IL-1 Gene Family Research Articles

Abstract 5373: Decoding the role of intrinsic pancreatic cellular signaling in shaping the immunosuppressive tumor microenvironment

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) has the highest mortality rate among major cancers, with over 50% of patients having liver metastases at diagnosis. Conventional T-cell-based immunotherapies are ineffective against PDAC liver metastases due to the intricate hepatic tumor microenvironment (TME) that hinders effective immune responses. Thus, it is vital to promote the influx of cytotoxic lymphocytes and prime antitumor immunity. PDAC cells exhibit plasticity and intrinsic cellular heterogeneity, leading to varying immune cell infiltrates and immune responses. Concurrently, early metastases to the liver create an immunosuppressive environment. Our study employs multiple PDAC models, originating from a congenic array of distinct PDAC cell clone tumors with varying degrees of T cell infiltration to delineate the signaling pathways that lead to immunosuppression in the pancreas and liver. Methods: The compendium of congenic PDAC murine cell clones mirrors immune heterogeneity observed in tumors, spanning from low to high T cell infiltration. To overcome a scarcity of tumor-specific neoepitopes in PDAC, we engineered PDAC cell clones expressing chicken ovalbumin (OVA) for antigen-specific T and B cell responses. By subcutaneous and intraportal vein injection of PDAC cell clones into mice, we induce primary pancreatic and metastatic liver tumors to explore immune phenotypes and assess anti-tumor adaptive immune responses, including myeloid and lymphoid cell subsets. Results: In vivo engraftment of PDAC clones showed varied CD4+/CD8+ T cells and polymorphonuclear myeloid-derived suppressor cells, accentuated with immunogenic OVA antigen expression. While all OVA+ PDAC lines elicited antigen-specific immune responses, only ‘TIL high’ tumors were eradicated, while ‘TIL low’ clones persisted in vivo. Additionally, ‘TIL low’ clones formed liver metastases upon intraportal injection, despite OVA expression. RNAseq unveiled differential expression in IL-1 family cytokines and receptors, chemokine-like factor gene superfamily, parathyroid hormone relevant function and metabolism. Current investigations include altering these pathways to re-program the tumor microenvironment and induce local immune suppression. Conclusion: Using diverse PDAC models, we created tumors with immune heterogeneity and altered transcriptional profiles. We identified validated differentially expressed immune altering cytokines, such as CXCL1 and CSF3, and potential novel IL-1 family genes and parathyroid signaling genes. The altered TME conferred protection against tumor antigen-specific immunity in certain clones, both in subcutaneous and liver metastasis models. Ongoing studies will assess the impact of these genes in the distinct environments, offering avenues for immunotherapeutic strategies targeting pancreatic cancer liver metastasis. Citation Format: Bushangqing Liu, Ethan Agritelley, Daniel Nussbaum, Junping Wei, Gangjun Lei, Melissa Gajda, Zachary Hartman. Decoding the role of intrinsic pancreatic cellular signaling in shaping the immunosuppressive tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5373.

The interactions between interleukin-1 family genes: IL1A, IL1B, IL1RN, and obesity parameters

BackgroundObesity has been recognized as a worldwide growing problem, producing many pathologies including the promotion of “proinflammatory state.” The etiology of human obesity is still only partially understood; however, the genetic background has been proved. Its nature is complex, and currently, it appears that the combined effects of the interactions among multiple genes should receive more attention. Due to the fact that obesity promotes proinflammatory conditions, in this study, we investigated the genetic polymorphism of IL-1 family genes in healthy people with normal and elevated body mass index (BMI) and fat %.ResultsThe single-nucleotide polymorphisms (SNPs) within the IL1A -889C > T (rs1800587), IL1B + 3954 T > C (rs1143634), and IL1RN -87G > A (rs2234677) genes alone were associated neither with BMI nor fat % values in tested group. The associations between SNP–SNP interaction and BMI for the IL1B × IL1RN interactions were significant for dominant model (p = 0.02) and codominant model (p = 0.03). The same SNP-SNP interaction (IL1B × IL1RN) was associated also with fat % for codominant (p = 0.01) and recessive (p = 0.002) models.ConclusionsThis study further confirmed that IL-1 family genes are involved in genetic background of obesity. It has been shown that interaction IL1B × IL1RN was associated with both BMI and fat % with rare T allele protecting form higher values. Thus, even if certain polymorphisms in single genes of IL-1 family cannot be defined as related to obesity in examined population, the genetic interrelationships should be analyzed.

Immune-Inhibitory Gene Expression is Positively Correlated with Overall Immune Activity and Predicts Increased Survival Probability of Cervical and Head and Neck Cancer Patients.

