Jill Suttles

Macrophages Sequentially Change Their Functional Phenotype in Response to Changes in Microenvironmental Influences

Recent studies have described the development of distinct functional subsets of macrophages in association with cancer, autoimmune disease, and chronic infections. Based on the ability of Th1 vs Th2 cytokines to promote opposing activities in macrophages, it has been proposed that macrophages develop into either type 1 inflammatory or type 2 anti-inflammatory subsets. As an alternative to the concept of subset development, we propose that macrophages, in response to changes in their tissue environment, can reversibly and progressively change the pattern of functions that they express. As demonstrated herein, macrophages can reversibly shift their functional phenotype through a multitude of patterns in response to changes in cytokine environment. Macrophages display distinct functional patterns after treatment with IFN-γ, IL-12, IL-4, or IL-10 and additional functional patterns are displayed depending on whether the cytokine is present alone or with other cytokines and whether the cytokines are added before or concomitantly with the activating stimulus (LPS). Sequential treatment of macrophages with multiple cytokines results in a progression through multiple functional phenotypes. This ability to adapt to changing cytokine environments has significant in vivo relevance, as evidenced by the demonstration that macrophage functional phenotypes established in vivo in aged or tumor-bearing mice can be altered by changing their microenvironment. A concept of functional adaptivity is proposed that has important implications for therapeutic targeting of macrophages in chronic diseases that result in the dominance of particular functional phenotypes of macrophages that play a significant role in disease pathology.

Lack of macrophage fatty-acid-binding protein aP2 protects mice deficient in apolipoprotein E against atherosclerosis

The adipocyte fatty-acid–binding protein, aP2, has an important role in regulating systemic insulin resistance and lipid metabolism. Here we demonstrate that aP2 is also expressed in macrophages, has a significant role in their biological responses and contributes to the development of atherosclerosis. Apolipoprotein E (ApoE)-deficient mice also deficient for aP2 showed protection from atherosclerosis in the absence of significant differences in serum lipids or insulin sensitivity. aP2-deficient macrophages showed alterations in inflammatory cytokine production and a reduced ability to accumulate cholesterol esters when exposed to modified lipoproteins. Apoe−/− mice with Ap2+/+ adipocytes and Ap2−/− macrophages generated by bone-marrow transplantation showed a comparable reduction in atherosclerotic lesions to those with total aP2 deficiency, indicating an independent role for macrophage aP2 in atherogenesis. Through its distinct actions in adipocytes and macrophages, aP2 provides a link between features of the metabolic syndrome and could be a new therapeutic target for the prevention of atherosclerosis.

Functional plasticity of macrophages: reversible adaptation to changing microenvironments

There has been substantial research activity in the past decade directed at phenotyping macrophage lineages and defining macrophage functional subsets or patterns of activity. The emphasis over the past 2–3 years has been to divide macrophage functional patterns into type 1 (Th1‐driven) or type 2 (Th2‐driven) functions. However, a huge array of environmental factors (including cytokines, chemokines, pattern recognition receptors, hormones) differentially regulates macrophage response patterns, resulting in the display of numerous distinct, functional phenotypes. Upon stimulation, a macrophage does not display just a single set of functions but rather displays a progression of functional changes in response to the progressive changes in its microenvironment. The remarkable ability of monocytes and tissue macrophages to adapt to changes in their microenvironment challenges the thesis that macrophages displaying unique tissue‐specific or response‐specific, functional patterns represent distinct lineages. With the exception of mature osteoclasts and mature dendritic cells, evidence supporting stable differentiation as the basis for macrophage functionl heterogeneity is equivocal. The concept of whether macrophages develop into functional subsets as opposed to continuously adapting their functional pattern in response to the changing environment of a progressive inflammatory response is important to resolve from the perspectives of therapeutic targeting and understanding the role of macrophages in disease pathogenesis.

Adenosine 5′-Monophosphate-Activated Protein Kinase Promotes Macrophage Polarization to an Anti-Inflammatory Functional Phenotype

Herein, we demonstrate a role of AMP-activated protein kinase (AMPK) as a potent counterregulator of inflammatory signaling pathways in macrophages. Stimulation of macrophages with anti-inflammatory cytokines (i.e., IL-10 and TGFβ) resulted in the rapid phosphorylation/activation of AMPK, whereas stimulation of macrophages with a proinflammatory stimulus (LPS) resulted in AMPK dephosphorylation/inactivation. Inhibition of AMPKα expression by RNA interference dramatically increased the mRNA levels of LPS-induced TNF-α, IL-6, and cyclooxygenase-2. Likewise, expression of a dominant negative AMPKα1 in macrophages enhanced TNF-α and IL-6 protein synthesis in response to LPS stimulation, while diminishing the production of IL-10. In contrast, transfection of macrophages with a constitutively active form of AMPKα1 resulted in decreased LPS-induced TNF-α and IL-6 production, and heightened production of IL-10. In addition, we found that AMPK negatively regulated LPS-induced IκB-α degradation and positively regulated Akt activation, accompanied by inhibition of glycogen synthase kinase β and activation of CREB. Thus, AMPK directs signaling pathways in macrophages in a manner that suppresses proinflammatory responses and promotes macrophage polarization to an anti-inflammatory functional phenotype.

