Team:CPU CHINA/Demonstrate
Demonstration
Outline
Plasmid 1: TLR1, TLR2 and CD14
Plasmid 2: Granulysin
Plasmid 3: mir-let-7f and PD-L1-targeting peptide
Results
The recognition ability of immune-like cells to M.tuberculosis (Mtb)
The plasmid 1 is responsible for the expression of TLR1/2 and CD14, which could recognize the antigen of Mtb, activate the downstream pathway of HEK293 to produce NF-κB and thus turn on the transcription of the next plasmids. The three receptors of plasmid 1 were first characterized with WB assay. As Figure.1A showed, there were striking bands presenting in the transfected group, which demonstrated that TLR1/2 and CD14 both expressed on the membrane of HEK293 after the transfection. Flow cytometry was employed to further verify the expression, and the similar results were obtained (Figure.1B). IκB is the inhibitor of NF-κB by masking of its nuclear localization sequence. Phosphorylation promotes ubiquitination and degradation of IκB, enabling the NF-κB to translocate to the nucleus and activate transcription. To evaluate the activation of NF-κB, the increased level of phosphorylated IκB (p-IκB) and decreased level of IκB were generally determined. After antigen added, it could be observed that lighter color was presented in the IκB band and deeper color was presented in the p-IκB group with WB assay (Figure.1C). In short, we determined that the transfection of plasmid 1 enabled HEK293 cells to recognize Mtb and successfully activate downstream NF-κB.
Responsive secretion and the bactericidal effect of granulysin
The promising candidate for bactericidal effect, granulysin, was screened out by mathematical model . After stimulated by Mtb, the activation of intracellular NF-κB would activate the NF-κB inducible promotor, then responsively make our immune-like cells produce and secrete granulysin. In in vitro experiment, antigen of Mtb was used to stimulate our immune-like cells. Subsequently, the secretion of granulysin was detected by ELISA assay and we explored the secretion condition over time in 4 the divided groups. For Figure.2A, compared with other three groups, there was a significant elevation only in the group which had transfection and stimulation at the same time. While nearly no variation was found in other groups over time, a certain extent of increase presented in the fourth group over time. Results indicated that plasmid 2 did function as expected in immune-like cells and the ability of ideal responsive promotion may secure the safety in vivo.
The extracted supernatant containing granulysin, from three groups, was separately incubated with Mycobacterium smegmatis and we further evaluated the bactericidal effect through measuring the absorption at different time. In about 24 hours, the effect started to emerge in the stimulated transfection group and the bacteria survival rate plunged to a half at 40 h (Figure.2B). The amount of bacteria was controlled under a steady level since then. It demonstrated that our immune-like cells could achieve satisfying bactericidal effect after recognizing Mtb. To further determine the safety of granulysin to normal human cells, MTT assay was conducted to examine the survival rate of HEK293 cells after incubated with the supernatant containing different concentration of granulysin for 12 hours. The value of survival rate was steady, about 100%, proved the good safety of granulysin.
Intracellular treatment with targeting exosomes containing hsa-let-7f
To characterize the reprogrammed targeting exosomes and verify its ability of enhancing bactericidal efficacy of macrophages, exosomes were extracted from the supernatant with extraction kit ExoQuick-TC™(SBI). First, electronic microscope was employed to display the morphology and probable size of the exosomes (Figure.3A). Then, in order to further characterize exosomes, the diameter was measured by DLS (Figure.3A). Generally, exosome particle size is within the range of 50-150 nm in diameter, and the measured results, about 101.97nm, correspond to this range, indicating that what we extracted is indeed exosomes.
HA tag was introduced to the C-terminal of PD-L1-targrting peptide in plasmid-3 to help examine its expression on the membrane of exosomes with WB assay. As Figure.3B showed, the successful expression of HA could be found in the transfected group, and the band presented at a desirable site of about 45 kD as expected. There is no existence of this peptide in control group, suggesting PD-L1-targeting ability has been attached to the exosomes.
In this system, exosomes secreted from immune-like cells would carry the supply of miRNA hsa-let-7f, so the exosomal RNA was extracted for reverse transcription and then detection of let-7f amount by Real-time Quantitative PCR Detecting System (QPCR) (Figure.3C). The results demonstrate that the amount of let-7f after transfection is 174 times greater than that of control group, indicating that a considerable amount of hsa-let-7f miRNA was successfully encapsulated in the exosomes.
At last, we verified the effect of miRNA-carrying exosomes on the enhancement of the microbicidal ability of infected macrophages. Exosomes were incubated with murine RAW264.7 cells for 48 hours. After redundant exosomes being removed, the RNA extraction from the macrophages was conducted to be quantified by qPCR to monitor changes in the level of IL1β, IL6, TNFα, MCP1 and MIP2 genes (Figure.3D). Elevated expression levels of these gene improve the inflammation and enhance microbicidal efficacy [1]. The results showed that exosomes significantly increased IL1β, IL6 and MIP2 expression, while the level of MCP1 and TFNα was only lifted for a little bit. This demonstrates that our miRNA-carrying exosomes did and enhance microbicidal efficacy of macrophages and activate immunological function.
Reference
[1] Taams, L.S. (2018). Inflammation and immune resolution. Clin Exp Immunol 193, 1-2.