LPG promotes binding of TLR signaling proteins and NF-κB nuclear translocation.

<p>(A and B) Western blot shows TLR1 and TLR6 in NK cells. (C and D) Immunoprecipitations were performed with anti-TLR2 in non-stimulated (lane a) and LPG-stimulated NK cells after 1 h of co-incubation (lane b). (C) Immunoprecipitates were subjected to Western blotting and probed with anti-TLR1 and (D) anti-TLR6 antibodies, respectively. A representative immunoblot from four different experiments is shown. (E) NK cell lysates in non-stimulated and LPG-stimulated NK cells were immunoprecipitated with anti-TLR2, anti-MyD88, anti-IRAK-1 and anti-TRAF-6 antibodies and Western blotted with anti-MyD88, anti-IRAK-1, anti-TRAF-6 and anti-IKK-γ antibodies. (F) Nuclear translocation of p50 and p65 NF-κB isoforms were analyzed in non-stimulated and LPG-stimulated NK cells. (G) Phosphorilation of pIKK-α/β and pIκB-α were analyzed in non-stimulated NK cells (Non-stim) or in cells stimulated with PMA (15 min) or LPG (15, 30, 45 and 60 min). A representative immunoblot from two different experiments is show.</p

TNF-α production by NK cells.

<p>(A) TNF-α production in peripheral blood [control subjects (n = 21), LCL patients (n =  28) and DCL patients (n = 6)]. (B) Analysis of TNF-α production of LCL patients according to gender [female (n = 11) and male (n = 17)]. (C) Double immunohistochemistry (CD57⁺/TNF-α⁺) staining of lesions of LCL and DCL patients showed dark green staining induced by the combination of a green substrate (Stay Green/AP) used for NK cells and DAB (brown) used for TNF-α staining. □ Non-stimulated NK cells, ▪ LPG-stimulated NK cells. Mean ±SEM is shown. *p≤0.05 was considered significant. Scale bar  = 50 µm. Double positive cells CD57⁺/TNF-α⁺ (black arrows) and single positive cells CD57^-/TNF-α⁺ (red arrows).</p

TLR1, TLR2 and TLR6 expression on NK cells in lesions of 3 LCL and 3 DCL patients.

<p>(A) Double immunohistochemistry staining of TLR expression (TLR1, TLR2 and TLR6) on NK cells (CD57). (B) Number of NK cells expressing TLRs per mm². Results are the mean ±SEM. Scale bar  = 50 µm. Black arrows show double positive cells.</p

IFN-γ production by NK cells.

<p>(A) IFN-γ production of peripheral blood NK cells [control subjects (n = 21), LCL patients (n =  28) and DCL patients (n = 6)]. Analysis in LCL patients [female (n = 11) and male (n = 17)] according to: (B) gender; (C) disease duration; (D) age. (E) Double immunohistochemical labelling (CD57⁺/IFN-γ⁺) in lesions of patients (LCL and DCL) showed redish-brown staining generated by the combination of a red AP substrate used for NK cells and DAB Black used for IFN-γ staining. □ Non-stimulated NK cells. ▪ LPG-stimulated NK cells. Mean ±SEM is shown. *p≤0.05 was considered significant. Scale bar  = 50 µm. Black arrows show double positive cells.</p

TLR1, TLR2 and TLR6 expression on NK-cell subsets (CD56^bright and CD56^dim) of patients and controls.

<p>(A) Representative flow cytometry dot plot (TLR2 vs. CD56) and histogram of TLR2 expression in NK cells. (B) This gate was subsequently analyzed for TLR2 expression on NK-cell subsets. Dot plot (CD16⁺/CD56⁺) and histogram for CD56^bright (blue box) and CD56^dim (green box) NK cells. (C) TLR1 expression. (D) TLR2 expression. (E) TLR6 expression. □ Non-stimulated NK cells; ▪ LPG-stimulated NK cells. Lane 1: CD56^dim; lane 2: CD56^bright NK cells. Cell surface expression is indicated by MFI. Results are the mean ±SEM. *Significant differences were observed between NK cell subsets of LCL and DCL patients for all 3 TLRs in LPG-stimulated and non-stimulated cells. LCL patients (n = 9), DCL patients (n = 4) and controls (n = 4).</p

Transcript expression by quantitative real time PCR in non-stimulated (NS) and LPG-stimulated NK cells (S).

<p>TLR2, IFN-γ and TNF-α gene expression in NK cells of healthy controls (n = 5), LCL (n = 6) and DCL (n = 3) patients.</p><p><b>1</b>. C_S vs C_NS; <b>2</b>. LCL_S vs LCL_NS; <b>3</b>. DCL_S vs DCL_NS; <b>4</b>. LCL_NS vs C_NS; <b>5</b>. DCL_NS vs C_NS; <b>6</b>. DCL_NS vs LCL_NS; <b>7</b>. LCL_S vs C_S; <b>8</b>. DCL_S vs C_S; <b>9</b>. DCL_S vs LCL_S. C: healthy controls, LCL: patients with localized cutaneous leishmaniasis, DCL: patients with diffuse cutaneous leishmaniasis. *p<0.05 significant differences.</p><p>Transcript expression by quantitative real time PCR in non-stimulated (NS) and LPG-stimulated NK cells (S).</p

Analysis of TLR2 expression on NK cells.

<p>(A) Representative flow CD56⁺/CD3^- expression in membranes of NK cells; right image: NK cells were gated and analyzed for TLR2 (TLR2⁺/CD56⁺) expression. (B) Representative histograms showing the MFI levels of TLR2 expression on NK cells from control and patients (LCL and DCL). (C) Quantitation of TLR2 expression on NK cells. □ Non-stimulated cells, ▪ LPG-stimulated cells. Data are representative of healthy controls (n = 21), LCL patients (n = 28) and DCL patients (n = 6). These results are the mean ±SEM. *p≤0.05 was considered significant.</p

IFNγ production by CD8 from LCL and DCL patients.

<p>A) PBMC were non-stimulated (CNT), stimulated with PMA-Ionomycin during 4 h or with MOi during 24 h. Density diagrams of IFNγ vs. CD8 from 10 LCL and 4 DCL patients are shown. B) CD8⁺IFNγ⁺ percentage analysis from 10 LCL and 4 DCL patients are shown. C) ELISA results of IFNγ production in supernatants of the co-incubation CD8-MOi from 9 LCL and 4 DCL patients are shown (<i>p</i><0.01).</p

Apoptosis in LCL and DCL patient lesions.

<p>Frozen tissue sections of LCL (A) and DCL (B) were stained with TUNEL. Two representative photographs are shown. C) Double staining of LCL patient tissue: TUNEL in peroxidase (brown), CD68 in phosphatase (red). Black arrows show TUNEL⁺ cells, red arrows show TUNEL⁺ CD68⁺ cells. D). Positive percentage analysis for TUNEL of 7 LCL and 5 DCL patients is shown. Three fields were counted, 200 cells per field (<i>p</i><0.05). Bar  = 20 µm.</p

Lymphocyte proliferation in LCL and DCL patients.

<p>A) CFDA-labeled PBMC were incubated with PBS (CNT), MOi or Con A [5 µM] for seven days. CFDA^low cell percentages of total CD8 are shown. Representative dotplots from 8 LCL and 4 DCL patients are shown. B). Proliferation analysis of CD8 from 8 LCL and 4 DCL patients, stimulated with PBS (black bars), MOi (white bars) and Con A (striped bars) are shown (<i>p</i><0.01).</p