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B Kinase 2/
Deficiency Controls Expansion of Autoreactive T Cells and Suppresses Experimental Autoimmune Encephalomyelitis1
,
* Hertie Institute for Clinical Brain Research, Tübingen, Germany;
Center for Neurologic Diseases, Harvard Medical School, Boston, MA 02115;
Stanford University School of Medicine, Stanford, CA 94305;
Veterans Affairs Health Care System, Palo Alto, CA 94304;
¶ Millennium Pharmaceuticals, Cambridge, MA 02139; and
|| CBR Institute for Biomedical Research, Harvard Medical School, Boston, MA 02115
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Abstract |
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B family of transcription factors plays a pivotal role in T cell activation and survival during (auto) immune responses. IB kinase 2/
(IKK2) is part of the IB kinase complex, a central component of the intracellular signaling pathway mediating NF-B activation. We studied the role of IKK2 in autoantigen-specific T cell activation and induction of autoimmune disease using mice that lack this kinase specifically in T cells (IKK2
T cell mice). We found highly impaired myelin-oligodendrocyte-glycoprotein (MOG)35–55-specific T cell activation in vitro and complete resistance to MOG35–55-induced experimental autoimmune encephalomyelitis (EAE) in IKK2T cell C57BL/6 mice in vivo. By contrast, transgenic expression of a pathogenic MOG35–55-specific TCR (2D2 TCR) rendered IKK2T cell mice susceptible to MOG35–55-induced EAE and restored in vitro MOG35–55-specific T cell responses, indicating an expansion defect in IKK2-deficient T cells. Treatment with the IKK2-inhibitory compound PS-1145 reduced MOG35–55-specific proliferation and cytokine production of 2D2 transgenic spleen cells in vitro and diminished clinical signs of EAE in vivo. Our data underscore the potential of therapeutic IKK inhibition in autoimmune diseases. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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B transcription factor family (1). Activation of NF-B takes place during both innate and adaptive immune responses. Different stimulatory pathways in lymphocytes (e.g., signaling via TNFR1 and cross-linking of the TCR) lead to the activation of NF-B through an intracellular signaling cascade and enhanced transcriptional activity of a plethora of genes, including genes that encode proinflammatory as well as antiapoptotic proteins that promote survival of resting and activated immune cells (1, 2, 3).
In most resting cells, NF-B family members are kept inactive through association with the inhibitor of NF-B (IB) proteins. During activation of NF-B through the ‘canonical pathway’, IB
is phosphorylated by the IB kinase (IKK)4 complex and thereby targeted for degradation by the proteasome. The IKK complex consists of two kinases, IKK1 (IKK) and IKK2 (IKK), which deliver the kinase activity, and the regulatory subunit NF-B essential modulator (NEMO/IKK
). Processing of p100 to p52 by the alternative pathway of NF-B activation is mediated by IKK1 and NF-B-inducing kinase (4). Furthermore, IKK-independent activation of NF-B has been demonstrated in Ikk1/Ikk2–/– cells (5).
The consequences of IKK2 deficiency in T cells have been studied in CD4cre/Ikk2FL (IKK2T cell) mice (6, 7, 8). TCR- or TNFR-mediated NF-B activation was reduced in IKK2-deficient compared with wild-type (wt) T cells, but not absent; this was shown likely to be due to IKK activity of an IKK complex consisting of NEMO and IKK1 only. In IKK2T cell mice, early T cell development in the thymus occurs normally, but peripheral CD4 and CD8 T cell numbers are reduced by 20 and 50%, respectively, and a defect in generation of regulatory (TR), NK-like, and memory T cells was detected (7). Functional analysis of IKK2-deficient T cells showed normal in vitro proliferation in response to polyclonal activation, but peptide-specific T cell responses were strongly impaired (8).
In this study, we investigate the relevance of IKK2 for autoantigen-specific activation of T cells and induction of a T cell-dependent autoimmune disease, myelin-oligodendrocyte-glycoprotein (MOG)35–55-induced experimental autoimmune encephalomyelitis (EAE) in C57BL/6 mice. Previous studies using knockout mice for the NF-B subunits NF-B1 and c-Rel had uncovered important roles for these transcription factors in EAE (9, 10), suggesting that interference with their activation pathways should have protective effects. We show here that T cells require IKK2 activity in a cell-autonomous manner for full activation in response to MOG35–55 and that IKK2T cell C57BL/6 mice are completely protected against the induction of EAE by immunization with this autoantigen. However, we could reconstitute the EAE susceptibility of these mice by introducing a transgenic MOG35–55-specific TCR and conclude that IKK2 controls the expansion of autoreactive T cells.
