IL-1β Release Research Articles

Differential effects of acute and chronic hydrocortisone treatment on pyroptosis

Pyroptosis is a programmed and inflammation-inducing cell death that occurs predominantly in macrophages. It is characterized by the inflammasome-mediated activation of caspase-1, leading to cell lysis. During pyroptosis, pro-inflammatory mediators such as IL-1β are released extracellularly to further recruit and activate other immune cells. Thus, pyroptosis plays a crucial role in the prevention of the spread of pathogens. The clinically applied synthetic glucocorticoid, hydrocortisone (HC), has strong immunoregulatory properties. It may act as an immunosuppressive agent by negatively regulating pro-inflammatory gene transcription but has also shown immune-sensitizing properties. The conditions that determine the immunosuppressive or immune-sensitizing actions of HC during an infection are not fully clear. We hypothesized that the outcome may differ depending on the onset and duration of its administration. Therefore, we investigated the impact of acute (treatment upon infection) and chronic (24 h pre-treatment before infection) HC treatment on pyroptosis induction and execution in THP-1 macrophage-like cells. The focus was on pyroptosis-associated signaling pathways, inflammasome assembly and activation, IL-1β, and cell death. Physiological HC concentration and HC deprivation were used as controls. Compared to the physiological concentration, cells displayed augmented inflammasome activation and IL-1β release following acute HC treatment. Conversely, the whole pyroptosis machinery was suppressed by chronic HC administration.These in vitro investigations demonstrate pro-inflammatory actions of acute HC exposure and the immunosuppressive effects of chronic treatment. These differential effects on pyroptosis emphasize the importance of individualized HC medication in patients upon infection, and suggest the inclusion of IL-1β as a marker for current immune capacities.

Circulating Extracellular Vesicle-Mediated Immune Activation After Resuscitation From Cardiac Arrest in Swine

Objective: Systemic inflammation is a well-established contributor to morbidity and mortality in patients that are initially resuscitated from sudden cardiac arrest (SCA). However, our mechanistic understanding of immune activation in this context remains limited, impeding the development of targeted anti-inflammatory treatments. To address this knowledge gap, we tested the hypothesis that post-resuscitation immune cell activation is mediated, at least in part, by circulating extracellular vesicles (EVs). Methods: Swine (n=3) were subjected to 10 minutes of electrically-induced SCA followed by CPR with defibrillation and epinephrine (0.015 mg/kg iv) to achieve return of spontaneous circulation (ROSC). Blood samples were collected before SCA and 4-hours post-ROSC for isolation of plasma EVs from clarified plasma using 100kD filters, which were subsequently quantified by Nano-particle Tracking Analysis (ZetaView). Naïve PBMCs from healthy swine were cultured with basal media (negative control), lipopolysaccharide (LPS; positive control), or EVs isolated from pre-SCA or post ROSC plasma that were stained with lipophilic dye (DiD) to facilitate assessment of EV uptake (n=3 per group). After 24 hours in culture, PBMC activation was assessed via surface marker expression (flow cytometry) and inflammatory cytokine release (ELISA). Results: Compared with exposure to pre-SCA EVs and basal media, PBMCs exposed to post-ROSC EVs exhibited a significant shift toward an inflammatory phenotype, with enrichment of inflammatory dendritic cells (CD172+CD16+), inflammatory granulocytes (CD172+CD163-), and inflammatory macrophages (CD14+CD163+) in both DiD+ and DiD- cell populations (all p<0.05 vs. pre-SCA EVs). Although TNF-α, IFN-α, and IFN-γ were not detected after EV co-culture, a near-significant increase in IL-1β release was observed in PBMCs exposed to post-ROSC EVs vs. pre-SCA EVs (5.72±2.4 vs. 2.21±0.7 pg/mL; p=0.068). Conclusion: Our findings suggest that circulating plasma EVs may contribute to immune cell activation following systemic ischemia/reperfusion injury associated with resuscitation from SCA. Further studies to delineate mechanisms underlying EV-mediated pro-inflammatory shifts in immune cell phenotype and potentiation of IL-1β release may reveal novel therapeutic targets to mitigate post-resuscitation inflammation and improve patient outcomes following SCA. Supported by funding from the NIH and US Department of Veteran's Affairs. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Characterizing inflammasome activation by bacterial amyloid curli complexes from biofilms in dendritic cells and its role in SLE

Abstract Bacterial infections are a crucial environmental trigger for flares in Systemic lupus erythematosus (SLE). Although these infections are common in lupus patients, a precise mechanism of action remains unknown. Bacteria protect themselves from stresses by embedding in an extracellular matrix to form biofilms. Biofilms from bacterial species such as E. coli and Salmonella contain an amyloid protein called curli. Previous work from our labs has shown that curli/DNA complexes can accelerate lupus-like disease in mice, and increased levels of anti-curli antibodies are associated with flares in SLE patients, making these complexes important in autoimmunity. We had previously found that curli/DNA is a potent trigger of immune activation in dendritic cells (DCs) and macrophages. In macrophages, curli/DNA was also reported to induce IL-1b production through activation of the NLRP3 inflammasome. Here we expand these studies to delineate how curli/DNA complexes regulate IL-1b in murine and human DCs. Using curli/DNA alone or in combination with inflammasome stimuli, we found that curli/DNA stimulate pro-IL1b expression and inflammasome assembly, leading to caspase 1 activation, cleavage and release of mature IL-1B by DCs. However, we observed no cell death post inflammasome activation, suggesting a hyperactivated state in stimulated DCs. A deeper knowledge of this molecular mechanism might shed light on the role of inflammasome activation by bacterial infections in escalating SLE.

