IL-1β Production Research Articles

Antigen Presenting Cell activator protein 1 (AP-1) complex contributes to Salt-sensitive Blood Pressure

Salt-sensitivity of blood pressure (SSBP) affects 50% of the hypertensive and 25% of the normotensive population and is a major independent risk factor for cardiovascular morbidity and mortality. We previously found that SSBP is associated with activation of the NLRP3 inflammasome in antigen presenting cells (APCs) via increased oxidated stress and isolevuglandin (IsoLG) formation, but the underlying mechanisms are unknown. The activator protein 1 (AP-1) (FOS/JUN) has been implicated in activation of the NLRP3 inflammasome but its role in SSBP is not known. We hypothesized that AP-1 transcription factor in APCs senses elevated sodium and contributes to SSBP. We further hypothesize that antioxidant flavonoid, diosmetin prevents AP-1 activation and SSBP. Using bulk RNA-sequencing in human monocytes, we found elevated sodium increases expression of the AP-1 gene family when compared to normal sodium concentration, including cFOS (2378.18 ± 480.7 vs 6494.09 ± 945.55, p= 0.0009), FOSB (53.63 ± 17. 55 vs 131.06 ± 10.30, p= 0.397), cJUN (7313.90 ± 984.93 vs 11370.09 ± 1286.35, p= 0.2563) JUNB (4218.4.36 ± 199.17 vs 570.45 ± 200.91, p= 0.0445) and JUND (3309. 63 ± 270.64 vs 8057.90 ± 1043.05, p= 0.00006). In additional experiments, we enrolled people with hypertension and phenotype them for SSBP using an established in-patient protocol of salt-loading/depletion and performed single-cell transcriptomic analyses in vivo on peripheral blood mononuclear cells (PBMCs). We found that expression of the FOSB and JUNB genes are more sensitive in concert with blood pressure in salt-sensitive (SS) but not salt-resistant (SR) humans. Moreover, diosmetin attenuated high salt-induced hypertension, 123.5 ± 4.1 vs 146.8 ± 2.5 mm Hg in controls, **p< 0.01). In addition, diosmetin also reduced high salt-induced inflammation including APC production of IsoLG-protein adducts, IL-1β, and NLRP3 inflammasome expression. These findings reveal a role for immune cell AP-1 (FOS/JUN) signaling in salt-sensitive hypertension and diosmetin may provide a potential therapeutic target for treatment of SSBP. 1R01HL144941-01A1. 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.

NKCC1-dependent K+ influx inhibits NLRP3 inflammasome activation and prevents intestinal epithelial cell injury

Background: Loss of function mutations in SLC12A2, the gene encoding the Na+-K+-2Cl− cotransporter (NKCC1) are associated with autoinflammatory and severe chronic inflammation in multiple organs. NLRP(s) are a family of cytosolic sensors that initiate the innate immune response. Once activated, NLRP(s) form inflammasomes, which are multi-protein complex that sense danger signals, damaged and infected cells. Assembly of the inflammasome mediates caspases-1 activation and the production of the proinflammatory cytokines IL-1β and IL-18 and initiates an inflammatory cell death process called pyroptosis. Aberrant activation of NLRP3 and other inflammasomes leads to a variety of autoimmune and chronic inflammatory diseases. Here we ask whether loss of NKCC1 function affects inflammasome activation in intestinal epithelial cells. Methods: Wild-type intestinal epithelial cells (IEC) and CRISPR/Cas9 NKCC1-deleted clones were tested for inflammasome activation. WT and NKCC1-deficient clones were treated with nigericin, ATP or ouabain to activate the inflammasome. Conditioned media, whole cell lysate and fixed cells were collected at different time points and analyzed by immunoblotting, immunofluorescence, and ELISA (to measure cytokine release). Inflammasome-induced pyroptosis was assessed by measuring LDH release in the supernatant and gasdermine D cleavage by immunoblotting. Results: Loss of NKCC1 function induces inflammasome activation by increasing IL-1β secretion in intestinal epithelial cells. Prolong inflammatory cytokine secretion led to increased pyroptosis-mediated cell death. Loss of NKCC1 mediates NLRP3 inflammasome activation by compromising K+ influx. NLRP3 agonists increase the interaction and localization of WNK1 and SPAK kinases with the NLRP3 inflammasome complex. Conclusions: NKCC1 regulates inflammatory cytokines secretion. Loss of NKCC1 inhibits K+ and Cl− inward fluxes, which in turn activate NLRP3 inflammasome. Loss of NKCC1 function is a previously unknown mechanism that causes aberrant inflammasome activation. GM118944 DK093501. 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.

