Cytokines In Supernatants Research Articles

Immunomodulatory effect of mesenchymal stem cells-derived extracellular vesicles to modulate the regulatory T cells and Th1/Th2 imbalance in peripheral blood mononuclear cells of patients with allergic rhinitis.

Mesenchymal stem cell-derived extracellular vesicles (MSC-EVs) have shown promising immunomodulatory capabilities for a variety of clinical conditions. However, the potential regulatory mechanisms of MSC-EVs in allergic rhinitis (AR) remain unexplored. The present study was designed to investigate the immunomodulatory effect of MSC-EVs in patients with AR. Peripheral blood mononuclear cells (PBMCs) were isolated from AR patients. The number of peripheral CD4+Foxp3+IL-17+, CD4+Foxp3+IL-17- and CD4+Foxp3-IL-17+ T cells in healthy controls and AR patients were evaluated using flow cytometry. Therapeutic effect of MSC-EVs was determined by detecting IFN-γ, IL-4, IL-17 and IL-10 cytokines in supernatant by ELISA and flow cytometry. The mean fluorescence intensity (MFI) was calculated in PBMCs for IL-10, IL-17 and TGF-β on T cells after MSC-EVs treatment. Bioinformatic analysis of microRNA was performed by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. CD4+Foxp3+IL-17+ T cells expression in PBMCs was higher in the AR group and the balance of Treg/Th17 was tilted towards Th17 cells. Supernatant from AR patients revealed that MSC-EVs treatment upregulated IL-10 and IFN-γ, and downregulated IL-4 and IL-17. EVs treatment effectively re-established Th1(CD4+IFN-γ+cells)/Th2(CD4+IL-4+cells) balance, reduced CD4+IL-17+ and increased CD4+IL-10+ and CD4+TGF-β+ cells. The MFI of IL-10 and TGF-β in CD4+CD25+CD127- T cells were higher, whereas lower levels of IL-17 were observed. Bioinformatic analysis revealed that the TGF-β, Wnt signalling pathways and STAT5 transcription factor might mechanistically support the immunomodulatory effect of MSC-EVs. This study presents the immunomodulatory effect of MSC-EVs in PBMCs from AR patients. The results provide a new therapeutic strategy for AR.

Th17 and T Regulatory Cytokines in Serum, Lesional Skin, and Stimulated Peripheral Blood Mononuclear Cell Culture Supernatants from Type 1 Leprosy Reaction Patients

There are conflicting reports regarding the roles of T helper-17 (Th17) and T regulatory (Treg) cells in type 1 leprosy reactions (T1Rs). Also, literature on the correlation of immunological parameters with a validated scoring system and the effect of treatment on cytokines is lacking. Adult patients with untreated T1R and nonreactional spectrum–matched controls were included in the study for comparison of levels of Th17 and Treg pathway cytokines in serum, skin lesions (reactional), and peripheral blood mononuclear cells (PBMCs) culture supernatants. Venous blood samples were collected at baseline and after resolution of reaction (post treatment with nonsteroidal anti-inflammatory drugs [NSAIDs] or steroids) for serum cytokine estimation and PBMC stimulation assays, and lesional (reactional) skin biopsy for cytokine messenger RNA (mRNA) estimation. Thirty-two cases of T1R were recruited (23 patients completed follow-up). Serum levels of cytokines were not significantly different between cases and controls or between pre- and post-treatment samples. Tissue mRNA and Mycobacterium leprae (M. leprae) antigen–stimulated PBMC culture supernatant levels of Interleukin (IL)-17A, IL-17F, IL-6, and IL-23 were significantly higher in T1R than in controls. Levels of IL-10 and Transforming Growth Factor-beta (TGF-β) were comparable among the two groups. The levels of all cytokines were significantly reduced after treatment. There was no significant difference in magnitude of the fall between those treated with steroids versus NSAIDs. This study suggests heightened Th17 response in T1R, with a prominent inability of the regulatory cytokines IL-10 and TGF-β to control the associated inflammation. The dynamics of change after resolution of T1R were comparable between NSAID and oral steroid treatment groups.

Evaluation of the Anti-Inflammatory/Immunomodulatory Effect of Teucrium montanum L. Extract in Collagen-Induced Arthritis in Rats.

