Tripartite motif 13 orchestrates endoplasmic reticulum-associated degradation and endoplasmic reticulum-phagy to modulate dendritic cell-mediated immune responses in sepsis.

LL-37 transports immunoreactive cGAMP to activate STING signaling and enhance interferon-mediated host antiviral immunity.

Cancer cell-intrinsic mechanisms affecting radiation immunomodulation could be exploited to optimize systemic effects of localized radiation. Radiation-induced DNA damage is sensed by cyclic GMP-AMP synthase (cGAS), which ultimately activates stimulator of interferon (IFN) genes (STING). Resultant expression of soluble mediators such as CCL5 and CXCL10 can facilitate recruitment of dendritic cells and immune effector cells into the tumor. The primary objectives of this study were to determine the baseline expression levels of cGAS and STING in OSA cells and evaluate the dependence of OSA cells on STING signaling for eliciting radiation-induced expression of CCL5 and CXCL10. cGAS and STING expression, and CCL5/CXCL10 expression in control cells, STING-agonist treated cells, and cells treated with 5 Gy ionizing radiation were assessed utilizing RTqPCR, Western blot, and ELISA. U2OS and SAOS-2 OSA cells were deficient in STING relative to human osteoblasts (hObs), while SAOS-2-LM6 and MG63 OSA cells expressed equivalent amounts of STING compared to hObs. A dependence on baseline or induced STING expression was observed for STING-agonist, and radiation-induced, expression of CCL5 and CXCL10. This finding was confirmed by performing siRNA knockdown of STING in MG63 cells. These results show that STING signaling is necessary for radiation-induced expression of CCL5 and CXCL10 in OSA cells. Additional studies are necessary to determine whether STING expression in OSA cells in vivo alters immune cell infiltrates after radiation exposure. These data may also have implications for other potentially STING-dependent characteristics such as resistance to oncolytic virus cytotoxicity.

M6PR upregulation by berberine attenuates hepatic senescence via sorting STING into endosome for degradation.

Dendritic cells (DCs) induce peripheral Tcell tolerance, but cell-intrinsic signaling cascades governing their stable tolerogenesis remain poorly defined. Janus Kinase 1 (JAK1) transduces cytokine-receptor signaling, and JAK inhibitors (Jakinibs), including JAK1-specific filgotinib, break inflammatory cycles in autoimmunity. Here, we report in heterogeneous DC populations of multiple secondary lymphoid organs that JAK1 promotes peripheral Tcell tolerance during experimental autoimmune encephalomyelitis (EAE). Mice harboring DC-specific JAK1 deletion exhibit elevated peripheral CD4+ Tcell expansion, less regulatory Tcells (Tregs), and worse EAE outcomes, whereas adoptive DC transfer ameliorates EAE pathogenesis by inducing peripheral Tregs, programmed cell death ligand 1 (PD-L1) dependently. This tolerogenic program is substantially reduced upon the transfer of JAK1-deficient DCs. DC-intrinsic IFN-γ-JAK1-STAT1 signaling induces PD-L1, which is required for DCs to convert CD4+ Tcells into Tregs invitro and attenuated upon JAK1 deficiency and filgotinib treatment. Thus, DC-intrinsic JAK1 promotes peripheral tolerance, suggesting potential unwarranted DC-mediated effects of Jakinibs in autoimmune diseases.

ObjectiveChronic pancreatitis (CP) is an inflammatory disease with progressive fibrosis leading to exocrine and endocrine dysfunction. Currently, there are no approved effective therapies for CP. Stimulator of interferon genes (STING)...

