Arundhati Ghosh

STING Contributes to Antiglioma Immunity via Triggering Type I IFN Signals in the Tumor Microenvironment

Ohkuri, Ghosh, Kosaka, and colleagues show that a STING-mediated DNA-sensor signaling is involved in IFN induction in the sterile microenvironment of brain tumor that enhances antitumor immunity, providing a proof-of-concept for developing STING agonists for cancer immunotherapy. Although type I IFNs play critical roles in antiviral and antitumor activity, it remains to be elucidated how type I IFNs are produced in sterile conditions of the tumor microenvironment and directly affect tumor-infiltrating immune cells. Mouse de novo gliomas show increased expression of type I IFN messages, and in mice, CD11b+ brain-infiltrating leukocytes (BIL) are the main source of type I IFNs that are induced partially in a STING (stimulator of IFN genes)-dependent manner. Consequently, glioma-bearing StingGt/Gt mice showed shorter survival and lower expression levels of Ifns compared with wild-type mice. Furthermore, BILs of StingGt/Gt mice showed increased CD11b+ Gr-1+ immature myeloid suppressor and CD25+ Foxp3+ regulatory T cells (Treg) and decreased IFNγ-producing CD8+ T cells. CD4+ and CD8+ T cells that received direct type I IFN signals showed lesser degrees of regulatory activity and increased levels of antitumor activity, respectively. Finally, intratumoral administration of a STING agonist (cyclic diguanylate monophosphate; c-di-GMP) improved the survival of glioma-bearing mice associated with enhanced type I IFN signaling, Cxcl10 and Ccl5, and T-cell migration into the brain. In combination with subcutaneous OVA peptide vaccination, c-di-GMP increased OVA-specific cytotoxicity of BILs and prolonged their survival. These data demonstrate significant contributions of STING to antitumor immunity via enhancement of type I IFN signaling in the tumor microenvironment and suggest a potential use of STING agonists for the development of effective immunotherapy, such as the combination with antigen-specific vaccinations. Cancer Immunol Res; 2(12); 1199–208. ©2014 AACR.

OASL – a new player in controlling antiviral innate immunity

The cellular innate immune system plays a crucial role in mounting the initial resistance to virus infection. It is comprised of various pattern-recognition receptors that induce type I interferon production, which further shapes the adaptive immunity. However, to overcome this resistance and promote replication, viruses have evolved mechanisms to evade this host innate immune response. Here we discuss a recently described mechanism of boosting the innate immunity by oligoadenylate synthetase-like (OASL) protein, which can potentially be used to overcome viral evasion and enhance innate immunity.

Shaping the Murine Macrophage Phenotype: IL-4 and Cyclic AMP Synergistically Activate the Arginase I Promoter

Arginase I is a marker of murine M2 macrophages and is highly expressed in many inflammatory diseases. The basis for high arginase I expression in macrophages in vivo is incompletely understood but likely reflects integrated responses to combinations of stimuli. Our objective was to elucidate mechanisms involved in modulating arginase I induction by IL-4, the prototypical activator of M2 macrophages. IL-4 and 8-bromo-cAMP individually induce arginase I, but together they rapidly and synergistically induce arginase I mRNA, protein, and promoter activity in murine macrophage cells. Arginase I induction by IL-4 requires binding of the transcription factors STAT6 and C/EBPβ to the IL-4 response element of the arginase I gene. Chromatin immunoprecipitation showed that the synergistic response involves binding of both transcription factors to the IL-4 response element at levels significantly greater than in response to IL-4 alone. The results suggest that C/EBPβ is a limiting factor for the level of STAT6 bound to the IL-4 response element. The enhanced binding in the synergistic response was not due to increased expression of either STAT6 or C/EBPβ but was correlated primarily with increased nuclear abundance of C/EBPβ. Our findings also suggest that induction of arginase I expression is stochastic; that is, differences in induction reflect differences in probability of transcriptional activation and not simply differences in rate of transcription. Results of the present study also may be useful for understanding mechanisms underlying regulated expression of other genes in macrophages and other myeloid-derived cells in health and disease.

Differential Effects of Phenethyl Isothiocyanate and D,L-Sulforaphane on TLR3 Signaling

Naturally occurring isothiocyanates (ITCs) from cruciferous vegetables are widely studied for their cancer chemopreventive effects. In this study, we investigated the effects of ITCs on TLR signaling, and found that the two most promising ITCs, phenethyl ITCs (PEITC) and D,L-sulforaphane (SFN), have differential effects on dsRNA-mediated innate immune signaling through TLR3. PEITC preferentially inhibited TLR3-mediated IFN regulatory factor 3 (IRF3) signaling and downstream gene expression in vivo and in vitro, whereas SFN caused inhibition of TLR3-mediated NF-κB signaling and downstream gene expression. Mechanistically, PEITC inhibited ligand (dsRNA)-dependent dimerization of TLR3, resulting in inhibition of signaling through IFN regulatory factor 3. In contrast, SFN did not disrupt TLR3 dimerization, indicating that it affects further downstream pathway resulting in NF-κB inhibition. To examine the biological significance of these findings in the context of antitumor activities of these compounds, we used two approaches: first, we showed that dsRNA-mediated apoptosis of tumor cells via TLR3 was inhibited in the presence of PEITC, whereas this response was augmented by SFN treatment; second, in a separate assay measuring anchorage-independent growth and colony formation by immortalized fibroblasts, we made similar observations. Again in this study, PEITC antagonized dsRNA-mediated inhibition of colony formation, whereas SFN enhanced the inhibition. These results indicate biologically relevant functional differences between two structurally similar ITCs and may provide important insights in therapeutic development of these compounds targeted to specific cancer.

