Interleukin-6 Bioactivity Research Articles

A novel immunomodulating peptide with potential to complement oligodeoxynucleotide-mediated adjuvanticity in vaccination strategies

The identification of adjuvants to improve vaccination efficacy is a major unmet need. One approach is to augment the functionality of dendritic cells (DCs) by using Toll-like receptor-9 (TLR9) agonists such as cytosine-phosphate-guanine oligodeoxynucleotides (CpG ODNs) as adjuvants. Another approach is adjuvant selection based on production of bioactive interleukin-12 (IL-12). We report a D-peptide isomer, designated D-15800, that induces monocyte differentiation to the DC phenotype in vitro and more effectively stimulates IL-12p70 production upon T cell receptor (TCR) activation than the L-isomer. In the absence of TCR activation and either IL-12p70 or interleukin-2 production, only D-15800 activates CD4+ T and natural killer cells. In the presence of CpG ODN, D-15800 synergistically enhances production of interferon-alpha (IFN-α). Taken together with its biostability in human serum and depot retention upon injection, co-delivery of D-15800 with TLR9 agonists could serve to improve vaccine efficacy.

Riboswitch-controlled IL-12 gene therapy reduces hepatocellular cancer in mice.

Hepatocellular carcinoma (HCC) and solid cancers with liver metastases are indications with high unmet medical need. Interleukin-12 (IL-12) is a proinflammatory cytokine with substantial anti-tumor properties, but its therapeutic potential has not been realized due to severe toxicity. Here, we show that orthotopic liver tumors in mice can be treated by targeting hepatocytes via systemic delivery of adeno-associated virus (AAV) vectors carrying the murine IL-12 gene. Controlled cytokine production was achieved in vivo by using the tetracycline-inducible K19 riboswitch. AAV-mediated expression of IL-12 led to STAT4 phosphorylation, interferon-γ (IFNγ) production, infiltration of T cells and, ultimately, tumor regression. By detailed analyses of efficacy and tolerability in healthy and tumor-bearing animals, we could define a safe and efficacious vector dose. As a potential clinical candidate, we characterized vectors carrying the human IL-12 (huIL-12) gene. In mice, bioactive human IL-12 was expressed in a vector dose-dependent manner and could be induced by tetracycline, suggesting tissue-specific AAV vectors with riboswitch-controlled expression of highly potent proinflammatory cytokines as an attractive approach for vector-based cancer immunotherapy.

OP17 IBD ulcers are characterized by bioactive interleukin-1 and transcriptomic hallmarks of stromal cell state reprogramming

Abstract Background The programs that perpetuate the inflammation and prevent epithelial repair in Inflammatory Bowel Disease (IBD) remain unclear. Interleukin (IL)-1 plays a role in the maintenance of mucosal homeostasis but also in IBD. Expansion of IL-1 expressing myeloid cells is a hallmark of IBD tissue, including severe and anti-TNF unresponsive disease, and IL-1 expression is highly localized to ulcer beds. However, little is known about the presence of bioactive IL-1 proteins in the cell-free mucosal environment of IBD, nor whether IL-1-driven programs affect epithelial regeneration in IBD ulcers. Methods We established a highly sensitive assay (~500-fold greater than ELISA) to assess bioactive extracellular IL-1 from previously cryopreserved IBD biopsies. We assessed a cohort of Crohn’s Disease (CD, n=23), Ulcerative Colitis (UC, n=18) and non-IBD (n=17) patients, with biopsies from paired inflamed/uninflamed regions (primarily colon but also ileal). We assessed IL-1 bioactivity, including IL-1α vs IL-1β contributions, and performed RNA-seq of samples’ matched cellular compartments. An ulcer-associated gene signature was interrogated in a single-cell (sc)RNA-seq cohort (n=42) of very early onset (VEO)IBD including monogenic disorders, as well as publicly available IBD bulk RNA-seq datasets and scRNA-seq of mouse models of colitis. Results IL-1α and IL-1β bioactivity corresponded with disease and ulcer severity in CD and UC. The most extreme signals were seen in select CD patients with deep ulceration. IL-1α was the predominant contributor to total IL-1 bioactivity in most patients although several with ulcers displayed IL-1β predominance. IL-1 bioactivity correlated with IL-1 transcripts in matched RNA-seq, and weighted gene co-expression network analysis revealed a compelling ulcer-specific module. This module contained transcription factors and genes related to cell state, validated in publicly available datasets (e.g. RISK cohort of ileal CD and scRNA-seq of murine DSS colitis), that when interrogated in a scRNA-seq dataset of VEOIBD (including patients with IL-10RA mutations characterized by deep ulcers), suggests an orchestrated IL-1-driven myeloid/stromal response to epithelial ulceration involving cell state reprogramming and loss of key myofibroblast populations. Conclusion Mucosal ulceration in IBD is associated with bioactive IL-1α and IL-1β proteins, and transcriptomic evaluation of the same correlates with bioactive signal. An ulcer-associated gene module, validated in other datasets, sheds light on IL-1 biology in intestinal epithelial repair. Results strongly suggest the IL-1 signaling pathway being an attractive and precision-based therapeutic target in subsets of IBD patients.

