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HIV-1 evades immune detection by the cGAS-STING cytosolic DNA-sensing pathway during acute infection. STING is a critical mediator of type I IFN production, and STING agonists such as cGMP-AMP (cGAMP) and other cyclic dinucleotides elicit potent immune and antitumor response. In this article, we show that administration of cGAMP, delivered by an ultra–pH-sensitive nanoparticle (NP; PC7A), in human PBMCs induces potent and long-acting antiretroviral response against several laboratory-adapted and clinical HIV-1 isolates. cGAMP-PC7A NP requires endocytosis for intracellular delivery and immune signaling activation. cGAMP-PC7A NP-induced protection is mediated through type I IFN signaling and requires monocytes in PBMCs. cGAMP-PC7A NPs also inhibit HIV-1 replication in HIV+ patient PBMCs after ex vivo reactivation. Because pattern recognition receptor agonists continue to show more clinical benefits than the traditional IFN therapy, our data present important evidence for potentially developing cGAMP or other STING agonists as a new class of immune-stimulating long-acting antiretroviral agents.

Summary STING is an endoplasmic reticulum (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 and function of vacuolar ATPase complex. Late-stage STING vesicles are sorted to Rab7-positive endolysosomes for degradation. Based on analysis of existing structures, we also identified the helix amino acid 281 (aa281)–297 as a motif required for trafficking-mediated STING degradation. Immuno-electron microscopy (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 cyclic guanosine monophosphate (GMP)-AMP (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.

Summary Type I interferon (IFN) response is commonly recognized as the main signaling activity of STING. Here, we generate the Sting1S365A/S365A mutant mouse that precisely ablates IFN-dependent activities while preserving IFN-independent activities of STING. StingS365A/S365A mice protect against HSV-1 infection, despite lacking the STING-mediated IFN response. This challenges the prevailing view and suggests that STING controls HSV-1 infection through IFN-independent activities. Transcriptomic analysis reveals widespread IFN-independent activities of STING in macrophages and T cells, and STING activities in T cells are predominantly IFN independent. In mouse tumor models, T cells in the tumor experience substantial cell death that is in part mediated by IFN-independent activities of STING. We found that the tumor induces STING-mediated cell death in T cells to evade immune control. Our data demonstrate that mammalian STING possesses widespread IFN-independent activities that are important for restricting HSV-1 infection, tumor immune evasion and likely also adaptive immunity.

STING (stimulator of interferon genes) 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 STING protein 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-linked polyQ protein aggregates. Ectopically expressed polyQ proteins in vitro or endogenous polyQ proteins in Huntington's disease 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 maintain tissue immune homeostasis.

Three-prime repair exonuclease 1 knockout (Trex1-/-) mice suffer from systemic inflammation caused largely by chronic activation of the cyclic GMP-AMP synthase-stimulator of interferon genes-TANK-binding kinase-interferon regulatory factor 3 (cGAS-STING-TBK1-IRF3) signaling pathway. We showed previously that Trex1-deficient cells have reduced mammalian target of rapamycin complex 1 (mTORC1) activity, although the underlying mechanism is unclear. Here, we performed detailed metabolic analysis in Trex1-/- mice and cells that revealed both cellular and systemic metabolic defects, including reduced mitochondrial respiration and increased glycolysis, energy expenditure, and fat metabolism. We also genetically separated the inflammatory and metabolic phenotypes by showing that Sting deficiency rescued both inflammatory and metabolic phenotypes, whereas Irf3 deficiency only rescued inflammation on the Trex1-/- background, and many metabolic defects persist in Trex1-/-Irf3-/- cells and mice. We also showed that Leptin deficiency (ob/ob) increased lipogenesis and prolonged survival of Trex1-/- mice without dampening inflammation. Mechanistically, we identified TBK1 as a key regulator of mTORC1 activity in Trex1-/- cells. Together, our data demonstrate that chronic innate immune activation of TBK1 suppresses mTORC1 activity, leading to dysregulated cellular metabolism.

