Article Figures & Data
Figures
- Figure 1 Glatiramer acetate induces time-dependent phosphorylation of Akt in monocytes
(A) Monocytes were stimulated for 30 or 60 minutes with 50 μg/mL glatiramer acetate (GA) or forskolin (FK), a labdane diterpene, which is an agonist of the enzyme adenylyl cyclase, and serves as a positive control for induction of intracellular cyclic adenosine 3′,5′-monophosphate (cAMPi). Monocytes were lysed and cAMPi was determined by enzyme immunoassay. Values are expressed as the mean ± SD (n = 3). Data are representative of 3 independent experiments. (B, C) Monocytes were stimulated with 50 μg/mL GA for the indicated time periods prior to cell lysis. (B) Cell lysates were then subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis and immunoblotted with phospho-specific antibodies to serine-473 of Akt1 (phospho-Akt1 [Ser473]). The same blot was stripped and probed for total Akt. Relative ratio represents the fold induction of Akt1 (Ser473) phosphorylation by GA treatment. (C) Endogenous levels of phospho-Akt1 (Ser473) protein were detected by a solid phase sandwich ELISA. The magnitude of absorbance is proportional to the quantity of phospho-Akt1 (Ser473) protein. *p < 0.05, **p < 0.01, ***p < 0.001 as determined by Student t test. Results shown are representative of 2 separate experiments.
- Figure 2 Glatiramer acetate treatment induced M2 differentiation through a MyD88-independent pathway
(A) As described previously,3 M2 monocytes were treated in the presence or absence of glatiramer acetate (GA) for 6 days. They were then stimulated with lipopolysaccharide (LPS), Poly(I:C), or Pam3CSK4 for 24 hours. (B) Wild-type (WT) monocytes cultured in the presence or absence of GA were stimulated with LPS (100 ng/mL) for the indicated duration. Cell lysate proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and membranes were probed for phosphorylated IκBα (Ser32/36). Data are representative of 2 separate experiments. (C) Human peripheral blood monocytes were preincubated for 1 hour with or without 50 μg/mL GA and then cultured for 24 hours in the presence or absence of Poly(I:C) (10 μg/mL) or Pam3CSK4 (100 ng/mL). Tumor necrosis factor (TNF) (left panels) and interleukin (IL)–6 (right panels) secretion was quantitated in cell supernatants by ELISA. Results are presented as mean ± SD (n = 3); **p < 0.01, ***p < 0.001 by Student t test. Data presented are representative of 3 independent experiments. MyD88 = myeloid differentiation primary response gene 88; TRIF = Toll-IL-1 receptor domain–containing adaptor inducing interferon-β.
- Figure 3 TRIF deficiency abrogates the immunomodulatory effects of glatiramer acetate treatment on cytokines and experimental autoimmune encephalomyelitis
Wild-type (WT) and Toll-IL-1 receptor domain–containing adaptor inducing interferon-β (TRIF)–deficient mice treated with glatiramer acetate (GA) or vehicle (n = 5 mice/group) were injected IP with (A) lipopolysaccharide (LPS) (100 ng/kg) or (B) Pam3CSK4 (100 ng/kg). (C) Myeloid differentiation primary response gene 88 (MyD88)–deficient mice were injected IP with Poly(I:C) (10 μg/kg). Serum levels of tumor necrosis factor (TNF) and interleukin (IL)–6 were quantitated by ELISA 5 hours after injection. Results are presented as the mean ± SEM (n = 3) of 2 experiments that provided similar results; *p < 0.05, **p < 0.01, ***p < 0.001 as determined by Student t test. (D) On day 0, C57BL/6J WT (left) or TRIF-deficient mice (right) mice were immunized with MOG peptide (p35-55; 50 μg). GA (250 μg) was administered once (SC in incomplete Freund's adjuvant [IFA]) on the same day as immunization (day 0). Control mice received a single SC injection of IFA. For all experimental autoimmune encephalomyelitis experiments, mean disease score ±SEM is shown. *p < 0.05 as determined by Mann-Whitney U test. Results shown are representative of 3 independent experiments.