MOV10 Provides Antiviral Activity against RNA Viruses by Enhancing RIG-I–MAVS-Independent IFN Induction

Moloney leukemia virus 10, homolog (MOV10) is an IFN-inducible RNA helicase, associated with small RNA-induced silencing. In this article, we report that MOV10 exhibits antiviral activity, independent of its helicase function, against a number of positive- and negative-strand RNA viruses by enhancing type I IFN induction. Using a number of genome-edited knockout human cells, we show that IFN regulatory factor 3–mediated IFN induction and downstream IFN signaling through IFN receptor was necessary to inhibit virus replication by MOV10. MOV10 enhanced IFN regulatory factor 3–mediated transcription of IFN. However, this IFN induction by MOV10 was unique and independent of the known retinoic acid–inducible gene I/mitochondrial antiviral-signaling protein–mediated RNA-sensing pathway. Upon virus infection, MOV10 specifically required inhibitor of κB kinase ε, not TANK-binding kinase 1, for its antiviral activity. The important role of MOV10 in mediating antiviral signaling was further supported by the finding that viral proteases from picornavirus family specifically targeted MOV10 as a possible innate immune evasion mechanism. These results establish MOV10, an evolutionary conserved protein involved in RNA silencing, as an antiviral gene against RNA viruses that uses an retinoic acid–inducible gene I–like receptor–independent pathway to enhance IFN response.

Protective role of STING against gliomagenesis: Rational use of STING agonist in anti-glioma immunotherapy

We recently reported that STING contributes to antiglioma immunity by triggering type I IFN induction in glioma microenvironment. Moreover, intratumoral administration of STING agonist improved the efficacy of peptide vaccination in a mouse glioma model, suggesting the rational use of STING agonists in the immunotherapy of brain tumor.

Innate immune signaling through differential RIPK1 expression promote tumor progression in head and neck squamous cell carcinoma.

Head and neck squamous cell carcinoma (HNSCC) is a devastating disease for which new treatments, such as immunotherapy are needed. Synthetic double-stranded RNAs, which activate toll-like receptor 3 (TLR3), have been used as potent adjuvants in cancer immunotherapy by triggering a proapoptotic response in cancer cells. A better understanding of the mechanism of TLR3-mediated apoptosis and its potential involvement in controlling tumor metastasis could lead to improvements in current treatment. Using paired, autologous primary and metastatic HNSCC cells we previously showed that metastatic, but not primary tumor-derived cells, were unable to activate prosurvival NF-κB in response to p(I):p(C) resulting in an enhanced apoptotic response. Here, we show that transcriptional downregulation of receptor-interacting serine/threonine-protein kinase 1 (RIPK1) in metastatic HNSCC cells causes a loss of TLR3-mediated NF-κB signaling, resulting in enhanced apoptosis. Loss of RIPK1 strongly correlates with metastatic disease in a cohort of HNSCC patients. This downregulation of RIPK1 is possibly mediated by enhanced methylation of the RIPK1 promoter in tumor cells and enhances protumorigenic properties such as cell migration. The results described here establish a novel mechanism of TLR3-mediated apoptosis in metastatic cells and may create new opportunities for using double stranded RNA to target metastatic tumor cells.

Gasdermin D Restrains Type I Interferon Response to Cytosolic DNA by Disrupting Ionic Homeostasis

SUMMARY Inflammasome‐activated caspase‐1 cleaves gasdermin D to unmask its pore‐forming activity, the predominant consequence of which is pyroptosis. Here, we report an additional biological role for gasdermin D in limiting cytosolic DNA surveillance. Cytosolic DNA is sensed by Aim2 and cyclic GMP‐AMP synthase (cGAS) leading to inflammasome and type I interferon responses, respectively. We found that gasdermin D activated by the Aim2 inflammasome suppressed cGAS‐driven type I interferon response to cytosolic DNA and Francisella novicida in macrophages. Similarly, interferon‐&bgr; (IFN‐&bgr;) response to F. novicida infection was elevated in gasdermin D‐deficient mice. Gasdermin D‐mediated negative regulation of IFN‐&bgr; occurred in a pyroptosis‐, interleukin‐1 (IL‐1)‐, and IL‐18‐independent manner. Mechanistically, gasdermin D depleted intracellular potassium (K+) via membrane pores, and this K+ efflux was necessary and sufficient to inhibit cGAS‐dependent IFN‐&bgr; response. Thus, our findings have uncovered an additional interferon regulatory module involving gasdermin D and K+ efflux. Graphical Abstract Figure. No caption available. HIGHLIGHTSInflammasome‐activated gasdermin D limits type I interferon responses to cytosolic DNAGasdermin D targets cGAS activation to inhibit IFN‐&bgr; response to cytosolic DNADepletion of intracellular K+ by gasdermin D is responsible for limiting cGAS signalingK+ efflux is sufficient to inhibit cGAS‐dependent type I interferon responses &NA; Gasdermin D is a pore‐forming protein, which upon activation by inflammasome complexes mediates pyroptotic cell death and IL‐1 release. Banerjee et al. demonstrate a previously unknown regulatory role for gasdermin D‐driven K+ efflux in reining in cGAS‐dependent type I interferon response to cytosolic DNA.