No immune responses by the expression of the yeast Ndi1 protein in rats - PubMed (original) (raw)

No immune responses by the expression of the yeast Ndi1 protein in rats

Mathieu Marella et al. PLoS One. 2011.

Abstract

Background: The rotenone-insensitive internal NADH-quinone oxidoreductase from yeast, Ndi1, has been shown to work as a replacement molecule for complex I in the respiratory chain of mammalian mitochondria. In the so-called transkingdom gene therapy, one major concern is the fact that the yeast protein is foreign in mammals. Long term expression of Ndi1 observed in rodents with no apparent damage to the target tissue was indicative of no action by the host's immune system.

Methodology/principal findings: In the present study, we examined rat skeletal muscles expressing Ndi1 for possible signs of inflammatory or immune response. In parallel, we carried out delivery of the GFP gene using the same viral vector that was used for the NDI1 gene. The tissues were subjected to H&E staining and immunohistochemical analyses using antibodies specific for markers, CD11b, CD3, CD4, and CD8. The data showed no detectable signs of an immune response with the tissues expressing Ndi1. In contrast, mild but distinctive positive reactions were observed in the tissues expressing GFP. This clear difference most likely comes from the difference in the location of the expressed protein. Ndi1 was localized to the mitochondria whereas GFP was in the cytosol.

Conclusions/significance: We demonstrated that Ndi1 expression did not trigger any inflammatory or immune response in rats. These results push forward the Ndi1-based molecular therapy and also expand the possibility of using foreign proteins that are directed to subcellular organelle such as mitochondria.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1

Figure 1. H&E staining of coronal sections of rat muscles.

Rats were injected with either PBS, rAAV-GFP, or rAAV-NDI1 in the skeletal muscles. Muscles (tibialis anteriors) were collected at 1 week (1w) or 1 month (1 m) and were stained with Hematoxylin-Eosin (H&E) solutions and infiltrated cells were counted. (A) Representative images of muscle sections from each group after H&E staining. (B) Comparison of the number of infiltrated cells. The histogram shows the number of small mononuclear cells per field of view in the sections separated by at least 90 µm (n = 4 for rats, n = 6 for muscle sections from each animal). Results are expressed as mean ± SD. ** p<0.01 from the respective control (Student's t-test).

Figure 2

Figure 2. Evaluation of infiltrated cells in the rat brain.

Rats were injected with PBS (control), rAAV-GFP, or rAAV-NDI1 in the striatum of rat brain. The brain samples were collected 1 month post-injection. (A) Representative images of brain sections from each group after H&E staining. (B) Comparison of the number of infiltrated cells. The histogram shows the number of infiltrating cells per field of view in the sections separated by at least 90 µm (n = 4 for rats, n = 6 for brain sections from each animal). Results are expressed as mean ± SD. ** p<0.01 and * p<0.05 from the respective control (Student's t-test).

Figure 3

Figure 3. Representative images of macrophage staining of tissue sections from rat muscles.

Rats were injected with PBS, rAAV-GFP, or rAAV-NDI1 in the skeletal muscles and muscle samples were collected 1 month post-injection. Coronal sections of the muscle from the animals injected with PBS (Control), rAAV-NDI1 or rAAV-GFP were stained with anti-CD11b antibody (A, D, and G, red) or with anti-Ndi1 antibody (E, green). Muscle sections from the rats injected with rAAV-GFP were examined for the fluorescence of the GFP protein (H, green). A green channel image for the control (B) is also shown. Merged images of the red and green channels are also shown (C, F, and I) Scale bar = 75 µm.

Figure 4

Figure 4. Immune cells distribution within the injected area of the rat muscle.

Rats received an injection of rAAV-GFP or rAAV-NDI1 in the skeletal muscles. Muscle samples were harvested either 1 week (A) or 1 month (B) after the injection. Immunohistochemistry was performed on coronal sections of rat muscles expressing the GFP and the Ndi1 protein. Antibodies against immunological marker proteins, CD3, CD4, CD8, CD11b, were used and positively stained cells were counted (n = 4 for rats, n = 6 for brain sections from each animal). Results are expressed as mean ± SD. ** p<0.01 and * p<0.05 from the respective control (Student's t-test).

Figure 5

Figure 5. Evaluation of the presence of antibodies against Ndi1 protein in the rat serum.

Rats were injected with rAAV-NDI1 either in the skeletal muscles or the brain. One group of rat received an injection of purified Ndi1 protein in the tail vein. Blood samples of each group of rats (n = 4) were drawn and the presence of anti-Ndi1 antibodies was assessed by using ELISA. A 96 wells plate was coated with purified Ndi1 protein and the sera were tested from the following groups (serum dilution in parenthesis): a, Ndi1 protein injected into tail vein (1/5000); b, PBS injected muscle, 1 week (1/100); c, rAAV-NDI1 injected muscle, 1 week (1/100); d, PBS injected muscle, 1 month (1/100); e, rAAV-NDI1 injected muscle, 1 month (1/100); f, PBS injected brain, 1 week (1/100), g; rAAV-NDI1 injected brain, 1 week (1/100); h, PBS injected brain, 1 month (1/100); i, rAAV-NDI1 injected brain 1, month (1/100). Data are presented as mean ± SD.

References

    1. Janssen RJ, Van den Heuvel LP, Smeitink JA. Genetic defects in the oxidative phosphorylation (OXPHOS) system. Expert Rev Mol Diagn. 2004;4:143–56. - PubMed
    1. Marella M, Seo BB, Yagi T, Matsuno-Yagi A. Parkinson's disease and mitochondrial complex I: a perspective on the Ndi1 therapy. J Bioenerg Biomembr. 2009;41:493–7. - PMC - PubMed
    1. Yagi T, Seo BB, Nakamaru-Ogiso E, Marella M, Barber-Singh J, et al. Possibility of transkingdom gene therapy for Complex I diseases. Biochim Biophys Acta. 2006;1757:708–14. - PubMed
    1. Yagi T, Seo BB, Nakamaru-Ogiso E, Marella M, Barber-Singh J, et al. Can a Single Subunit Yeast NADH Dehydrogenase (Ndi1) Remedy Diseases Caused by Respiratory Complex I Defects? Rejuvenation Res. 2006;9:191–7. - PubMed
    1. Marella M, Seo BB, Nakamaru-Ogiso E, Greenamyre JT, Matsuno-Yagi A, et al. Protection by the NDI1 Gene against Neurodegeneration in a Rotenone Rat Model of Parkinson's Disease. PLoS ONE. 2008;3:e1433. - PMC - PubMed

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