XTX301, a Tumor-Activated Interleukin-12 Has the Potential to Widen the Therapeutic Index of IL12 Treatment for Solid Tumors as Evidenced by Preclinical Studies.

IL12 is a proinflammatory cytokine, that has shown promising antitumor activity in humans by promoting the recruitment and activation of immune cells in tumors. However, the systemic administration of IL12 has been accompanied by considerable toxicity, prompting interest in researching alternatives to drive preferential IL12 bioactivity in the tumor. Here, we have generated XTX301, a tumor-activated IL12 linked to the human Fc protein via a protease cleavable linker that is pharmacologically inactivated by an IL12 receptor subunit beta 2 masking domain. In vitro characterization demonstrates multiple matrix metalloproteases, as well as human primary tumors cultured as cell suspensions, can effectively activate XTX301. Intravenous administration of a mouse surrogate mXTX301 demonstrated significant tumor growth inhibition (TGI) in inflamed and non-inflamed mouse models without causing systemic toxicities. The superiority of mXTX301 in mediating TGI compared with non-activatable control molecules and the greater percentage of active mXTX301 in tumors versus other organs further confirms activation by the tumor microenvironment-associated proteases in vivo. Pharmacodynamic characterization shows tumor selective increases in inflammation and upregulation of immune-related genes involved in IFNγ cell signaling, antigen processing, presentation, and adaptive immune response. XTX301 was tolerated following four repeat doses up to 2.0 mg/kg in a nonhuman primate study; XTX301 exposures were substantially higher than those at the minimally efficacious dose in mice. Thus, XTX301 has the potential to achieve potent antitumor activity while widening the therapeutic index of IL12 treatment and is currently being evaluated in a phase I clinical trial.

Tmem178 Negatively Regulates IL-1β Production Through Inhibition of the NLRP3 Inflammasome.

Inflammasomes modulate the release of bioactive interleukin (IL)-1β. Excessive IL-1β levels are detected in patients with systemic juvenile idiopathic arthritis (sJIA) and cytokine storm syndrome (CSS) with mutated and unmutated inflammasome components, raising questions on the mechanisms of IL-1β regulation in these disorders. To investigate how the NLRP3 inflammasome is modulated in sJIA, we focused on Transmembrane protein 178 (Tmem178), a negative regulator of calcium levels in macrophages, and measured IL-1β and caspase-1 activation in wild-type (WT) and Tmem178-/- macrophages after calcium chelators, silencing of Stim1, a component of store-operated calcium entry (SOCE), or by expressing a Tmem178 mutant lacking the Stromal Interaction Molecule 1 (Stim1) binding site. Mitochondrial function in both genotypes was assessed by measuring oxidative respiration, mitochondrial reactive oxygen species (mtROS), and mitochondrial damage. CSS development was analyzed in Perforin-/- /Tmem178-/- mice infected with lymphocytic choriomeningitis virus (LCMV) in which inflammasome or IL-1β signaling was pharmacologically inhibited. Human TMEM178 and IL1B transcripts were analyzed in data sets of whole blood and peripheral blood monocytes from healthy controls and patients with active sJIA. TMEM178 levels are reduced in whole blood and monocytes from patients with sJIA while IL1B levels are increased. Accordingly, Tmem178-/- macrophages produce elevated IL-1β compared with WT cells. The elevated intracellular calcium levels after SOCE activation in Tmem178-/- macrophages induce mitochondrial damage, release mtROS, and ultimately promote NLRP3 inflammasome activation. In vivo, inhibition of inflammasome or IL-1β neutralization prolongs Tmem178-/- mouse survival in LCMV-induced CSS. Down-regulation of TMEM178 levels may represent a marker of disease activity and help identify patients who could benefit from inflammasome targeting.

