The present study demonstrates that the local environment in the CNS is critical in the initiation of autoimmune encephalitis. Using luciferase reporter mice and in vivo bioluminescence imaging, we discovered that astrocytes, microglia, and Smad-dependent TGF-β signaling are activated early in the CNS, several days before clinical onset of disease. The adjuvant CFA is likely sufficient to initiate these early events, leading to increased production of TGF-β1 in astrocytes and microglia. Consequently, TGF-β signaling is activated in neurons and later in infiltrating T cells. The inhibition of TGF-β signaling delayed and ameliorated progression of autoimmune encephalomyelitis by significantly reducing the accumulation of T cells in the CNS but not by altering the priming of peripheral autoreactive T cells or the expansion of specific CD4+ T cell subsets.
In autoimmune inflammatory disease, autoreactive T cells typically generated in lymphoid organs not only need to infiltrate but also need to successfully accumulate in the target organ to cause disease. TGF-β1 can participate in all these processes by regulating T cell proliferation, differentiation, and apoptosis as well as chemotaxis and homing (7
). These site-specific actions may explain the seemingly paradoxical effects of TGF-β1 in inhibiting or promoting inflammatory responses and autoimmunity. Indeed in EAE, TGF-β1 was initially discovered to be immunosuppressive and to inhibit disease (13
) but is now recognized as a critical factor in the genesis of autoreactive, IL-17–producing T cells (17
). The current study demonstrates that TGF-β1 in the CNS may be critical in promoting disease. We propose that CNS-produced TGF-β1 creates a permissive environment for the accumulation of pathogenic T cells and propagation of an autoimmune response. Our findings parallel observations in an experimental model of arthritis, where administering TGF-β1 locally in the joint exacerbates the inflammatory response and aggravates disease (41
), but systemic inoculation inhibits inflammation (42
). Similarly, blocking endogenous TGF-β1 by systemic injection of anti–TGF-β1 neutralizing antibody exacerbates arthritis (43
), but local blockage of TGF-β1 ameliorates ongoing inflammation (44
Pharmacological inhibition of TGF-β signaling with the ALK5 kinase inhibitor IN-1130 resulted in delayed disease onset and overall less disease in MOG35–55
–immunized mice (Figure and Table ). It was shown recently that transgenic mice with CD4+
T cells expressing a dominant-negative form of the TGF-β type II receptor and thus, not responsive to TGF-β1, failed to generate TH17 cells and did not develop EAE (20
). Interestingly, inhibition of TGF-β signaling with IN-1130 did not alter priming of MOG35–55
–specific T cells nor their production of IL-17 or other cytokines involved in the generation of TH17 cells (Supplemental Figure 5). This suggests IN-1130 was not sufficient to interfere with TGF-β signaling at the dendritic cell–T cell interface (20
) and to block the generation of autoreactive TH17 cells in the periphery. IN-1130 also did not change the percentage of CD4+
T cell subsets in the CNS or the periphery (Figures and ). Nevertheless, IN-1130 treatment strongly inhibited GFAP- and SBE-dependent reporter gene transcription (Figure ), reduced TGF-β1 and IL-6 mRNA levels in the CNS (Figure ), reduced the percentage of CD4+
cells with activated TGF-β signaling in spinal cord and spleen (Figure ), and most importantly, potently suppressed the accumulation of T cells in the CNS (Figure and Table ). These results strongly indicate that the TGF-β–dependent effects observed after IN-1130 treatment go beyond the generation of TH17 cells and involve what we believe are novel immune-modulatory effects of TGF-β1 in the CNS. This is also an example of the modulation of the immune system by the brain (23
). Neurons have a crucial role in the regulation of the T cell response. Function of Tregs has recently been shown to be modulated by neuronal signals (22
), and interestingly, IL-6 is a potent inhibitor of Treg differentiation (17
). Finally, the modulation of weight loss (Figure and Table ) may indicate an interaction between the immune system and the hypothalamic pituitary axis, which is critical in leptin regulation (45
). Leptin is both a fat modulator and a Th1 cytokine (proinflammatory).
