IL-1β Antagonist Research Articles

The effect of dental gel with IL-1β antagonist on indicators of nitrosative stress and antioxidant system in rats with experimental chronic generalized periodontitis

The effect of dental gel with IL-1β antagonist on indicators of nitrosative stress and antioxidant system in rats with experimental chronic generalized periodontitis

Targeting IL-1 controls refractory pityriasis rubra pilaris.

Pityriasis rubra pilaris (PRP) is a rare inflammatory skin disease with a poorly understood pathogenesis. Through a molecularly driven precision medicine approach and an extensive mechanistic pathway analysis in PRP skin samples, compared to psoriasis, atopic dermatitis, healed PRP, and healthy controls, we identified IL-1β as a key mediator, orchestrating an NF-κB-mediated IL-1β-CCL20 axis, including activation of CARD14 and NOD2. Treatment of three patients with the IL-1 antagonists anakinra and canakinumab resulted in rapid clinical improvement and reversal of the PRP-associated molecular signature with a 50% improvement in skin lesions after 2 to 3 weeks. This transcriptional signature was consistent with in vitro stimulation of keratinocytes with IL-1β. With the central role of IL-1β underscoring its potential as a therapeutic target, our findings propose a redefinition of PRP as an autoinflammatory keratinization disorder. Further clinical trials are needed to validate the efficacy of IL-1β antagonists in PRP.

Ligandability Assessment of IL-1β by Integrated Hit Identification Approaches.

Human interleukin-1β (IL-1β) is a pro-inflammatory cytokine that plays a critical role in the regulation of the immune response and the development of various inflammatory diseases. In this publication, we disclose our efforts toward the discovery of IL-1β binders that interfere with IL-1β signaling. To this end, several technologies were used in parallel, including fragment-based screening (FBS), DNA-encoded library (DEL) technology, peptide discovery platform (PDP), and virtual screening. The utilization of distinct technologies resulted in the identification of new chemical entities exploiting three different sites on IL-1β, all of them also inhibiting the interaction with the IL-1R1 receptor. Moreover, we identified lysine 103 of IL-1β as a target residue suitable for the development of covalent, low-molecular-weight IL-1β antagonists.

Optimisation of the composition of safe dental gel with IL-1β antagonist for the treatment of inflammatory periodontal diseases

Aim. The study aims to develop the composition of the oromucosal gel with the IL-1β interleukin antagonist for complex treatment of inflammatory periodontal diseases. Materials and methods. Experimental studies were conducted based on the Department of Medicines Technology of Zaporizhzhia State Medical and Pharmaceutical University and the Training Medical and Laboratory Center of Zaporizhzhia State Medical and Pharmaceutical University of the Ministry of Health of Ukraine. As an active component, in the recipe of oromucosal gel, used antagonist of the interleukin IL-1B. Excipients: D-panthenol (the plasticizer), carboxymethylcellulose sodium salt (the viscosity modifier and mucoadhesive component), Tween-80 (the enhancer absorption), benzalkonium chloride (the preservative), sodium hydrophosphate + citric acid (the phosphate buffer solution), purified water. The experiments used existing and auxiliary ingredients of pharmaceutical purity, which were obtained from Sinbias LLC, Istok-Plus LLC, LLC “Mobile Medical”. For the design of the experiment, the methodology of the response surface (Box-Behnken Design) is used: Na CMC, Tween-80, D-panthenol; and four answers: pH, viscosity test, system type, mucoadhesive properties. Research methods: rheological research (viscosity test, amplitude test, frequency test, mucoadhesive test, thixotropy test) performed in oscillation mode on the modular compact Rheometer Anton Paar MCR 302 (CP50-1 SN71317), which, compared to cylindrical devices, requires a much smaller amount of gel sample and allows the planned tests in the oscillation mode, the temperature in the experiments was provided with a built-in thermostat (Peltier Temperature Control, C-PTD 200). Pharmaco-toxicological studies were conducted on 46 white outbred rats of both sexes, weighing 160–180 grams, which were received from Vivarium by the Institute of Pharmacology and Toxicology. The studies were performed on enough animals, all manipulations were carried out by the provisions on the use of animals in biomedical experiments. The results of the study were calculated using the standard statistical package of the Statistica for Windows 13 (StatSoft Inc., No. JPZ804I382130ARCN10-J), as well as SPSS 16.0, Microsoft Office Excel 2003. Results. According to the design of the experiment, it is established that the components of the system do not change the value of pH in experimental oromucosal gels (6.5500 ± 0.0334). The Anova for Quadratic Model statistical analysis data certify the significant impact of Na CMC, and Tween-80 factors on the viscous characteristics of oromucosal gels (F-value > p-value). Comparison of mucoadhesive characteristics of the studied samples of oromucosal gels was made using dynamic mechanical analysis and the results of statistical analysis of ANOVA for Quadratic Model highlight the significant influence of Na CMC, D-panthenol, Tween-80. Optimized oromucosal gel test data is obtained that its structure is restored after the applied effort, namely the restoration of the structure after 10 seconds occurs by 69.5 %, after 30 s by 76.1 %, after 180 s by 85.4 %, after 180 s which allows to predict the stability of the dosage form, both after manufacture and after use. Conclusions. The composition of the oromucosal gel of anti-inflammatory action with the IL-1β interleukin antagonist for the complex treatment of inflammatory diseases of periodontal is developed. The optimized composition of the oromucosal gel obtained has satisfactory performance of kinetic stability and thixotropic properties. The developed gel for dentistry meets all the requirements for harmlessness and safety for dosage forms of this group toxicity, lack of local irritant and allergic action.

