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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

NMO IgG induces expression of inflammatory and reactive astrocyte genes in mouse astroglia. a–f Gene expression was assessed by microarray analysis of astroglia after 24 h of stimulation with 100 μg/mL NMO IgG (NMO) or control IgG (CON). Changes in expression were calculated by comparison to untreated cultures. a A heatmap reveals robust up- and downregulation of numerous genes only in cells stimulated with NMO IgG. Of 22640 genes detected on the microarray, 3628 differed between NMO and CON IgG stimulation at p < 0.05. Fold changes for these genes are mapped on a log2 scale, with values downregulated to <−0.5-fold in green and values upregulated to >+0.5 shown in red. Note that because only significantly changed genes are mapped, there is a discontinuity between the upregulated and downregulated genes. b A subset of chemokine and cytokine genes are shown on a log2 scale, with downregulation <−2-fold in green and upregulation >+2-fold shown in red. White represents zerofold change relative to untreated samples. c A subset of genes encoding canonical NFκB-dependent factors are shown on a log2 scale, with downregulation <−2-fold in green and upregulation >+2-fold shown in red. d A subset of NFκB-dependent stress response genes sorted by gene name on log2 scale, with downregulation <−2-fold in green and upregulation >+2-fold shown in red. White represents zerofold change relative to untreated samples. e A published reactive astrocyte transcriptional response pattern (“reactive”) [18] was compared to the changes induced by astroglial stimulation with NMO IgG or CON IgG. These factors were mapped on a log2 scale with <0-fold change shown in white and >+5-fold induction shown in red. f Published data reporting the astrocyte transcriptional response to LPS, middle cerebral artery occlusion (“MCAO”), or PBS [18] were compared to our data for NMO IgG or CON IgG stimulation. The heatmap shows all genes detected on our array; genes with fold change values between −0.26 and +0.26 on a log2 scale following NMO IgG stimulation are excluded from the figure (discontinuity in the NMO lane). Genes downregulated <−2-fold are in green, unchanged genes are black, and genes upregulated >+2-fold are shown in red. A hierarchical cluster analysis showing Euclidean distance and average linkage score was performed in Gitools. The published data used for these comparisons were accessed via the GEO database at NCBI. g The NFκB canonical pathway was identified as a top response pathway (p = 4.14E-07) using the Ingenuity Pathway Analysis package. Top upstream regulators in this pathway were identified as Stat1 (z = 5.530), MyD88 (z = 5.603), Ripk2 (z = 2.486), and IRF3 (z = 2.804). Likewise, IFNγ (z = 9.203), IFNβ1 (z = 2.412), CSF2 (z = 2.789), and TNFα (z = 2.121) were identified as top response factors possibly involved in NFκB activation following NMO IgG stimulation. The microarray data were generated in two separate experiments performed with triplicate samples; the purified IgG used for these two experiments were derived from separate patient serum pools (Additional file 1: Table S1). The initial inclusion criteria for detection on the microarray were based on Illumina Beadchip significance calls. Genes exhibiting significant differences between NMO IgG- and CON IgG-stimulated samples were identified using Storey’s positive false-discovery rate for multiple hypothesis testing
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Fig2: NMO IgG induces expression of inflammatory and reactive astrocyte genes in mouse astroglia. a–f Gene expression was assessed by microarray analysis of astroglia after 24 h of stimulation with 100 μg/mL NMO IgG (NMO) or control IgG (CON). Changes in expression were calculated by comparison to untreated cultures. a A heatmap reveals robust up- and downregulation of numerous genes only in cells stimulated with NMO IgG. Of 22640 genes detected on the microarray, 3628 differed between NMO and CON IgG stimulation at p < 0.05. Fold changes for these genes are mapped on a log2 scale, with values downregulated to <−0.5-fold in green and values