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Supplementary Figures 1-3

S1. VSV-hIFNβ treatment followed by RT caused S-phase cell cycle arrest of prostate cancer cells. S2. VSV treatment in combination with radiation therapy increases the GFP or Luciferase expression in vitro and in vivo. S3. C57BL/6 CD8 Re-challenge study with VSV-mIFNβ+RT followed by CD8 depletion.

Mycobacterium tuberculosis (Mtb) infection remains a major public health concern. The STING (stimulator of interferon genes) pathway contributes to the cytosolic surveillance of host cells. Most studies on the role of STING activation in Mtb infection have focused on macrophages. Moreover, a detailed investigation of the role of STING during Mtb infection in vivo is required. Here, we deciphered the involvement of STING in the activation of dendritic cells (DCs) and the host response to Mtb infection in vivo. In DCs, this adaptor molecule was important for Ifn-β expression and IL-12 production as well as for the surface expression of the activation markers CD40 and CD86. We also documented that Mtb DNA induces STING activation in murine fibroblasts. In vivo Mtb aerogenic infection induced the upregulation of the STING and cGAS (cyclic GMP-AMP synthase) genes, and Ifn-β pulmonary expression was dependent on both sensors. However, mice deficient for STING or cGAS presented a similar outcome to wild-type controls, with no major alterations in body weight gain, bacterial burden, or survival. Lung inflammation, proinflammatory cytokine production, and inflammatory cell recruitment were similar in STING- and cGAS-deficient mice compared to wild-type controls. In summary, although the STING pathway seems to be crucial for DC activation during Mtb infection, it is dispensable for host protection in vivo.

Recombinant vesicular stomatitis virus expressing interferon-β (VSV-IFN-β) has demonstrated antitumor activity in vitro and in vivo. In preparation for clinical testing in human squamous cell carcinoma (SCC) of the head and neck, we conducted preclinical studies of VSV-IFN-β in syngeneic SCC models.In vitro, VSV-IFN-β (expressing rat or mouse interferon [IFN]-β)-induced cytotoxicity and propagated in rat (FAT-7) or mouse (SCC-VII) SCC cells during normoxia and hypoxia. In vivo, intratumoral administration of VSV-rat-IFN-β or VSV-human-IFN-β in FAT-7 bearing or non-tumor bearing immunocompetent rats did not result in acute organ toxicity or death.VSV-r-IFN-β replicated predominantly in tumors and a dose dependent anti-VSV antibody response was observed. Intratumoral or intravenous administration of VSV-IFN-β resulted in growth delay and improved survival compared with controls.The above data confirm safety and feasibility of VSV-IFN-β administration in immunocompetent animals and support its clinical evaluation in advanced human head and neck cancer.

The innate immune system is critical for the early detection of invading pathogens and for initiating cellular host defense counter measures, which include the production of type I interferon (IFN). Immune dysfunction develops in patients with many cancer types and may contribute to tumor progression and failure of immunotherapy. This study will investigate the role of a newly identified IFN-mediated innate immunity pathway in immune surveillance, and the inactivation of a critical component of the pathway, STING, as a mechanism for immuo-escape in the development and progression of breast cancer. We propose a pilot study to test this hypothesis, that innate immunity pathway involved STING plays a role in purging pre-neoplastic cells and suppression of STING is a mechanism for escape of breast cancer cells from immune surveillance. Over the course of the study, we determined that STING was absent from many human breast cancer cell lines, at both the RNA and protein level. This caused the lack of IFN beta; secretion in response to intracellular DNA. In human breast cancer tissue, we observed the presence of human retroviral elements and up-regulation of a number of immune response genes, as compared to normal human tissue.

Polyomaviruses (PyV) are nonenveloped DNA viruses that persist as low-level asymptomatic infections in a variety of vertebrates, including humans. In immunosuppressed individuals, the human BK and JC PyVs may cause transplant-associated nephropathy and progressive multifocal leukoencephalopathy, respectively. Mouse polyomavirus (MPyV) allows investigation of PyV immunity in a natural host. After caveolae-/clathrin-mediated endocytosis, PyV virions traffic in microtubule-guided microvesicles from endosomes to the ER, where uncoating is initiated. Unknown is whether complete uncoating occurs in the cytosol, exposing the viral DNA to cytosolic DNA sensors, before it is transported to the nucleus where viral replication and assembly occur. Here, we report that persistent MPyV infection in mice is associated with chronically elevated IFN-β transcripts, and that WT primary mouse embryo fibroblasts (MEFs) infected with MPyV upregulate IFN-β, TNF-α and ISG (PKR and MDA-5) mRNAs. However, MPyV infection failed to induce expression for these transcripts in MEFs from STING-/- mice. Interestingly, MPyV acute infection activated NK cells in WT, but not in IFNαβR-/- mice or in STING-deficient (Goldenticket) mice. Together, these data suggest that MPyV-induced activation of NK cells requires Type I IFN and a STING-dependent cytosolic DNA sensing pathway(s). Moreover, these data provide strong evidence that PyV DNA is exposed in the cytosol and activate innate immune sensors.