Immune gene polymorphisms associated with poor response to platelet transfusion and recombinant factor VII administration in Glanzmann thrombasthenia.

Poor response to platelet and recombinant factor VII administration is a major problem in patients with Glanzmann Thrombasthenia (GT). The risk factors associated with poor response to treatment in these patients are unknown. Some genetic variations of cytokines may contribute to therapy resistance. We evaluated, for the first time, whether genetic polymorphisms on cytokine genes are related to poor treatment response in GT patients. We enrolled 30 patients with GT (15 resistant and 15 non-resistant) and 100 healthy controls. Gene polymorphisms of IL-10 and TNF-α were analysed using TaqMan Realtime PCR, and IL-1, IL-1R1 and IL-1RN were investigated with the RFLP method. In-silico analyses were performed to predict the potential impact of these polymorphisms. In the resistant group, all patients had a variant of the IL-10 gene at the -1082 position (rs1800896), with a GG genotype that was significantly more frequent than the non-resistant group. Analysis between healthy controls and GT patients revealed a probable correlation between rs3783550, rs3783553, rs3917356 and rs2234463 and GT. The In-silico study indicated that TNF-α rs1800629 and IL-10 rs1800896 polymorphisms result in different allelic expressions which may contribute to poor response to therapy. These findings suggest that polymorphisms in the IL-10 and IL-1 receptor antagonist genes may play a role in poor therapy response in GT patients. In addition, some polymorphisms in IL-1α, IL1-β, IL-1R1 and IL-R antagonists might be involved in the GT progression.

IL-1β Antagonist Receptor Peptide Associated with Photobiomodulation Accelerates Diabetic Wound Tissue Repair.

Chronic hyperglycemia caused by diabetes mellitus (DM) slows down the healing process due to prolonged inflammation which impedes the regeneration progression. Photobiomodulation (PBM) is considered a non-pharmacological intervention and has anti-inflammatory and biostimulatory effects that accelerate the healing process. Currently found IL-1β inhibitors are difficult to implement due to their cytotoxic potential, excessive amounts, and invasive administration, and therefore, the application of this peptide in diabetic wounds represents a promising intervention to help resolve the inflammatory response. This study aimed to investigate the effect of an IL-1β inhibitor molecule associated with PBM irradiation in a model of epithelial injury in diabetic mice. After the induction of the DM model with streptozotocin (STZ), the skin lesion model was implemented through surgical excision. Sixty C57BL/6 mice divided into five experimental groups (n = 12) were used: excisional wound (EW), DM + EW, DM + EW + DAP 1-2 (inhibitor peptide), DM + EW + PBM, and DM + EW + PBM + DAP 1-2. Treatment started 12h after wound induction and was performed daily for 5 days. Twenty-four hours after the last application, the animals were euthanized and the outer edge of the wound was removed. The results obtained demonstrate that the DM + EW + PBM + DAP 1-2 group caused a reduction in the levels of pro-inflammatory cytokines, an increase in anti-inflammatory cytokines, and an increase in TGF-β and maintenance of the cellular redox state with a consequent reduction in levels of inflammatory infiltrate and concomitant stimulation of type III collagen gene expression, as well as a decrease in the size of the wound in square centimeter 6 days after the injury. Only the combination of therapies was able to favor the process of tissue regeneration due to the development of an approach capable of acting at different stages of the regenerative process, through the mechanisms of action of interventions on the inflammatory process by avoiding its stagnation and stimulating progression of regeneration.

