Increased Production Of IL-1β Research Articles

Hyperactive Dendritc Cells Redirect Aged Antitumor Immunity.

Aging is one of the biggest risk factors for cancer development. More than 85% of all cancers occur in individuals above 55 years old, often accompanied by age-associated immune defects. Previous studies on the tumor microenvironment during aging have identified several factors, such as the roles of fibroblasts, immunosuppression, and metastasis. However, the aging-associated defects in antitumor immunity, particularly with regard to T cells, remain underexplored. Recent findings by Zhivaki and colleagues suggest that age-related immune defects affecting antitumor responses involve reduced levels of CD8+ T cells and compromised dendritic cell (DC) functions such as antigen presentation and migration. Their study demonstrates that a hyperactive DC vaccine can restore DC functions in older mice. Furthermore, these hyperactive DCs, characterized by increased IL1β production and better migratory capability to the lymph node, promote the development of cytolytic CD4+ T cells exhibiting Th1-like phenotypes. This research reveals mechanisms underlying the response to hyperactive DC vaccines in older mice and highlights the critical role of cytolytic CD4+ T cells as substitutes for CD8+ T cells in driving antitumor immunity and achieving long-term tumor control in older mice.

Spontaneous and Stimulated Production of Cytokines by Blood Cells Ex Vivo as a Biomarker of Initially High or Low Hypoxia Resistance in Rats.

Spontaneous and stimulated production of cytokines by peripheral blood cells obtained from the caudal vein of male Wistar rats was assessed before testing their resistance to oxygen deficiency in a decompression chamber. To study the spontaneous production of cytokines, heparinized blood cells were incubated in a culture medium (24 h, 5% CO2, 37°C) and the content of proinflammatory cytokines IL-6 and TNFα and anti-inflammatory IL-10 in the culture medium was assessed by ELISA. To stimulate cytokine production, blood cells were incubated for 24 h with LPS, phytohemagglutinin, and concanavalin A at final concentrations of 2, 4, and 4 μg, respectively. Two weeks after blood sampling, individual resistance of the animals to hypoxia in a decompression chamber was determined. In animals with low resistance to hypoxia, the levels of spontaneous production of all three cytokines were significantly higher, while after stimulation, the level of IL-1β increased by more than 2 times. The animals with spontaneous production of IL-10>50 pg/ml, IL-6>10 pg/ml, and TNFα>10 pg/ml, as well as with the increase in IL-1β production by more than 2 times upon stimulation were classified as low-resistant. At IL-10<15 pg/ml, IL-6<9 pg/ml, and TNFα<7 pg/ml, as well as the absence of the increase in IL-1β production upon stimulation, they were classified as high-resistant. The identified features of spontaneous and stimulated production of cytokines can be used as non-invasive biomarkers to determine the resistance to hypoxia without exposure to sublethal hypoxia in a decompression chamber.

