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Immunometabolic Circuits in Infection for Advancing Host Directed Therapies
In This Article
Summary
Here, a protocol for designing, constructing, and delivering tetracycline-controlled based inducible synthetic circuit is presented. The effect of the synthetic circuits on parasite elimination and cytokine expression can be studied by channelizing the presented pipeline. This protocol is relevant for researchers studying synthetic circuit-based therapeutics in infectious diseases.
Abstract
Immuno-metabolism is a pivotal determinant in the progression of leishmaniasis. Synthetic biology-based approach has garnered significant attention as a step toward therapeutic intervention targeting host-associated factors that drive leishmaniasis. Synthetic biology entails the engineering of genetic components in an orthogonal and modular manner to precisely modulate biological systems, imparting novel functions to cells. In the presented study, elucidation of a systematic pipeline for the development of an inducible tetracycline-controlled (TetON)-based synthetic circuit was aimed at delivering succinate dehydrogenase as a therapeutic agent to facilitate the elimination of intracellular Leishmania parasites. The outlined protocol describes the designing of a synthetic circuit and its subsequent validation. The proposed strategy also concentrates on the incorporation of synthetic circuits in the plasmid backbone as a delivery vehicle. Additionally, delivery machinery employing polyplexes-based nano-particles for the delivery of synthetic circuits was used in murine macrophage cell lines without compromising the cellular morphology. Standardization of the method was conducted for selecting transfected cells and determining optimal induction concentration for synthetic circuit expression. Observations reveal a distinct reduction in intracellular parasite burden in transfected cells compared to infected cells. Pro-inflammatory cytokines were expressed post-infection in synthetic circuit transfected and induced cells as a mechanism to promote parasite elimination. This underscores the synthetic biology-based method as a potent approach in leishmaniasis by targeting host factors associated with disease progression.
Introduction
The advancement of holistic, integrative medicine is a significant link that has been identified between metabolic alterations in immune cells and their ability to modulate immune cell phenotype in infectious diseases. Immuno-metabolism offers fresh perspectives for elucidating the pathogenesis of prevalent infectious diseases in humans. Moreover, it provides promising techniques for the development of vaccines and drugs by identifying novel targets for intervention1. Metabolic reprogramming of immune cells is identified across various infectious diseases, including leishmaniasis. Leishmania spp. is responsible for causing leishmaniasi....
Protocol
1. Cell culture of RAW264.7 cells
NOTE: Use low passage numbers for experimentation. RAW264.7 cell line was procured from National Cell repository of Biotechnology Research Innovation Council-National Centre for cell Sciences (BRIC-NCCS), Pune. As RAW264.7 cell line is a macrophage cell line it may start differentiating with increasing passages. After reviving the cells use the cell line for transfection after 2 passages.
- Seed 5 x 105 cells of the RAW264.......
Representative Results
The arrangement of genetic parts forming the synthetic circuit for expressing the synthetic circuit is shown in (Figure 1A). The corresponding parts were cloned in pEGFP-N1 vector in an orthogonal and modular fashion, which is represented in Figure 1B. The simulation of the synthetic circuit revealed oscillatory dynamics in both SDHA and the tetracycline repressor. This observation suggests a reciprocal expression pattern characterized by periodic wave functions.......
Discussion
The obtained results underscore the efficiency of the pipeline, which has been channelized for the design and implementation of a synthetic circuit. The construction of this synthetic circuit was a step towards developing precision medicine for infectious diseases like leishmaniasis. Through the in silico assembly of genetic parts and deterministic simulation, the distinguished modulatory effect of the TetON system was monitored. The effect of the circuit, notably, might have led to the reciprocal bistable expre.......
Acknowledgements
The authors would like to thank the Director of the Biotechnology Research and Innovation Council- National Centre for Cell Science (BRIC-NCCS), Pune for supporting our research and the BRIC-NCCS Bioinformatics Facility.