Background: Limited immunotherapy options are approved for the treatment of cervical cancer and only 10–25% of patients respond effectively to checkpoint inhibition monotherapy. To aid the development of novel therapeutic immune targets, we aimed to explore survival-associated immune biomarkers and co-expressed immune networks in cervical cancer. Methods: Using The Cancer Genome Atlas (TCGA) Cervical Squamous Cell Carcinoma (CESC) data (n = 304), we performed weighted gene co-expression network analysis (WGCNA), and determined which co-expressed immune-related genes and networks are associated with survival probability in CESC patients under conventional therapy. A “Pan-Immune Score” and “Immune Suppression Score” was generated based on expression of survival-associated co-expressed immune networks and immune suppressive genes, which were subsequently tested for association with survival probablity using the TCGA Head Neck Squamous Cell Carcinoma (HNSCC) data (n = 528), representing a second SCC cancer type. Results: In CESC, WGCNA identified a co-expression module enriched in immune response related genes, including 462 genes where high expression was associated with increased survival probability, and enriched for genes associated with T cell receptor, cytokine and chemokine signaling. However, a high level of expression of 43 of the genes in this module was associated with decreased survival probability but were not enriched in particular pathways. Separately, we identified 20 genes associated with immune suppression including inhibitory immune checkpoint and regulatory T cell-related genes, where high expression was associated with increased survival probability. Expression of these 20 immune suppressive genes (represented as “Immune Suppression Score”) was highly correlated with expression of overall survival-associated immune genes (represented as “Pan-Immune Score”). However, high expression of seven immune suppression genes, including TWEAK-R, CD73, IL1 family and TGFb family genes, was significantly associated with decreased survival probability. Both scores also significantly associated with survival probability in HNSCC, and correlated with the previously established “Immunophenoscore.” Conclusion: CESC and HNSCC tumors expressing genes predictive of T cell infiltrates (hot tumors) have a better prognosis, despite simultaneous expression of many immune inhibitory genes, than tumors lacking expression of genes associated with T cell infiltrates (cold tumors) whether or not these tumor express immune inhibitory genes.

The association between IL-1 family gene polymorphisms and colorectal cancer: A meta-analysis.

Colorectal cancer (CRC) is a major public health problem given its high incidence and mortality. This study focuses on examining the associations between IL-1α, IL-1β, and IL-1RN polymorphisms and colorectal cancer susceptibility. A systematic literature search of PubMed, Embase, Web of Science, CNKI (China National Knowledge Infrastructure) and Wan Fang databases was conducted to identify relevant studies. Relevant data were extracted from the original included studies. The correlation was demonstrated based on the odds ratio (OR) and corresponding 95% confidence intervals (95% CIs). Publication bias was investigated by Egger's line regression test and Begg's funnel plot. Eighteen independent studies involving 6218 cases and 10160 controls were eligible for this pooled analysis. Overall, the result revealed that the IL-1α rs3783553 polymorphism was significantly associated with an increased risk of CRC (G vs. C, OR=1.02, 95% CI=0.90-1.15, I2=51%, P=0.78; GG vs. CC, OR=1.97, 95% CI=1.04-3.74, I2=70%, P=0.04; GC vs. CC, OR=1.75, 95% CI=1.12-2.75, I2=42%, P=0.01; GG+GC vs. CC, OR=1.85, 95% CI=1.08-3.18, I2=63%, P=0.03; and GG vs. GC+CC, OR=1.28, 95% CI=1.04-1.58, I2=39%, P=0.02), and significance was also noted for IL-1RN VNTR under the dominant model (22+2L vs. LL, OR=1.49, 95% CI=1.01-2.19, I2=77%, P=0.045) and allelic contrast model (2 vs. L, OR=1.28, 95% CI=1.00-1.64, I2=58.6%, P=0.047). For IL-1β+31C/T, significance was observed in the dominant model (CC+CT vs. TT, OR=0.83, 95% CI=0.69-0.99, I2=52%, P=0.034) and the heterozygous model (CT vs. TT, OR=0.80, 95% CI=0.65-0.98, I2=60%, P=0.04). For IL-1β+511 C/T, a significant association was noted in four gene models (CT vs. TT, OR=0.72, 95% CI=0.63-0.83, I2=0%, P<0.001; CC+CT vs. TT, OR=0.74, 95% CI=0.65-0.84, I2=0%, P<0.001; CC vs. TT, OR=0.77, 95% CI=0.65-0.91, I2=30.9%, P=0.003; C vs. T, OR=0.87, 95% CI=0.80-0.95, I2=38%, P=0.001), but a significant relationship was not found in the recessive model (CC vs. CT+TT, OR=1.09, 95% CI=0.86-1.38, I2=57.1%, P=0.25). In addition, borderline statistical significance was noted between IL-1β+3954 Ins/Del and CRC in the homozygous model, but no significance was identified for IL-1β+3737 G/A, Il-1β+1464 G/C, and IL-1RN+2018T/C under all five genetic models. In the subgroup analysis of ethnic groups, significant associations with CRC were found for IL-1β+31 (CC vs. TT: OR=0.82, 95% CI=0.67-0.99, I2=20.2%, P=0.04; CT vs. TT: OR=0.62, 95% CI=0.47-0.82, I2=0%, P<0.001; CC+CT vs. TT: OR=0.69, 95% CI=0.55-0.87, I2=29.7%, P=0.001), IL-1β+511 (CT vs. TT, OR=0.65, 95% CI=0.55-0.77, I2=0%, P<0.001; CC+CT vs. TT, OR=0.67, 95% CI=0.58-0.78, I2=0%, P<0.001; C vs. T, OR=0.83, 95% CI=0.75-0.92, I2=49.6%, P<0.001) and IL-1RN+2018T/C in the allelic contrast model (T vs. C, OR=0.66, 95% CI=0.44-0.98, I2=0%, P=0.04) among Asians but not in Caucasians. A significant association between IL-1β+1464 G/C polymorphisms in Caucasians was observed under the recessive model (OR=0.87, 95% CI=0.77-0.98, I2=45%, P=0.03). The current meta-analysis demonstrated that IL-1α rs3783553, IL-1β+31C/T, IL-1β+511C/T, and IL-1RN VNTR are critical genes for CRC susceptibility.

