IL-6 Secretion Research Articles

The Protective Role of Baicalin in the Regulation of NLRP3 Inflammasome in Different Diseases.

The NACHT, LRR, and PYD domains-containing protein 3 (NLRP3) inflammasome consists of pro-caspase-1, NLRP3 and apoptosis-related speckle-like protein (ASC). It can detect multiple microorganisms, endogenous danger signals and environmental stimulus including adenosine triphosphate (ATP), urate, cholesterol crystals, and so on, thereby forming activated NLRP3 inflammasome. During the course of the activation of NLRP3 inflammasome, pro-caspase-1 is transformed into activated caspase-1 that results in the maturation and secretion of interleukin-1beta (IL-1β) and IL-18. The dysfunction of NLRP3 inflammasome participates in multiple diseases such as liver diseases, renal diseases, nervous system diseases and diabetes. Baicalin is the primary bioactive component of Scutellaria baicalensis, which has been used since ancient times. Baicalin has many types of biological functions, such as anti-bacterial, anti-tumor and antioxidant. More and more evidence suggests that baicalin regulation of NLRP3 inflammasome is involved in different diseases. However, the mechanism is still elusive. Here, we reviewed the progress of baicalin regulation of NLRP3 inflammasome in many kinds of diseases to lay a foundation for future researches.

4-Anilinoquinazoline Derivatives as the First Potent NOD1-RIPK2 Signaling Pathway Inhibitors at the Nanomolar Range.

Inflammation is a defense mechanism that restores tissue damage and eliminates pathogens. Among the pattern recognition receptors that recognize danger or pathogenic signals, nucleotide oligomerization domains 1 and 2 (NOD1/2) have been identified to play an important role in innate immunity responses, and inhibition of NOD1 could be interesting to treat severe infections and inflammatory diseases. In this work, we identified the first selective NOD1 versus NOD2 pathway inhibitors at the nanomolar range based on a 4-anilinoquinazoline scaffold. We demonstrated that NOD1 inhibition occurs through the inhibition of receptor interacting protein kinase 2 (RIPK2), which is involved in its downstream signaling pathways. Compound 37 demonstrates no cytotoxicity, a selectivity for RIPK2 over epithelial and vascular endothelial growth factor receptors (EGFR/VEGFR), and a capacity to reduce pro-inflammatory cytokine IL-8 secretion. The structure of the RIPK2-compound 37 complex was resolved by crystallography. The 4-anilinoquinazoline scaffold offers novel perspectives to design NOD1-RIPK2 signaling inhibitors.

Cloperastine Reduces IL-6 Expression via Akt/GSK3/Nrf2 Signaling in Monocytes/Macrophages and Ameliorates Symptoms in a Mouse Sepsis Model Induced by Lipopolysaccharide.

Cloperastine (CLP) is a drug with a central antitussive effect that is used to treat bronchitis. Therefore, we have attempted to examine the anti-inflammatory effects of CLP. CLP reduced the secretion of interleukin (IL)-6, a pro-inflammatory cytokine, from RAW264.7 monocyte/macrophage-linage cells treated with lipopolysaccharide (LPS). IL-6 is a biomarker of sepsis and has been suggested to exacerbate its symptoms. We found that the intraperitoneal administration of CLP reduced IL-6 levels in the lungs and also improved hypothermia in mice with LPS-induced sepsis. CLP ameliorated kidney pathologies such as congestion and increased the survival rate of mice administered with a lethal dose of LPS. To reveal the mechanisms underlying the anti-inflammatory function of CLP, we analysed the intracellular signaling in LPS-treated RAW264.7 cells. CLP induced the phosphorylation of protein kinase B (Akt) and glycogen synthase kinase 3 (GSK3) and also increased the amount of nuclear factor erythroid-2-related factor 2 (Nrf2) in RAW264.7 cells with/without LPS. Wortmannin, an inhibitor of phosphoinositide 3-kinase (PI3K), reduced the upregulated phosphorylation levels of Akt and GSK3 and the increased amount of Nrf2. It also halted the reduction of IL-6 secretion caused by CLP. These results suggest that CLP has an anti-inflammatory function via Akt/GSK3/Nrf2 signaling and could be a candidate drug for the treatment of inflammatory diseases, including sepsis.

Inhibitory Effect of Luteolin on Spike S1 Glycoprotein-Induced Inflammation in THP-1 Cells via the ER Stress-Inducing Calcium/CHOP/MAPK Pathway.

