IL36G Human

What is IL-36γ and how does it relate to the broader cytokine family?

IL-36γ, previously known as interleukin-1 family member 9 (IL1F9), belongs to the IL-1 superfamily of cytokines. It is one of four IL-36 isoforms: three with agonist activity (IL-36α, IL-36β, IL-36γ) and one with antagonist activity (IL-36 receptor antagonist, IL-36Ra). All IL-36 isoforms bind to the IL-36 receptor (IL-36R). When IL-36γ binds to IL-36R, it recruits the IL-1 receptor accessory protein (IL-1RAcP) and activates downstream signaling pathways mediated by nuclear transcription factor kappa B and mitogen-activated protein kinase pathways. This pathway plays a pivotal role in both innate and adaptive immune responses .

Where is IL-36γ primarily expressed in human tissues?

IL-36γ is predominantly expressed in epithelial tissues, particularly in the skin, gut, and lung. In skin tissue, IL-36γ shows a distinct expression pattern, being mainly expressed in epidermal granular layer keratinocytes with minimal expression in basal layer keratinocytes . Research has demonstrated that various cell types can produce IL-36γ in response to inflammatory stimuli, including pulmonary macrophages, interstitial fibroblasts, and alveolar epithelial cells .

What stimuli can induce IL-36γ expression?

Multiple stimuli can induce IL-36γ expression in various cell types:

• Proinflammatory cytokines: Interferon-gamma, tumor necrosis factor-alpha, and interleukin-1β can stimulate IL-36γ expression in keratinocytes

• Pathogen-Associated Molecular Patterns (PAMPs): Various microbial components can trigger IL-36γ production

• Bacterial stimuli: Heat-killed bacteria like Klebsiella pneumoniae can induce substantial upregulation of IL-36γ mRNA in pulmonary macrophages and fibroblasts

• Cigarette smoke compounds: Cigarette smoke condensates have been shown to induce IL-36γ production in lung cells

How is IL-36γ activated, and which proteases are involved in its processing?

Unlike many cytokines, IL-36γ requires proteolytic cleavage of its N-terminus for full biological activity, yet this activation is inflammasome-independent (unlike IL-1β). Several proteases have been identified that can process IL-36γ:

• Endogenous proteases: Cathepsin S can specifically cleave IL-36γ

• Exogenous proteases from pathogens:

  • Bacterial proteases: Streptococcus pyogenes secretes SpeB protease, which can cleave pro-IL-36γ into its active form

  • Fungal proteases: Several fungal pathogens produce proteases capable of activating IL-36γ

Experimental validation of proteolytic activation often employs SDS-PAGE analysis to observe truncation patterns and liquid chromatography-mass spectrometry (LC-MS) to identify specific cleavage sites, as demonstrated with SpeB which generates the IL-36γ S18 form .

What methods are used to measure IL-36γ activity in experimental settings?

Researchers employ multiple approaches to assess IL-36γ activity:

• ELISA-based detection: Quantification of IL-36γ protein in cell lysates and supernatants

• Biological activity assays: A common approach involves treating IL-36γ-sensitive cells (such as HaCaT keratinocytes) with processed IL-36γ and measuring secondary cytokine production (particularly IL-8)

• Inhibition assays: Using IL-36 receptor antagonist (IL-36RA) to confirm specificity of observed effects

• qPCR analysis: Measuring relative concentrations of IL-36γ mRNA from cell lysates at various time points after stimulation

A typical experimental workflow involves exposing cells to stimuli, collecting supernatants and lysates, measuring IL-36γ levels, and then using the collected IL-36γ in secondary assays with reporter cells to assess biological activity.

How does IL-36γ discriminate between pathogenic and commensal microbes?

IL-36γ functions as a sophisticated discriminator between harmful pathogens and harmless microbes through a two-step mechanism:

• Induction phase: Both pathogenic and commensal microbes can induce intracellular pro-IL-36γ expression in epithelial cells

• Release and activation phase: Only pathogenic microbes trigger:

  • Release of pro-IL-36γ through pathogen-induced cell damage

  • Processing to the mature, potent form via pathogen-derived proteases

This was demonstrated experimentally with Aspergillus fumigatus and Streptococcus pyogenes (pathogens) versus Staphylococcus epidermidis (commensal). When cells were treated with live pathogens, significant IL-36γ release was observed in the supernatant, while the commensal S. epidermidis failed to induce IL-36γ release despite upregulating its expression .

What is IL-36γ's role in inflammatory skin conditions, particularly psoriasis?

IL-36γ has been strongly implicated in psoriasis pathogenesis:

• Biomarker potential: Both IL-36γ mRNA and protein are elevated in psoriatic lesions and have been used as biomarkers to differentiate between eczema and psoriasis

• Genetic evidence: Mutations affecting the IL-36 pathway, particularly in IL-36Ra, are associated with generalized pustular psoriasis (GPP), a rare but severe form of psoriasis

• Mechanistic role: Uncontrolled activation of the IL-36 pathway leads to excessive inflammatory responses in the skin, promoting the characteristic features of psoriasis

Experimental approaches to study IL-36γ in psoriasis include immunohistochemistry of skin biopsies, gene expression analysis, and mouse models of psoriasiform dermatitis, such as imiquimod-induced dermatitis .

How does IL-36γ contribute to inflammatory lung diseases?

