NOD2-mediated Suppression of CD55 on Neutrophils Enhances C5a Generation During Polymicrobial Sepsis

<div><p>Nucleotide-binding oligomerization domain (NOD) 2 is a cytosolic protein that plays a defensive role in bacterial infection by sensing peptidoglycans. C5a, which has harmful effects in sepsis, interacts with innate proteins. However, whether NOD2 regulates C5a generation during sepsis remains to be determined. To address this issue, cecal ligation & puncture (CLP)-induced sepsis was compared in wild type and Nod2^−/− mice. Nod2^−/− mice showed lower levels of C5a, IL-10, and IL-1β in serum and peritoneum, but higher survival rate during CLP-induced sepsis compared to wild type mice. Injection of recombinant C5a decreased survival rates of Nod2^−/− mice rate during sepsis, whereas it did not alter those in wild type mice. These findings suggest a novel provocative role for NOD2 in sepsis, in contrast to its protective role during bacterial infection. Furthermore, we found that NOD2-mediated IL-10 production by neutrophils enhanced C5a generation by suppressing CD55 expression on neutrophils in IL-1β-dependent and/or IL-1β-independent manners, thereby aggravating CLP-induced sepsis. SB203580, a receptor-interacting protein 2 (RIP2) inhibitor downstream of NOD2, reduced C5a generation by enhancing CD55 expression on neutrophils, resulting in attenuation of polymicrobial sepsis. Therefore, we propose a novel NOD2-mediated complement cascade regulatory pathway in sepsis, which may be a useful therapeutic target.</p></div

Design and Optimization of an α‑Helical Bundle Dimer Cell-Penetrating Peptide for <i>In Vivo</i> Drug Delivery

To deliver membrane-impermeable drugs into eukaryotic cells, a lot of cell-penetrating peptides (CPPs) were discovered. Previously we designed an amphipathic α-helical peptide which dimerizes itself via its two C-residues. This bis-disulfide-linked dimeric bundle, LK-3, has remarkable cell-penetrating ability at nanomolar concentration, which is an essential prerequisite for CPP. In an effort to optimize the sequence of LK-3, we adjusted its length and evaluated changes in the dimerization rate. We found that a 10-amino-acid monomer has the fastest dimerization rate and subsequently modified its hydrophobic and hydrophilic residues to construct a small peptide library. The evaluation of cell permeability of these derivatives showed that their cell-penetrating ability is comparable to that of the LK-3, except V- or H-containing ones. In this library, diLR10 was found to display fast nanomolar cell membrane penetration, low toxicity, and ease of production. The methotrexate (MTX) conjugate of diLR10, MTX-diLR10, has a 19-fold increased efficacy over MTX in MDA-MB-231 cells and efficiently deflates lesions in a rheumatoid arthritis (RA) in vivo mouse model

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 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

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

<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

Histological characteristics of <i>ALK</i>-rearranged tumors.

<p>A, cribriform formation; B, mucin-containing goblet cells; C, mucin-containing signet-ring cell; D, psammoma body; E, cholesterol cleft.</p

Multivariate analysis: Factors significantly associated with <i>ALK</i> rearrangement on logistic regression analysis.

<p>Multivariate analysis: Factors significantly associated with <i>ALK</i> rearrangement on logistic regression analysis.</p