Clinical spectrum of Cryopyrin-associated Periodic Syndrome associated with a single nucleotide variant of the NLRP3 gene in a Puerto Rican family

Cryopyrin-associated Periodic Syndromes (CAPS) are a group of autoinflammatory disorders of varying degrees of severity that include familial cold autoinflammatory syndrome (FCAS), Muckle-Wells syndrome (MWS), and neonatal-onset multisystem inflammatory disease (NOMID, or as Chronic Infantile Neurological, Cutaneous, and Articular (CINCA). There is one case per one million inhabitants in the United States. Cryopyrinopathies arise from mutations in the NOD-like receptor protein 3 (NLRP3) gene, which assembles with the cysteine protease caspase-1 to form the NLP3 and releases several proinflammatory cytokines, including IL-1β. An autosomal dominant GOF mutation of the NLRP3 gene causes the overproduction of IL-1β, inducing excessive inflammation and even permanent organ damage. We describe a Puerto Rican family of three with Cryopyrin-associated Periodic Syndrome and varying degrees of severity that received Canakinumab. A 40-year-old mother and her two sons, ages 26 and 20, presented for an evaluation of cold-induced maculopapular rash, arthralgia, and lower extremity swelling. Their symptoms initially presented at birth and continued into their adulthood. The episodes started briefly after exposure to cool temperatures and lasted hours to days. High fevers and chills often accompany episodes. They all presented with chronic leukocytosis and high inflammatory laboratory values. The oldest son showed the most intense, enduring flares and severe complications. At age 1, he was diagnosed with neuroblastoma of the right adrenal gland and achieved remission with a combination of surgical resection, chemotherapy, and radiotherapy. He subsequently required multiple hospitalizations due to episodes of high fevers as a child. He also developed severe leptomeningeal inflammation at age 12 and underwent an exploratory craniotomy that yielded normal findings. The three family members underwent genetic testing two decades after the children were born, revealing a heterozygous missense mutation. They had a single nucleotide variant of the NPL3 gene c.913G>A (p. Asp305Asn) that has been linked to CAPS. Severe inflammatory neurologic symptoms and familial cold urticaria were the clues for the diagnostic suspicion of CAPS. IL-1β antagonists have been demonstrated to be effective in substantially reducing inflammatory markers and thus decreasing inflammation-related signs and symptoms. Residence in the tropics could have contributed to delayed diagnosis.Table 1General characteristics of the Cryopyrin-associated Periodic SyndromesFCASMWSNOMID/CINCAGenes and pattern of inheritanceNLRP3 gene ADNLRP3 gene ADNLRP3 gene ADEpidemiology Age of onset1:1 million.1Infancy1:1 million1Infancy1:1 million1Neonatal/early infancyCutaneous NeurologicUrticaria-like rash Headaches with fever after cold exposureUrticaria-like rash Headaches with fever during flares. Uncommon to have other CNS symptoms. 3Urticaria-like rash Headaches, fever, chronic aseptic meningitis, and high CNS pressure. Many develop mental or cognitive impairment.AuditoryMild hearing lossSensorineural hearing loss starting in adolescenceSensorineural hearing loss from infancy/childhoodOphthalmicConjunctivitis during flaresConjunctivitis during flaresPapilledema, uveitis, iritis, conjunctivitis, and vision lossLymphaticNot notedMay be notedMay have enlarged lymph nodesJointsArthralgiaArthralgia, stiffness, and swelling during flaresArthralgia, stiffness, and swelling during flaresLaboratory valuesHigh ESR, CRP, SAA, and leukocytosis with flaresHigh ESR, CRP, SAA, and leukocytosis with flaresChronically elevated ESR, CRP, SAA, and leukocytosisFCAS, Familial cold autoinflammatory syndrome; MWS Muckle Wells Syndrome; CINCA/NOMID, chronic infantile neurologic, cutaneous, and articular syndrome/neonatal-onset multisystem inflammatory disease.

Single-cell sequencing reveals the immune microenvironment landscape related to anti-PD-1 resistance in metastatic colorectal cancer with high microsatellite instability

BackgroundThe objective response rate of microsatellite instability-high (MSI-H) metastatic colorectal cancer (mCRC) patients with first-line anti-programmed cell death protein-1 (PD-1) monotherapy is only 40–45%. Single-cell RNA sequencing (scRNA-seq) enables unbiased analysis of the full variety of cells comprising the tumor microenvironment. Thus, we used scRNA-seq to assess differences among microenvironment components between therapy-resistant and therapy-sensitive groups in MSI-H/mismatch repair-deficient (dMMR) mCRC. Resistance-related cell types and genes identified by this analysis were subsequently verified in clinical samples and mouse models to further reveal the molecular mechanism of anti-PD-1 resistance in MSI-H or dMMR mCRC.MethodsThe response of primary and metastatic lesions to first-line anti-PD-1 monotherapy was evaluated by radiology. Cells from primary lesions of patients with MSI-H/dMMR mCRC were analyzed using scRNA-seq. To identify the marker genes in each cluster, distinct cell clusters were identified and subjected to subcluster analysis. Then, a protein‒protein interaction network was constructed to identify key genes. Immunohistochemistry and immunofluorescence were applied to verify key genes and cell marker molecules in clinical samples. Immunohistochemistry, quantitative real-time PCR, and western blotting were performed to examine the expression of IL-1β and MMP9. Moreover, quantitative analysis and sorting of myeloid-derived suppressor cells (MDSCs) and CD8+ T cells were performed using flow cytometry.ResultsTumor responses in 23 patients with MSI-H/dMMR mCRC were evaluated by radiology. The objective response rate was 43.48%, and the disease control rate was 69.57%. ScRNA-seq analysis showed that, compared with the treatment-resistant group, the treatment-sensitive group accumulated more CD8+ T cells. Experiments with both clinical samples and mice indicated that infiltration of IL-1β-driven MDSCs and inactivation of CD8+ T cells contribute to anti-PD-1 resistance in MSI-H/dMMR CRC.ConclusionsCD8+ T cells and IL-1β were identified as the cell type and gene, respectively, with the highest correlation with anti-PD-1 resistance. Infiltration of IL-1β-driven MDSCs was a significant factor in anti-PD-1 resistance in CRC. IL-1β antagonists are expected to be developed as a new treatment for anti-PD-1 inhibitor resistance.

