The Merkel Cell Polyomavirus T-Antigens and IL-33/ST2-IL1RAcP Axis: Possible Role in Merkel Cell Carcinoma.

Merkel cell polyomavirus (MCPyV) is a causal factor in Merkel cell carcinoma (MCC). The oncogenic potential is mediated through its viral oncoproteins large T-antigen (LT) and small T-antigen (sT). Cytokines produced by tumor cells play an important role in cancer pathogenesis, and viruses affect their expression. Therefore, we compared human cytokine and receptor transcript levels in virus positive (V+) and virus negative (V-) MCC cell lines. Increased expression of IL-33, a potent modulator of tumor microenvironment, was observed in V+ MCC cell lines when compared to V- MCC-13 cells. Transient transfection studies with luciferase reporter plasmids demonstrated that LT and sT stimulated IL-33, ST2/IL1RL1 and IL1RAcP promoter activity. The induction of IL-33 expression was confirmed by transfecting MCC-13 cells with MCPyV LT. Furthermore, recombinant human cytokine domain IL-33 induced activation of MAP kinase and NF-κB pathways, which could be blocked by a ST2 receptor antibody. Immunohistochemical analysis demonstrated a significantly stronger IL-33, ST2, and IL1RAcP expression in MCC tissues compared to normal skin. Of interest, significantly higher IL-33 and IL1RAcP protein levels were observed in MCC patient plasma compared to plasma from healthy controls. Previous studies have demonstrated the implication of the IL-33/STL2 pathway in cancer. Because our results revealed a T-antigens-dependent induction of the IL-33/ST2 axis, IL-33/ST2 may play a role in the tumorigenesis of MCPyV-positive MCC. Therefore, neutralizing the IL-33/ST2 axis may present a novel therapeutic approach for MCC patients.

Construction of A375·S2 Melanoma Cell Line with High Sensibility to IL-1 by Overexpressing IL-1 Receptor.

We described an operation that co-overexpress interleukin receptor 1 (IL-1R1) and its co-receptor (IL-1R1AcP) genes in wild-type A375·S2 cells in order to increase their sensibility to IL-1. Firstly, laser scanning confocal microscope observed that IL-1R1 could be expressed on the surface of A375·S2 cells. qPCR was performed to estimate the ratio of two genes and result showed the ratio was almost 4.57:1. Then two genes were linked to vectors and co-transfected into A375·S2 cells. qPCR and Western blotting showed the protein content improved markedly. Finally, MTS assay was executed and the sensitivity of A375·S2 cells that co-transfected receptors to IL-1ß increased significantly. Another MTS assay showed the cell activity variation changed significantly (P < 0.05) and the reliability of the experiment was high, indicating that cell line established in this study could be further used for the activity test of IL-1Ra. Supplementary Information: The online version contains supplementary material available at 10.1007/s12088-022-01027-8.

Special aspects of interleukin-33 and the IL-33 receptor complex.

Interleukin-33 (IL-33) is an unconventional member of the IL-1 family: it is a dual function cytokine. Many different cell types, tissue cells and leukocytes, produce IL-33 either constitutively or after stimulation and release it by a poorly defined molecular mechanism. Free IL-33 acts as a classical cytokine by binding to target cells expressing receptors for IL-33 minimally consisting of ST2 and IL-1RAcP. Depending on the target cell type IL-33 will stimulate cell-type specific signal transduction mechanisms and thereby change the biosynthetic profile of the respective cell. In addition, it is stored in the nucleus of cells and may be released after cell stress, death by injury or necrosis, acting as an alarmin by orchestrating a sterile inflammation. Furthermore, IL-33 has intracrine functions in the cell producing it, which are independent of IL-33 receptors. Intracellular IL-33 is predominantly found in the nucleus associated to the chromatin and may exert gene regulatory function by yet poorly defined mechanisms. It is the aim of this review to address two basic biological aspects of the IL-33/IL-33 receptor system. First, to summarize the current understanding of the fate and function of intracellular IL-33, and second, to discuss recent advances in the knowledge of the molecular composition, function and regulation of the IL-33 receptor complex, including initial signaling mechanisms.

