Cancer and the science of innate immunity

Monocyte ontogeny

In mice, monocytes consist of at least two functionally and phenotypically distinct subsets. Classic monocytes, or inflammatory monocytes, are Ly6C ^high (GR-1 ⁺ ) with proinflammatory properties, migrating to injured ^, or infected tissues from the blood or bone marrow via signaling through chemokine receptor CCR2. Nonclassical Ly6C ^neg (GR-1 ^neg ), or patrolling, monocytes reside in the vasculature and crawl along the luminal vessel surfaces of endothelial cells to surveil tissues during the resolution of inflammation. Ly6C ^neg monocytes enter into tissues during the latter phase of infection, via CX3CR1 signaling, exerting a tissue repair immune regulatory program. Ly6C ^hi monocytes have a short-lived half-life of 20 hours but constitute the steady-state precursors to the blood-residing Ly6C ^neg migratory pool such that the abundance of Ly6C ^hi monocytes dictates the pool of Ly6C ^neg progeny. Monocytes are thought to be derived from the common monocyte progenitor (cMoP), a myeloid precursor population found in the bone marrow in mice, and phenotypically defined as CLEC12A ^hi CD64 ^hi subsets in humans. Progenitor populations were originally thought to derive in a hierarchical progression from common myeloid progenitor (CMP)- granulocyte-monocyte progenitor (GMP)- monocyte-macrophage dendritic cell progenitor (MDP)-cMoP-monocyte. However, it has been demonstrated that under specific inflammatory contexts a bifurcation in the pathway occurred whereby CMP arose independently from MDP into GMP-derived neutrophil-like monocytes whereas MDP differentiated into monocyte-derived DCs. Thus, distinct ontogenies likely underlie observed functional heterogeneity in monocyte populations. Besides originating from the bone marrow, large pools of monocytes reside in the spleen and lungs, where they are maintained and are recruited en masse in response to inflammation, injury, or other systemic insults.

Most studies investigating monocyte origins and deployment throughout the body have used murine models. Human counterparts exist and have both similar and distinct functional attributes compared with their murine counterparts, which are outlined in Table 4.1 . In humans, monocytes represent 10% of the nucleated cells in the blood, whereas in mice monocytes represent 4% of nucleated blood cells. In humans, monocytes are defined based on CD14 and CD16 expression. CD14 ⁺⁺ CD16 ⁻ monocytes are more similar in function and phenotype to Ly6C ^hi monocytes in mice, including having high expression of CCR2 and CD115 while also having lower CD16 expression levels. On the other hand, CD14 ⁺ CD16 ⁺ monocytes also appear to have similar attributes to Ly6C ^neg monocytes, including having elevated CX3CR1 expression. Despite these similarities, important differences between murine and human monocytes have been identified. For example, comprehensive transcriptional and flow cytometric profiling of murine monocytes and their human equivalents revealed that murine monocytes had high PPARγ expression signatures, whereas human monocytes had opposed patterns of surface receptors responsible for recognition and uptake of apoptotic cells and phagocytosis.

Monocyte function: Differentiation during homeostasis and inflammation

Monocytes are recruited in waves, where short-lived Ly6C ^hi monocytes contribute to early inflammation and Ly6C ^neg monocytes specialize in tissue repair and immune regulation. Ly6C ^neg monocytes are longer-lived, with a half-life of up to 2 weeks. Upon entering tissues, and depending on the signals present within a given tissue environment, monocytes will either act as terminally differentiated effectors, as outlined previously, or differentiate into tissue macrophage and dendritic cell populations.

In steady-state conditions, little evidence exists to indicate that classic Ly6C ^hi monocytes contribute significantly to the pool of tissue-resident macrophages. ^{, ,} In the context of pathologic inflammation, Ly6C ^hi monocytes repopulate both tissue-resident macrophage and dendritic cell pools (for in-depth review, see Wynn et al. ). One interesting exception to this paradigm is the lamina propria myeloid cells residing in the intestines. Using macrophage depleted conditions, monocyte fate mapping, adoptive transfer of monocytes, and parabiosis experiments, Varol et al. and Bogunovic et al. both demonstrated that circulating inflammatory Ly6C ^hi monocytes replenish the pool of immune-regulatory CX3CR1 ⁺ macrophages but not of CD103 ⁺ lamina propria migratory dendritic cell subsets. ^, This process occurs during homeostasis and is thought to differ from other steady-state macrophage pools because of the low-level tonic signaling by the commensal microbiota. Follow-up studies demonstrated that signals from the commensal microbiota were essential for monocyte-driven repopulation of luminal macrophages, because antibiotic treatment significantly depleted monocyte recruitment into the intestines, whereas germ-free mice exhibited a significant reduction in luminal macrophage levels. Thus, the chronic signals derived by the gut microbiota were central for continued recruitment of Ly6C ^hi monocytes into the lamina propria. During acute inflammation, such as during colitis, Ly6C ^hi monocytes respond to bacterial products and give rise to proinflammatory effectors that eventually differentiate into migratory antigen-presenting dendritic cell subsets. On the other hand, noninflammatory patrolling monocytes in both mice and humans directly give rise to migratory dendritic cell subsets in homeostatic settings. This occurs in the absence of acute inflammation, where Ly6C ^hi monocytes recirculate back to the bone marrow and are converted into Ly6C ^neg populations that replenish depleted dendritic cell populations in mucosal, but not splenic, tissues.