Interleukin-1 Ligands and Receptors in Lumpfish (Cyclopterus lumpus L.): Molecular Characterization, Phylogeny, Gene Expression, and Transcriptome Analyses.

The interleukin (IL)-1 family play a fundamental role as immune system modulators. Our previous transcriptome-analyses of leukocytes from lumpfish (Cyclopterus lumpus L.) showed that IL-1β was among the most highly upregulated genes following bacterial exposure. In the present study, we characterized IL-1 signaling pathways, identified and characterized four ligands of the IL-1 family in lumpfish; IL-1β type I and type II, IL-18, and the novel IL-1 family members (nIL-1F), both at mRNA and gene levels. The two IL-1β in lumpfish is termed IL-1β1 (type II) and IL-1β2 (type I). Furthermore, a comprehensive phylogenetic analysis of 277 IL-1 ligands showed that nIL-1F, in common with IL-1β, likely represents an ancestral gene, as representatives for nIL-1F were found in cartilaginous and lobe-finned fish, in addition to teleosts. This shows that nIL-1F is not exclusively present in teleosts as previously suggested. Our analyses of exon-intron structures, intron phases, phylogeny and synteny clearly show the separation of IL-1β into groups; type I and type II, which likely is a result of the third whole genome duplication (3R WGD). The phylogenetic analysis shows that most teleosts have both type I and type II. Furthermore, we have determined transcription levels of the IL-1 ligands in leukocytes and 16 different tissues, and their responses upon in vitro stimulation with seven different ligands. In addition, we have identified the IL-1 receptors IL-1R1, IL-1R2, IL-1R4 (ST2/IL-33 receptor/IL-1RL), IL-1R5 (IL-18R1), and partial sequences of DIGIRR and IL-1R3 (IL-RAcP). Identification of immune molecules and description of innate responses in lumpfish is interesting for comparative and evolutionary studies and our study constitutes a solid basis for further functional analyses of IL-1 ligands and receptors in lumpfish. Furthermore, since lumpfish are now farmed in large numbers to be used as cleaner fish for removal of sea lice on farmed salmon, in-depth knowledge of key immune molecules, signaling pathways and innate immune responses is needed, as the basis for design of efficient immune prophylactic measures such as vaccination.

The Roles of IL-1 Family Cytokines in the Pathogenesis of Systemic Sclerosis.

The IL-1 family consists of 11 cytokines, 7 ligands with agonist activity (IL-1α, IL-1β, IL-18, IL-33, IL-36α, IL-36β, IL-36γ) and four members with antagonistic activities [IL-1 receptor antagonist (IL-1Ra), IL-36Ra, IL-37, IL-38]. Recent articles have described that most members of IL-1 family cytokines are involved in the process of innate and adaptive immunity as well as fibrosis in systemic sclerosis (SSc). IL-1 family gene polymorphisms, abnormal expression of IL-1 and its potential role in the fibrosis process have been explored in SSc. IL-33 and IL-18 have also been discussed in the recent years. IL-33 may contribute to the fibrosis of SSc, while IL-18 remains to be researched to confirm its role in fibrosis process. There is a lack of study on the pathophysiological roles of IL-36, IL-37, and IL-38 in SSc, which might provide us new study area. Here, we aim to give a brief overview of IL-1 family cytokines and discuss their pivotal roles in the pathogenesis of SSc.

Recent insights into apoptosis and toxic autophagy: The roles of MDA-7/IL-24, a multidimensional anti-cancer therapeutic.

