In vitro cytokine expression analysis by droplet microfluidics

Droplet microfluidics provides a miniaturized platform to conduct biological assays. We previously developed a droplet microfluidic chip assay for screening cancer cells against chemical drugs and chimeric antigen receptor T (CAR-T) cells, respectively. In this study, we investigated chip application on a cytokine expression assay using MCF7 breast cancer reporter cells engineered by fusing green fluorescent protein (GFP) to the C-terminus of endogenous interleukin-6 (IL6) gene. Combined tumor necrosis factor alpha (TNFalpha) treatment and serum-free medium starvation stimulated IL6-GFP expression and enhanced GFP fluorescence. Our data showed that on-chip assay recapitulates the cellular response in vitro, although absolute quantification of IL6 induction could not be accomplished. The demonstration of multi-timepoint IL6 expression analysis paves the way for our future study on tumor response to immune attack via cytokine signaling.

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SOCS1/SOCS3 Immune Axis Modulates Synthetic Perturbations in IL6 Biological Circuit for Dynamical Cellular Response

10.1101/2020.08.31.276634 ◽

2020 ◽

Author(s):

Bhavnita Soni ◽

Shailza Singh

Keyword(s):

Signaling Pathway ◽

Cellular Response ◽

Inflammatory Cytokine ◽

Cytokine Expression ◽

Janus Kinase ◽

Cytokine Signaling ◽

Macrophage Phenotype ◽

Regulatory Circuit ◽

Stat Pathway

AbstractMacrophage phenotype plays a crucial role in the pathogenesis of Leishmanial infection. Pro-inflammatory cytokines are the key regulators that eliminate the infection induced by Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway. Suppressor of cytokine signaling (SOCS) is a well-known negative feedback regulator of JAK/STAT pathway. However, change in expression levels of SOCS in correlation with the establishment of infection is not well understood. Mathematical modeling of IL6 signaling pathway have helped identified the role of SOCS1 in establishment of infection. Furthermore, the ratio of SOCS1 and SOCS3 has been quantified both in silico as well as in vitro, indicating an immune axis which governs the macrophage phenotype during L. major infection. The ability of SOCS1 protein to inhibit the JAK/STAT1 signaling pathway and thereby decreasing pro-inflammatory cytokine expression makes it a strong candidate for therapeutic intervention. Using synthetic biology approaches, peptide based immuno-regulatory circuit have been designed to target the activity of SOCS1 which can restore pro-inflammatory cytokine expression during infection.

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T helper 1 to T helper 2 shift in cytokine expression: an autoregulatory process in superantigen-associated psoriasis progression?

Journal of Medical Microbiology ◽

10.1099/jmm.0.003939-0 ◽

2009 ◽

Vol 58 (2) ◽

pp. 180-184 ◽

Cited By ~ 18

Author(s):

Sarika Jain ◽

Iqbal R. Kaur ◽

Shukla Das ◽

S. N. Bhattacharya ◽

Anjani Singh

Keyword(s):

Interleukin 2 ◽

Cytokine Expression ◽

T Helper ◽

T Helper 1 ◽

Th2 Response ◽

Peripheral Blood Mononuclear ◽

Necrosis Factor Alpha ◽

Duration Of Disease ◽

Th1 Immune Responses

Psoriasis is an inflammatory skin disorder characterized by increased activation of CD4+ T lymphocytes, and systemic and local overexpression of pro-inflammatory cytokines such as interleukin 2 (IL-2), gamma interferon (IFN-γ), IL-6 and tumour necrosis factor alpha, indicating that immunopathogenesis of the disease is T helper 1 (Th1) mediated. Several studies suggest a pivotal role of bacterial superantigens in the initiation and/or exacerbation of this illness. This study was conducted to assess the systemic Th1/Th2 imbalance in Indian psoriasis patients presenting with variable duration of disease by studying systemic superantigen-stimulated peripheral blood mononuclear cell (PBMC) cytokine expression. PBMCs were isolated and stimulated in vitro with superantigens (streptococcal pyrogenic exotoxin A and staphylococcal enterotoxin B), and the cytokines released (IFN-γ for a Th1 response, and IL-4 and IL-10 for a Th2 response) were assayed. In contrast to controls, psoriasis patients in the early course of disease were characterized by significantly increased expression of the pro-inflammatory cytokine IFN-γ, whilst a shift towards IL-10 secretion (Th2 response) was observed in those presenting with increased duration of disease. These observations suggest a possible shift from a Th1 to a Th2 cytokine response with superantigen-associated progression for the duration of psoriasis, perhaps as an adaptive process by the immune system in an attempt to downregulate abnormal inflammatory Th1 immune responses.

