Stable downregulation of MyD88 in murine melanoma cells.

<p><b>(A-B)</b> Representative immunohistochemistry images of tissue samples from primary and <b>(C-D)</b> metastatic human melanomas stained with antiMyD88 antibody (brown). The nuclei were counter-stained with Hematoxylin (blue). All images were obtained from The Human Protein Atlas; <a href="http://www.proteinatlas.org/ENSG00000172936-MYD88/cancer" target="_blank">http://www.proteinatlas.org/ENSG00000172936-MYD88/cancer</a>. Scale bar = 100μμm. <b>(E)</b> Quantitative RT-PCR analysis of MyD88 mRNA expression in B16 cells stably transduced with lentiviruses to express either scrambled (SCR) or MyD88-specific shRNAs (shMyD88-A and -B). Data represent fold increase ± SD after normalization with GAPDH mRNA (** p<0.001). <b>(F)</b> Protein expression of MyD88 was determined by In-Cell Western analysis. Representative output image showing whole-cell fluorescence from duplicate wells of a 96-well plate captured with an Odyssey infrared scanner. α-tubulin signal was used for normalization. <b>(G)</b> Graph represents the fold change in normalized signal intensity values respect to SCR cells. Data are shown as means ± SEM (** p<0.001; * p<0.05).</p

MyD88-knockdown cells showed a decreased tumor growth <i>in vivo</i>.

<p>Non-transduced B16 cells and B16 cells expressing SCR or MyD88-specific shRNAs were injected subcutaneously into C57BL/6 mice. Growth curves <b>(A)</b> and individual weights represented as scatter dot plot <b>(B)</b> of generated tumors. Results are shown as mean ± SEM (* p<0.01; n = 7 per group). <b>(C)</b> Representative photograph of the excised tumors. <b>(D)</b> Flow cytometric analysis of murine cell populations in B16-derived tumors obtained with the indicated shMyD88 and SCR cells. Representative dot plots showing percentages of vascular endothelial cells (CD31+) and myeloid subsets (within CD45+ gate). <b>(E)</b> Quantification of intra-tumor composition of indicated cell populations. Data presented as mean ± SEM (* p<0.01).</p

Downregulation of MyD88 alters the secretion of angiogenic mediators.

<p>Murine angiogenesis antibody arrays were probed with conditioned media collected from B16 cells. <b>(A)</b> Blot images from SCR and shMyD88 (shA and shB) cells. <b>(B)</b> Quantitative analysis of array data for down- and up-regulated proteins. Results shown are representative of two independent experiments <b>(C)</b> Representative photomicrographs of <i>in vitro</i> tube formation assay. Microvascular endothelial cells (HMEC-1) were seeded onto matrigel-coated slides and incubated with culture medium (media) and B16 conditioned media from the indicated cell lines <b>(D)</b> Quantitation of HMEC-1 branch points. Values are expressed as percentages ± SEM respect to those obtained with supernatants from SCR cells (* p<0.05).</p

Knockdown of MyD88 did not compromise B16 cell growth.

<p>Experiments were performed in B16 cells expressing either SCR or MyD88-specific shRNAs and incubated in the absence (mock) or presence of LPS (1 μg/mL) as indicated. <b>(A)</b> Cell proliferation evaluated in cells cultured at 10% serum for 48 h. The data are presented as percentage over SCR control cells ± SD. <b>(B)</b> Cell cycle distribution of B16 cells in exponential growth phase was analyzed by flow cytometry. Cell cycle profiles (top) and percentages of cells in G0/G1, S, and G2/M phases (bottom) are shown. <b>(C)</b> Cell viability of B16 cells exposed to doxorubicin (Doxo; 1 μM) or cisplatin (Cis-Pt; 20 μμg/ml) for 48 h was determined using an MTS assay. Survival of cells treated with vehicle (DMSO) was established as 100%.</p

Aging Impairs the Ability of Conventional Dendritic Cells to Cross-Prime CD8⁺ T Cells upon Stimulation with a TLR7 Ligand

<div><p>The aging process is accompanied by altered immune system functioning and an increased risk of infection. Dendritic cells (DCs) are antigen-presenting cells that play a key role in both adaptive and innate immunity, but how aging affects DCs and their influence on immunity has not been thoroughly established. Here we examined the function of conventional DCs (cDCs) in old mice after TLR7 stimulation, focusing on their ability to cross-prime CD8⁺ T cells. Using polyU, a synthetic ssRNA analog, as TLR7 ligand and OVA as an antigen (Ag) model, we found that cDCs from old mice have a poor ability to stimulate a CD8⁺ T cell-mediated cytotoxic response. cDCs from old mice exhibit alterations in Ag-processing machinery and TLR7 activation. Remarkably, CD8α⁺ cDCs from old mice have an impaired ability to activate naïve CD8⁺ T cells and, moreover, a lower capacity to mature and to process exogenous Ag. Taken together, our results suggest that immunosenescence impacts cDC function, affecting the activation of naïve CD8⁺ T cells and the generation of effector cytotoxic T cells.</p></div

Ag degradation in cDCs is affected by aging.