Monocytes are versatile cell types and, similar to other myeloid-derived counterparts, exhibit sensitivity to the environment in which they reside. Functionally, monocytes exhibit plasticity and have a broad functional repertoire and differentiation potential that is dictated by the signals present in the tissues that they are recruited into. These properties endow monocytes with the potential to elicit robust inflammatory responses or participate in tissue remodeling and repair. Understanding the basis for monocyte fate and function in specific inflammatory contexts will be important for targeting these functionally diverse subsets for the treatment of various diseases, including cancer.

Macrophage ontogeny

Macrophages populate tissues throughout the body in distinct waves during embryogenesis, with inputs from the yolk sac, fetal liver hematopoietic stem cells, or bone marrow. The first wave originates from the yolk sac, where erythroid–myeloid precursors either populate tissues directly or establish a niche of embryonic hematopoietic stem cells in the fetal liver. Monocytes originating from the fetal liver constitute the second wave of macrophage seeding into embryonic tissues. With the exception of the intestinal and cardiac macrophages, the latter of which are renewed by adult monocytes during aging, tissue macrophages are able to self-renew into adulthood. Brain microglia are the only population of tissue macrophages seeded exclusively by yolk sac embryonic precursors, whereas heart macrophages, liver Kupfer cells, and skin Langerhans cells are seeded by both the yolk sac and fetal liver. ^, Alveolar macrophages, kidney macrophages, and red-pulp macrophages are seeded only by fetal liver monocytes. ^,

Macrophage cellular functions: Tissue-specific functions and the spectrum model of activation

Macrophages are considered professional phagocytes, a cell that specializes in phagocytosis, deriving from the Greek words phago (to devour) and cytos (cell). Phagocytosis is a cellular process in which innate immune cells engulf and ingest large materials (greater than 0.5 μm in size) such as dead and dying cells, pathogens, or other particulate materials. Phagocytosis by macrophages and other professional phagocytes is essential for immune homeostasis and host defense, a process that was first described by Elie Metchnikoff, who received a Nobel Prize for his work in 1908 (for in-depth review, see Gordon ). Phagocytosis occurs in multiple phases beginning with sensing of the particle to be ingested through receptors on the cell surface, ^, internalization of the particle through the plasma membrane and into a distinct cellular compartment termed the phagosome , and fusion of the phagosome with lysosomes, an organelle responsible for degradation of phagocytosed macromolecules.

Tissue-resident macrophages exhibit multiple specialized functions for distinct tissue compartments and are identified by specific transcription factors that are induced in response to the tissue microenvironment. Several functionally nonredundant macrophage populations reside in the spleen. Macrophages residing in the red pulp are regulated by the transcription factor PU.1, enabling localization to red pulp for phagocytosis of damaged and dying red blood cells scavenging released iron to maintain iron homeostasis. On the other hand, marginal zone splenic macrophages are dependent on LXRa signaling, where they specialize in the clearance of apoptotic cells while also regulating selective uptake of apoptotic cells by CD8a dendritic cells, a subset in the spleen responsible for regulating autoreactivity to self-antigens. Alveolar macrophages express the transcription factor B lymphoid transcription repressor BTB and CNC homology 2 (Bach2), which enables lipid handling and clearance of surfactant in the alveolar spaces of the lungs. They also facilitate gas exchange and prevent against hypoxia during infection.

Although there are multiple additional examples of tissue specification for macrophages, these studies underscore that macrophage function and phenotype are dictated by the signals present within the tissues that they reside. The advent of single-cell technologies to measure differences in gene expression, protein production, and chromatin regulation have enabled discovery of specific signaling pathways contributing to macrophage heterogeneity during health and disease. This concept is central to the heterogeneity of macrophages observed in disease settings such as cancer. Through transcriptional and epigenetic profiling of tissue-resident macrophages, Lavin et al. identified that aside from lineage-specific transcription factors, tissue-specific microenvironmental signals regulated macrophage identity and function through the induction of core transcription factors and specific chromatin modifications. Tissue signals were so robust that transplanted macrophage precursors or mature macrophages were effectively epigenetically “reprogrammed” when transferred into new tissue environments, exemplifying macrophage functional and transcriptional plasticity. Macrophage plasticity and the ability to be reprogrammed in response to changes in the microenvironment are key considerations when developing anticancer therapies, as will be outlined in the following.

The conceptual framework of macrophage activation has long maintained that macrophages can be polarized into two opposing states: M1, or classically activated inflammatory macrophages, and M2, or alternatively activated immune regulatory macrophages. Studies evaluating macrophage response to acute infection, allergies, obesity, and asthma supported the paradigm that macrophages existed in one of two polarized states. However, this model did not account for the specialized functions of tissue macrophages, macrophages during sterile inflammation and tissue repair, and resolution of inflammation, nor did it encompass macrophage functional changes during chronic and complex disease states such as autoimmunity, chronic infection, and cancer. This paradigm has evolved coincident with the work related to macrophage ontogeny, with elegant studies demonstrating that macrophages have a broad functional repertoire with multiple, distinct, activation programs. Using ex vivo–derived human macrophages, Xue et al. differentiated macrophages in 29 distinct conditions ranging from stimuli associated with M1 and M2 activation axis to stimuli associated with free fatty acids, high-density lipoprotein, or combinations associated with chronic inflammation. Using transcriptomics and network analysis, it was found that in conditions diverging from the classical M1 and M2 paradigm, macrophages had a broad spectrum of activation signatures. These studies support the framework that macrophage activation is multidimensional, where macrophages receive input from tissue environments and external stimuli such as sterile insult, pathogens, or cancer, culminating in a broad range of activation phenotypes. The multispectral model is highlighted in Fig. 4.1 .