REVIEW

Tissue perspective on macrophages

Yasutaka Okabe & Ruslan Medzhitov

Macrophages are essential components of mammalian tissues. Although historically known mainly for their in host defense and the clearance of apoptotic cells, macrophages are now increasingly recognized as serving many roles in tissue development, and repair. In addition, tissue-resident macrophages have many tissue-specific functional characteristics, which are a reflection of distinct gene-expression programs. Here we discuss the emerging views of biology from evolutionary, developmental and homeostatic perspectives.

Macrophages are multifunctional types present in most tissues in help in the development of new perspectives on macrophages and their vertebrates. Macrophage-like cells, or ‘hemocytes’, are also found in roles in normal biology and . some invertebrate lineages, including arthropods. Macrophages are best known for their role in immunity, which was first elucidated by Tissue-resident macrophages are integral tissue components Eli Metchnikoff in the late nineteenth century1. That initial discovery It has long been appreciated that macrophages are present in most if not focused on the phagocytic activity of macrophages, which is impor- all tissues in mammals12 and probably in other vertebrates. Studies have tant not only for host defense against but also for a variety revealed several surprising characteristics of these macrophages. First, of ‘housekeeping’ functions, such as the removal of apoptotic cells tissue-resident macrophages comprise cell derived from and remodeling of the (ECM). Studies are now three sources: yolk sac, fetal , and hematopoietic stem cells in the beginning to highlight a much more general role for macrophages in marrow13,14. The relative contributions of embryonic sources ver- vertebrate biology, including their roles in systemic , cold sus hematopoietic sources to resident macrophages varies by tissue, with , tissue homeostasis and development2,3. The normal devel- some tissues being populated mainly by yolk sac–derived macrophages opment and function of some tissues and organs, including the , (for example, Langerhans cells in the and in the brain) ovaries and bone, is critically dependent on macrophages that reside in and other tissues having mainly bone marrow–derived macrophages (for these organs2,3. It is now appreciated that in addition to their phagocytic example, the intestine)15. Another major insight has been provided the America, Inc. All rights reserved. America, Inc. © 201 6 Nature activity, macrophages can function as an important source of growth fac- realization that yolk sac–derived macrophages (and presumably fetal tors for other cell types within tissues4. Tissue-resident macrophages also liver–derived macrophages) can be maintained for the lifespan of the sense tissue damage and orchestrate tissue-repair responses5. Consistent by continuous self-renewal16. It is not yet clear whether all

npg with such a broad array of essential functions, macrophages are also tissue-resident macrophages can enter the cell cycle or whether small reported to be important contributors to various pathological conditions fractions of these self-renew by asymmetric cell division (similar to stem that reflect the derailment of these functions. Indeed, macrophages are cells). This distinction is important because of its implications for under- now well established as having critical roles in atherosclerosis6, osteopo- standing the functional heterogeneity of tissue macrophages. Finally, rosis7, obesity and type 2 diabetes8,9, fibrosis10 and cancer11. Many addi- studies of gene-expression and enhancer activity in different tissue- tional macrophage functions and corresponding will probably resident macrophages have revealed distinct tissue-specific transcrip- be discovered in the near future. Such advances are starting to transform tional and epigenetic programs17–19 that clearly suggest that resident the view of macrophages from specialized anti-microbial defenders to macrophages are specialized to perform many tissue-specific functions. a cell type with many essential functions in tissue development and Indeed, it has long been recognized that tissue-resident macro- homeostasis in complex metazoans. Thus, science is coming full circle phages are heterogeneous populations in terms of their phenotypes and back to the Metchnikoff’s original view of macrophages as essential com- functions20. Some of their functions are specialized for the anatomi- ponents of metazoan biology. There is a growing need, therefore, for the cal location in which they reside3,21. For examples, (bone development of new conceptual frameworks to integrate macrophage macrophages) are highly specialized in bone resorption7; alveolar functions with mammalian development, and tissue biology. macrophages are responsible for the recycling of surfactant molecules, In this Review, we will present and discuss several concepts that might which are produced by alveolar epithelial cells22; microglia, the brain macrophages, have a role in the development and function of the cen- Howard Hughes Medical Institute, Department of Immunobiology, Yale tral nervous system through synaptic pruning and provision of neu- University School of , New Haven, Connecticut, USA. Correspondence rotrophic factors23; and splenic red-pulp macrophages are important should be addressed to R.M. ([email protected]). in the processing of and iron from senescent red cells24. These examples illustrate the finding that tissue-specialized functions Received August 30; accepted October 9; published online 17 December 2015; of macrophages are tightly associated with normal tissue physiology. doi:10.1038/ni.3320 Accordingly, abnormality of tissue-resident macrophage functions is

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Figure 1 The concept of primary and accessory cell types. (a) Progressive division of labor leads to the of multiple specialized cell types. a For example, a neuron (left) is specialized in the transmission of electrical and chemical signals, whereas a sperm cell (right) is specialized on sexual Neural function Pluripotent . (b) Partition of primary and supportive functions leads stem cell to the evolution of accessory cell types: left, myelin sheaths formed by Reproductive Schwann cells (green) perform accessory functions that enable neurons function to perform saltatory conduction of action potential; right, Sertoli cells (green) provide a spermatogonial stem-cell niche, which ensures the differentiation of spermatogonia into mature sperm cells (red arrows). Action Sperm cell potential (c) Client cells (osteoblasts, alveolar epithelial cells and neurons) and Neuron tissue-specific accessory functions provided by macrophages (osteoclasts, alveolar macrophages and microglia). In bone tissue (left), the bone- remodeling process requires cooperation between osteoblast-mediated bone generation and -mediated bone resorption. The homeostasis of pulmonary surfactant in (middle) is maintained by the balance of the of surfactant and by alveolar type 2 epithelial cells and the removal of these lipids and proteins, mediated by alveolar b macrophages. During development of the (right), microglia-mediated synaptic pruning of enables the establishment of brain architecture and connectivity.

often linked to various . Deficiency in osteoclast develop- Action potential ment is characterized by osteopetrosis caused by insufficient bone 7 Neuron resorption . Alveolar macrophage deficiency in humans and mice results Schwann cells in pulmonary alveolar proteinosis caused by progressive accumulation Sertoli cell Spermatogonia of surfactant25,26. Mice deficient in the transcription factor Spi-C, which have defective development of splenic red-pulp macrophages, develop a disorder in iron homeostasis characterized by iron overload in splenic c Synaptic 24 Osteoclast Alveolar macrophage Microglia red pulp . These findings illustrate the critical role of tissue-resident pruning macrophages in the maintenance of tissue homeostasis. This diversity of functions of tissue macrophages has not yet been fully appreciated from evolutionary and developmental perspectives. In the following sections, we discuss two general principles that might help Bone resorption Surfactant clearance conceptualize the broader view of macrophages as essential components Axonal pruning of tissues of complex metazoans. Bone Pulmonary surfactant remodeling homeostasis Cell-type diversification in metazoan evolution

