Recombinant Mouse Interleukin-22 protein (Il22) (Active)

What is the molecular structure of recombinant mouse IL-22 protein?

Recombinant mouse IL-22 is a 179 amino acid residue protein with a putative 33 amino acid signal peptide that is cleaved to generate a 147 amino acid mature protein. The commercially available recombinant protein typically spans amino acids 25-179. The protein shares approximately 79% amino acid sequence identity with human IL-22 and 22% with IL-10. The mouse IL-22 gene is located on chromosome 10 and exists as either a single copy or duplicated gene (IL-TIF alpha and IL-TIF beta with >98% sequence homology) depending on the mouse strain, with duplication occurring in C57B1/6, FVB, and 129 strains .

What are the key biological functions of IL-22 in mouse models?

IL-22 plays critical roles in modulating tissue responses during inflammation and is essential for the regeneration of epithelial cells to maintain barrier function after injury and prevent further tissue damage. Unlike most cytokines, IL-22 has no direct effect on immune cells. It signals through a heterodimeric receptor composed of the specific receptor IL-22RA1 (present on non-immune cells in multiple organs) and the shared subunit IL-10RB. This interaction activates the tyrosine kinases JAK1 and TYK2, which then activate STAT3. Consequently, IL-22 promotes cell survival and proliferation through STAT3, ERK1/2, and PI3K/AKT pathways, and promotes phosphorylation of GSK3B and CTTN .

How is IL-22 expression regulated in mouse tissues during inflammation?

IL-22 is produced by normal mouse T cells upon Concanavalin A (Con A) activation. Its expression is also induced in various organs following lipopolysaccharide (LPS) injection, suggesting its involvement in inflammatory responses. Type 3 Innate Lymphoid Cells (ILC3) are a significant source of IL-22, as demonstrated in lupus-prone MRL/lpr mice . The expression pattern indicates that IL-22 serves as an important mediator in the inflammatory cascade, bridging adaptive and innate immune responses in tissue inflammation scenarios.

What are the optimal conditions for using recombinant mouse IL-22 in in vitro experiments?

For in vitro experiments with mouse primary cells or cell lines, the recommended effective dose (ED50) of recombinant mouse IL-22 is typically 60-300 pg/mL . The protein should be reconstituted according to manufacturer specifications, usually in sterile filtered PBS containing at least 0.1% carrier protein such as BSA. When stimulating cells in culture, pretreatment with the protein for 24-48 hours is common in many experimental protocols. For instance, when testing IL-22's effects on kidney epithelial cells, researchers typically treat cells with recombinant IL-22 for 30 minutes to 24 hours, with phosphorylation of STAT3 observable within 30 minutes and changes in chemokine expression (CCL2, CXCL10) detectable within several hours .

How should researchers design experiments to study IL-22 signaling pathways?

When investigating IL-22 signaling pathways, researchers should consider:

• Receptor expression analysis: Confirm expression of IL-22RA1 and IL-10RB in target cells before experiments

• Pathway inhibitor controls: Include STAT3 pathway inhibitors (e.g., C188-9) as controls

• Time-course experiments: Monitor both immediate (minutes to hours) and delayed (12-48 hours) responses

• Phosphorylation analysis: Use Western blot or flow cytometry to detect phosphorylated STAT3, which is a key mediator of IL-22 signaling

• Downstream target measurement: Assess expression of known IL-22-responsive genes such as CCL2 and CXCL10

Western blot analysis should be performed to detect phosphorylated STAT3 at 15, 30, 60, and 120 minutes post-stimulation, while qPCR for target genes should be performed at 6, 12, and 24 hours to capture the complete signaling cascade.

What functional assays can be used to assess IL-22 biological activity in research settings?

Several functional assays can be employed to assess IL-22 biological activity:

• Cell proliferation/survival assays: MTT or WST-1 assays with epithelial cell lines

• Wound healing assays: Scratch assays with epithelial monolayers

• Chemokine induction: qPCR or ELISA measurement of CCL2, CXCL10 expression

• Transwell migration assays: Using supernatant from IL-22-stimulated cells to assess immune cell recruitment

• STAT3 activation: Phospho-flow cytometry or Western blot for phosphorylated STAT3

• Barrier function: Transepithelial electrical resistance (TEER) measurements in epithelial cultures

In transwell assays, supernatant from primary kidney epithelial cells treated with recombinant IL-22 has been shown to recruit significantly more macrophages compared to controls, demonstrating IL-22's role in promoting immune cell chemotaxis through indirect mechanisms .

How does IL-22 function in mouse models of inflammatory bowel disease?

