, serum lipoproteins, particularly low density lipoprotein (LDL), accumulate in the arterial intima through binding to matrix proteoglycans (Skålén et al., 2002) and undergo oxidative modification. Blood monocytes are recruited into these regions of the intima, differentiate into macrophages or DCs, and internalize oxidatively modified lipoproteins via scavenger receptors or by macropinocytosis (for review see Moore and Freeman, 2006). BM-derived cells accumulate in early mouse atherosclerotic lesions (Mullick et al., 2008), and monocyte recruitment persists in advanced lesions (Swirski et al., 2006; Tacke et al., 2007).Circulating monocytes consist of at least two subpopulations, with distinct expression of cell-surface markers, chemokine receptors, and functions during inflammatory responses (Grage-Griebenow et al., 2001; Geissmann et al., 2003). In mice, the Ly-6C^high subset is elevated during hypercholesterolemia and is preferentially recruited to atherosclerotic lesions (Swirski et al., 2007; Tacke et al., 2007). Ly-6C^high monocytes express abundant L-selectin and multiple chemokine receptors, including CCR2, but relatively low levels of CX3CR1. Ly-6C^low monocytes express higher levels of CX3CR1, and low to nondetectable levels of most chemokine receptors, L-selectin and Ly-6C. Thus, it is intriguing that recruitment of Ly-6C^high monocytes to lesions is dependent on CCR2, CCR5, and CX3CR1, whereas recruitment of Ly-6C^low monocytes is independent of CX3CR1 and dependent on CCR5, which is up-regulated in hypercholesterolemic mice (Tacke et al., 2007).Fatty streaks are composed almost entirely of myeloid foam cells, and their formation and lateral expansion are critically dependent on monocytes/macrophages. This was demonstrated using osteopetrotic mice that have a point mutation that disrupts M-CSF and are deficient in circulating monocytes, tissue macrophages, and osteoclasts. Osteopetrotic mice are highly protected from atherosclerosis in the setting of hypercholesterolemia (Smith et al., 1995; Qiao et al., 1997), and these studies implicate monocytes and their progeny as key cells in lesion development. In contrast to M-CSF, deficiency of GM-CSF has no significant effects on circulating monocytes or tissue macrophages, and variable effects on mouse atherosclerotic lesion formation (Ditiatkovski et al., 2006; Shaposhnik et al., 2007). An interesting feature in the setting of GM-CSF deficiency is a decrease of CD11c⁺ cells in lesions (Shaposhnik et al., 2007).The biology of macrophages and DCs in atherosclerotic lesions is complex. These cells can proliferate or undergo apoptosis, and both processes have been observed primarily in advanced human and experimental lesions (Rosenfeld and Ross, 1990; Orekhov et al., 1998; Merched et al., 2003) and in a vascular occlusion model in hypercholesterolemic apolipoprotein E–deficient (Apoe^−/−) mice (Lessner et al., 2002). The prevailing opinion is that monocyte recruitment accounts for the formation of early lesions, and the relationship of proliferation to monocyte recruitment is not understood. A key impediment has been the lack of a sensitive approach to study cell proliferation and monocyte recruitment in early lesions. We used BrdU pulse labeling combined with en face confocal microscopy of the mouse aorta to detect cell proliferation and Ly-6C^high monocyte recruitment, and localize them with respect to lipid accumulation in the intima. We found that intimal cell proliferation begins in nascent lesions preferentially at the periphery, increases as lesions progress in parallel to monocyte recruitment, primarily involves DCs, and is dependent on GM-CSF but not monocyte recruitment.     

Monocyte subsets differentially employ CCR2, CCR5, and CX3CR1 to accumulate within atherosclerotic plaques