P-593 Inflammaging in the ovary: focus on foamy cells generation

Abstract Study question Could the dysregulation of the inflammatory response in the aged ovary be associated with foamy cells generation and be ameliorated using an alternative therapeutic treatment? Summary answer Aged ovarian macrophages show an inflammatory profile associated with incorporation of lipid droplets that can be reduced after culture with platelet-rich plasma from fertile women. What is known already The concept of ‘inflammaging’ represents the inflammation associated to aging, which is systemic, chronic and linked to the increase of pro-inflammatory mediators. This microenvironment is associated with the presence of ovarian foamy macrophages, a unique subset with intracytoplasmic lipids accumulation. Here, we investigate the mechanisms underlying the exacerbated inflammatory response during ovarian aging, focusig on the generation of foamy macrophages and their impact in oocyte quality. Since, oocytes develop in the context of ovarian follicles, we obtained follicular fluid (FF) from women with different ages, to investigate ovarian immune microenvironment and their modulation after in vitro platelet-rich plasma (PRP) treatment. Study design, size, duration Mononuclear cells were recovered from FF from women of different ages and IL-1β production and macrophages and lymphocytes profiles tested by immune-staining and FACS analysis. To test foamy cells generation, we measured surface and intracellular CD68 expression (scavenger receptor involved in lipid accumulation). Immune cells profiles were correlated with clinic parameters as number of oocytes, M2 and atretic follicles. Finally, isolated ovarian macrophages were treated with PRP and modulation of macrophage profile was tested. Participants/materials, setting, methods We studied FF (n = 70) from women from ovodonation (21-29y), with cryopreservation desire (30-35y) and ART indication (36-42y). The San Isidro Ethics Committee, Buenos Aires, Argentina, approved this study. Mononuclear cells were isolated using Ficoll-Hypaque and macrophages profile (IL-1β+, CD68+) and the novel lymphocyte regulatory subpopulation (CD3+CD4-CD8-) tested by flow cytometry. Lipid droplets were measured using BODIPY 493/503 probe. Ovarian macrophages were isolated by adherence and treated with PRP (obtained from fertile women) for 5 days. Main results and the role of chance FF macrophages from aged women (35-42y) showed a significant increase in IL-1β production in comparison with the youngest group (21-29y), that also correlates negatively with the number of M2 oocytes recovered. Focusing on macrophage characterization, FF from aged women displayed an increased frequency of CD68+ macrophages in comparison with young women. Since CD68 shuttles between the cytoplasm and the plasmatic membrane to incorporate lipids, we evaluated lipid accumulation in FF macrophages to generate foamy cells. Interestingly, we found an increase of lipid droplets in FF macrophages from aged women in comparison with the younger group. Therefore, we discriminated surface and intracellular CD68 detection and found higher intracellular CD68 expression in aged macrophages, correlating with lipid droplets accumulation. Then, we isolated ovarian macrophages from aged women and treated with fertile PRP to evaluate its modulatory effects and observed a reduction in the frequency of CD68+IL-1β cells in comparison with those cultures without treatment. Finally, we focused on a novel T lymphocyte regulatory subpopulation, CD3+CD4-CD8- (DNT), that is emerging as effector cells capable of mediating immune tolerance in the female reproductive system. We found that FF from aged women displayed a reduced frequency of DNT, in comparison with FF from younger women. Limitations, reasons for caution The present results were obtained using FF samples from women from different ages. Even though the samples represent the ovarian microenvironment, it is difficult to study the human ovary per se and further studies are necessary to elucidate whether these mechanisms operate similarly in vivo. Wider implications of the findings We demonstrated that dysregulation of the inflammatory response during aging leads to the generation of foamy cells and reduction of DNT cells associated to the regulation of the immune ovarian microenvironment impacting in oocyte quality. In vitro PRP treatment displayed immunomodulatory effects, suggesting its relevance as a potential therapeutic treatment. Trial registration number not applicable

Probing Isosteric Replacement for Immunoadjuvant Design: Bis-Aryl Triazole Trehalolipids are Mincle Agonists.

Herein, we report the modular synthesis and immunological activity of seven bis-aryl triazole trehalolipids (1a-1g) as Brartemicin analogs. The compounds comprised one or two octyloxy (C8) alkyl chains and were synthesized using the venerable CuAAc reaction between the respective aryl acetylenes and a trehalose diazide. A Mincle reporter cell assay revealed that all lipidated analogs activated Mincle. Two compounds, 1c and 1d, produced strong Mincle-dependent immune responses in vitro. The activity was dependent on the degree of alkylation and regiochemistry, with 1c and 1d showing significantly increased IL-1β production in vitro compared to monoalkylated compounds and dialkylated compounds lacking ortho substitution. Molecular docking of 1c positioned the triazole in proximity to Arg-183, which may offer additional interactions that could explain the binding affinity for this class of ligand. These findings demonstrate the capability of triazole-linked Brartemicin analogs as Mincle-mediated Th1/Th17 vaccine adjuvants.

Impaired autophagy in myeloid cells aggravates psoriasis-like skin inflammation through the IL-1β/CXCL2/neutrophil axis

