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Cytokines are small secreted proteins that among many functions also play key roles in the orchestration of inflammation in host defense and disease. Over the past years, a large number of biologics have been developed to target cytokines in disease, amongst which soluble receptor fusion proteins have shown some promise in pre-clinical studies. We have previously shown proof-of-concept for the therapeutic targeting of interleukin (IL)-33 in airway inflammation using a newly developed biologic, termed IL-33trap, comprising the ectodomains of the cognate receptor ST2 and the co-receptor IL-1RAcP fused into a single-chain recombinant fusion protein. Here we extend the biophysical and biological characterization of IL-33trap variants, and show that IL-33trap is a stable protein with a monomeric profile both at physiological temperatures and during liquid storage at 4°C. Reducing the N-glycan heterogeneity and complexity of IL-33trap via GlycoDelete engineering neither affects its stability nor its inhibitory activity against IL-33. We also report that IL-33trap specifically targets biologically active IL-33 splice variants. Finally, we document the generation and antagonistic activity of a single-chain IL-4/13trap, which inhibits both IL-4 and IL-13 signaling. Collectively, these results illustrate that single-chain soluble receptor fusion proteins against IL-4, IL-13, and IL-33 are novel biologics that might not only be of interest for research purposes and further interrogation of the role of their target cytokines in physiology and disease, but may also complement monoclonal antibodies for the treatment of allergic and other inflammatory diseases. [ABSTRACT FROM AUTHOR]

The emergence of IL-33 as a key molecular player in the development and propagation of widespread inflammatory diseases, including asthma and atopic dermatitis, has established the need for effective IL-33–neutralizing biologics. Here we describe the development and validation of a new antagonist of IL-33, termed IL-33trap, which combines the extracellular domains of the IL-33 receptor (ST2) and its coreceptor, IL-1 receptor accessory protein, into a single fusion protein. We produced and purified recombinant IL-33trap from human cells and analyzed its IL-33–binding affinity and IL-33 antagonistic activity in cultured cells and mice. IL-33trap activity was also benchmarked with a recombinant soluble ST2 corresponding to the naturally occurring IL-33 decoy receptor. Finally, we studied the effect of IL-33trap in the Alternaria alternata mouse model of allergic airway inflammation. In vitro IL-33trap binds IL-33 and inhibits IL-33 activity to a much stronger degree than soluble ST2. Furthermore, IL-33trap inhibits eosinophil infiltration, splenomegaly, and production of signature cytokines in splenic lymphocytes and lung tissue on IL-33 injection. Finally, administration of IL-33trap at the time of allergen challenge inhibits inflammatory responses in a preclinical mouse model of acute allergic airway inflammation. IL-33trap is a novel IL-33 antagonist that outperforms the natural IL-33 decoy receptor and shows anti-inflammatory activities in a preclinical mouse model of acute allergic airway inflammation when administered at the time of allergen challenge. [ABSTRACT FROM AUTHOR]

Interleukin (IL)-33 is a cytokine that is released from epithelial and endothelial cells at barrier surfaces upon tissue stress or damage to operate as an alarmin. IL-33 has been primarily implicated in the induction of T helper (Th) 2 type immune responses. Therefore, IL-33 has attracted a lot of interest as a potential therapeutic target in asthma and other allergic diseases. Over the years, it has become clear that IL-33 has a much broader activity and also contributes to Th1 immunity, expanding the possibilities for therapeutic modulation of IL-33 activity to multiple inflammatory diseases. However, more recently IL-33 has also been shown to mediate immunosuppression and tissue repair by activating regulatory T cells (Treg) and promoting M2 macrophage polarization. These pleiotropic activities of IL-33 illustrate the need for a tight molecular regulation of IL-33 activity, and have to be taken into account when IL-33 or its receptor is targeted for therapeutic modulation. Here we review the multiple molecular mechanisms that regulate IL-33 activity and describe how IL-33 can shape innate and adaptive immune responses by promoting Th1, Th2 and Treg function. Finally, we will discuss the possibilities for therapeutic modulation of IL-33 signaling as well as possible safety issues. [ABSTRACT FROM AUTHOR]

