Limits...
NFκB signaling drives pro-granulocytic astroglial responses to neuromyelitis optica patient IgG.

Walker-Caulfield ME, Guo Y, Johnson RK, McCarthy CB, Fitz-Gibbon PD, Lucchinetti CF, Howe CL - J Neuroinflammation (2015)

Bottom Line: Astrocytes expressing the aquaporin-4 water channel are a primary target of pathogenic, disease-specific immunoglobulins (IgG) found in patients with neuromyelitis optica (NMO).This signaling resulted in the release of pro-granulocytic chemokines and was inhibited by the clinically relevant proteasome inhibitors bortezomib and PR-957.We propose that the astrocytic NFκB-dependent inflammatory response to stimulation by NMO IgG represents one of the earliest events in NMO pathogenesis, providing a target for therapeutic intervention upstream of irreversible cell death and tissue damage.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurology, Mayo Clinic, 200 First St. SW, Rochester, MN, 55905, USA.

ABSTRACT

Background: Astrocytes expressing the aquaporin-4 water channel are a primary target of pathogenic, disease-specific immunoglobulins (IgG) found in patients with neuromyelitis optica (NMO). Immunopathological analyses of active NMO lesions highlight a unique inflammatory phenotype marked by infiltration of granulocytes. Previous studies characterized this granulocytic infiltrate as a response to vasculocentric complement activation and localized tissue destruction. In contrast, we observe that granulocytic infiltration in NMO lesions occurs independently of complement-mediated tissue destruction or active demyelination. These immunopathological findings led to the hypothesis that NMO IgG stimulates astrocyte signaling that is responsible for granulocytic recruitment in NMO.

Methods: Histopathology was performed on archival formalin-fixed paraffin-embedded autopsy-derived CNS tissue from 23 patients clinically and pathologically diagnosed with NMO or NMO spectrum disorder. Primary murine astroglial cultures were stimulated with IgG isolated from NMO patients or control IgG from healthy donors. Transcriptional responses were assessed by microarray, and translational responses were measured by ELISA. Signaling through the NFκB pathway was measured by western blotting and immunostaining.

Results: Stimulation of primary murine astroglial cultures with NMO IgG elicited a reactive and inflammatory transcriptional response that involved signaling through the canonical NFκB pathway. This signaling resulted in the release of pro-granulocytic chemokines and was inhibited by the clinically relevant proteasome inhibitors bortezomib and PR-957.

Conclusions: We propose that the astrocytic NFκB-dependent inflammatory response to stimulation by NMO IgG represents one of the earliest events in NMO pathogenesis, providing a target for therapeutic intervention upstream of irreversible cell death and tissue damage.

No MeSH data available.


Related in: MedlinePlus

The proteasome inhibitor bortezomib and the immunoproteasome inhibitor PR-957 effectively inhibit NFκB-dependent pro-granulocytic responses induced by NMO IgG. a Lysates (30 μg per lane) from unstimulated cells (UNT), following stimulation for 60 min with 100 μg/mL CON IgG (CON) or NMO IgG (NMO), or following stimulation with NMO IgG after pretreatment with bortezomib (BRT 1 μM or 2.5 μM) or PR-957 (PR 1 μM or 10 μM) were probed by immunoblot with antibodies for either pIκB-α or IκB-α. As expected, pIκB-α accumulated in BRT- or PR-treated cells due to impaired proteasome and immunoproteasome function. Blot is representative of 2 independent experiments. b Nuclear translocation of NFκB p65 (green) in GFAP-labeled cells (red) was assessed after stimulation for 60 min with 100 μg/mL NMO IgG alone or following 60-min stimulation with NMO IgG after pretreatment for 2 h with either bortezomib (BRT; 1 μM) or PR-957 (PR; 1 μM). Scale bar: 50 μM. p65 translocation was robustly blocked by both inhibitors. Panel is representative of 2 independent experiments performed in duplicate. c The fold inhibition of CCL5, CCL2, CXCL1, and CXCL2 release induced after stimulation of cells for 24 h with 100 μg/mL of NMO IgG in the presence of BRT (1 μM or 2.5 μM) or PR (1 μM or 10 μM) was assessed by ELISA. Both BRT and PR were effective inhibitors of CCL5, CCL2, and CXCL1 responses with little inhibition of CXCL2, as previously observed for MG132. Fold inhibition was calculated as in Fig. 4. Findings are representative of two separate experiments performed in triplicate. Error bars show the 95 % confidence interval. p < 0.001 from two-way ANOVA comparing chemokines and inhibitors used for treatment
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC4590277&req=5