Loss of myeloid-KLF2 mediates LPS-induced mortality in neonatal pups by NLRP3 inflammasome signaling and altered neutrophil aging

Abstract Preterm neonates have disrupted myeloid cell activation and aging. KLF2 inhibits myeloid cell activation by repressing NF-kB, which controls NLRP3 transcription. In our myeloid-KLF2 deleted (MKO) murine model, postnatal day (P) 4 pups have higher LPS-induced mortality than controls but 100% survival in both groups after LPS at P12. P4 and P12 reflect preterm and term physiology, respectively. We aimed to to determine the role of neutrophil-NLRP3 and neutrophil aging in LPS-induced mortality in P4 MKO pups. Serum IL-1β was measured 4h post-LPS injection. BM neutrophils were treated with LPS and nigericin for NLRP3 protein expression in lysate and IL-1b levels in the supernatant. Daily MCC950 injections (P1-P4) were used to inhibit NLRP3 before LPS injection on P4. BM neutrophils were treated with LPS ex vivo and flow-sorted to examine CXCR4/CD62L expression. MKO pups have increased serum IL-1β, neutrophil-NLRP3 expression, and IL-1b release, P4>P12. In vivo NLRP3 inhibition rescued survival in P4 MKO and controls. P4 MKO have reduced BM and higher circulating neutrophils. MKO BM neutrophils are more aged (CD62Llo), but CXCR4lo, affecting aged neutrophil homing to the BM. Myeloid-KLF2 loss increases LPS mortality via NLRP3 activation in P4 pups. It accelerates neutrophil aging, but leads to persistence of aged neutrophils in the circulation by reducing CXCR4 expression. Future mechanistic studies will focus on KLF2's regulation of NLRP3 and CXCR4 expression at P4 and P12.

Honokiol Exhibits Anti-NLRP3 Inflammasome and Antimicrobial Properties in Neisseria gonorrhoeae-Infected Macrophages.

The NOD-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome, crucial in infectious and inflammatory diseases by regulating IL-1β, presents a target for disease management. Neisseria gonorrhoeae causes gonorrhea in over 87 million people annually, with previous research revealing NLRP3 inflammasome activation in infected macrophages. No natural products have been reported to counteract this activation. Exploring honokiol, a phenolic compound from Chinese herbal medicine, we investigated its impact on NLRP3 inflammasome activation in N. gonorrhoeae-infected macrophages. Honokiol's impact on the protein expression of pro-inflammatory mediators was analyzed using ELISA and Western blotting. The generation of intracellular H2O2 and mitochondrial reactive oxygen species (ROS) was detected through specific fluorescent probes (CM-H2DCFDA and MitoSOX, respectively) and analyzed by flow cytometry. Mitochondrial membrane integrity was assessed using specific fluorescent probes (MitoTracker and DiOC2(3)) and analyzed by flow cytometry. Additionally, the effect of honokiol on the viability of N. gonorrhoeae was examined through an in vitro colony-forming units assay. Honokiol effectively inhibits caspase-1, caspase-11 and GSDMD activation and reduces the extracellular release of IL-1β, NLRP3, and apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) in N. gonorrhoeae-infected macrophages. Detailed investigations have demonstrated that honokiol lowers the production of H2O2 and the phosphorylation of ERK1/2 in N. gonorrhoeae-infected macrophages. Importantly, the phosphorylation of JNK1/2 and p38 and the activation of NF-κB remain unaffected. Moreover, honokiol reduces the N. gonorrhoeae-mediated generation of reactive oxygen species within the mitochondria, preserving their integrity. Additionally, honokiol suppresses the expression of the pro-inflammatory mediator IL-6 and inducible nitric oxide synthase induced by N. gonorrhoeae independently of NLRP3. Impressively, honokiol exhibits in vitro anti-gonococcal activity against N. gonorrhoeae. Honokiol inhibits the NLRP3 inflammasome in N. gonorrhoeae-infected macrophages and holds great promise for further development as an active ingredient in the prevention and treatment of symptoms associated with gonorrhea.

