Name: Immunometabolic Circuits in Infection for Advancing Host Directed Therapies (Video) | JoVE Journal
Uploaded: 2024-09-13T14:00:00.000+00:00
Duration: 11 min 12 s
Description: Explore synthetic circuit design for Leishmania major infection using tetracycline-controlled inducible systems. This protocol demonstrates parasite elimination and cytokine modulation in host-directed immunotherapy research.

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5 minutes

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.

Designing Tetracycline-Controlled Synthetic Circuits for Leishmaniasis Therapy

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<li>Cheung, S. T., Shakibakho, S., So, E. Y., Mui, A. L. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Transfecting+RAW264.7+cells+with+a+luciferase+reporter+gene%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">Transfecting RAW264.7 cells with a luciferase reporter gene.</a> J Vis Exp. (100), (2015).</li>
<li>Fus-Kujawa, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+overview+of+methods+and+tools+for+transfection+of+eukaryotic+cells+in+vitro%5BTitle%5D)+AND+%22Front+Bioeng+Biotechnol%22%5BJournal%5D)">An overview of methods and tools for transfection of eukaryotic cells in vitro.</a> Front Bioeng Biotechnol. 9, 701031(2021).</li>
<li>Das, A. T., Tenenbaum, L., Berkhout, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tet-On+systems+for+doxycycline-inducible+gene+expression%5BTitle%5D)+AND+%22Curr+Gene+Ther%22%5BJournal%5D)">Tet-On systems for doxycycline-inducible gene expression.</a> Curr Gene Ther. 16 (3), 156-167 (2016).</li>
<li>De Boeck, J., Verfaillie, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Doxycycline+inducible+overexpression+systems%3A+how+to+induce+your+gene+of+interest+without+inducing+misinterpretations%5BTitle%5D)+AND+%22Mol+Biol+Cell%22%5BJournal%5D)">Doxycycline inducible overexpression systems: how to induce your gene of interest without inducing misinterpretations.</a> Mol Biol Cell. 32 (17), 1517-1522 (2021).</li>
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The authors have nothing to disclose.

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 expression of TetR and SDHA/GFP. The expression of SDHA was periodic, which might indicate that only in the presence of doxycycline its expression was increased. To observe potential changes in expression patterns over a period, a time unit of 100 was selected. Results identify that the synthetic circuit showed periodic behavior for TetR and SDHA/GFP, likely attributable to its orthogonal nature21. The assembly of genetic parts from the iGEM parts registry played a pivotal role, as it provided genetic components necessary for constructing synthetic circuits22. Modular arrangement of genetic parts is essential as translation and functioning of the circuit might get affected, which may lead to inefficient expression and modulatory effect in cells23.
The insert size of the synthetic circuit was approximately 1.3 kb to ensure the size of the vector is not too large, which may potentially reduce the transformation efficiency24. Selecting an appropriate vector is a critical step as it must possess genetic parts, which are essential to increase the copy number in the bacterial system and yield high plasmid copy, and its expression machinery must adapt well in the mammalian system25. The pEGFP-N1 vector is well-characterized in the RAW264.7 cell line and possesses a compact plasmid structure with a multiple cloning site26. Lab-standardized buffer solutions were employed for plasmid isolation to ensure the production of highly concentrated and pure plasmid, which was validated through spectrophotometric estimation and AGE. Insertion of the synthetic circuit in the pEGFP-N1 vector was validated through restriction digestion, and the samples were analyzed on AGE and sequencing. AGE results represented a 1.3 kb band of insert, 4.7 kb band of pEGFP-N1 vector, and 6 kb band of the entire synthetic circuit. 
Transfection using 1:1, 1:2, and 2:1 ratio of DNA: PEI ratios was performed, where the observed ratio of 1:1 worked best without disrupting the cell morphology (data not shown). It is recommended to use 400 &#181;g/mL - 1000 &#181;g/mL concentration of G418 for selection of transfected cells27. A 500 &#181;g/mL of G418 was used for the selection of transfected cells as it effectively suppressed the growth of non-transfected cells without compromising cell growth or morphology of transfected cells (data not shown). Given the importance of cell phenotype in transfection efficiency, particularly in macrophage cell lines, which can undergo differentiation and become challenging to transfect, a low passage number of the RAW264.7 cell line was used for transformation28. Cell passage numbers might also be a critical factor in transfection efficiency. A high passage number may lead to inefficient transfection29. Dox is the preferred inducer for the Tet-ON induction system because of its high binding efficiency to TetR, favorable tissue distribution, low toxicity, and a half-life of 24 h30. Post transfection, cells were induced with dox for 24 h, and they were observed for GFP expression. For RAW264.7 cells, the concentration of dox used for synthetic circuit induction was 1 &#181;g/mL which falls in the recommended range of 1-2 &#181;g/mL31 (data not shown). However, the regulation of SDHA concentration post-induction was undetermined. Overall, the determination and standardization of cell passage number, DNA: PEI concentration, mammalian transfection selection marker concentration, and doxycycline induction concentration are crucial factors in the transfection process.
Following the induction of the synthetic circuit post-infection at different time points, a significant reduction in intracellular parasite burden was observed. A comparison of parasite loads between infected and IMTI samples revealed a lower burden in cells induced with the synthetic circuit, likely attributable to SDHA expression. As SDHA expression is associated with NO production, IMTI cells were observed to have an irregular and rough morphological form, which occurs in the RAW264.7 cell line when NO is produced32. IL-10 and IL-12 levels post 6 h infection were highest as both cytokines have been reported to be upregulated at this time point, which further undergoes phenotypic modification to produce IL-10 more than IL-1219. As IL-10 and IL-12 levels did not significantly alter post-transfection and in IMTI samples, cytokine profiling was performed to detect pro-inflammatory and anti-inflammatory cytokine responses. 
Expression levels of TNF-&#945;, IFN-&#947;, and TGF-&#946; were identified in 6 h infection, IMT, and 24 h IMTI samples. To eliminate intracellular parasites, TNF-&#945; and IFN-&#947; are principally essential as they induce oxidative bursts through NO production. M2 polarized macrophages express TGF-&#946;, which downregulates pro-inflammatory cytokines33. The 24 h IMTI sample showed increased TNF-&#945; and IFN-&#947; expression than TGF-&#946;, which suggests that the macrophages, upon transfection and infection with L. major, might have polarized to pro-inflammatory cytokine expressing phenotype. The study, therefore, provides insight into the phenotypic alterations induced upon transfection with a synthetic circuit and highlights infection dynamics. The effect of the synthetic circuits as a therapeutic can be investigated as their induction had increased pro-inflammatory cytokine expression significantly as compared to anti-inflammatory cytokine. Thus, the study not only established the synthetic circuit construction and delivery pipeline but also demonstrated its therapeutic potential in a leishmaniasis model through the upregulation of pro-inflammatory cytokines in Leishmania-challenged host cells.