Hypoxia-regulated secretion of IL-12 enhances antitumor activity and safety of CD19 CAR-T cells in the treatment of DLBCL

CD19-targeted chimeric antigen receptor-modified T (CD19 CAR-T) cell therapy has been demonstrated as one of the most promising therapeutic strategies for treating B cell malignancies. However, it has shown limited treatment efficacy for diffuse large B cell lymphoma (DLBCL). This is, in part, due to the tumor heterogeneity and the hostile tumor microenvironment. Human interleukin-12 (IL-12), as a potent antitumor cytokine, has delivered encouraging outcomes in preclinical studies of DLBCL. However, potentially lethal toxicity associated with systemic administration precludes its clinical application. Here, an armed CD19 CAR expressing hypoxia-regulated IL-12 was developed (CAR19/hIL12ODD). In this vector, IL-12 secretion was restricted to hypoxic microenvironments within the tumor site by fusion of IL-12 with the oxygen degradation domain (ODD) of HIF1α. Invitro, CAR19/hIL12ODD-T cells could only secrete bioactive IL-12 under hypoxic conditions, accompanied by enhanced proliferation, robust IFN-γ secretion, increased abundance of CD4+, and central memory Tcell phenotype. Invivo, adoptive transfer of CAR19/hIL12ODD-T cells significantly enhanced regression of large, established DLBCL xenografts in a novel immunodeficient Syrian hamster model. Notably, this targeted and controlled IL-12 treatment was without toxicity in this model. Taken together, our results suggest that armed CD19 CARs with hypoxia-controlled IL-12 (CAR19/hIL12ODD) might be a promising and safer approach for treating DLBCL.

Effects of AIM2 and IFI16 on Infectious Diseases and Inflammation.

Both absent in melanoma 2 (AIM2) and interferon-inducible protein 16 (IFI16) are intracellular innate immune receptors that recognize double-stranded DNA released during pathogenic infection, leading to the assembly of the inflammasome. The assembly of the inflammasome results in the secretion of bioactive interleukin (IL)-1β and IL-18 and induces cell death through an inflammatory process called pyroptosis. Although the AIM2 inflammasome is generally harmful in the context of some aseptic inflammatory illnesses, it plays a protective role in infectious diseases. During inflammatory processes, there is competition between IFI16 and AIM2. In this review, we explore the impacts of IFI16 and AIM2 in infectious disease and aseptic inflammation, respectively, and how they compete.

Engineering an active immunotherapy for personalized cancer treatment and prevention of recurrence.

Breast cancer has been shown to be resistant to immunotherapies. To overcome this challenge, we developed an active immunotherapy for personalized treatment based on a smart nanovesicle. This is achieved by anchoring membrane-bound bioactive interleukin 2 (IL2) and enriching T cell-promoting costimulatory factors on the surface of the dendritic cell-derived small extracellular vesicles. This nanovesicle also displays major histocompatibility complex-bound antigens inherited from tumor lysate-pulsed dendritic cell. When administrated, the surface-bound IL2 is able to guide the nanovesicle to lymphoid organs and activate the IL2 receptor on lymphocytes. Furthermore, it is able to perform antigen presentation in the replacement of professional antigen-presenting cells. This nanovesicle, named IL2-ep13nsEV, induced a strong immune reaction to rescue 50% of the mice in our humanized patient-derived xenografts, sensitized cancer cells to immune checkpoint inhibitor treatment, and prevented the recurrence of resected tumors. This paradigm presents a feasible strategy for the treatment and prevention of metastatic breast cancer.