BackgroundIt is established that pulmonary disorders are comorbid with metabolic disorders such as obesity. Previous studies show that the stimulator of interferon genes (STING) signaling plays crucial roles in obesity-induced chronic inflammation via TANK-binding kinase 1 (TBK1) pathways. However, it remains unknown whether and how the STING signaling is implicated in the inflammatory processes in the lung in obesity.MethodsHuman lung tissues were obtained from obese patients (n = 3) and controls (n = 3). Mice were fed with the high-fat diet or regular control diet to establish the diet-induced obese (DIO) and lean mice, and were treated with C-176 (a specific STING inhibitor) or vehicle respectively. The lung macrophages were exposed to palmitic acid (PA) in vitro. The levels of STING singaling and metabolic inflammation factors were detected and anlyzed.ResultsWe find that STING+/CD68+ macrophages are increased in lung tissues in patients with obesity. Our data also show that the expressions of STING and the levels of proinflammatory cytokines are increased both in lung tissues and bronchoalveolar lavage fluid (BALF) in obesity compared to controls, and inhibition of the STING blunted the obesity-induced lung inflammation. Mechanistically, our data demonstrate that the STING signaling pathway is involved in the PA-induced inflammation through the STING-TBK1-IRF3 (interferon regulatory factor 3)/NF-κB (nuclear factor kappa B) pathways in the lung macrophages.ConclusionsOur results collectively suggest that the STING signaling contributes to obesity-associated inflammation by stimulating proinflammatory processes in lung macrophages, one that may serve as a therapeutic target in ameliorating obesity-related lung dysfunctions.

Background: Activation of stimulator of interferon genes (STING) has shown great potential to enhance anti-tumor effect and reshape the tumor microenvironment (TME). Instead, the impact of TME on STING signaling remains elusive. Leveraging patient-derived samples at single-cell resolution from our clinical trial (NCT04507841), we find that TGF-β significantly attenuates STING signaling and downstream type I interferons production, which is related to the accelerated STING trafficking and degradation. Mechanistically, we identify AP3D1, a subunit of the AP-3 complex, could response to TGF-β and accelerate STING signaling termination in the TME. Methods and results: Utilizing unique samples with HRD precision stratification and PARPi treatment information from our clinical trial (NCT04507841), and employing cutting-edge technologies such as single-cell sequencing and multiplex immunohistochemistry to reveal the complex immune microenvironment, we identified TGF-β suppresses the STING signaling pathway and the production of downstream type I IFN. Additionally, through RT-PCR, western blot, and immunofluorescence co-localization, we discovered that TGF-β promotes STING transport from Golgi to lysosome, accelerating STING degradation. To elucidate the specific mechanism by which TGF-β promotes STING transport, we employed techniques including the STING-APEX2 based proximity biotin labeling system, phosphorylation mass spectrometry and IP-MS, our analysis revealed and verified that TGF-β activates MEK to facilitate phosphorylation of adaptor related protein complex 3 (AP-3) subunit delta 1 (AP3D1) at the S9 site, thereby enhancing the interaction between AP3D1 and STING and accelerating the degradation of STING. Furthermore, we conducted validation experiments in multiple mouse models to confirm the effectiveness of peptides that target the inhibition of AP3D1-STING binding at the AP3D1 R26 site and the blockade of AP3D1 phosphorylation at the S9 site. When administered in combination with PARP inhibitors and STING agonists, these peptides demonstrated satisfactory therapeutic outcomes in upregulating STING signaling, reducing tumor burden, and inducing anti-tumor immune responses, all without evident toxicity. Conclusions: Our work identified TGF-β as an exogenous STING regulator that accelerates AP3D1-mediated STING trafficking and degradation, providing an unprecedented insight into crosstalk between the TME and the STING signaling. Furthermore, our in-depth dissection of mechanisms offered conceptual strategies for new drug discovery and combined pharmacotherapy in cases where STING agonists fail to provide anticipated efficacy currently. Citation Format: Xingzhe Liu, Yuanjia Wen, Jiahao Liu, Guannan Li, Huayi Li, Xiaofei Jiao, Mengshi Luo, Yuewen Zhang, Xuejiao Zhao, Gorden B. Mills, Ding Ma, Guangnian Zhao, Qinglei Gao, Yong Fang. TGF-β accelerates STING signaling termination in the tumor microenvironment by enhancing AP3D1-mediated STING trafficking [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2695.