upregulated to >+0.5 shown in red. Note that because only significantly changed genes are mapped, there is a discontinuity between the upregulated and downregulated genes. b A subset of chemokine and cytokine genes are shown on a log2 scale, with downregulation <−2-fold in green and upregulation >+2-fold shown in red. White represents zerofold change relative to untreated samples. c A subset of genes encoding canonical NFκB-dependent factors are shown on a log2 scale, with downregulation <−2-fold in green and upregulation >+2-fold shown in red. d A subset of NFκB-dependent stress response genes sorted by gene name on log2 scale, with downregulation <−2-fold in green and upregulation >+2-fold shown in red. White represents zerofold change relative to untreated samples. e A published reactive astrocyte transcriptional response pattern (“reactive”) [18] was compared to the changes induced by astroglial stimulation with NMO IgG or CON IgG. These factors were mapped on a log2 scale with <0-fold change shown in white and >+5-fold induction shown in red. f Published data reporting the astrocyte transcriptional response to LPS, middle cerebral artery occlusion (“MCAO”), or PBS [18] were compared to our data for NMO IgG or CON IgG stimulation. The heatmap shows all genes detected on our array; genes with fold change values between −0.26 and +0.26 on a log2 scale following NMO IgG stimulation are excluded from the figure (discontinuity in the NMO lane). Genes downregulated <−2-fold are in green, unchanged genes are black, and genes upregulated >+2-fold are shown in red. A hierarchical cluster analysis showing Euclidean distance and average linkage score was performed in Gitools. The published data used for these comparisons were accessed via the GEO database at NCBI. g The NFκB canonical pathway was identified as a top response pathway (p = 4.14E-07) using the Ingenuity Pathway Analysis package. Top upstream regulators in this pathway were identified as Stat1 (z = 5.530), MyD88 (z = 5.603), Ripk2 (z = 2.486), and IRF3 (z = 2.804). Likewise, IFNγ (z = 9.203), IFNβ1 (z = 2.412), CSF2 (z = 2.789), and TNFα (z = 2.121) were identified as top response factors possibly involved in NFκB activation following NMO IgG stimulation. The microarray data were generated in two separate experiments performed with triplicate samples; the purified IgG used for these two experiments were derived from separate patient serum pools (Additional file 1: Table S1). The initial inclusion criteria for detection on the microarray were based on Illumina Beadchip significance calls. Genes exhibiting significant differences between NMO IgG- and CON IgG-stimulated samples were identified using Storey’s positive false-discovery rate for multiple hypothesis testing

Mentions: The transcriptional response to astroglial stimulation with NMO IgG (NMO) for 24 h was measured using an Illumina mouse WG-6 v2.0 Beadchip (Fig. 2). Compared to cells stimulated with IgG isolated from healthy controls (CON), 3628 genes of the 22,640 genes detected on the array were significantly altered by NMO IgG (Fig. 2a), suggesting a strong transcriptional response to stimulation. A key response included the upregulation of numerous C-C and C-X-C motif chemokine genes, including CCL2, 3, 4, 5, 6, 7, and 9 and CXCL1, 2, 4, 8, 10, 12, and 16 (Fig. 2b). Consistent with our previous findings [7], CCL5 was upregulated 60-fold in astrocytes stimulated with NMO IgG relative to CON IgG. Other significantly upregulated genes of interest were cytokines such as IL-1α, IL-1β, IL-6, and TNFα, suggesting the induction of a broad inflammatory program in astrocytes stimulated with NMO IgG. Genes for several B cell factors, such as B cell activating factor (BAFF), a proliferation inducting ligand (APRIL), and glucocorticoid-induced tumor necrosis factor receptor-related ligand (GITRL) were also upregulated following stimulation with NMO IgG, suggesting a potential interaction between IgG-stimulated astrocytes and localized support for B cell function within the CNS. A large number of canonical NFκB-dependent and NFκB-associated transcription factors (Fig. 2c) and stress response genes (Fig. 2d) were also upregulated in astrocytes stimulated