The anti-inflammatory/immunomodulatory effects of Teucrium montanum L. (TM), a plant distributed in the Mediterranean region, have been insufficiently examined. The effects of the TM ethanol extract were tested in a rat collagen-induced arthritis (CIA) model of rheumatoid arthritis. LC-MS was used for the phytochemical analysis of the TM extract. Dark Agouti rats were immunized with bovine type II collagen (CII) in incomplete Freund's adjuvant for CIA, and treated with 100 or 200 mg/kg of TM extract daily via oral administration. Clinical and histopathological evaluations and a flow cytometric analysis of the phenotypic and functional characteristics of splenocytes and draining lymph node cells were performed. The cytokines in the paw tissue culture supernatants and anti-CII antibodies in serum were determined by ELISA. The TM extract, with the dominant components verbascoside and luteolin 7-O-rutinoside, reduced the arthritic score and ankle joint inflammation in CIA rats, promoted the antioxidant profile in serum, and lowered pro-inflammatory TNF-α, IL-6 and IL-1β production. It suppressed the activation status of CD11b+ cells by lowering CD86, MHCII and TLR-4 expression, and promoted the Th17/T regulatory cell (Tregs) balance towards Tregs. A lower frequency of B cells was accompanied by a lower level of anti-CII antibodies in treated rats. These findings imply the favorable effect of TM extract on the clinical presentation of CIA, suggesting its anti-inflammatory/immunomodulatory action and potential therapeutic effect.

Interleukin-27 Regulates Adaptative Immune Responses Associated With Control of Parasite Replication in Canine Leishmaniasis.

Interleukin 27 (IL-27) is a cytokine that regulates susceptibility to Leishmania infantum infection in humans and experimental models. This cytokine has not yet been described in canine leishmaniasis (CanL). Therefore, we investigated whether IL-27 has a regulatory role in CanL. The EBI3 and p28 subunits of IL-27 were measured in splenic leukocytes culture supernatant from dogs with CanL and compared to control dogs. We also correlated EBI3 and p28 levels with IL-21, anti-L. infantum antibodies and parasite loads. We performed functional assays followed by IL-27 blockade and measured parasite loads, production of cytokines in splenic leukocytes culture supernatant, and the expression of PD-1, CTLA-4, phospho-Stat-1/3, T-bet, GATA3 and nitric oxide production (NO). Both IL-27 subunits increased in the supernatant of dogs with CanL compared to control dogs. EBI3 and p28 levels showed a moderate positive correlation with IL-21 (r = 0.67, p < 0.0001 and r = 0.45, p < 0.012, respectively), and the EBI3 subunit was positively associated with anti-L. infantum IgG antibodies (r = 0.38, p < 0.040) and parasite load (r = 0.47, p < 0.009). IL-27 and IL-21 participate of immune responses in CanL. IL-27 may be associated with the failure of immunity to control parasite replication via upregulation of the expression of PD-1, CTLA-4, T-bet and NO in splenic leukocytes from dogs with CanL. These findings suggest that the pathways regulated by IL-27 are involved in CanL pathogenesis in the host, and may be targets for new therapies.

Milk fat globule-epidermal growth factor-VIII–derived oligopeptide 3 (MOP3) attenuates inflammation and improves survival in hepatic ischemia/reperfusion injury

IntroductionHepatic ischemia/reperfusion injury is a severe clinical condition leading to high mortality as the result of excessive inflammation, partially triggered by released damage-associated molecular patterns. Extracellular cold-inducible RNA-binding protein is a new damage-associated molecular pattern. Current clinical management of hepatic ischemia/reperfusion injury is limited to supportive therapy, necessitating the development of novel and effective treatment strategies. Milk fat globule-epidermal growth factor-VIII–derived oligopeptide 3 is a newly invented oligopeptide originating from milk fat globule-epidermal growth factor-VIII. This peptide acts as an opsonic compound that specifically binds to extracellular cold-inducible RNA-binding protein to facilitate its clearance by phagocytes, thereby attenuating inflammation. In this study, we hypothesized that milk fat globule-epidermal growth factor-VIII–derived oligopeptide 3 attenuated hepatic ischemia/reperfusion injury by inhibiting extracellular cold-inducible RNA-binding protein–induced inflammation in Kupffer cells. MethodsWe treated Kupffer cells isolated from male C57BL/6 mice with extracellular cold-inducible RNA-binding protein and various doses of milk fat globule-epidermal growth factor-VIII–derived oligopeptide 3 for 4 hours, then measured cytokines in the culture supernatants. In addition, mice underwent 70% hepatic ischemia for 60 minutes immediately followed by the intravenous administration of either vehicle or milk fat globule-epidermal growth factor-VIII–derived oligopeptide 3. Blood and ischemic liver tissues were collected 24 hours later, and inflammatory markers including cytokines, liver enzymes, chemokines, myeloperoxidase activity, and Z-DNA-binding protein 1 were measured. Hepatic tissue damage and cell death were evaluated histologically. Survival rates were monitored for 10 days posthepatic ischemia/reperfusion. ResultsThe release of interleukin-6 and tumor necrosis factor-α from extracellular cold-inducible RNA-binding protein–challenged Kupffer cells was significantly reduced by milk fat globule-epidermal growth factor-VIII–derived oligopeptide 3 in a dose-dependent manner. In hepatic ischemia/reperfusion mice, milk fat globule-epidermal growth factor-VIII–derived oligopeptide 3 treatment significantly decreased serum levels of extracellular cold-inducible RNA-binding protein, interleukin-6, tumor necrosis factor-α, aspartate aminotransferase, alanine aminotransferase, and lactate dehydrogenase. Milk fat globule-epidermal growth factor-VIII–derived oligopeptide 3 treatment also significantly reduced mRNA levels of interleukin-6, tumor necrosis factor-α, interleukin-1β, Z-DNA-binding protein 1, and chemokine macrophage inflammatory protein-2, as well as myeloperoxidase activity in hepatic tissues. Histologic evaluation demonstrated that treatment with milk fat globule-epidermal growth factor-VIII–derived oligopeptide 3 significantly attenuated tissue damage and cell death in the liver of hepatic ischemia/reperfusion mice. Milk fat globule-epidermal growth factor-VIII–derived oligopeptide 3 treatment significantly improved the survival rate of hepatic ischemia/reperfusion mice. ConclusionMilk fat globule-epidermal growth factor-VIII–derived oligopeptide 3 significantly attenuated inflammation and liver tissue damage and improved survival after hepatic ischemia/reperfusion. Thus, milk fat globule-epidermal growth factor-VIII–derived oligopeptide 3 holds promise as a potential future therapeutic strategy for hepatic ischemia/reperfusion injury.

