Interferon-γ signaling in human iPSC-derived neurons recapitulates neurodevelopmental disorder phenotypes

Abstract

Maternal immune activation increases the risk of neurodevelopmental disorders. Elevated cytokines, such as interferon-γ (IFN-γ), in offspring's brains play a central role. IFN-γ activates an antiviral cellular state, limiting viral entry and replication. Moreover, IFN-γ is implicated in brain development. We tested the hypothesis that IFN-γ signaling contributes to molecular and cellular phenotypes associated with neurodevelopmental disorders. Transient IFN-γ treatment of neural progenitors derived from human induced pluripotent stem cells increased neurite outgrowth. RNA sequencing analysis revealed that major histocompatibility complex class I (MHCI) genes were persistently up-regulated through neuronal differentiation-an effect that was mediated by IFN-γ-induced promyelocytic leukemia protein (PML) nuclear bodies. Critically, IFN-γ-induced neurite outgrowth required both PML and MHCI. We also found evidence that IFN-γ disproportionately altered the expression of genes associated with schizophrenia and autism, suggesting convergence between genetic and environmental risk factors. Together, these data implicate IFN-γ signaling in neurodevelopmental disorder etiology.

Fig. 1. IFN-γ treatment of NPCs leads to enhanced neurite outgrowth in post-mitotic neurons.

(A) Schematic representation of the experimental timeline of iPSC differentiation and IFN-γ treatment strategy. NPCs received IFN-γ (25 ng/ml) daily in cell culture media from D17 to D20 before terminal plating on D21 and examination of neurite outgrowth. (B) Automated tracing of βIII-tubulin–stained neurites on D26, D30, D35, and D40 untreated (UNTR) and IFN-gamma treated cells carried out with CellInsight high content screening operated by HCS Studio Software. (C) Fluorescence images of βIII-tubulin and Hoechst staining acquired with CellInsight. (D to G) Graphs show the time courses of neuronal morphological properties including neurite total length per cell (D), neurite average length per cell (E), branch point count per cell (F), and neurite count per cell (G) in three control male cell lines, M1, M2, and M3. D26 untreated: n = 8 independent biological replicates, 6382 cells analyzed; D26 IFN-γ: n = 8 independent biological replicates, 7122 cells analyzed; D30 untreated: n = 9 independent biological replicates, 5651 cells analyzed; D30 IFN-γ: n = 9 independent biological replicates, 7741 cells analyzed; D35 untreated: n = 7 independent biological replicates, 4250 cells analyzed; D35 IFN-γ: n = 7 independent biological replicates, 4733 cells analyzed; D40 untreated: n = 4 independent biological replicates, 2792 cells analyzed; D40 IFN-γ: n = 4 independent biological replicates, 2872 cells analyzed. Data generated with CellInsight high content screening operated by HCS Studio Software. Results are presented as means ± SEM. Two-way RM ANOVA with Sidak’s multiple comparison adjustment method. *P < 0.05.

Fig. 2. RNA sequencing analysis reveals a widespread and persistent transcriptomic response of human NPCs and neurons to IFN-γ.

(A) Schematic representation of the experimental conditions. (B) Principal components analysis biplot of all samples. The first principal component segregates conditions by time point, while the second separates conditions by recent treatment. Cell lines are represented by point shape: M1 = square, M2 = cross, M3 = circle. PC, principal component. (C) Heatmap of all DEGs clustered by row and column. Replicates are collapsed by condition. (D to G) Volcano plots with selected genes annotated. The dotted red line represents the threshold for statistical significance of P_adj = 0.05. Red dots identify genes of the MHCI protein complex. (H) Cleveland plot of selected enriched GO terms from the up-regulated gene sets illustrating statistical significance and fold enrichment (FE).

Fig. 3. Relevance of IFN-γ-dependent gene expression changes to SZ and ASD.

(A) Enrichment of SZ and ASD risk genes and (B) DEGs detected in the brains of patients with SZ and ASD among our IFN-γ-responding genes. Log₂ odds ratio (OR) is represented by color and statistical significance is indicated by asterisks (*FDR < 0.05, ***FDR < 0.001, and ****FDR < 0.0001). Fisher’s exact test BH corrected for multiple comparisons.

Fig. 4. PML bodies persistently increase following IFN-γ treatment, regulate MHCI gene transcription, and associate spatially with HLA-B transcription.

