A localizing nanocarrier formulation enables multi-target immune responses to multivalent replicating RNA with limited systemic inflammation.

RNA vaccines possess significant clinical promise in counteracting human diseases caused by infectious or cancerous threats. Self-amplifying replicon RNA (repRNA) has been thought to offer the potential for enhanced potency and dose sparing. However, repRNA is a potent trigger of innate immune responses in vivo, which can cause reduced transgene expression and dose-limiting reactogenicity, as highlighted by recent clinical trials. Here, we report that multivalent repRNA vaccination, necessitating higher doses of total RNA, could be safely achieved in mice by delivering multiple repRNAs with a localizing cationic nanocarrier formulation (LION). Intramuscular delivery of multivalent repRNA by LION resulted in localized biodistribution accompanied by significantly upregulated local innate immune responses and the induction of antigen-specific adaptive immune responses in the absence of systemic inflammatory responses. In contrast, repRNA delivered by lipid nanoparticles (LNPs) showed generalized biodistribution, a systemic inflammatory state, an increased body weight loss, and failed to induce neutralizing antibody responses in a multivalent composition. These findings suggest that in vivo delivery of repRNA by LION is a platform technology for safe and effective multivalent vaccination through mechanisms distinct from LNP-formulated repRNA vaccines.

Self-assembled single-crystal bimodal porous GaN exhibiting a petal effect: application as a sensing platform and substrate for optical devices.

This paper investigates the petal effect (hydrophobicity and strong adhesion) observed on single-crystal bimodal porous GaN (porous GaN), which has almost the same electrical properties as bulk GaN. The water contact angles of porous GaN were 100°-135° despite the intrinsic hydrophilic nature of GaN. Moreover, it was demonstrated that the petal effect of porous GaN leads to the uniform attachment of water solutions, enabling highly uniform and aggregation-free attachment of chemicals and quantum dots. These results indicate that porous GaN can be applied in quantum dot light-emitting diodes and as an analytical substrate.

A food-responsive switch modulates TFEB and autophagy, and determines susceptibility to coxsackievirus infection and pancreatitis.

Almost a billion people worldwide are chronically undernourished. Herein, using a mouse model of coxsackievirus B3 (CVB3) infection, we report that a single day of food restriction (FR) markedly increases susceptibility to attenuated enterovirus infection, replication, and disease. These "pro-viral" effects, which are rapidly-reversed by the restoration of food, are mediated by several genes whose expression is altered by FR, and which support CVB3 replication. Central to this is TFEB, a protein whose expression and activation status are rapidly increased by FR. TFEB, which regulates the transcription of >100 genes involved in macroautophagy/autophagy and lysosomal biogenesis, responds similarly to both FR and CVB3 infection and plays a pivotal role in determining host susceptibility to CVB3. We propose that, by upregulating TFEB, FR generates an intracellular environment that is more hospitable to the incoming virus, facilitating its replication. This interplay between nutritional status and enterovirus replication has implications for human health and, perhaps, for the evolution of these viruses.Abbreviations: Atg/ATG: autophagy-related; CAR: Coxsackievirus and adenovirus receptor; Cas9: CRISPR associated protein 9; Cre: recombinase that causes recombination; CRISPR: clustered regularly interspaced short palindromic repeats; Ctsb/CTSB: cathepsin B; CVB3: coxsackievirus B3; DsRedCVB3: a recombinant CVB3 that encodes the Discosoma red fluorescent protein; EL: elastase; FR: food restriction; GFP: green fluorescent protein; gRNA: guide RNA; HBSS: Hanks Buffered Salt Solution; LYNUS: lysosomal nutrient sensing machinery; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MFI: mean fluorescence intensity; MOI: multiplicity of infection; MTOR: mechanistic target of rapamycin kinase; Nluc: nanoluciferase; NlucCVB3: a recombinant CVB3 encoding nanoluciferase; pfu: plaque-forming unit(s); p.i.: post infection; rCVB: recombinant coxsackievirus B3; RPS6KB/p70S6K: ribosomal protein S6 kinase; RT: room temperature; siRNA: small interfering RNA; TFEB: transcription factor EB; tg: transgenic; TUBB: ß-tubulin; UNINF: uninfected; wrt: with respect to; WT: wild type.

Hepatocytes trap and silence coxsackieviruses, protecting against systemic disease in mice.