The Fatty Acid-binding Protein, aP2, Coordinates Macrophage Cholesterol Trafficking and Inflammatory Activity: MACROPHAGE EXPRESSION OF aP2 IMPACTS PEROXISOME PROLIFERATOR-ACTIVATED RECEPTOR γ AND IκB KINASE ACTIVITIES*

Fatty acid-binding proteins are cytosolic fatty acid chaperones, and the adipocyte isoform, aP2, plays an important role in obesity and glucose metabolism. Recently, this protein has been detected in macrophages where it strongly contributes to the development of atherosclerosis. Here, we investigated the role of aP2 in macrophage biology and the molecular mechanisms underlying its actions. We demonstrate that aP2-deficient macrophages display defects in cholesterol accumulation and alterations in pro-inflammatory responsiveness. Deficiency of aP2 alters the lipid composition in macrophages and enhances peroxisome proliferator-activated receptor γ activity, leading to elevated CD36 expression and enhanced uptake of modified low denwsity lipoprotein. The increased peroxisome proliferator-activated receptor γ activity in aP2-deficient macrophages is also accompanied by a significant stimulation of the liver X receptor α-ATP-binding cassette transporter A1-mediated cholesterol efflux pathway. In parallel, aP2-deficient macrophages display reduced IκB kinase and NF-κB activity, resulting in suppression of inflammatory function including reduced cyclooxygenase-2 and inducible nitric-oxide synthase expression and impaired production of inflammatory cytokines. Our results demonstrate that aP2 regulates two central molecular pathways to coordinate macrophage cholesterol trafficking and inflammatory activity.

Role of Leukotriene B4 Receptors in the Development of Atherosclerosis: Potential Mechanisms

Objective—Leukotriene B4 (LTB4), a potent leukocyte chemoattractant, is known to promote several inflammatory diseases, including atherosclerosis. We sought to determine mechanisms through which LTB4 modulates atherosclerosis in cell lines expressing LTB4 receptors, BLT-1, and in mice deficient in BLT-1 as well as macrophage cell lines derived from BLT-1+/+ and BLT-1−/− mice. Methods and Results—Analysis of global changes in gene expression induced by LTB4 in rat basophilic leukemia cells (RBL-2H3) expressing the human BLT-1 showed highest-fold increase in expression of fatty acid translocase/CD36 and the chemokine MCP1/JE/CCL2, which are critical in atherogenesis. To determine the importance of BLT-1 in atherogenesis, we crossed BLT-1-null mice with apolipoprotein (apo)-E-deficient mice, which develop severe atherosclerosis. Deletion of BLT-1 significantly reduced the lesion formation in apo-E−/− mice only during initiating stages (4 and 8 weeks) but had no effect on the lesion size in mice fed atherogenic diet for 19 weeks. Macrophage cell lines from BLT-1-deficient mice expressed the low-affinity LTB4 receptor, BLT-2, and exhibited chemotaxis to LTB4. Conclusions—The effects of LTB4 in atherosclerosis are likely mediated through the high-affinity BLT-1 and the low-affinity BLT-2 receptors. LTB4 promotes atherosclerosis by chemo-attracting monocytes, by providing an amplification loop of monocyte chemotaxis via CCL2 production, and by converting monocytes to foam cells by enhanced expression of CD36 and fatty acid accumulation.

Immunosenescence and macrophage functional plasticity: dysregulation of macrophage function by age-associated microenvironmental changes

Summary:  The macrophage lineage displays extreme functional and phenotypic heterogeneity, which appears to be because, in large part, of the ability of macrophages to functionally adapt to changes in their tissue microenvironment. This functional plasticity of macrophages plays a critical role in their ability to respond to tissue damage and/or infection and to contribute to clearance of damaged tissue and invading microorganisms, to recruitment of the adaptive immune system, and to resolution of the wound and of the immune response. Evidence has accumulated that environmental influences, such as stromal function and imbalances in hormones and cytokines, contribute significantly to the dysfunction of the adaptive immune system. The innate immune system also appears to be dysfunctional in aged animals and humans. In this review, the hypothesis is presented and discussed that the observed age‐associated ‘dysfunction’ of macrophages is the result of their functional adaptation to the age‐associated changes in tissue environments. The resultant loss of orchestration of the manifold functional capabilities of macrophages would undermine the efficacy of both the innate and adaptive immune systems. The macrophages appear to maintain functional plasticity during this dysregulation, making them a prime target of cytokine therapy that could enhance both innate and adaptive immune systems.

