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Glatiramer acetate treatment negatively regulates type I interferon signaling.

Molnarfi N, Prod'homme T, Schulze-Topphoff U, Spencer CM, Weber MS, Patarroyo JC, Lalive PH, Zamvil SS - Neurol Neuroimmunol Neuroinflamm (2015)

Bottom Line: Furthermore, GA did not provide clinical benefit in TRIF-deficient mice.Consequently, nuclear translocation of ATF-2 and IRF3, components of the IFN-β enhanceosome, was impaired.Consistent with these observations, GA inhibited production of IFN-β in vivo in WT mice, but did not modulate proinflammatory cytokine production by monocytes from IFNAR1-deficient mice.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurology and Program in Immunology (N.M., T.P., U.S.-T., C.M.S., J.C.P., S.S.Z.), University of California, San Francisco; the Institute of Neuropathology and Department of Neurology (M.S.W.), University Medical Center, Georg-August University, Göttingen, Germany; the Department of Pathology and Immunology (P.H.L.), Faculty of Medicine, University of Geneva; and the Department of Neurosciences (P.H.L.), Division of Neurology, University Hospital of Geneva, Switzerland. N.M. is currently affiliated with the Department of Pathology and Immunology, Faculty of Medicine, University of Geneva, and Department of Neurosciences, Division of Neurology, University Hospital of Geneva, Switzerland. T.P. is currently affiliated with Momenta Pharmaceuticals, Cambridge, MA. U.S.-T. is currently affiliated with Silence Therapeutics GmbH, Berlin, Germany. J.C.P. is currently affiliated with Vedanta Biosciences, Inc., Cambridge, MA.

ABSTRACT

Objective: Glatiramer acetate (GA; Copaxone), a disease-modifying therapy for multiple sclerosis (MS), promotes development of anti-inflammatory (M2, type II) monocytes that can direct differentiation of regulatory T cells. We investigated the innate immune signaling pathways that participate in GA-mediated M2 monocyte polarization.

Methods: Monocytes were isolated from myeloid differentiation primary response gene 88 (MyD88)-deficient, Toll-IL-1 receptor domain-containing adaptor inducing interferon (IFN)-β (TRIF)-deficient, IFN-α/β receptor subunit 1 (IFNAR1)-deficient, and wild-type (WT) mice and human peripheral blood. GA-treated monocytes were stimulated with Toll-like receptor ligands, then evaluated for activation of kinases and transcription factors involved in innate immunity, and secretion of proinflammatory cytokines. GA-treated mice were evaluated for cytokine secretion and susceptibility to experimental autoimmune encephalomyelitis.

Results: GA-mediated inhibition of proinflammatory cytokine production by monocytes occurred independently of MyD88 and nuclear factor-κB, but was blocked by TRIF deficiency. Furthermore, GA did not provide clinical benefit in TRIF-deficient mice. GA inhibited activation of p38 mitogen-activated protein kinase, an upstream regulator of activating transcription factor (ATF)-2, and c-Jun N-terminal kinase 1, which regulates IFN regulatory factor 3 (IRF3). Consequently, nuclear translocation of ATF-2 and IRF3, components of the IFN-β enhanceosome, was impaired. Consistent with these observations, GA inhibited production of IFN-β in vivo in WT mice, but did not modulate proinflammatory cytokine production by monocytes from IFNAR1-deficient mice.

Conclusion: Our results demonstrate that GA inhibits the type I IFN pathway in M2 polarization of monocytes independently of MyD88, providing an important mechanism connecting innate and adaptive immune modulation in GA therapy and valuable insight regarding its potential use with other MS treatments.

No MeSH data available.


Related in: MedlinePlus

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.
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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.

