Secretion Of Interleukin Research Articles

Abstract C043: Alprazolam abrogates the secretion of proinflammatory cytokines in PDAC CAFs

Abstract The present study sets out to determine how commonly prescribed triazolobenzodiazepines (triazoloBZDs) modulate the tumor microenvironment in pancreatic ductal adenocarcinoma (PDAC), and how this can be leveraged therapeutically to improve patient outcomes. PDAC is one of the most lethal cancer subtypes with a 5-year survival rate of ∼13%. The poor prognosis of this disease is, in part, attributable to the dense, fibrotic tumor microenvironment (TME), and lack of durable responses to standard-of-care treatment options. A recent study from our lab reported that over 40% of PDAC patients at Roswell Park are prescribed benzodiazepines (BZDs) to manage anxiety, nausea, and other disease-related side effects. Importantly, survival outcomes in these patients varied in a compound-specific manner, as the usage of the n-unsubstituted BZD lorazepam was associated with poorer outcomes, while usage of the n-substituted triazoloBZD alprazolam was associated with improved progression-free survival. In vitro and in vivo data from the aforementioned study suggested that these findings may be a result of BZD-specific regulation of interleukin-6 (IL6), a proinflammatory cytokine with a known role in PDAC pathogenesis. More specifically, alprazolam-treated cancer-associated fibroblasts (CAFs) exhibited a potent reduction in IL6 transcription and secretion, while lorazepam treatment increased IL6 production. Based on these data, we hypothesized that triazoloBZDs may uniquely reduce intratumoral inflammation to alleviate immunosuppression in PDAC. We now find that triazoloBZDs such as alprazolam suppress the secretion of several proinflammatory cytokines (IL6, CCL2, CXCL12, IL8) by CAFs, the stromal components of the PDAC TME responsible for fibrosis and exclusion of immune infiltrates. Furthermore, we have observed that this phenotype is likely a result of triazoloBZD-mediated inhibition of the platelet- activating factor (PTAFR) signaling cascade. Investigations are currently underway to determine the impact of triazoloBZDs on regulating the immune landscape of the PDAC TME in vivo. Altogether, these findings provide a basis to inform clinical selection of BZDs for symptom management and present a novel therapeutic opportunity to repurpose FDA-approved compounds for treatment of PDAC. Citation Format: Hunter D. Reavis, Arwen A. Tisdale, Kathryn E. Maraszek, Michael E. Feigin. Alprazolam abrogates the secretion of proinflammatory cytokines in PDAC CAFs [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr C043.

Functional characterization of OR51B5 and OR1G1 in human lung epithelial cells as potential drug targets for non-type 2 lung diseases

Abstract Background Hypersensitivity to odorants like perfumes can induce or promote asthma with non-type 2 inflammation for which therapeutic options are limited. Cell death of primary bronchial epithelial cells (PBECs) and the release of the pro-inflammatory cytokines interleukin-6 (IL-6) and IL-8 are key in the pathogenesis. Extra-nasal olfactory receptors (ORs) can influence cellular processes involved in asthma. This study investigated the utility of ORs in epithelial cells as potential drug targets in this context. Methods We used the A549 cell line and primary bronchial epithelial cells using air–liquid interface culture system (ALI-PBECs). OR expression was investigated by RT-PCR, Western blot, and Immunofluorescence. Effects of OR activation by specific ligands on intracellular calcium concentration, cAMP, Phospholipase C (PLC), cell viability, and IL-6 and IL-8 secretion were analyzed by calcium imaging, enzyme immunoassays, Annexin V/ propidium iodide -based fluorescence-activated cell staining or by ELISA, respectively. Results By screening A549 cells, the OR51B5 agonists Farnesol and Isononyl Alcohol and the OR1G1 agonist Nonanal increased intracellular Ca2 + . OR51B5 and OR1G1 mRNAs and proteins were detected. Both receptors showed a preferential intracellular localization. OR51B5- but not OR1G1-induced Ca2 + dependent on both cAMP and PLC signaling. Farnesol, Isononyl Alcohol, and Nonanal, all reduced cell viability and induced IL-8 and IL-6 release. The data were verified in ALI-PBECs. Conclusion ORs in the lung epithelium might be involved in airway-sensitivity to odorants. Their antagonism could represent a promising strategy in treatment of odorant-induced asthma with non-type 2 inflammation. Graphical Abstract

More powerful dysregulation of Helicobacter pylori East Asian-type CagA on intracellular signalings.

