Recombinant Human Interleukin-33 protein (IL33), partial (Active)

Basic Research Questions

• How is recombinant human IL-33 expressed and purified for research applications?

  Recombinant human IL-33 can be expressed in several expression systems, with the most common being:

  Expression System	Advantages	Typical Yield	Applications
  E. coli	Cost-effective, high yield, simpler purification	High	In vitro bioassays, structural studies
  HEK293 cells	Post-translational modifications, higher bioactivity	Moderate	Complex cellular assays, in vivo studies

  The typical purification workflow includes:

  1. Cell lysis by sonication (for E. coli) or gentle detergent treatment (for mammalian cells)

  2. Clarification of lysate by centrifugation

  3. Affinity chromatography (typically Ni-affinity for His-tagged proteins)

  4. Size exclusion chromatography for final purification

  The purity of recombinant IL-33 should be ≥95% as assessed by SDS-PAGE and HPLC, with endotoxin levels kept below 0.1 ng/μg (1 EU/μg) for in vitro and in vivo applications .

• What are the standard methods for assessing the biological activity of recombinant IL-33?

  Several bioassays are employed to determine the biological activity of recombinant human IL-33:

  • Proliferation assay using murine D10S cells: The ED50 (effective dose for 50% maximum response) typically ranges from 0.03-0.24 ng/mL

  • Cytokine production assay: Measuring IL-5 and IL-13 secretion from Th2-polarized lymphocytes following IL-33 stimulation

  • Cell migration/scratch assay: Evaluating the wound-healing capability of keratinocytes and fibroblasts after IL-33 treatment

  • NF-κB reporter assay: Monitoring activation of NF-κB signaling pathway downstream of IL-33/ST2 receptor engagement

  The optimal dilutions for each application should be determined empirically for each laboratory and specific experimental setup .

Advanced Research Questions

• How does proteolytic processing affect the activity of IL-33?

  IL-33 activation through proteolytic processing is a complex regulatory mechanism that significantly impacts its biological function:

  • Full-length IL-33 (amino acids 1-270) has lower bioactivity compared to the cleaved mature forms

  • Processing of IL-33 by various proteases can increase its alarmin activity up to ~60-fold

  • Multiple processed forms of IL-33 with apparent molecular weights of ~18, 20, 22, and 23 kDa have been detected in human tissues

  Recent research has revealed that in addition to immune cell-derived proteases (neutrophil elastase, cathepsin G), environmental allergen proteases and endogenous calpains from damaged airway epithelial cells can process full-length IL-33 . This processing appears to be a sensor for both the proteolytic and oxidative microenvironment during tissue injury, facilitating rapid activation and subsequent inactivation to regulate the duration of IL-33's alarmin function .

  Importantly, oxidation of cysteine residues in IL-33 can lead to its degradation by allergen proteases, suggesting a regulatory mechanism for limiting excessive IL-33 signaling .

• What metabolic changes are induced by IL-33 in immune cells?

  IL-33 induces profound metabolic reprogramming in various immune cell types:

  Cell Type	Metabolic Changes	Functional Outcome	Key Regulators
  ILC2s	Enhanced glycolysis and OXPHOS, increased lipid uptake	Proliferation, IL-5/IL-13 production	HIF-1α, STAT3, PPARγ, Arginase-1
  Mast cells	Increased glycolysis and OXPHOS, with glycolysis being critical	IL-6/TNF production, neutrophil recruitment	ERK, NF-κB
  Macrophages	UCP2-dependent mitochondrial rewiring	Reduced ROS, preserved Krebs cycle, GATA3-dependent M2 differentiation	GATA3

  In IL-33-activated ILC2s, HIF-1α accumulation results in enhanced glycolytic capacity and attenuated mitochondrial respiration . HIF-1α drives the expression of the glycolytic enzyme pyruvate kinase M2 (PKM2) and increases glycolytic metabolite pyruvate, which plays a central role in controlling ILC2 homeostasis .

  IL-33 also activates mTOR, a key metabolic checkpoint that influences nutrient uptake and utilization in immune cells . Through the PI3K-AKT pathway, IL-33 can activate mTORC1, which is crucial for ILC2 metabolism and function .

• How does IL-33 influence epigenetic regulation in immune cells?

  IL-33 induces specific epigenetic modifications that shape immune cell function:

  • In ILC2s, IL-33-mediated STAT3 activation increases S-adenosylmethionine (SAM) levels, a major methyl donor during DNA or histone methylation, dramatically increasing H3K4me3 (a permissive histone mark) at Il5 and Il13 gene loci

  • Conversely, through the PKM2-pyruvate metabolic checkpoint, IL-33-induced glycolysis can lead to decreased H3K4me3 modification at the Il1rl1 (ST2) locus, creating a negative feedback loop controlling IL-33-mediated ILC2 maturation

  • IL-33-induced GATA3 phosphorylation in Treg and Th2 cells leads to histone H3K4 methylation and H3K9 acetylation at Th2 cytokine genes

  These findings demonstrate that IL-33 mediates immune cell function not only through direct transcriptional activation but also through metabolite-dependent epigenetic reprogramming, creating complex regulatory networks that fine-tune immune responses.