Wu et al. show that STING gain-of-function disease mutant disrupts calcium homeostasis and chronically activates ER stress as well as the unfolded protein response (UPR) through a newly identified “UPR motif,” leading to T cell death and lung disease., STING gain-of-function mutations cause lung disease and T cell cytopenia through unknown mechanisms. Here, we found that these mutants induce chronic activation of ER stress and unfolded protein response (UPR), leading to T cell death by apoptosis in the StingN153S/+ mouse and in human T cells. Mechanistically, STING-N154S disrupts calcium homeostasis in T cells, thus intrinsically primes T cells to become hyperresponsive to T cell receptor signaling–induced ER stress and the UPR, leading to cell death. This intrinsic priming effect is mediated through a novel region of STING that we name “the UPR motif,” which is distinct from known domains required for type I IFN signaling. Pharmacological inhibition of ER stress prevented StingN153S/+ T cell death in vivo. By crossing StingN153S/+ to the OT-1 mouse, we fully restored CD8+ T cells and drastically ameliorated STING-associated lung disease. Together, our data uncover a critical IFN-independent function of STING that regulates calcium homeostasis, ER stress, and T cell survival., Graphical Abstract

TANK-binding kinase 1 (TBK1) is a serine/threonine protein kinase that plays a crucial role in innate immunity. Enhanced TBK1 function is associated with autoimmune diseases and cancer, implicating the potential benefit of therapeutically targeting TBK1. In this article, we examined a recently identified TBK1 inhibitor Compound II on treating autoimmune diseases. We found that Compound II is a potent and specific inhibitor of TBK1-mediated IFN response. Compound II inhibited polyinosinic-polycytidylic acid–induced immune activation in vitro and in vivo. Compound II treatment also ameliorated autoimmune disease phenotypes of Trex1−/− mice, increased mouse survival, and dampened the IFN gene signature in TREX1 mutant patient lymphoblasts. In addition, we found that TBK1 gene expression is elevated in systemic lupus erythematosus patient cells, and systemic lupus erythematosus cells with high IFN signature responded well to Compound II treatment. Together, our findings provided critical experimental evidence for inhibiting TBK1 with Compound II as an effective treatment for TREX1-associated autoimmune diseases and potentially other interferonopathies.

Mycoplasmas are a common cause of pneumonia in humans and animals, and attempts to create vaccines have not only failed to generate protective host responses, but they have exacerbated the disease. Mycoplasma pulmonis causes a chronic inflammatory lung disease resulting from a persistent infection, similar to other mycoplasma respiratory diseases. Using this model, Th1 subsets promote resistance to mycoplasma disease and infection, whereas Th2 responses contribute to immunopathology. The purpose of the present study was to evaluate the capacity of cytokine-differentiated dendritic cell (DC) populations to influence the generation of protective and/or pathologic immune responses during M. pulmonis respiratory disease in BALB/c mice. We hypothesized that intratracheal inoculation of mycoplasma Ag–pulsed bone marrow–derived DCs could result in the generation of protective T cell responses during mycoplasma infection. However, intratracheal inoculation (priming) of mice with Ag-pulsed DCs resulted in enhanced pathology in the recipient mice when challenged with mycoplasma. Inoculation of immunodeficient SCID mice with Ag-pulsed DCs demonstrated that this effect was dependent on lymphocyte responses. Similar results were observed when mice were primed with Ag-pulsed pulmonary, but not splenic, DCs. Lymphocytes generated in uninfected mice after the transfer of either Ag-pulsed bone marrow–derived DCs or pulmonary DCs were shown to be IL-13+ Th2 cells, known to be associated with immunopathology. Thus, resident pulmonary DCs most likely promote the development of immunopathology in mycoplasma disease through the generation of mycoplasma-specific Th2 responses. Vaccination strategies that disrupt or bypass this process could potentially result in a more effective vaccination.

SummarySTING is an ER-associated membrane protein that is critical for innate immune sensing of pathogens. STING-mediated activation of the IFN-I pathway through the TBK1/IRF3 signaling axis involves both cyclic-dinucleotide binding and its translocation from the ER to vesicles. However, how these events are coordinated, and the exact mechanism of STING activation, remain poorly understood. Here, we found that the Shigella effector protein IpaJ potently inhibits STING signaling by blocking its translocation from the ER to ERGIC, even in the context of dinucleotide binding. Reconstitution using purified components revealed STING translocation as the rate-limiting event in maximal signal transduction. Furthermore, STING mutations associated with autoimmunity in humans were found to cause constitutive ER exit and to activate STING independent of cGAMP binding. Together, these data provide compelling evidence for an ER retention and ERGIC/Golgi-trafficking mechanism of STING regulation that is subverted by bacterial pathogens and is deregulated in human genetic disease.