Apoptosis and autophagy play seminal roles in maintaining organ homeostasis. Apoptosis represents canonical type I programmed cell death. Autophagy is viewed as pro-survival, however, excessive autophagy can promote type II cell death. Defective regulation of these two obligatory cellular pathways is linked to various diseases, including cancer. Biologic or chemotherapeutic agents, which can reprogram cancer cells to undergo apoptosis- or toxic autophagy-mediated cell death, are considered effective tools for treating cancer. Melanoma differentiation associated gene-7 (mda-7) selectively promotes these effects in cancer cells. mda-7 was identified more than two decades ago by subtraction hybridization showing elevated expression during induction of terminal differentiation of metastatic melanoma cells following treatment with recombinant fibroblast interferon and mezerein (a PKC activating agent). MDA-7 was classified as a member of the IL-10 gene family based on its chromosomal location, and the presence of an IL-10 signature motif and a secretory sequence, and re-named interleukin-24 (MDA-7/IL-24). Multiple studies have established MDA-7/IL-24 as a potent anti-cancer agent, which when administered at supra-physiological levels induces growth arrest and cell death through apoptosis and toxic autophagy in a wide variety of tumor cell types, but not in corresponding normal/non-transformed cells. Furthermore, in a phase I/II clinical trial, MDA-7/IL-24 administered by means of a non-replicating adenovirus was well tolerated and displayed significant clinical activity in patients with multiple advanced cancers. This review examines our current comprehension of the role of MDA-7/IL-24 in mediating cancer-specific cell death via apoptosis and toxic autophagy.

D-103 Cancer terminator viruses: A strategy for targeting metastatic cancers

Our laboratory pioneered subtraction hybridization and used this elegant and effective gene identification strategy to clone novel genes of physiological relevance. This methodology defined differentially regulated genes in an unbiased manner that are modified when metastatic human melanoma cells are pharmacologically induced to terminally differentiate and lose irreversibly oncogenic potential following treatment with IFN-band the protein kinase C activator mezerein. This approach, called DISH (differentiation induction subtraction hybridization), identified melanoma differentiation associated (mda) genes relevant to growth control, cancer development, tumor progression, and cancer cell survival. One intriguing gene identified using the DISH scheme was mda-7, which based on chromosomal location, secretory properties and structure has been classified as interleukin-24 (IL-24). mda-7/IL-24 is a novel member of the IL-10 gene family that promotes apoptosis and toxic autophagy in a broad-spectrum of human tumors, exerts potent “bystander” antitumor activity, inhibits tumor angiogenesis, stimulates the immune system and synergizes with conventional therapies (including chemotherapy, radiation, monoclonal antibodies and immunomodulators) to promote selective tumor destruction. mda-7/IL-24 displays profound anti-cancer activity in preclinical animal models (including nude, syngeneic and transgenic mice) and following intratumoral administration using a type 5 adenovirus in a Phase I/II clinical trial in patients with advanced cancers. To enhance the therapeutic efficacy of mda-7/IL-24, we generated a novel class of adenoviruses, called Cancer Terminator Viruses (CTVs), that selectively replicate in cancer cells and simultaneously produce mda-7/IL-24, Ad.5-CTV. A further iteration of this virus employs a chimera between Ad.5 and Ad.3, Ad.5/3, that employs Coxsackie-Adenovirus Receptors (CAR) as well as desmoglien and CD46 to infect cells, thereby increasing its infectivity in cancer cells with reduced CAR. Ad.5/3-CTV displays profound anti-cancer activity in pre-clinical animal models, including GBM, melanoma, and prostate, colorectal and breast cancer. A clinical trial is in preparation to test the effect of the Ad.5/3-CTV in patients with recurrent glioma. To facilitate the delivery of adenoviruses systemically, avoiding immune clearance and liver trapping, we have refined the ultrasound-targeted microbubble-destruction (UTMD) approach. This strategy has allowed us to deliver therapeutic viruses and proteins intravenously to internal target organs, such as the prostate, to evoke pre-clinical antitumor activity. To enhance further the therapeutic efficacy of the UTMD approach, we are employing microbubbles that contain targeting ligands on their surface permitting accumulation in the tumor vasculature, that is, decorated microbubbles. Employing the same cancer-selective promoter used in the original CTV, the rat progression elevated gene-3 promoter (PEG-Prom), we also developed genetic-based imaging approaches for metastases. These approaches use the PEG-Prom (or another cancer-selective promoter) to drive an imaging gene (eg, luciferase or HSV-Tk) linked to a delivery vehicle PEI. These nanoparticle vectors when combined with bioluminescence imaging and SPECT-CT permit non-invasive imaging of tumors and metastases in pre-clinical animal models. As a further iteration of our CTV we have used 2 cancer-selective and a non-selective promoter to generate first in class tripartite Ads that selectively replicate in cancer cells, produce an imaging agent in cancer cells (Lucirerase or HSV-Tk) and simultaneously produce the therapeutic immunogenic cytokine mda-7/IL-24, Ad.Tri-CTV. These “theranostic viruses” permit tumor and metastasis imaging and treatment through application of a single vector. Overall, the novel reagents and methodologies we have developed are providing paradigm shifts in how cancers will be detected and treated in the future.

