Signals mediated by transforming growth factor- initiate autoimmune encephalomyelitis, but chronic inflammation is needed to sustain disease

Marc Veldhoen, Richard J Hocking, Richard A Flavell & Brigitta Stockinger

Division of Molecular Immunology, Medical Research Council National Institute for Medical Research, London NW7 1AA, UK.

It is unclear whether TGF-, a critical differentiation factor for T cells producing interleukin 17 (T_H-17 cells), is required for the initiation of experimental autoimmune encephalomyelitis (EAE) in vivo. Here we show that mice whose T cells cannot respond to TGF- signaling lack T_H-17 cells and do not develop EAE despite the presence of T helper cell type 1 infiltrates in the spinal cord. Local but not systemic antibody blockade of TGF- prevented T_H-17 cell differentiation and the onset of EAE. The pathogen stimulus zymosan, like mycobacterium, induced T_H-17 cells and initiated EAE, but the disease was transient and correlated with reduced production of interleukin 23. These data show that TGF- is essential for the initiation of EAE and suggest that disease progression may require ongoing chronic inflammation and production of interleukin 23.

Given the paradigm shift associated with the discovery of the subset of T cells producing interleukin 17 (T_H-17 cells) and its involvement in autoimmune diseases such as experimental autoimmune encephalomyelitis (EAE), which is a mouse model for multiple sclerosis, elucidation of the physiological mechanisms underlying the development of T_H-17 cells in vivo and the rules for their participation in autoimmune pathology are of crucial importance. A connection between transforming growth factor- (TGF-) and T_H-17 cells has been confirmed not only in vitro as initially described but also in vivo¹. That was based on the observation that mice overexpressing TGF- in T cells after activation have more-severe EAE and more T_H-17 cells. Given that the experimental system was not devoid of TGF-, it could not be used to address the issue of whether TGF- is essential for the development of T_H-17 cells as well as EAE.

EAE is induced in mice by immunization with encephalitogenic antigens such as myelin oligodendrocyte glycoprotein (MOG) and depends on the inclusion of complete Freund's adjuvant (CFA) containing heat-killed mycobacteria². Pathogenesis is characterized by inflammation of the central nervous system with spinal cord involvement caused by the infiltration of inflammatory cells, including T cells and neutrophils, and C57BL/6 mice show a primary progressive pattern. EAE has historically been considered a disease mediated by T helper type 1 cells (T_H1 cells). Indeed, analysis of the central nervous system has shown infiltration of T cells producing interferon- (IFN-) among other cells. However, the demonstration that mice lacking T_H1-associated molecules, such as IFN-, IFN-R, interleukin-12 receptor -2 (IL-12R2), IL-12p35 (refs. 3, 4, 5, 6, 7) and IL-18 (ref. 8) develop severe EAE calls into question the essential function of T_H1 in this autoimmune disease. In contrast, it seems that T_H-17 cells^{9, 10} are crucial to the autoimmune pathology of EAE and rheumatoid arthritis, given the absence of disease in mice lacking IL-17 or IL-23 (refs. 11,12). The differentiation of the T_H-17 subset from naive CD4⁺ T cells requires stimulation in the presence of IL-6 and TGF-1 and is amplified by tumor necrosis factor (TNF) and IL-1¹³.

Here we show that TGF- signaling in T cells is required for the initiation of EAE, as mice with defective TGF- signaling in T cells¹⁴ neither developed the T_H-17 subset nor succumbed to EAE, despite the fact that they had exacerbated T_H1 responses. Furthermore, only interruption of local but not of systemic TGF- signaling prevented the development of T_H-17 cells and onset of EAE. That suggests that the induction of T_H-17 cells is promoted by production of TGF- in close proximity, most likely contributed by dendritic cells (DCs) during activation of T cells, whereas other sources of TGF- in the body are more likely to dampen already ongoing immune responses. Close-range action of TGF- by antigen-presenting cells is also supported by our finding that mycobacteria induced the differentiation of T_H-17 cells in a TGF--dependent way without the requirement for exogenous addition of TGF-. Similarly, another pathogen-derived molecule, zymosan, which is a fungal cell wall component, also elicited T_H-17 cell differentiation and supported the initiation of MOG-induced EAE in the absence of CFA or mycobacteria. However, in contrast to mycobacteria, zymosan did not sustain chronic inflammation or the production of IL-23. As a consequence, mice with EAE induced by zymosan and MOG had a notable reversal of symptoms after the acute phase of disease. That suggests that different pathogen-derived molecules have distinct effects on DCs, which subsequently influence CD4 effector T cell differentiation, and emphasizes the complex control mechanisms involved in the maintenance and progression of immune pathology.