Further emphasizing the importance of TGF-β1 in the CNS during EAE, the overproduction of a constitutively active form of TGF-β1 in astrocytes of transgenic mice resulted in an acceleration and exacerbation of disease after immunization with spinal cord homogenate (16
) or MOG35–55
peptide (Supplemental Table 1). Again, T cell priming and production of cytokines after recall stimulation with MOG35–55
in culture were not affected by this tissue-restricted overexpression of TGF-β1 (Supplemental Figure 3). In a related experiment, astrocyte-restricted overexpression of TGF-β1 in a mouse model for Alzheimer disease resulted in increased T cell accumulation in the brain after immunization with the peptide that accumulates in the brains of these mice (46
), indicating that the effect of TGF-β1 on cerebral accumulation of T cells is not restricted to myelin-specific cells. Because infiltrating T cells in EAE brains showed prominent activation of TGF-β signaling (Figures and ), it is tempting to speculate that at least some of the disease-attenuating effect observed in mice with TGF-β signaling–deficient T cells (20
) is related to their inability to respond to TGF-β1 in the CNS.
How TGF-β1 regulates T cell function and facilitates accumulation in the CNS requires further study. TGF-β1 is strongly chemotactic for monocytes (47
), neutrophils (48
), and T cells (49
) and induces the production of chemokines such as monocyte chemoattractant protein 1 (MCP-1) and several chemokine receptors (8
). Besides this effect on chemotaxis, TGF-β1 might increase homing to the brain by increasing adhesion molecules on infiltrating cells and the vasculature (8
). Interestingly, unmanipulated GFAP-pTGF-β1 mice, which produce significant amounts of bioactive TGF-β1 in their brains, only slowly accumulate T cells in the brain with aging (46
). This suggests that, at least in the absence of autoimmune T cells, TGF-β1 does not significantly increase chemotaxis or homing of T cells. Instead, TGF-β1 may increase the survival of infiltrating T cells via activation of antiapoptotic and prosurvival pathways (7
). In the context of an immune reaction, such a function would obviously be ill-fated.
CFA is required to induce EAE, and we show that it is sufficient to activate TGF-β1 production from glial cells and TGF-β signaling in the CNS (Supplemental Figure 2). The effect of CFA is likely due to the presence of Mycobacterium tuberculosis
, which strongly activates monocytes to produce TGF-β1 in cell culture (50
) and can also convert latent TGF-β1 to its bioactive form (51
). Accordingly, these bacteria may be responsible for the observed increase in TGF-β1 production in microglia and astrocytes in our model (Figure ). M. tuberculosis
can also stimulate IL-17 production in T cells (52
) and induce TH17 cells in a TGF-β1–dependent fashion (20
). Given the observation that LPS is also a strong inducer of TGF-β signaling in the CNS (31
) and has been shown to promote relapses in EAE (53
), it is tempting to speculate that bacterial infections may similarly promote relapses in MS patients (1
) by creating a more permissive, proinflammatory environment in the CNS. Activation of glial cells by microbial signals is mediated primarily through pattern recognition receptors, of which TLRs are key participants (54
). TLRs recognize microbial structural motifs referred to as pathogen-associated molecular patterns (PAMP). One TLR molecule, TLR4, binds LPS from Gram-negative bacteria. The engagement of the TLRs initiates TLR signaling cascade, leading to the activation of NF-κB (57
), which in turn induces the transcription of proinflammatory cytokines, chemokines, and upregulation of cell surface molecules. The critical role of the canonical pathway of NF-κB in EAE was shown when this pathway of NF-κB signaling was targeted in astrocytes (24
). Our studies show that TGF-β signaling in the brain is activated by these innate immune triggers and may have a critical role in setting the conditions for local immune disease.
Our study investigates an important but often neglected aspect of autoimmune encephalomyelitis by exploring the conditions that facilitate the accumulation of autoimmune T cells in the CNS. We propose that TGF-β1 has a key role in creating a permissive environment several days before the onset of clinical disease in EAE by activating TGF-β signaling first in CNS cells and then in infiltrating T cells. The influence of TGF-β signaling in glial cells of the nervous system on the immune system is striking here. Inhibition of TGF-β signaling counteracts these events and therefore may have therapeutic benefits in the early phases of autoimmune inflammation in the CNS.