Chronic infection by Helicobacter pylori strains expressing cytotoxin-associated gene A (CagA) are the strongest risk factor for gastric cancer. CagA can be classified into East Asian-type and Western-type (CagAE and CagAW), with CagAE being more closely associated with gastric cancer. This study aimed to investigate the impact of CagAE on intracellular signaling pathways to explain its high oncogenicity. Mutant H. pylori strains expressing either CagAE or CagAW were generated by transforming CagAE/W-expression plasmid into CagA-deleted G27 strain (G27ΔCagA). In human gastric epithelial cells (GES-1) infection, CagAE induced more severe cytopathic changes, including higher interleukin-8 (IL-8) secretion, reduced cell viability, more pronounced "hummingbird phenotype" alterations, and increased cell migration and invasion compared to CagAW. Transcriptome analysis revealed that CagAE had a stronger effect on the up-regulation of key intracellular processes, including tumor necrosis factor-ɑ (TNF-ɑ) signal pathway via nuclear factor kappa-B (NF-κB), inflammatory response, interferon-γ (IFN-γ) response, hypoxia, ultraviolet (UV) response, and Kirsten Rat Sarcoma Viral Oncogene Homolog (KRAS) signaling. A significant upregulation of hypoxia-related genes was a notable feature of CagAE. GES-1 cells infected with CagAE exhibited more severe intracellular hypoxia and higher levels of reactive oxygen species (ROS) than those infected with CagAW. Inhibition of hypoxia-inducible factor-1α (HIF-1α), which blocks hypoxia signaling, mitigated CagAE-induced cell migration, emphasizing the role of hypoxia in mediating CagAE effects. The study provides transcriptome evidence of CagA-associated intracellular regulation during H. pylori infection, demonstrating that CagAE exerts stronger effects on intracellular signaling than CagAW. These findings offer insights into the heightened carcinogenic potential of CagAE in H. pylori-induced gastric cancer.

Evaluation of mucosal barrier disruption due to Staphylococcus lugdunensis and Staphylococcus epidermidis exoproteins in patients with chronic rhinosinusitis.

Chronic rhinosinusitis (CRS) is a persistent inflammatory condition of the sinus mucosa. While Staphylococcus aureus has been shown to play a significant role in mucosal barrier disruption in CRS patients, coagulase-negative staphylococci (CoNS) such as Staphylococcus epidermidis and Staphylococcus lugdunensis are also implicated in CRS pathophysiology. This study investigates the effects of exoproteins secreted by planktonic and biofilm forms of clinical isolates of S. epidermidis and S. lugdunensis on the nasal epithelial barrier. Thirty-one clinical isolates of CoNS were grown in planktonic and biofilm forms, and their exoproteins were concentrated. The epithelial barrier structure was assessed by measuring transepithelial electrical resistance (TEER) and the permeability of fluorescein isothiocyanate-dextran. Toxicity and inflammatory response were also studied. Our findings demonstrate that exoproteins from all planktonic forms of S. lugdunensis disrupted the mucosal barrier, whereas only nine of 16 biofilm-derived exoproteins had similar effects. Conversely, 11 of 15 exoproteins from planktonic S. epidermidis significantly disrupted barrier integrity; however, biofilm exoproteins did not. The study also showed that some exoproteins from planktonic S. epidermidis significantly reduced cell viability, while exoproteins from planktonic and biofilm forms of S. lugdunensis and biofilm S. epidermidis did not induce any statistically significant change in cell viability. Notably, four of 16 biofilm exoproteins from S. lugdunensis induced higher interleukin-6 (IL-6) secretion, whereas none of the S. epidermidis isolates showed a significant increase in IL-6 secretion. Our results suggest that CoNS exoproteins may contribute to CRS etiopathogenesis.

Amino acid deletions at positions 893 and 894 of cytotoxin-associated gene A protein affect Helicobacter pylori gastric epithelial cell interactions.

