We arrived in mid January in Guangzhou, China for PVRI’s 8th Annual World Congress. Having already started our New Year in the Western world, our visit to Guangzhou occurred just prior to China’s turn into its new zodiac calendar year: the year of the sheep. And as customary, beautiful red envelopes embossed with golden signs were displayed and available for purchase everywhere - waiting in anticipation to be filled with money, to be given as gifts of good fortune to family and friends for the coming new year.
As I was reading through the meeting’s program, enjoying the warm weather of Southern China compared to the icy conditions of the Eastern US, I became excited about a session on macrophages. While the drug industry, in large, is still targeting the vasodilatory pathways for the treatment of patients with pulmonary hypertension, these same patients, sadly, despite current drugs, still succumb to disease. Targeting this route alone, in my opinion, is clearly not the path to a cure. Dr. Rubin Tuder already observed and reported decades ago on the presence of immune cells in the plexiform lesions of patients with pulmonary hypertension. His work, in part, led me to focus our research on the role of the immune system in pulmonary vascular disease.
I thus happily volunteered to write about this very interesting, highly informative, and inspiring session on “Macrophage diversity in inflammation-related pulmonary vascular remodeling”. The session included five presentations, with the following titles 1) The many faces of macrophage activation (Dr. Limin Zheng, Director for Tumor Biotherapy, Sun Yat-sen University, Guangzhou, China) 2) ‘Immune cell cross-talk with endothelium and vascular smooth muscle’ 3) ‘Macrophages and monocytes in experimental pulmonary hypertension’ 4) The macrophage in human pulmonary hypertension— a case of scleroderma and 5) Putting the brakes on macrophages in pulmonary hypertension (Dr. John Rogers, Professor in Anesthesiology and Critical Care Medicine at Johns Hopkins University, US). Since PVRI only received authorization to video-record from two of the presenters, I will cover only these two talks here: the key-note address presented by Dr. Zheng and the final presentation of the session by Dr. Rogers.
Macrophage “Basics”– With its roots in the Greek - macro meaning ‘large’ and phage meaning ‘to devour’ - the term at its simplest describes a large cell that is able to devour cellular content. Elie Metchnikoff, a Russian bacteriologist, was credited with the discovery of the macrophage in 1884, sharing the Nobel Price in Physiology or Medicine with Paul Ehrlich in 1908 “in recognition of their work on immunity”. Though originally described only based on its phagocytotic function, the macrophage is now thought of as one of the most plastic cells in the hematopoietic system, and its complexity is still not fully understood.
The journey of the macrophage begins as a bone marrow derived cell called a monocyte. As it matures the monocyte enters the blood stream with the majority of monocytes taking up residence in specific sites, silently waiting at their final destination for an activation signal. In this fashion alveolar macrophages reside in the lung, while Kupffer cells are the resident macrophages of the liver, Langerhans cells in the skin, microglia cells in the nervous system, and osteoclasts in the bone. However, a much smaller group of monocytes remains in the blood, freely circulating, and waiting for chemotactic clues to guide them to the site of infection where they transform into a macrophage, invade the tissue and do their job. While the life span of a monocyte is rather shortlived (maximum of about 3 days), a transformed macrophage can live for months, even years. Macrophages have been studied for over 100 years. A PubMed search using the keyword “macrophage” resulted in numerous reports demonstrating the importance and influence of this cell in biology and disease (see Fig 1).
Another quick online search revealed the existence of a journal carrying just the single-worded title: ‘Macrophage’ –a peer-reviewed open access journal. Scientists even created a community based website dedicated to macrophages (http:// www.macrophages.com/) – a centralized resource connecting scientists worldwide in their common interest on macrophage biology.
While far from new to immunologists, I was happy to hear and learn how the macrophage is now also entering our world of pulmonary vascular disease, including one of my main interests, the proliferative disorder of pulmonary hypertension. Keynote Presentation by Dr. Zheng
In his keynote address “The Many Faces of Macrophage Activation”, Dr. Zheng clearly demonstrated the plasticity and diversity of the macrophage using examples from his field of tumor immunology and cancer immunotherapy. He divided his talk into three parts:
1) an introduction to monocytes/macrophages,
2) a presentation of data on how macrophages contribute and promote disease and tumor progression, and
3) a presentation of data to support macrophages’ ability to change function based on location and additional environmental factors. Dr. Zheng pointed out that the potential of our increasing knowledge hopefully will allow us to redirect the “negative” macrophage function in one area of the tumor to its positive function in a separately distinct area within the same tumor.