TGF- signaling is essential for T_H-17 cell differentiation

TGF-1 has a nonredundant function in the differentiation of T_H-17 cells that extends beyond its known function in the downregulation of T_H1 and T_H2 cell differentiation, as T_H-17 cell differentiation in the presence of antibodies to IFN-, to IL-12 and IL-23, and to IL-4 is blocked by the addition of antibody to TGF- (anti-TGF-)¹³. TGF--transgenic mice expressing TGF- under control of the Il2 promoter develop EAE with greater severity and have more T_H-17 cells¹⁵, confirming the link between TGF- and IL-17. However, that system cannot be used to address the issue of whether TGF- signaling is obligatory for the initiation of EAE and whether interference in this process prevents EAE. To investigate that, we used transgenic mice expressing a dominant negative form of TGF- receptor II under control of the Cd4 promoter (called 'CD4dnTGFRII mice' here)¹⁴ so that T cells from such mice cannot respond to TGF- signals.

We first tested whether flow cytometry–sorted naive CD4⁺ T cells from CD4dnTGFRII mice generated T_H-17 cells when stimulated in vitro. Naive T cells from transgenic mice, in contrast to nontransgenic littermates, had a strong 'bias' for T_H1 responses, with more than 50% IFN--producing T cells after stimulation in culture together with DCs even in the absence of an inflammatory stimulus (Fig. 1, left). However, T cells from CD4dnTGFRII mice did not differentiate into T_H-17 cells even in the presence of lipopolysaccharide (LPS) and TGF-, whereas those from nontransgenic littermates developed a sizeable fraction of T_H-17 cells in those conditions (Fig. 1, right).

Figure 1. Naive CD4 T cells from CD4dnTGFRII mice do not generate TH-17 cells. Flow cytometry for IL-17 and IFN- in naive CD4+ T cells from CD4dnTGFRII mice (a) or C57BL/6 mice (b), sorted by flow cytometry and stimulated with anti-CD3 in the presence of bone marrow–derived DCs alone (left) or bone marrow–derived DCs plus LPS and TGF-1 (right); intracellular cytokine staining was done 6 d after activation. Numbers in quadrants indicate percent positive cells in each. Data are representative of two independent experiments. Full Figure and legend (38K)

Mycobacteria affect T_H-17 cell differentiation via TGF-

EAE induction requires the inclusion of CFA and mycobacteria with the antigen, and molecules from microbial pathogens such as Borrelia burgdorferi or Mycobacterium tuberculosis can stimulate IL-17 expression in human and mouse CD4⁺ T cells^{16, 17}. To test how mycobacteria affect T cell differentiation and what molecules are involved in this process, we sorted naive CD4⁺ T cells by flow cytometry and cultured them in the presence of bone marrow–derived DCs with either LPS or mycobacteria (Fig. 2). Stimulation of DCs with mycobacteria promoted T_H-17 cell differentiation without the need for exogenous TGF-1, whereas LPS stimulated T_H-17 cell generation only when exogenous TGF-1 was added (Fig. 2a,c). Nevertheless, mycobacteria-dependent T_H-17 cell differentiation was absolutely dependent on TGF-1 and IL-6, as neutralizing antibodies to those cytokines abrogated T_H-17 cell generation (Fig. 2f,h). TGF-1 is a pleiotropic cytokine produced by nearly every cell type in the body¹⁸. In vitro work has indicated that DCs, the most important cell type to activate naive T cells, express sufficient TGF-1 themselves to drive T_H-17 cell differentiation, if the production of IL-12 and subsequent T_H1 cell differentiation is reduced by blockade of the p40 subunit shared by IL-12 and IL-23 and of IFN-¹³. The results obtained with mycobacterial stimulation of DCs emphasize that different pathogen-derived molecules have distinct effects on DCs that subsequently influence CD4 effector T cell differentiation.

