Macrophages are critical effectors of antibody therapies for cancer

Macrophages are innate immune cells that derive from circulating monocytes, reside in all tissues, and participate in many states of pathology. Macrophages play a dichotomous role in cancer, where they promote tumor growth but also serve as critical immune effectors of therapeutic antibodies. Macrophages express all classes of Fcγ receptors, and they have immense potential to destroy tumors via the process of antibody-dependent phagocytosis. A number of studies have demonstrated that macrophage phagocytosis is a major mechanism of action of many antibodies approved to treat cancer. Consequently, a number of approaches to augment macrophage responses to therapeutic antibodies are under investigation, including the exploration of new targets and development of antibodies with enhanced functions. For example, the interaction of CD47 with signal-regulatory protein α (SIRPα) serves as a myeloid-specific immune checkpoint that limits the response of macrophages to antibody therapies, and CD47-blocking agents overcome this barrier to augment phagocytosis. The response of macrophages to antibody therapies can also be enhanced with engineered Fc variants, bispecific antibodies, or antibody-drug conjugates. Macrophages have demonstrated success as effectors of cancer immunotherapy, and further investigation will unlock their full potential for the benefit of patients.     

Macrophages are critical effectors of antibody therapies for cancer

Macrophages are innate immune cells that derive from circulating monocytes, reside in all tissues, and participate in many states of pathology. Macrophages play a dichotomous role in cancer, where they promote tumor growth but also serve as critical immune effectors of therapeutic antibodies. Macrophages express all classes of Fcγ receptors, and they have immense potential to destroy tumors via the process of antibody-dependent phagocytosis. A number of studies have demonstrated that macrophage phagocytosis is a major mechanism of action of many antibodies approved to treat cancer. Consequently, a number of approaches to augment macrophage responses to therapeutic antibodies are under investigation, including the exploration of new targets and development of antibodies with enhanced functions. For example, the interaction of CD47 with signal-regulatory protein α (SIRPα) serves as a myeloid-specific immune checkpoint that limits the response of macrophages to antibody therapies, and CD47-blocking agents overcome this barrier to augment phagocytosis. The response of macrophages to antibody therapies can also be enhanced with engineered Fc variants, bispecific antibodies, or antibody-drug conjugates. Macrophages have demonstrated success as effectors of cancer immunotherapy, and further investigation will unlock their full potential for the benefit of patients.     

Origin,production and molecular determinants of macrophages for their therapeutic targeting

Macrophages, the most heterogeneous cells of the hematopoietic system and the giant eaters of the immune system that present either as tissue-resident cells or infiltrated immune cells, eliminate foreign pathogens and microbes and also play different physiological roles to maintain the body's immune response. In this review, we basically provide a broad overview of macrophages from their origin, functional diversity to M1-M2 polarization, specialized markers, and their role as important therapeutic targets in different diseases based on the current research and evidence. Apart from this, we have precisely discussed about tumor-associated macrophages (TAMs) and their role in tumor progression and newly discovered lesser-known markers of TAMs that could be used as potential therapeutic targets to treat life-threatening diseases. It is really very important to understand the diversity of macrophages to develop TAM-modulating strategies to activate our own immune system against diseases and to overcome immune resistance.     

Characterisation and trophic functions of murine embryonic macrophages based upon the use of a Csf1r-EGFP transgene reporter

All solid organs contain resident monocyte-derived cells that appear early in organogenesis and persist throughout life. These cells are critical for normal development in some organs. Here we report the use of a previously described transgenic line, with EGFP driven by the macrophage-restricted Csf1r (c-fms) promoter, to image macrophage production and infiltration accompanying organogenesis in many tissues. Using microarray analysis of FACS-isolated EGFP-positive cells, we show that fetal kidney, lung and brain macrophages show similar gene expression profiles irrespective of their tissue of origin. EGFP-positive cells appeared in the renal interstitium from 12 days post coitum, prior to nephrogenesis, and maintain a close apposition to renal tubules postnatally. CSF-1 added to embryonic kidney explants increased overall renal growth and ureteric bud branching. Expression profiling of tissue macrophages and of CSF-1-treated explants showed evidence of the alternate, pro-proliferative (M2) activation profile, including expression of macrophage mannose receptor (CD206), macrophage scavenger receptor 2 (Msr2), C1q, CD163, selenoprotein P, CCL24 and TREM2. This response has been associated with the trophic role of tumour-associated macrophages. These findings suggest a trophic role of macrophages in embryonic kidney development, which may continue to play a similar role in postnatal repair.     

