Stimulator Of Interferon Genes Deficiency Research Articles

Cytosolic DNA-STING-NLRP3 axis is involved in murine acute lung injury induced by lipopolysaccharide.

The role of NOD‐like receptor protein 3 (NLRP3)‐mediated pyroptosis in acute lung injury (ALI) has been well identified previously. Stimulator of interferon genes (STING) is an indispensable adaptor protein, which could regulate inflammation and pyroptosis during infection; however, its role in lipopolysaccharide (LPS)‐induced ALI remains obscure. This study aimed to explore whether STING participated in the development of LPS‐induced ALI as well as the underlying mechanism. We confirmed that LPS significantly enhanced the expression and phosphorylation of STING in lung tissue and primary macrophages from mice. STING deficiency relieved inflammation and oxidative stress in LPS‐treated murine lungs and macrophages. Meanwhile, STING deficiency also abolished the activation of NLRP3 inflammasome and pyroptosis; however, NLRP3 overexpression by adenovirus offset the beneficial effects of STING deficiency in macrophages treated with LPS. Additionally, the level of mitochondrial DNA (mt‐DNA) significantly increased in macrophages after LPS treatment. Intriguingly, although exogenous mt‐DNA stimulation did not influence the level of STING, it could still trigger the phosphorylation of STING as well as pyroptosis, inflammation, and oxidative stress of macrophages. And the adverse effects induced by mt‐DNA could be offset after STING was knocked out. Furthermore, the inhibition of the sensory receptor of cytosolic DNA (cyclic GMP‐AMP synthase, cGAS) also blocked the activation of STING and NLRP3 inflammasome, meanwhile, it alleviated ALI without affecting the expression of STING after LPS challenge. Furthermore, cGAS inhibition also blocked the production of cGAMP induced by LPS, indicating that mt‐DNA and cGAS could activate STING‐NLRP3‐mediated pyroptosis independent of the expression of STING. Finally, we found that LPS upregulated the expression of transcription factor c‐Myc, which subsequently enhanced the activity of STING promoter and promoted its expression without affecting its phosphorylation. Collectively, our study disclosed that LPS could activate STING in a cytosolic DNA‐dependent manner and upregulate the expression of STING in a c‐Myc‐dependent manner, which cooperatively contribute to ALI.

STING couples with PI3K to regulate actin reorganization during BCR activation.

The adaptor protein, STING (stimulator of interferon genes), has been rarely studied in adaptive immunity. We used Sting KO mice and a patient's mutated STING cells to study the effect of STING deficiency on B cell development, differentiation, and BCR signaling. We found that STING deficiency promotes the differentiation of marginal zone B cells. STING is involved in BCR activation and negatively regulates the activation of CD19 and Btk but positively regulates the activation of SHIP. The activation of WASP and accumulation of F-actin were enhanced in Sting KO B cells upon BCR stimulation. Mechanistically, STING uses PI3K mediated by the CD19-Btk axis as a central hub for controlling the actin remodeling that, in turn, offers feedback to BCR signaling. Overall, our study provides a mechanism of how STING regulates BCR signaling via feedback from actin reorganization, which contributes to positive regulation of STING on the humoral immune response.

STING is an essential regulator of heart inflammation and fibrosis in mice with pathological cardiac hypertrophy via endoplasmic reticulum (ER) stress

Pathological cardiac hypertrophy is characterized by myocyte enlargement and cardiac dysfunction. However, the pathogenesis for this disease is still poorly understood. Stimulator of interferon genes (STING) could meditate inflammation and immune response in various kinds of diseases. In this work, we demonstrated that STING was critical for pressure overload-induced cardiac hypertrophy. Results showed that STING expression was up-regulated in human and mouse hypertrophic hearts. STING knockout attenuated cardiac hypertrophy induced by aortic banding (AB). The effects of STING deficiency on the improvement of cardiac hypertrophy and dysfunction were associated with the restrained macrophage infiltration, inflammatory response and fibrosis. Moreover, ER stress was detected in hearts of AB-operated mice, as evidenced by the increased expression of phospho-protein kinase RNA-like endoplasmic reticulum kinase (PERK), phospho-eukaryotic initiation factor 2 alpha (eIF2α) and phospho-inositol-requiring kinase (IRE)-1α. Importantly, these proteins were restrained in mice with STING knockout after AB surgery. What’s more, angiotensin II (Ang II)-induced STING could be accelerated by ER stress activator, while being markedly abolished by the ER stress inhibitor. We then found that whether co-treated with or without transforming growth factor-beta 1 (TGF-β1), cardiac fibroblasts cultured in the conditional medium (CM) from Ang II-incubated cardiomyocytes with STING knockdown exhibited significantly reduced fibrosis, as displayed by the clearly down-regulated expression of α-SMA, Collagen type I (Col I) and Collagen type III (Col III). Therefore, we defined STING as an important signal contributing to cardiac hypertrophy closely associated with ER stress.

