In Vivo Zymosan Treatment Induces IL15-Secreting Macrophages and KLRG1-Expressing NK Cells in Mice.

Beta-glucan (ß-glucan) is a natural polysaccharide produced by fungi, bacteria, and plants. Although it has been reported that ß-glucan enhances innate immune memory responses, it is unclear whether different types of ß-glucans display similar immune effects. To address this issue, we employed zymosan (ß-1,3-glycosidic linkage) and pustulan (ß-1,6-glycosidic linkage) to investigate their in vivo effects on innate memory immune responses. We examined the changes of innate memory-related markers in macrophages and natural killer (NK) cells, two immune cell types that display innate memory characteristics, at two different time points (16 h and 7 days) after ß-glucan stimulation. We found that short-term (16 h) zymosan treatment significantly induced macrophages to upregulate IL15 production and increased surface IL15Rα expression on NK cells. In addition, long-term (7 days) zymosan treatment significantly induced macrophages to upregulate the expression of innate memory-related markers (e.g., TNFα, HIF1α, and mTOR) and induced NK cells to express enhanced levels of KLRG1, known as an innate memory-like marker. Our results provide support that zymosan can be an effective adjuvant to promote innate memory immune responses, providing a bridge between innate and adaptive immune cells to enhance various immune responses such as those directed against tumors.

Phosphorothioate-linked guanine/cytosine-based stem-loop oligonucleotides induce the extracellular release of mitochondrial DNA from peritoneal B1a cells.

It has been previously demonstrated that phosphorothioate-linked GpC-based stem-loop oligonucleotides (GC-SL ODN) induce the release of mitochondrial DNA (mtDNA) from chronic lymphocytic leukemia (CLL) B cells. Although CLL B cells are believed to originate from CD5+ B cells because of their phenotypic similarities, it remains unclear whether GC-SL ODN can stimulate CD5+ B1 cells to secrete mtDNA. To explore this possibility, we compared the frequency of the mtDNA-producing population among peritoneal cells after GC-SL ODN treatment. We found that mtDNA-releasing cells are enriched for peritoneal CD19+ B cells upon GC-SL ODN challenge. Among peritoneal CD19+ B cells, the CD5+ B1a subpopulation was a primary cellular source of mtDNA secretion in GC-SL ODN-elicited immune responses. GC-SL ODN-stimulated mtDNA release by B1a cells was positively regulated by MyD88 and TRIF signaling pathways. In vivo GC-SL ODN treatment increased lipopolysaccharide-induced activation of innate immune cells such as NK cells, suggesting the immune-enhancing effects of mtDNA secretion. Furthermore, the loop size formed by GC-SL ODNs was a critical factor in inducing mtDNA release by B1a cells. Taken together, our results identified GC-SL ODN as promising biomaterials for enhancing immune responses.

CD1d-independent NK1.1+ Treg cells are IL2-inducible Foxp3+ T cells co-expressing immunosuppressive and cytotoxic molecules.

Regulatory T cells (Treg) play pivotal roles in maintaining self-tolerance and preventing immunological diseases such as allergy and autoimmunity through their immunosuppressive properties. Although Treg cells are heterogeneous populations with distinct suppressive functions, expression of natural killer (NK) cell receptors (NKR) by these cells remains incompletely explored. Here we identified that a small population of Foxp3+CD4+ Treg cells in mice expresses the NK1.1 NKR. Furthermore, we found that rare NK1.1+ subpopulations among CD4+ Treg cells develop normally in the spleen but not the thymus through CD1d-independent pathways. Compared with NK1.1- conventional Treg cells, these NK1.1+ Treg cells express elevated Treg cell phenotypic hallmarks, pro-inflammatory cytokines, and NK cell-related cytolytic mediators. Our results suggest that NK1.1+ Treg cells are phenotypically hybrid cells sharing functional properties of both NK and Treg cells. Interestingly, NK1.1+ Treg cells preferentially expanded in response to recombinant IL2 stimulation in vitro, consistent with their increased IL2Rαß expression. Moreover, DO11.10 T cell receptor transgenic NK1.1+ Treg cells were expanded in an ovalbumin antigen-specific manner. In the context of lipopolysaccharide-induced systemic inflammation, NK1.1+ Treg cells downregulated immunosuppressive molecules but upregulated TNFα production, indicating their plastic adaptation towards a more pro-inflammatory rather than regulatory phenotype. Collectively, we propose that NK1.1+ Treg cells might play a unique role in controlling inflammatory immune responses such as infection and autoimmunity.

