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Oligodeoxynucleotide IMT504: Effects on Central Nervous System Repair Following Demyelination

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Abstract

Multiple sclerosis (MS) is an immune-mediated central nervous system (CNS) disease characterized by demyelination resulting from oligodendrocyte loss and inflammation. Cuprizone (CPZ) administration experimentally replicates MS pattern-III lesions, generating an inflammatory response through microgliosis and astrogliosis. Potentially remyelinating agents include oligodeoxynucleotides (ODN) with a specific immunomodulatory sequence consisting of the active motif PyNTTTTGT. In this work, the remyelinating effects of ODN IMT504 were evaluated through immunohistochemistry and qPCR analyses in a rat CPZ-induced demyelination model. Subcutaneous IMT504 administration exacerbated the pro-inflammatory response to demyelination and accelerated the transition to an anti-inflammatory state. IMT504 reduced microgliosis in general and the number of phagocytic microglia in particular and expanded the population of oligodendroglial progenitor cells (OPCs), later reflected in an increase in mature oligodendrocytes. The intracranial injection of IMT504 and intravenous inoculation of IMT504-treated B lymphocytes rendered comparable results. Altogether, these findings unveil potentially beneficial properties of IMT504 in the regulation of neuroinflammation and oligodendrogenesis, which may aid the development of therapies for demyelinating diseases such as MS.

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Data Availability

The datasets generated during the current study are not publicly available but are available from the corresponding author on reasonable request.

Abbreviations

ANOVA:

Analysis of variance

Arg1:

Arginase 1

BSA:

Bovine serum albumin

CC:

Corpus callosum

CD24:

Cluster of differentiation 24

CD38:

Cluster of differentiation 38

CD59:

Cluster of differentiation 59

CD68:

Cluster of differentiation 68

CICUAL:

Comité Institucional para el Cuidado y Uso de Animales de Laboratorio

CIOMS:

Council for International Organizations for Medical Science

CNS:

Central nervous system

CpG:

Unmethylated cytosine-guanosine dinucleotides

CPZ:

Cuprizone

Ct:

Cycle threshold

GAPDH:

Glyceraldehyde-3-phosphate dehydrogenase

IFNα:

Interferon alpha

Iba1:

Ionized calcium-binding adaptor molecule 1

ICLAS:

International Council for Laboratory Animal Science

IL-1β:

Interleukin-1β

IL 10:

Interleukin 10

IL 35:

Interleukin 35

IHC:

Immunohistochemistry

iNOS:

Inducible nitric oxide synthase

IC:

Intracranial injection

IV:

Injected into the tail vein

LB:

Lymphocyte B

MAG:

Myelin-associated glycoprotein

MBP:

Myelin basic protein

MHC I:

Major histocompatibility complex I

MHC II:

Major histocompatibility complex II

mRNA:

Messenger ribonucleic acid

MS:

Multiple sclerosis

NG2:

Neural/glial antigen 2

NSC:

Neural stem cell

ODN:

Oligodeoxynucleotide

OL:

Oligodendrocyte

OPC:

Oligodendrocyte precursor cell

PBS:

Phosphate-buffered saline

PBST:

PBS containing 0.1% Triton

PDGFRα:

Platelet-derived growth factor receptor alpha

qPCR:

Quantitative polymerase chain reaction

RNA:

Ribonucleic acid

RRID:

Research Resource Identifiers

SC:

Via subcutaneous

SEM:

Standard error of the mean

SNPs:

Single-nucleotide polymorphisms

SS:

Saline solution

SVZ:

Subventricular zone

TGF-β:

Transforming growth factor beta

Wnt:

Blending of the name of the Drosophila segment polarity gene Wingless and the name of the vertebrate homolog, integrated or int-1

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Funding

Supported by grants from ANPCyT (PICT 2017-0890), Universidad de Buenos Aires (20020160100050BA) and CONICET (PIP0567), Argentina.

