Immune Cell Cytokine Secretion Research Articles

Ultrasimple Single-Cell Detection of Multiple Cytokines by a Nanowell Chip Integrated with Encoded Microarrays.

Cytokine production is often regarded as the marker of immune cells' activation status. The spectrum and temporal secretion of cytokines are dramatically varied between cell phenotypes and even within the same phenotype. Multiparameter analysis of individual immune cell's cytokine secretion has always been a challenging and complicated process that needs special facilities in a laboratory setting. Herein, we present an ultrasimple method with high sensitivity and high robustness to quantify cytokine expression at the single-cell resolution. A microchip is developed based on poly(dimethylsiloxane) nanowells on sticky tape, while each nanowell is integrated with a DNA-antibody convertible microarray. Only pipetting is needed for the whole single-cell analysis process. The sensitivity of the assay is evaluated by measuring various concentrations of six recombinant cytokine proteins, which was found comparable to conventional methods. Once single cells are loaded to nanowells and incubated there, a Fluorinert FC-40 is used to isolate nanowells; so, cytokines from those cells are captured by separate microarrays. The rest of the sandwich enzyme-linked immunosorbent assay detection process is also executed simply by pipetting of various reagents. This method is validated by measuring cytokine production from hundreds of single cells. It has simplified a typically sophisticated multiplex single-cell assay into an instrument-free, point-of-detection technology, and thus it may find a broad utility in clinical diagnostics.

Sphingosine-1-Phosphate (S1P) Signaling through S1P Receptor 1 (S1PR1) in Macrophage Contributes to Pain in Sickle Cell Disease

Sickle cell disease (SCD) is the most common autosomal recessive hemolytic disorder affecting more than 100,000 African-Americans and millions around the world. The severe health complication of patients with SCD is chronic pain, which is the leading cause of SCD patient morbidity and hospitalization. Our proposed research builds on the results from our nonbiased high-throughput metabolomics screen, which revealed erythrocyte sphingosine-1-phosphate (S1P), a bioactive lipid, is significantly elevated in the circulation of humans and mice with SCD. We identified that erythrocyte-sphingosine kinase 1 (Sphk1), the enzyme that generates S1P, promote erythrocyte sickling and the genetic deletion of Sphk1 significantly reduce pain symptoms in SCD mice. S1P is a pleiotropic molecule that signals via five g-protein coupled receptors (GPCRs) known as S1P receptors (S1PR1-5). S1PR1 is widely expressed in many cell types, including neurons and immune cells. We treated SCD mice with FDA approved drug, FTY720, to specifically target S1PR1. We demonstrated that mice had improved mechanical and thermal pain sensitivity. Notably, using flow cytometry, we found that the percentage of macrophages infiltrated to skin and percentage of S1PR1 expression in macrophages in the skin were significantly induced in SCD mice compared to the controls. This implies that elevated S1PR1 in macrophage contributes to chronic pain in SCD mice. To test this possibility, we treated SCD mice with clodronate liposomes to deplete tissue macrophages. We identified that mice had reduced sensitivity to mechanical and thermal stimuli assessments. Altogether, we provide both genetic and pharmacological evidence that elevated plasma S1P signaling via S1PR1 likely on macrophages contributes to chronic pain in SCD. Additionally, S1PR1 is known to mediate pro-inflammatory cytokine secretion in immune cells. We identified that SCD mice have elevated levels of interleukin-6 (IL-6), a pro-inflammatory cytokine, and is likely involved in SCD pain. To study the role of IL-6 in SCD pain, we generated SCD-IL-6-/- mice and observed improved mechanical and thermal pain sensitivity in these mice. From these evidences, we hypothesize that Sphk1 mediate S1P-S1PR1 activation in macrophages and IL-6 induction to promote SCD pain. Our work addresses a knowledge gap regarding an inflammatory mediated mechanism involved in SCD pain and novel therapies to counteract pain in SCD. DisclosuresNo relevant conflicts of interest to declare.

