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Critical Reviews in Immunology, 32(06):463-488 (2012)

M1 and M2 Macrophages: Oracles of Health and Disease


Charles D. Mills
BioMedical Consultants, 16930 197th St. N, Marine, MN, 55047, Phone: 651 600 6519, Fax: 651 600 6519, Mills002@umn.edu

ABSTRACT: The purpose of immunology is simple. Cure or prevent disease. M1/M2 is useful because it is simple. M1/M2 describes the two major and opposing activities of macrophages. M1 activity inhibits cell proliferation and causes tissue damage while M2 activity promotes cell proliferation and tissue repair. Remarkably, the molecules primarily responsible for these Fight (NO) or Fix (Ornithine) activities both arise from arginine, and via enzymatic pathways (iNOS and arginase) that down regulate each other. The names M1 and M2 were chosen because M1 and M2 macrophages promote Th1 and Th2 responses, respectively. Products of Th1 and Th2 responses (e.g., IFN-, IL-4) also down regulate M2 and M1activity, respectively. Thus, M1/M2 demonstrated the importance of Innate Immunity and how it is linked to Adaptive Immunity in a beautifully counterbalanced system. Civilization and increased longevity present new disease challenges such as cancer and atherosclerosis that do not display classical foreign antigens. And, these diseases are often associated with (or caused by) M1- or M2- type responses that were formerly useful for fighting infections, but now are inappropriate in our increasingly germ-free societies. In turn, there is considerable potential for modulating M1 or M2 Innate responses in modern diseases to achieve better health. Finally, since M1 and Th1 (or M2 and Th2) often work in concert to produce characteristic immune responses and disease pathologies, it is recommended that Immune Type 1 or 2 (IT1, IT2) would be a simpler and unifying terminology going forward. KEY WORDS: Cancer, M1, M2, Macrophages, Atherosclerosis, Innate Immunity, Autoimmunity, Stress, Obesity ABBREVIATIONS: ACTH: Adrenocorticotropic Hormone; DAMP: Damage Associated Molecular Patterns; EGF: Epidermal Growth Factor; Human Immunodeficiency Virus; IFN-g: Interferon Gamma; IL: Interleukin; NO: Nitric Oxide; PAMP: Pathogen Associated Molecular Patterns; SCID: Severe Combined Immunodeficient; SLE: Systemic Lupus Erythematosus; TGF-b: Transforming Growth Factor Beta; TNF-a: Tumor Necrosis Factor Alpha; VEGF: Vascular Endothelial Growth Factor

I. SCOPE AND GOAL

of clinical applications. Others have expertly reviewed the molecular biology, surface markers The greatest successes of immunology (i.e., and other aspects of macrophages.3-6 Therefore, smallpox and polio) occurred in a simpler time, my goal is somewhat different. It is to explain when little was known of how the immune Innate Immunity mainly from an M1/M2 persystem operated. On the other hand, now the spective, and to identify diseases where changing immune system (and macrophages in particular) the M1/M2 balance has therapeutic potential. often seems vexingly complex. M1/M2 changed M1/M2 is, of course, an oversimplification of that by describing Innate Immunity in simpler the many other cells and processes involved in terms, and helped show that Innate Immunity Innate Immunity. However, I felt the usefulness controls Adaptive Immunity, not vice versa.1,2 of describing Innate Immunity in terms of its These were fundamental changes in under- core Fight or Fix functions would outweigh standing the immune system and have a myriad what is lost by omission.
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II. HISTORY: VACCINES AND THE FALL AND RISE OF MACROPHAGES

attention was paid to the simpler cells (macrophages) that were mainly viewed as Servants, performing tasks (such as killing pathogens and cleaning up debris) under the direction of the A. B.C. (Before Cells): Simple - Curative Masters (T cells). It was a time of an Adaptive Those who dont know history are destined to repeat it. Dictatorship. Edmund Burke (1769) My own belief in the magic of Adaptive Immunology began with a simple observation. Immunity was typical and instructive. I (like many Edward Jenner noticed during outbreaks of others in the 70s and 80s), thought the next great smallpox that milkmaids often survived.7 The cows immunologic triumph would be a vaccine against had skin lesions that resembled those dying of our biggest health problemcancer. In my case, smallpox (later found to be a similar virus cow- I studied mouse tumors that were immunogenic pox). So, he took some material from these skin (elicit a tumor-specific response in the host), and lesions and put it on the skin of a boy without found that elevating tumor-specific cytolytic T smallpox. Later, the boy was immune to smallpox. cell responses could cause tumor regression.8,9 Skin is an important word here because if Jenner Others tried to identify antigens that are specific had administered Compox orally, or injected it to human tumors that could be recognized by T intravenously, immunity would not have resulted cells.10 Much effort has also been expended trying or death would have occurred. Smallpox eradica- to identify monoclonal antibodies that specifically tion became a triumph of immunology. Unknow- recognize cancer cells.11 Whereas potential exists ingly, Jenner had discovered that humans have an in these areas, the problem in cancer and many effective system for recognizing pathogens on the other diseases goes beyond classical T or B cell skin (Innate Immunity) that can result in specific specificity. protection (Adaptive Immunity). As will become apparent later, the ability of Innate Immunity to protect exposed surfaces like the skin and lungs, C. Paradise (Specificity) Lost: Innate and other characteristics of Innate Immunity, Immunity Found are critical to survival. However, scientists first focused on the Holy Grail of immunology Dreams of a cancer vaccine have faded because Specificity. the majority of cancer cells do not express antigens that can be made to be immunogenic. Also, most of modern civilizations other important diseases B. A.D. (Adaptive Dictatorship): (e.g., atherosclerosis, autoimmunity) also do not Macrophages as Servants express classical foreign antigens. Does this mean that the immune system will continue mainly The ability to simply isolate a causative agent (e.g., as a weapon against exogenous pathogens (i.e., smallpox or polio), inject it (or something similar), bacteria, parasites, viruses)? The good news is no. and have the body take care of the rest with specific The immune system does play an important role immunity was a remarkable scientific advance. In in most modern diseases, just in a different way turn, immunologists naturally focused on figuring than against pathogens. Additionally, some current out what constituted specificity to try and cure problem pathogens (or vaccines made from them) other diseases. In some cases, T cells were found elicit a response, but it is ineffective. For example, to be critical for protection (e.g., Tuberculosis); HIV naturally elicits an antibody response, but in others, B cells/antibody were found to be most a cytolytic T cell response seems required for effective (e.g., Diphtheria). During this quest, scant immunity.12 Therefore, understanding how to
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improve health requires a better understanding of how immune responses work. It has also led to recognition of the critical importance of Innate Immunity and the primary cells that mediate it, macrophagesthe simpler cells.

