Involvement Of The Kallikrein-Kinin System In Neuro-Inflammation

Abstract

Introduction: Traumatic brain injury (TBI) is a major cause of disability and mortality worldwide. It can occur as a primary acute injury resulting from an exterior mechanical force or a secondary injury, which can become chronic, leading to neuro-inflammation and increase in production of reactive oxygen species which will lead to brain damage. TBI-associated inflammation leads to the activation of microglial cells in an uncontrolled manner where the brain-blood barrier (BBB) increases and becomes prone to immune cells infiltration. Bradykinin is an inflammatory peptide of the Kallikrein-kinin system (KKS) that results from the cleavage of high molecular weight kininogen through plasma kallikrein (PKall). PKall contributes to increased BBB permeability by acting through protease-activated receptor-2 (PAR-2). However, studies have also demonstrated that Bradykinin can have neuroprotective and anti-apoptotic effects by acting directly through its receptors expressed on neurons. Therefore, the exact effect of KKS after TBI remains unclear. Thus, in this project we will study the contribution of the KKS in inducing neuro-inflammation and production of reactive oxygen species (ROS) in mice exposed to TBI and we will study the effect of PKall on microglial cell line cells in order to assess how it can induce their pro-inflammatory cytokine production through the ROS pathway. Methods: TBI was induced in mice by controlled cortical impact (CCI). The expression profile of components of the KKS, inflammatory cytokines and oxidative genes were measured in the cortex of the brain, 2, 7- and 30-days post TBI, and compared to sham mice. To determine if PKall will activate microglial cells to induce oxidative stress, N9 microglial cells were stimulated with PKall in the presence and absence of aprotinin, a PKall inhibitor. The NBT (Nitroblue Tetrazolium Test) assay was used to determine the effect of PKall on ROS generation in N9, microglial cells. In addition, to determine the role of oxidative stress, in modulating the PKall response in microglial cell activation, N9 cells were stimulated with PKall in the presence and absence of N-acetyl-l-cysteine (NAC) which is a chelating agent that will inhibit ROS. Results: Our results showed an increase in the expression of components of the KKS, activation of immune resident cells, inflammation and ROS production in mice sacrificed after 2 and 7 days post injury compared to control mice. This was shown through PCR (polymerase chain reaction) where the expression of PKall (*P=0.021), Interleukin-6 (IL-6) (**P=0.01), integrin alpha M (CD11) (**P=0.002), Nadph oxidase 4 (NOX4) (*P=0.042) increased significantly during the acute phase of after TBI showing an association between the KKS, microglia and NOX’s. Indeed, microglial cells increased their mRNA expression of NOX1(*P=0.021) and NOX4 (*P=0.021) after their treatment with PKall. NBT assay also showed an increase in ROS production in response to PKall treatment. Conclusion: Our data demonstrate that the expression of PKall was significantly increased in the injured brain in response to TBI at 2 and 7 days post injury. In addition, TBI was also associated with increased expression of inflammatory and oxidative stress genes and activation of microglia. PKall treatment of microglial cells resulted in the increased expression of inflammatory and oxidative stress genes and the activation of microglial cells. Taken together, these findings implicate a role for PKall in TBI and could be a target for interventional therapy.