Anti-inflammatory And Immunomodulatory Mechanisms Of Bone Marrow Mesenchymal Stem Cells Transplantation In Experimental Traumatic Brain Injury

Traumatic brain injury (TBI) is a major cause of mortality and morbidity among the population worldwide. The inflammatory response is regarded as a key factor in the secondary injury cascade following TBI. Activation of the inflammatory cascade is mediated by the release of pro-and anti-inflammatory cytokines. TBI induces a strong inflammatory response characterized by the recruitment of peripheral leukocytes into the cerebral parenchyma and the activation of resident immune cells. The infiltration of neutrophils, monocytes, and lymphocytes to the injured site directly affects neuronal survival and death. Moreover, activated microglia migrate to injured sites and release cytokines, chemotactic cytokines, reactive oxygen species, nitric oxide, proteases and other factors with cytotoxic effects, which may in turn exacerbate neuronal death.However, these immune cells and inflammatory mediators can also have neuroprotective effects in TBI. For example, T lymphocytes may contribute to later repair processes in brain injury, pro-inflammatory cytokines such as interleukin (IL)-1,IL-6, and tumor necrosis factor (TNF)-a have both deleterious and beneficial effects on neural cells, and microglia can remove cell debris, promote tissue remodeling and exert numerous neuroprotective effects under certain condition. Of these, TBI-induced inflammation appears to be a key factor in secondary brain damage, which suggests that anti-inflammatory or immunoregulatory strategies could provide effective treatments for the management of TBI-induced pathology.Previous studies have shown beneficial effects of mesenchymal stem cells (MSCs) transplantation in central nervous system (CNS) injuries, including TBI, stroke, and spinal cord injury animal models. The main findings of these studies suggested that MSCs improved neurological functional recovery, decreased apoptosis, increased endogenous cell proliferation, promoted angiogenesis and reduced lesion size. The potential mechanisms whereby transplanted MSCs might exert beneficial effects in CNS injury include their ability to migrate to injured tissues, transdifferentiation to replace damaged neural cells, and the production of growth factors by MSCs. However, recent evidence indicates that the therapeutic effect of MSCs transplantation may not be through direct cell replacement, but via modulating the host microenvironment. MSCs can secrete a variety of bioactive molecules such as trophic factors and anti-apoptotic molecules, which may provide the main mechanism responsible for their therapeutic effect.More recently, many studies demonstrated that MSCs possess immunomodulatory properties. MSCs can directly inhibit the proliferation of T lymphocytes and microglial cells, and can modulate the cytokine-secretion profile of dendritic cells (DC) and monocytes and/or macrophages. MSCs are also known to inhibit basal and formyl-methionyl-leucyl-phenylalanine-stimulated production of reactive oxygen species by neutrophils. In experimental autoimmune encephalomyelitis models, MSCs inhibited myelin-specific T cells and induced peripheral tolerance. The immunosuppressive effect of transplanted MSCs has also been demonstrated in acute, severe graft-versus-host disease and in multiple system atrophy. In addition, MSCs can induce peripheral tolerance and migrate to injured tissues, where they can inhibit the release of pro-inflammatory cytokines and promote the survival of damaged cells. For example, the therapeutic benefit of MSCs transplantation has been observed in acute lung injury, myocardial infarction, acute renal failure, cerebral ischemia and Alzheimer’s disease (AD). Furthermore, some studies have found an inflammation-modulatory function for transplanted stem cells. One study demonstrated anti-inflammatory effects of human cord blood cells in a rat model of stroke. Another study reported that intravenous nerve stem cells (NSCs), administered during the hyperacute stage in stroke, could modulate innate cerebral inflammatory responses by interacting with peripheral inflammatory systems.These studies indicate the feasibility of using MSCs to reduce cerebral inflammation and modulate the immune response after TBI. However, few studies have focused simultaneously on the effects of MSCs on inflammation-associated cytokines and immune cells in CNS injury, especially in an experimental TBI model. In this study, we therefore investigated the anti-inflammatory and immunomodulatory properties of MSCs in TBI-induced neuroinflammation using systemic bone marrow MSCs transplantation in a rat TBI model.Chapter I Isolation, culture and characterization of Sprague Dawley rats’bone marrow MSCsObjectiveTo establish the protocol to isolation, culture and identification of MSCs.MethodsMSCs were generated from the bone marrow of SD rats. When the cells reached90%confluent, adherent cells were trypsinized, harvested, and expanded. The expanded cells from passages three-eight were used for further testing or transplantation. MSCs were assessed by flow cytometry analysis of positive CD44, CD90and CD105, the negative hematopoietic markers CD14, CD34, CD45, and