Mentions: MyD88 and TRIF represent the 2 essential adapters of innate immune signaling. Previous data demonstrated that GA treatment was associated with reduction of proinflammatory cytokine production and STAT1 phosphorylation in monocytes following stimulation with LPS, a TLR4 agonist.3 All TLRs utilize MyD88 except TLR3, which depends exclusively on TRIF.8 TLR4 signals via both MyD88 and TRIF. Therefore, it was possible that GA could interfere with MyD88 or TRIF. We employed distinct TLR agonists in order to distinguish between these 2 possibilities. First, we observed that GA itself did not alter baseline or ligand-induced expression of TLR2, TLR3, or TLR4 (figure e-1 at Neurology.org/nn), indicating that GA did not influence the capacity to sense TLR agonists. As shown in figure 2A and figure e-2, activation of GA-treated monocytes by the TLR2 ligands, Pam3CSK4 or LTA, did not alter TNF and IL-6 secretion. In contrast, secretion of these proinflammatory cytokines was reduced when GA-treated monocytes were activated by the TLR3 ligand, Poly(I:C), or the TLR4 ligand, LPS. These results therefore suggested that GA inhibited TRIF-dependent signaling and was independent of MyD88. Similar to monocytes from WT mice, monocytes from MyD88-deficient mice exhibited reduced IL-6 and TNF levels following GA treatment (figure 2A). Engagement of MyD88 activates NF-κB. As shown in figure 2B, GA exposure did not impair phosphorylation and degradation of the inhibitor protein IκBα following LPS treatment. GA exposure also did not significantly alter DNA binding of p50 or p65 NF-κB subunits (data not shown). These results suggest that the NF-κB pathway is dispensable for the immunologic effects of GA on monocytes. Further, GA did not reduce proinflammatory cytokine secretion in TRIF-deficient monocytes (figure 2A), providing further support that inhibition of TRIF-dependent signaling is required for GA-mediated reduction of proinflammatory cytokine secretion. As GA treatment of murine monocytes suppressed proinflammatory cytokine production by Poly(I:C), but not Pam3CSK4, we tested how GA-treated human monocytes responded to these 2 TLR ligands. GA treatment of human peripheral blood monocytes decreased TNF and IL-6 secretion induced by Poly(I:C), but not Pam3CSK4 (figure 2C). Thus, as with murine monocytes, these results indicate that GA inhibits proinflammatory cytokine production by human monocytes by blocking TRIF-mediated signaling.


Glatiramer acetate treatment negatively regulates type I interferon signaling.

Molnarfi N, Prod'homme T, Schulze-Topphoff U, Spencer CM, Weber MS, Patarroyo JC, Lalive PH, Zamvil SS - Neurol Neuroimmunol Neuroinflamm (2015)

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.
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Related In: Results  -  Collection

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Show All Figures
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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.
Mentions: MyD88 and TRIF represent the 2 essential adapters of innate immune signaling. Previous data demonstrated that GA treatment was associated with reduction of proinflammatory cytokine production and STAT1 phosphorylation in monocytes following stimulation with LPS, a TLR4 agonist.3 All TLRs utilize MyD88 except TLR3, which depends exclusively on TRIF.8 TLR4 signals via both MyD88 and TRIF. Therefore, it was possible that GA could interfere with MyD88 or TRIF. We employed distinct TLR agonists in order to distinguish between these 2 possibilities. First, we observed that GA itself did not alter baseline or ligand-induced expression of TLR2, TLR3, or TLR4 (figure e-1 at Neurology.org/nn), indicating that GA did not influence the capacity to sense TLR agonists. As shown in figure 2A and figure e-2, activation of GA-treated monocytes by the TLR2 ligands, Pam3CSK4 or LTA, did not alter TNF and IL-6 secretion. In contrast, secretion of these proinflammatory cytokines was reduced when GA-treated monocytes were activated by the TLR3 ligand, Poly(I:C), or the TLR4 ligand, LPS. These results therefore suggested that GA inhibited TRIF-dependent signaling and was independent of MyD88. Similar to monocytes from WT mice, monocytes from MyD88-deficient mice exhibited reduced IL-6 and TNF levels following GA treatment (figure 2A). Engagement of MyD88 activates NF-κB. As shown in figure 2B, GA exposure did not impair phosphorylation and degradation of the inhibitor protein IκBα following LPS treatment. GA exposure also did not significantly alter DNA binding of p50 or p65 NF-κB subunits (data not shown). These results suggest that the NF-κB pathway is dispensable for the immunologic effects of GA on monocytes. Further, GA did not reduce proinflammatory cytokine secretion in TRIF-deficient monocytes (figure 2A), providing further support that inhibition of TRIF-dependent signaling is required for GA-mediated reduction of proinflammatory cytokine secretion. As GA treatment of murine monocytes suppressed proinflammatory cytokine production by Poly(I:C), but not Pam3CSK4, we tested how GA-treated human monocytes responded to these 2 TLR ligands. GA treatment of human peripheral blood monocytes decreased TNF and IL-6 secretion induced by Poly(I:C), but not Pam3CSK4 (figure 2C). Thus, as with murine monocytes, these results indicate that GA inhibits proinflammatory cytokine production by human monocytes by blocking TRIF-mediated signaling.