Previous research suggests that hepatocytes catabolize chemical toxins but do not remove microbial agents, which are filtered out by other liver cells (Kupffer cells and endothelial cells). Here we show that, contrary to current understanding, hepatocytes trap and rapidly silence type B coxsackieviruses (CVBs). In genetically wildtype mice, this activity causes hepatocyte damage, which is alleviated in mice carrying a hepatocyte-specific deletion of the coxsackievirus-adenovirus receptor. However, in these mutant mice, there is a dramatic early rise in blood-borne virus, followed by accelerated systemic disease and increased mortality. Thus, wild type hepatocytes act similarly to a sponge for CVBs, protecting against systemic illness at the expense of their own survival. We speculate that hepatocytes may play a similar role in other viral infections as well, thereby explaining why hepatocytes have evolved their remarkable regenerative capacity. Our data also suggest that, in addition to their many other functions, hepatocytes might be considered an integral part of the innate immune system.

Biphasic and cardiomyocyte-specific IFIT activity protects cardiomyocytes from enteroviral infection.

Viral myocarditis is a serious disease, commonly caused by type B coxsackieviruses (CVB). Here we show that innate immune protection against CVB3 myocarditis requires the IFIT (IFN-induced with tetratricopeptide) locus, which acts in a biphasic manner. Using IFIT locus knockout (IFITKO) cardiomyocytes we show that, in the absence of the IFIT locus, viral replication is dramatically increased, indicating that constitutive IFIT expression suppresses CVB replication in this cell type. IFNß pre-treatment strongly suppresses CVB3 replication in wild type (wt) cardiomyocytes, but not in IFITKO cardiomyocytes, indicating that other interferon-stimulated genes (ISGs) cannot compensate for the loss of IFITs in this cell type. Thus, in isolated wt cardiomyocytes, the anti-CVB3 activity of IFITs is biphasic, being required for protection both before and after T1IFN signaling. These in vitro findings are replicated in vivo. Using novel IFITKO mice we demonstrate accelerated CVB3 replication in pancreas, liver and heart in the hours following infection. This early increase in virus load in IFITKO animals accelerates the induction of other ISGs in several tissues, enhancing virus clearance from some tissues, indicating that-in contrast to cardiomyocytes-other ISGs can offset the loss of IFITs from those cell types. In contrast, CVB3 persists in IFITKO hearts, and myocarditis occurs. Thus, cardiomyocytes have a specific, biphasic, and near-absolute requirement for IFITs to control CVB infection.

Self-Assembled Single-Crystalline GaN Having a Bimodal Meso/Macropore Structure To Enhance Photoabsorption and Photocatalytic Reactions.

This paper describes the self-assembled fabrication of single-crystal GaN with a bimodal pore (meso/macropore) size distribution (BiPS-GaN). A 4.7 µm-thick BiPS-GaN layer was grown spontaneously using halogen-free vapor phase epitaxy in conjunction with boron impurity doping (>1 × 1019 atoms/cm3) on a GaN template fabricated via metalorganic chemical vapor deposition (MOCVD-GaN). The boron impurity acted as a surfactant, and its segregation generated a dense (>1 × 1010 cm-2), homogeneous distribution of mesopores with sizes of 30-40 nm in GaN during growth. In addition, macropores with sizes of 0.1-2 µm were produced by the fusion of mesopores in close proximity to one another. As a result, BiPS-GaN exhibited a high density of both meso- and macropores, all aligned in the vertical direction (that is, along the c axis). BiPS-GaN showed good electroconductivity and almost the same high degree of crystallinity as the MOCVD-GaN template. Furthermore, the hybrid meso/macropore structure of BiPS-GaN imparted excellent photoabsorption properties and allowed this material to work as an efficient support for a nanosized IrO x catalyst. The photocurrent density in BiPS-GaN was enhanced by as much as a factor of 5 compared to planar GaN by effective absorption due to the hybrid meso/macropore structure of BiPS-GaN. Moreover, the oxygen generation efficiency of BiPS-GaN with the IrO x catalyst was approximately doubled, compared to that of BiPS-GaN without IrO x, while maintaining long-term stability. These results demonstrate that BiPS-GaN fabricated in this facile manner has significant potential in applications such as photoelectrochemical reactions and catalysis.

