The immunomodulatory properties of equine mesenchymal stem cells (MSCs) are important for their therapeutic potential and for their facilitating role in their escape from immune recognition, which may also be influenced by donor–recipient major histocompatibility complex (MHC) matching/mismatching and MHC expression level. Factors such as inflammation can modify the balance between regulatory and immunogenic profiles of equine MSCs, but little is known about how the exposure to the immune system can affect these properties in equine MSCs. In this study, we analyzed the gene expression and secretion of molecules related to the immunomodulation and immunogenicity of equine MSCs, either non-manipulated (MSC-naive) or stimulated by pro-inflammatory cytokines (MSC-primed), before and after their exposure to autologous or allogeneic MHC-matched/-mismatched lymphocytes, either activated or resting. Cytokine priming induced the immunomodulatory profile of MSCs at the baseline (MSCs cultured alone), and the exposure to activated lymphocytes further increased the expression of interleukin 6 (IL6), cyclooxygenase 2, and inducible nitric oxide synthase, and IL6 secretion. Activated lymphocytes were also able to upregulate the regulatory profile of MSC-naive to levels comparable to cytokine priming. On the contrary, resting lymphocytes did not upregulate the immunomodulatory profile of equine MSCs, but interestingly, MSC-primed exposed to MHC-mismatched lymphocytes showed the highest expression and secretion of these mediators, which may be potentially linked to the activation of lymphocytes upon recognition of foreign MHC molecules. Cytokine priming alone did not upregulate the immunogenic genes, but MSC-primed exposed to activated or resting lymphocytes increased their MHC-I and MHC-II expression, regardless of the MHC-compatibility. The upregulation of immunogenic markers including CD40 in the MHC-mismatched co-culture might have activated lymphocytes, which, at the same time, could have promoted the immune regulatory profile aforementioned. In conclusion, activated lymphocytes are able to induce the equine MSC regulatory profile, and their effects seem to be additive to the priming action. Importantly, our results suggest that the lymphocyte response against MHC-mismatched MSC-primed would promote further activation of their immunomodulatory ability, which eventually might help them evade this reaction. Further studies are needed to clarify how these findings might have clinical implications in vivo, which will help developing safer and more effective therapies.

Prion diseases are chronic and fatal neurodegenerative diseases characterized by the accumulation of disease-specific prion protein (PrPSc), spongiform changes, neuronal loss, and gliosis. Growing evidence shows that the neuroinflammatory response is a key component of prion diseases and contributes to neurodegeneration. Toll-like receptors (TLRs) have been proposed as important mediators of innate immune responses triggered in the central nervous system in other human neurodegenerative diseases, including Alzheimer’s disease, Parkinson’s disease, and amyotrophic lateral sclerosis. However, little is known about the role of TLRs in prion diseases, and their involvement in the neuropathology of natural scrapie has not been studied. We assessed the gene expression of ovine TLRs in four anatomically distinct brain regions in natural scrapie-infected sheep and evaluated the possible correlations between gene expression and the pathological hallmarks of prion disease. We observed significant changes in TLR expression in scrapie-infected sheep that correlate with the degree of spongiosis, PrPSc deposition, and gliosis in each of the regions studied. Remarkably, TLR4 was the only gene upregulated in all regions, regardless of the severity of neuropathology. In the hippocampus, we observed milder neuropathology associated with a distinct TLR gene expression profile and the presence of a peculiar microglial morphology, called rod microglia, described here for the first time in the brain of scrapie-infected sheep. The concurrence of these features suggests partial neuroprotection of the hippocampus. Finally, a comparison of the findings in naturallyinfected sheep versus an ovinized mouse model (tg338 mice) revealed distinct patterns of TLRgene expression.