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Single-cell secretion analysis reveals a dual role for IL-10 in restraining and resolving the TLR4-induced inflammatory response.

ABSTRACT: Following Toll-like receptor 4 (TLR4) stimulation of macrophages, negative feedback mediated by the anti-inflammatory cytokine interleukin-10 (IL-10) limits the inflammatory response. However, extensive cell-to-cell variability in TLR4-stimulated cytokine secretion raises questions about how negative feedback is robustly implemented. To explore this, we characterize the TLR4-stimulated secretion program in primary murine macrophages using a single-cell microwell assay that enables evaluation of functional autocrine IL-10 signaling. High-dimensional analysis of single-cell data reveals three tiers of TLR4-induced proinflammatory activation based on levels of cytokine secretion. Surprisingly, while IL-10 inhibits TLR4-induced activation in the highest tier, it also contributes to the TLR4-induced activation threshold by regulating which cells transition from non-secreting to secreting states. This role for IL-10 in restraining TLR4 inflammatory activation is largely mediated by intermediate interferon (IFN)-β signaling, while TNF likely mediates response resolution by IL-10. Thus, cell-to-cell variability in cytokine regulatory motifs provides a means to tailor the TLR4-induced inflammatory response.

SUBMITTER: Alexander AF

PROVIDER: S-EPMC8995750 | biostudies-literature | 2021 Sep

REPOSITORIES: biostudies-literature

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Single-cell secretion analysis reveals a dual role for IL-10 in restraining and resolving the TLR4-induced inflammatory response.

Alexander Amanda F AF Kelsey Ilana I Forbes Hannah H Miller-Jensen Kathryn K

Cell reports 20210901 12

Following Toll-like receptor 4 (TLR4) stimulation of macrophages, negative feedback mediated by the anti-inflammatory cytokine interleukin-10 (IL-10) limits the inflammatory response. However, extensive cell-to-cell variability in TLR4-stimulated cytokine secretion raises questions about how negative feedback is robustly implemented. To explore this, we characterize the TLR4-stimulated secretion program in primary murine macrophages using a single-cell microwell assay that enables evaluation of ...[more]

PMID: 34551303

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Project description:Microglia respond to CNS injuries and diseases with complex reactions, often called "activation." A pro-inflammatory phenotype (also called classical or M1 activation) lies at one extreme of the reactivity spectrum. There were several motivations for this study. First, bacterial endotoxin (lipopolysaccharide, LPS) is the most commonly used pro-inflammatory stimulus for microglia, both in vitro and in vivo; however, pro-inflammatory cytokines (e.g., IFNγ, TNFα) rather than LPS will be encountered with sterile CNS damage and disease. We lack direct comparisons of responses between LPS and such cytokines. Second, while transcriptional profiling is providing substantial data on microglial responses to LPS, these studies mainly use mouse cells and models, and there is increasing evidence that responses of rat microglia can differ. Third, the cytokine milieu is dynamic after acute CNS damage, and an important question in microglial biology is: How malleable are their responses? There are very few studies of effects of resolving cytokines, particularly for rat microglia, and much of the work has focused on pro-inflammatory outcomes. Here, we first exposed primary rat microglia to LPS or to IFNγ+TNFα (I+T) and compared hallmark functional (nitric oxide production, migration) and molecular responses (almost 100 genes), including surface receptors that can be considered part of the sensome. Protein changes for exemplary molecules were also quantified: ARG1, CD206/MRC1, COX-2, iNOS, and PYK2. Despite some similarities, there were notable differences in responses to LPS and I+T. For instance, LPS often evoked higher pro-inflammatory gene expression and also increased several anti-inflammatory genes. Second, we compared the ability of two anti-inflammatory, resolving cytokines (IL-4, IL-10), to counteract responses to LPS and I+T. IL-4 was more effective after I+T than after LPS, and IL-10 was surprisingly ineffective after either stimulus. These results should prove useful in modeling microglial reactivity in vitro; and comparing transcriptional responses to sterile CNS inflammation in vivo.

Project description:BackgroundABCA1 is known to suppress proinflammatory cytokines.ResultsABCA1 activates PKA and up-regulates anti-inflammatory cytokine IL-10. Elevated PKA transforms macrophages to M2-like phenotype. Disrupting lipid rafts by statins MCD, and filipin recuperates ABCA1 phenotype and likely functions downstream of ABCA1.ConclusionBy modulating cholesterol, ABCA1 activates PKA. This generates M2-like macrophages.SignificanceABCA1 does not simply suppress inflammatory response. It promotes M2-like activation and facilitates resolution. Nonresolving inflammatory response from macrophages is a major characteristic of atherosclerosis. Macrophage ABCA1 has been previously shown to suppress the secretion of proinflammatory cytokine. In the present study, we demonstrate that ABCA1 also promotes the secretion of IL-10, an anti-inflammatory cytokine critical for inflammation resolution. ABCA1(+/+) bone marrow-derived macrophages secrete more IL-10 but less proinflammatory cytokines than ABCA1(-/-) bone marrow-derived macrophages, similar to alternatively activated (M2) macrophages. We present evidence that ABCA1 activates PKA and that this elevated PKA activity contributes to M2-like inflammatory response from ABCA1(+/+) bone marrow-derived macrophages. Furthermore, cholesterol lowering by statins, methyl-?-cyclodextrin, or filipin also activates PKA and, consequently, transforms macrophages toward M2-like phenotype. Conversely, cholesterol enrichment suppresses PKA activity and promotes M1-like inflammatory response. As the primary function of ABCA1 is cholesterol removal, our results suggest that ABCA1 activates PKA by regulating cholesterol. Indeed, forced cholesterol enrichment in ABCA1-expressing macrophages suppresses PKA activation and elicits M1-like response. Collectively, these findings reveal a novel protective process by ABCA1-activated PKA in macrophages. They also suggest cholesterol lowering in extra-hepatic tissues by statins as an anti-inflammation strategy.

Dataset Information

Single-cell secretion analysis reveals a dual role for IL-10 in restraining and resolving the TLR4-induced inflammatory response.

Publications

Single-cell secretion analysis reveals a dual role for IL-10 in restraining and resolving the TLR4-induced inflammatory response.

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