Chronic Intake of a Marginally Low Copper Diet Impairs In Vitro Activities of Lymphocytes and Neutrophils from Male Rats despite Minimal Impact on Conventional Indicators of Copper Status

We investigated the impact of chronic intake of a diet marginally low in Cu on traditional indicators of Cu status and in vitro activities of splenic mononuclear cells and neutrophils. Pregnant Sprague-Dawley rats were fed diets containing either 2.8 (LCu) or 6.7 [adequate (ACu)]mg Cu/kg from midgestation through lactation. Weaned male and female offspring were fed the same diets as their dams until 6 months of age. Dietary Cu level did not alter growth, heart weight, the concentrations of Cu or the activities of cuproenzymes in serum and most tissues. In contrast, splenic mononuclear cells (MNC) from males, but not females, fed the LCu diet were less responsive to in vitro mitogenic activation and generated less interleukin-2 bioactivity than cultures prepared from males fed the ACu diet. Repletion with the ACu diet for 2 wk restored in vitro activities of splenic MNC to control levels. The phorbol myristate acetate-induced generation of superoxide anion by elicited neutrophils from male rodents fed the LCu diet was only 60% that of cells from the ACu diet group. Brain Cu in adult rats fed LCu diet was irreversibly decreased compared with those fed ACu diet. These data show that the in vitro activities of T lymphocytes and neutrophils isolated from adult male rats chronically fed a diet marginally low in Cu were significantly suppressed without marked alterations in the traditional indicators of Cu status.

Landscape and age dynamics of immune cells in the Egyptian rousette bat.

Bats harbor high-impact zoonotic viruses often in the absence of disease manifestation. This restriction and disease tolerance possibly rely on specific immunological features. In-depth molecular characterization of cellular immunity and imprinting of age on leukocyte compartments remained unexplored in bats. We employ single-cell RNA sequencing (scRNA-seq) and establish immunostaining panels to characterize the immune cell landscape in juvenile, subadult, and adult Egyptian rousette bats (ERBs). Transcriptomic and flow cytometry data reveal conserved subsets and substantial enrichments of CD79a+ B cells and CD11b+ Tcells in juvenile animals, whereas neutrophils, CD206+ myeloid cells, and CD3+ Tcells dominate as bats reach adulthood. Despite differing frequencies, phagocytosis of circulating and tissue-resident myeloid cells and proliferation of peripheral and splenic lymphocytes are analogous in juvenile and adult ERBs. We provide a comprehensive map of the immune landscape in ERBs and show age-imprinted resilience progression and find that variability in cellular immunity only partly recapitulates mammalian archetypes.

Curcuma phaeocaulis Inhibits NLRP3 Inflammasome in Macrophages and Ameliorates Nanoparticle-Induced Airway Inflammation in Mice.

The activation of NLRP3 results in the assembly of inflammasome that regulates caspase-1 activation and the subsequent secretion of bioactive interleukin (IL)-1β. Excessive activation of the NLRP3 inflammasome is mechanistically linked to diverse pathophysiological conditions, including airway inflammation. Here, we discovered that Curcuma phaeocaulis can suppress caspase-1 activation and processing of pro-IL-1β into mature cytokine in macrophages stimulated with NLRP3 inflammasome activators, such as SiO2 or TiO2 nanoparticles. Furthermore, in the bronchoalveolar lavage fluids of animals administered the nanoparticles, the in vitro effects of C. phaeocaulis translated into a decrease in IL-1β levels and cell infiltration. Demethoxycurcumin (DMC) and curcumin were found to be responsible for the inflammasome inhibitory activity of C. phaeocaulis. Interestingly, in contrast to the previously reported higher antioxidant- and NFκB-inhibitory activities of curcumin, DMC exhibited approximately two-fold stronger potency than curcumin against nanoparticle induced activation of NLRP3 inflammasome. In the light of these results, both compounds seem to act independently of their antioxidant- and NFκB-inhibitory properties. Although how C. phaeocaulis inhibits nanoparticle-activated NLRP3 inflammasome remains to be elucidated, our results provide a basis for further research on C. phaeocaulis extract as an anti-inflammatory agent for the treatment of disorders associated with excessive activation of NLRP3 inflammasome.

Controlled release of bioactive IL-2 from visible light photocured biodegradable elastomers for cancer immunotherapy applications

Biodegradable elastomeric controlled-release poly (decane-co-tricarballylate) (PDET) based matrices capable of maintaining the stability and bioactivity of Interleukin-2 (IL-2) through the utilization of visible-light curing and solvent-free loading of the cytokine are reported. The elastomeric devices were fabricated by intimately mixing lyophilized IL-2 powder with the acrylated prepolymer before photocrosslinking. The bioactivity of the released protein was assessed by its ability to stimulate the proliferation of the C57BL/6 mouse cytotoxic T lymphocyte, and its concentration was analysed using ELISA. The influence of changes in the polymer's physicochemical and mechanical properties on IL-2 release kinetics and bioactivity were also studied. The increase in the device's surface area and the incorporation of trehalose in the loaded lyophilized mix increased the IL-2 release rate with drug release proceeding via typical zero-order release kinetics. Moreover, the decrease in the degree of acrylation of the prepared devices increased the IL-2 release rate. The bioactivity assay showed that IL-2 retained over 94% of its initial bioactivity throughout 28 days of the release period. A new protein delivery vehicle composed of biodegradable PDET elastomers was demonstrated to be promising and effective for linear, constant, and sustained osmotic-driven release of bioactive IL-2 and other sensitive proteins and hormones.