BackgroundPsoriasis is an inflammatory skin disease characterized by the hyperproliferative epidermal keratinocytes and significant immune cells infiltration, leading to cytokines production such as IL-1β, TNF-α, IL-23, and IL-17. Recent study highlights the critical role of IL-1β in the induction and activation of pathogenic Th17 and IL-17-producing γδ T cells, contributing to psoriasis. However, the mechanism underlying IL-1β dysregulation in psoriasis pathogenesis is unclear. Autophagy regulates IL-1β production and has a pleiotropic effect on inflammatory disorders. Previous studies showed controversial role of autophagy in psoriasis pathogenesis, either pro-inflammatory in autophagy-deficient keratinocyte or anti-inflammatory in pharmacologically autophagy-promoting macrophages. Thus, the direct role of autophagy and its therapeutic potential in psoriasis remains unclear.MethodsWe used myeloid cell-specific autophagy-related gene 7 (Atg7)-deficient mice and determined the effect of autophagy deficiency in myeloid cells on neutrophilia and disease pathogenesis in an imiquimod-induced psoriasis mouse model. We then assessed the pathogenic mechanism focusing on immune cells producing IL-1β and IL-17 along with gene expression profiles associated with psoriasis in mouse model and public database on patients. Moreover, therapeutic potential of IL-1β blocking in such context was assessed.ResultsWe found that autophagy deficiency in myeloid cells exacerbated neutrophilic inflammation and disease pathogenesis in mice with psoriasis. This autophagy-dependent effect was associated with a significant increase in IL-1β production from myeloid cells, particularly macrophages, Cxcl2 expression, and IL-17 A producing T cells including γδ T cells. Supporting this, treatment with systemic IL-1 receptor blocking antibody or topical saccharin, a disaccharide suppressing pro-IL-1β expression, led to the alleviation of neutrophilia and psoriatic skin inflammation linked to autophagy deficiency. The pathophysiological relevance of this finding was supported by dysregulation of autophagy-related genes and their correlation with Th17 cytokines in psoriatic skin lesion from patients with psoriasis.ConclusionsOur results suggest that autophagy dysfunction in myeloid cells, especially macrophages, along with IL-1β dysregulation has a causal role in neutrophilic inflammation and psoriasis pathogenesis.

Abstract 3980: Investigating the role of host obesity in promoting immunotherapy resistance

Abstract Obesity is linked to an increased risk of various cancers, including renal cell carcinoma (RCC). Many RCC patients have obesity at diagnosis and treatment initiation, and our lab has found that this significantly impairs immunotherapy outcomes. Both preclinical and clinical reports, from our lab and others, have demonstrated that obesity reduces the efficacy of CD8+ T cell-dependent immunotherapy by increasing pro-tumorigenic IL-1β. Combining anti-PD-1 based therapy with anti-IL-1β triggers the recruitment of CD8+ T cells and improves the efficacy of anti-PD-1 based immunotherapy. However, the mechanistic links between obesity and increased IL-1β production remains unknown. Our objective is to identify mechanisms promoting IL-1 β-driven immunosuppression within renal tumors of obese mice. In our in vivo studies, we utilized a BALB/c mouse model of diet-induced obesity (DIO) and lean controls in combination with the Renca-Luc RCC cell line. Using flow cytometry and western blot, we respectively assessed leukocyte populations producing IL-1β and tumor energetics. Flow cytometry revealed that intra-tumoral IL-1β is produced primarily by tumor associated macrophages (TAMS). Immunoblotting demonstrated that increased IL-1β production is linked to altered mitochondrial function and ATP production. These early findings support the hypothesis that obesity driven IL-1β expression is mediated by tumor-associated macrophages and that mitochondrial dysfunction drives immune suppression. Understanding these molecular and immune-related mechanisms may pave the way for novel strategies to reduce immunosuppression in obese RCC patients, thus improving immunotherapeutic efficacy and overall clinical outcomes. Citation Format: Henry Nnaemeka Ogbonna, Zachary Roberts, Haley Kvarnberg, Francesca Dempsey, Patrick N. Song, Anna G. Sorace, Lyse Norian. Investigating the role of host obesity in promoting immunotherapy resistance [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 3980.

Apolipoprotein E (ApoE) orchestrates adipose tissue inflammation and metabolic disorders through NLRP3 inflammasome

Obesity is a metabolic disorder characterized by the hypertrophy expansion of adipose tissue, resulting in dysregulated energy metabolism, and accompanied by chronic low-grade inflammation. Adipose tissue macrophages (ATMs), a principal component of inflammation, respond to microenvironment signals and modulate adipose tissue remodeling and metabolic processes situation-specific. However, the mechanisms governing how the organism maintains equilibrium between its chronic inflammation and metabolism still need to be understood. Here, we describe a novel role of apolipoprotein E (ApoE), which associated with lipid particles, in maintaining fat deposition and system metabolic inflammation. Using human samples and mouse models, we show that ApoE is robustly downregulated in obese individuals, db/db mice, and mice of high-fat diet (HFD) feeding and increased in obese subjects with diabetes. Furthermore, we found that ApoE deficiency mice globally prevented obesity by restraining adipose tissue expansion and improved systemic glucose tolerance and insulin sensitivity. However, macrophage contributed to metabolic inflammation due to increased IL-1β production in adipose tissue from ApoE-/- mice induced by HFD. Our results suggest that the role of ApoE in regulating obesity and obesity-associated glucose dysregulation is inconsistent. Mechanistically, ApoE modulates of the NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3) inflammasome priming and activation step. Thus, our studies might provide new sights into ApoE, which is required for obesity-induced hyperglycemia, hyperinsulinism, and adaptive inflammation responses but diminishes the tolerance towards a subsequent metabolic inflammatory challenge. Our study shed new light on the integral role of apolipoprotein APOE in immunometabolism and adipose tissue homeostasis.