HLA-DR.ResultsMSCs were isolated from SD rats’bone marrow and maintained in culture for several passages. Before intravenous transplantation, third-and eighth-passage cells were characterized, and flow cytometry analysis confirmed that the cells at transplantation were positive for CD44(99.01%), CD90(99.28%), and CD105(97.71%), and had low expression of CD14(0.79%), CD34(0.78%), CD45(0.67%) and HLA-DR (1.11%).ConclusionMSCs have the similar morphologies and phenotype with classic MSCs, and also have very strong proliferation ability, were promising cell types for future studies.Chapter Ⅱ Temporal changes of inflammation-associated immune cells and cytokines in rat cortex after experimental traumatic brain injuryObjectiveTraumatic brain injury (TBI) induces an intense inflammatory response, which can persist for days to months, resulting in severe secondary brain injury. However, few information is available related to the underlying mechanisms and effects of inflammation-associated cells and cytokines in the injured brain, particularly in long-term studies. MethodsIn the present study, temporal changes of peripherally infiltrating neutrophils, lymphocytes and resident astrocytes and microglia were analyzed after TBI using immunohistochemistry throughout a4-week period. Meanwhile, inflammation-associated cytokines in the injured brain were measured by multiplex assays.ResultsThe numbers of microglia and astrocytes peaked at the7th day after TBI. MPO+neutrophils and CD3+lymphocytes peaked at the1st and3rd day after TBI, respectively. Cytokines IL-1a, TNF-a, IL-17, IL-10and TGF-β1were increased at the1st and3rd day after TBI, followed by a decrease and then a subsequent increase to day28. Chemokines, including MCP-1, MIP-2and RANTES, increased and peaked at the1st day after TBI, and then gradually decreased. The IL-1β increased at the1st day after TBI and peaked at the3rd day post-injury. The level of IL-6did not increase until7d after TBI, and peaked at the28th day. Interferon (IFN)-γ levels significantly increased from14d to28d, and peaked at the21st day. IL-4was significantly elevated beginning at the14th day after injury, and maintained a significantly high level through day28.ConclusionsBoth the inflammation-correlated cells including astrocytes, microglia, neutrophils, and lymphocytes and the secreted cytokines such as IL-1, tNF-α, IL-1β, IL-4, IL-6, IL-10, IL-17, TNF-a, IFN-γ, RANTES, MCP-1, MIP-2and TGF-β1, showed temporal changes along with the time extension after TBI. Chapter III Anti-inflammatory and immunomodulatorymechanisms of mesenchymal stem cell transplantation in experimental traumatic brain injuryObjectivePrevious studies have shown beneficial effects of mesenchymal stem cells (MSCs) transplantation in central nervous system (CNS) injuries, including traumatic brain injury (TBI). Potential repair mechanisms involve transdifferentiation to replace damaged neural cells and production of growth factors by MSCs. However, few studies have simultaneously focused on the effects of MSCs on immune cells and inflammation-associated cytokines in CNS injury, especially in an experimental TBI model. In this study, we investigated the anti-inflammatory and immunomodulatory properties of MSCs in TBI-induced neuroinflammation by systemic transplantation of MSCs into a rat TBI model.MethodsMSCs were transplanted intravenously into rats at the2nd hour after TBI. Modified neurologic severity score (mNSS) tests were performed to measure behavioral outcomes on days1,3,7,14,21and28after TBI. The effect of MSC treatment on neuroinflammation was analyzed by immunohistochemical analysis of astrocytes, microglia/macrophages, neutrophils and T lymphocytes and by measuring cytokine levels [interleukin (IL)-1α,IL-1β,IL-4, IL-6, IL-10, IL-17, tumor necrosis factor-a (TNF-a), interferon-γ (IFN-γ), regulated upon activation normal T cell expressed and secreted factor (RANTES), macrophage chemotactic protein-1(MCP-1), macrophage inflammatory protein2(MIP-2) and transforming growth factor-β1(TGF-β1)] in brain homogenates. The immunosuppression-related factors TNF-a stimulated gene/protein6(TSG-6) and nuclear factor-κB (NF-κB) were examined by reverse transcription-polymerase chain reaction and western blotting.ResultsTreatment with MSCs significantly lowered mNSS from days3-28compared with the PBS group. There was no significant difference in scores between the MSC-and PBS-treated groups only at the24th hour post-TBI. The PBS group had a significantly higher brain water content than the sham-injured control group. MSC treatment significantly reduced brain water content compared with the PBS group. Intravenous MSC transplantation after TBI was associated with a lower density of microglia/macrophages and peripheral infiltrating leukocytes at the injury site, reduced levels of pro-inflammatory cytokines, and increased anti-inflammatory cytokines, and enhanced expression of TSG-6and suppress activation of the NF-κB signaling pathway.ConclusionsThe results of this study suggest that MSCs have the ability to modulate inflammation-associated immune cells and cytokines in the TBI-induced cerebral inflammatory responses, possibly mediated by enhanced expression of TSG-6, which may suppress activation of the NF-κB signaling pathway.