....Materials
| Name | Company | Catalog Number | Comments |
| 10 μL tips | Tarson | 521050 | |
| 10X restriction buffer | BioLabs | R0146S | Restriction digestion |
| 1mL tips | Tarson | 521016 | |
| 1mL tube | Eppendorf | 30121023 | |
| 200 μL tips | Tarson | 521010 | |
| 25cm2 flask | Corning | 430639 | For cell culture |
| 4',6-diamidino-2-phenylindole | ThermoFischer Scientific | D1306 | nuclear staining |
| 50mL tube | Corning | 352070 | |
| 60mm plates | Falcon | 353002 | |
| 6X TrackIt Cyan/Orange Loading buffer | ThermoFischer Scientific | 10488085 | Loading samples for AGE |
| 96 well coverslip bottom plate | ThermoFischer Scientific | 160376 | For confocal imaging and transfection |
| 96 well micro test plate flat bottom wells | Tarson | 941196 | |
| Agarose | Sigma | A9539 | For AGE |
| Agarose gel electrophoresis assembly | GeNei | 93 | For AGE |
| Bacterial laminar flow | ESCO Lifesciences | 2120765 | For transformation |
| Calcium chloride | Merck | C4901 | Buffer /Reagent preparation |
| Cell culture laminar air flow | ThermoFischer Scientific | 1323TS | For cell culture |
| Cell scraper | Genetix | 90020 | For cell culture |
| Cell spreader | Fischer Scientific | 12822775 | |
| Centrifuge for 1.5mL tubes | Eppendorf | EP5404000537 | |
| Centrifuge for 50mL falcon | ThermoFischer Scientific | 75004210 | |
| Chlorofrom | Fischer Scientific | 67-66-3 | For RNA isolation |
| CO2 incubator | ThermoFischer Scientific | 3110 | For cell culture |
| Cuvette | Eppendorf | 6138000018 | Estimation of plasmid concentration |
| CYTIVA IQ 500 gel imager | Cytiva | 29655893 | For AGE |
| DEPC TREATED H2O 1000 ML | ThermoFischer Scientific | 750023 | For RNA isolation |
| Doxycycline | Merck | 33429 | For induction of transfected cells |
| Dry heating block | Labnet | For transformation and inactivation of restriction enzymes | |
| Dulbecco's Modified Eagle Medium | National centre for cell science | For cell culture | |
| Ethanol | Sigma-Aldrich | 1.00983 | Buffer /Reagent preparation |
| Ethidium bromide | Sigma | E7637 | For AGE |
| Ethylenediamine tetraacetic acid | Fischer Scientific | 12635 | Buffer /Reagent preparation |
| Evos brightfield microscope | ThermoFischer Scientific | AMEX1000 | |
| ExPasy | https://web.expasy.org/translate/ | for contruction of synthetic circuit | |
| Fetal Bovine Serum | Gibco | 16000-044 | For cell culture |
| FV31S-SW software | Olypmus | Image acquisition | |
| Geneticin | Gibco | 1563411 | for transfection |
| Gibson assembly method for cloning | BIOMATIK | Construction of synthetic circuit | |
| Graphpad prism | Graphpad | Statistical analysis | |
| High-Capacity cDNA Reverse Transcription Kit | ThermoFischer Scientific | 4368814 | for cDNA synthesis |
| Isopropanol | Fischer Scientific | 13825 | Buffer /Reagent preparation |
| Kanamycin | Sigma | 60615 | For transformation and colony selection |
| Luria Bertini broth | Himedia | M1245 | For culturing E.coli |
| Magnesium chloride | Fischer Scientific | BP214 | Buffer /Reagent preparation |
| Micro Filt Vertical Laminar Air Flow | Microfilt | ||
| MicroAmp Fast 8-Tube Strip, 0.1 mL | ThermoFischer Scientific | 4358293 | For qRT-PCR |
| MicroAmp Optical 8-Cap Strips | ThermoFischer Scientific | 4323032 | For qRT-PCR |
| Microwave oven | LG | MC-7649DW | For AGE |
| Mm00434228_m1 (IFN-γ) | ThermoFischer Scientific | 4331182 | Taqman probes |