Nature America, Inc. All rights reserved. America, Inc. © 201 6 Nature One well-appreciated tendency in metazoan evolution is the progres- Bone generation Surfactant production sive ‘division of labor’ between different tissues and the correspond- ing cell types that make up those tissues. For example, adipocytes

npg are specialized for triglyceride storage; epithelial cells are specialized for absorptive and barrier functions; neurons are specialized for the Osteoblast Alveolar epithelial cell Neuron transduction of electrical signals; and sperm cells are specialized for sexual reproduction (Fig. 1a). The purpose of the division of labor is cells, etc.) delegate some of their functions to accessory cells. A familiar to optimize the performance of the functions for which each cell type analogy would be a professional athlete or musician delegating most is specialized. Thus, in more-primitive metazoans (such as the phyla of their chores to housekeepers and personal assistants. The former Porifera and Cnidaria), with only a few cell types available, cells are less can optimize their professional activity (primary function), while the specialized and are often multifunctional27. For example, in Hydrozoa latter enable optimal performance of the primary functions of their (of the phylum Cnidaria), the surface layer is made of epitheliomus- clients. It should be noted, however, that only non–cell-autonomous cular cells, which combine barrier functions and contractile functions functions can be delegated, while cell-autonomous functions cannot in a single cell type28. These functions are segregated into epithelial (thus, of the two functions ‘eating’ and ‘cooking’, only the latter can cells and muscle cells in more-complex metazoans. Subsequent pro- be delegated to someone else). Similarly, immunological defense and gressive division of labor led to further diversification of cell types, scavenging functions and the production of ECM can be delegated to each specialized for fewer functions. Thus, in mammals there are doz- specialized accessory cells (macrophages and , respectively), ens of epithelial cell types specialized for various absorptive, secre- while the synthesis of intracellular proteins and metabolites cannot be tory and sensory functions. This progressive specialization has been delegated to other cells. accompanied by the emergence of many new cell types in the phyla With the emergence of many specialized parenchymal cell types Nematoda, Arthropoda and Chordata. Another consequence of the in arthropods and especially in vertebrates, accessory cell types have emergence of many specialized cell types is the segregation of primary emerged as well. Parenchymal cell types specialized in performing pri- functions and accessory (or supportive) functions. This segregation mary functions delegate supportive functions to accessory cell types, occurs because the cells specialized for primary functions (i.e., tissue- such as macrophages, fibroblasts, , Schwann cells and oligo- specific parenchymal cells, such as hepatocytes, neurons, epithelial dendrocytes. All these cell types perform a variety of accessory functions

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Figure 2 Two types of signals that define the functional states of a cell. Signals reporting tissue identity _` (a) When progenitor cells migrate into tissues (tissues a and b), ‘tissue- identity’ signals (a and b) present in the tissue environment induce tissue- Signals reporting functional demand X Y specific characteristics or differentiation of the cells (states A and B). (b) Signals reporting functional demand (X and Y) induce reversible functional polarization (states Cʹ and Cʺ). (c) Overall phenotypes of cells are a dictated by the combination of tissue-identity signals and signals reporting Progenitor functional demand. Migration Migration

Tissue_ Tissue ` that support the corresponding ‘client’ cells: the tissue-specific paren- chymal cell types. For example, Schwann cells, which wrap around the _` axons of motor and sensory neurons, perform an accessory function (myelination of axons) that enables superior performance of the primary function (action potential propagation in the axon) of their client cell State AState B (the neuron) (Fig. 1b). In seminiferous tubules, Sertoli cells perform accessory roles in support of the differentiation and migration of their client cells, the spermatogonia (Fig. 1b). According to the same prin- ciple, many tissue-specific functions of resident macrophages can be considered as supporting the various primary functions performed by their client cell types, such as osteoblasts, alveolar epithelial cells and neurons (Fig. 1c). Tissue-resident macrophages thus perform a variety b of accessory functions, some of which are tissue specific, to support the Progenitor primary functions of the corresponding parenchymal client cell types. Migration Migration While some accessory cell functions are highly specialized and are needed only for specific client cells (as in the example of Schwann cells Tissue_ Tissue ` and neurons), other accessory functions are generic (housekeeping) and are needed in most tissues for most client cells (for example, the removal of apoptotic cells). Certain accessory functions, such as tissue repair and host defense, need to be performed only on demand. Tissue- X Y X Y resident macrophages can perform these functions when the challenge is small enough. When the challenge ( or injury) is too great to be handled by resident macrophages, they recruit specialized acces- sory cells, such as and inflammatory . Thus, State Cv State C´´ State Cv State Cw while the function of resident macrophages is analogous to that of butlers, who handle many common everyday chores, neutrophils and other inflammatory cell types are analogous to plumbers or firefight- Nature America, Inc. All rights reserved. America, Inc. © 201 6 Nature ers, who are better equipped to deal with specific problems, but the c presence of such cells in tissues is beneficial only when these specific Progenitor problems arise. Migration Migration

npg As macrophages and stromal cells are programmed to support the functions of their client cells, they perform these supportive functions Tissue_ Tissue `

even when their clients are cancer cells. Although tumors are in many _ ` ways abnormal tissues, they are still dependent on various supportive functions of accessory cells, particularly macrophages, stromal cells and X X endothelial cells29–31. These cells are responsible for the production of Y Y growth factors and ECM, the delivery of oxygen and nutrients, and many other essential functions on which tumor cells depend. The provision of these functions by accessory cell types explains their critical role in 11,30 tumor growth . Depending on their cell type of origin, cancer cells State Av State Aw State Bv State Bw might need different tissue-specific accessory functions, in addition to the generic housekeeping functions. This, in turn, might dictate the functional specialization of macrophages and stromal cells in different The concept of functional demand tumor types. The dependence of both normal client cells and cancer The perspective described above might help to explain the pervasive client cells on accessory functions provided by macrophages is an impor- presence of macrophages in mammalian tissues and their diverse, tissue- tant area of future investigation. specific phenotypes and functions. How these tissue-specific functional In summary, the progressive specialization of cells during metazoan programs are established is a question of central importance in mac- evolution has been accompanied by a decrease in their functional auton- rophage biology. One possibility is that these functional phenotypes omy. Some of the functions have been delegated to accessory cells, such are developmentally and genetically hard-wired. Intuitively, this sce- as macrophages, which ‘complement’ the functional repertoire of their nario seems unlikely, as it is difficult to imagine that so many details of client cells (including cancer cells) and ‘support’ the performance of the macrophage phenotypes are specified by pre-defined genetic programs, primary functions. especially if the possibility that macrophages within each tissue are

