LPS-mediated engagement of TLR4 in <i>i</i>NKT cells aggravates <i>Saccharopolyspora rectivirgula</i> (SR)-induced hypersensitivity pneumonitis (HP).

<p>HP was induced by inoculating the SR antigen nasally. (A) The levels of SR-specific IgG in serum, and IL-4 and IFN-γ transcripts in the lungs of B6 or TLR4^−/− mice were analyzed seven days after the first nasal inoculation of SR antigen using ELISA and real-time PCR, respectively. (B–D) B6, CD1d^−/−, and CD1d^−/− mice adoptively transferred with sorted <i>i</i>NKT cells (1×10⁵ cells) from WT B6 or TLR4^−/− mice were inoculated nasally with SR antigens. Sorted <i>i</i>NKT cells from B6 or TLR4^−/− mice were incubated with LPS or PBS for 30 min before adoptive transfer into CD1d^−/− mice. (B) These mice were sacrificed three weeks after induction of HP, and SR-specific IgG levels in serum were determined. (C) IL-4 and (D) IFN-γ levels were measured in the bronchoalveolar lavage fluid from B6, CD1d^−/−, and CD1d^−/− mice adoptively transferred with sorted <i>i</i>NKT cells from B6 or TLR4^−/− mice seven days after inoculation of the first SR antigen by ELISA. (E) Histological examination of the lungs was performed 7 days after first SR treatment (×100). (A–D) Data are from a representative of three repeated experiments. (n = 4 in A, n = 3 in B, C, D; *p<0.05, **p<0.01, ***p<0.001).</p

Direct Engagement of TLR4 in Invariant NKT Cells Regulates Immune Diseases by Differential IL-4 and IFN-γ Production in Mice

<div><p>During interaction with APCs, invariant (<em>i</em>) NKT cells are thought to be indirectly activated by TLR4-dependently activated APCs. However, whether TLR4 directly activates <em>i</em>NKT cells is unknown. Therefore, the expression and function of TLR4 in <em>i</em>NKT cells were investigated. Flow cytometric and confocal microscopic analysis revealed TLR4 expression on the surface and in the endosome of <em>i</em>NKT cells. Upon LPS stimulation, <em>i</em>NKT cells enhanced IFN-γ production, but reduced IL-4 production, in the presence of TCR signals, depending on TLR4, MyD88, TRIF, and the endosome. However, enhanced TLR4-mediated IFN-γ production by <em>i</em>NKT cells did not affect IL-12 production or CD1d expression by DCs. Adoptive transfer of WT, but not TLR4-deficient, <em>i</em>NKT cells promoted antibody-induced arthritis in CD1d^−/− mice, suggesting that endogenous TLR4 ligands modulate <em>i</em>NKT cell function in arthritis. Furthermore, LPS-pretreated WT, but not TLR4-deficient, <em>i</em>NKT cells suppressed pulmonary fibrosis, but worsened hypersensitivity pneumonitis more than untreated WT <em>i</em>NKT cells, indicating that exogenous TLR4 ligands regulate <em>i</em>NKT cell functions in pulmonary diseases. Taken together, we propose a novel direct activation pathway of <em>i</em>NKT cells in the presence of TCR signals via endogenous or exogenous ligand-mediated engagement of TLR4 in <em>i</em>NKT cells, which regulates immune diseases by altering IFN-γ and IL-4 production.</p> </div

LPS-mediated direct engagement in <i>i</i>NKT cells enhances IFN-γ production, but reduces IL-4 production in the presence of TCR engagement.