• What are the challenges in quantifying IL-33 in biological samples and how can they be overcome?

  Accurate quantification of IL-33 in biological samples faces several challenges:

  • Interference from endogenous binding partners, especially soluble ST2 (sST2), causes under-quantitation in commercial IL-33 assays

  • IL-33 can exist in multiple forms (full-length, various cleaved products) with different bioactivities

  • IL-33 may be rapidly degraded or modified in biological samples

  A modified method for accurate IL-33 quantification includes:

  1. Acid dissociation of serum samples to release IL-33 from endogenous binding partners

  2. Addition of detection reagent simultaneously with the capture step

  3. Specific focus on reduced (active) forms of IL-33

  This method increases soluble ST2 tolerance to >1000 ng/ml and provides a lower limit of quantification (LLOQ) of 6.25 pg/ml for reduced IL-33 in human serum .

  Interestingly, analysis of over 300 samples from individuals with and without asthma and with different smoking status revealed no significant difference in serum IL-33 levels, highlighting the importance of proper quantification methods for accurate clinical interpretation .

• How can recombinant IL-33 be applied in wound healing research?

  Recombinant IL-33 shows promising applications in wound healing research, particularly for diabetic wounds:

  • In streptozotocin (STZ)-induced diabetic mice, exogenous administration of recombinant mature IL-33 (rhmatIL-33) accelerated wound healing compared to untreated diabetic mice

  • At the cellular level, rhmatIL-33 accelerated scratch-healing of keratinocytes and fibroblasts in a dose-dependent manner

  • In diabetic mice, endogenous IL-33 mRNA is decreased after injury, unlike the upregulation seen in wild-type mice

  The mechanisms of IL-33-mediated wound healing include:

  1. Increasing endogenous IL-33 mRNA levels in diabetic tissue

  2. Elevating ILC2 cells in the wounds of both diabetic and non-diabetic mice

  3. Improving transcript levels of YM1, a marker of M2 macrophages, suggesting accelerated transformation of macrophages from M1 to M2 phenotype

  For wound healing experiments, rhmatIL-33 (10 μL, 100 μg/mL) is typically administered locally at the wound site once daily for seven days, with wound contraction measured as:

  Wound contraction (%) = (day 0 wound area − wound area on a particular day)/day 0 wound area × 100

• What are the dual intracellular and extracellular functions of IL-33?

  IL-33 exhibits unique dual functionality as both an intracellular nuclear factor and an extracellular cytokine:

  • Intracellular (nuclear) functions:

    • Acts as a chromatin-associated nuclear factor with transcriptional repressor properties

    • May sequester nuclear NF-κB/RELA, lowering expression of its targets

    • Is constitutively and abundantly expressed in quiescent endothelial cells

  • Extracellular (cytokine) functions:

    • Binds to IL1RL1/ST2 receptor, activating NF-κB and MAPK signaling pathways

    • Induces secretion of T-helper type 2-associated cytokines

    • Activates mast cells, basophils, eosinophils, and natural killer cells

    • Acts as a chemoattractant for Th2 cells

    • Functions as an 'alarmin' that amplifies immune responses during tissue injury

  This dual functionality makes IL-33 a unique cytokine with context-dependent roles in inflammation, tissue homeostasis, and immune regulation.

• How do different recombinant IL-33 variants compare in research applications?

  Different recombinant IL-33 variants have distinct characteristics that affect their utility in research:

  Variant	Amino Acids	Expression System	Key Properties	Optimal Applications
  Full-length	1-270	Mammalian cells	Nuclear localization, lower extracellular bioactivity	Studies of nuclear functions, cell transfection
  Mature (partial)	112-270	E. coli or mammalian	High receptor-binding activity, no nuclear localization	In vitro bioassays, receptor studies
  "Super-active" processed forms	Various (e.g., 95-270)	Mammalian cells	Up to 60-fold higher activity than full-length	Studies of inflammatory responses

  The choice of which variant to use depends on the specific research question. For studies focusing on receptor activation and downstream signaling, the mature form (amino acids 112-270) expressed in E. coli is often sufficient and cost-effective . For more complex studies examining IL-33's role in physiological or pathological contexts, mammalian-expressed variants with proper post-translational modifications may provide more relevant results .

  Researchers should carefully consider the expression system, purification method, and specific amino acid boundaries when selecting recombinant IL-33 for their experiments, as these factors can significantly impact experimental outcomes.