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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 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.
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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.

Mentions: IFN-β is a key cytokine induced via the TRIF-dependent pathway that contributes to both innate and adaptive immune responses.4–6 Therefore, we analyzed expression of IFN-β in monocytes treated with GA. As anticipated, we observed a reduction of IFN-β secretion by GA-treated WT monocytes (figure 4A). Serum IFN-β was also decreased in GA-treated mice that were subsequently challenged with LPS (figure 4B).


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 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.
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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.
Mentions: IFN-β is a key cytokine induced via the TRIF-dependent pathway that contributes to both innate and adaptive immune responses.4–6 Therefore, we analyzed expression of IFN-β in monocytes treated with GA. As anticipated, we observed a reduction of IFN-β secretion by GA-treated WT monocytes (figure 4A). Serum IFN-β was also decreased in GA-treated mice that were subsequently challenged with LPS (figure 4B).

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