Helicobacter pylori (H. pylori) persistently colonizes the human gastric mucosa in more than 50% of the global population, leading to various gastroduodenal diseases ranging from chronic gastritis to gastric carcinoma. Cytotoxin-associated gene A (CagA) protein, an important oncoprotein, has highly polymorphic Glu-Pro-Ile-Tyr-Ala segments at the carboxyl terminus, which play crucial roles in pathogenesis. Our previous study revealed a significant association between amino acid deletions at positions 893 and 894 and gastric cancer. To investigate the impact of amino acid deletions at positions 893 and 894 on CagA function. We selected a representative HZT strain from a gastric cancer patient with amino acid deletions at positions 893 and 894. The cagA gene was amplified and mutated into cagA-NT and cagA-NE (sequence characteristics of strains from nongastric cancer patients), cloned and inserted into pAdtrack-CMV, and then transfected into AGS cells. The expression of cagA and its mutants was examined using real-time polymerase chain reaction and Western blotting, cell elongation via cell counting, F-actin cytoskeleton visualization using fluorescence staining, and interleukin-8 (IL-8) secretion via enzyme-linked immunosorbent assay. The results revealed that pAdtrack/cagA induced a more pronounced hummingbird phenotype than pAdtrack/cagA-NT and pAdtrack/cagA-NE (40.88 ± 3.10 vs 32.50 ± 3.17, P < 0.001 and 40.88 ± 3.10 vs 32.17 ± 3.00, P < 0.001) at 12 hours after transfection. At 24 hours, pAdtrack/cagA-NE induced significantly fewer hummingbird phenotypes than pAdtrack/cagA and pAdtrack/cagA-NT (46.02 ± 2.12 vs 53.90 ± 2.10, P < 0.001 and 46.02 ± 2.12 vs 51.15 ± 3.74, P < 0.001). The total amount of F-actin caused by pAdtrack/cagA was significantly lower than that caused by pAdtrack/cagA-NT and pAdtrack/cagA-NE (27.54 ± 17.37 vs 41.51 ± 11.90, P < 0.001 and 27.54 ± 17.37 vs 41.39 ± 14.22, P < 0.001) at 12 hours after transfection. Additionally, pAdtrack/cagA induced higher IL-8 secretion than pAdtrack/cagA-NT and pAdtrack/cagA-NE at different times after transfection. Amino acid deletions at positions 893 and 894 enhance CagA pathogenicity, which is crucial for revealing the pathogenic mechanism of CagA and identifying biomarkers of highly pathogenic H. pylori.

Alamandine, a protective component of the renin-angiotensin system, reduces cellular proliferation and interleukin-6 secretion in human macrophages through MasR–MrgDR heteromerization

Alamandine (ALA) exerts protective effects similar to angiotensin (Ang) (1–7) through Mas-related G protein-coupled receptor type D receptor (MrgDR) activation, distinct from Mas receptor (MasR). ALA induces anti-inflammatory effects in mice but its impact in human macrophages remains unclear. We aimed to investigate the anti-inflammatory effects of ALA in human macrophages. Interleukin (IL)-6 and IL-1β were measured by ELISA in human THP-1 macrophages and human monocyte-derived macrophages exposed to lipopolysaccharide (LPS). Consequences of MasR–MrgDR heteromerization were investigated in transfected HEK293T cells. ALA decreased IL-6 and IL-1β secretion in LPS-activated THP-1 macrophages. The ALA-induced decrease in IL-6 but not in IL-1β was prevented by MasR blockade and MasR downregulation, suggesting MasR–MrgDR interaction. In human monocyte-derived M1 macrophages, ALA decreased IL-1β secretion independently of MasR. MasR–MrgDR interaction was confirmed in THP-1 macrophages, human monocyte-derived macrophages, and transfected HEK293T cells. MasR and MrgDR formed a constitutive heteromer that was not influenced by ALA. ALA promoted Akt and ERK1/2 activation only in cells expressing MasR–MrgDR heteromers, and this effect was prevented by MasR blockade. While Ang-(1–7) reduced cellular proliferation in MasR −but not MrgDR- expressing cells, ALA antiproliferative effect was elicited in cells expressing MasR–MrgDR heteromers. ALA also induced an antiproliferative response in THP-1 cells and this effect was abolished by MasR blockade, reinforcing MasR–MrgDR interaction. MasR–MrgDR heteromerization is crucial for ALA-induced anti-inflammatory and antiproliferative responses in human macrophages. This study broaden our knowledge of the protective axis of the RAS, thus enabling novel therapeutic approaches in inflammatory-associated diseases