with NMO IgG. Interestingly, the transcript for RELB, the chief transcription factor associated with the alternative NFκB signaling pathway, was strongly upregulated, suggesting that multiple NFκB pathways may be involved in the glial response to NMO IgG over time. Of the stress genes induced by NMO IgG, lipocalin 2 (Lcn2) and ceruloplasmin (Cp) are canonical reactive astrocyte response factors that were strongly upregulated. Indeed, Lcn2 was increased 40-fold in astrocytes stimulated with NMO IgG relative to CON IgG. The induction of a reactive program is further supported by comparison of the NMO IgG-induced response to previously published microarray data from Barres and colleagues [18] characterizing the astrocyte reactome (Fig. 2e). Of note, NMO IgG stimulation induced only a subset of the reactive genes induced by lipopolysaccharide (LPS), suggesting that the NMO-specific response shares some downstream signaling events with LPS-induced reactivity, but does not utilize the same upstream initiators. This conclusion is further supported by comparison of the entire transcriptional response pattern elicited in our system by NMO IgG or CON IgG to the published response induced by LPS, middle cerebral artery occlusion (MCAO), or a phosphate-buffered saline (PBS) control (Fig. 2f) [18]. While NMO IgG stimulation clearly induced a subset of the genes that are also induced by LPS or MCAO, a unique pattern of activation exists in response to the autoantibody. Hierarchical cluster analysis confirmed that LPS and MCAO induced reactive responses that are more closely related to each other than to the NMO IgG-induced response, but that the NMO IgG-induced response is unique from the controls (CON and PBS) (Fig. 2f). Finally, Ingenuity Pathway Analysis [19] revealed that NFκB signaling was a top canonical pathway engaged by stimulation with NMO IgG (Fig. 2g), highlighting the role of this pathway in the observed inflammatory and stress response. We conclude that stimulation of astroglial cultures with NMO IgG induces a distinctive reactive, inflammatory, pro-granulocytic response.Fig. 2


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)

NMO IgG induces expression of inflammatory and reactive astrocyte genes in mouse astroglia. a–f Gene expression was assessed by microarray analysis of astroglia after 24 h of stimulation with 100 μg/mL NMO IgG (NMO) or control IgG (CON). Changes in expression were calculated by comparison to untreated cultures. a A heatmap reveals robust up- and downregulation of numerous genes only in cells stimulated with NMO IgG. Of 22640 genes detected on the microarray, 3628 differed between NMO and CON IgG stimulation at p < 0.05. Fold changes for these genes are mapped on a log2 scale, with values downregulated to <−0.5-fold in green and values upregulated to >+0.5 shown in red. Note that because only significantly changed genes are mapped, there is a discontinuity between the upregulated and downregulated genes. b A subset of chemokine and cytokine genes are shown on a log2 scale, with downregulation <−2-fold in green and upregulation >+2-fold shown in red. White represents zerofold change relative to untreated samples. c A subset of genes encoding canonical NFκB-dependent factors are shown on a log2 scale, with downregulation <−2-fold in green and upregulation >+2-fold shown in red. d A subset of NFκB-dependent stress response genes sorted by gene name on log2 scale, with downregulation <−2-fold in green and upregulation >+2-fold shown in red. White represents zerofold change relative to untreated samples. e A published reactive astrocyte transcriptional response pattern (“reactive”) [18] was compared to the changes induced by astroglial stimulation with NMO IgG or CON IgG. These factors were mapped on a log2 scale with <0-fold change shown in white and >+5-fold induction shown in red. f Published data reporting the astrocyte transcriptional response to LPS, middle cerebral artery occlusion (“MCAO”), or PBS [18] were compared to our data for NMO IgG or CON IgG stimulation. The heatmap shows all genes detected on our array; genes with fold change values between −0.26 and +0.26 on a log2 scale following NMO IgG stimulation are excluded