The RAAS plays a pivotal role in the regulation of autoimmunity. We have recently reported that its inhibition with ACE inhibitors and AT1R blockers suppresses autoreactive Th1 and Th17 cells, promotes antigen-specific CD4+
Tregs, and inhibits the canonical NF-κB1 transcription factor complex, while activating the alternative NF-κB2 pathway. We also showed that the RAAS may be critically involved in Th1/Th17-mediated autoimmune diseases and that use of an ACE inhibitor, lisinopril, and of an AT1R-inhibitor, CA, is capable of reversing relapsing-remitting EAE in SJL mice (15
). Concurrently, Stegbauer et al. showed similar effects of the RAAS on immune cells, adding a special role for a reduced APC migration caused by downregulation of the chemokines CCL2, CCL3, and CXCL10 (39
Here we focused on the role of AT1R on CNS-resident cells during chronic-progressive EAE and identified what we believe to be a novel role of Ang II during neuroinflammation. We demonstrated that astrocytes, microglia, and neurons express AT1R on high levels and are responsive to it by initiating the upregulation and activation of TGF-β. Previously, we and others had shown that AT1R is also expressed by infiltrating macrophages and epithelial cells, and it is highly upregulated in the murine and human CNS during neuroinflammation caused by PLP139–151
– or MOG35–55
–induced autoimmunity or by viral encephalitis (15
). As shown in Supplemental Figure 3, we observed that AT1R expression is upregulated after immunization and before clinical symptoms (day 7) and is sustained throughout the peak of disease (day 14) and into disease chronicity (day 21).
TGF-β is a highly pleiotropic and multifunctional molecule that plays pivotal roles in embryogenesis, carcinogenesis, tissue development and maintenance, and especially in the immune response. It is involved in various pathologic states as diverse as fibrosis, cancer, atherosclerosis, muscular dystrophy, and Marfan disease (16
). Of the 3 members of the TGF-β family (TGF-β1–TGF-β3), TGF-β1 is the predominant isoform expressed in the immune system (42
). Almost every mammalian cell holds the capacity to secrete TGF-β as well as respond to it via its dimerizing receptors TGF-βRI/TGF-βRII, which then phosphorylate SMAD2/3 (19
). TGF-β influences the development, homeostasis, tolerance, and immune response of T cells (19
). It is now well established that TGF-β, together with IL-2, leads to induction of CD4+
Treg cells, whereas, in combination with IL-6, it induces RORγt expression and therefore causes CD4+
T cells to become aggressive Th17 cells (17
Ang II–dependent overproduction of TGF-β is a well-known pathophysiological mechanism in animal models of pulmonary, cardiac, and renal fibrosis (2
). In vitro studies demonstrate similar mechanisms in cardiac fibroblasts and smooth muscle cells as well as renal tubular and mesangial cells (29
). We show here that, also in the CNS, total production of TGF-β is increased by Ang II and blocked by CA treatment. Our in vitro data suggests that microglial cells are the main producers of TGF-β in the CNS, being highly responsive to Ang II. In autoimmunity, the role of TGF-β still remains controversial, and we must differentiate carefully under which circumstances we alter TGF-β-signaling, especially where we modulate the signaling, how we influence it, at which state of inflammation, and in which context we intervene. During the onset of EAE, high levels of TGF-β signaling occur normally in brains and spinal cords (18
). Systemic injections of TGF-β ameliorate EAE in SJL mice (26
) and also clinical symptoms in a rat model of rheumatoid arthritis (43
), while reciprocally, systemic inhibition of TGF-β by a blocking antibody aggravates the disease (44
). Knockout of TGF-β leads to massive multifocal systemic autoimmune diseases in mice (45
), and knockout mice of the TGF-β activator TSP-1 suffer from similar albeit weaker symptoms (22
). But TGF-β does not behave like a unidirectionally immunosuppressive cytokine, as it was initially perceived. Its involvement in Th17 induction is probably only one part of the explanation. Indeed, current studies showed that blocking TGF-β locally in the inflamed joints of the same rat model of rheumatoid arthritis could reverse inflammation and clinical symptoms (46
). Its inhibition in the brain, using a synthetic inhibitor of TGF-βRI, results in a delay of onset as well as in an amelioration of EAE (18
), very similar to what we observed when treating with CA. Also TSP-1–knockout mice show a weaker course of EAE compared with WT animals (24
). Inhibiting TSP-1 with the peptide LSKL is even more specific, as it particularly blocks the binding site between TSP-1 and the LLC and does not interfere with the multiple collateral functions of TSP-1 (38
). Thus, we prove that TSP-1 is an important activator of TGF-β in the brain, and its inhibition in EAE leads to delay and amelioration of the disease.