SO2 activates Th17 cells through the JAK1,2/STAT3 signaling pathway

ObjectiveTo investigate the effect of SO2 on Th1/Th2/Th17 cells in allergic rhinitis (AR) and the role of JAK1, 2/STAT3 signaling pathways.To Provide potential directions for the treatment of AR. MethodsFifteen AR patients were enrolled as the experimental group, while 15 healthy volunteers served as the normal control group. After collecting venous blood, peripheral blood mononuclear cells (PBMCs) were isolated and cultured, followed by the addition of SO2 derivatives and the JAK inhibitor Ruxolitinib. Flow cytometry was employed to assess alterations in the Th1/Th2 and Th17/Treg cell balance upon stimulation with SO2 and Ruxolitinib. qRT-PCR was utilized to detect the expression of Th1-related cytokines IL-2 and IFN-γ, Th2-related cytokines IL-4 and IL-5, Th17-related cytokines IL-17A and RORγt, as well as genes JAK1, JAK2, and STAT3. Flow cytometric cytokine analysis was conducted for quantitative assessment of the expression levels of inflammation-related cytokines in PBMC culture supernatants after stimulation. In addition, we stimulated the Jurkat T lymphocyte cell line with SO2 derivatives, added Ruxolitinib as an inhibitor, and used Western blot analysis to further determine the effects of SO2 on Th cells and the role of the JAK1,2/STAT3 signaling pathway in this process. ResultsStimulation with SO2 derivatives upregulated the expression levels of Th2 cells and associated cytokines, as well as Th1 cells and associated cytokines. both AR patients and healthy individuals displayed increased percentages of Th17 cells and Th17/Treg ratios in PBMCs. The expression of IL-17A, RORγt, and IL-6 was also elevated. Under SO2 stimulation, the expression of JAK1, JAK2, STAT3, and RORγt in Jurkat cells increased. Moreover, after the application of Ruxolitinib, the JAK/STAT signaling pathway was inhibited. This led to a reduction in Th17 cells and IL-17A levels in both AR patients and healthy individuals, as well as a decrease in RORγt expression in Jurkat cells. Additionally, the expression of IL-5 decreased in healthy individuals. ConclusionSO2 exposure exacerbated Th1/Th2/Th17 inflammation in AR patients and induced Th1 and Th17 inflammation in healthy individuals. The stimulatory effect of SO2 on Th17 cell differentiation could be inhibited by Ruxolitinib. This suggests that the Th17 inflammation induced by SO2 stimulation may be related to the activation of the JAK/STAT signaling pathway, and this has been confirmed in the Jurkat cell line.

Expression of Transcriptional Factors of T Helper Differentiation (T-bet, GATA-3, RORγt, and FOXP3), MIF Receptors (CD44, CD74, CXCR2, 4, 7), and Th1, Th2, and Th17 Cytokines in PBMC from Control Subjects and Rheumatoid Arthritis Patients.