Immunostimulating factor isolated from actinobacillus actinomycetemcomitans stimulates monocytes and inflammatory macrophages

Immunostimulating factor (ISTF) isolated from Actinobacillus actinomycetemcomitans which has been described previously, is distinct from lipopolysaccharide and induces proliferation of B cells. This study was undertaken to investigate whether ISTF might enhance the stimulation of other immune cells. Immunohistochemically, ISTF exhibited a profound stimulating effect on macrophages and dendritic cells as well as B cells in the spleen of BALB/c mice. ISTF was also recognized for its capacity to induce direct activation of mouse macrophages to produce IL-6, TNF-alpha, and NO and MHC class II expression. Therefore, it is postulated that ISTF stimulates macrophages and possibly other cells to produce a wide variety of proinflammatory mediators, which may be involved in the chronicity and tissue destruction of periodontal disease.     

Three matrix metalloproteinases are required in vivo for macrophage migration during embryonic development

Macrophages are essential in development, repair and pathology of a variety of tissues via their roles in tissue remodelling, wound healing and inflammation. These biological functions are also associated with a number of human diseases, for example tumour associated macrophages have well defined functions in cancer progression. Xenopus embryonic macrophages arise from a haematopoietic stem cell population by direct differentiation and act as the main mechanism of host defence, before lymphoid cells and a circulatory system have developed. This function is conserved in mouse and human development. Macrophages express a number of matrix metalloproteinases (MMPs), which are central to their function. MMPs are a large family of zinc-dependent endoproteases with multiple roles in extracellular matrix remodelling and the modulation of signalling pathways. We have previously shown MMP-7 to be expressed by Xenopus embryonic macrophages. Here we investigate the role of MMP-7 and two other MMPs (MMP-18 and MMP-9) that are also expressed in the migrating macrophages. Using morpholino (MO) mediated knockdown of each of the MMPs we demonstrate that they are necessary for normal macrophage migration in vivo. The loss-of-function effect can be rescued using the specific MMPs, altered to be resistant to morpholinos but not by overexpression of the other MMPs. Double and triple morpholino knockdowns further suggest that these MMPs act combinatorily to promote embryonic macrophage migration. Thus, our results imply that these three MMPs have distinct functions, which together are crucial to mediate macrophage migration in the developing embryo. This demonstrates conclusively that MMPs are required for normal macrophage cell migration in the whole organism.     

Live imaging of wound angiogenesis reveals macrophage orchestrated vessel sprouting and regression

Wound angiogenesis is an integral part of tissue repair and is impaired in many pathologies of healing. Here, we investigate the cellular interactions between innate immune cells and endothelial cells at wounds that drive neoangiogenic sprouting in real time and in vivo. Our studies in mouse and zebrafish wounds indicate that macrophages are drawn to wound blood vessels soon after injury and are intimately associated throughout the repair process and that macrophage ablation results in impaired neoangiogenesis. Macrophages also positively influence wound angiogenesis by driving resolution of anti‐angiogenic wound neutrophils. Experimental manipulation of the wound environment to specifically alter macrophage activation state dramatically influences subsequent blood vessel sprouting, with premature dampening of tumour necrosis factor‐α expression leading to impaired neoangiogenesis. Complementary human tissue culture studies indicate that inflammatory macrophages associate with endothelial cells and are sufficient to drive vessel sprouting via vascular endothelial growth factor signalling. Subsequently, macrophages also play a role in blood vessel regression during the resolution phase of wound repair, and their absence, or shifted activation state, impairs appropriate vessel clearance.     

Bone Marrow-derived Macrophage Production

Macrophages are critical components of the innate and adaptive immune responses, and they are the first line of defense against foreign invaders because of their powerful microbicidal activities. Macrophages are widely distributed throughout the body and are present in the lymphoid organs, liver, lungs, gastrointestinal tract, central nervous system, bone, and skin. Because of their repartition, they participate in a wide range of physiological and pathological processes. Macrophages are highly versatile cells that are able to recognize microenvironmental alterations and to maintain tissue homeostasis. Numerous pathogens have evolved mechanisms to use macrophages as Trojan horses to survive, replicate in, and infect both humans and animals and to propagate throughout the body. The recent explosion of interest in evolutionary, genetic, and biochemical aspects of host-pathogen interactions has renewed scientific attention regarding macrophages. Here, we describe a procedure to isolate and cultivate macrophages from murine bone marrow that will provide large numbers of macrophages for studying host-pathogen interactions as well as other processes.     