The global SARS-CoV-2 outbreak has escalated into a critical public health emergency, with the spike glycoprotein S1 subunit of SARS-CoV-2 (spike-S1) linked to inflammation in lung tissue and immune cells. Luteolin, a flavone with anti-inflammatory properties, shows promise, but research on its effectiveness against long-COVID-related inflammation and spike protein-induced responses remains limited. This study aims to elucidate the underlying mechanisms of inflammation in THP-1 cells induced by the spike-S1. Additionally, it seeks to assess the potential of luteolin in mitigating inflammatory responses induced by the spike-S1 in a THP-1 macrophage model. The gene expression profiles of spike-S1 in THP-1 cells were analyzed by transcriptome sequencing. The inhibitory effect of luteolin on ER stress and inflammation in spike-S1-induced THP-1 cells was investigated using Western blotting, RT-PCR, and ELISA. The candidate genes (CAMK2A, SIGLEC7, PPARGC1B, SEC22B, USP28, IER2, and TIRAP) were upregulated in the spike-S1-induced THP-1 group compared to the control group. Among these, calcium/calmodulin-dependent protein kinase II alpha (CAMK2A) was identified as the most promising molecule in spike-S1-induced THP-1 cells. Our results indicate that the spike S1 significantly increased the expression of ER-stress markers at both gene and protein levels. Luteolin significantly reduced ER stress by decreasing the expression of ER-stress marker genes and ER-stress marker proteins (p < 0.01). Additionally, luteolin exhibited anti-inflammatory properties upon spike S1-induction in THP-1 cells by significantly suppressing IL-6, IL-8, and IL-1β cytokine secretion in a dose-dependent manner (p < 0.05). Furthermore, our results revealed that luteolin exhibited the downregulation of the MAPK pathway, as evidenced by modulating the phosphorylation of p-ERK1/2, p-JNK and p-p38 proteins (p < 0.05). The results from this study elucidate the mechanisms by which the spike S1 induces inflammation in THP-1 cells and supports the use of naturally occurring bioactive compounds, like luteolin, against inflammation-related SARS-CoV-2 infection.

Inflammasome activation links enteric Salmonella Typhimurium infection to a rapid, cytokine-dependent increase in intestinal mucin release

ABSTRACT The host restricts Salmonella enterica serovar Typhimurium infection of the gut via inflammasome-dependent sloughing of infected epithelial cells. Here we determined that concurrent caspase 1/11-dependent release of the goblet cell-derived mucin, Muc2, into the intestinal lumen also controls Salmonella burdens in infected mice. The increased release of mucins from goblet cells in the cecum and nearby proximal colon, and the subsequent thickening of the protective mucus barrier layer in the distal colon, were all dependent on the cytokines interleukin (IL)-18 and IL-22, as deficiencies in either cytokine resulted in reduced mucin secretion. Supplementation of IL-18 into IL-22 deficient mice restored mucin secretion, indicating that IL-22 acted upstream of IL-18 secretion during infection. In contrast, IL-18 and IL-22 independent signaling through Nlrp6 underlies only a modest, infection-induced increase in mucin secretion from goblet cells in the distal colon. These findings reveal that inflammasome signaling orchestrates multiple levels of protection centered on the intestinal epithelium, including pyroptosis and expulsion of infected enterocytes, as well as the release of mucins by goblet cells in the cecum and along the length of the colon. Our studies underscore the pivotal, multi-faceted role of inflammasome signaling in promoting host defense at the intestinal mucosal surface.

Acylated Anthocyanins From Black Carrots and Their Related Phenolic Acids Diminish Priming and Activation of the NLRP3 Inflammasome in THP-1 Monocytes.

Excessive activation of the nucleotide-binding oligomerization domain-like receptor pyrin domain-containing protein 3 (NLRP3) inflammasome contributes to chronic inflammation. Thus, targeting NLRP3 inflammasome activation by anthocyanins may prevent inflammatory diseases. Therefore, the present study determines the influence of a black carrot extract (BCE) with high amounts of acylated anthocyanins and their related phenolic acids on the NLRP3 inflammasome. THP-1 monocytes are pretreated with a BCE, cyanidin-3-glucoside (C3G), or hydroxycinnamic acids. NLRP3 inflammasome assembly is initiated by priming THP-1 monocytes with lipopolysaccharide and/or activating the NLRP3 inflammasome with nigericin. Flow cytometry is used to assess apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) speck formation, as well as ASC and NLRP3 protein expression. Caspase-1 activity is measured using a bioluminescent assay, and cytokine concentrations are determined by enzyme-linked immunosorbent assays (ELISA). C3G and phenolic acids diminish ASC and NLRP3 protein expression. In addition, C3G and phenolic acids attenuate ASC speck formation. Furthermore, the BCE and C3G decline caspase-1 activity. Consistently, IL-1β and IL-18 secretion are reduced upon NLRP3 inflammasome activation. The present study shows that a BCE with high amounts of acylated anthocyanins and their related phenolic acids diminish priming and activation of the NLRP3 inflammasome in THP-1 monocytes.