Interaction of Der p 2 with Toll-like Receptor 4 and its Effect on Cytokine Secretion

Der p 2, which is a major allergen of house dust mite, plays an important role in the pathogenesis of allergic disease. There is controversy regarding whether Der p 2 binds to Toll-like receptor 4 (TLR4), and its inflammatory effect has not yet been elucidated. In the current study, we examined the interaction of Der p 2 with TLR4 and the effect of Der p 2 on cytokine release in THP-1 cells and lymphocytes. Among house dust mite extracts, recombinant TLR4 protein interacted with Der p 2. The overall structure of Der p 2 is characteristic of the immunoglobulin superfamily and contains ten β-strands, forming a β-cup fold with two anti-parallel β-sheets, and a short 310 helix. The two sheets can be separated, further allowing the formation of a large internal pocket, which is narrow and suitable for binding large flat molecules such as lipid-like molecules. Der p 2 caused increased secretion of IL-6, IL-8, and MCP-1, which are neutrophil survival factors, in human monocytic THP-1 cells in a time-dependent manner. Der p 2 also induces the release of cytokines in normal and allergic lymphocytes. Supernatant after treatment with Der p 2 inhibited neutrophil apoptosis. In coculture of lymphocytes with neutrophils, Der p 2 inhibited spontaneous apoptosis of allergic neutrophils. In summary, Der p 2 binds to TLR4 and induces an inflammatory response such as cytokine secretion in immune cells. These findings may enable elucidation of allergy pathogenesis by specific allergen of house dust mite.

Cationic Lipid and Bacterial Lipopolysaccharides Both Activate Toll-Like Receptor 4 Pathways via Different Binding Regions

DiC14-amidine is a cationic lipid which induces pro-inflammatory cytokine secretion in immune cells upon interaction with the Toll-like receptor 4 (TLR4)/Myeloid Differentiation factor-2 (MD-2) membrane bound-complex, the natural sensor of bacterial lipopolysaccharides (LPS) [1-3].The aim of the present work is to characterize the interaction between diC14-amidine and the TLR4/MD2 receptor complex. Taking advantage of the species-dependent activity of TLR4 agonists [4], we compared the TLR4 agonist activity of diC14-amidine in four different species in order to map domains in TLR4 and MD2 that are important for diC14-amidine TLR4-agonist activity. We demonstrate that, while LPS is an agonist in all species, diC14-amidine is a full agonist for human, mouse and cat receptors, but a poor agonist for horse. Using chimeric constructs made from human and horse TLR4 and single mutants, we identify two regions in the human TLR4 that modulates the agonist activity of diC14-amidine. Interestingly, these regions in TLR4 are different from the previously identified bacterial lipopolysaccharides binding domains [5]. How ligand binding to two different regions of the same receptor affects the cellular pathways is currently under investigation.[1] Tanaka, T. et al. (2008) Eur.J.Immunol. 38: 1351-1357.[2] Lonez, C. Et al. (2008) Prog Lipid Res. 47:340-7.[3] Lonez, C; et al. (2012) Adv Drug Deliv Rev. 64:1749-58.[4] Walsh, C. et al. (2008) J.Immunol. 181: 1245-1254.[5] Park, B. S. et al. (2009) Nature 458: 1191-1195.

Multiple Cytotoxic Factors and JAK/STAT Pathways Are Involved in the Enhanced Antitumor Function of CIK Cells Induced by IL-21