NF-B-inhibiting compounds such as pyrrolidine thiocarbamate (PDTC), or peptides mimicking the NEMO-binding domain of IKK proteins were shown to have beneficial effects in EAE (11, 12). Our findings in IKK2T cell mice, together with the observation that lack of IKK2 in neuroectodermal-derived CNS cells reduces EAE severity (13), makes IKK2 a promising target for therapeutic intervention in autoimmune demyelinating disease. We therefore evaluated the possible therapeutic value of IKK2 inhibition in vitro and in vivo using an IKK-inhibitory compound, PS-1145. The results of those experiments indicated that autoantigen-specific T cells responses are well suppressed in vitro and that PS-1145 exerts a beneficial effect in EAE when administered during the induction and at onset of the disease.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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The generation and genotyping of CD4cre/Ikk2FL mice has been described previously (6). Mice were housed at the Thorn Research Building animal facility of the Brigham and Women’s Hospital (Boston, MA) and the animal facility of the Verfügungsgebäude of the University of Tübingen (Tübingen, Germany). EAE was induced by s.c. immunization in both flanks of age-matched female mice using 70–100 µg of MOG35–55 peptide (MEVGWYRSPFSRVVHLYRNGK) dissolved in PBS and emulsified with an equivalent volume of CFA supplemented with 4 mg/ml Mycobacterium tuberculosis H37RA (Difco). On days 0 and 2, each mouse was treated with 150 ng of pertussis toxin (List Biological Laboratories) i.v. The severity score of clinical disease was assessed as: 0 = no paralysis; 1 = limp tail; 2 = limp tail and weak gait; 3 = hind limb paralysis; 4 = fore limb paralysis; 5 = death, or moribund animal. Mice were given water and food on the bottom of the cage when they were paralyzed.
Animal experiments were conducted as approved by local authorities according to the animal experimentation protocol of V. K. Kuchroo (United States) and of B. Greve and R. Weissert (Germany).
MOG35–55-specific TCR (2D2)-transgenic mice were typed by flow cytometry as described in (15). 2D2-transgenic IKK2T cell mice were generated by breeding 2D2-transgenic and CD4cre-transgenic Ikk2FL mice; subsequent matings included mice that contained one transgene of 2D2 and CD4cre and variable numbers of loxP-flanked Ikk2 alleles. Mice containing each one transgenic allele of 2D2 and CD4cre together with two loxP-flanked Ikk2 alleles are termed 2D2 CD4cre tg Ikk2FL or 2D2 tg IKK2T cell mice; those mice lacking the 2D2 or CD4cre transgene or did not contain two loxP-flanked Ikk2 alleles were used as appropriate wt littermate controls.
Histopathology
Brains and spinal cords were removed from the mice between days 15 and 35 postimmunization (p.i.) and fixed in 10% phosphate-buffered formalin. Paraffin-embedded sections were stained with Luxol fast blue-H&E for light microscopy. Histological disease was quantitated by counting the inflammatory foci in meninges and parenchyma, as described (16).
In vitro proliferation and cytokines
Mice were injected s.c. at five sites with 100 µg of MOG35–55 in CFA. Ten days later, cells from draining lymph nodes were prepared, and 5 x 105 cells/well in 96-well plates were reactivated in the presence of various concentration of MOG35–55 in complete cell medium (CM; RPMI 1640 supplemented with 10% FCS, 2 mM L-glutamine, 1000 U/ml penicillin/1 µg/ml streptomycin, and sodium pyruvate). Cell cultures were pulsed after 48 h with 1 µCi/well [3H]thymidine, and incorporation of radioactivity was assessed 18 h later in a Beckman scintillation counter (model LS5000; Beckman Instruments). The concentrations of cytokines were measured in cell culture supernatants 48 h after activation as previously described (17). For enumeration of IL-17-producing cells, we used a mouse IL-17 ELISPOT assay according to the manufacturer’s instructions (eBioscience). Plates were counted using an automated ELISPOT reader (from AID).
For in vitro proliferation experiments using 2D2 TCR-transgenic T cells, whole spleen cells (SPC) from unimmunized 2D2-transgenic mice were prepared, and 5 x 105 cells were reactivated in the presence of various concentration of MOG35–55 in complete CM. Proliferation and cytokine production were measured as described above. Additionally, for flow cytometry, cells were reactivated at 5 x 106 cells/well in 24-well plates for different time periods, harvested, washed twice in PBS, and stained with anti-CD4 FITC and propidium iodide (PI), both from BD Pharmingen.