The Innate Immune Sensor Sting Promotes Donor CD8+ T Cell Activation and Recipient APC Death Early after Preclinical Allogeneic Hematopoietic Stem Cell Transplantation

Graft-versus-host disease (GVHD) remains a significant cause of morbidity and mortality in patients receiving allogeneic hematopoietic stem cell transplants (aHSCTs). Pre-HSCT chemoradiation results in the death of dividing cells and release of endogenous danger signals. These molecules drive the activation of antigen presenting cells (APCs) and the differentiation of allo-reactive donor T cells. We identified a role for Stimulator of Interferon Genes (STING), an innate immune sensor, in GVHD using pre-clinical MHC-matched unrelated donor (MUD) HSCT models. Here we show that STING rapidly promotes donor CD8⁺ T cell activation and recipient APC death early after aHSCT. Post MUD HSCT, we previously found a >2x reduction in IFNβ, TNFα and IL-6 mRNA in STING^−/− vs WT recipient colonic mRNA expression (48 hrs) as well as decreased weight loss, GVHD scores and skin pathology (6 wks) vs WT. Chimeric studies showed that STING loss in non-hematopoietic cells induced this effect. Conversely, we recently found a single early (<D14), but not late (>D100), dose of the STING agonist DMXAA increased GVHD scores and lethality in WT, but not STING^−/−, recipients. Thus, the activation of this pathway can promote GVHD following MUD HSCT. Furthermore, mice homozygous for a murine homologue of a human allele associated with diminished STING activity (STING^HAQ/HAQ) also exhibited reduced GVHD after MUD HSCT, suggesting potential clinical importance. Interestingly, our findings that STING deficiency ameliorates GVHD in MUD HSCT (Bader CS, et al, in revision <i>Sci. Transl. Med.</i>) contrasts reported observations that STING deficiency can exacerbate GVHD after MHC-mismatched (MMUD) HSCT. Since CD4⁺ and CD8⁺ T cells are central in MMUD and MUD GVHD, respectively, we transplanted MMUD BALB/c BM + CD8⁺ T cells into B6-WT and STING^−/− mice and notably, found that STING^−/- recipients now developed reduced GVHD clinical scores, skin pathology and frequencies of activated T cells 8 wks post-HSCT vs WT. We also found that STING^−/− mice had greater numbers of recipient splenic CD11b⁺CD11c⁺ APCs and these cells expressed reduced MHC I protein 1 day after MMUD B6 into BALB/c aHSCT vs WT (Fig. A, B). Moreover, STING^−/− recipient spleens contained lower numbers of donor CD8⁺ T cells producing IFNγ and TNFα (Fig. C), supporting the hypothesis that STING contributes to early activation of donor CD8⁺ T cells and loss of recipient APCs. Next, to identify if reduced host MHC II⁺ APCs affected donor CD4⁺ T cell activation, B6-Nur77^GFP T cells were used to explicitly monitor T cell receptor signaling. Indeed, STING^−/− spleens had greater numbers of donor Nur77^GFP CD4⁺ T cells expressing GFP, CD69 and IFNγ 6 days post-HSCT (Fig. D). Overall, these data provide a mechanism by which STING could promote CD8⁺ T cell-mediated GVHD yet diminish CD4⁺-mediated GVHD. Ongoing studies are exploring the use of STING agonists to augment GVL following aHSCT.