FuKe QianJin capsule alleviates endometritis via inhibiting inflammation and pyroptosis through modulating TLR4/ NF-κB /NLRP3 pathway

Ethnopharmacological relevanceFuke Qianjin Capsule (FKC), a traditional Chinese medicine commonly employed for treating endometritis, lacks reported treatment mechanisms. Aim of the studyThe aim of the present study was to explore the role and mechanism of FKC in lipopolysaccharide (LPS)-induced endometritis. Materials and methodsThe main active ingredients of FKC were identified via high-performance liquid chromatography (HPLC) in conjunction with standard substances. Prior to endometritis induction, Sprague Dawley female rats received FKC for 7 days. The endometritis model was established through an intrauterine injection of 1 mg/kg LPS. Concurrently, an LPS-induced RAW264.7 cell inflammation model was utilized, in which the cells were treated with serum containing Fuke Qianjin Capsule. Pathological alterations in the endometrium were assessed via H&E staining and transmission electron microscopy (TEM). The contents of MPO in uterine tissues, and NO release in cells, along with the secretion of IL-18, IL-1β, IL-6, and TNF-α in both tissues and cells, were determined via assay kits. The mRNA levels of Nlrp3, Caspase-1, Gsdmd, and Il-1β in uterine tissues and cells were analyzed via qPCR. The protein levels of TLR4, p65, p-P65, NLRP3, Caspase-1, GSDMD, and IL-1β in these samples were evaluated through Western blot analysis. Immunofluorescence was used to assess the protein levels of p-P65 and NLRP3 in uterine tissues and cells. ResultsFive primary active components of FKC were identified. Treatment with FKC in vivo mitigated endometrial pathological damage and significantly decreased the levels of MPO, IL-18, IL-1β, IL-6, and TNF-α, as well as the levels of Nlrp3, Caspase-1, Gsdmd, and Il-1β mRNA in tissue samples. Treatment with FKC inhibited the expression of TLR4, p-P65, NLRP3, Caspase-1, GSDMD, and IL-1β, as well as reduced NLRP3 protein fluorescence intensity, and inhibited P65 phosphorylation. In vitro findings demonstrated that FKC-containing serum reduced IL-18, IL-1β, IL-6, and TNF-α levels, as well as reduced Nlrp3, Caspase-1, Gsdmd, and Il-1β mRNA levels. In addition, FKC-containing serum inhibited the protein expression of TLR4, p-P65, NLRP3, Caspase-1, GSDMD, and IL-1β. FKC-containing serum also reduced NLRP3 protein fluorescence intensity and suppressed P65 phosphorylation. ConclusionFKC reverses the LPS induced NLRP3 inflammasome activation, and mitigates inflammation and pyroptosis through the modulation of the TLR4/NF-κB/NLRP3 pathway, thereby alleviating endometritis.

Regulation of the NLRP3 inflammasome by autophagy and mitophagy.

The NLRP3 inflammasome is a multiprotein complex that upon activation by the innate immune system drives a broad inflammatory response. The primary initial mediators of this response are pro-IL-1β and pro-IL-18, both of which are in an inactive form. Formation and activation of the NLRP3 inflammasome activates caspase-1, which cleaves pro-IL-1β and pro-IL-18 and triggers the formation of gasdermin D pores. Gasdermin D pores allow for the secretion of active IL-1β and IL-18 initiating the organism-wide inflammatory response. The NLRP3 inflammasome response can be beneficial to the host; however, if the NLRP3 inflammasome is inappropriately activated it can lead to significant pathology. While the primary components of the NLRP3 inflammasome are known, the precise details of assembly and activation are less well defined and conflicting. Here, we discuss several of the proposed pathways of activation of the NLRP3 inflammasome. We examine the role of subcellular localization and the reciprocal regulation of the NLRP3 inflammasome by autophagy. We focus on the roles of mitochondria and mitophagy in activating and regulating the NLRP3 inflammasome. Finally, we detail the impact of pathologic NLRP3 responses in the development and manifestations of pulmonary disease.

