The purpose of the present study was to explore the so far unexplained role of the innate and of the adaptive cellular immune response in the context of AT1 inhibition after TBI. This study investigated for the first time the effect of candesartan treatment in neutrophil depleted as well as in RAG1−/− mice. The results suggest that one possible neuroprotective mechanism of AT1 inhibition may be mediated by neutrophils. Initially, the roles of neutrophils and lymphocytes in the development of secondary brain damage after TBI were analyzed. To this end, we successfully achieved sustained neutrophil depletion by treatment with the specific anti-Ly6G antibody. Furthermore, RAG1 deficiency, that results in a lack of mature B- and T-cells, showed significant lymphopenia. The present data show, that reduction of immune cells of the innate and of the adaptive immune system, neutropenia and lymphopenia, each independently lead to reduced brain damage after TBI. Furthermore, the present results demonstrate that posttraumatic AT1 inhibition reduces histological brain damage and limits immune response in control antibody treated mice with normal neutrophil count and in lymphopenic RAG1-deficient mice. In neutrophil depleted mice, however, AT1 inhibition had no effect on brain damage and neuroinflammation.
4.1 Experimental setting and blood cell count
The aim of the present study was to investigate the neuroprotective mechanisms that is mediated by AT1 inhibition and the effects of neutropenia and lymphopenia. To this end we performed the experiments with our established CCI model (4, 6, 8, 29, 33, 34). The perioperative setting and the conditions are standardized and physiological values, perioperative temperature and blood pressure showed to be stable (6). For the depletion of neutrophils the novel selective antibody against Ly6G (clone 1A8) was chosen, in order to selectively and significantly reduce the number of neutrophils without affecting other cell types (35–37). In contrast, the widely used less specific antibody for experimental neutrophil depletion anti-Gr1 (clone RB6-8C5) not only reduces Ly6G-specific cells (neutrophils, Gr1+/Ly6G+), but also decreases the number of other lines of WBC with Ly6-receptors (dendritic cells and subpopulations of monocytes and CD8 T-lymphocytes) (35, 38). Therefore, in the present study a sustained selective neutrophil depletion during the whole observation period after TBI was successfully achieved by repetitive application of the specific monoclonal antibody anti-Ly6G, with a dosage, based on recent studies (36, 39, 40). For the assessment of the effects of lymphopenia in TBI the well-established method with RAG1-deficient mice was chosen (41, 42). Activated RAG1 plays a key role in VDJ-recombination. RAG1−/− have small lymphatic organs without mature B- and T-lymphocytes (43) and in the present study they showed to have a significant lymphopenia. The observation times of each study were chosen with respect to the maximum brain tissue infiltration of the two immune cell types. After an early posttraumatic liberation of neutrophils from bone marrow and other reservoirs (44) neutrophils are the dominant blood cell population 24 hours after TBI (45). They infiltrate early after TBI into hippocampal and cortical brain tissue (between 4 and 72 hours) and reach a maximum of parenchymal infiltration at 1 day after TBI (46, 47). Therefore, in study A we applied the observation time lengths of 4 and 24 hours post CCI. Lymphocytes invade into cerebral tissue from the third day on after injury (6, 46). In study B, therefore, we chose the observation time lengths of 1 and 5 days after TBI. To facilitate comparability of the effect of candesartan between neutropenic and lymphopenic mice, we decided to use the observation time length of 72 hours after TBI in studies C and D, respectively (46). In order to ensure timely accurate hematologic analyses and interpretation and to minimize pre-analytic errors a mouse-species-appropriate practical hematologic instrumentation was performed with consistent collection method and appropriate anticoagulation (EDTA) (48). The retroorbital sinus as site of blood withdrawal corresponds more with central blood samples than other peripheral sites (49). The present blood cell count results are consistent with recent data (48). The quality can be considered adequate, as the platelet counts are at normal murine levels and cell clumping occurred rarely (48). Within the first hours after TBI, there was a reduction of WBC. Due to trauma-associated neutrophil sequestration, a transient leukopenia is part of the inflammatory reaction after TBI (50). In control antibody (IgG2a) treated mice WBC count normalized one day after TBI. In anti-Ly6G treated mice the initial decrease of WBC was more distinct and sustaining with lasting relatively low WBC to day 3 after TBI. However, at each time point, WBC counts were within physiological range (48). In naïve wild type mice lymphocytes are the predominant leukocytes (70 – 80 % of WBC). Neutrophils are the most common granulocytes in naïve mice and generally comprise 20 – 30 % of WBC count in mice (48). After TBI neutrophils are the most abundant cell population in circulation and cause increased expression of oxidative enzymes (44). In the present study, in Ctrl mice, as a response to TBI, there was a shift from lymphocyte dominated WBC to a significant and sustaining elevation of the neutrophil fraction (44, 45). In ND mice, in contrast, instead of a posttraumatic increase of neutrophils, there was neutropenia. From 24 hours on after TBI, the fraction of neutrophils was lower in ND mice than in naïve mice, alongside reduced absolute WBC numbers and a compensatory elevation of lymphocytes. After TBI monocyte counts decreased in all groups, without affection by the selective