The Innate Immune Sensor Sting Promotes Donor CD8+ T Cell Activation and Recipient APC Death Early after Preclinical Allogeneic Hematopoietic Stem Cell Transplantation

Graft-versus-host disease (GVHD) remains a significant cause of morbidity and mortality in patients receiving allogeneic hematopoietic stem cell transplants (aHSCTs). Pre-HSCT chemoradiation results in the death of dividing cells and release of endogenous danger signals. These molecules drive the activation of antigen presenting cells (APCs) and the differentiation of allo-reactive donor T cells. We identified a role for Stimulator of Interferon Genes (STING), an innate immune sensor, in GVHD using pre-clinical MHC-matched unrelated donor (MUD) HSCT models. Here we show that STING rapidly promotes donor CD8+ T cell activation and recipient APC death early after aHSCT. Post MUD HSCT, we previously found a >2x reduction in IFNβ, TNFα and IL-6 mRNA in STING−/− vs WT recipient colonic mRNA expression (48 hrs) as well as decreased weight loss, GVHD scores and skin pathology (6 wks) vs WT. Chimeric studies showed that STING loss in non-hematopoietic cells induced this effect. Conversely, we recently found a single early ( D100), dose of the STING agonist DMXAA increased GVHD scores and lethality in WT, but not STING−/−, recipients. Thus, the activation of this pathway can promote GVHD following MUD HSCT. Furthermore, mice homozygous for a murine homologue of a human allele associated with diminished STING activity (STINGHAQ/HAQ) also exhibited reduced GVHD after MUD HSCT, suggesting potential clinical importance. Interestingly, our findings that STING deficiency ameliorates GVHD in MUD HSCT (Bader CS, et al, in revision Sci. Transl. Med.) contrasts reported observations that STING deficiency can exacerbate GVHD after MHC-mismatched (MMUD) HSCT. Since CD4+ and CD8+ T cells are central in MMUD and MUD GVHD, respectively, we transplanted MMUD BALB/c BM + CD8+ T cells into B6-WT and STING−/− mice and notably, found that STING−/- recipients now developed reduced GVHD clinical scores, skin pathology and frequencies of activated T cells 8 wks post-HSCT vs WT. We also found that STING−/− mice had greater numbers of recipient splenic CD11b+CD11c+ APCs and these cells expressed reduced MHC I protein 1 day after MMUD B6 into BALB/c aHSCT vs WT (Fig. A, B). Moreover, STING−/− recipient spleens contained lower numbers of donor CD8+ T cells producing IFNγ and TNFα (Fig. C), supporting the hypothesis that STING contributes to early activation of donor CD8+ T cells and loss of recipient APCs. Next, to identify if reduced host MHC II+ APCs affected donor CD4+ T cell activation, B6-Nur77GFP T cells were used to explicitly monitor T cell receptor signaling. Indeed, STING−/− spleens had greater numbers of donor Nur77GFP CD4+ T cells expressing GFP, CD69 and IFNγ 6 days post-HSCT (Fig. D). Overall, these data provide a mechanism by which STING could promote CD8+ T cell-mediated GVHD yet diminish CD4+-mediated GVHD. Ongoing studies are exploring the use of STING agonists to augment GVL following aHSCT.

STING-IRF3 contributes to lipopolysaccharide-induced cardiac dysfunction, inflammation, apoptosis and pyroptosis by activating NLRP3

Mountainous evidence suggests that inflammation, cardiomyocyte apoptosis and pyroptosis are involved in the development of sepsis and sepsis-induced cardiomyopathy (SIC). Stimulator of interferon genes (STING) is an indispensable molecule that could regulate inflammation and immune response in multiple diseases. However, the role of STING in cardiovascular disease, especially SIC remains unclear. This study was designed to investigate the potential molecular mechanisms of STING in lipopolysaccharide (LPS)-induced cardiac injury using STING global knockout mice. In wild type mice and cardiomyocytes, LPS stimulation triggered the perinuclear translocation of STING, which further bound to Type-I interferons (IFN) regulatory factor 3 (IRF3) and phosphorylated IRF3. Phosphorylated (P-) IRF3 subsequently translocated into nucleus and increased the expression of NOD-like receptor protein 3 (NLRP3). Knockout of STING in mice significantly improved survival rate and cardiac function, apart from suppressing myocardial and serum inflammatory cytokines, apoptosis, as well as cardiomyocyte pyroptosis. In vitro experiments revealed that NLRP3 overexpression by adenovirus could offset protective effects of STING knockdown in LPS-induced cardiomyocytes. Additionally, LPS stimulation also promoted the production of intracellular reactive oxygen (ROS), which further induced the NLRP3 translocation to the cytoplasm from the nucleus. Dissociative TXNIP could directly interact with cytoplasmic NLRP3 and form inflammasome, eventually triggering cardiomyocyte injury. Collectively, our findings disclose that STING deficiency could alleviate LPS-induced SIC in mice. Hence, targeting STING in cardiomyocytes may be a promising therapeutic strategy for preventing SIC.

