IL-6 Mouse, His

What is IL-6 and what are its primary functions in mouse models?

IL-6 is a cytokine with diverse biological functions in immunity, tissue regeneration, and metabolism. In mice, IL-6 functions as a potent lymphoid cell growth factor affecting B-lymphocytes, T-lymphocytes, and hybridoma cells . It plays crucial roles in regulating the immune response, acute-phase reactions, and hematopoiesis . IL-6 is primarily expressed at sites of acute and chronic inflammation, where it is secreted into the serum and induces transcriptional inflammatory responses through the IL-6 receptor alpha .

Recent studies have revealed unexpected functions of IL-6 in neuronal protection. Mayo Clinic researchers found that neurons in the spinal cords of mice lacking IL-6 were degenerating dramatically, supporting a neuron-protection role for IL-6 .

How does IL-6 signaling work in mouse models?

IL-6 signaling in mice involves a complex cascade that begins when IL-6 binds to the IL-6 receptor (IL-6R). This receptor consists of two components:

• IL-6R alpha (CD126) - the binding subunit

• IL-6ST/gp130 - the signaling subunit

Three distinct signaling mechanisms have been identified:

• Classic signaling: Membrane-bound IL-6R and IL-6ST interact, primarily mediating regenerative and anti-inflammatory effects

• Trans-signaling: Soluble IL-6R (sIL6R) binds IL-6, and this complex then activates IL-6ST on cells that don't express membrane-bound IL-6R

• Cluster signaling: Membrane-bound IL-6:IL-6R complexes on transmitter cells activate IL-6ST receptors on neighboring receiver cells

What are the structural characteristics of recombinant mouse IL-6 with His-tag?

Recombinant mouse IL-6 with His-tag is typically a 21.7 kDa protein containing 188 amino acid residues . The His-tag is added to facilitate purification and detection. High-quality recombinant mouse IL-6 proteins exhibit ≥80-98% purity as determined by SDS-PAGE and HPLC analysis, with endotoxin levels less than 0.1 ng per μg (1EU/μg) . These proteins are typically expressed in mammalian expression systems such as HEK 293 cells to ensure proper folding and post-translational modifications .

What specialized mouse models are available for IL-6 research?

Several specialized mouse models have been developed for IL-6 research:

IL6-DIO-KO mouse: A conditional reversible IL-6 knockout mouse model using double-inverted, open-reading-frame (DIO) technology. This model lacks IL-6 production in all cells, which can be selectively restored by Cre recombinase activity .

LGL-IL6 transgenic mouse: A conditional human IL-6 transgenic mouse that can express human IL-6 when activated by Cre recombinase drivers .

IL6-DIO-KO Cx3cr1-CreER mice: A specialized model where IL-6 expression is restored specifically in microglia through breeding IL6-DIO-KO mice with Cx3cr1-CreER mice and subsequent tamoxifen administration .

How is the IL6-DIO-KO mouse model generated and validated?

The IL6-DIO-KO mouse model is generated using double-inverted, open-reading-frame (DIO) technology. This creates a mouse line with loss of IL6 expression in all cells that can be restored by the action of Cre recombinase .

Generation protocol:

• The IL6 gene is engineered with a double-inverted orientation

• This configuration prevents expression of functional IL-6 protein

• When Cre recombinase is present, it recognizes specific sites flanking the inverted sequence and restores the correct orientation

• This enables expression of IL-6 only in cells where Cre is active

Validation methods:

• Confirming loss of IL-6 expression in the initial knockout state

• Demonstrating recovery of IL-6 expression in specific cell types after Cre-mediated recombination

• Functional testing in disease models such as experimental autoimmune encephalomyelitis (EAE)

What are the advantages of conditional IL-6 mouse models compared to global knockouts?

Conditional IL-6 mouse models offer several significant advantages over global knockouts:

• Cell-specific analysis: They allow investigation of IL-6 functions from specific cellular sources, which is critical as IL-6 regulates disease in a source-specific manner .

• Recovery-of-function paradigm: Models like IL6-DIO-KO enable researchers to study the sufficiency (not just necessity) of IL-6 from specific cell types .

• Temporal control: When combined with inducible Cre systems (like CreER), they permit temporally controlled IL-6 expression or deletion.

