Life-threatening influenza pneumonitis in a child with inherited IRF9 deficiency

Hernandez, Nicholas; Melki, Isabelle; Jing, Huie; Habib, Tanwir; Huang, Susie S.Y.; Danielson, Jeffrey; Kula, Tomasz; Drutman, Scott; Belkaya, Serkan; Rattina, Vimel; Lorenzo-Diaz, Lazaro; Boulai, Anais; Rose, Yoann; Kitabayashi, Naoki; Rodero, Mathieu P.; Dumaine, Cecile; Blanche, Stéphane; Lebras, Marie-Noëlle; Leung, Man Chun; Mathew, Lisa Sara; Boisson, Bertrand; Zhang, Shen-Ying; Boisson-Dupuis, Stephanie; Giliani, Silvia; Chaussabel, Damien; Notarangelo, Luigi D.; Elledge, Stephen J.; Ciancanelli, Michael J.; Abel, Laurent; Zhang, Qian; Marr, Nico; Crow, Yanick J.; Su, Helen C.; Casanova, Jean-Laurent

Due to the inability of P's cells to form functional ISGF3, we hypothesized that her cells would display a profound inability to up-regulate ISGs following IFN stimulation. In contrast to control cells, P's F-SV40s and B-LCLs did not substantially up-regulate MX1, IFIT1, or OAS1 expression in response to type I IFN, as assessed by quantitative RT-PCR (qRT-PCR) and WB (Fig. 3 , A-D). Stable transfection of P's F-SV40 cells with WT IRF9 was able to rescue the impaired induction of ISGs (Fig. 3 E) . Similarly, U2A cells stably transfected with Δex7 were not able to induce transcription of either classic ISG, compared with WT IRF9 (Fig. 3 F) . CXCL9 expression was used throughout these qRT-PCR experiments to demonstrate the ability of all cells to respond to type II IFN. However, the response to type I IFN is substantially broader than four or five genes, with potentially hundreds of genes displaying altered expression levels (Mostafavi et al., 2016) . To characterize fully the transcriptional responses of P's cells to type I IFN, we performed mRNA sequencing (mRNA-seq) time points. GAP DH was used as a loading control. Representative results of three independent experiments are shown. (D) Transcription levels of MX1, IFIT1, IFIT3, and CXCL9 assessed by qRT-PCR of B-LCL cells treated with 1,000 U/ml of IFN-α2b, -β, or -γ for 2 h. Cells were from three healthy controls (T1, T2, and T3), an IRF9-deficient patient (IRF9 −/− ), her mother (IRF9 +/− ), and STAT1-deficient (STAT1 −/− ), STAT2-deficient (STAT2 −/− ), IRF7-deficient (IRF7 −/− ), and IFN GR2-deficient (IFN GR2 −/− ) patients. Representative results four independent experiments are shown. (E) Transcription levels of MX1, IFIT1, and CXCL9 assessed by qRT-PCR in F-SV40 cells from a healthy control (C1), P's mother (IRF9 +/− ), and P (IRF9 −/− ) stably transfected with luciferase as a control (Luc) or indicated IRF9 variants (WT: WT IRF9, green: reported loss-of-function variants, blue: variants found in-house, red: patient variant). Cells were stimulated with 1,000 U/ml of IFN-α2b, -β, or -γ for 2 or 8 h. Representative results of four independent experiments are shown. (F) Similar to E, qRT-PCR analysis of MX1, IFIT1, and CXCL9 expression in parental HT1080 cells and U2A cells. Cells were stimulated with 1,000 U/ml of IFN-α2b, -β, or -γ for 2 or 8 h. Representative results of three independent experiments are shown. analysis of P's B-LCL cells and primary fibroblasts following in vitro stimulation with IFN-α2b. P's B-LCL cells (Fig. 4, B and D-G) and primary fibroblasts (Fig. 4 , A, C, and E-G) demonstrated an inability to regulate a broad array of genes, including ISGs but also negatively regulated transcripts (data not shown). However, the response in P's cells was not abolished altogether. Indeed, out of all transcripts that passed our filter criteria for being IFN-α2b-regulated in P's B-LCL cells and primary fibroblasts, 118 and 267 transcripts, respectively, were also found to be regulated by IFN in the Interferome database v2.01 (Fig. 4 , C and D). Furthermore, many of the up-regulated ISGs were significantly less induced than those same ISGs were in healthy control cells (Fig. 4, E and F) . Interestingly, however, some ISGs actually were induced at higher levels in IRF9-deficient cells when compared with healthy controls (Fig. 4 , E and F). Furthermore, these difference were not necessarily due to a reduction in basal transcript levels, since, of those genes that are more highl