Anti-cytokine therapies in T1D: Concepts and strategies.

Therapeutic targeting of proinflammatory cytokines is clinically beneficial in several autoimmune disorders. Several of these cytokines are directly implicated in the pathogenesis of type 1 diabetes, suggesting opportunities for design of clinical trials in type 1 diabetes that incorporate selective cytokine blockade as a component of preventative or interventional immunotherapy. The rationale and status of inhibitory therapy directed against IL-1, TNF, IL-12, IL-23, and IL-6 are discussed, towards a goal of using cytokine inhibition as a therapeutic platform to establish an in vivo milieu suitable for modulating the immune response in T1D.

Pro- and anti-inflammatory roles of interleukin (IL)-33, IL-36, and IL-38 in inflammatory bowel disease.

Interleukin-33 (IL-33), IL-36, and IL-38 are members of the IL-1 cytokine family. The expression of each cytokine has been reported to be increased in the inflamed mucosa of patients with inflammatory bowel disease (IBD). IL-33 and IL-36 have been studied for pro- and anti-inflammatory functions, and IL-38 has been characterized as an anti-inflammatory cytokine by antagonizing the IL-36 receptor (IL-36R). IL-33 is a nuclear cytokine constitutively expressed by certain cell types such as epithelial, endothelial, and fibroblast-like cells and released on necrotic cell death. IL-33 mainly induces type 2 immune response through its receptor suppression tumorigenicity 2 (ST2) from Th2 cells and type 2 innate lymphoid cells (ILC2s), but also by stimulating Th1 cells, regulatory T cells, and CD8+ T cells. IL-36 cytokines consist of three agonists: IL-36α, IL-36ß, and IL-36γ, and two receptor antagonists: IL-36R antagonist (IL-36Ra) and IL-38. All IL-36 cytokines bind to the IL-36R complex and exert various functions through NF-κB and mitogen-activated protein kinase (MAPK) pathways in inflammatory settings. IL-33 and IL-36 also play a crucial role in intestinal fibrosis characteristic manifestation of CD. In this review, we focused on the current understanding of the pro- and anti-inflammatory roles of IL-33, IL-36, and IL38 in experimental colitis and IBD patients.

Constitutive photomorphogenic protein 1 ubiquitinates interleukin-1 receptor accessory protein in human liver cancer.

BACKGROUND: Constitutive photomorphogenic protein 1 (COP1) plays a pivotal role in the development and progression of several human cancers and is reported to be upregulated in liver cancer. However, the role of COP1 in human liver cancer is unclear. METHODS: We analyzed the COP1 expression in normal liver and liver cancer tissue samples using western blot and immunohistochemical analysis. We overexpressed and silenced COP1 in HepG2 and Huh7 cells and analyzed the effect on liver cancer cell proliferation. Additionally, COP1 was used as a bait to screen COP1-interacting proteins in a human cDNA library in a yeast two-hybrid screen and the results were confirmed with co-immunoprecipitation (co-IP) assays. Moreover, immunofluorescence staining was performed to assess co-localization. The protein levels of COP1 and mIL1RAcP were determined in clinical samples. RESULTS: COP1 was upregulated in liver cancer samples compared to that in normal tissue samples. COP1 overexpression promoted proliferation of liver cancer cells, while COP1 knockdown exerted the opposite effect. Yeast two-hybrid screen identified interleukin-1 receptor accessory protein (IL1RAP) as a potential COP1-interacting protein. Co-IP assays further confirmed that COP1 interacts with both preIL1RAP and membrane-bound form of IL1RAP (mIL1RAP). Furthermore, COP1 upregulated mIL1RAP protein levels and promoted nuclear translocation and activation of the nuclear factor kappa B (NF-κB) (p50/p65) dimer. Additionally, we demonstrated that COP1 regulated mIL1RAP expression through K63-linked polyubiquitination, suggesting that COP1 plays a role in stabilizing mIL1RAP. Finally, the protein levels of COP1 and mIL1RAcP were found to be positively correlated in clinical samples. CONCLUSION: COP1 regulates IL1RAP, which in turn results in activation of the NF-κB signaling. Our findings suggest that the COP1/IL1RAP/NF-κB axis promotes proliferation of liver cancer cells and is a potential target for the treatment of liver cancer.