Oncolytic virus immunotherapy induces immunogenic cell death and overcomes STING deficiency in melanoma

ABSTRACTSuccessful immunotherapy for melanoma depends on the recruitment of effector CD8+ T cells to the tumor microenvironment. Factors contributing to T cell regulation in melanoma have recently been recognized, including the stimulator of interferon genes (STING). Agents that can activate STING or enhance T cell infiltration into established tumors have become an important focus for further clinical development. Talimogene laherparepvec (T-VEC) is an oncolytic herpes simplex virus, type 1 (HSV-1) encoding granulocyte-macrophage colony stimulating factor (GM-CSF) and is approved for the treatment of melanoma and has shown therapeutic activity in murine tumors known to express high levels of STING. The mechanism of action for T-VEC has not been fully elucidated but is thought to include induction of immunogenic cell death (ICD) and activation of host anti-tumor immunity. Thus, we sought to investigate how T-VEC mediates anti-tumor activity in a melanoma model. To determine if T-VEC induced ICD we established the relative sensitivity of a panel of melanoma cell lines to T-VEC oncolysis. Following T-VEC infection in vitro, melanoma cell lines released of HMGB1, ATP, and translocated ecto-calreticulin. To identify potential mediators of this effect, we found that melanoma cell sensitivity to T-VEC was inversely related to STING expression. CRISPR/Cas9-STING knockout was also associated with increased T-VEC cell killing. In the D4M3A melanoma, which has low expression of STING and is resistant to PD-1 blockade therapy, T-VEC was able to induce therapeutic responses in both injected and non-injected tumors and demonstrated recruitment of viral- and tumor-antigen specific CD8+ T cells, and induction of a pro-inflammatory gene signature at both injected and non-injected tumors. These data suggest that T-VEC induces ICD in-vitro and promotes tumor immunity and can induce therapeutic responses in anti-PD-1-refractory, low STING expressing melanoma.

The Innate Immune Sensor Sting Promotes CD8+ T Cell-Mediated Gvhd after Preclinical Allogeneic Hematopoietic Stem Cell Transplantation

Graft-versus-host disease (GVHD) remains a significant cause of morbidity and mortality in patients receiving allogeneic hematopoietic stem cell transplants (aHSCTs). Pre-HSCT conditioning typically consists of irradiation and drug administration resulting in the death of rapidly dividing cells. Damage to host tissues initiates a cytokine storm, promoting activation and expansion of donor anti-host alloreactive T cells. Cell death following conditioning has promoted the hypothesis that sensors of cytoplasmic DNA damage in GVHD target tissues contribute to cytokine production. We identified a role for Stimulator of Interferon Genes (STING), an innate immune sensor, in GVHD using pre-clinical MHC-matched (MUD) aHSCT models. Here, we show that the STING pathway promotes CD8+ T cell-mediated GVHD after MHC matched and MHC-mismatched aHSCT. To assess a role for STING after MHC-matched aHSCT, we transplanted C3H.SW BM + unfractionated T cells into irradiated B6-WT or STING−/− recipients. STING−/− recipients developed reduced weight loss, GVHD scores and skin pathology 6 wks post-HSCT. Chimeric studies demonstrated that the absence of STING in non-hematopoietic cells was responsible for the amelioration of GVHD. Therefore, to test STING signaling in non-hematopoietic intestinal cells, we generated intestinal organoid cultures which upregulated IFNβ, TNFα, IL-6, CXCL10 and MHC class I mRNA 6 hrs after stimulation with the highly specific STING agonist DMXAA, supporting the notion that STING can contribute to GI inflammation in vivo (Fig. 1A). To evaluate the potential impact of STING in the clinical setting, we examined MHC-matched recipients homozygous for a human allele associated with diminished STING activity and found STINGHAQ/HAQ mice exhibited diminished GVHD vs WT. Since MHC-matched GVHD is primarily mediated by CD8+ T cells, we next asked if CD4+ T cells were dispensable for the reduction of GVHD in STING−/− recipients. Indeed, GVHD was reduced in STING−/− recipients after transplant of C3H.SW BM and CD8+ T cells (Fig. 1B,C). Notably, STING deficiency was recently reported to worsen GVHD after MHC-mismatched aHSCT (Fischer J, et al, Sci. Transl. Med. 2017). Since CD4+ T cells are dominant during MHC-mismatched GVHD, we transplanted BALB/c BM + CD8+ T cells into B6-WT and STING−/− mice. Again, STING−/- recipients developed lower GVHD clinical scores, reduced skin pathology and had lower frequencies of activated T cells 8 wks post-HSCT vs WT, supporting a role for STING in the promotion of CD8+ T cell-mediated GVHD – consistent with this pathways upregulation of MHC class I (Fig. 1D,E). In total, these studies demonstrate that STING plays an important role in regulating aHSCT and promotes CD8+ T cell-mediated GVHD regardless of the genetic disparity between donor and recipient. The findings support the notion that this pathway may provide a target for new strategies to ameliorate GVHD.