Immunity against Brucella abortus depends on the recognition of pathogen-associated molecular patterns (PAMPs) by pattern recognition receptors (PRRs). Signaling pathways triggered by Brucella DNA involves TLR9, AIM2 and possibly STING. Previous studies reported by our research group showed that B. abortus-induced IFN-b is dependent on STING. Herein, we demonstrated that STING is required for Brucella abortus control in vivo. STING KO mice showed enhanced susceptibility at three- and six-weeks post-infection compared to wild-type animals. Furthermore, we observed reduced levels of IFN-g production in spleen cells of STING KO compared to wild-type that corroborates with enhanced susceptibility. Moreover, additional results showed that STING plays an important role in the response to infection in vitro. Macrophages derived from STING KO mice showed reduction in IL-12 and IL-1b cytokine production, when infected with the bacteria or stimulated with bacterial DNA. Diminished production of IL-1b in KO macrophages implicates STING as possible regulator of inflammasome activation. Additionally, murine embryonic fibroblasts (MEFs) from STING KO mice produced much less IL-6 when stimulated with Brucella DNA compared to wild-type MEFs. In summary, we demonstrated the importance of STING as a cytosolic sensor during Brucella abortus infection and its contribution to bacterial control in vivo.

Activation of Stimulator of Interferon genes (STING) adaptor causes the release of IFN αβ in most of the cells, an activity that is tightly controlled to avoid continuous immune activation that could lead to autoimmunity. Here we show that STING activators can be used systemically to suppress experimental autoimmune encephalitis following myelin oligodendrocyte (MOG) immunization through STING-IFN αβ pathway. DNA nanoparticles (DNPs) attenuated MOG-specific effector responses in CNS and spleen. Therapeutic responses to DNPs are dependent on STING adaptor and IFN αβ receptors as they were abolished in mice lacking those genes. Additionally, indoleamine 2,3 dioxygenase enzyme (IDO) is crucial for therapeutic responses to DNPs as the loss of IDO1 gene in hematopoietic cells and IDO inhibition by 1-methyl-D-tryptophan had ablated those responses. Cyclic diguanylate monophosphate (c-diGMP), a direct STING activator also attenuated EAE progression and disease severity. Thus, Systemic administration of DNPs and c-diGMP can attenuate EAE through STING-IFN αβ-IDO regulatory pathway that is activated in certain immune cells subsets to overcome CNS specific autoimmunity. These findings emphasize a previously unappreciated role of STING activators to attenuate autoimmunity.

Patients defective in the 3’ repair exonuclease 1 (Trex1) suffer from Aicardi-Goutieres Syndrome (AGS) which instigates lethal encephalitis characterized by high levels of type I IFN production being present in the cerebrospinal fluid. Trex1 deficient mice exhibit a median life span of approximately 9 weeks since as yet uncharacterized self-DNA, presumably normally digested by Trex1, activates intracellular DNA sensors which triggers cytokine production and causes lethal inflammatory aggravated myocarditis. STING is essential for triggering cytoplasmic DNA-dependent innate immune signaling that comprises the production of type I IFN. Loss of TREX1 was observed to augment ssDNA and dsDNA-mediated induction of type I IFN in a STING-dependent manner. The replication of HSV1 was greatly reduced in hTERT-BJ1 or MEF lacking TREX1 due to elevated STING-dependent cytokine production. Loss of STING was observed to significantly extend the lifespan of Trex1-/- due to prevention of myocarditis. Microarray analysis showed dramatically reduced levels of proinflammatory genes in the hearts of Sting-/- Trex1-/-, Sting-/+ Trex1-/- compared to Trex1-/- mice. Anti-nuclear autoantibody (ANA) observed to be highly prevalent in the sera of Trex1-/- mice was almost completely absent in the sera of Sting-/- Trex1-/- mice. These data indicates that STING is responsible for pro-inflammatory gene induction in Trex1 deficient mice and plausibly may be involved in AGS.

How the cell recognizes cytosolic DNA including DNA based microbes to trigger host defense related gene activation remains to be fully resolved. Here, we demonstrate that STING (for Stimulator of Interferon Genes), an endoplasmic reticulum (ER) translocon associated transmembrane protein, acts to detect cytoplasmic DNA species. STING homodimers were able to complex with self (apoptotic, necrotic) or pathogen related ssDNA and dsDNA and were indispensible for HSV-1-mediated transcriptional activation of a wide array of innate immune and pro-inflammatory genes in addition to type I IFN. Our data indicates that STING instigates cytoplasmic DNA-mediated cellular defense gene transcription and facilitates adoptive responses that are required for protection of the host. In contrast, chronic STING activation may manifest inflammatory responses and possibly autoimmune disease triggered by self-DNA.