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 leishmaniasis, of which Leishmania major (L. major) causes cutaneous leishmaniasis (CL). L. major parasites elicit a significant impact on the metabolism of host cells, particularly in macrophages, for proliferation and survival as intracellular parasites2. Macrophages can typically polarize into either classically activated (M1) or alternatively activated (M2) subsets where M1 cells possess high pro-inflammatory cytokine secretion and production of nitric oxide (NO) through the metabolism of nitrogen species. M2 cells exhibit elevated levels of anti-inflammatory cytokines and display high arginase activity through nitrogen metabolism3. Thus, both phenotypes not only differ through their immune response but also in their metabolic pathways.
Systems biology of leishmaniasis aims to identify molecular targets for designing novel therapeutics through drug designing and development. Reconstruction of signaling networks and metabolic pathways aids in understanding leishmaniasis as a holistic model and draws insight for the identification of the principal component driving the diseased state. Mathematical modeling is one of the most favored applied analytical approaches for understanding the complexities of leishmaniasis4. Immunometabolic networks and mathematical models enabled the identification of succinate dehydrogenase (SDHA) as a key component of the M1 phenotype, which is expressed to eliminate the parasite5. Synthetic biology has applications in diagnostics, vaccine development and therapeutics in leishmaniasis. Metabolism targeting immune response modulation may be achieved through synthetic biology tools. For modulation of inositol phosphoryl ceramide synthase which regulates sphingolipid metabolism and alters host immune signaling, a genetic circuit that possessed bistable behavior was used to synthetically tune the intrinsic robustness of L. major infection model6,7. Hence, systems and synthetic biology can provide a model-based genetic engineering platform for the development of precision medicine in the Leishmania model system.
M1 phenotype is marked by the expression of immuno-metabolic signaling networks where the production of inflammatory cytokines and metabolic pathways synergistically enhances parasite elimination. M1 polarization signifies the production of pro-inflammatory cytokines such as Interleukin-12 (IL-12), interferon-gamma (IFN-&#947;), and Tumor necrosis factor alpha (TNF-&#945;). Metabolic pathways that are highly enriched in the M1 phenotype include glycolysis, pentose phosphate pathway, and truncated tricarboxylic acid cycle (TCA cycle)5. The truncated TCA cycle directs parasite elimination machinery through increased succinate and citrate levels, which guide the production of NO3. Accumulation of succinate by SDHA leads to activation of transcription factor hypoxia-inducible factor 1-alpha (HIF-1&#945;), which induces the production of IL-1&#946;8. In the M2 phenotype, fatty acid oxidation, increase in glutamine utilization, and enhanced oxidative phosphorylation of glucose are observed along with the production of anti-inflammatory cytokines such as Interleukin-10 (IL-10), Transforming growth factor beta (TGF-&#946;), Interleukin-4 (IL-4) and Interleukin-13 (IL-13)3. M2 exhibits elongated mitochondria as a distinctive feature, leading to enhanced efficiency in energy generation. They utilize fatty acids, glucose, and glutamine as substrates to fuel the TCA cycle, thereby generating ATP through oxidative phosphorylation at a higher rate to promote parasite growth and proliferation9. SDHA enzyme responsible for succinate accumulation through truncated TCA can not only promote NO production but also initiate a succinate-dependent oxygen consumption through electron transport10. In Leishmania-infected macrophages, SDHA gets downregulated by 2.17 fold, indicating aerobic respiration might also be downregulated11. In order to increase succinate consumption to induce pro-inflammatory cytokine-mediated NO production and promote parasite elimination, expression of SDHA is essential.
The synthetic circuit offers precision-targeted therapies that may alter the diseased system to achieve a bistable oscillation that may restore healthy phenotype at the spatiotemporal level. The synthetic circuit delivery and expression make the system dynamic and robust. One pivotal factor contributing to the advancement of synthetic biology lies in the ability to assemble genetic parts and elements in a modular fashion to enable the generation of intricate architectures and tailored functionalities12. Constraints associated with traditional expression techniques have spurred the emergence of tetracycline operon-based inducible plasmid synthetic circuits, thus allowing rapid evaluation of plasmid and easy delivery across various cell types13. The tetracycline-responsive repressor (TetR) protein, when existing as a dimer, exhibits strong binding affinity to the tet operon (TetO), effectively suppressing transcription. However, upon the interaction of its ligand, doxycycline (dox), TetR undergoes a structural alteration, resulting in its dissociation from TetO, consequently allowing transcription to proceed unimpeded. This circuit can exhibit protein translation control14. Therefore, through the defined methodology, the designed tetracycline operon-based inducible plasmid synthetic circuit was enabled to express SDHA in the pEGFP-N1 vector. Evaluation of the transformation efficiency of the circuit and its yield provided insights into the competence of delivery. Through restriction digestion of the vector, the determination of the cloning efficiency was confirmed. Further, a transfected RAW264.7 mouse macrophage cell line derived from peritoneal macrophages of Balb/c mice was observed for doxycycline dose-dependent green fluorescent protein (GFP) expression in transfected cells. Additionally, the parasite elimination effect in transfected cells was corroborated with an expression of the synthetic circuit. Moreover, induction and expression of pro-inflammatory cytokines were observed, which was directed through an increase in the time point of infection in the synthetic circuit transfected and induced cells, which was directly associated with the functioning of the synthetic circuit.

