BoNT/A used TLR2 for NO (A) and TNFα (B) production in the RAW264.7 mouse macrophage cell line.

<p>The levels of NO (A) and TNFα (B) were assessed in RAW264.7 cells stimulated with BoNT/A after preincubation with polyclonal anti-TLR2 (50 μg/ml) or anti-TLR4 (20 μg/ml). TLR2, TLR4, IgG1, and IgG2a indicate the counterpart antigen against which the respective antibody was used to pretreat RAW264.7 cells. Mouse anti-IgG1 or rat anti-IgG2a was used as a negative control. Anti-TLR2 had a significant (<i>p</i><0.05) inhibitory effect on both NO and TNFα production by RAW264.7 cells stimulated with BoNT/A but anti-TLR4 had no noticeable effect.</p

Dose-dependent production of NO (A), TNFα (B), and IL-6 (C) in BoNT/A-treated RAW264.7 cells.

<p>Cells were incubated with BoNT/A (0, 1, 2, 5, and 10 nM) or 1 μg/ml LPS as a positive control for 24 h at 37°C. Culture supernatants were collected, and the levels of NO, TNFα, and IL-6 were measured. Values are the mean ± SD of three replicates for each group.</p

Microarray data analysis.

<p>(A) Hierarchical clustering (performed using ArrayAssist) shows subsets of RAW264.7 macrophage genes that are specifically regulated in response to 1 or 5 nM BoNT/A over time (0 to 10 h). Bright green indicates very low signal values, bright red represents very high signal values, and black represents OR median signal values. B1T and B5T represent the concentration of BoNT/A added to RAW264.7 cells (1 or 5 nM), respectively. (B) Venn diagram showing up- and down-regulated genes after 1 or 5 nM BoNT/A. The intersections of the circles indicate the number of genes in common between treatments. The dotted horizontal line separates the up- and down-regulated genes. The cutoffs used were a fold change of at least 2.0 and an adjusted <i>p</i> value of <0.05. (C) Time-dependent gene expression changes in BoNT/A-stimulated RAW264.7 cells. Fold changes of 60 genes commonly induced by 1 and 5 nM BoNT/A were recorded according to treatment time.</p

BoNT/A inhibited LPS-induced production of TNFα and NO in a dose- and time-dependent manner in RAW264.7 macrophages.

<p>RAW264.7 cells were treated with 0 to 1 nM BoNT/A for 24 h (A and B) or with 1 nM BoNT/A for 0 to 24 h and then stimulated with or without 1 μg/ml LPS (C and D). Culture media were collected at 24 h to measure NO (A and C) and TNFα (B and D) concentrations using the Griess reaction and sandwich ELISA, respectively. Three independent experiments were performed, and the data shown are the mean ± SD.</p

TLR2 KO mice show decreased expression of TNFα and IL-6 cytokines upon BoNT/A stimulation.

<p>WT (black bar; n = 6) and TLR2 KO mice (white bar; n = 6) were used to isolate bone marrow monocytes. Differentiated BMDM cells and subjected to BoNT/A treatment. Culture supernatants were analyzed for TNFα (A) and IL6 (B). All data were given as means ± SD.</p

BoNT/A inhibited the production of LPS-induced pro-inflammatory mediators in RAW264.7 cells.

<p>A and B, production of NO and proinflammatory cytokines in LPS-stimulated RAW264.7 cells with or without BoNT/A pretreatment. RAW264.7 cells were pretreated with 1 nM BoNT/A or 1 nM BoNToxoid/A for 24 h, and then stimulated with or without 1 μg/ml LPS. Culture media were collected at 24 h to measure NO (A), TNFα, IL-6, and IL-12 (B) concentrations using the Griess reaction and sandwich ELISA, respectively. Three independent experiments were performed, and the data are the mean ± S.D. *, <i>p</i><0.05 vs. LPS alone. C and D, quantification of mRNA expression of <i>Tnf</i> and iNOS in LPS-stimulated RAW264.7 macrophages with or without BoNT/A pretreatment. Total RNAs were isolated, and mRNA levels of iNOS and TNFα were analyzed with qRT-PCR. GAPDH expression was used as an internal control for RT-PCR. Representative results of three independent experiments are shown.</p

Microscopic analysis of binding of FITC-labeled PSP1 and annexin V to apoptotic cells.