Monocytes participate critically in atherosclerosis. There are 2 major subsets expressing different chemokine receptor patterns: CCR2(+)CX3CR1(+)Ly-6C(hi) and CCR2(-)CX3CR1(++)Ly-6C(lo) monocytes. Both C-C motif chemokine receptor 2 (CCR2) and C-X(3)-C motif chemokine receptor 1 (CX3CR1) are linked to progression of atherosclerotic plaques. Here, we analyzed mouse monocyte subsets in apoE-deficient mice and traced their differentiation and chemokine receptor usage as they accumulated within atherosclerotic plaques. Blood monocyte counts were elevated in apoE(-/-) mice and skewed toward an increased frequency of CCR2(+)Ly-6C(hi) monocytes in apoE(-/-) mice fed a high-fat diet. CCR2(+)Ly-6C(hi) monocytes efficiently accumulated in plaques, whereas CCR2(-)Ly-6C(lo) monocytes entered less frequently but were more prone to developing into plaque cells expressing the dendritic cell-associated marker CD11c, indicating that phagocyte heterogeneity in plaques is linked to distinct types of entering monocytes. CCR2(-) monocytes did not rely on CX3CR1 to enter plaques. Instead, they were partially dependent upon CCR5, which they selectively upregulated in apoE(-/-) mice. By comparison, CCR2(+)Ly-6C(hi) monocytes unexpectedly required CX3CR1 in addition to CCR2 and CCR5 to accumulate within plaques. In many other inflammatory settings, these monocytes utilize CCR2, but not CX3CR1, for trafficking. Thus, antagonizing CX3CR1 may be effective therapeutically in ameliorating CCR2(+) monocyte recruitment to plaques without impairing their CCR2-dependent responses to inflammation overall.     

Inflammatory arthritis increases mouse osteoclast precursors with myeloid suppressor function

Increased osteoclastic bone resorption leads to periarticular erosions and systemic osteoporosis in RA patients. Although a great deal is known about how osteoclasts differentiate from precursors and resorb bone, the identity of an osteoclast precursor (OCP) population in vivo and its regulatory role in RA remains elusive. Here, we report the identification of a CD11b^–/loLy6C^hi BM population with OCP activity in vitro and in vivo. These cells, which can be distinguished from previously characterized precursors in the myeloid lineage, display features of both M1 and M2 monocytes and expand in inflammatory arthritis models. Surprisingly, in one mouse model of RA (adoptive transfer of SKG arthritis), cotransfer of OCP with SKG CD4⁺ T cells diminished inflammatory arthritis. Similar to monocytic myeloid-derived suppressor cells (M-MDSCs), OCPs suppressed CD4⁺ and CD8⁺ T cell proliferation in vitro through the production of NO. This study identifies a BM myeloid precursor population with osteoclastic and T cell–suppressive activity that is expanded in inflammatory arthritis. Therapeutic strategies that prevent the development of OCPs into mature bone-resorbing cells could simultaneously prevent bone resorption and generate an antiinflammatory milieu in the RA joint.     

ATP11B inhibits breast cancer metastasis in a mouse model by suppressing externalization of nonapoptotic phosphatidylserine

Cancer metastasis is the cause of the majority of cancer-related deaths. In this study, we demonstrated that no expression or low expression of ATP11B in conjunction with high expression of PTDSS2, which was negatively regulated by BRCA1, markedly accelerates tumor metastasis. Further analysis revealed that cells with low ATP11B expression and high PTDSS2 expression (ATP11B^loPTDSS2^hi cells) were associated with poor prognosis and enhanced metastasis in breast cancer patients in general. Mechanistically, an ATP11B^loPTDSS2^hi phenotype was associated with increased levels of nonapoptotic phosphatidylserine (PS) on the outer leaflet of the cell membrane. This PS increase serves as a global immunosuppressive signal to promote breast cancer metastasis through an enriched tumor microenvironment with the accumulation of myeloid-derived suppressor cells and reduced activity of cytotoxic T cells. The metastatic processes associated with ATP11B^loPTDSS2^hi cancer cells can be effectively overcome by changing the expression phenotype to ATP11B^hiPTDSS2^lo through a combination of anti-PS antibody with either paclitaxel or docetaxel. Thus, blocking the ATP11B^loPTDSS2^hi axis provides a new selective therapeutic strategy to prevent metastasis in breast cancer patients.     

Ly-6Chi monocytes dominate hypercholesterolemia-associated monocytosis and give rise to macrophages in atheromata

Macrophage accumulation participates decisively in the development and exacerbation of atherosclerosis. Circulating monocytes, the precursors of macrophages, display heterogeneity in mice and humans, but their relative contribution to atherogenesis remains unknown. We report here that the Ly-6C(hi) monocyte subset increased dramatically in hypercholesterolemic apoE-deficient mice consuming a high-fat diet, with the number of Ly-6C(hi) cells doubling in the blood every month. Ly-6C(hi) monocytes adhered to activated endothelium, infiltrated lesions, and became lesional macrophages. Hypercholesterolemia-associated monocytosis (HAM) developed from increased survival, continued cell proliferation, and impaired Ly-6C(hi) to Ly-6C(lo) conversion and subsided upon statin-induced cholesterol reduction. Conversely, the number of Ly-6C(lo) cells remained unaffected. Thus, we believe that Ly-6C(hi) monocytes represent a newly recognized component of the inflammatory response in experimental atherosclerosis.     