<table><tbody><tr><td>10 &amp;mu;L tips</td><td>Tarson</td><td>521050</td><td></td></tr><tr><td>10X restriction buffer</td><td>BioLabs</td><td>R0146S</td><td>Restriction digestion</td></tr><tr><td>1mL tips</td><td>Tarson</td><td>521016</td><td></td></tr><tr><td>1mL tube</td><td>Eppendorf</td><td>30121023</td><td></td></tr><tr><td>200 &amp;mu;L tips</td><td>Tarson</td><td>521010</td><td></td></tr><tr><td>25cm2 flask</td><td>Corning</td><td>430639</td><td>For cell culture</td></tr><tr><td>4&#039;,6-diamidino-2-phenylindole</td><td>ThermoFischer Scientific</td><td>D1306</td><td>nuclear staining</td></tr><tr><td>50mL tube</td><td>Corning</td><td>352070</td><td></td></tr><tr><td>60mm plates</td><td>Falcon</td><td>353002</td><td></td></tr><tr><td>6X TrackIt Cyan/Orange Loading buffer</td><td>ThermoFischer Scientific</td><td>10488085</td><td>Loading samples for AGE</td></tr><tr><td>96 well coverslip bottom plate</td><td>ThermoFischer Scientific</td><td>160376</td><td>For confocal imaging and transfection</td></tr><tr><td>96 well micro test plate flat bottom wells</td><td>Tarson</td><td>941196</td><td></td></tr><tr><td>Agarose</td><td>Sigma</td><td>A9539</td><td>For AGE</td></tr><tr><td>Agarose gel electrophoresis assembly</td><td>GeNei</td><td>93</td><td>For AGE</td></tr><tr><td>Bacterial laminar flow</td><td>ESCO Lifesciences</td><td>2120765</td><td>For transformation</td></tr><tr><td>Calcium chloride</td><td>Merck</td><td>C4901</td><td>Buffer /Reagent preparation</td></tr><tr><td>Cell culture laminar air flow</td><td>ThermoFischer Scientific</td><td>1323TS</td><td>For cell culture</td></tr><tr><td>Cell scraper</td><td>Genetix</td><td>90020</td><td>For cell culture</td></tr><tr><td>Cell spreader</td><td>Fischer Scientific</td><td>12822775</td><td></td></tr><tr><td>Centrifuge for 1.5mL tubes</td><td>Eppendorf</td><td>EP5404000537</td><td></td></tr><tr><td>Centrifuge for 50mL falcon</td><td>ThermoFischer Scientific</td><td>75004210</td><td></td></tr><tr><td>Chlorofrom</td><td>Fischer Scientific</td><td>67-66-3</td><td>For RNA isolation</td></tr><tr><td>CO2 incubator</td><td>ThermoFischer Scientific</td><td>3110</td><td>For cell culture</td></tr><tr><td>Cuvette</td><td>Eppendorf</td><td>6138000018</td><td>Estimation of plasmid concentration</td></tr><tr><td>CYTIVA IQ 500 gel imager</td><td>Cytiva</td><td>29655893</td><td>For AGE</td></tr><tr><td>DEPC TREATED H2O 1000 ML</td><td>ThermoFischer Scientific</td><td>750023</td><td>For RNA isolation</td></tr><tr><td>Doxycycline</td><td>Merck</td><td>33429</td><td>For induction of transfected cells</td></tr><tr><td>Dry heating block</td><td>Labnet</td><td></td><td>For transformation and inactivation of restriction enzymes</td></tr><tr><td>Dulbecco&#039;s Modified Eagle Medium</td><td>National centre for cell science</td><td></td><td>For cell culture</td></tr><tr><td>Ethanol</td><td>Sigma-Aldrich</td><td>1.00983</td><td>Buffer /Reagent preparation</td></tr><tr><td>Ethidium bromide</td><td>Sigma</td><td>E7637</td><td>For AGE</td></tr><tr><td>Ethylenediamine tetraacetic acid</td><td>Fischer Scientific</td><td>12635</td><td>Buffer /Reagent