Inhibition of the uric acid efflux transporter ABCG2 enhances stimulating effect of soluble uric acid on IL-1β production in murine macrophage-like J774.1 cells.

Soluble uric acid (UA) absorbed by cells through UA transporters (UATs) accumulates intracellularly, activates the NLRP3 inflammasome and thereby increases IL-1β secretion. ABCG2 transporter excludes intracellular UA. However, it remains unknown whether ABCG2 inhibition leads to intracellular accumulation of UA and increases IL-1β production. In this study, we examined whether genetic and pharmacological inhibition of ABCG2 could increase IL-1β production in mouse macrophage-like J774.1 cells especially under hyperuricemic conditions. We determined mRNA and protein levels of pro-IL-1β, mature IL-1β, caspase-1 and several UATs in culture supernatants and lysates of J774.1 cells with or without soluble UA pretreatment. Knockdown experiments using an shRNA against ABCG2 and pharmacological experiments with an ABCG2 inhibitor were conducted. Extracellularly applied soluble UA increased protein levels of pro-IL-1β, mature IL-1β and caspase-1 in the culture supernatant from lipopolysaccharide (LPS)-primed and monosodium urate crystal (MSU)-stimulated J774.1 cells. J774.1 cells expressed UATs of ABCG2, GLUT9 and MRP4, and shRNA knockdown of ABCG2 increased protein levels of pro-IL-1β and mature IL-1β in the culture supernatant. Soluble UA increased mRNA and protein levels of ABCG2 in J774.1 cells without either LPS or MSU treatment. An ABCG2 inhibitor, febuxostat, but not a urate reabsorption inhibitor, dotinurad, enhanced IL-1β production in cells pretreated with soluble UA. In conclusion, genetic and pharmacological inhibition of ABCG2 enhanced IL-1β production especially under hyperuricemic conditions by increasing intracellularly accumulated soluble UA that activates the NLRP3 inflammasome and pro-IL-1β transcription in macrophage-like J774.1 cells.

Genetic inhibition of CARD9 accelerates the development of atherosclerosis in mice through CD36 dependent-defective autophagy

Caspase recruitment-domain containing protein 9 (CARD9) is a key signaling pathway in macrophages but its role in atherosclerosis is still poorly understood. Global deletion of Card9 in Apoe-/- mice as well as hematopoietic deletion in Ldlr-/- mice increases atherosclerosis. The acceleration of atherosclerosis is also observed in Apoe-/-Rag2-/-Card9-/- mice, ruling out a role for the adaptive immune system in the vascular phenotype of Card9 deficient mice. Card9 deficiency alters macrophage phenotype through CD36 overexpression with increased IL-1β production, increased lipid uptake, higher cell death susceptibility and defective autophagy. Rapamycin or metformin, two autophagy inducers, abolish intracellular lipid overload, restore macrophage survival and autophagy flux in vitro and finally abolish the pro-atherogenic effects of Card9 deficiency in vivo. Transcriptomic analysis of human CARD9-deficient monocytes confirms the pathogenic signature identified in murine models. In summary, CARD9 is a key protective pathway in atherosclerosis, modulating macrophage CD36-dependent inflammatory responses, lipid uptake and autophagy.

Particulate air pollution exaggerates diet-induced insulin resistance through NLRP3 inflammasome in mice

