SNX8 mediates IFNγ-triggered noncanonical signaling pathway and host defense against Listeria monocytogenes Jin Weia,b, Wei Guoa,b, Huan Liana,b, Qing Yanga,b, Heng Lina,b, Shu Lia,b,1, and Hong-Bing Shua,b,1 aMedical Research Institute, Wuhan University, Wuhan, China, 430071; and bCollege of Life Sciences, Wuhan University, Wuhan, China, 430071 Edited by George R. Stark, Lerner Research Institute, The Cleveland Clinic Foundation, Cleveland, OH, and approved November 2, 2017 (received for review July 29, 2017) IFNγ is a cytokine that plays a key role in host defense against the activation of these genes in response to IFNγ. The mechanisms intracellular pathogens. In addition to the canonical JAK-STAT1 and biological functions of this IFNγ-triggered noncanonical sig- pathway, IFNγ also activates an IKKβ-mediated noncanonical sig- naling pathway remain enigmatic. naling pathway that is essential for induction of a subset of down- Sorting nexin 8 (SNX8) belongs to the sorting nexin protein stream effector genes. The molecular mechanisms and functional family, which is involved in endocytosis, endosomal sorting, and significance of this IFNγ-triggered noncanonical pathway remains signaling (19). It has been shown that SNX8 is a β-amyloid (Aβ) enigmatic. Here, we identified sorting nexin 8 (SNX8) as an impor- toxicity enhancer and associated with Alzheimer’s disease (20, tantcomponentoftheIFNγ-triggered noncanonical signaling 21). A few of sorting nexin family proteins form the retromer pathway. SNX8-deficiency impaired IFNγ-triggered induction of a complex with VPS26-VPS29-VPS35 heterotrimer that has been − − subset of downstream genes. Snx8 / mice infected with Listeria implicated in membrane recruitment and formation of recycling monocytogenes exhibited lower serum cytokine levels and higher tubules (22, 23). In addition, SNXs play an important role in bacterial loads in the livers and spleens, resulting in higher lethal- modulating the degradation of receptors through endocytic ity. Mechanistically, SNX8 interacted with JAK1 and IKKβ and promoted pathways (19, 24). Whether and how SNX8 is involved in cellular their association. IFNγ induced JAK1-mediated phosphorylation of signaling events are unclear. SNX8 at Tyr95 and Tyr126, which promoted the recruitment of IKKβ In this study, we identified SNX8 as a key adaptor in the IFNγ- to the JAK1 complex. SNX8-deficiency impaired IFNγ-induced oligomer- triggered noncanonical signaling pathway. SNX8 deficiency im- β ization and autophosphorylation of IKK at Ser177, which is critical for paired expression of a subset of downstream genes induced by − − selective induction of downstream genes. Our findings suggest that IFNγ. Snx8 / mice were more susceptible to lethal infection by γ SNX8 acts as a link for IFN -triggered noncanonical signaling pathway, Listeria. SNX8 interacted with JAK1 and IKKβ and selectively which induces a subset of downstream genes important for host de- promoted IKKβ dimerization/oligomerization and autophos- fense against L. monocytogenes infection. phorylation. Moreover, JAK1-mediated phosphorylation of SNX8 at Tyr95 and Tyr126 was required for its recruitment of IKKβ interferon | SNX8 | noncanonical | IKK | phosphorylation to JAK1. These findings reveal molecular mechanisms of IFNγ-triggered and SNX8-mediated noncanonical signaling path- FNγ is a cytokine that plays pivotal roles in host defense to way that is important for host defense to microbial infection. Imicrobial infection. IFNγ activates macrophages and other cell types, leading to production of various cytokines, phagosomal Significance maturation, autophagy, and bactericidal activity (1). Individuals γ with partial or complete defects in the IFN signaling pathways IFNγ is a cytokine that induces various downstream genes for have increased susceptibility to Listeria monocytogenes as well as host defense against pathogens. How IFNγ induces down- other bacterial species (2). γ stream genes via the classical JAK1-STAT1 pathway is well The canonical IFN -triggered signaling pathway is character- understood, but how IFNγ induces a subset of downstream ized by JAK-mediated phosphorylation of STAT1 (3). The β γ genes via the kinase IKK remains enigmatic. We identified binding of IFN homodimer to IFNGR1 and IFNGR2 results in SNX8 protein as an important component of this IFNγ-triggered spatial proximity of JAK1 and JAK2, leading to phosphorylation noncanonical pathway. SNX8 deficiency impairs IFNγ-triggered of IFNGR1 and JAKs. The phosphorylation of IFNGR1 at induction of a subset of downstream genes, rendering the Tyr440 by JAKs provides a docking site for the recruitment of mouse more susceptible to infection of intracellular bacteria STAT1, and the phosphorylated JAKs subsequently