Regulatory effect of IL-38 on NF-κB pathway in systemic lupus erythematosus.

BACKGROUND: IL-38 is a newly identified cytokine that exhibits immunosuppression effects. However, there are few studies focusing on the effects and mechanisms of IL-38 in the systemic lupus erythematosus (SLE). AIM: We investigated the effects and mechanisms of IL-38 on NF-κB signaling pathway in SLE. METHODS: Levels of IL-38, IL-36R, IL-1RAcP, IKKα/ß, NF-κB, TNF-α and anti-dsDNA antibody levels in peripheral blood of SLE patients, and in peripheral blood and kidney tissues of MRL/lpr mice, were examined with real-time PCR, ELISA, Western blot and immunohistochemistry. Pathological changes of kidney were detected with PAS staining. Recombinant human IL-38 protein and IL-38 siRNA were used to intervene the PBMCs of SLE patients and MRL/lpr mice. RESULTS: The mRNA and protein levels of IL-38 in peripheral blood of SLE patients decreased and were positively correlated. The mRNA and protein levels of IKKα/ß, NF-κB, and TNF-α increased, especially in patients with active SLE. There was a negative correlation between IL-38 and the levels of IKKα/ß, NF-κB and TNF-α in SLE patients. In vitro experiments showed that the levels of IKKα/ß, NF-κB and TNF-α, and anti-dsDNA antibodies decreased in PBMCs of SLE patients after treatment with human recombinant IL-38 protein. These effects were reversed after IL-38 siRNA intervention. Consistent results were obtained on IL-38, IKKα/ß, NF-κB, and TNF-α in MRL/lpr lupus mice after treatment with IL-38 protein or IL-38 shRNA. Additionally, kidney function (reflected by creatinine and blood urea nitrogen), anti-dsDNA antibody, complement C3, and urinary protein levels decreased after treatment with IL-38 protein but increased after IL-38 shRNA treatment. PAS staining showed IL-38 protein treatment induced mild hyperplasia of glomerular mesangial cells and a small amount of lymphocyte infiltration. However, these were aggravated after IL-38 shRNA treatment. CONCLUSION: IL-38 may be involved in the occurrence and development of SLE by regulating the NF-κB signaling pathway. This study only discussed the relationship between IL-38 and NF-κB, and more biological functions of IL-38 need to be further studied.

Glycan-mediated functional assembly of IL-1RI: structural insights into completion of the current description for immune response.

Interleukin 1 Receptor type I (IL-1RI) is a multi-domain transmembrane receptor that triggers the inflammatory response. Understanding its detailed mechanism of action is crucial for treating immune disorders. IL-1RI is activated upon formation of its functional assembly that occurs by binding of the IL-1 cytokine and the accessory protein (Il-1RAcP) to it. X-ray crystallography, small-Angle X-ray Scattering and molecular dynamics simulation studies showed that IL-1RI adopts two types of 'compact' and 'extended' conformational states in its dynamical pattern. Furthermore, glycosylation has shown to play a critical role in its activation process. Here, classical and accelerated atomistic molecular dynamics were carried out to examine the role of full glycosylation of IL-1RI and IL-1RAcP in arrangement of the functional assembly. Simulations showed that the 'compact' and 'extended' IL-1RI form two types of 'cytokine-inaccessible-non-signaling' and 'cytokine-accessible-signaling' assemblies with the IL-1RacP, respectively that are both abiding in the presence of glycans. Suggesting that the cytokine binding to IL-1RI is not required for the formation of IL-1RI-IL-1RAcP complex and the 'compact' complex could act as a down-regulatory mechanism. The 'extended' complex is maintained by formation of several persistent hydrogen bonds between the IL-1RI-IL-1RAcP inter-connected glycans. Taken together, it was shown that full glycosylation regulates formation of the IL-1RI functional assembly and play critical role in cytokine biding and triggering the IL-1RI involved downstream pathways in the cell.Communicated by Ramaswamy H. Sarma.