IL-12 Rapidly Alters the Functional Profile of Tumor-Associated and Tumor-Infiltrating Macrophages In Vitro and In Vivo

Tumor-associated macrophages (TAMs) play a major role in promoting tumor growth and metastasis and in suppressing the antitumor immune response. Despite the immunosuppressive environment created by the tumor and enforced by tumor-associated macrophages, treatment of tumor-bearing mice with IL-12 induces tumor regression associated with appearance of activated NK cells and activated tumor-specific CTLs. We therefore tested the hypothesis that IL-12 treatment could alter the function of these tumor-associated suppressor macrophages. Analysis of tumor-infiltrating macrophages and distal TAMs revealed that IL-12, both in vivo and in vitro, induced a rapid (<90 min) reduction of tumor supportive macrophage activities (IL-10, MCP-1, migration inhibitory factor, and TGFβ production) and a concomitant increase in proinflammatory and proimmunogenic activities (TNF-α, IL-15, and IL-18 production). Similar shifts in functional phenotype were induced by IL-12 in tumor-infiltrating macrophages isolated from the primary tumor mass and in TAMs isolated from lung containing metastases, spleen, and peritoneal cavity. Therefore, although TAMs display a strongly polarized immunosuppressive functional profile, they retain the ability to change their functional profile to proinflammatory activities given the appropriate stimulus. The ability of IL-12 to initiate this functional conversion may contribute to early amplification of the subsequent destructive antitumor immune response.

Functional plasticity of macrophages: in situ reprogramming of tumor-associated macrophages

The extent to which the functional heterogeneity of Mφs is dependent on the differentiation of functional sublineages remains unresolved. One alternative hypothesis proposes that Mφs are functionally plastic cells, which are capable of altering their functional activities progressively in response to progressively changing signaling molecules generated in their microenvironment. This “functional plasticity” hypothesis predicts that the functionally polarized Mφs in chronic pathologies do not represent Mφ sublineages but rather, are mutable phenotypes sustained by chronic signaling from the pathological environment. Solid TAMφs are chronically polarized to provide activities that support tumor growth and metastasis and suppress adaptive immune responses. In support of the functional plasticity hypothesis, administration of slow‐release microsphere‐encapsulated IL‐12 successfully reprogrammed TAMφs in situ, reducing Mφ support of tumor growth and metastasis and enhancing Mφ proimmunogenic activities. Increased knowledge of how Mφ function is regulated and how polarized Mφs can be reprogrammed in situ will increase our ability to control Mφ function in a variety of pathological states, including cancer and chronic inflammatory disease.

Anti‐apoptotic signalling by the Dot/Icm secretion system of L. pneumophila

The Dot/Icm type IV secretion system of Legionella pneumophila triggers robust activation of caspase‐3 during early and exponential stages of proliferation within human macrophages, but apoptosis is delayed till late stages of infection, which is novel. As caspase‐3 is the executioner of the cell, we tested the hypothesis that L. pneumophila triggers anti‐apoptotic signalling within the infected human macrophages to halt caspase‐3 from dismantling the cells. Here we show that during early and exponential replication, L. pneumophila‐infected human monocyte‐derived macrophages (hMDMs) exhibit a remarkable resistance to induction of apoptosis, in a Dot/Icm‐dependent manner. Microarray analyses and real‐time PCR reveal that during exponential intracellular replication, L. pneumophila triggers upregulation of 12 anti‐apoptotic genes that are linked to activation of the nuclear transcription factor kappa‐B (NF‐κB). Our data show that L. pneumophila induces a Dot/Icm‐dependent sustained nuclear translocation of the p50 and p65 subunits of NF‐κB during exponential intracellular replication. Bacterial entry is essential both for the anti‐apoptotic phenotype of infected hMDMs and for nuclear translocation of the p65. Using p65–/– and IKKα–/–β–/– double knockout mouse embryonic fibroblast cell lines, we show that nuclear translocation of NF‐κB is required for the resistance of L. pneumophila‐infected cells to apoptosis‐inducing agents. In addition, the L. pneumophila‐induced nuclear translocation of NF‐κB requires the activity of IKKα and/or IKKβ. We conclude that although the Dot/Icm secretion system of L. pneumophila elicits an early robust activation of caspase‐3 in human macrophages, it triggers a strong anti‐apoptotic signalling cascade mediated, at least in part by NF‐κB, which renders the cells refractory to external potent apoptotic stimuli.