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Figure 3 Macrophage phenotype is a product ab of differentiation and polarization. (a) In differentiation, cooperation between macrophage lineage–differentiation factors (for example, Progenitor Progenitor M-CSF) and tissue-identity signals (signal 1 M-CSF M-CSF or signal 2) irreversibly controls the terminal M-CSF differentiation of tissue macrophage subsets + + Signal 1 Irreversible Signal 2 Irreversible (type 1 or type 2). (b) In polarization, mature differentiation differentiation macrophages maintain the ability to generate reversible polarization states (type 3 or type 4) depending on the availability of functional Reversible Reversible polarization polarization demand signals (signal 3 or signal 4).

Macrophage Macrophage Macrophage Macrophage Macrophage type 1 type 2 type 3 Signal 3 Signal 4 type 4

functionally heterogeneous, depending on their precise location and Different tissues have diverse functional needs: macrophages in the tissue state, is taken into account. Therefore, an alternative perspec- red pulp of the spleen remove senescent erythrocytes, while macro- tive might need to be developed to account for the generation of tissue phages in alveoli eliminate excess surfactant phospholipids and pro- specificity and functional diversity of macrophages. teins. The corresponding functional programs are induced in these Two developmental paradigms seem to be applicable to the develop- macrophages by dedicated transcription factors that operate as sensors ment and specialization of tissue-resident macrophages (Fig. 2a). The of specific functional needs: Bach1 in red-pulp macrophages senses first is the paradigm of cells: here, stem cells derived from heme, a product of erythrocyte degradation36, while PPAR-g in alveolar the neural crest migrate to various tissues, where they receive a local macrophages senses surfactant lipids37. The phenotypes of these mac- combination of signaling molecules (including members of the BMP, rophages can thus be explained by their response to these particular FGF, Wnt and Hedgehog families) that determine their terminal dif- functional demands. Extrapolating this view would suggest that many ferentiation into various cell types, including chondrocytes, sensory other tissue-specific functional phenotypes of macrophages could be neurons and melanocytes32. This principle has some parallels with the due to the sensing of corresponding functional demands and induc- development of resident macrophages, whereby migratory progenitor tion of the appropriate transcriptional programs. When the functional cells (derived from the yolk sac, fetal liver or bone marrow) home to demands cannot be met by the available number of macrophages, their different tissues to acquire tissue-specific differentiation programs. This local proliferation (through increased local production of the cytokine is thought to occur in response to locally produced and mostly unchar- M-CSF) or recruitment (through expression of the appropriate chemo- acterized signals (we refer to these as ‘tissue-identity signals’ here). This kines) ensures adequate supply of these cells within the tissue. Sensors of developmental mechanism is highly plastic and adjustable; variable char- functional demands might thus orchestrate not only the direct responses acteristics, such as tissue location, shape and size, do not need to be of macrophages to these demands but also the expression of signals that pre-specified for the generation of tissue-resident macrophages with expand, recruit and position macrophages in the appropriate locations. phenotypic characteristics matching the tissues in which they reside. We suggest that many aspects of the development and function of Two known examples that seem to follow this paradigm are the differ- tissue-resident macrophages can best be described by a variation on the entiation of microglia and peritoneal macrophages in response to local themes of the two paradigms described above. First, migratory progeni- Nature America, Inc. All rights reserved. America, Inc. © 201 6 Nature production of TGF-b in the brain and retinoic acid in the omentum, tor cells populate different tissues, where they may receive ‘tissue-iden- respectively33,34. tity’ signals that promote their local differentiation into tissue-resident A second developmental paradigm that we believe is applicable to macrophages. Second, resident macrophages receive signals that indicate

npg the specialization of tissue-resident macrophages is exemplified by local functional demands, which induce diverse functional programs angiogenesis and the development of the peripheral nervous system. In and associated phenotypes of macrophages, as well as their precise loca- both cases, the ‘details’ of the final product (that is, the vascularization tion and number within tissues (Fig. 2c). or innervation of tissues) occurs in a manner that is not pre-specified Functional demands can be transient or continuous, and can be com- by genetic programs. Instead, local provision of growth and survival mon or tissue specific. Accordingly, these translate into transcriptional factors establishes vascularization or innervation of all of the target tis- programs in tissue-resident macrophages that are transient, stable or sues, with appropriate positioning and density of micro- or permanent, corresponding to cellular activation, polarization and differ- neurons, respectively. This developmental principle is also characterized entiation, respectively. The phenotypes of tissue-resident macrophages by plasticity and evolvability: it can easily accommodate new phenotypic are mostly a combination of differentiation and polarization programs. variants, such as changes in body size, without the need for a genetic Differentiation is a stable and irreversible transition from progenitor cell rewiring of the entire system35. to one of several possible cell types under the control of external signals Angiogenesis, in particular, clearly illustrates the key aspect of this that promote distinct differentiation paths (Fig. 3a). In contrast, polar- developmental paradigm: the new blood vessels develop ‘on demand’. ization is a stable and reversible program that is induced on demand. The demand for micro-vessels is dictated by oxygen availability, which in The signals that report functional demand promote stable phenotypic turn is detected by a dedicated sensor, the hypoxia-inducible transcrip- changes through induction of the corresponding gene-expression pro- tion factor HIF-1a. More generally, this paradigm can be formulated grams (Fig. 3b). Once the functional demand is met, the signal reporting as follows: a functional demand is detected by a sensor that promotes it and the corresponding polarization programs subside, which results an increase in the number or activity of cells that meet that demand. in a reversal of the polarization to the initial state, ready to respond Thus, macrophages derived from common migratory progenitor cells to other functional demands. The phenotypes of tissue-specific mac- might sense functional demands in the different tissues in which they rophages can thus be viewed as a combination of differentiation and reside and then might activate the corresponding functional polariza- polarization programs. Indeed, the transplantation of resident peritoneal tion programs (Fig. 2b). macrophages into the alveolar cavity results in gene-expression changes