Air particulate matter 2.5 (PM2.5) has been demonstrated to exaggerate insulin resistance in both human and animal studies. However, the exact molecular mechanisms remain elusive. This study sought to assess the role of NLRP3 inflammasome in PM2.5 exposure-induced insulin resistance and explore the underlying mechanisms. Wild-type (WT), Nlrp3−/−, Tlr4Lps-d, or Nrf2−/− mice, on a normal diet or high-fat diet (HFD), were exposed to PM2.5 or filtered air (FA) in a whole-body exposure facility. Priming (first signal) and assembly (second signal) of NLRP3 inflammasome activation were assessed by measuring the transcription of Nlrp3/Il-1β and detecting the activity of caspase-1 and secretion of IL-1β. We found PM2.5 exposure exaggerated insulin resistance and increased IL-1β production in the HFD-fed WT mice, but not Nlrp3−/− mice. Gene expressions of Nlrp3 and Il-1β in the lungs and peritoneal macrophages were upregulated in WT mice exposed to PM2.5. When stimulated with LPS (first signal) or monosodium urate (second signal), PM2.5 exposure was able to enhance the activity of caspase-1 and IL-1β secretion, suggesting that PM2.5 may serve as a stimulus of either the first or second signal for NLRP3 inflammasome activation. Effects of PM2.5 on caspase-1 activation and IL-1β secretion were partially blocked in Tlr4Lps-d mice. Reactive oxygen species (ROS), co-localization of NLRP3 and mitochondria, and secondary lysosomes in macrophages were increased after PM2.5 exposure, while deficiency of antioxidant gene Nrf2 in mice significantly enhanced PM2.5-induced secretion of IL-1β. Imaging flow cytometry and transmission electron microscopy demonstrated an engulfment of PM2.5 particles by macrophages, while suppression of phagocytosis by cytochalasin D abolished PM2.5-induced transcription of Nlrp3/Il-1β. Our results demonstrated a critical role of NLRP3 inflammasome in PM2.5 exaggerated insulin resistance, and multiple pathways in the first and second signals of NLRP3 inflammasome activation may be involved.

Food additive succinate exacerbates experimental autoimmune encephalomyelitis accompanied by increased IL-1β production

Food additive succinate exacerbates experimental autoimmune encephalomyelitis accompanied by increased IL-1β production

BPOZ-2 is a negative regulator of the NLPR3 inflammasome contributing to SARS-CoV-2-induced hyperinflammation.

Inflammation play important roles in the initiation and progression of acute lung injury (ALI), acute respiratory distress syndrome (ARDS), septic shock, clotting dysfunction, or even death associated with SARS-CoV-2 infection. However, the pathogenic mechanisms underlying SARS-CoV-2-induced hyperinflammation are still largely unknown. The animal model of septic shock and ALI was established after LPS intraperitoneal injection or intratracheal instillation. Bone marrow-derived macrophages (BMDMs) from WT and BPOZ-2 KO mouse strains were harvested from the femurs and tibias of mice. Immunohistology staining, ELISA assay, coimmunoprecipitation, and immunoblot analysis were used to detect the histopathological changes of lung tissues and the expression of inflammatory factors and protein interaction. We show a distinct mechanism by which the SARS-CoV-2 N (SARS-2-N) protein targets Bood POZ-containing gene type 2 (BPOZ-2), a scaffold protein for the E3 ubiquitin ligase Cullin 3 that we identified as a negative regulator of inflammatory responses, to promote NLRP3 inflammasome activation. We first demonstrated that BPOZ-2 knockout (BPOZ-2 KO) mice were more susceptible to lipopolysaccharide (LPS)-induced septic shock and ALI and showed increased serum IL-1β levels. In addition, BMDMs isolated from BPOZ-2 KO mice showed increased IL-1β production in response to NLRP3 stimuli. Mechanistically, BPOZ-2 interacted with NLRP3 and mediated its degradation by recruiting Cullin 3. In particular, the expression of BPOZ-2 was significantly reduced in lung tissues from mice infected with SARS-CoV-2 and in cells overexpressing SARS-2-N. Importantly, proinflammatory responses triggered by the SARS-2-N were significantly blocked by BPOZ-2 reintroduction. Thus, we concluded that BPOZ-2 is a negative regulator of the NLPR3 inflammasome that likely contributes to SARS-CoV-2-induced hyperinflammation.

Increased Inflammasome Activation Is Associated with Aging and Chronic Myelomonocytic Leukemia Disease Severity.

Aging causes chronic low-grade inflammation known as inflamm-aging. It is a risk factor for several chronic disorders, including chronic myelomonocytic leukemia (CMML), a hematological malignancy that is most prevalent in older people. Recent studies suggest a critical role for the NLRP3 (NOD-, LRR- and pyrin domain-containing protein 3) inflammasome in inflamm-aging. However, the mechanisms involved in NLRP3 activation in aging and its involvement in CMML progression are not fully understood. In this study, we report that aging increases IL-1β production upon NLRP3 activation in human CD14+ monocytes. Interestingly, we found that the TLR1/2 agonist Pam3CSK4 directly activates the NLRP3 inflammasome in monocytes from older but not from younger healthy donors. Furthermore, we observed a dichotomous response to NLRP3 inflammasome activation in monocytes from a small cohort of CMML patients, and some patients produced high levels of IL-1β and some patients produced low levels of IL-1β compared with older healthy donors. Intriguingly, CMML patients with heightened NLRP3 activation showed increased treatment dependency and disease severity. Collectively, our results suggest that aging causes increased sensitivity to NLRP3 inflammasome activation at a cellular level, which may explain increased inflammation and immune dysregulation in older individuals. Furthermore, NLRP3 inflammasome activation was dysregulated in a small cohort of CMML patients and was positively correlated with disease severity.