<p>Persistence of OVA protein in cell lysates of total (A) or CD8α⁺ (B) cDCs purified from young and old mice was determined by Western blot after 1 hour pulse loading with 0.625 mg/mL OVA plus 20 μg/mL polyU/DO (time 0, 0h) and 4h chase. Actin was used for loading control. The control line contains total cell lysates of untouched splenic DCs. Densitometric analysis of Western blots (right) is expressed as the ratio of integrated optical density (IOD) at time 0 relative to IOD at chase time. (C) Percentages of total live and dead cDCs after 1 hour pulse loading and 4h chase. Data represent the mean ± SEM of duplicate cultures and are representative of 2 independent experiments. ***p < 0.001.</p

Differential phosphorylation of IκB-α in cDCs from young and old mice after TLR7 stimulation.

<p>(A) Pooled splenic cDC isolated from 3 young or old mice were incubated with 20 μg/mL polyU/DO for the indicated time. Cells were then lysed, and the level of phosphorylation of IκB-α was evaluated by Western blot using an anti-pIκB-α antibody. β-actin levels were used for loading control. (B) IOD of phosphorylated IκB-α normalized to β-actin. Data represent mean ± SEM of 2 pooled independent experiments.</p

DC maturation is affected by aging.

<p>(A and B) Young and old mice were intravenously injected with 100 μg polyU/DO. Eighteen hours later, expression levels of CD86, CD40, MHC II (A) and PDL-1 (B) were determined in DC subsets by flow cytometry. Total (C) or CD8α⁺ (D) cDCs purified from the spleen of young and old mice were stimulated with 20 μg/mL polyU/DO and then supernatants were assayed for cytokine production by ELISA. (D) Spleen DC subsets from young and old mice were purified and total RNA of 1x10⁶ DCs was extracted. Relative mRNA levels for <i>Tlr7</i> were quantified by qPCR and normalized to <i>Hprt1</i>. Data show the mean ± SEM. Results are representative of 3 independent experiments (3–4 mice/age group/experiment). *p < 0.05, **p < 0.01, ***p < 0.001.</p

Aged splenic cDCs have impaired ability to cross-prime naïve CD8⁺ T cells <i>in vitro</i>.

<p>Total (A-D) or CD8α⁺ (E) cDCs purified from young and old mice were incubated with 1 mg/mL OVA mixed with 20 μg/mL polyU/DO for 90 minutes. Additional cDCs from young and old mice were incubated with RPMI or OVA as control. cDCs were then washed and cultured for 3 days with CFSE-labeled CD8β⁺ T cells isolated from the spleen of OT-I mice at different DC:T cell ratios. After culture, T cell proliferation and CD25 expression were analyzed by flow cytometry. (A) Representative histograms of T cell proliferation are shown from 1:1 ratio. (B, E) Percentages of proliferating T cells, (C) CD25 expression and (D) IFN-γ content in culture supernatants, determined by ELISA. Data show the mean ± SEM. *p < 0.05, **p < 0.01. Results are representative of 3 independent experiments (3–4 mice/age group/experiment).</p

Helminth Antigens Enable CpG-Activated Dendritic Cells to Inhibit the Symptoms of Collagen-induced Arthritis through Foxp3+ Regulatory T Cells

<div><p>Dendritic cells (DC) have the potential to control the outcome of autoimmunity by modulating the immune response. In this study, we tested the ability of <em>Fasciola hepatica</em> total extract (TE) to induce tolerogenic properties in CpG-ODN (CpG) maturated DC, to then evaluate the therapeutic potential of these cells to diminish the inflammatory response in collagen induced arthritis (CIA). DBA/1J mice were injected with TE plus CpG treated DC (T/C-DC) pulsed with bovine collagen II (CII) between two immunizations with CII and clinical scores CIA were determined. The levels of CII-specific IgG2 and IgG1 in sera, the histological analyses in the joints, the cytokine profile in the draining lymph node (DLN) cells and in the joints, and the number, and functionality of CD4+CD25+Foxp3+ T cells (Treg) were evaluated. Vaccination of mice with CII pulsed T/C-DC diminished the severity and incidence of CIA symptoms and the production of the inflammatory cytokine, while induced the production of anti-inflammatory cytokines. The therapeutic effect was mediated by Treg cells, since the adoptive transfer of CD4+CD25+ T cells, inhibited the inflammatory symptoms in CIA. The <em>in vitro</em> blockage of TGF-β in cultures of DLN cells plus CII pulsed T/C-DC inhibited the expansion of Treg cells. Vaccination with CII pulsed T/C-DC seems to be a very efficient approach to diminish exacerbated immune response in CIA, by inducing the development of Treg cells, and it is therefore an interesting candidate for a cell-based therapy for rheumatoid arthritis (RA).</p> </div