from the figure (discontinuity in the NMO lane). Genes downregulated <−2-fold are in green, unchanged genes are black, and genes upregulated >+2-fold are shown in red. A hierarchical cluster analysis showing Euclidean distance and average linkage score was performed in Gitools. The published data used for these comparisons were accessed via the GEO database at NCBI. g The NFκB canonical pathway was identified as a top response pathway (p = 4.14E-07) using the Ingenuity Pathway Analysis package. Top upstream regulators in this pathway were identified as Stat1 (z = 5.530), MyD88 (z = 5.603), Ripk2 (z = 2.486), and IRF3 (z = 2.804). Likewise, IFNγ (z = 9.203), IFNβ1 (z = 2.412), CSF2 (z = 2.789), and TNFα (z = 2.121) were identified as top response factors possibly involved in NFκB activation following NMO IgG stimulation. The microarray data were generated in two separate experiments performed with triplicate samples; the purified IgG used for these two experiments were derived from separate patient serum pools (Additional file 1: Table S1). The initial inclusion criteria for detection on the microarray were based on Illumina Beadchip significance calls. Genes exhibiting significant differences between NMO IgG- and CON IgG-stimulated samples were identified using Storey’s positive false-discovery rate for multiple hypothesis testing
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Fig2: NMO IgG induces expression of inflammatory and reactive astrocyte genes in mouse astroglia. a–f Gene expression was assessed by microarray analysis of astroglia after 24 h of stimulation with 100 μg/mL NMO IgG (NMO) or control IgG (CON). Changes in expression were calculated by comparison to untreated cultures. a A heatmap reveals robust up- and downregulation of numerous genes only in cells stimulated with NMO IgG. Of 22640 genes detected on the microarray, 3628 differed between NMO and CON IgG stimulation at p < 0.05. Fold changes for these genes are mapped on a log2 scale, with values downregulated to <−0.5-fold in green and values upregulated to >+0.5 shown in red. Note that because only significantly changed genes are mapped, there is a discontinuity between the upregulated and downregulated genes. b A subset of chemokine and cytokine genes are shown on a log2 scale, with downregulation <−2-fold in green and upregulation >+2-fold shown in red. White represents zerofold change relative to untreated samples. c A subset of genes encoding canonical NFκB-dependent factors are shown on a log2 scale, with downregulation <−2-fold in green and upregulation >+2-fold shown in red. d A subset of NFκB-dependent stress response genes sorted by gene name on log2 scale, with downregulation <−2-fold in green and upregulation >+2-fold shown in red. White represents zerofold change relative to untreated samples. e A published reactive astrocyte transcriptional response pattern (“reactive”) [18] was compared to the changes induced by astroglial stimulation with NMO IgG or CON IgG. These factors were mapped on a log2 scale with <0-fold change shown in white and >+5-fold induction shown in red. f Published data reporting the astrocyte transcriptional response to LPS, middle cerebral artery occlusion (“MCAO”), or PBS [18] were compared to our data for NMO IgG or CON IgG stimulation. The heatmap shows all genes detected on our array; genes with fold change values between −0.26 and +0.26 on a log2 scale following NMO IgG stimulation are excluded from the figure (discontinuity in the NMO lane). Genes downregulated <−2-fold are in green, unchanged genes are black, and genes upregulated >+2-fold are shown in red. A hierarchical cluster analysis showing Euclidean distance and average linkage score was performed in Gitools. The published data used for these comparisons were accessed via the GEO database at NCBI. g The NFκB canonical pathway was identified as a top response pathway (p = 4.14E-07) using the Ingenuity Pathway Analysis package. Top upstream regulators in this pathway were identified as Stat1 (z = 5.530), MyD88 (z = 5.603), Ripk2 (z = 2.486), and IRF3 (z = 2.804). Likewise, IFNγ (z = 9.203), IFNβ1 (z = 2.412), CSF2 (z = 2.789), and TNFα (z = 2.121) were identified as top response factors possibly involved in NFκB activation following NMO IgG stimulation. The microarray data were generated in two separate experiments performed with triplicate samples; the purified IgG used for these two experiments were derived from separate patient serum pools (Additional file 1: Table S1). The initial inclusion criteria for detection on the microarray were based on Illumina Beadchip significance calls. Genes exhibiting significant differences between NMO IgG- and CON IgG-stimulated samples were identified using Storey’s positive false-discovery rate for multiple hypothesis testing