Historically immunologists grouped macrophages phenotypically into 2 subsets: M1-like activated macrophages representing classical activation, and M2-like activated macrophage – activated by IL-4 and mainly responsible for T cell remodeling within tissue, like the heart and vasculature. Although well supported and understood in mice, this simplified view is not fully supported to hold true in humans. Markers like iNOS activation and arginase are important in the murine macrophage biology, but these markers seem to be absent in human macrophage biology. And, like all science, also macrophage biology evolves as new evidences are discovered.
Using atherosclerosis as an example, Dr. Zheng pointed out the macrophage’s well-established contributory role to disease. In atherosclerosis macrophages were believed to have no ability to proliferate. However, increasing recent evidence suggests that indeed, these aortic macrophages do proliferate as shown by several groups using Ki67 staining and BrdU uptake. What pathways lead to macrophage activation? Based on the classical view, a macrophage is the result of a differentiated monocyte. On the other hand, there also exists the potential of macrophage self-renewal.
Dr. Zheng continued to demonstrate the diversity of macrophage function with examples from his field in tumor immunology. A tumor infiltrating macrophage is able to cause inflammation, producing IL12, to act immunosuppressive, activating arginase and producing IL10. It is able to promote angiogenesis and tumor growth, as well as invasiveness of the cells. Dr. Zheng presented data of positive macrophage staining throughout the entire tumor. However, positive staining for macrophage activation alone, was limited only to the peritumoral stroma, while the rest of the tumor contained immunosuppressive macrophages expressing IL4. How does the tumor educate the macrophage? In vitro experiments revealed a temporal component. The macrophage, once activated, changes over time into an immunosuppressive state including elevated IL-10 expression. This is comparable to Lipopolysaccharide (LPS) tolerance in humans, where we observe an early strong inflammatory response that switches over time.
The questions that then arise are: Is macrophage activation a host defense mechanism? And what role does it play in tumor progression? Dr. Zheng explained that a subset of T- cells, expressing IL-17, strongly promote angiogenesis with an observed positive correlation between CD68+ and IL-17+ cells in the peritumoral stroma, however, without any apparent association in the intratumoral region. To further elucidate this potential link, his team treated macrophages with tumor-supernatant, waited six days to allow them to change into the immunosuppressive phenotype, and then examined their ability to trigger a Th17 T- cell expansion. Repeating these studies with activated macrophages showed that activated cells, in comparison to immunosuppressive ones, are superior in causing this Th17 response and expressing elevated levels of interferon-Υ. We begin to get a glimpse of the complexity of the macrophage and its changing roles based on its position and environment.
In addition, autocrine cytokines produced by the tumor itself, like TNFα and IL-10, transiently lead to increased expression of Programmed-death 1 ligand (PDL1) in macrophages and is highly enriched in the peritumoral stroma of patients. PDL1 is thought to protect tumor cells from being targeted by the immune system. To further complicate our understanding of macrophage biology, there can be large differences between a macrophage’s in vivo biology compared to its in vitro behavior. For example, indoleamine 2,3 dioxygenase expression (IDO) is strongly expressed in macrophages and monocytes in human tumor tissue in vivo but is undetectable in macrophages used for in vitro studies. Why? The macrophage reacts and interacts very dynamically to and with its environment. It fine-tunes when and where it needs to be immunosuppressive versus activated. This plasticity seems to be one major requirement to be a well-functioning immune cell. The in vitro loss of IDO expression in macrophages, for example, is due to an absence of T-cells in cell culture in laboratory bench experiments. When viewed in a broader context, these findings, give us important insights. The knowledge hopefully allows us in the future to take advantage of the plasticity of the macrophage by shifting its state to the one that is desirable for us to help prevent and cure disease.
Concluding Presentation by Dr. Rogers
Dr. Rogers concluded the macrophage session with an excellent presentation entitled, “Putting the brakes on macrophages and immune response in pulmonary hypertension”. He highlighted current progress on targeted immune therapy for the treatment of pulmonary hypertension. In agreement with my personal view, Dr. Rogers pointed out that targeting the Nitric Oxide (NO) pathway alone does not present a successful treatment or cure for patients with pulmonary hypertension – though still continuously exploited by industry. As Dr. Zheng had explained earlier, Dr. Rogers also pointed to the well-established role of macrophages in the vascular disease atherosclerosis. In atherosclerosis several subsets of macrophages have been identified, displaying clearly the continuous and plastic phenotype a macrophage can inhabit.