In our system, TGF-β is blocked at the site of inflammation by influencing resident CNS cells. Using luciferase reporter mice and in vivo bioluminescence imaging as well as immunohistochemistry, we showed that CA potently inhibits TGF-β signaling in the brains and spinal cords of mice with EAE. This approach contributes to attenuation of the immune response, especially at the onset of disease. However, when using AT1R inhibitors, the baseline of TGF-β signaling was not altered. In fact, we were only able to reduce the immense upregulation of TGF-β signaling that occurred during the onset phase of neuroinflammation. One could hypothesize that other molecules account for the baseline activation of TGF-β, whereas the Ang II–responsive TSP-1 is responsible for its high activation during neuroinflammation. This might explain why inhibitors of AT1R and ACE do not cause common side effects related to TGF-β inhibition. We showed that the main paracrine responders to TGF-β via pSMAD are T cells, neurons, and microglia but not astrocytes.
It is important to scrutinize the complex system of activation that distinguishes TGF-β from most other cytokines (19
). Briefly, the homodimer TGF-β is secreted in its inactive form, noncovalently bound to LAP. It forms the LLC with latent TGF-β–binding protein (LTBP) and binds via LTBP to extracellular matrix proteins. TGF-β can be activated by cleavage from the LAP, which can be easily simulated in vitro by acidification or heat. In vivo, a variety of molecules can act as activators of TGF-β, including proteases, such as plasmin and matrix metalloproteinases, reactive oxygen species, the integrins αv
, and TSP-1 (19
). In the CNS, TSP-1 has been described to take a special role in the context of glioma, being responsible for over 50% of TGF-β activation caused by glioma cells (47
), and its expression appears to correlate with the malignancy of gliomas (48
). TSP-1 has been shown to be upregulated by Ang II in different tissues, like the heart, kidney (20
), and muscle (21
), in which it can be inhibited by blockade of AT1R. This is of importance in several pathologic conditions like cardiac and renal fibrosis (2
) or Marfan disease (21
). We show here that this signaling pathway is active in the CNS as well and therefore, AT1R inhibition with CA leads to a decrease of active TGF-β.
From our in vitro data in these experiments, it is mainly astrocytes that showed a high degree of upregulation of a TGF-β–activating agent in response to Ang II. In vivo, we demonstrated AT1R controlled upregulation of TSP-1 in the CNS during EAE by more than 9 fold. This result is supported by proteomic studies from human brain samples of chronic active MS lesions where TSP-1 is among the most highly upregulated proteins, compared with normal brain tissue (23
). In turn, with CA treatment TSP-1 is suppressed very effectively, a mechanism that adequately explains the decrease of pSMAD signaling we observed. In inflamed murine spinal cords, TSP-1 is highly expressed by neurons, astrocytes, and microglia but not by the infiltrating T cells themselves. Moreover, we find TSP-1 mainly in the vicinity of inflammatory infiltrates, suggesting a paracrine mechanism of TSP-1 induction. This underscores again the cross-talk between the immune system and CNS cells. The augmentation of active TGF-β would be protective of neural tissue. However, in this context, it does not silence but rather promotes inflammation, probably caused by the distinct constellation of infiltrating lymphocytes and their cytokines.