In mouse models of ulcerative colitis, IL-22 regulates pro-inflammatory pathways involved in microbial recognition, cancer, and immune cell chemotaxis, most prominently those involving CXCR2+ neutrophils. IL-22-mediated transcriptional regulation of CXC-family neutrophil-active chemokine expression is highly conserved across species, depends on STAT3 signaling, and is functionally important in recruiting CXCR2+ neutrophils into colonic tissue. The magnitude of enrichment of IL-22-regulated transcripts in colonic biopsies correlates with neutrophil infiltration in ulcerative colitis patients . When designing experiments to study IL-22 in colitis models, researchers should consider using organoid cultures alongside in vivo models to comprehensively assess epithelial-specific effects.

What is the role of IL-22 in lupus nephritis models, and how can this be studied?

In lupus nephritis models using MRL/lpr mice, IL-22 secreted primarily by Type 3 Innate Lymphoid Cells (ILC3) has been shown to play a pathogenic role. IL-22 binding to IL-22R on kidney epithelial cells activates the STAT3 signaling pathway, enhancing chemokine secretion (particularly CCL2 and CXCL10) and promoting macrophage infiltration into the kidney. Both IL-22 knockout and IL-22R knockout MRL/lpr mice exhibit decreased macrophage infiltration in the kidney, reduced proteinuria, improved renal function, and less severe pathological impairment compared to control mice. These mice also demonstrate milder lymphadenopathy and splenomegaly, suggesting IL-22 may also influence systemic immune responses .

For studying IL-22 in lupus nephritis, researchers should:

• Compare wild-type, IL-22 KO, and IL-22R KO mice in the MRL/lpr background

• Monitor survival, skin lesions, proteinuria, and renal function

• Assess immune complex deposition and complement activation in kidney tissue

• Quantify infiltrating immune cells using flow cytometry and immunohistochemistry

• Measure autoantibody production and serum complement levels

How do knockout/knockin approaches compare with antibody neutralization for studying IL-22 function in vivo?

Both genetic approaches (knockout/knockin) and antibody neutralization have distinct advantages and limitations when studying IL-22 function:

Genetic approaches (IL-22 KO or IL-22R KO):

• Advantages: Complete absence of target protein; no off-target effects from therapeutic agents; allows study of developmental effects

• Limitations: May trigger compensatory mechanisms during development; cannot study temporal effects easily; strain-specific variations exist (e.g., IL-22 gene duplication in C57B1/6, FVB, and 129 strains)

Antibody neutralization:

• Advantages: Can be administered at specific time points; dose-dependent inhibition possible; more translatable to therapeutic approaches

• Limitations: Incomplete neutralization; potential off-target effects; antibody immunogenicity in long-term studies

In lupus nephritis studies, both IL-22 KO and anti-IL-22 monoclonal antibody treatment have shown similar protective effects in MRL/lpr mice, confirming the pathogenic role of IL-22 . For comprehensive understanding, researchers should consider using both approaches - genetic models for mechanistic insights and antibody neutralization for therapeutic potential assessment.

What expression systems are optimal for producing high-quality recombinant mouse IL-22?

Recombinant mouse IL-22 can be produced in various expression systems, each with distinct characteristics affecting protein quality and function:

• E. coli expression systems: Yield high amounts of protein but may lack proper post-translational modifications. Commercial E. coli-derived mouse IL-22 proteins typically contain N-terminal methionine and span Leu34-Val179 .

• Mammalian expression systems (HEK293): Produce proteins with proper folding and post-translational modifications, resulting in higher biological activity. Commercial HEK293-derived mouse IL-22 proteins typically achieve ≥95% purity with endotoxin levels ≤0.005 EU/μg .

For research requiring high biological activity and minimal endotoxin contamination, mammalian expression systems are preferred, particularly for in vivo studies and primary cell culture experiments.

How can researchers accurately quantify biologically active IL-22 in experimental samples?

Accurate quantification of biologically active IL-22 in experimental samples requires multiple complementary approaches:

• ELISA: Measures protein concentration but not necessarily biological activity

• Bioactivity assays: Cell-based assays measuring STAT3 phosphorylation in responsive cell lines

• Western blot: For semi-quantitative analysis with specificity verification

• Mass spectrometry: For precise identification and absolute quantification

• Flow cytometry: For intracellular detection in specific cell populations

When designing quantification experiments, researchers should include appropriate standards and controls, and consider the specific context of their research question. For instance, detection of IL-22 in tissue lysates may require different approaches than detection in cell culture supernatants.

What are common pitfalls in experimental design when studying IL-22 signaling, and how can they be avoided?

Several common pitfalls occur in IL-22 signaling studies:

• Failure to confirm receptor expression: Always verify IL-22RA1 and IL-10RB expression in target cells, as IL-22 effects are dependent on receptor presence.

• Cross-reactivity issues: When using anti-IL-22 antibodies, validate specificity, as IL-22 shares structural similarities with other IL-10 family cytokines.

• Overlooking strain differences: Consider that some mouse strains (C57B1/6, FVB, 129) have duplicated IL-22 genes (IL-TIF alpha and IL-TIF beta), which may affect knockout strategies and interpretation of results .