Association analysis of interleukin-1 single nucleotide polymorphisms in viral hepatitis of Iraqi patients

Hepatitis is a liver inflammation most commonly caused by one of the five types of hepatitis viruses; A, B (HBV), C (HCV), D and E, in which interleukin-1 (IL-1) is suggested to play an important role in initiation and regulation of immune response against these viruses. Accordingly, the association between five IL1 gene family single nucleotide polymorphisms (SNPs: IL1A−889; IL1B−511; IL1B+3962; IL1Rpsti 1970; and IL1RAmspa 11,100) and viral hepatitis was investigated in HBV and HCV Iraqi patients (71 patients each). The SNPs were detected by the polymerase chain reaction-specific sequence primer (PCR-SSP) method. The results revealed a significant positive association between HBV and IL1Rpsti 1970 SNP. The T allele of this SNP and its homozygous genotype (TT) were associated with an increased risk to develop HBV. In addition, an increased risk of HBV was also associated with three TT haplotypes (IL1A−889-IL1B−511, IL1B−511-IL1B+3962 and IL1B+3962-IL1Rpsti 1970). In contrast, none of the investigated SNP alleles, genotypes or haplotypes was associated with HCV. In conclusion, T allele (and its homozygous genotype) of IL-1Rpst-I 1970 SNP and haplotypes containing such allele (IL1B+3962-IL1Rpsti 1970) are suggested to be risk factors for HBV infection, while no such conclusion could be reached in HCV infection.

Neutralization of IL-18 by IL-18 binding protein ameliorates bleomycin-induced pulmonary fibrosis via inhibition of epithelial-mesenchymal transition

Idiopathic pulmonary fibrosis (IPF) is a fatal parenchymal lung disease with limited effective therapies. Interleukin (IL)-18 belongs to a rather large IL-1 gene family and is a proinflammatory cytokine, which acts in both acquired and innate immunity. We have previously reported that IL-18 play an important role in lipopolysaccharide-induced acute lung injury in mice. Persistent inflammation often drives fibrotic progression in the bleomycin (BLM) injury model. However, the role of IL-18 in pulmonary fibrosis (PF) is still unknown. IL-18 binding protein (IL-18BP) is able to neutralize IL-18 biological activity and has a protective effect against renal fibrosis. The aim of this study was to investigate the effects of IL-18BP on BLM-induced PF. In the present study, we found that IL-18 was upregulated in lungs of BLM-injured mice. Neutralization of IL-18 by IL-18BP improved the survival rate and ameliorated BLM-induced PF in mice, which was associated with attenuated pathological changes, reduced collagen deposition, and decreased content of transforming growth factor-β1 (TGF-β1). We further demonstrated that IL-18BP treatment suppressed the BLM-induced epithelial mesenchymal transition (EMT), characterized by decreased α-smooth muscle actin (α-SMA) and increased E-cadherin (E-cad) in vivo. In addition, we provided in vitro evidence demonstrating that IL-18 promoted EMT through upregulation of Snail-1 in A549 cells. In conclusion, our findings raise the possibility that the increase of IL-18 is involved in the development of BLM-induced PF through modulating EMT in a Snail-1-dependent manner. IL-18BP may be a worthwhile candidate option for PF therapy.

Interleukin-18 binding protein attenuates lipopolysaccharide-induced acute lung injury in mice via suppression NF-κB and activation Nrf2 pathway

Interleukin (IL)-18 belongs to a rather large IL-1 gene family and is a proinflammatory cytokine. IL-18 plays important roles in lung injury. IL-18 binding protein (IL-18BP), a natural antagonist of IL-18, binds IL-18 with high affinity. IL-18BP is able to neutralize IL-18 biological activity and has a protective effect against renal fibrosis. The aim of this study was to evaluate the potential protective effect of IL-18BP on lipopolysaccharide (LPS)-induced acute lung injury (ALI) in mice and to illuminate the underlying mechanisms. Results indicated that pretreatment with IL-18BP significantly attenuated LPS-induced pulmonary pathological injury. Meanwhile, IL-18BP pretreatment markedly inhibited infiltration of inflammatory cell and release of inflammatory factor in ALI mice in vivo and in primary macrophages after LPS insult in vitro. IL-18BP treatment dramatically reduced oxidative stress through increasing superoxide dismutase (SOD) and glutathione (GSH) contents, and decreasing the levels of malondialdehyde (MDA) and reactive oxygen species (ROS) in LPS-induced ALI mice and primary macrophages. Additionally, IL-18BP was also observed to markedly decreased the activation of nuclear factor-kappa B (NF-κB) and upregulated the nuclear factor erythroid 2-related factor 2 (Nrf2). Taken together, IL-18BP possessed protective effect against LPS-induced ALI, which might be associated with its regulation of NF-κB and Nrf2 activities. The results rendered IL-18BP worthy of further development into a pharmaceutical drug for the treatment of ALI.

Association of IL33 and IL1RAP Polymorphisms With Acute Anterior Uveitis.