<p>(A, B) Sorted <i>i</i>NKT cells from B6 or TLR4^−/− mice (1×10⁵/well) were stimulated using coated anti-CD3 (5 µg mL⁻¹) + CD28 mAbs (5 µg mL⁻¹) in culture plates, LPS (5 µg mL⁻¹), or LPS (5 µg mL⁻¹) + anti-CD3 (5 µg mL⁻¹) + CD28 mAbs (5 µg mL⁻¹) for 24 h. (A) The amounts of IL-4 and IFN-γ in the culture supernatant were measured by ELISA. (B) T-bet or GATA-3 mRNA expression were analyzed by real-time PCR. (C) B6, CD1d^−/−, and TLR4^−/− mice were injected i.p. with α-GalCer (1 µg in 300 µl PBS), LPS (25 µg in 300 µl PBS), or α-GalCer (1 µg) + LPS (25 µg in 300 µl PBS). Serum IL-4 levels were monitored 2 h later, and serum IFN-γ levels were measured by ELISA 24 h after injection of these reagents. (D) <i>i</i>NKT cells were co-cultured with irradiated or un-irradiated bone marrow-derived DCs (BMDCs) from WT or TLR4^−/− mice in the presence of LPS and/or α-GalCer for 24 h. The levels of IL-12 in culture supernatant and CD1d expression on BMDCs were estimated. Numbers in diagrams represent mean fluorescence intensity. (A–D) Data are presented as the means ± SD of three mice in each group. Similar results were obtained from either two (D) or three (A–C) independent experiments. (*p<0.05, **p<0.01 and ***p<0.001).</p

TLR4 in <i>i</i>NKT cells plays a crucial role in promoting antibody-induced joint inflammation.

<p>(A) Sorted WT or TLR4-deficient <i>i</i>NKT cells (3×10⁵cells mouse⁻¹) were adoptively transferred into CD1d^−/− mice one day before K/BxN serum transfer (n = 3 per group). Clinical scores and ankle thickness were then monitored (*p<0.05 and **p<0.01, ***p<0.001). (B) The amounts of IL-4, IFN-γ and TGF-β1 were measured relative to GAPDH by real-time PCR in the joint tissues of B6, CD1d^−/−, and CD1d^−/− mice administered sorted WT or TLR4-deficient <i>i</i>NKT cells 10 days after K/BxN serum transfer. (C) Histological examination of the joints was performed 7 days after K/BxN serum transfer (×100). Data are representative of three independent experiments. (n = 4 in each group; *p<0.05, **p<0.01, ***p<0.001).</p

LPS-mediated engagement of TLR4 in <i>i</i>NKT cells suppresses bleomycin-induced pulmonary fibrosis.

<p>(A) Lungs were removed from B6 or TLR4^−/− mice 7 or 21 days after an intratracheal injection of bleomycin (2 mg/kg), and the levels of hydroxyproline, and IL-4 and IFN-γ transcripts were determined. (B) Hydroxyproline content in the lungs of B6, CD1d^−/−, and CD1d^−/− mice adoptively transferred with sorted WT, TLR4-deficient <i>i</i>NKT cells, LPS-pretreated WT <i>i</i>NKT, or LPS-pretreated TLR4-deficient <i>i</i>NKT cells was determined 21 days after bleomycin injection. The increased hydroxyproline content in the lungs of experimental groups are expressed as a percentage. Data are indicated as mean ± SEM of six mice in each group. (C) The transcript levels of TGF-β1, IFN-γ, and IL-4 were determined by quantitative analysis relative to GAPDH using real-time PCR in the lungs of B6, CD1d^−/−, and CD1d^−/− mice adoptively transferred with sorted WT <i>i</i>NKT or TLR4-deficient <i>i</i>NKT cells seven days after intratracheal injection of bleomycin. Data are representative of three repeated experiments. (n = 3 in each group; *p<0.05, **p<0.01, ***p<0.001).</p

Gold Nanoparticles as Nucleation-Inducing Reagents for Protein Crystallization

Protein crystallization is a necessary but time-consuming and difficult step in structure determination by crystallography. The rate-limiting step of crystallization is nucleation, where protein monomers in solution cluster in an ordered fashion to form a stable nucleus. Here, we propose the use of gold nanoparticles to accelerate nucleation and enhance crystal formation. We tested whether gold nanoparticles can facilitate nucleation by interacting with target proteins and inducing favorable clustering. We used differently sized gold nanospheres and gold nanostars to crystallize hen egg-white lysozyme. Our results indicated that gold nanoparticles significantly increased the number of crystallization conditions (by about 20%). Spherical and larger gold particles were found to be more efficient. Furthermore, the use of gold nanoparticles did not have any adverse effect on data collection or structure determination. Our findings indicate that the use of nanoparticles as protein nucleation-inducing reagents can greatly accelerate structure determination by X-ray crystallography

<i>i</i>NKT cells constitutively express TLR4 on the cell surface and in the endosomal compartment.