• Endotoxin contamination: Ensure recombinant proteins have low endotoxin levels (<0.005 EU/μg), as endotoxin can independently activate inflammatory pathways .

• Timing of measurements: IL-22 effects can be rapid (phosphorylation events within minutes) or delayed (gene expression changes over hours), so design time-course experiments accordingly.

To avoid these pitfalls, researchers should include appropriate positive and negative controls, validate reagents thoroughly, perform time-course experiments, and consider using multiple complementary approaches to confirm findings.

How can researchers effectively study the differential effects of IL-22 on various tissue-specific epithelial cells?

To study tissue-specific effects of IL-22, researchers should:

• Isolate primary epithelial cells from different organs (intestine, kidney, liver, skin) using tissue-specific isolation protocols

• Characterize IL-22 receptor expression on each cell type using flow cytometry or qPCR

• Perform comparative transcriptomic analysis (RNA-seq) on different epithelial cell types after IL-22 stimulation to identify tissue-specific gene signatures

• Use tissue-specific conditional knockout models of IL-22RA1 to examine in vivo relevance

• Employ organoid cultures from different organs to maintain tissue-specific architecture

For example, when studying kidney epithelial cells versus colonic epithelial cells, researchers have observed that IL-22 induces CCL2 and CXCL10 in kidney cells, promoting macrophage recruitment , while in colonic cells, IL-22 regulates neutrophil-active chemokines, promoting neutrophil infiltration . These tissue-specific differences highlight the context-dependent nature of IL-22 signaling.

What approaches can be used to study the interplay between IL-22 and other cytokines in complex inflammatory environments?

Studying cytokine interplay requires sophisticated experimental designs:

• Combinatorial cytokine stimulation: Treat cells with IL-22 alone or in combination with other cytokines (IL-17, TNFα, IL-1β) to identify synergistic or antagonistic effects

• Multi-parametric flow cytometry: To simultaneously detect multiple phosphorylated signaling molecules

• Single-cell RNA sequencing: To identify cell-specific responses in heterogeneous populations

• Conditional knockout systems: Use Cre-lox systems to delete IL-22RA1 in specific cell types while exposing them to complex cytokine environments

• In vivo cytokine blockade combinations: Combine anti-IL-22 antibodies with blockade of other cytokines

In inflammatory bowel disease models, the relationship between IL-22 and other cytokines (particularly IL-17) has been shown to significantly influence disease progression. Similarly, in lupus nephritis models, the interplay between IL-22 and type I interferons impacts disease severity .

How can researchers investigate the therapeutic potential of targeting IL-22 in autoimmune and inflammatory diseases?

To investigate IL-22 as a therapeutic target, researchers should:

• Compare prophylactic versus therapeutic interventions: Administer anti-IL-22 antibodies before disease onset (prophylactic) or after disease establishment (therapeutic)

• Develop tissue-targeted delivery systems: To specifically target IL-22 signaling in affected tissues

• Explore combination therapies: Test IL-22 blockade alongside standard-of-care treatments

• Monitor biomarkers of IL-22 activity: Develop assays to measure IL-22-regulated genes as pharmacodynamic markers

• Consider biphasic effects: Design studies to account for potential protective versus pathogenic roles of IL-22 depending on disease stage

How should researchers interpret seemingly contradictory findings regarding IL-22 function in different disease models?

Contradictory findings regarding IL-22 function are common due to its context-dependent effects. When interpreting such findings, researchers should consider:

• Disease stage: IL-22 may be protective during tissue repair but pathogenic during active inflammation

• Genetic background: Different mouse strains (e.g., C57BL/6 vs. MRL/lpr) may show different IL-22 responses

• Cellular source of IL-22: T cell-derived versus ILC3-derived IL-22 may have different implications

• Target tissue receptor expression: Variation in IL-22RA1 expression across tissues affects response

• Co-existing cytokine milieu: Presence of other cytokines may synergize with or antagonize IL-22 effects

For example, in inflammatory bowel disease, IL-22 has been reported to have both protective effects (promoting epithelial barrier repair) and pathogenic effects (enhancing neutrophil recruitment) . These seemingly contradictory roles likely reflect the complex biology of IL-22 rather than experimental artifacts.

What statistical approaches are most appropriate for analyzing IL-22-related experimental data in complex disease models?

For complex IL-22 experimental data, researchers should consider:

• Mixed-effects models: To account for both fixed effects (treatment) and random effects (individual variation)

• Longitudinal data analysis: For time-course experiments monitoring disease progression

• Multivariate analysis: To examine relationships between multiple IL-22-regulated outcomes

• Network analysis: To understand IL-22's position within broader cytokine networks

• Machine learning approaches: For identifying patterns in complex datasets with multiple parameters

When comparing IL-22 KO, IL-22R KO, and control mice, researchers typically use one-way ANOVA for comparison among all groups, with post-hoc tests to identify specific group differences. For time-course data, repeated measures ANOVA or mixed-effects models are more appropriate .