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Kinetics of MSC-based enzyme therapy for immunoregulation.

ABSTRACT: BACKGROUND:Mesenchymal stromal cells (MSC) demonstrate innate and regulatory immunologic functions and have been widely explored for cell therapy applications. Mechanisms by which MSCs achieve therapeutic effects are theorized, though appropriate dosing and duration of these mechanisms in vivo warrant deeper investigation. One, rapid immunosuppressive function of MSCs is through ectoenzyme expression of CD73 and CD39 which cooperatively hydrolyze inflammatory, extracellular adenosine triphosphate (ATP) to anti-inflammatory adenosine. Extracellular ATP has a key role in autoimmune and inflammatory diseases, which administered MSCs have the potential to modulate in a timescale that is befitting of shorter acting therapeutic function. METHODS:In vitro experiments were performed to determine the hydrolysis rates of ATP by MSCs. Through kinetic modeling from experimental results, the rate at which a single cell can metabolize ATP was determined. Based on these rates, the ability of MSCs to downregulate inflammatory signaling pathways was prospectively validated using model system parameters with respect to two different mechanisms: extracellular ATP stimulates lymphocytes to suppress proliferation and induce apoptosis and with co-stimulation, it stimulates monocytes to release pro-inflammatory IL-1?. MSCs were co-cultured with immune cells using transwell inserts and compared to immune cell only groups. RESULTS:Hydrolysis of ATP was efficiently modeled by first-order enzyme kinetics. For in vitro culture, the rate at which a single cell can hydrolyize ATP is 8.9 nmol/min. In the presence of extracellular ATP, cocultures of MSCs reduced cytotoxicity and allows for proliferation of lymphocytes while limiting IL-1? secretion from monocytes. CONCLUSIONS:Such use of these models may allow for better dosing predictions for MSCs to be used therapeutically for chronic inflammatory diseases such as rheumatoid arthritis, diabetes, pancreatitis, and other systemic inflammatory response syndromes. For the first time, the effect of MSCs on ATP hydrolysis in immune cell response is quantitatively analyzed on a cell-molecule basis by modeling the hydrolysis as an enzyme-substrate reaction. The results also give insight into MSCs' dynamic response mechanisms to ameliorate effects of extracellular ATP whether it be through positive or negative regulation.

SUBMITTER: Burr A

PROVIDER: S-EPMC6693124 | biostudies-literature | 2019 Aug

REPOSITORIES: biostudies-literature

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Kinetics of MSC-based enzyme therapy for immunoregulation.

Burr Alexandra A Parekkadan Biju B

Journal of translational medicine 20190813 1

<h4>Background</h4>Mesenchymal stromal cells (MSC) demonstrate innate and regulatory immunologic functions and have been widely explored for cell therapy applications. Mechanisms by which MSCs achieve therapeutic effects are theorized, though appropriate dosing and duration of these mechanisms in vivo warrant deeper investigation. One, rapid immunosuppressive function of MSCs is through ectoenzyme expression of CD73 and CD39 which cooperatively hydrolyze inflammatory, extracellular adenosine tri ...[more]

PMID: 31409424

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Project description:CONSPECTUS: The active site of an enzyme is surrounded by a fluctuating environment of protein and solvent conformational states, and a realistic calculation of chemical reaction rates and kinetic isotope effects of enzyme-catalyzed reactions must take account of this environmental diversity. Ensemble-averaged variational transition state theory with multidimensional tunneling (EA-VTST/MT) was developed as a way to carry out such calculations. This theory incorporates ensemble averaging, quantized vibrational energies, energy, tunneling, and recrossing of transition state dividing surfaces in a systematic way. It has been applied successfully to a number of hydrogen-, proton-, and hydride-transfer reactions. The theory also exposes the set of effects that should be considered in reliable rate constants calculations. We first review the basic theory and the steps in the calculation. A key role is played by the generalized free energy of activation profile, which is obtained by quantizing the classical potential of mean force as a function of a reaction coordinate because the one-way flux through the transition state dividing surface can be written in terms of the generalized free energy of activation. A recrossing transmission coefficient accounts for the difference between the one-way flux through the chosen transition state dividing surface and the net flux, and a tunneling transmission coefficient converts classical motion along the reaction coordinate to quantum mechanical motion. The tunneling calculation is multidimensional, accounting for the change in vibrational frequencies along the tunneling path and shortening of the tunneling path with respect to the minimum energy path (MEP), as promoted by reaction-path curvature. The generalized free energy of activation and the transmission coefficients both involve averaging over an ensemble of reaction paths and conformations, and this includes the coupling of protein motions to the rearrangement of chemical bonds in a statistical mechanically correct way. The standard deviations of the transmissions coefficients provide information on the diversity of the distribution of reaction paths, barriers, and protein conformations along the members of an ensemble of reaction paths passing through the transition state. We first illustrate the theory by discussing the application to both wild-type and mutant Escherichia coli dihydrofolate reductase and hyperthermophilic Thermotoga maritima dihydrofolate reductase (DHFR); DHFR is of special interest because the protein conformational changes have been widely studied. Then we present shorter discussions of several other applications of EA-VTST/MT to transfer of protons, hydrogen atoms, and hydride ions and their deuterated analogs. Systems discussed include hydride transfer in alcohol dehydrogenase, xylose isomerase, and thymidylate synthase, proton transfer in methylamine dehydrogenase, hydrogen atom transfer in methylmalonyl-CoA mutase, and nucleophilic substitution in haloalkane dehalogenase and two-dimensional potentials of mean force for potentially coupled proton and hydride transfer in the ?-oxidation of butyryl-coenzyme A catalyzed by short-chain acyl-CoA dehydrogenase and in the pyruvate to lactate transformation catalyzed by lactate dehydrogenase.

Dataset Information

Kinetics of MSC-based enzyme therapy for immunoregulation.

Publications

Kinetics of MSC-based enzyme therapy for immunoregulation.

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