• Reduced compensatory mechanisms: Global knockouts may develop compensatory mechanisms during development that mask the true function of IL-6.

• Disease relevance: Cell-specific models better mimic therapeutic interventions that might target IL-6 signaling in specific cell types rather than systemically.

How can IL-6 mouse models be used to study neuroinflammatory diseases?

IL-6 mouse models have proven valuable for studying neuroinflammatory diseases:

Experimental Autoimmune Encephalomyelitis (EAE):
The IL6-DIO-KO mouse crossed with Cx3cr1-CreER mice provides a powerful tool to investigate microglial IL-6 contribution to EAE, a popular mouse model of multiple sclerosis. Research has shown that selective recovery of microglial IL-6 was sufficient to initiate mild paralyzing symptoms related to EAE and regulate the inflammatory cascade .

Allergic Rhinitis and Olfactory Dysfunction:
IL-6 has been implicated in olfactory dysfunction (OD) in allergic rhinitis (AR). Studies using AR mice with OD showed significantly increased IL-6 levels in the nasal mucosa and olfactory bulb, positively correlating with the expression of olfactory bulb microglia marker Iba-1 and the severity of OD .

Neuronal Protection:
Research has demonstrated that IL-6 plays a crucial protective role for neurons. In healthy mice, IL-6 is typically not detected in the brain, but upon viral infection, astrocytes rapidly produce IL-6. Mice lacking IL-6 showed significantly higher mortality rates (~60%) compared to IL-6-positive mice (~9%) after viral infection .

What is the optimal protocol for using His-tagged recombinant mouse IL-6 in cell culture experiments?

Reconstitution and Storage Protocol:

• Reconstitute lyophilized protein in sterile water or appropriate buffer according to manufacturer specifications

• For short-term storage (1-2 weeks), store at 4°C

• For long-term storage, prepare aliquots and store at -20°C or -80°C

• Avoid repeated freeze-thaw cycles to maintain protein integrity

Experimental Considerations:

• Purity: Use high-purity (≥80-98%) recombinant protein with endotoxin levels <0.1 ng/μg

• Concentration: Typical working concentrations range from 0.1-100 ng/mL; perform dose-response experiments to determine optimal concentration for your specific application

• Functional validation: Confirm bioactivity through proliferation assays with IL-6-dependent cell lines

• Controls: Include positive controls (known IL-6 responsive cells) and negative controls (IL-6R blocking antibodies)

How can I evaluate IL-6 signaling pathway activation in mouse tissue samples?

Several techniques can be employed to evaluate IL-6 signaling pathway activation:

Biochemical Analysis:

• Western blot for phosphorylated STAT3 (pSTAT3), the primary transcription factor activated by IL-6 signaling

• Quantification of SOCS3 expression, an IL-6-induced negative regulator

• Measurement of acute phase proteins (e.g., SAA, CRP) in liver or serum

Histological Methods:

• Immunohistochemistry for pSTAT3 localization in tissue sections

• Dual immunofluorescence for co-localization of pSTAT3 with cell-type markers

• In situ hybridization for IL-6 target gene expression

Flow Cytometry:

• Intracellular staining for pSTAT3 in specific cell populations

• Surface staining for IL-6R and gp130 expression levels

• Multiplex analysis of downstream signaling molecules

What controls should be included when working with IL-6 conditional mouse models?

When working with IL-6 conditional mouse models, a comprehensive set of controls is essential:

• Genotype controls:

  • Wild-type littermates as primary control group

  • IL6-DIO-KO mice without Cre (to control for the genetic modification itself)

  • Cre-expressing mice without IL6-DIO-KO (to control for Cre effects)

  • Heterozygous mice (IL6-DIO-Het) to assess gene dosage effects

• Induction controls (for tamoxifen-inducible systems):

  • Vehicle-treated Cre+/IL6-DIO-KO mice (to control for tamoxifen effects)

  • Tamoxifen-treated Cre-/IL6-DIO-KO mice (to control for non-specific recombination)

  • Appropriate timing controls (e.g., 7 weeks post-tamoxifen for Cx3cr1-CreER )

• Experimental controls:

  • Age-matched mice (10-16 weeks old is typical for EAE studies )

  • Sex-matched cohorts (to account for sex-specific differences in IL-6 signaling)

  • Treatment controls specific to the disease model (e.g., adjuvant-only control for EAE)

How can I validate cell-specific IL-6 expression/deletion in conditional mouse models?