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Synthetic Perturbations in IL6 Biological Circuit Induces Dynamical Cellular Response

Molecules ◽

10.3390/molecules27010124 ◽

2021 ◽

Vol 27 (1) ◽

pp. 124

Author(s):

Bhavnita Soni ◽

Shailza Singh

Keyword(s):

Synthetic Biology ◽

Cellular Response ◽

Leishmania Major ◽

Early Stage ◽

Macrophage Polarization ◽

Janus Kinase ◽

Cytokine Signaling ◽

Macrophage Phenotype ◽

Stat Pathway

Macrophage phenotype plays a crucial role in the pathogenesis of Leishmanial infection. Pro-inflammatory cytokines signals through the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway that functions in parasite killing. Suppression of cytokine signaling (SOCS) is a well-known negative feedback regulator of the JAK/STAT pathway. However, change in the expression levels of SOCSs in correlation with the establishment of infection is not well understood. IL6 is a pleotropic cytokine that induces SOCS1 and SOCS3 expression through JAK-STAT signaling. Mathematical modeling of the TLR2 and IL6 signaling pathway has established the immune axis of SOCS1 and SOCS3 functioning in macrophage polarization during the early stage of Leishmania major infection. The ratio has been quantified both in silico and in vitro as 3:2 which is required to establish infection during the early stage. Furthermore, phosphorylated STAT1 and STAT3 have been established as an immunological cross talk between TLR2 and IL6 signaling pathways. Using synthetic biology approaches, peptide based immuno-regulatory circuits have been designed to target the activity of SOCS1 which can restore pro-inflammatory cytokine expression during infection. In a nutshell, we explored the potential of synthetic biology to address and rewire the immune response from Th2 to Th1 type during the early stage of leishmanial infection governed by SOCS1/SOCS3 immune axis.

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Pathogenesis of Yersinia pestis Infection in BALB/c Mice: Effects on Host Macrophages and Neutrophils

Infection and Immunity ◽

10.1128/iai.73.11.7142-7150.2005 ◽

2005 ◽

Vol 73 (11) ◽

pp. 7142-7150 ◽

Cited By ~ 101

Author(s):

Roman A. Lukaszewski ◽

Dermot J. Kenny ◽

Rosa Taylor ◽

D. G. Cerys Rees ◽

M. Gill Hartley ◽

...

Keyword(s):

Yersinia Pestis ◽

Fluorescent Protein ◽

Virulent Strain ◽

Late Phase ◽

Flow Cytometric Analysis ◽

Annexin V ◽

Intracellular Bacteria ◽

Necrosis Factor Alpha ◽

Green Fluorescent

ABSTRACT The pathogenesis of infection with Yersinia pestis, the causative agent of plague, was examined following subcutaneous infection of BALB/c mice with a fully virulent strain expressing green fluorescent protein. Plate culturing, flow cytometry, and laser confocal microscopy of spleen homogenates throughout infection revealed three discernible stages of infection. The early phase was characterized by the presence of a small number of intracellular bacteria mostly within CD11b+ macrophages and Ly-6G+ neutrophils. These bacteria were not viable, as determined by plate culturing of spleen homogenates, until day 2 postinfection. Between days 2 and 4 postinfection, a plateau phase was observed, with bacterial burdens of 103 to 104 CFU per spleen. Flow cytometric analysis revealed that there was even distribution of Y. pestis within both CD11b+ macrophage and Ly-6G+ neutrophil populations on day 2 postinfection. However, from day 3 postinfection onward, intracellular bacteria were observed exclusively within splenic CD11b+ macrophages. The late phase of infection, between days 4 and 5 postinfection, was characterized by a rapid increase in bacterial numbers, as well as escape of bacteria into the extracellular compartment. Annexin V staining of spleens indicated that a large proportion of splenic neutrophils underwent rapid apoptosis on days 1 and 2 postinfection. Fewer macrophages underwent apoptosis during the same period. Our data suggest that during the early stages of Y. pestis infection, splenic neutrophils are responsible for limiting the growth of Y. pestis and that splenic macrophages provide safe intracellular shelters within which Y. pestis is able to grow and escape during the later stages of infection. This macrophage compliance can be overcome in vitro by stimulation with a combination of gamma interferon and tumor necrosis factor alpha.

