Threonine Phosphorylation of STAT1 Restricts Interferon Signaling and Promotes Innate Inflammatory Responses

Since its discovery over three decades ago, signal transducer and activator of transcription 1 (STAT1) has been extensively studied as a central mediator for interferons (IFNs) signaling and antiviral defense. Here, using genetic and biochemical assays, we unveil Thr748 as a conserved IFN-independent phosphorylation switch in Stat1, which restricts IFN signaling and promotes innate inflammatory responses following the recognition of the bacterial-derived toxin lipopolysaccharide (LPS). Genetically-engineered mice expressing phospho-deficient threonine748–to-alanine (T748A) mutant Stat1 are resistant to LPS–induced lethality. Of note, T748A mice exhibited undisturbed IFN signaling, as well as total expression of Stat1. Further, the T748A point-mutation of Stat1 recapitulates the safeguard effect of the genetic ablation of Stat1 following LPS-induced lethality, indicating that the Thr748 phosphorylation contributes inflammatory functionalities of Stat1. Mechanistically, LPS-induced Toll-like receptor 4 endocytosis activates a cell-intrinsic I?B kinase (IKK)–mediated Thr748 phosphorylation of Stat1, which promotes macrophages inflammatory response while restricting the IFN and anti-inflammatory responses. Depletion of macrophages restores the sensitivity of the T748A mice to LPS-induced lethality. Together, our study indicates a phosphorylation-dependent functional dichotomy of Stat1 in innate immune responses: IFN phospho-tyrosine dependent, and inflammatory phospho-threonine dependent. Better understanding of the Thr748 phosphorylation of Stat1 may uncover novel pharmacologically targetable molecules and offer better treatment modalities for sepsis, a disease that claims millions of lives annually. Overall design: To investigate the effect of the Thr748 phosphorylation of Stat1 on macrophages function following LPS stimulation, we performed RNA-seq analysis of bone marrow derived macrophages (BMDMs) and peritoneal cavity macrophages (PCMs) from wildtype (Wt) and Stat1T78A mutant littermates. Two biological replicates of 2× 105 of BMDMs or 1× 106 of PCMs with or without LPS treatment were lysed in 0.5 ml of TRIzol Reagent (Thermo Fisher). RNA-seq analysis was performed by Genome Information Research Center (GIRC, Osaka University). In brief, total RNA was extracted and preparation of cDNA libraries was performed using a TruSeq stranded mRNA sample prep kit (Illumina, catalog no. 20020594) according to the manufacturer's instructions. Sequencing was performed on an Illumina HiSeq 2500 platform in the 75-base single-end mode. Illumina Casava1.8.2 software was used for basecalling. Sequenced reads were mapped to the mouse reference genome sequences (mm10) using TopHat version 2.0.13 in combination with Bowtie2 version 2.2.3 and SAMtools version 0.1.19. The fragments per kilobase of exons per million mapped fragments (FPKM) was calculated using Cuffnorm version 2.2.1. RNA-seq read files were processed and analyzed on Galaxy platform (https://usegalaxy.org/). Qualities of reads were evaluated by fastQC (v0.73), and reads were mapped onto GRCm38/mm10 genome using HISAT2 (v2.2.1) with default setting after the trimming of adaptor sequences. Count files were generated by use of featureCounts (v2.0.3), and differentially expressed genes were investigated using DESeq2 (v2.11.40.7).