Fig5: The proteasome inhibitor bortezomib and the immunoproteasome inhibitor PR-957 effectively inhibit NFκB-dependent pro-granulocytic responses induced by NMO IgG. a Lysates (30 μg per lane) from unstimulated cells (UNT), following stimulation for 60 min with 100 μg/mL CON IgG (CON) or NMO IgG (NMO), or following stimulation with NMO IgG after pretreatment with bortezomib (BRT 1 μM or 2.5 μM) or PR-957 (PR 1 μM or 10 μM) were probed by immunoblot with antibodies for either pIκB-α or IκB-α. As expected, pIκB-α accumulated in BRT- or PR-treated cells due to impaired proteasome and immunoproteasome function. Blot is representative of 2 independent experiments. b Nuclear translocation of NFκB p65 (green) in GFAP-labeled cells (red) was assessed after stimulation for 60 min with 100 μg/mL NMO IgG alone or following 60-min stimulation with NMO IgG after pretreatment for 2 h with either bortezomib (BRT; 1 μM) or PR-957 (PR; 1 μM). Scale bar: 50 μM. p65 translocation was robustly blocked by both inhibitors. Panel is representative of 2 independent experiments performed in duplicate. c The fold inhibition of CCL5, CCL2, CXCL1, and CXCL2 release induced after stimulation of cells for 24 h with 100 μg/mL of NMO IgG in the presence of BRT (1 μM or 2.5 μM) or PR (1 μM or 10 μM) was assessed by ELISA. Both BRT and PR were effective inhibitors of CCL5, CCL2, and CXCL1 responses with little inhibition of CXCL2, as previously observed for MG132. Fold inhibition was calculated as in Fig. 4. Findings are representative of two separate experiments performed in triplicate. Error bars show the 95 % confidence interval. p < 0.001 from two-way ANOVA comparing chemokines and inhibitors used for treatment

Mentions: Although MG132, a constitutive proteasome inhibitor, was the least specific NFκB inhibitor that we examined, it showed effective suppression of NMO IgG-induced NFκB signaling and concomitant chemokine release. Considering the successful use of proteasome inhibitors in other diseases, we examined the possible therapeutic relevance of proteasome inhibition in NMO. We tested bortezomib, a dipeptide boronate, approved for treatment of multiple myeloma [20], and the highly selective immunoproteasome inhibitor PR-957, a tripeptide epoxyketone, shown to be efficacious in mouse models of rheumatoid arthritis [21] and SLE [22]. As with MG132, treatment with either bortezomib or PR-957 before stimulation with NMO IgG resulted in the cellular accumulation of pIκB-α (Fig. 5a) due to inhibition of proteasome-mediated degradation. Critically, however, treatment with bortezomib or PR-957 suppressed the NMO IgG-induced nuclear accumulation of NFκB (Fig. 5b) and robustly inhibited the release of CCL2, CCL5, and CXCL1 (Fig. 5c). Indeed, the suppression of CCL5 release by PR-957 approached 99 % inhibition. As with the other NFκB inhibitors, neither bortezomib nor PR-957 inhibited CXCL2 production. These data suggest that inhibition of either the proteasome or immunoproteasome has profound effects on astroglial responses to NMO IgG, leading us to conclude that such inhibition may serve as a therapeutically relevant strategy for suppressing early pathogenic events in NMO.Fig. 5


NFκB signaling drives pro-granulocytic astroglial responses to neuromyelitis optica patient IgG.