AAU (acute anterior uveitis) is the most common entity of uveitis characterized by acute vision loss and violent sore eyes. IL-33 and IL-1RacP have been found to play crucial roles in the innate immune system. In the present study, we investigated the association of IL33 and IL1RAP genes with AAU. A total of 549 AAU patients and 1080 unrelated healthy controls were recruited for this study. Ten single nucleotide polymorphisms (SNPs) were genotyped using Sequenom Mass ARRAY technology. Our findings demonstrated that IL1RAP-rs3773978 significantly associated with AAU and could serve as a genetic risk marker in Chinese AAU patients. A significantly increased frequency of the A allele and AA homozygosity of IL1RAP-rs3773978 was observed in AAU patients compared with that in controls (p=0.001, pc=0.01, OR=1.282, 95% CI 1.106 to 1.487; p=0.0003, pc=0.003, OR=1.647, 95% CI 1.255 to 2.163, respectively). Further stratification analyses showed that the genetic correlation may differ depending on HLA-B27 status, AS (ankylosing spondylitis) status, attack times and laterality status. Our findings provide new insights that enhance the current knowledge of uveitis genetics by demonstrating the specific functional roles of IL1RAP and other IL-1 family genes in uveitis.

Lentivirus‑mediated MDA7/IL24 expression inhibits the proliferation of hepatocellular carcinoma cells.

MDA7/IL24 is a member of the IL-10 gene family that functions as a cytokine. Notably, supra-physiological endogenous MDA7 levels have been indicated to suppress tumor growth and induce apoptosis in different cancer types. In the present study, MDA7 roles were investigated during the proliferation of hepatocellular carcinoma (HCC) cells and the molecular mechanisms underlying this process. A lentiviral vector expressing MDA7/IL24 (LV-MDA7/IL24) was constructed and used to infect HCC SMMC-7721 cells. The expression levels of MDA7/IL24 in these cells were determined using RT-qPCR and western blot analysis. The effects of LV-MDA7/IL24 on cell proliferation were analyzed using MTT and colony formation assays. Furthermore, the influence of LV-MDA7/IL24 on cell apoptosis and cell cycle distribution were detected using flow cytometry. The underlying molecular mechanisms were investigated using microarray and western blot analysis. The expression of MDA7/IL24 was confirmed to be significantly increased in the cells infected with LV-MDA7/IL24 compared with that the negative-control infected group. Lentivirus-mediated MDA7/IL24 expression was found to inhibit HCC cell proliferation and colony formation, and it also induced cell arrest and apoptosis. Microarray analysis and western blotting results indicated that multiple cancer-associated pathways and oncogenes are regulated by MDA7/IL24, including cell cycle regulatory and apoptosis activation pathway. In conclusion, it was determined that MDA7/IL24 inhibits the proliferation and reduces the tumorigenicity of HCC cells by regulating cell cycle progression and inducing apoptosis, indicating that it may be used as a potential prognostic and therapeutic target in HCC.

111 Cornification of normal epidermal keratinocytes is preceded by the expression of specific interleukin-1 family members

The interleukin (IL)-1 family of cytokines includes IL-1α and IL-1β which are ubiquitous inducers of inflammation and other interleukins, that are expressed in a cell type-specific manner. Here, we investigated the expression of IL-1 family genes during terminal differentiation of normal human epidermal keratinocytes and the association of these genes with skin phenotypes during evolution. While IL1A and IL1B were downregulated, IL36 isoforms as well as IL37 and IL38 genes were strongly upregulated during differentiation of primary human keratinocytes. Western blot analysis showed that the anti-inflammatory cytokine IL-37 was highly abundant at the protein level in differentiated keratinocytes in vitro and in normal human epidermis where immunohistochemical staining revealed expression specifically in the granular layer. The IL36A, IL36B, IL37, and IL38 genes have been lost during the evolutionary degeneration of the epidermal cornification program in fully aquatic mammals. Our results suggest that the formation of a normal stratum corneum is associated with the expression of IL-1 family cytokines, including the anti-inflammatory protein IL-37.

Genetic analysis of innate immunity in Behcet’s disease identifies an association with IL-37 and IL-18RAP

Interleukin-1 (IL-1) and the IL-1 receptor (IL-1R) family play an important role in the pathogenesis of inflammatory diseases. This study aimed to investigate the association between single nucleotide polymorphisms (SNP) of IL-1 and IL-1R family genes with Vogt-Koyanagi-Harada (VKH) and Behcet’s disease (BD) in Han Chinese. The case-control study was divided into two stages and included 419 VKH cases, 1063 BD cases and 1872 healthy controls. The MassARRAY platform (Sequenom), iPLEX Gold Assay and TaqMan SNP assays were used to score genotypes of 24 SNPs. The expression of IL-37 and IL-18Rap was measured by ELISA and real-time PCR in genotyped healthy individuals. A significantly lower frequency of the AG genotype, and a higher frequency of the GG genotype and G allele of IL-37/rs3811047 were observed in BD as compared to controls. AA genotype and A allele frequency of IL-18RAP/rs2058660 was significantly decreased in BD as compared to controls. Functional studies performed in healthy controls showed that rs3811047 AG genotype carriers had a higher IL-37 gene expression in peripheral blood mononuclear cells (PBMCs) than GG carriers. GG carriers showed a higher cytokine expression as compared to AG carriers. No association was detected between the tested SNPs and VKH.