Abstract 1116: IL-33 Promotes Cardiovascular Lesion Development In A Murine Model Of Kawasaki Disease By Boosting IL-1β Production

Kawasaki Disease (KD), a pediatric acute febrile systemic vasculitis, is the leading cause of acquired heart diseases in children. Coronary artery aneurysms (CAAs) occur in up to 25% of untreated children, which is reduced to 5% with intravenous immunoglobulin (IVIG) treatment. However, up to 20% of KD patients are refractory to IVIG and at higher risk of developing CAAs. This highlights the need to characterize the immune mechanisms mediating KD to develop more efficient therapies. IL-1β plays a key role in KD pathogenesis. IL-33, a member of the IL-1 cytokine family, is released upon inflammation and tissue damage and exerts its effects by binding to its receptor ST2 ( Il1rl1 ). Circulating levels of IL-33 are elevated in KD patients during the acute phase of the disease. However, if and how IL-33 contributes to cardiovascular lesion development in KD remains unknown. Using the Lactobacillus casei cell wall extract (LCWE) murine model of KD vasculitis, we observed increased Il33 mRNA expression in hearts and abdominal aorta aneurysms of LCWE-injected mice. While ST2 was detected by immunofluorescence in the hearts of both control and LCWE-injected mice, its expression was increased in the abdominal aortas of LCWE-injected mice. In contrast, IL-33 expression was only increased in inflamed cardiovascular tissues of LCWE-injected mice. Single-cell RNA-sequencing and flow cytometric analysis of abdominal aortas, as well as spatial transcriptomics of heart tissues from LCWE-injected mice, indicated that Il33 transcripts were expressed primarily by stromal cells, such as fibroblasts, endothelial cells, and vascular smooth muscle cells. On the other hand, Il1rl1 transcripts were highly expressed by tissue infiltrating immune cells, such as macrophages, eosinophils, and neutrophils. Blocking IL-33, using either Il33 -/- mice or an anti-IL-33 antibody, significantly attenuated LCWE-induced KD vasculitis. In vitro, IL-33 treatment of LCWE-stimulated bone marrow-derived macrophages boosted their IL-1β production. Overall, our results indicate that IL-33, produced by stromal cells, may promote LCWE-induced KD vasculitis by increasing the release of IL-1β by tissue infiltrating immune cells, and this axis could therapeutically be targeted in KD.

First-in-human safety, tolerability, and pharmacokinetic results of DFV890, an oral low-molecular-weight NLRP3 inhibitor.

This first-in-human study evaluated the safety, tolerability, single- and multiple-dose pharmacokinetic profiles with dietary influence, and pharmacodynamics (PD) of DFV890, an oral NLRP3 inhibitor, in healthy participants. In total, 122 participants were enrolled into a three-part trial including single and 2-week multiple ascending oral doses (SAD and MAD, respectively) of DFV890, and were randomized (3:1) to DFV890 or placebo (SAD [3-600 mg] and MAD [fasted: 10-200 mg, once-daily or fed: 25 and 50 mg, twice-daily]). DFV890 was generally well-tolerated. Neither deaths nor serious adverse events were reported. A less than dose-proportional increase in exposure was observed with the initially used crystalline suspension (3-300 mg); however, an adjusted suspension formulation using spray-dried dispersion (SDD; 100-600 mg) confirmed dose-proportional increase in exposure. Relative bioavailability between crystalline suspension and tablets, and food effect were evaluated at 100 mg. Under fasting conditions, Cmax of the tablet yielded 78% compared with the crystalline suspension, and both formulations showed comparable AUC. The fed condition led to a 2.05- and 1.49-fold increase in Cmax and AUC0-last compared with the fasting condition. The median IC50 and IC90 for ex-vivo lipopolysaccharide-stimulated interleukin IL-1β release inhibition (PD) were 61 (90% CI: 50, 70) and 1340 ng/mL (90% CI: 1190, 1490). Crystalline tablets of 100 mg once-daily or 25 mg twice-daily were sufficient to maintain ~90% of the IL-1β release inhibition over 24 h at steady state. Data support dose and formulation selection for further development in diseases, in which an overactivated NLRP3 represents the underlying pathophysiology.