- Figure 4 Glatiramer acetate treatment negatively regulates IFN-β production by targeting components of the IFN-β enhanceosome
(A) Wild-type (WT) monocytes differentiated in the presence or absence of glatiramer acetate (GA) were stimulated with lipopolysaccharide (LPS) (100 ng/mL) for 24 hours. Interferon (IFN)–β secretion was quantitated in cell culture supernatants by ELISA. Results are representative of 2 experiments. Data are presented as mean ± SEM; *p < 0.05, **p < 0.01 as determined by Student t test. (B) WT mice (n = 3/group) treated with GA or vehicle were injected IP with LPS (100 ng/kg). Serum levels of IFN-β were quantitated by ELISA, 5 hours following injection. Data are representative of 2 separate experiments. (C) Interferon-α/β receptor subunit-1 (IFNAR1)–deficient monocytes differentiated in the presence or absence of GA were stimulated with LPS at the indicated dose for 24 hours. Tumor necrosis factor (TNF) and interleukin-6 secretion was quantitated in cell supernatants by ELISA. Results are representative of 3 independent experiments (n = 3/group). (D) Monocytes generated in the presence or absence of GA were stimulated with LPS (100 ng/mL) or Poly(I:C) (10 μg/mL) for the indicated duration. IRF3 binding activity in nuclear extracts was measured with TransAM IRF3. (E) Monocytes generated in the presence or absence of GA were stimulated with LPS (100 ng/mL) for the indicated duration. Cell lysate proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and membranes were probed for phosphorylated SAPK/JNK (Thr183/Tyr185) and total GAPDH. Data are representative of 3 separate experiments. (F) Monocytes differentiated in the presence or absence of GA and stimulated with 100 ng/mL LPS for the indicated duration. DNA binding of ATF-2 was quantitated from nuclear extracts. (G) Monocytes generated in the presence or absence of GA were stimulated with LPS (100 ng/mL) for the indicated duration, and whole-cell lysates were subjected to SDS-PAGE and Western blot analysis for phosphorylated p38 MAPK (Thr180/Tyr182) and total STAT1. Data are representative of 3 separate experiments.
- Figure 5 TLR-dependent signaling pathways inhibited by glatiramer acetate
Myeloid differentiation primary response gene 88 (MyD88) and Toll-IL-1 receptor domain–containing adaptor inducing interferon-β (TRIF) are 2 major mediators of Toll-like receptor (TLR) signaling that cooperate in promoting innate immune responses. Engagement of TLR2 by Pam3CSK4 or lipoteichoic acid (LTA) initiates the MyD88-dependent pathway resulting in the transcription of proinflammatory cytokines via the induction (black lines) of nuclear factor (NF)–κB. Stimulation of TLR3 by Poly(I:C) induces the recruitment of TRIF, which in turn triggers expression of interferon (IFN)–β by activating interferon regulatory factor 3 (IRF3), a substrate of c-Jun N-terminal kinase 1 (JNK1).15 Engagement of TLR4 by lipopolysaccharide (LPS) triggers both MyD88-dependent and TRIF-dependent signaling. Both TLR3 and TLR4, but not TLR2, initiate an IFN-β-positive feedback loop that amplifies the initial response through the IFNAR–signal transducers and activators of transcription 1 (STAT1) signaling axis. The differentiation of M2 monocytes occurred independently of MyD88 and phosphorylation and degradation of the NF-κB inhibitor, IκBα. In contrast, TRIF-dependent TLR signaling was negatively regulated in M2 monocytes. Phosphorylation of JNK1 and p38 MAPK and subsequent nuclear translocation (DNA binding) of IRF3 and ATF-2, 2 components of the IFN-β enhanceosome, were inhibited. GA-mediated M2 monocyte differentiation is associated with inhibition of STAT1 phosphorylation.3 Copyright Xavier Studio, reprinted with permission.
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