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The STING-STAT6 pathway drives Cas9-induced host response in human monocytes.

ABSTRACT: Cas9 (CRISPR associated protein 9) is an RNA-guided DNA endonuclease enzyme derived from Streptococcus that has been widely used for genome editing in a variety of organisms, including humans. Here, we report that exogenous Cas9 protein can elicit an inflammatory immune response through the release of MIP3?, CD40L, and MPO in primary human peripheral blood mononuclear cells and human monocytic cell lines (THP1). Inhibition of the STING-STAT6 pathway blocks Cas9-induced proinflammatory mediator release. These results suggest that targeting the STING-STAT6 axis may prevent host immune responses in human gene therapy with the CRISPR-Cas9 system.

SUBMITTER: Kang R

PROVIDER: S-EPMC6290677 | biostudies-literature | 2018 Nov

REPOSITORIES: biostudies-literature

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The STING-STAT6 pathway drives Cas9-induced host response in human monocytes.

Kang Rui R Zhu Shan S Zeh Herbert H Tang Daolin D

Biochemical and biophysical research communications 20181020 1

Cas9 (CRISPR associated protein 9) is an RNA-guided DNA endonuclease enzyme derived from Streptococcus that has been widely used for genome editing in a variety of organisms, including humans. Here, we report that exogenous Cas9 protein can elicit an inflammatory immune response through the release of MIP3α, CD40L, and MPO in primary human peripheral blood mononuclear cells and human monocytic cell lines (THP1). Inhibition of the STING-STAT6 pathway blocks Cas9-induced proinflammatory mediator r ...[more]

PMID: 30348526

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Project description:The clinicopathological features of the hamster model of visceral leishmaniasis (VL) closely mimic active human disease. Studies in humans and hamsters indicate that the inability to control parasite replication in VL could be related to ineffective classical macrophage activation. Therefore, we hypothesized that the pathogenesis of VL might be driven by a program of alternative macrophage activation. Indeed, the infected hamster spleen showed low NOS2 but high arg1 enzyme activity and protein and mRNA expression (p<0.001) and increased polyamine synthesis (p<0.05). Increased arginase activity was also evident in macrophages isolated from the spleens of infected hamsters (p<0.05), and arg1 expression was induced by L. donovani in primary hamster peritoneal macrophages (p<0.001) and fibroblasts (p<0.01), and in a hamster fibroblast cell line (p<0.05), without synthesis of endogenous IL-4 or IL-13 or exposure to exogenous cytokines. miRNAi-mediated selective knockdown of hamster arginase 1 (arg1) in BHK cells led to increased generation of nitric oxide and reduced parasite burden (p<0.005). Since many of the genes involved in alternative macrophage activation are regulated by Signal Transducer and Activator of Transcription-6 (STAT6), and because the parasite-induced expression of arg1 occurred in the absence of exogenous IL-4, we considered the possibility that L. donovani was directly activating STAT6. Indeed, exposure of hamster fibroblasts or macrophages to L. donovani resulted in dose-dependent STAT6 activation, even without the addition of exogenous cytokines. Knockdown of hamster STAT6 in BHK cells with miRNAi resulted in reduced arg1 mRNA expression and enhanced control of parasite replication (p<0.0001). Collectively these data indicate that L. donovani infection induces macrophage STAT6 activation and STAT6-dependent arg1 expression, which do not require but are amplified by type 2 cytokines, and which contribute to impaired control of infection.

Project description:BackgroundDespite its potential utility in delivering direct tumor killing and in situ whole-cell tumor vaccination, tumor cryoablation produces highly variable and unpredictable clinical response, limiting its clinical utility. The mechanism(s) driving cryoablation-induced local antitumor immunity and the associated abscopal effect is not well understood.MethodsThe aim of this study was to identify and explore a mechanism of action by which cryoablation enhances the therapeutic efficacy in metastatic tumor models. We used the subcutaneous mouse model of the rhabdomyosarcoma (RMS) cell lines RMS 76-9STINGwt or RMS 76-9STING-/-, along with other murine tumor models, in C57BL/6 or STING-/- (TMEM173-/- ) mice to evaluate local tumor changes, lung metastasis, abscopal effect on distant tumors, and immune cell dynamics in the tumor microenvironment (TME).ResultsThe results show that cryoablation efficacy is dependent on both adaptive immunity and the STING signaling pathway. Contrary to current literature dictating an essential role of host-derived STING activation as a driver of antitumor immunity in vivo, we show that local tumor control, lung metastasis, and the abscopal effect on distant tumor are all critically dependent on a functioning tumor cell-intrinsic STING signaling pathway, which induces inflammatory chemokine and cytokine responses in the cryoablated TME. This reliance extends beyond cryoablation to include intratumoral STING agonist therapy. Additionally, surveys of gene expression databases and tissue microarrays of clinical tumor samples revealed a wide spectrum of expressions among STING-related signaling components.ConclusionsTumor cell-intrinsic STING pathway is a critical component underlying the effectiveness of cryoablation and suggests that expression of STING-related signaling components may serve as a potential therapy response biomarker. Our data also highlight an urgent need to further characterize tumor cell-intrinsic STING pathways and the associated downstream inflammatory response evoked by cryoablation and other STING-dependent therapy approaches.

Dataset Information

The STING-STAT6 pathway drives Cas9-induced host response in human monocytes.

Publications

The STING-STAT6 pathway drives Cas9-induced host response in human monocytes.

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