Figure 2. Mycobacteria stimulate TH-17 cell differentiation without added TGF-1. Flow cytometry for IL-17 and IFN- in naive CD4+ T cells from C57BL/6 mice sorted by flow cytometry and stimulated with anti-CD3 in the presence of bone marrow–derived DCs and either LPS (left) or M. tuberculosis H37Ra (right); intracellular cytokine staining was done 6 d after activation. (a,b) No additional treatment. (c,d) TGF-1 added at a concentration of 0.5 ng/ml. (e–h) Anti-IL-6 (e,f) or anti-TGF-1,2,3 (g,h) added at a concentration of 10 g/ml. Numbers in quadrants indicate percent positive cells in each. Data are representative of three independent experiments. Full Figure and legend (75K)

Absence of T_H-17 cells and EAE in CD4dnTGFRII mice

We next immunized CD4dnTGFRII mice and littermate control mice with a MOG peptide consisting of amino acids 33–55, emulsified in incomplete Freund's adjuvant (IFA), and supplemented this with 250 g of M. tuberculosis strain H37Ra. The crucial involvement of TGF- signaling in the initiation of EAE was emphasized by the finding that CD4dnTGFRII mice did not develop any signs of EAE or developed mild signs only (Fig. 3a). In contrast, all nontransgenic littermates of CD4dnTGFRII mice succumbed to EAE pathology within 12–14 d of immunization. Flow cytometry of cells isolated from spinal cords showed that samples from CD4dnTGFRII mice contained T_H1 T cells, albeit fewer than those from control littermates, but no T_H-17 cells, whereas nontransgenic littermates had extensive infiltrates of both T cell subsets (Fig. 3b). These data showed conclusively that T_H-17 cells, not T_H1 cells, were responsible for initiating EAE.

Figure 3. Blockade of TGF- signaling and local but not systemic anti-TGF treatment prevent the development of EAE. (a) Clinical scores of CD4dnTGFRII mice (TG) and littermate controls (Ctrl) immunized with MOG emulsified in IFA plus M. tuberculosis H37Ra; the development of clinical signs of EAE was monitored daily. Data represent mean ( s.d.) clinical scores of six mice per group. (b) Absolute numbers of infiltrating T cells (on day 18) producing IFN- (TH1) or IL-17 (TH-17) in the spinal cords of CD4dnTGFRII mice and littermate controls. ND, not detectable. (c) Clinical scores of C57BL/6 mice immunized with MOG emulsified in IFA plus M. tuberculosis H37Ra, with 100 g isotype control antibody (Isotype ctrl) or 100 g anti-TGF- administered locally (c; loc) or systemically (d; sys) at the time of immunization; the development of clinical signs of EAE was monitored daily. Data represent mean ( s.d.) clinical scores of eight mice per group (c,d). Data are representative of three (a,b) or two (c,d) independent experiments. Full Figure and legend (53K)

Local but not systemic TGF- blockade inhibits EAE

We next studied the effect of neutralizing antibodies to TGF- on the induction of EAE. Studies of an experimentally induced arthritis model in rats have demonstrated that local administration of anti-TGF- blocks acute inflammation, whereas local administration of the cytokine TGF- increases its severity^{19, 20}. Although that observation predated the discovery of the T_H-17 subset, it nevertheless is consistent with the predicted function of TGF- in the induction of T_H-17 cells. Inclusion of anti-TGF- in the antigen emulsion blocked the development of EAE, whereas all mice immunized with isotype control antibody in the antigen emulsion developed severe symptoms of EAE (Fig. 3c). In contrast, all mice that received intravenous injection of anti-TGF-1,2,3 at the time of immunization developed EAE with even slightly higher severity (Fig. 3d). Local injection of anti-TGF- interferes with TGF- signaling at the DC–T cell interface and probably prevents T_H-17 cell differentiation. Systemic injection of anti-TGF-, in contrast, probably blocks the action of TGF- released by other cell types, including regulatory T cells, and may therefore dampen potential regulatory responses^{21, 22, 23, 24, 25} but not affect the generation of T_H-17 cells.