For in vitro studies, PS-1145 was dissolved in DMSO at 10 mM and subsequently diluted in complete CM to the final concentration. For in vivo studies, a 0,5% methylcellulose/0,2% Tween 80 solution was used as vehicle for PS-1145.
Statistics
ANOVA and the post hoc Tukey-Kramer test were used to compare multiple group means and Student’s t test was applied when comparing two group means. A p value of <0.05 was assumed to indicate significance.
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Results |
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IKK2-deficient T cells proliferate as well as wt T cells in response to polyclonal stimulation (anti-CD3, Con A) (6). However, recall responses to the protein Ags keyhole limpet hemocyanin and OVA were shown to be impaired (8). We tested the ability of IKK2-deficient T cells to proliferate in response to autoantigen. We used MOG35–55 in our experiments because this peptide has been mapped as the immunodominant epitope in T cell responses against the CNS-specific MOG protein in C57BL/6 mice (H-2b) (18).
Ex vivo-isolated IKK2-deficient T cells did not proliferate in response to MOG35–55 (up to 100 µg of MOG35–55 per ml) 10 days after in vivo immunization, and no IL2 and IFN- production could be detected in culture supernatants (Fig. 1A). Furthermore, the numbers of IL-17-producing cells were greatly reduced after 48 h of restimulation with MOG35–55 as demonstrated by ELISPOT (Fig. 1, B and C). However, we detected some low level proliferation and IFN- production in IKK2-deficient T cells in one experiment after restimulation with 200 µg of MOG35–55 per ml (data not shown).
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Next, we tested the effect of T cell-specific ablation of IKK2 in a T cell-dependent autoimmune disease, MOG35–55-induced EAE in C57BL/6 mice. In two independent experiments, we did not find any clinical signs of EAE in IKK2T cell mice, whereas wt littermates developed severe EAE (Fig. 2). It has been shown that not only is the development of clinical EAE dependent on trafficking of cells into the CNS, but additionally reactivation of migrating cells is required to establish disease (19) To investigate whether autoantigen-specific IKK2-deficient T cells still may be able to establish subclinical inflammation in the CNS, we examined brain sections of immunized animals for signs of histological disease. We did not find any parenchymal inflammation in MOG35–55-immunized IKK2T cell mice, only one animal showed mild inflammation in the meninges (Table I).
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C57BL/6 mice transgenic for a MOG35–55 peptide-specific TCR (2D2) have been described recently (15). T cells of 2D2 transgenic mice are >90% MOG35–55 specific. These cells proliferate extensively and secrete large amounts of IFN- in response to MOG35–55 stimulation. Furthermore, 2D2 mice develop optic neuritis and, to a lesser extent, spontaneous EAE (15). To examine the peptide-specific T cell responses in IKK2-deficient cells in more detail and independent of immunizations with peptide/CFA, we generated 2D2-transgenic IKK2T cell mice as described in Materials and Methods.
We found a significant, dose-dependent proliferation of SPC derived from 2D2tg-IKK2T cell mice in response to MOG35–55 (Fig. 3A). T cells from nontransgenic wt littermates did not proliferate. When we measured cytokine production in culture supernatants we found a dichotomous pattern: Although 2D2tg-IKK2-deficient T cells produced only very low amounts of IL-2 that could not be further enhanced by high-dose peptide stimulation, there was comparable IFN- production in T cells from 2D2tg-IKK2T cell and control mice (Fig. 3, B and C).
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T cell mice
Because we found a substantial peptide-specific response in 2D2tg-IKK2-deficient T cells we tested these mice for their susceptibility to EAE. We immunized 2D2tg-IKK2T cell mice with MOG35–55 and two doses of pertussis toxin. This immunization regimen has been shown to induce severe EAE in 2D2tg mice (15). In contrast to the IKK2T cell littermates, 2D2tg-IKK2T cell mice developed severe EAE upon immunization comparable with that of C57BL/6 wt and 2D2tg mice (Fig. 4A). There was a slight delay in the onset of disease in wt littermates and 2D2tg-IKK2T cell mice compared with 2D2tg mice (Table I). However, 2D2tg mice and 2D2tg-IKK2T cell mice did not differ in their maximal disease scores. As seen in the previous experiments, IKK2T cell mice were resistant to disease induction.