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from a ‘peritoneal’ macrophage signature to an ‘alveolar’ macrophage tion and require minimal changes in transcriptional control circuits. signature, yet some of the gene-expression signature of peritoneal mac- Indeed, such transitions require only that the expression of transcrip- rophages is retained19. This example illustrates the distinction between tion factors that control polarization programs becomes stable. This gene-expression programs corresponding to irreversible components can be achieved either through a positive auto-regulatory loop (as is (differentiation) and reversible components (polarization) of the spe- the case with the transcription factor MyoD, which activates its own cialization of tissue-specific macrophages. transcription) or through the establishment of a stable chromatin state The transitions of a given program from reversible polarization to (for example, through the effect of histone methyl transferases of the irreversible differentiation presumably occur repeatedly during evolu- Trithorax group, which promote gene activity though methylation of

acb d Tissue-identity Functional- Tissue-identity Functional- M-CSF M-CSF signal M-CSF demand signal M-CSF signal demand signal _ _ X X _ _ X X _ X _ X

PU.1 PU.1 TF _ PU.1 TF XTPU.1 TF _ F X

PU.1 PU.1 TF _ PU.1 TF X PU.1 TF _ TF X

Gene U Gene _ Gene X Gene AX PU.1 PU.1 TF _ PU.1 TF X PU.1 TF _ TF X

Gene Uv Gene _v Gene X´ Gene AXv PU.1 PU.1 TF _ PU.1 TF X PU.1 TF _ TF X

Gene Uw Gene _w Gene Xw Gene AXw PU.1 PU.1 TF _ PU.1 TF X PU.1 TF _ TF X

Universal module Module _ Module XModule _X

Tissue-identity Functional- efM-CSF signal demand signal _ X ++_ X Module Cooperative modules _ X aZ

Module Z

Module _X Module Functional-requirement Y modules Nature America, Inc. All rights reserved. America, Inc. © 201 6 Nature Module X Module X ophage (number of genes)

Module npg _ Module Module Tissue-identity PU.1 Module uni ` a modules

PU.1 TF _ Module _ ogram of macr

PU.1 TF X Module X

PU.1 TF _ TF X Module _X Module Common macrophage anscriptional pr uni Module Module lineage module Tr uni uni

_`a

Tissue

Figure 4 Transcriptional modules that control macrophage phenotypes. (a) Transcriptional master regulators of the macrophage lineage (such as PU.1) control genes (Universal module) that are commonly expressed in all macrophage subsets. (b,c) Signals that report tissue identity (a) or functional demand (X) regulate specific transcription programs (module a or X), through the cooperation between lineage master regulator transcription factors (such as PU.1) and signal-dependent transcription factors (TF a or TF X). (d) Multiple signals (a and X) present in the environment can be integrated for cooperative regulation of the transcription module (module aX). (e) The overall phenotype of tissue-resident macrophages is dictated by a combination of macrophage lineage–determination factors (such as M-CSF), signals that report tissue identity (a) and signals that report functional demand (X). Module uni, universal module. (f) The transcriptional landscape of tissue-resident macrophage subsets. The universal transcription module (gray) is expressed in all tissue-resident macrophage subsets. Tissue-identity modules (yellow) are constitutively induced by the signals present in tissue environments. Functional-polarization modules (green) are transiently or stably expressed depending on the functional demand; the same functional-polarization modules (module Y) can be induced in multiple tissues. Multiple tissue-identity signals and/or functional-demand signals can cooperatively induce transcriptional programs (cooperative modules; purple).