High-CBD Extract (CBD-X) in Asthma Management: Reducing Th2-Driven Cytokine Secretion and Neutrophil/Eosinophil Activity.

Asthma is a chronic inflammatory disorder of the airways affecting over 10% of the global population. It is characterized by airway inflammation, mucus hypersecretion, and bronchial hyperresponsiveness, driven predominantly by type 2 helper T cells (Th2) and type 2 innate lymphoid cells (ILC2s) in a subset of patients. However, a significant portion of asthmatic individuals present with "type 2-low" asthma that is often refractory to standard inhaled corticosteroid (ICS) therapy. Therefore, developing innovative therapeutic strategies has become essential. Recent studies have highlighted cannabidiol (CBD) as a promising anti-inflammatory agent capable of modulating immune responses. This study investigates the therapeutic potential of a high-CBD extract (CBD-X) in asthma. We evaluated the effects of CBD-X on cells involved in asthma pathogenesis using primary human Th2 cells, neutrophils, and asthma mouse model. Our findings indicate that CBD-X extract inhibits Th2 differentiation and reduces the secretion of IL-5 and IL-13, which are crucial cytokines in asthma. Additionally, CBD-X significantly reduces pro-inflammatory cytokines IL-8 and IL-6 in neutrophils and impairs their migration, a critical step in airway inflammation. In a murine asthma model, CBD-X administration led to marked downregulation of IgE and pro-asthmatic cytokines, along with reduced leukocyte, eosinophil, and neutrophil infiltration in lung tissues. These results suggest that CBD-X extract could offer a novel and complementary approach to managing both type 2-high and type 2-low asthma by targeting key inflammatory pathways and modulating immune cell behavior.

Heme Oxygenase-1 Protected Against Severe Acute Pancreatitis by Inhibiting Inflammatory Response

Abstract Background Activation of NLPR3 inflammasome promotes the maturation and secretion of IL-1β and IL-18, leading to a series of inflammatory reactions, while inhibition of NLRP3 inflammasome alleviates the severity of Severe Acute Pancreatitis (SAP). An inducible enzyme responsible for heme decomposition, heme oxygenase-1 (HO-1), has anti-inflammatory, antioxidant, and anti-proliferative effects. HO-1 activity profoundly affects the host ability to forbear infection by reducing tissue damage or affecting resistance and increasing the capacity to pathogen load. We postulated that hemin, a strong HO-1 inducer, could decrease NLRP3 inflammasome activation, which would alleviate the severity of SAP and acute lung injury caused by pancreatitis. Methods By administering intraperitoneal injections of caerulein (Cae) and lipopolysaccharide (LPS), the SAP rat model was created. Then, the SAP rats were pretreated with Hemin or zinc protoporphyrin IX (Znpp, a HO-1 inhibitor) to stimulate or inhibit the HO-1 enzyme respectively, and the effects and mechanisms were investigated. Results The pancreas and lung tissue of the SAP rats suffered considerable pathological damage after Cae and LPS injection, with significant increases of amylase, lipase, IL-1β and IL-18 levels in the serum. Hemin pretreatment decreased IL-1β and IL-18 release in the serum and prevented pancreatic and pulmonary damage. Hemin dramatically reduced oxidative stress, downregulated the expression of NLRP3, ASC, and Caspase-1, and elevated HO-1 expression. On the contrary, there were no discernible changes between the SAP control and Znpp treated groups. Conclusion These results showed that hemin prevented Cae and LPS-induced lung and pancreatic injury through suppression of the inflammatory response. The impact of hemin on the activity of the NLRP3 inflammasome was depending critically on HO-1 activity. The protective role and mechanism HO-1 against the acute and severe inflammatory responses may provide a novel and effective therapeutic approach for SAP treatment.