Unravelling the broader complexity of IL-6 involvement in health and disease.

The classification of interleukin-6 (IL-6) as a pro-inflammatory cytokine undervalues the biological impact of this cytokine in health and disease. With broad activities affecting the immune system, tissue homeostasis and metabolic processes, IL-6 displays complex biology. The significance of these involvements has become increasingly important in clinical settings where IL-6 is identified as a prominent target for therapy. Here, clinical experience with IL-6 antagonists emphasises the need to understand the context-dependent properties of IL-6 within an inflammatory environment and the anticipated or unexpected consequences of IL-6 blockade. In this review, we will describe the immunobiology of IL-6 and explore the gamut of IL-6 bioactivity affecting the clinical response to biological drugs targeting this cytokine pathway.

NLRP3 inflammasome as a key driver of vascular disease.

Nucleotide-binding oligomerization domain-like receptor family pyrin domain containing 3 (NLRP3) is an intracellular innate immune receptor that recognizes a diverse range of stimuli derived from pathogens, damaged or dead cells, and irritants. NLRP3 activation causes the assembly of a large multiprotein complex termed the NLRP3 inflammasome, and leads to the secretion of bioactive interleukin (IL)-1β and IL-18 as well as the induction of inflammatory cell death termed pyroptosis. Accumulating evidence indicates that NLRP3 inflammasome plays a key role in the pathogenesis of sterile inflammatory diseases, including atherosclerosis and other vascular diseases. Indeed, the results of the Canakinumab Anti-inflammatory Thrombosis Outcome Study trial demonstrated that IL-1β-mediated inflammation plays an important role in atherothrombotic events and suggested that NLRP3 inflammasome is a key driver of atherosclerosis. In this review, we will summarize the current state of knowledge regarding the role of NLRP3 inflammasome in vascular diseases, in particular in atherosclerosis, vascular injury, aortic aneurysm, and Kawasaki disease vasculitis, and discuss NLRP3 inflammasome as a therapeutic target for these disorders.

Molecular Biomarkers of Neovascular Age-Related Macular Degeneration With Incomplete Response to Anti-Vascular Endothelial Growth Factor Treatment.

The standard treatment for neovascular age-related macular degeneration (nAMD) consists of intravitreal anti-vascular endothelial growth factors (VEGF). However, for some patients, even maximal anti-VEGF treatment does not entirely suppress exudative activity. The goal of this study was to identify molecular biomarkers in nAMD with incomplete response to anti-VEGF treatment. Aqueous humor (AH) samples were collected from three groups of patients: 17 patients with nAMD responding incompletely to anti-VEGF (18 eyes), 17 patients affected by nAMD with normal treatment response (21 eyes), and 16 control patients without any retinopathy (16 eyes). Proteomic and multiplex analyses were performed on these samples. Proteomic analyses showed that nAMD patients with incomplete anti-VEGF response displayed an increased inflammatory response, complement activation, cytolysis, protein-lipid complex, and vasculature development pathways. Multiplex analyses revealed a significant increase of soluble vascular cell adhesion molecule-1 (sVCAM-1) [ p = 0.001], interleukin-6 (IL-6) [ p = 0.009], bioactive interleukin-12 (IL-12p40) [ p = 0.03], plasminogen activator inhibitor type 1 (PAI-1) [ p = 0.004], and hepatocyte growth factor (HGF) [ p = 0.004] levels in incomplete responders in comparison to normal responders. Interestingly, the same biomarkers showed a high intercorrelation with r2 values between 0.58 and 0.94. In addition, we confirmed by AlphaLISA the increase of sVCAM-1 [ p < 0.0001] and IL-6 [ p = 0.043] in the incomplete responder group. Incomplete responders in nAMD are associated with activated angiogenic and inflammatory pathways. The residual exudative activity of nAMD despite maximal anti-VEGF treatment may be related to both angiogenic and inflammatory responses requiring specific adjuvant therapy. Data are available via ProteomeXchange with identifier PXD02247