<p>(A) Apoptotic H460 cells in a chamber slide were incubated with 20 μg of PSP1, control peptide, or annexin V for 30 min. The cells were washed and fixed with 4% PFA before staining with DAPI. The confocal images were taken by laser confocal microscopy. (B) Normal H460 cells were subjected to incubation with the same amount of peptide or annexin V.</p

<i>In vivo</i> imaging of tumors after apoptosis induction.

<p>(A) Cy7.5-labelled PSP1peptide, control peptide, or annexin V was systematically injected into the tail vein of tumor-bearing nude mice treated with camptothecin 24 hrs before injection. The same amount of annexin V was also injected into non-camptothecin-treated tumor-bearing mice as a control. The homing of the peptides was examined after the indicated times using an optical imaging system. The white dotted circles indicate the tumors. (B) Histological examination of tumor tissues after homing of PSP1 and annexin V, performed under a fluorescence microscope. Tumor cell apoptosis was observed by TUNEL staining.</p

Botulinum Neurotoxin Type A Induces TLR2-Mediated Inflammatory Responses in Macrophages

<div><p>Botulinum neurotoxin type A (BoNT/A) is the most potent protein toxin and causes fatal flaccid muscle paralysis by blocking neurotransmission. Application of BoNT/A has been extended to the fields of therapeutics and biodefense. Nevertheless, the global response of host immune cells to authentic BoNT/A has not been reported. Employing microarray analysis, we performed global transcriptional profiling of RAW264.7 cells, a murine alveolar macrophage cell line. We identified 70 genes that were modulated following 1 nM BoNT/A treatment. The altered genes were mainly involved in signal transduction, immunity and defense, protein metabolism and modification, neuronal activities, intracellular protein trafficking, and muscle contraction. Microarray data were validated with real-time RT-PCR for seven selected genes including <i>tlr2</i>, <i>tnf</i>, <i>inos</i>, <i>ccl4</i>, <i>slpi</i>, <i>stx11</i>, and <i>irg1</i>. Proinflammatory mediators such as nitric oxide (NO) and tumor necrosis factor alpha (TNFα) were induced in a dose-dependent manner in BoNT/A-stimulated RAW264.7 cells. Increased expression of these factors was inhibited by monoclonal anti-Toll-like receptor 2 (TLR2) and inhibitors specific to intracellular proteins such as c-Jun N-terminal kinase (JNK), extracellular signal–regulated kinase (ERK), and p38 mitogen–activated protein kinase (MAPK). BoNT/A also suppressed lipopolysaccharide-induced NO and TNFα production from RAW264.7 macrophages at the transcription level by blocking activation of JNK, ERK, and p38 MAPK. As confirmed by TLR2-/- knock out experiments, these results suggest that BoNT/A induces global gene expression changes in host immune cells and that host responses to BoNT/A proceed through a TLR2-dependent pathway, which is modulated by JNK, ERK, and p38 MAPK.</p></div

Structure of PSP1 peptide and binding of FITC-labeled PSP1 and annexin V to apoptotic cells.

<p>(A) PEP-FOLD simulations give the structure of the PSP1 peptide (magenta; hydroxyl group: red/white). PS (white) is displayed with its head group (phosphate: yellow; amide: blue) toward PSP1. (B) PS exposure on the cell membrane of apoptotic cells is examined by FACS analysis based on shifting following binding to annexin V-Alexa Fluor 488 (X-axis) and PI (Y-axis). (C) The indicated amount of FITC-CLSYYPSYC or FITC-annexin V was incubated with apoptotic H460 cells (red) or normal H460 cells (black) at 4°C for 1 hr. Control peptide (grey); FITC-NSSSVDK. (D) 10 μg/ml of FITC-PSP1 peptide (CLS<b>YY</b>PSYC) and mutants FITC-PSP1 (AA-PSP1; CLS<b>AA</b>PSYC. FF-PSP1; CLS<b>FF</b>PSYC) were incubated with apoptotic H460 cells (red) or normal H460 cells (black). (E) Bar chart indicates the FITC labeled peptide binding (under bar in D) to apoptotic H460 (red) vs. normal H460 cells (black). The results are presented as the means + s.d. (n = 3 independent examinations per group) **P<0.01.</p