Involvement of the p38 MAPK-NLRC4-Caspase-1 Pathway in Ionizing Radiation-Enhanced Macrophage IL-1β Production.

Macrophages are abundant immune cells in the tumor microenvironment and are crucial in regulating tumor malignancy. We previously reported that ionizing radiation (IR) increases the production of interleukin (IL)-1β in lipopolysaccharide (LPS)-treated macrophages, contributing to the malignancy of colorectal cancer cells; however, the mechanism remained unclear. Here, we show that IR increases the activity of cysteine-aspartate-specific protease 1 (caspase-1), which is regulated by the inflammasome, and cleaves premature IL-1β to mature IL-1β in RAW264.7 macrophages. Irradiated RAW264.7 cells showed increased expression of NLRC4 inflammasome, which controls the activity of caspase-1 and IL-1β production. Silencing of NLRC4 using RNA interference inhibited the IR-induced increase in IL-1β production. Activation of the inflammasome can be regulated by mitogen-activated protein kinase (MAPK)s in macrophages. In RAW264.7 cells, IR increased the phosphorylation of p38 MAPK but not extracellular signal-regulated kinase and c-Jun N-terminal kinase. Moreover, a selective inhibitor of p38 MAPK inhibited LPS-induced IL-1β production and NLRC4 inflammasome expression in irradiated RAW264.7 macrophages. Our results indicate that IR-induced activation of the p38 MAPK-NLRC4-caspase-1 activation pathway in macrophages increases IL-1β production in response to LPS.

Development of a cell line-based in vitro assay for assessment of Diphtheria, Tetanus and acellular Pertussis (DTaP)-induced inflammasome activation

Safety and potency assessment for batch release testing of established vaccines still relies partly on animal tests. An important avenue to move to batch release without animal testing is the consistency approach. This approach is based on thorough characterization of the vaccine to identify critical quality attributes that inform the use of a comprehensive set of non-animal tests to release the vaccine, together with the principle that the quality of subsequent batches follows from their consistent production. Many vaccine antigens are by themselves not able to induce a protective immune response. The antigens are therefore administered together with adjuvant, most often by adsorption to aluminium salts. Adjuvant function is an important component of vaccine potency, and an important quality attribute of the final product. Aluminium adjuvants are capable of inducing NLRP3 inflammasome activation. The aim of this study was to develop and evaluate an in vitro assay for NLRP3 inflammasome activation by aluminium-adjuvanted vaccines. We evaluated the effects of Diphtheria-Tetanus-acellular Pertussis combination vaccines from two manufacturers and their respective adjuvants, aluminium phosphate (AP) and aluminium hydroxide (AH), in an in vitro assay for NLRP3 inflammasome activation. All vaccines and adjuvants tested showed a dose-dependent increase in IL-1β production and a concomitant decrease in cell viability, suggesting NLRP3 inflammasome activation. The results were analysed by benchmark dose modelling, showing a similar 50% effective dose (ED50) for the two vaccine batches and corresponding adjuvant of manufacturer A (AP), and a similar ED50 for the two vaccine batches and corresponding adjuvant of manufacturer B (AH). This suggests that NLRP3 inflammasome activation is determined by the adjuvant only. Repeated freeze-thaw cycles reduced the adjuvant biological activity of AH, but not AP. Inflammasome activation may be used to measure adjuvant biological activity as an important quality attribute for control or characterization of the adjuvant.