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histone H3 on Lys4). Thus, when a given functional demand is constitu- a tively present in a given tissue, the corresponding polarization program Deviation of may become ‘fixed’ as a differentiation program. In these cases, the tran- regulated variable Controller Signal from set point scription factors that control polarization programs become equivalent to lineage-determining master regulators, while the signals that report on functional demand become equivalent to tissue-identity signals that Regulated variable or induce differentiation. In fact, tissue-identity signals are presumably ‘evolutionarily derived’ from the functional-demand signals that are Pancreatic associated with specific tissues. Thus, all macrophages can participate Hepatocyte b alpha-cell in the of apoptotic cells on demand; however, tingible-body Low glucose macrophages are specialized for the phagocytosis of massive amounts concentration Glucagon of apoptotic B cells in the germinal center38. Similarly, PPAR-g can be upregulated on demand in most macrophages to control metabo- lism, but it is specifically induced by the cytokine GM-CSF in alveolar Glucose macrophages to ensure efficient handling of surfactant lipoproteins37. Tissue-identity and functional-demand signals are not always equiva- Macrophage Vascular lent, however. Thus, retinoic acid induces expression of the transcription c endothelial cell factor GATA-6 only in precursors of peritoneal macrophages but not in Hypoxia VEGF-A progenitors of bone marrow macrophages, because the Gata6 locus is silenced in the latter34. Thus, retinoic acid as a tissue-identity signal can induce the GATA-6 program only in competent cells. [ O2 ] In general, it appears that tissue-identity signals tend to induce dif- ferentiation programs, while functional-demand signals tend to induce Figure 5 The macrophage as a homeostatic controller. (a) In a homeostatic polarization programs, although there are probably many variations on control circuit, the value of a regulated variable is monitored by a Controller. this theme. In response to deviation of the regulated variable from the set point, the Controller produces a signal that acts on the Plant, which in turn can bring Control of the specialization of tissue-resident macrophages the value of the regulated variable closer to its set point(s). (b) During systemic homeostasis, pancreatic alpha cells sense low concentrations Gene-expression analyses and functional studies of tissue-resident mac- of glucose in the blood; this is followed by production of the peptide rophages have provided valuable insights into their transcriptional and glucagon, which acts on hepatocytes to induce glycogenolysis and epigenetic profiles. The emerging view is that gene expression in macro- gluconeogenesis. (c) Macrophages act as Controllers of tissue homeostasis phages is made up of distinct transcriptional modules that are controlled by sensing hypoxia and produce VEGF-A to promote angiogenesis, which by hierarchically arranged transcriptional regulators17–19,34,39. At the increases the supply of oxygen (O2) to the hypoxic area. top of the are the lineage-determining master regulators, such as PU.1 and CEBPb, which control the gene-expression module com- ing development and might account for the difficulty in recapitulating mon to all macrophages (Fig. 4a). PU.1, in particular, has been found differentiation programs in vitro. to occupy and engage macrophage-specific gene enhancers, making Transcription factors that are induced by tissue-identity signals (such Nature America, Inc. All rights reserved. America, Inc. © 201 6 Nature them accessible to other transcriptional regulators40,41. For example, as GATA-6) control tissue-specific gene-expression modules34,44,45. peritoneal macrophages and microglia have been shown to have PU.1 These gene-expression modules specify different tissue-resident mac- bound to enhancer regions of genes transcribed in a tissue-specific rophage subsets, such as intestinal macrophages, Kupffer cells, microglia, 18 46 npg manner . Expression of PU.1 and the universal macrophage-specific osteoclasts and alveolar macrophages . Indeed, deletion of particular gene-expression module is maintained by the CSF-1 receptor and its transcription factors leads to deficiency in specific tissue-resident mac- ligands, including M-CSF and interleukin 34 (refs. 4,42). This generic rophage subsets, such as Spi-C in splenic red-pulp macrophages24, LXRa macrophage gene-expression program characterizes macrophage pro- in splenic marginal zone macrophages47, PPAR-g in lung alveolar macro- genitor cells before their tissue-specific diversification. Expression of phages37 and NR4A1 in thymic macrophages48. This might suggest that PU.1 controls the differentiation of more-primitive progenitor cells into the induction of these transcription factors by tissue-identity signals is macrophages. As noted above, differentiation is generally stable and responsible for the terminal differentiation of these tissue-resident mac- irreversible: withdrawal of M-CSF results in macrophage rather rophage subsets. However, it is also formally possible that rather than than dedifferentiation. regulating differentiation programs, these transcription factors control The next (second) level of hierarchy is controlled by transcription the recruitment of macrophages to a particular anatomical location or factors that are induced by tissue-specific signals (‘tissue-identity’ the expression of genes encoding products used as markers of a particu- signals) (Fig. 4b). The two known examples that fit ‘tissue-identity’ lar macrophage subset. It is curious, in this context, that many of the signals are TGF-b and retinoic acid in the brain and peritoneum, commonly used ‘cell-type markers’ are integrins and chemokine recep- respectively33,34. Neither TGF-b nor retinoic acid is unique to any tors—that is, gene products that control cell localization in specific tis- particular tissue, however, which would suggest that the ‘identity sig- sue compartments. Thus, the definitive distinction between true loss of nal’ is probably a combination of signals received in a specific order. lineage determination versus other effects of the deletion of a transcrip- Indeed, in the case of neural-crest-cell differentiation, it is a combi- tion factor can be complicated. However, there are several special cases nation of several signals (including those from members of the BMP, that illustrate an irreversible terminal differentiation program, such as Wnt, FGF and Hedgehog families), acting in a specific succession, that osteoclast differentiation, which is characterized by multi-nucleated controls the final differentiation programs43. This ‘non-commutative’ giant cells formed by cell- of mononuclear precursor cells7,46. property of differentiation signals (A + B ≠ B + A) might explain how The next (third) level of hierarchy is characterized by transcription a small number of signaling pathways generate diverse cell types dur- modules regulated by functional demands. These can be tissue specific

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or can be shared by macrophages in several tissues (Fig. 4c). Expression These parameters are known as ‘regulated variables’. In systemic homeo- of these modules can also be dependent on the previous provision of stasis, regulated variables were defined by Walter Cannon in 1929 and tissue-identity signals (Fig. 4d). In other words, some transcriptional include the concentration of glucose and calcium in blood and core programs can be dictated by a combination of multiple signals that are body temperature49. Formally, all homeostatic systems have two com- present in the same tissue environment (module aX and module gZ, Fig. ponents: Controllers and . Controllers monitor the values of regu- 4e,f). This can be exemplified by the peritoneal macrophage–specific lated variables, while Plants can change these values. A familiar example expression of genes encoding arginase-1, 1 and proteinase 3, of this in engineered systems would be a thermostat (the Controller) which requires both retinoic acid and an unidentified omentum-derived and a furnace (the Plant) that maintain room temperature (the regu- factor for induction34. lated variable). Controllers communicate to the Plant through a signal Together, the combination of tissue-derived signals seems to induce they produce if the value of a regulated variable differs from the desired multiple gene-expression modules, which accounts for the overall struc- or ‘set-point’ value (Fig. 5a). In systemic homeostasis, Controllers are ture of the phenotype of tissue-resident macrophages (Fig. 4e). In this endocrine organs that produce in response to deviations in context, some of the gene modules can be induced by the same func- regulated variables from their set points. Hormones act on their target tional demand across several tissue macrophage subsets (module Y, Fig. tissues (Plants), which then bring these values closer to their set points. 4f). For example, heme induces expression of the transcription factor Thus, in the case of glucose homeostasis, pancreatic alpha and beta cells Spi-C in both red-pulp macrophages and bone marrow macrophages function as Controllers and produce glucagon and insulin that affect the for their functional specialization36. Plants (muscle, liver and fat) to change the concentration of glucose in blood in the desired direction50 (Fig. 5b). Macrophages and tissue homeostasis To describe tissue homeostasis, we need to define the quantitative The concept of functional demand is applicable to a variety of biological characteristics that are maintained at the tissue level (i.e., the regulated contexts ranging from acute cellular responses to certain types of devel- variables), the components that monitor these variables (Controllers) opmental processes that are not hard-wired. It is particularly useful in and the components that can change their values (Plants). In contrast to describing biological processes at the tissue level, where the majority of their characterization in systemic homeostasis, the regulated variables, the control mechanisms remain poorly understood. Controllers and Plants in tissue homeostasis are not well characterized51. ‘Tissue homeostasis’ is a term often used to refer to a normal (not Examples of the variables that are maintained homeostatically include cell inflamed or damaged) tissue state. A more precise definition of tissue number and composition per tissue compartment; ECM density, com- homeostasis can help to unveil many fundamental characteristics of position and stiffness; and the volume, oxygen concentration, pH, tem- tissue biology that have been largely unstudied. ‘Homeostasis’ refers to perature and osmolarity of interstitial fluids51. Homeostatic Controllers maintenance of the stability of some critical parameters of the system. that monitor these parameters are tissue-resident macrophages,

Table 1 Signals monitored by macrophages and their receptors Signal source Sensor or receptor Ref. Lipopolysaccharide, flagellin, CpG DNA TLRs 54 b-glucan, mannose CLRs 55 Flagellin, pore-forming NLRs 56 Virus Double-stranded RNA, single-stranded RNA TLR3 and TLR7; RLRs 54