Heterogeneity Among Ly-49C Natural Killer (NK) Cells: Characterization of Highly Related Receptors with Differing Functions and Expression Patterns

Ly-49C is a member of the polymorphic family of murine NK cell inhibitory receptors. The 5E6 antibody that defines a subset of NK cells responsible for the rejection of parental H-2^d bone marrow by F₁ mice has been shown previously to react with Ly-49C. Here, the 5E6 antibody was found to detect two Ly-49C-related molecules in B6 mice. Two cDNA clones were isolated from B6 NK cells, one identical to previously reported Ly-49C^B6 and the other a novel cDNA. The deduced amino acid sequence of the latter differs from that of Ly-49C^BALB at only 4 residues, whereas the previously reported Ly-49C^B6 differs at 22 residues. Flow cytometric analyses of COS cells transfected with the two cDNAs showed that the 5E6 antibody binds to both Ly-49 molecules, while another anti-Ly-49C antibody, 4LO3311, binds to the newly described Ly-49C but not the previously reported Ly-49C^B6. Two-color flow cytometric analysis detected 5E6⁺4LO3311⁻ as well as 5E6⁺4LO3311⁺ subsets of NK cells from B6, but not BALB/c, mice. The level of Ly-49C expression on B6 NK cells detected by the 4LO3311 antibody was substantially lower than that on BALB/c NK cells. Binding specificity of the novel Ly-49C^B6 was indistinguishable from that of Ly-49C^BALB, whereas no binding was detectable with previously reported Ly-49C^B6. These results demonstrate that the newly described Ly-49C^B6, not the previously reported Ly-49C^B6, is the probable B6 allelic form of Ly49C. The previously reported Ly-49C^B6 must be encoded by a separate gene and should be renamed Ly-49I. The implication of these results with respect to the role of Ly-49C in hybrid resistance is discussed.     

On-site education of VEGF-recruited monocytes improves their performance as angiogenic and arteriogenic accessory cells