Abstract 1049: Long Noncoding RNA Regulation Of Inflammation In Atherosclerosis

Atherosclerosis is a chronic inflammatory disease driven by cholesterol deposition in the artery wall. Resolution of atherosclerotic inflammation and plaque regression are important clinical goals. As long noncoding RNAs (lncRNAs) are key regulators of gene expression, there is an intense interest in understanding their roles in atherosclerosis and their potential as therapeutic targets. To identify lncRNAs regulated during plaque progression and regression, we performed single cell RNA-sequencing of CD45 + immune cells isolated from aortic arch plaques of Ldlr –/– mice under conditions of plaque progression (16 wks Western diet; WD) or regression (16 wks WD + 4 wks apoB ASO). We screened for lncRNAs acting in cis to modify the expression of proximal protein-coding genes and identified Caslr ( C tsc a nti s ense l nc R NA) , a novel lncRNA transcribed from the opposite strand of Cathepsin C gene ( Ctsc ) encoding a pro-atherosclerotic lysosomal protease. Caslr–Ctsc pair is upregulated in macrophages of progressing plaques and in response to pro-inflammatory stimuli ( i.e . LPS, IL-6) in vitro , and conversely downregulated during atherosclerosis regression and in response to anti-inflammatory stimulus ( i.e. IL-4). LncRNA knockdown approaches combined with transcriptomic analyses and functional assays showed that Caslr regulates Ctsc expression and associated inflammatory responses, including NF-κB signaling and inflammasome-dependent IL-1β release. RNA purification strategies combined with mass-spectrometry revealed that Caslr interacts with spliceosome-associated proteins to coordinate splicing of Ctsc mRNA. Notably, Caslr shares functional homology with a human lncRNA, CASLR, proximal to the CTSC gene, which regulates CTSC expression and protease activity. Human CASLR and CTSC are highly expressed in the blood of hyperlipidemic patients and their expression is coordinately decreased upon aggressive cholesterol lowering by anti-PCSK9 treatment. Collectively, our data identify Caslr as an evolutionary conserved lncRNA acting in cis to drive the expression of cathepsin C with downstream consequences on inflammatory signaling in plaque macrophages, and suggest that targeting this lncRNA may be beneficial in treating atherosclerosis.

High glucose reduces Nrf2-dependent cRAGE release and enhances inflammasome-dependent IL-1β production in monocytes: the modulatory effects of EGCG

Soluble receptor for advanced glycation end products (sRAGE) acts as a decoy sequestering of RAGE ligands, thus preventing the activation of the ligand-RAGE axis linking human diseases. However, the molecular mechanisms underlying sRAGE remain unclear. In this study, THP-1 monocytes were cultured in normal glucose (NG, 5.5 mmol/L) and high glucose (HG, 15 mmol/L) to investigate the effects of diabetes-relevant glucose concentrations on sRAGE and interleukin-1β (IL-1β) secretion. The modulatory effects of epigallocatechin gallate (EGCG) in response to HG challenge were also evaluated. HG enhanced intracellular reactive oxygen species (ROS) generation and RAGE expression. The secretion of sRAGE, including esRAGE and cRAGE, was reduced under HG conditions, together with the downregulation of a disintegrin and metallopeptidase 10 (ADAM10) and nuclear factor erythroid 2-related factor 2 (Nrf2) nuclear translocation. Mechanistically, the HG effects were counteracted by siRAGE and exacerbated by siNrf2. Chromatin immunoprecipitation results showed that Nrf2 binding to the ADAM10 promoter and HG interfered with this binding. Our data reinforce the notion that RAGE and Nrf2 might be sRAGE-regulating factors. Under HG conditions, the treatment of EGCG reduced ROS generation and RAGE activation. EGCG-stimulated cRAGE release was likely caused by the upregulation of the Nrf2-ADAM10 pathway. EGCG inhibited HG-mediated NLRP3 inflammasome activation at least partly by stimulating sRAGE, thereby reducing IL-1β release.

P2X7 receptor knockout does not alter renal function or prevent angiotensin II-induced kidney injury in F344 rats