METTL3 mediates m6A modification of lncRNA CRNDE to promote ATG10 expression and improve brain ischemia/reperfusion injury through YTHDC1

BackgroundIschemia/reperfusion (I/R) injury is a severe brain disorder with currently limited effective treatments. This study aims to explore the role of N6-methyladenosine (m6A) modification and associated regulatory factors in I/R to identify potential therapeutic targets.MethodsWe utilized a middle cerebral artery occlusion (MCAO) rat model and SH-SY5Y cells subjected to oxygen-glucose deprivation/reoxygenation (OGD/R) to assess m6A levels and investigate the impact of METTL3 overexpression on long non-coding RNA (lncRNA) CRNDE expression. The effects of silencing lncRNA CRNDE on the interaction between YTHDC1 and ATG10 mRNA, as well as the stability of ATG10 mRNA, were evaluated. Additionally, apoptosis rates, pro-inflammatory and anti-inflammatory factor levels, ATG10 expression, and autophagic activity were analyzed to determine the effects of METTL3. The reverse effects of YTHDC1 overexpression were also examined.ResultsMCAO rats and OGD/R-treated SH-SY5Y cells exhibited reduced m6A levels. METTL3 overexpression significantly inhibited lncRNA CRNDE expression. Silencing lncRNA CRNDE mitigated OGD/R-induced apoptosis and inflammation in SH-SY5Y cells, while enhancing autophagy and stabilizing ATG10 mRNA. METTL3 overexpression decreased cell apoptosis, reduced the levels of pro-inflammatory cytokines TNF-α, IL-1β, IL-6, and increased IL-10 secretion. Furthermore, METTL3 overexpression upregulated ATG10 expression and promoted autophagy. Conversely, lncRNA CRNDE overexpression negated these effects.ConclusionThe inhibition of lncRNA CRNDE affects the interaction between YTHDC1 and ATG10 mRNA and stabilizes ATG10 mRNA, mediated by METTL3 overexpression. These findings suggest that targeting lncRNA CRNDE to reduce apoptosis, inhibit inflammation, increase ATG10 expression, and enhance autophagy could offer new therapeutic strategies for I/R injury.

Protective Effects of a Dihydrodiazepine Against Endotoxin Shock Through Suppression of TLR4/NF-κB/IRF3 Signaling Pathways.

Sepsis and septic shock are life-threatening systemic inflammatory conditions and among the most frequent causes of morbidity and mortality globally. Preclinical evidence has identified a number of diazepine-based compounds with therapeutic potential in inflammatory diseases. However, the potential anti-inflammatory properties of diazepines in the overwhelming immune response during sepsis have been rarely examined. Thus, the current study aimed to identify a new diazepine compound with therapeutic potential in sepsis. Assessing the inflammatory response of macrophages to Lipopolysaccharides (LPS) in vitro identified 2-[7-(trifluoromethyl)-2,3-dihydro-1H-1,4-diazepin-5-yl]phenol (2-TDDP) as a potential anti-inflammatory agent. It reduced secretion of Interleukin-1β (IL-1β), IL-6, IL-12p70, IL-18, Tumor necrosis factor-α (TNF-α), Interferon-γ (IFN-γ), IFN-β, and increased the secretion of IL-10. In a mouse model of LPS-induced endotoxin shock, 2-TDDP reduced mortality and attenuated inflammation-induced tissue injury in the spleen, liver, kidney, and lung. This was accompanied by reduced serum levels of IL-1β, IL-6, IL-12p70, TNF-α, IFN-γ, IFN-β, and increased levels of IL-10. Importantly, 2-TDDP suppressed the Toll-like receptor 4 (TLR4)/Nuclear factor-κB (NF-κB) and TLR4/Interferon regulatory factor 3 (IRF3) signaling pathways through a reduction in the expression of TLR4, Myeloid differentiation primary response 88 (MyD88), P65, and TNF receptor-associated factor 3 (Traf3). Moreover, 2-TDDP suppressed the expression of CD86, Programmed death-ligand 1 (PD-L1) and C5a receptor (C5aR), but not Major histocompatibility complex II (MHCII). Analysis of splenic lymphocyte populations revealed a decrease in the number of CD4+, CD8+, and B cells. Collectively, these findings introduced the dihydrodiazepine 2-TDDP as a new anti-inflammatory agent with potent therapeutic potential in endotoxin shock, paving an avenue for future clinical application.