tocompatibility antigens so foreign antigens are presented to T cells in the context of self.20,21 This sophistication allows T cells to develop specific responses, not only to exogenous pathogens, but also self cells expressing alterations of self because of viral infection or oncogenic transformation. One of these T cell responses causes B III. MACROPHAGES cells to produce antibody (although B recognition of some microbial components is T cell indepenA. Basic Definition dent). Another T cell response (discovered at the Trudeau Institute by Mackaness and colleagues) A macrophage is a cell that samples the environ- causes further amplification of the macrophage ment, often by engulfing (phagocytosing) mate- response (termed macrophage activation) that is rial. So, Metchnikov picked a good name in big necessary to kill many intracellular pathogens.3,22 eater.13 Metchnikov had other pioneering ideas Thus, throughout evolution, macrophages (or about immunity and health as well.14 Sampling macrophage like cells) perform critical functions is a primordial function (amoebas do it) that origi- that are directly (Innate Response) or indirectly nally served to help simple organisms find food, and (Adaptive Response) necessary for survival. to avoid using other like members of the species as food (preservation of the species)!15,16 Later on in evolution, sampling became more sophisticated, B. Macrophages Living in Different mainly through the use of Toll receptors17,18 that Locations recognize pathogens by what are called Pathogen Associated Molecular Patterns (PAMP). More Depending on where they reside in the body, recently, Damage Associated Molecular Patterns macrophage-like cells have different names (e.g., (DAMP) have been identified that also allow mac- microglial cells [brain], Kuppfer cells [liver], alveorophages to detect damaged or effete self-tissue.19 lar macrophages [lung], peritoneal macrophages Once a human macrophage recognizes what [peritoneum], foam cells [blood vessel plaques], it is dealing with, it selects a response appropriate Langherhan cells [skin]). The presence of these for the circumstance. Macrophages have a battery macrophage-like cells in different places is essential, of responses available, many of which are present not only for defense, but also for fetal development far back in evolution (e.g., digestion, NO and and adult life (In contrast, T and B cells are not oxygen radical production, factors that promote required for life under germ-free conditions).15 proliferation and angiogenesis or matrix deposi- There are no macrophage knock out mice. At the tion).3,15 Essentially though, a macrophage does same time, the functions of these cells in different one of two things. It either decides to Fight by locations all depend on sampling the environment turning on a killing program (e.g., a pathogen), or for self/non-self like classical macrophages. For Fix using a repair program (e.g., wound healing). example, resident tissue macrophages in the skin If the material is foreign, human macrophages often encounter tissue damage signals (wounds) (also other mammals and birds) have evolved a and make a Fight response. Alveolar macrophages second, more sophisticated, system to commu- more commonly encounter inhaled material that nicate with T cells that goes beyond recognition they engulf to Fix the problem. Intestinal macroof non-self to recognition of subtle variations of phages encounter ingested material (and resident self. To achieve this, macrophages (and dendritic bacteria, etc.), and sampling in this environment cells and B cells) uniquely possess Class II his- helps Fix the immune system so it doesnt respond
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inappropriately (preserve tolerance). To perform these somewhat different daily functions, cells in these various places have recognizable differences in the portfolios of genes and products they express.4,5,23 However, all these cells make the same Innate Fight or Fix decisions (and all employ similar intracellular processes to do so). Therefore, for the purpose of this review it is most useful to view all these cells as macrophages. Two other important cells of note will not be covered separately here. Monocytes live in the blood, and are important because they are the precursors that supply the extravascular spaces with new macrophages.24,25 At the same time, these circulating cells have no known function, and macrophages derived from monocytes in vitro do not necessarily recapitulate in vivo macrophages.26 Myeloid-derived suppressor cells (MDSC) are present in certain diseases (e.g., cancer), and are an interesting lineage/group of cells which suppress immune responses.6,27,28 MDSC includes macrophages, but they will not be covered as a separate group of cells for the purpose of this review.

IV. M1 AND M2 MACROPHAGES A. M1/M2: Fight or FixThe Arginine Fork in the Road

As mentioned earlier, when macrophages are sampling and encounter something (be it a pathogen or damaged tissue), their receptors trigger a decision to Fight (kill it), or Fix (repair it). M1 and M2 biochemically defined molecules that macrophages produce to perform these diametrically opposed functions.1,2 Fight is mediated by macrophages preferentially producing NO (Nitric Oxide) that inhibits cell proliferation,31 while Fix is mediated by macrophages producing Ornithine that promotes proliferation and repair (through polyamines and collagen).2,32-34 Both NO (and Citrulline) and Ornithine (and Urea) result from the enzymatic cleavage of terminal nitrogen linkages of Arginine in slightly different ways: Arginine: O2CCHNH3 C3H6 NH C (NH)2 iNOS: O2CCHNH3 C3H6 NH C - NH C. Macrophages Wearing (Citrulline) + NO (Nitric Oxide) Different Outfits Arginase: O2CCHNH3 C3H6 NH (Ornithine) + C (NH)2 (Urea) Dendritic cells are unusual looking cells compared Importantly, NO or Ornithine production is to macrophages. But, evidence suggests that they favored in a macrophage because intermediates arise from the same precursor as macrophages in in each enzyme pathway inhibit the opposing bone marrow.29 Despite their distinctive morphol- pathway.35 A more elegantly simple mechanism for ogy, location in T cell areas of lymph nodes, and making a Fight or Fix response than this Arginine enhanced antigen-presenting capacity (on a cell per Fork in the Road2 is difficult to envisage. That cell basis), current evidence indicates that dendritic arginine metabolism is a central pathway in maccells do not perform any unique functions, or have rophages is indicated by the fact that the concenany unique markers, compared to macrophages.29 tration of this amino acid at sites of inflammation Like macrophages, they sample the environment, often declines to undetectable levels.36 make innate responses, and present antigens. Often, There are other changes in macrophages that dendritic cells and macrophages are separated in typically occur in concert with NO production articles and in immunologists minds that can be (e.g., increased Class II expression and production counterproductive. So, I will leave that debate to of IL-12/23 and IL-8) or Ornithine production others30 and consider dendritic cells as a special- (e.g., increased TGF-b, IL-10, IFN a/b, chitinases, ized subset of macrophages for the purpose of matrix metaloproteinases, and scavenger recepthis review. tors).3-6 As will be seen, these different molecules
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set in motion very different types of immune responses. M2 is the default activity in resident macrophages.1 And, TGF-b appears to be the most important cytokine for maintaining M2 because macrophages produced it endogenously, and it strongly inhibits NO production.1,37 There are several other macrophage cytokines (e.g., TNFa, IL-1b, IL-6) that have important physiologic effects such as fever and joint pain.3 However, the production of these inflammatory cytokines does not directly correlate with NO or Ornithine production (the very presence of macrophages at a site is inflammatory). In this connection, M1 and M2 refer to two different opposing activities.1 It does not mean there are only two types of macrophages, as will be discussed below. However, NO or Ornithine are the most characteristic molecules of macrophage Fight or Fix responses. This fact has made M1 and M2 very useful and has largely replaced the older terms classically and alternatively activated. B. M1/M2: Masters, not Servants, of T Cells As discussed, M1 and M2 refer to NO or Ornithine macrophage activity. However, the names M1 and M2 were specifically chosen because of two other sentinel findings: 1) M1 and M2 macrophage activities exist without T or B cell influence; 2) M1 and M2 macrophages stimulate Th1 and Th2 responses, respectively.1 The experiments that established M1/M2 and the sea change it helped bring about in our understanding of immune responses are discussed below. 1. The Dark Ages: The Th1/Th2 Paradigm. As discussed earlier, the fascination with vaccines and specificity allowed an Adaptive Dictator to control immunology. In turn, it came to be believed that differences in disease resistance between
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individuals could be attributed solely to differences in T or B cell responses.38-40 In particular, it was thought that one type of T cell response was responsible for activating macrophages (Th1), and another for stimulating antibody production (Th2). The perceived importance of T cells and Th1/Th2 can be traced to differences in resistance between C57Bl/6 and Balb/c mice to the Leishmania parasite.41 C57Bl/6 mice are more resistant and that correlates with increased T cell production of IFN- that activates macrophages to produce NO and kill the parasite.42 In contrast, Leishmania infection in Balb/c mice causes their T cells to produce more IL-4 that stimulates antibody production, but which does not kill the parasite. Actually, Th1 and Th2 propensities are not disease specific because we found that spleen cells of C57Bl/6 T mice make more IFN-, and BALB/c spleen cells more IL-4, in response to polyclonal stimulation also.1 Thus, according to the existing dogma, C57Bl/6 mice were Th1 and BALB/c Th2-type mice. However, as will be seen, other experiments in the lab were beginning to suggest that differences in T cell cytokine production between these mice were not due to T cells alone. 2. The Age of Enlightenment: Macrophage Renaissance The first observation that cast doubt on the validity of the Th1/Th2 paradigm was that isolated C57Bl/6 macrophages are more readily activated to produce NO by IFN- or Lipopolysaccharide stimulation than BALB/c macrophages.1,43,44 The results with Lipopolysaccharide were particularly informative because it demonstrated differences in macrophage Toll receptor reactivity (TLR4), a T cell independent response. Therefore, these findings suggested that differences in disease susceptibility between individuals could result from inherent differences in macrophage responsiveness. However, another explanation could have been that C57Bl/6 and BALB/c macrophages exhibited these differences because they had already been primed in vivo in