<p>(A) TLR4 expression was analyzed on gated α-GalCer/CD1d tetramer^-CD3⁺ T cells, α-GalCer/CD1d tetramer⁺<i>i</i>NKT cells, and F4/80⁺ macrophages from B6 (solid line) or TLR4^−/− mice (gray) compared with an isotype-matched control IgG (dotted line) by flow cytometric analysis. Numbers in diagrams represent mean fluorescence intensity (top for control, middle for TLR4^−/− mice, bottom for B6 mice). (B) Sorted <i>i</i>NKT cells and F4/80⁺ macrophages were stained with anti-TLR4 mAb (green) or isotype-matched control IgG, and DAPI (blue) (C) Sorted <i>i</i>NKT cells were stained with anti-TLR4 mAb or isotype-matched control IgG (red), and EEA-1 (early endosome marker; green) and DAPI (blue). (D) CD14 expression was analyzed on gated α-GalCer/CD1d tetramer⁺<i>i</i>NKT cells and F4/80⁺ macrophages from B6 mice (solid line) as compared with an isotype-matched IgG control (gray). Data are representative of three independent experiments.</p

Tunable Plasmonic Cavity for Label-free Detection of Small Molecules

Owing to its high sensitivity and high selectivity along with rapid response time, plasmonic detection has gained considerable interest in a wide variety of sensing applications. To improve the fieldwork applicability and reliability of plasmonic detection, the integration of plasmonic nanoparticles into optical devices is desirable. Herein, we propose an integrated label-free detection platform comprising a plasmonic cavity that allows sensitive molecular detection via either surface-enhanced Raman scattering (SERS) or plasmon resonance energy transfer (PRET). A small droplet of metal ion solution spontaneously produces a plasmonic cavity on the surface of uncured poly­(dimethylsiloxane) (PDMS), and as PDMS is cured, the metal ions are reduced to form a plasmonic antennae array on the cavity surface. Unique spherical feature and the integrated metallic nanoparticles of the cavity provide excellent optical functions to focus the incident light in the cavity and to rescatter the light absorbed by the nanoparticles. The optical properties of the plasmonic cavity for SERS or PRET are optimized by controlling the composition, size, and density of the metal nanoparticles. By using the cavity, we accomplish both 1000-fold sensitive detection and real-time monitoring of reactive oxygen species secreted by live cells via PRET. In addition, we achieve sensitive detection of trace amounts of toxic environmental molecules such as 5-chloro-2-methyl-4-isothiazolin-3-one/2-methyl-4-isothiazol-3-one (CMIT/MIT) and bisphenol A, as well as several small biomolecules such as glucose, adenine, and tryptophan, via SERS

Immunofluorescence studies: Representative photographs showing excessive P-LKB1/LC3-II (+) expression and markedly diminished CTFβ(+)-cells under treatment with cilostazol (10 μM) in the activated N2aSwe cells, as compared with vehicle-treated group.

<p>Immunofluorescence studies: Representative photographs showing excessive P-LKB1/LC3-II (+) expression and markedly diminished CTFβ(+)-cells under treatment with cilostazol (10 μM) in the activated N2aSwe cells, as compared with vehicle-treated group.</p

Comparison of cilostazol- and resveratrol-stimulated P-LKB1 and P-AMPKα expressions in N2a cells that LKB1 is detectable, and in HeLa cells that lack LKB1.

<p><b>A</b>. Immunoblot of P-LKB significantly increased after pretreating N2a cells with cilostazol (CSZ, 10 μM) or resveratrol (RES, 20 μM), whereas P-LKB expression was not appeared in HeLa cells. <b>B</b>. The significant increases in P-AMPK expression by cilostazol (CSZ, 10 μM) or resveratrol (Res, 20 μM) were not observed in HeLa cells, whereas they were obviously identified in N2a cells. Results are expressed as the means ± SDs of 4 experiments. ***<i>P</i> < 0.001 vs. vehicle (Veh). <b>C</b>. Proposed signal pathways for the neuroprotective effect of cilostazol against Aβ-induced neurotoxicity: Cilostazol upregulates autophagy through activating SIRT1/LKB1/AMPK1α signal pathways and depletes intracellular Aβ and APP-CTFβ accumulation, and thereby results in decreased neurotoxicity.</p