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

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 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

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

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

<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

Electrospun Nanofibrous Sheets for Selective Cell Capturing in Continuous Flow in Microchannels

Electrospun nanofibrous meshes were surface-modified for selective capturing of specific cells from a continuous flow in PDMS microchannels. We electrospun nanofibrous mats composed of poly­(ε-carprolactone) (PCL) and amine-functionalized block copolymers composed of PCL and poly­(ethylenimine) (PEI). A mixture of biotinylated PEG and blunt PEG was chemically tethered to the nanofibrous mats via the surface-exposed amines on the mat. The degree of biotinylation was fluorescently and quantitatively assayed for confirming the surface-biotinylation levels for avidin-specific binding. The incorporation level of avidin gradually increased when the blend ratio of biotinylated PEG on the mat increased, confirming the manipulated surfaces with various degree of biotinylation. Biotinylated cells were incubated with avidin-coated biotinylated mats and the specific binding of biotinylated cells was monitored in a microfluidic channel with a continuous flow of culture medium, which suggests efficient and selective capturing of the biotinylated cells on the nanofibrous mat

Atom Transfer Radical Polymerization of Multishelled Cationic Corona for the Systemic Delivery of siRNA

We propose an effective siRNA delivery system by preparing poly­(DAMA-HEMA)-multilayered gold nanoparticles using multiple surface-initiated atom transfer radical polymerization processes. The polymeric multilayer structure is characterized by transmission electron microscopy, matrix-associated laser desorption/ionization time-of-flight mass spectrometry, UV–vis spectroscopy, Fourier transform infrared spectroscopy, dynamic light scattering, and ζ-potential. The amount of siRNA electrostatically incorporated into the nanoparticle can be tuned by the number of polymeric shells, which in turn influences the cellular uptake and gene silencing effect. In a bioreductive environment, the interlayer disulfide bond breaks to release the siRNA from the degraded polymeric shells. Intravenously injected c-Myc siRNA-incorporated particles accumulate in the tumor site of a murine lung carcinoma model and significantly suppress the tumor growth. Therefore, the combination of a size-tunable AuNP core and an ATRP-functionalized shell offers control and versatility in the effective delivery of siRNA

RNA ISH scores for <i>GREM1</i> in basal cell carcinomas subtypes.

<p>RNA ISH scores for <i>GREM1</i> in basal cell carcinomas subtypes.</p

<i>GREM1</i> expression in normal skin and scar tissue.

<p>RNA in situ hybridization (ISH) for <i>GREM1</i> and immunohistochemical analysis for α-SMA was performed on the normal skin (n = 6) and scar tissues (n = 10). (A–C) Dermal fibroblasts of normal skin were negative for <i>GREM1</i> or α-smooth muscle actin (α-SMA). (D–F) Scar tissue fibroblasts were positive for both <i>GREM1</i> and α-SMA. (G) RNA ISH scores for <i>GREM1</i> in normal skin and scar tissues. Scale bar: 40 μm (A and D), 20 μm (B, C, E, and F).</p