anti-Ly6G. RAG1−/− mice showed to be leukopenic and lymphopenia is compensated by elevated neutrophils. In all groups a short-term elevation of hemoglobin and hematocrit in the first hours after TBI, due to pre-traumatic fasting, were normalized in the following observation periods. Platelets were within physiological range in all groups at all time points without affection by treatment. AT1 inhibition had no effect on posttraumatic blood cell count in wild type, neutropenic and RAG1−/− mice. In the present study the selective AT1 blocker candesartan was chosen, that crosses the BBB (16). Previous TBI studies demonstrated that neuroprotection and brain damage reduction were achieved at low doses of candesartan that did not affect blood pressure (8), as a decline in blood pressure immediately after TBI may worsen outcome (51). Furthermore, clinical studies showed beneficial effects of candesartan, independent of the blood pressure lowering effect (52). Therefore, in the present study low dose candesartan (0.1 mg/kg) was applied that has been shown not to affect blood pressure previously (4, 8, 12, 17). In order to achieve a sustaining AT1 inhibition, treatment was started 30 minutes after TBI and then repeated daily (8). As a result, in the present study, during the whole observation time, posttraumatic blood pressure was not affected by candesartan. The peritraumatic body weight was assessed as a surrogate parameter of well-being and intake of food and water. Neither neutrophil depletion, nor candesartan treatment, had any effect on postoperative body weight loss, that had a maximum on day 2, with recovery on days 3 and 5.
4.2 Depletion of neutrophils and lymphopenia lead to reduced brain damage and inflammation
Neutrophils are an abundant proinflammatory population of circulating leukocytes that are usually among the first responders to tissue injuries in the periphery and the central nervous system (53). After trauma neutrophil sequestration plays a major role in multiple organ failure (50). In the early phase after TBI, neutrophils are the dominating immune cells that infiltrate damaged brain tissue (47), causing an increase of brain damage (46). Evidence suggests that depletion of neutrophils may have beneficial effects in the early phase after TBI. Recent studies have revealed that depletion of neutrophils with anti-Gr-1 antibodies reduced neuroinflammation and brain tissue loss up to seven days after TBI (54). Actually, in the present study, neutrophil depletion reduced brain damage 24 hours after TBI compared to control group. Alongside the damage reduction, there was a reduction of inflammatory TNFα levels.
Although T cells play diverse roles in adaptive immune responses and the regulation of inflammation, their role in TBI pathogenesis is unresolved (55). Our recent data indicated that cerebral T cell infiltration aggravated neuroinflammation but did not increase lesion volume after TBI (56, 57). In a previous closed-head injury study no difference in any pathologic or neurologic parameters was observed between wild-type and RAG1−/− mice up to 7 days after closed head injury (41). The authors concluded, that adaptive immunity is not of crucial importance for initiating and sustaining inflammatory neuropathology after closed head injury (41). In another TBI (aseptic cerebral injury) study, however, Fee and colleagues demonstrated that CD4+ T lymphocytes contribute to the severity of the acute phase of TBI and that brain injury is attenuated in RAG1−/− mice compared to wild type animals (42). In accordance with these results, in the present study, RAG1 deficiency lead to reduced lesion volumes and neuroinflammation 1 and 5 days after TBI.
However, 24 hours after TBI, neutrophil depletion showed to be more effective in reduction of lesion volume (33 % compared to non-depleted wild type) than lymphopenia (17 % compared to wild type). It has been shown that acute posttraumatic cerebral infiltration of neutrophils is more distinct, compared to infiltration of lymphocytes (46). Therefore, it is possible that a reduction of neutrophil infiltration may have a stronger anti-inflammatory effect in the first days after TBI, than a reduction of lymphocyte infiltration. This may explain why in studies with neutrophil depletion brain damage and inflammation are consistently reduced (54) and why in studies with RAG1 deficiency the results are inconsistent (41, 42, 56).
4.3 AT1 inhibition reduced histological brain damage and microglial activation in non-neutrophil depleted mice and RAG1 deficient mice, whereas candesartan treatment failed to be neuroprotective in neutrophil depleted mice
Growing amount of evidence shows that the entire cellular immune response (granulocytes, monocytes, lymphocytes, etc.) is modulated by the RAS (58, 59). Angiotensin II, via AT1 is one of the most important inducers and perpetuators of cerebral inflammation and oxidative stress (7, 60). AT1 is widely expressed in the mature central nervous system, mainly in neurons, endothelial and smooth muscle cells, astrocytes and microglia, important modulators of neuroinflammation (9, 61). AT1 is also expressed on migrating immune cells, like neutrophils, macrophages and T-cells. AT1 activation initiates subsequent production of chemokines, cytokines, and adhesion molecules, which contribute to the immigration of these activated immune cells into the lesion (7, 62–65). They induce inflammatory responses and release high levels of ROS by activation of NADPH oxidase. AT1 signaling modulates the NADPH oxidase complex activation and induces transcription of several pro-inflammatory cytokines by activation of NF-κB dependent transcription (15, 66). This leads to subsequent stimulation of several different kinases that themselves participate in the propagation of inflammatory responses and apoptotic pathways (17, 67–70).