STING signalling protects against chronic pancreatitis by modulating Th17 response

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)...

The Innate Immune Sensor Sting Regulates Intestinal Inflammation and GVHD after Allogeneic Hematopoietic Stem Cell Transplantation in Knock-out and Human Allele Knock-in Recipient Mice

Graft-versus-host disease (GVHD) remains a significant cause of morbidity and mortality in patients receiving allogeneic hematopoietic stem cell transplants (allo-HSCTs). Pre-HSCT conditioning typically consists of irradiation and drug administration resulting in the death of rapidly dividing cells. Damage to host tissues initiates a cytokine storm, promoting activation and expansion of donor anti-host alloreactive T cells. Cell death following conditioning has promoted the hypothesis that sensors of cytoplasmic DNA damage in GVHD target tissues contribute to cytokine production. We identified a role for Stimulator of Interferon Genes (STING), an innate immune sensor, in GVHD using pre-clinical MHC-matched (MUD) allo-HSCT models. Our studies show that the STING pathway rapidly regulates cytokine production in the intestinal tract and non-hematopoietic cells can contribute to these responses. Using mice expressing a human STING allele associated with decreased STING activity (Patel S, et al, J Immunol. 2016), we demonstrate its potential clinical importance.To assess STING involvement immediately post-HSCT, cytokine mRNA expression was examined 48 hrs after transplant of C3H.SW bone marrow (BM) + T cells into irradiated B6-WT or STING-/- recipients. Colonic tissue from STING-/- recipients had >2x reduction in IFNβ, TNFα and IL-6 mRNA vs. WT. On day 10 post-transplant, colons from STING-/- recipients exhibited reduced inflammation and overall pathology scores than WT. MHC-matched STING-/- HSCT recipients also experienced decreased weight loss, GVHD scores and skin pathology 6 weeks post-HSCT vs. WT. Chimeric studies demonstrated that the absence of STING in non-hematopoietic cells was responsible for the amelioration of GVHD. Therefore, to test STING signaling in non-hematopoietic intestinal cells, we generated intestinal organoid cultures. Intestinal organoids upregulated IFNβ, TNFα, IL-6 and CXCL10 mRNA 6hrs after stimulation with the highly specific STING agonist DMXAA, supporting the notion that STING in intestinal tissues can contribute to inflammation in vivo. Interestingly, expression of these cytokines returned to baseline levels 24 hrs after stimulation (Fig. 1A). Next, we posited that if the absence of the STING pathway in recipients ameliorated GVHD after MHC-matched HSCT, pathway stimulation would exacerbate GVHD. B6-WT mice were injected with DMXAA immediately prior to HSCT with donor C3H.SW BM + T cells. Administration of a single dose of DMXAA increased expression of IFNβ, TNFα and IL-6 mRNA in colon tissue 48 hrs after transplant (Fig. 1B). Importantly, DMXAA treatment of WT - but not STING-/- - recipients significantly increased GVHD scores and lethality post-HSCT. To evaluate the potential impact of STING in the clinical setting, we evaluated recipients after transplant of C3H.SW BM + T cells into mice homozygous for a human allele associated with diminished STING activity (HAQ-MPYS knock-in mice, termed B6N-STINGHAQ/HAQ here) and found that STINGHAQ/HAQ mice contained a lower frequency of donor T cells expressing an activated phenotype (CD44hiCD62Llo) vs. WT recipients and the former also exhibited diminished GVHD (Fig. 1C,D). In contrast to STING knock-out recipients completely lacking protein, these results indicate that reduced STING activity can also affect GVHD.Interestingly, our findings that STING deficiency ameliorates GVHD in MHC-matched allo-HSCT contrast reported observations that STING activation can exacerbate GVHD after MHC-mismatched HSCT (Fischer J, et al, Sci. Transl. Med. 2017). We are currently investigating how the STING pathway regulates CD4+ and CD8+ T cell mediated GVHD and initial findings may provide insight into understanding the pathway's involvement in MHC-matched vs. mismatched allo-HSCT. In total, our studies demonstrate that STING plays an important role in regulating allo-HSCT and suggest this pathway can provide a target for new therapeutic strategies to ameliorate GVHD. DisclosuresLevy:Allergan: Consultancy; Capricor Therapeutics: Consultancy; HEAT Biologics: Consultancy, Membership on an entity's Board of Directors or advisory committees; Pelican Therapeutics: Consultancy; OccuRx: Research Funding; Shire: Research Funding.