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.

Amplifying protection against acute lung injury: Targeting both inflammasome and cGAS-STING pathway by Lonicerae Japonicae Flos-Forsythiae Fructus drug pair

ObjectiveAcute lung injury (ALI) is characterized by inflammation and currently lacks an efficacious pharmacological intervention. The medicine combination of Lonicerae Japonicae Flos (LJF) and Forsythiae Fructus (FF) demonstrates combined properties in its anti-infective, anti-inflammatory, and therapeutic effects, particularly in alleviating respiratory symptoms. In previous studies, Chinese medicine has shown promising efficacy in lipopolysaccharides (LPS)-induced ALI. However, there have been no reports of LJF and FF pairing for lung injury. The aim of this study is to compare the efficacy of herb pair Lonicerae Japonicae Flos-Forsythiae Fructus (LF) with LJF or FF alone in the treatment of ALI, and to explore whether LJF and FF have a combined effect in the treatment of lung injury, along with the underlying mechanism involved. MethodsA total of 36 mice were divided into six groups (control, model, LJF, FF, LF, dexamethasone) based on the treatments they received after undergoing sham-operation/LPS tracheal instillation. H&E staining and pulmonary edema indexes were used to evaluate lung injury severity. Alveolar exudate cells (AECs) were counted based on cell count in bronchoalveolar lavage fluid (BALF), and neutrophil percentage in BALF was measured using flow cytometry. Myeloperoxidase (MPO) activity in BALF was measured using enzyme-linked immunosorbent assay (ELISA), while the production of IL-1β, TNF-α, and IL-6 in the lung and secretion level of them in BALF were detected by quantitative polymerase chain reaction (qPCR) and ELISA. The effect of LJF, FF, and LF on the expression of Caspase-1 and IL-1β proteins in bone marrow derived macrophages (BMDMs) supernatant was assessed using Western blot method under various inflammasome activation conditions. In addition, the concentration of IL-1β and changes in lactatedehydrogenase (LDH) release levels in BMDMs supernatant after LJF, FF, and LF administration, respectively, were measured using ELISA. Furthermore, the effects of LJF, FF and LF on STING and IRF3 phosphorylation in BMDMs were detected by Western blot, and the mRNA changes of IFN-β, TNF-α, IL-6 and CXCL10 in BMDMs were detected by qPCR. ResultsLF significantly attenuated the damage to alveolar structures, pulmonary hemorrhage, and infiltration of inflammatory cells induced by LPS. This was evidenced by a decrease in lung index score and wet/dry weight ratio. Treatment with LF significantly reduced the total number of neutrophil infiltration by 75% as well as MPO activity by 88%. The efficacy of LF in reducing inflammatory factors IL-1β, TNF-α, and IL-6 in the lungs surpasses that of LJF or FF, approaching the effectiveness of dexamethasone. In BMDMs, the co-administration of 0.2 mg/mL of LJF and FF demonstrated superior inhibitory effects on the expression of nigericin-stimulated Caspase-1 and IL-1β, as well as the release levels of LDH, compared to individual treatments. Similarly, the combination of 0.5 mg/mL LJF and FF could better inhibit the phosphorylation levels of STING and IRF3 and the production of IFN-β, TNF-α, IL-6, and CXCL10 in response to ISD stimulation. ConclusionThe combination of LJF and FF increases the therapeutic effect on LPS-induced ALI, which may be mechanistically related to the combined effect inhibition of cyclic-GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) and NOD-like receptor family protein 3 (NLRP3) inflammasomes pathways by LJF and FF. Our study provides new medicine candidates for the clinical treatment of ALI.