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a Th1- or Th2-dominant environment, respectively. To eliminate the influence of T (or T and B) cells, resident peritoneal macrophages were removed from Nude and SCID C57Bl/6 and BALB/c mice, and their M1 (NO) and M2 (Ornithine) activity was examined. It was found that macrophages from Nude or SCID C57Bl/6 mice exhibited M1dominant activity, while those from Nude or SCID BALB/c mice exhibited M2 - dominant activity. Indeed, it appeared that M1 and M2 differences in these mice were greater than in their normal counterparts. This was a surprising and important result because it indicated that propensities for M1 or M2 dominant responses (and disease susceptibilities) can be independent of T cells.1 The foregoing results also brought up the very intriguing possibility that differences in M1 and M2 responses between individuals could be responsible for differences in T cell cytokine production. To test this hypothesis, macrophages from SCID C57Bl/6 or BALB/c were again removed. Then, each was mixed with macrophage-depleted CB6 (C57Bl/6 X BALB/c F1 progeny) spleen cells (for histocompatibility). The polyclonal T cell activator, Con A, was then added to stimulate T cell cytokine production. It was found that SCID C57Bl/6 macrophages (M1) stimulated CB6F1 spleen cells to produce IFN-, while SCID BALB/c macrophages (M2) stimulated TGF-b production (TGF-b is also Th2-related; serum-free conditions did not allow IL-4 production). Since the T cells were the same, these results demonstrated that M1 and M2 macrophage- dominant responses stimulated T cells to make Th1- or Th2-dominant cytokine responses.1 Results in other experimental systems had also shown that antigen-presenting cells (whether called macrophages or dendritic cells) can influence that type of T cell response that occurs.45,46 However, the experiments in SCID mice described above were fundamentally different because they proved that macrophages can drive T cell responses without prior T cell influence. In turn, M1/M2 showed that the Th1/Th2 paradigm was insufficient to explain how immune responses work. As will be seen later, if one looks at the design and function of Innate

Immunity, it makes sense that macrophages are the central controlling element in immune responses. 3. M1/M2: Redefining Macrophage Activation Whereas I tried to inject some humor by describing an Adaptive Dictatorship, M1/M2 did help change our concept of what macrophage activation means that is worth explaining. Again, M1 and M2 activities do not require T cell influence. This fact is particularly important for understanding M2 macrophage activity. M2 (Ornithine production) is the normal default program present in resident macrophages. And, M2 macrophage activity that is similar (or identical) to that of resident tissue macrophages is found in sterile wounds and other circumstances where there is no T cell component.3,36 In contrast, the concept of alternatively activated macrophages arose mainly from the observation that addition of the T cell cytokines IL-4 or IL13 in vitro resulted in some changes in macrophages.47-49 More recent results suggest that alternatively activated macrophages are not a distinct in vivo phenotype, but instead most closely resemble resident macrophages (M2) in their gene and protein expression.4,23 Thus, alternatively activated and M2 are not synonyms because M2 (and M1) are T cell independent macrophage activities. The older perception that macrophages depend on T cell stimulation to be functional also lead to the additional sub typing of M2 macrophages into M2a, b and c based on in vitro stimulation with: IL-4/13; Ig and Toll receptor agonists; and IL-10 or TGF-b, and glucocorticoid hormones, respectively.50 Like alternatively activated, macrophages treated in these or other ways in vitro do have quantitatively different patterns of activity (e.g., phagocytosis), gene expression and cell surface markers.50 Macrophage M1 and M2 activity can be elevated by T cell-derived cytokines. However, M1/M2 most importantly means that macrophage propensities precede and direct T cell responses, not vice versa. Also, NO or Ornithine production is
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the clearest and most functional way to categorize opposing macrophage activities in Innate Immunity (or in directing Adaptive Immunity). C. M1/M2 and Macrophage Plasticity M1 or M2 describes macrophage activity committed to Fighting or Fixing, or stimulating Th1 or Th2 responses as described.1,2 Plasticity describes macrophages as a continuum of phenotypes.51,52 The two ways of describing macrophages, while seemingly at odds, are not. M1/M2 is useful because it describes succinct macrophage functions that influence the outcome of inflammation in polar opposite ways. And, it is important to understand that M1 and M2 specifically defines inflammatory circumstances where NO or Ornithine production predominates.1 Predominate is the key word because all inflammatory circumstances are clearly a mix of M1 and M2 responses. One need not look further than cultures of macrophages to know that there is a mixture of different cells (Figure 1).1 Some have interpreted M1 and M2 as meaning there are only two types of macrophages. Actually, in the original description of M1 and M2, plasticity was the word used to allow for the possibility that individual macrophages might produce varying amounts of NO or Ornithine.1 It does appear, however, that all macrophages produce one or the other. The important point then is that M1/M2 describes polar opposite responses, and plasticity describes the ability of macrophages to adapt incrementally (or continuously) to achieve M1 or M2 predominate activity, or varying mixtures of these responses. It may be useful to think of M1 as a school bus and M2 as a Ferrari; and plasticity as SUVs, minivans and compact cars. Using this analogy, if you needed to transport 40 people across the country, and two other people across the country quickly, what would you do? All the vehicles above transport people, but only the school bus and the Ferrari are ideal for these jobs. Similarly, macrophages that produce NO or Ornithine are ideally suited for certain circumstances. On the
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other hand, there may be circumstances where neither NO or Ornithine production is needed for a desired macrophage function, like phagocytosis. Going back to the analogy then, if one needed to transport seven people across the country, an intermediate type vehicle such as a minivan would be ideal. Intermediate-type macrophages with maximum phagocytic activity would likewise be ideal, regardless of NO or Ornithine production (if such cells exist). Macrophages with intermediate activities brings up a very important point relevant to both M1/M2 and plasticity. In either case, it is difficult to known from the study of populations of cells what is going on at the single cell level. For example, does an individual macrophage, when maximally activated (e.g., IFN- and LPS), fully turn on NO production and shut down the other pathway, or is there plasticity in this and the many other molecules macrophages produce. Even the study of individual cells does not answer this question because analysis at any point in time is a snapshot. For example, if a macrophage is committed to becoming pure M1, it can be caught transitioning there from M2, and appear to be an intermediate cell. Thus, by definition, there is plasticity. That said, plasticity is not likely to be fully bidirectional. In particular, once an M2 macrophage (or population) becomes M1 (NO-producing), it does not appear to revert back to M2. Although not proven, this makes intuitive sense because NO is toxic to macrophages also. Perhaps it depends on how much NO is induced intracellularly. As we learn more about the physiology of individual macrophages perhaps new distinct phenotypes will be discovered. In the meantime, M1/M2 and plasticity both have important and different roles in helping us understand macrophage functions. D. Beyond Plasticity: Are Macrophages Pluripotent? Regardless of how one views or labels macrophage activity, evidence keeps appearing to suggest that

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FIGURE 1. Demonstration of the dramatic effects of serum on functions and morphology of macrophages. TGF- appears primarily responsible. Reprinted by permission, J. of Immunology (Mills, C.D., K. Kincaid, J. A. Alt, M. J. Heilman, A. H. Hill. 2000. M-1/M-2 Macrophages and the Th1/Th2 Paradigm. J. Immunol. 164:6166.)

macrophages can transform into other cells.53-57 My own experience is that unstimulated macrophages, or, particularly, macrophages cultured with TGF-b exhibit a fibroblast-like appearance after a few days in vitro (Figure 1E). That these macrophages may be transitioning into fibroblasts (or some other cell) is supported by the fact that their phagocytic activity is markedly reduced.1 Having a cell in most tissues of the body (like macrophages) with pluripotent activity would be an advantage in times of tissue damage (e.g., spinal cord injury) or tissue deterioration (e.g., Alzheimers). Therefore, although additional results are needed to determine the potential pluripotency of macrophages, I think this is an area worthy of further investigation.