Validating cell-specific IL-6 expression or deletion requires a multi-faceted approach:

Genetic Validation:

• PCR genotyping to confirm presence of the IL6-DIO-KO allele and Cre transgene

• qRT-PCR on FACS-sorted cell populations to quantify IL-6 mRNA levels in target vs. non-target cells

Protein-Level Validation:

• Immunohistochemistry with co-staining for IL-6 and cell-type markers

• ELISA on supernatants from isolated cell populations after stimulation

• Intracellular cytokine staining and flow cytometry analysis

Functional Validation:

• Cell-specific responses to stimulation (e.g., LPS challenge)

• Phenotypic analysis in disease models (e.g., EAE clinical scores, histopathology)

• Phospho-STAT3 staining to assess IL-6 signaling activation in target tissues

What is the optimal protocol for inducing and evaluating EAE in IL-6 conditional mouse models?

The protocol for inducing and evaluating EAE in IL-6 conditional mouse models involves several key steps:

Preparation Phase:

• For Cx3cr1-CreER/IL6-DIO-KO mice, administer tamoxifen (typically 1-2 mg/day for 5 consecutive days) when mice are 10-16 weeks old

• Allow 7 weeks post-tamoxifen treatment for complete microglial turnover and IL-6 expression

EAE Induction:

• Immunize mice with myelin oligodendrocyte glycoprotein 35-55 peptide (MOG 35-55) emulsified in complete Freund's adjuvant

• Administer pertussis toxin on days 0 and 2 post-immunization

• Monitor mice daily for clinical symptoms using a standardized scale

Evaluation Methods:

• Clinical assessment: Daily scoring of neurological deficits (0-5 scale)

• Histopathological analysis:

  • Demyelination evaluation in spinal cord sections

  • CD3+ T cell infiltration quantification

  • Gliosis assessment using markers like GFAP and Iba1

• Immunological assessment:

  • Cytokine profiling from serum and CNS

  • Flow cytometry analysis of infiltrating immune cells

  • qRT-PCR for inflammatory markers

How can I explain contradictory results between different IL-6 mouse models?

Contradictory results between different IL-6 mouse models can occur for several reasons:

Source-Specific Effects:
IL-6 regulates disease in a source-specific manner, as demonstrated by varied results with conditional cell-specific IL-6 KO mice . For example, IL-6 from microglia versus astrocytes or peripheral immune cells may have different or even opposing effects.

Temporal Considerations:

• Developmental versus adult IL-6 deletion may produce different phenotypes

• Acute versus chronic IL-6 deficiency can trigger different compensatory mechanisms

• The timing of IL-6 manipulation relative to disease induction is critical

Genetic Background Issues:

• Strain background differences can significantly influence phenotypes

• Backcrossing status (number of generations on C57BL/6 background)

• Presence of passenger mutations linked to the modified IL-6 locus

Methodological Variations:

• Disease induction protocols (e.g., EAE immunization methods)

• Housing conditions and microbiome differences

• Analysis timepoints and methods

What are common technical challenges when working with His-tagged recombinant mouse IL-6?

Researchers often encounter several challenges when working with His-tagged recombinant mouse IL-6:

Protein Quality Issues:

• Batch-to-batch variation in activity

• Endotoxin contamination affecting experimental outcomes

• Protein aggregation or degradation during storage

Experimental Design Challenges:

• Determining appropriate concentration for specific cell types

• Accounting for the His-tag's potential interference with protein activity

• Protein stability in different culture media compositions

Detection Problems:

• Cross-reactivity between anti-His antibodies and endogenous proteins

• Interference of the His-tag with certain IL-6 epitopes

• Limited sensitivity of detection methods for low IL-6 concentrations

Troubleshooting Strategies:

• Always perform quality control testing on new batches

• Include positive controls (known IL-6 responsive cells)

• Consider removing the His-tag for certain applications if it interferes with function

• Store properly and avoid repeated freeze-thaw cycles

How can I distinguish between direct and indirect effects of IL-6 in conditional mouse models?