P2X7 receptors (P2X7) mediate immune and endothelial cell responses to extracellular ATP. Acute pharmacological blockade increases renal blood flow (RBF) and glomerular filtration rate (GFR), suggesting that P2X7 receptor activation promotes tonic vasoconstriction. P2X7 expression is increased in kidney disease and blockade/knockout is renoprotective. Here, we generated a novel P2X7 global knockout (KO) rat on the F344 background, hypothesising enhanced RBF and protection from chronic angiotensin II (ANG II)-induced injury. CRISPR/Cas9 technology introduced an early stop codon into exon 2 of P2rx7. In male and female KO and WT rats, renal P2X7 expression was measured by RTqPCR (n=4-5/group) and western blotting (n=3/group). Bone marrow-derived macrophages (BMDM) were used to assess P2X7 function by measuring interleukin (IL)-1β release (ELISA) after stimulation with lipopolysaccharide (LPS) (1μg/mL, 4h) and ATP (3mM, 1h) (n=5-6/group). In vivo kidney function was assessed in anesthetized rats (thiopental, 50mg/kg, i.p.) at baseline and following serial arterial ligation to induce acute pressure natriuresis (n=9-10/group). Arterial blood pressure (BP) was measured directly, and urine was collected via tube cystotomy. RBF was measured using a Transonic Doppler flow probe placed around the main renal artery, and cortical and medullary flux by laser Doppler needle probes. GFR was determined by FITC-inulin clearance. Urinary sodium was measured by sodium-selective electrode analysis. In a separate experiment, BP was recorded over five consecutive days in conscious male KO and WT rats via implanted radiotelemetry devices (n=7-8/group). Finally, male KO and WT rats (n=9-10/group) were infused with ANG II (250ng/kg/min, s.c.) for 6 weeks. At end, one renal artery was isolated and vascular reactivity to phenylephrine, acetylcholine and sodium nitroprusside determined by wire myography. Bladder urine was collected to measure albumin/creatinine ratio (n=4-7/group), and kidneys were harvested for histological analyses (n=6/group). Data were analysed with unpaired t-test (two groups compared) or two-way ANOVA (two variables), with p<0.05 statistically significant. P2rx7 mutation was confirmed by genome sequencing (Sanger). P2rx7 mRNA abundance was partially decreased in kidney from male (-54%) and female (-25%) KO vs WT. P2X7 protein was detected in kidneys from WT but not from KO rats. In BMDM from WT rats, LPS+ATP stimulation induced IL-1β release; this response was markedly suppressed in KO BMDM (males: 2577±140 vs 413±175pg/mL, p<0.0001; females: 1889±394 vs 265±121pg/mL, p<0.0001). In vivo BP, renal haemodynamics and tubular sodium handling were not different between genotypes for either sex (P>0.05). Radiotelemetry confirmed no difference in BP between KO and WT rats. Following chronic ANG II infusion, KO and WT rats had no renal artery dysfunction but showed similar increases in albuminuria (P=0.3587), renal perivascular fibrosis (Picrosirius Red, P=0.5393), cortical interstitium CD68-positive area (p=0.6546) and tubular casts (Periodic acid-Schiff, P=0.8803), compared with naïve controls. Contrary to our hypothesis, global genetic deletion of P2X7 did not affect renal hemodynamics and we found no significant role for P2X7 in the modulation of tubular sodium reabsorption. Our data do not support a major role of P2X7 in causing renal vascular and tubular injury. This work was supported by Diabetes UK grant 17/0005685, The British Heart Foundation(FS/15/60/31510; RE/18/5/34216), the Medical Research Council (MR/S01053X), and the University of Edinburgh College of Medicine & Veterinary Medicine PhD Studentship. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Abstract 1115: Novel Mechanism For Extrahepatic Inflammation In Progressive Familial Intrahepatic Cholestasis 1

Background: Human mutations in ATP8b1, a member of the P4-type ATPase family, cause Progressive Familial Intrahepatic Cholestasis I (PFIC1). PFIC1 patients often show extrahepatic inflammatory manifestations such as atherosclerosis, pancreatitis, and hearing loss. Even after liver transplantation, which rescues PFIC1 hepatic symptoms, the extrahepatic inflammatory symptoms persist. The mechanism of how ATP8b1 regulates inflammation is not clear. Pyroptosis executor Gasdermin D (GsdmD) plays a major role in promoting inflammation. GsdmD can be cleaved via a caspase-1/Nlrp3 dependent canonical inflammasome pathway, or a caspase-11 (in mice) or caspase-4/5 (in humans) dependent non-canonical inflammasome pathway. Goal: To decipher the mechanism for ATP8b1-mediated regulation of inflammasome activity in immune cells. Methods & Results: We employed Crispr-Cas9 generated homozygous ATP8b1 knockout human monocytes/macrophages to determine the status of inflammasome activity. Human monocytes/macrophages lacking ATP8b1 released significantly more IL-1β upon exposure to LPS (1μg/ml) + Nigericin (5 mM) vs. control cells (mean±SD, N=3-6, p<0.0001 by t-test.) Interestingly, ATP8b1 -/- cells showed GsdmD cleavage with LPS priming alone without a secondary signal, and markedly increased IL-1β release vs. WT cells (N=3-6, mean±SD, p<0.001). Mechanistically, LPS gained cytoplasmic access in ATP8b1 -/- immune cells, leading to cleavage of GsdmD in an NLRP3-independent, and caspase 4/5 dependent non-canonical inflammasome. The ATP8b1 -/- immune cells showed impaired lysosomal acidification, though the lysosomal number and lysosomal membrane integrity were not compromised. ATP8b1 -/- immune cells showed defects in inflammasome-resolving pathways such as phagocytosis and efferocytosis. Atomic force microscopy of ATP8b1 -/- cells showed a reduced order state of the plasma membrane with Young’s modulus of elasticity for THP-1 ATP8b1 -/- (Ey= 296±10.42 Pa) vs. WT macrophages (Ey =680±25.27 Pa, N =30, mean±SD, p<0.0001). Conclusions: ATP8b1 negatively regulates GsdmD cleavage via a non-canonical inflammasome pathway. GsdmD may serve as a therapeutic target for resolving inflammation in PFIC1 patients.

Obesity-associated inflammatory macrophage polarization is inhibited by capsaicin and phytolignans.

