The type I interferon-alpha mediates a more severe neurological disease in the absence of the canonical signaling molecule interferon regulatory factor 9

Abstract

Type I interferons (IFN) are crucial in host defense but also are implicated as causative factors for neurological disease. Interferon regulatory factor (IRF9) is involved in type I IFN-regulated gene expression where it associates with STAT1:STAT2 heterodimers to form the transcriptional complex ISGF3. The role of IRF9 in cellular responses to type I IFN is poorly defined in vivo and hence was examined here. While transgenic mice (termed GIFN) with chronic production of low levels of IFN-alpha in the CNS were relatively unaffected, the same animals lacking IRF9 [GIFNxIRF9 knock-out (KO)] had cataracts, became moribund, and died prematurely. The brain of GIFNxIRF9 KO mice showed calcification with pronounced inflammation and neurodegeneration whereas inflammation and retinal degeneration affected the eyes. In addition, IFN-gamma-like gene expression in the CNS in association with IFN-gamma mRNA and increased phosphotyrosine-STAT1 suggested a role for IFN-gamma. However, GIFNxIRF9 KO mice deficient for IFN-gamma signaling developed an even more severe and accelerated disease, indicating that IFN-gamma was protective. In IRF9-deficient cultured mixed glial cells, IFN-alpha induced prolonged activation of STAT1 and STAT2 and induced the expression of IFN-gamma-like genes. We conclude that (1) type I IFN signaling and cellular responses can occur in vivo in the absence of IRF9, (2) IRF9 protects against the pathophysiological actions of type I IFN in the CNS, and (3) STAT1 and possibly STAT2 participate in alternative IRF9-independent signaling pathways activated by IFN-alpha in glial cells resulting in enhanced IFN-gamma-like responses.

Figure 1.

Survival of GIFN mice is reduced in the absence of STAT1 or IRF9. Survival was significantly longer in GIFNxIRF9 KO mice compared with GIFNxSTAT1 KO mice (p < 0.0001, χ² logrank test). While IFNGR-null GIFNxIRF9 KO mice were not viable (*), IFNGR-haploinsufficiency resulted in a significantly shorter survival of these animals compared with GIFNxIRF9 KO mice (p < 0.0001, χ² logrank test).

Figure 2.

GIFN mice deficient for IRF9 develop ocular disease. A–C, In contrast to GIFN mice, whose eyes appeared normal (A), the eyes of GIFNxIRF9 KO mice showed cataracts (B). Histological examination of the eyes of GIFNxIRF9 KO mice showed destruction of the retinal architecture (C, arrows) and increased cellularity in the vitreous body (C, arrowhead). Infiltrating CD4⁺ (D, arrows) and CD8⁺ T cells (E, arrows) were present in the retina and the vitreous body, and in the lens dysplastic bladder-like fibers were present (F, arrow). No overt pathological changes were observed in the eyes of GIFN mice (G), whereas the eyes of GIFNxSTAT1 KO mice showed increased cellularity of the vitreous body (H, arrowheads) and few infiltrating cells in the retinal layers (H, arrows). C, G, H, Original magnification 50×; D, E, 400×; F, 1000×.

Figure 3.

Marked pathological changes in the CNS of GIFNxSTAT1 KO and GIFNxIRF9 KO mice. A, B, The medial–ventral forebrain of 12-week-old GIFN mice showed no obvious pathological changes. C, D, In contrast, the medial–ventral forebrain of similarly aged GIFNxIRF9 KO mice showed areas of calcifying necrosis (C, arrow) with loss of neurons and leukocyte infiltrates that accumulated around blood vessels (D) but also diffusely infiltrated the parenchyma. In addition to lymphocytes (D, white arrowheads), monocytes, and macrophages/microglia, some neutrophils (D, inset, arrow) were present. Endothelial cells in the GIFNxIRF9 KO mice appeared hypertrophic and nuclei were increased and broadened in size (D, black arrowhead). E, F, Areas of calcifying necrosis (E, arrow) with neuronal loss and significant numbers of infiltrating leukocytes and microglia/macrophages were also present in the medial–ventral forebrain of GIFNxSTAT1 KO mice. Lymphocytes (F, white arrowheads) and neutrophils (F, inset, arrow) were seen and endothelial cells of blood vessels were activated (F, black arrowheads). A, C, E, Original magnification 400×; B, D, F, original magnification 750×.