Factors that elevate amyloid-β (Aβ) peptide levels are associated with an increased risk for Alzheimer's disease. Insulysin has been identified as one of several proteases potentially involved in Aβ degradation based on its hydrolysis of Aβ peptides in vitro . In this study, in vivo levels of brain Aβ40 and Aβ42 peptides were found to be increased significantly (1.6- and 1.4-fold, respectively) in an insulysin-deficient gene-trap mouse model. A 6-fold increase in the level of the γ-secretase-generated C-terminal fragment of the Aβ precursor protein in the insulysin-deficient mouse also was found. In mice heterozygous for the insulysin gene trap, in which insulysin activity levels were decreased ≈50%, brain Aβ peptides were increased to levels intermediate between those in wild-type mice and homozygous insulysin gene-trap mice that had no detectable insulysin activity. These findings indicate that there is an inverse correlation between in vivo insulysin activity levels and brain Aβ peptide levels and suggest that modulation of insulysin activity may alter the risk for Alzheimer's disease.

The Francisella FTT0831c/FTL_0325 gene encodes amino acid motifs to suggest it is a lipoprotein and that it may interact with the bacterial cell wall as a member of the OmpA-like protein family. Previous studies have suggested that FTT0831c is surface exposed and required for virulence of Francisella tularensis by subverting the host innate immune response (M. Mahawar et al., J. Biol. Chem. 287:25216–25229, 2012). We also found that FTT0831c is required for murine pathogenesis and intramacrophage growth of Schu S4, but we propose a different model to account for the proinflammatory nature of the resultant mutants. First, inactivation of FTL_0325 from live vaccine strain (LVS) or FTT0831c from Schu S4 resulted in temperature-dependent defects in cell viability and morphology. Loss of FTT0831c was also associated with an unusual defect in lipopolysaccharide O-antigen synthesis, but loss of FTL_0325 was not. Full restoration of these properties was observed in complemented strains expressing FTT0831c in trans , but not in strains lacking the OmpA motif, suggesting that cell wall contact is required. Finally, growth of the LVS FTL_0325 mutant in Mueller-Hinton broth at 37°C resulted in the appearance of membrane blebs at the poles and midpoint, prior to the formation of enlarged round cells that showed evidence of compromised cellular membranes. Taken together, these data are more consistent with the known structural role of OmpA-like proteins in linking the OM to the cell wall and, as such, maintenance of structural integrity preventing altered surface exposure or release of Toll-like receptor 2 agonists during rapid growth of Francisella in vitro and in vivo .

Trex1−/− mice suffer from systemic inflammation caused largely by chronic activation of the cGAS-STING-TBK1-IRF3 signaling pathway. We showed previously that Trex1-deficient cells have reduced mTORC1 activity, although the underlying mechanism is unclear. Here, we performed detailed metabolic analysis in Trex1−/− mice and cells that revealed defects in mitochondrial respiration, reduced adiposity, increased energy expenditure and glycolysis. We also found that Trex1−/− mice and cells exhibit chronically suppressed mTORC1 activity. Furthermore, we provide genetic evidence corroborated with cellular and biochemical validation to show that metabolic dysregulation in Trex1−/− mice is caused by STING-dependent activation of TBK1, but not the downstream IRF3-mediated signaling and inflammation, and that TBK1 binds to the mTORC1 complex and inhibits its activity in Trex1−/− cells. Our data thus establish chronic STING-dependent activation of TBK1 as a novel regulatory axis of mTORC1 and cellular metabolism.