Obesity is often accompanied by increased adipose tissue inflammation, a process that is partially driven by adipose tissue-resident macrophages. In this study, we explored the potential for plant-derived dietary compounds to exert anti-inflammatory effects in macrophages that alleviate obesity-associated adipocyte dysfunction. Capsaicin (CAP), schisandrin A (SA), enterodiol (END), and enterolactone (ENL) treatment polarized J774 macrophages to an "M2" or anti-inflammatory phenotype and inhibited responses to stimulation with lipopolysaccharide (LPS). Furthermore, these compounds blocked inflammasome activation when administered just before ATP-induced NLRP3 activation, as evidenced by the abrogation of IL-1β release in mouse macrophages and human peripheral blood monocytes. The addition of CAP, SA, or ENL during the differentiation of bone marrow-derived macrophages was also sufficient to inhibit LPS-induced IL-6 and TNFα production. Finally, CAP, END, and ENL treatment during differentiation of 3T3-L1 adipocytes induced an adiponectin-high phenotype accompanied by increases in thermogenic gene expression, and conditioned media from these adipocytes inhibited LPS-induced production of IL-1β, IL-6, and TNFα from J774 macrophages. These polarizing effects were partially mediated by the elevated adiponectin and decreased syndecan-4 in the adipocyte-conditioned media. These results implicate the contribution of plant-derived dietary components to the modulation of macrophages and adipocytes in obesity.NEW & NOTEWORTHY The utility of food-based products to prevent or alleviate chronic conditions such as obesity and its associated comorbidities is an attractive approach. Capsaicin, schisandrin A, enterodiol, and enterolactone, phytochemicals present in traditional medicinal food, decreased proinflammatory cytokine production from macrophages that, in turn, reduced obesity-associated adipocyte dysfunction. These results implicate the contribution of plant-derived dietary components to the modulation of macrophages and adipocytes in obesity.

Development of clobetasol-loaded biodegradable nanoparticles as an endodontic intracanal medicament.

The aim of current study is the development and optimization of biodegradable polymeric nanoparticles (NPs) to be used in the field of Endodontics as intracanal medication in cases of avulsed teeth with extended extra-oral time, utilizing PLGA polymers loaded with the anti-inflammatory drug clobetasol propionate (CP). CP-loaded nanoparticles (CP-NPs) were prepared using the solvent displacement method. CP release profile from CP-NPs was assessed for 48 h against free CP. Using extracted human teeth, the degree of infiltration inside the dentinal tubules was studied for both CP-NPs and CP. The anti-inflammatory capacity of CP-NPs was evaluated invitro measuring their response and reaction against inflammatory cells, in particular against macrophages. The enzyme-linked immunosorbent assay (ELISA) was used to examine the cytokine release of IL-1β and TNF-α. Optimized CP-NPs displayed an average size below 200 nm and a monomodal population. Additionally, spherical morphology and non-aggregation of CP-NPs were confirmed by transmission electron microscopy. Interaction studies showed that CP was encapsulated inside the NPs and no covalent bonds were formed. Moreover, CP-NPs exhibited a prolonged and steady release with only 21% of the encapsulated CP released after 48 h. Using confocal laser scanning microscopy, it was observed that CP-NPs were able to display enhanced penetration into the dentinal tubules. Neither the release of TNF-α nor IL-1β increased in CP-NPs compared to the LPS control, displaying results similar and even less than the TCP after 48 h. Moreover, IL-1β release in LPS-stimulated cells, decreased when macrophages were treated with CP-NPs. In the present work, CP-NPs were prepared, optimized and characterized displaying significant increase in the degree of infiltration inside the dentinal tubules against CP and were able to significantly reduce TNF-α release. Therefore, CP-NPs constitute a promising therapy for the treatment of avulsed teeth with extended extra-oral time.

Novel Isoalantolactone-Based Derivatives as Potent NLRP3 Inflammasome Inhibitors: Design, Synthesis, and Biological Characterization.

The NLRP3 inflammasome has been recognized as a promising therapeutic target in drug discovery for inflammatory diseases. Our initial research identified a natural sesquiterpene isoalantolactone (IAL) as the active scaffold targeting NLRP3 inflammasome. To improve its activity and metabolic stability, a total of 64 IAL derivatives were designed and synthesized. Among them, compound 49 emerged as the optimal lead, displaying the most potent inhibitory efficacy on nigericin-induced IL-1β release in THP-1 cells, with an IC50 value of 0.29 μM, approximately 27-fold more potent than that of IAL (IC50: 7.86 μM), and exhibiting higher metabolic stability. Importantly, 49 remarkably improved DSS-induced ulcerative colitis in vivo. Mechanistically, we demonstrated that 49 covalently bound to cysteine 279 in the NACHT domain of NLRP3, thereby inhibiting the assembly and activation of NLRP3 inflammasome. These results provided compelling evidence to further advance the development of more potent NLRP3 inhibitors based on this scaffold.

Mycobacterium tuberculosis PE_PGRS38 Enhances Intracellular Survival of Mycobacteria by Inhibiting TLR4/NF-κB-Dependent Inflammation and Apoptosis of the Host.