CCL21 Induces Plasmacytoid Dendritic Cell Migration and Activation in a Mouse Model of Glioblastoma

Dendritic cells (DCs) are professional antigen-presenting cells that are traditionally divided into two distinct subsets: myeloid DCs (mDCs) and plasmacytoid DCs (pDCs). pDCs are known for their ability to secrete large amounts of cytokine type I interferons (IFN- α). In our previous work, we have demonstrated that pDC infiltration promotes glioblastoma (GBM) tumor immunosuppression through decreased IFN-α secretion via TLR-9 signaling and increased suppressive function of regulatory T cells (Tregs) via increased IL-10 secretion, resulting in poor overall outcomes in mouse models of GBM. Further dissecting the overall mechanism of pDC-mediated GBM immunosuppression, in this study, we identified CCL21 as highly upregulated by multiple GBM cell lines, which recruit pDCs to tumor sites via CCL21-CCR7 signaling. Furthermore, pDCs are activated by CCL21 in the GBM microenvironment through intracellular signaling of β-arrestin and CIITA. Finally, we found that CCL21-treated pDCs directly suppress CD8+ T cell proliferation without affecting regulatory T cells (Tregs) differentiation, which is considered the canonical pathway of immunotolerant regulation. Taken together, our results show that pDCs play a multifaced role in GBM immunosuppression, and CCL21 could be a novel therapeutic target in GBM to overcome pDC-mediated immunosuppression.

Ginseng fruit rare saponins (GFRS) improved inflammatory response: In vitro and in vivo assessment

Inflammation is the body's protective immune response to tissue damage. Ginseng has a long history of medicinal use, and its active ingredient ginsenosides have anti-inflammatory effects. Ginseng fruit rare saponins (GFRS) is a transformation product of ginseng saponins and rich in a variety of rare saponins. We used HPLC-DAD method to study GFRS rare saponins with ginsenoside F4, R-Rg3, SRg3, Rk1, Rg6, Rg5, Rk3 and Rh4. However, there is no study on the use of GFRS to reduce skin inflammation. This study enriched the action pathway of GFRS through network pharmacology and revealed the anti-inflammatory effect of GFRS for the first time. In vitro experiments showed that GFRS could significantly reduce the release of NO in lipopolysaccharide (LPS) -induced RAW264.7 cells and HaCaT cells, and reduce the secretion and expression of inflammation-related factors Interleukin-6 (IL-6), Tumor necrosis factor-α (TNF-α) and Interleukin-17 A (IL-17 A), thereby reducing cell inflammatory damage. In the imiquimod (IMQ) -induced mouse inflammatory model, the therapeutic effect of GFRS on the pathogenesis of psoriasis-like dermatitis was studied. In vivo experiments showed that the skin erythema, scales, thickness and inflammatory infiltration of GFRS-treated mice were reduced, and the psoriasis area severity index score was significantly lower than that of IMQ group. GFRS restored IMQ-induced spleen size and reduced the secretion and expression of TNF-α, IL-6, Interferon-γ (IFN-γ) and IL-17 A in serum. In summary, our results demonstrate that GFRS alleviates IMQ-induced dermatitis symptoms, effectively reduces the secretion of inflammatory factors, and inhibits IL-17 A expression.

Modulation of IFN-γ induced macrophage inflammatory responses via indomethacin-loaded NLCs for OA management

Macrophages are the main cells present in the synovial membrane. They play an important role in the development and progression of osteoarthritis (OA). After the establishment of the disease macrophages mostly adopt a pro-inflammatory secretory phenotype (OA phenotype) further inducing cartilage degradation. Indomethacin (IND) is a non-steroidal anti-inflammatory drug (NSAID) able to inhibit the synthesis of prostaglandins mediated by both cyclooxygenase isoforms depicting a potent anti-inflammatory capacity. However, the lack of specificity and short half-like of free drugs within the joint cavity limits its utility in controlling inflammation after intra-articular administration. This study aims at developing IND loaded glycosylated nanostructured lipid carriers (NLCs) to selectively target macrophages and promote their reprogramming to an anti-inflammatory phenotype. This approach focused on the local administration of the NLCs, offers a promising therapeutic strategy for treating OA by modulating the inflammatory environment within the joint. NLCs will be designed by combining experimental and in silico docking analyses, and thoroughly characterized to obtain drug delivery systems with high stability and suitable physicochemical properties. The proposed mannose-functionalized systems exhibited adequate particle sizes (≈ 70 nm) and positive surface charges (> 20 mV) to be efficiently retained in the joint cavity. Moreover, the developed NLCs demonstrated effective and specific uptake by OA-like macrophages leading to a significant decrease in the secretion of the pro-inflammatory cytokines IL-6, IL-8 and TNF-α similarly to the free drug. Therefore, these systems effectively reprogrammed OA-associated macrophages to adopt a more regenerative phenotype, offering a promising strategy for managing inflammation in OA.