In accordance with recent studies the present study showed that repeated posttraumatic treatment with the selective AT1 inhibitor candesartan resulted 3 days after TBI in a reduced histological brain damage in mice with normal posttraumatic neutrophil count. AT1 inhibition additionally decreased microglial activation and pro-inflammatory signaling in these mice (8, 12). In the present study AT1 blockade mediated brain damage reduction was accompanied by a decrease of microglial activation, respectively. Activated microglia were reduced by app. 14 % by candesartan and by app. 7 % by neutrophil depletion compared to vehicle and control antibody treatment. Therefore, earlier findings in candesartan treated mice, where number of neutrophils and activated microglia were reduced 3 days after TBI (4, 17) are confirmed in the present study. The pleiotropic cytokine TNFα is produced in microglia, astrocytes and neurons. It is involved in BBB-dysfunction and transmigration of WBC into brain tissue and induces neuronal loss via microglial activation and phagoptosis of neurons (2, 71–73). It has been demonstrated earlier that microglial release of TNFα is reduced by AT1 antagonists (7, 74). After an early upregulation in the first 8 hours after TBI (72) TNFα decreases significantly in the following time (75). This kinetic could explain that in the present study there is only a reduction of TNFα 4 hours after TBI in ND mice. One of the most potent key pro-inflammatory cytokines is IL1β. It plays a major role in leukocyte adhesion, BBB-dysfunction, brain edema and apoptosis as well as in the induction of other pro-inflammatory cytokines. Clinical studies showed a correlation between increased IL1β levels and elevated ICP with worse prognosis (76). Reduced activity of IL1β showed improved neurological outcomes and reduced infiltration of neutrophils (72, 77–79). Cerebral IL1β expression increases in the first hour after TBI and reaches highest levels 12 and 24 hours after experimental TBI, and remains elevated up to 48 hours (80). In the present study there is a strong tendency towards lower IL1β expression in candesartan treated control-antibody mice. However, the lack of effect of RAG1 deficiency or neutropenia on IL1β expression in the present study may be due to low cytokine mRNA levels 3 days after CCI (15). In a recent study, we could demonstrate reduced cytokine expression by AT1 inhibition 12 hours after TBI (8). Several studies have shown that the cytokine IL6 is up-regulated after TBI with consecutive increased microglia activation and neurological impairment (2, 81). Clinical studies postulate a correlation between elevated IL6 serum levels, ICP increase and severity of TBI (82). Furthermore, IL6 regulates migration of neutrophils during acute inflammation (83). In the present study IL6 is reduced by AT1 inhibition three days after TBI in control antibody treated mice, whereas in neutropenic mice, candesartan did not affect IL-6 expression.
In a recent study we could demonstrate that posttraumatic AT1 inhibition improved neurological recovery, reduced histological brain damage and limited immune response in young adult and aged mice. We postulated that the protective effect is attributed to a diminished microglia activation and increased anti-inflammatory microglia polarization. One major finding was that neutrophil infiltration was largely reduced (4). As AT1 is expressed on circulating neutrophils and lymphocytes (64, 84), several studies indicate that infiltrations of both immune cell types, neutrophils and lymphocytes are reduced by AT1 blockade (85, 86). However, in our recent study perilesional T-cell immigration was not affected by AT1 inhibition (4). Neuroprotective mechanisms of AT1 inhibition in the acute phase after TBI may therefore be independent of adaptive lymphocyte reaction. A recent study showed that expression of CD62L on human neutrophils is modulated by AT1 receptors, on pathways involving extracellular signal-regulated kinases 1 and 2 (ERK1/2) mitogen-activated protein kinase (MAPK), phosphatidylinositol 3-kinase, and calcineurin (87), leading to reduced transmigration of neutrophils. Recent studies revealed that AT1 inhibition leads to down regulation of important recruitment proteins like ICAM1 in endothelial cells and CD11b/CD18 on WBC and the post traumatic increase of BBB permeability. Consecutively, the infiltration of immune cells is largely reduced by AT1 inhibition (85, 86, 88, 89). A recent murine cerebral transcriptomic analysis after TBI showed strong alterations of gene transcription by candesartan treatment. The authors point to a role for candesartan in altering many different aspects of the response to TBI, particularly those involved with cellular response to stress, extracellular matrix alterations and the innate immune response (90). Therefore, AT1 inhibition may have a direct and modulating anti-inflammatory effect on invading neutrophils and resident activated microglia (17). The reduction of the cerebral inflammatory response of the innate immune system with reduced microglial activation as well as decreased infiltration of neutrophils may be a putative protective mechanism of AT1 inhibition mediated anti-inflammation and neuroprotection (4).