E. The Power and Pitfalls of In Vitro Immunology in the Discovery of M1/M2. The ability to analyze leukocytes in vitro has unique advantages, but can also cause conflicting results and conclusions because conditions dont resemble those occurring in situ or in vivo. Appreciating these pitfalls can lead to a better understanding of macrophages and other leukocytes. As an example, awareness of the composition of different culture medias played a key role in elucidating the NO and Ornithine pathways in macrophages. And, it also allowed experiments to be performed showing that M1/M2 activities can control the type of T cell response as discussed below.
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The majority of immunologists still use fetal bovine serum (or other serum supplements) in culture media. Fetal serum is used to avoid complications of heterophile and other antibodies present in adult serum. However, serum is obtained by lysing platelets, which contain a lot of TGF-b. And, TGF-b is the strongest inhibitor of M1 activity (NO production) and promoter of M2 activity (Ornithine production).2,35,37 Serum contains over 500 pg/ml TGF-b that is similar to the concentration found in wounds, and which is sufficient to markedly inhibit macrophage NO production.1,36 As shown in Figure 1, the effects of TGF-b on macrophage morphology and functions are dramatic. And, the unknown presence of this cytokine in vitro has lead to some erroneous conclusions. For example, inhibition of NO production by TGF-b in serum resulted in the belief that macrophages require two signals to become activated (e.g., IFN- and LPS).58 The use of serum-free media (actually contains albumin and other necessary nutrients) allowed us to show that either signal is sufficient.1 The use of serum (e.g., TGF-b) or other culture additives can lead to other types of pitfalls in analyzing macrophage functions. For example, since macrophages succumb to their own NO produced in vitro,59 culture in serum-free (no TGF-b) media elevates NO production, and decrease macrophage viability after three days.59 Therefore, if one then adds IFN-g or LPS to macrophages, one would come to the incorrect conclusion that culture in serum-free media decreased their ability to produce NO compared to serum-containing media. However, if one had added live bacteria at the beginning of culture, there would have been many fewer bacteria three days later in serum-free media compared to serum-containing media. Similar misinterpretations have come from pre culturing macrophages in IL-4 (that also decreases NO production in macrophages). Because serum-free media promoted M1, and serum supplementation (TGF-b) promoted M2 activity, T cell responses were also altered in important ways. Serum-free media stimulated a Th1-dominant T cell response
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(IFN- production), while serum stimulated a Th2-dominant T cell response (IL-4 and TGFb production). Recognition of this difference allowed us to show that M1 and M2 dominant responses can drive T cells toward Th1 and Th2 responses.1 Therefore, if a cell does not (or does) exhibit an activity in vitro, it cannot necessarily be concluded that the cell performs the same way in vivo. F. Human M1 and M2 Macrophages A particularly pertinent example of the tail wagging the dog (trying to make conclusions about in vivo immunology from in vitro results) is continuing debates about whether human macrophages display M1 or M2 characteristics.60-62 These debates stem mainly from observations that human monocytes do not produce much NO or Ornithine. But, compared to what? Mouse monocytes dont produce much NO or Ornithine either! Moreover, why would one expect monocytes (no known functions) to produce NO or Ornithine anyway? As will be discussed later, civilization has decreased infections (and wounds) in humans, so it seems reasonable to postulate that human macrophages produce somewhat less NO (or Ornithine) than other species. But, there is abundance evidence of the expression of both the iNOS and arginase pathways in different human disease conditions.63-65 This makes sense because these two enzyme pathways are present in every other vertebrate (and many invertebrate) species.15,33 Obviously, it is more difficult to study macrophages in humans than in rodents or other species. However, it is important to measure NO and Ornithine production in human macrophages because they are the best indicators of Fight or Fix activities. As a side note here, one might alternatively measure iNOS or arginase enzyme activities, or the genes that code for these enzymes as an indicator of M1/M2. It should be kept in mind, however, that enzyme levels or gene expression in general are indirect measures of what really matters, the functional products of macrophages, like NO or Ornithine. This fact is

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sometimes forgotten when results with enzyme or gene expression in macrophages are interpreted as direct evidence of variations in functions. V. M1/M2 MACROPHAGES IN NNATE IMMUNITY: DESIGN, DANGER, AND DIRECTION Having identified the basic functions of macrophages, and what M1/M2 represents, it is useful to describe how the design and the different responses of Innate Immunity protect a host. A. Design: Protect the Vascular System Humans (and other vertebrates) have evolved separate neural, endocrine, circulatory and respiratory organs connected by a vasculature system. The importance of protecting these organs caused the human Innate immune system to evolve a powerful three-tier system of protection designed to keep pathogens out of the vascular system.15,16 The first and most important tier is located near surfaces exposed to the environment (skin, lungs and the gut). Macrophages are the predominant cells in these areas.3 As macrophages sample events, self or non-self material is recognized. Also, epithelial cells secrete defensins at these surfaces. In parallel with these events, the second tier of protection begins with a natural fluid pressure gradient that carries antigens and other materials away from the local site into lymph channels and into draining lymph nodes (or similar type collections of lymphocytes). Here, material is sampled by macrophages again, and T and B cell proliferation occurs if foreign antigens are detected. Finally, the third tier of Innate protection comes from macrophages that reside in organs themselves (e.g., microglia). That the Innate System is designed to keep pathogens out of the vasculature (and thus the organs) was demonstrated by experiments in the 1960s. Introduction of live organisms (or antigens) intravenously rather than subcutaneously

was ineffective at generating immunity, and resulted in death or tolerance, respectively.66 As mentioned earlier then, by placing cowpox on the skin, Jenner inadvertently discovered much about how Innate Immunity is designed. HIV is a clinically relevant example of the importance of the route of exposure because it is very poorly infective, unless introduced directly into the blood. Of course, the clinical term for what happens when the immune system fails to keep pathogens out the vasculature is sepsis, which is often fatal. B. Danger: The Wake Up Call Any breach of exposed surfaces (skin, lung, intestine) is potentially dangerous. Polly Matzinger coined the fitting term Danger to describe the critical nature of the events that occur immediately after a breach.67 Speed and Overwhelming Force are important in response to Danger, regardless of the nature of threat. 1. Speed Why must macrophages respond quickly? One bacterium can become over 2 million in one day. One white cell can become two in one day! So, to overcome this overwhelming prokaryotic proliferative advantage, resident macrophages respond within minutes to molecules such as prostaglandins released from damaged cells in a skin wound. And, inside of an hour, macrophages are phagocytosing material and signaling other Innate cells to enter the area from the vasculature.25 2. Overwhelming Force Former US Defense Secretary, Colin Powell, used this term to describe successful warfare, and it aptly describes the goal of the initial Innate local response. Because macrophages need time to figure out if pathogens are present in the wound,
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the first Biologic Priority of the initial Danger response (416 hours) is simply to try to sterilize the area.2,36,68 Recruited neutrophils, macrophages (and sometimes Mast Cells) are the predominate cells that do this through the secretion NO and other oxygen radicals. Not surprisingly, there is collateral damage to normal tissue. The circumstances in the lung, intestines, or in organs, are normally somewhat different from a skin wound because the tissue damage is microscopic, not macroscopic. For example, macrophages in the lung are normally encountering and phagocytosing inhaled material, not damaged lung cells. Regardless of the location of Danger, macrophages are critically required to determine in what Direction the immune response should proceed. C. Direction: What Type of Danger is Present?
Know when to hold em, know when to fold emKenny Rogers (song lyric from The Gambler).