Canakinumab as first-line biological therapy in Still's disease and differences between the systemic and the chronic-articular courses: Real-life experience from the international AIDA registry.

Interleukin (IL)-1 inhibitors are largely employed in patients with Still's disease; in cases with refractory arthritis, IL-6 inhibitors have shown to be effective on articular inflammatory involvement. The aim of the present study is to assess any difference in the effectiveness of the IL-1β antagonist canakinumab prescribed as first-line biologic agent between the systemic and the chronic-articular Still's disease. Data were drawn from the retrospective phase of the AutoInflammatory Disease Alliance (AIDA) international registry dedicated to Still's disease. Patients with Still's disease classified according to internationally accepted criteria (Yamaguchi criteria and/or Fautrel criteria) and treated with canakinumab as first-line biologic agent were enrolled. A total of 26 patients (17 females, 9 males; 18 patients developing Still's disease after the age of 16 years) were enrolled; 16 (61.5%) patients suffered from the systemic pattern of the disease; 10 (38.5%) patients suffered from the chronic-articular type. No differences were observed between the systemic and the chronic-articular Still's disease in the frequency of complete response, of flares after the start of canakinumab (p = 0.701) and in the persistence in therapy (p = 0.62). No statistical differences were observed between the two groups after 3 months, 12 months and at the last assessment in the decrease of: the systemic activity score (p = 0.06, p = 0.17, p = 0.17, respectively); the disease activity score on 28 joints (p = 0.54, p = 0.77, p = 0.98, respectively); the glucocorticoid dosage (p = 0.15, p = 0.50, and p = 0.50, respectively); the use of concomitant disease modifying anti-rheumatic drugs (p = 0.10, p = 1.00, and p = 1.00, respectively). No statistically significant differences were observed in the decrease of erythrocyte sedimentation rate (p = 0.34), C reactive protein (p = 0.48), and serum ferritin levels (p = 0.34) after the start of canakinumab. Canakinumab used for Still's disease has been effective in controlling both clinical and laboratory manifestations disregarding the type of disease course when used as first-line biotechnological agent. These excellent results might have been further enhanced by the early start of IL-1 inhibition.

Down-regulated Th17 cells in a patient with familial Mediterranean fever associated with amyloid A amyloidosis in the treatment of canakinumab.

Autoinflammatory diseases are innate immune-mediated inflammatory disorders, unlike autoimmune diseases, which are characterised by abnormalities in adoptive immunity, although autoimmune and autoinflammatory diseases have certain similar clinical features. Familial Mediterranean fever (FMF), the most common monogenic autoinflammatory disease, is associated with mutations in the MEFV gene that encodes pyrin, which results in inflammasome activation and uncontrolled production of interleukin (IL)-1β. Regular use of colchicine, the primary drug for FMF treatment, prevents febrile attacks and reduces the long-term risk of subsequent complications of amyloid A (AA) amyloidosis. However, a minority of FMF patients develop colchicine resistance, and anti-IL-1β treatment with canakinumab, which is a genetically modified human IgG subclass type 1 (IgG1) monoclonal antibody specific for human IL-1β, was beneficial in inhibiting inflammation in such patients. Here, we present a patient with FMF associated with AA amyloidosis, who was treated with canakinumab and demonstrated down-regulated Th17 cells and activated Th17 cells (from 21.4% to 12.8%, and from 1.45% to 0.83%, respectively) in peripheral blood, as shown by immunophenotyping via multicolour flow cytometry and by disease activity and improved laboratory inflammatory surrogate markers-C-reactive protein (CRP) and serum AA protein (SAA). CRP had values within normal limits, but SAA did not (Spearman's rank correlation coefficient; ρ = 0.133). We report that SAA and IL-1β may differentiate Th17 cells from CD4+-naïve T cells, and we discuss interactions between autoinflammation and autoimmunity as a model based on this case, through modes of action with IL-1β and SAA. This report is the first demonstrating that an IL-1β antagonist may reduce Th17 cells in FMF as a therapeutic option.