Histone deacetylase-2 controls IL-1β production through the regulation of NLRP3 expression and activation in tuberculosis infection

SummaryHistone deacetylases (HDACs) are critical immune regulators. However, their roles in interleukin-1β (IL-1β) production remain unclear. By screening 11 zinc-dependent HDACs with chemical inhibitors, we found that HDAC1 inhibitor, 4-(dimethylamino)-N-[6-(hydroxyamino)-6-oxohexyl]-benzamide (DHOB), enhanced IL-1β production by macrophage and dendritic cells upon TLR4 stimulation or Mycobacterium tuberculosis infection through IL-1β maturation via elevated NLRP3 expression, increased cleaved caspase-1, and enhanced ASC oligomerization. DHOB rescued defective IL-1β production by dendritic cells infected with M. tuberculosis with ESAT-6 deletion, a virulence factor shown to activate NLRP3 inflammasome. DHOB increased IL-1β production and NLRP3 expression in a tuberculosis mouse model. Although DHOB inhibited HDAC activities of both HDAC1 and HDAC2 by direct binding, knockdown of HDAC2, but not HDAC1, increased IL-1β production and NLRP3 expression in M. tuberculosis-infected macrophages. These data suggest that HDAC2, but not HDAC1, controls IL-1β production through NLRP3 inflammasome activation, a mechanism with a significance in chronic inflammatory diseases including tuberculosis.

Antimicrobial Triclosan (TCS) Increases Production of Interleukin 1 beta (IL‐1β) in Human Immune Cells

Triclosan (TCS) is an antimicrobial compound that is widely used in personal hygiene products such as mouthwash and toothpaste. TCS can be ingested or absorbed through the skin and has been found in human blood, breast milk, and urine. Interleukin‐1 beta (IL‐1β) is an important pro‐inflammatory cytokine produced by immune cells such as monocytes and lymphocytes, and plays a critical role in immune response regulation, tissue repair, and cellular growth. Overproduction of IL‐1β can contribute to chronic inflammation and inflammatory diseases such as rheumatoid arthritis and multiple sclerosis. IL‐1β has also been shown to stimulate tumor development. In a previous study, we showed that TCS at concentrations between 0.05‐5 µM increased the secretion of IL‐1β from immune cells within 24 h. Here we will examine whether the increase in IL‐1 β secretion stimulated by TCS is due to an increase in the cellular production of IL‐1β or only due to release of existing stores of the cytokine. Human peripheral blood mononuclear cells (PBMCs) were exposed to TCS at concentrations of 0‐5 µM. The cellular production (combination of secreted and intracellular levels) of IL‐1β was measured at 10 minutes, 30 minutes, 6 hours, and 24 hours. Secreted levels were measured in supernatants from exposed cells using enzyme‐linked immunosorbent assay (ELISA) and intracellular levels were measured by lysing the exposed cell pellet followed by Western Blot. Results indicate the production of IL‐1β was increased by exposure to one or more concentration of TCS at each length of exposure. The greatest increase in IL‐1β production was seen at 6 h, where all TCS exposures caused quite substantial increases in IL‐1β production in all donors. The magnitude of these increases varied dependent on the donor. These results indicate that TCS has the capacity to increase cellular production of the important pro‐inflammatory cytokine IL‐1β from immune cells. TCS‐induced increases in IL‐1 β production may serve to produce unwarranted inflammation and the pathologies associated with it.

Oleamide-Mediated Polarization of M1 Macrophages and IL-1β Production by Regulating NLRP3-Inflammasome Activation in Primary Human Monocyte-Derived Macrophages.

Macrophages are a type of innate immune cell that activates the NLRP3 inflammasome, causing the release of the cytokine IL-1β, which is a crucial mediator of the inflammatory response. NLRP3 activation that is dysregulated worsens a variety of inflammatory and autoimmune diseases, as well as neurodegenerative diseases. Oleamide is an endogenous fatty acid amide that was first determined as a sleep-inducing molecule and later shown to have wide-ranging beneficial effects on the central nervous system. How oleamide influences human macrophage polarization and NLRP3-inflammasome activation remains unclear. The effect of oleamide on macrophage polarization was explored using an in vitro culture of primary human monocyte-derived macrophages (MDMs) supplemented with human serum-containing media. Cellular and molecular mechanisms of oleamide-regulated MDMs polarization were also investigated. Results showed that oleamide promoted naïve macrophages (M0) toward the M1 phenotype by upregulating M1-associated genes (IL-1β, iNOS, CXCL10), along with downregulation of M2-associated genes (Arg-1, CD206, CCL22). Cell surface expression indicated that oleamide enhanced CD80 expression in M0 naïve macrophages and hider CD206 and CD163 expression in M2 macrophages. Higher production of IL-1β cytokine was observed but with no alteration in IL-6 and TNF-α levels by MDMs and differentiated THP-1 models. Whether oleamide functioned as a second signal that activated the NLRP3 inflammasome and mediated IL-1β production was further investigated using LPS-primed MDMs followed by oleamide treatment that induced activation of inflammasome-related proteins including NLRP3, ASC, cleaved casp-1, and cleaved IL-1β. These findings suggested that oleamide promoted M1 macrophage polarization and increased IL-1β production by activating the NLRP3 inflammasome in primary MDMs. This research reveals a new function for oleamide as well as prospective targets for treating NLRP3-related inflammatory disorders.