The role of cardiac resident macrophage in cardiac aging

Advancements in longevity research have provided insights into the impact of cardiac aging on the structural and functional aspects of the heart. Notable changes include the gradual remodeling of the myocardium, the occurrence of left ventricular hypertrophy, and the decline in both systolic and diastolic functions. Macrophages, a type of immune cell, play a pivotal role in innate immunity by serving as vigilant agents against pathogens, facilitating wound healing, and orchestrating the development of targeted acquired immune responses. Distinct subsets of macrophages are present within the cardiac tissue and demonstrate varied functions in response to myocardial injury. The differentiation of cardiac macrophages according to their developmental origin has proven to be a valuable strategy in identifying reparative macrophage populations, which originate from embryonic cells and reside within the tissue, as well as inflammatory macrophages, which are derived from monocytes and recruited to the heart. These subsets of macrophages possess unique characteristics and perform distinct functions. This review aims to summarize the current understanding of the roles and phenotypes of cardiac macrophages in various conditions, including the steady state, aging, and other pathological conditions. Additionally, it will highlight areas that require further investigation to expand our knowledge in this field.     

A distinct subset of podoplanin (gp38) expressing F4/80+ macrophages mediate phagocytosis and are induced following zymosan peritonitis

Macrophages are important tissue resident cells that regulate the dynamics of inflammation. However, they are strikingly heterogeneous. During studies looking at podoplanin (gp38) expression on stromal cells in the murine spleen and peritoneal cavity we unexpectedly discovered that podoplanin was expressed on a subset of F4/80⁺ macrophages; a subset which we have termed fibroblastic macrophages (FM). These cells function as phagocytes in vitro as measured by bead mediated phagocytosis assays. FM also exist at high frequency in the peritoneal cavity and in zymosan induced peritonitis in vivo. These FM represent a unique subgroup of F4/80⁺ macrophages and their presence in the inflamed peritoneum suggests that they play a role in zymosan induced peritonitis.     

Are Macrophages in Tumors Good Targets for Novel Therapeutic Approaches?

The development of cancer has been an extensively researched topic over the past few decades. Although great strides have been made in cancer prevention, diagnosis, and treatment, there is still much to be learned about cancer’s micro-environmental mechanisms that contribute to cancer formation and aggressiveness. Macrophages, lymphocytes which originate from monocytes, are involved in the inflammatory response and often dispersed to areas of infection to fight harmful antigens and mutated cells in tissues. Macrophages have a plethora of roles including tissue development and repair, immune system functions, and inflammation. We discuss various pathways by which macrophages get activated, various approaches that can regulate the function of macrophages, and how these approaches can be helpful in developing new cancer therapies.     

Macrophages in haemopoietic and other tissues of the developing mouse detected by the monoclonal antibody F4/80.

Macrophages are widely distributed in lymphohaemopoietic and other tissues of the normal and diseased adult, where they play an important role in host defence and repair. Although the development of haemopoiesis has been well studied in several species, the ontogeny of the mononuclear phagocyte system remains poorly understood. We have used a highly specific mAb, F4/80, to examine the distribution of mature macrophages in the developing mouse, with special reference to their presence in the haemopoietic microenvironment. Monocytes and macrophages were first seen in embryos on day 10 in the yolk sac and liver as well as in mesenchyme. In liver, spleen and bone marrow, there was expansion of this population associated with the initiation of haemopoiesis on days 11, 15 and 17, respectively. Macrophages in these sites formed part of the haemopoietic stroma and their extensively spread plasma membrane processes could be seen making intimate contacts with clusters of differentiating haemopoietic cells. F4/80+ cells were widely dispersed in undifferentiated mesenchymal tissue in organs such as lung, kidney and gut. Numbers of F4/80-labelled cells increased concomitantly with organ growth and local mitoses were evident, as well as actively phagocytic macrophages. Our studies establish that macrophages are among the earliest haemopoietic cells to be produced during development and that they are relatively abundant in fetal tissues in the absence of overt inflammatory stimuli. Their distribution is correlated with the sequential migration of haemopoiesis and they constitute a prominent component of the stroma in fetal liver, spleen red pulp and bone marrow. Apart from a role in haemopoietic cellular interactions, their highly developed endocytic and biosynthetic activities suggest that macrophages contribute major undefined functions during growth, turnover and modelling of fetal tissues.     

