Over the past decade, mesoporous silica nanoparticles (MSNs) smaller than 200 nm with a high colloidal stability have been extensively studied for systemic drug delivery. Although small molecule delivery via MSNs has been successful, the encapsulation of large therapeutic biomolecules, such as proteins or DNA, is limited due to small pore size of the conventional MSNs obtained by soft-templating. Here, we report the synthesis of mesoporous silica nanoparticles with extra-large pores (XL-MSNs) and their application to in vivo cytokine delivery for macrophage polarization. Uniform, size-controllable XL-MSNs with 30 nm extra-large pores were synthesized using organic additives and inorganic seed nanoparticles. XL-MSNs showed significantly higher loadings for the model proteins with different molecular weights compared to conventional small pore MSNs. XL-MSNs were used to deliver IL-4, which is an M2-polarizing cytokine and very quickly degraded in vivo, to macrophages and polarize them to anti-inflammatory M2 macrophages in vivo. XL-MSNs induced a low level of reactive oxygen species (ROS) production and no pro-inflammatory cytokines in bone marrow-derived macrophages (BMDMs) and in mice injected intravenously with XL-MSNs. We found that the injected XL-MSNs were targeted to phagocytic myeloid cells, such as neutrophils, monocytes, macrophages, and dendritic cells. Finally, we demonstrated that the injection of IL-4-loaded XL-MSNs successfully triggered M2 macrophage polarization in vivo, suggesting the clinical potential of XL-MSNs for modulating immune systems via targeted delivery of various cytokines.

Retinoic acid-inducible gene I (RIG-I) recognizes specific molecular patterns of viral RNAs for inducing type I interferon. The C-terminal domain (CTD) of RIG-I binds to double-stranded RNA (dsRNA) with the 5′-triphosphate (5′-PPP), which induces a conformational change in RIG-I to an active form. It has been suggested that RIG-I detects infection of influenza A virus by recognizing the 5′-triphosphorylated panhandle structure of the viral RNA genome. Influenza panhandle RNA has a unique structure with a sharp helical bending. In spite of extensive studies of how viral RNAs activate RIG-I, whether the structural elements of the influenza panhandle RNA confer the ability to activate RIG-I signaling has been poorly explored. Here, we investigated the dynamics of the influenza panhandle RNA in complex with RIG-I CTD using NMR spectroscopy and showed that the bending structure of the panhandle RNA negates the requirement of a 5′-PPP moiety for RIG-I activation.

Cyclic GMP-AMP synthase (cGAS) is a DNA-sensing enzyme in the innate immune system. Recent studies using core-cGAS lacking the N terminus investigated the mechanism for binding of double-stranded (ds) DNA and synthesis of 2',3'-cyclic GMP-AMP (cGAMP), a secondary messenger that ultimately induces type I interferons. However, the function of the N terminus of cGAS remains largely unknown. Here, we found that the N terminus enhanced the activity of core-cGAS in vivo. Importantly, the catalytic activity of core-cGAS decreased as the length of double-stranded DNA (dsDNA) increased, but the diminished activity was restored by addition of the N terminus. Furthermore, the N terminus de-oligomerized the 2 : 2 complex of core-cGAS and dsDNA into a 1 : 1 complex, suggesting that the N terminus enhanced the activity of core-cGAS by facilitating formation of a monomeric complex of cGAS and DNA.

The stimulator of IFN genes (STING)-mediated DNA-sensing pathway plays an important role in the innate immune response to pathogen infection, autoimmunity, and cancer; however, its regulatory mechanism has not been fully elucidated, and we do not yet know whether the STING pathway is counter-regulated by other innate immune pathways. Here, we show that the NLRP3-activating agonists, ATP and nigericin, prevent STING pathway activation in association with mitochondrial fragmentation; however, the suppression of the STING pathway and mitochondria fission were not dependent on NLRP3 or potassium efflux. Although nigericin-induced mitochondria fission was rescued by knockdown of either dynamin-related protein 1 or TBC1 domain family member 15 (TBC1D15), which are two distinct mitochondria fission regulators, only TBC1D15 restored the activity of the STING pathway, which indicates that inflammasome-activating signals curtail STING pathway activation via TBC1D15. Finally, we found that deficiency of mitofusin (MFN) 1, a mediator of mitochondrial fusion, inhibited STING pathway activation, which leads to a decrease in the induction of IFN-b and its inducible gene, ISG56, in conjunction with diminished activation of the signaling molecules, TANK-binding kinase 1 and IFN regulatory factor 3, that are downstream of STING. These results highlight the crucial role of MFN1 in maintaining the competency of the STING pathway. Collectively, our findings reveal that mitochondrial dynamics regulators modulate the activation of the STING signaling pathway.-Kwon, D., Park, E., Kang, S.-J. Stimulator of IFN genes-mediated DNA-sensing pathway is suppressed by NLRP3 agonists and regulated by mitofusin 1 and TBC1D15, mitochondrial dynamics mediators. FASEB J. 31, 4866-4878 (2017). www.fasebj.org

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