No-reflow phenomenon and reperfusion injury. Mechanisms and treatment

Currently, one of the key methods of treating a patient with ST-elevation myocardial infarction is to restore blood flow to the infarct-related artery as quickly, completely and steadily as possible. However, in some cases, it is not possible to achieve adequate myocardial reperfusion, despite the restoration of coronary blood flow. This phenomenon was named no-reflow. Due to the lack of a unified approach to the diagnosis of no-reflow, its occurrence varies widely – from 2 to 44 %. Failure to achieve adequate myocardial perfusion leads to a higher mortality rate – from 7.4 to 30.3 %, as well as to more aggressive remodeling of the myocardium. For a long time, distal embolization in percutaneous coronary intervention was considered one of the leading mechanisms. However, the routine use of protective devices did not show a pronounced effect on the outcome and prognosis, although it is justified in certain clinical situations. Ischemic injury directly plays a significant role due to overload of cardiomyocytes with calcium, cellular edema, necrosis and apoptosis, which is significantly aggravated by myocardial reperfusion and forms obstruction at the level of the microcirculatory bed. More data is being accumulated about immune-mediated injury through activation of cellular immunity, intense inflammation and thrombosis in situ. Despite the success in the animal experiment, the clinical use of certain groups of drugs showed an ambiguous results. According to the latest recommendations European Society of Cardiology / European Association for Cardio-Thoracic Surgery (ESC / EACTS) 2018, GPIIb / IIIa platelet receptor inhibitors are recommended in the case of no-reflow. Besides this, according to the literature nicorandil and sodium nitroprusside, as well as IL-1β antagonists, seem to be promising. As a non-drug therapy, selective intracoronary hypothermia also has shown its effectiveness and safety in a pilot study. To date, it is clear that the no-reflow phenomenon is a manifestation of a complex cascade of reactions, including ischemic, reperfusion and immune-related injury, as well as distal embolization. Considering its significant contribution to the frequency of adverse outcomes and late complications, it seems necessary to introduce unified approaches to the diagnosis, prevention and treatment of no-reflow, which requires high-quality clinical studies.

What’s new on the front-line of gout pharmacotherapy?

ABSTRACT Introduction Gout is the most common form of inflammatory arthritis affecting millions of persons around the world. Painful flares and tophaceous deposits are debilitating, reduce quality of life and put strain on health-care systems. Areas Covered This review provides an overview of the treatment of gout flares and lowering serum urate. First line agents are discussed with emphasis on emerging evidence. Novel therapies are also covered. Expert opinion Lifestyle modifications form a part of gout prevention. NSAIDs, colchicine, and glucocorticoids are first line agents for gout flares. The IL-1β antagonists are highly effective for arresting flares but their cost-effectiveness render them salvage therapies. Allopurinol is an agent of first choice for urate lowering therapy. In Southeast Asian and Black populations, screening for HLA*B58:01 mutation is a cost-effective approach to decrease the occurrence of allopurinol hypersensitivity syndrome. Febuxostat is another efficacious urate lowering therapy, but has received a U.S. FDA black box warning for cardiovascular safety. Novel uricosurics are a class for continued drug development; verinurad and arhalofenate are agents with future promise. For patients with recalcitrant gout, pegloticase is effective. Its immunogenicity significantly threatens the achievement of sustained urate lowering responses. Abrogating pegloticase’s immunogenicity with immunomodulatory co-therapy may lend to sustained efficacy.

Abstract 13589: The Mechano-Signaling Roles of the Adaptor Protein Nck1 in Atherogenic Endothelial Activation

Rational: Alterations in hemodynamic shear stress (SS) at atherosclerosis-prone sites promotes endothelial activation, characterized by nuclear factor-κB (NF-κB)-driven expression of cell adhesion molecules that mediate leukocyte homing. In addition, proinflammatory cytokines (e.g. IL-1β) promote NF-κB-dependent endothelial activation. While the recent CANTOS trial using an IL-1β antagonist highlights the potential for treating atherogenic inflammation beyond lipid lowering therapies alone, our understanding of how endothelial activation contributes to this effect remains limited. Methodology & Results: We identified the signaling adaptor Nck1 as a critical regulator of atherogenic endothelial activation. Endothelial cells deficient in Nck1 lack SS-induced NF-κB activation and proinflammatory gene expression. Additionally, we demonstrated an interaction between Nck1 and IL-1β signaling in response to SS, showing that SS activates the IL-1β receptor (IL1R1) signaling partner IRAK-1 in a Nck1-dependent manner both in vitro and in vivo . IRAK-1 depletion inhibited the proinflammatory response to SS. Mechanistically, point mutation analysis suggest a critical role for the Nck1 SH2 domain (phosphotyrosine binding domain) in mediating these effects. Consistent with our cell culture studies, Global Nck1 knockout mice show reduced atherosclerosis characterized by diminished macrophage and smooth muscle incorporation and bone marrow chimeras suggest non-hematopoietic Nck1 mediates this response. Conclusions: A recent GWAS analysis linked Nck1 to coronary artery disease, underscoring its importance to human disease. Therefore, our data suggest that Nck1 mediates the formation of IL-1-related signaling complexes to induce atherogenic endothelial activation, and selective inhibition of Nck1 or its critical domains will allow efficient inhibition of atherosclerosis in humans.