preparation</td></tr><tr><td>Evos brightfield microscope</td><td>ThermoFischer Scientific</td><td>AMEX1000</td><td></td></tr><tr><td>ExPasy</td><td>https://web.expasy.org/translate/</td><td></td><td>for contruction of synthetic circuit</td></tr><tr><td>Fetal Bovine Serum</td><td>Gibco</td><td>16000-044</td><td>For cell culture</td></tr><tr><td>FV31S-SW software</td><td>Olypmus</td><td></td><td>Image acquisition</td></tr><tr><td>Geneticin</td><td>Gibco</td><td>1563411</td><td>for transfection</td></tr><tr><td>Gibson assembly method for cloning</td><td>BIOMATIK</td><td></td><td>Construction of synthetic circuit</td></tr><tr><td>Graphpad prism</td><td>Graphpad</td><td></td><td>Statistical analysis</td></tr><tr><td>High-Capacity cDNA Reverse Transcription Kit</td><td>ThermoFischer Scientific</td><td>4368814</td><td>for cDNA synthesis</td></tr><tr><td>Isopropanol</td><td>Fischer Scientific</td><td>13825</td><td>Buffer /Reagent preparation</td></tr><tr><td>Kanamycin</td><td>Sigma</td><td>60615</td><td>For transformation and colony selection</td></tr><tr><td>Luria Bertini broth</td><td>Himedia</td><td>M1245</td><td>For culturing &lt;em&gt;E.coli&lt;/em&gt;</td></tr><tr><td>Magnesium chloride</td><td>Fischer Scientific</td><td>BP214</td><td>Buffer /Reagent preparation</td></tr><tr><td>Micro Filt Vertical Laminar Air Flow</td><td>Microfilt</td><td></td><td></td></tr><tr><td>MicroAmp Fast 8-Tube Strip, 0.1 mL</td><td>ThermoFischer Scientific</td><td>4358293</td><td>For qRT-PCR</td></tr><tr><td>MicroAmp Optical 8-Cap Strips</td><td>ThermoFischer Scientific</td><td>4323032</td><td>For qRT-PCR</td></tr><tr><td>Microwave oven</td><td>LG</td><td>MC-7649DW</td><td>For AGE</td></tr><tr><td>Mm00434228_m1 (IFN-&amp;gamma;)</td><td>ThermoFischer Scientific</td><td>4331182</td><td>Taqman probes</td></tr><tr><td>Mm00443258_m1 (TNF)</td><td>ThermoFischer Scientific</td><td>4331182</td><td>Taqman probes</td></tr><tr><td>Mm01321739_m1(TGFB)</td><td>ThermoFischer Scientific</td><td>4331182</td><td>Taqman probes</td></tr><tr><td>Mouse ELISA Kit IL-10</td><td>ThermoFischer Scientific</td><td>BMS614</td><td>For ELISA</td></tr><tr><td>Mouse ELISA Kit IL-12</td><td>ThermoFischer Scientific</td><td>BMS616</td><td>For ELISA</td></tr><tr><td>Multiskan Sky with Touch Screen + &amp;mu;Drop Plate</td><td>ThermoFischer Scientific</td><td>51119600DP</td><td>For ELISA</td></tr><tr><td>MX-M Microplate Mixers 96-well Cell Culture Plate Mixer Adjustable 0-1500 rpm Laboratory Shaker Agitator</td><td>DIAB</td><td></td><td></td></tr><tr><td>National centre for Biotechnology Information</td><td>https://www.ncbi.nlm.nih.gov</td><td></td><td>for contruction of synthetic circuit</td></tr><tr><td>Neubauer&#039;s chamber</td><td>Marienfeld superior</td><td>640010</td><td>For cell count</td></tr><tr><td>Non-vented 25cm&lt;sup&gt;2 &lt;/sup&gt;flask</td><td>Corning</td><td>353014</td><td>For culturing parasite</td></tr><tr><td>NotI</td><td>BioLabs</td><td>R3189M</td><td>Restriction enzyme</td></tr><tr><td>Nuclease free water</td><td>Biodesign</td><td>1QIA</td><td>Reagent preparation</td></tr><tr><td>Olympus Cell Sens Dimension Desktop 2.3 