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Expression Analysis of the Yersiniabactin Receptor GenefyuA and the Heme Receptor hemR ofYersinia enterocolitica In Vitro and In Vivo Using the Reporter Genes for Green Fluorescent Protein and Luciferase

Infection and Immunity ◽

10.1128/iai.69.12.7772-7782.2001 ◽

2001 ◽

Vol 69 (12) ◽

pp. 7772-7782 ◽

Cited By ~ 25

Author(s):

Christoph A. Jacobi ◽

Sebastian Gregor ◽

Alexander Rakin ◽

Jürgen Heesemann

Keyword(s):

Green Fluorescent Protein ◽

Expression Analysis ◽

Peritoneal Cavity ◽

Fluorescent Protein ◽

Reporter Genes ◽

Infection Model ◽

Mouse Infection Model ◽

Green Fluorescent

ABSTRACT The enteropathogenic Yersinia enterocolitica strains have several systems for scavenging iron from their environment. We have studied the expression of the fyuA gene, which encodes the outer membrane receptor for the siderophore yersiniabactin (Ybt), and the hemR gene, which encodes the receptor for heme, using the reporter genes gfp (encoding green fluorescent protein) and luc (encoding firefly luciferase). To study gene expression in vitro as well as in vivo, we have constructed several translational reporter gene fusions to monitor simultaneously expression of fyuA andhemR or expression of one gene by agfp-luc tandem reporter. Results of the in vitro expression analysis (liquid media) indicated that fyuA and hemR are strongly derepressed under iron starvation conditions, resulting in strong fluorescence and/or luminescence at 27°C. In the in vivo BALB/C mouse infection model, tissue-specific expression of fyuA andhemR reporter fusions was observed. Surprisingly,fyuA and hemR reporter constructs were weakly expressed by yersiniae located in the liver and intestinal lumen, whereas strong expression was found for yersiniae in the peritoneal cavity and moderate expression was found in the spleen. Strikingly, yersiniae carrying fyuA orhemR reporter fusions exhibited threefold-stronger signals when grown in the peritoneal cavity of mice than those growing under iron derepression in vitro. This hyperexpression suggests that besides Fur derepression, additional activators may be involved in the enhanced expression of fyuA and hemRunder peritoneal growth conditions. Differential expression of thefyuA and hemR reporter fusions could not be observed, suggesting similar regulation of fyuA andhemR in the mouse infection model.

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Inhibition of miR-665 alleviates lipopolysaccharide-induced inflammation via up-regulation of SOCS7 in chondrogenic ATDC5 cells

Tropical Journal of Pharmaceutical Research ◽

10.4314/tjpr.v19i10.7 ◽

2020 ◽

Vol 19 (10) ◽

pp. 2067-2072

Author(s):

Qiaoyi Ning ◽

Yiting He ◽

Wukai Ma ◽

Fang Tang ◽

Ying Huang ◽

...

Keyword(s):