Mycobacterium tuberculosis (Mtb) ranks as the most lethal human pathogen, able to fend off repeated attacks by the immune system or medications. PE_PGRS proteins are hallmarks of the pathogenicity of Mtb and contribute to its antigenic diversity, virulence, and persistence during infection. M. smegmatis is a nonpathogenic mycobacterium that naturally lacks PE_PGRS and is used as a model to express Mtb proteins. PE_PGRS has the capability to evade host immune responses and enhance the intracellular survival of M. smegmatis. Despite the intense investigations into PE_PGRS proteins, their role in tuberculosis remains elusive. We engineered the recombinant M. smegmatis strain Ms-PE_PGRS38. The result shows that PE_PGRS38 is expressed in the cell wall of M. smegmatis. PE_PGRS38 contributes to biofilm formation, confers permeability to the cell wall, and shows variable responses to exogenous stresses. PE_PGRS38 downregulated TLR4/NF-κB signaling in RAW264.7 macrophages and lung tissues of infected mice. In addition, PE_PGRS38 decreased NLRP3-dependent IL-1β release and limited pathogen-mediated inflammasome activity during infection. Moreover, PE_PGRS38 inhibited the apoptosis of RAW264.7 cells by downregulating the expression of apoptotic markers including Bax, cytochrome c, caspase-3, and caspase-9. In a nutshell, our findings demonstrate that PE_PGRS38 is a virulence factor for Mtb that enables recombinant M. smegmatis to survive by resisting and evading the host's immune responses during infection.

P2X7 receptor knockout does not alter renal function or prevent angiotensin II-induced kidney injury in F344 rats

P2X7 receptors mediate immune and endothelial cell responses to extracellular ATP. Acute pharmacological blockade increases renal blood flow and filtration rate, suggesting that receptor activation promotes tonic vasoconstriction. P2X7 expression is increased in kidney disease and blockade/knockout is renoprotective. We generated a P2X7 knockout rat on F344 background, hypothesising enhanced renal blood flow and protection from angiotensin-II-induced renal injury. CRISPR/Cas9 introduced an early stop codon into exon 2 of P2rx7, abolishing P2X7 protein in kidney and reducing P2rx7 mRNA abundance by ~ 60% in bone-marrow derived macrophages. The M1 polarisation response to lipopolysaccharide was unaffected but P2X7 receptor knockout suppressed ATP-induced IL-1β release. In male knockout rats, acetylcholine-induced dilation of the renal artery ex vivo was diminished but not the response to nitroprusside. Renal function in male and female knockout rats was not different from wild-type. Finally, in male rats infused with angiotensin-II for 6 weeks, P2X7 knockout did not reduce albuminuria, tubular injury, renal macrophage accrual, and renal perivascular fibrosis. Contrary to our hypothesis, global P2X7 knockout had no impact on in vivo renal hemodynamics. Our study does not indicate a major role for P2X7 receptor activation in renal vascular injury.

INFLUENCES OF HEAT STRESS ON GLUTAMATE TRANSMISSION-DEPENDENT EXPRESSION LEVELS OF IL-1β and IL-18 IN BV-2 MICROGLIAL CELLS.

Objective: This study aimed to explore the impact of heat stress (HS) on glutamate transmission-dependent expression levels of interleukin-1β (IL-1β) and IL-18 in BV-2 microglial cells. Methods: BV-2 microglial cells were cultured in vitro , with cells maintained at 37°C serving as the control. The HS group experienced incubation at 40°C for 1 h, followed by further culturing at 37°C for 6 or 12 h. The experimental group was preincubated with glutamate, the glutamate antagonist riluzole, or the mGluR5 agonist, 2-chloro-5-hydroxyphenylglycine (CHPG), before HS. Glutamate content in BV-2 culture supernatant was assessed using colorimetric assay. Moreover, mRNA expression levels of EAAT3 and/or mGluR5 in BV-2 cells were determined via quantitative polymerase chain reaction. Interleukins (IL-1β and IL-18) in cell culture supernatant were measured using enzyme-linked immunosorbent assay. Western blot analysis was employed to assess protein levels of IL-1β and IL-18 in BV-2 cells. Results: HS induced a significant release of glutamate and increased the expression levels of mGluR5 and EAAT3 in BV-2 cells. It also triggered the expression levels and release of proinflammatory factors, such as IL-1β and IL-18, synergizing with the effects of glutamate treatment. Preincubation with both riluzole and CHPG significantly reduced HS-induced glutamate release and mitigated the increased expression levels and release of IL-1β and IL-18 induced by HS. Conclusion: The findings confirmed that microglia could be involved in HS primarily through glutamate metabolisms, influencing the expression levels and release of IL-1β and IL-18.

Clinical implications of innate immune exhaustion in cystic fibrosis.