P14.01 Impact of Stimulator of Interferon Genes (STING) Signaling on the Tumor Immune Microenvironment in Lung Adenocarcinoma

Lung adenocarcinoma (LUAD), the most prevalent histologic subtype of NSCLC, is one of the most fatal malignancies. Despite the weighted effect of stimulator of interferon genes (STING) signaling on innate and adaptive immunity, understanding its dynamic modulation and tumor microenvironment (TME) in LUAD poses a challenge. Herein, we investigated the correlation between STING signaling, TME, and the overall survival of LUAD patients utilizing data from The Cancer Genome Atlas (TCGA). SsGSEA, CIBERSORT, and ESTIMATE algorithms were applied before gene set enrichment analysis (GSEA) was used to screen Gene Ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) signaling pathways between low and high STING groups of LUAD. High STING signaling demonstrated increased levels of immunoregulatory and effector molecules, cytokines, and activated CD4/CD8 T cells. GSEA revealed that the high STING group from the LUAD cohort were mainly enriched in GO terms and KEGG pathways related to immune response. Additionally, both STING signaling and activated CD8+ T cells were positively related to overall survival. Our findings suggest the stimulation of the STING signaling pathway within the TME as a novel immunotherapeutic strategy for LUAD, indicating the potential combination therapy of STING agonists and immune checkpoint inhibitors. Funding Statement: This study was supported by the China Scholarship Council (grant number: 201806325017 and 201906240104). Declaration of Interests: The authors declare no conflicts of interest. Ethics Approval Statement: Lung Cancer Center, West China Hospital of Sichuan University gave ethical approval for the study.

Stimulator of interferon genes (STING) signaling has been extensively studied in inflammatory diseases and cancer, while its role in T cell responses to infection is unclear. Using Listeria monocytogenes strains engineered to induce different levels of c-di-AMP, we found that high STING signals impaired T cell memory upon infection via increased Bim levels and apoptosis. Unexpectedly, reduction of TCR signal strength or T cell-STING expression decreased Bim expression, T cell apoptosis, and recovered T cell memory. We found that TCR signal intensity coupled STING signal strength to the unfolded protein response (UPR) and T cell survival. Under strong STING signaling, Indoleamine-pyrrole 2,3-dioxygenase (IDO) inhibition also reduced apoptosis and led to a recovery of T cell memory in STING sufficient CD8 T cells. Thus, STING signaling regulates CD8 T cell memory fitness through both cell-intrinsic and extrinsic mechanisms. These studies provide insight into how IDO and STING therapies could improve long-term T cell protective immunity.