Walker-Caulfield ME, Guo Y, Johnson RK, McCarthy CB, Fitz-Gibbon PD, Lucchinetti CF, Howe CL - J Neuroinflammation (2015)

The proteasome inhibitor bortezomib and the immunoproteasome inhibitor PR-957 effectively inhibit NFκB-dependent pro-granulocytic responses induced by NMO IgG. a Lysates (30 μg per lane) from unstimulated cells (UNT), following stimulation for 60 min with 100 μg/mL CON IgG (CON) or NMO IgG (NMO), or following stimulation with NMO IgG after pretreatment with bortezomib (BRT 1 μM or 2.5 μM) or PR-957 (PR 1 μM or 10 μM) were probed by immunoblot with antibodies for either pIκB-α or IκB-α. As expected, pIκB-α accumulated in BRT- or PR-treated cells due to impaired proteasome and immunoproteasome function. Blot is representative of 2 independent experiments. b Nuclear translocation of NFκB p65 (green) in GFAP-labeled cells (red) was assessed after stimulation for 60 min with 100 μg/mL NMO IgG alone or following 60-min stimulation with NMO IgG after pretreatment for 2 h with either bortezomib (BRT; 1 μM) or PR-957 (PR; 1 μM). Scale bar: 50 μM. p65 translocation was robustly blocked by both inhibitors. Panel is representative of 2 independent experiments performed in duplicate. c The fold inhibition of CCL5, CCL2, CXCL1, and CXCL2 release induced after stimulation of cells for 24 h with 100 μg/mL of NMO IgG in the presence of BRT (1 μM or 2.5 μM) or PR (1 μM or 10 μM) was assessed by ELISA. Both BRT and PR were effective inhibitors of CCL5, CCL2, and CXCL1 responses with little inhibition of CXCL2, as previously observed for MG132. Fold inhibition was calculated as in Fig. 4. Findings are representative of two separate experiments performed in triplicate. Error bars show the 95 % confidence interval. p < 0.001 from two-way ANOVA comparing chemokines and inhibitors used for treatment
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4590277&req=5

Fig5: The proteasome inhibitor bortezomib and the immunoproteasome inhibitor PR-957 effectively inhibit NFκB-dependent pro-granulocytic responses induced by NMO IgG. a Lysates (30 μg per lane) from unstimulated cells (UNT), following stimulation for 60 min with 100 μg/mL CON IgG (CON) or NMO IgG (NMO), or following stimulation with NMO IgG after pretreatment with bortezomib (BRT 1 μM or 2.5 μM) or PR-957 (PR 1 μM or 10 μM) were probed by immunoblot with antibodies for either pIκB-α or IκB-α. As expected, pIκB-α accumulated in BRT- or PR-treated cells due to impaired proteasome and immunoproteasome function. Blot is representative of 2 independent experiments. b Nuclear translocation of NFκB p65 (green) in GFAP-labeled cells (red) was assessed after stimulation for 60 min with 100 μg/mL NMO IgG alone or following 60-min stimulation with NMO IgG after pretreatment for 2 h with either bortezomib (BRT; 1 μM) or PR-957 (PR; 1 μM). Scale bar: 50 μM. p65 translocation was robustly blocked by both inhibitors. Panel is representative of 2 independent experiments performed in duplicate. c The fold inhibition of CCL5, CCL2, CXCL1, and CXCL2 release induced after stimulation of cells for 24 h with 100 μg/mL of NMO IgG in the presence of BRT (1 μM or 2.5 μM) or PR (1 μM or 10 μM) was assessed by ELISA. Both BRT and PR were effective inhibitors of CCL5, CCL2, and CXCL1 responses with little inhibition of CXCL2, as previously observed for MG132. Fold inhibition was calculated as in Fig. 4. Findings are representative of two separate experiments performed in triplicate. Error bars show the 95 % confidence interval. p < 0.001 from two-way ANOVA comparing chemokines and inhibitors used for treatment
Mentions: Although MG132, a constitutive proteasome inhibitor, was the least specific NFκB inhibitor that we examined, it showed effective suppression of NMO IgG-induced NFκB signaling and concomitant chemokine release. Considering the successful use of proteasome inhibitors in other diseases, we examined the possible therapeutic relevance of proteasome inhibition in NMO. We tested bortezomib, a dipeptide boronate, approved for treatment of multiple myeloma [20], and the highly selective immunoproteasome inhibitor PR-957, a tripeptide epoxyketone, shown to be efficacious in mouse models of rheumatoid arthritis [21] and SLE [22]. As with MG132, treatment with either bortezomib or PR-957 before stimulation with NMO IgG resulted in the cellular accumulation of pIκB-α (Fig. 5a) due to inhibition of proteasome-mediated degradation. Critically, however, treatment with bortezomib or PR-957 suppressed the NMO IgG-induced nuclear accumulation of NFκB (Fig. 5b) and robustly inhibited the release of CCL2, CCL5, and CXCL1 (Fig. 5c). Indeed, the suppression of CCL5 release by PR-957 approached 99 % inhibition. As with the other NFκB inhibitors, neither bortezomib nor PR-957 inhibited CXCL2 production. These data suggest that inhibition of either the proteasome or immunoproteasome has profound effects on astroglial responses to NMO IgG, leading us to conclude that such inhibition may serve as a therapeutically relevant strategy for suppressing early pathogenic events in NMO.Fig. 5