High glucose mediates NLRP3 inflammasome activation via upregulation of ELF3 expression

Microtubule affinity regulating kinase 4 (MARK4) plays a crucial role in the regulation of NOD-like receptor pyrin domain 3 (NLRP3) inflammasome activation, which leads to the generation of bioactive interleukin (IL)-1β and IL-18. E74-like ETS transcription factor 3 (ELF3) participates in endothelial inflammatory processes. We hypothesized that ELF3 modulates MARK4 expression in vascular endothelial cells, thus contributing to high glucose-mediated NLRP3 inflammasome activation. Plasma IL-1β, IL-18, NLRP3 inflammasome and MARK4 expression was increased in diabetic patients and rats. An in vitro study indicated that high glucose increased IL-1β and IL-18 expression and activated the NLRP3 inflammasome via upregulation of MARK4 in human umbilical vein endothelial cells (HUVECs). Furthermore, high glucose increased ELF3 expression. ELF3 downregulation reversed the effects of high glucose treatment. Accordingly, the effects of ELF3 overexpression were similar to those of high glucose treatment and were counteracted by siMARK4. Furthermore, ELF3 was found to interact with SET8. High glucose inhibited SET8 expression and histone H4 lysine 20 methylation (H4K20me1), a downstream target of SET8. Overexpression of SET8 inhibited high glucose-induced MARK4 expression and NLRP3 inflammasome activation. The effects of shSET8 were similar to those of high glucose treatment and were counteracted by siMARK4. A mechanistic study found that ELF3 and H4K20me1 were enriched in the MARK4 promoter region. si-ELF3 attenuated MARK4 promoter activity and augmented the inhibitory effect of SET8 on MARK4 promoter activity. Furthermore, SET8 downregulation and ELF3 upregulation were confirmed in diabetic patients and rats. In conclusion, ELF3 interacted with SET8 to modulate MARK4 expression, which participated in hyperglycaemia-mediated endothelial NLRP3 inflammasome activation.

Neurons and astrocytes of the chicken hypothalamus directly respond to lipopolysaccharide and chicken interleukin-6

In 4–5-month-old chicken, intravenous injections of bacterial lipopolysaccharide (LPS) induced a dose-dependent fever response and a pronounced increase of circulating interleukin-6 (IL-6). To assess a possible role for IL-6 in the brain of birds, a hypothalamic neuro-glial primary culture from 1-day-old chicken was established. Each well of cultured hypothalamic cells contained some 615 neurons, 1350 astrocytes, and 580 microglial cells on average. Incubation of chicken hypothalamic primary cultures with 10 or 100 µg/ml LPS induced a dose-dependent release of bioactive IL-6 into the supernatant. Populations of hypothalamic neurons (4%) and astrocytes (12%) directly responded to superfusion with buffer containing 10 µg/ml LPS with a transient increase of intracellular calcium, a sign of direct cellular activation. Stimulation of hypothalamic cultures with buffer containing 50 ng/ml chicken IL-6 induced calcium signaling in 11% of neurons and 22% of astrocytes investigated. These results demonstrate that IL-6 is produced in the periphery and in the hypothalamus in response to LPS in chicken. The observed cellular responses of hypothalamic cells to chicken IL-6 indicate that this cytokine may readily be involved in the manifestation of fever in the avian hypothalamus.

Amino acid residues involved in the heparin-binding activity of murine IL-12 in the context of an antibody-cytokine fusion protein

