IL-10 Human, Sf9

What is IL-10 Human, Sf9?

IL-10 Human produced in Sf9 Baculovirus cells is a single glycosylated polypeptide chain containing 166 amino acids (19-178 aa) with a molecular mass of 19.4kDa. It is typically fused to a 6 amino acid His tag for purification purposes. IL-10, also known as human cytokine synthesis inhibitory factor (CSIF), is an anti-inflammatory cytokine encoded by the IL10 gene in humans .

What is the receptor complex for IL-10?

The IL-10 receptor complex consists of two IL-10 receptor-1 and two IL-10 receptor-2 proteins, forming a tetrameric structure. When IL-10 binds to this receptor complex, it initiates STAT3 signaling through the phosphorylation of the cytoplasmic domains of IL-10 receptor 1 and IL-10 receptor 2 via JAK1 and Tyk2 kinases .

How does the structure of IL-10 relate to its function?

IL-10's structure allows it to bind and activate its receptor complex, triggering intracellular signaling pathways primarily through STAT3 and to a lesser extent STAT1. The structural features of IL-10 determine its binding affinity to IL-10Rα (high affinity) and IL-10Rβ (extremely low affinity), which influences downstream signaling . This differential binding is crucial for its pleiotropic effects - suppressing inflammatory responses in myeloid cells while potentially enhancing certain functions in T cells.

How does the IL-10-induced STAT signaling pathway differ from other cytokine signaling pathways?

IL-10 signaling is distinctive in several ways:

• Receptor composition: IL-10 signals through a tetrameric receptor complex consisting of two IL-10Rα and two IL-10Rβ chains .

• STAT activation profile: IL-10 predominantly activates STAT3, with secondary activation of STAT1, creating a specific ratio of activated STATs that contributes to its unique effects .

• Temporal dynamics: IL-10 typically induces sustained STAT3 activation compared to some other cytokines like IL-6 .

• Cell-type specificity: The effects of IL-10 vary dramatically between cell types (anti-inflammatory in myeloid cells, potentially pro-inflammatory in T cells), despite using the same core signaling machinery .

What is the relationship between STAT1 and STAT3 activation in IL-10 signaling?

The relationship between STAT1 and STAT3 activation in IL-10 signaling is complex:

How does the competition between STATs for receptor binding sites affect IL-10 function?

Competition between STATs for receptor binding sites significantly impacts IL-10 function:

• STAT molecules compete for a limited number of phospho-Tyr motifs in the intracellular domains of cytokine receptors .

• The relative abundance of STAT1 versus STAT3 in cells can shift the signaling output toward different gene expression programs .

• In conditions where STAT1 levels are elevated (e.g., in IFN-rich environments or SLE), IL-10 may induce a more STAT1-biased response, potentially altering its functional effects .

• Mathematical modeling suggests that receptor and STAT concentrations critically contribute to shaping cytokine responses .

• This competition mechanism provides a basis for understanding how the same signaling pathway can produce different outcomes in different cellular contexts or disease states .

How does IL-10 exert both anti-inflammatory and pro-inflammatory effects in different cell types?

IL-10's dual effects stem from different signaling outcomes in distinct cell populations:

• In myeloid cells (monocytes, macrophages, dendritic cells):

  • IL-10 predominantly activates STAT3

  • Suppresses production of pro-inflammatory cytokines (TNF-α, IL-1β, IL-6)

  • Inhibits antigen presentation and co-stimulatory molecule expression

  • Downregulates MHC class II expression

• In T cells, particularly CD8+ T cells:

  • IL-10 activates both STAT3 and STAT1

  • STAT1 activation promotes IFN-γ production

  • Enhances cytolytic activity through increased granzyme B expression

  • Induces expression of other pro-inflammatory factors like IL-9 and IL-17F

The balance between these opposing functions depends on the cellular context, the relative expression of receptors and STATs, and the inflammatory environment .

What are the key gene expression changes induced by IL-10 in different immune cell populations?

