Team:CPU CHINA/Design
From our visit to Nanjing CDC , we found that tuberculosis (TB) patients have a higher risk of relapse within three months after recovery. In this case, regular check is essential for patients. But the existing methods are facing the problem of false negative results and bad medication adherence after patients being discharged from hospitals, which may cause delayed treatment and eventually leads to the deterioration of patients’ condition. What’s more, as we mentioned in the Background, resistance against existing TB drugs has not been well addressed.
In order to put forward a therapeutic strategy to solve all the above problems, we come up with an idea to employ cells capable of long circulation, to be more specific, mesenchymal stem cells to design the immune-like cells. This immune-like cells would display lasting function of surveillance and protection after being injected into the patient’s body, which can help the patients pull through the high recurrence risk period. After recognizing Mycobacterium tuberculosis (Mtb) in vivo, these immune-like cells can release granulysin to destroy Mtb in the blood circulation; in the meantime, they can secret targeting exosomes containing miRNA to rescue the infected macrophages. Since the mechanism of granulysin and miRNA against Mtb differs from that of existing antibacterial drugs, it is not easy to confer resistance and thus provide a new strategy to combat the drug-resistant Mtb.
Input
Our cells are expected to express Toll-like receptors (TLRs) and CD14 to recognize Mtb. TLR exists in all kinds of immune cells related with innate immunity. This receptor can recognize antigens on the surface of pathogen like lipoteichoic acid (LTA), lipoarabinomannan (LAM) and lipoprotein[1]. Lipopolysaccharide (LPS) receptor CD14, a kind of leukocyte differentiation antigen found on the surface of monocytes, macrophages and other immune cells, can co-recognize and bind the LAM on the surface of Mtb[2].
Through mathematical modeling, we screened out TLR1 and TLR2 from Toll-like receptor family as the most crucial roles in the recognition of Mtb. When these two receptors are successfully expressed on the membrane of selected normal cells, they will form heterodimers to identify Mtb with the help of CD14.
The composite part(BBa_K2976013) consisting of these three parts is assumed to have a promising ability to recognize Mtb. After recognition, a series of signal transduction will happen in “immune-like” cells, which ends with activation of the NF-κB transcriptional factor, and then we use an NF-κB inducible promoter[3] to responsively promote downstream pathway designed: granulysin against extracellular Mtb and transformative exosomes against intracellular Mtb.
Output
Our artificial immune-like cells achieve signal output responsively by secreting granulysin and macrophage-targeting exosome containing hsa-let-7f (Figure.1). We designed this dual treatment strategy because Mtb is a parasitic bacteria presenting extracellularly as well as intracellularly, mostly in macrophage[4].
Extracellular Treatment with Granulysin
Granulysin is an immunologically active substance secreted by cytotoxic T lymphocytes and natural killer cells [5], which damages the cell membrane of bacillus by electrostatic interaction, hydrogen bonding and van der Waals force [6] . It has been observed in clinical trials that granulysin is quite competent for the treatment of tuberculosis[7]. In our system, granulysin kills extracellular Mtb by damaging membrane integrity and disrupting lipid metabolism of the bacillus (Figure.2), and it has a desirable safety to human cells for its lack of cell wall. However, due to this property, granulysin fails to act the same function on the infected macrophages as well. To conquer this drawback, we further designed the strategy to target intracellular bacteria.
Intracellular Treatment with Targeting Exosomes Containing miRNA let-7f
When Mtb infects macrophages, miRNA hsa-let-7f, a miRNA inside macrophages decreases. In our design, exogenous supplementation of let-7f increases the level of endogenous let-7f and further up regulates NF-κB. The upregulation of NF-κB can boost the secretion of cytokines, chemokines and NO to kill intracellular Mtb[8].
Exosomes are extracellular vesicles secreted by human cells, which are known for the ability of infusion with cell membrane and capable of carrying miRNA [8]. LAMP2 is a membrane protein that is highly expressed on exosome membrane, of which our modification could endow exosomes with macrophage-targeting ability. Since macrophages infected with Mtb will highly express PD-L1[9], we fuse LAMP2 with PD-L1-targeting peptide(Figure.3) and add glycosylation protection, which help avoid excision of signal peptides, to increase their expression on exosomes [10]. We made let-7f delivered by designed targeting exosomes (Figure.4) to macrophages infected by Mtb. With the utilization of such targeting exosomes, let-7f could be increased in infected macrophages and the treatment of intracellular Mtb could be realized (Figure.5).
GNSTM is a glycosylation protection sequence. HA is a Western Blot tag for protein purification. PD-L1 targeting peptide and GNSTM, LAMP-2B are linked by GS-rich flexible linkers.
To summarize, after recognizing Mtb by TLR and CD14, our immune-like cells secrete granulysin and targeting exosomes carrying let-7f to fight against extracellular and intracellular Mtb respectively.
References
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