Figure 4.

Leukocytes in the CNS of GIFNxSTAT1 KO and GIFNxIRF9 KO mice consisted of T cells, macrophages/monocytes, and neutrophils. A–E, The medial–ventral forebrain of 12-week-old GIFN mice showed no reactive astrocytosis (A) or infiltrating CD4⁺ (B) or CD8⁺ T cells (C), neutrophils (D), or macrophages (E). F–I, Pronounced reactive astrocytosis (F) was present in the medial–ventral forebrain of GIFNxIRF9 KO mice, and leukocyte infiltrates consisted of CD4⁺ (G) and CD8⁺ (H) T cells and some neutrophils (I). J, Pronounced microglia/macrophage accumulation was evident. K–O, In GIFNxSTAT1 KO mice reactive astrocytosis (K) was similar to GIFNxIRF9 KO mice. Significant numbers of infiltrating CD4⁺ (L) and CD8⁺ (M) T cells, neutrophils (N), and microglia/macrophage accumulation (O) were observed in the medial–ventral forebrain of GIFNxSTAT1 KO mice. Original magnification 400×.

Figure 5.

Deficiency of IRF9 in GIFN mice resulted in IFN-γ-like gene expression and increased pY-STAT1 and pY-STAT2 levels. A, RPAs with RNA from the medial–ventral forebrain showed increased expression of type I IFN-regulated genes and transcription factors in GIFN mice. In contrast to this, GIFNxIRF9 KO mice showed pronounced expression of genes associated with an IFN-γ-like response as well as of proinflammatory cytokines. Increased mRNA levels for proinflammatory cytokines were present in the CNS of GIFNxSTAT1 KO mice. GIFNxIRF9 KOxIFNGR HT mice also showed increased expression of genes characteristic of an IFN-γ-like response. B, Quantification of the autoradiographs was performed by densitometry. Values were normalized to the housekeeping gene L32 and shown as mean ± SEM. C, Immunoblot analysis of protein lysates from the medial–ventral forebrain showed increased levels for pS-STAT1 and STAT1 but not pY-STAT1 in GIFN mice compared with WT mice. In contrast, pS-STAT1, pY-STAT1, and pY-STAT2, as well as total STAT1 and STAT2, were increased in the CNS of GIFNxIRF9 KO and GIFNxIRF9 KOxIFNGR HT mice. D, Bar graphs showing quantification of immunoblot results for phosphorylated and total STAT1 and STAT2. For each genotype, three independent samples were analyzed. Protein levels were normalized to GAPDH levels and means ± SEM are shown. *p < 0.05, **p < 0.01, ***p < 0.001 compared with GIFN mice by one-way ANOVA.

Figure 6.

IFN-α induces prolonged nuclear accumulation of pY-STAT1 and pY-STAT2 in IRF9-deficient MGCs. Immunoblot analysis of nuclear protein lysates from untreated and IFN-α-treated MGCs shows a transient accumulation of pY-STAT1 and pY-STAT2 in WT cells and prolonged accumulation in IRF9-deficient cells.

Figure 7.

IFN-α induces IFN-γ-like gene expression in IRF9-deficient MGCs. A, RPA analysis shows a delayed expression of genes that are characteristic of an IFN-γ-like response in IRF9-deficient cells compared with WT cells. B, Quantification of the autoradiographs was performed by densitometry. Values were normalized to the housekeeping gene L32 shown as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 compared with respective untreated controls by one-way ANOVA.