An antibody-cytokine fusion protein, composed of the murine single-chain cytokine interleukin-12 (IL-12) genetically fused to a human IgG3 specific for the human tumor-associated antigen HER2/neu maintains antigen binding, cytokine bioactivity, and IL-12 heparin-binding activity. This latter property is responsible for the binding of the cytokine to glycosaminoglycans (GAGs) on the cell surface and the extracellular matrix and has been implicated in modulating IL-12 bioactivity. Previous studies indicate that the p40 subunit of human and murine IL-12 is responsible for the heparin-binding activity of this heterodimeric cytokine. In the present study we used bioinformatic analysis and site-directed mutagenesis to develop a version of the antibody-(IL-12) fusion protein without heparin-binding activity. This was accomplished by replacing the basic arginine (R) and lysine (K) residues in the cluster of amino acids 254–260 (RKKEKMK) of the murine IL-12 p40 subunit by the neutral non-polar amino acid alanine (A), generating an AAAEAMA mutant fusion protein. ELISA and flow cytometry demonstrated that the antibody fusion protein lacks heparin-binding activity but retains antigen binding. A T-cell proliferation assay showed IL-12 bioactivity in this construct. However, the IL-12 bioactivity is decreased compared to its non-mutated counterpart, which is consistent with an ancillary role of the heparin-binding site of IL-12 in modulating its activity. Thus, we have defined a cluster of amino acid residues with a crucial role in the heparin-binding activity of murine IL-12 in the context of an antibody-cytokine fusion protein.

Cytokine Secretion and Pyroptosis of Thyroid Follicular Cells Mediated by Enhanced NLRP3, NLRP1, NLRC4, and AIM2 Inflammasomes Are Associated With Autoimmune Thyroiditis.

Inflammasomes, which mediate maturation of interleukin-1β (IL-β) and interleukin-18 (IL-18) and lead to pyroptosis, have been linked to various autoimmune disorders. This study investigated whether they are involved in the pathogenesis of autoimmune thyroiditis (AIT). We collected thyroid tissues from 50 patients with AIT and 50 sex- and age-matched controls. Serum levels of free T3, free T4, thyrotropin, thyroid peroxidase antibody (TPOAb), and thyroglobulin antibody (TgAb) were measured by electrochemiluminescent immunoassays. Expression of several inflammasome components, the NOD-like receptor (NLR) family pyrin domain containing 1 (NLRP1), NLRP3, CARD-domain containing 4 (NLRC4), absent in melanoma 2 (AIM2), the apoptosis-associated speck-like protein that contains a caspase recruitment domain (ASC), caspase-1, IL-1β, and IL-18 was determined by real-time PCR and western blot. Immunohistochemistry was used to localize the expression of NLRP1, NLRP3, NLRC4, and AIM2. The Nthy-ori 3-1 thyroid cell line was stimulated with tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), interleukin-17A, interleukin-6, and poly(dA:dT). The levels of IL-18 and IL-1β in the cell supernatant were measured by enzyme-linked immunosorbent assay, and lactate dehydrogenase was quantified by absorptiometry. ASC specks were examined by confocal immunofluorescence microscopic analysis. Cell death was examined by flow cytometry, and the N-terminal domain of gasdermin D was detected by western blot analysis. Expression of NLRP1, NLRP3, NLRC4, AIM2, ASC, caspase-1, pro IL-1β, pro IL-18, mRNA, and protein was significantly increased in thyroid tissues from patients with AIT, and enhanced posttranslational maturation of caspase-1, IL-18 and IL-1β was also observed. Expression of NLRP1, NLRP3, NLRC4, and AIM2 was localized mainly in thyroid follicular cells adjacent to areas of lymphatic infiltration. The thyroid mRNA level of NLRP1 and ASC was correlated to the serum TPOAb and TgAb levels in the AIT group. TNF-α and IFN-γ had a priming effect on the expression of multiple inflammasome components in thyroid cells. IFN-γ was found to strengthen poly(dA:dT)-induced cell pyroptosis and bioactive IL-18 release. Our work has demonstrated for the first time that multiple inflammasomes are associated with AIT pathogenesis. The identified NLRP3, NLRP1, NLRC4, AIM2 inflammasomes and their downstream cytokines may represent potential therapeutic targets and biomarkers of AIT.

Research progress on the role of NLRP3 inflammasome in ocular diseases

Nucleotide-binding oligomerization domain-like receptor pyrin domain containing-3 (NLRP3) inflammasome is a type of multi-protein complex within the cell, the activation of which promotes the release of bioactive interleukin(IL)-1β and IL-18 and contributes to inflammatory progress. In recent years, the research on the role of NLRP3 inflammasome in the pathogenesis and development of ocular surface and fundus diseases has made considerable progress. This review summarizes current understanding in the structure, ocular expression, and mechanisms of activation of NLRP3 inflammasome, as well as the contribution of this protein complex to eye diseases, thereby revealing the theoretical possibility that NLRP3 inflammasome might serve as a novel therapeutic target for the related ocular diseases.(Chin J Ophthalmol, 2018, 54: 396-400).