TAT-IL-24-KDEL-induced apoptosis is inhibited by survivin but restored by the small molecular survivin inhibitor, YM155, in cancer cells.

Interleukin-24 (IL-24) is a cytokine belonging to the IL-10 gene family. This cytokine selectively induces apoptosis in cancer cells, without harming normal cells, through a mechanism involving endoplasmic reticulum (ER) stress response. TAT-IL-24-KDEL is a fusion protein that efficiently enters the tumor cells and locates in the ER. Here we report that TAT-IL-24-KDEL induced apoptosis in human cancer cells, mediated by the ER stress cell death pathway. This process was accompanied by the inhibition of the transcription of an antiapoptotic protein, survivin. The forced expression of survivin partially protected cancer cells from the induction of apoptosis by TAT-IL-24-KDEL, increased their clonogenic survival, and attenuated TAT-IL-24-KDEL-induced activation of caspase-3/7. RNA interference of survivin markedly sensitized the transformed cells to TAT-IL-24-KDEL. Survivin was expressed at higher levels among isolated clones that resistant to TAT-IL-24-KDEL. These observations show the important role of survivin in attenuating cancer-specific apoptosis induced by TAT-IL-24-KDEL. The pharmacological inhibition of survivin expression by a selective small-molecule survivin suppressant YM155 synergistically sensitized cancer cells to TAT-IL-24-KDEL-induced apoptosis in vitro and in vivo. The combined regimen caused significantly higher activation of ER stress and dysfunction of mitochondria than either treatment alone. As survivin is overexpressed in a majority of cancers, the combined TAT-IL-24-KDEL and YM155 treatment provides a promising alternative to the existing therapies.

Abstract 594: Long Non-coding RNA Regulation of the Interleukin-1 Gene Family Cluster

Long non-coding RNAs (lncRNAs) are a poorly understood class of regulatory RNAs, known to be highly species specific and capable of binding DNA, RNA, and/or protein to regulate transcriptional and epigenetic networks. Although thousands of lncRNAs have been identified in disease-related screens, only a few have been functionally characterized. Here we identify a primate-specific lncRNA, CHROME, located on human chr2 in a locus associated with premature cardiovascular disease, which regulates the expression of the IL-1 family of cytokines. Using RNA fluorescence in situ hybridization (FISH) and cell fractionation, we show that CHROME is abundant in the nucleus. Furthermore, its expression is induced in primary human macrophages treated with pro-inflammatory Toll-like receptor (TLR) agonists. To investigate CHROME function, we used RNA immunoprecipitation (RIP) to map RNA-protein interactions, and found that CHROME has strong histone binding affinity, suggesting a potential role in gene regulation. Notably, repression of CHROME using anti-sense oligonucleotides leads to a decrease in expression of genes in the IL-1 family gene cluster located nearby on the q arm of chromosome 2, including IL1A , IL1B and IL1RL1 . These data suggest that CHROME may act as a scaffold for chromatin modification of the IL-1 locus, thereby regulating transcription of these potent inflammatory cytokines. This hypothesis is currently being tested using ChIRP (chromatin isolation by RNA purification) coupled with high throughput DNA sequencing and mass spectrometry. As studies support a causal role for IL-1α and IL-1β in the promotion of atherosclerosis, further characterization of CHROME and its regulatory network may uncover novel opportunities for therapeutic intervention in cardiovascular diseases, and other inflammatory diseases in which IL-1 cytokines predominate.

Interleukin-33 in tumorigenesis, tumor immune evasion, and cancer immunotherapy.

Interleukin-33 (IL-33) is a member of the IL-1 gene family and mainly expressed in the nucleus of tissue lining cells, stromal cells, and activated myeloid cells. IL-33 is considered a damage-associated molecular pattern (DAMP) molecule and plays an important role in many physiological and pathological settings such as tissue repair, allergy, autoimmune disease, infectious disease, and cancer. The biological functions of IL-33 include maintaining tissue homeostasis, enhancing type 1 and 2 cellular immune responses, and mediating fibrosis during chronic inflammation. IL-33 exerts diverse functions through signaling via its receptor ST2, which is expressed in many types of cells including regulatory T cells (Treg), group 2 innate lymphoid cells (ILC2s), myeloid cells, cytotoxic NK cells, Th2 cells, Th1 cells, and CD8(+) T cells. Tumor development results in downregulation of IL-33 in epithelial cells but upregulation of IL-33 in the tumor stroma and serum. The current data suggest that IL-33 expression in tumor cells increases immunogenicity and promotes type 1 antitumor immune responses through CD8(+) T cells and NK cells, whereas IL-33 in tumor stroma and serum facilitates immune suppression via Treg and myeloid-derived suppressor cell (MDSC). Understanding the role of IL-33 in cancer immunobiology sheds lights on targeting this cytokine for cancer immunotherapy.