The macrophage migration inhibitory factor (MIF) plays a pivotal role in the development of rheumatoid arthritis (RA). Previous research indicates that MIF can trigger the expression of cytokine profiles associated with Th1, Th2, and Th17 responses in peripheral blood mononuclear cells (PBMC) from both RA patients and control subjects (CS). Despite these, few studies to date precisely elucidate the molecular mechanisms involved. The present study aimed to associate the expression of Th differentiation TF (T-bet, GATA-3, RORγt) with MIF receptors (CD44, CD74, CXCR2, 4, 7) and Th1, Th2, and Th17 cytokines in PBMC from CS and RA patients. PBMC from both groups was cultured for 24 h. The expression of the canonical and non-canonical MIF receptors and the TF was determined by flow cytometry. Additionally, multiplex bead analysis was employed to assess the levels of cytokines in the culture supernatants. The findings revealed that T CD4+ lymphocytes in the CS group exhibited a heightened expression of CD74 (p<0.05), whereas RA patients displayed an elevated expression of CXCR7 (p<0.001). Furthermore, T CD4+ lymphocytes from RA patients exhibited greater expression of GATA3, RORγt, and FOXP3, along with elevated levels of pro-inflammatory cytokines compared to the CS group (p<0.001). These results indicate that CD74 is more prominently expressed in PBMC from the CS group, whereas CXCR7 is more expressed in PBMC from RA patients. We also noted an increased secretion of Th17 profile cytokines in RA, potentially influenced by the activation of FOXP3 via CD74 and RORγt through CXCR7 using the endocytic pathway.

Correlation of Immunomodulatory Cytokines with Tumor Volume and Cerebrospinal Fluid in Vestibular Schwannoma Patients.

Sporadic vestibular schwannomas (VSs) often exhibit slow or negligible growth. Nevertheless, some VSs increase significantly in volume within a few months or grow continuously. Recent evidence indicates a role of inflammation in promoting VS growth. Therefore, our study aimed to identify cytokines, which are associated with larger VSs. The expression of different cytokines in VS tumor samples and VS primary cultures was investigated. Additionally, the concentration of cytokines in cell culture supernatants of VS primary cultures and cerebrospinal fluid (CSF) of VS patients and healthy controls were determined. Correlation analysis of cytokine levels with tumor volume, growth rate, Koos grade, age, and hearing was examined with Spearman's-rank test. The mRNA expression of CC-chemokine ligand (CCL) 18, growth differentiation factor (GDF) 15, and interferon regulatory factor 4 correlated positively with tumor volume. Moreover, the amount of GDF15 in the cell culture supernatant of primary cells correlated positively with tumor volume. The concentrations of the cytokines CCL2, CCL5, and CCL18 and transforming growth factor beta (TGFB) 1 in the CSF of the patients were significantly different from those in the CSF controls. Inhibition of immune cell infiltration could be a putative approach to prevent and control VS growth.

Protective Effects of 7S,15R-Dihydroxy-16S,17S-Epoxy-Docosapentaenoic Acid (diHEP-DPA) against Blue Light-Induced Retinal Damages in A2E-Laden ARPE-19 Cells.

The purpose of this study was to investigate the protective effects of 7S,15R-dihydroxy-16S,17S-epoxy-docosapentaenoic acid (diHEP-DPA) in retinal pigment epithelial (RPE) cell damage. ARPE-19 cells, a human RPE cell line, were cultured with diHEP-DPA and Bis-retinoid N-retinyl-N-retinylidene ethanolamine (A2E), followed by exposure to BL. Cell viability and cell death rates were determined. Western blotting was performed to determine changes in apoptotic factors, mitogen-activated protein kinase (MAPK) family proteins, inflammatory proteins, and oxidative and carbonyl stresses. The levels of pro-inflammatory cytokines in the culture medium supernatants were also measured. Exposure to A2E and BL increased the ARPE-19 cell death rate, which was alleviated by diHEP-DPA in a concentration-dependent manner. A2E and BL treatments induced apoptosis in ARPE-19 cells, which was also alleviated by diHEP-DPA. Analysis of the relationship with MAPK proteins revealed that the expression of p-JNK and p-P38 increased after A2E and BL treatments and decreased with exposure to diHEP-DPA in a concentration-dependent manner. DiHEP-DPA also affected the inflammatory response by suppressing the expression of inflammatory proteins and the production of pro-inflammatory cytokines. Furthermore, it was shown that diHEP-DPA regulated the proteins related to oxidative and carbonyl stresses. Taken together, our results provide evidence that diHEP-DPA can inhibit cell damage caused by A2E and BL exposure at the cellular level by controlling various pathways involved in apoptosis and inflammatory responses.

α-IRAK-4 Suppresses the Activation of RANK/RANKL Pathway on Macrophages Exposed to Endodontic Microorganisms.