Distinguishing between direct and indirect effects of IL-6 requires careful experimental design:

In vivo approaches:

• Use cell-specific IL-6 expression models like IL6-DIO-KO crossed with cell-specific Cre lines

• Analyze the temporal sequence of events following IL-6 restoration

• Employ bone marrow chimeras to distinguish between effects of IL-6 from hematopoietic versus non-hematopoietic sources

• Use IL-6R conditional knockout mice to identify direct target cells

In vitro approaches:

• Establish co-culture systems with IL-6-producing and responding cells

• Use transwell systems to separate direct cell-cell interactions from soluble factors

• Apply IL-6 receptor blocking antibodies to specific cell populations

• Perform gene expression profiling to identify direct STAT3 target genes versus secondary responses

Molecular approaches:

• ChIP-seq for STAT3 binding sites to identify direct transcriptional targets

• Use rapid inhibition of protein synthesis to distinguish primary from secondary transcriptional responses

• Single-cell RNA-seq to identify cell-specific responses to IL-6 signaling

• Phospho-flow cytometry to measure immediate signaling events in specific cell populations

How can IL-6 mouse models contribute to understanding neurodegenerative diseases?

IL-6 mouse models offer valuable insights into neurodegenerative diseases:

Neuronal Protection Mechanisms:
Research has revealed that IL-6 plays a critical neuroprotective role. In healthy mice, IL-6 is typically absent from the brain, but upon viral infection, astrocytes rapidly produce IL-6. Mice lacking IL-6 showed dramatically degenerating neurons in the spinal cord and higher mortality rates (~60%) compared to IL-6-positive mice (~9%) after viral infection .

Neuroinflammatory Regulation:
The IL6-DIO-KO Cx3cr1-CreER mouse model enables specific investigation of microglial IL-6 contributions to neuroinflammation. Studies have shown that microglial IL-6 is sufficient to initiate mild paralyzing symptoms in EAE and regulate inflammatory cascades .

Olfactory System Research:
IL-6 has been implicated in olfactory dysfunction in allergic rhinitis through olfactory bulb microglia-mediated neuroinflammation. Increased IL-6 levels in the olfactory bulb correlate with microglia marker Iba-1 expression and the severity of olfactory dysfunction .

What are the emerging applications of IL-6R alpha conditional mouse models?

IL-6R alpha conditional mouse models are revealing new insights into IL-6 signaling:

Central Nervous System Function:
IL-6R alpha in the central nervous system regulates energy and glucose homeostasis through "trans-signaling" mechanisms . This highlights the metabolic importance of IL-6 signaling beyond its inflammatory roles.

T-Cell Differentiation:
IL-6R drives naive CD4+ T cells to the Th17 lineage through "cluster signaling" by dendritic cells . This mechanism is distinct from classic IL-6 signaling and represents an important immunoregulatory pathway.

Tissue Regeneration:
IL-6R plays a protective role during liver injury and is required for maintenance of tissue regeneration . Conditional IL-6R models help distinguish between regenerative and inflammatory IL-6 functions.

Therapeutic Target Validation:
Cell-specific IL-6R knockout models are valuable for validating potential therapeutic approaches targeting specific aspects of IL-6 signaling while preserving beneficial functions.

How can IL-6 mouse models and recombinant proteins be used to study the different signaling modes of IL-6?

Researchers can employ several strategies to study the different signaling modes of IL-6:

Classic Signaling Studies:

• Use cells expressing both membrane-bound IL-6R and gp130

• Apply blocking antibodies specific to membrane-bound IL-6R

• Analyze IL-6 signaling in IL-6R-rich tissues like liver and immune cells

• Use recombinant mouse IL-6 protein with membrane IL-6R-expressing cells

Trans-Signaling Studies:

• Use soluble IL-6R (sIL-6R) in combination with recombinant IL-6

• Study IL-6 functions in tissues with low IL-6R but high gp130 expression

• Design experiments comparing responses to IL-6 alone versus IL-6 plus sIL-6R

• Employ the designer cytokine Hyper-IL-6 (fusion protein of IL-6 and sIL-6R)

Cluster Signaling Studies:

• Establish co-culture systems with IL-6R-positive "transmitter" cells and gp130-positive "receiver" cells

• Perform immunohistochemistry to identify cell clusters in tissues

• Use conditional IL-6R knockout models to disrupt specific cellular interactions

• Study dendritic cell interactions with naive CD4+ T cells