Immunological and pathological consequences of coxsackievirus RNA persistence in the heart.

Type B coxsackieviruses (CVB) can cause myocarditis and dilated cardiomyopathy (DCM), a potentially-fatal sequela that has been correlated to the persistence of viral RNA. Herein, we demonstrate that cardiac RNA persistence can be established even after an inapparent primary infection. Using an inducible Cre/lox mouse model, we ask: (i) Does persistent CVB3 RNA cause ongoing immune activation? (ii) If T1IFN signaling into cardiomyocytes is ablated after RNA persistence is established, is there any change in the abundance of persistent CVB3 RNA and/or does cytopathic infectious virus re-emerge? (iii) Does this loss of T1IFN responsiveness by cardiomyocytes lead to the recurrence/exacerbation of myocarditis? Our findings suggest that persistent enteroviral RNAs probably do not contribute to ongoing myocardial disease, and are more likely to be the fading remnants of a recent, possibly sub-clinical, primary infection which may have set in motion the process that ultimately ends in DCM.

Lypd8 promotes the segregation of flagellated microbiota and colonic epithelia.

Colonic epithelial cells are covered by thick inner and outer mucus layers. The inner mucus layer is free of commensal microbiota, which contributes to the maintenance of gut homeostasis. In the small intestine, molecules critical for prevention of bacterial invasion into epithelia such as Paneth-cell-derived anti-microbial peptides and regenerating islet-derived 3 (RegIII) family proteins have been identified. Although there are mucus layers providing physical barriers against the large number of microbiota present in the large intestine, the mechanisms that separate bacteria and colonic epithelia are not fully elucidated. Here we show that Ly6/PLAUR domain containing 8 (Lypd8) protein prevents flagellated microbiota invading the colonic epithelia in mice. Lypd8, selectively expressed in epithelial cells at the uppermost layer of the large intestinal gland, was secreted into the lumen and bound flagellated bacteria including Proteus mirabilis. In the absence of Lypd8, bacteria were present in the inner mucus layer and many flagellated bacteria invaded epithelia. Lypd8(-/-) mice were highly sensitive to intestinal inflammation induced by dextran sulfate sodium (DSS). Antibiotic elimination of Gram-negative flagellated bacteria restored the bacterial-free state of the inner mucus layer and ameliorated DSS-induced intestinal inflammation in Lypd8(-/-) mice. Lypd8 bound to flagella and suppressed motility of flagellated bacteria. Thus, Lypd8 mediates segregation of intestinal bacteria and epithelial cells in the colon to preserve intestinal homeostasis.

A critical role for PKR complexes with TRBP in Immunometabolic regulation and eIF2α phosphorylation in obesity.

Aberrant stress and inflammatory responses are key factors in the pathogenesis of obesity and metabolic dysfunction, and the double-stranded RNA-dependent kinase (PKR) has been proposed to play an important role in integrating these pathways. Here, we report the formation of a complex between PKR and TAR RNA-binding protein (TRBP) during metabolic and obesity-induced stress, which is critical for the regulation of eukaryotic translation initiation factor 2 alpha (eIF2α) phosphorylation and c-Jun N-terminal kinase (JNK) activation. We show that TRBP phosphorylation is induced in the setting of metabolic stress, leading to PKR activation. Suppression of hepatic TRBP reduced inflammation, JNK activity, and eIF2α phosphorylation and improved systemic insulin resistance and glucose metabolism, while TRBP overexpression exacerbated the impairment in glucose homeostasis in obese mice. These data indicate that the association between PKR and TRBP integrates metabolism with translational control and inflammatory signaling and plays important roles in metabolic homeostasis and disease.

Generation of colonic IgA-secreting cells in the caecal patch.

Gut-associated lymphoid tissues are responsible for the generation of IgA-secreting cells. However, the function of the caecal patch, a lymphoid tissue in the appendix, remains unknown. Here we analyse the role of the caecal patch using germ-free mice colonized with intestinal bacteria after appendectomy. Appendectomized mice show delayed accumulation of IgA(+) cells in the large intestine, but not the small intestine, after colonization. Decreased colonic IgA(+) cells correlate with altered faecal microbiota composition. Experiments using photoconvertible Kaede-expressing mice or adoptive transfer show that the caecal patch IgA(+) cells migrate to the large and small intestines, whereas Peyer's patch cells are preferentially recruited to the small intestine. IgA(+) cells in the caecal patch express higher levels of CCR10. Dendritic cells in the caecal patch, but not Peyer's patches, induce CCR10 on cocultured B cells. Thus, the caecal patch is a major site for generation of IgA-secreting cells that migrate to the large intestine.