Objective: To investigate the effects of lipopolysaccharide (LPS)-activated stimulator of interferon genes (STING) signaling on the biological behavior of periodontal ligament cells and its mechanism of action. Methods: Human periodontal ligament cells (hPDLC) were divided into the PBS group and the LPS group by stimulated with phosphate-buffered saline (PBS) and LPS derived from Porphyromonas gingivalis (ATCC 33277) for 12 hours, respectively. The intracellular distribution of 8-hydroxydeoxyguanosine (8-OHdG), a marker of DNA damage, and the activation level of STING signaling were detected by immunofluorescence. The source of intracellular double-stranded DNA was detected by live-cell probes. The levels of osteogenic-related proteins, such as special protein 7 (SP7), bone morphogenetic protein 2 (BMP2), cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS), and STING were detected by Western blotting. The cell supernatants of the PBS group and the LPS group were collected, and the levels of inflammatory cytokines, such as interleukin (IL)-1β, IL-6, and interferon (IFN)-β, were detected by double-antibody sandwich enzyme-linked immunosorbent assay (ELISA). A total of 12 cgas knockout mice and 12 littermate wild-type mice were constructed. The maxillary second molars of the mice were ligated with silk or sham surgery, respectively. After 7 days of modeling, the mice were divided into littermate control sham surgery group, littermate control periodontitis group, cgas knockout sham surgery group, and cgas knockout periodontitis group, with 6 mice in each group. Micro-CT was used to collect image data, and three-dimensional reconstructions were performed on the maxillary samples of each group. The distance from the cemento-enamel junction to the alveolar bone crest (CEJ-ABC), bone volume fraction (BV/TV), and bone mineral density (BMD) in the model area were statistically analyzed using CTAn and CTVOX software. Frozen sectioning was used to obtain sections of the maxillary molars of each group of mice, and the signal intensities of cGAS and STING proteins were detected by immunofluorescence. Results: Immunofluorescence results showed that the fluorescence signal intensity of 8-OHdG outside the nucleus in the LPS group (4.09±0.24) was significantly higher than that in the PBS group (1.00±0.10) (t=20.33, P<0.001). The co-localization signal of mitochondrial marker TOM20 and 8-OHdG (8.56±0.53) were significantly higher than that of PBS group (1.00±0.09) (t=24.37, P<0.001). Live cell DNA probe detection showed that the signal intensity of double-stranded DNA in LPS group (3.23±0.12) was significantly stronger than that in PBS group (1.00±0.17) (t=18.30, P<0.001). Immunofluorescence demonstrated a significant increase in STING expression in hPDLC of the LPS group (t=6.42, P<0.001), and it was colocalized with the Golgi marker GM130. ELISA results showed that the abundance of IL-6, IFN-β, and IL-1β in the supernatant of the LPS group were higher than those of the PBS group (t=12.44, t=11.38, t=9.48, all P<0.001). Animal experiments confirmed that compared with the sham operation group [(207.61±38.09) and (238.97±45.90) μm], the CEJ-ABC in the periodontitis group [(420.31±35.32) and (405.16±35.51) μm] were increased (P<0.01), while the CEJ-ABC in the cgas knockout periodontitis group [(295.11±35.43) and (309.15±32.22) μm] were significantly lower than those in the control periodontitis group of the same litter (P<0.01). Compared with the sham operation group (45.84±6.41), the STING fluorescence signal in the periodontitis group (152.44±6.86) was significantly increased (P<0.001). Compared with the control periodontitis group of the same litter, the STING signal in the cgas knockout periodontitis group was significantly reduced (88.31±9.70) (P<0.001). Conclusions: LPS stimulation can activate the STING signal by generating mitochondrial-derived double-stranded DNA, stimulating hPDLC to secrete inflammatory cytokines and impairing osteogenic differentiation potential. Suppressing STING activation in animal models can reduce bone destruction in periodontitis.

SummarySignaling through stimulator of interferon genes (STING) leads to the production of type I interferons (IFN-Is) and inflammatory cytokines. A gain-of-function mutation in STING was identified in an autoinflammatory disease (STING-associated vasculopathy with onset in infancy; SAVI). The expression of cyclic GMP-AMP, DNA-activated cGAS-STING pathway, increased in a proportion of patients with SLE. The STING signaling pathway may be a candidate for targeted therapy in SLE. Here, we demonstrated that disruption of STING signaling ameliorated lupus development in Fcgr2b-deficient mice. Activation of STING promoted maturation of conventional dendritic cells and differentiation of plasmacytoid dendritic cells via LYN interaction and phosphorylation. The inhibition of LYN decreased the differentiation of STING-activated dendritic cells. Adoptive transfer of STING-activated bone marrow-derived dendritic cells into the FCGR2B and STING double-deficiency mice restored lupus phenotypes. These findings provide evidence that the inhibition of STING signaling may be a candidate targeted treatment for a subset of patients with SLE.

The activation of stimulator of interferon genes (STING) signaling induces the production of type I interferons (IFNs), which play critical roles in protective innate immunity for the host to defend against viral infections. Therefore, achieving sustained or enhanced STING activation could become an antiviral immune strategy with potential broad-spectrum activities. Here, we discovered that various clinically used microtubule-destabilizing agents (MDAs) for the treatment of cancer showed a synergistic effect with the activation of STING signaling in innate immune response. The combination of a STING agonist cGAMP and a microtubule depolymerizer MMAE boosted the activation of STING innate immune response and showed broad-spectrum antiviral activity against multiple families of viruses. Mechanistically, MMAE not only disrupted the microtubule network, but also switched the cGAMP-mediated STING trafficking pattern and changed the distribution of Golgi apparatus and STING puncta. The combination of cGAMP and MMAE promoted the oligomerization of STING and downstream signaling cascades. Importantly, the cGAMP plus MMAE treatment increased STING-mediated production of IFNs and other antiviral cytokines to inhibit viral propagation in vitro and in vivo. This study revealed a novel role of the microtubule destabilizer in antiviral immune responses and provides a previously unexploited strategy based on STING-induced innate antiviral immunity.