Abstract MP06: The Signaling Roles Of The Adaptor Protein Nck1 In Atherosclerosis

Rational: Alterations in hemodynamic shear stress (SS) at atherosclerosis-prone sites promotes endothelial activation, characterized by nuclear factor-κB (NF-κB)-driven expression of cell adhesion molecules that mediate leukocyte homing. In addition, proinflammatory cytokines (e.g. IL-1β) promote NF-κB-dependent endothelial activation. While the recent CANTOS trial using an IL-1β antagonist highlights the potential for treating atherogenic inflammation beyond lipid lowering therapies alone, our understanding of how endothelial activation contributes to this effect remains limited. Methodology & Results: We identified the signaling adaptor Nck1 as a critical regulator of atherogenic endothelial activation. Endothelial cells deficient in Nck1 lack SS-induced NF-κB activation and proinflammatory gene expression. Additionally, we demonstrated an interaction between Nck1 and IL-1β signaling in response to SS, showing that SS activates the IL-1β receptor (IL1R1) signaling partner IRAK-1 in a Nck1-dependent manner both in vitro and in vivo . Mechanistically, point mutation analysis suggest a critical role for the Nck1 SH2 domain (phosphotyrosine binding domain) in mediating these effects. Nck1 affinity pulling down and mass spectrometry identified multiple downstream effectors of IL-1 pathway. In vivo, Global Nck1 knockout mice show reduced atherosclerosis characterized by diminished macrophage and smooth muscle incorporation and bone marrow chimeras suggest non-hematopoietic Nck1 mediates this response. Conclusions: A recent GWAS analysis linked Nck1 to coronary artery disease, underscoring its importance to human disease. Therefore, our data suggest that Nck1 mediates the formation of IL-1-related signaling complexes to induce atherogenic endothelial activation, and selective inhibition of Nck1 or its critical domains will allow efficient inhibition of atherosclerosis in humans.

NF-κB Regulation of LRH-1 and ABCG5/8 Potentiates Phytosterol Role in the Pathogenesis of Parenteral Nutrition-Associated Cholestasis.

Chronically administered parenteral nutrition (PN) in patients with intestinal failure carries the risk for developing PN-associated cholestasis (PNAC). We have demonstrated that farnesoid X receptor (FXR) and liver X receptor (LXR), proinflammatory interleukin-1 beta (IL-1β), and infused phytosterols are important in murine PNAC pathogenesis. In this study we examined the role of nuclear receptor liver receptor homolog 1 (LRH-1) and phytosterols in PNAC. In a C57BL/6 PNAC mouse model (dextran sulfate sodium [DSS] pretreatment followed by 14 days of PN; DSS-PN), hepatic nuclear receptor subfamily 5, group A, member 2/LRH-1 mRNA, LRH-1 protein expression, and binding of LRH-1 at the Abcg5/8 and Cyp7a1 promoter was reduced. Interleukin-1 receptor-deficient mice (Il-1r-/- /DSS-PN) were protected from PNAC and had significantly increased hepatic mRNA and protein expression of LRH-1. NF-κB activation and binding to the LRH-1 promoter were increased in DSS-PN PNAC mice and normalized in Il-1r-/- /DSS-PN mice. Knockdown of NF-κB in IL-1β-exposed HepG2 cells increased expression of LRH-1 and ABCG5. Treatment of HepG2 cells and primary mouse hepatocytes with an LRH-1 inverse agonist, ML179, significantly reduced mRNA expression of FXR targets ATP binding cassette subfamily C member 2/multidrug resistance associated protein 2 (ABCC2/MRP2), nuclear receptor subfamily 0, groupB, member 2/small heterodimer partner (NR0B2/SHP), and ATP binding cassette subfamily B member 11/bile salt export pump (ABCB11/BSEP). Co-incubation with phytosterols further reduced expression of these genes. Similar results were obtained by suppressing the LRH-1 targets ABCG5/8 by treatment with small interfering RNA, IL-1β, or LXR antagonist GSK2033. Liquid chromatography-mass spectrometry and chromatin immunoprecipitation experiments in HepG2 cells showed that ATP binding cassette subfamily G member 5/8 (ABCG5/8) suppression by GSK2033 increased the accumulation of phytosterols and reduced binding of FXR to the SHP promoter. Finally, treatment with LRH-1 agonist, dilauroyl phosphatidylcholine (DLPC) protected DSS-PN mice from PNAC. This study suggests that NF-κB regulation of LRH-1 and downstream genes may affect phytosterol-mediated antagonism of FXR signaling in the pathogenesis of PNAC. LRH-1 could be a potential therapeutic target for PNAC.