Adult neovascularization relies on the recruitment of monocytes to the target organ or tumor and functioning therein as a paracrine accessory. The exact origins of the recruited monocytes and the mechanisms underlying their plasticity remain unclear. Using a VEGF-based transgenic system in which genetically tagged monocytes are conditionally summoned to the liver as part of a VEGF-initiated angiogenic program, we show that these recruited cells are derived from the abundant pool of circulating Ly6C^hi monocytes. Remarkably, however, upon arrival at the VEGF-induced organ, but not the naive organ, monocytes undergo multiple phenotypic and functional changes, endowing them with enhanced proangiogenic capabilities and, importantly, with a markedly increased capacity to remodel existing small vessels into larger conduits. Notably, monocytes do not differentiate into long-lived macrophages, but rather appear as transient accessory cells. Results from transfers of presorted subpopulations and a novel tandem transfer strategy ruled out selective recruitment of a dedicated preexisting subpopulation or onsite selection, thereby reinforcing active reprogramming as the underlying mechanism for improved performance. Collectively, this study uncovered a novel function of VEGF, namely, on-site education of recruited “standard” monocytes to become angiogenic and arteriogenic professional cells, a finding that may also lend itself for a better design of angiogenic therapies.Unlike developmental neovascularization that makes extensive use of resident mesenchymal progenitor cells (angioblasts), adult neovascularization typically takes place via sprouting angiogenesis, as the pool of tissue angioblasts has been exhausted by adulthood. Likewise, the relative contribution of bone marrow–derived endothelial progenitor cells (EPCs) to the neovasculature is still debated and may vary in different experimental settings (Ruzinova et al., 2003; Peters et al., 2005; Purhonen et al., 2008) Adult angiogenesis is mediated by locally induced angiogenic factors, primarily VEGF, but is also assisted by complementary activities by myeloid cells, recruited to the angiogenic site in a process known as cell-assisted angiogenesis (Lin et al., 2001; De Palma et al., 2003; Grunewald et al., 2006; Jin et al., 2006). In a tumor milieu, for example, the essential proangiogenic role played by recruited monocytes and intratumoral macrophages has been demonstrated using different approaches to prevent their recruitment, as well as by their on-site ablation (De Palma et al., 2003; De Palma et al., 2005; Lin et al., 2006). Refractoriness of the tumor vasculature to VEGF inhibition has been attributed to compensatory factors provided by recruited myeloid cells (Shojaei et al., 2007, 2009).Most, if not all angiogenic responses in the adult are promoted by VEGF, which is either induced as a result of stochastic genetic changes in tumors or by environmental cues, primarily hypoxia. The incidental nature of VEGF induction has prompted the proposition that recruitment of accessory cells might be required for efficient VEGF-initiated neovascularization. Indeed, we have previously shown that an ongoing VEGF stimulus is required for perivascular positioning of incoming monocytes and for their retention in this strategic location from which they can exert a paracrine accessory role (Grunewald et al., 2006).Extravasated monocytes were shown to participate in the process of arteriogenesis, i.e., remodeling of existing small vessels into larger vessels via promotion of in-wall proliferation of endothelial cells, in the context of the compensatory response to vessel occlusion (collateralization) (Cai and Schaper, 2008). However, the proposition that monocytes summoned by VEGF foster the generation of large conduits as an indispensable part of a natural, VEGF-promoted adult neovascularization requires further study.Monocytes represent a most versatile and dynamic cell population composed of subsets that differ in phenotype, size, and morphology and gene expression profiles (Geissmann et al., 2003; Ingersoll et al., 2010). Originating in the bone marrow and released to the peripheral circulation as short-lived, nondividing cells, monocytes are believed to serve as a transient reservoir of myeloid precursors that can be summoned to sites of injury to aid in the maintenance of tissue homeostasis. Two discrete subsets of blood monocytes have been identified, distinguishable in the mouse by their membrane expression of Ly6C and the chemokine receptor CCR2 (Geissmann et al., 2003; Palframan et al., 2001). Adoptive transfers of Ly6C^lo and Ly6C^hi blood monocytes established that the two subsets differ with respect to their respective fate and function in the periphery (Geissmann et al., 2003; Landsman and Jung, 2007; Varol et al., 2009). Ly6C^hi monocytes are efficiently recruited to sites of inflammation, but in absence of the latter give rise to Ly6C^lo cells (Varol et al., 2007). Ly6C^lo monocytes have been proposed to be involved in tissue remodeling, e.g., after myocardial infarction, and to replenish resident macrophage populations (Arnold et al., 2007; Landsman and Jung, 2007; Nahrendorf et al., 2007).Given the heterogeneity of monocytes and their descendants, the question arises whether proangiogenic and proarteriogenic monocytes recruited by VEGF represent a preexisting subpopulation committed to perform these specialized functions in the periphery. Alternatively, acquisition of these properties could reflect post-recruitment education governed by cues prevailing in the particular tissue milieu. In the case of pro-angiogenic Tie2-expressing monocytes (TEMs), for example, it has been suggested on the basis of gene expression signature kinships that they represent a monocyte subset committed to perform a distinct, pro-angiogenic extravascular function in the tumor microenvironment (Pucci et al., 2009).The notion of macrophage education has been proposed to explain the multiple, often opposing functions of tumor-associated macrophages (TAMs; Qian and Pollard, 2010). Thus, polarized expression of either pro- or antitumor functions has been argued to result from inherent macrophage plasticity and to be determined by the particular microenvironmental signals the cells are exposed to, culminating in selective tuning of TAM functions within a spectrum encompassing the M1 and M2 extremes (Sica et al., 2008). Yet, given the complexity of the tumor microenvironment, it has been difficult to exclude alternative mechanisms generating functional heterogeneity, including preexisting minor populations, selective and/or sequential recruitment or on-site variant selection. Moreover, it remained possible that extravasated monocytes do not necessarily need to differentiate into macrophages but might fulfill these specialized functions in the periphery as short-lived, reprogrammed monocytes.To distinguish between different mechanisms responsible for generating angiogenic and arteriogenic monocytes, we took advantage of a unique transgenic system that allows conditional summoning of genetically tagged monocytes by VEGF to a target organ of choice and subsequently retrieving them for analysis of changes in gene expression and functional performance.     