Decidual macrophages and their roles at the maternal-fetal interface

The semi-allogeneic fetus, whose genome consists of maternally and paternally inherited alleles, must coexist with an active maternal immune system during its 9 months in utero. Macrophages are the second most abundant immune cell at the maternal-fetal interface, although populations and functions for these populations remain ill defined. We have previously reported two distinct subsets of CD14(+) decidual macrophages found to be present in first trimester decidual tissue, 20 percent CD11c(HI) and 68 percent CD11c(LO). Interestingly, CD11c(HI) decidual macrophages express genes associated with lipid metabolism, inflammation, and antigen presentation function and specifically upregulate CD1 molecules. Conversely, CD11c(LO) decidual macrophages express genes associated with extracellular matrix formation, muscle regulation, and tissue growth. The large abundance of CD11c(HI) decidual macrophages and their ability to process antigens more efficiently than CD11c(LO) macrophages suggests that CD11c(HI) macrophages may be important antigen processing and presenting cells at the maternal-fetal interface, while CD11c(LO) macrophages may perform necessary homeostatic functions during placental construction. Thus, macrophage heterogeneity may be an important and necessary division of labor that leads to both an induction of maternal immune cell tolerance to fetal antigens as well as basic homeostatic functions in human pregnancy.     

Zoledronic acid modulates antitumoral responses of prostate cancer-tumor associated macrophages

Macrophages are considered a key component of the immunosuppressive environment present in solid tumors, where they support tumor growth through the production of pro-angiogenic factors and active suppression of effector immune responses. Zoledronic acid (ZA), an aminobisphosphonate clinically approved for treatment of symptomatic skeletal events, has recently been shown to have immunomodulatory properties that can be exploited in cancer immunotherapy. Here, we utilize an in vitro model of prostate cancer cell-macrophage interaction to dissect the effect of ZA, on the function of prostate cancer tumor-associated macrophages (PC-TAM). We show that prostate cancer cells recruit macrophages, which in turn express a variety of proangiogenic and immunosuppressive mediators. ZA selectively suppressed the expression of MMP-9 by PC-TAM, whereas the expression of other mediators was not limited. PC-TAM treated with ZA, on the other hand, could effectively drive the proliferation of activated Tgammadelta lymphocytes, which lysed bisphosphonate-pulsed prostate cancer cells. Moreover, ZA boosted the production of type-1 cytokines by PC-TAM in response to immunomodulators such as IL-12 and polyI:C, which are known to polarize macrophages towards an anti-tumoral M1 phenotype. Overall, we provide evidence that ZA shifts the balance of PC-TAM from a tumor promoting to a tumor-eliminating phenotype and also suggest a potential use of this pharmacological agent as an immunotherapeutic adjuvant.     

An unexpected role for hypoxic response: oxygenation and inflammation

The eradication of invading microorganisms depends initially on innate immunity mechanisms that preexist in all individuals and act within minutes of infection. Pathogen spread is often countered by an inflammatory response that recruits more effector molecules and cells of the innate immune system from local blood vessels, while inducing clotting farther downstream so that pathogens cannot spread throughout the blood. If a microorganism crosses an epithelial barrier and begins to replicate in the tissues of the host, it is, in some cases, immediately recognized by the mononuclear phagocytes, or macrophages, that reside in tissues. Macrophages mature continuously from circulating monocytes that leave the circulation to migrate into tissues throughout the body. The second major family of phagocytes, the neutrophils or polymorphonuclear leukocytes (PMNs) are short-lived cells that are abundant cells in the blood but are not present in healthy tissues. Both phagocytic cell types play a key role in innate immunity because they can recognize, ingest and destroy many pathogens without the aid of an adaptive immune response. This infiltration of neutrophils and later macrophages to the site of bacterial infection is tightly linked with the need of these immune defense cells to respond to the tissue microenvironment.     