Pseudomonas aeruginosa Elicits Sustained IL-1β Upregulation in Alveolar Macrophages from Lung Transplant Recipients

<h3>Purpose</h3> Clinical infection or colonization of the lung, specifically with <i>Pseudomonas aeruginosa</i> (<i>PsA</i>), is associated with increased incidence of lung allograft injury in lung transplant recipients (LTRs) and with an increase in BAL (bronchoalveolar lavage) pro-inflammatory cytokines. However, the effect of bacterial colonization on sustained innate immune activation and the mechanisms involved are not well understood. We sought to determine the mechanisms underlying persistent pro-inflammatory alveolar macrophage response to <i>PsA</i> stimulation. <h3>Methods</h3> We stimulated THP-1 derived macrophages and BAL-derived alveolar macrophages from LTRs with bacteria representative of infectious pathogens and upper respiratory commensals for up to 96 hours. Using a high dimensional flow cytometry panel, we evaluated macrophage responses including expression of costimulatory molecules (CD80, CD86, CD40); co-inhibitor molecules (B7-H1), pro-inflammatory cytokines (TNF-α, IL-6, IL-8, IL-1β), and anti-inflammatory mediators (IL-10, IL-1RA, and TGF-β). <h3>Results</h3> We observed an upregulation of pro-inflammatory cytokines (TNF-α, IL-6, IL-8, IL-1β) following stimulation by <i>PsA</i> compared to stimulation with other pathogens (<i>Staphylococcus aureus</i>) and oral-commensal bacteria (<i>Prevotella melaninogenica, Streptococcus pneumoniae</i>) in THP-1 macrophages. IL-1 β elevations were sustained for 96 hours after <i>PsA</i> exposure. Alveolar macrophages from LTRs undergoing surveillance bronchoscopy demonstrated elevated levels of IL-6 and showed a similar IL-1β responses to that observed in THP-1 macrophages when exposed to <i>PsA</i>. <h3>Conclusion</h3> <i>PsA</i> induces a sustained increase in IL-1β production in alveolar macrophages from LTRs. Given the prior described role of IL-1β in lung allograft injury and CLAD, further investigations are ongoing to determine clinical correlates of allograft injury with <i>PsA</i> and IL-1β.

Inhibition of the NLRP3 inflammasome by progesterone is attenuated by abnormal autophagy induction in endometriotic cyst stromal cells: implications for endometriosis.

The NOD-like receptor pyrin domain containing 3 (NLRP3) inflammasome is a cytosolic multi-protein complex that induces inflammation and is known to be regulated negatively by autophagy. Previous studies reported an abnormal induction of autophagy linked to progesterone resistance in human endometriotic cells. Therefore, an aberrant autophagy induction response to progesterone might contribute to the altered inflammatory response observed in endometriotic tissues. To evaluate this hypothesis, we elucidate whether regulation of the NLRP3 inflammasome by ovarian steroids is mediated by autophagy in human endometrial stromal cells (normal endometrial stromal cells (NESCs)) from patients with uterine leiomyoma (presumed normal) and whether abnormal autophagy induction in endometriotic cyst stromal cells (ECSCs) affects NLRP3 inflammasome-induced interleukin-1β (IL-1β) production. Our results show that estrogen enhanced NLRP3 inflammasome activation in NESCs, resulting in increased IL-1β production. Progesterone decreased NLRP3 inflammasome activity with an increase in autophagy induction in estrogen-treated NESCs. Inhibition of NLRP3 inflammasome activity by progesterone was blocked by autophagy inhibition. However, progesterone failed to change NLRP3 inflammasome activity and autophagy induction in estrogen-treated ECSCs. In contrast, dienogest, a specific progesterone receptor agonist, reduced NLRP3 inflammasome-mediated IL-1β production through autophagy induction in ECSCs. Furthermore, autophagy induction was decreased and NLRP3 inflammasome activity was increased in endometriotic tissues, which was reversed by preoperative administration of dienogest. In conclusion, our results suggest that progesterone inhibits NLRP3 inflammasome activation through autophagy in endometrial stromal cells. However, this inhibitory effect is attenuated in endometriotic stromal cells due to an aberrant autophagic response to progesterone, which could lead to an altered inflammatory response in endometriosis.