Periapical lesions are common pathologies affecting the alveolar bone, often initiated by intraradicular lesions resulting from microbial exposure to dental pulp. These microorganisms trigger inflammatory and immune responses. When endodontic treatment fails to eliminate the infection, periapical lesions persist, leading to bone loss. The RANK/RANKL/OPG pathway plays a crucial role in both the formation and the destruction of the bone. In this study, the objective was to inhibit the RANK/RANKL pathway in vitro within exposed Thp-1 macrophages to endodontic microorganisms, specifically Enterococcus faecalis, which was isolated from root canals of 20 patients with endodontic secondary/persistent infection, symptomatic and asymptomatic, and utilizing an α-IRAK-4 inhibitor, we introduced endodontic microorganisms and/or lipoteichoic acid from Streptococcus spp. to cellular cultures in a culture plate, containing thp-1 cells and/or PBMC from patients with apical periodontitis. Subsequently, we assessed the percentages of RANK+, RANKL+, and OPG+ cells through flow cytometry and measured the levels of several inflammatory cytokines (IL-1β, TNF-α, IL-6, IL-8, IL-10, and IL-12p70) in the cellular culture supernatant through a CBA kit and performed analysis by flow cytometry. A significant difference was observed in the percentages of RANK+RANKL+, OPG+ RANKL+ cells in thp-1 cells and PBMCs from patients with apical periodontitis. The findings revealed significant differences in the percentages of the evaluated cells, highlighting the novel role of the IRAK-4 inhibitor in addressing this oral pathology, apical periodontitis, where bone destruction is observed.

The immun function of dendritic cells in SFTS patients

ObjectivesSevere fever with thrombocytopenia syndrome (SFTS) is a newly emerging infectious disease caused by a novel bunyavirus in which host immune system suppression is thought to be crucial in disease development. Dendritic cells (DCs) are professional antigen-presenting cells (APCs) critical for initiation and orchestration of the immune response. And it have been suggested that functionally impaired DCs may mediate the suppression of host-specific T-cell immune responses and thus facilitate viral persistence and disease progression.This study was designed to improve the in vitro culture method for DCs and investigate the different immunologic functions of DCs between SFTS patients and healthy people. MethodsAll confirmed SFTS patients (N = 10) were recruited from the Jinan Infectious Diseases Hospital in 2019; routine laboratory parameters were collected. The frequencies, phenotypes were analyzed by flow cytometry. And the levels of 8 cytokines in the cell culture supernatant were detected by flow cytometry. ResultsOn day 8 of the incubation period, cells were harvested and analyzed by flow cytometry. There were significant differences in the rates of CD1a-, CD83-positive cells between SFTS patients and healthy people (all P < 0.05). The detection of 8 cytokines in the culture supernatant showed that the expressions of IFN-α and IFN-γ in the culture supernatant of DC cells in SFTS patients were lower than those in normal people (P < 0.05, P < 0.01). ConclusionsThe present results indicate that DCs may be functionally impaired in SFTS. A decreased level of circulating mDCs was closely correlated with SFTS progression.

The Cytokine Levels of Cord Blood- and Wharton's Jelly-Derived Mesenchymal Stem Cells from Early to Late Passages.

Mesenchymal stem cells (MSCs) are promising for clinical studies owing to their self-renewal, multipotency, trophic, and immunomodulatory properties. This study aimed to investigate the cytokine levels of human umbilical cord blood (CB) and Wharton's Jelly-(WJ) derived MSCs relevant to immune modulation on different passage levels in vitro. Umbilical CB MSCs were isolated using the ficoll-paque gradient method, and WJ-MSCs were isolated by the explant method. After isolation, the MSCs were characterized using flow cytometry. The supernatant cytokine levels (interferon-gamma (IFN-γ), interleukin 4 (IL-4), interleukin 17 (IL-17)) of MSCs at each passage were evaluated using the ELISA assay. MSCs exhibited different cytokine levels with each passage number. In WJ-MSC culture supernatants, IL-17 levels significantly increased at P4 and P5 compared to the first passage (p < 0.005), while the other passages showed a decrease. IFN-γ levels increased at passage P1 and P4 and decreased at other passages (p < 0.005). IL-4 levels significantly increased only at passage P3 (p < 0.005). In CB-MSC culture supernatants, IL-17 and IL-4 cytokines decreased compared to P0, while IFN-γ cytokine increased from P0 (p < 0.005). The changing ratio of cytokine levelsfor both CB-MSCs and WJ-MSCs were similarly maintained from early to late passages. More research is needed to understand the immunomodulatory functions of MSCs.

The Influence of Sulfation Degree of Glycosaminoglycan-Functionalized 3D Collagen I Networks on Cytokine Profiles of In Vitro Macrophage-Fibroblast Cocultures.