While testing various pathogen-derived molecules for their effect on DC-driven differentiation of T_H-17 cells, we noted that zymosan, a cell wall component of Saccharomyces cerevisiae, had an even greater ability than mycobacteria to drive T_H-17 cell differentiation without the need for exogenous TGF- (Fig. 4a). The -glucan components of zymosan trigger arthritis in SKG mice through dectin-1-dependent activation of DCs²⁶. Kinetic analysis of cytokine induction in DCs exposed to zymosan showed that they had less IL-12p35 mRNA but more mRNA of the p40 subunit shared by IL-12 and IL-23 (Supplementary Fig. 1 online). Thus, the smaller amount of IL-12 mRNA induced after stimulation with zymosan may compromise T_H1 cell differentiation and instead facilitate TGF--mediated induction of T_H-17 cells.

Figure 4. Zymosan induces TH-17 cells and the initiation of EAE but cannot sustain disease progression. (a) Flow cytometry for IL-17 and IFN- in naive CD4 T cells from C57BL/6 mice sorted by flow cytometry and stimulated with anti-CD3 in the presence of bone marrow–derived DCs and 10 g/ml of zymosan; intracellular cytokine staining was done 6 d after activation. Numbers in quadrants indicate percent positive cells in each. (b) Clinical scores of C57BL/6 mice immunized with MOG emulsified in IFA plus 250 g M. tuberculosis H37Ra (Mtb) or 500 g zymosan (Zym); the development of clinical signs of EAE was monitored daily. Data represent mean ( s.d.) clinical scores of eight mice per group. (c) Absolute numbers of T cells producing IFN- (TH1) or IL-17 (TH-17), assessed in spinal cords from mice immunized with MOG and mycobacteria (filled bars) or mice immunized with MOG and zymosan (open bars), at 42 d after immunization. At that time point, mice immunized with MOG and mycobacteria had EAE of grades 2 and 3; mice immunized with MOG and zymosan were disease free. Right, mean fluorescent intensity (MFI) values for intracellular IL-17. P values, two-tailed Mann-Whitney test. (d,e) Real-time PCR of IL-6 mRNA (d) and IL-23p19 mRNA (e) in DCs from spleens and draining lymph nodes (LN) of nonimmunized mice (None) or mice immunized with mycobacteria or zymosan as described in b; cells purified by magnetic-activated cell sorting were analyzed at day 18 (d18) and day 42 (d42) after immunization. Expression is normalized to that of the housekeeping gene Hprt. Data are representative of two independent experiments. Full Figure and legend (56K)

The demonstration that TGF- and IL-6 are the main factors determining the lineage differentiation of T_H-17 cells^{13, 15, 27} seems to 'sideline' IL-23, which is neither required for in vitro nor in vivo generation of these cells. Nevertheless, that cytokine was first to be linked to T_H-17 cells²⁸, and it is apparent that mice lacking IL-23 (p19-deficient) are protected from the induction of arthritis and EAE. Furthermore, therapy with anti-IL-23 can ameliorate EAE²⁹.