(A and B) Increased PML nuclear body expression following acute IFN-γ treatment: D18 U, n = 354 cells; D18T, n = 286 cells. Two-tailed Mann-Whitney test. (C and D) PML body expression was persistently increased following IFN-γ treatment: D30 UU, n = 236 cells; D30 TU, n = 264 cells; D30 UT, n = 231 cells; and D30 TT, n = 307 cells. Kruskal-Wallis test with Dunn’s multiple comparison adjustment. (E) Treatment schematic. (F and G) As₂O₃ (As) blocked IFN-γ-induced PML body expression: D18 UNTR, n = 57 cells; D18 IFN-γ, n = 65 cells; D18 As, n = 64 cells; and D18 IFN-γ + As, n = 54 cells. D30 UNTR, n = 104 cells; D30 IFN-γ, n = 129 cells; D30 As, n = 119 cells; and D30 As + IFN-γ, n = 105 cells. Kruskal-Wallis test with Dunn’s multiple comparison test. (H) IFN-γ-induced HLA-B, HLA-C, and B2M expression is blocked by As. One-way ANOVA with Tukey’s multiple comparison test. (I to K) Expression of HLA-B pre-mRNA and PML in IFN-γ-treated NPCs (D18): UNTR, n = 57 cells; IFN-γ, n = 65 cells; As, n = 64 cells; and IFN-γ + As, n = 54 cells. Kruskal-Wallis test with Dunn’s multiple comparison adjustment. (L to N) Colocalization of HLA-B pre-mRNA and PML in D18 and 30 IFN-γ-treated cells: D18, n = 67 cells; D30, n = 58 cells. Kruskal-Wallis test with Dunn’s multiple comparison test. Three control cell lines throughout. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001; ns, not significant.

Fig. 5. MHCI proteins are enriched in neuronal growth cones in a PML-dependent manner, and disruption of PML prevents IFN-γ-dependent neurite outgrowth.

(A and B) iSIM super-resolution images of actin and MHCI in D30 neurons, with MHCI observable in cell bodies, neurites, and growth cones in UNTR, IFN-γ, As, and As + IFN-γ treatment conditions. (C to F) Quantification of MHCI and actin in D30 neurons in neurites and growth cones (GC). UNTR, n = 52 cells; IFN-γ, n = 82 cells; As, n = 55 cells; and As + IFN-γ, n = 59 cells; three control cell lines. (C and E) One-way ANOVA with Tukey’s multiple comparison test. (D and F) RM two-way ANOVA with Sidak’s multiple comparison adjustment. (G) Fluorescence images of βIII-tubulin and Hoechst staining acquired with CellInsight high content screening system operated by HCS Studio Software. (H) Automated tracing of βIII-tubulin–stained neurites in D30 UNTR, IFN-γ, As, and As + IFN-γ conditions carried out with CellInsight high content screening system. (I) Graph showing neurite total length per cell in D30 UNTR, IFN-γ, As, and As + IFN-γ conditions. n = 9 independent biological replicates. UNTR, 5038 cells analyzed; IFN-γ, 5668 cells analyzed; As, 5169 cells analyzed; and As + IFN-γ, 3210 cells analyzed; three control cell lines. RM one-way ANOVA with Tukey’s multiple comparison test. Data generated with CellInsight high content screening operated by HCS Studio Software. Results are presented as means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Fig. 6. Loss of B2M prevents MHCI cell surface expression and IFN-γ-dependent neurite outgrowth.

(A) Confocal images of B2M-, MHCI-, and Hoechst-stained D18 NPCs, UNTR, IFN-γ, IFN-γ + shRNA scrambled, and IFN-γ + shRNA B2M knockdown treatment conditions. (B and C) Quantification of B2M and MHCI in D18 UNTR, IFN-γ, IFN-γ + shRNA scrambled, and IFN-γ + shRNA B2M knockdown. n = 3 independent biological replicates; >10,000 cells per condition; three control cell lines. One-way ANOVA with Tukey’s multiple comparison correction. (D) Confocal images of B2M-, MHCI-, and Hoechst-stained D18 MS3 HLA null UNTR and IFN-γ-treated NPCs. (E and F) Quantification of B2M and MHCI in D18, MS3 HLA null UNTR, and IFN-γ-treated NPCs. MS3 HLA null UNTR: n = 3 biological replicates; >10,000 cells analyzed. MS3 HLA null IFN-γ: n = 3 biological replicates; >10,000 cells analyzed. Paired t test. (G) Fluorescence images of βIII-tubulin– and Hoechst-stained neurons and automated tracing of βIII-tubulin–stained neurites in D30 MS3 HLA null UNTR and IFN-γ neurons. (H) Neurite total length per cell in D30 MS3 HLA null UNTR and IFN-γ neurons. n = 5 independent biological replicates. MS3 HLA null UNTR, >10,000 cells analyzed; MS3 HLA null IFN-γ, >10,000 cells analyzed. Paired t test. All images and data generated using Opera Phenix high content screening system. (I) Schematic depicting our proposed model for IFN-γ-induced PML and MHCI-dependent neurite outgrowth. Results are presented as means ± SEM. **P < 0.01, and ****P < 0.0001.