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T cell mice had the highest numbers of inflammatory lesions in the CNS, although this difference, as compared with 2D2-transgenic mice and wt littermates, was not significant (Table I, Expt. 3). Again, the numbers of lesions in the CNS of immunized IKK2T cell mice were low, and lesions were found predominantly in the meninges. To exclude the possibility that the disease observed in 2D2tg-IKK2T cell mice was due to the presence of IKK2-expressing 2D2tg T cells that somehow escaped Cre-mediated recombination, we FACS purified peripheral T cells of these animals after induction of disease and assessed the recombination status of the Ikk2 alleles by Southern blot. Peripheral T cells isolated from 2D2tg-IKK2T cell mice, similar to T cells from their IKK2T cell littermates, were devoid functional Ikk2 alleles (Fig. 4B), demonstrating that 2D2tg-IKK2-deficient T cells are responsible for EAE-induction. Thus, our data show that the introduction of a pathogenic MOG35–55-specific TCR into IKK2T cell mice renders these mice highly susceptible to induction of EAE. Pharmacological IKK inhibition of T cell activation in vitro
Given the reduced IL-2 secretion and proliferation in 2D2tg-IKK2-deficient T cells in response to MOG35–55, we investigated whether we could inhibit the activation of autoantigen-specific T cells using an IKK2-inhibitory compound in vitro. For these experiments, we used the recently described IKK2-specific inhibitor PS-1145 (20). PS-1145 inhibits IKK2 in a specific and dose-dependent manner in a multiple myeloma cell line (20). We demonstrated inhibition of MOG35–55-induced proliferation of spleen cells (SPC) from 2D2tg mice using PS-1145 in a dose-dependent manner in vitro. Additionally, the extent of inhibition was also dependent on the amount of peptide used for the stimulation (Fig. 5A). Low concentrations of PS-1145 (1 µM) showed some inhibition in cells stimulated with intermediate amounts of MOG35–55 (5 µg/ml) but no effect at a high dose of the peptide (50 µg/ml). High concentrations (10 µM) of PS-1145 inhibited proliferation of 2D2tg cells independent of the concentration of MOG35–55. Similarly, PS-1145 also inhibited the secretion of cytokines (both IL-2 and IFN-) from the same SPC cultures (Fig. 5A). PS-1145 also inhibited IFN- production, whereas 2D2tg-IKK2-deficient SPC secreted IFN- in response to MOG35–55 comparable with SPC derived from 2D2tg mice.
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Effects of PS-1145 in vivo on MOG35–55-induced EAE
We next tested the effects of administration of PS-1145 during induction and effector phase of EAE in C57BL/6 mice. For investigation of the effects of IKK2 inhibition during the induction phase and early effector phase of EAE, we administered 1 mg of PS-1145 or vehicle alone orally daily, beginning 1 day before immunization. No major effects on the disease course were observed when PS-1145 was given until day 11 p.i. (data not shown); however, treatment with PS-1145 until day 21 p.i. resulted in overall disease reduction in the PS-1145-treated group compared with the vehicle-only treated group (average disease score, p = 0.01; Fig. 6A and Table II). We observed a delay in the onset of disease in the PS-1145-treated group; however, this difference did not reach statistical significance (p = 0.08; Table II).
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To test whether PS-1145 acts via modulation of peripheral T cell responses, mice were immunized with MOG35–55 and subsequently treated with PS-1145 or vehicle orally daily and in vitro responses of lymph node cells (LNC) against MOG35–55 were measured at day 10 p.i. We found almost complete suppression of IL-17 responses in LNC cultures from mice treated with 1 mg of PS-1145 per day (Fig. 6C). Also, proliferative responses (Fig. 6D) as well as IL-2 and IFN- responses (data not shown) were suppressed. Similar findings were obtained when mice were treated with 0.5 mg of PS-1145/day (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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T cell mice are completely resistant to MOG35–55-induced EAE; 2) expression of a MOG-specific TCR renders 2D2tg-IKK2T cell mice susceptible to MOG35–55-induced EAE and SPCs from these mice produce normal amounts of IFN- but not IL-2 in response to MOG35–55; and 3) pharmacological inhibition of IKK2 leads to reduced autoantigen-specific T cell responses in vitro and in vivo but has only transient effects in vivo on MOG35–55-induced EAE in C57BL/6 mice.