Bottom Line: Furthermore, GA did not provide clinical benefit in TRIF-deficient mice.Consequently, nuclear translocation of ATF-2 and IRF3, components of the IFN-β enhanceosome, was impaired.Consistent with these observations, GA inhibited production of IFN-β in vivo in WT mice, but did not modulate proinflammatory cytokine production by monocytes from IFNAR1-deficient mice.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurology and Program in Immunology (N.M., T.P., U.S.-T., C.M.S., J.C.P., S.S.Z.), University of California, San Francisco; the Institute of Neuropathology and Department of Neurology (M.S.W.), University Medical Center, Georg-August University, Göttingen, Germany; the Department of Pathology and Immunology (P.H.L.), Faculty of Medicine, University of Geneva; and the Department of Neurosciences (P.H.L.), Division of Neurology, University Hospital of Geneva, Switzerland. N.M. is currently affiliated with the Department of Pathology and Immunology, Faculty of Medicine, University of Geneva, and Department of Neurosciences, Division of Neurology, University Hospital of Geneva, Switzerland. T.P. is currently affiliated with Momenta Pharmaceuticals, Cambridge, MA. U.S.-T. is currently affiliated with Silence Therapeutics GmbH, Berlin, Germany. J.C.P. is currently affiliated with Vedanta Biosciences, Inc., Cambridge, MA.

ABSTRACT

Objective: Glatiramer acetate (GA; Copaxone), a disease-modifying therapy for multiple sclerosis (MS), promotes development of anti-inflammatory (M2, type II) monocytes that can direct differentiation of regulatory T cells. We investigated the innate immune signaling pathways that participate in GA-mediated M2 monocyte polarization.

Methods: Monocytes were isolated from myeloid differentiation primary response gene 88 (MyD88)-deficient, Toll-IL-1 receptor domain-containing adaptor inducing interferon (IFN)-β (TRIF)-deficient, IFN-α/β receptor subunit 1 (IFNAR1)-deficient, and wild-type (WT) mice and human peripheral blood. GA-treated monocytes were stimulated with Toll-like receptor ligands, then evaluated for activation of kinases and transcription factors involved in innate immunity, and secretion of proinflammatory cytokines. GA-treated mice were evaluated for cytokine secretion and susceptibility to experimental autoimmune encephalomyelitis.

Results: GA-mediated inhibition of proinflammatory cytokine production by monocytes occurred independently of MyD88 and nuclear factor-κB, but was blocked by TRIF deficiency. Furthermore, GA did not provide clinical benefit in TRIF-deficient mice. GA inhibited activation of p38 mitogen-activated protein kinase, an upstream regulator of activating transcription factor (ATF)-2, and c-Jun N-terminal kinase 1, which regulates IFN regulatory factor 3 (IRF3). Consequently, nuclear translocation of ATF-2 and IRF3, components of the IFN-β enhanceosome, was impaired. Consistent with these observations, GA inhibited production of IFN-β in vivo in WT mice, but did not modulate proinflammatory cytokine production by monocytes from IFNAR1-deficient mice.

Conclusion: Our results demonstrate that GA inhibits the type I IFN pathway in M2 polarization of monocytes independently of MyD88, providing an important mechanism connecting innate and adaptive immune modulation in GA therapy and valuable insight regarding its potential use with other MS treatments.

No MeSH data available.


Related in: MedlinePlus