default (resident) M2 mode. Specifically, Ornithine, EGF, VEGF, and other growth factors are produced that are necessary for repair and resolution of the wound.69 Also, wound signals such as TGF-b and adenosine (from fibroblasts and other cells) are important in maintaining M2 activity.69 In parallel, phagocytosis is important to clear and digest damaged tissue and other debris. T cells are not noticeably involved in wound healing.70,71 Therefore, Damage Danger provides an example of the difference between M/1/M2 and Th1/Th2 because M2 macrophages heal the wound without the need for alternative activation by T cells.47 2. Localized Danger: Real AlarmM1 Macrophage Containment If pathogens are detected through PAMP, then a different set of signals is received3-6,20 and resident (and newly recruited) M2 macrophages become M1 macrophages within a day of infection. Also, IFN-g is produced by NK and other Innate cells at this time to further elevate the M1 response.71 The intestine is somewhat different. Macrophages there are routinely bathed in bacteria (and parasites before civilization), but unknown unique elements in that environment have evolved to routinely suppress M1 activation to allow nutritionally beneficial symbiosis. Similarly, ingestion of food (or other antigens) results in beneficial oral tolerance compared to exposure at other sites.72 Outside of a sterile operating room, most skin injuries (or air breathed, food eaten) contain potential pathogens. Therefore, it would seem that there is daily Localized Danger, but M1 activation is normally sufficient to contain pathogens without T cell involvement. 3. Definite Danger: M1/M2 Induction of Adaptive Immunity If the pathogen is not eliminated at the first tier of defense within a few days, antigens or pathogens

This lyric about poker playing colorfully describes the critical nature of the decisions macrophages make in determining what Direction to proceed if there are pathogens present, if they can be contained, or if Adaptive Immunity is needed. 1. Damage Danger: False AlarmM2 Macrophage Repair During the initial Danger phase, macrophages are also determining what Direction to take based on the nature of the threat. The extent of damage is determined by receptors for DAMP (damage associated molecular patterns). Concurrently, other receptors recognize pathogens by PAMP (pathogen associated molecular patterns). If no pathogens are detected, the Biologic Priority switches from sterilization to repair. Monocytes recruited from the blood primarily handle this process.5 Because there are no new Danger signals or pathogens present, the recruited macrophages remain in the
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enter the lymph channel and travel to the second tier - the draining lymph nodes. There, macrophages (and/or dendritic cells) present antigens and Class II antigens together because T cells are too stupid to do it on their own (Innate Immunity humor). This event results in clonal proliferation of specific T cells.73 And, as mentioned earlier, it is now clear that if the pathogen stimulates an M1 dominant response, then macrophages send signals to T cells (IL-12/23) to make a Th1 response (primarily IFN-g) that further elevates the M1 response. M1 also seems to drive the more recently discovered Th17 response associated with autoimmunity.74 On the other hand, if macrophages remain as M2 (because some pathogens subvert M1 responses, to be discussed), then a Th2 response (e.g., IL-4/13, TGF-b, IL-10) occurs and results in antibody production.75 And, the cytokines produced during Th1 or Th2 responses reciprocally down regulate M2 and M1 macrophage responses so one response (that may be optimally protective) can dominate.48 In this regard, only birds and mammals make true antibody responses.(15) Presumably, antibody responses evolved to better handle pathogens by utilizing the circulation to provide more effective systemic protection and/or because antibody is more effective against certain pathogens than macrophages. Also, antibody may have also evolved to handle pathogens that can subvert M1/Th1 responses (to be discussed). The foregoing description of how Innate Immunity functions was based on a host encountering a pathogen for the first time. If there is preexisting T cell memory (a higher frequency of specific T cells), or existing antibody from a prior exposure, then Adaptive Immunity will play a slightly different role. For example, preexisting antibody might be sufficient to halt a pathogen without requiring a secondary antibody response. Nonetheless, Innate Immunity still functions as the initial gatekeeper to identify threats and respond accordingly. And, as will be seen later, M1/Th1 and M2/Th2 responses each have important and different roles in preventing (or causing) many modern diseases.

4. M1 Danger: Immunoregulation and Damage by NO Up until now, the protective functions of M1 responses (NO, antigen presentation) have been described, but M1 also has important immunoregulatory functions.2 NO produced by what are often called suppressor macrophages diffuses freely in all directions, and can inhibit T cells, or whatever other normal cells it encounters (recall collateral damage in the early wound response).76,77 Therefore, NO creates a conundrum during immune responses. For example, T cell production of IFN-g (that elevates NO production in macrophages) is required for protection against many major infections. However, it is clear in some circumstances that the NO produced then inhibits the T cell response in vivo.78 The suppressive effects of macrophage NO production can cause different outcomes depending on the amount produced, the timing, and the disease involved. For example, during a response to a pathogen (like Leishmania, where macrophage NO is required), if there is a lot of NO produced early, then T cell activation and proliferation can be strongly inhibited and the pathogen grows. If sufficient NO is produced during T cell amplification, then the pathogen is eliminated. If surfeit NO is produced during T cell amplification, the T cell response can be partially inhibited and a chronic infection may result. There are examples of all three of these circumstances with pathogens.2 It has not yet been determined if the inhibitory effect of NO evolved to immunoregulate overzealous T cell responses, or is just an unintended side effect. Either way, there are other diseases (particularly viruses), where only a T cell response (e.g., cytolytic) is curative. In these circumstances, concomitant NO production is clearly inhibitory what I termed an effector deflector.2,78 Aside from these different immunoregulatory effects, NO can also exacerbate certain diseases directly because the cure is worse than the disease. Specifically, excessive NO production can cause
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more damaging effects in the lungs, intestines and the brain than the pathogen itself. There are numerous examples of this phenomenon with bacteria (Mycobacterium), parasites (Toxoplasma ghondi) and viruses (HIV).2 Finally, another perplexing element of the NO conundrum is the ability of M1 macrophages to stimulate a Th1 response, as described earlier. It would seem that if an NO producing macrophage presented antigen to a T cell, it would kill it. Therefore, it seems like something is going on during antigen presentation that remains unknown. The foregoing results make it apparent that consideration of both the protective and destructive effects of an M1 response need to be taken into consideration when designing vaccines or other therapeutic strategies to be discussed later. VI. M1/M2 MACROPHAGES: BIDIRECTIONAL COMMUNICATION WITH NEURAL AND ENDOCRINE SYSTEMS A. Introduction
No man is an island John Donne (1624).