Macrophages are very responsive to external stimuli, such as chemokines, adhesion molecules, or other inflammatory mediators. Using these to our advantage might allow us to use macrophages as new therapeutic target that hopefully will result in better treatment options for our patients. Dr. Rogers described several approaches currently used by institutions and companies, i.e. the creation of small molecule inhibitors, the targeting of transcription factors, and lastly the creation of antibodies. As of now ten antibodies are available for therapy in the US cumulating in over 400 clinical trials. The approach is relatively easy. In the cancer field companies have already progressed from a single target antibody to create bi-specific antibodies: for example targeting VEGF in addition to Interleukin-6. Dr. Rogers described various methods of antibody production, such as creating mouse monoclonal antibodies followed by humanization to allow for higher affinity. Another more recent approach is to begin the work using a humanized mouse. While there are over 590 pulmonary hypertension clinical trials targeting the vasodilatory prostacyclin pathway, only 21 trials have targets outside the traditional research field, like the immune system.
As Dr. Rogers pointed out, lungs from patients can display fibrosis in addition to elevated levels of a large range of chemo- and cytokines. Furthermore, fibrosis is a Th2 macrophage response. Since targeting an entire pathway is difficult and often leads to extensive side effects, targeting of specific cells would thus be a better option. As part of his work, he has previously identified that the protein hypoxia-induced mitogenic factor (HIMF) is upregulated by a Th2 stimulus as well as hypoxia and is furthermore strongly present in the pulmonary vasculature. HIMF belongs to the FIZZ/resistin/RELMbeta protein family, has adipokine and insulin resistance properties, and may thus mediate vascular pathology associated with obesity and metabolic syndrome. Dr. Rogers observed “massive release” of HIMF into cell media after hypoxic exposure as well as its upregulation in the vasculature itself. Additionally, HIMF/Resistin seems to be particularly relevant in the lung, only being upregulated under specific stresses, and could thus present a good target to minimize side effects. Dr. Rogers presented findings that blocking HIMF leads to attenuate disease in rodent models of pulmonary hypertension using measures of pulmonary remodeling, right ventricular systolic pressure, Fulton index, and cardiac output to evaluate severity of disease. Furthermore, injection of HIMF into mice will induce experimental pulmonary hypertension and is associated with an increase in Interleukin-6 (IL-6) production. Overexpression of IL-6 itself has already been reported to lead to experimental pulmonary hypertension in mouse models. On the other hand, the lack of HIMF in null mice leads to attenuated disease compared to wildtype controls.
Originally HIMF was described as chemokine mediating myeloid cell chemotaxis. In this pulmonary context HIMF has been shown to lead to IL- 4 dependent macrophage recruitment as injection of HIMF itself showed no effect in mice lacking IL- 4. Dr. Rogers’ team further observed that tail vein administration of HIMF into mice caused significant whole heart hypertrophy after 16 weeks. In more relevant cases of human scleroderma, heart biopsies revealed increased levels of Resistin-like molecule (RELM β), rising with heart failure but dropping with unloading. Dr. Roger’s team is currently exploring the role of RELM β in patients with pulmonary hypertension and he shared at this meeting his latest findings on RELM β’s increased expression in pulmonary plexiform lesions, in dendritic cells as well as in pulmonary vascular smooth muscle cells. Furthermore, serum resistin levels can be used to predict mortality in human PAH (IPAH and SSc PAH) and show correlation with the 6 min walk test. Based on all of these supporting data, Dr. Rogers was excited to share with the group that he is currently in the process of creating therapeutic antibodies and he hopes to push his project as fast as possible from the bench to bedside.
The Take-Home Lesson
This session on macrophages in pulmonary vascular disease was highly informative and the science presented by all the participants was clearly on the cutting edge of how to approach pulmonary disease.
What did we learn?
1) The potential for a single immune cell, the macrophage, to play a significant role in pulmonary vascular disease, a disease not traditionally thought of as being immunemediated.
2) The macrophage, and most likely all immune cells, possess tremendous plasticity.
3) As our understanding of this plasticity increases it might allow is to use the macrophage as therapeutic tool, so that we can artificially shift its phenotype and use its natural function at the time and location we want to.
I know that my fellow colleagues in the PVRI Committee for Young Clinicians and Scientists were as excited as I was about the session on macrophages in vascular disease, and I hope that this excitement will carry over into our research and give us a closer look of the role immune system in pulmonary hypertension.
Thank you all for organizing this meeting in China: XieXie – Thank you.