Bottom Line: Astrocytes expressing the aquaporin-4 water channel are a primary target of pathogenic, disease-specific immunoglobulins (IgG) found in patients with neuromyelitis optica (NMO).This signaling resulted in the release of pro-granulocytic chemokines and was inhibited by the clinically relevant proteasome inhibitors bortezomib and PR-957.We propose that the astrocytic NFκB-dependent inflammatory response to stimulation by NMO IgG represents one of the earliest events in NMO pathogenesis, providing a target for therapeutic intervention upstream of irreversible cell death and tissue damage.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurology, Mayo Clinic, 200 First St. SW, Rochester, MN, 55905, USA.

ABSTRACT

Background: Astrocytes expressing the aquaporin-4 water channel are a primary target of pathogenic, disease-specific immunoglobulins (IgG) found in patients with neuromyelitis optica (NMO). Immunopathological analyses of active NMO lesions highlight a unique inflammatory phenotype marked by infiltration of granulocytes. Previous studies characterized this granulocytic infiltrate as a response to vasculocentric complement activation and localized tissue destruction. In contrast, we observe that granulocytic infiltration in NMO lesions occurs independently of complement-mediated tissue destruction or active demyelination. These immunopathological findings led to the hypothesis that NMO IgG stimulates astrocyte signaling that is responsible for granulocytic recruitment in NMO.

Methods: Histopathology was performed on archival formalin-fixed paraffin-embedded autopsy-derived CNS tissue from 23 patients clinically and pathologically diagnosed with NMO or NMO spectrum disorder. Primary murine astroglial cultures were stimulated with IgG isolated from NMO patients or control IgG from healthy donors. Transcriptional responses were assessed by microarray, and translational responses were measured by ELISA. Signaling through the NFκB pathway was measured by western blotting and immunostaining.

Results: Stimulation of primary murine astroglial cultures with NMO IgG elicited a reactive and inflammatory transcriptional response that involved signaling through the canonical NFκB pathway. This signaling resulted in the release of pro-granulocytic chemokines and was inhibited by the clinically relevant proteasome inhibitors bortezomib and PR-957.

Conclusions: We propose that the astrocytic NFκB-dependent inflammatory response to stimulation by NMO IgG represents one of the earliest events in NMO pathogenesis, providing a target for therapeutic intervention upstream of irreversible cell death and tissue damage.

No MeSH data available.


Related in: MedlinePlus