phosphory- Listeria monocytogenes. Mechanistically, IFNγ induces phos- late STAT1 at Tyr701. Phosphorylated STAT1 forms homo- phorylation of SNX8, which promotes the recruitment of IKKβ dimers, which translocate to the nucleus and bind to the β γ to the JAK1 complex and subsequent activation of IKK . Our conserved IFN -activated sites (GASs) on the promoters of the findings suggest that SNX8 acts as a link for IFNγ-triggered IFN-stimulated genes (ISGs) to initiate the transcription of these noncanonical signaling pathway and host defense against in- downstream target genes (4, 5). In addition to the phosphory- tracellular bacteria infection. lation of Tyr701 of STAT1, IFNγ induces phosphorylation of STAT1 at Ser727, which is mediated by CDK8, PI3K, and the Author contributions: J.W., S.L., and H.-B.S. designed research; J.W., W.G., H. Lian, Q.Y., downstream protein kinase C family members PKC-δ and PKC-e, and H. Lin performed research; J.W., Q.Y., S.L., and H.-B.S. analyzed data; S.L. and H.-B.S. and this phosphorylation is critical for the full activation of supervised research; and J.W. and H.-B.S. wrote the paper. STAT1 (6–10). In addition to the well-known IFNγ-JAK-STAT1 The authors declare no conflict of interest. canonical pathway, IFNγ activates additional signal pathways This article is a PNAS Direct Submission. β (11, 12). IKK , a master activator of inflammatory response, is Published under the PNAS license. required to mediate the transcriptional induction of a subset of 1To whom correspondence may be addressed. Email: [email protected] or shuh@whu. IFNγ-stimulated genes, such as guanylate binding proteins (GBPs) edu.cn. – and several chemokines (CXCL9, CXCL10, and CXCL11) (1, 13 18). This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. The phosphorylation of STAT1 is necessary but not sufficient for 1073/pnas.1713462114/-/DCSupplemental. 13000–13005 | PNAS | December 5, 2017 | vol. 114 | no. 49 www.pnas.org/cgi/doi/10.1073/pnas.1713462114 Downloaded by guest on September 24, 2021 Results SNX8 Positively Regulates IFNγ-Triggered Signaling. It has been demonstrated that IFNγ stimulation induces the expression of IFN-regulatory factor 1 (IRF1) gene whose promoter contains two conserved STAT1-binding sites (25). To identify additional molecules involved in IFNγ-triggered signaling, we screened ∼10,000 individual human cDNA clones for their ability to ac- tivate the IRF1 promoter by reporter assays in 293 cells. These efforts led to the identification of SNX8, which activated the IRF1 promoter and dramatically potentiated IFNγ-triggered activation of the IRF1 promoter (Fig. 1A). Further experiments indicated that SNX8 potentiated IFNγ-triggered activation of the IRF1 promoter in a dose-dependent manner (Fig. 1A). We next examined the role of endogenous SNX8 in regulation of IFNγ-triggered signaling. We made two SNX8 RNAi con- structs that could markedly inhibit the expression of endogenous SNX8 (Fig. 1B). Knockdown of SNX8 inhibited IFNγ-triggered activation of the IRF1 promoter (Fig. 1B). In addition, knock- down of SNX8 inhibited transcription of a subset of IFNγ- induced downstream genes in THP1 cells, such as GBP1, CXCL9, and CXCL10 (Fig. 1C). In these experiments, knockdown of SNX8 moderately inhibited transcription of IRF1 at an early time point (1 h) but not later time points after IFNγ stimulation, whereas knockdownofSNX8hadnomarkedeffectsontranscriptionof other examined IFNγ-inducible genes, including SOCS1 and STAT1 (Fig. 1C). We then confirmed these observations using SNX8- knockout HeLa. Consistently, SNX8 deficiency inhibited IFNγ- induced transcription of GPB1, CXCL9, CXCL10,andIRF1 but not SOCS1 or STAT1 genes (Fig. 1D). Moreover, SNX8 deficiency reduced IFNγ-triggered phosphorylation of STAT1 at Tyr701 but not Ser727 (Fig. 1E). These data suggest that SNX8 is essential for IFNγ-induced transcription of a subset of downstream genes. SNX8 Is Essential for IFNγ-Triggered Signaling in Murine Cells. HumanSNX8consistsof465aminoacid(aa)residuesand Fig. 1. Identification of SNX8 as a positive regulator of IFNγ-triggered sig- shares 88.9% sequence identity with its murine ortholog. To in- γ naling. (A) Expression screens for clones that activate the IRF1 promoter. The vestigate the functions of SNX8 in IFN -triggered signaling, we 293 cells were transfected with the IRF1 reporter plasmids and ∼10,000 in- generated SNX8-deficient mice (Fig. S1A). Immunoblot analysis dividual cDNA clones (Left histograph shows a few representative clones) or −/− confirmed that SNX8 was undetectable in Snx8 mouse lung increased SNX8 plasmid (Right histograph) for 24 h. Cells were left untreated fibroblasts (MLFs) and mouse embryonic fibroblasts (MEFs) (Fig. or treated with IFNγ for 12 h before