IL-36γ appears to be a critical amplifier of neutrophilic lung inflammation:

• In chronic obstructive pulmonary disease (COPD): IL-36α and IL-36γ concentrations are elevated both locally in bronchoalveolar lavage fluid (BALF) and systemically in plasma of long-term smokers (LTS) with and without COPD compared to healthy non-smokers

• During infections: IL-36γ acts as a key upstream amplifier of neutrophilic lung inflammation during viral and bacterial infections

• Cellular mechanisms: IL-36γ promotes activation of neutrophils, macrophages, and fibroblasts, particularly through cooperation with GM-CSF and viral mimics like poly(I:C)

Mouse models with IL-36 receptor deficiency exposed to cigarette smoke or cigarette smoke plus H1N1 influenza virus demonstrate attenuated lung inflammation compared to wild-type controls, providing strong evidence for IL-36's role in these conditions .

What is known about IL-36γ in COVID-19 and other infectious diseases?

IL-36 cytokines, including IL-36γ, play significant roles in infectious diseases:

• COVID-19: IL-36 has been implicated in COVID-19 pathogenesis, though the specific mechanisms require further investigation

• Bacterial infections: IL-36γ contributes to the inflammatory response during bacterial pneumonia, with experimental models showing its importance in host defense against pathogens like Klebsiella pneumoniae

• Fungal infections: IL-36γ can be induced and activated during fungal infections, particularly with pathogenic fungi like Aspergillus fumigatus

The dual nature of IL-36γ in infections merits careful study—it appears necessary for effective host defense but can contribute to pathological inflammation when dysregulated.

What cell and tissue models are most appropriate for studying IL-36γ functions?

Based on current research, several model systems have proven valuable for IL-36γ research:

The choice of model system should be guided by the specific research question, with consideration of species differences in the IL-36 pathway.

What are the recommended protocols for measuring IL-36γ expression and activation in human samples?

Based on published methodologies, a comprehensive approach to studying IL-36γ should include:

• mRNA expression analysis:

  • qPCR with specific primers for IL36G

  • Time-course experiments (e.g., 0, 6, and 18 hours post-stimulation)

• Protein detection:

  • ELISA for quantification in cell lysates, supernatants, and biological fluids

  • Western blotting for detecting processing/cleavage events

  • Immunohistochemistry for tissue localization

• Activation assays:

  • Incubation of recombinant pro-IL-36γ with candidate proteases

  • SDS-PAGE analysis to detect truncation

  • LC-MS to identify specific cleavage sites

  • Biological activity assays using IL-36γ-responsive cells (e.g., HaCaT) with IL-8 or other cytokine readouts

• Clinical sample collection:

  • BALF collection and sonication for lung studies

  • Plasma preparation for systemic measurements

  • Normalization strategies for comparative analyses

When comparing patient populations (e.g., smokers vs. non-smokers, disease vs. healthy), matching for age, sex, and other confounding factors is essential for meaningful results.

What are the key unresolved questions regarding IL-36γ signaling mechanisms?

Despite significant advances, several fundamental aspects of IL-36γ biology remain unclear:

• Complete protease repertoire: While some proteases that activate IL-36γ have been identified, the full complement of enzymes capable of processing IL-36γ in different physiological and pathological contexts remains unknown.

• Signaling crosstalk: How IL-36γ signaling integrates with other inflammatory pathways, particularly other IL-1 family members, requires further elucidation. Studies suggest cooperation with GM-CSF and poly(I:C), but the molecular mechanisms need clarification .

• Cell-type specific responses: Different cell types express varying levels of IL-36R and may respond differently to IL-36γ stimulation. Comprehensive mapping of cellular responsiveness would advance our understanding of tissue-specific effects.

• Negative regulation: While IL-36Ra antagonizes IL-36 signaling, other potential negative regulators and feedback mechanisms controlling IL-36γ responses warrant investigation.

How might therapeutic targeting of the IL-36 pathway be approached in inflammatory diseases?

Based on current understanding of IL-36γ biology, several therapeutic approaches could be considered:

• Direct cytokine inhibition:

  • Neutralizing antibodies against IL-36γ

  • Soluble IL-36R to act as a decoy receptor

• Receptor antagonism:

  • IL-36Ra mimetics or recombinant IL-36Ra

  • Small molecule inhibitors of IL-36R

• Protease inhibition:

  • Targeting specific proteases that activate IL-36γ, such as cathepsin S inhibitors

  • Pathogen-specific protease inhibitors for infectious contexts

• Downstream signaling inhibition:

  • Targeting the NF-κB and MAPK pathways activated by IL-36γ

The choice of approach would depend on the specific disease context, with generalized pustular psoriasis representing an obvious candidate given the established genetic and biological links to IL-36 pathway dysregulation .

What technological advances might enhance IL-36γ research in the near future?

Emerging technologies likely to impact IL-36γ research include:

• Single-cell analyses:

  • Single-cell RNA sequencing to identify specific cell populations producing or responding to IL-36γ

  • Mass cytometry to characterize IL-36 pathway components at the protein level

• Advanced tissue imaging:

  • Multiplexed immunofluorescence to visualize IL-36γ production and signaling in tissue context

  • Intravital microscopy to observe IL-36γ dynamics in living tissues

• CRISPR-based approaches:

  • Precise genetic manipulation of IL-36 pathway components

  • High-throughput CRISPR screens to identify novel regulators

• Structural biology:

  • Cryo-EM structures of IL-36γ in complex with its receptor

  • Structural analysis of IL-36γ processing by various proteases

• Improved animal models:

  • Humanized mouse models that better recapitulate human IL-36 biology

  • Tissue-specific conditional knockout/knockin models to dissect cell-specific roles

Integration of these technologies with conventional approaches will likely accelerate understanding of IL-36γ's functions in health and disease.