(210d) Chance and Circumstance Govern Macrophage Functional Diversity

Macrophages play a critical role in maintaining the balance between homeostasis and protective inflammation by adopting either immunostimulatory (M1) or immunosuppressive (M2) phenotypes. Tumors and pathogens manipulate this balance by inducing the production of immunosuppressive stimuli, which shifts macrophages into a M2 phenotype, supporting tumor growth and pathogen survival. In addition, M2 macrophages have been reported to convert to an M1 phenotype given certain stimuli, which is termed “plasticity”. Despite the centrality of these phenomena to diverse disease processes, the mechanisms regulating macrophage “decision-making” vis-à-vis polarization remain poorly understood. Here, we describe several novel systems-level insights into how such dysfunctional immune network states are established, maintained, and potentially may be disrupted.

Since most macrophage polarization studies to date involve applying coherent stimuli (either pro-M1 or pro-M2 stimuli alone), and these stimuli rarely exist independently in vivo, we first investigated how macrophages integrate contradictory signals to “calculate” a response. Macrophages were exposed to combinatorial variations in dose of IL-10 and IL-12 (pro-M2 and pro-M1 stimuli, respectively), and macrophages were “activated” using bacterial endotoxin. Surprisingly, the presence of IL-10 blocked previously-described IL-12-mediated conversion of M2 cells to an M1 phenotype. Moreover, M2-type responses increased with IL-10 dose and were largely independent of IL-12 co-treatment. We also investigated a competition between IL-10 and the canonical pro-M1 stimulus, IFNγ. Interestingly, IFNγ specifically suppressed the production of additional IL-10 by M2 cells, potentially disrupting a key positive feedback loop involved with maintaining the M2 state. This suggests that IFNγ may enable the restoration of an immune network state that promotes M1 polarization and immunological clearance or control of cancer.

We also investigated whether incoherent stimuli induce heterogeneous responses among different macrophages within a single population. Using flow cytometric profiling, we observed that under some conditions, distinct M1 and M2 cells were induced within a single population. The probability of polarization towards an M2 state increased with IL-10 dose and was independent of IL-12 co-treatment. Other cells remained non-responsive to activation, and this probability of activation was unaffected by cytokine treatment. These data represent the first evidence to date that macrophage polarization may be a stochastic process and suggest that separate regulatory mechanisms govern activation and polarization. Subsequent investigations focus on determining whether plasticity is also heterogeneous, and if so, how this is dependent upon a cell’s prior polarization history. This system-level analysis of macrophage function provides new insights into innate immune function and will help to formulate therapeutic strategies for treating the many chronic diseases characterized by local immune dysfunction.