software</td><td>Olypmus</td><td></td><td>Image processing</td></tr><tr><td>Olympus FV 3000</td><td>Olympus</td><td></td><td>For confocal imaging</td></tr><tr><td>OPTI-MEM media</td><td>Gibco</td><td>31985070</td><td>media for Transfection</td></tr><tr><td>pEGFP-N1 vector cloned with synthetic circuit</td><td>Biomatik</td><td></td><td></td></tr><tr><td>Penicillin</td><td>ThermoFischer Scientific</td><td>15070063</td><td>For cell culture</td></tr><tr><td>Polyethylenimine</td><td>MERCK</td><td>764965</td><td>for transfection</td></tr><tr><td>Potassium Acetate</td><td>Fischer Scientific</td><td>YBP364500</td><td>Buffer /Reagent preparation</td></tr><tr><td>Potassium Chloride</td><td>Fischer Scientific</td><td>BP366</td><td>Buffer /Reagent preparation</td></tr><tr><td>Potassium Phosphate Monobasic</td><td>Sigma</td><td>P5655</td><td>Buffer /Reagent preparation</td></tr><tr><td>Power supply pack</td><td>BIORAD</td><td>1645070</td><td>For AGE</td></tr><tr><td>Registry of Standard Biological parts</td><td>https://parts.igem.org/Main_Page</td><td></td><td>for contruction of synthetic circuit</td></tr><tr><td>RNAiso Plus</td><td>Takara</td><td>38220090</td><td>For RNA isolation</td></tr><tr><td>RNase A</td><td>ThermoFischer Scientific</td><td>12091021</td><td>Buffer /Reagent preparation</td></tr><tr><td>Roswell Park Memorial Institute Medium</td><td>National centre for cell science</td><td></td><td>For culturing parasite</td></tr><tr><td>Shaker incubator</td><td>REMI</td><td>CIS 24 plus</td><td>For culturing &lt;em&gt;E.coli&lt;/em&gt;</td></tr><tr><td>Sodium chloride</td><td>Qualigens</td><td>Q27605</td><td>Buffer /Reagent preparation</td></tr><tr><td>Sodium Dodecyl Sulfate</td><td>Sigma-Aldrich</td><td>L3771</td><td>Buffer /Reagent preparation</td></tr><tr><td>Sodium Hydroxide</td><td>Fischer Scientific</td><td>15895</td><td>Buffer /Reagent preparation</td></tr><tr><td>Sodium Phosphate Dibasic</td><td>Sigma</td><td>S5136</td><td>Buffer /Reagent preparation</td></tr><tr><td>Spectrophotometer</td><td>Eppendorf</td><td></td><td>Estimation of plasmid concentration</td></tr><tr><td>Spin win</td><td>Trason</td><td>1020</td><td></td></tr><tr><td>StepOne plus Real Time PCR system</td><td>Life technologies</td><td>272006777</td><td>For qRT-PCR</td></tr><tr><td>StepOne software version 2.3</td><td>Life technologies</td><td></td><td>For qRT-PCR</td></tr><tr><td>TaqMan Fast Universal PCR Master Mix (2X), no AmpErase UNG</td><td>ThermoFischer Scientific</td><td>4366072</td><td></td></tr><tr><td>Tinker cell</td><td></td><td></td><td>Synthetic circuit construction and simulation</td></tr><tr><td>TrackIt 1 Kb Plus DNA ladder</td><td>ThermoFischer Scientific</td><td>10488085</td><td>For AGE</td></tr><tr><td>Tris (hydroxymethyl) aminomethane hydrochloride</td><td>Himedia</td><td>MB029</td><td>Buffer /Reagent preparation</td></tr><tr><td>Tris-acetate-EDTA</td><td>SERVA</td><td>42549.1</td><td>Buffer /Reagent preparation</td></tr><tr><td>Trypan blue</td><td>Sigma</td><td>T8154</td><td>For cell count</td></tr><tr><td>XhoI</td><td>BioLabs</td><td>R0146S</td><td>Restriction enzyme</td></tr></tbody></table>