The influence of interleukin-4 on transendothelial transport of low-density lipoproteins

BACKGROUND: Cytokines are important participants in the atherosclerotic process. The product of T-helpers and mast cells, interleukin-4, causes activation of endothelial cells and induces polarization of macrophages into anti-inflammatory M2 phenotype. AIM: The aim was to study the influence of interleukin-4 on the activation of transendothelial transport of low-density lipoproteins. MATERIALS AND METHODS: Low-density lipoproteins transendothelial transport was evaluated in a two-chamber model on a monolayer of human endothelial cell line EA.hy926. For that, 200 mkg/ml of low-density lipoproteins, and 1, or 10, or 100 ng/ml of interleukin-4, or 10, or 50 ng/ml of interleukin-6, or 50 ng/ml of tumor necrosis factor, or 10 ng/ml of interleukin-4 and 50 ng/ml of tumor necrosis factor, or a phosphate buffer saline were added to the cells on 24 h. The integrity of endothelial monolayer was controlled by the passage through 1 mkg/ml of fluorescein Na. The levels of mRNA and proteins in the cells were determined by reverse transcription polymerase chain reaction and Western blotting, the activity of matrix metalloproteinases-1, -2 and -9 in the culture medium — by zymography, interleukin-6 and -8 concentrations — by enzyme-linked immunosorbent assay. RESULTS: Interleukin-4 had no effect on the passage of low-density lipoproteins through the monolayer of endothelial cells in basal and tumor necrosis factor stimulation conditions. At the same time, interleukin-4 suppressed interleukin-8 secretion by cells and increased their production of interleukin-6. The latter also did not cause the changes in the permeability of endothelial monolayer. In addition, interleukin-4 did not affect the expression of CAV, SCARB1, ACVRL1 genes, encoding proteins involved in low-density lipoproteins transendothelial transport. An addition of interleukin-4 or -6 to the cells did not change the activity of the matrix metalloproteinases. CONCLUSIONS: Interleukin-4 does not affect the transendothelial transport of low-density lipoproteins, while modulating the production of other cytokines by endothelial cells. In the future, it is necessary to establish the effect of a wider range of cytokines produced by intimal cells on low-density lipoproteins transendothelial transport, as well as to clarify the molecular mechanisms of influence of tumor necrosis factor on this process in order to identify new targets for atherosclerosis therapy.

Immune Effect of Co-Culture of Dendritic Cells and Cytokine-Induced Killer Cells on Prostate Cancer Cells.

It was to explore the immune outcome of co-culture of dendritic cells (DC) and cytokine-induced killer cells (CIK) on prostate cancer (PCa) cells. Peripheral blood mononuclear cells (PBMCs) were extracted from healthy blood donors. DC and CIK cells were induced and co-cultured. The proliferation and phenotypic changes of DC, CIK, and DC-CIK cells/groups were observed. Model rats were constructed by injecting PC3 PCa cells into the abdominal cavity. The successful 50 cases were divided into a negative control group, a chemotherapy group, a DC group, a CIK group, and a DC-CIK treatment group (each consisting of 10 rats) to observe tumor progression. The secreted concentrations of interleukin-12 (IL-12) ((103.67 ± 2.77) pg/mL) and interferon-γ (IFN-γ) ((730.09 ± 23.52) pg/mL) were higher in DC-CIK group as against DC and CIK groups; the proliferation of CIK was higher in DC-CIK group as against CIK within 12 to 20 days of culture. The positive rate (PR) of CD3+/ CD56+ and CD8+ was higher and that of CD45RA+ was lower in DC-CIK group as against CIK.The killing rate of the DC-CIK group was higher than that of the DC and CIK groups at a target effect ratio of 10:1/20:1/50:1 (P < 0.05). After the treatment, the body weight of rats in the chemotherapy group, DC group, and CIK group was significantly reduced (P < 0.05), while no significant changes were observed in the negative control group and DC-CIK group (P > 0.05). After 25 days of treatment, the tumor size in the DC-CIK group was significantly smaller compared to the negative control group, chemotherapy group, DC group, and CIK group; the necrotic area of the tumor tissue in the DC-CIK group was also significantly larger than that in the negative control group, chemotherapy group, DC group, and CIK group (P < 0.05). Co-culture of DC and CIK is excellent in enhancing the proliferation of CIK cells, increasing the secretion of IL-12 and IFN-γ, and enhancing the activity of immune cells and anti-tumor ability, showing its potential in anti-PCa tumor immunotherapy.