Peptidomics-based identification of antihypertensive and antidiabetic peptides from sheep milk fermented using Limosilactobacillus fermentum KGL4 MTCC 25515 with anti-inflammatory activity: in silico, in vitro, and molecular docking studies.

This study investigated the synthesis of bioactive peptides from sheep milk through fermentation with Limosilactobacillus fermentum KGL4 MTCC 25515 strain and assessed lipase inhibition, ACE inhibition, α-glucosidase inhibition, and α-amylase inhibition activities during the fermentation process. The study observed the highest activities, reaching 74.82%, 70.02%, 72.19%, and 67.08% (lipase inhibition, ACE inhibition, α-glucosidase inhibition, and α-amylase inhibition) after 48h at 37°C, respectively. Growth optimization experiments revealed that a 2.5% inoculation rate after 48h of fermentation time resulted in the highest proteolytic activity at 9.88mg/mL. Additionally, fractions with less than 3kDa of molecular weight exhibited superior ACE-inhibition and anti-diabetic activities compared to other fractions. Fermentation of sheep milk with KGL4 led to a significant reduction in the excessive production of NO, TNF-α, IL-6, and IL-1β produced in RAW 267.4 cells upon treatment with LPS. Peptides were purified utilizing SDS-PAGE and electrophoresis on 2D gels, identifying a maximum number of proteins bands ranging 10-70kDa. Peptide sequences were cross-referenced with AHTPDB and BIOPEP databases, confirming potential antihypertensive and antidiabetic properties. Notably, the peptide (GPFPILV) exhibited the highest HPEPDOCK score against both α-amylase and ACE.

Discovery of sesquiterpenoids from the roots of Chloranthus henryi Hemsl. var. hupehensis (Pamp.) K. F. Wu and their anti-inflammatory activity by IKBα/NF-κB p65 signaling pathway suppression

Phytochemical analysis of Chloranthus henryi var. hupehensis roots led to the identification of a new eudesmane sesquiterpenoid dimer, 18 new sesquiterpenoids, and three known sesquiterpenoids. Among the isolates, 1 was a rare sesquiterpenoid dimer that is assembled by a unique oxygen bridge (C11-O-C8′) of two highly rearranged eudesmane-type sesquiterpenes with the undescribed C16 carbon framework. (+)-2 and (−)-2 were a pair of new skeleton dinorsesquiterpenoids with a remarkable 6/6/5 tricyclic ring framework including one γ-lactone ring and the bicyclo[3.3.1]nonane core. Their structures were elucidated using spectroscopic data, single-crystal X-ray diffraction analysis, and quantum chemical computations. In the LPS-induced BV-2 microglial cell model, 17 suppressed IL-1β and TNF-α expression with EC50 values of 6.81 and 2.76 µM, respectively, indicating its excellent efficacy in inhibiting inflammatory factors production in a dose dependent manner and without cytotoxicity. In subsequent mechanism studies, compounds 3, 16, and 17 could reduce IL-1β and TNF-α production by inhibiting IKBα/p65 pathway activation.

Cardiac hypertrophy that affects hyperthyroidism occurs independently of the NLRP3 inflammasome.

Cardiac hypertrophy (CH) is an adaptive response to maintain cardiac function; however, persistent stress responses lead to contractile dysfunction and heart failure. Although inflammation is involved in these processes, the mechanisms that control cardiac inflammation and hypertrophy still need to be clarified. The NLRP3 inflammasome is a cytosolic multiprotein complex that mediates IL-1β production. The priming step of NLRP3 is essential for increasing the expression of its components and occurs following NF-κB activation. Hyperthyroidism triggers CH, which can progress to maladaptive CH and even heart failure. We have shown in a previous study that thyroid hormone (TH)-induced CH is linked to the upregulation of S100A8, leading to NF-κB activation. Therefore, we aimed to investigate whether the NLRP3 inflammasome is involved in TH-induced CH and its potential role in CH pathophysiology. Hyperthyroidism was induced in NLRP3 knockout (NLRP3-KO), Caspase-1-KO and Wild Type (WT) male mice of the C57Bl/6J strain, aged 8-12 weeks, by triiodothyronine (7μg/100g BW, i.p.) administered daily for 14 days. Morphological and cardiac functional analysis besides molecular assays showed, for the first time, that TH-induced CH is accompanied by reduced NLRP3 expression in the heart and that it occurs independently of the NLRP3 inflammasome and caspase 1-related pathways. However, NLRP3 is important for the maintenance of basal cardiac function since NLRP3-KO mice had impaired diastolic function and reduced heart rate, ejection fraction, and fractional shortening compared with WT mice.