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The importance of T_H-17 cells in autoimmune diseases such as EAE has received considerable attention, although dogma emphasizes the involvement of T_H1 cells, based on the composition of inflammatory infiltrates in the central nervous system and the finding that adoptive transfer of T_H1 clones^{31, 32, 33}, and even T_H2 clones in certain conditions³⁴, can induce EAE. It is not apparent, however, whether the participation of T_H-17 cells can be ruled out in those experiments, as the presence of T_H-17 cells in central nervous system or spinal cord has not been investigated. The situation is complicated by the fact that a multitude of cytokines seem to be involved in EAE. However, on closer inspection, many of those cytokines have a conspicuous connection with the T_H-17 subset. IL-6 is important in EAE, as demonstrated by the resistance of IL-6-deficient mice to EAE³⁵ and blockade of EAE with neutralizing antibodies to IL-6 (ref. 36). However, IL-6 is also an obligatory differentiation factor for the T_H-17 subset, and IL-6-deficient mice do not generate T_H-17 cells^{13, 15}. IL-6 is produced mainly by macrophages and DCs, and its production is under positive feedback by IL-17 (ref. 37). Although a published report has stated that T_H-17 cells produce IL-6 themselves¹², in our experience they do not make that cytokine, as assessed by RT-PCR as well as intracellular staining (data not shown). TNF likewise is important in EAE³⁸, and its blockade with antibodies and TNF receptor–immunoglobulin fusion proteins can prevent disease in a chronic-relapsing EAE model³⁹. However, like IL-6, TNF is involved in the differentiation of T_H-17 cells, and its production is enhanced by positive feedback from IL-17 (ref. 40). Another proinflammatory cytokine linked to EAE is osteopontin^{41, 42}. Although some studies consider osteopontin a key cytokine for T_H1 immune responses, as mice deficient in this cytokine have lower IFN- responses^{41, 43}, that is not universally accepted⁴⁴. Furthermore, it is notable that osteopontin-deficient mice have suppressed TNF responses⁴⁵. It is possible that T_H1 and T_H-17 cells may have divergent functions in the acute and chronic stages of EAE, as IL-6 and IL-17 are increased in chronic but not in acute lesions of patients with multiple sclerosis⁴⁶. However, acute lesions have increased expression of granulocyte colony-stimulating factor (G-CSF), a cytokine that is upregulated substantially by IL-17 and is associated with an increase in neutrophil progenitors⁴⁷.

Our data have provided unequivocal evidence not only that TGF- is obligatory for the development of EAE but also that this disease does not manifest when T_H-17 cells are absent, despite the infiltration of T_H1 cells in the peripheral nervous system. Notably, CD4dnTGFRII mice immunized with MOG had fewer T_H1 cells in their spinal cords than did control littermates, although such mice have increased proportions of activated T cells in their lymphoid organs that spontaneously secrete T_H1 cytokines¹⁴. That indicates that T_H1 cells may be recruited to the spinal cord in large numbers only subsequent to T_H-17 cell infiltration.

The complex and seemingly paradoxical functions of TGF- in the regulation of immune responses are emphasized by the different outcomes of local and systemic blockade of this cytokine. The fact that T_H-17 cell differentiation and EAE induction was blocked only when anti-TGF- was present in the emulsion containing the antigen and Toll-like receptor stimulus emphasizes the idea that T_H-17 cell induction is promoted by production of TGF- in close proximity, most likely contributed by DCs during T cell activation, whereas other sources of TGF-, which are widely available in the body, are more likely to dampen already ongoing immune responses. The availability of DC-derived TGF- seems to critically depend on the type of pathogen stimulus those cells receive. Much attention has been focused on IL-23 induction in DCs or macrophages exposed to different triggers⁴⁹, but it is now apparent that that is not the main factor determining T_H-17 cell differentiation. Our data have shown that zymosan, like mycobacteria, stimulates DCs in a way that supports the TGF--dependent differentiation of T_H-17 cells without the requirement for additional exogenous TGF-. It is unknown whether different pathogens induce TGF- to varying degrees in DCs or whether modulation of IL-12 induction would dampen T_H1 cell differentiation, thereby allowing even small amounts of endogenous TGF- to drive T_H-17 cell differentiation. The different effects of various pathogens on DC activation, resulting in distinct CD4 effector T cell differentiation, is probably crucial not only in immune responses to pathogens but also in the induction of immune pathology or autoimmunity.

DC responses elicited by zymosan involve Toll-like receptor 2 and dectin-1 (ref. 50) and induce the production of proinflammatory cytokines including IL-12, although analysis of the p40 subunit shared by IL-23 did not allow discrimination between IL-12 and IL-23. In fact, our data have indicated that zymosan stimulates more IL-23 than IL-12 mRNA. TGF- production elicited by zymosan has been described⁵¹, but the induction of T_H-17 cells was not tested in that study, and the suggestion that zymosan is responsible for the induction of regulatory T cells and tolerance⁵¹ is at variance with its profound effect on induction of arthritis²⁶ as well as our data on the development of EAE. Zymosan also induces IL-10, but modulates IL-10 receptor expression on antigen-presenting cells exposed to zymosan so that the proinflammatory host defense is not compromised, whereas IL-10 responsiveness on other cells remains intact⁵².