Phagocytic activity and proinflammatory activation potential of smooth muscle cells of the Tunica intima of human aorta under experimental conditions

The aorta is the largest blood vessel in the body, which reaches a diameter of about 3 cm and is responsible for transporting oxygen-enriched arterial blood from the heart to tissues and organs. The aortic wall consists of three layers: the inner Tunica intima, the middle Tunica media and the outer Tunica adventitia. The layers of the aortic wall have a diverse cellular composition and include smooth muscle cells (SMCs), fibroblasts, endothelial cells, etc. Functional disorders in the cells of the intima of the human aorta can lead to various cardiovascular diseases (СVD), such as aneurysm and, as a result, dissection or rupture of the thoracic aorta. The cellular and molecular mechanisms of CVD development remain not fully understood, therefore, the study of the functional characteristics of various cell populations that make up the human aorta is an urgent task today. The aim is to evaluate the inflammatory response formed by cells that are part of the Tunica intima of the human aorta during phagocytosis of latex particles and internalization of low-density lipoproteins (LDL) to study their role in the development of aneurysms. The experiments were performed on smooth muscle cells (SMCs) isolated from the intima of the human aorta in patients with aneurysm. Phagocytic activity was studied by adding latex beads to the SMCs of Tunica intima, the ability to internalize LDL was evaluated using the BDP 630/650 dye and a biochemical method, the assessment of the ability to pro- and anti-inflammatory activation was studied using enzyme-linked immunosorbent assay (ELISA). Our results demonstrated that the absorption of latex beads and LDL stimulates the activation of interleukin secretion by smooth muscle cells that are part of the Tunica intima of the aorta, namely the proinflammatory cytokines IL-6 and IL-8. This fact may indicate that in the conditions of the body, the human aortic intima SMCs phenotype may switch from contractile to secretory or macrophage-like, which indicates the participation of this phenotypic cell transition in the process of aneurysm development.

Hemozoin induces malaria via activation of DNA damage, p38 MAPK and neurodegenerative pathways in a human iPSC-derived neuronal model of cerebral malaria

Malaria caused by Plasmodium falciparum infection results in severe complications including cerebral malaria (CM), in which approximately 30% of patients end up with neurological sequelae. Sparse in vitro cell culture-based experimental models which recapitulate the molecular basis of CM in humans has impeded progress in our understanding of its etiology. This study employed healthy human induced pluripotent stem cells (iPSCs)-derived neuronal cultures stimulated with hemozoin (HMZ) - the malarial toxin as a model for CM. Secretome, qRT-PCR, Metascape, and KEGG pathway analyses were conducted to assess elevated proteins, genes, and pathways. Neuronal cultures treated with HMZ showed enhanced secretion of interferon-gamma (IFN-γ), interleukin (IL)1-beta (IL-1β), IL-8 and IL-16. Enrichment analysis revealed malaria, positive regulation of cytokine production and positive regulation of mitogen-activated protein kinase (MAPK) cascade which confirm inflammatory response to HMZ exposure. KEGG assessment revealed up-regulation of malaria, MAPK and neurodegenerative diseases-associated pathways which corroborates findings from previous studies. Additionally, HMZ induced DNA damage in neurons. This study has unveiled that exposure of neuronal cultures to HMZ, activates molecules and pathways similar to those observed in CM and neurodegenerative diseases. Furthermore, our model is an alternative to rodent experimental models of CM.