Isoliquiritigenin limits inflammasome activation of macrophage via docking into Syk to alleviate murine non-alcoholic fatty liver disease.

Isoliquiritigenin (ISL) is a chalcone-type flavonoid derived from the root of licorice with antioxidant, anti-inflammatory, anti-tumour and neuroprotective properties. ISL has been proven to downregulate the productions of IL-1β, TNF-α and IL-6 by macrophages. However, detailed molecular mechanisms of this modulation remain elusive. Here, ISL suppressed Syk phosphorylation and CD80, CD86, IL-1β, TNF-α and IL-6 expressions in lipopolysaccharide-stimulated macrophages exvivo. ApoC3-transgenic (ApoC3TG) mice had more activated macrophages. ISL was also able to downregulate the inflammatory activities of macrophages from ApoC3TG mice. Administration of ISL inhibited Syk activation and inflammatory activities of macrophages in ApoC3TG mice invivo. The treatment of ISL further alleviated MCD-induced non-alcoholic fatty liver disease (NAFLD) in wild-type and ApoC3TG mice, accompanied by less recruitment and activation of liver macrophages. Due to the inhibition of Syk phosphorylation, ISL-treated macrophages displayed less production of cytoplasmic ROS, NLRP3, cleaved-GSDMD and cleaved-IL-1β, suggesting less inflammasome activation. Finally, the molecular docking study demonstrated that ISL bound to Syk directly with the Kd of 1.273 × 10-8 M. When the Syk expression was knocked down by its shRNA, the inhibitory effects of ISL on activated macrophages disappeared, indicating that Syk was at least one of key docking-molecules of ISL. Collectively, ISL could alleviate MCD-induced NAFLD in mice involved with the inhibition of macrophage inflammatory activity by the blockade of Syk-induced inflammasome activation.

Proximity proteomics reveals UCH-L1 as an essential regulator of NLRP3-mediated IL-1β production in human macrophages and microglia

Activation of the NACHT, LRR, and PYD domains-containing protein 3 (NLRP3) inflammasome complex is an essential innate immune signaling mechanism. To reveal how human NLRP3 inflammasome assembly and activation are controlled, in particular by components of the ubiquitin system, proximity labeling, affinity purification, and RNAi screening approaches were performed. Our study provides an intricate time-resolved molecular map of different phases of NLRP3 inflammasome activation. Also, we show that ubiquitin C-terminal hydrolase 1 (UCH-L1) interacts with the NACHT domain of NLRP3. Downregulation of UCH-L1 decreases pro-interleukin-1β (IL-1β) levels. UCH-L1 chemical inhibition with small molecules interfered with NLRP3 puncta formation and ASC oligomerization, leading to altered IL-1β cleavage and secretion, particularly in microglia cells, which exhibited elevated UCH-L1 expression as compared to monocytes/macrophages. Altogether, we profiled NLRP3 inflammasome activation dynamics and highlight UCH-L1 as an important modulator of NLRP3-mediated IL-1β production, suggesting that a pharmacological inhibitor of UCH-L1 may decrease inflammation-associated pathologies.

Inflammasome-Independent Mechanism of NLRP3 Is Critical for Platelet GPIb-IX Function and Thrombosis.