Cell-cell interactions between fibroblasts and immune cells, like macrophages, are influenced by interaction with the surrounding extracellular matrix during wound healing. In vitro hydrogel models that mimic and modulate these interactions, especially of soluble mediators like cytokines, may allow for a more detailed investigation of immunomodulatory processes. In the present study, a biomimetic extracellular matrix model based on fibrillar 3D collagen I networks with a functionalization with heparin or 6-ON-desulfated heparin, as mimics of naturally occurring heparan sulfate, was developed to modulate cytokine binding effects with the hydrogel matrix. The constitution and microstructure of the collagen I network were found to be stable throughout the 7-day culture period. A coculture study of primary human fibroblasts/myofibroblasts and M-CSF-stimulated macrophages was used to show its applicability to simulate processes of progressed wound healing. The quantification of secreted cytokines (IL-8, IL-10, IL-6, FGF-2) in the cell culture supernatant demonstrated the differential impact of glycosaminoglycan functionalization of the collagen I network. Most prominently, IL-6 and FGF-2 were shown to be regulated by the cell culture condition and network constitution, indicating changes in paracrine and autocrine cell-cell communication of the fibroblast-macrophage coculture. From this perspective, we consider our newly established in vitro hydrogel model suitable for mechanistic coculture analyses of primary human cells to unravel the role of extracellular matrix factors in key events of tissue regeneration and beyond.

The immunoreactive signature of monocyte-derived dendritic cells from patients with Down syndrome.

The clinical spectrum of Down syndrome (DS) ranges from congenital malformations to premature aging and early-onset senescence. Excessive immunoreactivity and oxidative stress are thought to accelerate the pace of aging in DS patients; however, the immunological profile remains elusive. We investigated whether peripheral blood monocyte-derived dendritic cells (MoDCs) in DS patients respond to lipopolysaccharide (LPS) distinctly from non-DS control MoDCs. Eighteen DS patients (age 2-47 years, 12 males) and 22 controls (age 4-40 years, 15 males) were enrolled. CD14-positive monocytes were immunopurified and cultured for 7 days in the presence of granulocyte-macrophage colony-stimulating factor and IL-4, yielding MoDCs in vitro. After the LPS-stimulation for 48 hours from days 7 to 9, culture supernatant cytokines were measured by multiplex cytokine bead assays, and bulk-prepared RNA from the cells was used for transcriptomic analyses. MoDCs from DS patients produced cytokines/chemokines (IL-6, IL-8, TNF-α, MCP-1, and IP-10) at significantly higher levels than those from controls in response to LPS. RNA sequencing revealed that DS-derived MoDCs differentially expressed 137 genes (74 upregulated and 63 downregulated) compared with controls. A gene enrichment analysis identified 5 genes associated with Toll-like receptor signaling (KEGG: hsa04620, P = 0.00731) and oxidative phosphorylation (hsa00190, P = 0.0173) pathways. MoDCs obtained from DS patients showed higher cytokine or chemokine responses to LPS than did control MoDCs. Gene expression profiles suggest that hyperactive Toll-like receptor and mitochondrial oxidative phosphorylation pathways configure the immunoreactive signature of MoDCs in DS patients.

Immunomodulatory and Anticancer Effects of Fridericia chica Extract-Loaded Nanocapsules in Myeloid Leukemia.

Nanocapsules provide selective delivery and increase the bioavailability of bioactive compounds. In this study, we examined the anticancer and immunomodulatory potential of Fridericia chica (crajiru) extract encapsulated in nanocapsules targeting myeloid leukemias. Nanocapsules containing crajiru (nanocapsules-CRJ) were prepared via interfacial polymer deposition and solvent displacement. Size and polydispersity were measured by dynamic light scattering. Biological assays were performed on leukemia cell lines HL60 and K562 and on non-cancerous Vero cells and human PBMC. The anticancer activity was evaluated using cytotoxicity and clonogenic assays, while the immunomodulatory activity was evaluated by measuring the levels of pro- and anti-inflammatory cytokines in PBMC supernatants treated with concentrations of nanocapsules-CRJ. Nanocapsules-CRJ exhibited significant cytotoxic activity against HL60 and K562 cells at concentrations ranging from 0.75 to 50 μg/mL, with the greatest reductions in cell viability observed at 50 μg/mL (p < 0.001 for HL60; p < 0.01 for K562), while not affecting non-cancerous Vero cells and human PBMCs. At concentrations of 25 μg/mL and 50 μg/mL, nanocapsules-CRJ reduced the formation of HL60 and K562 colonies by more than 90% (p < 0.0001). Additionally, at a concentration of 12 μg/mL, nanocapsules-CRJ induced the production of the cytokines IL-6 (p = 0.0002), IL-10 (p = 0.0005), IL-12 (p = 0.001), and TNF-α (p = 0.005), indicating their immunomodulatory potential. These findings suggest that nanocapsules-CRJ hold promise as a potential therapeutic agent with both cytotoxic and immunomodulatory properties.

Human mesenchymal stromal cells ameliorate cisplatin-induced acute and chronic kidney injury via TSG-6.