Repeated α-GalCer Administration Induces a Type 2 Cytokine-Biased iNKT Cell Response and Exacerbates Atopic Skin Inflammation in Vα14Tg NC/Nga Mice.

We have previously shown that Vα14 TCR Tg (Vα14Tg) NC/Nga (NC) mice contain increased numbers of double-negative (DN) invariant natural killer T (iNKT) cells that protect against spontaneous development of atopic dermatitis (AD). iNKT cells can regulate immune responses by producing various cytokines such as IFNγ and IL4 rapidly upon stimulation with α-galactosylceramide (α-GalCer), a prototypical iNKT cell agonist. However, the precise role of α-GalCer-activated iNKT cells in AD development remains unclear. Therefore, we examined whether repeated activation of iNKT cells with α-GalCer can regulate the pathogenesis of AD in Vα14Tg NC mice. We found that Vα14Tg NC mice injected repeatedly with α-GalCer display exacerbated AD symptoms (e.g., a higher clinical score, IgE hyperproduction, and increased numbers of splenic mast cells and neutrophils) compared with vehicle-injected Vα14Tg NC mice. Moreover, the severity of AD pathogenesis in α-GalCer-injected Vα14Tg NC mice correlated with increased Th2 cells but reduced Th1 and Foxp3+ Treg cells. Furthermore, the resulting alterations in the Th1/Th2 and Treg/Th2 balance were strongly associated with a biased expansion of type 2 cytokine-deviated iNKT cells in α-GalCer-treated Vα14Tg NC mice. Collectively, our results have demonstrated the adverse effect of repeated α-GalCer treatment on skin inflammation mediated by type 2 immunity.

Chromatin Regulator SRG3 Overexpression Protects against LPS/D-GalN-Induced Sepsis by Increasing IL10-Producing Macrophages and Decreasing IFNγ-Producing NK Cells in the Liver.

We previously showed that ubiquitous overexpression of the chromatin remodeling factor SWItch3-related gene (SRG3) promotes M2 macrophage differentiation, resulting in anti-inflammatory responses in the experimental autoimmune encephalomyelitis model of multiple sclerosis. Since hepatic macrophages are responsible for sepsis-induced liver injury, we investigated herein the capacity of transgenic SRG3 overexpression (SRG3ß-actin mice) to modulate sepsis in mice exposed to lipopolysaccharide (LPS) plus d-galactosamine (d-GalN). Our results demonstrated that ubiquitous SRG3 overexpression significantly protects mice from LPS/d-GalN-induced lethality mediated by hepatic M1 macrophages. These protective effects of SRG3 overexpression correlated with the phenotypic conversion of hepatic macrophages from an M1 toward an M2 phenotype. Furthermore, SRG3ß-actin mice had decreased numbers and activation of natural killer (NK) cells but not natural killer T (NKT) cells in the liver during sepsis, indicating that SRG3 overexpression might contribute to cross-talk between NK cells and macrophages in the liver. Finally, we demonstrated that NKT cell-deficient CD1d KO/SRG3ß-actin mice are protected from LPS/d-GalN-induced sepsis, indicating that NKT cells are dispensable for SRG3-mediated sepsis suppression. Taken together, our findings provide strong evidence that SRG3 overexpression may serve as a therapeutic approach to control overwhelming inflammatory diseases such as sepsis.

