RESUMEN

IL-1 family cytokines act as apical initiators of inflammation in many settings and can promote the production of a battery of inflammatory cytokines, chemokines and other inflammatory mediators in diverse cell types. IL-36α, IL-36ß and IL-36γ, which belong to the extended IL-1 family, have been implicated as key initiators of skin inflammation in psoriasis. IL-36γ is highly upregulated in lesional skin from psoriatic individuals, and heritable mutations in the natural IL-36 receptor antagonist result in a severe form of psoriasis. IL-36 family cytokines are initially expressed as inactive precursors that require proteolytic processing for activation. The neutrophil granule-derived protease elastase proteolytically processes and activates IL-36α and IL-36γ, increasing their biological activity ~ 500-fold, and also robustly activates IL-1α and IL-33 through limited proteolytic processing. Consequently, inhibitors of elastase activity may have potential as anti-inflammatory agents through antagonizing the activation of multiple IL-1 family cytokines. Using in silico screening approaches, we have identified small-molecule inhibitors of elastase that can antagonize activation of IL-36γ by the latter protease. The compounds reported herein may have utility as lead compounds for the development of inhibitors of elastase-mediated activation of IL-36 and other IL-1 family cytokines in inflammatory conditions, such as psoriasis.

RESUMEN

Sterile inflammation is initiated by molecules released from necrotic cells, called damage-associated molecular patterns (DAMPs). Members of the extended IL-1 cytokine family are important DAMPs, are typically only released through necrosis, and require limited proteolytic processing for activation. The IL-1 family cytokines, IL-36α, IL-36ß, and IL-36γ, are expressed as inactive precursors and have been implicated as key initiators of psoriatic-type skin inflammation. We have recently found that IL-36 family cytokines are proteolytically processed and activated by the neutrophil granule-derived proteases, elastase, and cathepsin G. Inhibitors of IL-36 processing may therefore have utility as anti-inflammatory agents through suppressing activation of the latter cytokines. We have identified peptide-based pseudosubstrates for cathepsin G and elastase, based on optimal substrate cleavage motifs, which can antagonize activation of all three IL-36 family cytokines by the latter proteases. Human psoriatic skin plaques displayed elevated IL-36ß processing activity that could be antagonized by peptide pseudosubstrates specific for cathepsin G. Thus, antagonists of neutrophil-derived proteases may have therapeutic potential for blocking activation of IL-36 family cytokines in inflammatory conditions such as psoriasis.

Asunto(s)

Inflamación/metabolismo , Interleucina-1/metabolismo , Neutrófilos/enzimología , Péptido Hidrolasas/metabolismo , Antiinflamatorios/uso terapéutico , Catepsina G/metabolismo , Células HeLa , Humanos , Neutrófilos/efectos de los fármacos , Elastasa Pancreática/metabolismo , Psoriasis/tratamiento farmacológico , Psoriasis/metabolismo , Piel/efectos de los fármacos , Piel/metabolismo , Piel/patología

RESUMEN

Recent evidence has strongly implicated IL-36 cytokines as key initiators of inflammation in the skin barrier. IL-36 cytokines belong to the extended IL-1 family and, similar to most members of this family, are expressed as inactive precursors that require proteolytic processing for activation. Because the proteases responsible for activation of members of the IL-36 subfamily have not been reported, we have developed a method for the production of biologically active IL-36 through introduction of a caspase cleavage motif, DEVD, within the N-termini of these cytokines. Here, we show that DEVD-modified IL-36α, IL-36ß and IL-36γ cytokines were highly soluble and were readily processed and activated by caspase-3. Caspase-3-processed IL-36 family cytokines exhibited robust biological activity on a range of responsive cell types, including primary keratinocytes. We also generated specific polyclonal antibodies against all three IL-36 family members through immunization with purified recombinant IL-36 cytokines. The modified forms of IL-36 described herein will be useful for production of large quantities of biologically active IL-36 for structure and function studies on these important proinflammatory cytokines.