NF-B has been shown to be highly activated at sites of inflammation in autoimmune diseases such as rheumatoid arthritis, inflammatory bowel disease, multiple sclerosis, psoriasis, asthma, and chronic inflammatory demyelinating polyneuropathy (2). In animal models of autoimmune diseases, notably rheumatoid arthritis, but also colitis, or allergen-induced asthma, enhanced NF-B activity has been detected (2). IKK2 is an essential part of the IB kinase complex that mediates activation of NF-B via the canonical pathway. Although IKK2-deficient T cells are able to respond normally to polyclonal stimulation, there is a defect in Ag-specific responses and T cell-dependent B cell responses, indicating a defect in T cell priming (6, 8).
We evaluated T cell recall responses to the MOG35–55 self-Ag in LNC cultures from immunized mice and found severely impaired T cell responses in the cultures derived from IKK2T cell mice compared with control mice. Specifically, upon stimulation with MOG35–55, we observed a near absence of proliferative responses, of IL-2 and IFN- production and of IL-17-producing Th17 cells in MOG35–55-stimulated cultures from immunized IKK2T cell mice, whereas vigorous responses could be detected in cultures from immunized control mice.
Our disease induction data show that the residual MOG35–55-specific T cell responses that we could detect in response to very high doses of Ag are not sufficient to induce EAE in IKK2T cell mice. Impaired T cell-mediated B cell help cannot contribute to the absence of disease in IKK2T cell mice, because the EAE induction protocol used here is strictly T cell dependent (21, 22).
The importance of single Rel/NF-B proteins for the inducibility of EAE has been demonstrated previously. However, c-Rel or NF-B1-deficient mice still developed EAE with clinical incidences of 16 and 37.5%, respectively (9, 10). The effect of c-Rel deficiency has been further examined using bone marrow chimeras, and it was shown that c-Rel deficiency in bone marrow cells was essential and sufficient for EAE resistance (10). However, it remains possible that c-Rel deficiency in dendritic cells may contribute to EAE resistance.
In contrast, in our model exclusively T cells lacked IKK2, and only 1 of 19 IKK2T cell mice showed mild, transient clinical EAE (score 0.5 for a maximum time period of 24 h) after immunization with MOG35–55. The modulation of NF-B activity caused by the absence of IKK2 specifically in T cells is therefore more effective in preventing EAE than the absence of single NF-B proteins in all tissues.
Interestingly, we could reconstitute T cell responsiveness toward MOG35–55 in IKK2-deficient T cells by transgenic expression of a pathogenic MOG peptide-specific TCR (2D2 TCR). IKK2-deficient 2D2tg T cells proliferated less and produced reduced amounts of IL-2, but in contrast similar amounts of IFN-, when compared with control T cells. Therefore, IKK2 deficiency impairs expansion of T cells, and the reduced IL-2 production likely contributes to this defect. When the defect in T cell expansion was overcome by expression of a MOG35–55-specific transgenic TCR, 2D2tg-IKK2T cell mice readily developed disease after immunization, reflecting the IKK2-independent production of IFN-.
In our EAE experiments, we found a tendency for an even higher lesion load in 2D2 IKK2T cell mice than in 2D2tg mice. These findings might be explained by lower numbers of TR cells in IKK2T cell mice (6, 7). Once the numbers of pathogenic T cells is sufficient to induce disease, the lack of TR cells in those animals might lead to an even higher degree of inflammation.
Targeting of the NF-B pathway is discussed as an option for treatment of inflammatory diseases and cancer. Different agents currently in use as anti-inflammatory and immunosuppressive therapeutics, such as glucocorticoids and nonsteroidal anti-inflammatory drugs, have been shown to directly or indirectly interfere with the NF-B pathway (23, 24). Manipulations of the NF-B pathway have previously been shown to be beneficial in animal models of rheumatoid arthritis and inflammatory bowel disease (2). Administration of PDTC, a potent antioxidant and inhibitor of NF-B, which is under intensive investigation as a potential anticancer agent, partly prevents myelin basic protein-induced EAE in Lewis rats (11). However, PDTC most probably does not selectively inhibit NF-B activation but likely interferes in general with the ubiquitinin-proteasome pathway (25). Furthermore, functional and morphological alterations of peripheral nerves have been observed after 15-day low-dose treatment with PDTC in rats (26), which might render this agent not suitable for clinical purposes.
A promising approach for NF-B targeting is to modulate pathways leading to NF-B activation rather than nonselectively blocking NF-B (24). Blocking interaction between NEMO and IKK components using peptides mimicking the NEMO-binding domain of IKK proteins reduces the incidence of adoptively transferred myelin basic protein-induced EAE in the SJL mouse and, even administered after onset of the disease, ameliorates the disease course. In contrast to the findings in adoptively transferred EAE, application of these peptides did not prevent actively induced EAE, but led to reduced mean peak EAE scores (12).