Just as we have seen that M1/M2 responses act in concert with Th1/Th2 responses, it is also important to recognize that the immune system is not a stand-alone organ. One only need be reminded of how it feels to have the flu (or getting sick after stressing out about a grant renewal!) to understand that the immune system is interconnected with the neural and endocrine systems. So, although it is not my expertise, I thought a brief discussion of how these systems influence each other is important. B. Normal Stress and Beneficial M1/M2 Responses The neural and endocrine systems help to maintain homeostatic balance by reacting to stress.79 The fundamental objective of a stress response
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is likely to promote survival of the individual via neurotransmitters and hormones that mobilize energy from stores (e.g., glucose) to allow increased physical and mental activity (e.g., running from a dog), and increased immunologic activity (e.g., if the dog bites your head). Processive stressors are those that elicit the fight-or-flight reaction from facets within the hosts environment that are perceived by the host as potential dangers, but do not cause harm directly.80 When a host senses a threat, the pituitary gland responds involuntarily by releasing a surge of ACTH, which acts on the adrenal glands to release several stress hormones, including epinephrine and cortisol. In contrast, systemic stressors are those that actually pose a threat to homeostasis, such as extreme pain, dehydration or injury. These stressors require much less cognitive processing than processive stressors and often occur simultaneously with processive stressors. Systemic stressors initially activate the same type of neural and endocrine responses as processive stressors. As we saw earlier, the Innate Immune system makes its own response to stress in the Danger response. And, this response communicates with the neural and endocrine systems in several ways. For example, macrophages produce molecules such as IL-1, IL-6, and TNF-a that cause the brain to increase body temperature and create a feeling of malaise. These effects are beneficial because fever helps kill microorganisms, and malaise signals a host to sequester itself (e.g., in the house) until normal function is restored (dog bite heals). I used the example of a dog bite in the head because it illustrates that macrophages (in this case microglial cells) are located in most organs, and form the third tier in the Design of the Innate Immunity. In addition, microglial cells are in constant bidirectional communication with astrocytes that is necessary for normal brain function.81 Microglial cells, like macrophages elsewhere, can make M1 responses to deal with an infection (dog head bite), or an M2 response to heal a head injury (sterile dog head bite). Thus, the neural, endocrine, and immune

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systems have evolved important bidirectional communication pathways that serve to optimize host performance and survival under conditions of processive or systemic stress. C. Abnormal Stress and Detrimental M1/ M2 Responses Unlike normal stress that is acute and beneficial, chronic or severe stress degrades the neural, endocrine and immune network. In particular, prolonged elevation of cortisol depresses Innate Immunity. One example is life-long spouses. If one dies, the other often dies shortly after, in part, because of depressed immunity.82 Another important effect of chronic stress is that it specifically decreases the M1/M2 ratio.83 Chronic inflammation also can be deleterious because byproducts of Innate Immunity, such as glutamate, cause decreases in serotonin and dopamine that maintain a healthy mood. Schizophrenia, depression, and other behavioral disorders can be exacerbated by chronic inflammation.84 Although macrophages are the primary link to the neural and endocrine systems, if Innate Immunity stimulates Adaptive Immunity, T cells produce similar molecules as macrophages that also degrade the neural and endocrine systems.82 Civilization has markedly affected the type of stress we experience, and has contributed to changes in the balance of M1/M2 as described in the following section. VII. M1/M2 MACROPHAGES IN MODERN CIVILIZATION As it was instructive to understand how Innate Immunity works, it is useful to appreciate that Civilization has changed Innate Immunity. Civilization has roughly doubled humans lifespan. Many modern diseases (cancer, atherosclerosis, autoimmunity) are the result of these two changes. Civilization (in evolutionary terms) is a recent event. Therefore, the immune system has not had much time to adapt. In addition, most modern

diseases occur later in life. Therefore, there is little evolutionary pressure (breeding preferences) for the immune system to adapt. Below are some changes Progress (P) has brought in the input that Innate Immunity receives that have changed the balance of M1and M2. A. Predation to Pets Until fairly recently, the biggest threat to man was being wounded or killed by tigers, or snakes (predators), or other humans.85 Now, we only have pets! Loss of predation/acute stress responses and decreased infections have decreased M1 responses. A useful comparison of early and modern man is mice versus men. Mouse macrophages have a stronger M1 component because they are exposed to predators and more infectious agents.60,61 Net Result: Decreased M1/M2 ratio. B. Pestilence to Purity The major reason for the decrease in infectious disease (and M1) in the last couple of centuries has been increased sanitation. In addition, food sources have been simplified and sanitized. Approximately 70% of the worlds food now comes from 6 grains and one animal (cows), and more of the food consumed is sterile. This has reduced the variety and quantity of the intestinal floral which also decreases M1 responses.85 In a similar manner, antibiotics and vaccines have decreased infectious diseases resulting in decreased M1 responses. Because products of M1 and M2 responses mutually inhibit each other, M2 responses also have increased. Net Result: Decreased M1/M2 ratio. C. Paradise to Pressure One might not think of earl mans existence as paradise. However, industrialization, increased population density and other factors have altered
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the type of neuroendocrine stress we experience. For example, housing and transportation have decreased acute stress (e.g., being attacked while sleeping or walking). In addition, crowding, jobs, financial pressure, etc., have elevated chronic stress. As discussed, both of these changes in the type of stress experienced alter the M1/M2 balance.82 Net Result: Decreased M1/M2 ratio. D. Petite to Pounds The major increase in obesity in civilized countries in the last century has caused excess circulating energy (glucose, fatty acids) that fools the immune system into making chronic Danger responses on vessel walls.86 Energy Imbalance and Metabolic Syndrome are terms used to describe this important new civilization-induced replacement for infection-induced M1 responses to be discussed in the next section. Net Result: Increased M1/M2 ratio In summary, Civilization has markedly decreased major infections, and brought other changes to make modern human Innate Immunity M2 dominant. M1 and M2 responses that evolved to protect early man are now often inappropriate in modern man because of damage they cause during increased lifespan. Diseases associated with inappropriate M1 and M2 responses, and how one might alter them for better health is discussed in the following section. VIII. M1/M2 BALANCES IN MODERN DISEASES AND STRATEGIES FOR HEALTH. The causes and cures of modern diseases can be complex. At the same time, many diseases exhibit inappropriate M1/M2 balances that play a role in disease pathologies. M1/M2 came about mainly from my studies of cancer and wounds, and are the conditions I know best from an immunologic perspective.2,87,88 And, there is considerable
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therapeutic potential for changing the M1/M2 balance in cancer.89,90 Also, the immunologic circumstances that occur in cancer are applicable to other diseases involving inappropriate M1/M2 balances. So, I will use cancer (and its relationship to a wound) as the central model to develop some general ideas about how to develop effective therapeutic strategies. A. Background to Cancer The history of diseases can be instructive. This is particularly true in cancer. Cancer is with us today because it occurs mostly later in life, and therefore does not affect breeding; there is little evolutionary pressure against cancer. Most other modern diseases (e.g., atherosclerosis, allergy, autoimmunity) are also with us for the same reason. Cells populating tumors were observed by Virchow in the 1800s. Around the turn of the 20th century, the primary cells that populate tumors came to be called macrophages. About the same time, Dr. Coley had observed that life-threatening infections in cancer patients sometimes resulted in complete tumor regression. So, he developed Coleys Toxin (a bacteria mix) and used it to successfully cause complete cancer regression in some patients.91 However, there was unacceptable mortality and Coleys Toxin has been mostly forgotten. Later on, during the Adaptive Dictatorship, it came to be believed in the 1960s and 70s that cancer develops now and then, and the immune system specifically recognizes and eliminates it.92 As appealing as this notion was, a majority of cancers studied did not express unique antigens. A telling blow to the Immunosurveillance theory was the observation that T cell deficient mice (Nude) exhibited nearly the same incidence of cancer as normal mice.93,94 Additionally, it has long been observed that invertebrates and other lower organism that do not possess Adaptive Immunity do not have a higher cancer incidence than humans.16 So, what are macrophages doing in tumors, and what