Nature America, Inc. All rights reserved. America, Inc. © 201 6 Nature DNA AIM2, cGAS 57,58 b-glucan TLRs 54 b-glucan, mannose CLRs 55

npg Dead cell Apoptotic cell TAMs, TIMs 59,60

Necrotic cell (ATP, HMGB1, uric acid crystal, etc.) NLRP3, TLRs, P2X7, P2Y2 56,54,61 Tissue microenvironment Hypoxia HIF-1a 52 pH GPR65 62 Heat TRPV2, TRPM2 63,64 Osmolarity NLRP3, NLRC4, TRPV2, NFAT5 56,63,65, 66 Alum, asbestos, silica NLRP3 56 Metabolite Nucleotides and nucleosides Purinergic receptors 61 Heme Bach1 36 Vitamin A Retinoic acid receptors 34 Vitamin D Vitamin D receptor 67 Fatty acid PPAR-g, CD36, SR-A 68,69,70 Omega-3 fatty acid GPR120 71 b-hydroxybutyrate GPR109a 72 Sphingosine 1-phosphate Sphingosine 1-phosphate receptors 73

Extracellular matrix Fibronectin Integrin a5b1 74 Proteoglycans SR-A 75 DDRs, LAIR1, mannose receptor 76,77,78

Laminin Integrin a6b1 79 Hyaluronic acid CD44, Lyve-1, TLR2 and TLR4 80,81,82 This table is not comprehensive and has been simplified for clarity.

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afferents of the autonomic nervous system, C-fiber and mast tional repertoire of macrophages far beyond their ‘classical’ functions cells. These cell types monitor various aspects of the tissue microenvi- as anti-microbial . Metchnikoff would have been pleased to ronment, which is another way of saying that they monitor the values know that his favorite cell type is finally getting the attention it deserves. of the regulated variables. Thus, macrophages can sense hypoxia and produce the growth factor VEGF-A to induce angiogenesis52 (Fig. 5c); ACKNOWLEDGMENTS We thank members of the Medzhitov laboratory for discussions, and L. Kopp they can sense hyper-osmolarity and produce VEGF-C to induce lym- for reading the manuscript. Supported by The Blavatnik Family Foundation, phangiogenesis to promote lymphatic drainage53; and they can sense Else Kröner-Fresenius-Stiftung, the US National Institutes of Health (AI046688, unknown characteristics of the ECM to produce growth factors and AI089771 and CA157461) and the Howard Hughes Medical Institute (all for cytokines to induce the production or degradation of the ECM. research in the Medzhitov laboratory). Thus, tissue-resident macrophages function as an equivalent of endo- COMPETING FINANCIAL INTERESTS crine cells: both function as homeostatic Controllers monitoring differ- The authors declare no competing financial interests. ent regulated variables, either at the tissue level (macrophages) or at the organismal level (endocrine cells). While endocrine cells produce hor- Reprints and permissions information is available online at http://www.nature.com/ reprints/index.html. mones in response to the deviation of regulated variables, macrophages produce paracrine signals that act locally to maintain homeostasis. These 1. Tauber, A.I. Metchnikoff and the phagocytosis theory. Nat. Rev. Mol. Cell Biol. 4, signals include growth factors, angiogenic factors, vasoactive signals 897–901 (2003). and various inflammatory mediators. The last example highlights the 2. Wynn, T.A., Chawla, A. & Pollard, J.W. Macrophage biology in development, homeostasis fundamental parallels between homeostatic control systems and inflam- and . Nature 496, 445–455 (2013). 3. Gordon, S., Pluddemann, A. & Martinez Estrada, F. Macrophage heterogeneity in tis- 50 matory control systems . sues: phenotypic diversity and functions. Immunol. Rev. 262, 36–55 (2014). There are two special features of tissue homeostasis that need to be 4. Pollard, J.W. Trophic macrophages in development and disease. Nat. Rev. Immunol. 9, 259–270 (2009). highlighted for clarity. First, in some cases, tissue macrophages can 5. Mantovani, A. et al. The chemokine system in diverse forms of macrophage activation function as both Controllers and Plants; that is, they can both ‘moni- and polarization. Trends Immunol. 25, 677–686 (2004). tor’ and ‘change’ homeostatic variables (in systemic homeostasis, these 6. Moore, K.J. & Tabas, I. Macrophages in the pathogenesis of atherosclerosis. Cell 145, 341–355 (2011). functions are typically separated and performed by endocrine cells 7. Boyle, W.J., Simonet, W.S. & Lacey, D.L. Osteoclast differentiation and activation. and their target tissues, respectively). For example, macrophages sense Nature 423, 337–342 (2003). and phagocytose apoptotic cells; sense and modify ECM; and sense 8. Chawla, A., Nguyen, K.D. & Goh, Y.P. Macrophage-mediated inflammation in metabolic disease. Nat. Rev. Immunol. 11, 738–749 (2011). and kill bacteria. Second, some aspects of tissue homeostasis cannot 9. Olefsky, J.M. & Glass, C.K. Macrophages, inflammation, and insulin resistance. Annu. be sensed directly and are instead monitored through various proxies. Rev. Physiol. 