Structure-based decoupling of the pro- and anti-inflammatory functions of interleukin-10

Cryo-EM helps engineer enhanced IL-10 Interleukin-10 (IL-10) binds to the IL-10 receptor (IL-10R), comprising two high-affinity α chains and two low-affinity β chains. Depending on the cellular context of its recognition, IL-10 can either suppress or activate immune responses. This pleiotropic behavior has complicated efforts to use IL-10 as an anti-inflammatory agent. Saxton et al. generated a high-affinity version of IL-10 (super-10), which allowed them to visualize IL-10 in complex with both IL-10Rα and IL-10Rβ by cryo–electron microscopy (cryo-EM). This enabled them to engineer additional variants of IL-10 with variable IL-10Rβ–binding affinities. Some of the partial agonists exhibited biased actions on myeloid cells, which exhibited anti-inflammatory properties without concomitant CD8 T cell activation. These findings may serve as a blueprint for future enhanced cytokine–based therapeutics. Science, this issue p. eabc8433 The cryo-EM structure of the IL-10 receptor complex enables the rational design of myeloid-selective agonists. INTRODUCTION Interleukin-10 (IL-10) is an important immunoregulatory cytokine that acts to suppress and terminate inflammatory immune responses, largely through the inhibition of monocyte and macrophage activation. Polymorphisms in genes encoding IL-10 and IL-10 receptor (IL-10R) subunits are associated with autoimmune diseases, most notably inflammatory bowel disease (IBD). IL-10 has consequently garnered substantial clinical interest for use as an anti-inflammatory immune modulating agent. However, IL-10 has shown limited therapeutic efficacy, due in part to its pleiotropic nature and its capacity to also elicit proinflammatory effects, including the stimulation of interferon-γ (IFN-γ) and granzyme B production by CD8+ T cells. RATIONALE We hypothesized that obtaining structural information for the complete IL-10 receptor complex would enable the rational design of IL-10 analogs with enhanced functional specificity and improved therapeutic utility. Mechanistically, IL-10 functions as a secreted homodimer that engages two copies of a heterodimeric receptor complex comprising the private receptor subunit, IL-10Rα, and the shared subunit, IL-10Rβ. The IL-10–dependent dimerization of IL-10Rα and IL-10Rβ in turn initiates activation of the transcription factor STAT3, which mediates the diverse biological effects of IL-10. However, structural information for the complete IL-10 receptor signaling complex is lacking, due in part to the extremely low affinity of IL-10 for IL-10Rβ. RESULTS To overcome this limitation, we first used yeast display-based directed evolution to engineer a “super-10” variant with greatly enhanced affinity for IL-10Rβ, enabling assembly of the hexameric IL-10–IL-10Rα–IL-10Rβ complex. Using this stabilized complex, we then determined the structure of the complete IL-10 receptor complex at 3.5 Å resolution by cryo–electron microscopy (cryo-EM). The structure revealed how IL-10 engages IL-10Rβ to initiate signal transduction, and also uncovered the molecular basis for a mutation in IL-10Rβ associated with early-onset IBD. In addition, the structure provided an engineering blueprint for the design of IL-10 variants with which we could pharmacologically probe the nature of IL-10’s functional pleiotropy. Characterization of these IL-10 variants revealed that the plasticity of IL-10 signaling varies extensively across immune cell types, inversely correlating with the level of IL-10Rβ expression. In particular, we found that myeloid cells exhibit robust STAT3 activation in response to IL-10 variants across a wide range of IL-10Rβ–binding affinities, whereas IL-10 signaling in lymphocytes was highly tunable. Several engineered IL-10 variants exploited these differences to elicit myeloid-biased signaling in both cell lines and human peripheral blood mononuclear cells (PBMCs). Functionally, these variants effectively inhibited inflammatory monocyte and macrophage activation in vivo and promoted survival in a mouse model of sepsis, thus retaining the major anti-inflammatory effects of IL-10. However, they showed significantly reduced capacity to stimulate proinflammatory gene expression in CD8+ T cells and failed to potentiate IFN-γ or granzyme B production, thereby uncoupling the major opposing functions of IL-10. CONCLUSION The cryo-EM structure of the IL-10 receptor complex yields key insights into the mechanisms of IL-10 signaling and functional pleiotropy. Characterization of IL-10–derived partial agonists and super-agonists revealed that modulating receptor affinity can “tune” IL-10 signaling in a cell type–selective manner, exploiting differences in IL-10 response thresholds between immune cell populations. These results suggest a model in which the differential expression of the shared receptor subunit IL-10Rβ results in distinct IL-10 response thresholds across immune cell populations, enhancing the functional specificity of IL-10. By exploiting these natural features of IL-10 signaling, we engineered IL-10 variants exhibiting myeloid cell selectivity, which effectively uncoupled the pro- and anti-inflammatory functions of IL-10. These findings provide a conceptual framework for the development of improved cytokine-based therapeutics. Engineering IL-10. The structure of the IL-10 receptor complex is shown on a cell surface with “engineers” depicted as welders making alterations to IL-10 at the receptor-binding interface that was the focus of Saxton et al. ILLUSTRATION: ERIC SMITH AND CHRIS GARCIA Interleukin-10 (IL-10) is an immunoregulatory cytokine with both anti-inflammatory and immunostimulatory properties and is frequently dysregulated in disease. We used a structure-based approach to deconvolute IL-10 pleiotropy by determining the structure of the IL-10 receptor (IL-10R) complex by cryo–electron microscopy at a resolution of 3.5 angstroms. The hexameric structure shows how IL-10 and IL-10Rα form a composite surface to engage the shared signaling receptor IL-10Rβ, enabling the design of partial agonists. IL-10 variants with a range of IL-10Rβ binding strengths uncovered substantial differences in response thresholds across immune cell populations, providing a means of manipulating IL-10 cell type selectivity. Some variants displayed myeloid-biased activity by suppressing macrophage activation without stimulating inflammatory CD8+ T cells, thereby uncoupling the major opposing functions of IL-10. These results provide a mechanistic blueprint for tuning the pleiotropic actions of IL-10.