Only viable <i>B</i>. <i>abortus</i>, independently of its virulence factors, is able to inhibit MHC-I expression.

<p>(A-E, Panels i and iii) THP-1 cells were infected with <i>B</i>. <i>abortus</i> WT (A), <i>btpA</i> (B), <i>btpB</i> (C), <i>btpAbtpB</i> (D) and <i>Bpe159</i> (E) at different MOI in the presence of IFN-γ for 2 h, washed and cultured in the presence of IFN-γ for 48 h. (A-E, Panels ii and iv) At the same time, heat-killed (HK) bacteria were used to treat THP-1 cells in the presence of IFN-γ for 48 h. MHC-I expression was assessed by flow cytometry. Bars represent the arithmetic means ± SEM of five experiments. MFI, mean fluorescence intensity. **<i>P</i><0.01; ***<i>P</i><0.001 <i>vs</i>. IFN-γ-treated.</p

<i>B</i>. <i>abortus</i> RNA mimics MHC-I intracellular retention in Golgi apparatus mediated by <i>B</i>. <i>abortus</i> infection.

<p>(A) Confocal micrographs of THP-1 cells infected with <i>B</i>. <i>abortus</i> or treated with <i>B</i>. <i>abortus</i> RNA in the presence of IFN-γ for 48 h. MHC-I expression was determined with a primary anti-human MHC-I Ab (W6/32) and Alexa 546-labelled secondary Ab (red). (B) Quantification of MHC-I retention. Data are expressed as percentage of cells with MHC-I retained ± SEM of three independent experiments. The number of cells counted per experimental group was 200. (C) Confocal micrographs of THP-1 cells treated with <i>B</i>. <i>abortus</i> RNA in the presence of IFN-γ for 48 h. MHC-I expression was determined with a primary anti-human MHC-I Ab (W6/32) and Alexa 546-labelled secondary Ab (red). Subcellular localization markers were detected using mAbs specific for EEA1 (early endosomes), LAMP-2 (late endosomes/lysosomes), GM130 (Golgi) and calnexin (ER) followed by Alexa 488-labelled secondary Ab (green). White arrow shows co-localization (yellow staining). Results are representative of three independent experiments. (D) Quantification of co-localization of MHC-I with the subcellular compartments. Data are expressed as percentage of cells with MHC-I co-localized with indicated compartment ± SEM of three independent experiments. The number of cells counted per experimental group was 200. ***<i>P</i><0.001 <i>vs</i>. IFN-γ-treated; ^ΔΔΔ<i>P</i><0.001 <i>vs</i>. the other subcellular compartments.</p

Proposed model for the MHC-I surface down-regulation mechanism mediated by <i>B</i>. <i>abortus</i>.

<p>1. Infection of human monocytes/macrophages with <i>B</i>. <i>abortus</i> induces the release of its RNA and RNA degradation products into the <i>Brucella</i>-containing endosomes. 2. These molecules via TLR8 induce the secretion of EGF-like ligands such as EGF and TGF-α which bind ErbB receptors on the cell surface causing their activation. 3. These effects finally cause the retention of MHC-I molecules within the Golgi apparatus. 4. MHC-I molecules are therefore unable to reach the cell surface and present bacterial Ags to CD8⁺ T cells. 5. Inhibition of Ag presentation enables the bacteria to hide inside human monocytes/macrophages and avoid the cytotoxic CD8⁺ T cell responses.</p

<i>B</i>. <i>abortus</i> RNA degradation products are also able to retain MHC-I within the Golgi apparatus.

<p>(A) Confocal micrographs of THP-1 cells treated with <i>B</i>. <i>abortus</i> RNA or RNase I-treated <i>B</i>. <i>abortus</i> RNA in the presence of IFN-γ for 48 h. MHC-I expression was determined with a primary anti-human MHC-I Ab (W6/32) and Alexa 546-labelled secondary Ab (red). Golgi apparatus was detected using a mAb specific for GM130 followed by Alexa 488-labelled secondary Ab (green). White arrows show co-localization (yellow staining). Cells treated only with RNase I were used as negative controls. Results are representative of three independent experiments.</p

<i>B</i>. <i>abortus</i> RNA degradation products are also capable of inhibiting the IFN-γ-induced expression of MHC-I.

<p>(A) RNA from <i>B abortus</i> was purified and treated with DNase, Proteinase K (PK) or <i>E</i>. <i>coli</i> RNase I. Each treatment was visualized by 1% agarose gel electrophoresis. (B and C) THP-1 cells were stimulated with DNase (B) or PK (C)–treated <i>B</i>. <i>abortus</i> RNA in the presence of IFN-γ for 48 h. Cells treated with DNase or PK alone were used as negative controls. Cells treated with <i>B</i>. <i>abortus</i> RNA were used as positive controls. (D and E) THP-1 cells were treated with RNase I-treated <i>B</i>. <i>abortus</i> RNA in the presence of IFN-γ for 48 h. Cells treated only with RNase I were used as negative controls. Cells treated with <i>B</i>. <i>abortus</i> RNA were used as positive controls. MHC-I was assessed by flow cytometry. Bars represent the arithmetic means ± SEM of five experiments. MFI, mean fluorescence intensity. *<i>P</i><0.05; **<i>P</i><0.01; ***<i>P</i><0.001 <i>vs</i>. IFN-γ-treated. ^##<i>P</i><0.01; ^###<i>P</i><0.001 <i>vs</i>. negative controls.</p

<i>B</i>. <i>abortus</i> RNA-mediated MHC-I inhibition correlates with diminished Ag presentation to MHC-I-restricted CD8⁺ T cells.

<p>BMM from WT (A) and TLR7 KO (B) mice were treated with different doses of <i>B</i>. <i>abortus</i> RNA in the presence of mIFN-γ for 48 h. Then cells were washed and incubated with 20 ng/ml of 257–264 OVA peptide (SIINFEKL) for 20 min at 37°C. BMM from WT (A) and TLR7 KO (B) mice were washed and cultured for 0, 4, 6 and 18 h at 37°C with B3Z cells, a T cell hybridoma specific for OVA-K^b, which carries a β-galactosidase construct driven by NF-AT elements from the IL-2 promoter. T cell activation was measured using a colorimetric assay for LacZ activity with <i>o</i>-nitrophenyl-P-D-galactoside as a substrate. Background absorbance values obtained for BMM cultured in the absence of OVA were subtracted. ***<i>P</i><0.001 <i>vs</i>. mIFN-γ-treated.</p

<i>B</i>. <i>abortus</i> RNA is able to down-modulate MHC-I on primary cultures of monocytes/macrophages.

<p><b>(A and B)</b> Peripheral blood-isolated human monocytes (A) and murine bone marrow-derived macrophages (BMM) (B) were treated with different doses of <i>B</i>. <i>abortus</i> RNA. MHC-I expression was assessed by flow cytometry. Bars represent the arithmetic means ± SEM of five experiments. MFI, mean fluorescence intensity; mIFN-γ, murine IFN-γ. ***<i>P</i><0.001 <i>vs</i>. IFN-γ-treated.</p