COVID19-associated cardiomyocyte dysfunction, arrhythmias and the effect of Canakinumab.

BackgroundCardiac injury associated with cytokine release frequently occurs in SARS-CoV-2 mediated coronavirus disease (COVID19) and mortality is particularly high in these patients. The mechanistic role of the COVID19 associated cytokine-storm for the concomitant cardiac dysfunction and associated arrhythmias is unclear. Moreover, the role of anti-inflammatory therapy to mitigate cardiac dysfunction remains elusive.Aims and methodsWe investigated the effects of COVID19-associated inflammatory response on cardiac cellular function as well as its cardiac arrhythmogenic potential in rat and induced pluripotent stem cell derived cardiomyocytes (iPS-CM). In addition, we evaluated the therapeutic potential of the IL-1β antagonist Canakinumab using state of the art in-vitro confocal and ratiometric high-throughput microscopy.ResultsIsolated rat ventricular cardiomyocytes were exposed to control or COVID19 serum from intensive care unit (ICU) patients with severe ARDS and impaired cardiac function (LVEF 41±5%; 1/3 of patients on veno-venous extracorporeal membrane oxygenation; CK 154±43 U/l). Rat cardiomyocytes showed an early increase of myofilament sensitivity, a decrease of Ca2+ transient amplitudes and altered baseline [Ca2+] upon exposure to patient serum. In addition, we used iPS-CM to explore the long-term effect of patient serum on cardiac electrical and mechanical function. In iPS-CM, spontaneous Ca2+ release events were more likely to occur upon incubation with COVID19 serum and nuclear as well as cytosolic Ca2+ release were altered. Co-incubation with Canakinumab had no effect on pro-arrhythmogenic Ca2+ release or Ca2+ signaling during excitation-contraction coupling, nor significantly influenced cellular automaticity.ConclusionSerum derived from COVID19 patients exerts acute cardio-depressant and chronic pro-arrhythmogenic effects in rat and iPS-derived cardiomyocytes. Canakinumab had no beneficial effect on cellular Ca2+ signaling during excitation-contraction coupling. The presented method utilizing iPS-CM and in-vitro Ca2+ imaging might serve as a novel tool for precision medicine. It allows to investigate cytokine related cardiac dysfunction and pharmacological approaches useful therein.

Transport study of interleukin-1 inhibitors using a human in vitro model of the blood-brain barrier

The proinflammatory cytokine Interleukin-1 (IL-1), with its two isoforms α and β, has important roles in multiple pathogenic processes in the central nervous system. The present study aimed to evaluate and compare the blood-to-brain distribution of anakinra (IL-1 receptor antagonist), bermekimab (IL-1α antagonist) and canakinumab (IL-1β antagonist).A human in vitro model of the blood-brain barrier derived from human umbilical cord blood stem cells was used, where isolated CD34+ cells co-cultured with bovine pericytes were matured into polarized brain-like endothelial cells. Transport rates of the three test items were evaluated after 180 ​min incubation at concentrations 50, 250 and 1250 ​nM in a transwell system. We report herein that anakinra passes the human brain-like endothelial monolayer at a 4-7-fold higher rate than the monoclonal antibodies tested. Both antibodies had similar transport rates at all concentrations. No dose-dependent effects in transport rates were observed, nor any saturation effects at supraphysiological concentrations. The larger propensity of anakinra to pass this model of the human blood-brain barrier supports existing data and confirms that anakinra can reach the brain compartment at clinically relevant concentrations. As anakinra inhibits the actions of both IL-1α and IL-1β, it blocks all effects of IL-1 downstream signaling. The results herein further add to the growing body of evidence of the potential utility of anakinra to treat neuroinflammatory disorders.

Spinal interleukin-1β induces mechanical spinal hyperexcitability in rats: Interactions and redundancies with TNF and IL-6.