Polymerase chain reaction analysis of Amblyomma americanum adults, nymphs, and larvae from Aberdeen Proving Ground, MD (APG), revealed a very high prevalence of a spotted fever group (SFG) rickettsia. Restriction fragment length polymorphism (RFLP) and sequence analysis identified "Rickettsia amblyommii." This organism is not yet described or well studied, and its pathogenicity is unknown; however, investigations of the organism are warranted because of its high prevalence in A. americanum. This tick is extremely abundant at military training facilities in the south, central, and Mid-Atlantic United States, and many soldiers experience multiple concurrent tick bites. Bites by R. amblyommii-infected A. americanum may account for rates of SFG rickettsia seropositivity that are higher than reported rates of Rocky Mountain spotted fever (RMSF) cases from the same location. Seroconversion to SFG rickettsia following bites of A. americanum may suggest that R. amblyommii is infectious in humans. Subclinical infection in the numerous A. americanum tick bite victims could contaminate donated blood and compromise immunodeficient recipients. Detection of R. amblyommii in questing A. americanum larvae suggests transovarial transmission. The absence of R. rickettsii, the agent of RMSF, in A. americanum may be due to transovarial interference by R. amblyommii. The likelihood of pathogen transmission by larvae is magnified by their habit of mass attack. The very small size of the larvae is also a risk factor for pathogen transmission. High R. amblyommii prevalence in populations of A. americanum presage co-infection with other A. americanum-borne pathogens. A. americanum nymphs and adults from APG were found to be co-infected with R. amblyommii and Borrelia lonestari, Ehrlichia chaffeensis and Ehrlichia ewingii, respectively, and larval pools were infected with both R. amblyommii and B. lonestari. Co-infections can compound effects and complicate diagnosis of tick-borne disease.

We used a nested PCR with Borrelia flagellin gene ( flaB ) primers and DNA sequencing to determine if Borrelia lonestari was present in Amblyomma americanum ticks removed from military personnel and sent to the Tick-Borne Disease Laboratory of the U.S. Army Center for Health Promotion and Preventive Medicine. In our preliminary investigation, we detected Borrelia sequences in 19 of 510 A. americanum adults and nymphs from Ft. A. P. Hill, Va. During the 2001 tick season, the flaB primers were used to test all A. americanum samples as they were received, and 29 of 2,358 A. americanum samples tested individually or in small pools were positive. PCRs with 2,146 A. americanum samples in 2002 yielded 26 more Borrelia -positive samples. The positive ticks in 2001 and 2002 were from Arkansas, Delaware, Kansas, Kentucky, Maryland, New Jersey, North Carolina, Tennessee, and Virginia. The last positive sample of the 2001 season was a pool of larvae. To further investigate larval infection, we collected and tested questing A. americanum larvae from Aberdeen Proving Ground, Md.; 4 of 33 pools (40 larvae per pool) were positive. Infection of unfed larvae provides evidence of the maintenance of B. lonestari by means of transovarial transmission. Sequence analysis revealed that the amplicons were identical to sequences of the B. lonestari flaB gene in GenBank. Despite the low prevalence of infection, the risk of B. lonestari transmission may be magnified because A. americanum is often abundant and aggressive, and many tick bite victims receive multiple bites.

Mycoplasmas cause chronic respiratory diseases in animals and humans, and vaccine development has been problematic. T cell responses were shown to confer protection as well as promote immunopathology in murine mycoplasma pneumonia. Because T cells play a critical role, the role of antigen presenting cells (APC) was examined as they likely influence either an increase in disease severity or promote protective immunity. The roles of APC, such as dendritic cells (DC) and macrophages, in mycoplasma disease are currently unknown. In this study, we examined the ability pulmonary APC populations and their contribution to T cell responses during disease pathogenesis. Both macrophages and DC increased in the lungs of mice after infection. These cell populations showed different patterns of cytokine mRNA expression, supporting the idea that these cells have different impacts on immunity in response to infection. In fact, DC from the lungs of infected mice were most capable of stimulating mycoplasma-specific T helper (Th) cell responses in vitro. In vivo, DC cells were co-localized with Th cells in inflammatory lesions in the lungs of mycoplasma-infected mice. Intratracheal inoculation of mycoplasma antigen-pulsed DC resulted in increased disease severity after subsequent infection. Thus, DC appear to be the major APC population responsible for pulmonary T cell stimulation in mycoplasma-infected mice, and these DC likely contribute to responses impacting disease pathogenesis.