Abstract 1059Adoptive transfer of activated T and NK cells has had significant clinical benefits in certain tumor models. Cytokine induced killer (CIK) cells are a group of cells that possess both T and NK cell like recognition of target cells. These cells are generated after extensive ex vivo manipulation of PBMCs. Maintenance of not only CIK cells but other activated effector T and NK cells in culture is vital for their effective transfer and development following adoptive immunotherapy. IL-21 is the newest member of the common γ chain family which has been shown to increase cytotoxic factors and cytokine secretion in immune cells without over stimulation. Such qualities make IL-21 a suitable agent in immunotherapy of tumors. IL-21 has shown effective antitumor function and is currently going clinical trials for tumors such as renal cell carcinoma, melanoma and lymphoma. Our previous experiment showed that like in T cells and NK cells, IL-21 significantly improves the cytotoxicity of CIK cell on K562 cells and primary leukemic cells from patients. Although proliferation of cells in a CIK cell pool was not observed we found that it helped maintain and grow the CD3+ and CD56+ phenotype. Our present experiment aims to explain the mechanism through which IL-21 promotes CIK cells survival and cell cytotoxicity.In our experiment, blood from healthy donors was collected and PBMCs were transformed into CIK cells following 14 days of culture using appropriate methods. The cells were then stimulated with IL-21 for a defined period of time and subjected to MTT assays to measure cellular viability and cytotoxicity to K562 cells. To elucidate the mechanism of action of IL-21, CIK cells were checked for the level of mRNA expression of perforin, Granzyme B, FasL, INF-γ, TNF-α,Granzyme A,NKG2D, TNF-β using RT-PCR. Furthermore the expression of significantly important cytotoxic factors and cytokines was measured through flow cytometry and ELISA. Western blot was performed to check the involvement of JAK/STAT pathway following stimulation.We found that IL-21 doesn’t enhance in vitro proliferation of CIK cells, but does increase the number of cells expressing the CD3+/CD56+ phenotype. IL-21 can also significantly increase the cytotoxic potential of CIK cells to K562 cells. It does so with significantly increased production of perforin which increased almost 2 folds from (0.5592±0.1457) to (0.9831±0.1265); Granzyme B also by almost 2 folds from (0.4084±0.1589) to (0.7319±0.1639) and FasL which increased by almost 2 folds from (0.4015±0.2842) to (0.7381±0.2568). Increase in secretion of cytokines such as INF-γ was observed from (25.8±6.1)ng/L to (56.0±2.3)ng/L; and TNF-α from (5.64±0.61)ug/L to (15.14±0.93)ug/L while no significant difference was observed in the expression of Granzyme A,TNF-β and NKG2D. Measurement of IL-21R receptor on CIK cell surface following IL-21 stimulation caused a more than two folds increase in expression of IL-21R from 1.88% to 4.25%. We further affirm that JAK/STAT is actively involved in IL-21 signalling. STAT3 and STAT5b could be potential signalling mechanisms taking part in IL-21 enhanced cytotoxic potential of CIK cells.Using this information we have concluded that increased expression of perforin, Granzyme B, FasL, IFN-γ and TNF-α plays a significant role in IL-21 enhanced cytotoxic potential of CIK cells and STAT-3 and STAT-5b signaling pathway are involved in the processes. Our data indicate that IL-21 is a potent enhancer of antitumor function of CIK cells. As CIK cells and IL-21 have both been shown to increase patient survival or tumor free periods in certain hematological malignancies using them in conjunction might be therapeutically more beneficial. Disclosures:No relevant conflicts of interest to declare.

In vitro characterization of the immunotoxic potential of several perfluorinated compounds (PFCs)