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Authors and Affiliations

  1. Facultad de Farmacia y Bioquímica, Departamento de Química Biológica, Universidad de Buenos Aires, Junín 956, C1113AAD, Buenos Aires, Argentina

    Patricia A. Mathieu, Yim Rodriguez Sampertegui, Alexis Silva Silva & Ana M. Adamo

  2. CONICET, Instituto de Química y Fisicoquímica Biológicas (IQUIFIB), Universidad de Buenos Aires Junín 956, C1113AAD, Buenos Aires, Argentina

    Patricia A. Mathieu, Yim Rodriguez Sampertegui, Alexis Silva Silva & Ana M. Adamo

  3. Instituto de Ciencia y Tecnología Dr. César Milstein (CONICET-Fundación Pablo Cassará), Saladillo 2468, C1440FFX, Buenos Aires, Argentina

    Fernanda Elias

  4. Facultad de Farmacia y Bioquímica, Departamento de Fisicomatemática, Universidad de Buenos Aires, Junín 956, C1113AAD, Buenos Aires, Argentina

    María de Luján Calcagno

  5. Immunotech S.A., Buenos Aires, Argentina

    Ricardo López

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  1. Patricia A. Mathieu
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Contributions

P.A.M. and Y.R.S. performed most of the experiments. F.E. performed the isolation and culture of LB. A.S.S carried out microglial and OPC culture experiments. P.A.M. and A.M.A. designed and supported all the experiments. M.L.C contributed with statistical analysis. R.L. collaborated with the discussion of this paper. A.M.A. wrote the manuscript.

Corresponding author

Correspondence to Ana M. Adamo.

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Ethics Approval

Experiments were carried out following the guidelines of the Institutional Committee for the Care and Use of Laboratory Animals, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, approved by Council Directive (CICUAL; CUDAP:EXP-FYB 0043071/2019). This study was exploratory and was not pre-registered.

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Supplementary Information

ESM 1

Figure S1: Analysis of LB purity. LBs were isolated from rat spleen, incubated with CD45RA:RPE, and analyzed by flow cytometry to confirm LB purity. Histogram shows the CD45 positive cells. Percentage of LBs was determined through the FlowJo 7.6. software. Representative image shows that 97.5% cells correspond to CD45 LBs. (TIF 434 kb)

ESM 2

Figure S2: Characterization of CPZ demyelination. A, Body weight of control animals treated with saline solution (Control-SS) and CPZ animals treated with saline solution (CPZ-SS) or IMT504 (CPZ-IMT504) at the different experimental time points. Values are expressed as the mean ± SEM. B, Representative images of Sudan black staining of the CC of control and CPZ-demyelinated animals. Statistical analysis was done as described in Materials and Methods; different letters indicate significant differences, p<0.05. (TIF 6416 kb)

ESM 3

Figure S3: Stimulation of HEK 293-hTLR9 cells with ODNs 2006 and IMT504. Cells were treated with 15 μg/ml 2006 and IMT504 or with culture medium (negative control, NC) for 24 h. Cells were lysed, and NFkB activation was measured relative to the NC. Results are expressed as the average activation index with respect to the NC ± standard deviation. Statistical analysis was done using Student’s t-tests as described in Materials and Methods. (***p < 0.0001). (TIF 975 kb)

ESM 4

Figure S4: Effects of SC treatment with IMT504 in control animals. Quantification of A, Iba-1+, B, CD68+Iba-1+, C, PDGFRα+ and D, MAG+ cells per area in the CC of control animals treated with saline solution (Control-SS) or IMT504 (Control-IMT504). Values are expressed as the mean ± SEM. qPCR analyses of E, iNOS, F, IL-1β, G, Arg1, H, TGF- β, I, PDGFRα, J, MAG and K, MBP transcript levels in the CC of control animals treated with saline solution (Control-SS) or IMT504 (Control-IMT504). Results expressed as RQ fold changes regarding controls (dotted lines). Values are expressed as the mean ± SEM. Statistical analysis was done as described in Materials and Methods; different letters indicate significant differences, p<0.05. (TIF 33747 kb)

ESM 5

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Mathieu, P.A., Sampertegui, Y.R., Elias, F. et al. Oligodeoxynucleotide IMT504: Effects on Central Nervous System Repair Following Demyelination. Mol Neurobiol (2023). https://doi.org/10.1007/s12035-023-03825-7

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