Jingjing Tang

Targeted delivery of proapoptotic peptides to tumor-associated macrophages improves survival

Significance Tumor-associated macrophages (TAMs) are cells of our innate immune system that have been associated with poor prognosis in many types of cancers. When polarized toward the anti-inflammatory state, TAMs promote immune evasion and angiogenesis, thereby driving tumor growth. Using a peptide library selection strategy, we identified a sequence, called M2pep, that preferentially binds to anti-inflammatory murine macrophages. We then used M2pep to carry a proapoptotic peptide to TAMs by i.v. delivery and demonstrated that selective reduction of TAMs resulted in improved survival in tumor-bearing mice. These results suggest that a molecular-targeting approach for TAM depletion is a promising adjunct strategy to add to the arsenal of anticancer therapies. Most current cancer therapies focus on killing malignant cells, but these cells are often genetically unstable and can become resistant to chemotherapy. Tumor-associated macrophages (TAMs) facilitate disease progression by promoting angiogenesis and tumor cell growth, as well as by suppressing the adaptive immune response. TAMs are therefore potential targets for adjuvant anticancer therapies. However, resident macrophages are critical to host defense, and preferential ablation of TAMs remains challenging. Macrophage activation is broadly categorized as classically activated, or M1, and alternatively activated, or M2, and TAMs in the tumor microenvironment have been shown to adopt the anti-inflammatory, M2-like phenotype. To date, there are no methods for specific molecular targeting of TAMs. In this work, we report the discovery of a unique peptide sequence, M2pep, identified using a subtractive phage biopanning strategy against whole cells. The peptide preferentially binds to murine M2 cells, including TAMs, with low affinity for other leukocytes. Confocal imaging demonstrates the accumulation of M2pep in TAMs in vivo after tail vein injection. Finally, tail vein injection of an M2pep fusion peptide with a proapoptotic peptide delays mortality and selectively reduces the M2-like TAM population. This work therefore describes a molecularly targeted construct for murine TAMs and provides proof of concept of this approach as an anticancer treatment. In addition, M2pep is a useful tool for murine M2 macrophage identification and for modulating M2 macrophages in other murine models of disease involving M2 cells.

Blockade of Platelet-Derived Growth Factor or Its Receptors Transiently Delays but Does Not Prevent Fibrous Cap Formation in ApoE Null Mice

Platelet-derived growth factor (PDGF) is a potent stimulant of smooth muscle cell migration and proliferation in culture. To test the role of PDGF in the accumulation of smooth muscle cells in vivo, we evaluated ApoE -/- mice that develop complex lesions of atherosclerosis. Fetal liver cells from PDGF-B-deficient embryos were used to replace the circulating cells of lethally irradiated ApoE -/- mice. One month after transplant, all monocytes in PDGF-B -/- chimeras are of donor origin (lack PDGF), and no PDGF-BB is detected in circulating platelets, primary sources of PDGF in lesions. Although lesion volumes are comparable in the PDGF-B +/+ and -/- chimeras at 35 weeks, lesions in PDGF-B -/- chimeras contain mostly macrophages, appear less mature, and have a reduced frequency of fibrous cap formation as compared with PDGF-B +/+ chimeras. However, after 45 weeks, smooth muscle cell accumulation in fibrous caps is indistinguishable in the two groups. Comparison of elicited peritoneal macrophages by RNase protection assay shows an altered cytokine and cytokine receptor profile in PDGF-B -/- chimeras. ApoE -/- mice were also treated for up to 50 weeks with a PDGF receptor antagonist that blocks all three PDGF receptor dimers. Blockade of the PDGF receptors similarly delays, but does not prevent, accumulation of smooth muscle and fibrous cap formation. Thus, elimination of PDGF-B from circulating cells or blockade of PDGF receptors does not appear sufficient to prevent smooth muscle accumulation in advanced lesions of atherosclerosis.

The Absence of Platelet-Derived Growth Factor-B in Circulating Cells Promotes Immune and Inflammatory Responses in Atherosclerosis-Prone ApoE−/− Mice

Both innate and adaptive immunity contribute to the progression of inflammatory-fibrotic lesions of atherosclerosis. Although platelet-derived growth factor (PDGF)-B has been investigated as a stimulant of smooth muscle cells in vascular diseases, its effects on the immune response during disease have not been evaluated in vivo. We used hematopoietic chimeras generated after lethal irradiation of ApoE-/- recipients to test the role of PDGF in atherosclerosis. Monocyte accumulation in early atherosclerotic lesions increased 1.9-fold in ApoE-/-/PDGF-B-/- chimeras. Lymphocytes from null chimeras showed a 1.6- to 2.0-fold increase in the number of activated CD4(+) T cells and a 2.5-fold elevation of interferon-gamma-secreting CD4(+) T cells on ex vivo challenge with modified low-density lipoprotein. Splenocyte transcript levels were also altered with a twofold decrease in interleukin-10 and 1.7- and 3.0-fold increases in interleukin-18 and CCR 5, respectively. These cellular and molecular changes were consistent with a shift to a proinflammatory phenotype in null chimeras. Our data also demonstrated for the first time the presence of a recently discovered family of negative regulators of innate and adaptive immunity, the suppressors of cytokine signaling (SOCS), in developing atherosclerotic lesions. Thus, our studies identify two independent negative immune regulatory pathways-PDGF-B and SOCS-that may help limit lesion expansion.