Molecular Basis of Selective Cytokine Signaling Inhibition by Antibodies Targeting a Shared Receptor.

Interleukin-1 (IL-1) family cytokines are potent mediators of inflammation, acting to coordinate local and systemic immune responses to a wide range of stimuli. Aberrant signaling by IL-1 family cytokine members, however, is linked to myriad inflammatory syndromes, autoimmune conditions and cancers. As such, blocking the inflammatory signals inherent to IL-1 family signaling is an established and expanding therapeutic strategy. While several FDA-approved IL-1 inhibitors exist, including an Fc fusion protein, a neutralizing antibody, and an antagonist cytokine, none specifically targets the co-receptor IL-1 receptor accessory protein (IL-1RAcP). Most IL-1 family cytokines form productive signaling complexes by binding first to their cognate receptors - IL-1RI for IL-1α and IL-1ß; ST2 for IL-33; and IL-36R for IL-36α, IL-36ß and IL-36γ - after which they recruit the shared secondary receptor IL-1RAcP to form a ternary cytokine/receptor/co-receptor complex. Recently, IL-1RAcP was identified as a biomarker for both AML and CML. IL-1RAcP has also been implicated in tumor progression in solid tumors and an anti-IL1RAP antibody (nadunolimab, CAN04) is in phase II clinical studies in pancreatic cancer and non-small cell lung cancer (NCT03267316). As IL-1RAcP is common to all of the abovementioned IL-1 family cytokines, targeting this co-receptor raises the possibility of selective signaling inhibition for different IL-1 family cytokines. Indeed, previous studies of IL-1ß and IL-33 signaling complexes have revealed that these cytokines employ distinct mechanisms of IL-1RAcP recruitment even though their overall cytokine/receptor/co-receptor complexes are structurally similar. Here, using functional, biophysical, and structural analyses, we show that antibodies specific for IL-1RAcP can differentially block signaling by IL-1 family cytokines depending on the distinct IL-1RAcP epitopes that they engage. Our results indicate that targeting a shared cytokine receptor is a viable therapeutic strategy for selective cytokine signaling inhibition.

Structure-based Development of Human Interleukin-1ß-Specific Antibody That Simultaneously Inhibits Binding to Both IL-1RI and IL-1RAcP.

Interleukin-1ß (IL-1ß) is a potent pleiotropic cytokine playing a central role in protecting cells from microbial pathogen infection or endogenous stress. After it binds to IL-1RI and recruits IL-1 receptor accessory protein (IL-1RAcP), signaling culminates in activation of NF-κB. Many pathophysiological diseases have been attributed to the derailment of IL-1ß regulation. Several blocking reagents have been developed based on two mechanisms: blocking the binding of IL-1ß to IL-1RI or inhibiting the recruitment of IL-1RAcP to the IL-1ß initial complex. In order to simultaneously fulfill these two actions, a human anti-IL-1ß neutralizing antibody IgG26 was screened from human genetic phage-display library and furthered structure-optimized to final version, IgG26AW. IgG26AW has a sub-nanomolar binding affinity for human IL-1ß. We validated IgG26AW-neutralizing antibodies specific for IL-1ß in vivo to prevent human IL-1ß-driving IL-6 elevation in C56BL/6 mice. Mice underwent treatments with IgG26AW in A549 and MDA-MB-231 xenograft mouse cancer models have also been observed with tumor shrank and inhibition of tumor metastasis. The region where IgG26 binds to IL-1ß also overlaps with the position where IL-1RI and IL-1RAcP bind, as revealed by the 26-Fab/IL-1ß complex structure. Meanwhile, SPR experiments showed that IL-1ß bound by IgG26AW prevented the further binding of IL-1RI and IL-1RAcP, which confirmed our inference from the result of protein structure. Therefore, the inhibitory mechanism of IgG26AW is to block the assembly of the IL-1ß/IL-1RI/IL-1RAcP ternary complex which further inhibits downstream signaling. Based on its high affinity, high neutralizing potency, and novel binding epitope simultaneously occupying both IL-1RI and IL-1RAcP residues that bind to IL-1ß, IgG26AW may be a new candidate for treatments of inflammation-related diseases or for complementary treatments of cancers in which the role of IL-1ß is critical to pathogenesis.