230 - The Innate Immune Sensor Sting Can Augment or Ameliorate Graft-Versus-Host Disease Dependent on the Genetic Disparity between Donors and Recipients

Abstract Graft-versus-host disease (GVHD) remains a significant cause of morbidity and mortality in patients receiving allogeneic hematopoietic stem cell transplants (allo-HSCTs). Pre-HSCT conditioning typically consists of irradiation and drug administration which results in the death of rapidly dividing cells and the initiation of a cytokine storm, promoting activation and expansion of donor anti-host alloreactive T cells in HSCT recipients. However, the precise mechanism of cytokine production remains unclear. Cell death following pre-transplant conditioning has promoted the hypothesis that sensors of cytoplasmic DNA damage in GVHD target tissues contribute to cytokine production. One such sensor is Stimulator of Interferon Genes (STING) which following activation induces phosphorylation of IRF3 and IκBα. Although STING activation was recently reported to worsen GVHD after MHC-mismatched allo-HSCT (Fischer J, et al, Sci. Transl. Med. 2017), STING involvement in MHC-matched allo-HSCT has not yet been thoroughly evaluated. Here, using B6-STING knock-out recipients and either MHC-mismatched or matched (“MUD”) donor strains we corroborate that STING deficiency worsens - but in contrast - ameliorates GVHD in the former and latter pre-clinical mouse models, respectively. To evaluate the effect of STING deficiency on GVHD after MHC-mismatched allo-HSCT, BALB/c BM + T cells were transplanted into 11.5Gy irradiated B6-WT and B6-STING-/- recipients. Consistent with previously reported results using B6-STINGgt/gt mice, STING-/- recipients experienced increased weight loss and GVHD clinical scores post-HSCT as well as decreased survival relative to WT recipients (Fig. 1A). Notably, when irradiation was given on day 0 instead of day -1 using the same donor/recipient combination no significant difference in GVHD was observed between WT and STING-/- recipients (Fig. 1B & 1E). We next investigated the role of STING after clinically relevant models of MHC-matched allo-HSCT. To assess for a role by STING immediately after HSCT, we examined cytokine mRNA expression 48 hrs. after transplant of LP/J BM + T cells → 8.5Gy irradiated B6-WT and B6-STING-/- recipients. Colonic tissue from STING-/- recipients had reproducibly >2x reduction in IFNβ, TNFα and IL-6 mRNA compared to WT (Fig. 1C). Importantly, IFNβ is one of the major downstream effector molecules produced as a result of IRF3 phosphorylation, consistent with the notion that STING activation occurs shortly after conditioning and/or allo-HSCT. Notably, LN examination at this time demonstrated fewer donor CD8+ T cells and CD8+CD44hiCD62Llo cells in STING-/- vs WT recipients. In contrast to STING-/- recipients of MHC-mismatched HSCT, MHC-matched STING-/- HSCT recipients experienced decreased weight loss and GVHD clinical scores vs. WT mice. STING-/- recipients contained 20-50% fewer donor lymphocytes and a significantly reduced frequency of activated donor CD4+ and CD8+ T cells in lymphoid tissues on D7. Analysis of peripheral LN and spleen 1-3 mo. post-HSCT revealed a similar profile and a higher frequency of naive T cells - consistent with their decreased clinical signs of disease. Histological examination of recipient skin also showed higher pathology scores in WT recipients relative to STING-/- recipients. These findings were corroborated using a second MUD model, i.e. C3H.SW → B6 (Fig. 1D). Furthermore, measurement of serum cytokines 6 wks. post-transplant showed that B6-STING-/- recipients had higher levels of the immunosuppressive cytokine IL-10. The effect of donor T cell dose was also examined. Increased donor T cell numbers resulted in enhanced GVHD clinical scores in STING-/- recipients, however, the GVHD remained less severe than that observed in WT. Notably, HSCT studies using fully reconstituted chimeric B6-CD45.1↔B6-STING-/- indicated that STING expression in non-hematopoietic tissues is important for the development of GVHD. In total, this is the first report we are aware of in which the same pathway appears to differentially impact the outcome of allogeneic HSCT based upon the genetic disparity across the transplant. These findings reveal that STING's contribution to the development of GVHD apparently differs depending on the presence or absence of an MHC-disparity between donors and recipients as well as the timing of the pathway's activation and HSCT. Download : Download high-res image (232KB) Download : Download full-size image Disclosures Levy: Capricor Therapeutics: Consultancy; OccuRx: Research Funding; Shire: Research Funding; Allergan: Consultancy.

