Activated STING in a vascular and pulmonary syndrome

Abstract

Background: The study of autoinflammatory diseases has uncovered mechanisms underlying cytokine dysregulation and inflammation.

Methods: We analyzed the DNA of an index patient with early-onset systemic inflammation, cutaneous vasculopathy, and pulmonary inflammation. We sequenced a candidate gene, TMEM173, encoding the stimulator of interferon genes (STING), in this patient and in five unrelated children with similar clinical phenotypes. Four children were evaluated clinically and immunologically. With the STING ligand cyclic guanosine monophosphate-adenosine monophosphate (cGAMP), we stimulated peripheral-blood mononuclear cells and fibroblasts from patients and controls, as well as commercially obtained endothelial cells, and then assayed transcription of IFNB1, the gene encoding interferon-β, in the stimulated cells. We analyzed IFNB1 reporter levels in HEK293T cells cotransfected with mutant or nonmutant STING constructs. Mutant STING leads to increased phosphorylation of signal transducer and activator of transcription 1 (STAT1), so we tested the effect of Janus kinase (JAK) inhibitors on STAT1 phosphorylation in lymphocytes from the affected children and controls.

Results: We identified three mutations in exon 5 of TMEM173 in the six patients. Elevated transcription of IFNB1 and other gene targets of STING in peripheral-blood mononuclear cells from the patients indicated constitutive activation of the pathway that cannot be further up-regulated with stimulation. On stimulation with cGAMP, fibroblasts from the patients showed increased transcription of IFNB1 but not of the genes encoding interleukin-1 (IL1), interleukin-6 (IL6), or tumor necrosis factor (TNF). HEK293T cells transfected with mutant constructs show elevated IFNB1 reporter levels. STING is expressed in endothelial cells, and exposure of these cells to cGAMP resulted in endothelial activation and apoptosis. Constitutive up-regulation of phosphorylated STAT1 in patients' lymphocytes was reduced by JAK inhibitors.

Conclusions: STING-associated vasculopathy with onset in infancy (SAVI) is an autoinflammatory disease caused by gain-of-function mutations in TMEM173. (Funded by the Intramural Research Program of the National Institute of Arthritis and Musculoskeletal and Skin Diseases; ClinicalTrials.gov number, NCT00059748.).

Figure 1. Clinical Features of Stimulator of Interferon Genes (STING)–Associated Vasculopathy with Onset in Infancy (SAVI)

Panel A shows the typical facial distribution of telangiectatic lesions on the nose and cheeks of Patient 1, who has SAVI. Panel B shows violaceous, scaling, atrophic plaques on the hands of Patient 6. Panel C shows histologic features of vascular inflammation in a skin-biopsy sample from a clinically involved area depicting a dense neutrophilic infiltrate with karyorrhexis throughout the vessel wall (hematoxylin and eosin); fibrin deposits are seen in the lumen of a severely damaged vessel. In Panel D, a high-resolution computed tomographic image of the lung of Patient 5 shows interstitial lung disease.

Figure 2. Mutations in TMEM173, the Gene Encoding STING

Panel A shows the family pedigrees of the six children with the mutations N154S, V155M, and V147L in TMEM173. Solid symbols indicate Patients 1 through 6, open symbols unaffected relatives, squares male persons, circles female persons, and slashes deceased persons. For the persons for whom the TMEM173 genotype was determined, H denotes heterozygous mutated gene, NA not available, and NM nonmutated gene. Panel B shows the genomic structure with the centromere in red triangles and the location of the TMEM173 locus shown by a red line. Also shown is the gene structure (National Center for Biotechnology Information Reference Sequence [RefSeq] number, NM_198282) with the exons shown as blue boxes. The mutations were clustered in a small region of exon 5. Electropherograms of the three de novo mutations are shown (which are named under the plots, along with the predicted amino acid substitutions) for Patients 1, 2, and 4; Patients 3, 5, and 6 had the same mutation as Patient 1. The mutation detected in Patient 6 is probably somatic.

Figure 3. The STING–Interferon-β Pathway

STING, an endoplasmic reticulum transmembrane protein, forms homodimers and functions as an adaptor for cytosolic DNA sensing. STING is activated by the binding of cyclic guanosine monophosphate–adenosine monophosphate (cGAMP), a second messenger that is synthesized by cyclic GMP–AMP synthase (cGAS), a family member of nucleotidyltransferases that is activated on its recognition and binding of double-stranded DNA (dsDNA). Binding of cGAMP to the STING homodimer activates interferon regulatory factor 3 (IRF-3) through TANK-binding kinase 1 (TBK1) and leads to the induction of interferon-β. In patients with SAVI, constitutively activated STING leads to increased transcription of the type 1 interferon gene, IFNB1, which encodes interferon-β. Binding of inter feron-β to its receptor activates Janus kinases (JAKs), including JAK1 and tyrosine kinase 2 (TYK2), which in turn phosphory-late the receptor. This process allows the binding of the DNA-binding proteins signal transducers and activators of transcription 1 (STAT1) and 2 (STAT2) to the receptor, whereupon they become phosphorylated (P). Phosphorylation allows them to dimerize, and the dimer translocates to the nucleus, where it up-regulates transcription of interferon-response genes, including interferon regulatory factor 7–dependent transcription of type 1 interferon genes. The synthesis and release of interferons and their binding to interferon receptor further up-regulate STING and the transcription of other proinflammatory cytokine genes in a positive feedback loop. JAK inhibition blocks the loop, resulting in a decrease in STAT1 phosphorylation and transcription of its target genes in vitro.

