IFNγ-stimulated dendritic cell extracellular vesicles can be nasally administered to the brain and enter oligodendrocytes

Extracellular vesicles secreted from IFNγ-stimulated rat dendritic cells (referred to here as IFNγ-DC-EVs) contain miRNAs which promote myelination (including but not limited to miR-219), and preferentially enter oligodendrocytes in brain slice cultures. IFNγ-DC-EVs also increase myelination when nasally administered to naïve rats. While we can infer that these extracellular vesicles enter the CNS from functional studies, here we demonstrate biodistribution throughout the brain after nasal delivery by way of imaging studies. After nasal administration, Xenolight DiR-labelled IFNγ-DC-EVs were detected 30 minutes later throughout the brain and the cervical spinal cord. We next examined cellular uptake of IFNγ-DC-EVs by transfecting IFNγ-DC-EVs with mCherry mRNA prior to nasal administration. mCherry-positive cells were found along the rostrocaudal axis of the brain to the brainstem. These cells morphologically resembled oligodendrocytes, and indeed cell-specific co-staining for neurons, astrocytes, microglia and oligodendrocytes showed that mcherry positive cells were predominantly oligodendrocytes. This is in keeping with our prior in vitro results showing that IFNγ-DC-EVs are preferentially taken up by oligodendrocytes, and to a lesser extent, microglia. To confirm that IFNγ-DC-EVs delivered cargo to oligodendrocytes, we quantified protein levels of miR-219 mRNA targets expressed in oligodendrocyte lineage cells, and found significantly reduced expression. Finally, we compared intranasal versus intravenous delivery of Xenolight DiR-labelled IFNγ-DC-EVs. Though labelled IFNγ-DC-EVs entered the CNS via both routes, we found that nasal delivery more specifically targeted the CNS with less accumulation in the liver. Taken together, these data show that intranasal administration is an effective route for delivery of IFNγ-DC-EVs to the CNS, and provides additional support for their development as an EV-based neurotherapeutic that, for the first time, targets oligodendrocytes.</p

Truncated MSP1-42 proteins possess disulfide sensitive conformation.

<p>Immunoblots of recombinant proteins separated under reducing (lanes 1) and non-reducing (lanes 2) conditions and probed with conformational sensitive anti-MSP1-19 monoclonal antibodies. Panel A: Construct 33-A – 33-C and MSP1-19 probed with mAb 12.8; Panel B: Constructs 33-A – 33-C and MSP1-19 probed with mAb 2.2; and Panel C: Constructs 33-A – 33-C and MSP1-19 probed with mAb 5.2 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024782#pone.0024782-Hui4" target="_blank">[47]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024782#pone.0024782-Leung1" target="_blank">[48]</a>.</p

Sequence and location of previously identified and predicted T cell epitopes in truncated Constructs 33-A - 33-K.

<p>Sequence and location of previously identified and predicted T cell epitopes in truncated Constructs 33-A - 33-K.</p

Anti-Construct 33-C Antibodies Interfere with Inhibitory Anti-MSP1-42 Antibodies.

<p>Anti-Construct 33-C Antibodies Interfere with Inhibitory Anti-MSP1-42 Antibodies.</p

Primer Sequences for the Construction of Truncated MSP1-42 Constructs.

<p>Primer Sequences for the Construction of Truncated MSP1-42 Constructs.</p

ELISA antibody responses against MSP1-19 in Swiss Webster mice immunized with recombinant truncated MSP1-42 proteins.

<p>Panel A, percent responsiveness of mice immunized with Constructs 33-A – 33-K after the first booster injection (grey) and after the second booster injection (black). Panel B, antibody titers of mice vaccinated with Constructs 33-A – 33-K. Results of tertiary bleeds are shown. Horizontal bars indicate mean antibody titers. ANOVA (p<0.05) indicated that the levels of antibody titers differed among groups. Asterisk indicates a significant difference (Turkey post-hoc test, p<0.05) between Construct 33-D and all other vaccination groups.</p