Methotrexate promotes the release of granulocyte–macrophage colony-stimulating factor from rheumatoid arthritis fibroblast-like synoviocytes via autocrine interleukin-1 signaling

BackgroundActivated fibroblast-like synoviocytes (FLS) are drivers of synovitis and structural joint damage in rheumatoid arthritis (RA). Despite the use of disease-modifying drugs, only about 50% of RA patients reach remission in real-world settings. We used an unbiased approach to investigate the effects of standard-of-care methotrexate (MTX) and a Janus kinase inhibitor, tofacitinib (TOFA), on gene expression in RA-FLS, in order to identify untargeted disease mediators.MethodsPrimary RA-FLS were activated by stimulation with interleukin-1β (IL-1β) or platelet-derived growth factor + IL-1β in the presence or absence of MTX or TOFA, with or without additional inhibitors. Co-cultures of synovial cells were performed in direct and indirect systems. Cells were collected for RNA sequencing or qPCR, and supernatants were analyzed for protein concentrations.ResultsSix thousand three hundred fifty genes were differentially expressed, the majority being upregulated, in MTX-treated activated RA-FLS and 970 genes, the majority being downregulated, in TOFA-treated samples. Pathway analysis showed that MTX had largest effects on ‘Molecular mechanisms of cancer’ and TOFA on ‘Interferon signaling’. Targeted analysis of disease-associated genes revealed that MTX increased the expression of cell cycle-regulating genes but also of pro-inflammatory mediators like IL-1α (IL1A) and granulocyte–macrophage colony-stimulating factor, GM-CSF (CSF2). The MTX-promoted expression of CSF2 in activated RA-FLS peaked at 48 h, could be mediated via either NF-κB or AP-1 transcription factors, and was abrogated by IL-1 inhibitors (IRAK4 inhibitor and anakinra). In a co-culture setting, MTX-treatment of activated RA-FLS induced IL1B expression in macrophages.ConclusionsMTX treatment induces secretion of IL-1 from activated RA-FLS which by autocrine signaling augments their release of GM-CSF. This unexpected effect of MTX might contribute to the persistence of synovitis.

Complement factor H is an ICOS ligand modulating Tregs in the glioma microenvironment.

The survival rate of glioma patients has not significantly increased in recent years despite aggressive treatment and advances in immunotherapy. The limited response to treatments is partially attributed to the immunosuppressive tumor microenvironment, where regulatory T cells (Tregs) play a pivotal role in immunological tolerance. In this study, we investigated the impact of complement factor H (FH) on Tregs within the glioma microenvironment and found that FH is an ICOS ligand. The binding of FH to this immune checkpoint molecule promoted the survival and function of Tregs and induced the secretion of TGF-beta (TGF-β) and IL-10, while also suppressing T-cell proliferation. We further demonstrated that cancer cells in human and mouse gliomas directly produce FH. Database investigations revealed that upregulation of FH expression was associated with the presence of Tregs and correlated with worse prognosis for glioma patients. We confirmed the effect of FH on glioma development in a mouse model, where FH knockdown was associated with decrease in number of ICOS+ Tregs and demonstrated a tendency of prolonged survival (p=0.064). Since the accumulation of Tregs represents a promising prognostic and therapeutic target, evaluating FH expression should be considered when assessing the effectiveness of and resistance to immunotherapies against glioma.

The impact of cryopreservation on cytokine secretion and polyfunctionality in human PBMCs: a comparative study.

Human peripheral blood mononuclear cells (hPBMCs) are widely used in fundamental research and clinical applications as studying their responses to in vitro activation is an effective way to uncover functional alterations and disease associated phenotypes. However, the availability of samples in large numbers at a specific time and location remains challenging, hence they often might preferably be collected and cryopreserved for later analysis. While the effect of cryopreservation on viability and cell surface expression is well established, changes in activity and cytokine secretion still lead to conflicting results as it is often measured in bulk or within the cells. Here, we used our platform for dynamic single-cell multiplexed cytokine secretion measurement and compared it to a traditional intracellular cytokine staining to quantify the effect of cryopreservation on cytokine secretion and expression of individual hPBMCs. Following stimulation with LPS or anti-CD3/CD28 antibodies for up to 36 or 72 h incubation, we observed distinct alterations in cytokine responses due to cryopreservation when comparing to fresh samples, but also remarkable consistencies for some cytokines and parameters. In short, the frequencies of cytokine-secreting cells in cryopreserved samples were lower for IL-6 (LPS), IL1-β (CD3/CD28) and IFN-γ (CD3/CD28), while the frequency and dynamics of IL-8 secretion were strongly impacted in all cases. We observed a large disconnect between cytokine expression and secretion for TNF-α, where the expression dramatically increased after cryopreservation, but actual secretion was, in comparison, remarkably stable. The polyfunctionality of single cells was altered by cryopreservation in specific co-secreting populations led by the effects on IL-6 or IL-8 secretion. Among immune cells, cryopreservation seemed to affect lymphocytes and monocytes differently as effects appeared early on in lymphocytes while generally observed in later time points in monocytes. Together, this study offers an in-depth quantitative insight into the biological behavior of immune cells in response to cryopreservation and stimulation, further providing some insights into conflicting results in theliterature as well as guidelines for researchers planning to assess cytokine-secreting from frozen hPBMCs in immunological research or clinical applications.

