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

Abstract

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-10more » 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.« less

Authors:

Saxton, Robert A.  ^[1];

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• Search DOE PAGES for ORCID "0000-0002-9376-3984"

Tsutsumi, Naotaka  ^[1];

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• Search DOE PAGES for ORCID "0000-0002-3617-7145"

Su, Leon L.  ^[2];

• Search DOE PAGES for author "Su, Leon L."
• Search DOE PAGES for ORCID "0000-0001-7654-9997"

Abhiraman, Gita C.  ^[3];

• Search DOE PAGES for author "Abhiraman, Gita C."
• Search DOE PAGES for ORCID "0000-0002-0546-4133"

Mohan, Kritika  ^[2];

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• Search DOE PAGES for ORCID "0000-0002-7163-6423"

Henneberg, Lukas T.  ^[2];

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• Search DOE PAGES for ORCID "0000-0003-3477-4541"

Aduri, Nanda G.  ^[4];

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• Search DOE PAGES for ORCID "0000-0003-4301-7446"

Gati, Cornelius  ^[4];

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• Search DOE PAGES for ORCID "0000-0003-3693-7009"

Garcia, K. Christopher  ^[5]

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• Search DOE PAGES for ORCID "0000-0001-9273-0278"

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+ Show Author Affiliations

1. Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA 94305, USA., Howard Hughes Medical Institute, Stanford University School of Medicine, Stanford, CA 94305, USA.
2. Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA 94305, USA.
3. Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA 94305, USA., Program in Immunology, Stanford University School of Medicine, Stanford, CA 94305, USA.
4. Department of Structural Biology, Stanford University School of Medicine, Stanford, CA 94305, USA., Biosciences Division, SLAC National Accelerator Laboratory, Menlo Park, CA 94025, USA.
5. Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA 94305, USA., Howard Hughes Medical Institute, Stanford University School of Medicine, Stanford, CA 94305, USA., Department of Structural Biology, Stanford University School of Medicine, Stanford, CA 94305, USA.

Publication Date:

Thu Mar 18 00:00:00 EDT 2021

Research Org.:

SLAC National Accelerator Lab., Menlo Park, CA (United States)

Sponsoring Org.:

USDOE Office of Science (SC), Basic Energy Sciences (BES); National Institutes of Health (NIH); USDOE Laboratory Directed Research and Development (LDRD) Program; Stanford Medical Scientist Training Program

OSTI Identifier:

1771371

Alternate Identifier(s):

OSTI ID: 1781077

Grant/Contract Number:  

AC02-76SF00515; U54CA244711; 5R01CA177684; R37-AI51321; T32GM007365

Resource Type:

Published Article

Journal Name:

Science

Additional Journal Information:

Journal Name: Science Journal Volume: 371 Journal Issue: 6535; Journal ID: ISSN 0036-8075

Publisher:

American Association for the Advancement of Science (AAAS)

Country of Publication:

United States

Language:

English

Subject:

60 APPLIED LIFE SCIENCES