immunometabolic circuits in infection for advancing host directed therapies

Immunometabolic Circuits in Infection for Advancing Host Directed Therapies

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.

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. The increase in SDHA concentration may be attributed to the induction of doxycycline, while the expression of TetR may result from declining levels of doxycycline (Figure 1C). Given the inducible oscillatory expression of SDHA and TetR observed in silico, a strategy for delivery of synthetic circuit through pEGFP-N1 plasmid vector was designed and implemented.
The iGEM parts identified for the study include BBa_K1493803 for TetO, BBa_K4344028 for the Kozak sequence, BBa_C0040 for TetR, and BBa_K1537016 for P2A, respectively. The NCBI accession ID for SDHA is NM_025848. These components were assembled to construct a doxycycline-inducible TetON synthetic circuit, spanning a total of 1317 base pairs (Supplementary File 1). As TetO functions as a DNA regulatory element, its sequence was not translated. Sequence downstream of the Kozak sequence, extending from 5' TetR to the 3' end of P2A, positioned downstream of SDHA, was chosen for in silico validation of translation. Observation revealed the absence of internal stop codons, ensuring uninterrupted translation across the entire sequence length (Supplementary File 2). The regulatory parts of a synthetic circuit comprising 1317 bp were inserted between NotI and XhoI restriction sites, which are 5' upstream of EGFP (Figure 1B). Sequencing results highlight that the insert sequence was cloned in the plasmid (Supplementary File 3).
The introduction of an immuno-metabolic circuit into the E. coli DH5α strain led to the growth of transformed colonies exhibiting resistance to kanamycin, the antibiotic employed as a selective marker during bacterial transformation. The abundant presence of transformed colonies on the selection plate was higher, which suggests that the competent cells displayed a high potential for transformation, indicative of the efficiency of the transformation protocol utilized (Figure 1D).
A single colony was isolated and seeded in 10 mL of LB supplemented with kanamycin for isolation of plasmid. The plasmid yield derived from a 10 mL culture is detailed in Table 1. A standard method for plasmid isolation was employed for this procedure. Buffer solutions were freshly prepared the day prior to plasmid isolation, and incubation conditions were optimized to maximize plasmid yield. The obtained plasmid concentration exceeded 1 µg/µL, with 260/280 and 260/230 ratios surpassing 1.8 and 2.0, respectively. These findings indicate that the isolated plasmids were of high purity and devoid of contaminants such as proteins, EDTA, carbohydrates, and phenol.
The electrophoretic mobility shift for the isolated synthetic circuit was evaluated. Restriction digestion was performed using NotI and XhoI restriction enzymes, as those were the restriction sites where the synthetic circuit was inserted. Two bands were observed on 1% agarose gel, one band near 1500 bp and one between 4 kb and 5 kb. Given that the insert size is 1317 bp, the band at 1500 bp likely corresponds to the insert. The pEGFP-N1 vector has a size of 4.7 kb. Hence, the band near 5 kb is presumed to be the pEGFP-N1 vector. Consequently, an inference was drawn that a complete and intact synthetic circuit was present within the delivery vector. Additionally, an approximately 6 kb band was detected for the undigested intact synthetic circuit, indicating that the pEGFP-N1 vector harbors an insert of nearly 1.3 kb within the plasmid vector (Figure 1E).
<img alt="Synthetic gene circuit diagram with plasmid map, bacterial culture plates, gel electrophoresis, data chart." class="xfigimg" src="/files/ftp_upload/66980/66980fig01.jpg" data-altupdatedat="1788304762000" data-alt="Figure 1"> 
Figure 1: Designing and validation of synthetic circuit. (A) Representation of synthetic circuits by the assembly of genetic parts and regulatory components. (B) Deterministic simulation graph showing periodic expression of SDHA and TetR at 100-time units. (C) Synthetic circuit transformed colonies of E. coli DH5α resistant to kanamycin. The unit is 100-time unit as it is a deterministic simulation. (D) Agarose gel electrophoresis of intact and restriction digested synthetic circuit using NotI and XhoI restriction enzymes. <a href="https://www.jove.com/files/ftp_upload/66980/66980fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Concentration of plasmid (μg/mL)</td>
			<td>260/280</td>
			<td>260/230</td>
		</tr>
		<tr>
			<td>1248.5</td>
			<td>1.95</td>
			<td>2.45</td>
		</tr>
		<tr>
			<td>1123.1</td>
			<td>1.89</td>
			<td>2.43</td>
		</tr>
		<tr>
			<td>1155.8</td>
			<td>1.87</td>
			<td>2.41</td>
		</tr>
		<tr>
			<td>1056.8</td>
			<td>1.94</td>
			<td>2.47</td>
		</tr>
	</tbody>
</table>
Table 1: Yield of synthetic circuit construct obtained after plasmid isolation. The table shows the concentration of plasmid in µg/mL, their 260/280 and 260/230 ratio.
