Direct intrathecal implantation of mesenchymal stromal cells leads to enhanced neuroprotection via an NFkappaB-mediated increase in interleukin-6 production

Abstract

Mesenchymal stromal cell (MSC) therapy has shown promise for the treatment of traumatic brain injury (TBI). Although the mechanism(s) by which MSCs offer protection is unclear, initial in vivo work has suggested that modulation of the locoregional inflammatory response could explain the observed benefit. We hypothesize that the direct implantation of MSCs into the injured brain activates resident neuronal stem cell (NSC) niches altering the intracerebral milieu. To test our hypothesis, we conducted initial in vivo studies, followed by a sequence of in vitro studies. In vivo: Sprague-Dawley rats received a controlled cortical impact (CCI) injury with implantation of 1 million MSCs 6 h after injury. Brain tissue supernatant was harvested for analysis of the proinflammatory cytokine profile. In vitro: NSCs were transfected with a firefly luciferase reporter for NFkappaB and placed in contact culture and transwell culture. Additionally, multiplex, quantitative PCR, caspase 3, and EDU assays were completed to evaluate NSC cytokine production, apoptosis, and proliferation, respectively. In vivo: Brain supernatant analysis showed an increase in the proinflammatory cytokines IL-1alpha, IL-1beta, and IL-6. In vitro: NSC NFkappaB activity increased only when in contact culture with MSCs. When in contact with MSCs, NSCs show an increase in IL-6 production as well as a decrease in apoptosis. Direct implantation of MSCs enhances neuroprotection via activation of resident NSC NFkappaB activity (independent of PI3 kinase/AKT pathway) leading to an increase in IL-6 production and decrease in apoptosis. In addition, the observed NFkappaB activity depends on direct cell contact.

FIG. 1.

Mesenchymal stromal cells (MSCs) double labeled: red with quantum dots, green with green fluorescent protein. Nuclei stained with 4′,6-diamidino-2-phenylindole (DAPI). (A) Fifty-eight percent of the MSCs were located in the ventricles. (B) MSCs located around the needle track transversing the cerebral cortex in the penumbra (10× magnification).

FIG. 2.

Nuclear factor kappa B (NFκB) firefly luciferase activity divided by Renilla luciferase activity measured in neuronal stem cell (NSC)/mesenchymal stromal cell (MSC) co-cultures. (A) Difference is found between unstimulated contact cultures and transwell cultures. (B) Difference is found between neuronal stem cells (NSCs) transfected with NFκB alone and NSCs transfected with IKKβ.

FIG. 3.

Neuronal stem cells (NSCs) in direct contact culture with mesenchymal stromal cells (MSCs; stained red with conjugated CD29-PE antibody). Interleukin (IL)-6 secretion is localized to the NSC (stained green with IL-6 antibody with FITC secondary). Nuclei stained with 4′,6-diamidino-2-phenylindole (DAPI) blue (40× magnification).

FIG. 4.

Real-time quantitative polymerase chain reaction (qPCR) for interleukin 6 (IL-6) production from mesenchymal stromal cell (MSC) and neuronal stem cell (NSC) monocultures as well as NSC/MSC direct contact co-cultures after flow cytometric sorting based upon MSC CD29 and Stro-1 positivity. Minimal IL-6 production is observed with MSC and NSC monocultures; however, NSC/MSC co-cultures show a significant increase (*P = 0.002) in IL-6 production.

FIG. 5.

(A) EDU assay shows an increase in neuronal stem cell (NSC) proliferation when stimulated with tumor necrosis factor alpha (TNFα); however, mesenchymal stem cell (MSC) co-culture has no effect. (B and C) Flow cytometric histogram overlays for NSC caspase 3 expression. Gray area (isotype control). Transparent overlay (apoptotic fraction). (B) NSCs cultured alone. (C) NSCs/MSC contact culture. *P < 0.05.

FIG. 6.

Nuclear factor kappa B (NFκB) pathway detailing the point of action for the dominant negatives vectors IKKβ, AKT, and the PI3 kinase inhibitor. Activation of the neuronal stem cell (NSC) NFκB pathway leads to increased IL-6 production and decreased apoptosis thereby enhancing neuroprotection.