Pathological responses of glial cells in spinal cord injury and rheumatoid arthritis

Scientists have considered glia as mere passive allies of neurons for a long time. As a consequence, their functions have been greatly underestimated. Major discoveries made in the last three decades have changed our view on glial cells and it is now accepted that they play important roles in health and disease. In this thesis, we have investigated the role of 3 glial cell types - astrocytes, microglia and ependymal cells - in spinal cord injury (SCI), rheumatoid arthritis (RA) and in pain-related processes.

In Study I, we have delineated the mechanisms that regulate interleukin-6 (IL-6) expression and secretion in adult rat astrocyte cultures. We found that the PI3K-mTORAKT pathway negatively regulates IL-6 expression and that IL-6 secretion is calcium (Ca2+)-dependent. Interestingly, we observed that astrocytes express IL-6 in vivo after SCI, however IL-6 levels decline after 2-3 weeks. Since induction of IL-6 in reactive astrocytes could be beneficial due to the regenerative properties of this cytokine, we treated adult rats 2 weeks after SCI with mTOR inhibitors, torin2 and rapamycin, to boost astrocytic IL-6 secretion by blocking the PI3K-mTOR-AKT pathway and increasing cytosolic Ca2+, respectively. This combinatorial treatment led to a transient improvement in mechanical hypersensitivity during the treatment period.

In Study II, we have established an adult ex vivo model of SCI, to facilitate the study of cellular processes that are difficult to address using animal models. In particular, we focused on assessing the ependymal cell response to injury in our model, which is based on adult mouse spinal cord cultured tissue slices. Interestingly, we found that, ependymal cells become activated, proliferate, migrate out of the ependymal layer and differentiate in a manner that fundamentally resembles their response to injury in vivo. Moreover, we show that these cells can respond to external adenosine triphosphate (ATP) stimulation and that some of them have spontaneous Ca2+ activity. We believe that this model is a useful platform to study and modulate ependymal cell responses and could contribute to the development of novel treatment avenues for SCI.

In Study III, we have investigated mechanisms that may participate in central sensitization in the context of RA. Here, we report for the first time the presence of disease associated autoantibodies known as ACPA (anti-citrullinated protein antibodies) in the cerebrospinal fluid (CSF) of a subset of RA patients. Moreover, we show that intrathecal injection of such antibodies into the CSF of mice led to pain-like behavior, while injection of other antibodies from RA patients or from healthy individuals did not. Furthermore, we show that co-stimulation of human astrocytes in culture with ACPA and interleukin-1beta (IL-1ß) led to IL-6 secretion in these cells, an effect that was blocked upon addition of an Fc-gamma receptor 1 (FcγRI) inhibitor. These findings support the notion that ACPA may enter the central nervous system (CNS) of RA patients, act on glial cells and activate pathways that could contribute to centrally mediated pain.

In Study IV, we have investigated differences between male and female spinal microglia in the context of arthritis-induced persistent pain. We focused on the late phase of the collagen type-II antibody induced arthritis (CAIA) animal model, which occurs after joint inflammation has resolved and it is characterized by persistent mechanical hypersensitivity and spinal glial activation. We found that intrathecal delivery of minocycline, often described as a microglial inhibitor, was able to revert CAIA-induced pain in male, but not female mice. Moreover, using flow cytometry we found that females had lower dorsal horn spinal microglial relative numbers as compared to males. Furthermore, genome-wide RNA sequencing results pointed to several transcriptional differences between male and female microglia, while no convincing differences were identified between control and CAIA groups. Taken together, these results suggest that during the late phase of the CAIA model changes in microglial gene expression might be highly localized or short-lasting, and that the sexually dimorphic response to minocycline might additionally involve other factors such as changes in protein expression or epigenetic modifications.

In summary, this thesis expands our understanding of mechanisms that are important in glial cell responses to pathological events and opens new avenues to explore the modulation of glial cells. The ultimate hope is that continued efforts will result in the discovery of suitable targets for therapy in individuals with spinal cord injury, rheumatoid arthritis and chronic pain.

List of scientific papers

I. Codeluppi S, Fernandez-Zafra T, Sandor K, Kjell J, Liu Q, Abrams M, Olson L, Gray NS, Svensson CI, Uhlén P. Interleukin-6 secretion by astrocytes is dynamically regulated by PI3K-mTOR-Ca2+ signalling. PLoS One. (2014) Mar 25;9(3):e92649.
https://doi.org/10.1371/journal.pone.0092649

II. Fernandez-Zafra T, Codeluppi S, Uhlén P. An ex vivo spinal cord injury model to study ependymal cells in adult mouse tissue. Exp Cell Res. (2017) Aug 15;357(2):236-242.
https://doi.org/10.1016/j.yexcr.2017.06.002

III. Le Maître E, Fernandez-Zafra T, Revathikumar P, Estelius J, Rogoz K, Sandor K, Lundberg K, Hansson M, Amara K, Kosek E, Khademi M, Andersson M, Malmström V, Klareskog L, Svensson CI, Lampa P. Central nervous system autoimmunity in rheumatoid arthritis: Anti-citrullinated peptide antibodies activate human astroglial cells and induce pain behaviour in mice. [Manuscript]

IV. Fernandez-Zafra T, Agalave N, Sandor K, Gao T, Su J, Jurczak A, Estelius J, Lampa J, Wiesenfeld-Hallin Z, Xu XJ, Denk F, Svensson CI. Exploring the transcriptome of resident spinal microglia after collagen antibody-induced arthritis. [Manuscript]