We demonstrate impaired autoantigen-driven expansion of IKK2-deficient T cells, although IKK2 is not essential for T cell development and survival. Moreover, lack of IKK2 in T cells leads to complete inhibition of EAE. In addition, lack of IKK2 or NEMO in CNS cells also has a protective effect during EAE-induction in mice and has no influence on brain development in the mouse (13). Taken together, selective targeting of IKK2 seems to be a promising therapeutic option in EAE in that it controls both autoreactive T cells and the response of the target tissue.
In our experiments, we used the -carbolin derivative PS-1145, which inhibits IKK2- and NEMO-dependent canonical NF-B activation but does not interfere with p100 processing of the alternative pathway (27), in vivo and in vitro (20, 28). We were able to suppress autoantigen-dependent T cell proliferation and cytokine secretion by PS-1145 in a dose-dependent manner in vitro, and we show that this suppression was not due to PS-1145 toxicity. The extent of the observed suppression of T cell proliferation and cytokine production is unexpected, given that IKK2-deficient 2D2 transgenic T cells proliferate and produce IFN- in response to stimulation with MOG35–55. One possible explanation of this discrepancy is that PS-1145-inhibited IKK2 remains part of the IKK complex as a dominant negative protein. This possibility is supported by our previous demonstration that replacement of endogenous IKK2 by a kinase-inactive IKK2 compromises T cell development much more substantially than ablation of IKK2 (6). This suggested that in IKK2-deficient T cells, but not in T cells expressing kinase-inactive IKK2, NEMO-IKK1 complexes are formed, which display IKK activity and are able to induce residual NF-B activity.
In a previous report, a single dose of PS-1145 (50 mg/kg body weight) resulted in a 60% decrease of serum TNF- levels after in vivo LPS challenge (28). We used the same route and a similar dose of PS-1145 in our EAE experiments for a time period of up to 23 days. Administration of PS-1145 during the induction and early effector phase of EAE resulted in an ameliorated disease course in the treated mice compared with control mice. However, when PS-1145 was given after onset of EAE, the disease course was not significantly altered. In vivo administration of PS-1145 led to almost completely suppressed ex vivo responses in draining lymph node T cells 10 days after immunization with MOG35–55, indicating that oral administration of a NF-B inhibitor can efficiently interfere with the activation of autoreactive T cells in vivo. In contrast, the fact that the PS-1145 therapy regimen used by us cannot completely suppress EAE development shows that we did not achieve a complete systemic in vivo inhibition of T cell activation and that residual T cell responses are sufficient to induce clinical disease at later time points in our EAE experiments. In addition, PS-1145 might not cross the blood-brain barrier, or the amounts of PS-1145 that are able to penetrate into the brain are insufficient to block ongoing disease.
In summary, taken together with the observation that EAE is suppressed in absence of IKK2 in neuroectodermal-derived CNS cells (13), our results strongly suggest IKK2 as a target for therapeutic interventions in inflammatory CNS disease. Further work is required to examine the influence of selective IKK2 inhibition in vivo in EAE models reflecting other pathomechanisms (e.g., B cell-dependent EAE) and using IKK2-inhibitory compounds with different pharmacokinetics.
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Acknowledgment |
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Disclosures |
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Footnotes |
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1 This study was supported by grants from the Deutsche Forschungsgemeinschaft GR1925/1-1 and 2-1 (to B.G.) and National Institutes of Health Grant AI057947 (to K.R.). R.W. holds Heisenberg Fellowship We 1947/4-2 from the Deutsche Forschungsgemeinschaft. 
2 Current address: MedImmune, One MedImmune Way, Gaithersburg, MD 20878.
3 Address correspondence and reprint requests to Dr. Marc Schmidt-Supprian, The CBR Institute for Biomedical Research, 200 Longwood Avenue, Boston, MA 02115. E-mail address: supprian{at}cbr.med.harvard.edu
4 Abbreviations used in this paper: IKK, IB kinase; NEMO, NF-B essential modulator; wt, wild type; TR, T regulatory; MOG, myelin-oligodendrocyte-glycoprotein; EAE, experimental autoimmune encephalomyelitis; PDTC, pyrrolidine thiocarbamate; CM, cell medium; SPC, spleen cells; PI, propidium iodide; LNC, lymph node cell.
Received for publication July 10, 2006. Accepted for publication April 16, 2007.
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References |
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