Platelets link thrombosis and inflammation, but how platelets handle the endogenous intraplatelet inflammatory machinery is less well understood. NACHT, LRR, and PYD domain-containing protein 3 (NLRP3) is the central component of the interleukin (IL)-1-producing inflammasome. Elucidating the cell type-specific mechanism of NLRP3 in platelets may improve our understanding of thrombotic diseases. Ferric chloride-induced mesenteric arteriole thrombosis models, tail bleeding models, and microfluidic whole-blood perfusion were used to study thrombosis and hemostasis. Additionally, we utilized aggregometry, flow cytometry, immunoprecipitation, and western blotting to investigate glycoprotein (GP)Ib-IX-mediated platelet function and signaling. NLRP3-/- mice exhibited severely impaired thrombosis and hemostasis, whereas apoptosis-associated speck-like protein containing a CARD (ASC)-/-, caspase-1-/-, and Nlrp3 A350V/+ CrePF4 mice did not exhibit such changes. NLRP3-/- platelets exhibited reduced adhesion to injured vessel walls and collagen and impaired von Willebrand factor (vWF)-dependent translocation and rolling behavior. NLRP3 deficiency decreased botrocetin-induced platelet aggregation and the phosphorylation of key signaling molecules in the GPIb-IX pathway. Mechanistically, decreased cAMP/PKA activity led to reduced phosphorylation of NLRP3, thereby enabling the interaction between NLRP3 and filamin A. This interaction accelerated the dissociation of filamin A from GPIbα, which allowed a 14-3-3ζ-dependent increase in GPIb-IX affinity to vWF. Finally, platelet NLRP3 was found to largely regulate thrombotic disease models, such as models of stroke and deep vein thrombosis. NLRP3 promoted the function of the major platelet adhesion receptor GPIb-IX without involving NLRP3 inflammasome assembly or IL-1β production.

Sex-Specific Differences in Cytokine Production Capacity in Patients with Gout Compared to Controls

Gout, an inflammatory disease orchestrated by interleukin-1β activation and release, is more prevalent in men. The clinical profiles of patients with gout report differences by sex. This study aims to investigate sex-specific cytokine profiles in circulation and in stimulated peripheral blood mononuclear cells (PBMCs) of patients with gout and controls. Participants included in the gout group met the criteria of the American College of Rheumatology/European League Against Rheumatism (ACR/EULAR). The control group included individuals with varying levels of serum urate and absence of gout. PBMCs were stimulated in vitro for 24 h with various TLR ligands. Cytokines were determined in culture supernatants and plasma. Plasma IL-1Ra and high-sensitivity C-reactive protein (hsCRP) were higher in men with gout compared to men without gout whereas no significant differences in circulating cytokines were observed in women. PBMCs of patients with gout showed higher cytokine production of IL-1β, IL-1Ra, and TNF-α following 24 h stimulation, predominantly observed in women. We identified sex-specific cytokine production in gout in response to in vitro stimulation. While men with gout had higher levels of circulating cytokines, stimulated PBMCs of women with gout show an enhanced capacity for cytokine production. These data may suggest potentially different regulatory mechanisms of inflammation in men and women with gout.

Differential Response of Human Dendritic Cells upon Stimulation with Encapsulated or Non-Encapsulated Isogenic Strains of Porphyromonas gingivalis.

During periodontitis, the extracellular capsule of Porphyromonas gingivalis favors alveolar bone loss by inducing Th1 and Th17 patterns of lymphocyte response in the infected periodontium. Dendritic cells recognize bacterial antigens and present them to T lymphocytes, defining their activation and polarization. Thus, dendritic cells could be involved in the Th1 and Th17 response induced against the P. gingivalis capsule. Herein, monocyte-derived dendritic cells were obtained from healthy individuals and then stimulated with different encapsulated strains of P. gingivalis or two non-encapsulated isogenic mutants. Dendritic cell differentiation and maturation were analyzed by flow cytometry. The mRNA expression levels for distinct Th1-, Th17-, or T-regulatory-related cytokines and transcription factors, as well as TLR2 and TLR4, were assessed by qPCR. In addition, the production of IL-1β, IL-6, IL-23, and TNF-α was analyzed by ELISA. The encapsulated strains and non-encapsulated mutants of P. gingivalis induced dendritic cell maturation to a similar extent; however, the pattern of dendritic cell response was different. In particular, the encapsulated strains of P. gingivalis induced higher expression of IRF4 and NOTCH2 and production of IL-1β, IL-6, IL-23, and TNF-α compared with the non-encapsulated mutants, and thus, they showed an increased capacity to trigger Th1 and Th17-type responses in human dendritic cells.