An Unexpected Role for Hypoxic Response: Oxygenation and Inflammation

The eradication of invading microorganisms depends initially on innate immunity mechanisms that preexist in all individuals and act within minutes of infection. Pathogen spread is often countered by an inflammatory response that recruits more effector molecules and cells of the innate immune system from local blood vessels, while inducing clotting farther downstream so that pathogens cannot spread throughout the blood. If a microorganism crosses an epithelial barrier and begins to replicate in the tissues of the host, it is, in some cases, immediately recognized by the mononuclear phagocytes, or macrophages, that reside in tissues. Macrophages mature continuously from circulating monocytes that leave the circulation to migrate into tissues throughout the body. The second major family of phagocytes, the neutrophils or polymorphonuclear leukocytes (PMNs) are short-lived cells that are abundant cells in the blood but are not present in healthy tissues. Both phagocytic cell types play a key role in innate immunity because they can recognize, ingest and destroy many pathogens without the aid of an adaptive immune response. This infiltration of neutrophils and later macrophages to the site of bacterial infection is tightly linked with the need of these immune defense cells to respond to the tissue microenvironment.     

Distinct Features of Brain-Resident Macrophages: Microglia and Non-Parenchymal Brain Macrophages

Tissue-resident macrophages play an important role in maintaining tissue homeostasis and innate immune defense against invading microbial pathogens. Brain-resident macrophages can be classified into microglia in the brain parenchyma and non-parenchymal brain macrophages, also known as central nervous system-associated or border-associated macrophages, in the brain-circulation interface. Microglia and non-parenchymal brain macrophages, including meningeal, perivascular, and choroid plexus macrophages, are mostly produced during embryonic development, and maintained their population by self-renewal. Microglia have gained much attention for their dual roles in the maintenance of brain homeostasis and the induction of neuroinflammation. In particular, diverse phenotypes of microglia have been increasingly identified under pathological conditions. Single-cell phenotypic analysis revealed that microglia are highly heterogenous and plastic, thus it is difficult to define the status of microglia as M1/M2 or resting/activated state due to complex nature of microglia. Meanwhile, physiological function of non-parenchymal brain macrophages remain to be fully demonstrated. In this review, we have summarized the origin and signatures of brain-resident macrophages and discussed the unique features of microglia, particularly, their phenotypic polarization, diversity of subtypes, and inflammasome responses related to neurodegenerative diseases.     

Role and function of macrophages in the metabolic syndrome

Macrophages are key innate immune effector cells best known for their role as professional phagocytes, which also include neutrophils and dendritic cells. Recent evidence indicates that macrophages are also key players in metabolic homoeostasis. Macrophages can be found in many tissues, where they respond to metabolic cues and produce pro- and/or anti-inflammatory mediators to modulate metabolite programmes. Certain metabolites, such as fatty acids, ceramides and cholesterol crystals, elicit inflammatory responses through pathogen-sensing signalling pathways, implicating a maladaptation of macrophages and the innate immune system to elevated metabolic stress associated with overnutrition in modern societies. The outcome of this maladaptation is a feedforward inflammatory response leading to a state of unresolved inflammation and a collection of metabolic pathologies, including insulin resistance, fatty liver, atherosclerosis and dyslipidaemia. The present review summarizes what is known about the contributions of macrophages to metabolic diseases and the signalling pathways that are involved in metabolic stress-induced macrophage activation. Understanding the role of macrophages in these processes will help us to develop therapies against detrimental effects of the metabolic syndrome.     

Isolation of Murine Peritoneal Macrophages to Carry Out Gene Expression Analysis Upon Toll-like Receptors Stimulation

During infection and inflammation, circulating monocytes leave the bloodstream and migrate into tissues, where they differentiate into macrophages. Macrophages express surface Toll-like receptors (TLRs), which recognize molecular patterns conserved through evolution in a wide range of microorganisms. TLRs play a central role in macrophage activation which is usually associated with gene expression alteration. Macrophages are critical in many diseases and have emerged as attractive targets for therapy. In the following protocol, we describe a procedure to isolate murine peritoneal macrophages using Brewer’s thioglycollate medium. The latter will boost monocyte migration into the peritoneum, accordingly this will raise macrophage yield by 10-fold. Several studies have been carried out using bone marrow, spleen or peritoneal derived macrophages. However, peritoneal macrophages were shown to be more mature upon isolation and are more stable in their functionality and phenotype. Thus, macrophages isolated from murine peritoneal cavity present an important cell population that can serve in different immunological and metabolic studies. Once isolated, macrophages were stimulated with different TLR ligands and consequently gene expression was evaluated.