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.

Evaluation of STK17B as a cancer immunotherapy target utilizing highly potent and selective small molecule inhibitors.

The serine/threonine kinase 17B (STK17B) is involved in setting the threshold for T cell activation and its absence sensitizes T cells to suboptimal stimuli. Consequently, STK17B represents an attractive potential target for cancer immunotherapy. To assess the potential of STK17B as an immuno-oncology target, we developed potent and selective tool compounds from starting points in Blueprint Medicines Corporation's proprietary kinase inhibitor library. To characterize these molecules, enzyme and cellular assays for STK17A and STK17B were established to drive chemistry optimization. Mass spectrometry-based phosphoproteomics profiling with tool inhibitors led to the identification of Ser19 on myosin light chain 2 as STK17B substrate, which is then developed into a flow cytometry-based pharmacodynamic readout of STK17B inhibition both in vitro and in vivo. In a mouse T cell activation assay, STK17B inhibitors demonstrated the ability to enhance interleukin-2 (IL-2) production. Similarly, treatment with STK17B inhibitors resulted in stronger cytokine secretion in human T cells activated using a T cell bispecific antibody. Subsequent chemistry optimization led to the identification of a highly selective and orally bioavailable tool compound, BLU7482. In vivo, STK17B inhibition led to dose-dependent modulation of myosin light chain 2 phosphorylation and enhanced priming of naïve T cells, as determined by upregulation of CD69, IL-2 and interferon-γ secretion. In line with increased T cell activation, treatment with STK17B inhibitor enhanced antitumor activity of anti-PD-L1 antibody in the MCA205 model. In summary, we successfully identified and optimized STK17B kinase inhibitors which led to increased T cell responses in vitro and in vivo. This allowed us to evaluate the potential of STK17B inhibition as an approach for cancer immunotherapy.

Mimicking and in vitro validating chronic inflammation in human gingival fibroblasts

ObjectiveThe aim of this study was to identify and validate in vitro conditions that may mimic the translational, cytokine and chemokine profiles observed in human inflamed gingiva in vivo. DesignPrimary human gingiva fibroblast cells (HFIB-G) were cultured under serum starvation conditions (0 – 10 %), supplemented with increasing lipopolysaccharide (LPS) concentrations (0.1, 1, or 10 µg/ml) from two bacterial strains E. coli and P. gingivalis and 0.1, 1, or 10 ng/ml recombinant interleukin 1β (IL-1β), alone or in combinations. The levels of cytokines/chemokines were measured in the cell culture medium by Luminex, and gene expression was quantified by Affymetrix microarrays at 24, 48 and 72 h. ResultsInflammation markers were not elevated after stimulation with P. gingivalis LPS, while E. coli LPS and IL-1β individually increased the secretion of interleukin 6 (IL-6) and monocyte chemoattractant protein-1 (MCP-1) to the cell culture medium. IL-1β administration also increased the secretion of several factors, including tumor necrosis factor (TNFα). However, the combination of 1 µg/ml E. coli LPS, 1 ng/ml IL-1β and serum starvation led to increased secretion of IL-6, TNFα, in addition to other factors found in inflamed tissue. Gene expression analyses revealed that this combination not only enhanced the expression interleukins/chemokines genes but also T helper cell signaling and matrix metalloproteinases. ConclusionSerum reduction in cell culture medium together with the administration of E. coli LPS and IL-1β resulted in gene expression and secreted cytokine/chemokine profiles similar to that found in vivo during chronic inflammation.

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.

Cabozantinib-encapsulated and maytansine-conjugated high-density lipoprotein for immunotherapy in colorectal cancer

