SUMMARY
STING is an ER-associated transmembrane protein that turns on and quickly turns off downstream signaling as it translocates from the ER to vesicles. How STING signaling is attenuated during trafficking remains poorly understood. Here, we show that trafficking-mediated STING degradation requires ER-exit, function of vacuolar ATPase complex, and late stage STING vesicles are sorted to Rab7-positive endolysosomes for degradation. Based on analysis of existing structures, we also identified the helix aa281-297 as a motif required for trafficking-mediated STING degradation. Immuno-EM reveals the size and clustering of STING vesicles and topology of STING on the vesicle. Importantly, blockade of trafficking-mediated STING degradation using bafilomycin A1 specifically enhanced cGAMP-mediated immune response and anti-tumor effect in mice. Together, our findings provide biochemical and imaging evidence for STING degradation by the lysosome, and pinpoint trafficking-mediated STING degradation as a previously unanticipated therapeutic target for enhancing STING signaling in cancer therapy.
Mice with a mutated form of RNase H2 found in patients with the neuroinflammatory
Aicardi-Goutières Syndrome develop a lethal, cGAS–STING–dependent
disease.
Aberrant nucleic acids generated during viral replication are the main trigger for antiviral immunity, and mutations disrupting nucleic acid metabolism can lead to autoinflammatory disorders. Here we investigated the etiology of X-linked reticulate pigmentary disorder (XLPDR), a primary immunodeficiency with autoinflammatory features. We discovered that XLPDR is caused by an intronic mutation that disrupts expression of POLA1, the gene encoding the catalytic subunit of DNA polymerase-α. Unexpectedly, POLA1 deficiency results in increased type I interferon production. This enzyme is necessary for RNA:DNA primer synthesis during DNA replication and strikingly, POLA1 is also required for the synthesis of cytosolic RNA:DNA, which directly modulates interferon activation. Altogether, this work identified POLA1 as a critical regulator of the type I interferon response.
STING is an important innate immune protein, but its homeostatic regulation at the resting-state is unknown. Here, we identified TOLLIP as a stabilizer of STING through direct interaction to prevent its degradation.
Tollip
-deficiency results in reduced resting-state STING protein level in nonhematopoietic cells and tissues and renders STING protein unstable in immune cells, leading to severely dampened STING signaling capacity. The competing degradation mechanism of resting-state STING requires IRE1α and lysosomes. TOLLIP mediates clearance of Huntington’s disease (HD)-linked polyQ protein aggregates. Ectopic expression of polyQ protein in vitro or naturally occurring polyQ proteins in HD mouse striatum sequester TOLLIP away from STING, leading to reduced STING protein and dampened immune signaling.
Tollip
−/−
also ameliorates STING-mediated autoimmune disease in
Trex1
−/−
mice. Together, our findings reveal that resting-state STING protein level is strictly regulated by a constant tug-of-war between ‘stabilizer’ TOLLIP and ‘degrader’ IRE1α-lysosome that together maintains tissue immune homeostasis.
The signaling adapter protein STING is crucial for the host immune response to cytosolic DNA and cyclic dinucleotides. Under basal conditions, STING resides on the endoplasmic reticulum (ER), but upon activation, it traffics through secretory pathway to cytoplasmic vesicles, where STING activates downstream immune signaling. Classical STING activation and trafficking are triggered by binding of cyclic dinucleotide ligands. STING signaling can also be activated by gain-of-function mutations that lead to constitutive trafficking of STING. These gain-of-function mutations are associated with several human diseases such as STING-associated vasculopathy with onset in infancy (SAVI), systemic lupus erythematosus (SLE), or familial chilblain lupus (FCL). This dynamic activation pathway presents a challenge to study. We describe methods here for measuring ligand-dependent and ligand-independent activation of STING signaling in HEK293T cells. We also describe a retroviral-based reconstitution assay to study STING protein trafficking and activation in immune competent cells such as mouse embryonic fibroblasts (MEF), which avoids the use of plasmid DNA. These methods will expedite research regarding STING trafficking and signaling dynamics in the settings of infection and autoimmune diseases.
SARS-CoV-2 infection leads to a broad range of outcomes and immune responses, with the development of neutralizing antibodies generally correlated with protection against reinfection. Here, we have characterized both neutralizing activity and T cell responses in a cluster of subjects with mild disease linked to a single spreading event. Surprisingly, we observed sex-specific associations between spike- and particularly nucleoprotein-specific T cell responses and neutralization, with pro-inflammatory cytokines being linked to higher titers only in males. Using single cell immunoprofiling, which provided matched transcriptome and T-cell receptor (TCR) profiles in restimulated CD4 + and CD8 + cells from these subjects, we identified differences in type I IFN signaling that may underlie this difference in antibody generation. Finally, we also identified several TCRs associated with cytokine producing T cells. Altogether, our work maps the breadth of immunological outcomes of SARS-CoV2 infections and highlight the potential role of sex-specific feedback loops during the generation of neutralizing antibodies.
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