Both spinal tumor necrosis factor (TNF) and interleukin-6 (IL-6) contribute to the development of "mechanical" spinal hyperexcitability in inflammatory pain states. Recently, we found that spinal sensitization by TNF was significantly reduced by blockade of spinal IL-6 signaling suggesting that IL-6 signaling is involved in spinal TNF effects. Here, we explored whether spinal interleukin-1β (IL-1β), also implicated in inflammatory pain, induces "mechanical" spinal hyperexcitability, and whether spinal IL-1β effects are related to TNF and IL-6 effects. We recorded the responses of spinal cord neurons to mechanical stimulation of the knee joint in vivo and used cellular approaches on microglial and astroglial cell lines to identify interactions of IL-1β, TNF, and IL-6. Spinal application of IL-1β in anesthetized rats modestly enhanced responses of spinal cord neurons to innocuous and noxious mechanical joint stimulation. This effect was blocked by minocycline indicating microglia involvement, and significantly attenuated by interfering with IL-6 signaling. In the BV2 microglial cell line, IL-1β, like TNF, enhanced the release of soluble IL-6 receptor, necessary for spinal IL-6 actions. Different to TNF, IL-1β caused SNB-19 astrocytes to release interleukin-11. The generation of "mechanical" spinal hyperexcitability by IL-1β was more pronounced upon spinal TNF neutralization with etanercept, suggesting that concomitant TNF limits IL-1β effects. In BV2 cells, TNF stimulated the release of IL-1Ra, an endogenous IL-1β antagonist. Thus, spinal IL-1β has the potential to induce spinal hyperexcitability sharing with TNF dependency on IL-6 signaling, but TNF also limited IL-1β effects explaining the modest effect of IL-1β.

MECHANISMS OF ATHEROGENESIS AND ITS INTENSIFICATION IN PATIENTS WITH RHEUMATOID ARTHRITIS

The article describes the role of cholesterol crystals (CS) in the mechanisms of development in the inner lining of arteries of inflammation – granulomatosis, induced by foreign bodies. The smallest CS crystals are found already in early atherosclerotic lesions. They are a factor in the initiation and exacerbation of atherosclerosis (At), cause cell damage and apoptosis. The formation of crystals within the necrotic nuclei of plaques can lead to an increase in their volume and to rupture. It has been shown that damage to the membranes of macrophages phagolysosomes by absorbed CS crystals leads to the inclusion of protein complexes – inflammasomes – in the inflammatory process, which trigger the inflammatory signaling cascade and are responsible for the secretion of pro-inflammatory cytokines. Inflammasomes NLRP3 are necessary for the process of atherogenesis; their activation is a link between the metabolism of cholesterol and inflammation involving macrophages. Unlike At, RA is manifested by autoimmune inflammation and immunocomplex vasculitis. When these diseases are combined, the effects of proinflammatory cytokines add up, an increase in the severity of inflammation, increased tissue damage and progression of atherosclerotic arterial lesions. The accelerated development of At in RA patients is facilitated by a combination of the action of traditional risk factors for atherogenesis and damage to the walls (endothelium) of arteries by immune complexes, complement, neutrophils and lymphocytes with an increase in their permeability to the lipid factor. The deposition of immune complexes in the capillaries of plaques leads to damage to their walls, destabilization of plaques and the development of acute cardiovascular events. The intensification of lipid accumulation and inflammation in the plaques of RA patients is confirmed in the section. Anticytokine drugs, primarily TNF-α and IL-1β antagonists, are pathogenetically substantiated agents for the progression of At in patients with RA. Statins remain a widely used class of drugs. They, in addition to hypolipidemic, immunomodulatory and anti-inflammatory effects, affect the crystallization of cholesterol, dissolve crystals and stabilize plaques.

Radiation-induced IL-1β expression and secretion promote cancer cell migration/invasion via activation of the NF-κB–RIP1 pathway

Here, we demonstrate that interleukin-1β (IL-1β) contributes to the γ-ionizing radiation (IR)-induced increase of migration/invasion in A549 lung cancer cells, and that this occurs via RIP1 upregulation. We initially observed that the protein expression and secreted concentration of IL-1β were increased upon exposure of A549 cells to IR. We then demonstrated that IR-induced IL-1β is located downstream of the NF-κB–RIP1 signaling pathway. Treatments with siRNA and specific pharmaceutical inhibitors of RIP1 and NF-κB suppressed the IR-induced increases in the protein expression and secreted concentration of IL-1β. IL-1Ra, an antagonist of IL-1β, treatment suppressed the IR-induced epithelial-mesenchymal transition (EMT) and IR-induced invasion/migration in vitro. These results suggest that IL-1β could regulate IR-induced EMT. We also found that IR could induce the expression of IL-1β expression in vivo and that of IL-1 receptor (R) I/II in vitro and in vivo. The IR-induced increases in the protein levels of IL-1 RI/II and IL-1β suggest that an autocrine loop between IL-1β and IL-1 RI/II might play important roles in IR-induced EMT and migration/invasion. Based on these collective results, we propose that IR concomitantly activates NF-κB and RIP1 to trigger the NF-κB–RIP1–IL-1β–IL-1RI/II–EMT pathway, ultimately promoting metastasis.