Bisphenol A (BPA) Exposure In Utero Leads to Immunoregulatory Cytokine Dysregulation in the Mouse Mammary Gland: A Potential Mechanism Programming Breast Cancer Risk.

Bisphenol-A (BPA) is a ubiquitous estrogen-like endocrine disrupting compound (EDC). BPA exposure in utero has been linked to breast cancer and abnormal mammary gland development in mice. The recent rise in incidence of human breast cancer and decreased age of first detection suggests a possible environmental etiology. We hypothesized that developmental programming of carcinogenesis may involve an aberrant immune response. Both innate and adaptive immunity play a role in tumor suppression through cytolytic CD8, NK, and Th1 T-cells. We hypothesized that BPA exposure in utero would lead to dysregulation of both innate and adaptive immunity in the mammary gland. CD1 mice were exposed to BPA in utero during gestation (days 9-21) via osmotic minipump. At 6weeks, the female offspring were ovariectomized and estradiol was given at 8weeks. RNA and protein were extracted from the posterior mammary glands, and the mRNA and protein levels were measured by PCR array, qRT-PCR, and western blot. In mouse mammary tissue, BPA exposure in utero significantly decreased the expression of members of the chemokine CXC family (Cxcl2, Cxcl4, Cxcl14, and Ccl20), interleukin 1 (Il1) gene family (Il1β and Il1rn), interleukin 2 gene family (Il7 receptor), and interferon gene family (interferon regulatory factor 9 (Irf9), as well as immune response gene 1 (Irg1). Additionally, BPA exposure in utero decreased Esr1 receptor gene expression and increased Esr2 receptor gene expression. In utero exposure of BPA resulted in significant changes to inflammatory modulators within mammary tissue. We suggest that dysregulation of inflammatory cytokines, both pro-inflammatory and anti-inflammatory, leads to a microenvironment that may promote disordered cell growth through inhibition of the immune response that targets cancer cells.

Aberrant Expression of IL-22 Receptor 1 and IL-22 Stimulation Increase the Activation of STAT3 and ERK1/2 in ABC-DLBCL Cells

BACKGROUND: Interleukin-22 (IL-22) was recently discovered as a T-cell derived cytokine that belongs to the IL-10 gene family. It has been shown that IL-22 can promote cell proliferation and activate several oncogenic pathways including STAT3, MAPK and AKT signaling. IL-22 exerts its function by binding to its heterodimeric receptor complex which is composed of IL-22 receptor (R) 1 and IL-10R2. IL-22R1 is restricted and barely detected in normal immune cells including B and T cells. In the current study, we sought to determine whether IL-22 plays a part in the development of diffuse large B-cell lymphoma (DLBCL).METHODS: Plasma IL-22 level was measured in newly diagnosed patients with DLBCL and normal individuals by ELISA assay. We also assessed the expression of IL-22R1 in FFPE sample and DLBCL cell lines (TMD8, OCI-LY3, SUDHL16, OCI-LY1) by immunohistochemistry and immunofluorescence staining respectively. Immunoblotting was performed to determine changes of signaling pathways in cell lines upon the treatment with IL-22 recombinant protein.RESULTS: The plasma IL-22 concentration was significantly increased in newly diagnosed DLBCL patients (n=12) compared with normal individuals (n=26, P= 0.0177). IL-22R1 protein was expressed in all of the four DLBCL cell lines, while not all but some of the primary tumor samples expressed it on membranes of tumor cells. Using immunoblotting, we demonstrated a substantial increase of pSTAT3 level with the treatment of IL-22 in ABC-DLBCL cell lines TMD8 and OCI-LY3, while no pSTAT3 was detectable in GCB-DLBCL cell lines SUDHL16 and OCI-LY1. Interestingly, upon IL-22 stimulation, the p-ERK1/2 level was increased shortly and quickly returned to the normal level in TMD8 cells, and steadily increased in LY3 cells during 30 min of treatment. However, there was no change about p-ERK1/2 in SUDHL16 and OCI-LY1 cells after IL-22 treatment.CONCLUSIONS: Our current study has shown the aberrant expression of IL-22R1 in DLBCL. And recombinant IL-22 protein increased the activation of STAT3 and ERK1/2 in ABC rather than GCB-DLBCL cell lines. For the first time, we suggested the importance of IL-22 and its receptor IL-22R1 pathway in ABC-DLBCL, which warrants further studies.Figure 1. The expression of IL-22R1 in DLBCL cell lines was detected by using immunofluorescence staining. DisclosuresNo relevant conflicts of interest to declare.