IL-10 induces distinct gene expression profiles in different immune cells:

• In myeloid cells:

  • Upregulates anti-inflammatory mediators (SOCS3)

  • Downregulates pro-inflammatory cytokines and chemokines

  • Suppresses genes involved in antigen presentation

• In CD8+ T cells:

  • Upregulates genes associated with cytolytic function (GZMB)

  • Enhances expression of pro-inflammatory cytokines (IFNG)

  • Induces IL9, IL17F, and other T cell-associated genes

  • Downregulates certain chemokines (CCL1, CCL17, CCL22)

• IL-10 can also induce TGF-β expression, which synergistically acts with IL-10 to induce IL-10 secretion from regulatory T cells, contributing to its anti-inflammatory functions .

What phenotypes are observed in IL-10 deficient models, and what do they tell us about IL-10 function?

IL-10 deficient models exhibit multiple inflammatory phenotypes that reveal IL-10's crucial role in immune regulation:

These models demonstrate that IL-10 serves as a critical brake on immune responses, limiting inflammatory damage while potentially constraining pathogen clearance.

What are the most effective methods to measure IL-10 activity in experimental settings?

Several approaches can be used to measure IL-10 activity:

• Phospho-STAT3/STAT1 detection: Measuring phosphorylation of STAT3 and STAT1 in target cells using flow cytometry, western blotting, or ELISA-based methods .

• Reporter cell assays: Using cells expressing the IL-10 receptor complex and a STAT3-responsive reporter gene.

• Functional assays: Measuring IL-10's ability to inhibit pro-inflammatory cytokine production by LPS-stimulated monocytes/macrophages .

• Target gene expression: Quantifying the expression of IL-10-induced genes like SOCS3 by qRT-PCR .

• T cell function assays: Assessing IL-10's effect on CD8+ T cell activation, IFN-γ production, and granzyme B expression .

How can researchers effectively study IL-10 signaling dynamics in different cell types?

To study IL-10 signaling dynamics across different cell types, researchers should:

• Isolate specific cell populations using magnetic or fluorescence-activated cell sorting.

• Perform time-course experiments measuring STAT1 and STAT3 phosphorylation at multiple time points (e.g., 5, 15, 30, 60, 120 minutes) .

• Quantify receptor expression levels (IL-10Rα and IL-10Rβ) on target cells using flow cytometry .

• Measure the kinetics of target gene expression using qRT-PCR or RNA-seq .

• Employ mathematical modeling to understand the relationship between receptor occupancy, STAT activation, and downstream effects .

• Use inhibitors of specific pathway components (e.g., JAK inhibitors like Tofacitinib) to dissect the contribution of different signaling molecules .

How should researchers approach contradictions in IL-10 experimental data?

When faced with contradictory IL-10 experimental results, researchers should:

• Examine cell type differences:

  • IL-10 has opposing effects in different cells; ensure you're comparing the same cell populations

  • Consider the activation/differentiation state of cells, which affects IL-10 responsiveness

• Evaluate concentration-dependent effects:

  • IL-10 may have different effects at different concentrations

  • Perform full dose-response curves rather than single concentrations

• Consider temporal factors:

  • Short-term versus long-term IL-10 exposure can yield opposite results

  • Analyze kinetics with appropriate time points

• Assess the cytokine environment:

  • IL-10 effects are modified by the presence of other cytokines

  • Document and control the full cytokine milieu in your system

• Validate reagent quality:

  • Different sources of recombinant IL-10 may have varying bioactivity

  • Bacterial versus insect cell (Sf9) versus mammalian cell-derived IL-10 may behave differently

How do alterations in STAT expression levels affect IL-10 responses in inflammatory diseases?

Changes in STAT expression significantly impact IL-10 responses in inflammatory diseases:

• In systemic lupus erythematosus (SLE):

  • Patients show increased expression of STAT1 (and to a lesser extent STAT3)

  • This correlates with biased STAT1 responses when cells are stimulated with IL-10

  • The imbalance shifts IL-10 signaling toward more pro-inflammatory outcomes

  • Similar effects can be reproduced experimentally by priming cells with IFNα to increase STAT1 levels

• In other inflammatory/autoimmune conditions:

  • Chronic exposure to certain cytokines alters the STAT1:STAT3 ratio

  • These changes can convert IL-10 from an anti-inflammatory to a pro-inflammatory mediator

  • The altered signaling may contribute to disease pathogenesis and treatment resistance

• Therapeutic implications:

  • Partial inhibition of JAK activation using sub-saturating doses of inhibitors like Tofacitinib can specifically reduce STAT1 activation by IL-10

  • This approach might restore normal IL-10 responses in conditions with elevated STAT1 expression

How can the opposing functions of IL-10 be decoupled for therapeutic applications?

