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Q: Dr. Leitinger, I am very excited that you agreed to comment on your outstanding study that was published in Circulation Research in September 2010. It is one of those where we start to wonder why did not think of this earlier. In essence, your group identified a new macrophage phenotype that can be found in atherosclerotic lesions, which you designated “Mox” because of its induction and role in oxidative stress handling. Please tell us more about the highlights of your study.
A: Thank you for your interest and the opportunity to talk about our findings. We definitely believe that the identification of this new macrophage phenotype is a major step forward toward understanding the chronic inflammatory state that can be seen in oxidatively damaged tissues. In my opinion, the complex pathology that drives the formation of atherosclerotic lesions cannot be explained by the oversimplified model of only 2 major macrophage phenotypes (M1 and M2). The specific gene expression pattern and biological functions of the Mox phenotype may help explain some of the pathological features that are seen in advanced atherosclerosis and even be indicative of plaque instability. We were surprised to find that in established murine atherosclerotic lesions about one third of all lesion macrophages were of the Mox phenotype, readily distinguishable from M1 or M2 macrophages. We also found that Nrf2-dependent gene expression was a distinctive feature of Mox macrophages.
Q: What sparked the search for this phenotype? What is intuition or were, for instance, wondering what would further happen to the monocytes/macrophages attracted and continued to be exposed to oxidized phospholipids (OxPAPC).
A: We started from the premise that the immunological tissue microenvironment determines phenotypic polarization of macrophages. If we consider the microenvironment in chronically inflamed tissue, especially in atherosclerotic lesions, it is comprised of various oxidatively modified molecules, including lipid oxidation products. Therefore, we were curious about the response of infiltrating macrophages, once they encounter oxidized lipids in chronically inflamed tissues. To examine this question, we incubated macrophages with oxidized phospholipids, which are abundant in atherosclerotic lesions, and compared gene expression patterns with those of conventional M1 or M2 macrophages. We were surprised to see that macrophages that encounter oxidized lipids respond in a quite distinctive manner, resulting in the described novel phenotype (Mox). That does not mean that oxidized phospholipids are the only stimuli that can induce the Mox phenotype. Our finding that Mox are characterized by upregulation of a set of Nrf2-dependent redox-regulatory genes points to the possibility that many different factors that upset the redox balance in macrophages can induce this phenotype. We therefore concluded that the Mox phenotype is characteristic of general oxidative tissue damage and thus may be representative for chronically inflamed tissues other than atherosclerotic lesions as well.
Q: The first step, as I understand, was to take resting macrophages and to leave them untreated or treated with IFNgamma + LPS for the M1 phenotype and IL-4 for the M2 phenotype and OxPAPC for an oxidative phenotype (Mox). Was the latter hypothetical at the start?
A: We initially hypothesized that oxidized phospholipids would drive the macrophages towards an M1-type polarization. We were surprised to see the striking difference to the conventional M1 phenotype. However, there is some overlap in expression of inflammatory genes between M1 and Mox, indicating that the Mox phenotype contributes to the “low grade” inflammation seen in chronically inflamed tissues.
Q: Is this differentiation pattern limited to bone marrow-derived macrophages? Moreover, can only be undifferentiated macrophages differentiate into Mox or can M1 and M2 phenotype turned into Mox? In other words, are these macrophages separated out from the beginning and the exposure of OxPAPC reveals them or is re-differentiation possible and essentially any macrophage can turn this way?
A: These are excellent questions, which we also tried to address in our study. As it turns out, the Mox phenotype (as well as M1 or M2 phenotypes) can be readily produced in vitro from various kinds of macrophages, including Thp-1 monocytes, Raw cells, murine bone marrow-derived and peritoneal macrophages. To address your second question we treated M1 or M2 macrophages with oxidized phospholipids and surprisingly they switched their phenotype into the Mox phenotype. This was not only demonstrated by upregulation of Mox-specific genes, but also by downregulation of M1 genes. These data show the remarkable plasticity of tissue macrophages, being able to respond to changes in the microenvironment by adapting gene expression patterns and switching to the appropriate phenotype. These findings also imply that the relative abundance of macrophage phenotypes changes during lesion development and progression.