Association of Common Variants in the IL-33/ST2 Axis with Ischemic Stroke.

BACKGROUND: Recent studies have reported that the levels of serum interleukin-33 (IL- 33) and its receptor, suppression of tumorigenicity 2 (ST2), are potential biomarkers for susceptibility of cardiovascular diseases. However, the genetic association of the IL-33/ST2 axis with cardiovascular diseases remains controversial. OBJECTIVE: We aimed to investigate the association between common variants in the IL-33/ST2 axis and ischemic stroke in the Han Chinese population. METHODS: We consecutively enrolled 1166 patients with ischemic stroke and 1079 age- and gender- matched controls. Eight single nucleotide polymorphisms (SNPs) within IL-33/ST2 axis were genotyped using the improved Multiple Ligase Detection Reaction platform. We analyzed the association between the tested SNPs and ischemic stroke at both the genotype and haplotype levels. RESULTS: Binary logistic regression analysis indicated that rs10435816 (additive model: odds ratio [OR]=0.72, 95% confidence interval [CI], 0.54-0.95; recessive model: OR=0.72, 95%CI, 0.56- 0.94) was associated with a decreased risk of ischemic stroke after adjustment of confounding factors. Subgroup analysis indicated that rs10435816 (additive model: OR=0.61, 95%CI, 0.41-0.89; recessive model: OR=0.56, 95%CI, 0.40-0.80), rs7025417 (additive model: OR=0.57, 95%CI, 0.39-0.83), rs11792633 (additive model: OR=0.66, 95%CI, 0.46-0.95; recessive model: OR=0.67, 95%CI, 0.49-0.93), and rs7044343 (additive model: OR=0.69, 95%CI, 0.48-0.97; recessive model: OR=0.67, 95%CI, 0.49-0.91) were associated with a decreased risk of large-artery atherosclerosis stroke after adjustment of confounding factors. CONCLUSION: Our findings suggested an association between common variants in the IL-33/ST2 axis and a decreased risk of ischemic stroke in the Han Chinese population.

Long-term interleukin-33 treatment delays disease onset and alleviates astrocytic activation in a transgenic mouse model of amyotrophic lateral sclerosis.

Inflammation is a prominent feature of the neuropathology of amyotrophic lateral sclerosis (ALS). Emerging evidence suggests that inflammatory cascades contributing to the disease progression are not restricted to the central nervous system (CNS) but also occur peripherally. Indeed, alterations in T cell responses and their secreted cytokines have been detected in ALS patients and in animal models of ALS. One key cytokine responsible for the shift in T cell responses is interleukin-33 (IL-33), which stimulates innate type 2 immune cells to produce a large amount of Th2 cytokines that are possibly beneficial in the recovery processes of CNS injuries. Since the levels of IL-33 have been shown to be decreased in patients affected with ALS, we sought to determine whether a long-term recombinant IL-33 treatment of a transgenic mouse model of ALS expressing G93A-superoxide dismutase 1 (SOD1-G93A) alters the disease progression and ameliorates the ALS-like disease pathology. SOD1-G93A mice were treated with intraperitoneal injections of IL-33 and effects on disease onset and inflammatory status were determined. Spinal cord (SC) neurons, astrocytes and T-cells were exposed to IL-33 to evaluate the cell specific responses to IL-33. Treatment of SOD1-G93A mice with IL-33 delayed the disease onset in female mice, decreased the proportion of CD4+ and CD8 + T cell populations in the spleen and lymph nodes, and alleviated astrocytic activation in the ventral horn of the lumbar SC. Male SOD1-G93A mice were unresponsive to the treatment. In vitro studies showed that IL-33 is most likely not acting directly on neurons and astrocytes, but rather conveying its effects through peripheral T-cells. Our results suggest that strategies directed to the peripheral immune system may have therapeutic potential in ALS. The effect of gender dimorphisms to the treatment efficacy needs to be taken into consideration when designing new therapeutic strategies for CNS diseases.