IL-33 plays a major role in the pathogenesis of allergic diseases such as asthma and atopic dermatitis. On its release from lung epithelial cells, IL-33 primarily drives type 2 immune responses, accompanied by eosinophilia and robust production of IL-4, IL-5, and IL-13. However, several studies show that IL-33 can also drive a type 1 immune response. We sought to determine the role of A20 in the regulation of IL-33 signaling in macrophages and IL-33–induced lung immunity. We studied the immunologic response in lungs of IL-33–treated mice that specifically lack A20 in myeloid cells. We also analyzed IL-33 signaling in A20-deficient bone marrow–derived macrophages. IL-33–induced lung innate lymphoid cell type 2 expansion, type 2 cytokine production, and eosinophilia were drastically reduced in the absence of macrophage A20 expression, whereas neutrophils and interstitial macrophages in lungs were increased. In vitro , IL-33–mediated nuclear factor kappa B activation was only weakly affected in A20-deficient macrophages. However, in the absence of A20, IL-33 gained the ability to activate signal transducer and activator of transcription 1 (STAT1) signaling and STAT1-dependent gene expression. Surprisingly, A20-deficient macrophages produced IFN-γ in response to IL-33, which was fully STAT1-dependent. Furthermore, STAT1 deficiency partially restored the ability of IL-33 to induce ILC2 expansion and eosinophilia in myeloid cell–specific A20 knockout mice. We reveal a novel role for A20 as a negative regulator of IL-33–induced STAT1 signaling and IFN-γ production in macrophages, which determines lung immune responses. [ABSTRACT FROM AUTHOR]

Immune checkpoint blockade (ICB) has heralded a new era in cancer therapy. Research into the mechanisms underlying response to ICB has predominantly focused on T cells; however, effective immune responses require tightly regulated crosstalk between innate and adaptive immune cells. Here, we combine unbiased analysis of blood and tumors from metastatic breast cancer patients treated with ICB with mechanistic studies in mouse models of breast cancer. We observe an increase in systemic and intratumoral eosinophils in patients and mice responding to ICB treatment. Mechanistically, ICB increased IL-5 production by CD4+ T cells, stimulating elevated eosinophil production from the bone marrow, leading to systemic eosinophil expansion. Additional induction of IL-33 by ICB-cisplatin combination or recombinant IL-33 promotes intratumoral eosinophil infiltration and eosinophil-dependent CD8+ T cell activation to enhance ICB response. This work demonstrates the critical role of eosinophils in ICB response and provides proof-of-principle for eosinophil engagement to enhance ICB efficacy. [Display omitted] • Eosinophils increase in patients with metastatic TNBC who respond to ICB • Eosinophils are critical for ICB response by enhancing CD8+ T cell activation • Accumulation of eosinophils is driven by CD4+ T cells, IL-5, and IL-33 • Proof-of-principle evidence for eosinophil engagement to enhance ICB-response Immunotherapy is only effective in a proportion of breast cancer patients. Blomberg et al. discover that eosinophils, a myeloid immune cell type, are expanded in responders. They show that eosinophil accumulation depends on CD4+ T cells and eosinophil-stimulating molecules IL-5 and IL-33. Eosinophils enhance activation of CD8+ T cells and improve immunotherapy response. [ABSTRACT FROM AUTHOR]

The progressive and sight-threatening disease, age-related macular degeneration (AMD), is a growing public health concern due to ageing demographics, with the highest unmet medical need for the advanced stage of dry AMD, geographic atrophy. The pathogenesis underlying AMD is driven by a complex interplay of genetic and environmental factors. There is ample evidence that inflammation is strongly involved in AMD development. Interleukin-33 (IL-33) has been proposed to be critically involved in retinal degeneration, but a protective role in eye pathophysiology was also demonstrated. The current study investigated the therapeutic potential of IL-33trap, a novel IL-33-neutralizing biologic, in dry AMD/geographic atrophy and, based on controversial data regarding the protective versus detrimental functions of IL-33 in neovascularization, evaluated the risk of progression to wet AMD by IL-33 neutralization. Repeated intravitreal (IVT) injections of IL-33trap in the mouse laser-induced choroidal neovascularization model did not exacerbate neovascularization or leakage, while it significantly inhibited inflammatory cell infiltration in the retinal pigment epithelium and choroid. On the contrary, IVT treatment with IL-33trap significantly induced retinal inflammation and could not prevent retinopathy induction in the mouse sodium iodate (NaIO 3) model. Overall, these data suggest a complex and dichotomous role of IL-33 in eye pathology and indicate that IL-33 neutralization is not able to prevent onset and progression of dry AMD pathogenesis. • IL-33trap reduces inflammation during laser-induced choroidal neovascularization. • Leakage and choroidal neovascularization are not affected by IL-33 neutralization. • IL-33 neutralization induces an inflammatory response in a dry AMD mouse model. • IL-33trap does not protect mice from sodium iodate-induced retinopathy. [ABSTRACT FROM AUTHOR]