Advanced colorectal cancer (CRC) responds poorly to current adjuvant therapies, partially due to its immunosuppressive intestinal microenvironment. We found that myeloid-derived suppressor cells (MDSCs) were enriched in orthotopic tumors due to treatment-induced succinate release, which activated tuft cells and upregulated interleukin 25 (IL-25) and interleukin 13 (IL-13). We engineered a cabozantinib (Cabo)-encapsulated and maytansine (DM1)-conjugated synthetic high-density lipoprotein (ECCD-sHDL) to modulate the tumor microenvironment. DM1 induced immunogenic cell death and promoted the maturation of dendritic cells. Meanwhile, Cabo alleviated DM1-induced succinate release, preventing tuft cell activation, downregulating IL-25 and IL-13 secretion, and reducing intratumoral MDSC infiltration. ECCD-sHDL increased the densities of active cytotoxic T lymphocytes (CTLs) and M1 macrophages in the tumors, effectively inhibiting tumor growth and metastasis, thereby prolonging survival in murine CRC models. Our study sheds light on the mechanism of treatment-induced immunosuppression in orthotopic CRC and demonstrates that this combinatorial therapy could be an effective treatment for CRC.

Toxic effect of polycyclic aromatic hydrocarbons (PAHs) on co-culture model of human alveolar epithelial cells (A549) and macrophages (THP-1)

Polycyclic aromatic hydrocarbons (PAHs) are particulate matter bound environmental contaminants known to cause adverse effects on human health. The toxicity of carcinogenic PAH such as benzo[a]pyrene (BaP) has been extensively investigated, whereas other PAHs have received less attention. The present work investigated the toxic effects of three less investigated PAHs with distinct molecular weights in comparison to BaP on co-culture model of human epithelial lung cells (A549) and macrophages (THP-1). Due to the involvement of more than one cell type in the response to PAH exposure, the new co-culture model is considered to be suitable for the prediction of undesired toxicological effects of PAHs. To do so, the co-culture was established and exposed to 0–400 µM of phenanthrene (PHE), fluoranthene (FLA), and, benzo [ghi] perylene (BghiP) for 24 h. Subsequently, cytotoxicity, micronucleus formation, and cytokine excretion were analyzed. The results revealed that the viability of A549 cells decreased after being exposed to increasing concentrations of PAHs. The formation of micronuclei in binucleated cells (BNC) was found more frequently in cells treated with PAHs in comparison to the untreated group, indicating the genotoxic effect of these compounds. Moreover, an exposure to PAHs enhanced the pro-inflammatory cytokine, i.e., interleukin-6 secretion, while diminished the anti-inflammatory cytokine, i.e., interleukin-10. In summary, PAHs possess negative effects on A549 and THP-1 co-culture model, implying an adverse effect on human health when coming into contact with these chemicals via respiration.

TRIM29 controls enteric RNA virus-induced intestinal inflammation by targeting NLRP6 and NLRP9b signaling pathways

Infections by enteric virus and intestinal inflammation are recognized as a leading cause of deadly gastroenteritis, and NLRP6 and NLRP9b signaling control these infection and inflammation. However, the regulatory mechanisms of the NLRP6 and NLRP9b signaling in enteric viral infection remain unexplored. In this study, we found that the E3 ligase TRIM29 suppressed type III interferon (IFN-λ) and interleukin-18 (IL-18) production by intestinal epithelial cells (IECs) when exposed to polyinosinic:polycytidylic acid (poly I:C) and enteric RNA viruses. Knockout of TRIM29 in IECs was efficient to restrict intestinal inflammation triggered by the enteric RNA viruses, rotavirus in suckling mice, and the encephalomyocarditis virus (EMCV) in adults. This attenuation in inflammation was attributed to the increased production of IFN-λ and IL-18 in the IECs and more recruitment of intraepithelial protective Ly6A+CCR9+CD4+ T cells in small intestines from TRIM29-deficient mice. Mechanistically, TRIM29 promoted K48-linked ubiquitination, leading to the degradation of NLRP6 and NLRP9b, resulting in decreased IFN-λ and IL-18 secretion by IECs. Our findings reveal that enteric viruses utilize TRIM29 to inhibit IFN-λ and inflammasome activation in IECs, thereby facilitating viral-induced intestinal inflammation. This indicates that targeting TRIM29 could offer a promising therapeutic strategy for alleviating gut diseases.