| Mm00443258_m1 (TNF) | ThermoFischer Scientific | 4331182 | Taqman probes |
| Mm01321739_m1(TGFB) | ThermoFischer Scientific | 4331182 | Taqman probes |
| Mouse ELISA Kit IL-10 | ThermoFischer Scientific | BMS614 | For ELISA |
| Mouse ELISA Kit IL-12 | ThermoFischer Scientific | BMS616 | For ELISA |
| Multiskan Sky with Touch Screen + μDrop Plate | ThermoFischer Scientific | 51119600DP | For ELISA |
| MX-M Microplate Mixers 96-well Cell Culture Plate Mixer Adjustable 0-1500 rpm Laboratory Shaker Agitator | DIAB | ||
| National centre for Biotechnology Information | https://www.ncbi.nlm.nih.gov | for contruction of synthetic circuit | |
| Neubauer's chamber | Marienfeld superior | 640010 | For cell count |
| Non-vented 25cm2 flask | Corning | 353014 | For culturing parasite |
| NotI | BioLabs | R3189M | Restriction enzyme |
| Nuclease free water | Biodesign | 1QIA | Reagent preparation |
| Olympus Cell Sens Dimension Desktop 2.3 software | Olypmus | Image processing | |
| Olympus FV 3000 | Olympus | For confocal imaging | |
| OPTI-MEM media | Gibco | 31985070 | media for Transfection |
| pEGFP-N1 vector cloned with synthetic circuit | Biomatik | ||
| Penicillin | ThermoFischer Scientific | 15070063 | For cell culture |
| Polyethylenimine | MERCK | 764965 | for transfection |
| Potassium Acetate | Fischer Scientific | YBP364500 | Buffer /Reagent preparation |
| Potassium Chloride | Fischer Scientific | BP366 | Buffer /Reagent preparation |
| Potassium Phosphate Monobasic | Sigma | P5655 | Buffer /Reagent preparation |
| Power supply pack | BIORAD | 1645070 | For AGE |
| Registry of Standard Biological parts | https://parts.igem.org/Main_Page | for contruction of synthetic circuit | |
| RNAiso Plus | Takara | 38220090 | For RNA isolation |
| RNase A | ThermoFischer Scientific | 12091021 | Buffer /Reagent preparation |
| Roswell Park Memorial Institute Medium | National centre for cell science | For culturing parasite | |
| Shaker incubator | REMI | CIS 24 plus | For culturing E.coli |
| Sodium chloride | Qualigens | Q27605 | Buffer /Reagent preparation |
| Sodium Dodecyl Sulfate | Sigma-Aldrich | L3771 | Buffer /Reagent preparation |
| Sodium Hydroxide | Fischer Scientific | 15895 | Buffer /Reagent preparation |
| Sodium Phosphate Dibasic | Sigma | S5136 | Buffer /Reagent preparation |
| Spectrophotometer | Eppendorf | Estimation of plasmid concentration | |
| Spin win | Trason | 1020 | |
| StepOne plus Real Time PCR system | Life technologies | 272006777 | For qRT-PCR |
| StepOne software version 2.3 | Life technologies | For qRT-PCR | |
| TaqMan Fast Universal PCR Master Mix (2X), no AmpErase UNG | ThermoFischer Scientific | 4366072 | |
| Tinker cell | Synthetic circuit construction and simulation | ||
| TrackIt 1 Kb Plus DNA ladder | ThermoFischer Scientific | 10488085 | For AGE |
| Tris (hydroxymethyl) aminomethane hydrochloride | Himedia | MB029 | Buffer /Reagent preparation |
| Tris-acetate-EDTA | SERVA | 42549.1 | Buffer /Reagent preparation |
| Trypan blue | Sigma | T8154 | For cell count |
| XhoI | BioLabs | R0146S | Restriction enzyme |
References
- Chen, J. Y., Zhou, J. K., Pan, W. Immunometabolism: Towards a better understanding the mechanism of parasitic infection and Immunity. Front Immunol. 12, 661241 (2021).
- Ferreira, C., Estaquier, J., Silvestre, R.
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