Stimulator of Interferon Genes Deficiency in Acute Exacerbation of Idiopathic Pulmonary Fibrosis.

The stimulator of interferon genes (STING) is a key adaptor protein mediating innate immune defense against DNA viruses. To investigate the role of STING in acute exacerbation of idiopathic pulmonary fibrosis (AE-IPF), we isolated primary peripheral blood mononuclear cells (PBMCs) from patients and healthy controls (HCs). Raw264.7 and A549 cells were infected with herpes simplex virus type 1 (HSV-1). Mice with bleomycin-induced lung fibrosis were infected with HSV-1 to stimulate acute exacerbation of the lung fibrosis. Global gene expression profiling revealed a substantial downregulation of interferon-regulated genes (downstream of STING) in the AE-IPF group compared with the HC and stable IPF groups. The PBMCs of the AE-IPF group showed significantly reduced STING protein levels, increased levels of endoplasmic reticulum (ER) stress markers, and elevated apoptosis. HSV-1 infection decreased STING expression and stimulated the ER stress pathways in Raw264.7 and A549 cells in a time- and dose-dependent manner. HSV-1 infection exacerbated the bleomycin-induced lung injury in mice. In the primary bone marrow-derived macrophages of mice treated with bleomycin and HSV-1, STING protein expression was substantially reduced; ER stress was stimulated. Tauroursodeoxycholic acid, a known inhibitor of ER stress, partially reversed those HSV-1-mediated adverse effects in mice with bleomycin-induced lung injury. STING levels in PBMCs increased after treatment in patients showing improvement but remained at low levels in patients with deterioration. Viral infection may trigger ER stress, resulting in STING deficiency and AE-IPF onset.

The Innate Immune Sensor Sting Can Augment or Ameliorate Graft-Versus-Host Disease Dependent on the Genetic Disparity between Donors and Recipients