Lung disease progression in people with cystic fibrosis (pwCF) varies from one individual to another. Different immunological characteristics have been suggested to explain this variation, and we hypothesised that lung capacity may be associated with the innate immune response in pwCF. In an exploratory study, we aimed to investigate potential links between the innate immune response and lung function in pwCF using the standardised immune function assay TruCulture. In a single-centre study with combined cross-sectional and longitudinal data before and after intravenous antibiotics, blood was sampled from Pseudomonas aeruginosa-infected pwCF. Whole blood was analysed by TruCulture to reveal the unstimulated and stimulated cytokine release. Tobit regressions and Spearman's correlations were used to estimate the associations between lung function and cytokine release. We included 52 pwCF in the cross-sectional study and 24 in the longitudinal study. In the cross-sectional study, we found that compared to a healthy population, the release of toll-like receptor (TLR)3, TLR4- and TLR7/8-stimulated interferon-γ, and interleukin (IL)-12p40 was reduced. Although TLR3-stimulated IL-1β and IL-6 release increased with lung function, overall, cytokine release did not correlate well with lung function. In the longitudinal study, the cytokine release was modified by antibiotic treatment, but the cytokine release before antibiotic treatment did not associate with changes in lung function after treatment. The stimulated cytokine release could not predict lung function levels or changes in pwCF, but our data indicate that pwCF experience exhaustion in the innate immune response after years of chronic bacterial infection.

Bile acids attenuate hepatic inflammation during ischemia/reperfusion injury

Background & AimsPersistent cholestasis has been associated with poor prognosis after orthotopic liver transplantation (OLT). In this study, we aimed to investigate how the accumulation of tauro-beta-muricholic acid (TβMCA), resulting from the reprogramming of bile acid (BA) metabolism during liver ischemia/reperfusion (IR) stress, attenuates liver inflammation. MethodsIngenuity Pathway Analysis (IPA) was performed using transcriptome data from a murine hepatic IR model. Three different models of hepatic IR (liver warm IR, bile duct separation-IR, common bile duct ligation-IR) were employed. We generated AAV-transfected mice and CD11b-DTR mice to assess the role of BAs in regulating the myeloid S1PR2-GSDMD axis. Hepatic BA levels were analyzed using targeted metabolomics. Finally, the correlation between the reprogramming of BA metabolism and hepatic S1PR2 levels was validated through RNA-seq of human OLT biopsies. ResultsWe found that BA metabolism underwent reprogramming in murine hepatocytes under IR stress, leading to increased synthesis of TβMCA, catalyzed by the enzyme CYP2C70. The levels of hepatic TβMCA were negatively correlated with the severity of hepatic inflammation, as indicated by the serum IL-1β levels. Inhibition of hepatic CYP2C70 resulted in reduced TβMCA production, which subsequently increased serum IL-1β levels and exacerbated IR injury. Moreover, our findings suggested that TβMCA could inhibit canonical inflammasome activation in macrophages and attenuate inflammatory responses in a myeloid-specific S1PR2-GSDMD-dependent manner. Additionally, Gly-βMCA, a derivative of TβMCA, could effectively attenuate inflammatory injury in vivo and inhibit human macrophage pyroptosis in vitro. ConclusionsIR stress orchestrates hepatic BA metabolism to generate TβMCA, which attenuates hepatic inflammatory injury by inhibiting the myeloid S1PR2-GSDMD axis. Bile acids have immunomodulatory functions in liver reperfusion injury that may guide therapeutic strategies. Impact and implicationsOur research reveals that liver IR stress triggers reprogramming of BA metabolism. This functions as an adaptive mechanism to mitigate inflammatory injury by regulating the S1PR2-GSDMD axis, thereby controlling the release of IL-1β from macrophages. Our results highlight the crucial role of bile acids in regulating hepatocyte-immune cell crosstalk, which demonstrates an immunomodulatory function in liver reperfusion injury that may guide therapeutic strategies targeting bile acids and their receptors.

The inflammasome pathway is activated by dengue virus non-structural protein 1 and is protective during dengue virus infection.

Dengue virus (DENV) is a medically important flavivirus causing an estimated 50-100 million dengue cases annually, some of whom progress to severe disease. DENV non-structural protein 1 (NS1) is secreted from infected cells and has been implicated as a major driver of dengue pathogenesis by inducing endothelial barrier dysfunction. However, less is known about how DENV NS1 interacts with immune cells and what role these interactions play. Here we report that DENV NS1 can trigger activation of inflammasomes, a family of cytosolic innate immune sensors that respond to infectious and noxious stimuli, in mouse and human macrophages. DENV NS1 induces the release of IL-1β in a caspase-1 dependent manner. Additionally, we find that DENV NS1-induced inflammasome activation is independent of the NLRP3, Pyrin, and AIM2 inflammasome pathways, but requires CD14. Intriguingly, DENV NS1-induced inflammasome activation does not induce pyroptosis and rapid cell death; instead, macrophages maintain cellular viability while releasing IL-1β. Lastly, we show that caspase-1/11-deficient, but not NLRP3-deficient, mice are more susceptible to lethal DENV infection. Together, these results indicate that the inflammasome pathway acts as a sensor of DENV NS1 and plays a protective role during infection.