Macrophage ADAM17 deficiency augments CD36-dependent apoptotic cell uptake and the linked anti-inflammatory phenotype

Rationale: Apoptotic cell phagocytosis (efferocytosis) is mediated by specific receptors and is essential for resolution of inflammation. In chronic inflammation, apoptotic cell clearance is dysfunctional and soluble levels of several apoptotic cell receptors are elevated. Reports have identified proteolytic cleavage as a mechanism capable of releasing soluble apoptotic cell receptors, but the functional implications of their proteolysis are unclear. Objective: To test the hypothesis that ADAM17-mediated cleavage of apoptotic cell receptors limits efferocytosis in vivo. Methods and Results: In vivo comparison of macrophage efferocytosis in wild-type and Adam17-null hematopoietic chimeras demonstrates that ADAM17 deficiency leads to a 60% increase in efferocytosis and an enhanced anti-inflammatory phenotype in a model of peritonitis. In vitro uptake of phosphatidylserine liposomes identifies the dual-pass apoptotic cell receptor CD36 as a major contributor to enhanced efferocytosis, and CD36 surface levels are elevated on macrophages from Adam17-null mice. Further, temporal elevation of CD36 expression with inflammation may also contribute to its impact. Soluble CD36 from macrophage-conditioned media comprises 2 species based on Western blotting, and mass spectrometry identifies 3 N-terminal peptides that represent probable cleavage sites. Levels of soluble CD36 are decreased in Adam17-null conditioned media, providing evidence for involvement of ADAM17 in CD36 cleavage. Importantly, enhanced efferocytosis in vivo by macrophages lacking ADAM17 is CD36 dependent and accelerates macrophage clearance from the peritoneum, thus promoting resolution of inflammation and highlighting the impact of increased apoptotic cell uptake. Conclusions: Our studies demonstrate the importance of ADAM17-mediated proteolysis for in vivo efferocytosis regulation and suggest a possible mechanistic link between chronic inflammation and defective efferocytosis.

Osteopontin mediates macrophage chemotaxis via α4 and α9 integrins and survival via the α4 integrin.

Osteopontin (OPN) is highly expressed by macrophages and plays a key role in the pathology of several chronic inflammatory diseases including atherosclerosis and the foreign body reaction. However, the molecular mechanism behind OPN regulation of macrophage functions is not well understood. OPN is a secreted molecule and interacts with several integrins via two domains: the RGD sequence binding to αv‐containing integrins, and the SLAYGLR sequence binding to α4β1, α4β7, and α9β1 integrins. Here we determined the role of OPN in macrophage survival, chemotaxis, and activation state. For survival studies, OPN treated‐bone marrow derived macrophages (BMDMs) were challenged with growth factor withdrawal and neutralizing integrin antibodies. We found that survival in BMDMs is mediated primarily through the α4 integrin. In chemotaxis studies, we observed that migration to OPN was blocked by neutralizing α4 and α9 integrin antibodies. Further, OPN did not affect macrophage activation as measured by IL‐12 production. Finally, the relative contributions of the RGD and the SLAYGLR functional domains of OPN to leukocyte recruitment were evaluated in an in vivo model. We generated chimeric mice expressing mutated forms of OPN in myeloid‐derived leukocytes, and found that the SLAYGLR functional domain of OPN, but not the RGD, mediates macrophage accumulation in response to thioglycollate‐elicited peritonitis. Collectively, these data indicate that α4 and α9 integrins interacting with OPN via the SLAYGLR domain play a key role in macrophage biology by regulating migration, survival, and accumulation. J. Cell. Biochem. 114: 1194–1202, 2013.

Adam17-dependent shedding limits early neutrophil influx, but does not alter early monocyte recruitment to inflammatory sites

TNF-α-converting enzyme (TACE, herein denoted as Adam17) proteolytically sheds several cell-surface inflammatory proteins, but the physiologic importance of the cleavage of these substrates from leukocyte subsets during inflammation is incompletely understood. In this study, we show that Adam17-null neutrophils have a 2-fold advantage in their initial recruitment during thioglycollate-induced peritonitis, and they roll slower and adhere more readily in the cremaster model than wild-type neutrophils. Although CD44 and ICAM-1 are both in vitro substrates of Adam17, their surface levels are not altered on Adam17-null neutrophils. In contrast, L-selectin levels are elevated up to 10-fold in Adam17-null circulating neutrophils, and their accelerated peritoneal influx, slower rolling, and increased adhesion in the cremaster muscle are dependent on L-selectin. Analysis of mixed chimeras shows that enhanced L-selectin levels and accelerated influx were both cell-intrinsic properties of neutrophils lacking Adam17. In contrast, Adam17-null monocytes display no acceleration of infiltration into the peritoneum in spite of elevated L-selectin surface levels, and their peritoneal influx was independent of L-selectin. Therefore, our data demonstrate substrate and myeloid cell-type specificity of Adam17-mediated cleavage of its substrates, and show that neutrophils and monocytes use distinct mechanisms for infiltration of tissues.