A viral RNA structural element alters host recognition of nonself RNA.

Although interferon (IFN) signaling induces genes that limit viral infection, many pathogenic viruses overcome this host response. As an example, 2'-O methylation of the 5' cap of viral RNA subverts mammalian antiviral responses by evading restriction of Ifit1, an IFN-stimulated gene that regulates protein synthesis. However, alphaviruses replicate efficiently in cells expressing Ifit1 even though their genomic RNA has a 5' cap lacking 2'-O methylation. We show that pathogenic alphaviruses use secondary structural motifs within the 5' untranslated region (UTR) of their RNA to alter Ifit1 binding and function. Mutations within the 5'-UTR affecting RNA structural elements enabled restriction by or antagonism of Ifit1 in vitro and in vivo. These results identify an evasion mechanism by which viruses use RNA structural motifs to avoid immune restriction.

Ifit1 inhibits Japanese encephalitis virus replication through binding to 5' capped 2'-O unmethylated RNA.

The interferon-inducible protein with tetratricopeptide (IFIT) family proteins inhibit replication of some viruses by recognizing several types of RNAs, including 5'-triphosphate RNA and 5' capped 2'-O unmethylated mRNA. However, it remains unclear how IFITs inhibit replication of some viruses through recognition of RNA. Here, we analyzed the mechanisms by which Ifit1 exerts antiviral responses. Replication of a Japanese encephalitis virus (JEV) 2'-O methyltransferase (MTase) mutant was markedly enhanced in mouse embryonic fibroblasts and macrophages lacking Ifit1. Ifit1 bound 5'-triphosphate RNA but more preferentially associated with 5' capped 2'-O unmethylated mRNA. Ifit1 inhibited the translation of mRNA and thereby restricted the replication of JEV mutated in 2'-O MTase. Thus, Ifit1 inhibits replication of MTase-defective JEV by inhibiting mRNA translation through direct binding to mRNA 5' structures.

CREBH determines the severity of sulpyrine-induced fatal shock.

Although the pyrazolone derivative sulpyrine is widely used as an antipyretic analgesic drug, side effects, including fatal shock, have been reported. However, the molecular mechanism underlying such a severe side effect is largely unclear. Here, we report that the transcription factor CREBH that is highly expressed in the liver plays an important role in fatal shock induced by sulpyrine in mice. CREBH-deficient mice were resistant to experimental fatal sulpyrine shock. We found that sulpyrine-induced expression of cytochrome P450 2B (CYP2B) family genes, which are involved in sulpyrine metabolism, in the liver was severely impaired in CREBH-deficient mice. Moreover, introduction of CYP2B in CREBH-deficient liver restored susceptibility to sulpyrine. Furthermore, ectopic expression of CREBH up-regulated CYP2B10 promoter activity, and in vivo knockdown of CREBH in wild-type mice conferred a significant resistance to fatal sulpyrine shock. These data demonstrate that CREBH is a positive regulator of CYP2B in response to sulpyrine administration, which possibly results in fatal shock.

A cluster of interferon-γ-inducible p65 GTPases plays a critical role in host defense against Toxoplasma gondii.

Interferon-γ (IFN-γ) is essential for host defense against intracellular pathogens. Stimulation of innate immune cells by IFN-γ upregulates ∼2,000 effector genes such as immunity-related GTPases including p65 guanylate-binding protein (Gbp) family genes. We show that a cluster of Gbp genes was required for host cellular immunity against the intracellular parasite Toxoplasma gondii. We generated mice deficient for all six Gbp genes located on chromosome 3 (Gbp(chr3)) by targeted chromosome engineering. Mice lacking Gbp(chr3) were highly susceptible to T. gondii infection, resulting in increased parasite burden in immune organs. Furthermore, Gbp(chr3)-deleted macrophages were defective in IFN-γ-mediated suppression of T. gondii intracellular growth and recruitment of IFN-γ-inducible p47 GTPase Irgb6 to the parasitophorous vacuole. In addition, some members of Gbp(chr3) restored the protective response against T. gondii in Gbp(chr3)-deleted cells. Our results suggest that Gbp(chr3) play a pivotal role in anti-T. gondii host defense by controlling IFN-γ-mediated Irgb6-dependent cellular innate immunity.