The number of T_H-17 cells infiltrating the spinal cords of mice that had lost signs of paralysis 42 d after the injection of MOG and zymosan was not statistically different from the number of cells in mice injected with MOG and mycobacteria. Given the distinct link between T_H-17 cells and EAE, the presence of this T cell population despite the absence of clinical signs of EAE might seem unexpected. However, it is possible that the function of the persisting T_H-17 cells in the spinal cords of mice immunized with MOG and zymosan was compromised in the face of decreasing IL-23 mRNA. That was indicated by the detection of less IL-17 by intracellular staining of spinal cord T cells from mice immunized with MOG and zymosan than for mice immunized with MOG and mycobacteria and suggests that IL-23 may not be simply a survival factor for T_H-17 cells but may also maintain their effector function. The implication is that sustained T_H-17 cell–driven autoimmune reactions depend on an ongoing source of inflammation that provides proinflammatory IL-23. Although local blockade of TGF- signaling at the site of T cell activation effectively compromises the differentiation of T_H-17 cells and initiation of EAE, that intervention would be difficult to extrapolate to humans, for whom the site of T cell activation for initiation of multiple sclerosis is unknown. Global blockade of TGF- has detrimental effects and does not prevent the differentiation of T_H-17 cells. In contrast, blockade of chronic inflammation and in particular IL-23 production could be successful in interrupting the progression to full-blown disease or could even cause reversion of disease, in agreement with published data on the effect of anti-IL-23 in a chronic relapsing model of EAE²⁹.

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Transgenic CD4dnTGFRII mice on a C57BL/6 background as well as wild-type C57BL/6 mice were kept in specific pathogen–free conditions, and all animal experiments were done according to institutional guidelines and UK Home Office regulations.

Mice were injected subcutaneously at two sites with 100 l of an emulsion of IFA containing 250 g MOG peptide (amino acids 35–55) and either 250 g heat-killed M. tuberculosis strain H37Ra or 500 g zymosan. For local blockade of TGF-, 100 g anti-TGF-1,2,3 (1D11) was included in the emulsion; the control group received 100 g isotype control antibody instead. The anti-TGF- 1D11 hybridoma was a gift from A. Cooke (University of Cambridge, Cambridge, UK); the antibody was purified according to standard procedures. Care was taken not to denature the antibody during preparation of the emulsion. Mice received 200 ng Bordetella pertussis (Calbiochem) intraperitoneally on the day of immunization and 2 d later. EAE was assessed daily, and clinical scores were assigned according to the following criteria: 0, unaffected; 1, flaccid tail; 2, impaired righting reflex and/or gait; 3, partial hindlimb paralysis; 4, total hind limb paralysis; 5, total hindlimb paralysis with partial forelimb paralysis. UK Home Office regulations require 'culling' of mice that have reached stage 5.

Naive T cells sorted by flow cytometry (CD4⁺CD25^-CD44^lo) were cultured together with DCs derived from the culture of bone marrow with granulocyte-monocyte colony-stimulating factor as described¹³, in the presence of anti-CD3 plus 50 ng/ml of LPS, 10 g/ml of mycobacteria or 10 g/ml of zymosan. TGF-1 was added at a concentration of 0.5 ng/ml and blocking antibodies were added at a concentration of 10 g/ml. For measurement of intracellular IFN- or IL-17, T cells were restimulated with 500 ng/ml of phorbol dibutyrate and 500 ng/ml of ionomycin in the presence of brefeldin A on day 6 after stimulation and were stained according to the manufacturer's protocol (Becton Dickinson).

M.V. did the experiments; R.J.H. assisted with RT-PCR; R.A.F. provided the CD4dnTGF RII mice; and B.S. wrote the paper.

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