Figure 4. Functional Data Showing Gain-of-Function Mutation in TMEM173 in Patients with SAVI

In Panel A, a heat map shows increased expression of interferon-regulated genes in 4 patients with SAVI, as compared with another interferon-mediated autoinflammatory disease, the chronic atypical neutrophilic dermatosis with lipodystrophy and elevated temperature (CANDLE) syndrome (11 patients), the interleukin-1 mediated neonatal-onset multisystem inflammatory disease (NOMID; 5 patients), and 18 healthy controls. Up-regulated genes are shown in red, and down-regulated genes in blue. Asterisks indicate samples obtained at different time points from Patient 1, and plus signs indicate samples obtained at different time points from Patient 3. The circle indicates a sample from Patient 4, and the triangle a sample from Patient 6. Panel B shows the result of a quantitative reverse-transcriptase–polymerase-chain-reaction assay of IFNB1 messenger RNA (mRNA) in peripheral-blood mononuclear cells (PBMCs) from 3 patients with SAVI and 7 relatives or controls, with (+) and without (−) cGAMP stimulation. Shown are normalized IFNB1 mRNA levels against the average value for healthy controls in the absence of stimulation. P1, P3, and P6 denote Patients 1, 3, and 6, respectively. The healthy controls included Patient 1's sibling, father, and mother (HC1, HC2, and HC3, respectively), Patient 3's female sibling and mother (HC4 and HC5, respectively), and two persons who were unrelated to the patients (HC6 and HC7). Errors bars indicate the standard error for triplicates in the assay. Asterisks denote P = 0.01 for the comparison between unstimulated samples from patients and unstimulated samples from controls; no significant increase in IFNB1 mRNA levels was seen after cGAMP stimulation in the patients, as compared with controls. Panel C shows cotransfection of HEK293T cells with a TMEM173 construct (2 ng of plasmid DNA), either the nonmutated gene or a gene with a disease-associated mutation (N154S, V155M, or V147L) or loss-of-function mutation (V155R), in addition to an IFNB1 luciferase reporter construct, performed to assess IFNB1 reporter activity with and without cGAMP stimulation. A green fluorescent protein expressing plasmid was also contransfected as control. The reporter activity was normalized to the result of nonmutant construct without cGAMP stimulation. The three constructs carrying the putative disease-causing mutations, but not the nonmutated constructs, constitutively activated IFNB1 transcription, which was further up-regulated with cGAMP stimulation. The cells with the nonmutated construct responded to cGAMP stimulation in a dose-dependent manner, whereas the cells with the loss-of-function mutation V155R had minimal responses only at the highest cGAMP concentrations. The cells with the control transfection did not respond to all tested cGAMP concentrations. I bars indicate standard errors.

Figure 5. Analysis of Dermal Vascular Endothelial-Cell Activation

The images show tissue immunofluorescence staining of skin-biopsy samples from Patient 2 and a healthy control. Endothelial-cell marker CD31 (green) was costained with intercellular adhesion molecule 1 (ICAM-1, red) and coagulation marker tissue factor (TF, red). Shown is a damaged endothelial-cell layer in Patient 2 with loss of continuous staining of CD31 in the endothelial lining (lower panels) and a preserved endothelial-cell layer in healthy controls (upper panels). Both ICAM-1 and TF were absent in control cells but were up-regulated in the endothelial cells from Patient 2. Nuclei were stained blue with 4′,6-diamidine-2-phenylidole dihydrochloride (DAPI). The scale bars represent 20 μm.

Figure 6. Suppression of Constitutively Phosphorylated STAT1 (pSTAT1) with JAK Inhibitors in a Patient with SAVI

The upper panel shows data from a healthy control, and the middle and lower panels data from Patient 6. Peripheral-blood mononuclear cells from the patient with SAVI and from the healthy control were treated with and without the indicated JAK inhibitors for 4 hours. STAT1 phosphorylation in CD4 T cells and CD19 B cells was analyzed by means of intracellular staining of pSTAT1 and the respective cell-surface marker. The healthy-control data are representative of experiments with samples from five healthy controls.