Mentions: The transcriptional response to astroglial stimulation with NMO IgG (NMO) for 24 h was measured using an Illumina mouse WG-6 v2.0 Beadchip (Fig. 2). Compared to cells stimulated with IgG isolated from healthy controls (CON), 3628 genes of the 22,640 genes detected on the array were significantly altered by NMO IgG (Fig. 2a), suggesting a strong transcriptional response to stimulation. A key response included the upregulation of numerous C-C and C-X-C motif chemokine genes, including CCL2, 3, 4, 5, 6, 7, and 9 and CXCL1, 2, 4, 8, 10, 12, and 16 (Fig. 2b). Consistent with our previous findings [7], CCL5 was upregulated 60-fold in astrocytes stimulated with NMO IgG relative to CON IgG. Other significantly upregulated genes of interest were cytokines such as IL-1α, IL-1β, IL-6, and TNFα, suggesting the induction of a broad inflammatory program in astrocytes stimulated with NMO IgG. Genes for several B cell factors, such as B cell activating factor (BAFF), a proliferation inducting ligand (APRIL), and glucocorticoid-induced tumor necrosis factor receptor-related ligand (GITRL) were also upregulated following stimulation with NMO IgG, suggesting a potential interaction between IgG-stimulated astrocytes and localized support for B cell function within the CNS. A large number of canonical NFκB-dependent and NFκB-associated transcription factors (Fig. 2c) and stress response genes (Fig. 2d) were also upregulated in astrocytes stimulated with NMO IgG. Interestingly, the transcript for RELB, the chief transcription factor associated with the alternative NFκB signaling pathway, was strongly upregulated, suggesting that multiple NFκB pathways may be involved in the glial response to NMO IgG over time. Of the stress genes induced by NMO IgG, lipocalin 2 (Lcn2) and ceruloplasmin (Cp) are canonical reactive astrocyte response factors that were strongly upregulated. Indeed, Lcn2 was increased 40-fold in astrocytes stimulated with NMO IgG relative to CON IgG. The induction of a reactive program is further supported by comparison of the NMO IgG-induced response to previously published microarray data from Barres and colleagues [18] characterizing the astrocyte reactome (Fig. 2e). Of note, NMO IgG stimulation induced only a subset of the reactive genes induced by lipopolysaccharide (LPS), suggesting that the NMO-specific response shares some downstream signaling events with LPS-induced reactivity, but does not utilize the same upstream initiators. This conclusion is further supported by comparison of the entire transcriptional response pattern elicited in our system by NMO IgG or CON IgG to the published response induced by LPS, middle cerebral artery occlusion (MCAO), or a phosphate-buffered saline (PBS) control (Fig. 2f) [18]. While NMO IgG stimulation clearly induced a subset of the genes that are also induced by LPS or MCAO, a unique pattern of activation exists in response to the autoantibody. Hierarchical cluster analysis confirmed that LPS and MCAO induced reactive responses that are more closely related to each other than to the NMO IgG-induced response, but that the NMO IgG-induced response is unique from the controls (CON and PBS) (Fig. 2f). Finally, Ingenuity Pathway Analysis [19] revealed that NFκB signaling was a top canonical pathway engaged by stimulation with NMO IgG (Fig. 2g), highlighting the role of this pathway in the observed inflammatory and stress response. We conclude that stimulation of astroglial cultures with NMO IgG induces a distinctive reactive, inflammatory, pro-granulocytic response.Fig. 2

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