Signal strength of STING activation determines cytokine plasticity and cell death in human monocytes

Abstract

The cyclic GMP-AMP synthase (cGAS)/stimulator of interferon genes (STING) pathway is a cytosolic sensor of microbial and host-derived DNA and plays a key role in innate immunity. Activation of STING by cyclic dinucleotide (CDN) ligands in human monocytes induces a type I interferon response and production of pro-inflammatory cytokines associated with the induction of massive cell death. In this study we have re-evaluated the effect of signal strength of STING activation on the cytokine plasticity of human monocytes. CDN (2′3′c-GAMP) and non-CDN (diABZI, MSA-2) STING ligands in the range of EC50 concentrations (15 μM 2′3′c-GAMP, 100 nM diABZI, 25 μM MSA-2) induced IFN-β, IP-10, and large amounts of IL-1β and TNF-α, but no IL-10 or IL-19. Interestingly, LPS-induced production of IL-10 and IL-19 was abolished in the presence of diABZI or MSA-2, whereas IL-1β and TNF-α were not inhibited. Surprisingly, we observed that tenfold lower (MSA-2, i.e. 2.5 μM) or 100-fold lower (diABZI, i.e. 1 nM) concentrations strongly stimulated secretion of anti-inflammatory IL-10 and IL-19, but little of IL-1β and TNF-α. Induction of IL-10 was associated with up-regulation of PRDM1 (Blimp-1). While cytokine secretion stimulated by the higher concentrations was accompanied by apoptosis as shown by cleavage of caspase-3 and PARP-1, the low concentrations did not trigger overt cell death yet induced cleavage of gasdermin-D. Our results reveal a previously unrecognized plasticity of human monocytes in their signal strength-dependent production of pro- versus anti-inflammatory cytokines upon STING activation.

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“…To further evaluate that 29f indeed activated type I IFN response, we measured the cytokine secretion (i.e., human IP-10 and IFN-β) by enzyme-linked immunosorbent assay (ELISA) for quantitative measurements. IFN-β and IP-10 (CXCL10; interferon-gamma induced protein) are widely used as biomarkers of STING activation because the increase of secreted IFN-β and IP-10 in monocytes triggers monocyte homing, promotes the infiltration of T cells, and eventually suppresses tumor growth. , 29f in the presence of a marginal amount of cGAMP induced considerable extracellular secretion of IP-10 and IFN-β in THP-1 cells (Figure A). To further confirm the activation of type I IFN response by 29f , we investigated the expression levels of representative ISGs in THP-1 cells.…”

“…Once cGAMP binds STING at the endoplasmic reticulum (ER), STING is translocated from the ER via the ER−Golgi intermediate compartment (ERGIC) to the Golgi, where STING recruits TANK binding kinase 1 (TBK1) and interferon regulatory factor 3 (IRF3). 8 Activated STING boosts autophosphorylation of TBK1, which subsequently phosphorylates IRF3. Eventually, phosphorylated IRF3 is translocated to the nucleus, resulting in the production and release of cytokines such as interferon-β (IFN-β).…”

“…Stimulator of interferon genes (STING) is a major regulator of innate immunity inducing type I interferon response by recognizing cytosolic double-stranded DNA (dsDNA) from damaged nuclei, mitochondria, or pathogens and is ubiquitously expressed in both immune cells and nonimmune cells. , In response to cytosolic DNA, 2′,3′-cyclic GMP–AMP (cGAMP), a natural STING ligand, is endogenously synthesized by cyclic GMP–AMP synthase (cGAS) in mammalian cells. Once cGAMP binds STING at the endoplasmic reticulum (ER), STING is translocated from the ER via the ER–Golgi intermediate compartment (ERGIC) to the Golgi, where STING recruits TANK binding kinase 1 (TBK1) and interferon regulatory factor 3 (IRF3) . Activated STING boosts autophosphorylation of TBK1, which subsequently phosphorylates IRF3.…”

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“…Mechanistically, apoptosis, pyroptosis, ferroptosis, necroptosis, and PANoptosis have all been reported in STING-mediated cell death [25][26][27][28][29][30][31]42,43 . Different cell types and STING agonists used likely contributed to the inconsistency and complexity.…”

“…We first used the synthetic non-CDNs STING agonist diABZI 41 to induce lymphocyte death because diABZI induces cell death without the needs for lipid transfection or detergent for cell permeabilization 28,42 and diABZI is in clinical trials (NCT05514717).…”