Hypoxia-Induced Inflammation in In Vitro Model of Human Blood-Brain Barrier: Modulatory Effects of the Olfactory Ensheathing Cell-Conditioned Medium.

Hypoxia compromises the integrity of the blood-brain barrier (BBB) and increases its permeability, thereby inducing inflammation. Olfactory ensheathing cells (OECs) garnered considerable interest due to their neuroregenerative and anti-inflammatory properties. Here, we aimed to investigate the potential modulatory effects of OEC-conditioned medium (OEC-CM) on the response of human brain microvascular endothelial cells (HBMECs), constituting the BBB, when exposed to hypoxia. HBMECs were utilized to establish the in vitro BBB model. OECs were isolated from mouse olfactory bulbs, and OEC-CM was collected after 48h of culture. The effect of OEC-CM treatment on the HBMEC viability was evaluated under both normoxic and hypoxic conditions at 6h, 24h, and 30h. Western blot and immunostaining techniques were employed to assess NF-κB/phospho-NF-κB expression. HIF-1α, VEGF-A, and cPLA2 mRNA expression levels were quantified using digital PCR. ELISA assays were performed to measure PGE2, VEGF-A, IL-8 secretion, and cPLA2 specific activity. The in vitro formation of HBMEC capillary-like structures was examined using a three-dimensional matrix system. OEC-CM attenuated pro-inflammatory responses and mitigated the HIF-1α/VEGFA signaling pathway activation in HBMECs under hypoxic condition. Hypoxia-induced damage of the BBB can be mitigated by novel therapeutic strategies harnessing OEC potential.

Features of subset composition and functional activity of blood lymphocytes in tick-borne infections of different etiologies

Aim. To perform a comparative assessment of subset composition and functional activity of peripheral blood lymphocytes in patients with tick-borne encephalitis (TBE) and ixodid tick-borne borreliosis (ITBB) in the acute phase of the disease.Materials and methods. The study involved 22 patients with febrile and meningeal TBE, 15 patients with ITBB with and without erythema, and 11 healthy controls. Subset composition of blood lymphocytes was determined by flow cytometry. The blast transformation assay was applied to assess lymphocyte proliferation. Cytokine-producing activity of cells was studied in 24-hour incubated mononuclear cell cultures. Cytokine concentrations (interleukin (IL)-2, IL-4, IL-10, interferon (IFN)γ) were determined in the supernatants by the enzyme-linked immunosorbent assay (ELISA).Results. Patients with TBE demonstrated an increase in the proportion of helper – inducer T-cells, a pronounced decrease in the proportion and absolute count of cytotoxic T cells, and low T lymphocyte count compared to the control values. The study in ITBB patients revealed an increase in the helper – inducer T-cell count and the proportion of NK-cells, a decrease in the cytotoxic T cell count, and the T lymphocyte count comparable to normal values. The most significant decrease in the levels of phytohemagglutinin-induced lymphocyte proliferation was found in patients with TBE. Patients of both groups showed a decrease in IL-2 secretion in the mononuclear cell culture, a rise in IL-4 and IL-10 production, and IFNγ production levels comparable to control values.Conclusion. The study of TBE patients revealed relative lymphocytopenia with changes in the subset composition of lymphocytes characterized by an increase in the proportion of helper – inducer T-cells and a decrease in the absolute cytotoxic T lymphocyte count. Patients with ITBB demonstrated an increase in the proportion of NK-cells and a more pronounced imbalance in the T-helper / cytotoxic T lymphocyte ratio. Changes in the functional phenotype of lymphocytes, regardless of the etiology of tick-borne infection, were characterized by reduced proliferative reserve, low IL-2 secretion, increased IL-4 and IL-10 production, and depressed reactivity of lymphocytes with respect to IFNγ secretion.