We have previously shown that PFOA and PFOS directly suppress cytokine secretion in immune cells, with different mechanisms of action. In particular, we have demonstrated a role for PPAR-α in PFOA-induced immunotoxicity, and that PFOS has an inhibitory effect on LPS-induced I-κB degradation. These studies investigate the immunomodulatory effects of four other PFCs, namely PFBS, PFOSA, PFDA, and fluorotelomer using in vitro assays. The release of the pro-inflammatory cytokines IL-6 and TNF-α was evaluated in lipolysaccharide (LPS)-stimulated human peripheral blood leukocytes (hPBL) and in the human promyelocytic cell line THP-1, while the release of IL-10 and IFN-γ was evaluated in phytohemagglutinin (PHA)-stimulated hPBL. All PFCs suppressed LPS-induced TNF-α production in hPBL and THP-1 cells, while IL-6 production was suppressed by PFOSA, PFOS, PFDA and fluorotelomer. PFBS, PFOSA, PFOS, PFDA and fluorotelomer inhibited PHA-induced IL-10 release, while IFN-γ secretion was affected by PFOSA, PFOS, PFDA and fluorotelomer. Leukocytes obtained from female donors appear to be more sensitive to the in vitro immunotoxic effects of PFCs when their responses are compared to the results obtained using leukocytes from male donors. Mechanistic investigations demonstrated that inhibition of TNF-α release in THP-1 cells occurred at the transcriptional level. All PFCs, including PFOA and PFOS, decreased LPS-induced NF-κB activation. With the exception of PFOA, none of the PFCs tested was able to activate PPARα driven transcription in transiently transfected THP-1 cells, excluding a role for PPARα in the immunomodulation observed. PFBS and PFDA prevented LPS-induced I-κB degradation. Overall, these studies suggest that PFCs affect NF-κB activation, which directly suppresses cytokine secretion by immune cells. Our results indicate that PFOA is the least active of the PFCs examined followed by PFBS, PFDA, PFOS, PFOSA and fluorotelomer.

Anti-Inflammatory Activity of Curcumin in Macrophages Stimulated by Lipopolysaccharides from Porphyromonas gingivalis

Background:Porphyromonas gingivalis, a major periodontopathic bacterium, is necessary for periodontitis to take place. The lipopolysaccharide (LPS) of P. gingivalis stimulates cytokine secretion in immune cells, and thereby initiates the inflammation related to periodontitis. Macrophages are the important ones of the immune cells that are prominent at inflammatory periodontal sites. Curcumin, a major curcumanoid found in the spice turmeric, exhibits anti-inflammatory properties. The aim of this study was to investigate the anti-inflammatory effect and the mechanism of action of curcumin in macrophages stimulated by P. gingivalis LPS. Methods: RAW264.7 cells pre-treated with various concentrations of curcumin were stimulated by P. gingivalis LPS. TNF-α and IL-1β expressions were separately detected by RT-PCR and ELISA. Next, activation of NF-ĸB-dependent transcription was examined by luciferase assay. Results: Curcumin dose-dependently inhibited TNF-α and IL-1β gene expression and protein synthesis in RAW264.7 cells stimulated with P. gingivalis LPS. P. gingivalis LPS activated NF-ĸB-dependent transcription in RAW264.7 cells, which were down-regulated by pre-treatment with curcumin as well. Conclusion: Our data suggest that curcumin can inhibit P. gingivalis LPS-induced cytokine expression, and that this could be due to the inhibition of the NF-ĸB pathway.

VIP InhibitsPorphyromonas gingivalisLPS-induced Immune Responses in Human Monocytes

Lipopolysaccharide (LPS) from the Gram-negative pathogen Porphyromonas gingivalis (Pg) stimulates cytokine secretion in immune cells, and thereby initiates the inflammation associated with periodontitis. Modulation of pro-inflammatory cytokine activity is a plausible therapeutic target in periodontal disease. Vasoactive intestinal peptide (VIP) has a role in immunoregulation, and has been identified as a molecule with therapeutically beneficial immunosuppressive effects in inflammatory and autoimmune conditions. We aimed to investigate the effect of VIP on immune responses induced by Pg LPS in vitro. VIP (10(-8) M) significantly (P < 0.05) inhibits TNF-alpha production by human monocytic THP1 cells stimulated with Pg LPS. In parallel, we showed that VIP inhibits nuclear translocation of NFkappaB and c-Jun in a time-dependent manner, but does not decrease the expression of CD14 receptors. This is the first report to show the potential of VIP as an immunomodulator of Pg-stimulated inflammatory pathways in human monocytes.