NLRP3 Inflammasome in Acute and Chronic Liver Diseases.

NLRP3 (NOD-, LRR-, and pyrin domain-containing protein 3) is an intracellular complex that upon external stimuli or contact with specific ligands, recruits other components, forming the NLRP3 inflammasome. The NLRP3 inflammasome mainly mediates pyroptosis, a highly inflammatory mode of regulated cell death, as well as IL-18 and IL-1β production. Acute and chronic liver diseases are characterized by a massive influx of pro-inflammatory stimuli enriched in reactive oxygen species (ROS) and damage-associated molecular patterns (DAMPs) that promote the assemblage and activation of the NLRP3 inflammasome. As the major cause of inflammatory cytokine storm, the NLRP3 inflammasome exacerbates liver diseases, even though it might exert protective effects in regards to hepatitis C and B virus infection (HCV and HBV). Here, we summarize the current knowledge concerning NLRP3 inflammasome function in both acute and chronic liver disease and in the post liver transplant setting, focusing on the molecular mechanisms involved in NLRP3 activity.

Electroacupuncture alleviates the symptom of depression in mice by regulating the cGAS-STING-NLRP3 signaling.

To investigate the therapeutic effect of electroacupuncture (EA) on chronic and unpredictable mild stress (CUMS)-induced depression in mice and the underlying mechanism. Male C57BL/6 mice were randomly divided into 6 groups: Control, CUMS, CUMS+EA-placebo, CUMS+EA, CUMS+ ad-NC, CUMS+ ad-cGAS-shRNA. CUMS was utilized to establish the depression model in mice. The behavioral changes were determined by the forced swimming, open field, and sucrose preference experiments. The pathological changes in the hippocampus tissue were evaluated by HE staining. The release of TNF-α, IL-1β, IL-6, 5-HT, and NE in the hippocampus tissue was determined by ELISA. IBA-1 expression detected by the immunofluorescence was used to represent the activity of microglia. Western blot and RT-PCR were utilized to measure the expression of Bax, bcl-2, cGAS, STING, TBK1, IRF3, and NLRP3. The depression behavior in CUMS mice was significantly alleviated by the treatment of EA and cGAS-shRNA, accompanied by ameliorated hippocampus pathological changes, declined production of TNF-α, IL-1β, and IL-6, elevated secretion of 5-HT and NE, and inhibition on the activity of microglia. Furthermore, significantly elevated expression level of Bax, cGAS, STING, TBK1, IRF3, and NLRP3 and declined expression level of bcl-2 were observed in the CUMS+EA and CUMS+ ad-cGAS-shRNA groups. EA significantly mitigated the symptom of depression in mice, which was closely associated with the repressed neuroinflammation, increased monoamine concentration, inactivated microglia, and inhibited cGAS-STING-NLRP3 signaling.

Kahweol Inhibits Pro-Inflammatory Cytokines and Chemokines in Tumor Necrosis Factor-α/Interferon-γ-Stimulated Human Keratinocyte HaCaT Cells.