72, 219–246 (2010). For example, tissue damage is sensed via signals, such as extracellular 10. Wynn, T.A. & Barron, L. Macrophages: master regulators of inflammation and fibrosis. Semin. Liver Dis. 30, 245–257 (2010). ATP, low pH or ECM proteolytic fragments, that function as a proxy of 11. Noy, R. & Pollard, J.W. Tumor-associated macrophages: from mechanisms to therapy. damage. Although these signals are monitored, they are obviously not Immunity 41, 49–61 (2014). 12. Gordon, S. et al. Localization and function of tissue macrophages. Ciba Found. Symp. maintained; they are simply used as an indication of tissue damage. The 118, 54–67 (1986). same principle applies to the sensing of pathogens; thus, macrophages 13. Epelman, S., Lavine, K.J. & Randolph, G.J. Origin and functions of tissue macrophages. sensing local bacterial infection through various pathogen-associated Immunity 41, 21–35 (2014). 14. Ginhoux, F. & Jung, S. Monocytes and macrophages: developmental pathways and molecular patterns produce tumor- factor, interleukin 1 and tissue homeostasis. Nat. Rev. Immunol. 14, 392–404 (2014). chemokines to induce the recruitment of neutrophils that then kill the 15. Varol, C., Mildner, A. & Jung, S. Macrophages: development and tissue specialization. Nature America, Inc. All rights reserved. America, Inc. © 201 6 Nature bacteria. Finally, it should be noted that the deviation of a homeostatic Annu. Rev. Immunol. 33, 643–675 (2015). 16. Sieweke, M.H. & Allen, J.E. Beyond stem cells: self-renewal of differentiated macro- variable from its set point is equivalent to functional demand, while the phages. Science 342, 1242974 (2013). proxies of homeostatic deviations are equivalent to signals that report 17. Gautier, E.L. et al. Gene-expression profiles and transcriptional regulatory pathways that underlie the identity and diversity of mouse tissue macrophages. Nat. Immunol. npg on functional demands. 13, 1118–1128 (2012). The full range of signals that are monitored by tissue-resident mac- 18. Gosselin, D. et al. Environment drives selection and function of enhancers controlling rophages is unknown, as is the full spectrum of macrophage responses tissue-specific macrophage identities. Cell 159, 1327–1340 (2014). 19. Lavin, Y. et al. Tissue-resident macrophage enhancer landscapes are shaped by the to these signals. We have summarized some of the known examples of local microenvironment. Cell 159, 1312–1326 (2014). these signals and their macrophage receptors to highlight their diversity 20. Gordon, S. & Taylor, P.R. and macrophage heterogeneity. Nat. Rev. Immunol. (Table 1). 5, 953–964 (2005). 21. Davies, L.C., Jenkins, S.J., Allen, J.E. & Taylor, P.R. Tissue-resident macrophages. Nat. Immunol. 14, 986–995 (2013). Conclusions and perspectives 22. Hussell, T. & Bell, T.J. Alveolar macrophages: plasticity in a tissue-specific context. In this Review we have highlighted several emerging principles of mac- Nat. Rev. Immunol. 14, 81–93 (2014). 23. Parkhurst, C.N. et al. Microglia promote learning-dependent synapse formation through rophage biology. Macrophages are essential components of mammalian brain-derived neurotrophic factor. Cell 155, 1596–1609 (2013). tissues in which they perform a variety of accessory functions. Some of 24. Kohyama, M. et al. Role for Spi-C in the development of red pulp macrophages and these functions are ‘instructed’ by locally produced tissue-identity sig- splenic iron homeostasis. Nature 457, 318–321 (2009). 25. Dranoff, G. et al. Involvement of -macrophage colony-stimulating factor in nals; others are performed in response to specific functional demands. pulmonary homeostasis. Science 264, 713–716 (1994). The combinations of these signals and the corresponding gene-expres- 26. Suzuki, T. et al. Pulmonary macrophage transplantation therapy. Nature 514, 450–454 (2014). sion programs give rise to diverse tissue-specific phenotypes of macro- 27. Arendt, D. The evolution of cell types in : emerging principles from molecular phages. The accessory functions performed by macrophages include studies. Nat. Rev. Genet. 9, 868–882 (2008). functional complementation of their client cells. Macrophages also func- 28. Hickman, C.P., Roberts, L.S. & Larson, A. in Integrated Principles of (McGraw- Hill Science, 2009). tion as Controllers (and in some cases, Plants) of tissue homeostasis. 29. Egeblad, M., Nakasone, E.S. & Werb, Z. Tumors as organs: complex tissues that inter- Both types of functions rely on the macrophage’s ability to sense the face with the entire organism. Dev. Cell 18, 884–901 (2010). tissue microenvironment and to promote homeostasis, stress adaptation 30. Hanahan, D. & Coussens, L.M. Accessories to the crime: functions of cells recruited to the tumor microenvironment. Cancer Cell 21, 309–322 (2012). and defense from noxious challenges, such as infection and injury. The 31. Hanahan, D. & Weinberg, R.A. Hallmarks of cancer: the next generation. Cell 144, emerging genomic and functional data are starting to reveal a rich func- 646–674 (2011).