In the control and infected cells, the observed fluorescence intensity was low for GFP. However, transfection of the synthetic circuit with a 1:1 ratio of plasmid and PEI with 500 µg/mL G418 and induction with 1 µg/µL dox was observed to induce high GFP expression in IMT cells. In 6 h IMTI cells, GFP expression was also significantly higher, although GFP expression was lower than in the IMT sample (Figure 2A,B). Further, an increase in GFP expression was observed in 12 h IMTI, 18 h IMTI, and 24 h IMTI samples. This increase may be due to an increase in the time of infection, which might have induced higher metabolic alternations in macrophages by residential parasite17. Intracellular SDHA levels might be affected due to an increase in the time of infection18. As dox was supplemented to these IMTI samples, SDHA levels through synthetic circuits might have increased, thus leading to an increase in the expression of SDHA.
DAPI staining was done to identify the number of residential parasites in the macrophages. In 6 h L. major infected macrophages, the observed number of intracellular parasites was 6 cells per macrophage, which is higher as compared to the 6 h IMTI sample, which had 1-2 intracellular parasites per macrophage. Post transfection and dox induction, a significant reduction in intracellular parasite load was observed at different time points of infection (Figure 2C). Observations suggested that synthetic circuits may have induced parasite-eliminating effects by stimulating SDHA expression.
<img alt="RAW264.7 cell infection analysis; fluorescence microscopy, intensity bar graph; DAPI, 488nm channels." class="xfigimg" src="/files/ftp_upload/66980/66980fig02.jpg" data-altupdatedat="1788304762000" data-alt="Figure 2"> 
Figure 2: Confocal laser scanning microscopy. Images of Control, infected, IMT, and IMTI samples. (A) Expression analysis of GFP in all experimental samples with a scale bar of 50 µm for control, 6 h infected and IMT, 20 µm for 6 h IMTI, 10 µm for 12 h IMTI, 18 h IMTI, 24 h IMTI respectively. The channels used were DIC, DAPI, GFP, and merged images of all channels. (B) One-way ANOVA with Tukey's correction test of all experimental samples (n=3) for GFP expression (p &lt; 0.05 is considered significant) Error bars represent standard deviation. (C) One-way ANOVA with Tukey's correction test analysis of parasite load in 100 macrophage cells for samples from (A) (n=3) was performed (p &lt; 0.05 is considered significant). Error bars represent standard deviation. <a href="https://www.jove.com/files/ftp_upload/66980/66980fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Detection of IL-10 and IL-12 levels in the media of all the samples revealed that the concentration of IL-10 and IL-12 were highest at 6 h post-infection with downregulation in cytokine levels post 24 h infection. There was no significant difference in IL-10 and IL-12 levels upon transfection and induction of synthetic circuits. Also, there was a non-significant difference in IL-10 and IL-12 levels in IMTI time point samples (Figure 3). The 6 h infected sample has already been reported to show expression of IL-10 and IL-1219,20, observing a non-significant difference in IL-10 and IL-12 made us investigate the effect of synthetic circuit on other cytokines.
<img alt="Bar chart of IL-10 and IL-12 levels; cytokine concentration analysis; p-value&lt;0.0001." class="xfigimg" src="/files/ftp_upload/66980/66980fig03.jpg" data-altupdatedat="1788304762000" data-alt="Figure 3"> 
Figure 3: ELISA of IL-10 and IL-12 to identify their secretory levels in media of different samples. Samples (n=2) include control, 6 h infected, 24 h infected, IMT, 6 h IMTI, 12 h IMTI, 18 h IMTI, 24 h IMTI. Two-way ANOVA with Tukey's correction test analysis was performed (p &lt;.05 is considered significant). Error bars represent standard deviation. <a href="https://www.jove.com/files/ftp_upload/66980/66980fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Detection of transcript levels of TNF-α, IFN-γ, and TGF-β in 6 h infected, IMT, and 24 h IMTI samples revealed that a significant increase in TNF-α to 2.5-fold was observed in 24 h IMTI sample as compared to infection and IMT samples (Figure 4). In the 24 h IMTI sample, a 35-fold increase in IFN-γ levels was observed as compared to infection and IMT samples. TGF-β levels, although they showed significant upregulation to 1.5-fold in the 24 h IMTI sample, the relative fold change was lower as compared to TNF-α and IFN-γ, suggesting that pro-inflammatory cytokines are significantly upregulated in the 24 h IMTI sample.
<img alt="Bar chart comparing relative fold changes in TNF-α, IFN-γ, TGF-β for different infection treatments." class="xfigimg" src="/files/ftp_upload/66980/66980fig04.jpg" data-altupdatedat="1788304762000" data-alt="Figure 4"> 
Figure 4: qRT-PCR analysis of cytokines. (A) TNF-α, (B) IFN-γ, and (C) TGF-β for 6 h infected, IMT and 24 h IMTI samples (n=2). One-way ANOVA with Tukey's correction test was performed (p &lt; 0.05 is considered significant). Error bars represent standard deviation. <a href="https://www.jove.com/files/ftp_upload/66980/66980fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplementary File 1: Nucleotide sequence of genetic parts assembled to form the synthetic circuit. <a href="https://www.jove.com/files/ftp_upload/66980/Supplementary%20file%201_RE.docx" target="_blank">Please click here to download this File.</a>
Supplementary File 2: Translated sequence of synthetic circuit expressing TetR, P2A, and SDHA. <a href="https://www.jove.com/files/ftp_upload/66980/Supplementary%20file%202_RE.docx" target="_blank">Please click here to download this File.</a>
Supplementary File 3: Sequencing result of the cloned synthetic circuit in pEGFP-N1 plasmid. <a href="https://www.jove.com/files/ftp_upload/66980/Supplementary%20file%203_RE.docx" target="_blank">Please click here to download this File.</a>