Atopic dermatitis (AD), marked by intense itching and eczema-like lesions, is a globally increasing chronic skin inflammation. Kahweol, a diterpene that naturally occurs in coffee beans, boasts anti-inflammatory, antioxidative, and anti-cancer properties. This research explores the anti-inflammatory action of kahweol on HaCaT human keratinocytes stimulated by tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ), focusing on key signal transduction pathways. Our results demonstrate that kahweol markedly reduces the production of IL-1β, IL-6, C-X-C motif chemokine ligand 8, and macrophage-derived chemokine in TNF-α/IFN-γ-activated HaCaT cells. Furthermore, it curtails the phosphorylation of key proteins in the mitogen-activated protein kinase (MAPK) pathways, including c-Jun N-terminal kinase, extracellular signal-regulated kinase, and p38. Additionally, kahweol impedes the phosphorylation and nuclear translocation of the NF-κB p65 subunit and constrains its DNA-binding capability. It also hampers the phosphorylation, nuclear translocation, and DNA-binding activities of signal transducer and activator of transcription 1 (STAT1) and STAT3. Collectively, these findings suggest that kahweol hinders the generation of cytokines and chemokines in inflamed keratinocytes by inhibiting the MAPK, NF-κB, and STAT cascades. These insights position kahweol as a promising agent for dermatological interventions, especially in managing inflammatory skin conditions such as AD.

TLR8-activating miR-146a-3p is an intermediate signal contributing to fetal membrane inflammation in response to bacterial LPS.

Preterm birth is the largest contributor to neonatal morbidity and is often associated with chorioamnionitis, defined as inflammation/infection of the fetal membranes (FMs). Chorioamnionitis is characterised by neutrophil infiltration of the FMs and is associated with elevated levels of the neutrophil chemoattractant, interleukin (IL)-8 and the proinflammatory cytokine, IL-1β. While FMs can respond to infections through innate immune sensors, such as toll-like receptors (TLRs), the downstream mechanisms by which chorioamnionitis arises are not fully understood. A novel group of non-classical microRNAs (miR-21a, miR-29a, miR-146a-3p, Let-7b) function as endogenous danger signals by activating the ssRNA viral sensors TLR7 and TLR8. In this study, the pro-inflammatory roles of TLR7/TLR8-activating miRs were examined as mediators of FM inflammation in response to bacterial lipopolysaccharide (LPS) using an in vitro human FM explant system, an in vivo mouse model of pregnancy, and human clinical samples. Following LPS exposure, miR-146a-3p was significantly increased in both human FM explants and wild-type mouse FMs. Expression of miR-146a-3p was also significantly elevated in FMs from women with preterm birth and chorioamnionitis. FM IL-8 and inflammasome-mediated IL-1β production in response to LPS was dependent on miR-146a-3p and TLR8 downstream of TLR4 activation. In wild-type mice, LPS exposure increased FM IL-8 and IL-1β production and induced preterm birth. In TLR7-/-/TLR8-/- mice, LPS exposure was able to initiate but not sustain preterm birth, and FM inflammation was reduced. Together, we demonstrate a novel signalling mechanism at the maternal-fetal interface in which TLR8-activating miR-146a-3p acts as an intermediate danger signal to drive FM inflammasome-dependent and -independent mechanisms of inflammation and, thus, may play a role in chorioamnionitis and subsequent preterm birth.

The Biologic IRL201805 Alters Immune Tolerance Leading to Prolonged Pharmacodynamics and Efficacy in Rheumatoid Arthritis Patients.

A homologue of binding immunoglobulin protein/BiP-IRL201805 alters the function of immune cells in pre-clinical in vivo and in vitro studies. The aim of the study was to select biomarkers that clearly delineate between RA patients who respond to IRL201805 and placebo patients and reveal the immunological mode of action of IRL201805 driving the extended pharmacodynamics observed in responding patients. Biomarkers that distinguished between responding patients and placebo patients included downregulation of serum interferon-γ and IL-1β; upregulation of anti-inflammatory mediators, serum soluble CTLA-4, and intracellular monocyte expression of IDO; and sustained increased CD39 expression on CD3+CD4+CD25hi CD127lo regulatory T cells. In the responding patients, selected biomarkers verified that the therapeutic effect could be continuous for at least 12 weeks post-infusion. In secondary co-culture, pre-infusion PBMCs cultured 1:1 with autologous PBMCs, isolated at later time-points during the trial, showed significantly inhibited IL-6 and IL-1β production upon anti-CD3/CD28 stimulation demonstrating IRL201805 alters the function of immune cells leading to prolonged pharmacodynamics confirmed by biomarker differences. IRL201805 may be the first of a new class of biologic drug providing long-term drug-free therapy in RA.