Testing the Role of Myeloid Cell Glucose Flux in Inflammation and Atherosclerosis

Inflammatory activation of myeloid cells is accompanied by increased glycolysis, which is required for the surge in cytokine production. Although in vitro studies suggest that increased macrophage glucose metabolism is sufficient for cytokine induction, the proinflammatory effects of increased myeloid cell glucose flux in vivo and the impact on atherosclerosis, a major complication of diabetes, are unknown. We therefore tested the hypothesis that increased glucose uptake in myeloid cells stimulates cytokine production and atherosclerosis. Overexpression of the glucose transporter GLUT1 in myeloid cells caused increased glycolysis and flux through the pentose phosphate pathway but did not induce cytokines. Moreover, myeloid-cell-specific overexpression of GLUT1 in LDL receptor-deficient mice was ineffective in promoting atherosclerosis. Thus, increased glucose flux is insufficient for inflammatory myeloid cell activation and atherogenesis. If glucose promotes atherosclerosis by increasing cellular glucose flux, myeloid cells do not appear to be the key targets.

Metalloproteinase-mediated Shedding of Integrin β2 Promotes Macrophage Efflux from Inflammatory Sites

Background: Efflux of macrophages limits inflammation. Results: Macrophage integrin αMβ2 is cleaved during exiting from inflammatory sites, released αMβ2 retains ligand binding capabilities, and inhibition of its metalloproteinase-mediated cleavage impairs macrophage efflux. Conclusion: Metalloproteinase-mediated proteolysis of integrin β2 promotes macrophage efflux from inflammatory sites. Significance: Regulated proteolysis of integrin β2 during inflammation demonstrates the potential of this mechanism to contribute to resolution of inflammation. Macrophage exiting from inflammatory sites is critical to limit the local innate immune response. With tissue insult, resident tissue macrophages rapidly efflux to lymph nodes where they modulate the adaptive immune response, and inflammatory macrophages attracted to the site of injury then exit during the resolution phase. However, the mechanisms that regulate macrophage efflux are poorly understood. This study has investigated soluble forms of integrin β2 whose levels are elevated in experimental peritonitis at times when macrophages are exiting the peritoneum, suggesting that its proteolytic shedding may be involved in macrophage efflux. Both constitutive and inducible metalloproteinase-dependent shedding of integrin β2 from mouse macrophages are demonstrated. Soluble integrin β2 is primarily released as a heterodimeric complex with αM that retains its ability to bind its ligands intracellular adhesion molecule-1, fibrin, and collagen and thus may serve as a soluble antagonist. In a model of accelerated exiting, administration of a metalloproteinase inhibitor prevents macrophage efflux by 50% and impedes loss of macrophage integrin β2 from the cell surface. Exiting of peritoneal macrophages in mice lacking integrin β2 is accelerated, and antibody disruption of integrin β2-substrate interactions can reverse 50% of the metalloprotease inhibitor blockade of macrophage exiting. Thus, our study demonstrates the ability of metalloproteinase-mediated shedding of integrin β2 to promote macrophage efflux from inflammatory sites, and the release of soluble integrin heterodimers may also limit local inflammation.

Hematopoietic Fas deficiency does not affect experimental atherosclerotic lesion formation despite inducing a proatherogenic state.

The Fas death receptor (CD95) is expressed on macrophages, smooth muscle cells, and T cells within atherosclerotic lesions. Given the dual roles of Fas in both apoptotic and nonapoptotic signaling, the aim of the present study was to test the effect of hematopoietic Fas deficiency on experimental atherosclerosis in low-density lipoprotein receptor-null mice (Ldlr(-/-)). Bone marrow from Fas(-/-) mice was used to reconstitute irradiated Ldlr(-/-) mice as a model for atherosclerosis. After 16 weeks on an 0.5% cholesterol diet, no differences were noted in brachiocephalic artery lesion size, cellularity, or vessel wall apoptosis. However, Ldlr(-/-) mice reconstituted with Fas(-/-) hematopoietic cells had elevated hyperlipidemia [80% increase, relative to wild-type (WT) controls; P < 0.001] and showed marked elevation of plasma levels of CXCL1/KC, CCL2/MCP-1, IL-6, IL-10, IL-12 subunit p70, and soluble Fas ligand (P < 0.01), as well as systemic microvascular inflammation. It was not possible to assess later stages of atherosclerosis because of increased mortality in Fas(-/-) bone marrow recipients. Our data indicate that hematopoietic Fas deficiency does not affect early atherosclerotic lesion development in Ldlr(-/-) mice.