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.

Immuno-metabolism is a pivotal determinant in the progression of leishmaniasis. Synthetic biology-based approach has garnered significant attention as a ...

immunometabolic-circuits-infection-for-advancing-host-directed

Research

JoVE Journal

Bioengineering

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The present protocol describes an experimental workflow that allows for the ex vivo analysis of human T cell stimulation in an allogenic co-culture system with pre-treated tumor cells.

Analysis of Human T Cell Activity in an Allogeneic Co-Culture Setting of Pre-Treated Tumor Cells

This article outlines a versatile method to create cell-derived tissue rings by cellular self-assembly.  Smooth muscle cells seeded into ring-shaped agarose wells aggregate and contract to form robust three-dimensional (3D) tissues within 7 days.  Millimeter-scale tissue rings are conducive to mechanical testing and serve as building blocks for tissue assembly.

Directed Cellular Self-Assembly to Fabricate Cell-Derived Tissue Rings for Biomechanical Analysis and Tissue Engineering

This protocol demonstrates methods used to establish 2D and 3D environments in custom-designed electrotactic chambers, which can track cells in vivo/ex vivo using time-lapse recording at the single cell level, in order to investigate galvanotaxis/electrotaxis and other cellular responses to direct current (DC) electric fields (EFs).

Electric Field-controlled Directed Migration of Neural Progenitor Cells in 2D and 3D Environments

Explore synthetic circuit design for Leishmania major infection using tetracycline-controlled inducible systems. This protocol demonstrates parasite elimination and cytokine modulation in host-directed immunotherapy research.

Immunometabolic Circuits in Infection for Advancing Host Directed Therapies

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