Heteroglycan of Annona muricata leaves: Extraction, characterization, modulation of the inflammatory response in raw 264.7 macrophages and peripheral Blood mononuclear cells

The natural products contained in Annona muricata leaves are characterized by their ability to effectively enhance the immune response without exhibiting toxicity. The immunomodulatory effect of natural polysaccharides is considered one of the most important bioactive functions. In this study, we investigated the antioxidant activities and immunological properties of heteroglycans extracted from A. muricata leaves using hot water followed by ethanol precipitation. The polysaccharide was characterized for its physicochemical properties (UV spectroscopy, FTIR, gas chromatography), antioxidant potential (scavenging properties, chelating ability, reducing capability, and protection against DNA damage), immunomodulatory activities, and production of pro-inflammatory cytokines using peripheral blood mononuclear cells (PBMCs) and RAW 264.7 cells. The results indicated that the heteroglycans consisted of Glu-Man-GalA-GluA-Xyl and GluA-Man-Glu, respectively, for low and high molecular weight water-soluble polysaccharides coded as LWPC and HWPC. They exhibited strong metal chelating and scavenging activities. Additionally, each sample provided protection against DNA damage induction, with LWPC exhibiting up to 73.1 % protection. LWPC and HWPC significantly down-regulated the expression of pro-inflammatory cytokines TNF-α, IL-1β, IL-6, and COX-2, as well as NO production. At 200 µg/mL, only LWPC increased the production of IL-1β and IL-6 in PBMCs. Therefore, LWPC and HWPC hold promise for inhibiting radical-induced and inflammatory disorders.

Minor Hydroxylated Triterpenoids Produced in Engineered Yeast by the Enzymes OSC and CYP716s from the Plant Enkianthus chinensis and Their Anti-Inflammatory and Hepatoprotective Activities.

Triterpenoids are a type of specialized metabolites that exhibit a wide range of biological activities. However, the availability of some minor triterpenoids in nature is limited, which has hindered our understanding of their pharmacological potential. To overcome this limitation, heterologous biosynthesis of triterpenoids in yeast has emerged as a promising and time-efficient production platform for obtaining these minor compounds. In this study, we analyzed the transcriptomic data of Enkianthus chinensis to identify one oxidosqualene cyclase (EcOSC) gene and four CYP716s. Through heterologous expression of these genes in yeast, nine natural pentacyclic triterpenoids, including three skeleton products (1-3) produced by one multifunctional OSC and six minor oxidation products (4-9) catalyzed by CYP716s, were obtained. Of note, we discovered that CYP716E60 could oxidize ursane-type and oleanane-type triterpenoids to produce 6β-OH derivatives, marking the first confirmed C-6β hydroxylation in an ursuane-type triterpenoid. Compound 9 showed moderate inhibitory activity against NO production and dose-dependently reduced IL-1β and IL-6 production at the transcriptional and protein levels. Compounds 1, 2, 8, and 9 exhibited moderate hepatoprotective activity with the survival rates of HepG2 cells from 61% to 68% at 10 μM.

Anti-Inflammatory Salicin Derivatives from the Barks of Salix tetrasperma.

The genus Salix L. is traditionally used in folk medicine to alleviate pain caused by various kinds of inflammation. In the present study, 10 undescribed salicin derivatives along with 5 known congeners were isolated from the barks of Salix tetrasperma, and their structures were elucidated by spectroscopic analyses, single-crystal X-ray diffraction, electronic circular dichroism (ECD) calculations, and chemical conversions. Compounds 4-6 significantly inhibited NO production in lipopolysaccharide (LPS)-induced RAW 264.7 macrophages, and the most active 4 obviously suppressed the production of IL-1β and IL-6 and decreased iNOS and COX-2 expression in a dose-dependent manner. Further Western blotting analysis revealed that the anti-inflammatory mechanism of 4 is possibly mediated through the MAPK and NF-κB signaling pathways.