Tangeretin inhibits airway inflammatory responses by reducing early growth response 1 (EGR1) expression in mice exposed to cigarette smoke and lipopolysaccharide

BackgroundTangeretin, a natural polymethoxyflavone compound, possesses potent anti-inflammatory activity that improves respiratory inflammation in chronic obstructive pulmonary disease (COPD). However, the molecular mechanisms underlying the anti-COPD effects of tangeretin remain unclear. In this study, we aimed to investigate the key molecular mechanisms by which tangeretin suppresses COPD-related inflammatory responses. MethodsWe conducted the investigation in phorbol-12-myristate-13-acetate (PMA)-stimulated human airway epithelial cells (in vitro) and cigarette smoke (CS)/lipopolysaccharide (LPS)-exposed mice (in vivo). ResultsTangeretin decreased the release of inflammatory mediators, including tumor necrosis factor-α (TNF-α), interleukin 6 (IL-6), and mucin 5AC (MUC5AC), by suppressing early growth response 1 (EGR1) expression in vitro. Tangeretin and EGR1 small interfering ribonucleic acid (siRNA) combination showed a synergistic reduction in MUC5AC and TNF-α secretion. Tangeretin administration significantly inhibited the levels of reactive oxygen species (ROS) production, elastase activity, TNF-α, IL-6, and monocyte chemoattractant protein-1 (MCP-1) secretion, and macrophage and neutrophil numbers in the bronchoalveolar lavage fluid of CS/LPS-exposed mice. Tangeretin also prevented CS/LPS-induced abnormal pathological changes and excessive MUC5AC and EGR1 expression in lung tissue. ConclusionComprehensively, tangeretin inhibits the lung inflammatory response associated with COPD by reducing EGR1 expression in PMA-induced human epithelial cells and in a CS/LPS-stimulated mouse model. This study shows that tangeretin has anti-COPD properties and can be a promising alternative (or complementary) treatment for inflammatory lung disease.

Injectable Oxygen-Carrying Microsphere Hydrogel for Dynamic Regulation of Redox Microenvironment of Wounds.

The delayed healing of infected wounds can be attributed to the increased production of reactive oxygen species (ROS) and consequent damages to vascellum and tissue, resulting in a hypoxic wound environment that further exacerbates inflammation. Current clinical treatments including hyperbaric oxygen therapy and antibiotic treatment fail to provide sustained oxygenation and drug-free resistance to infection. To propose a dynamic oxygen regulation strategy, this study develops a composite hydrogel with ROS-scavenging system and oxygen-releasing microspheres in the wound dressing. The hydrogel itself reduces cellular damage by removing ROS derived from immune cells. Simultaneously, the sustained release of oxygen from microspheres improves cell survival and migration in hypoxic environments, promoting angiogenesis and collagen regeneration. The combination of ROS scavenging and oxygenation enables the wound dressing to achieve drug-free anti-infection through activating immune modulation, inhibiting the secretion of pro-inflammatory cytokines interleukin-6, and promoting tissue regeneration in both acute and infected wounds of rat skins. Thus, the composite hydrogel dressing proposed in this work shows great potential for dynamic redox regulation of infected wounds and accelerates wound healing without drugs.

Four new diarylheptanoids and two new terpenoids from the fruits of Alpinia oxyphylla and their anti-inflammatory activities

Four previously unreported diarylheptanoids (1a/1b-2a/2b), one undescribed sesquiterpenoid (8), one new diterpenoid (12), and twelve known analogs were isolated from the fruits of Alpinia oxyphylla. The structural elucidation of these compounds was achieved through a comprehensive analysis of spectroscopic data, single-crystal X-ray diffraction, electronic circular dichroism (ECD), and modified Mosher's method. Enantiomeric mixtures (1a/1b, 2a/2b, 3a/3b, 4a/4b, and 5a/5b) were separated on a chiral column using acetonitrile-water mixtures as eluents. Among them, compounds 3a/3b and 4a/4b were isolated as optically pure enantiomers in the initial chiral separation. Furthermore, most of the isolates were evaluated for their inhibitory effects against the production of nitric oxide (NO) and interleukin-6 (IL-6) in lipopolysaccharide (LPS)-induced RAW264.7 macrophages. Interestingly, 2 and 4 showed significant inhibitory activities against NO production with IC50 values of 33.65 and 9.88 μmol·L-1 (hydrocortisone: IC50 34.26 μmol·L-1), respectively. Additionally, they also partially reduced the secretion of IL-6.