ATF6beta is a host cellular target of the Toxoplasma gondii virulence factor ROP18.

The ROP18 kinase has been identified as a key virulence determinant conferring a high mortality phenotype characteristic of type I Toxoplasma gondii strains. This major effector molecule is secreted by the rhoptries into the host cells during invasion; however, the molecular mechanisms by which this kinase exerts its pathogenic action remain poorly understood. In this study, we show that ROP18 targets the host endoplasmic reticulum-bound transcription factor ATF6ß. Disruption of the ROP18 gene severely impairs acute toxoplasmosis by the type I RH strain. Because another virulence factor ROP16 kinase modulates immune responses through its N-terminal portion, we focus on the role of the N terminus of ROP18 in the subversion of host cellular functions. The N-terminal extension of ROP18 contributes to ATF6ß-dependent pathogenicity by interacting with ATF6ß and destabilizing it. The kinase activity of ROP18 is essential for proteasome-dependent degradation of ATF6ß and for parasite virulence. Consistent with a key role for ATF6ß in resistance against this intracellular pathogen, ATF6ß-deficient mice exhibit a high susceptibility to infection by ROP18-deficient parasites. The results reveal that interference with ATF6ß-dependent immune responses is a novel pathogenic mechanism induced by ROP18.

A novel in vivo inducible dendritic cell ablation model in mice.

Dendritic cells (DCs) are involved in T cell activation via their uptake and presentation of antigens. In vivo function of DCs was analyzed using transgenic mouse models that express diphtheria toxin receptor (DTR) or the diphtheria toxin-A subunit (DTA) under the control of the CD11c/Itgax promoter. However, CD11c+ cells are heterogeneous populations that contain several DC subsets. Thus, the in vivo function of each subset of DCs remains to be elucidated. Here, we describe a new inducible DC ablation model, in which DTR expression is induced under the CD11c/Itgax promoter after Cre-mediated excision of a stop cassette (CD11c-iDTR). Crossing of CD11c-iDTR mice with CAG-Cre transgenic mice, expressing Cre recombinase under control of the cytomegalovirus immediate early enhancer-chicken beta-actin hybrid promoter, led to the generation of mice, in which DTR was selectively expressed in CD11c+ cells (iDTRDelta mice). We successfully deleted CD11c+ cells in bone marrow-derived DCs in vitro and splenic CD11c+ cells in vivo after DT treatment in iDTRDelta mice. This mouse strain will be a useful tool for generating mice lacking a specific subset of DCs using a transgenic mouse strain, in which the Cre gene is expressed by a DC subset-specific promoter.

NFATc1 mediates Toll-like receptor-independent innate immune responses during Trypanosoma cruzi infection.

Host defense against the intracellular protozoan parasite Trypanosoma cruzi depends on Toll-like receptor (TLR)-dependent innate immune responses. Recent studies also suggest the presence of TLR-independent responses to several microorganisms, such as viruses, bacteria, and fungi. However, the TLR-independent responses to protozoa remain unclear. Here, we demonstrate a novel TLR-independent innate response pathway to T. cruzi. Myd88(-/-)Trif(-/-) mice lacking TLR signaling showed normal T. cruzi-induced Th1 responses and maturation of dendritic cells (DCs), despite high sensitivity to the infection. IFN-gamma was normally induced in T. cruzi-infected Myd88(-/-)Trif(-/-) innate immune cells, and further was responsible for the TLR-independent Th1 responses and DC maturation after T. cruzi infection. T. cruzi infection induced elevation of the intracellular Ca(2+) level. Furthermore, T. cruzi-induced IFN-gamma expression was blocked by inhibition of Ca(2+) signaling. NFATc1, which plays a pivotal role in Ca(2+) signaling in lymphocytes, was activated in T. cruzi-infected Myd88(-/-)Trif(-/-) innate immune cells. T. cruzi-infected Nfatc1(-/-) fetal liver DCs were impaired in IFN-gamma production and DC maturation. These results demonstrate that NFATc1 mediates TLR-independent innate immune responses in T. cruzi infection.