(625o) Stochastic Origins of Macrophage Polarization

Innate immune cells such as macrophages play a central role in regulating local immune networks, and are involved in many chronic diseases ranging from cancer to autoimmune diseases such as diabetes. However, the process by which these networks are coordinated and regulated remains poorly understood. To maintain a balance between homeostasis and protective inflammation, macrophages can adopt either immunostimulatory (M1) or immunosuppressive (M2) phenotypes, which are referred to as functional polarization. Tumors manipulate this balance by inducing the production of immunosuppressive stimuli (such as IL-10) in the tumor microenvironment, which shifts macrophages into a tumor-promoting M2 phenotype. Such local immune dysfunction is recognized as one of the major barriers to cancer immunotherapy. Macrophages are also functionally plastic, meaning that they can interconvert between functional states. In particular, it has been reported that tumor-associated macrophages (TAMs) can be converted from a M2 phenotype to a M1 phenotype following the application of stimuli such as interleukin-12 (IL-12). To date, macrophage polarization has been studied in the context of coherent stimuli (either pro-M1 or pro-M2 stimuli alone). However, immunostimulatory and immunosuppressive stimuli rarely exist independently in vivo, especially at tumor sites; macrophages’ responses to such contradictory inputs are not known.

The primary goal of this study was to determine how macrophages “calculate” a response when presented with contradictory stimuli.  Macrophages were pre-polarized by incubation with combinations of IL-10 and IL-12, and then cells were “activated” with bacterial endotoxin (or lipopolysaccharide, LPS), which is known to enhance either M1- or M2-type responses in pre-polarized cells. Cellular responses were assessed using a quantitative real-time PCR (QPCR) panel. Contrary to previous reports, the presence of IL-10 prevented IL-12-mediated promotion of an M1 phenotype. Moreover, M2-type responses increased with IL-10 dose and were largely independent of IL-12 co-treatment. From these bulk measurements, one cannot determine whether these trends indicate the presence of intermediate polarization states or, instead, a heterogeneous mixture of M1- and M2-polarized cells. Therefore, to investigate polarization at the individual cell level, we next analyzed cells for characteristic M1 and M2 markers by flow cytometry. Using this methodology, we observed M1 and M2 co-existing in single populations. The probability of polarization towards an M2 state increased with IL-10 dose, and this probability was independent of IL-12 co-treatment. Interestingly, some cells remained non-responsive to LPS-mediated activation, and this probability of activation was not modulated by cytokine pretreatment. These data represent the first evidence to date that macrophage polarization is a stochastic process.

This intriguing and exciting discovery motivates many questions, including the following: Do individual macrophages exhibit functional plasticity? Do some cells possess an intrinsic bias for polarization to a particular phenotype? How might polarization bias and plasticity be interrelated? To investigate such questions, we have developed a fluorescent reporter-based platform for dynamically tracking macrophage polarization states in individual cells over time. This system incorporates a microfluidic platform for precisely controlling and modulating the extracellular environment. These tools allow us to probe and investigate macrophage polarization and plasticity at an unprecedented level of detail, allowing us to address such fundamental questions as those described above. Here, we present the results of investigations into the stochastic origins of macrophage polarization. Such new insights promise to transform our understanding of local immune responses and should identify novel therapeutic targets and strategies, which may overcome existing barriers to treatment for cancer and other diseases involving chronic immune dysfunction.