Inflammatory Cytokines ◽

Luciferase Reporter ◽

Cytokine Signaling ◽

Necrosis Factor Alpha ◽

Inflammatory Injury ◽

Expression Levels ◽

Over Expression ◽

Qrt Pcr ◽

Lps Treatment

Purpose: To examine the effect and mechanism of action of miR-665 in osteoarthritis.Methods: An in vitro inflammatory injury model of osteoarthritis was established using chondrogenic ATDC5 cells with lipopolysaccharide (LPS) treatment. The expression levels of inflammatory cytokines were determined by enzyme-linked immunosorbent assays (ELISAs) and by quantitative real-time polymerase chain reaction (qRT-PCR). A binding target for miR-665 was predicted using TargetScan and then evaluated using a dual-luciferase reporter assay.Results: Treatment with LPS significantly up-regulated the inflammatory cytokine expressions of interleukin-1β (IL-1β), IL-6, and tumor necrosis factor-alpha (TNF-α), in ATDC5 cells (p < 0.01), and the expression of miRNA-665 was significantly increased in LPS-treated ATDC5 cells (p < 0.01).Knockdown of miR-665 down-regulated the expression levels of these inflammatory cytokines. Suppressor of cytokine signaling 7 (SOCS7) was identified as a target of miR-665. Data from qRT-PCR and western-blot analyses indicated that SOCS7 expression was promoted by miR-665 inhibition and inhibited by miR-665 over-expression. LPS treatment significantly decreased the expression of SOCS7 protein in ATDC5 cells (p < 0.01), and over-expression of SOCS7 attenuated the LPS-stimulated inflammatory injury. In addition, over-expression of miR-655 enhanced the inflammatory injury and reversed the protective effect of SOCS7 against LPS-stimulated inflammation.Conclusion: Inhibition of miR-665 alleviated LPS-stimulated inflammatory injury in ATDC5 cells via the up-regulation of SOCS7, suggesting a potential therapeutic target for osteoarthritis. Keywords: MiR-665, Lipopolysaccharide, Inflammation, SOCS7, Chondrogenic, ATDC5

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Inhibition of the PI3K/AKT Pathway Reduces Tumor Necrosis Factor-Alpha Production in the Cellular Response to Wear Particles In Vitro

Artificial Organs ◽

10.1111/j.1525-1594.2012.01568.x ◽

2013 ◽

Vol 37 (3) ◽

pp. 298-307 ◽

Cited By ~ 15

Author(s):

Jian-bin Huang ◽

Yue Ding ◽

Dong-sheng Huang ◽

An-jing Liang ◽

Wei-ke Zeng ◽

...

Keyword(s):

Tumor Necrosis Factor ◽

Cellular Response ◽

Tumor Necrosis ◽

Akt Pathway ◽

Wear Particles ◽

Necrosis Factor Alpha ◽

Alpha Production ◽

Factor Alpha ◽

Necrosis Factor

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A poly (saccharide-ester-urethane) scaffold for mammalian cell growth

Cellular and Molecular Biology ◽

10.14715/cmb/2021.67.3.15 ◽

2021 ◽

Vol 67 (3) ◽

pp. 113-117

Author(s):

Maykel González-Torres ◽

Marymar Becerra-González ◽

Gerardo Leyva-Gómez ◽

Enrique Lima ◽

Oswaldo González Mendoza ◽

...

Keyword(s):

Cell Growth ◽

Cell Lines ◽

Mammalian Cell ◽

Mammalian Cells ◽

Cellular Response ◽

Fluorescent Protein ◽

Membrane Integrity ◽

Dermal Fibroblasts ◽

Mammalian Cell Lines

Chitosan and poly(3-hydroxybutyrate) are non-toxic, biodegradable, and biocompatible polymers extensively used in regenerative medicine. However, it is unknown whether the chemical combination of these polymers can produce a biomaterial that induces an appropriate cellular response in vitro in mammalian cells. This study aimed to test the ability of a novel salt-leached polyurethane scaffold of chitosan grafted with poly(3-hydroxybutyrate) to support the growth of three mammalian cell lines of different origin: a) HEK-293 cells, b) i28 mouse myoblasts, and c) human dermal fibroblasts. The viability of the cells was assessed by either evaluation of their capacity to maintain the expression of the green fluorescent protein by adenoviral transduction or by esterase activity and plasma membrane integrity. The results indicated that the three cell lines attached well to the scaffold; however, when i28 cells were induced to differentiate, they did not produce morphologically distinct myofibers, and cell growth ceased. In conclusion, the findings reveal that, altogether, these observations suggest that this foam scaffold supports cell growth and proliferation but may not apply to all cell types. Hence, one crucial question yet to be resolved is a poly (saccharide-ester-urethane) derivative with a nano-topography that elicits a similar cellular response for different biological environments.

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