Graft-versus-host disease (GVHD) remains a significant cause of morbidity and mortality in patients receiving allogeneic hematopoietic stem cell transplants (allo-HSCTs). Pre-HSCT conditioning typically consists of irradiation and drug administration which results in the death of rapidly dividing cells and the initiation of a cytokine storm, promoting activation and expansion of donor anti-host alloreactive T cells in HSCT recipients. However, the precise mechanism of cytokine production remains unclear. Cell death following pre-transplant conditioning has promoted the hypothesis that sensors of cytoplasmic DNA damage in GVHD target tissues contribute to cytokine production. One such sensor is Stimulator of Interferon Genes (STING) which following activation induces phosphorylation of IRF3 and IκBα. Although STING activation was recently reported to worsen GVHD after MHC-mismatched allo-HSCT (Fischer J, et al, Sci. Transl. Med. 2017), STING involvement in MHC-matched allo-HSCT has not yet been thoroughly evaluated. Here, using B6-STING knock-out recipients and either MHC-mismatched or matched (“MUD”) donor strains we corroborate that STING deficiency worsens - but in contrast - ameliorates GVHD in the former and latter pre-clinical mouse models, respectively.To evaluate the effect of STING deficiency on GVHD after MHC-mismatched allo-HSCT, BALB/c BM + T cells were transplanted into 11.5Gy irradiated B6-WT and B6-STING-/- recipients. Consistent with previously reported results using B6-STINGgt/gt mice, STING-/- recipients experienced increased weight loss and GVHD clinical scores post-HSCT as well as decreased survival relative to WT recipients (Fig. 1A). Notably, when irradiation was given on day 0 instead of day -1 using the same donor/recipient combination no significant difference in GVHD was observed between WT and STING-/- recipients (Fig. 1B & 1E).We next investigated the role of STING after clinically relevant models of MHC-matched allo-HSCT. To assess for a role by STING immediately after HSCT, we examined cytokine mRNA expression 48 hrs. after transplant of LP/J BM + T cells → 8.5Gy irradiated B6-WT and B6-STING-/- recipients. Colonic tissue from STING-/- recipients had reproducibly >2x reduction in IFNβ, TNFα and IL-6 mRNA compared to WT (Fig. 1C). Importantly, IFNβ is one of the major downstream effector molecules produced as a result of IRF3 phosphorylation, consistent with the notion that STING activation occurs shortly after conditioning and/or allo-HSCT. Notably, LN examination at this time demonstrated fewer donor CD8+ T cells and CD8+CD44hiCD62Llo cells in STING-/- vs WT recipients. In contrast to STING-/- recipients of MHC-mismatched HSCT, MHC-matched STING-/- HSCT recipients experienced decreased weight loss and GVHD clinical scores vs. WT mice. STING-/- recipients contained 20-50% fewer donor lymphocytes and a significantly reduced frequency of activated donor CD4+ and CD8+ T cells in lymphoid tissues on D7. Analysis of peripheral LN and spleen 1-3 mo. post-HSCT revealed a similar profile and a higher frequency of naïve T cells - consistent with their decreased clinical signs of disease. Histological examination of recipient skin also showed higher pathology scores in WT recipients relative to STING-/- recipients. These findings were corroborated using a second MUD model, i.e. C3H.SW → B6 (Fig. 1D). Furthermore, measurement of serum cytokines 6 wks. post-transplant showed that B6-STING-/- recipients had higher levels of the immunosuppressive cytokine IL-10. The effect of donor T cell dose was also examined. Increased donor T cell numbers resulted in enhanced GVHD clinical scores in STING-/- recipients, however, the GVHD remained less severe than that observed in WT. Notably, HSCT studies using fully reconstituted chimeric B6-CD45.1↔B6-STING-/- indicated that STING expression in non-hematopoietic tissues is important for the development of GVHD.In total, this is the first report we are aware of in which the same pathway appears to differentially impact the outcome of allogeneic HSCT based upon the genetic disparity across the transplant. These findings reveal that STING's contribution to the development of GVHD apparently differs depending on the presence or absence of an MHC-disparity between donors and recipients as well as the timing of the pathway's activation and HSCT. [Display omitted] DisclosuresLevy:Capricor Therapeutics: Consultancy; OccuRx: Research Funding; Shire: Research Funding; Allergan: Consultancy.

STING-IRF3 Triggers Endothelial Inflammation in Response to Free Fatty Acid-Induced Mitochondrial Damage in Diet-Induced Obesity.

Metabolic stress in obesity induces endothelial inflammation and activation, which initiates adipose tissue inflammation, insulin resistance, and cardiovascular diseases. However, the mechanisms underlying endothelial inflammation induction are not completely understood. Stimulator of interferon genes (STING) is an important molecule in immunity and inflammation. In the present study, we sought to determine the role of STING in palmitic acid-induced endothelial activation/inflammation. In cultured endothelial cells, palmitic acid treatment activated STING, as indicated by its perinuclear translocation and binding to interferon regulatory factor 3 (IRF3), leading to IRF3 phosphorylation and nuclear translocation. The activated IRF3 bound to the promoter of ICAM-1 (intercellular adhesion molecule 1) and induced ICAM-1 expression and monocyte-endothelial cell adhesion. When analyzing the upstream signaling, we found that palmitic acid activated STING by inducing mitochondrial damage. Palmitic acid treatment caused mitochondrial damage and leakage of mitochondrial DNA into the cytosol. Through the cytosolic DNA sensor cGAS (cyclic GMP-AMP synthase), the mitochondrial damage and leaked cytosolic mitochondrial DNA activated the STING-IRF3 pathway and increased ICAM-1 expression. In mice with diet-induced obesity, the STING-IRF3 pathway was activated in adipose tissue. However, STING deficiency (Stinggt/gt ) partially prevented diet-induced adipose tissue inflammation, obesity, insulin resistance, and glucose intolerance. The mitochondrial damage-cGAS-STING-IRF3 pathway is critically involved in metabolic stress-induced endothelial inflammation. STING may be a potential therapeutic target for preventing cardiovascular diseases and insulin resistance in obese individuals.