Cisplatin is widely used in tumor chemotherapy, but nephrotoxicity is an unavoidable side effect of cisplatin. Several studies have demonstrated that mesenchymal stromal cells (MSCs) ameliorate cisplatin-induced kidney injury, but the underlying mechanisms are unknown. In this study, the cisplatin-induced kidney injury mouse model was established by subjecting a single intraperitoneal injection with cisplatin. One hour before cisplatin injection, the mice received human bone marrow MSCs (hBM-MSCs) with or without siRNA-transfection, recombinant human tumor necrosis factor-α-stimulated gene/protein 6 (rhTSG-6), or PBS through the tail vein. In addition, cisplatin-stimulated HK-2 cells were treated with hBM-MSCs or rhTSG-6. Human BM-MSCs treatment remarkably ameliorated cisplatin-induced acute and chronic kidney injury, as evidenced by significant reductions in serum creatinine (Scr), blood urea nitrogen, tubular injury, collagen deposition, α-smooth muscle actin accumulation, as well as inflammatory responses, and by remarkable increased anti-inflammatory factor expression and Treg cells infiltration in renal tissues. Furthermore, we found that only a few hBM-MSCs engrafted into damaged kidney and that the level of human TSG-6 in the serum of mice increased significantly following hBM-MSCs administration. Moreover, hBM-MSCs significantly increased the viability of damaged HK-2 cells and decreased the levels of inflammatory cytokines in the culture supernatant. However, the knockdown of the TSG-6 gene in hBM-MSCs significantly attenuated their beneficial effects in vivo and in vitro. On the contrary, treated with rhTSG-6 achieved similar beneficial effects of hBM-MSCs. Our results indicate that systemic administration of hBM-MSCs alleviates cisplatin-induced acute and chronic kidney injury in part by paracrine TSG-6 secretion.

Molecular mechanism of Chang Shen Hua volatile oil modulating brain cAMP-PKA-CREB pathway to improve depression-like behavior in rats

BackgroundDepression is a common and complex mental illness that manifests as persistent episodes of sadness, loss of interest, and decreased energy, which might lead to self-harm and suicide in severe cases. Reportedly, depression affects 3.8 % of the world's population and has been listed as one of the major global public health concerns. In recent years, aromatherapy has been widely used as an alternative and complementary therapy in the prevention and treatment of depression; people can relieve anxiety and depression by sniffing plant aromatic essential oils. Acorus tatarinowii and Panax ginseng essential oils in Chang Shen Hua volatile oil (CSHVO) are derived from Acorus tatarinowii and Panax ginseng, respectively, the main medicines in the famous Chinese medicine prescription Kai Xin San (KXS), Then, these oils are combined with the essential oil of Albizia julibrissin flower to form a new Chinese medicine inhalation preparation, CSHVO. KXS has been widely used in the treatment of depression; however, whether CSHVO can ameliorate depression-like behavior, its pharmacological effects, and the underlying mechanisms of action are yet to be elucidated. Study design and MethodsA rat model of chronic and unpredictable mild stimulation (CUMS) combined with orphan rearing was treated with CSHVO for 4 weeks. Using behavioral tests (sucrose preference, force swimming, tail suspension, and open field), the depression-like degree was evaluated. Concurrently, brain homogenate and serum biochemistry were analyzed to assess the changes in the neurotransmitters and inflammatory and neurotrophic factors. Furthermore, tissue samples were collected for histological and protein analyses. In addition, network pharmacology and molecular docking analyses of the major active compounds, potential therapeutic targets, and intervention pathways predicted a role of CSHVO in depression relief. Subsequently, these predictions were confirmed by in vitro experiments using a corticosterone (CORT)-induced PC12 cell damage model. ResultsCSHVO inhalation can effectively improve the weight and depression-like behavior of depressed rats and regulate the expression of inflammatory factors and neurotransmitters. Hematoxylin-eosin, Nissl, and immunofluorescence staining indicated that compared to the model group, the pathological damage to the brain tissues of rats in the CSHVO groups was improved. The network pharmacological analysis revealed that 144 CSHVO active compounds mediate 71 targets relevant to depression treatment, most of which are rich in the cAMP signaling and inflammatory cytokine pathways. Protein-protein interaction analysis showed that TNF, IL6, and AKT are the core anti-depressive targets of CSHVO. Molecular docking analysis showed an adequate binding between the active ingredients and the key targets. In vitro experiments showed that compared to the model group, the survival rate of PC12 cells induced by CSHVO intervention was increased, the apoptosis rate was decreased, and the expression of inflammatory cytokines in the cell supernatant was improved. Western blot analysis and immunofluorescence staining confirmed that CSHVO regulates PC12 cells in the CORT model through the cAMP-PKA-CREB signaling pathway, and pretreatment with PKA blocker H89 eliminates the protective effect of CSHVO on CORT-induced PC12 cells. ConclusionsCSHVO improves CORT-induced injury in the PC12 cell model and CUMS combined with orphan rearing-induced depression model in rats. The antidepressant mechanism of CSHVO is associated with the modulation of the cAMP-PKA-CREB signaling pathway.