Cardiac fibroblasts mediate IL-17A–driven inflammatory dilated cardiomyopathy

Inflammatory dilated cardiomyopathy (DCMi) is a major cause of heart failure in individuals below the age of 40. We recently reported that IL-17A is required for the development of DCMi. We show a novel pathway connecting IL-17A, cardiac fibroblasts (CFs), GM-CSF, and heart-infiltrating myeloid cells with the pathogenesis of DCMi. Il17ra^−/− mice were protected from DCMi, and this was associated with significantly diminished neutrophil and Ly6Chi monocyte/macrophage (MO/MΦ) cardiac infiltrates. Depletion of Ly6Chi MO/MΦ also protected mice from DCMi. Mechanistically, IL-17A stimulated CFs to produce key chemokines and cytokines that are critical downstream effectors in the recruitment and differentiation of myeloid cells. Moreover, IL-17A directs Ly6Chi MO/MΦ in trans toward a more proinflammatory phenotype via CF-derived GM-CSF. Collectively, this IL-17A–fibroblast–GM-CSF–MO/MΦ axis could provide a novel target for the treatment of DCMi and related inflammatory cardiac diseases.Inflammatory dilated cardiomyopathy (DCMi) is among the most common causes of noncongenital heart failure in individuals under the age of 40 (Dimas et al., 2009). There has been only limited success with symptomatic therapy in chronic DCMi patients, leaving cardiac transplantation the only cure for end stage heart failure secondary to DCMi (Pietra et al., 2012). Autoimmunity to heart tissue is often involved in the pathogenesis of DCMi (Čiháková and Rose, 2008; Cooper, 2009). In an effort to investigate the immunopathologic mechanism responsible for DCMi in humans, we have adopted a mouse model of experimental autoimmune myocarditis (EAM). EAM is induced by immunization of genetically susceptible BALB/c mice with a peptide derived from the cardiac myosin heavy chain α (MyHCα_614-629). Immunized mice develop myocarditis characterized by inflammatory infiltration peaking about day 21, and subsequently progress to DCMi around day 40 to day 70, characterized by cardiac fibrosis and impairment of cardiac function (Čiháková et al., 2004).EAM is a CD4⁺ T helper cell–dependent disease (Smith and Allen, 1991, 1993). One of the CD4⁺ T helper cell subsets, Th17 cells, has been observed to infiltrate the heart during EAM (Baldeviano et al., 2010), and has been reported to be critical in autoimmunity (Korn et al., 2009). Furthermore, patients with DCMi have increased numbers of Th17 cells in their blood and an elevated level of Th17 cytokines in serum, suggesting that Th17 cells are involved in the pathogenesis of DCMi (Ding et al., 2010; Yuan et al., 2010). When we examined whether the hallmark Th17 cytokine, IL-17A, drives the pathogenesis of myocarditis, we discovered that Il17a^−/− mice were completely protected from the development of DCMi, although they had myocardial inflammation comparable in overall severity to WT controls (Baldeviano et al., 2010). Thus, IL-17A is dispensable for early stage myocarditis but required for the progression to DCMi. These results indicated a critical role of IL-17A in driving cardiac damage and fibrosis during the development of DCMi. Similar profibrotic functions of IL-17A have been reported in cirrhosis (Lan et al., 2009) and fibrotic lung injury (Wilson et al., 2010) models.Monocytes (MOs) and macrophages (MΦs) are key effector cells during inflammatory processes (Gordon and Taylor, 2005) including myocarditis and DCMi. MO/MΦs comprise about half of all heart-infiltrating inflammatory cells at the peak of EAM and play important roles in the pathogenesis (Čiháková et al., 2008; Barin et al., 2012). Monocytes arise from hematopoietic stem cells and form distinct subpopulations. In mouse, the two monocyte subsets, CCR2^hiCX3CR1^loLy6C^hi and CCR2^loCX3CR1^hiLy6C^lo monocytes, infiltrate sites of inflammation responding to different chemokine signals and differentiate into inflammatory MΦs guided by local cytokine signals (Gordon and Taylor, 2005; Shi and Pamer, 2011). The balance between MO/MΦ subsets and their differentiation is critical in determining the pathogenic outcome in immune responses (Wynn et al., 2013). In this paper, while examining the pathogenic mechanisms of IL-17A–dependent DCMi, we describe a novel immunological pathway connecting IL-17A with MO/MΦs that drives DCMi development.     