Stimulator of interferon genes (STING) is an endoplasmic reticulum (ER) signaling adaptor that is essential for the host immune response triggered by DNA pathogens. Precise regulation of STING is crucial for maintaining a balanced immune response and preventing harmful autoinflammation. Activation of STING requires its translocation from the ER to the Golgi apparatus. However, the mechanisms that maintain STING in its resting state remain largely unclear. Here, we find that deficiency of the ancient ubiquitous protein 1 (AUP1) causes spontaneous activation of STING and enhances the expression of type I interferons (IFNs) under resting conditions. Furthermore, deficiency of UBE2G2, a cofactor of AUP1, also promotes the abnormal activation of STING. AUP1 deficiency significantly enhances STING signaling induced by DNA virus, and AUP1 deficiency exhibits increased resistance to DNA virus infection in vitro and in vivo. Mechanistically, AUP1 may form a complex with UBE2G2 to interact with STING, preventing its exit from the ER membrane. Notably, infection with the RNA virus vesicular stomatitis virus (VSV) promotes the accumulation of lipid droplets (LDs) and AUP1 proteins. Additionally, AUP1 deficiency markedly inhibits the replication of VSV because AUP1 deficiency reduces lipid accumulation and alters the expression of lipid metabolism genes, such as carnitine palmitoyltransferase 1A (CPT1A), monoglyceride lipase (MGLL), and sterol regulatory element-binding transcription factor 1 (SREBF1). This study uncovers the essential roles of AUP1 in the STING signaling pathway and lipid metabolism pathway, highlighting its dual role in regulating virus replication.IMPORTANCEThe stimulator of interferon genes (STING) signaling cascade plays an essential role in coordinating innate immunity against DNA pathogens and autoimmunity. Precise regulation of the innate immune response is essential for maintaining homeostasis. In this study, we demonstrate that ancient ubiquitous protein 1 (AUP1) and UBE2G2 act as negative regulators of the innate immune response by targeting STING. Notably, AUP1 interacts with STING to retain STING in the endoplasmic reticulum (ER), preventing STING translocation and thereby limiting STING signaling in the resting state. In addition, deficiency of AUP1 markedly inhibits the replication of DNA virus and RNA virus. Our findings provide new insights into the regulation of STING signaling and confirm AUP1 has a dual role in regulating virus replication.

Proinflammatory hyperactivation of Kupffer cells (KCs) is foremost involved in the pathogenesis of sepsis-induced liver injury. Our previous study found that stimulator of interferon genes (STING) signaling was activated in KCs in response of lipopolysaccharide (LPS) and knocking down dynamin-related protein 1 (DRP1) in KCs effectively inhibited the activation of STING signaling and the subsequent production of proinflammatory cytokines. In this study, we demonstrated that in vivo treatment with mitochondrial division inhibitor 1 (Mdivi-1), a selective inhibitor of DRP1, alleviated cecal ligation and puncture (CLP)-induced liver injury with the improvement of liver pathology and function. Moreover, we found that STING in liver was mainly concentrated in KCs and STING signaling was significantly activated in KCs after CLP. The STING deficiency effectively ameliorated liver injury and decreased the mortality of septic mice, which were reversely worsened by the enhanced activation of STING with DMXAA. The further study showed that Mdivi-1 markedly attenuated STING signaling activation in KCs and inhibited systemic inflammatory response. Importantly, DMXAA application in CLP mice blunted Mdivi-1's liver protection effect. Taken together, our study confirmed Mdivi-1 effectively alleviated CLP-induced liver injury partially through inhibiting STING signaling activation in KCs, which provides new insights and a novel potential pharmacological therapeutic target for treating septic liver injury.