Inflammatory Response to Wear Particles: Comparisons Between Zirconia and Titanium In Vitro.

To compare the inflammatory responses of peripheral blood mononuclear cells (PBMCs) subjected to titanium (Ti) and/or zirconia (Zr) particles while growing on Ti or Zr discs. In total, 240 discs were fabricated at a size of 2 mm in height and 5 mm in diameter. Of the 240 discs, 120 discs were made of Ti (commercially pure [CP] grade 4), and 120 discs were made of Zr (3 mol% yttria-stabilized zirconia polycrystals [3Y-TZP]). The PBMCs were cultured on the two-disc materials, and particles with a size up to 20 mm Ti (99.5% Ti) and 0.1 to 0.2 mm Zr (3Y-TZP) were added to the cultures. The concentration levels of inflammatory cytokines in culture supernatants were measured through Bio-Plex assay (Bio-Rad Laboratories), and light microscopic analysis was performed to detect cell attachment and characterize particle shape and cell-particle interaction. The inflammatory responses of PBMCs were generally higher when cells were cultured on a Ti surface compared to a Zr surface. In addition, higher cytokine levels were seen when cells were cultured in the presence of Ti particles compared to Zr particles when no discs were used. However, there were only significantly increased levels for three cytokines (MCP-1, IFN-γ, and TNF-α) when particles were added to Ti discs. Higher release of neutrophil extracellular traps (NETs) from neutrophils were seen in presence of Zr particles compared to Ti particles. A reduction in cell death was observed in the presence of Zr particles compared to Ti particles and unstimulated control samples. The type of growth material and presence of particle affects PBMCs in vitro. Cells seeded on Ti discs and together with Ti particles generated higher levels of inflammatory cytokines compared to the Zr counterparts.

Bacterial peptidoglycan drives synovial inflammation in osteoarthritis

Abstract Peptidoglycan (PG) is a potent inflammatory mediator. We have previously demonstrated that PG is present in the synovial tissue of osteoarthritis (OA) patients but its role in OA inflammation remains unknown. We hypothesized that PG in synovial tissue drives inflammation in OA. Intraoperative synovial tissue and synovial fluid samples were obtained from 56 consecutive patients with OA, with no prior history of infection. PG in synovial tissue was detected by immunohistochemical (IHC) staining of serial sections of 10 mm2 of tissue with anti-PG antiserum. PG was found in 33/56 (59%) of the tissue samples using IHC, with a median of eight PG occurrences per 10 mm2 in PG-positive samples. Tissue inflammation and fibrosis were assessed by histopathology. Cellular localization was further characterized in three PG-positive tissue samples by immunofluorescent microscopy (IFM). PG was localized to cells demonstrating mononuclear and fibroblastic morphology primarily in and around areas of vascularization and inflammation. CD68+ macrophages and to a lesser degree CD90+ synovial sublining fibroblasts. In multiplex assay analysis of synovial fluid cytokine levels, IL-6 and PG abundance were positively correlated. Fibroblasts and macrophages stimulated with PG in vitro secreted high levels of innate cytokines, paricularly IL-6 when measured by supernatant cytokine quantification. Together these data support the hypothesis that PG in synovial tissue drives joint inflammation in OA.

Characterization of CD4+ t cell subsets during Chlamydia muridarum genital infection

Characterization of CD4+ T cell subsets in a stressed mouse model during Chlamydia muridarum genital infection. Katrina Davis, Tesfaye Belay. Department of Applied Sciences and Mathematics. Chlamydia genital infection caused by Chlamydia trachomatis is highly prevalent in low socioeconomic status populations. Whether stress is a risk factor for the high incidence of chlamydia remains to be investigated. CD4+ T cells are the major immune cells that fight chlamydia genital infection; however, the dynamics of CD4+ T cells during stressful conditions and chlamydia infection are unclear. The profiles of CD4+ T cell signature cytokines: T helper 1 (IL-12, Interferon-gamma (IFN-γ), Thelper2 (IL-4, IL-5, IL-13), Thelper17(IL-17), and Treg (IL-10). Mice were stressed for 5 minutes daily for 21 days and infected with 1x 105 IFU/ml of Chlamydia muridarum intravaginally. Non-stressed mice were infected as controls. Mice were sacrificed 7 days post-infection, the spleen and lymph nodes were harvested, and T cells were purified and proliferated for 72 h. Determination of the levels of cytokines in culture supernatants of T cells from stressed and non-stressed mice are underway for poster presentation. Supported by the NIH grant # 1R15AI124156-01 awarded to BSU