Ubiquitous Overexpression of Chromatin Remodeling Factor SRG3 Exacerbates Atopic Dermatitis in NC/Nga Mice by Enhancing Th2 Immune Responses.

The SWItch (SWI)3-related gene (SRG3) product, a SWI/Sucrose Non-Fermenting (SNF) chromatin remodeling subunit, plays a critical role in regulating immune responses. We have previously shown that ubiquitous SRG3 overexpression attenuates the progression of Th1/Th17-mediated experimental autoimmune encephalomyelitis. However, it is unclear whether SRG3 overexpression can affect the pathogenesis of inflammatory skin diseases such as atopic dermatitis (AD), a Th2-type immune disorder. Thus, to elucidate the effects of SRG3 overexpression in AD development, we bred NC/Nga (NC) mice with transgenic mice where SRG3 expression is driven by the ß-actin promoter (SRG3ß-actin mice). We found that SRG3ß-actin NC mice exhibit increased AD development (e.g., a higher clinical score, immunoglobulin E (IgE) hyperproduction, and an increased number of infiltrated mast cells and basophils in skin lesions) compared with wild-type NC mice. Moreover, the severity of AD pathogenesis in SRG3ß-actin NC mice correlated with expansion of interleukin 4 (IL4)-producing basophils and mast cells, and M2 macrophages. Furthermore, this accelerated AD development is strongly associated with Treg cell suppression. Collectively, our results have identified that modulation of SRG3 function can be applied as one of the options to control AD pathogenesis.

Integration of sputter-deposited multiferroic CoFe2O4-BiFeO3 nanocomposites on conductive La0.7Sr0.3MnO3 electrodes.

The structure, magnetic and ferroelectric properties of sputtered epitaxial CoFe2O4-BiFeO3 (CFO-BFO) nanocomposite thin films grown on La0.7Sr0.3MnO3 (LSMO) layers on (001) oriented SrTiO3 (STO) substrates and on STO-buffered Si are described. The as-grown LSMO thin films were smooth and poorly conductive but the resistivity was reduced and the surfaces roughened after annealing. Cosputtered CFO and BFO on STO formed vertically aligned nanostructures consisting of epitaxial spinel CFO pillars within a perovskite BFO matrix, but the rough surface of the annealed LSMO film promoted additional CFO pillar orientations. A reorientation of the CFO magnetic easy axis to an in-plane direction occurred as the LSMO became thicker due to changes in the strain state of the CFO pillars. The LSMO underlayer enabled the ferroelectric response of the BFO to be measured. Nanocomposites were grown onto LSMO/SrTiO3/Si which provides a path towards large scale integration of electrically contacted nanocomposites on Si.

Magnetic Phase Formation in Self-Assembled Epitaxial BiFeO3-MgO and BiFeO3-MgAl2O4 Nanocomposite Films Grown by Combinatorial Pulsed Laser Deposition.

Self-assembled epitaxial BiFeO3-MgO and BiFeO3-MgAl2O4 nanocomposite thin films were grown on SrTiO3 substrates by pulsed laser deposition. A two-phase columnar structure was observed for BiFeO3-MgO codeposition within a small window of growth parameters, in which the pillars consisted of a magnetic spinel phase (Mg,Fe)3O4 within a BiFeO3 matrix, similar to the growth of BiFeO3-MgFe2O4 nanocomposites reported elsewhere. Further, growth of a nanocomposite with BiFeO3-(CoFe2O4/MgO/MgFe2O4), in which the minority phase was grown from three different targets, gave spinel pillars with a uniform (Mg,Fe,Co)3O4 composition due to interdiffusion during growth, with a bifurcated shape from the merger of neighboring pillars. BiFeO3-MgAl2O4 did not form a well-defined vertical nanocomposite in spite of having lower lattice mismatch, but instead formed a two-phase film with in which the spinel phase contained Fe. These results illustrate the redistribution of Fe between the oxide phases during oxide codeposition to form a ferrimagnetic phase from antiferromagnetic or nonmagnetic targets.