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mechanism had Coley (and others) been able to stimulate that caused cancer regression? The relationship between cancer and wound repair provides salient insight. B. Innate Immunity and Cancer: M2 Promoted Growth As mentioned, macrophages are the primary leukocytes in tumor beds. M1 macrophage activity can clearly kill tumor cells31,95 and the presence of intratumor Fight M1 macrophages is very favorable for survival.96 However, evidence indicates that intratumor macrophages are primarily M2 Fix dominant.6,97-99 What goes wrong? Cancer and wounds may seem odd bedfellows, but are intimately linked. Cancer has accurately been called wounds that do not heal.100,101 Consider back to the discussion of Damage Danger present in a wound. Initially, there is an M1 response that serves to sterilize the area. However, if the wound is sterile, an M2 -dominant response occurs to provide Ornithine (polyamines, collagen), EGF, VEGF and other growth factors that are required for proliferation, repair and resolution. That this environment is conducive to cancer is evidenced by the preferential appearance of tumors at sites of wound repair,102-104 and is oft called the Seed and Soil Hypothesis.105 Cancer is analogous to a sterile wound in that there are no (or insignificant) specific antigens, as mentioned. Therefore, having M2 macrophages in a tumor is not simply neutral, but can promote growth.106 That M2 macrophages are actively involved in stimulating cancer growth is evidenced by decreased growth rates in hosts depleted of macrophages.107 In this regard, a tumor can only grow to about one million cells before it requires new blood vessel support (about the size of the period at the end of this sentence). But cancers dont normally produce angiogenic factors, and so require M2 macrophages to provide it.108,109 Whereas a wound naturally heals, cancer keeps proliferating and pushes normal tissue aside,

resulting in new Danger signals and signaling more monocytes to enter from the circulation. Returning to the wound analogy, why dont newly recruited macrophages keep making the early M1 response that would kill cancer cells? Cancer cells have evolved active strategies to subvert Innate Immunity by preventing curative M1 responses from developing. Cancer cells produce a variety a factors such as TGF-b and prostaglandin E2 that inhibit M1 (NO) Fight activity, and keep macrophages in M2 (Ornithine) Fix mode.110 Also, cancer cells stimulate metaloproteinases in macrophages to aid in the breakdown of matrix allowing continued encroachment into normal tissue. Thus, cancer is mainly a disease that is not recognized by T cells, and whose growth and metastasis are aided by keeping macrophages in M2- wound healing mode. An unrelated, but interesting possible connection between cancer and M2 macrophages is the influence of stress. As discussed earlier, chronic stress is more of an element in modern life than acute stress and chronic stress has been shown to decrease the M1/M2 ratio.83 Although it is difficult to establish cause and effect, there is a wealth of evidence associating failure to handle stress (antisocial behavior, depression, etc.) with increased cancer incidence and growth rates. Sometimes this connection is referred to as the Melancholy Personality in cancer.111 1. M2 Promotion of Infectious Diseases Many pathogens, like cancer, have evolved mechanisms to actively keep M2 macrophages from transitioning to M1. For example, Mycobacterium , Leishmania and HIV all subvert macrophage physiology for their own survival and benefit.3,112,113Some do this by expressing an antigen that fools macrophages into thinking they have encountered self (preserving M2 activity), while others cause macrophages to secrete cytokines (e.g., TGF-b or IL-10) that prevent M1 activation.3,6
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There are human cancers that do express unique tumor antigens (e.g., Melanoma) and so are foreign like infectious diseases.114 However, as with infectious diseases, the same problem remains. Cancer cells subvert M1 responses to M2. And, because Innate Immunity is required for T cell responses, the inhibition of M1 prevents the development of an effective anti cancer Th1 response. This phenomenon was observed many years ago in my laboratory using the P815 Mastocytoma in mice. P815 elicits a tumor-specific cytolytic T cell and an activated macrophage response in a host, but the responses are insufficient to cause tumor rejection.9,87 It can be seen in Figure 2 that if a host is specifically preimmunized against the P815 tumor, and rechallenged with tumor, there is robust M1 activity (NO) in the tumor bed and the tumor is rejected. However, in a nave mouse, the P815 decreases M1 and increases M2 activity allowing progressive tumor growth in much the same manner as some pathogens.

3. Therapy of M2-Dominant Diseases

Once cancer appears, transiently boosting M1 (and/ or inhibiting M2) responses locally or systemically seems to have great potential.90,119 Because allergy is often localized and seasonal, there seems even greater therapeutic potential for transiently increasing the M1/M2 ratio in the airways.120 Although many autoimmune conditions seem to result from overzealous M1 responses (e.g., MS, inflammatory bowel disease, arthritis),3 SLE appears to mainly result from excessive M2- mediated autoantibody production, and could be a good candidate for increasing the M1/M2 ratio also.121 Finally, as discussed, certain infectious agents subvert M1 to M2 as recently discussed by others.3 There are interesting new strategies to increase the M1/M2 ratio, including injected and inhaled adjuvants, as well as probiotics and other materials that work orally. Some of the most promising agents are Toll receptor agonists. However, it is beyond the scope of this review so readers are referred elsewhere for that information.119,120,122-125 2. M2 Promotion of Allergy Increasing the M1/M2 ratio in cancer seems logical. However, the vexing Two-Edged Sword Cancer may seem unrelated to allergy. However, nature of immune responses comes into play in consider both conditions in light of civilization- cancer and other diseases as discussed below. induced M2- dominant immune systems. Allergy and Asthma are clearly increasing because of increased M2/Th2 responses, and/or decreased C. M1 Oxidants and Cancer: M1/Th1 responses.3,115,116 Decreased infections Accumulation of Damage combined with increased hygiene have lead to the popular Hygiene Hypothesis to explain increased For cancer to appear, a normal cell must lose growth allergy.117 For example, people raised on a farm control become immortal. And, unlike a growing (dirtier) environment have lower incidences of tumor, it appears that overactive M1 responses over allergy.118 Although somewhat different, cancer the course of a lifetime may not only not routinely actively promotes M2 responses for its growth. eliminate new cancer, but can sometimes cause it. And, recall that the only well documented cases That is, expression of NO and other oxygen radicals of cancer regression occurred when hosts had mas- can be carcinogenic/mutagenic.126 Lung cancer sive infections.91 Looked at in this light, allergy seems the best example. Continued inhalation of and cancer are both conditions exacerbated by tar and other materials causes resident alveolar a decreased M1/M2 ratio. A summary of some M2 macrophages to chronically sense Danger diseases where an M1/M2 imbalance is important resulting in an M1 response with attendant local is shown in Figure 3. damage. In line with this wounding there is
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a continuous cycle of M2 repair. And because humans have limited capacity to regenerate tissue, repair is imperfect (e.g., scarring), and lung function declines. In addition, continuing M2 repair creates a fertile soil for the seed much like it does in non-healing wounds.100 Sun-induced skin damage resulting in skin cancers, or cancer of the bowel from chronic inflammation, are two other examples where chronic M1 oxidant damage are implicated. Thus, M1 responses that were required for survival in early man (and which are beneficial against existent cancer) can result in accumulation of damage over time and cause cancer. 1. M1 Responses and Autoimmunity As in cancer, the accumulation of M1 Oxidant damage (and associated M2 repair) seem to play an important role in many autoimmune conditions such as MS, inflammatory bowel disease, arthritis and Alzheimers disease.3,127.128 This might seem at odds with the fact that M1 responses overall are less common in modern civilization, as discussed

earlier. However, as in cancer, accumulation of exposure because of increased longevity is important because most autoimmune conditions develop over many years. These conditions also resemble most cancers because although there may sometimes be a viral or microbial vector initially involved, once established, they do not express classical foreign antigens. Therefore, Innate, not Adaptive, Immunity is primarily responsible.3 2. M1 Responses and Atherosclerosis In the last several years, it has come to be appreciated that macrophages are the primary leukocyte component and main cause of atherosclerotic plaques.86,129,130 And again, like cancer, the endothelial lining of vessels does not display classical foreign antigens. So, why are macrophages causing reactions in the blood vessel walls resulting in plaque formation? The answer seems to lie in chronic excess energy availability, resulting from what I called Pounds (obesity). Adipose tissue somehow resembles a wound and attracts mac-