The high coexpression of AT1R, TSP-1, and TGF-β in neurons is striking and seems to be higher in the vicinity of lymphocytic infiltrates. Neurons appear to be tightly involved in this regulating mechanism via the RAAS, and their direct role must be subject to extensive further investigations.
From these experiments, we hypothesized the following events (Figure ). During neuroinflammation, microglia and astrocytes, the main resident immunomodulatory cells in the CNS, are stimulated by Ang II via the AT1R (Figure , part i). In microglial cells, this initiates the production of TGF-β (Figure , part ii), whereas in astrocytes, Ang II mainly upregulates TSP-1 (Figure , part iii), which in turn cleaves off LAP and therefore activates latent TGF-β (Figure , part iv). An increase in active TGF-β levels in the brain creates a permissive niche in the CNS, allowing T cells to obtain a more inflammatory phenotype (Figure , part v), and therefore worsens EAE. CA treatment inhibits this cascade at the beginning by blocking the AT1R (Figure , part vi), while LSKL interferes in the binding between TSP-1 and TGF-β and therefore blocks its activation (Figure , part vii).
Schematic pathway of Ang II–TGF-β interaction in the CNS.
Due to the multifunctional character of Ang II, we have to keep in mind that inhibition of TGF-β is not the only immunomodulatory mechanism of AT1R inhibitors. Most likely it happens synergistically, with the shift from the canonical to the alternative NF-κB1 pathway and the induction of Tregs (15
). Further research will be needed to elucidate how other mechanistic pathways of Ang II contribute to its T cell–dependent effect on neuroinflammation, e.g., the production of reactive oxygen species by NADPH oxidase and the induction of TNF (25
) or interferon-γ (13
). Also, the unique features of neurons in this context will be of great interest and further investigations are currently in progress.
We show the clinical impact of Ang II–dependent TGF-β activation by blocking the TGF-β–activating cascade at these 2 separate points: binding of Ang II to the AT1R and activation of TGF-β by TSP-1. Both result in a delayed onset and an amelioration of the disease. The role of AT1R was confirmed by showing a similar delayed disease onset in Agtr1–/–
mice in comparison with WT mice. Interestingly, later clinical scores in this experiment measure up to the levels of WT mice. While humans only have 1 AT1R, mice express 2 AT1Rs (AT1Ra and AT1Rb). Only the predominant isoform, AT1Ra, is knocked out in the Agtr1–/–
EAE study, so we could hypothesize that later on in the disease, AT1Rb is able to compensate for the absence of the typical AT1Ra immunomodulating function, whereas CA constantly inhibits both isoforms. The role for the 2 murine AT1R isoforms in autoimmunity remains somewhat controversial, as Stegbauer et al. reported a slight aggravation of disease in Agtr1–/–
mice, although their data support our findings with pharmacologic AT1R inhibition (39
Taken together, we demonstrate here extensive cross-talk among resident CNS cells, infiltrating T cells, and the endocrine RAAS pathway. Treatment with AT1R inhibitors delays the onset and ameliorates EAE by influencing neurons, astrocytes, and microglia to downregulate TGF-β and TSP-1, which are normally upregulated early during inflammation. pSMAD signaling in the CNS can be dramatically inhibited by treatment with CA. We show that inhibition of TSP-1 itself is beneficial in EAE as well, indicating that TSP-1 is the major activator of TGF-β during EAE. TGF-β is known to be highly multifunctional and dependent on the location and surrounding milieu in which it functions, and further studies are needed to elucidate exactly why inhibition of TGF-β in the brain results in this perhaps counterintuitive outcome. Finally, using AT1R inhibitors as treatment for MS would be highly appealing, because they are already well-known and widely-used antihypertensive drugs, with tolerable safety profiles. These findings add to the impetus to try this promising approach for patients suffering from MS.