16 VOLUME 17 NUMBER 1 JANAURY 2016 NATURE IMMUNOLOGY REVIEW

32. Knecht, A.K. & Bronner-Fraser, M. Induction of the neural crest: a multigene process. DNA sensor that activates the type I interferon pathway. Science 339, 786–791 Nat. Rev. Genet. 3, 453–461 (2002). (2013). 33. Butovsky, O. et al. Identification of a unique TGF-b-dependent molecular and func- 59. Nagata, S., Hanayama, R. & Kawane, K. Autoimmunity and the clearance of dead tional signature in microglia. Nat. Neurosci. 17, 131–143 (2014). cells. Cell 140, 619–630 (2010). 34. Okabe, Y. & Medzhitov, R. Tissue-specific signals control reversible program of local- 60. Lemke, G. & Rothlin, C.V. Immunobiology of the TAM receptors. Nat. Rev. Immunol. ization and functional polarization of macrophages. Cell 157, 832–844 (2014). 8, 327–336 (2008). 35. Gerhart, J. & Kirschner, M. The theory of facilitated variation. Proc. Natl. Acad. Sci. 61. Desai, B.N. & Leitinger, N. Purinergic and calcium signaling in macrophage function USA 104 Suppl 1, 8582–8589 (2007). and plasticity. Front. Immunol. 5, 580 (2014). 36. Haldar, M. et al. Heme-mediated SPI-C induction promotes monocyte differentiation 62. Lattin, J. et al. G--coupled receptor expression, function, and signaling in into iron-recycling macrophages. Cell 156, 1223–1234 (2014). macrophages. J. Leukoc. Biol. 82, 16–32 (2007). 37. Schneider, C. et al. Induction of the nuclear receptor PPAR-g by the cytokine GM-CSF 63. Link, T.M. et al. TRPV2 has a pivotal role in macrophage particle binding and phago- is critical for the differentiation of fetal monocytes into alveolar macrophages. Nat. cytosis. Nat. Immunol. 11, 232–239 (2010). Immunol. 15, 1026–1037 (2014). 64. Kashio, M. et al. Redox signal-mediated sensitization of transient receptor potential 38. Hanayama, R. et al. Autoimmune disease and impaired uptake of apoptotic cells in melastatin 2 (TRPM2) to temperature affects macrophage functions. Proc. Natl. MFG-E8-deficient mice. Science 304, 1147–1150 (2004). Acad. Sci. USA 109, 6745–6750 (2012). 39. Gorgani, N.N., Ma, Y. & Clark, H.F. Gene signatures reflect the marked heterogeneity 65. Compan, V. et al. Cell volume regulation modulates NLRP3 inflammasome activation. of tissue-resident macrophages. Immunol. Cell Biol. 86, 246–254 (2008). Immunity 37, 487–500 (2012). 40. Lawrence, T. & Natoli, G. Transcriptional regulation of macrophage polarization: 66. Ip, W.K. & Medzhitov, R. Macrophages monitor tissue osmolarity and induce enabling diversity with identity. Nat. Rev. Immunol. 11, 750–761 (2011). inflammatory response through NLRP3 and NLRC4 inflammasome activation. Nat. 41. Glass, C.K. Genetic and genomic approaches to understanding macrophage identity Commun. 6, 6931 (2015). and function. Arterioscler. Thromb. Vasc. Biol. 35, 755–762 (2015). 67. Liu, P.T. et al. Toll-like receptor triggering of a vitamin D-mediated human antimi- 42. Mossadegh-Keller, N. et al. M-CSF instructs myeloid lineage fate in single haemato- crobial response. Science 311, 1770–1773 (2006). poietic stem cells. Nature 497, 239–243 (2013). 68. Ganeshan, K. & Chawla, A. Metabolic regulation of immune responses. Annu. Rev. 43. Sauka-Spengler, T. & Bronner-Fraser, M. A gene regulatory network orchestrates Immunol. 32, 609–634 (2014). neural crest formation. Nat. Rev. Mol. Cell Biol. 9, 557–568 (2008). 69. Hansson, G.K. & Hermansson, A. The in atherosclerosis. Nat. 44. Rosas, M. et al. The transcription factor Gata6 links tissue macrophage phenotype Immunol. 12, 204–212 (2011). and proliferative renewal. Science 344, 645–648 (2014). 70. Moore, K.J., Sheedy, F.J. & Fisher, E.A. Macrophages in atherosclerosis: a dynamic 45. Gautier, E.L. et al. Gata6 regulates aspartoacylase expression in resident peritoneal balance. Nat. Rev. Immunol. 13, 709–721 (2013). macrophages and controls their survival. J. Exp. Med. 211, 1525–1531 (2014). 71. Oh, D.Y. et al. GPR120 is an omega-3 fatty acid receptor mediating potent anti- 46. Stout, R.D. & Suttles, J. Functional plasticity of macrophages: reversible adaptation inflammatory and insulin-sensitizing effects. Cell 142, 687–698 (2010). to changing microenvironments. J. Leukoc. Biol. 76, 509–513 (2004). 72. Rahman, M. et al. The b-hydroxybutyrate receptor HCA2 activates a neuroprotective 47. A-Gonzalez, N. et al. The nuclear receptor LXRa controls the functional specialization subset of macrophages. Nat. Commun. 5, 3944 (2014). of splenic macrophages. Nat. Immunol. 14, 831–839 (2013). 73. Weigert, A., Weis, N. & Brune, B. Regulation of macrophage function by sphingosine- 48. Tacke, R. et al. The transcription factor NR4A1 is essential for the development of 1-phosphate. Immunobiology 214, 748–760 (2009). a novel macrophage subset in the thymus. Sci. Rep. 5, 10055 (2015). 74. McCutcheon, J.C. et al. Regulation of macrophage phagocytosis of apoptotic neutro- 49. Cannon, W.B. Organization for physiological homeostasis. Physiol. Rev. 9, 399–431 phils by adhesion to fibronectin. J. Leukoc. Biol. 64, 600–607 (1998). (1929). 75. Santiago-García, J., Kodama, T. & Pitas, R.E. The class A scavenger receptor binds 50. Kotas, M.E. & Medzhitov, R. Homeostasis, inflammation, and disease susceptibility. to proteoglycans and mediates adhesion of macrophages to the extracellular matrix. Cell 160, 816–827 (2015). J. Biol. Chem. 278, 6942–6946 (2003). 51. Chovatiya, R. & Medzhitov, R. Stress, inflammation, and defense of homeostasis. 76. Franco, C. et al. Discoidin receptor 1 on bone marrow-derived cells promotes Mol. Cell 54, 281–288 (2014). macrophage accumulation during atherogenesis. Circ. Res. 105, 1141–1148 (2009). 52. Lewis, C.E. & Pollard, J.W. Distinct role of macrophages in different tumor microen- 77. Leitinger, B. Transmembrane collagen receptors. Annu. Rev. Cell Dev. Biol. 27, vironments. Cancer Res. 66, 605–612 (2006). 265–290 (2011). 53. Machnik, A. et al. Macrophages regulate salt-dependent volume and blood pressure 78. Sturge, J., Todd, S.K., Kogianni, G., McCarthy, A. & Isacke, C.M. Mannose receptor by a vascular endothelial growth factor-C-dependent buffering mechanism. Nat. Med. regulation of macrophage cell migration. J. Leukoc. Biol. 82, 585–593 (2007). 15, 545–552 (2009). 79. Woo, H.J., Shaw, L.M., Messier, J.M. & Mercurio, A.M. The major non-integrin lam- 54. Takeuchi, O. & Akira, S. Pattern recognition receptors and inflammation. Cell 140, inin binding protein of macrophages is identical to binding protein 35 805–820 (2010). (Mac-2). J. Biol. Chem. 265, 7097–7099 (1990). 55. Geijtenbeek, T.B. & Gringhuis, S.I. Signalling through C-type lectin receptors: shaping 80. McKee, C.M. et al. Hyaluronan (HA) fragments induce chemokine gene expression in immune responses. Nat. Rev. Immunol. 9, 465–479 (2009). alveolar macrophages. The role of HA size and CD44. J. Clin. Invest. 98, 2403–2413 Nature America, Inc. All rights reserved. America, Inc. © 201 6 Nature 56. Lamkanfi, M. & Dixit, V.M. Mechanisms and functions of inflammasomes. Cell 157, (1996). 1013–1022 (2014). 81. Jang, J.Y. et al. Conditional ablation of LYVE-1+ cells unveils defensive roles of 57. Rathinam, V.A., Vanaja, S.K. & Fitzgerald, K.A. Regulation of inflammasome signal- lymphatic vessels in intestine and nodes. Blood 122, 2151–2161 (2013). ing. Nat. Immunol. 13, 333–342 (2012). 82. Termeer, C. et al. Oligosaccharides of Hyaluronan activate dendritic cells via toll-like npg 58. Sun, L., Wu, J., Du, F., Chen, X. & Chen, Z.J. Cyclic GMP-AMP synthase is a cytosolic receptor 4. J. Exp. Med. 195, 99–111 (2002).

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