Synthetic human monoclonal antibody targets hIL1 receptor accessory protein chain with therapeutic potential in triple-negative breast cancer.

High expression of Interluekin-1 receptor accessory protein chain (IL-1RAcP) and activated IL-1 signaling were found in some tumor types. IL-1RAcP is considered as the common accessory chain in the IL-1R family, and it is essential for the initiation of IL-1 signaling of all the receptor complexes that encompass it. Thus, the selection and characterization of human anti- IL-1RAcP single-chain antibody fragments variable (scFv) is the first step toward the construction of new anticancer monoclonal antibodies designed for optimal cancer therapy. Here, we found that IL-1RAcP expression was increased in both triple-negative breast cancer (TNBC) cell line cells and TNBC patient cohort, and correlated with shorter recurrence-free survival (RFS). In this study, we employed a human scFv-displaying phage library for the first establishment an antagonistic anti-IL-1RAcP human antibody, scFv 12H7. scFv 12H7 was found a high affinity and specificity binder of IL-1RAcP by a series assays, including EC50, IC50,KD values test and cell binding determination by flow cytometry and immunofluorescence. Also, scFv 12H7 was demonstrated bearing growth inhibitory activity of TNBC cells in vitro and in vivo. Mechanisms study showed that IL-1-activated-NF-κB pathway was significantly inhibited in TNBC cells by incubation with scFv 7H12 for 24 h. Crystal structure analysis, mutations introduction, and yeast two-hybrid assay showed that scFv 12H7 interacted with residues in the D1-D2 domain of IL-1RAcP, which further indicated that scFv 12H7 was a functional binding to IL-1RAcP and uncovered its structure mechanism. In conclusion, scFv 12H7 represent excellent therapeutic candidates for further preclinical and clinical development of TNBC therapy.

The role of the IL-33/IL-1RL1 axis in mast cell and basophil activation in allergic disorders.

Interleukin-33 (IL-33) is a recently discovered cytokine that belongs to the IL-1 superfamily and acts as an important regulator in several allergic disorders. It is considered to function as an alarmin, or danger cytokine, that is released upon structural cell damage. IL-33 activates several immune cells, including Th2 cells, mast cells and basophils, following its interaction with a cell surface heterodimer consisting of an IL-1 receptor-related protein ST2 (IL-1RL1) and IL-1 receptor accessory protein (IL-1RAcP). This activation leads to the production of a variety of Th2-like cytokines that mediate allergic-type immune responses. Thus, IL-33 appears to be a double-edged sword because, in addition to its important contribution to host defence, it exacerbates allergic responses, such as allergic rhinitis and asthma. A major purported mechanism of IL-33 in allergy is the activation of mast cells to produce a variety of pro-inflammatory cytokines and chemokines. In this review, we summarize the current knowledge regarding the genetics and physiology of IL-33 and IL-1RL1 and its association with different allergic diseases by focusing on its effects on mast cells and basophils.

Tollip-induced down-regulation of MARCH1.

In addition to their classical antigen presenting functions, MHC class II molecules potentiate the TLR-triggered production of pro-inflammatory cytokines. Here, we have addressed the effect of Tollip and MARCH1 on the regulation of MHC II trafficking and TLR signaling. Our results show that MARCH1-deficient mice splenocytes are impaired in their capacity to produce pro-inflammatory cytokines in response to poly(I:C) and that TLR3 and MHC II molecules interact in the endocytic pathway. Knocking down Tollip expression in human CIITA(+) HeLa cells increased expression of HLA-DR but reduced the proportion of MHC II molecules associated with the CLIP peptide. Truncation of the HLA-DR cytoplasmic tails abrogated the effect of Tollip on MHC class II expression. While overexpression of Tollip did not affect HLA-DR levels, it antagonized the function of co-transfected MARCH1. We found that Tollip strongly reduced MARCH1 protein levels and that the two molecules appear to compete for binding to MHC II molecules. Altogether, our results demonstrate that Tollip regulates MHC class II trafficking and that MARCH1 may represent a new Tollip target.