Recent structural and functional studies provide several approaches to decouple IL-10's opposing functions:

• Structure-based engineering:

  • Mutations in IL-10 that alter its binding to IL-10Rα or IL-10Rβ can create variants with selective activity profiles

  • For example, reducing IL-10's ability to activate STAT1 while preserving STAT3 activation could enhance anti-inflammatory effects while reducing pro-inflammatory T cell effects

• Cell-specific targeting:

  • Developing delivery systems that target IL-10 to specific cell populations

  • Creating fusion proteins that direct IL-10 activity to particular tissues or cell types

• Pathway modulation:

  • Using partial JAK inhibition (e.g., with sub-saturating doses of Tofacitinib) to selectively reduce STAT1 activation

  • Combining IL-10 with other modulators that bias the response toward specific outcomes

The ability to engineer IL-10 variants with selective activity on myeloid cells without stimulating IFN-γ production by T cells could unlock IL-10's full therapeutic potential in inflammatory diseases .

How can mathematical modeling enhance our understanding of IL-10 signaling dynamics?

Mathematical modeling offers powerful insights into IL-10 signaling:

• Predicting systems behavior:

  • Models can predict how changes in receptor expression, STAT levels, or inhibitor concentrations will affect signaling outcomes

  • This allows researchers to formulate testable hypotheses about complex signaling networks

• Understanding competition mechanisms:

  • Models reveal how STATs compete for binding to a limited number of receptor phospho-Tyr motifs

  • This competition is difficult to observe directly but has profound functional implications

• Explaining cell-type specificity:

  • Mathematical approaches can explain why the same cytokine (IL-10) produces different outcomes in different cell types

  • Models incorporate cell-specific parameters like receptor and STAT concentrations

• Optimization of therapeutic interventions:

  • Models can predict optimal dosing strategies for IL-10 or IL-10 modulators

  • They can identify the most effective targets within the pathway for therapeutic intervention

What protocols are most effective for studying IL-10 effects on human CD8+ T cells?

For studying IL-10 effects on human CD8+ T cells, researchers should:

• Isolate CD8+ T cells from human PBMCs using magnetic separation techniques (MACS) with CD8+ isolation kits .

• For studying IL-10's effects on IFN-γ and granzyme B production:

  • Seed isolated CD8+ T cells at 2×10^6 cells/ml in plates precoated with anti-CD3 antibody

  • Include soluble anti-CD28 antibody (5 μg/ml) for co-stimulation

  • Culture for 3 days to activate the cells

  • Collect and reseed cells at 10^6 cells/ml with or without 10 nM IL-10 (wild-type or variant)

  • Incubate for an additional 3 days

  • Restimulate with soluble anti-CD3 (2 μg/ml) for 4 hours

  • Measure IFN-γ, IL-9, and Granzyme B levels by ELISA

• For signaling studies:

  • Use freshly isolated or activated CD8+ T cells

  • Treat with IL-10 for short time periods (5-60 minutes)

  • Measure STAT1 and STAT3 phosphorylation by phospho-flow cytometry or western blotting

  • Compare wild-type IL-10 with engineered variants to assess signaling differences

What are the critical controls needed when studying IL-10 signaling in primary human cells?

When investigating IL-10 signaling in primary human cells, essential controls include:

• Receptor expression verification:

  • Confirm expression levels of IL-10Rα and IL-10Rβ on target cell populations

  • Account for donor-to-donor variability in receptor expression

• Cytokine specificity controls:

  • Include related cytokines that activate similar pathways (e.g., IL-6 for STAT3 activation)

  • Use receptor-blocking antibodies to confirm specificity

• Signaling pathway validation:

  • Include JAK inhibitors (e.g., Tofacitinib) to confirm that observed effects depend on canonical signaling

  • Measure multiple pathway components (e.g., JAK phosphorylation, STAT phosphorylation, target gene expression)

• Biological activity confirmation:

  • Verify that the IL-10 preparation has the expected biological activity

  • Include functional readouts like suppression of LPS-induced TNF-α in monocytes

• Time-course controls:

  • Include multiple time points to capture the dynamics of both rapid (phosphorylation) and delayed (transcriptional) responses

  • Ensure stable baseline measurements before cytokine addition