Q: The Mox phenotype is morphologically and functionally distinct. Does their lower chemoattracting and phagocytic capacity suggest that this is not their primary role? Are they less for immunological defense and more for something else? What is their primary role?
A: I can only speculate about the primary role of Mox macrophages. The fact that they express genes such as IL1 and COX-2, although to a lesser degree than M1 macrophages, indicates that the Mox phenotype contributes to the “low grade” inflammation seen in chronically inflamed tissues such as atherosclerotic lesions. This is further underlined by our finding that Mox induce chemotactic activity, which was much lower than that induced by M1, but still significantly higher than M0 (untreated). The reduced phagocytotic capacity of Mox indicates that they play a detrimental role in lesion development, probably even contributing to destabilization of lesions. However, much still needs to be learned about the biological features of the Mox phenotype, particularly about its role in lesion development.
Q: You performed extraordinary gene expression profiling and confirmed this with RT-PCR, pointing into the direction of oxidative stress (representative HO-1), ER stress (representative Trb3), and angiogenesis (VEGF). Regulating these processes is this the main role of the Mox macrophage for itself or other cells in the atherosclerotic plaque?
A: In Mox, a significant amount of energy is used for coping with oxidative stress, which may explain the reduced capacity to perform other biological functions. As we and others have shown, Nrf2-dependent gene expression in response to oxidative stress leads to prolonged cell survival in toxic environments. While the major functions of M1 and M2 macrophages are to boost the inflammatory response and to facilitate tissue regeneration, respectively, Mox macrophages may be involved in balancing the redox status in areas of oxidative tissue damage. However, we need to learn more about the biological functions of this phenotype to fully understand its properties.
Q: Which transcription factors did you identify that control the expression of these genes in Mox. Which ones are yet to be identified?
A: Based on gene expression analyses, we identified the redox-regulated transcription factor Nrf2 to be associated exclusively with Mox, but neither with M1 nor M2 macrophages. Although not shown in the current paper, we never observed activation of the NFB pathway by oxidized phospholipids. It will be interesting to see which transcription factors control the expression of inflammatory genes in Mox.
Q: Importantly, you were able to distinguish Mox from M1 and M2 macrophages in atherosclerotic plaques. How much is the relative contribution of these individual phenotypes to the total macrophage population in the atherosclerotic plaque? Is there any difference in the composition among different plaques types? Could different composition, for instance, a relative abundance of the Mox macrophage phenotype lead to a more “stable” plaque phenotype?
A: Based on the individual markers, we demonstrated that the relative abundance of macrophage phenotypes in established murine atherosclerotic lesions was approximately 40% for M1, 20% for M2 and 30% for Mox. For this analysis we used LDLR null mice that had been fed a high fat diet for 30 weeks. It will be interesting to see how the relative abundance of macrophage phenotypes changes during lesion development, and whether the abundance of a particular phenotype correlates with lesion stability.
Q: What controls the differentiation to Mox in atherosclerotic plaques in vivo and should and/or could it be influence by any intervention, e.g. pharmacological intervention?
A: Phenotypic polarization to the Mox phenotype is driven by sensors of oxidative tissue damage and oxidative stress. On the one hand, sensing of oxidized molecules by yet to be identified receptors may link oxidative damage to inflammatory gene expression. On the other hand, control of redox status by the Nrf2/Keap1 system may protect against uncontrolled cell death. Such receptors or “sensors” for oxidative tissue damage may turn out to be pharmacological targets, opening new possibilities for intervention.
Q: What are the next important questions and efforts to be done in this area?
A: We would like to see if the Mox phenotype also develops in other tissues that are chronically inflamed. Candidates are obese adipose tissue, rheumatoid synovium, chronically inflamed lung tissue, skin etc. Then we need to learn more about the biological functions of the Mox phenotype. Once we know whether Mox macrophages serve beneficial or detrimental functions in damaged tissue, we will be able to devise strategies for pharmacological intervention, either interfering with the development of this particular phenotype or manipulating the relative abundance of macrophage phenotypes.
A: Thank you, I will be happy to answer further questions via e-mail: firstname.lastname@example.org.
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