Cellular innate immune sensor pathways are essential for identifying invading pathogens and for initiating host defense responses. However, how cells recognize DNA based microbes and trigger innate immune gene activation remains to be fully resolved. Here, we demonstrate that STING, an endoplasmic reticulum associated transmembrane protein, acts as a receptor for cytosolic pathogen associated DNA. This event invoked STING trafficking from the ER through autophagy-related endosomes to promote the activation of interferon regulatory factors IRF- 3, 7 and NF-κB, which triggered the production of host defense genes. STING, predominantly found in endothelial cells, macrophages and dendritic cells, was found to be essential for protecting mice against lethal HSV-1 infection through production of type I interferon. However, while STING was found to be essential for host defense, chronic activation of STING was observed to be responsible for inflammatory disease in two murine model systems. For example, DNaseII-/- and Trex1-/- mice die prematurely due to self-DNA manifested cytokine production. Lethality was prevented in both models through loss of STING since cytosolic self-DNA failed to robustly trigger cytokine production through STING signaling pathways. Our data suggests that STING senses aberrant cytosolic DNA species and is responsible for initiating innate immune gene activation, though may play a key role in inflammation and autoimmune disease manifested through self-DNA.

Autoimmune diseases such as systemic lupus erythematosus (SLE) affect millions worldwide, the cause of which remains unknown. SLE and diseases such as Aicardi-Goutieres syndrome (AGS) are characterized by the overproduction of cytokines such as type I interferon (IFN), speculatively by chronic infection or self nucleic acids. Mice lacking DNAse II die during embryonic development through comparable autoimmune disease since phagocytosed DNA from apoptotic cells cannot be adequately digested and intracellular host DNA sensors are activated resulting in the production of a variety of cytokines including type I IFN. The cellular sensor responsible for triggering DNA-mediated inflammation aggravated autoimmune disease remains to be determined. However, we report here that STING (Stimulator of Interferon Genes) complexes with phagocytosed undigested DNA and controls innate immune signaling events that facilitate such events. DNase II-dependent autoimmune embryonic lethality was rescued by loss of STING function and polyarthritis completely prevented since cytosolic DNA failed to trigger cytokine production. Accordingly, loss of STING similarly alleviated Trex1-dependent lethal inflammatory myocarditis in mice, a model for AGS. Our data provides molecular insight into the causes of DNA-mediated inflammation-dependent autoimmune disorders and affords a new target that could plausibly be therapeutically controlled, to help prevent such diseases.

STING (TMEM173, MPYS,MITA) located in the endoplasmic reticulum (ER), plays a critical role in type I IFN responses to intracellular DNA. STING activates TBK1, resulting in phosphorylated IRF3 that regulates IFN-β transcription. Our previous work showing markedly enhanced LPS-induced IFN-β during ER stress implicated increased IRF3 recruitment to the ifnb1 promoter. In this study, utilizing STING-/- mouse embryonic fibroblasts (MEFs) and RNAi (in MEFs and macrophages) we determined that STING was essential for synergistic induction of IFN-β mRNA in response to LPS and ER stress (thapsigargin/TPG treatment). TPG treatment alone induced re-localization and clustering of STING and TBK1 in MEFs. Although ER stress resulted in nuclear translocation of IRF3 in a STING-independent manner, TPG and TPG+LPS-induced IRF3 phosphorylation (as detected by immunofluorescence and western blot respectively) depended upon STING. In comparison, STING was not required for LPS or LPS+TPG induced NF-κB phosphorylation. These results indicate that ER stress alone results in IRF3 nuclear translocation and phosphorylation. In mice, treatment with the ER stress inducer tunicamycin was sufficient for induction of serum-detectable IFN-β, comparable with low dose LPS. Our results also suggest that the synergistic IFN-β responses during LPS and ER stress involve STING-dependent IRF3 phosphorylation.

The literature for 1943 includes a number of excellent articles on delayed union of fractures and on nonunion, while little emphasis has been placed on malunion. In these articles, all of which are written from a clinical standpoint, attention has been directed about equally to study of the causes of delayed union and nonunion and to exposition of grafting technics, with statistical analyses of the results. Clinical Studies of Delayed Union and Nonunion. —In a comprehensive article, which merits careful study for full understanding and appreciation, Watson-Jones and Coltart520have analyzed a series of 804 fractures of the shaft of the tibia and femur. They state that the test of success in the treatment of a fracture is the quality of the end result rather than the time taken in achieving it. Time is not saved when recovery is accelerated at the cost of permanent disability; a disabled man