Peripheral monocyte–derived cells counter amyloid plaque pathogenesis in a mouse model of Alzheimer’s disease

Microglia, the parenchymal tissue macrophages in the brain, surround amyloid plaques in brains of individuals with Alzheimer’s disease (AD) but are ineffective at clearing amyloid to mitigate disease progression. Recent studies in mice indicate that microglia are derived exclusively from primitive yolk sac hematopoiesis and self-renew without contribution from ontogenically distinct monocytes/macrophages of definitive adult hematopoietic origin. Using a genetic fate-mapping approach to label cells of definitive hematopoietic origin throughout life span, we discovered that circulating monocytes contribute 6% of plaque-associated macrophages in aged AD mice. Moreover, peripheral monocytes contributed to a higher fraction of macrophages in the choroid plexus, meninges, and perivascular spaces of aged AD mice versus WT control mice, indicating enrichment at potential sites for entry into the brain parenchyma. Splenectomy, which markedly reduced circulating Ly6C^hi monocytes, also reduced abundance of plaque-associated macrophages of definitive hematopoietic origin, resulting in increased amyloid plaque load. Together, these results indicate that peripherally derived monocytes invade the brain parenchyma, targeting amyloid plaques to reduce plaque load.     

Myeloperoxidase and plasminogen activator inhibitor 1 play a central role in ventricular remodeling after myocardial infarction

Left ventricular (LV) remodeling after myocardial infarction (MI) results in LV dilation, a major cause of congestive heart failure and sudden cardiac death. Ischemic injury and the ensuing inflammatory response participate in LV remodeling, leading to myocardial rupture and LV dilation. Myeloperoxidase (MPO), which accumulates in the infarct zone, is released from neutrophils and monocytes leading to the formation of reactive chlorinating species capable of oxidizing proteins and altering biological function. We studied acute myocardial infarction (AMI) in a chronic coronary artery ligation model in MPO null mice (MPO(-/-)). MPO(-/-) demonstrated decreased leukocyte infiltration, significant reduction in LV dilation, and marked preservation of LV function. The mechanism appears to be due to decreased oxidative inactivation of plasminogen activator inhibitor 1 (PAI-1) in the MPO(-/-), leading to decreased tissue plasmin activity. MPO and PAI-1 are shown to have a critical role in the LV response immediately after MI, as demonstrated by markedly delayed myocardial rupture in the MPO(-/-) and accelerated rupture in the PAI-1(-/-). These data offer a mechanistic link between inflammation and LV remodeling by demonstrating a heretofore unrecognized role for MPO and PAI-1 in orchestrating the myocardial response to AMI.     

Tissue Inhibitor of Metalloproteinase‐2 Gene Delivery Ameliorates Postinfarction Cardiac Remodeling

Hypothesis: Adenoviral‐mediated (AdV‐T2) overexpression of TIMP‐2 would blunt ventricular remodeling and improve survival in a murine model of chronic ischemic injury. Methods: Male mice (n = 124) aged 10–14 weeks underwent either (1) left coronary artery ligation to induce myocardial infarction (MI group, n = 36), (2) myocardial injection of 6 × 10¹⁰ viral particles of AdV‐T2 immediately post‐MI (MI + T2 group, n = 30), (3) myocardial injection of 6 × 10¹⁰ viral particles of a control adenovirus (MI + Ct, n = 38), or 4) received no intervention (controls, n = 20). On post‐MI day 7, surviving mice (n = 79) underwent echocardiographic, immunohistochemical, and biochemical analysis. Results: In infarcted animals, the MI + T2 group demonstrated improved survival (p < 0.02), better preservation of developed pressure and ventricular diameter (p < 0.04), and the lowest expression and activity of MMP‐2 and MMP‐9 (p < 0.04) compared with MI and MI + Ct groups. All infarcted hearts displayed significantly increased inflammatory cell infiltration (p < 0.04 vs. control, MI, or MI + T2), with infiltration highest in the MI + Ct group and lowest in the MI + T2 group (p < 0.04). Conclusions: Adenoviral mediated myocardial delivery of the TIMP‐2 gene improves post‐MI survival and limits adverse remodeling in a murine model of MI. Clin Trans Sci 2011; Volume 4: 24–31