FIGURE 2. Evidence that intratumor macrophage arginine to NO or Ornithine is responsible for rejection or progression of cancer. Reprinted by permission, J. of Immunology Mills, C.D., J.D. Shearer, R. Evans, M.D. Caldwell. 1992. Macrophage arginine metabolism and the inhibition or stimulation of cancer. J. Immunol. 149:2709.
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FIGURE 3. M1- or M2-dominant immune responses are associated with specific diseases.

rophages that produce TNF-a, NO and other inflammatory mediators, and their production is proportional to the amount of adipose tissue.86 Along with this inflammation, obesity causes an inability of insulin to store glucose in fat (insulin resistance), resulting in obesity-onset diabetes (now called Type II). In turn, glucose and fatty acids increase in the circulation. This circumstance resembles what occurs in acute stress (recall early man being chased by a tiger) that shunts extra energy to the circulation to properly deal with a threat. However, in obesity this excess energy becomes chronic and fuels M1 responses. We showed some years ago that induction of diabetes in rats or mice results in elevated M1 responses, and which was corrected by insulin administration.131 Although it is not altogether clear, the increased concentration of glucose and fatty acids in the circulation seems to be the stimuli that fools macrophages into thinking there is Danger in the endothelial lining. Somewhat analogous to a smokers lung or
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a non-healing wound, the continuing deposition of fatty acids perpetuates a cycle of M1 damage and M2 repair resulting in an enlarging plaque. Thus, the problems associated with diabetes and atherosclerosis are increasingly being viewed as diseases of chronic M1 responses.132 3. Therapy of M1-Dominant Diseases Unlike tumor growth where both decreased M1 and increased M2 activity can exacerbate disease, diseases where M1 is contributory seem to result primarily from chronic low -level M1 damage. Because atherosclerosis is a disease of the vasculature and certain autoimmune conditions are localized to particular organs (e.g., inflammatory bowel disease), potential exists to specifically decrease M1 responses in the areas affected.125,133,134 How one might devise particular therapeutic strategies to simultaneously deal with M1 and M2related conditions are discussed below.

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D. Type, Territory, Timing and Tenacity of M1/M2 Therapy From the foregoing overview of M1/M2 imbalances in modern diseases, it is clear that no single therapeutic strategy suits all conditions. At the same time, diseases appear at different places in the body and at different times in life that opens up windows of opportunity for developing targeted immunologic interventions. However, success will depend on taking into account the Type, Territory (e.g., systemic or local), Timing and Tenacity of the therapy. As an example, it is widely believed that decreasing oxidant damage (e.g., M1) through nutrition and lifestyle changes is a successful Type of strategy. This seems logical in so far as chronic cellular damage results in aging and some cancers. Also, atherosclerosis would benefit from decreased M1 responses. The Territory to be covered would be systemic because damaging M1 responses are. As for Timing, this strategy makes sense in adults, after childhood diseases have waned, and when M1-mediated atherosclerosis appears. As for Tenacity, it is worth considering that M1 is already decreased, so a mild lifetime decrease in M1 might be appropriate. Decreasing M1 over a long period does again bring up the TwoEdged Sword nature of immune responses. That is, to the degree that Innate Immunity routinely eliminates cancer, decreasing M1 could result in greater incidences. By considering the Type, Territory, Timing, or Tenacity, more than one intervention could be employed in the same individual for another disease. For example, increasing the M1/M2 ratio is a different Type of strategy than discussed above that would be beneficial against growing cancer or allergies. However, the Territory treated could be limited in the case of localized cancer. Allergy is an even better example because the Territory is most often localized in the airways. Even if increasing the M1/M2 ratio needed to be systemic, the Timing of cancer treatment could be brief, or

seasonal with allergy. The Tenacity in the case of cancer might have to be high because of previous observations that regression only occurs if there is a substantial infection.91 Therefore, as one looks ahead to therapeutic strategies it is useful to view immune responses not as Two-Edged Swords, but different swords that can be wielded in different ways and at different times for better health. IX. SUMMARY AND PERSPECTIVE It is apparent that most diseases plaguing modern civilization result from inappropriate Innate immune responses, be it Type, Territory, Timing, or Tenacity. Most of these responses distill down to overactive or underactive M1 Fight or M2 Fix responses. It is also helpful to view todays human M1 and M2 responses as vestiges of a dirtier time when they were necessary for survival (early in lifeto reach breeding age). In turn, in our increasingly germ-free and long-lived societies it may be useful to somewhat decrease overall immunity (M1 and M2), towards what could be termed immunocivilization or M0. After all, we seem to be doing fine with decreased M1, and the level of M2 doesnt seem beneficial (and contributes to cancer and allergy, etc.). Given the central role of the immune system in modern disease pathologies, it is now important to include routine immunologic testing of individuals to assess who might benefit from an immunologic intervention before disease onset. In this regard, it is apparent that M1 and Th1 (or M2 and Th2) responses most often work in concert to create characteristic immune responses. Therefore, I think it would be useful in many circumstances to adapt a simpler, more inclusive terminology such as Immune Type 1, 2, or 0 (IT1, IT2, IT0) to describe an individuals immunologic status. In conclusion, viewing Innate Immunity mainly from an M1/M2 perspective has limitations as I said at the outset. However, it has become abundantly clear in the last few years that Innate
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Immunity controls both inflammatory and specific responses, and M1 and M2 activities are the essence of this control. Simple. ACKNOWLEDGMENTS I am grateful for the associations I have had over the years that have allowed me to look at the immune system from different angles. As my postdoctoral advisor, Robert North of the Trudeau Institute introduced me to macrophages (where, with his help, macrophage activation had been discovered).22,135 At Brown University, my association with Michael Caldwell and Jorge Albina opened my eyes to the importance of metabolism, specifically arginine, in macrophage physiology. I am grateful to my friend, John Hibbs, in Utah who discovered Nitric Oxide that helped me develop M1 and M2 macrophages. Finally, I am most fortunate to have had superior students and employees to supply fresh ideas, and my many friends and my children that make life rich and whole. I dedicate this article to two such friends who died before their time this year: Dr. Bob Stout (a fellow mac guy 51,52), and Dr. George Ebert (my life-long friend), who died suddenly while attending his mothers funeral. REFERENCES
1. Mills CD, Kincaid K, Alt JM, Heilman MJ, Hill AM. M-1/M-2 macrophages and the Th1/Th2 paradigm. J Immunol. 2000;164:616673. 2. Mills CD. Macrophage arginine metabolism to ornithine/urea or nitric oxide/citrulline: A life or death issue. Crit Rev Immunol. 2001;21:399425. 3. Murray PJ, Wynn TA. Protective and Pathogenic Functions of Macrophage Subsets. Nat Rev Immunol. 2011;11:72337. 4. Lawrence T, Natoli G. Transcriptional regulation of macrophage polarization: enabling diversity with identity. Nat Rev Immunol. 2011;11:75061. 5. Cassetta L, Cassol E, Poli G. Macrophage Polarization in Health and Disease. SciWorld
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6. 7. 8.

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